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Global tea scienceCurrent status and future needs

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BURLEIGH DODDS SERIES IN AGRICULTURAL SCIENCE

NUMBER 41

Global tea scienceCurrent status and future needs

Edited by Dr V. S. Sharma, formerly of the UPASI Tea Research Institute, India and Dr M. T. Kumudini Gunasekare, formerly Tea Research Institute, Sri Lanka

Published by Burleigh Dodds Science Publishing Limited82 High Street, Sawston, Cambridge CB22 3HJ, UKwww.bdspublishing.com

Burleigh Dodds Science Publishing, 1518 Walnut Street, Suite 900, Philadelphia, PA 19102-3406, USA

First published 2018 by Burleigh Dodds Science Publishing Limited© Burleigh Dodds Science Publishing, 2018. All rights reserved.

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ISBN 978-1-78676-160-6 (print)ISBN 978-1-78676-162-0 (online)ISBN 978-1-78676-163-7 (online)ISSN 2059-6936 (print)ISSN 2059-6944 (online)

Typeset by Deanta Global Publishing Services, Chennai, IndiaPrinted by Lightning Source

© Burleigh Dodds Science Publishing Limited, 2018. All rights reserved.

ContentsSeries list xi

Acknowledgements xv

Introduction xvi

Part 1 Tea Breeding and Germplasm

1 Ensuring the genetic diversity of tea plants 3Jian-Qiang Ma and Liang Chen, Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRI, CAAS), China

1 Introduction 32 Origins and distribution of tea 43 Status of tea genetic resources 44 Germplasm evaluation and characterization 85 Exploitation and utilization of genetic diversity 106 Future trends and conclusion 137 Where to look for further information 148 References 15

2 Mapping and exploiting the tea genome 21Xinchao Wang, Xinyuan Hao, Lu Wang and Yajun Yang, Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRI, CAAS), China

1 Introduction 212 Progress in genetic linkage map construction and qualitative

trait locus (QTL) identification for the tea plant 223 The progress of functional genomics in exploiting genes

associated with desirable traits 244 Progress in ‘omics’ research: overview and secondary metabolites 255 Progress in ‘omics’ research: stress response and dormancy 276 Conclusion and outlook 297 Where to look for further information 308 Acknowledgements 319 References 31

3 Advances in genetic modification of tea 37Mainaak Mukhopadhyay, University of Kalyani, India; and Tapan Kumar Mondal, National Bureau of Plant Genetic Resources, India

1 Introduction 372 Conventional tea propagation 373 The need for genetic transformation 384 Transformation systems 395 Methods of transformation 406 Conclusion and future trends 477 Where to look for further information 488 References 48

vi Contents


Part 2 Cultivation and Agronomy

4 Planting and cultivation of tea 53M. A. Wijeratne, Tea Research Institute, Sri Lanka

1 Introduction 532 Climatic requirements of tea 543 New planting of tea 554 Preparation of the planting hole 595 Planting of tea 616 Aftercare field operations 617 Establishment of shade trees and wind belts 658 Pruning 679 Harvesting of tea 72

10 Replanting 7911 Soil rehabilitation 8012 Future trends and conclusion 8113 Where to look for further information 8114 References 82

5 The effect of cultivation techniques on tea quality 85P. Okinda Owuor, Maseno University, Kenya

1 Introduction 852 Chemical quality parameters of tea 863 Cultivars and black tea quality 884 Environmental factors 915 Altitude and temperatures 936 Agronomic inputs and tea quality 957 Conclusion 1028 Where to look for further information 1029 Acknowledgement 102

10 References 102

6 The role of arbuscular mycorrhizal fungi in tea cultivation 113Shipra Singh and Anita Pandey, G. B. Pant National Institute of Himalayan Environment and Sustainable Development, India; and Lok Man S. Palni, Graphic Era University, India

1 Introduction 1132 AMF, tea and the tea rhizosphere 1153 Development of AMF-based bioformulation for tea plantations 1204 Plant growth promotion following inoculation with AMF consortia 1255 AMF inoculation, tea growth and tea quality 1266 Conclusion and future perspectives 1287 Where to look for further information 1308 Acknowledgements 1309 References 130

7 The role of microbes in tea cultivation 135P. N. Bhattacharyya and S. R. Sarmah, Tocklai Tea Research Institute, India

1 Introduction 1352 Soil microbial inoculants as biofertilzers: an overview 137


Contents vii

3 Nitrogen-fixing microbial biofertilizers 1394 Phosphate-solubilizing, potash-solubilizing and cellulose-degrading

microbial biofertilizers 1415 Microbial management of pests and diseases in tea 1446 Important interactions and mechanisms of action in the microbial

management of disease 1477 Tea pest management: microbiological approach 1508 Selection and characterization of microbial products for commercialization 1539 Conclusions, future prospects and challenges 155

10 Acknowledgements 15711 References 157

Part 3 Plant Protection

8 Diseases affecting tea plants 171G. D. Sinniah, Tea Research Institute, Sri Lanka

1 Introduction 1712 Foliar diseases affecting tea 1723 Stem diseases affecting tea 1784 Root diseases affecting tea 1825 Development of resistance: resistance of fungi to fungicides

and tea plants to diseases 1866 Recent advances in the management of tea diseases 1877 Advances in the molecular biology of tea diseases 1898 Disease forecasting for tea 1929 Conclusion 192

10 Future research needs 19211 Where to look for further information 19312 References 194

9 Insect pests of tea: shot hole borers, termites and nematodes 201Nalini C. Gnanapragasam, Former Deputy Director (Research), Tea Research Institute, Sri Lanka; currently Agricultural Tea Consultant - Malwatte Valley Plantations PLC, Sri Lanka

1 Introduction 2012 Shot hole borers 2063 Termites of tea: general comments 2134 Live wood termites 2135 Scavenging termites 2196 Nematodes 2227 Where to look for further information 2318 Acknowledgements 2329 References 232

10 Insect pests of tea: caterpillars and other seasonal, occasional and minor pests 241Nalini C. Gnanapragasam, Former Deputy Director (Research), Tea Research Institute, Sri Lanka; currently Agricultural Tea Consultant - Malwatte Valley Plantations PLC, Sri Lanka

1 Introduction 2412 Caterpillars and other seasonal pests 242

viii Contents


3 Sucking pests 2604 Occasional and minor pests 2775 Conclusion 2896 Acknowledgements 2907 References 291

11 Integrated pest management of insect, nematode and mite pests of tea 301Nalini C. Gnanapragasam, Former Deputy Director (Research), Tea Research Institute, Sri Lanka; currently Agricultural Tea Consultant - Malwatte Valley Plantations PLC, Sri Lanka

1 Introduction 3012 Detection methods 3033 Mechanical control 3054 Cultural control: cultivars and planting other crops 3055 Cultural control: soil, bush sanitation, nutrient management and

escape strategy 308

6 Biological control: botanicals and semiochemicals 3107 Biological control: predators, bacteria and viruses 3138 Chemical control 3169 IPM programmes on selected perennial pests 325

10 Conclusion and future trends 32811 Acknowledgements 33012 References 330

12 Pesticide residues in tea: challenges in detection and control 347A. K. Barooah, Tocklai Tea Research Institute, India

1 Introduction 3472 Measuring pesticide residues in tea 3483 Review of recent research on the extent of pesticide residues in tea 3494 Conventional methods for detecting residues in tea 3525 Advanced methods for detecting residues in tea 3546 Food safety standards for tea and the challenges of maintaining

maximum residue limits (MRLs) 3577 Strategies for reducing pesticide residues in tea 3658 Conclusion and future trends 3669 References 367

Part 4 Tea Chemistry and Phytochemicals

13 Instrumentation and methodology for the quantification of phytochemicals in tea 375Ting Zhang, China University of Geosciences and Huanggang Normal University, China; Xiaojian Lv, Yin Xu, Lanying Xu and Tao Long, Huanggang Normal University, China; Chi-Tang Ho, Rutgers University, USA; and Shiming Li, Huanggang Normal University, China and Rutgers University, USA

1 Introduction 3752 Phytochemicals in tea: bioactive compounds 3823 Phytochemicals in tea: flavour and colour compounds 388


Contents ix

4 Analytical techniques for tea characterization: overview and chromatic techniques 390

5 Analytical techniques for tea characterization: spectroscopic techniques 3936 Determination of compounds in tea: phenolic compounds and sugars 3957 Determination of compounds in tea: analysis of volatile compounds 4008 Determination of compounds in tea: other compounds and elements 4049 Diversified tea products 407

10 Summary 40811 References 412

14 The potential role for tea in combating chronic diseases 427Chung S. Yang, Rutgers University, USA

1 Introduction 4272 Chemical properties, bioavailability and biotransformation of tea

constituents 4283 Tea and cancer prevention 4314 Reduction of body weight, alleviation of metabolic syndrome and

prevention of diabetes 4345 Lowering of blood cholesterol, blood pressure and incidence of

cardiovascular diseases 4386 Neuroprotective effects of tea 4397 Conclusion 4418 Where to look for further information 4439 Acknowledgements 443

10 References 443

Part 5 Sustainability

15 Tea cultivation under changing climatic conditions 455Wenyan Han, Xin Li, Peng Yan, Liping Zhang and Golam Jalal Ahammed, Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRI, CAAS), China

1 Introduction 4552 Climate change and climatic variability 4563 Effects of climate change on the suitability of tea planting areas and

plucking duration 4584 Effects of climate change on tea production 4595 Effects of climate change on tea quality 4636 Adaptation and mitigation strategies 4647 Conclusion 4698 Where to look for further information 4699 Acknowledgements 470

10 References 470

16 Assessing and reducing the environmental impact of tea cultivation 473Thushari Lakmini Wijeratne, Tea Research Institute, Sri Lanka

1 Introduction 4732 The environmental impact of tea cultivation 4743 Making tea cultivation more sustainable 476

x Contents


4 Case studies: carbon sequestration and production 4785 Summary and future trends 4806 Where to look for further information 4817 References 481

17 Cultivation, production and marketing of organic tea 485Nikhil Ghosh Hajra, Organic Tea and Agri-horticultural Consulting, India

1 Introduction 4852 Establishing and maintaining a new organic tea plantation 4863 Maintenance of new and converted organic plantations 4884 Post-harvest and manufacturing practices 5035 Inspection and certification of organic tea 5046 Future prospects for organic tea cultivation 5057 Organic tea yield trends 5068 Major producing countries of organic tea 5079 Major markets for organic tea 510

10 Future trends and conclusion 51511 Where to look for further information 51512 Acknowledgements 51613 References 516

18 Supporting smallholders in tea cultivation 521Atik Dharmadi, Research Institute for Tea and Cinchona, Indonesia

1 Introduction 5212 Smallholders and their role in tea cultivation 5213 Problems facing smallholders 5224 Disseminating good agricultural practices and improving

market knowledge 5235 Organizing smallholders to improve their position in the market 5246 Case studies: Kenya and Sri Lanka 5277 Conclusions 5288 References 529

Index 531


Series listTitle Series number

Achieving sustainable cultivation of maize - Vol 1 001From improved varieties to local applications Edited by: Dr Dave Watson, CGIAR Maize Research Program Manager, CIMMYT, Mexico

Achieving sustainable cultivation of maize - Vol 2 002Cultivation techniques, pest and disease control Edited by: Dr Dave Watson, CGIAR Maize Research Program Manager, CIMMYT, Mexico

Achieving sustainable cultivation of rice - Vol 1 003Breeding for higher yield and quality Edited by: Prof. Takuji Sasaki, Tokyo University of Agriculture, Japan

Achieving sustainable cultivation of rice - Vol 2 004Cultivation, pest and disease managementEdited by: Prof. Takuji Sasaki, Tokyo University of Agriculture, Japan

Achieving sustainable cultivation of wheat - Vol 1 005Breeding, quality traits, pests and diseasesEdited by: Prof. Peter Langridge, The University of Adelaide, Australia

Achieving sustainable cultivation of wheat - Vol 2 006Cultivation techniquesEdited by: Prof. Peter Langridge, The University of Adelaide, Australia

Achieving sustainable cultivation of tomatoes 007Edited by: Dr Autar Mattoo, USDA-ARS, USA & Prof. Avtar Handa, Purdue University, USA

Achieving sustainable production of milk - Vol 1 008Milk composition, genetics and breedingEdited by: Dr Nico van Belzen, International Dairy Federation (IDF), Belgium

Achieving sustainable production of milk - Vol 2 009Safety, quality and sustainabilityEdited by: Dr Nico van Belzen, International Dairy Federation (IDF), Belgium

Achieving sustainable production of milk - Vol 3 010Dairy herd management and welfareEdited by: Prof. John Webster, University of Bristol, UK

Ensuring safety and quality in the production of beef - Vol 1 011SafetyEdited by: Prof. Gary Acuff, Texas A&M University, USA & Prof.James Dickson, Iowa State University, USA

Ensuring safety and quality in the production of beef - Vol 2 012QualityEdited by: Prof. Michael Dikeman, Kansas State University, USA

Achieving sustainable production of poultry meat - Vol 1 013Safety, quality and sustainabilityEdited by: Prof. Steven C. Ricke, University of Arkansas, USA

Achieving sustainable production of poultry meat - Vol 2 014Breeding and nutritionEdited by: Prof. Todd Applegate, University of Georgia, USA

Achieving sustainable production of poultry meat - Vol 3 015Health and welfareEdited by: Prof. Todd Applegate, University of Georgia, USA

xii Series list


Achieving sustainable production of eggs - Vol 1 016Safety and qualityEdited by: Prof. Julie Roberts, University of New England, Australia

Achieving sustainable production of eggs - Vol 2 017Animal welfare and sustainabilityEdited by: Prof. Julie Roberts, University of New England, Australia

Achieving sustainable cultivation of apples 018Edited by: Dr Kate Evans, Washington State University, USA

Integrated disease management of wheat and barley 019Edited by: Prof. Richard Oliver, Curtin University, Australia

Achieving sustainable cultivation of cassava - Vol 1 020Cultivation techniquesEdited by: Dr Clair Hershey, formerly International Center for Tropical Agriculture (CIAT), Colombia

Achieving sustainable cultivation of cassava - Vol 2 021Genetics, breeding, pests and diseasesEdited by: Dr Clair Hershey, formerly International Center for Tropical Agriculture (CIAT), Colombia

Achieving sustainable production of sheep 022Edited by: Prof. Johan Greyling, University of the Free State, South Africa

Achieving sustainable production of pig meat - Vol 1 023Safety, quality and sustainabilityEdited by: Prof. Alan Mathew, Purdue University, USA

Achieving sustainable production of pig meat - Vol 2 024Animal breeding and nutritionEdited by: Prof. Julian Wiseman, University of Nottingham, UK

Achieving sustainable production of pig meat - Vol 3 025Animal health and welfareEdited by: Prof. Julian Wiseman, University of Nottingham, UK

Achieving sustainable cultivation of potatoes - Vol 1 026Breeding, nutritional and sensory qualityEdited by: Prof. Gefu Wang-Pruski, Dalhousie University, Canada

Achieving sustainable cultivation of oil palm - Vol 1 027Introduction, breeding and cultivation techniquesEdited by: Prof. Alain Rival, Center for International Cooperation in Agricultural Research for Development (CIRAD), France

Achieving sustainable cultivation of oil palm - Vol 2 028Diseases, pests, quality and sustainabilityEdited by: Prof. Alain Rival, Center for International Cooperation in Agricultural Research for Development (CIRAD), France

Achieving sustainable cultivation of soybeans - Vol 1 029Breeding and cultivation techniquesEdited by: Prof. Henry Nguyen, University of Missouri, USA

Achieving sustainable cultivation of soybeans - Vol 2 030Diseases, pests, food and non-food usesEdited by: Prof. Henry Nguyen, University of Missouri, USA

Achieving sustainable cultivation of sorghum - Vol 1 031Genetics, breeding and production techniquesEdited by: Prof. Bill Rooney, Texas A&M University, USA


Series list xiii

Achieving sustainable cultivation of sorghum - Vol 2 032Sorghum utilisation around the worldEdited by: Prof. Bill Rooney, Texas A&M University, USA

Achieving sustainable cultivation of potatoes - Vol 2 033Production and storage, crop protection and sustainabilityEdited by: Dr Stuart Wale, Potato Dynamics Ltd, UK

Achieving sustainable cultivation of mangoes 034Edited by: Professor Víctor Galán Saúco, Instituto Canario de Investigaciones Agrarias (ICIA), Spain & Dr Ping Lu, Charles Darwin University, Australia

Achieving sustainable cultivation of grain legumes - Vol 1 035Advances in breeding and cultivation techniquesEdited by: Dr Shoba Sivasankar et al., CGIAR Research Program on Grain Legumes, ICRISAT, India

Achieving sustainable cultivation of grain legumes - Vol 2 036Improving cultivation of particular grain legumesEdited by: Dr Shoba Sivasankar et al., CGIAR Research Program on Grain Legumes, ICRISAT, India

Achieving sustainable cultivation of sugarcane - Vol 1 037Cultivation techniques, quality and sustainabilityEdited by: Prof. Philippe Rott, University of Florida, USA

Achieving sustainable cultivation of sugarcane - Vol 2 038Breeding, pests and diseasesEdited by: Prof. Philippe Rott, University of Florida, USA

Achieving sustainable cultivation of coffee 039Edited by: Dr Philippe Lashermes, Institut de Recherche pour le Développement (IRD), France

Achieving sustainable cultivation of bananas - Vol 1 040Cultivation techniquesEdited by: Prof. Gert Kema, Wageningen University, The Netherlands & Prof. André Drenth, University of Queensland, Australia

Global Tea Science 041Current status and future needsEdited by: Dr V. S. Sharma, Formerly UPASI Tea Research Institute, India & Dr M. T. Kumudini Gunasekare, Coordinating Secretariat for Science Technology and Innovation (COSTI), Sri Lanka

Integrated weed management 042Edited by: Emeritus Prof. Rob Zimdahl, Colorado State University, USA

Achieving sustainable cultivation of cocoa - Vol 1 043Genetics, breeding, cultivation and qualityEdited by: Prof. Pathmanathan Umaharan, Cocoa Research Centre – The University of the West Indies, Trinidad and Tobago

Achieving sustainable cultivation of cocoa - Vol 2 044Diseases, pests and sustainabilityEdited by: Prof. Pathmanathan Umaharan, Cocoa Research Centre – The University of the West Indies, Trinidad and Tobago

Water management for sustainable agriculture 045Edited by: Prof. Theib Oweis, Formerly ICARDA, Lebanon

Improving organic animal farming 046Edited by: Dr Mette Vaarst, Aarhus University, Denmark & Dr Stephen Roderick, Duchy College, Cornwall, UK

Improving organic crop cultivation 047Edited by: Prof. Ulrich Köpke, University of Bonn, Germany

xiv Series list


Managing soil health for sustainable agriculture - Vol 1 048FundamentalsEdited by: Dr Don Reicosky, USDA-ARS, USA

Managing soil health for sustainable agriculture - Vol 2 049Monitoring and managementEdited by: Dr Don Reicosky, USDA-ARS, USA

Rice insect pests and their management 050E. A. Heinrichs, Francis E. Nwilene, Michael J. Stout, Buyung A. R. Hadi & Thais Freitas

Improving grassland and pasture management in temperate agriculture 051Edited by: Prof. Athole Marshall & Dr Rosemary Collins, University of Aberystwyth, UK

Precision agriculture for sustainability 052Edited by: Dr John Stafford, Silsoe Solutions, UK

Achieving sustainable cultivation of temperate zone tree fruit and berries – Vol 1 053Physiology, genetics and cultivationEdited by: Prof. Gregory Lang, Michigan State University, USA

Achieving sustainable cultivation of temperate zone tree fruit and berries – Vol 2 054Case studiesEdited by: Prof. Gregory Lang, Michigan State University, USA

Agroforestry for sustainable agriculture 055Edited by: Prof. María Mosquera-Losada, University of Santiago de Compostela, Spain & Dr Ravi Prabhu, World Agroforestry Centre (ICRAF), Kenya

Achieving sustainable cultivation of tree nuts 056Edited by: Prof. Ümit Serdar, Ondokuz Mayis University, Turkey & Emeritus Prof. Dennis Fulbright, Michigan State University, USA

Assessing the environmental impact of sustainable agriculture 057Edited by: Prof. Bo P. Weidema, Aalborg University/2.-0 LCA Consultants, Denmark

Critical issues in plant health: 50 years of research in African agriculture 058Edited by: Dr. Peter Neuenschwander, IITA & Dr. Manuele Tamò, IITA

Achieving sustainable cultivation of vegetables – Vol 1 059Physiology, breeding, cultivation and qualityEdited by: Emeritus Prof. George Hochmuth, University of Florida, USA

Achieving sustainable cultivation of vegetables – Vol 2 060Case studiesEdited by: Emeritus Prof. George Hochmuth, University of Florida, USA


AcknowledgementsWe wish to acknowledge the following for their help in reviewing particular chapters:

• Chapter 16: Dr S. Marimuthu, General Manager, R & D, Parry-Agro Industries Ltd.


IntroductionTea is the most widely-consumed beverage in the world. Like other crops, tea cultivation faces a number of challenges. With the challenge of climate change and the competition for scarce resources, there is a need to make tea cultivation more efficient and sustainable. Cultivation of tea also needs to be more resilient to biotic and abiotic stresses, whether it be pests or more extreme weather (e.g. drought) associated with global warming.

Fortunately, there is a range of research addressing these challenges. Drawing on international expertise, this volume summarises global tea science by focusing on ways of improving the cultivation of tea at each step in the value chain, from breeding through to harvest. The volume emphasises the importance of interdisciplinary and collaborative research and summarises the key research trends in each area, putting them in the context of tea cultivation as a whole. It reviews the latest advances in understanding tea genetics and genetic diversity and how this has informed advances in conventional, marker-assisted and transgenic breeding techniques. Likewise, the volume summarises current best practices in cultivation techniques and control of pests and diseases, focusing on assessment of the environmental impact of tea cultivation.

Part 1 Tea Breeding and Germplasm

Part 1 reviews advances in tea breeding and issues concerning tea germplasm. The focus of Chapter 1 is on ensuring the genetic diversity of tea. Prolonged cross-pollination within and between populations of tea plants and related species in the ‘wild’ have produced considerable heritable variation, resulting in a high level of genetic diversity. A good understanding and management of this pool of genetic resource diversity is of vital importance to tea plant improvement, since it directly affects the potential for genetic gain through selection. The chapter provides an overview of the genetic diversity of the tea plant and its characterization and utilization. The chapter examines the origin and global distribution of tea cultivars, assessing the current status of tea genetic resources. The chapter explains the processes of tea germplasm evaluation and characterization and examines the exploitation and utilization of genetic diversity.

Developing the themes of Chapter 1, the focus of Chapter 2 is on mapping and exploiting the tea genome. As a dicotyledonous, perennial, evergreen and cross-pollinated woody plant, tea plant possesses a complex genetic background and high heterozygosity. Most of the genetic regulation information related to important traits is still unclear and many bottlenecks are hindering the mapping and exploiting of the tea genome. The chapter reviews progress in the construction of genetic linkage maps and the identification of qualitative trait loci (QTL) for the tea plant, as well as assessing the progress of functional genomics in exploiting genes associated with desirable traits. The chapter discusses the progress, challenges and potential advances in ‘omics’ for the tea plant.

Following on from Chapter 2’s emphasis on mapping the tea genome, Chapter 3 moves on to address advances in genetic modification of tea. Due to its botanical characteristics, genetic improvement of tea is slow. Its high gestation period, the difficulty of producing homozygous lines, and the non-availability of mutant genotypes and a mapping population are all hindrances to development. The chapter describes and evaluates the potential of genetic transformation as an alternative for varietal improvement of tea, via


Introduction xvii

the deployment of agrobacterium and particle bombardment. The chapter describes in detail progress global progress on research into transgenic tea.

Part 2 Cultivation and Agronomy

The second part of the volume discusses agronomics of tea plant and improvements in tea cultivation techniques. Chapter 4 examines the planting and cultivation of tea. Originating in natural forests characterized by warm and humid environmental conditions and nutrient rich soils, tea’s growth and yield largely depend on climatic and soil factors. Frequent removal of photosynthetically-active shoots (harvesting), and periodic removal of leaf-bearing branches (pruning), exert physiological stress on the bush, and it is crucial for sustainable productivity and profitability that the tea bush is provided with optimum conditions for normal growth. The chapter examines in detail the process of new planting, soil rehabilitation and re-planting, aftercare, field operations such as pruning, establishing shade trees and wind belts and harvesting. The chapter looks ahead to future trends, challenges and potential developments in this area.

Moving from Chapter 4’s overview of tea planting, Chapter 5 focuses on the contribution of agronomic cultivation techniques to improving tea quality. The profitability of tea production depends on whether the type of tea produced has the right quality that is acceptable to consumers. This chapter examines the environmental and agronomic factors underlying tea quality, addressing the chemical quality parameters of tea, the relationship between black tea quality and specific cultivars, the effect of environmental factors such as altitude and temperatures and the relationship between tea quality and agronomic inputs.

Chapter 6 examines the potential role of arbuscular mycorrhizal fungi (AMF) in tea cultivation. Continuous application of chemical fertilizers in tea gardens may increase tea production, but it adversely affects the quality of tea soils. There is therefore growing interest in rhizosphere associates of tea, including symbionts such as arbuscular mycorrhizal fungi (AMF). These colonize tea roots and support both plant growth and improvement of soil health. The chapter reviews the use of AMF-based bio-inoculants in tea cultivation, examining the range of AMF associated with tea and their effects on tea rhizosphere. The chapter considers the development of an AMF-based bioformulation for use in tea plantations and reviews the effects of using such a bioformulation on both tea plant growth and tea quality.

Chapter 7 focuses on the role of tea soil microflora in enhancing tea cultivation. Tea crops can suffer from nutrient deficiencies, attack by diverse pests and pathogens, and climatic stresses, which result in considerable crop losses. However, the application of synthetic chemicals to alleviate crop loss has had a detrimental impact on the tea ecosystem. Plant growth-promoting microorganisms (PGPMs) play an essential role in the maintenance of sustainable tea cultivation and ecosystem restoration, thereby promoting primary productivity and inducing systemic resistance of plants to diverse pests and diseases. The chapter summarizes and discusses recent progress regarding the understanding of tea soil microflora and its significance to tea plantations. It provides an overview of soil microbial inoculants as biofertilizers, as well as describing nitrogen-fixing, phosphate-solubilizing, potash-solubilizing and cellulose-degrading microbial biofertilizers. The chapter concludes that selection of microbial bioagents might form a reliable component in the management of significant tea diseases in order to achieve sustainable tea production.

xviii Introduction


Part 3 Plant Protection

The focus of the third part of the volume is on the protection of tea plants. Chapter 8 reviews diseases affecting tea plants. These include foliar diseases, stem diseases and root diseases. The chapter examines developments in disease resistance, including resistance of fungi to fungicides and the creation of disease-resistant tea plants. The chapter reviews recent advances in the management of tea diseases and advances in the molecular biology of tea diseases that may assist in increasing resistance.

Chapter 9 examines the impact of insect pests of tea. The tea plant is a perennial crop and every part of the plant is prone to infestation by some pests over its lifetime. The prevalence and occurrence of a pest is primarily determined by the specific agro-climatic conditions, the type of cultivar and the cultural practices adopted within a given specific location. The chapter describes the biology and ecology of important pests attacking tea in different tea growing areas of the world and the type of damage/injury induced, focusing on shot-hole borers, termites (both live wood and scavenging varieties) and nematodes.

Chapter 10 continues the focus on insect pests of tea, this time considering the impact of caterpillars and other seasonal pests, as well as sucking pests and occasional and minor pests. This class of pests causes damage to tea plants largely through feeding. The chapter considers a variety of factors associated with each of these pests, including their geographical distribution, appearance, the damage caused and their respective biology and ecology. The chapter looks ahead to future research into these pests, including understanding their behavior and habitats, their sensitivity to temperature, humidity and climate change in general.

Bringing together the themes of Chapters 9 and 10, the Chapter 11 considers the challenge of integrated pest management (IPM) of tea insect pests. The chapter describes the various strategies that are being used in different countries to manage pests of tea using integrated pest management programme (IPM) to ensure they do not reach economic injury levels. The chapter explores pest detection methods as well as methods of mechanical, biological, cultural and chemical control of insect and nematode pests of tea, and includes a number of detailed case studies describing the application of these methods in IPM.

Chapter 12 moves on to the problem of pesticides, addressing the measurement and reduction of pesticide residues in tea. Tea growers require pesticides to prevent crop loss due to pest attacks, which are aggravated by climate change. Since pesticides invariably leave residues, it is of the utmost importance that samples of traded tea are monitored to ensure compliance with food safety standards. The chapter reviews research into the extent of pesticide residues in tea, conventional methods for determining trace levels of multiple residues in tea and the problems with these methods, as well as advanced, rapid methods which are more suitable for ensuring food safety. The chapter also considers food safety standards in the EU and Japan, the challenge of maintaining maximum residue limits (MRLs) and methods of assessing the risk posed by pesticide residues, and strategies for reducing the residues in tea.

Part 4 Tea Chemistry and Phytochemicals

The focus of the fourth part of the volume is on the chemistry of tea and the role of phytochemicals. Chapter 13 examines qualitative and quantitative analysis of the


Introduction xix

phytochemical composition of tea. Tea contains many phytochemicals that demonstrate important physiological properties and health promoting benefits, such as polyphenols, amino acids, vitamins, carbohydrates, and purine alkaloids. Tea components are closely associated with tea variety, the growing conditions and regions of tea plants, and the plucking and processing of tea leaves. The chapter reviews the main chemical components in tea and the instrumental techniques to identify them. The chapter describes phytochemical bioactive compounds as well as flavour and colour compounds, before going on to consider analytical techniques for tea characterization, including chromatic, spectroscopic techniques. The chapter then examines the determination of phenolic compounds and sugars, volatile compounds and other compounds and elements.

Chapter 14 moves from the chemical analysis of compounds in tea to consider the potential beneficial effects of these compounds, specifically the role of tea in combating chronic diseases. The chapter considers the chemical properties, bioavailability and biotransformation of the constituent elements of tea, and assesses the connection between tea consumption and cancer prevention. The chapter considers the impact of tea on reduction of body weight, leading to alleviation of metabolic syndrome and preventing diabetes. It also considers claims that tea can lower blood cholesterol, blood pressure and incidence of cardiovascular diseases. Finally, the chapter considers potential neuroprotective effects of tea.

Part 5 Sustainability

The fifth part of the volume considers the challenge of making tea production sustainable. Chapter 15 considers the relationship between climate change and tea cultivation. Predicted climate change is likely to pose a major threat to normal tea cultivation. This chapter reviews the effects that climate change is likely to have on regions suitable for tea production and the duration of the plucking period. The chapter considers how tea production might be improved by temperature increases and CO2 elevation; it also discusses the negative impact of heavy rains, frosts, proliferation of pests and diseases and soil degradation. The chapter concludes that tea quality is likely to deteriorate due to the imbalance in the ratio of free amino acids to polyphenols. Appropriate planning for adaptation and mitigation needs to be developed and extended for sustainable development of the tea industry. The adaptation and mitigation strategies should operate at three levels: government policy, research and development for new technologies and techniques and community involvement and technology extension.

Complementing the preceding chapter’s focus on climate change and its impact on tea quality, Chapter 16 assesses the environmental impact of tea cultivation itself and prospects for reducing these impacts. Owing to its popularity, tea has become an important plantation crop in many countries. As a perennial crop occupying a large proportion of arable land, assessing its environmental impact would benefit the economy of tea growing countries immensely. This chapter reviews the impact of the tea industry on the environment and human activity. It covers life cycle assessment methodologies tailored to tea production, covering cultivation to final waste disposal, tea’s carbon footprint as well as other on and off-farm impacts caused by the tea industry. Further possible measures to minimize these impacts are also discussed. Two detailed case studies address the CO2 sink/source nature of tea plantations as the cultivation stage is one of the most significant contributors to the carbon footprint of tea.

xx Introduction


Chapter 17 moves on to consider the cultivation and marketing of organic tea. The chapter provides an overview of the development of organic and biodynamic tea production in different producing countries, exploring cultivation practices, the global market for and trade in organic tea, and research priorities. The chapter assesses the pattern of yield trend after conversion from conventional to organic production, providing a discussion of the development of target markets for organic tea, distribution channels and the volume of organic tea traded in the world market. The chapter examines the challenges of establishing and maintaining a new organic tea plantation, as well as the maintenance of new and converted organic plantations. It addresses post-harvest and manufacturing practices, inspection and certification of organic tea and the future prospects for organic tea cultivation. The chapter provides an overview of the major producing countries of organic tea and the major markets for this product.

Continuing the theme of sustainable forms of tea cultivation, the final chapter in the book, Chapter 18, considers the importance of supporting smallholders producing tea. The chapter describes how smallholder organization can be strengthened to support tea cultivation. Smallholders are weak in terms of productivity owing to low yields and lack of working capital in comparison with large state-owned and private plantations. The chapter describes how the transition from a smallholders’ group to a smallholder-owned company can be managed, and reviews existing literature on smallholder development and lessons that can be learned in this area. Finally, it examines the ways in which smallholder-owned companies compete and form partnerships.

Part 1

Tea Breeding and Germplasm

http://dx.doi.org/10.19103/AS.2017.0036.02© Burleigh Dodds Science Publishing Limited, 2018. All rights reserved.

Chapter 1

Ensuring the genetic diversity of tea plantsJian-Qiang Ma and Liang Chen, Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRI, CAAS), China

1 Introduction

2 Origins and distribution of tea

3 Status of tea genetic resources

4 Germplasm evaluation and characterization

5 Exploitation and utilization of genetic diversity

6 Future trends and conclusion

7 Where to look for further information

8 References

1 Introduction

Tea plant (Camellia sinensis (L.) O. Kuntze) is an evergreen, perennial and woody species, whose tender buds and leaves are used to prepare the beverage known worldwide as tea (Wight and Baruwa 1957). Tea is an important source of micronutrients for the daily diet in many countries. Recent studies have shown that tea’s bioactive components can provide desirable health benefits (Hayat et al. 2015) and have a protective effect against cancer, obesity and cardiovascular diseases (Khan and Mukhtar 2013). The consumption of products containing tea constituents has been increasing in recent years. Global production of tea increased from 3.06 million metric tons in 2001 to 5.29 million metric tons in 2015 (ITC 2016).

The tea plant belongs to the section Thea (L.) Dyer of the genus Camellia L. in the family Theaceae. Taxonomic systems for the section Thea established by individual scientists differ in that they consist of different numbers of species and varieties (Sealy 1958; Wight 1959; Chang 1984; Min 1992; Chen et al. 2000). It is beyond debate, however, that the major cultivated tea plant comprises two varieties: C. sinensis var. sinensis (the small-leaf variety) and C. sinensis var. assamica (Masters) Kitamura (the large-leaf variety). The variety of C. sinensis var. pubilimba Chang is also grown commercially in some regions of China and South Asia. In addition, a few wild relatives, such as C. taliensis (W. W. Smith) Melchior, C. tachangensis F. C. Zhang, C. crassicolumna Chang and C. gymnogyna Chang, are sometimes used locally.

Ensuring the genetic diversity of tea plants4


The tea plant shows strong self-incompatibility. Recent evidence suggests it is characterized by late-acting self-incompatibility, which might be under gametophytic control (Zhang et al. 2016). Long-term cross-pollination has produced great heritable variation within and between populations of the tea plant and its related species, resulting in a high level of genetic diversity. A good understanding and management of this pool of genetic diversity is of vital importance for the genetic improvement of tea plant, since it directly affects the potential for genetic gain through selection. This chapter is intended to provide an overview of the genetic diversity of the tea plant, and its characterization, exploitation and utilization.


The tea plant is usually considered to have originated in Southwest China, where it was first discovered and used as a medicinal drink as early as 2737 BC (Lu 1974; Chang 1981), although some believe its place of origin to have been regions of South China, Assam, northern Myanmar and Indochina (Kingdon-Ward 1950; Sealy 1958; Wight 1959; Yamamoto et al. 1997). About 80% of the species of the section Thea, including the tea plant and its wild relatives, have been found in China, according to Chang’s taxonomic system (Chang 1981; Yu 1986). Taking into consideration the quantity, distribution and morphological characteristics of wild germplasm and related species, Yu (1986) postulated the Yunnan province of China as a likely centre of origin of the tea plant similar to Min’s (1992) conclusion.

The first record of tea cultivation dates from the first century BC, when the tea plant was grown in the Sichuan province of China. The plant was later introduced to Japan from the Zhejiang province of China in the early ninth century. In India, the tea plant was first cultivated in 1834 since when seeds were brought from China, although the indigenous C. sinensis var. assamica was discovered in 1823. Tea cultivation and processing technology extended to Brazil from China in 1812. The tea plant was first introduced to Sri Lanka in 1824 by raising seeds brought from India. Tea cultivation in Transcaucasia started in 1883 using seeds from the Hubei province of China, and after that, the tea plant first reached Turkey in 1924. In Europe, successful cultivation of the tea plant began in Britain during 1768, from where it was introduced to Africa at the end of the nineteenth century. To date, there are more than 50 tea-producing countries, within the latitudinal range of 45°N to 34°S (Mondal 2014).


Genetic resources are reservoirs of genes and genotypes, which play an important role in promoting and sustaining agriculture. The collection and conservation of the cultivars, landraces and wild relatives of the tea plant will provide breeders and scientists with fundamental materials from which new cultivars are to be developed. To maintain a broad genetic base, most tea-producing countries have established a tea germplasm conservation system. There are roughly 20 000 accessions of tea germplasm preserved in the major tea-producing countries, including China, India, Japan, Sri Lanka, Kenya, Korea, Vietnam, Indonesia, Bangladesh and Turkey, which represent the majority of the world’s


Ensuring the genetic diversity of tea plants 5

tea plant genetic resources. In the following sections, the current status of tea germplasm collections in these countries will be described in more detail.

3.1 ChinaThe tea plant is native to China, which has the most abundant and diverse tea genetic resources in the world. In modern China, a project of targeted investigation and collection started in the 1980s after the tea plant was listed in the national plans for crop germplasm investigation and collection (Yao and Chen 2012). Permanent preservation centres – the China National Germplasm Tea Repository (CNGTR) and Menghai Branch – were established for ex situ conservation of tea germplasm in 1990. To date, the CNGTR has preserved about 3000 accessions, among which 2800 have been identified according to Chen’s taxonomic system (Chen et al. 2000). Most of the cultivated tea accessions belong to the three varieties, C. sinensis var. sinensis, var. assamica and var. pubilimba, while the majority of wild relatives are C. taliensis, C. tachangensis, C. crassicolumna and C. gymnogyna. Of the 3000 accessions, 11% are wild relatives, 60% landraces, 6% cultivars and 23% genetic materials. A core collection has been developed to improve germplasm utilization at the CNGTR (Liu 2008). Over the past decades, local government has increasingly emphasized in situ conservation. Several projects to protect famous landraces and wild relatives are underway. Additionally, techniques such as tissue culture and seed cryopreservation have been studied for in vivo conservation of tea germplasm, which has a great potential for the conservation of tea genetic resources (Wang et al. 1990, 1999).

3.2 IndiaThe first commercial tea garden in India was established using seeds originating from China in 1834 (Das et al. 2012). Subsequently, seeds were introduced from Burma, Cambodia, Vietnam and Japan, which resulted in the development of superior planting materials (Paul et al. 1997). In order to develop an Indian national tea plant gene bank, regional germplasm collection centres in at least three different geographical locations have been established: the Tea Research Association, Tocklai Experimental Station (TRA, TES) located in northeastern India, the United Planters’ Association of Southern India (UPASI), and the Council of Scientific and Industrial Research, Institute of Himalayan Bioresource Technology (CSIR-IHBT) located in northwestern India. It is estimated that more than 2100 and 1250 accessions are maintained at TES and UPASI, respectively (Das et al. 2012). The conserved germplasm has great morphological and genetic diversity, comprising diverse genotypes of ancient seedling populations and wild relatives. Many improved cultivars and seed varieties have been developed using these genetic resources.

3.3 JapanThe first record of the introduction of the tea plant to Japan dates to the ninth century, when two Japanese monks introduced seeds from China (Tanaka 2012). There is thus a long history of tea germplasm collection and utilization in Japan. Tea genetic resources in Japan are now being maintained as clone bushes within several national and regional research institutes. The NARO Institute of Fruit Tree and Tea Science (NIFTS) has constructed a global tea germplasm collection including more than 7800 accessions derived from fourteen countries over the past century, from which a core collection has been



generated (Taniguchi et al. 2014). The collection has been evaluated and characterized morphologically, chemically and molecularly, and indicates plentiful genetic diversity.

3.4 Sri LankaThe tea plant is an introduced crop in Sri Lanka, where no wild relatives or landraces can be found. During the early years of tea cultivation, seeds were mainly imported from India and China (Gunasekare 2012). After multiple generational hybridization of the seedlings, the offspring populations were of high heterozygosis with broad genetic variation. The collection and ex situ conservation of tea germplasm were initiated locally in 1986. At present, about 600 accessions are maintained in a field gene bank at the Tea Research Institute of Sri Lanka (Gunasekare et al. 2012). These genetic resources can be categorized into two groups, that is, beverage and non-beverage type. The beverage group consists of exotic and native improved cultivars, and breeding materials selected from old seedling populations. Some unique germplasm has been identified among these accessions, such as natural triploid cultivars ‘HS 10A’ and ‘GF 5’, and non-fermenting cultivar ‘TRI 9’. The non-beverage group contains related species such as C. sasanqua, C. japonica and C. rosaeflora, which may be utilized in interspecific hybridization programmes.

3.5 KenyaThe tea plant was reportedly first introduced into Kenya in 1904, when seeds of C. sinensis var. assamica from India were used to establish a plantation (Kamunya et al. 2012). In 1912, C. sinensis var. sinensis seeds were imported from Sri Lanka. The aforesaid pioneer seedlings were considered the main components of the country’s tea genetic resources. Studies have revealed, however, that Kenya’s tea genetic resources are in fact diverse (Wachira et al. 1995; Paul et al. 1997). Exotic tea germplasm collected from across the globe has extended the local gene pool. Tea germplasm collections in Kenya, for instance, contain seeds from popular Japanese cultivars such as ‘Yabukita’ and ‘Yutakamidori’. Additionally, several Camellia species, such as C. japonica, C. brevistyla, C. sasanqua and C. irrawadiensis, were also imported to access diversity from the secondary and tertiary gene pools of the tea plant. Currently, the Camellia Gene Bank of Tea Research Institute of Kenya conserves more than 250 accessions of the tea plant and related species (Kamunya et al. 2012).

3.6 KoreaThe tea plant was introduced to Korea from China more than two thousand years ago (Jeong and Park 2012). Seed propagation is the most frequent tea cultivation method in Korea. Currently, 80% of Korean tea plantations are still native seedling populations, which consist of genotypes with a broad range of phenotypic variation. Between 1994 and 1998, a total of 2300 tea germplasm samples were collected and preserved at the Boseong Tea Experiment Station (Jeong and Park 2012). The characteristics of this germplasm have been investigated and the data stored in a database for utilization during breeding (Kim 2008). Additionally, the Mokpo Experiment Station of the National Institute of Crop Science investigated a set of 700 accessions collected in 1988, from which some superior individuals were selected. More recently they characterized about 3000 individuals selected from a group of 15 000 tea plants nationwide (Jeong and Park 2012).



3.7 VietnamThe tea plant is a traditional Vietnamese crop. The first collection of tea genetic resources in Vietnam was initiated by the French in the 1880s. Presently, a total of 180 tea germplasm accessions have been collected and conserved at the Tea Research Stations in Phu Ho and Ha Giang (Ngoc 2012). Of these accessions, exotic germplasm introduced from ten countries accounts for 65% of the total. Most of the accessions have been preserved as seeds (68.8%) and the rest as cuttings. According to morphological characteristics (Chen et al. 2000), there are three main varieties: C. sinensis var. sinensis, var. assamica and var. pubilimba (also known as Shan tea in Vietnam).

3.8 IndonesiaThe tea plant is not native to Indonesia. The variety C. sinensis var. sinensis was first introduced to Indonesia as an ornamental plant from Japan in 1674, while the variety C. sinensis var. assamica was imported for commercial cultivation from Sri Lanka in 1877 (Sriyadi et al. 2012). Tea plant genetic resources in Indonesia are classified as either seed propagation plants or clonal plants. There are two types of seed propagation plants: plants with unknown or vague parentage and plants with a clear pedigree. The clonal plants include the first- and second-generation clones and later clonal generations, which are expected to be excellent clones. The first generation of clones consists of those plants bred by local breeders and exotic cultivars, some of which have been recommended for planters. A collection of 600 clones of the first generation is presently maintained at Pasir Sarongge and Simalungun experimental garden (Sriyadi et al. 2012).

3.9 BangladeshThe tea plant was first introduced to Bangladesh between 1840 and 1857, at the same time as its introduction to parts of northeast India, including Assam, Darjeeling, Terai and the Dooars (Khan 2012). Exchange of genetic resources among tea estates in these regions was fairly frequent until the first quarter of the twentieth century. Thus, the local tea germplasm consisted of various types from different sources. As time went on, however, the indigenous C. sinensis var. assamica was mainly used to develop new tea plantations. At present, the tea plants predominantly cultivated are of three basic types: C. sinensis var. sinensis, var. assamica and var. lasiocalyx. The Bangladesh Tea Research Institute has collected and conserved a total of 386 tea germplasm accessions, including 317 clones and 69 seed stocks. Of those accessions, 328 are local, whereas 58 are introduced. Using the aforementioned germplasm, the institute has developed 17 clonal cultivars and four seed stocks (Khan 2012).

3.10 TurkeyCultivation of the tea plant in Turkey commenced in 1924. Most of the tea plantations were established using seeds derived from Georgia (Ercisli et al. 2012). The Turkish tea plant has been characterized as C. sinensis var. sinensis. Seed propagation of the tea plant over a long period of time has produced great genetic variation within tea populations in the country. Native scientists began to select superior germplasm for tea breeding between 1965 and 1973, resulting in a set of 64 candidate clones (Ercisli et al. 2012). A germplasm



collection with these clones has been established and is conserved at the Ataturk Tea and Horticultural Research Institute.


Genetic diversity is fundamentally important for the survival of the species as it provides the necessary ability for populations to adapt to changing environments. The success of crop breeding programmes is based on the comprehension and availability of genetic variability for efficient selection. Continued efforts to evaluate and characterize tea germplasm have been made using various methods.

4.1 Germplasm evaluationSystematic evaluation of collections of tea germplasm has been performed in many tea-producing countries. For instance, Takeda (1994) and Taniguchi et al. (2014) assessed the variation of biochemical contents in the 1500 and 788 worldwide accessions of tea germplasm, respectively. In China, more than 1500 tea germplasm accessions have been appraised for agronomic and quality traits, biotic and abiotic stress tolerance and resistance using multidisciplinary approaches, and furthermore, several agricultural technique standards of the Ministry of Agriculture for tea germplasm evaluation have been released (Chen et al. 2007, 2011; Yao and Chen 2012). Economically important characteristics have been identified from these studies, such as high content of catechins (Jin et al. 2014a; Sabhapondit et al. 2012) and theanine (Song et al. 2015), low content of caffeine (Jin et al. 2014b), high yield and quality (Singh et al. 2013), biotic resistance (Liang et al. 2012), etc. Elite germplasm with the aforementioned desirable characters is more usable by breeders and scientists. Some unique tea plant germplasms have been successfully used in varietal development programmes. For example, ‘Baiye 1’, a Chinese cultivar with whitish colour and a significantly high content of free amino acids in young leaves in spring, was developed from an albino germplasm (Li et al. 2016). The natural low-caffeine cultivars ‘Kekecha 1’ and ‘Kekecha 2’ were developed from C. ptilophylla Chang germplasm identified in the Guangzhou province of China (Yang et al. 2011).

4.2 Characterization of genetic diversityMorphological and biochemical investigation is a traditional strategy used for characterization of genetic resources, based on the assessment of a range of phenotypic characteristics. A combination of morphological and anatomical descriptions was first used for the classification of three types of tea plant by Barua (1963), and further refined by Bezbaruah (1971). Since then, several morphological traits such as leaf geometry, flower structure and pollen morphology, as well as biochemical characteristics including the contents of essential elements, catechin, caffeine, theanine and terpene, have been identified and used to study phylogeny, classification and genetic diversity in tea plants (Mondal 2014). In China, a set of 111 descriptors have been used to facilitate the characterization, evaluation and management of tea germplasm (Chen et al. 2005). Morphological markers, however, present several limitations, as they may be affected by the subjectivity of investigators, changing environmental conditions, and limited variation



among germplasm collections. For instance, Piyasundara (2008) found only six descriptors proposed by the IPGRI (1997) were adequate to define the phenotypic variation of tea germplasm in Sri Lanka. Furthermore, characterization of some useful characteristics, such as flowering, may be restricted as they can be investigated only at a particular stage of development.

Isozyme polymorphism refers to allelic variations of alloenzymes or allozymes, which can be separated by polyacrylamide gel electrophoresis due to their structural differences. It has been used for genetic analysis as neutral or nearly neutral markers in a wide range of plant species (Brown 1979). The feasibility of this method as applied to the tea plant was validated by Nagato and Osone (1982). Subsequently, several studies have used different enzymes including peroxidase, esterase, tetrazolium oxidase, aspartate aminotransferase and alpha-amylase (Mondal 2014). The relatively low polymorphism of isozymes limits their utilization, however this limitation has been progressively overcome since the development of DNA-based molecular markers.

DNA-based molecular markers are nucleotide variations in a specific genome region, which may be a gene or DNA region without any known function. DNA-based molecular markers have several significant advantages compared with morphological and isozymic markers. For instance, they are accurate, highly polymorphic and environmentally stable, and in addition allow for early selection of specific characteristics with linked or functional markers, which is important and valuable for crops with a long juvenile phase such as the tea plant. Various marker systems have been developed and used for genetic studies, including RAPD (random amplified polymorphic DNA), AFLP (amplified fragment length polymorphism), ISSR (inter simple sequence repeat), SSR (simple sequence repeat or microsatellite), SNP (single nucleotide polymorphism), CAPS (cleaved amplified polymorphic sequence), specific cpDNA and ntDNA assays, and next-generation sequencing (NGS)-based assays. The first study on the use of molecular markers in tea plant was reported by Wachira et al. (1995) and Lee et al. (1995). Their work was soon followed by other researchers using different molecular marker systems (Ni et al. 2008; Mondal 2014; Kumar et al. 2016; Mukhopadhyay et al. 2016).

SSR is a tract of repetitive DNA consisting of tandemly repeated short motifs (1-6 nucleotides). It is a versatile and popular molecular marker due to its locus specificity, co-dominant inheritance, high level of reproducibility and polymorphism. Recently, several large-scale analyses of genetic diversity in tea germplasm were performed using SSR markers. Yao et al. (2012) assessed 450 Chinese tea accessions collected countrywide, including three C. sinensis varieties and some related species. A total of 409 alleles were detected, and the average values of gene diversity (H) and polymorphic information content (PIC) were estimated to be 0.64 and 0.61, respectively. A higher level of genetic diversity was observed in the accessions from Guangxi, Yunnan and Guizhou provinces, which are usually considered as the original centres of the tea plant. Wambulwa et al. (2016) evaluated a collection of 280 tea accessions collected from eight African countries using 23 SSR markers. A total of 297 alleles were detected, with an average estimated genetic diversity of 0.652. The population structure suggested two main genetic groups of African tea germplasm, which corresponds well to the two varieties C. sinensis var. sinensis and var. assamica. In another study, Taniguchi et al. (2014) analysed 788 tea germplasm derived from 14 countries using 23 SSR markers, and the results showed that 619 alleles were observed with an average of 0.85 for PIC value. The genetic diversity of germplasm from China, India and Sri Lanka was higher than that of germplasm from other countries.



Additionally, AFLP is another molecular marker frequently used in tea plant research. In a comprehensive study conducted by Raina et al. (2012), AFLP was employed to analyse the genetic diversity of 1644 Indian tea germplasm accessions, and a total of 412 AFLP loci were amplified by using seven primer pair combinations. Both PCoA and neighbour-Joining analysis based on genetic distance clustered the tea germplasm into six major groups with one group in each, and the results of structure analysis suggested that accessions of the same morphotype were not always of the same genetic ancestry and structure.

Although these previous studies have proved that molecular markers are powerful tools in the analysis of genetic diversity in the tea plant and related species, it is important to understand that different markers have different properties and will reflect different aspects of genetic diversity (Karp and Edwards 1995). In a comparative analysis of genetic diversity among a set of Chinese, Kenyan and Japanese tea germplasm, different values of gene diversity were detected using ISSR and SSR, which further affected the estimation of genetic distance and clustering analysis (Yao et al. 2009). Furthermore, the variation in marker numbers also has an effect on the results of genetic diversity analysis, even using the same molecular marker system (Fanizza et al. 1999). Therefore, it will be important to develop a set of universal molecular markers for the tea plant in the future.


Morphological, isozymic and molecular data on the variation, distribution and structure of genetic diversity provide the information necessary for further analysis of species taxonomy, origin and domestication, and germplasm management, core collection construction, cultivar improvement and protection, functional gene and allele mining, etc. The exploitation and utilization of genetic diversity data relating to the tea plant are discussed below.

5.1 TaxonomyTaxonomic information relating to tea genetic resources provides the necessary underpinning for germplasm management and parental selection in hybridization breeding programmes. The taxonomy of cultivated plants, however, is especially difficult (Harlan and de Wet 1971). Results of traditional taxonomic studies of the section Thea based on morphological traits are highly conflicting. The section has been considered to consist of five species and two varieties (Sealy 1958), 42 species and four varieties (Chang 1981, 1984), 12 species and six varieties (Min 1992), and five species and two varieties (Chen et al. 2000). The utilization of various molecular markers has provided new insights into phylogenetic analysis in recent years. By using a collection of RAPD data, Chen and Yamaguchi (2002) categorized the 24 species and varieties in the taxonomy of Chang and Bartholomew (1984) into two groups, that is, a 5-locule group and a 3-locule ovary group, which was largely consistent with the results of phytochemical, karyotypic and morphological classification. Yang et al. (2016) used the NGS-based genome-wide restriction site-associated DNA sequencing (RAD-Seq) approach to analyse a collection of cultivated and wild tea plants. The results showed that 18 tea accessions were clustered into six groups, corresponding with the traditional taxonomy, except one



accession which proved to be a semi-wild or transient landrace base on the analysis of genetic divergence.

The rational classification of the genus Camellia is yet more contentious and elusive, even using multidisciplinary approaches (Zhang and Ming 1999; Sen et al. 2000; Vijayan et al. 2009). Wachira et al. (1997) analysed genetic relationship at the sectional level using RAPD and STS, and the results were generally congruent with those detected by compatibility studies (Takeda 1990). Sharma et al. (2015) discovered that the classification of beveragial tea previously into 34 species using karyotype analysis (Sharma and Raina 2006) was not supported by their data derived from DNA assays of both mitochondrial and chloroplast genomes. Yang et al. (2013) sequenced the chloroplast genomes of seven individuals, representing six Camellia species, and used the whole chloroplast genome sequence variations for phylogenetic analysis; yet the result was not in agreement with that of any of the traditional classification systems. Regardless of the classification system used there is no doubt that more taxonomic work is necessary.

5.2 Origin and domesticationA good understanding of the origin and domestication of a crop species is important for the collection and utilization of genetic resources. Most scientists believe that the tea plant originated in Southwest China, where diverse wild relatives have been found in the natural forest (Yu 1986). Evidence from studies of genetic diversity has confirmed that Chinese tea germplasm has more genetic variation (Yao et al. 2009, Taniguchi et al. 2014), especially the germplasm from the original centres (Yao et al. 2012). Genetic parameters such as allele number, gene diversity and polymorphic information content, show a decreasing trend with increasing distance from the original centres, which might indicate how the tea plant spread in China (Yao et al. 2012). Tea cultivation started in India using the seeds of C. sinensis var. sinensis, and subsequently the variety var. assamica spread quickly due to its advantages in terms of local climate adaptation and character for producing black tea. Meegahakumbura et al. (2016) evaluated the genetic diversity of a set of 392 and Chinese and Indian tea germplasm samples consisting of three varieties (C. sinensis var. sinensis, var. assamica and var. lasiocalyx) using SSR markers. The results showed that tea germplasm from both countries had high levels of genetic variation, and could be clustered into three distinct genetic groups with significant pairwise genetic differentiation, which was consistent with their geographical distribution. This indicated that the varieties of var. sinensis, Chinese and Indian var. assamica were likely the result of three independent domestication events from three separate regions across China and India.

C. taliensis is a unique tea germplasm, found in subtropical mountain evergreen forests from the western Yunnan province of China to northern Myanmar. It has been cultivated traditionally throughout western Yunnan for at least hundreds of years. Zhao et al. (2014) used a collation of data obtained from wild, planted and recently domesticated populations to investigate the domestication and geographic origin of C. taliensis, based on the analysis of genetic diversity and population structure using 14 SSR. The results showed that C. taliensis had a moderately high level of overall genetic diversity, and domestication had a small, non-significant influence on the genetic diversity. A greater reduction in genetic diversity and stronger genetic drift were detected in the wild populations, indicating the loss of genetic diversity. The phylogenetic and structure analysis suggested planted C. taliensis might have been domesticated from the adjacent central forest of western Yunnan and then spread artificially.



5.3 Germplasm managementOn the basis of accurate evaluation of the range of genetic diversity within and between populations, scientists can adjust collection, management and breeding strategies to obtain maximum variation from the gene pools (Morikawa and Leggett 1990). In the case of Chinese tea germplasm, Yao et al. (2012) found a substantial amount of gene flow between adjacent populations; the overall genetic variation was mainly contributed by within-population variation (81%). The improved clonal cultivars had relatively low diversity and similar genetic make-up, which indicated a narrow genetic background caused by artificial selection. In Africa, Wambulwa et al. (2016) confirmed international germplasm exchange and movement among countries, and the fact that C. sinensis var. assamica germplasm separates into two groups. These may represent the two major breeding centres, corresponding to Southern Africa (Tea Research Foundation of Central Africa) and East Africa (Tea Research Institute of Kenya). The study suggested that some traits of var. assamica and their associated genes possibly underwent selection during geographic differentiation or due to local breeding preferences. This information is valuable for further collection and conservation of germplasm, as well as parental selection for hybridization.

5.4 Core collectionThe term ‘core collection’ refers to a subset of germplasm with a minimum repetitiveness and extensive coverage of the overall genetic diversity of a crop species and its wild relatives. Producing core collections is strategically important as it may decrease the cost of germplasm conservation and increase germplasm utilization efficiency. Construction of a worldwide core collection may help establish a standard system which may improve procedures of germplasm characterization and evaluation. By using allele number as a measure of genetic diversity, Taniguchi et al. (2014) developed a worldwide core collection and three mini-core collections of tea germplasm. The core collection comprises 192 tea accessions, and the three mini-core collections have 96, 48 and 24 accessions, respectively. The core collection fully contains the phenotypic variation of 788 tea accessions. In Sri Lanka, Gunasekare and Ranatunga (2008) assembled a preliminary core collection of tea genetic resources, including a total of 500 accessions from several countries. In China, a preliminary core collection consisting of 126 tea accessions was generated (Li and Jiang 2004), and further evaluated using RAPD markers (Li et al. 2005). Using a combination of parameters derived from morphological and molecular genetic diversity, Liu (2008) constructed a countrywide core collection of Chinese tea germplasm, including 360 accessions which largely covered the genetic variation of 2665 accessions.

5.5 Cultivar improvement and protectionThe genetic diversity preserved in germplasm collections is a precious resource for crop breeding. Individual selection from landraces and natural populations is one of the important methods in tea breeding. For instance, about 60% of the nationally registered cultivars in China have been developed from superior individuals selected from landraces. High levels of genetic variation between parents increase the possibility of creating the unique gene combinations necessary for new superior cultivars. The varieties C. sinensis var. sinensis and var. assamica are traditionally thought to be particularly suitable for the production of green tea and black tea, respectively. Through hybridization of two varieties,



several new tea cultivars suitable for producing both green tea and black tea have been developed.

New cultivars are a key element in stimulating the growth of the tea industry. It is estimated that more than 600 tea cultivars have been developed worldwide to date. Plant Variety Protection (PVP) or Plant Breeder’s Rights (PBR) are efficient strategies to promote the development and release of new cultivars. A good understanding of genetic variation is necessary to establish appropriate guidelines for the conduct of distinctness, uniformity and stability (DUS) tests which are the technical base of PVP and the scientific basis for the approval of PBR. Based on the systematic appraisal of 1500 tea accessions preserved in CNGTR, a set of 35 characteristics, including those of plant architecture, tender shoots, leaf blade, flower and so on, have been determined to be the testing characteristics in the UPOV-DUS test for tea plant (TG/238/1) (Chen et al. 2008). This guideline provides the foundation for a technical standard to examine the DUS of new tea cultivars, and may encourage the development of new cultivars and PVP for the tea plant.

5.6 Dissection of complex phenotypesThe major goal of tea breeding is to improve productivity, enhance tolerance to biotic and abiotic stress, and increase tea flavour and quality. Dissection of the genetic basis of these traits will provide the potential for accelerating the breeding process by developing new tools such as marker-assisted selection (MAS). However, only a few such studies have been undertaken on the tea plant (Tanaka 1996; Yao et al. 2010; Kamunya et al. 2010; Mphangwe et al. 2013; Ma et al. 2014; Bali et al. 2015; Elangbam et al. 2016; Tan et al. 2016; Jin et al. 2016). In a successful case, Shige et al. (1993) evaluated a set of tea germplasm, and identified a unique cultivar ‘Sayamakaori’ which was strongly resistant to mulberry scale. A linkage mapping study was subsequently performed using a segregation population derived from the cross of ‘Kana-Ck17’ and ‘Sayamakaori’. The mulberry scale-resistant gene MSR-1 was then detected, and the allele-specific marker was successfully developed and used for MAS (Tanaka et al. 2003, Tanaka 2005). In another case, Jin et al. (2016) evaluated the allelic variation of TCS1 (tea caffeine synthase 1) in a collection of tea cultivars and wild relatives, and subsequently detected a functional SNP affecting caffeine content using association mapping, and then validated it using site-directed mutagenesis. The CAPS marker developed from this locus may be applied for future MAS studies.


Efforts to collect and conserve germplasm have accelerated over the past decades, successfully expanding the gene pool and promoting the breeding of new tea cultivars. Despite this, however, there are still many challenges associated with the protection of tea germplasm genetic diversity. The wild relatives of the tea plant are threatened by habitat loss, due to the conversion of forest land for agricultural use and global climate change. Furthermore, the landraces, another important source of genetic variation, have been gradually been replaced by clonal cultivars, since the tea industry prefers improved cultivars with superior characteristics such as early sprouting, availability for machine harvest and better tea quality. It will therefore be crucial to collect and maintain valuable landraces and wild relatives of the tea plant in the future.



Research into the characterization and evaluation of germplasm has improved our understanding of the genetic diversity of the tea plant and its related species. The narrowness of the genetic diversity of improved tea cultivars will quite possibly lead to a decline in tea production and quality in the future. It will therefore be necessary to broaden the genetic base of future cultivars by introducing new genetic variations from other gene pools of wild relatives and landraces. It is challenging, however, to efficiently identify and exploit the valuable variation in the huge collections of genetic resources. Possible solutions include: (1) construction of core and mini-core collections; (2) development of accurate and large-scale phenotyping and genotyping methods; (3) utilization of multidisciplinary technologies such as linkage and association mapping, comparative genomics and molecular genetics to increase the efficiency of functional gene identification and allele mining; (4) development of public databases and promotion of data sharing among multidisciplinary scientists; and (5) germplasm exchange activities to exploit the full potential of the genetic diversity available globally in order to develop elite cultivars using systematic approaches.


OrganizationsChina: The Tea Research Institute of the Chinese Academy of Agricultural Sciences (TRICAAS, http://www.tricaas.com/English/) is the only national organization for tea research in China. The institute conducts research on a variety of topics including basic science, technological innovation and market analysis in the tea industry.

India: The Tocklai Experimental Station (TES) of the Tea Research Association (TRA, http://www.tocklai.net) focuses on all aspects of tea cultivation and processing with the aim to improve productivity and quality. The United Planters’ Association of Southern India (UPASI, http://www.upasi.org) is an apex body of planters of several cash crops in southern India, conducting comprehensive scientific research, and studies on market intelligence, industrial and public relations. The Institute of Himalayan Bioresource Technology is a constituent laboratory of Council of Scientific and Industrial Research (CSIR-IHBT, http://www.ihbt.res.in/en), with the goal to boost bioeconomy through sustainable utilization of Himalayan bioresources.

Japan: The Institute of Fruit Tree and Tea Science is a part of the National Agriculture and Food Research Organization (NIFTS, http://www.naro.affrc.go.jp/english/nifts/index.html) and conducts basic and innovative research in fruit tree and tea science to promote industrial development and the public good.

Sri Lanka: The Tea Research Institute of Sri Lanka (http://www.tri.lk), established in 1925, has been the only national organization in Sri Lanka for developing and expanding new technologies related to tea cultivation and processing.

Kenya: The Tea Research Institute (http://www.tearesearch.or.ke), previously known as the Tea Research Foundation of Kenya, was founded in 1980 to promote research related to tea plant and companion crops, and it focuses on development of new cultivars and technologies for the improvement of yield and quality of tea.

Database: The Chinese Crop Germplasm Information System (CCGIS, http://www.cgris.net/cgris_english.html) is a central repository for plant genetic resources information,



storing over 4 000 MB data on 410 000 accessions. The information, including number, distribution, and evaluation of tea germplasm conserved in the CNGTR, can be found on the database.

The Genetic Resources Center of the National Agriculture and Food Research Organization of Japan (https://www.naro.affrc.go.jp/english/ngrc/index.html) is an organization dedicated to collecting, evaluating, preserving, distributing and improving genetic resources. The Genebank has conserved more than 222 000 plant genetic resource accessions whose information can be retrieved from the database.

The whole genome of ‘Yunkang 10’, a diploid elite cultivar of Camellia sinensis var. assamica, has been sequenced and released. The information regarding genome assembly and gene annotation can be obtained from the Tea tree Genome Database (http://www.plantkingdomgdb.com/tea_tree/). The data provide the potential to develop new strategies and methods for germplasm evaluation and breeding.

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Yao, M. Z., Qiao, T. T., Ma, C. L., Jin, J. Q. and Chen, L. (2010). The association analysis of phenotypic traits with EST-SSR markers in tea plants. J. Tea Sci. 30(1), 45–51 (in Chinese).

Yu, F. L. (1986). Discussion on the originating place and the originating center of tea plants. J. Tea Sci. 6(1), 1–8 (in Chinese).

Zhang, C. C., Wang, L. Y., Wei, K., Wu, L. Y., Li, H. L., Zhang, F., Chen, H. and Ni, D. J. (2016). Transcriptome analysis reveals self-incompatibility in the tea plant (Camellia sinensis) might be under gametophytic control. BMC Genomics 17, 359.

Zhang, W. J. and Ming, T. (1999). A cytogeological study of genus Camellia. Plant Divers. 21(2), 1–3.Zhao, D. W., Yang, J. B., Yang, S. X., Kato, K. and Luo, J. P. (2014). Genetic diversity and domestication

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References

1 Chapter 1 Ensuring the geneticdiversity of tea plants

1 Introduction

Tea plant (Camellia sinensis (L.) O. Kuntze) is anevergreen, perennial and woody species,

whose tender buds and leaves are used to prepare thebeverage known worldwide as

tea (Wight and Baruwa 1957). Tea is an important source ofmicronutrients for the daily

diet in many countries. Recent studies have shown thattea’s bioactive components can

provide desirable health benefits (Hayat et al. 2015) andhave a protective effect against

cancer, obesity and cardiovascular diseases (Khan andMukhtar 2013). The consumption

of products containing tea constituents has been increasingin recent years. Global

production of tea increased from 3.06 million metric tonsin 2001 to 5.29 million metric

tons in 2015 (ITC 2016).

The tea plant belongs to the section Thea (L.) Dyer of thegenus Camellia L. in the family

Theaceae. Taxonomic systems for the section Theaestablished by individual scientists

differ in that they consist of different numbers of speciesand varieties (Sealy 1958; Wight

1959; Chang 1984; Min 1992; Chen et al. 2000). It is beyonddebate, however, that the

major cultivated tea plant comprises two varieties: C.sinensis var. sinensis (the small

leaf variety) and C. sinensis var. assamica (Masters)Kitamura (the large-leaf variety). The

variety of C. sinensis var. pubilimba Chang is also growncommercially in some regions of

China and South Asia. In addition, a few wild relatives,such as C. taliensis (W. W. Smith)

Melchior, C. tachangensis F. C. Zhang, C. crassicolumnaChang and C. gymnogyna Chang,

are sometimes used locally.

The tea plant shows strong self-incompatibility. Recentevidence suggests it is

characterized by late-acting self-incompatibility, whichmight be under gametophytic

control (Zhang et al. 2016). Long-term cross-pollinationhas produced great heritable

variation within and between populations of the tea plantand its related species, resulting

in a high level of genetic diversity. A good understandingand management of this pool

of genetic diversity is of vital importance for the geneticimprovement of tea plant, since

it directly affects the potential for genetic gain throughselection. This chapter is intended

to provide an overview of the genetic diversity of the teaplant, and its characterization,

exploitation and utilization.


The tea plant is usually considered to have originated inSouthwest China, where it was

first discovered and used as a medicinal drink as early as2737 BC (Lu 1974; Chang

1981), although some believe its place of origin to havebeen regions of South China,

Assam, northern Myanmar and Indochina (Kingdon-Ward 1950;

Sealy 1958; Wight 1959;

Yamamoto et al. 1997). About 80% of the species of thesection Thea, including the tea

plant and its wild relatives, have been found in China,according to Chang’s taxonomic

system (Chang 1981; Yu 1986). Taking into consideration thequantity, distribution and

morphological characteristics of wild germplasm and relatedspecies, Yu (1986) postulated

the Yunnan province of China as a likely centre of originof the tea plant similar to Min’s

(1992) conclusion.

The first record of tea cultivation dates from the firstcentury BC, when the tea plant

was grown in the Sichuan province of China. The plant waslater introduced to Japan from

the Zhejiang province of China in the early ninth century.In India, the tea plant was first

cultivated in 1834 since when seeds were brought fromChina, although the indigenous

C. sinensis var. assamica was discovered in 1823. Teacultivation and processing technology

extended to Brazil from China in 1812. The tea plant wasfirst introduced to Sri Lanka in

1824 by raising seeds brought from India. Tea cultivationin Transcaucasia started in 1883

using seeds from the Hubei province of China, and afterthat, the tea plant first reached

Turkey in 1924. In Europe, successful cultivation of thetea plant began in Britain during

1768, from where it was introduced to Africa at the end ofthe nineteenth century. To date,

there are more than 50 tea-producing countries, within the

latitudinal range of 45°N to

34°S (Mondal 2014).


Genetic resources are reservoirs of genes and genotypes,which play an important role

in promoting and sustaining agriculture. The collection andconservation of the cultivars,

landraces and wild relatives of the tea plant will providebreeders and scientists with

fundamental materials from which new cultivars are to bedeveloped. To maintain a

broad genetic base, most tea-producing countries haveestablished a tea germplasm

conservation system. There are roughly 20 000 accessions oftea germplasm preserved in

the major tea-producing countries, including China, India,Japan, Sri Lanka, Kenya, Korea,

Vietnam, Indonesia, Bangladesh and Turkey, which representthe majority of the world’s

tea plant genetic resources. In the following sections, thecurrent status of tea germplasm

collections in these countries will be described in moredetail.

3.1 China

The tea plant is native to China, which has the mostabundant and diverse tea genetic

resources in the world. In modern China, a project oftargeted investigation and collection

started in the 1980s after the tea plant was listed in thenational plans for crop germplasm

investigation and collection (Yao and Chen 2012). Permanentpreservation centres –

the China National Germplasm Tea Repository (CNGTR) andMenghai Branch – were

established for ex situ conservation of tea germplasm in1990. To date, the CNGTR has

preserved about 3000 accessions, among which 2800 have beenidentified according to

Chen’s taxonomic system (Chen et al. 2000). Most of thecultivated tea accessions belong

to the three varieties, C. sinensis var. sinensis, var.assamica and var. pubilimba, while

the majority of wild relatives are C. taliensis, C.tachangensis, C. crassicolumna and C.

gymnogyna. Of the 3000 accessions, 11% are wild relatives,60% landraces, 6% cultivars

and 23% genetic materials. A core collection has beendeveloped to improve germplasm

utilization at the CNGTR (Liu 2008). Over the past decades,local government has

increasingly emphasized in situ conservation. Severalprojects to protect famous landraces

and wild relatives are underway. Additionally, techniquessuch as tissue culture and seed

cryopreservation have been studied for in vivo conservationof tea germplasm, which has

a great potential for the conservation of tea geneticresources (Wang et al. 1990, 1999).

3.2 India

The first commercial tea garden in India was establishedusing seeds originating from China

in 1834 (Das et al. 2012). Subsequently, seeds wereintroduced from Burma, Cambodia,

Vietnam and Japan, which resulted in the development ofsuperior planting materials

(Paul et al. 1997). In order to develop an Indian nationaltea plant gene bank, regional

germplasm collection centres in at least three differentgeographical locations have

been established: the Tea Research Association, TocklaiExperimental Station (TRA, TES)

located in northeastern India, the United Planters’Association of Southern India (UPASI),

and the Council of Scientific and Industrial Research,Institute of Himalayan Bioresource

Technology (CSIR-IHBT) located in northwestern India. It isestimated that more than 2100

and 1250 accessions are maintained at TES and UPASI,respectively (Das et al. 2012). The

conserved germplasm has great morphological and geneticdiversity, comprising diverse

genotypes of ancient seedling populations and wildrelatives. Many improved cultivars

and seed varieties have been developed using these geneticresources.

3.3 Japan

The first record of the introduction of the tea plant toJapan dates to the ninth century,

when two Japanese monks introduced seeds from China (Tanaka2012). There is thus a

long history of tea germplasm collection and utilization inJapan. Tea genetic resources

in Japan are now being maintained as clone bushes withinseveral national and regional

research institutes. The NARO Institute of Fruit Tree andTea Science (NIFTS) has

constructed a global tea germplasm collection includingmore than 7800 accessions

derived from fourteen countries over the past century, fromwhich a core collection has been

generated (Taniguchi et al. 2014). The collection has beenevaluated and characterized

morphologically, chemically and molecularly, and indicatesplentiful genetic diversity.

3.4 Sri Lanka

The tea plant is an introduced crop in Sri Lanka, where nowild relatives or landraces can

be found. During the early years of tea cultivation, seedswere mainly imported from India

and China (Gunasekare 2012). After multiple generationalhybridization of the seedlings,

the offspring populations were of high heterozygosis withbroad genetic variation. The

collection and ex situ conservation of tea germplasm wereinitiated locally in 1986. At

present, about 600 accessions are maintained in a fieldgene bank at the Tea Research

Institute of Sri Lanka (Gunasekare et al. 2012). Thesegenetic resources can be categorized

into two groups, that is, beverage and non-beverage type.The beverage group consists

of exotic and native improved cultivars, and breedingmaterials selected from old seedling

populations. Some unique germplasm has been identifiedamong these accessions,

such as natural triploid cultivars ‘HS 10A’ and ‘GF 5’, andnon-fermenting cultivar ‘TRI 9’.

The non-beverage group contains related species such as C.sasanqua, C. japonica and

C. rosaeflora, which may be utilized in interspecifichybridization programmes.

3.5 Kenya

The tea plant was reportedly first introduced into Kenya in1904, when seeds of C. sinensis

var. assamica from India were used to establish aplantation (Kamunya et al. 2012). In 1912,

C. sinensis var. sinensis seeds were imported from SriLanka. The aforesaid pioneer

seedlings were considered the main components of thecountry’s tea genetic resources.

Studies have revealed, however, that Kenya’s tea geneticresources are in fact diverse

(Wachira et al. 1995; Paul et al. 1997). Exotic teagermplasm collected from across the

globe has extended the local gene pool. Tea germplasmcollections in Kenya, for instance,

contain seeds from popular Japanese cultivars such as‘Yabukita’ and ‘Yutakamidori’.

Additionally, several Camellia species, such as C.japonica, C. brevistyla, C. sasanqua and

C. irrawadiensis, were also imported to access diversityfrom the secondary and tertiary

gene pools of the tea plant. Currently, the Camellia GeneBank of Tea Research Institute of

Kenya conserves more than 250 accessions of the tea plantand related species (Kamunya

et al. 2012).

3.6 Korea

The tea plant was introduced to Korea from China more thantwo thousand years ago

(Jeong and Park 2012). Seed propagation is the mostfrequent tea cultivation method in

Korea. Currently, 80% of Korean tea plantations are stillnative seedling populations, which

consist of genotypes with a broad range of phenotypicvariation. Between 1994 and 1998,

a total of 2300 tea germplasm samples were collected andpreserved at the Boseong

Tea Experiment Station (Jeong and Park 2012). Thecharacteristics of this germplasm

have been investigated and the data stored in a databasefor utilization during breeding

(Kim 2008). Additionally, the Mokpo Experiment Station ofthe National Institute of Crop

Science investigated a set of 700 accessions collected in1988, from which some superior

individuals were selected. More recently they characterizedabout 3000 individuals

selected from a group of 15 000 tea plants nationwide(Jeong and Park 2012).

3.7 Vietnam

The tea plant is a traditional Vietnamese crop. The firstcollection of tea genetic resources

in Vietnam was initiated by the French in the 1880s.Presently, a total of 180 tea germplasm

accessions have been collected and conserved at the TeaResearch Stations in Phu Ho

and Ha Giang (Ngoc 2012). Of these accessions, exoticgermplasm introduced from ten

countries accounts for 65% of the total. Most of theaccessions have been preserved as

seeds (68.8%) and the rest as cuttings. According tomorphological characteristics (Chen

et al. 2000), there are three main varieties: C. sinensisvar. sinensis, var. assamica and var.

pubilimba (also known as Shan tea in Vietnam).

3.8 Indonesia

The tea plant is not native to Indonesia. The variety C.sinensis var. sinensis was first

introduced to Indonesia as an ornamental plant from Japanin 1674, while the variety

C. sinensis var. assamica was imported for commercialcultivation from Sri Lanka in 1877

(Sriyadi et al. 2012). Tea plant genetic resources inIndonesia are classified as either seed

propagation plants or clonal plants. There are two types ofseed propagation plants:

plants with unknown or vague parentage and plants with aclear pedigree. The clonal

plants include the first- and second-generation clones andlater clonal generations, which

are expected to be excellent clones. The first generationof clones consists of those plants

bred by local breeders and exotic cultivars, some of whichhave been recommended for

planters. A collection of 600 clones of the firstgeneration is presently maintained at Pasir

Sarongge and Simalungun experimental garden (Sriyadi et al.2012).

3.9 Bangladesh

The tea plant was first introduced to Bangladesh between1840 and 1857, at the same

time as its introduction to parts of northeast India,including Assam, Darjeeling, Terai

and the Dooars (Khan 2012). Exchange of genetic resourcesamong tea estates in these

regions was fairly frequent until the first quarter of thetwentieth century. Thus, the local tea

germplasm consisted of various types from different

sources. As time went on, however,

the indigenous C. sinensis var. assamica was mainly used todevelop new tea plantations.

At present, the tea plants predominantly cultivated are ofthree basic types: C. sinensis

var. sinensis, var. assamica and var. lasiocalyx. TheBangladesh Tea Research Institute has

collected and conserved a total of 386 tea germplasmaccessions, including 317 clones

and 69 seed stocks. Of those accessions, 328 are local,whereas 58 are introduced. Using

the aforementioned germplasm, the institute has developed17 clonal cultivars and four

seed stocks (Khan 2012).

3.10 Turkey

Cultivation of the tea plant in Turkey commenced in 1924.Most of the tea plantations were

established using seeds derived from Georgia (Ercisli etal. 2012). The Turkish tea plant

has been characterized as C. sinensis var. sinensis. Seedpropagation of the tea plant over

a long period of time has produced great genetic variationwithin tea populations in the

country. Native scientists began to select superiorgermplasm for tea breeding between

1965 and 1973, resulting in a set of 64 candidate clones(Ercisli et al. 2012). A germplasm

collection with these clones has been established and isconserved at the Ataturk Tea and

Horticultural Research Institute.


Genetic diversity is fundamentally important for the

survival of the species as it provides

the necessary ability for populations to adapt to changingenvironments. The success of

crop breeding programmes is based on the comprehension andavailability of genetic

variability for efficient selection. Continued efforts toevaluate and characterize tea

germplasm have been made using various methods.

4.1 Germplasm evaluation

Systematic evaluation of collections of tea germplasm hasbeen performed in many tea

producing countries. For instance, Takeda (1994) andTaniguchi et al. (2014) assessed

the variation of biochemical contents in the 1500 and 788worldwide accessions of tea

germplasm, respectively. In China, more than 1500 teagermplasm accessions have

been appraised for agronomic and quality traits, biotic andabiotic stress tolerance

and resistance using multidisciplinary approaches, andfurthermore, several agricultural

technique standards of the Ministry of Agriculture for teagermplasm evaluation have

been released (Chen et al. 2007, 2011; Yao and Chen 2012).Economically important

characteristics have been identified from these studies,such as high content of catechins

(Jin et al. 2014a; Sabhapondit et al. 2012) and theanine(Song et al. 2015), low content

of caffeine (Jin et al. 2014b), high yield and quality(Singh et al. 2013), biotic resistance

(Liang et al. 2012), etc. Elite germplasm with theaforementioned desirable characters is

more usable by breeders and scientists. Some unique teaplant germplasms have been

successfully used in varietal development programmes. Forexample, ‘Baiye 1’, a Chinese

cultivar with whitish colour and a significantly highcontent of free amino acids in young

leaves in spring, was developed from an albino germplasm(Li et al. 2016). The natural low

caffeine cultivars ‘Kekecha 1’ and ‘Kekecha 2’ weredeveloped from C. ptilophylla Chang

germplasm identified in the Guangzhou province of China(Yang et al. 2011).

4.2 Characterization of genetic diversity

Morphological and biochemical investigation is atraditional strategy used for

characterization of genetic resources, based on theassessment of a range of phenotypic

characteristics. A combination of morphological andanatomical descriptions was first

used for the classification of three types of tea plant byBarua (1963), and further refined

by Bezbaruah (1971). Since then, several morphologicaltraits such as leaf geometry,

flower structure and pollen morphology, as well asbiochemical characteristics including

the contents of essential elements, catechin, caffeine,theanine and terpene, have

been identified and used to study phylogeny, classificationand genetic diversity in tea

plants (Mondal 2014). In China, a set of 111 descriptorshave been used to facilitate

the characterization, evaluation and management of teagermplasm (Chen et al. 2005).

Morphological markers, however, present severallimitations, as they may be affected by

the subjectivity of investigators, changing environmentalconditions, and limited variation

among germplasm collections. For instance, Piyasundara(2008) found only six descriptors

proposed by the IPGRI (1997) were adequate to define thephenotypic variation of tea

germplasm in Sri Lanka. Furthermore, characterization ofsome useful characteristics, such

as flowering, may be restricted as they can be investigatedonly at a particular stage of

development.

Isozyme polymorphism refers to allelic variations ofalloenzymes or allozymes, which can

be separated by polyacrylamide gel electrophoresis due totheir structural differences. It

has been used for genetic analysis as neutral or nearlyneutral markers in a wide range of

plant species (Brown 1979). The feasibility of this methodas applied to the tea plant was

validated by Nagato and Osone (1982). Subsequently, severalstudies have used different

enzymes including peroxidase, esterase, tetrazoliumoxidase, aspartate aminotransferase

and alpha-amylase (Mondal 2014). The relatively lowpolymorphism of isozymes limits

their utilization, however this limitation has beenprogressively overcome since the

development of DNA-based molecular markers.

DNA-based molecular markers are nucleotide variations in aspecific genome region,

which may be a gene or DNA region without any knownfunction. DNA-based molecular

markers have several significant advantages compared withmorphological and isozymic

markers. For instance, they are accurate, highlypolymorphic and environmentally stable,

and in addition allow for early selection of specificcharacteristics with linked or functional

markers, which is important and valuable for crops with along juvenile phase such as the

tea plant. Various marker systems have been developed andused for genetic studies,

including RAPD (random amplified polymorphic DNA), AFLP(amplified fragment length

polymorphism), ISSR (inter simple sequence repeat), SSR(simple sequence repeat

or microsatellite), SNP (single nucleotide polymorphism),CAPS (cleaved amplified

polymorphic sequence), specific cpDNA and ntDNA assays, andnext-generation

sequencing (NGS)-based assays. The first study on the useof molecular markers in tea

plant was reported by Wachira et al. (1995) and Lee et al.(1995). Their work was soon

followed by other researchers using different molecularmarker systems (Ni et al. 2008;

Mondal 2014; Kumar et al. 2016; Mukhopadhyay et al. 2016).

SSR is a tract of repetitive DNA consisting of tandemlyrepeated short motifs (1-6

nucleotides). It is a versatile and popular molecularmarker due to its locus specificity,

co-dominant inheritance, high level of reproducibility andpolymorphism. Recently,

several large-scale analyses of genetic diversity in teagermplasm were performed using

SSR markers. Yao et al. (2012) assessed 450 Chinese teaaccessions collected countrywide,

including three C. sinensis varieties and some relatedspecies. A total of 409 alleles were

detected, and the average values of gene diversity (H) andpolymorphic information

content (PIC) were estimated to be 0.64 and 0.61,respectively. A higher level of genetic

diversity was observed in the accessions from Guangxi,Yunnan and Guizhou provinces,

which are usually considered as the original centres of thetea plant. Wambulwa et al.

(2016) evaluated a collection of 280 tea accessionscollected from eight African countries

using 23 SSR markers. A total of 297 alleles were detected,with an average estimated

genetic diversity of 0.652. The population structuresuggested two main genetic groups

of African tea germplasm, which corresponds well to the twovarieties C. sinensis var.

sinensis and var. assamica. In another study, Taniguchi etal. (2014) analysed 788 tea

germplasm derived from 14 countries using 23 SSR markers,and the results showed that

619 alleles were observed with an average of 0.85 for PICvalue. The genetic diversity

of germplasm from China, India and Sri Lanka was higherthan that of germplasm from

other countries.

Additionally, AFLP is another molecular marker frequentlyused in tea plant research. In

a comprehensive study conducted by Raina et al. (2012),AFLP was employed to analyse

the genetic diversity of 1644 Indian tea germplasmaccessions, and a total of 412 AFLP loci

were amplified by using seven primer pair combinations.Both PCoA and neighbour-Joining

analysis based on genetic distance clustered the teagermplasm into six major groups with

one group in each, and the results of structure analysissuggested that accessions of the

same morphotype were not always of the same geneticancestry and structure.

Although these previous studies have proved that molecularmarkers are powerful

tools in the analysis of genetic diversity in the tea plantand related species, it is

important to understand that different markers havedifferent properties and will reflect

different aspects of genetic diversity (Karp and Edwards1995). In a comparative analysis

of genetic diversity among a set of Chinese, Kenyan andJapanese tea germplasm,

different values of gene diversity were detected using ISSRand SSR, which further

affected the estimation of genetic distance and clusteringanalysis (Yao et al. 2009).

Furthermore, the variation in marker numbers also has aneffect on the results of genetic

diversity analysis, even using the same molecular markersystem (Fanizza et al. 1999).

Therefore, it will be important to develop a set ofuniversal molecular markers for the

tea plant in the future.


Morphological, isozymic and molecular data on thevariation, distribution and structure

of genetic diversity provide the information necessary forfurther analysis of species

taxonomy, origin and domestication, and germplasmmanagement, core collection

construction, cultivar improvement and protection,functional gene and allele mining,

etc. The exploitation and utilization of genetic diversitydata relating to the tea plant are

discussed below.

5.1 Taxonomy

Taxonomic information relating to tea genetic resourcesprovides the necessary

underpinning for germplasm management and parentalselection in hybridization

breeding programmes. The taxonomy of cultivated plants,however, is especially difficult

(Harlan and de Wet 1971). Results of traditional taxonomicstudies of the section Thea

based on morphological traits are highly conflicting. Thesection has been considered

to consist of five species and two varieties (Sealy 1958),42 species and four varieties

(Chang 1981, 1984), 12 species and six varieties (Min1992), and five species and two

varieties (Chen et al. 2000). The utilization of variousmolecular markers has provided new

insights into phylogenetic analysis in recent years. Byusing a collection of RAPD data,

Chen and Yamaguchi (2002) categorized the 24 species andvarieties in the taxonomy of

Chang and Bartholomew (1984) into two groups, that is, a5-locule group and a 3-locule

ovary group, which was largely consistent with the resultsof phytochemical, karyotypic

and morphological classification. Yang et al. (2016) usedthe NGS-based genome

wide restriction site-associated DNA sequencing (RAD-Seq)approach to analyse a

collection of cultivated and wild tea plants. The resultsshowed that 18 tea accessions

were clustered into six groups, corresponding with thetraditional taxonomy, except one

accession which proved to be a semi-wild or transientlandrace base on the analysis of

genetic divergence.

The rational classification of the genus Camellia is yetmore contentious and elusive,

even using multidisciplinary approaches (Zhang and Ming1999; Sen et al. 2000; Vijayan

et al. 2009). Wachira et al. (1997) analysed geneticrelationship at the sectional level

using RAPD and STS, and the results were generallycongruent with those detected by

compatibility studies (Takeda 1990). Sharma et al. (2015)discovered that the classification

of beveragial tea previously into 34 species usingkaryotype analysis (Sharma and Raina

2006) was not supported by their data derived from DNAassays of both mitochondrial

and chloroplast genomes. Yang et al. (2013) sequenced thechloroplast genomes of seven

individuals, representing six Camellia species, and usedthe whole chloroplast genome

sequence variations for phylogenetic analysis; yet theresult was not in agreement with

that of any of the traditional classification systems.Regardless of the classification system

used there is no doubt that more taxonomic work isnecessary.

5.2 Origin and domestication

A good understanding of the origin and domestication of acrop species is important for

the collection and utilization of genetic resources. Mostscientists believe that the tea plant

originated in Southwest China, where diverse wild relativeshave been found in the natural

forest (Yu 1986). Evidence from studies of geneticdiversity has confirmed that Chinese tea

germplasm has more genetic variation (Yao et al. 2009,Taniguchi et al. 2014), especially

the germplasm from the original centres (Yao et al. 2012).Genetic parameters such as

allele number, gene diversity and polymorphic informationcontent, show a decreasing

trend with increasing distance from the original centres,which might indicate how the tea

plant spread in China (Yao et al. 2012). Tea cultivationstarted in India using the seeds of

C. sinensis var. sinensis, and subsequently the varietyvar. assamica spread quickly due

to its advantages in terms of local climate adaptation andcharacter for producing black

tea. Meegahakumbura et al. (2016) evaluated the geneticdiversity of a set of 392 and

Chinese and Indian tea germplasm samples consisting ofthree varieties (C. sinensis var.

sinensis, var. assamica and var. lasiocalyx) using SSRmarkers. The results showed that tea

germplasm from both countries had high levels of geneticvariation, and could be clustered

into three distinct genetic groups with significantpairwise genetic differentiation, which

was consistent with their geographical distribution. Thisindicated that the varieties of

var. sinensis, Chinese and Indian var. assamica were likelythe result of three independent

domestication events from three separate regions acrossChina and India.

C. taliensis is a unique tea germplasm, found insubtropical mountain evergreen forests

from the western Yunnan province of China to northernMyanmar. It has been cultivated

traditionally throughout western Yunnan for at leasthundreds of years. Zhao et al. (2014)

used a collation of data obtained from wild, planted andrecently domesticated populations

to investigate the domestication and geographic origin ofC. taliensis, based on the

analysis of genetic diversity and population structureusing 14 SSR. The results showed that

C. taliensis had a moderately high level of overall geneticdiversity, and domestication had

a small, non-significant influence on the geneticdiversity. A greater reduction in genetic

diversity and stronger genetic drift were detected in thewild populations, indicating

the loss of genetic diversity. The phylogenetic andstructure analysis suggested planted

C. taliensis might have been domesticated from the adjacent

central forest of western

Yunnan and then spread artificially.

5.3 Germplasm management

On the basis of accurate evaluation of the range of geneticdiversity within and between

populations, scientists can adjust collection, managementand breeding strategies to

obtain maximum variation from the gene pools (Morikawa andLeggett 1990). In the case

of Chinese tea germplasm, Yao et al. (2012) found asubstantial amount of gene flow

between adjacent populations; the overall genetic variationwas mainly contributed by

within-population variation (81%). The improved clonalcultivars had relatively low diversity

and similar genetic make-up, which indicated a narrowgenetic background caused by

artificial selection. In Africa, Wambulwa et al. (2016)confirmed international germplasm

exchange and movement among countries, and the fact that C.sinensis var. assamica

germplasm separates into two groups. These may representthe two major breeding

centres, corresponding to Southern Africa (Tea ResearchFoundation of Central Africa)

and East Africa (Tea Research Institute of Kenya). Thestudy suggested that some traits of

var. assamica and their associated genes possibly underwentselection during geographic

collection and conservation of germplasm, as well asparental selection for hybridization.

5.4 Core collection

The term ‘core collection’ refers to a subset of germplasmwith a minimum repetitiveness

and extensive coverage of the overall genetic diversity ofa crop species and its wild

relatives. Producing core collections is strategicallyimportant as it may decrease the cost

of germplasm conservation and increase germplasmutilization efficiency. Construction

of a worldwide core collection may help establish astandard system which may improve

procedures of germplasm characterization and evaluation. Byusing allele number as a

measure of genetic diversity, Taniguchi et al. (2014)developed a worldwide core collection

and three mini-core collections of tea germplasm. The corecollection comprises 192 tea

accessions, and the three mini-core collections have 96, 48and 24 accessions, respectively.

The core collection fully contains the phenotypic variationof 788 tea accessions. In Sri

Lanka, Gunasekare and Ranatunga (2008) assembled apreliminary core collection of tea

genetic resources, including a total of 500 accessions fromseveral countries. In China, a

preliminary core collection consisting of 126 teaaccessions was generated (Li and Jiang

2004), and further evaluated using RAPD markers (Li et al.2005). Using a combination

of parameters derived from morphological and moleculargenetic diversity, Liu (2008)

constructed a countrywide core collection of Chinese teagermplasm, including 360

accessions which largely covered the genetic variation of2665 accessions.

5.5 Cultivar improvement and protection

The genetic diversity preserved in germplasm collections isa precious resource for

crop breeding. Individual selection from landraces andnatural populations is one of the

important methods in tea breeding. For instance, about 60%of the nationally registered

cultivars in China have been developed from superiorindividuals selected from landraces.

High levels of genetic variation between parents increasethe possibility of creating the

unique gene combinations necessary for new superiorcultivars. The varieties C. sinensis

var. sinensis and var. assamica are traditionally thoughtto be particularly suitable for the

production of green tea and black tea, respectively.Through hybridization of two varieties,

several new tea cultivars suitable for producing both greentea and black tea have been

developed.

New cultivars are a key element in stimulating the growthof the tea industry. It is

estimated that more than 600 tea cultivars have beendeveloped worldwide to date. Plant

Variety Protection (PVP) or Plant Breeder’s Rights (PBR)are efficient strategies to promote

the development and release of new cultivars. A goodunderstanding of genetic variation

is necessary to establish appropriate guidelines for theconduct of distinctness, uniformity

and stability (DUS) tests which are the technical base ofPVP and the scientific basis for the

approval of PBR. Based on the systematic appraisal of 1500tea accessions preserved in

CNGTR, a set of 35 characteristics, including those ofplant architecture, tender shoots,

leaf blade, flower and so on, have been determined to bethe testing characteristics in

the UPOV-DUS test for tea plant (TG/238/1) (Chen et al.2008). This guideline provides

the foundation for a technical standard to examine the DUSof new tea cultivars, and may

encourage the development of new cultivars and PVP for thetea plant.

5.6 Dissection of complex phenotypes

The major goal of tea breeding is to improve productivity,enhance tolerance to biotic

and abiotic stress, and increase tea flavour and quality.Dissection of the genetic basis of

these traits will provide the potential for acceleratingthe breeding process by developing

new tools such as marker-assisted selection (MAS). However,only a few such studies have

been undertaken on the tea plant (Tanaka 1996; Yao et al.2010; Kamunya et al. 2010;

Mphangwe et al. 2013; Ma et al. 2014; Bali et al. 2015;Elangbam et al. 2016; Tan et al.

2016; Jin et al. 2016). In a successful case, Shige et al.(1993) evaluated a set of tea

germplasm, and identified a unique cultivar ‘Sayamakaori’which was strongly resistant to

mulberry scale. A linkage mapping study was subsequentlyperformed using a segregation

population derived from the cross of ‘Kana-Ck17’ and‘Sayamakaori’. The mulberry scale

resistant gene MSR-1 was then detected, and theallele-specific marker was successfully

developed and used for MAS (Tanaka et al. 2003, Tanaka2005). In another case, Jin et al.

(2016) evaluated the allelic variation of TCS1 (teacaffeine synthase 1) in a collection of tea

cultivars and wild relatives, and subsequently detected afunctional SNP affecting caffeine

content using association mapping, and then validated itusing site-directed mutagenesis.

The CAPS marker developed from this locus may be appliedfor future MAS studies.


Efforts to collect and conserve germplasm have acceleratedover the past decades,

successfully expanding the gene pool and promoting thebreeding of new tea cultivars.

Despite this, however, there are still many challengesassociated with the protection of

tea germplasm genetic diversity. The wild relatives of thetea plant are threatened by

habitat loss, due to the conversion of forest land foragricultural use and global climate

change. Furthermore, the landraces, another importantsource of genetic variation, have

been gradually been replaced by clonal cultivars, since thetea industry prefers improved

cultivars with superior characteristics such as earlysprouting, availability for machine

harvest and better tea quality. It will therefore becrucial to collect and maintain valuable

landraces and wild relatives of the tea plant in the future.

Research into the characterization and evaluation of

germplasm has improved our

understanding of the genetic diversity of the tea plant andits related species. The narrowness

of the genetic diversity of improved tea cultivars willquite possibly lead to a decline in tea

production and quality in the future. It will therefore benecessary to broaden the genetic

base of future cultivars by introducing new geneticvariations from other gene pools of wild

relatives and landraces. It is challenging, however, toefficiently identify and exploit the

valuable variation in the huge collections of geneticresources. Possible solutions include:

(1) construction of core and mini-core collections; (2)development of accurate and large

scale phenotyping and genotyping methods; (3) utilizationof multidisciplinary technologies

such as linkage and association mapping, comparativegenomics and molecular genetics to

increase the efficiency of functional gene identificationand allele mining; (4) development of

public databases and promotion of data sharing amongmultidisciplinary scientists; and (5)

germplasm exchange activities to exploit the full potentialof the genetic diversity available

globally in order to develop elite cultivars usingsystematic approaches.


Organizations

China: The Tea Research Institute of the Chinese Academy ofAgricultural Sciences

(TRICAAS, http://www.tricaas.com/English/) is the onlynational organization for tea

research in China. The institute conducts research on avariety of topics including basic

science, technological innovation and market analysis inthe tea industry.

India: The Tocklai Experimental Station (TES) of the TeaResearch Association (TRA, http://www.

tocklai.net) focuses on all aspects of tea cultivation andprocessing with the aim to improve

productivity and quality. The United Planters’ Associationof Southern India (UPASI, http://

www.upasi.org) is an apex body of planters of several cashcrops in southern India, conducting

comprehensive scientific research, and studies on marketintelligence, industrial and public

relations. The Institute of Himalayan BioresourceTechnology is a constituent laboratory of

Council of Scientific and Industrial Research (CSIR-IHBT,http://www.ihbt.res.in/en), with the

goal to boost bioeconomy through sustainable utilization ofHimalayan bioresources.

Japan: The Institute of Fruit Tree and Tea Science is apart of the National Agriculture

and Food Research Organization (NIFTS,http://www.naro.affrc.go.jp/english/nifts/index.

html) and conducts basic and innovative research in fruittree and tea science to promote

industrial development and the public good.

Sri Lanka: The Tea Research Institute of Sri Lanka(http://www.tri.lk), established in 1925,

has been the only national organization in Sri Lanka fordeveloping and expanding new

technologies related to tea cultivation and processing.

Kenya: The Tea Research Institute(http://www.tearesearch.or.ke), previously known as the

Tea Research Foundation of Kenya, was founded in 1980 topromote research related

to tea plant and companion crops, and it focuses ondevelopment of new cultivars and

technologies for the improvement of yield and quality oftea.

Database: The Chinese Crop Germplasm Information System(CCGIS, http://www.cgris.

net/cgris_english.html) is a central repository for plantgenetic resources information,

storing over 4 000 MB data on 410 000 accessions. Theinformation, including number,

distribution, and evaluation of tea germplasm conserved inthe CNGTR, can be found on

the database.

The Genetic Resources Center of the National Agricultureand Food Research Organization

of Japan(https://www.naro.affrc.go.jp/english/ngrc/index.html) isan organization dedicated

to collecting, evaluating, preserving, distributing andimproving genetic resources. The

Genebank has conserved more than 222 000 plant geneticresource accessions whose

information can be retrieved from the database.

The whole genome of ‘Yunkang 10’, a diploid elite cultivarof Camellia sinensis var.

assamica, has been sequenced and released. The informationregarding genome

assembly and gene annotation can be obtained from the Teatree Genome Database

(http://www.plantkingdomgdb.com/tea_tree/). The dataprovide the potential to develop

new strategies and methods for germplasm evaluation andbreeding.

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2 Chapter 2 Mapping and exploiting thetea genome

1 Introduction

The tea plant (Camellia sinensis [L.] O. Kuntze) is aspecies complex comprising three

caffeine-containing taxa, namely, C. ssp. sinensis (‘Chinatype’), C. assamica ssp. assamica

(Masters) Wight (‘Assam type’) and C. assamica ssp.lasiocalyx (Planch. ex Wight) Wight

(‘Cambod type’). These freely hybridize with each other andwith other closely allied but

caffeine-free species of Camellia. The tea plant, which isa dicotyledonous, perennial and

evergreen woody plant, originated in Southwest China (Yu,1986). Its tender shoots are

processed into different types of tea for drinking and arerich in secondary metabolites

such as polyphenols, alkaloids and amino acids. Long-termconsumption of tea has been

associated with protection against different cancers,cardiovascular diseases, bacterial

infections, and obesity as well as improved sleep,relaxation and cognitive performance

(Wheeler and Wheeler, 2004; Yang et al., 2007, 2016;Jurado-Coronel et al., 2016; Suzuki

Sugihara et al., 2016; Turkozu and Sanlier, 2017). Tea hasbecome the most popular non

alcoholic beverage in the world. Secondary metabolites inthe tea plant are regulated by

various metabolic pathways, and these pathways involve anumber of genes composed

of complex regulatory networks to determine the contentsand components of different

metabolites in various cultivars. Hence, understanding theregulation mechanisms of

different metabolic pathways is very useful for breedingnew tea plant cultivars with

superior health-promoting and functional properties.

Moreover, being a perennial woody plant, the tea plant hasto cope with many biotic

or/and abiotic stresses, such as low or high temperatures,drought, infertility and pest

or disease stress during its life cycle. The defenceresponses of the tea plant are usually

controlled or regulated by a series of genes involved incomplex regulatory networks.

Understanding the molecular mechanism of stress response atthe transcription level is

very important in breeding new cultivars and cultivation oftea plants.

However, being a cross-pollinated woody plant, the teaplant is self-incompatible

and has a long gestation period and juvenile phase. Thesecharacteristics result in high

genome heterozygosity and unclear genetic informationrelated to important traits of the

tea plant. This means the results of breeding are hard topredict and specific characteristics

cannot be achieved directly. Moreover, the breeding cycleof the tea plant is very long,

usually up to 15 years. Developing improved, new tea plantcultivars using conventional

breeding techniques thus takes a long time. Identifyinggenes that control important traits

of the tea plant and their heritability are vital forimproving the efficiency of any breeding

programme. Another technological bottleneck in the geneticimprovement of tea plant is

the absence of a successful genetic transformation(transgenic) system. This hinders the

exploitation of key genes involved in the regulation ofdesirable traits in tea plants and has

resulted in the pace of research into the molecular biologyof the tea plant falling behind

other model plants.

Although researchers in this area have faced manydifficulties in the past two decades,

many advances in mapping the tea genome and identifyingfunctional genes related

to desirable traits in tea plants have still been made.Especially in the last decade, the

advent of high-throughput sequencing technology and therapid development of

molecular biology technologies and functional genomics havemade possible some

remarkable achievements in this area. Recent advances intea plant genome mapping

and exploitation are discussed in this chapter, with aparticular focus on genetic linkage

mapping, quantitative trait locus (QTL) identification andbroader ‘omics’ research.

2 Progress in genetic linkage map construction andqualitative trait locus (QTL) identification for the teaplant

The tea plant belongs to a large-genome species family andits diploid genome consists

of 15 chromosome pairs (2n = 30), with an estimated size of4.0 giga bases (Tanaka et

al., 2006). This genome size is bigger than that of manymodel plant species, such as

Arabidopsis thaliana (Arabidopsis Genome, 2000); rice (Yuet al., 2002); and woody plant

species such as poplar (Tuskan et al., 2006), grapevines(Jaillon et al., 2007), oil palms

(Singh et al., 2013) and Eucalyptus grandis (Myburg et al.,2014). It also has a complex

genome with a high ratio of repetitive sequences (Xia etal., 2017).

Due to the huge size and high complexity of the genome, theknowledge of tea plant

genetics and genomics is very limited, and no genome-widestudy on any members of

the Theaceae family has so far been carried out. Thesefactors have severely hindered

the use of molecular breeding and marker-assisted selectionto accelerate breeding and

crop improvement of the tea plant. In order to acceleratemolecular-assisted selection

of the tea plant, establishing a high-density geneticlinkage map is necessary. A genetic

linkage map is vital for mapping genes and QTLs controllingdesirable agronomic traits,

marker-assisted selection, comparative genomic research andis also a crucial tool for

the assembly of the genome sequence (Ma et al., 2015).Unfortunately, due to its self

incompatibility, the tea plant can only use F1 hybridpopulations and a pseudo-test cross

strategy to construct genetic maps (Ma et al., 2010).

The early genetic maps of the tea plant were constructedusing dominant molecular

markers, such as random amplified polymorphic DNA (RAPD),amplified fragment length

polymorphisms (AFLPs) or inter simple sequence repeats(ISSRs). Then, co-dominant

markers, such as simple sequence repeats (SSRs), cleavedamplified polymorphism

sequences (CAPSs) and single nucleotide polymorphisms(SNPs), were used to construct

genetic maps in the tea plant. The first genetic map of thetea plant was reported by Tanaka

(1996). This linkage map constituted six linkage groups and59 RAPD markers. However,

due to the mapping population (46 F1 plants) and the numberof markers being very small,

the linkage groups were not matched to the chromosome pairsand the mapping results

were very crude. Hackett et al. (2000) used 90F1-generation genotypes as the mapping

population and 126 RAPD and AFLP markers as mapping markersand constructed a

genetic map consisting of 15 linkage groups with an averagemap distance of 11.7 cM.

Thereafter, Huang et al. (2005) and Huang et al. (2006)constructed tea genetic maps using

AFLP, RAPD and ISSR markers based on 69 F1 genotypes and 94first-generation back

cross genotypes. The total lengths and the average mapdistances of the two maps were

2457.7 cM and 2545.3 cM, and 11.9 cM and 12.8 cM,respectively.

Kamunya et al. (2010) reported a genetic map which wasconstructed using 100 RAPD,

AFLP and SSR markers, and contained 30 (19 maternal and 11paternal) linkage groups.

The total length was 1411.5 cM with a mean interval of 14.1

cM between loci. The yield

related QTLs were detected based on yield data over theperiod 2003–07 across two

distinct growing sites, Timbilil and Kangaita in Kenya.Twenty-three putative QTLs were

detected, but no QTL was detected at either of theexperimental sites. Other authors also

reported on genetic maps, but the individuals in themapping population used were too

small to meet the demands of locating QTLs linked withimportant traits [see the review

of Ma et al. (2010)]. The first relatively high-densitygenetic map of the tea plant was

reported by Taniguchi et al. (2012). They used a populationof 54 F1 individuals for the

linkage analysis, and the core map constituted 15 linkagegroups with a total map length

of 1218 cM. The combined map contained 441 SSRs, 7 CAPSs, 2STSs and 674 RAPDs,

although the population used for mapping was still verysmall and several large gaps

between adjacent loci in some linkage groups existed. Thismap also has a limited utility

with regard to linking QTLs to traits.

Following the development of sequencing technologies,high-throughput molecular

markers were developed and used for linkage mapconstruction. Using a set of unigene

sequences generated from the tea plant transcriptome, Ma etal. (2014a) developed 1141

new SSR markers and, together with 368 previously reportedmarkers, a total of 1509 SSR

markers were used to screen useful markers. They then

constructed a moderately saturated

genetic map using 406 SSR markers, based on an F1population of 183 individuals. The

total length of this map was 1143.5 cM, with an averagelocus distance of 2.9 cM. Twenty

five QTLs controlling catechins content were identifiedover the course of two years,

and nine stable QTLs were validated and clustered into fourmain chromosome regions.

Furthermore, this team used the same F1 population anddeveloped a total of 6042

valid SNP markers using specific locus amplified fragmentsequencing and subsequently

mapped them into the previous SSR map. The final mapcontained 6448 molecular markers

distributed across 15 linkage groups. The total map lengthwas 3965 cM, with an average

inter-locus distance of 1.0 cM. This is the most saturatedmap to date (Ma et al., 2015).

Another team at the Tea Research Institute of the ChineseAcademy of Agricultural

Sciences (TRI, CAAS) (Tan et al., 2013, 2016) establishedan SSR linkage map using the

other F1 populations based on floral transcriptomesequencing. The map contained 483

SSR markers distributed across 15 linkage groups. The totalmap length was 1226.2 cM,

with an average inter-locus distance of 2.5 cM. FifteenQTLs associated with five phenotypic

traits (timing of spring bud flush, young shoot colour,mature leaf length, mature leaf width,

leaf shape index) were identified. Xu et al. (2016)identified eight anthracnose resistance

associated QTLs in six linkage groups using the same map.

3 The progress of functional genomics in exploiting genesassociated with desirable traits

Identification of the functional genes related to desirabletraits is the first step towards a

functional genomic study of the tea plant. In the earlystudies, genes were cloned based

on the homologous cloning method in the tea plant. Thefirst gene cloning was reported

by Takeuchi et al. (1994), and three full-length cDNAsencoding chalcone synthase

(CHS; EC 2.3.1.74) were isolated. Thereafter, many genesrelated to metabolism, stress

response, development and growth, such as, theβ-primeverosidase gene (Mizutani et al.,

2002), tubulin-encoding gene (Tua1) (Fang et al., 2006),ω-3 fatty acid desaturase genes

(CsFAD7 and CsFAD8) (Ma et al., 2014b) and theanthocyanidin reductase gene (CsANR)

(Thirugnanasambantham et al., 2014) were cloned andidentified through the homologous

cloning approach. However, the cloning efficiency ofhomologous cloning is very low, and

this method cannot be used for novel gene identification.Following the development

of sequencing technology, medium- and high-throughputtechnologies were used in

the identification and cloning of batch genes, includingeven some novel genes. Using

medium- and high-throughput strategies (such as suppressionsubtractive hybridization,

RNA-Seq), many genes or gene families, and even novelgenes, were identified and

cloned, and gene expressions were analysed in differentconditions or growth stages.

These results have helped uncover desirable traits in thetea plant (Krishnaraj et al., 2011;

Wang et al., 2014b,d; Yue et al., 2015; Cui et al., 2016).These research advances can be

found in the review of Mukhopadhyay et al. (2016).

Gene function validation is another key step in functionalgenomics. There are two

types of gene functional characterizations: homologicalidentification and heterological

identification. Homological identification is superior,although the tea plant is recalcitrant

to genetic engineering and is difficult to handle usinggenetic manipulation. It has

been reported that a couple of exogenous genes have beensuccessfully transformed

into tea plants and transgenic lines obtained (Mondal etal., 2001; Mohanpuria et al.,

2011; Singh et al., 2015). Unfortunately, the success rateof genetic transformation in

the tea plant is still low and most gene functionidentifications in the tea plant have

been validated through heterological expression in modelplants, such as A. thaliana

and tobacco (Fang et al., 2013; Mahajan and Yadav, 2014;Wang et al., 2014a,c, 2017;

Ding et al., 2015). Heterological expression can indirectlyprovide useful evidence to

validate the tea plant’s gene function, but many resultsstill need homological validation

in the tea plant. Establishment of an effective genetictransformation or manipulation

technology system is the key technological link for genefunction validation in the tea

plant.

4 Progress in ‘omics’ research: overview and secondarymetabolites

4.1 Overview

Biological traits are jointly controlled by many genes, andthese genes usually form a

network to accurately regulate the corresponding phenotypiccharacters. Traditional

gene cloning and expression analysis cannot completelyexplain complex traits. High

throughput sequencing technologies make the ‘omics’strategy an effective approach

in gene discovery in different species (Wang et al., 2009;Marguerat and Bahler, 2010).

The ‘omics’ strategy is also very helpful to those speciesfor which we lack full genome

information. Use of the ‘omics’ strategy to illustrate thecomplex biological traits in the tea

plant is very effective and economical and some usefulresults, as discussed below, have

already been obtained.

4.2 Secondary metabolites

The tea plant is rich in secondary metabolites, such aspolyphenol, caffeine and amino

acids, and the quality of tea is based on its metabolitecontents and their ratios.

Understanding the regulatory mechanism of these compoundsand metabolites is

very useful for the selection of elite compound-richcultivars. Secondary metabolites

are controlled or regulated by complex gene networks. Thus,it is hard to achieve

the desired results with the use of conventional methods.Shi et al. (2011) first used

RNA-Seq technology to identify the candidate genes involvedin major metabolic

pathways, and some of the unigenes were assigned toputative metabolic pathways.

These annotations are being used in targeted searches toidentify the major genes

associated with several primary metabolic pathways andnatural product pathways,

such as flavonoid, theanine and caffeine biosynthesispathways, that govern the quality

parameters of tea. Additionally, novel candidate genesinvolved in these secondary

pathways were discovered. Another team in TRI-CAAS (Li etal., 2015) performed RNA

sequencing on 13 different tissue samples from variousorgans and developmental

stages of tea plants, and 1719 unigenes were identified asbeing involved in the

secondary metabolic pathways. The expression changes of thegenes related to

flavonoid, caffeine and theanine biosyntheses were alsocharacterized, revealing the

dynamic nature of their regulation during plant growth anddevelopment. Moreover,

the possible transcription factors, encompassing 339transcription factors from 35

families, such as bHLH, MYB and NAC, as regulation networknodes for the biosynthesis

of flavonoid, caffeine and theanine, were highlighted.These research results revealed

the possible critical check points in the flavonoid,caffeine and theanine biosynthesis

pathways, and implicated the key transcription factors andrelated mechanisms in the

regulation of secondary metabolite biosynthesis in the teaplant.

Flavonoids are a group of tea polyphenol secondarymetabolites that includes mainly

flavones, flavonols, isoflavones, flavanones andanthocyanidins (Lin et al., 1996). Catechins

are the major components of flavonoids in tea plant leaves,and account for about 70–80%

total flavonoids (Singh et al., 1999). Catechins play animportant role in promoting human

health (Moore et al., 2009; Yu et al., 2014; Brown et al.,2011; Suzuki-Sugihara et al.,

2016), and its content in leaves influences tea quality andits health effects. The flavonoid

biosynthesis pathway is very complex and the understandingof this pathway has led to

numerous physiological, biochemical and genetic studies(Punyasiri et al., 2004; Rani

et al., 2012; Liu et al., 2012); however, the accurateregulation mechanism of flavonoid

biosynthesis, especially ester catechins in the tea plant,remains unclear. In order to

elucidate the mechanisms and critical genes that regulatecatechin biosynthesis, Wu et al.

(2014) compared the transcriptome of four cultivars withdifferent catechins content, and

identified 150 unigenes assigned to the flavonoidbiosynthetic pathway. Analysis results

indicated that expression profiles of these unigenes have a

strong correlation with tea

catechins content. The cultivar ‘Ruchengbaimaocha’, whichhas low contents of (−)-EC

and (−)-EGC, also has low expression levels of ANS-1,ANS-2, ANR-1 and ANR-2. Two

LAR genes (LRA-2 and LRA-3) have a higher expression levelin the ‘Ruchengbaimaocha’,

and its (+)-C and (+)-GC contents were higher than that ofthe other cultivars. The results

indicated that the isoform of the LAR genes may control(+)-C and (+)-GC generation in

the tea plant.

According to the structural differences at the 5ʹ positionin the B ring of catechins,

tea catechins can be classified as dihydroxylated (ECG, ECand C) and trihydroxylated

catechins (EGCG, EGC and GC). The ratio of dihydroxylatedto trihydroxylated catechins

(RDTC) is an important indicator of tea quality and is usedas a biochemical marker in the

study of genetic diversity (Jin et al., 2014; Wei et al.,2015). Flavonoid 3’-hydroxylase (F3’H)

and flavonoid 3’, 5’-hydroxylase (F3’, 5’ H) are keyenzymes involved in the biosynthesis

of dihydroxylated and trihydroxylated catechins (Toda etal., 2002). Wei et al. (2015)

compared the transcriptomes of two cultivars with differentcatechins content through

RNA-Seq and identified key F3’H and F3’5’H genes,CsF3’5’H1, CsF3’H1, CsF3’H2 and

CsF3’H3, from 109 909 unigenes, which have a closecorrelation with RDTC. This result was

then validated by real-time polymerase chain reaction (PCR)

and high-performance liquid

chromatography (HPLC) analysis in 13 cultivars, and it wasfound that the co-expression of

the four genes may play a key role in affecting RDTC in teaplant.

Anthocyanin is another important anti-oxidation compound inthe tea plant, and its

accumulation causes tea leaves to turn purple. However, themechanisms of anthocyanin

accumulation in the tea plant are still unclear. Wei et al.(2016) performed transcriptome

analysis of six green or purple shoot tea samples from anF1 population and found that

2193 unigenes showed significant differences in expressionlevels between green and

purple tea samples. Of these, 1143 were up-regulated and1050 were down-regulated in

the purple tea samples. Further analysis of differentiallyexpressed genes (DEGs) involved

in anthocyanin biosynthesis and transportation showed thatthe downstream biosynthetic

genes and anthocyanin transportation-related genes wereaffected to a large extent,

but the upstream biosynthetic genes were less or notsignificantly affected. Moreover,

28 transcriptional factors were isolated from the DEGs anda CsMYB gene, which is

very similar to AtPAP1 in Arabidopsis, and were consideredto play an important role in

controlling anthocyanin biosynthesis. ‘Zijuan’ is apurple-coloured tea plant cultivar rich

in anthocyanin. It has purple-coloured tender shoots andthe leaves become green with

growth and development. Gene expression profiles of‘Zijuan’ leaves were analysed at the

purple and green stages based on HiSeq platform. Finally,2250 differentially expressed

unigenes were identified. In an in-depth analysis, it wasfound that the anthocyanin

biosynthesis pathway and carbohydrate metabolism pathwaywere enriched through the

KEGG metabolic pathway analysis of the DEGs. Some keyenzyme genes, for example, the

sucrose synthase gene and the acetyl-coenzyme A carboxylasegene, were up-regulated in

purple leaves, and the catalytic product of these enzymesmay provide more intermediates

as substrates to promote anthocyanin accumulation (Li etal., 2017).

L-theanine is a unique non-protein amino acid and also thepredominant amino acid in

tea, accounting for about 50% of the total free amino acidsof the tea leaves. Numerous

enzymes, such as L-theanine synthetase (TS), glutaminesynthetase (GS), alanine

transaminase (ALT) and L-alanine decarboxylase (AIDA),participate in the metabolism

of theanine (Deng et al., 2008). L-theanine content is alsoregulated by some structural

genes (Deng et al., 2008, Li and de Silva, 2010). On thebasis of RNA-Seq data, Liu et

al. (2017) analysed the relationship between 17 genes thatencode enzymes (CsTS1,

CsTS2, CsGS1, CsGS2, CsGOGAT-Fe, CsGOGAT-NAD(P)H, CsGDH1,CsGDH2, CsALT,

CsSAMDC, CsADC, CsCuAO, CsPAO, CsNiR, CsNR, CsGGT1 andCsGGT3) involved in

theanine metabolism and theanine content of three teacultivars with different theanine

contents, and found that the theanine contents showed closecorrelations with related

gene expression levels among cultivars. They also foundthat among different tissues,

the transcript levels of CsTS2, CsGS1 and CsGDH2 showedpositive correlation with the

theanine contents, while the other genes showed negativecorrelation with the theanine

contents in most cases.

5 Progress in ‘omics’ research: stress response anddormancy

5.1 Stress response

As mentioned earlier, the tea plant has a long life cycle.It undergoes many types of

abiotic and biotic stresses, such as temperature stress,water stress, pest or disease stress,

etc. The tea plant can adapt to these stresses throughstress response; stress responses

are complex biological phenomena and their phenotypesdepend on complex network

regulations. The ‘omics’ strategy provides powerfulresearch tools to understand these

complex biological phenomena.

Low temperature is one of the most critical environmentalfactors that restricts plant

growth, survival and geographical distribution (Wang etal., 2012). Thus, finding a way

to improve tea plants’ resistance to low temperatures is ofgreat importance. Like other

perennial evergreen woody crops, the tea plant can improve

its cold resistance by

undergoing a so-called cold acclimation (CA) procedure.During the CA process, the

cold tolerance of the tea plants can be enhanced at lowertemperatures. Following

an increase in the temperature, the tea plant enters into ade-acclimation phase and

its cold tolerance is reduced (Wang et al., 2013). In orderto illustrate the molecular

mechanism of CA in tea plants, Wang et al. (2013) usedRNA-Seq in combination with

digital gene expression technologies to study thetranscriptome profiles in the tea plant,

and thereby gained a deeper insight into the molecularmechanism of CA. In total, 1770

differentially expressed transcripts were identified, ofwhich 1168 were up-regulated and

602 down-regulated. These transcripts are involved in coldsensor or signal transduction,

cold-responsive transcription, plasma membranestabilization, osmosensing-response

and enzyme detoxification. In addition, two pathways, thecarbohydrate metabolism

pathway and calcium signalling pathway, were identified andit was predicted that they

might play a vital role in the tea plants’ responses tocold stress. A putative mechanism

for a tea plant responding to low temperatures during theCA process was put forward

based on the global transcriptome changes. According tothese results, Yue et al. (2015)

further reported that during the CA period, sugarmetabolism-related genes exhibited

different expression patterns, in which the beta-amylasegene (CsBAM), invertase gene

(CsINV5) and raffinose synthase gene (CsRS2) engaged instarch, sucrose and raffinose

metabolism, respectively, were solidly up-regulated; theexpressions of sugar transporters

were stimulated in general except the down-regulations ofCsSWEET2, 3, 16, CsERD6.7

and CsINT2.

Drought stress (DS) is another important abiotic stressthat limits the growth and

development of the tea plant and affects its yield andquality. Liu et al. (2016) performed a

transcriptome analysis of the tea plant during the threestages of DS, and 5955 DEGs from

the three stages were identified. Among them, 3948 and 1673DEGs were up-regulated

under DS and RC (recovery after DS), respectively. Theyfound that genes involved in abscisic

acid, ethylene and jasmonic acid biosynthesis andsignalling were generally up-regulated

under DS and down-regulated during RC. In addition, proteinkinase genes, mannitol,

trehalose and sucrose synthesis-related genes andtranscription factor genes belonging

to 58 families were identified and were found to presentdifferent expression patterns at

different DS stages. Another experiment conducted by Tonyet al. (2016) showed that heat

shock proteins (HSP70), super oxide dismutase (SOD),catalase, peroxidase, calmoduline

like protein (Cam7) and galactinol synthase (Gols4)drought-related genes were shown to

be regulated differentially in tea plants when exposed towater stress. Moreover, HSP70

and SOD had higher expression levels in thedrought-tolerant cultivar relative to the

susceptible cultivar under drought conditions.

Ectropis obliqua, a chewing insect, is one of the mostcommon pests of the tea plant

in China. It always causes severe damage to the tea plantand leads to serious economic

loss. The tea plant can react by inducing an array of localand systemic defences to

protect itself against pest attacks (Li et al., 2008, Wanget al., 2016c). To elucidate

the resistance mechanism of tea plants in response toEctropis oblique, Wang et al.

(2016c) performed differential transcriptome analysis usingEctropis oblique damage

induced tea plants with undamaged plants as control.Finally, 949 up-regulated and 910

down-regulated genes were identified. These genes arerelated to signal transduction,

anti-insect responses, phenylpropanoid biosynthesis,herbivore-induced plant volatile

biosynthesis and caffeine biosynthesis. Furthermore, twopathways, the plant secondary

metabolites and the signalling pathways, may play animportant role in defence against

insects in the tea plant.

Anthracnose disease, caused by the Colletotrichum species,is one of the most

serious diseases of the tea plant in China and Japan(Yoshida and Takeda, 2006; Wang

et al., 2016b). It causes severe damage, resulting in yield

loss and quality decrease

in tea production. Different tea plant cultivars orgermplasms have different disease

resistance mechanisms (Yoshida and Takeda, 2006). Breedingresistant cultivars is

the most efficient and economical strategy to reduce damagefrom diseases, so

understanding the tea plant’s defence mechanisms isimportant for breeding resistant

cultivars (Wang et al., 2016a). Wang et al. (2016a)compared ‘ZC108’, an anthracnose

resistant cultivar, and its parent cultivar ‘LJ43’, asusceptible cultivar, by microarray

analysis and found many genes involved in secondarymetabolism-related pathways,

plant hormone biosynthesis and signalling andplant-pathogen interaction pathways

were up/down-regulated.

Blister blight disease is another serious tea plant diseasecaused by Exobasidium vexans

Massee, which significantly affects commercial productionin major tea-producing countries

(Priyadarshin et al., 2012; Sinniah et al., 2016).Transcriptomes blister blight-resistant

and -susceptible tea plant genotypes were analysed byJayaswall et al. (2016) and 149

defence-related transcripts/genes, includingdefence-related enzymes, resistance genes,

multidrug resistant transporters, transcription factors,retrotransposons, metacaspases and

chaperons, were observed in the resistant genotype. Theresults indicated these genes

may play important roles in defending against blister

blight disease.

5.2 Dormancy

Bud dormancy in winter is an adaptive mechanism that helpstea plants cope with

unfavourable environments, especially at low temperatures.Winter dormancy of the

tea plant is mainly affected by temperature and photoperiod(Barua, 1969), with other

factors such as hormone interaction and nutrient levelsalso influencing bud formation.

On the other hand, the budbreak time of the tea plant inspring is an important economic

trait that influences income levels in green tea-producingcountries, especially in China

(Wang et al., 2014b). Thus, illustrating the molecularmechanism of bud dormancy

and budbreak is very important for tea plant breeders, andis also very helpful for

uncovering the mechanism of the evergreen woody species.Paul et al. (2014) compared

the transcriptomes of two tea plants and a bud sampled inJuly (the period of active

growth, PAG) and December (winter dormancy), and a total of5204 DEGs were identified

between PAG and winter dormancy. Further analysis showedthe operation of the

mechanisms of winter dormancy involved in permitting thetea plant to tolerate winter

cold through up-regulating stress tolerance-associatedgenes and minimizing growth by

down-regulating the genes involved in growth, development,protein synthesis, DNA

processing and cell division. Moreover, they found that the

tea plant does not allow

leaf abscission due to the modulation of leafabscission-related genes during winter

dormancy. Hao et al. (2017) analysed the global geneexpression profiles of axillary buds

at the paradormancy, endodormancy, ecodormancy and budflush stages, and 16 125

DEGs were identified from the different samples. Gene setenrichment analysis indicated

that epigenetic regulation, phytohormone signallingpathways and callose-related

cellular communication regulation were the major mechanismsthat were involved in bud

dormancy and budbreak in the tea plant.

6 Conclusion and outlook

Here, we have reviewed the current status of tea plantgenome mapping and exploitation,

especially the advances in plant ‘omics’ related todesirable traits. Significant advances in

this area have been made. An increasing number of genesthat are related to secondary

metabolites, stress response, dormancy and so on wereidentified, their functions were

verified and the correlations between genes and relatedtraits were established. But, the

level of research relating to the tea plant is still farbehind that of other plant species.

The first genome sequence of the tea plant with a size of3.02-Gb was reported recently

by Xia et al. (2017). The genome provides the foundationfor revealing the genetic

basis of important desirable traits in the future and willaccelerate further studies on

the functional genomics of the tea plant. However, in thenear future, more emphasis

should be placed on the following issues to improve thelevel of tea plant genome

research: (1) Construction of a more accurate referencegenome and perfection of the

gene annotations. (2) Conducting more re-sequencings onsome trait-specific cultivars

and obtaining exact genetic regulation information ofdesirable traits. (3) Obtaining more

molecular markers and establishing a highly saturatedgenetic map to identify locations of

QTLs. (4) Establishing a high-efficiency genetictransformation system and/or gene editing

technology to fulfil gene function homologicalidentification and directional improvement

of tea plant traits.


In order to accelerate the development of tea plant genomeresearch, more attention

needs to be paid to these issues:

1 The published genome sequence of tea plant was sequencedby Illumina HiSeq2000 platform, due to its short readlength and the high repetition ratio of tea plant genomesequence; so this sequence is not good enough to be used asa high-quality reference genome. Following the fastdevelopment of sequencing technology, a third-generationsequencing technology such as single molecular sequencingtechnology can be used for tea plant genome sequencing andre-sequencing in the future for drawing high-qualityreference genome.

2 Though the published tea plant genome sequence wasn’tperfect enough, it was still the milestone of genomeresearch of tea plant. In order to obtain the exact geneticregulation information of desirable traits in tea plant, wecan do many re-sequencings of trait-specific cultivars and

help to annotate the unique genes in tea plant.

3 Many genetic maps of tea plant have been established, butthe density still isn’t saturated enough for elaborate QTLlocation. Although lots of molecular markers have beenidentified, the useful markers that have close linkage withdesirable traits are absent. Next, we can depend on theever-increasing sequence information to identify more andmore markers and construct large F 1 populations forestablishing a more saturated genetic map and find moreclosed linkage markers to benefit molecular assistantbreeding in tea plant.

4 Gene function homological identification is the keytechnological bottleneck in the development of tea plantmolecular biology research. We must improve the genetictransformation efficiency and develop new approaches suchas RNA silencing and gene editing technology (e.g. Cas9)to achieve the goal of molecular design breeding in teaplant.

8 Acknowledgements

This work was supported by the Chinese Academy ofAgricultural Sciences through an

Innovation Project for Agricultural Sciences and Technology(CAAS-ASTIP-2017-TRICAAS),

the Earmarked Fund for China Agriculture Research System(CARS-19) and the Major

Project of Agricultural Science and Technology in Breedingof Tea Plant Variety in Zhejiang

Province (2016C02053-4).

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3 Chapter 3 Advances in geneticmodification of tea

1 Introduction

Tea is one of the most important beverages that we consumevery frequently. Tea is

manufactured from the young tender leaves of Camelliasinensis (L.) O. Kuntze of family

Theaceae. However, cultivated taxa comprise three mainnatural hybrids such as China

type [C. sinensis (L.) O. Kuntze] or Assam type [Camelliaassamica (Masters)] and Cambod

or Southern type [C. assamica spp. lasiocalyx (Plan- chonex Watt.)]. Apart from that it

is believed that that some desirable traits such asanthocyanin pigmentation or special

quality characters of Darjeeling tea might have beenintroduced from wild species such

as Camellia irrawadiensis and Camellia taliensis (Wood andBarua, 1958). It is an open

pollinated woody plantation crop producing cheapernon-alcoholic drink that has high

medicinal value. Morphologically, it is an evergreen shrubcultivated in a wide range of

climate, starting from humid and sub-humid tropical,sub-tropical to temperate countries,

and grows largely on well-weathered acidic soils, with pHrange of 4.5–5.5 (Mukhopadhyay

and Mondal, 2014; Mukhopadhyay et al., 2016).

2 Conventional tea propagation

Seeds, vegetative cuttings and nursery graftings are theprincipal mode of propagation of

tea aside from budding or grafting of adult plants, whichis rarely practised. Conventional

tea breeding is difficult owing to its perennial life cycleand long gestation period

(4–5 years). Earlier seeds used to be the only commercialmethod of propagation.

Owing to its allogamous nature, the seed-borne plants arefound to be heterogeneous

which create problem to sustain their superiorcharacteristics. However, because of wide

variations of certain characteristic features, such asyield and quality, planters are forced to

shift to vegetative cuttings of elite genotypes (Mondal,2014). Additionally, non-availability

of dependable selection criteria is a major limitation oftea breeding. Hybridization in tea

breeding is done by natural or hand pollination; however,low reproducibility has rendered

this technique less acceptable. Alternatively, selection ofsuperior existing planting

materials is an age-old practice in tea breeding. Majorityof the tea clones have been

developed through selection in spite of unknown pedigreesof the clone (Mondal, 2014).

On the other hand, vegetative cutting is a much establishedway of breeding, but poor

rooting ability of some clones and requirement of ampleplanting materials within a short

period are few bottlenecks of this technique (Mondal,2011). Micrografting of in vitro

raised tea shoots on young seedlings raised root stock ofidentical maturity can reduce

the prolonged time period necessary for hardening, which isattributable to quicker root

growth and proliferation of the scion (Mondal et al., 2005).

Thus, the protracted breeding cycle and outbreeding natureof tea prevent the use

of conventional breeding methods to incorporate genes ortraits without altering the

rest of the genome. The only option to insert a gene ofdesirable trait into a proven

genotype is genetic transformation. Once a geneticallymodified plant is produced,

vegetative propagation offers faster multiplication withoutany changes in the transformed

genome. These two important advantages can be capitalizedin tea with regard to genetic

modification.

3 The need for genetic transformation

Because of various socio-economic reasons, extension fornew cultivation is limited. In a

number of countries, yield has declined significantly dueto ageing of the plantation. The

equilibrium between availability of land for expansion oftea cultivation and proportionate

increase in tea consumption are literally unattainable.Furthermore, going forward such a

scarcity of cultivable land would be predictably muchhigher. Hence, a boost in productivity

could be a pragmatic approach of increasing cropproduction. Tea being a monoculture

necessitates minimization of insect and disease linkedlosses that are currently estimated

to be 14% of the total production. Drought is another vitaland recurrent limiting factor of

tea cultivation in the tea-growing countries, which, at anaverage, incurs around 40% of

crop loss (Das et al., 2012). Apart from that, teacultivation being monoculture is always

amenable to pest attack; therefore, additional costs areincurred in the form of pesticides

applied for pest control. Immense applications ofpesticides intensify detrimental residues

apart from causing adverse effects on beneficial organismsand the environment. A lot of

species of insects, pathogens and weeds have already builtup resistance against existing

chemicals. In order to mitigate these complexities, novelexplorations in allied fields and

improvement of technological applications are the need ofthe hour, and development of

high-yielding clones that offer better quality and superiorstress tolerance ability would be

the most preferred variety. Because of perennial life cycleof tea plants, it often encounters

various biotic and abiotic stresses (Mukhopadhyay et al.,2012, 2013a,b). In addition to

these, winter dormancy, dormancy between flushes, quickerseed germination and growth

vis-à-vis development are some of the important charactersthat need to be manipulated.

However, long life cycle and outbreeding nature causes anumber of restrictions in the

direction of its genetic improvement. Furthermore,morphological distinctiveness does

not exhibit the in-built heritable difference within thecrop.

In tea natural haploids do not exist. Simultaneously, it isvery difficult to produce pure

line homozygous in vitro. Apart from that, limited mutantsare available for making

conventional crosses. Conventional tea breeding islaborious and difficult due to longer

gestation period, self-incompatibility and unavailabilityof discrete mutant against a variety

of biotic and abiotic stresses, little success of handpollination, short flowering time and

long duration for seed maturation.

Furthermore, marker-assisted selection is not possible dueto lack of robust genome

wide polymorphic DNA markers. Therefore, geneticimprovement in tea is extremely

essential to reveal the unidentified genetic variations.The continuous impoverishment

of tea gene pool has become a significant problem (Jain andNewton, 1990). Due to the

reduction of genetic variation, the variety gradually losesthe ability to overcome biotic

and abiotic stresses and also change the capability to copewith immediate challenges

like pathogen invasion. Thus detection and assessment ofgenetic erosion remains the first

priority in any major effort to arrest loss of geneticdiversity (Hammer and Teklu, 2008).

Therefore, abatement of genetic erosion along with geneticimprovement is unavoidable

to create and sustain superior clones.

Besides broadening the gene pool, genetic engineeringexploits a number of desirable

genes in a single event and reduces time required to insertnovel genes into elite varieties.

Investigation of transgenic crops intends to selectivelymodify, insert or eliminate a suitable

character of a plant that is appropriate with the need anddevelopment opportunities.

It offers incorporation of desirable characteristics fromthe unrelated species, apart from

the opportunity of introducing a desirable character fromclosely related species. After

successful completion of the transformation event, thetransformed plant serves as a

parent for utilization in future breeding programmes.

Thus, development and utilization of transgenic plants willbe an efficient method for

sustainable use of biotechnology for improvement of tea.

4 Transformation systems

Plant genetic transformation is a potent device tomanoeuvre the genome of an organism

for obtaining a desirable characteristic. There are morethan a few ways to insert foreign

genes into other plants, such as direct transformations,including microinjection,

electroporation, polyethylene glycol-mediated transfer andparticle bombardment.

Agrobacterium-mediated transfer is an indirecttransformation method. This indirect

transformation method has been extensively used in severalplant species due to several

advantages.

Agrobacterium-mediated genetic transformation is one of themost widely used techniques

of transgenic plant production. The natural ability of thephytopathogen for gene transfer to

other plants has been exploited. It is cheaper and simplerthan the direct transfer method

and permits little rearrangement of transgene and offersefficient insertion of transgene

into the genome of the desired plant (Sandal et al., 2007).Both direct and indirect DNA

delivery systems can be utilized to transform tea.Successful transformation technique via

Agrobacterium-mediated genetic transformation symbolizes anoteworthy step to prevail

over existing constraints in tea improvement programmes.Perhaps one of the major

limitations has been the lack of a suitable method foradventitious regeneration directly

from explants. Thus, successful genetic transformation oftea requires suitable explants,

minimization of bactericidal effect of polyphenols,Agrobacterium vir gene expression and

retention of shoot regeneration capacity of the transformedplants (Sandal et al., 2007).

On the basis of disease symptomology and host range, thegenus Agrobacterium

is classified into a number of species. Among them,Agrobacterium radiobacter is an

‘avirulent’, whereas Agrobacterium tumefaciens causes crowngall disease. Hairy root

disease is caused by Agrobacterium rhizogenes andAgrobacterium rubi causes cane

gall disease. Agrobacterium-mediated genetic transformationis carried out by a specific

plasmid, which is found in only a small percentage ofnatural populations of Agrobacterium.

However, curing a particular plasmid and replacing withanother type of tumorigenic

plasmid can alter disease symptoms (Gelvin, 2003).

5 Methods of transformation

The molecular basis of genetic transformation of plantcells by Agrobacterium is based on

transfer of a particular region of the plasmid (tumourinducing, Ti or root inducing and Ri

plasmid) from the soil bacterium and integration into thenuclear genome of the plant. In

general, Ti plasmids range from 200 to 800 bp in size ofthe plasmid, which is transferred

to the bacterium, is known as transferred DNA (T-DNA). TheT-regions on native Ti and Ri

plasmids usually correspond to less than 10% of the entireplasmid with a size of 10 to 30

kbp approximately. In general, Ti plasmids contain oneT-region (Barker et al., 1983). T-DNA

has two different kinds of genes; the oncogenic genes arethose that encode the enzymes

involved in the synthesis of hormones like auxin andcytokinin, which are accountable

for tumour formation. Apart from that, there are genes thatencode opine synthesis. The

opines are generated by condensation reactions betweenamino acids and sugars, and

are synthesized and exuded by the crown gall cells. Theseare utilized by the bacterium

as carbon and nitrogen sources. The opine catabolism genesare located outside the

T-DNA region. Additionally, the genes involved in T-DNAtransfer from Agrobacterium to

the host cell and the genes responsible forbacterium-to-bacterium conjugative transfer

of plasmid is located in the region that flanks T-DNA(Zupan and Zambrysky, 1995).

T-regions are delineated by the border sequences of T-DNA,which are 25 bp in length

and are extremely homologous sequences (Jouanin et al.,1989). The T-region is flanked

by 25 bp repeat sequences which are required to directT-DNA processing. Thus, any DNA

segments between these border sequences are transferred tothe plant cell (Zupan and

Zambrysky, 1995).

The export of the T-DNA region from the plasmid andconsequent insertion from the

bacterium to the host cell is generally controlled by theproducts encoded by the virulence

(vir) genes reside in the Ti plasmid (Hooykaas et al.,1984). These genes are regulated in

such a way that expression occurs simply under theinfluence of wounded plant cells. VirA

and VirG proteins function as sensory elements of atwo-component signal transduction

system. VirA senses the phenolic compounds discharged bywounded plant cells and as a

consequence autophosphorylated. In the next step, VirAphosphorylates VirG, which then

turns out to activate the transcription of vir genes. Uponstimulation, vir genes generate a

transfer intermediary. The intermediate is principally theT-strand, which is a single-stranded

copy of T-DNA (Stachel et al., 1986). This process alsorequires the participation of VirDl

and VirD2 (Filichkin and Gelvin, 1993). This is becausethey identify the border sequences

and generate border-specific endonuclease to cut the bottomstrand of both borders,

which are used as the sites for initiation and termination.VirD2 remains firmly attached to

the 5’ end of the T-strand subsequent to nicking and thusthe T-complex obtains a polar

nature that serves the similar purpose as leading end does.In order to make infection

successful, the T-strand must pass through a number ofbiological membranes and

associated spaces before its insertion in the host cellnucleus and virE locus plays a vital

role in the process. The virE locus encodes an induciblesingle-stranded nucleic acid

binding protein, entitled VirE2, which binds without anysequence specificity. VirE2 binds

with the T-strand, coats it entirely and renders resistanceagainst nucleolytic degradation.

VirE2 binding opens up the single-stranded DNA to such anextent that it can pass through

the biological membranes and get integrated into the hostchromosome.

Both Ti and Ri plasmids bear similarity in structure, inthe molecular mechanisms of

T-DNA processing and transformation to plant cells.However, oncogenesis in hairy root

disease varies considerably from crown gall disease. Crowngall tumours seldom regenerate

plants competent of rooting. Regenerated plants from thesetumours indicated deletion

of majority of T-DNA sequences or inactivation of geneexpression due to methylation of

T-DNA. However, hairy root tumours naturally regenerateplants, although with altered

morphology, but express T-DNA genes (Gelvin, 1990). A.rhizogenes transport T-DNA

through a type IV secretion system. Some A. rhizogenesstrains do not possess VirE2;

nevertheless, transfer of T-strands takes place with thehelp of the GALLS gene. Full-length

GALLS have nuclear localization sequences to target theproteins to the host cell nucleus.

Full-length GALLS contains ATP-binding and helicase motifsresembling TraA, which is

a transferase engaged in conjugation. Concentration offull-length GALLS builds up in

the nucleus following which ATP-dependent strandtransferase gathers T-strands in the

nucleus (Ream, 2009).

5.1 Agrobacterium tumefaciens

A. tumefaciens can transfer DNA to a wide group ofangiosperms, including a number

of dicots and monocots. Transformation by means of A.tumefaciens has been the most

acceptable technique in woody plants like C. sinensis.However, lower transformation

efficacy, along with recalcitrant regeneration system, isthe main cause of slow progress of

transgenic tea. This particular impediment is attributableto the presence of higher levels

of polyphenols in tea explants (Mondal, 2014). GUS(β-glucuronidase) assay indicates

that transient transformation efficiencies of tea leavesare, to a great extent, lesser than

that of tobacco, which is attributable to inhibition bycatechins present in tea leaves.

Lower transformation efficiencies also signified that otherbiochemical factors perhaps

exert inhibitory effects on transformation of tea (Song etal., 2014). However, genetic

transformation of tea plants through A. tumefaciens hasbeen attempted by several

workers using different explants such as in vitro leavesand somatic embryos (Matsumoto

and Fukui, 1998, 1999; Biao et al., 1998; Mondal et al.,1999, 2001b; Luo and Liang, 2000).

In case of tea, leaf explants are more desirable thancotyledon-derived somatic embryos,

particularly when it is essential to promote selected elitevarieties and in addition to

preserve its superior traits. Genetic transformation ofleaf explants facilitates introduction

of the desired genes for better adaptation, quality andyield into the selected genotypes

with simultaneous preservation of clonal fidelity (Sandalet al., 2007). Adventitious shoot

regeneration by using callus phase from in vitro leafexplants was reported by Sandal et al.

(2005). Adventitious shoot buds developed on leaf explantsdue to prolonged culture

on MS (Murashige and Skoog) medium supplemented with2,4-dichlorophenoxyacetic

acid (2,4-D). Gas chromatography analysis and histologicalstudies of the tissues during

developmental stages showed that specific levels of 2,4-Dpromoted morphogenesis.

It was also found that shoot buds grew on rhizogenic calliunder untraceable or insignificant

concentrations of tissue 2,4-D probably due to metabolismor detoxification. Alternatively,

shoot buds also developed after rhizogenic calli weretransferred to medium supplied with

lower concentration of 2,4-D (Sandal et al., 2005).

With an initial attempt of producing transgenic tea plantswith somatic embryos

as explants by Mondal et al. (2001b), stable transformedcalli from leaf explants were

developed by other groups (Matsumoto and Fukui, 1989, 1999;Biao et al., 1998; Mondal

et al., 1999; Siswanto and Chaidamsari, 1999). Among them,Siswanto and Chaidamsari

(1999) observed that pre-culturing of explants had noeffect on the transformation

efficiency. However, bacterial growth phase, cell density,co-cultivation periods and pH of

the medium were important factors to improve thetransformation efficiency considerably.

These parameters were optimized based on GUS activity thatemerged on the embryos

after a co-cultivation period of 48h. Intense GUS positivesignals were too perceived from

the leaf tissues of 1-year-old tea plants recovered viagermination of somatic embryos that

were resistant to kanamycin. On the other hand, noendogenous signal of GUS expression

was recorded in the untransformed somatic embryos ortissues. Acetosyringone was not

able to increase the transformation efficacy of tea (Mondalet al., 2001b); nevertheless, a

concentration of 500 µM enhanced transformation efficiencyin the same plant (Matsumoto

and Fukui, 1999). Although the reason is not clear,difference in genotype may contribute

to this result. Furthermore, transformation efficiency wasaffected by two more important

factors, namely efficiency of selection of transgenictissues on antibiotics such as kanamycin

and hygromycin, and an efficient application of antibioticsthat are bactericidal. It was

suggested that a dose of 50 mg l −1 kanamycin followed byan increase of concentration to

75 mg l −1 was the most advantageous for the effectiveselection of transformants for tea

somatic embryos (Mondal et al., 2001b). However, a dosageof 75 mg l −1 kanamycin for

selection of internode explants was found to be lethal forCamellia japonica (Tosca et al.,

1996). But when leaves were used as explants, 200 mg l −1kanamycin was observed to be

useful (Matsumoto and Fukui, 1999). Later on, thetransgenic microshoots were multiplied

in vitro and simultaneously micrografted on theseedling-derived young root stocks of the

same cultivar (Mondal and Parathi, 2003).

Inadequate systematic attempt has been made on theconsequence of bactericidal

antibiotics on tea transformation. The bactericidalantibiotic sporidex at 400 mg l −1 not

only ascertained a total control of the A. tumefaciensovergrowth but also established

poor effect on the growth retardation of the somaticembryos. Apart from sporidex,

carbenicillin was also found to be effective to controlbacterial growth. Thus, sporidex

together with carbenicillin was favoured due to reasonablelow price and easy availability

in medical stores (Mondal et al., 2001b). It has been foundthat the alteration of the

transformed in vitro leaves to calli formed a mucilage-likecompound enveloping the

calli that deter further development. ProbablyAgrobacterium overgrowth occurred

due to improper control after co-cultivation stage and thusa mucilage-like compound

developed on the explants (Siswanto and Chaidamsari, 1999).Steady transformations

in callus following molecular characterization bypolymerase chain reaction (PCR) and

Southern hybridization were reported by Matsumoto and Fukui(1998, 1999), and stable

integration of transgene after molecular characterizationthrough Southern hybridization

was confirmed (Mondal et al., 2001b). It has been observedthat 41.67% (5 out of 12)

kanamycin-resistant, GUS positives somatic embryos providedPCR amplification

products of 693 bp and 650 bp with neomycinphosphotransferase II (npt-II) and gus

gene-specific primers, respectively, indicating that themarker gene npt-II was linked

with gus as a solitary ‘T-DNA strand’ in the genomic DNA ofthe transformed plant.

Absence PCR amplification products in some lines may beaccredited to the presence of

‘false positives’ during antibiotic selection. Later on theleaves of the five independent

transgenic plants were subjected to Southern hybridization.Genomic DNA of all of the

five presumed transgenic lines digested by Pst I producedan internal transgene fragment

of 1.6 kb, which was hybridized to the probe (npt-II).

Additional smaller fragments of

some transgenic lines however specified a deletion ofnpt-II that presumably occurred

during transformation or subsequent regeneration. Diversebanding patterns observed

by Southern blotting may be attributed to manifoldinsertions, rearrangements and/or

deletions of the transgenes that were integrated in theregenerated plants, which is very

frequent to A. tumefaciens mediated transformation (Mercuriet al., 2000). A survival rate

of 80–90% was attained under greenhouse conditions.

In a separate experiment, vector containing Bt gene wasconstructed in order to

transform tea plants. Briefly, the plasmid having Bt gene,an intron (gus) and nptII was

transformed into Escherichia coli and inserted intoAgrobacterium strains via tri-parental

mating which was then used to transform the tea calli.Transient expression of gus in

transgenic calli and leaves of presumed transgenic plantswas identified. It was found

that 20 g l −1 and 50 g l −1 as optimal quantity ofhygromycin and kanamycin, respectively,

for screening tea leaves as in vitro explants.Nevertheless, no transgenic plants could be

generated (Luo and Liang, 2000).

Interestingly, secondary somatic embryos that were producedfrom the Agro-infected

primary embryos, found to be positive for nptII and gusAgenes, were later regenerated

into plantlets. The transformation frequency of A.tumefaciens with binary vector, based

on antibiotic selection, was found to be 3.3%. GUShistochemical assay, PCR and

Southern hybridization analysis of antibiotic-resistantplantlets were substantiated for

transfer of exogenous gene (Singh et al., 2014). PlasmidpBi121 contains a selectable

marker gene nptII, which provides kanamycin resistance, andβ-glucuronidase (uidA),

which is a reporter gene. This plasmid was used as binaryvector to co-cultivate with

A. tumefaciens. The maximum rate of transformation, thatis, 12 transformants g –1 fresh

weight [FW], was achieved with 5 d old tissues raised inliquid medium when co-cultivated

with Agrobacterium for 2 d in the same medium. Increasedrecovery of kanamycin

resistant tissues was observed when those were initiallygrown for 10 d on a medium

having 350 mgL –1 Timentin to thwart excessive bacterialgrowth but resistant tissues

obtained after 6 weeks on kanamycin-selection medium showedsteady uidA expression.

PCR and Southern blotting authenticated the integration ofgenes. Transgenic plants

were successfully regenerated from transformed tissuesfollowing co-culture (Jeyaramraja

and Meenakshi, 2005). In tea, leaf explants are preferredover cotyledon-derived somatic

embryos, when it is essential to improve the elite varietyand retain its superior traits. In

view of this, an Agrobacterium-mediated transformationmethod, based on the presence

of l-glutamine in the co-cultivation medium, was developed.

It was demonstrated that

transformation efficiency was increased through a shieldingaction of l-glutamine against

bactericidal effects of leaf polyphenols without disturbingthe expression of bacterial

virulence gene (Sandal et al., 2007).

It was seen that rate of Agrobacterium infection wasdependent on genotype of the tea

cultivars. In those cultivars, the extent of infection wasinfluenced by leaf wetness, micro

morphology and surface chemistry. Leaves with glabroussurface, high phenol content and

wax were found to be much more susceptible forAgrobacterium infection. Interestingly, it

was found that caffeine content of tea leaf promotedAgrobacterium infection. Conversely

caffeine-free wax inhibited both bacterial growth andinfectivity. Thus, this study indicated a

foundation for the screening of a clone or cultivar whichis most suitable for Agrobacterium

infection (Kumar et al., 2004).

Though Agrobacterium-mediated gene transformation is a keytechnology for achieving

introgression of transgene, intricate patent issues relatedto the use of Agrobacterium and

the necessity of creating transgenic plants can offercomplications in using the technology

for agricultural advancements in a number of plant species.Therefore, current studies

have proved that virus-based vectors are also efficient andcan be used for high transient

expression of foreign proteins in transgenic plants (Chunget al., 2005).

5.2 Agrobacterium rhizogenes

A. rhizogenes, the causal agent of the ‘hairy root’disease, is distinguished by its ability

to develop profuse root growth in the susceptible hosts atthe site of infection. This

phenomenon of root proliferation on hormone-free mediaunder in vitro conditions

has been comprehensively utilized in a wide range of plantspecies, especially for

the production of secondary metabolites. Zehra et al.(1996) were the first to use this

technique of transformation in tea, whereby in vitro leaves(35 days old) were co-cultured

with A. rhizogenes. The explants were then co-cultured withbacterial cells for a couple

of days. After removing bacterial solution, leaves werecultured on MS media for

inducing hairy roots. Mannopine formed in the roots werestudied by electrophoresis

to corroborate stable integration of the gene. In vitrodeveloped tea shoots of 4–6

months old were transformed by co-cultivation in liquidbasal MS media supplemented

with IBA and an antibiotic rifampicin. Roots were developedfrom the basal end in 66%

of explants, only after 32–45 days of culture, and it wasfollowed by hardening of the

micro shoots in nursery beds (Konwar et al., 1998). Leavesof transformed tea plants were

found to exhibit higher transformation efficiency (up to70%) in presence of 300 µM L –1

acetosyringone. It was also found that MS mediasupplemented with maltose and

indole-3-acetic acid accumulated higher quantity ofphenolic compounds. Amounts of

polyphenols and catechins were higher in transformed rootscompared to untransformed

leaf tissues. Thus it opened up an avenue of commercialexploitation of secondary

metabolites (John et al., 2009). To curtail the time periodrequired for regeneration of

tea, a quicker, well-organized and economic roottransformation system was developed.

The root elongation zone of tea seedlings (1 month old)were wounded and thereafter

inoculated with A. tumefaciens carrying an RNAi constructthat contained 376 bp of

caffeine synthase (CS) cDNA fragments in both sense andanti-sense directions with an

intron in between them. As a result, in young leaf, therewas a noticeable decrease in

caffeine and theobromine contents were noticed along withsuppressed expression of

CS gene upon root transformation by Agrobacteriuminfiltration. Thus, this study paved

a novel way for functional investigations of genes in woodyperennials such as C. sinensis

(Mohanpuria et al., 2010).

Tea explants are known to be recalcitrant toAgrobacterium-mediated transformation.

However, an efficiency of 15–20% was achieved in A.rhizogenes-induced formation of

transgenic roots from tea explants. It was suggested thatinhibition of gene transformation in

tea by catechins was prevailed over by using optimizedstrains of Agrobacterium. Probably

catechins released from the wounds of the tea explants werenot adequate to thwart

transformation of competent cells of the explants. This isbecause competence depends

on factors such as induction of mitogen-activated proteinkinases and modifications in

the hormonal level (Song et al., 2014). Hence, the newlyinvented techniques could be

exploited commercially to produce secondary metaboliteswhich will be an enormous

effort for a cash crop such as tea.

5.3 Biolistics

Biolistics or particle bombardment is a device by whichliving cells are bombarded with

genes coated in either gold or tungsten particles.Biolistics mediated transformation is an

unconventional method that has been effectively exercisedin production of transgenic

plants in an extensive range of species. An initialendeavour to understand the transient

expression was undertaken by Akula and Akula (1999).Somatic embryos of tea plants

were bombarded with gold particles coated a with plasmidvector that contained

marker and reporter genes under the control of 35S CaMV(cauliflower mosaic virus)

promoter. Various factors, such as the distance between themicroprojectile delivery

site and the target tissue, the state of target tissue toattain transient expression and

He (helium) pressure, were standardized. These conditionswere optimized following

a particular phase of bombardment based on p-glucuronidase(GUS) assay. Although

it was the beginning, still the way different protocolshave been standardized using

both Agrobacterium- and biolistic-mediated methods, itholds tremendous potential

for obtaining transgenic tea that contains functional genes(Mondal et al., 2004).

Transgenic tea plants expressing osmotin gene fromNicotiana tabacum were produced

using factors optimized for biolistics transformation.Altogether 4,500 somatic embryos

were bombarded with various combinations and finally 90independent, PCR-positive

lines were produced. Integration of osmotin gene in 26 outof 27 PCR-positive lines

were confirmed by Southern hybridization, but statisticalanalysis showed that efficiency

of transgene integration was appreciably influenced bytarget distance. The lines

obtained from somatic embryos bombarded with 1.0 µg plasmidDNA with a burst

pressure of 7.58 MPa along with 9-cm target distanceconfirmed expression of osmotin

(Saini et al., 2012).

Similarly, tender leaves of in vitro grown shoots of teawere bombarded with plasmid

DNA containing gus and nptII genes. Selection mediumcontaining kanamycin (1.71 µM)

showed 43.4% callusing after 5 weeks. Merely 3% of themregenerated into indirect

shoot buds and among them 46.67% putative transformantsexhibited gus gene

specific primers during PCR analysis. Alternatively, allthe putative transformants were

found to be positive with nptII-specific primers. In anassay with Southern hybridization,

having nptII as a specific probe, all the selectedPCR-positive plants showed steady

integration of nptII, and in polyhouse situation both thetransgenic and control plants

showed almost identical phenotypes. However, considerablylesser shoot height was

evidenced in transgenic plants. The reproductive nature ofthe transgenic plants was

Table 1 Summary of transgenic tea research

A. tumefaciens Antibiotic selection for Camellia specieswas reported — Tosca et al. (1996)

A. rhizogenes First Attempt to induce hairy root formationrolB Zehra et al. (1996)

A. rhizogenes Transformation technology was exploited topromote roots to facilitate hardening of themicropropagated tea plants rolB Konwar et al. (1998)

A. tumefaciens Preliminary study for gene transfer to teaplants gus-intron Matsumoto and Fukui (1998)

A. tumefaciens Standardization of somatic embryogenesisand transient expression of gus gene npt-II Mondal et al.(1999)

Particle

bombardment First attempt to standardize thebiolisticmediated transformation protocol npt-II Akula andAkula (1999)

A. tumefaciens Transgenic calli were produced utilizingthe phenolic inducer acetosyringone npt-II Matsumoto andFukui (1999)

A. tumefaciens Detailed study on Bt gene transformationwas reported bt Luo and Liang (2000)

A. tumefaciens Development of selection system forputative transformants npt-II Mondal et al. (2001d)

A. tumefaciens Production of transgenic plants fromtransformed somatic embryos npt-II and gus-intron Mondalet al. (2001b)

A. tumefaciens Tea leaves with glabrous surface havinglower phenol and wax content were identified to be moresuitable for infection npt-II Kumar et al. (2004)

A. tumefaciens

and particle

bombardment Green fluorescence protein gene wastransferred with organelle target signals gfp Kato et al.(2004)

A. tumefaciens

and particle

bombardment Attempt was made for standardization of theprotocol npt-II Wu et al. (2003)

A. tumefaciens Attempts to overcome the bactericidaleffect of tea leaf polyphenol npt-II Sandal et al. (2007)

A. tumefaciens Silencing of glutathione synthetase gene incallus gs Mohanpuria et al. (2008)

A. rhizogenes High production of catechin in hairy rootculture rolB John et al. (2009)

A. tumefaciens Caffeine-free plant production cs Mohanpuriaet al. (2011)

Particle

bombardment Production of transgenic tea expressingosmotin gene from Nicotiana tabacum osmotin Saini et al.(2014)

also affected because floral bud, flower and fruitabscission, and empty seed production

was much more in them while viability and germination wasdrastically lower than the

non-bombarded plants. However, the survival rate of thetransgenic seedlings was not

satisfactory (Sandal et al., 2015).

6 Conclusion and future trends

Needless to say genetic transformation of tea has immenseimportance. Being a woody

perennial with long gestation periods, conventionalbreeding is difficult. Additionally,

in absence of genome sequence, genomic resources,particularly DNA-based markers,

are also limited which further limits the marker assistantbreeding. Importantly natural

mutant of tea is also less. Collectively, it hindersdevelopment of mapping population as a

consequence, and discovery of quantitative trait loci(QTLs), genes and so on is also less.

In this situation, the genetic transformation is a betteralternative. Although the protocol

for genetic transformation has been standardized, notransgenic tea plant has been

developed till now. Several factors are associated with theslow progress of transgenic

research in tea. Robust transgenic protocol and involvementof limited publicly funded tea

research institute in the genetic transformation researchare two reasons. Unfortunately,

tea being an industry-oriented crop, private fundingtowards the transgenic tea research is

extremely low. Therefore, intensive research on thisdimension with larger funding will be

appropriate to develop transgenic tea.

It is noteworthy to mention that although transgenicapproach has immense potential

on tea in terms of genetic improvement, it has somenegative effect from the consumer’s

point of view. There are a few reports on probable toxin orallergen contamination in some

transgenic products due to the presence of antibiotic genesused as selection marker

in general. However, there are several scientificadvancements in this regard: (i) black

tea during the process of manufacturing is heated at 120°C,a temperature which will

degrade the toxin or foreign protein that might be presentin transgenic tea; (ii) there are

techniques to remove those marker genes, broadly called asmarker-free plant production.

A recent non-transgenic approach such as CRIPR/CAS9 will beappropriate to develop the

transgenic product.

A. tumefaciens Transformation of somatic embryo npt II andgus A Singh et al. (2014)

A. tumefaciens Effects of catechins onAgrobacteriummediated genetic transformation – Song et al.(2014)

Particle

bombardment Development of transgenic tea plant from leafexplants gus and npt-II Sandal et al. (2015)

Notes: rol B – rooting locus gene B, gus – glucuronidase,npt-II – neomycin phosphotransferase II, gfp – green

fluorescence protein, gs – glutathione synthetase, cs –caffeine synthase (adapted and modified from Mondal 2014).

Table 1 (Continued)


1. www.tocklai.net

2. tearesearch.or.ke

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4 Chapter 4 Planting and cultivation oftea

1 Introduction

Tea is a perennial tree crop grown in large commercialplantations of several 100 hectares

or as smallholdings of less than a hectare. Havingoriginated in natural forests characterized

by warm and humid conditions and nutrient-rich soils, itsgrowth and yield largely depend

on climatic and soil factors. Moreover, frequent removal ofphotosynthetically active

tea shoots (harvesting) and periodic removal ofleaf-bearing branches (pruning) exert

physiological stresses to the tea bush. Therefore,scientifically proven good agricultural

practices (GAP) should be adopted to provide optimumgrowing conditions for the

tea plant to ensure sustainable productivity andprofitability. Successful tea cultivation

depends chiefly on the following: • land selection, •land preparation, • soil and soil moisture conservation,• soil reconditioning, • tea planting, • supply ofnutrients, shade trees and green manure crops, • mulching(thatching), • bush formation (bringing into bearing), •harvesting, • pruning, • pest, disease and weedmanagement.

Planting and cultivation practices differ from one field,region or country to another

depending mainly on soil and climatic conditions, ongenetic materials (cultivar) selected

for planting and also on the field operation systems suchas mechanization and irrigation.

2 Climatic requirements of tea

Tea plants can adapt to a wide range of soil andenvironmental conditions. As a result,

commercial plantations are found from as far north asGeorgia with a latitude of 42°N to

as far south as Argentina with a latitude of 27°S (Watson,2008a). Temperature, rainfall,

solar radiation, relative humidity and wind are the mostimportant climatic factors affecting

the growth and yield of tea. These factors largelydetermine the crop distribution over

the year. Moreover, the succulence and maturity of teashoots, as well as dormancy and

chemical constituents in tea leaf, can be markedly affectedby these climatic factors and

hence are vital for determining the quality of the endproduct too.

2.1 Temperature

Tea grows well when the ambient temperature is around18–25°C. Very low (e.g. <13°C)

as well as high temperatures (e.g. >30°C) are undesirablefor tea growth. The base

temperature for tea shoot extension is considered to be12–13°C (Carr & Stephens, 1992).

The optimum temperature for tea growth can vary from around23 to 30°C (Amarathunga

et al., 1999; Carr, 1972; Carr & Stephens, 1992; Rahman,1988; Smith et al., 1993; Squire,

1979; Tanton, 1992). The analysis of data from Sri Lankahas shown that the growth of tea

shoots reduced when the ambient temperature rises above25–26°C in the low-country

tea-growing region (Wijeratne & Fordham, 1996). However,frost and hail damage also

adversely affect tea yield at low temperatures.

2.2 Rainfall

Rainfall is one of the most critical environmental factorsaffecting the growth and yield

of tea. A well-distributed rainfall of around 2500 mm peryear is ideal for rain-fed tea

cultivation and the growth is significantly affected whenthe rainfall is less than 1200 mm/

year (Watson, 2008a). Heavy rainfall can also limit rootgrowth, especially in low-lying

and flat lands where drainage and soil aeration arehampered. The optimum rainfall for

tea cultivation can vary depending on other climaticfactors such as temperature and soil

conditions. Recent studies in Sri Lanka have shown that thehighest tea yield is achieved

when the rainfall is between 223 and 417 mm/month indifferent agro-ecological regions

(Wijeratne et al., 2007).

2.3 Sunlight

The intensity and duration of sunshine hours has a directas well as indirect effect on the

growth and yield of tea. Lack of sunlight limitscarbohydrate assimilation (photosynthesis)

and encourages fungal diseases. Usually, sunlight is notconsidered a key limiting

factor for tea cultivation in the tropical region but canbe critical in temperate climatic

conditions. The growth of tea shoots is retarded due tolack of sunlight. The critical

number of sunshine hours for dormancy in tea has beenreported to be around 11 hrs

(Barua, 1969; Tanton, 1982; Herd & Squire, 1976). Lightsaturation of the tea leaf can

occur at a wide range of light intensity, that is, from 600to 1500 µmol/m 2 /s 2 and the

variation could be due to probable genotype X environmentalinteractions (De Costa

et al., 2007).

2.4 Relative humidity

Low humidity can create moisture stress conditions in teaplantations due to excess evapo

transpiration. High humidity predisposes tea bushes tofungal disease infestations. Tea

yield can be adversely affected when the vapour pressuredeficit (VPD) exceeds 2–2.3 kPa

(Hoshina et al., 1983; Carr & Stephens, 1992; Tanton,1992). However, Wijeratne and

Fordham (1996) reported that tea shoot growth at lowelevations in Sri Lanka reduced

when the VPD exceeded 1.2 kPa.

2.5 Wind

Wind modifies the micro-climate around tea bushes. Strongwinds are undesirable as

they shake young plants early in their development, stripoff tender foliage and damage

shade trees or tea bushes. Pests and diseases are alsospread by the wind. However, dry

winds accompanied by cold nights and bright sunlightreportedly contribute to flavour

development in some tea-growing regions of Sri Lanka(Watson, 2008a).

3 New planting of tea

3.1 Site selection

Tea plants prefer deep, permeable and well-drained acidic

soil with a pH in the range of

4.5–5.5. However, rockiness and gravelly soil can restricttea growth. Shallow soils (<50 cm)

with gravel content of more than 50% and rockiness above20% have been found to be

undesirable for tea cultivation (Watson, 2008a; Anon,2002). Also steep slopes in heavy

rainfall areas should not be used for tea cultivation owingto heavy soil erosion and nutrient

losses. A high organic matter content in soil is an addedadvantage for tea cultivation.

Further, loamy soil with equal proportions of sand, siltand clay is ideal for tea growth.

Usually, southern and western slopes are warmer due tosolar radiation than other slopes.

Hence, precautionary measures need to be taken to reduceexcess heat (e.g. provision

of dense shade) when such locations are used for teacultivation. By contrast, areas with

large rock outcrops should be avoided as they enhance solarradiation and warm up the

surrounding environment at midday. Most of the problemsrelated to low productivity of

tea lands could be avoided if the above factors areconsidered in land selection.

3.2 Land preparation

All vegetation should be dug up and removed from the field.Deep forking to remove

roots larger than pencil thickness and to a depth of about45 cm is necessary to prevent

root diseases. During this operation large stones and otherbarriers to root growth such

as hard pans can also be removed. These can then be

advantageously used to construct

bunds and terraces for soil conservation. Land clearing andpreparation needs to be done

before the onset of heavy rains and should start at theupper-most section of the slope

to minimize soil losses. The land should then be roughlylevelled to facilitate subsequent

operations (Fig. 1). Fine levelling may encourage soilerosion and therefore should be

avoided. Soil rehabilitation before replanting with tea ispreferred where soil conditions

are poor.

As manual land clearing and levelling is time consuming andcostly, earth moving

machines are best used to prepare the land, especially whenmanual workers are not

available in sufficient numbers. The use of such machinesalso reduces the time between

land clearance, establishment of soil conservation measuresand the planting of tea or

rehabilitation grasses.

3.3 Soil and soil moisture conservation measures

Soon after clearing the land, it is essential to constructsoil and moisture conservation

structures such as lateral (contour) and leader drains andcontour terraces where necessary.

Lateral drains act as rainfall-harvesting structures andcan also help remove excess water

from the land. The soils washed by run-off water are alsocollected in lateral drains built

across the slope. The surface run-off is also reduced bycontour terraces constructed using

rocks/stones, minimizing soil erosion. The excess waterflowing through lateral drains is

collected by the leader drains that are usually built onnatural drainage lines down the

slope to channel excess rainwater away from the land atnon-erosive velocities.

In Sri Lanka, the lateral drains are generally 45 cm wideand 45 cm deep. They are

connected to the leader drain with a gradient of about 1 in120 and are spaced 6–12 m

apart depending on the slope (closer spacing for steeperinclines) and rainfall intensity. In

Figure 1 Initial land preparation for tea cultivation.

Sri Lanka, the lateral drains are commonly of the ‘Lock andSpill’ type and are sometimes

provided with silt pits (Fig. 2) (Somaratne et al., 2008).The upper lips of the lateral drain

are strengthened and protected from collapse by planting adense row of grasses such

as Vetiveria zizanioides or Eragrostis curvula. The grassrow also acts as a live barrier

against soil erosion. In order to prevent damage to thewalls of the leader drain, the

lateral drains are opened into the leader drain in such away that two lateral drains are

not right opposite to each other. Soils that are collectedin lateral drains after heavy

rains should be removed (known as de-silting) to the upperside of the drains. In some

tea-growing regions such as those in North East India andKenya where flat lands with

heavy soils and waterlogging areas are planted with tea,deeper drains are built to remove

excess moisture from the root zone (Fig. 3).

The leader drains are constructed to receive large amountof water discharged by the

lateral drains so the side walls and bottom are paved withstones (Fig. 4). The width and

depth of the leader drain vary depending on the slope andamount of water discharged,

Figure 2 Contour drain (Lock and Spill type).

Figure 3 Deep drain for drainage of excess moisture.

but usually it is about 45–60 cm after stone paving. Thebottom of the drain is built in a

step-wise manner with a reverse slope (inward slope) toreduce the water flow speed.

Check dams built in the leader drain at frequent intervalscollect silt being washed away

by run-off water. The edges of leader drains can also beplanted with grasses.

Stone terraces (Fig. 5) are also built to help soilconservation in tea lands when sufficient

rocks/stones are available. They are also constructed alongthe contours and the upper

surface should preferably rise 45 cm above the soil surface.

In addition to the mechanical methods of soil conservationdescribed above, the

establishment of hedgerows known as sloping agriculturalland technology (SALT) is a

recognised biological method of soil conservation(Ekanayake, 1994). With this method,

two rows of leguminous plant species with fast-growing andcoppicing capabilities are

planted at closer spacings to form a hedge on the contourwhich breaks the slope of

the land and hinders surface run-off (Fig. 6). Leguminousspecies are selected from

those that are well adapted to the region and compete leastwith tea. Gliricidia sepium,

Erythrina lithosperma, Calliandra calothyrsus, Tithoniadiversifolia, Flemingia congesta

and Cassia spectabilis are some of the common species usedfor SALT hedgerows in Sri

Figure 4 Leader drain paved with stones.

Figure 5 Contour terraces (stone bunds).

Lanka. The use of plant species with pest repellentproperties is an added advantage.

The two rows of hedge are usually 45 cm apart and thespacing within the row is around

15–20 cm. Spacing between hedgerows can vary depending onthe slope of the land

and availability of planting material, but possibly about7–8 m apart. The hedgerows

should be regularly lopped above the canopy of tea bushesand the loppings used to

fill the space between the two rows of hedge as greenmanure/mulch for the tea plants.

Plant debris and stones can also be stacked within thehedge to form a strong bund or

barrier against soil erosion.

4 Preparation of the planting hole

The grass plants are removed at ground level preferably afew centimetres below the

soil surface using a mamoty (hoe) without uprooting and thegrass sods placed along the

contours leaving space between the rows to facilitate thedigging of planting holes for tea.

Planting holes of around 30 cm in diameter and 45 cm indepth are prepared along the

contour rows in Sri Lanka (Fig. 7). Alternatively, trenchesof the same diameter could be cut

where soils are less fertile, that is, gravelly, compactedand heavy. The size of the planting

hole varies in different regions, for instance, 23–45 cmdiameter holes are prepared in

different regions of India depending on soil conditions.

4.1 Spacing

The planting density of tea depends mainly on rowarrangement (single row or double

hedgerow system) and tea cultivar (spreading habits).Usually, around 12 000 plants

per ha is considered to be the optimum density for most ofthe popular tea cultivars

grown in tea-growing countries. Very low plant densitiesare found in old seedling tea

fields planted along the slope when tea was firstintroduced as a commercial plantation

crop in tea-growing nations. However, tea rows on slopesare best established on the

contours to minimize soil erosion and carry out fieldoperations more easily (Fig. 8).

In Sri Lanka, 120 cm between rows and 60 cm within the rowis the recommended

Figure 6 SALT hedge row established with Gliricidia spp.

distance for single row planting, ensuring about 12 500plants/ha on average,

allowing space for roads, footpaths and drains. Thespacings for double hedgerows

are 150 cm × 45 cm × 45 cm and 150 cm × 60 cm × 60 cm.Nevertheless, spacings

between two hedges from 130 to 180 cm and within the rowfrom 30 to 75 cm are also

adopted in India and China depending on the tea cultivarand to facilitate the use of

machines for harvesting and spraying. The double hedgerowplanting system is more

suitable for low-spreading tea cultivars and fields thatare mechanically harvested using

machines. However, such planting systems require bettersoil and climatic conditions

as closer planting can cause competition for moisturebetween plants and lead to

heavy drought casualties in warm temperatures and poor soilconditions (Wijeratne &

Ekanayake, 1990).

Figure 7 Tea planting holes.

Figure 8 Contour planting of tea.

5 Planting of tea

Soil moisture and humid conditions ensure the successfulestablishment of new plants.

Therefore, tea planting should take place when the soil ismoist and workable during

a period of wet weather. It can be continued into the earlystages of the monsoon and

completed well before the tail end of the monsoon season.When there are two monsoons,

the most certain one which is not followed by a long dryspell should be selected for

planting tea. However, the presence of such weatherconditions may not be as important if

irrigation facilities are provided from the beginning oftea cultivation. Fertile topsoil mixed

with compost or any other organic manure is used to fillthe planting holes or trenches. To

prevent subsidence or the subsequent sinking of plants,holes and trenches are partially

filled (about two-third) around 2–3 weeks prior to plantingand the soil is allowed some

time to settle with rains. Healthy and vigorously grownnursery plants of about 8–12

months of age should be selected for field planting, payingparticular attention to the

height, number of branches/leaves and maturity of thecollar region of the nursery plant.

For instance, a nursery plant of about 30 cm in height withtwo to three side branches

having around 15 leaves or more and with a well-developedroot system is recommended

for planting in Sri Lanka (Anon, 2009). Larger, more matureplants can be used for areas

where the soil and environmental conditions are marginal.

Planting should be done so that the top of the nursery bag(soil) is level with the ground

and no air pockets are left behind after filling the hole.Deep planting to cover the collar

region of the plant with soil may cause plants to die dueto collar rot or similar fungal

diseases. In areas with a history of pest or diseaseinfestations such as white grub or

nematodes, adequate precautionary measures should be takento control such infestations

by the application of appropriate chemicals at the time ofplanting. It is good practice to

support the plant by fixing two short sticks on either sideof the plant immediately after

planting to protect it from the wind.

6 Aftercare field operations

6.1 Mulching and cover crops

Soil erosion and profuse weed growth are common problems inyoung tea fields until

they form good ground cover. Mulching (thatching) and theplanting of cover crops

help reduce soil erosion and weed growth and also addorganic matter and plant

nutrients to young tea fields. Locally available plantmaterials such as the loppings of

rehabilitation grasses are commonly used to cover theinter-row spaces of tea (Fig. 9).

Additionally, partially decomposed agricultural waste andby-products such as paddy

husk, sawdust, tea refuse and coir dust can also be usedfor mulching provided their

C:N ratios are reduced to acceptable levels where necessaryby mixing with urea (Anon,

2011). However, such organic materials can providefavourable conditions for some

soil-borne pests such as white grubs (Holotrichiadisparillis) and, therefore, adequate

precautionary measures need to be taken to control suchpests. Mulching materials are

laid between rows a few inches away from the base of theplant, preferably before the

tail end of the rainy season to harvest rainwater andconserve moisture in the ground.

Green materials decompose very fast, especially undertropical conditions (warm

regions), hence mulching should be repeated a few times

until tea plants cover the

ground fully by themselves.

Instead of dead mulches applied in tea plantations, covercrops can also be grown to

cover the exposed ground between young tea plants (Fig.10). They should preferably

be low creeping legumes which provide little or nocompetition to young tea plants for

moisture and nutrients. Fast-growing creepers that have atendency to climb through

tea bushes are not suitable due to management implications.The growth of cover crops

needs to be adequately controlled during dry spells byslashing them and they should not

be allowed to grow within the manure circle of the teaplant.

Additionally, bushy-type green manure crops such asCrotalaria spp., Indigofera spp.

and Sesbania spp. are also grown in between tea rows (onein every few rows of tea) to

provide temporary shade during the early stages of growthand organic matter to young

tea fields in Sri Lanka and India (Fig. 11). However, theyshould be lopped regularly to

Figure 9 Mulching with Mana grass lopping in young tea.

Figure 10 Arachis pintoi grown as a covercrop in young tea.

control excessive growth and to minimize competitionbetween young tea and green

manure crops for moisture and nutrients. Nitrogen fixationby such leguminous plant

species is an additional advantage.

6.2 Training of young plants

Usually the training of tea plants begins in the nursery byeffecting different techniques

that stimulate branching habits. Consequently, well-grownnursery plants with several

branches are developed for field planting. Once nurseryplants are established in the

field and are adapted to the new environment, training forframe development should be

initiated. Development of a well-spread frame can beaccomplished by adopting one of

two common methods known as: • bending and pegging and •centering.

The former involves bending the main branches horizontallywith the support of pegs

stuck on the ground so that a set of new vertical branchesare developed upon the bent

branches and the ground cover by the plant is increased.With centring, the main stem

is severed at a given height to remove apical dominance andto promote the growth

of new lateral branches. The time allowed for free growthin the field before training

is undertaken varies depending on the method of training indifferent tea-growing

countries.

Frame formation of tea plants in China is achieved bycutting young tea plants in the

spring at a height of 15–25 cm in the first year, 20–25 cmin the second year and at

30–35 cm in the third year after planting. To minimizeabiotic stress to the plant, this

cutting is best carried out during wet weather.

Figure 11 Crotolaria spp. grown as temporary shade/greenmanure crop.

6.2.1 Bending and pegging

When the tea plants have been established in the field(about a month after planting),

well-developed lateral branches are bent to an angle ofabout 45° and the distal ends

are pegged to the ground without damaging the branches. Thegrowth of new shoots

can be further encouraged by removing the terminal portionof the bent branches. The

newly formed vertical shoots are bent and pegged again toform a wider frame or cut

across once or twice as necessary to increase the canopyspread. The tea bushes are

then harvested lightly until a well-grown canopy is formed.The operation of bending and

pegging should be done during wet weather only. As thismethod of training causes least

stress to the plant and allows the development of a sturdyand well-spread frame and root

system leading to early ground cover, harvesting can beginearlier than with the other

method. However, it is not very suitable for warm regionsprone to dry weather where sun

scorch and the development of fungal diseases such ascanker (Macrophoma theicola,

Phomopsis theae) are very common (Somaratne et al., 2008).Bending and pegging is less

popular among tea growers as it is more expensive as wellas being a time- and labour

intensive operation requiring close supervision andattention.

6.2.2 Centering

With this method, after their initial establishment teaplants are given a few cuts at different

heights to encourage the growth of more lateral branchesand then allowed a period of

free growth varying from 3 to 12 months depending on thecountry they are planted in.

For instance, in Sri Lanka, a period of free growth ofaround 12 months is preferred while

in India it is just 3–6 months, depending on climaticconditions. In Sri Lanka, the first cut

(centering) is made at a height of around 20–25 cm and thesecond at 35–40 cm, 4–6

months after the first cut (Fig. 12). Once the plants arerecovered sufficiently after the

second cut, the tips of the new shoots are removed(tipping) at a height of 45–50 cm.

In India and Kenya the centering is lower than in Sri Lankaoften under 20 cm, leaving

few side branches. Sometimes the plants are cut as low as10–15 cm to encourage bush

formation at a lower level. In the early stages aftertipping, tea bushes are only lightly

harvested increasing the canopy foliage.

Figure 12 Training of young tea by Centering method.

7 Establishment of shade trees and wind belts

Tea is naturally a shade-loving plant. Its canopy islight-saturated at around 60–70% of

full sunlight. Therefore, tea is inter-planted with shadetrees in commercial plantations.

Shade trees provide an environment conducive to the growthof tea plants by reducing

the temperature in warmer regions; reducing solar radiationto the optimum level for high

canopy photosynthesis; reducing transpiration; preventingscorching; minimizing wind

damage; suppressing weed growth; adding organic matter;serving as a diversionary

host for pests; and attracting beneficial predators ofthose pests. Usually, shade trees

are planted when soil is rehabilitated to provide shade tothe young tea plants. They are

commonly evergreen legumes characterized by deep rootsystems which compete least

with tea plants.

In Sri Lanka the planting of high and medium shade trees ina mixed stand is

recommended. The high shade species commonly grown at highand mid elevations

is Grevillea robusta and that at low elevation is Albiziamoluccana. Gliricidia sepium,

Calliandra calothyrsus, Erythrina lithosperma, Acaciapruinosa and A. decurrens are grown

as medium shade trees at different elevations depending onthe climatic conditions. High

shade trees are initially planted at a spacing of 6 m × 6 mand are thinned out to a final

spacing of 12 m × 12 m by the end of the third year afterplanting. Medium shade trees

are initially spaced at 3 m × 3.6 m and are thinned down to6 m × 7.2 m by the end of

the third year (Figs. 13 and 14). However, a higher densityof shade trees can be left in

tea fields with rocky outcrops and those on southern andwestern slopes due to exposure

to warmer temperatures. Shade trees are either raised innurseries from seed (e.g. Albizia

spp., Grevillea spp.) or planted directly in the fieldusing well-developed stumps or

vegetative cuttings (e.g. Gliricidia spp., Erythrina spp.).In addition to thinning after the tea

crop is well established, they are removed totally and arereplaced with new ones when

they are overgrown. When Albizia and Grevillea trees areabout 8 and 20 years old, new

plants should be introduced so that the old trees arereplaced at about 12 and 30 years,

respectively, for Albizia and Grevillea.

In North East India, Indigofera teysmannii (4.5 m × 4.5 m)and Leucaena leucocephala

are grown with tea as temporary shade for about 5–6 yearsafter planting. Albizia spp.,

Figure 13 Mix stand of shade at low elevations in Sri Lanka.

Acacia lenticularis and Derris robusta are the most commonhigh shade trees (Fig. 15). In

South India, Grevillea robusta (6 m × 6 m) and Acaciamearnsii are grown as high shade

trees. The spacing, method of planting and management ofshade trees vary in different

tea-growing regions.

Wind (shelter) belts are usually established where strongwinds are common at certain

times of the year (Fig. 16). Strong winds can cause damageto young tea (e.g. damage to

leaves, loosening the plant and damaging the bark at thebase of the plant) and mature tea

plants (e.g. breaking of shoots and desiccation), reducingcrop yields. In such areas, wind

belts are established at right angles to the wind directionbefore planting tea. Grevillea

Figure 14 Mix stand of shade at high elevations in SriLanka.

Figure 15 Shade in tea in North East India.

spp. (3.6 m) mixed with Hakea saligna or Acacia spp.(1.2–1.8 m) at high elevations and a

mixed stand of Fragrea fragrans (3.6 m) and Gliricidia spp.(1.2–1.8 m) at low elevations are

recommended as wind belts in Sri Lanka. The spacing betweentwo belts can vary from 45

to 60 m depending on the strength of the wind. Tea bushescan also be densely planted

and trained as a wind belt by allowing two to three rows togrow freely without harvesting

and pruning. Grevillea spp., Casuarina spp. and Acacia spp.are used to establish wind

belts in India (Arunachalam, 1995).

8 Pruning

Although left to nature the tea plant can grow to theheight of a big tree, about 10 m, its

growth is restricted to a low-grown bush by removingbranches and training, referred to

as ‘pruning’ in tea cultivation. Tea bushes are regularlypruned in order to maintain a high

yield of tea and restrict the growth of the tea canopy to aconvenient height for harvesting.

Tea pruning has three main objectives: • reducing theheight of the bush, • stimulating vegetative growth and •maintaining the health and vigour of the tea bush.

With continuous growth and added foliage, the pluckingsurface becomes higher making

harvesting less efficient. Due to repeated branching and anincrease in woody branches

as the bush ages, the vascular tissues become narrowerrestricting nutrient supply to the

growing shoots. This results in the production of fewer,smaller shoots, more flower buds

and profuse banjies in the canopy (Sharma, 2011).Additionally, a dense canopy after several

years of harvesting usually accumulates weak or debilitatedbranches and dead wood.

Consequently, the bush deteriorates warranting the removalof such unhealthy materials

by pruning (Fig. 17). Hence, tea pruning ensures effectiveuse of the land by improving

vegetative vigour, ground cover and the general health ofthe bush. The frequency of

Figure 16 Wind belt in India.

pruning depends largely on the condition of the field, typeof jat/cultivar and climatic

conditions. Generally, it varies from around 2 to 6 years.The length of the pruning cycle

also increases with a rise in elevation (due to a reductionin ambient temperature). The

growing period of tea bushes under temperate conditions isrestricted to a few months of

the year where the temperature and rainfall are conduciveto the growth of shoots. Under

such conditions, preparing the bush for the next flushingperiod also warrants a form of

pruning (trimming/skiffing) at the end of the annualharvesting period.

With the removal of leaf-bearing branches, tea pruningaffects the assimilation and

partitioning of dry matter to different parts of the bush.Hence, the quantity of starch

reserves in the root system and woody branches affects howwell the plant recovers

after pruning. Also, the vigour of the older dormant budson the mature stems left after

low pruning is less than that of the young wood. Therefore,the severity of pruning

or the depth of canopy removal largely determines the timetaken for the bushes to

recover after pruning. As the basal parts of the branchesare suddenly exposed to direct

sunlight after pruning, they are liable to sun scorch. Thismay reduce the number of

emerging buds and lead to poor frame development, wood rotand bush debilitation.

Therefore, the physiological condition of the tea bush atthe time of pruning and soil and

environmental conditions need to be studied carefullybefore embarking on a pruning

programme.

8.1 Styles of pruning

There are different pruning styles practised in differenttea-growing regions. Usually,

they can be categorized as rejuvenation, hard, medium andlight pruning depending

on the height or severity of pruning. The depth of canopyremoval is the highest under

rejuvenation pruning while it is less with light pruning.In Sri Lanka three different styles: • clean prune (25–40cm) • rim-lung prune (40–55 cm) and • cut-across prune(55–70 cm)

Figure 17 Pruned tea field in Kenya.

are commonly practised, with rim-lung prune being the bestfor achieving the objectives

described above (Fig. 18). Skiffing or trimming to reducethe depth of the canopy by a few

centimetres is also identified as a form of pruning, but itis more of a slashing or levelling

operation to help harvesting (Watson, 2008b). The firstfull pruning after planting is called

formative pruning. It is either a hard or medium prunedesigned to form a well-spread,

sturdy bush frame that supports crops for several years(Sharma, 2011).

The hardest form of pruning is the rejuvenation pruningwhich removes canopy branches

at a height of less than 30 cm from the ground. Generally,the height of hard pruning varies

from 25 to 45 cm while that of medium pruning varies from30 to 60 cm in different tea

growing countries. Tea bushes can be lightly pruned at aheight varying from 55 to 70 cm.

By pruning, the top of the canopy can be trained to be flator dome shaped specifically to

suit manual or mechanical harvesting.

Although a mean height is maintained over the field, thepruning height of individual

branches or bushes may vary depending on the topography ofthe land, vigour of the bush

and level of any pest or disease attack. Usually, thepruning level follows the slope of the

land to assist harvesting. However, it may vary indifferent tea-growing countries/regions

due to different systems of planting (double/hedgerowplanting, planting on terraces, etc.)

and the method of harvesting (manual or mechanical means).

The choice of the best style of pruning requires a clearunderstanding of the condition

of the tea bushes. Hard pruning to old wood frequentlyresults in more deaths due

to the exposure of senile buds, poor healing of pruningcuts and inadequacy of root

reserves for better recovery. Nevertheless, hard pruninglengthens the pruning cycle

and hence reduces the unproductive period of the bushduring its life cycle. With light

pruning, bushes can be ready to pluck within a short periodof time. However, it cannot be

practised for every cycle due to the need for cleaning ofdead/pest and disease attacked

wood, formation of ‘knots’ and a shorter period forharvesting before the next pruning.

Due to the production of more banji shoots and small shootsearly in the pruning cycle,

it is preferable to prune China hybrids a few centimetreslower than the Assam or Assam

hybrids (Sharma, 2011).

Usually, the labour requirement for manual pruning is about40–60 workers per

hectare. The efficiency can be significantly increased byusing pruning machines such as

brush cutters, two men-operated pruning machines andride-on type pruning machines

(Fig. 19).

Figure 18 Different styles of pruning (a. low/clean prune,b. medium/lung prune, c. high prune/

cut-across).

8.2 Preparation of tea bushes for pruning

As discussed elsewhere, the recovery of tea bushes afterpruning depends on the vigour

and health of the plants. As a large number of newlygrowing buds on the pruned

branches depend entirely on carbohydrate reserves (starchreserves), the volume of starch

reserves present in the bush determines the success of itsrecovery after pruning. Starch

reserves may not be an issue under temperate conditionswhere tea bushes are naturally

rested for 5–6 months after the flushing and harvestingperiod in the summer and early

autumn. However, under tropical conditions where tea bushesare continuously harvested

throughout the year, the level of starch reserves in rootsand bark are very low. Here, it is

preferable to withhold harvesting (known as resting) beforetea bushes are pruned so that

the starch reserves in the tea bush are augmented (Wieratneet al., 2002). The period of

resting depends on the availability of starch reserves inthe tea bushes and the type of

pruning to be undertaken, that is, the harder the pruningthe longer the period of resting.

For instance, a minimum of 6 weeks resting before pruningis recommended for all fields in

Sri Lanka. However, an extended period of resting (for afew months) and an improvement

of soil conditions by adding compost are prerequisites ofhard pruning styles such as

rejuvenation pruning to minimize deaths of tea bushes.

Environmental factors also determine the recovery of plants

after pruning. Ideally tea

bushes are pruned at a time that ensures the recoveryperiod coincides with wet weather

conditions. Pruning during dry weather often exposes livetissue to the hot sun and

moisture stress conditions which can delay recovery andresult in the formation of canker

and in turn wood rot, reducing the productivity andlifespan of the tea bushes.

8.3 Aftercare

In order to reduce drying the exposed bush frame, theremoved branches (tea prunings)

can be kept over the frame for about three days.Additionally, the frame is treated with

Figure 19 Pruning of tea by brush cutters.

hydrated lime (10%) after pruning (lime wash) to soften thebark and remove accumulated

moss on the woody stems to encourage bud break. Theapplication of protective paints

on the pruning cuts (wound dressing), removal of weeds,cleaning of drains, forking/soil

cultivation or burying of prunings in shallow trenches, andreplacing dead bushes with new

plants (infilling) are a few of the other GAP undertaken inpruning fields.

8.4 Tipping operations

Once the tea bushes have recovered adequately from pruningthey need to be brought

into plucking. Usually, tea shoots exhibit a ‘periodicgrowth’ habit, that is, having alternate

active and dormant phases of growth (see Section 9.1).However, the shoots growing on

pruned tea bushes display more vigorous growth than thosein plucking. They continue

Figure 20 Periodic (left) and aperiodic (right) growth oftea shoots (Note: shoot on the left produces a

new set of leaves from its terminal bud after lapse of adormant period whereas the shoot on the right

continues to produce a number of leaves without a break orcessation of growth of the terminal bud) .

Figure 21 Tea bush after tipping.

to produce a number of leaves before the apical/terminalbud becomes dormant (banji).

Therefore, such shoots are categorized as ‘aperiodicshoots’ (Fig. 20). Removal of these

aperiodic shoots at a specific height determined by thebush condition and height of

pruning is known as tipping. In order to sustain thesubsequent growth of shoots and

vigour of the tea bush, sufficient leaves need to be lefton the bush after tipping (Fig. 21).

The method and time of tipping vary in differenttea-growing countries and regions.

For instance, new shoots are tipped using a knife leavingfour to six leaves when they

have produced eight to ten leaves per shoot in Sri Lanka.In contrast, in India, the number

of leaves per shoot kept on the frame reduces from aroundfive to two with an increase

in the pruning height. Tipping-in material (shoots removedwhile tipping) contains three

to four leaves (Sharma, 2011). If the health and vigour oftea bushes are weak more

leaves need to be added to the canopy at the time oftipping. The tipping can also be

done at several stages, removing only those shoots thatqualify or are ready for tipping.

Alternatively, individual or groups of well-developedshoots can be plucked leaving the

required number of leaves in a few rounds. However, theminimum number of leaves per

shoot removed by tipping should usually be more than threeso that only well-grown

axillary buds are exposed to re-growth. If an inadequatenumber of leaves are left after

tipping, the frame development can be impaired and teayield reduced early in the

pruning cycle. Hence, timely tipping and leaving leaves onthe bush are prerequisites

for establishing a vigorous frame with sturdy brancheshelping to achieve higher yields.

Whatever the method used, the tipping operation shouldensure the development of a

level plucking surface above the pruned frame.

9 Harvesting of tea

The tender tea shoots with two to three succulent leavesare harvested at varying intervals

depending on their rate of growth. The harvesting of tea isalso known as plucking. Harvesting

policies viz. method, standard, severity and frequency ofharvesting directly influence the

yield, quality of end product and growth of the tea bush.These policies can vary from field

to field depending on the age and clone or jat. They alsodiffer from one estate to the other

depending on the resources and type of tea produced.Furthermore, plucking policies vary

Figure 22 Manual harvesting (North East India).

from one region to another depending on weather andclimatic conditions. Nevertheless,

the best plucking policy is the one that gives the highestproductivity at lowest cost while

ensuring the quality of the end product and vigour of thetea bush.

Tea is best harvested by hand (Fig. 22). However, due tothe high cost of labour and

scarcity, harvesting has now been mechanized in most of thetea-growing countries. For

instance, the labour requirement for manual harvesting oftea in Sri Lanka has been estimated

to be about 500–700 workers/ha/yr making up about 40% ofthe production costs. When

quality parameters are considered, the best quality tea isproduced from tender shoots

with two to three leaves that are free from physical damageand contamination (good

leaf). Tea shoots with mature (coarse) leaves, stalks anddamaged shoots are considered

as sub-standard shoots or bad leaf for processing. In orderto ensure a better quality end

product, more than 75–80% of good leaf is required.

Therefore, proper plucking policies should be employed sothat tender shoots are

harvested and transported to the factory carefully withminimum damage and delay.

9.1 Generation of tea shoots

A new tea shoot can be developed either from a dormantapical (terminal) bud known

as banji or an axillary bud. In harvesting tea, the apicaldormancy of a shoot is removed

by plucking, allowing the regeneration of shoots from theaxillary buds below the point

of plucking. Firstly, the axillary bud starts swelling andunfurls its two outer covers as

scale leaves (Fig. 23). They are also referred to as Janams(cataphylls). They often

fall off within a few days of opening. Next, an oval-shapedand blunt leaf without

apparent serration and veins known as a ‘fish leaf’ isproduced. Sometimes, the bud

may produce two such leaves of which the small oneimmediately above the scale

leaves is called janam or thumb leaf (Sharma, 2011). Thenthe terminal bud produces

Figure 23 Different leaf appendages and points (severity)of plucking.

several normal leaves before it ceases leaf production andexposes a dormant (banji

or wangi) bud. Banji is a very small bud compared to thatof an actively growing tea

shoot. Some hormonal interaction (Pethiyagoda, 1964;Ranganathan et al., 1983) and

lack of nutrient supply to the actively growing apices(Bond, 1945; Sharma, 2011)

are reported to temporarily halt the growth of the terminalor apical bud. Therefore,

environmental conditions, level of food reserve(Ranganathan et al., 1983) types and

levels of fertilizers (Kulasegaram and Kathiravetpillai,1972), and clonal characteristics

(Stephens and Carr, 1990) influence the formation ofdormant buds. After emerging

from its dormancy period this dormant apical bud produces anew set of similar leaf

appendages as given above. Therefore, a free-growing teashoot passes through

alternate active and dormant growth phases which is knownas periodic growth or

growth periodicity of tea.

Due to frequent regeneration of shoots, the pluckingsurface consists of a large

number of shoots at different stages of growth ranging fromgrowing axillary buds to

harvestable shoots with active and dormant buds (Fig. 24).

9.2 Manual (selective) harvesting

Usually, the top of the tea bush is trained to have a flator dome-shaped surface to assist

harvesting and enhance shoot production. This surface isknown as the plucking table

which consists of a large number of shoots at differentstages of growth ranging from

developing axillary buds to harvestable shoots with a fewleaves. Each of these groups

of shoots at the same stage (generally identified by thenumber of leaves) is called

a generation. In manual harvesting, the older couple ofgenerations are selectively

harvested as flush shoots with one to three leaves, leavingyounger generations known

as ‘arimbus’ (actively growing shoots with less than twoleaves; see Fig. 24) for later

rounds. However, all generations are removed when teabushes are harvested using

machines or other non-selective methods. Manual harvestingnot only gives a high

and sustainable yield, but it also ensures maximum

exploitation of shoot growth and

production of good quality tea.

Figure 24 Different types of tea shoots.

9.2.1 Severity of harvesting (plucking)

If a shoot is harvested leaving the most mature normal leaf(true leaf above the fish leaf)

it is called ‘single leaf or mother leaf plucking’ (Fig.23). Mother leaf plucking adds foliage

(maintenance foliage) to the bush to replace the dead(fallen) mature leaves at the bottom

of the canopy. The plucking to the fish leaf is termed as‘fish leaf plucking’ and plucking

below the fish leaf is called ‘janam leaf plucking’. It iscommon in tea estates in Sri Lanka

that single leaf and fish leaf plucking are practisedalternately during dry and wet weather

periods, respectively. Such a mixture of plucking combinesoptimum exploitation of shoot

growth with sustainable growth of the tea bush.

About 14–26% of the total dry matter produced by a tea bushis harvested as flush

shoots (Burgess, 1992; Magambo et al., 1988; Tanton, 1979)which is referred to as the

harvest index of tea. When individual shoots areconsidered, about 40–60% of the dry

mass of a harvestable shoot is removed by single or motherleaf plucking compared with

about 80% under fish leaf plucking (Wijeratne, 2001).Therefore, plucking policies, mainly

the severity and the standard of plucking, have asignificant impact on yield and the size

of the canopy.

The top layers of mother leaves act as the most efficientsite for carbohydrate assimilation.

Hence, it is extremely important to replace older motherleaves to sustain shoot growth

as younger generations of shoots are largely dependent onthe carbohydrates produced

by mature leaves. Around a 20 cm depth of healthymaintenance foliage is required to

support a sustainable yield of tea (Sharma, 2011).Therefore, continuous fish leaf plucking

without keeping well-established mother leaves leads to adeterioration in the health,

vigour and productivity of the tea bush. Moreover, fishleaf or janam leaf plucking to

remove shoots with many leaves (coarse leaf) can reduce thequality of tea due to the

inclusion of more fibrous tissues. Usually, the moisturecontent of internodes (stalks) is also

greater than that of leaves and apical buds, so theinclusion of more stalks reduces the

dry matter yield of tea. In order to harvest tea shootscomfortably and more efficiently,

the severity of harvesting should be regulated so that themaximum bush height does not

exceed about 110 cm at the end of the pruning cycle(Sharma, 2011).

9.2.2 Frequency of harvesting (plucking rounds)

The frequency of harvesting (plucking round) refers to thenumber of days between

successive harvests. It is defined in the field as the timetaken by the majority of shoots

remaining after the previous round (arimbu shoots) tobecome harvestable again. An

ideal plucking round can also be scientifically defined asthe number of days between

successive opening of leaves or leaf period (phyllochron)and varies widely according to

the environmental conditions. The ambient temperature isone of the most influential

environmental factors. Three critical temperaturesaffecting plant growth are identified as

the • base (minimum) temperature, • optimum temperatureand • ceiling (maximum) temperature which are specific tothe plant species or variety.

The rate of growth increases more or less linearly betweenbase and optimum temperatures

(Squire, 1990). The base temperature below which shootextension ceases has been

found to be around 12–13°C (Carr & Stephens, 1992). Thenumber of days taken to

unfurl a leaf is less under warm climatic conditions.Accordingly, the plucking round can

be scientifically defined based on mean daily temperatures(Sharma, 2011). Also, shoot

growth is significantly affected by moisture stressconditions. Therefore, plucking rounds

are generally shorter during wet weather and warmerclimates than during dry weather

and cooler climatic conditions. The leaf period of commontea cultivars in Sri Lanka in wet

weather has been reported to be about 5–6 days at highelevations and 4–5 days at low

elevations (Ekanayake, 1992; Wijeratne, 1994). However, itcan be extended to 8 days or

more during dry weather at low elevations (Wijeratne, 1994)with warmer conditions.

Although heavier shoots can be harvested with extendedrounds, the overall yield will

be lower due to harvesting a lesser number of shoots over agiven period. Moreover, it will

adversely affect the quality of the end product due to thepresence of more fibrous tissues

or coarse leaves in the harvest. Under warm and wet weatherconditions, tea shoots can

be manually harvested at 5–7 day rounds. When tea shootsare harvested by shears or

machines under similar environmental conditions, thefrequency of harvesting may extend

to about 2–3 weeks. However, under temperate growingconditions where tea bushes are

commonly harvested by machines, for example, Japan,harvesting is only done 3–4 times

during the growing season from April to October with aharvesting frequency of about 45

days.

9.2.3 Standards of harvesting

In tea cultivation, there are three plucking standardsviz., fine, medium and coarse. When

shoots with two leaves are harvested, it is called ‘fineplucking’. If the harvested shoots

consist of three leaves, it is considered as ‘mediumplucking’. ‘Coarse plucking’ implies the

removal of shoots with more than three leaves and/or coarseleaves (Fig. 25). Sometimes,

the definition is also based on the proportion of differentsize shoots in the harvest so

fine plucking refers to a harvest that contains a higherproportion of smaller shoots while

a lower proportion of such shoots results in either a

medium or coarse plucking standard.

Continuous fine plucking may give a low yield due toharvesting smaller shoots and an

Figure 25 Plucking standards (left – fine plucking, middle– medium plucking and right – coarse

plucking).

extension of the period of shoot growth (shoot replacementcycle). Owing to low worker

productivity, it is costly to maintain a fine pluckingstandard throughout the year. In

contrast, medium plucking gives a high yield with anacceptable leaf standard producing

better quality teas at lower cost.

9.3 Non-selective harvesting

In non-selective harvesting, as many generations of shootsas possible are harvested

without considering their size or level of maturity. Due toexposure of maintenance

foliage devoid of growing shoots, this practice issometimes known as ‘black plucking’. By

removing small immature shoots, non-selective harvestingcan adversely affect the source–

sink balance of the tea bush. Tea plucking machines andshears often remove tea shoots

non-selectively. Although non-selective harvesting can givea substantially higher yield for

a few months initially, there could be a marked reductionin the yield in the latter part of

the pruning cycle due to adverse effects on shoot growthand the physiology of the tea

bush. The degree of yield loss depends largely on theperiod of non-selective harvesting,

vigour and health of the tea bush, use of fertilizers andenvironmental conditions.

Under tropical conditions, a non-selectively harvested cropcan consist of shoots of

various degrees of maturity leading to the production ofpoor quality tea. However, such

problems are reduced when the growth of tea shoots isseasonal and harvesting is only

done during the few months of the growing season such as inChina and Japan.

9.4 Plucking after bringing into bearing and pruning

Tea bushes brought into bearing (plucking) and recoveredfrom pruning do not have

sufficient maintenance foliage to support the growth ofshoots. Under such conditions,

ground cover is also very poor. Therefore, it is necessaryto adopt a light plucking system

(mother leaf plucking) and add sufficient foliage to thecanopy during the early stages

of bringing into bearing and after pruning. The period oflight plucking depends on the

condition of the canopy.

9.5 Mechanical harvesting

Mechanical harvesting is primarily aimed at increasingworker productivity and the

extent harvested daily. It is often used as a solution fora shortage of labour. A socio

economically acceptable mechanical harvesting system shouldensure a high output

per worker, lower plucking cost, acceptable quality teashoots, good health and vigour

of the bush, minimum environmental impact and workercomfort. The most commonly

used mechanical aids for tea harvesting are shears,portable motorized machines (fuel

driven, pneumatic or battery powered) and self-propelled orride-on type tea harvesters

(Figs. 26 and 27). Tea harvesting machines require specialgrowing and ground conditions

for optimized output. Handheld harvesting machines could beoperated on tea lands

with a slope of less than 25% (Sharma, 2011). As describedelsewhere, tea harvesting

shears and machines are mostly non-selective in harvesting.However, the TRI Selective

Tea Harvester without prominent (long) handles introducedby the Tea Research Institute

of Sri Lanka is a selective tea harvesting shear(Wijeratne, 2001). The output of tea

harvesting shears and machines depends largely on the yieldpotential of the field and

length of the cutting blades. On average, the efficiency ofhandheld shears is reported to

be in the range of 50–100 kg/day while that of handheldmotorized machines varies from

about 100 kg/day for a small battery-operated machine to1000 kg/day for a two-person

large handheld machine. The output of aself-propelled/ride-on type machine can be as

high as 3000–5000 kg/day.

Generally mechanical harvesting results in a lower yieldthan manual harvesting. Ideally

shears should only be used 9–12 months after pruning.Selective hand plucking to mother

leaf during low cropping months minimizes the adverseimpact of shear harvesting on

growth and yield of tea (Pathiranage et al., 2016; Sharma,2011). Field observations in

Sri Lanka have shown that continuous mechanical harvestingcan reduce tea yield by

about 50% of the potential yield under manual harvesting.However, by restricting the

use of machines to high cropping months (sometimes referredto as rush crop) when the

environmental conditions are most conducive for shootgrowth, the yield loss can be

reduced to about 20–30%. Foliar application of nutrientscomprised of urea, magnesium

sulphate and zinc sulphate encourages shoot growth andminimizes the adverse impact of

mechanical harvesting on tea yield (Sharma, 2011).

Figure 27 Harvesting of tea using a fuel-driven machine inChina.

Figure 26 Harvesting of tea using shears in Sri Lanka(left) and India (right).

9.6 Leaf handling

Tender shoots can be easily damaged during harvesting andsubsequent processing,

packaging and transportation. Harvested shoots withphysical damage and contaminated

with extraneous matter such as sand reduce the quality ofthe produced tea. Therefore,

it is extremely important to ensure that the shoots are notphysically damaged or

contaminated with extraneous materials during harvestingand the allied operations and

transportation. Compaction of harvested leaf in bags andover-stacking of bags filled with

harvested leaf reduce aeration and increase leaftemperature. Harvested leaf exposed

to high temperatures produce poor quality teas(Samaraweera, 2008; Sharma, 2011)

and should be avoided. Tea leaf should, therefore, becarefully handled and transported

without delay to ensure a good quality end product.

10 Replanting

The economic life of the tea bush is generally consideredto be around 50 years. However,

some of the original tea plantations are still productiveeven after 75 years. In contrast,

the economic lifespan of vegetatively propagated teacultivars has often been less than

50 years (Samansiri et al., 2011). The productivity of teabushes varies during the pruning

cycle. In addition, they continue to decline as they age.Tea bushes die due to various biotic

and abiotic stresses reducing land productivity. Soildegradation as a result of continuous

cultivation and soil erosion, various pest and diseaseinfestations, and drought are some of

the major causes of debilitation and death of tea plants.Therefore, replanting is essential

to maintain their sustainability. Nevertheless, the reasonsfor bush debilitation should be

clearly identified and corrected before the land isreplanted. Further, the development

and introduction of new cropping systems and tea cultivarswith improved characteristics

such as high yield, high quality, and tolerance to variouspests, diseases and adverse

environmental conditions warrant the replanting of old tea.

The yield of a field is taken

as the yardstick when deciding whether it should bereplanted. Generally, those that are

below the mean yield of a plantation are earmarked forreplanting. It is also necessary to

weigh up the exercise before large-scale replanting isundertaken, as it is a costly affair and

takes several years to produce a tangible harvest and reapany benefit.

As described under Section 3.2 all vegetation including oldtea should be uprooted

and removed from the field. Ring-barking of old shade treesabout two years before

uprooting is desirable to deplete the carbohydrate reservesof roots and reduce the risk

of re-infestation of the new tea cultivation with rootdiseases. A thorough inspection of

the field for pest and disease attacks, especially the rootdiseases, nematode and termite

infestations, is essential and any affected patches must beisolated for special treatment.

Diseased tea bushes if any, together with two rings ofhealthy bushes on the perimeter,

should be carefully uprooted and burnt in situ so that nofragments of such bushes are

allowed to come in contact with the disease-free land.Areas with a history of pest and

disease infestation can be treated with a recommended soilfumigant before replanting.

Deep forking to remove roots larger than a pencil thicknessto a depth of about 45 cm

is necessary in areas where pest and disease infestationsare found. Land clearing and

preparation should be done before the onset of heavy rainsand should start from the

upper-most section of the slope as discussed elsewhere. Theland should then be roughly

and adequately levelled.

All soil and soil moisture conservation measures need to beestablished as described

under Section 3.3. Old terraces and drains may either beleft and improved or new

structures built as necessary. The soil should be improvedby planting rehabilitation

grasses as described below.

11 Soil rehabilitation

The soil condition of old tea lands generally deterioratesdue to various cultivation practices

over a long period of time and hence ideally should beimproved before replanting with

tea. The process is known as soil rehabilitation. Grassspecies such as Tripsacum laxum

(Guatemala grass) and Cymbopogon confertiflorus (managrass) are commonly used for

soil rehabilitation of tea lands (Fig. 28). They areclosely planted on contour rows and

fertilized to produce a large quantity of biomass within18–24 months before they are

removed to plant tea. Planting of T. laxum is done at 22–30cm in the row and 60–120 cm

between rows and C. confertiflorus at 10–15 cm in the rowand 60–90 cm between rows.

The planting density of grasses should be high on slopesand in areas with poor soil. It is

preferable to apply dolomite before planting grasses toimprove soil pH and alleviate Mg

deficiency. Grasses should then be regularly lopped beforeflowering and the loppings

spread in situ. Ideally shade trees should also be plantedalong with the grasses so that

they provide sufficient shade for the young tea plants.

Improving the soil’s physical and chemical properties byadding organic matter, soil

conservation, reducing the weed seed bank, eliminatingsoil-borne pest and disease

pathogens and allelopathic substances, and facilitating theearly establishment of shade

trees are some of the objectives of soil rehabilitation. Inthis way young tea plants can be

successfully established with minimum casualties.

After a period of rehabilitation, the grass tops areremoved leaving the root system

under ground. The cut grasses are retained as mulch and teaplanting holes are prepared

in between grass rows as described elsewhere. The otherfield practices are similar to

those described under Section 3 –6.

Figure 28 Soil rehabilitation with Mana grass: Inset –Guatemala grass.


Climate change, soil degradation and labour scarcity are afew of the main challenges

faced by the tea growers aiming to improve tea yield byadopting GAP. Additionally, cost

has also been an issue in commercial agriculture.Therefore, research and development

should be geared to finding appropriate solutions for thesechallenges.

Rising temperatures and carbon dioxide levels due to globalwarming are reported to

bring beneficial impacts to the tea lands at high elevationwhere the temperatures are

below the optimum for tea. However, low tea yields havebeen projected in the warmer

regions due to high temperature regimes and dry weatherconditions (Anon, 2016).

High temperatures and extreme rainfall (droughts and suddendownpours) accelerate

soil degradation and reduce soil fertility. Continuousapplication of chemicals including

chemical fertilizers also affects soil health over time.Therefore, development of new

tea cultivars suitable for varying ecological conditions,new cropping patterns, new

water harvesting techniques, low-cost soil and soilmoisture conservation measures and

irrigation techniques, and new shade tree species and newtechniques for reducing the soil

rehabilitation period are vital in preparing the teaplantations for the forthcoming decades.

The cultivation practices of tea are labour intensive andhence costly. The availability

of manual workers in tea-growing regions is decliningrapidly. Therefore, research and

development on the mechanization of labour-intensivecultivation practices is vital and

land preparation and holing especially on slopes, pruningand selective harvesting of

shoots should be made a high priority.

Consumers are becoming more and more health conscious anddemand food free from

chemical residues. Hence, GAP using less chemicals mayreceive greater attention in years

to come.

Good cultivation practices provide a strong foundation fora successful tea plantation. In

this way, land selection paying special attention to thesoil and climatic requirements of tea,

land preparation and soil rehabilitation improving the soiland soil moisture conservation

and facilitating planting and other field operations aswell as the selection of suitable

cultivars for planting are vital at the initial stages oftea cultivation. Training tea plants to

form a well-spread bush frame by different methods ofbringing into bearing and pruning

and implementing proper harvesting policies helps improveand sustain growth, yield and

quality of tea over a long period of time. Theestablishment and management of shade

trees and wind/shelter belts creates a conduciveenvironment close to the natural habitat

of tea which minimizes abiotic stresses and climate changeimpacts. Regular infilling

programmes to maintain bush density at an optimum levelarrests yield decline and

replanting of marginal or low-yielding fields to replaceunproductive tea bushes improves

profits. Mechanization of labour-intensive field operationsis essential to reduce costs and

manage plantations where labour is a scarce resource.


Further information on planting and harvesting tea,

including tea’s climatic requirements,

planting methods, bush management practices and both manualand mechanical

Amarathunga, M. K. S. L. D., Jayaratne, K. P. S. C. andWijeratne, M. A. (1999). Effect of ambient temperature andevaporation on yield of tea in Sri Lanka. In Proceedings ofthe 55th Annual sessions, Part 1, SLASS, Colombo, SriLanka.

Anon. (2002). Guidelines on Land Suitability Classificationfor Tea. Tea Research Institute of Sri Lanka, Talawakelle,Sri Lanka.

Anon. (2009). Tea Nursery Management. Tea ResearchInstitute of Sri Lanka, Talawakelle, Sri Lanka.

Anon. (2011). Drought Mitigation in Tea Plantations. TeaResearch Institute of Sri Lanka, Talawakelle, Sri Lanka.

Anon. (2016). Report of the working group on climate changeof the FAO Intergovernmental group on tea. Prepared by: R.M. Baghat, K. Z. Ahmed, N. Gupta, R. M. Baruah (India), M.A. Wijeratne (Sri Lanka), J. K. Bore, W. Nyabundi (Kenya),W. Han, X. Li, P. Yan and G. J. Ahammed. Food andAgriculture Organization of the United Nations, p. 86.

Arunachalam, K. (1995). A Handbook on Indian Tea.Arunachalam Associates, The Nilgiris, India, p. 271.

Barua, D. N. (1969). Seasonal dormancy in tea (Camelliasinensis L.). Nature 224: 514.

Bond, T. E. T. (1945). Studies in the vegetative growth andanatomy of the tea plant (Camellia thea Link) with specialreference to the phloem. II. Further analysis of flushingbehaviour. Annals of Botany 9: 183–216.

Burgess, P. J. (1992). Dry matter production andpartitioning by clone 6/8. Ngwazi Tea Research Institute,Quarterly Report, No. 9, pp. 11–16.

Carr, M. K. V. (1972). The climatic requirements of the teaplant: A review. Experimental Agriculture, 8: 1–14.

Carr, M. K. V. and Stephens, W. (1992). Climate weather andthe yield of tea. In Wilson, K. C. and Clifford, M. N.(Eds) Tea: Cultivation to Consumption. Chapman and Hall,London, pp. 87–135.

De Costa, W. A. J. M, Mohotti, J. A. and Wijeratne, M. A.(2007). Ecophysiology of tea. Brazilian Journal of PlantPhysiology 19(4): 299–332.

Ekanayake, P. B. (1992). Report of the Agronomy Division,Annual Report for 1990. Tea Research Institute of SriLanka, Talawakelle, Sri Lanka, pp. 45–57.

Ekanayake, P. B. (1994). Application of SlopingAgricultural Land Technology (SALT) in tea plantations.Tea Bulletin 14(1/2), 3–17.

Herd, E. M. and Squire, G. R. (1976). Observations on thewinter dormancy in tea. Journal of Horticultural Sciences51: 267–79.

Hoshina, T., Kosuge, N., Kozai, S. and Honjo, U. (1983).Influence of air temperature on Nitrogen uptake by the teaplant. Study of Tea 64: 24–8.

Jain, N. K. et al. (Eds). (2008). Economic Crisis in TeaIndustry. International Society of Tea Science StudiumPress, LLC, Houston, USA, p. 409.

Japanese Green Tea. Executive committee of the 4thInternational Conference on O-CHA (Tea) Culture andScience (Eds.), Shizuoka City, Japan, p. 52.

Kulasegaram, S. and Kathiravetpillai, A. (1972). Effect ofnutrition and hormones on growth and apical dominance intea (Camellia sinensis (L.) O. Kuntze). Journal ofHorticultural Sciences, 47: 11–24.

Magambo, M. J. S., Omolo, J. G. and Othieno, C. O. (1988).The effect of plant density on yield, harvest index anddry matter production. Bulletin, United PlantersAssociation of South India, No.: 40, pp. 7–14.

Pathiranage, S. R. W., Wijeratne, M. A. and De Costa, W. A.J. M. (2016). Physiological aspects governing yieldvariation under manual and mechanical harvesting of clonaltea. In Proceedings of the Sixth Symposium on PlantationCrop Research-Plantation Agriculture towards NationalProsperity. Coconut Research Institute of Sri Lanka,Lunuwila, Sri Lanka, pp. 93–100.

Pethiyagoda, U. (1964). Some observations on the dormancyof the tea bush. Tea Quarterly 35: 74–83.

Rahman, F. (1988). Physiology of the tea bush. Two and aBud 35: 1–14.

Ranganathan, V., Raman, K. and Natesan, S. (1983).Nutritional and physiological interactions with length ofplucking rounds and banjiness. Bulletin, United Planters’Association of south India, No.: 38, pp. 67–89.

Samansiri, B. A. D, Rajasinghe, J. C. K. and Mahindapala,K. G. J. P. (2011). Agronomic Profile of the CorporateSector Tea Plantations in Sri Lanka: A Diagnostic Study inthe Corporate Tea Sector. Tea Research Institute of SriLanka, 99p.

Samaraweera, D. S. A. (2008). Technology of tea processing.Revised by D. S. A. Samaraweera and M. T. Ziyad Mohamed.In Zoysa, A. K. N. (Ed.) Handbook on Tea. Tea ResearchInstitute of Sri Lanka, Talawakelle, Sri Lanka, pp.265–322.

Sharma, V. S. (2011). Pruning & Harvesting (Plucking), AManual of Tea Cultivation. International Society of TeaScience, pp. 41–62.

Sharma, V. S. (2011). A Manual of Tea Cultivation.International Society of Tea Science, Sree Sai AnjaliGraphics, Hyderabad, India, p. 148.

Smith, R. I., Harvey, F. J. and Canell, M. G. R. (1993).Clonal responses of tea shoot extension rate totemperature in Malawi. Experimental Agriculture, 29: 47–60.

Somaratne, A., Zoysa, A. K. N. and Rajasinghe, J. C. K.(2008). Land preparation, planting and early aftercare. InZoysa, A. K. N. (Ed.) Handbook on Tea. Tea ResearchInstitute of Sri Lanka, Talawakelle, Sri Lanka, pp. 70–85.

Squire, G. R. (1979). Weather physiology and seasonality oftea (Camellia sinensis) yields in Malawi. ExperimentalAgriculture, 15: 321–30.

Squire, G. R. (1990). The Physiology of Tropical CropProduction. CAB International, Wallingford, UK, p. 236.

Stephens, W. and Carr, M. K. V. (1990). Seasonal and clonaldifferences in shoot extension rates and numbers in tea(Camellia sinensis). Experimental Agriculture 26: 83–98.

Tanton, T. W. (1979). Some factors limiting yield of tea(Camellia sinensis). Experimental Agriculture 15: 187–91.

Tanton, T. W. (1982). Environmental factors affecting theyield of tea (Camellia sinensis). II. Effect of soiltemperature, day length and dry air. ExperimentalAgriculture, 18: 53–63.

Tanton, T. W. (1992). Tea crop physiology. In Wilson, K. C.and Clifford, M. N. (Eds) Tea: Cultivation to Consumption.Chapman and Hall, London, pp. 173–99.

Watson, M. (2008a). Climatic requirements and soil. InZoysa, A. K. N. (Ed.) Handbook on Tea (First edition). TeaResearch Institute, Talawakelle, Sri Lanka, pp. 10–15.

Watson, M. (2008b). Pruning of tea. Revised by M. A.Wijeratne. In A. K. N. Zoysa (Ed.) Handbook of Tea (Firstedition). Tea Research Institute, Talawakelle, Sri Lanka,pp. 105–16.

Wijeratne, M. A. (1994). Effect of climatic factors on thegrowth of tea (Camellia sinensis L.) in the Low countryWet Zone of Sri Lanka. PhD Thesis. Wye College Universityof London, UK, p. 199.

Wijeratne, M. A. (2001). Shoot Growth and Harvesting ofTea. Tea Research Institute of Sri Lanka, Talawakelle, SriLanka, p. 45.

Wijeratne, M. A., Aanadacumaraswamy, A., Amarathunga, M. K.S. L. D., Janaka Ratnasiri, Basnayake, B. R. S. B. andKalra, N. (2007). Assessment of impact of climate change onproductivity of tea (Camellia sinensis L.) plantation inSri Lanka. Journal of National Science Foundation of SriLanka 35(2): 119–26.

Wijeratne, M. A. and Fordham, R. (1996). Effect ofenvironmental factors on growth and yield of tea (Camelliasinensis L.) in the Low country wet Zone of Sri Lanka. SriLanka Journal of Tea Science 46: 21–34.

Wijeratne, M. A. and Ekanayake, P. B. (1990). Someobservations on practices to be adopted in minimizingdrought effects in new clearings of tea plantations. TeaBulletin 10(1): 15–22.

Wijeratne M. A. Premathunga P. and Karunaratne, W. R. M. M.(2002). Variation in root starch reserves of tea and itsimpact on recovery after pruning. Journal of PlantationCrops 30(1): 35–9.

Zoysa, A. K. N. (Ed.). (2008). Handbook on Tea (FirstEdition). The Tea Research Institute of Sri Lanka,Talawakelle, Sri Lanka, p. 384.

5 Chapter 5 The effect of cultivationtechniques on tea quality

1 Introduction

Tea (Camellia sinensis (L.) O. Kuntze) is an important cashcrop. The plant is commercially

exploited from as far north as 49° N in Outer Carpathiansto 33° S in Natal, South Africa 1 .

It grows at altitudes ranging from sea level in Japan andSri Lanka 2 to around to 2700 m

above mean sea level (amsl) in Kenya and Rwanda 3 . Teafrom C. sinensis is the most widely

consumed beverage in the world, after water 4 .Consequently, tea is the most important

crop species in the genus Camellia, which has been reportedto contain over 200 species

so far 5 . The plant is an important economic cash crop inmany countries, being a source

of gainful employment opportunities and generating foreignexchange revenues for the

producing countries. As any commercial cash crop, teaproduction should give economic

and/or profitable returns to farmers. The profitability intea production depends on whether

the type of tea produced has the right quality that isacceptable to consumers. The definition

of quality by stakeholders, however, depends on one’sposition in the world’s tea market.

Producers perceive good-quality tea as that which provideshighest profit at minimum cost

of production, while for consumers, good-quality teaproduces beverages with desirable

taste attributes at the right price. However, middlemen(blenders, packers, auctioneers and

traders) require tea from which they make maximum profits.Sensory evaluation is considered

the most practical and a fast method for evaluating qualityin tea trade. Usually, the black teas

are valued for appearance, liquor colour, strength,briskness, aroma, flavour 6–8 and ‘cuppage’.

These characteristics are mainly due to the polyphenoliccontents of plain black tea, especially

theaflavins (TFs) and thearubigins (TRs) 6–8 . However,sensory evaluation is criticized for being

subjective and influenced by many factors 8 outside teaquality such as individual taster’s

preference and lack of uniform scales in grading and and/ormarket forces 7 . The subjectivity

in the definition of good quality tea and unreliability ofpractical quality assessment method

makes it challenging in science to define the correctattributes for measuring black tea quality

objectively. In research on tea quality, it becomesimportant to develop objective standards

and definitions for identifying good quality black teas 9 .Despite the noted weaknesses of

sensory evaluation as a quality assessment tool, it remainsthe most practicable method in

the black tea trade. Thus, scientific evaluation of blacktea quality requires an assessment of

objectively and reproducibly measurable chemical parametersthat can be related to sensory

evaluation under stable market conditions.

2 Chemical quality parameters of tea

Black tea contains chemical components responsible for itscharacteristic colour, taste and

aroma. These compounds include TFs, which are responsible

for briskness and brightness,

and TRs, which give black tea its colour and thickness(mouth feel) 10 . In early studies 11 , TF

levels were recognized to be related to black tea quality.However, in subsequent studies

total TFs showed good relationships with market values orsensory evaluation of black teas

from some countries 12,13 ; but such relationships wereinsignificant for black tea from some

major producing countries 14,15 . Thus, for some countriestotal TF was suggested as the

objective measure of black tea quality 16,17 .

Black tea contains four major TFs (Fig. 1) with varyingastringencies 18 . Clonal evaluation

of black teas in Central Africa indicated total TFs as thebest predictor of high-quality

cultivars and the green leaf (–)-epicatechin (EC) contentshowed the highest relationship

with black tea TF levels 19 . The order of relationshipbetween the individual TFs was

TF-3’-G > TF-3-G > TF, while theaflavin digallate (TFDG)showed no significant relationship

with sensory evaluation 20 . The results demonstrated thatfor Central African tea, the

formation of the individual TFs could be used as aselection criterion in tea breeding or

clonal selection.

Figure 1 Structures of the major black tea theaflavins.

The lack of uniformity in the relationships between TF andsensory evaluation was in

part due to differences in compositions of the TF in theteas. When the astringencies of

the individual TFs were normalized, using a factortheaflavin digallate equivalent (TFDG

Eq.) 21 , good relationships were obtained between TFDG Eq.and sensory evaluations for

black teas from different tea-growing countries, even thosethat had shown insignificant

relationship 22 . This confirmed TF levels to be a usefulquality parameter for black tea

quality evaluation. Additionally, black tea containscaffeine, which contributes to briskness

and is the main factor responsible for the mild stimulatingeffect of tea. Thus, the levels of

TFs, TR and caffeine are the main chemical qualityparameters for measuring the quality of

plain black tea. However, during black tea processing, notall flavan-3-ols (catechins) are

oxidized. The residual flavan-3-ols 23–25 and possiblyother polyphenolic components of tea

leaf and black tea are astringent. Although theirastringency contributes to plain black tea

quality, such studies have not attracted much attention.

Black tea contains many volatile flavour compounds (VFCs)which are responsible

for giving aroma 26 . The aroma complex of black tea isresponsible for flavour. Some of

these compounds occur at low levels, below detection limitsof analytical instruments

used in quality determination, but affect the flavoursignificantly, as humans are more

sensitive and have a greater detection limits thananalytical instruments. However, many

of the compounds occur at levels that instruments candetect. These aroma compounds

(perceived by smell receptors) are classified as Group Iand II VFCs 27 . The Group I VFCs,

mainly green leaf volatiles, impart a green, grassy smellto black tea and are considered

undesirable to black tea quality. These Group I VFCs aremainly breakdown products

of unsaturated fatty acids in tea leaves 26 . Other VFCsimpart a sweet flowery aroma to

black tea 28 and are classified as Group II VFCs 27 . Theimportant factor as far as the tea

quality is concerned is the ratio of Group II to Group I,namely the flavour index (FI) 27,29,30 .

However, FI must be used with as a quality parameter withsome caution. Some VFCs

may occur in low concentrations, yet have a huge impact onaroma and vice versa 31 . For

example, methyl epijasmonate has 400 times more intensearoma than methyl jasmonate

at equivalent concentrations 32 . In addition, neuralresponse is not necessarily proportional

to concentrations as measured by gas chromatographicresponse 33 .

Based on sensory evaluation, Group I VFCs reduce black teaquality, while Group II VFCs

improve tea quality. The ratio of Group II:Group I VFC isreferred to as the FI and is a measure

of the aroma quality of black tea, while high TF, TR andcaffeine levels enhance tea quality.

Excessive amounts of TR, however, reduce TF levels and givea muddy and unpalatable taste

to black teas 10 . During black tea processing, it isimportant to increase TR levels only as long

as TF levels are also increasing. Linear regressionanalyses with sensory evaluation data have

shown the aforementioned parameters as objective indicatorsof black tea quality. Using

these parameters, studies have been conducted to assess howthe parameters change with

agronomic practices and environmental factors. However, itis recognized that while black

tea quality can be improved through proper agronomicpractices, manufacturing practices

must be optimized for quality black tea production 34 .Manufacturing practices without

adequate controls can destroy good leaf and produceinferior tea, while it is impossible to

improve low-quality leaf through proper manufacture.

In sensory evaluation, the quality of black tea refers toall characters such as colour,

brightness, appearance, liquoring properties, strength andaroma by which the black

tea may be judged, for its market value 35 . Black teaquality is therefore a summation of

its desirable attributes. In the tea trade, black teaquality is usually used to refer to the

presence of special desirable characters in the liquor11,36 required by the market at a given

time. The quality attributes and the special qualities,which may be present in some teas,

chemical quality parameters. It is important to understandhow agronomic practices impart

desirable chemical characteristics to black tea.

With increased productivity per unit area of land andfurther expansion of tea lands, the

supply of black tea appears to be outstripping demand andbuyers are becoming more

selective. Therefore, black tea quality has become a vitalaspect of tea production as most

of the time it is likely to determine the unit price oftea. Black tea chemical composition,

and hence quality, is influenced by factors both in thefield and factories 37 . These factors

can be broadly divided into controllable andnon-controllable factors. Controllable factors

include field cultural practices and, to some extent,genetics of plants and factory operation

practices. Genetic factors are classified as bothcontrollable and non-controllable. Tea

plants can have an economic lifespan of over a hundredyears. With such a long economic

lifespan, plantations shall be inherited from generation togeneration. The initial tea

plantations were mainly of seedling origin with diversequality potentials. The black tea

quality of seedling tea plantations that are selectedmainly based on vigour and hence

perceived yields is generally low. However, there are alsoseedling plantations producing

good-quality black tea. Plantations that are more recenthave used clonal materials or

seedling materials that were selected for yield rather thanquality. Planters with such crops

have few options for controlling the quality of theirproduce, as the genetic traits are fixed.

However, with the vast improvement in tea breeding andclonal selection, clonal materials

are now available that were purposely developed to producespecific high-quality black

teas. Current planters therefore have a choice to controlthe quality through the use of

selected cultivars with high black tea quality potentials.The environmental factors are

largely non-controllable. Most of the cultural practiceswhich affect black tea quality can

easily be controlled, while the environmental factors arethe most difficult to control 11,38 .

3 Cultivars and black tea quality

Farming for profit requires that plants with desirableeconomic attributes be exploited.

In tea production, considerable attention has been devotedto increasing tea production

through the mitigation of biotic and abiotic factors thatcould inhibit increased production

per unit area. Vigour and growth characteristics have beenused in the selection of both

seedling and clonal tea plants. However, apart from yields,producing black tea with

good quality leads to better prices and hence higherprofits. Unfortunately, there is no

relationship between yields and black tea quality 39 .Selection of tea plants for commercial

exploitation must therefore also address qualityrequirements.

3.1 Selection criteria for quality

Tea plants have different morphological features, some ofwhich have been thought to

be related to chemical composition. Leaf characters such aspubescence, colour and size

were reported to be related to black tea quality 15,36 .Yellow-leafed tea bushes produced

higher leaf extracts, high polyphenol contents and lowprotein nitrogen than very green

leafed tea bushes 36,40 . In colour development, theyellow-leafed bushes were better in

both colour intensity and brightness. The morphologicalfeatures have therefore been

used in selecting superior plants to be cloned. Thus,cultivar selection has for a long time

been recognized as a method of improving black tea quality.

Variety influences the tea leaf characteristics and finalinfusion chemical compositions 41 .

The chemical composition of the leaf, viz. the leafpolyphenols content, the flavour

compounds and caffeine content, are genetically influenced11,15,38 . The commercially

exploited C. sinensis varieties produce black tea ofdifferent chemical compositions

and quality 42 . Grown in the same environment with similaragronomic inputs, C. sinensis

var. Assamica produces teas with higher plain tea qualityparameters than variety

Sinensis. However, the variety Sinensis produces flavouryblack teas. Thus, the quality

of black tea can be controlled through the use of plantwith correct genetic makeup.

Tea varieties have different contents of total catechins(TCs). The levels are in the order

Var. Sinensis < Assamica < Pubilimba 43 . The Assamicavariety had the highest levels of

(−)-epicatechin gallate (ECG) and (−)-EC, whereas thePubilimba variety had the highest

levels of (−)-epigallocatechin gallate (EGCG),(+)-gallocatechin (GC), (+)-catechin (C) and

(−)-gallocatechin gallate (GCG) 43 . The levels of TCs werehighest in the ‘Cambod’ cultivar,

followed by Assamica cultivar and least in Sinensis variety44 . Carotenoid content of tea

leaves is dominated mainly by ß-carotene, lutein andzeaxanthin. China (Sinensis) varieties

contained more carotenoid compounds than Assam (Assamica)clones. During processing

the carotenoids degrade to yield desirable aroma volatilesin made black tea 45 . Even in

the same variety, the chemical composition 46,47 and hencequality vary with tea clones 48 .

In terms of elemental levels, the variety Sinensiscultivars exhibited higher Al and F than

Var. Assamica tea bushes 49 and the amount of Al and Freleased into tea liquor is directly

related to their levels in tea leaves. Within the samevariety, the concentrations of F and

Al in tea plants are significantly different among the teacultivars 50 . The F and Al levels in

black tea can be reduced through cultivar selection.

3.2 Selecting tea plants for quality

Grafting has been used to promote the survival of somedesirable clones from adverse

environmental conditions and to improve yields. However,grafting did not alter the

biochemical composition of green leaf and black tea 51,52 .Within a single variety, studies

have shown that different clones produce different qualityblack teas 53–55 . Proper breeding

and clonal selection for the desirable black tea qualityparameters is therefore one major

method of improving black tea quality. As a result, teabreeders and planters have invested

time in breeding and selecting clones for quality. The

selection process has, however,

been made difficult due to the lack of reliable selectioncriteria. Though early selection for

quality was based on chloroform test 56 , it does notalways correlate with black tea quality

and it only predicts the fermentation ability of clones 57. In clonal selection programmes,

clones have therefore been subjected to black tea qualityevaluation after it can produce

adequate leaves to process black tea. This has made clonalselection for black tea a long

process lasting for up to 12–16 years. The identified plantmust go through several stages

before it can produce adequate leaves for miniaturemanufacture 58–61 and before it can be

subjected to sensory evaluations and chemical analyses toevaluate the quality potential.

Thus, the process is normally long and tedious.

With advancement in analytical methods, it has been shownthat clonal black teas produce

a unique combination of the individual TFs 62,63 . This ledto the realization that some clones

had the potential to produce black teas with high TFDG Eq.even if the levels of total

TFs were similar or slightly lower 22,64 . Evaluation ofthe green leaf flavan-3-ols revealed

that the patterns of individual TFs was determined by thecomposition of the green leaf

flavan-3-ols 22,64,65 . The patterns and amounts of TCscould be used to predict plain black

tea quality 64 . This created a reliable method ofselecting cultivars at a single bush stage, as

the quality prediction needed very little leaf. Efforts are

now directed to understanding the

clonal variations in flavan-3-ol levels and factorsinfluencing the variations 66–68 . However,

the flavan-3-ol composition and their total amounts alonecannot be adequate parameters

in clonal selection in relation to black tea quality. Otherparameters including carotenoid

compounds and chlorophyll composition 69 , polyphenoloxidase (PPO) activity 55 and

caffeine content 55,67 have also been used as criteria inselecting clones. These traits are

genetically controlled 70 although there has been nosuccess in identifying the particular

genes controlling the composition of the various precursorconstituents responsible for the

chemical black tea quality precursors in the tea plants.However, recent parental crossing

studies have shown that the general combining abilityeffects were significant for black

tea quality traits but specific combining ability effectswere significant for fermentability

and pubescence 71 . The black tea traits were predominantlygoverned by an additive gene

effect with strong influence of maternal parent 71signifying the importance of the choice

of female parents in tea breeding programmes targetingblack tea quality. The black

tea quality-related traits are highly heritable and guidedbreeding and judicious clonal

selection would lead to tea quality improvement 71 .

Mixing the clones during black tea processing averages outthe effect of the low-quality

clones used 72 . Quality of the tea made from seedlings was

improved when blended with

clonal leaves 73 . However, such mixing of leaf shouldinvolve compatible leaves. Similarly,

mixing clonal leaf has the effect of averaging the qualityof resultant black teas 72 .

3.3 Other parameters influencing clonal tea quality

Despite the realization that the precursor black teaquality parameters are useful in

breeding/clonal selection for quality, these precursors areusually converted to black tea

quality parameters through enzymatic interventions. Levelsof the precursor chemicals

could be ideal, but in the absence of optimal levels andactivities of the relevant enzymes,

it would still not be possible to make better quality blackteas. Specific activities of PPO,

peroxidase (POD) and protease and the relative amounts offlavan-3-ols vary with clones

and shoot maturity 74 . PPO and phenylalanine ammonia lyase(PAL) activities in tea leaves

vary with clones resulting in variations in black teaquality 75 . PAL activity was highest in the

drought-tolerant ‘Assam’ cultivar UPASI-2, followed byUPASI-8 and UPASI-9, under non

stress conditions 76 . Indeed, the ratio of PPO tocatechins necessary to form TF has been

suggested as an important clonal selection criterion foroptimizing black tea quality 77 .

The PPO-to-substrate ratio can be employed in identifyingsuperior quality clones from

the existing tea germ plasm or clones under development.Also different tea clones show

different tea shoot lipoxygenase activities 78 .

Clonal variations in glycosidic precursors of volatilecompounds have been

observed and were directly correlated with the differenceobserved in the black tea 79 .

Glycosides are the precursors of the alcoholic aromacompounds of black tea, which are

hydrolysed by endogenous glycosidases during themanufacturing process. Eleven ß-D

glucopyranosides, 10 ß-primeverosides(6-O-ß-D-xylopyranosyl-ß-D-glucopyranoside)

with aglycons as the aforementioned alcohols and geranylß-vicianoside (6-O-ɑ-L

arabinopyranosyl-ß-D-glucopyranoside) were identified infresh tea leaves and their levels

varied with cultivars. Primeverosides were about 3-foldmore abundant than glucosides in

each cultivar investigated 80 . The level of primeverosidesdecreased greatly during black

tea manufacturing process, especially during the macerationand fermentation stages.

At the end of fermentation, primeverosides had almostdisappeared, but glucoside

levels were largely unchanged 81 . The results demonstratethat primeverosides are the

main black tea aroma precursors. The glycosidase activitiesremained at a high level

during withering in black tea processing but the activitieschanged during maceration

and fermentation processes confirming that hydrolysis ofthe glycosides mainly occurred

during these two stages to release the alcoholic aromacompounds 81 . In Malawi, the major

volatile constituents from some tea clones of China-type

characteristics were similar to

those reported for flavoury tea produced in India and SriLanka. Levels of the volatile

components varied with clones 82 .

Anthocyanins are secondary metabolites found in many plantspecies. They contribute

to the attractive colours of fruits, vegetables andflowers, imparting red, orange, purple,

violet and blue colours. Anthocyanins have potential healthbenefits and are widely used as

sources of synthetic colourants/dyes. In industries,anthocyanins are used as food colourants

and preservatives in the manufacture of cosmetics and, inthe pharmaceutical industry, in

tablet/capsule coatings, syrups and as health concentratesand in the making of red wine.

Tea cultivars produce different levels of anthocyanins.Five of the six most common natural

anthocyanidins were identified from purple coloured tealeaf in both black and green teas,

namely delphinidin, cyanidin, pelargonidin, peonidin andmalvidin. Malvidin levels were

higher than other anthocyanidins in tea. In addtion, twoanthocyanin glycosides, cyanidin

3-O-galactoside and cyanidin-3-O-glucoside were alsodetected in tea plants 83 .

4 Environmental factors

The environmental factors affecting the natural growth ofthe tea bushes have a great

effect on the quality of black tea 38,40 . Such factorsaffect leaf morphology, rate of shoot

growth and chemical composition of the leaf 11,37,39,84 .The leaf chemical composition

variations cause changes in the black tea quality whichvaries with the geographical area of

production 27,85 . The content of phenolic compounds andantioxidant properties of tea are

influenced by a variety of environmental factors 86 .Factors responsible for the differences

in the chemical composition have, however, not beencompletely documented but include

seasons, region of production, temperatures, rainfall/waterstress and shade/sun light

hours.

4.1 Seasons/times of year

As pointed out earlier, tea plant is economically exploitedfrom as far north as 49° N to 33° S 1 .

Where tea is grown further away from the equator, there arenormally distinct seasons with

large variations in factors responsible for growth. Forexample, temperatures vary widely. The

season in which tea is has a significant effect on blacktea quality. In Sri Lanka, for example,

certain seasons produce flavoury black teas 11,87 . Similarflavours have been observed in

Darjeeling, in northeast India, plantations of black teas79,88,89 . These are attributed to climatic

changes following the onset of rainfall and drastic changesin temperatures. Where tea is

grown closer to the equator, although there are clearrainfall distribution patterns, the

temperature variations are not high 90 . Under suchconditions, it is assumed that the black tea

quality of the same cultivar would be the same throughoutthe year. However, even along the

equator, although the changes in quality were not as high,black tea quality varied with time

of the year 91–95 . Despite lack of clear seasons, alongthe equator, rainfall and temperatures

varied depending on the time of the year. Such changes weretherefore attributed to the

variations in climatic factors 90 and growth patterns andparameters 96,97 . For plain black teas,

the effects produced at different times of the year are inpart due to changes in flavan-3-ol

composition 67 . However, it has been noted most changesobserved in black tea at different

times of the year have been related to the aroma quality 98.

These changes could be arising from differences in the leafchemical composition. For

example, tea polyphenol contents are higher in summer thanin spring green leaf 99,100 . In

China, the contents of EGCG, CG and TC in the autumn teawere significantly higher than

those from the spring tea, but no differences in thecontents of EGCG, CG and TC were

observed between spring and autumn teas grown at a highaltitude 101 . The contents of

(–)-epigallocatechin (EGC), (+)-catechin (C), (–)-GC andsimple catechins in the spring tea

leaves were significantly higher than those in the autumntea leaves grown at a low altitude,

but were not different from those in the autumn tea leavesgrown at a high altitude 101 . In

Australia, the levels of EGCG, ECG and CGs in the fresh teashoots were higher in the

warm months and lower during the cool months 102 .Accumulation of polyphenols was the

highest during summer, while that of amino acids andchlorophylls was higher in monsoon

periods 103 . Mechanisms that induce seasonal variations intea shoots may include one or all

three of the following environmental conditions: daylength, sunlight and/or temperature,

which vary markedly across seasons 102 . Seasonalvariations were also observed in the

quantity of bound volatiles in regional Kangra clone duringthree different growth flushes

of tea 79 . The seasonal variations in glycosidicprecursors of volatile compounds were

directly correlated to the difference in the black tea 79 .

In black tea, the quality fluctuated with seasons 100,104 .The variability in caffeine and tannins

is minimal, but variability in TF and TR was large,indicating better black tea quality in the

growing season 104 . The levels of black tea caffeine andcrude fibre contents of black tea

varied within different growing periods and at differenttimes within each shooting period in

Turkey 105 . TF, caffeine and water extract contents and FIwere the highest in dry season teas,

while the crude fibre content was low in dry season teas103 . In the Eastern Black Sea region of

Turkey, the levels of total phenolics and antioxidantactivities of clonal tea were lower in cool

months but the levels of total phenolics increased duringthe warmer months 106,107 . For Kangra

orthodox tea, TF, TR and caffeine contents were recordedmaximum during early flush and a

gradual decline with progress in seasons was observed, witha minimum during main flush and

slight improvement through backend flush 108 . TF and TRcontents, total colour and brightness

of orthodox black tea varied with seasons in Kangra teaplantations 89 . The rainy season teas

had low quality due to high chlorophyll content, while thesummer season teas were superior

in their total colour and brightness 89 . There were highproportions of provisionally identified

flavour components, linalool, geraniol, ß-ionone, methylsalicylate, phenyl acetaldehyde and

trans-Z-hexenal, and several unidentified components withtypical Kangra characteristics

recorded during early flush were in low levels orcompletely disappeared during the main

flush 108 . Flavour quality slightly improved in backendflush over main flush 108 .

Seasonal variation of minerals (N, P, K, Ca, Mg, Na, Fe,Cu, Mn and Zn) changed with

clones 106,107 . Mn and Fe levels varied with seasons inTurkey 109 . Black tea from the third

shooting period had the highest Mn content, while those ofthe first shooting period gave

the lowest Mn value. Fe values showed the opposite effect.There was also a seasonal

variation in F content 110 .

4.2 Geographical location of plantation

Changing environmental conditions influenced the tea leafand final infusion chemical

compositions, 41 leading to differences in quality ofblack teas from different parts of the

world 27,85 . Indeed some regions of the world produceunique high-quality black teas. This in

part has been attributed to cultivars used in the regions.It has been assumed that cultivars

with desirable attributes maintain those attributesirrespective of where the plants are grown.

As a result, there have been attempts to import good teacultivars across the tea-growing

regions. To maintain economic advantage of the goodattributes, owners of plants with good

attributes have resisted or minimized such exchanges. Dueto lack of the genetic material

exchange or minimal exchanges, there have been few studiesto compare the performance

of similar cultivars across different countries or betweenenvironments with large differences.

In one such attempt, similar clones were demonstrated toproduce black teas with different

black tea qualities when produced under similar agronomicinputs in Kenya and Malawi 111 .

The study demonstrated that while the exchange of geneticmaterials may be desirable in

tea production, selection of a cultivar for good attributesin one country does not guarantee

maintenance of such attributes when the cultivar is grownin different environments.

Within a country or neighbouring countries where climaticconditions are not very

diverse, changes in black tea quality have been observed tovary with geographical area

of production 92,112,113 . In northeast India, the chemicalparameters of the plain black tea

varied with the topography of the region of the plantation.Overall quality was highest in

Brahmaputra valley teas followed by Dooars region and Barakvalley teas. The high quality

was attributed to higher levels of TF, TR, BR and TSS.Barak valley and Dooars region teas

contained high residual catechin, indicating limitedoxidation during processing 114 . Such

variations were observed even with the use of the samecultivars 10,29,115 subjected to similar

agronomic and cultural practices 48,116 , even when theplantations are only within a radius

of 30 km 117–119 . Such variations were also observed ingrowth parameters and yields 120,121 .

Studies on the chemical quality precursors responsible forthe plain black tea quality

parameters demonstrate that the observed variations are inpart due to the changes in

flavan-3-ols composition 68,122 . In addition, precursorsof VFCs responsible for green grassy

aroma vary with the location of production 48,123–125 . Theobservations demonstrate that even

with the use of same cultivars, agronomic inputs andcultural practices, it is not possible to

replicate black tea quality in different environments.However, there are cultivars which are

very unstable under changes in the environment in terms ofyields 120,126 and flavan-3-ols 66,68 .

It is necessary that new cultivars be evaluated in theareas of intended production before

they are widely used by farmers. Only plant cultivars withhigh yield and quality potential

should be made available to the farmers in particularregions.

Some geographical locations are uniquely placed to produceblack teas with particular

attributes. For example, Darjeeling teas have unique

quality characteristics with a

reputation that largely is attributed to the location ofproduction. Indeed such quality

attributes have not been replicated elsewhere, even withthe use of same cultivars.

5 Altitude and temperatures

Commercial production of tea has been reported from sealevel 2 to up to 2700 m amsl 3 .

There are therefore wide variations in temperatures andother climatic factors under which

the plant thrives. The temperature of the environment underwhich tea is grown determines

the quality of made tea 87 . Provided moisture is notlimiting, high temperatures favour fast

growth and high yields 96,97 . Such fast growth leads tothe production of low-quality black

teas 87 . Generally, tea plants grown at high altitudewhere temperatures are normally low

are believed to make superior quality black tea. As aresult, some tea brands are marketed

as high grown to demonstrate their potential ashigh-quality black teas. However, lack

of well-planned trials across different altitudes fromclose to sea level to above 2000 m

amsl has made it difficult to conclusively support orrefute the belief. Such attempts are

compromised by other factors including large variations inenvironmental and climatic

factors across wide environments. When assessed overextremely wide geographical areas,

there were no significant relationships between black teaquality 29,112,127 or precursors of

black tea quality 67,123,124 and altitudes. However, when

such a study was conducted within

a radius of only 10 km and at altitudes ranging from 1940to 2180 m amsl, there was an

improvement in caffeine and black tea aroma with rise inaltitude 128 . For the polyphenolic

black tea quality parameters, in some cultivars, there wasquality improvement, while in

others, black tea quality declined with rise in altitude.Of the plain black tea precursors (–)

EGCG, catechin gallate (CG) and TC contents are higher inteas grown in a high altitude

than those grown at a low altitude in China 101 . Thevariation black tea quality with altitude

can be closely linked to variations in temperatures thatdecline with rise in altitude. In

Turkey, low-land teas contained more fluoride thanhigh-land teas 110 . The results confirm

the notion that high-grown teas make better quality blackteas than low-grown teas. In fact,

the high-grown teas are generally more flavoury, whilelow-grown teas are plainer.

5.1 Rainfall

Rainfall distribution can be a major factor in determiningthe productivity and quality of black

teas 129 . For example, in Kenya, the tea-growing areas canbe broadly divided into east and

west of the Great Rift Valley. Some major geographicalfeatures have caused major climatic

differences between the two zones. The presence of LakeVictoria in the western zone has

created a situation whereby the zone realizes warm and wetconditions during most of the

year. The western zone therefore realizes faster and even

growth of tea shoots most of the

year 130 . Fast growth rate and continuous production oflarge shoots are conducive to high

green leaf yields 39 . However, conditions which favourhigh yield potential often produce

low-flavour, and plain black tea quality 40 . There is ahigh potential for producing high tea

yields but plain black teas in the region 92,94,131 . Inthe east of the Rift Valley, there are two

distinct rainfall seasons separated by dry seasons. Duringthe dry season, there is a slow

growth rate and thus low yields 132 . These conditionsoffer a high potential for producing

good quality 35,93,95 . The dry season is alsocharacterized by cool night temperatures, which

are conducive to high-quality tea production. However, inthe wet seasons, the conditions

are close to those of the west of the Great Rift Valley. InChina 129 , tea grows faster by

up to 50% during the monsoon period compared to spring.Concurrently, the levels of

catechin and caffeine decrease, while total phenolicconcentrations and antioxidant activity

increased during the monsoon. The decrease in levels ofcatechins and caffeine leads to

reduced farmer preference of black tea based on sensoryparameters.

Soil moisture influences leaf characteristics. Fine andsmall leaves are produced under low

moisture condition 84,87,133 . Such leaves produce betterblack tea quality 35,39,87 . Dry seasons

lead to slower shoot growth rates and high-quality made tea35,39 . Tea grows faster during

rains; black teas from such tea plants generally have aflat taste and low quality 40 . Moisture is

thus an important factor determining made tea quality.Indeed, there is a close relationship

between soil moisture status and catechin content of greentea leaves 134 . Whereas TC, EC

and epigallocatechin levels were directly proportional tosoil water content or water index

status, EGCG and ECG levels did not respond to soilmoisture status. High level of water

deficiency reduced the tea polyphenol levels butdrought-tolerant cultivar showed minimum

variations suggesting that the levels of polyphenols can beused as selection criteria in

clonal selection 135 . Water deficit conditions/stressincreased levels of EC quinone (ECQ) and

EGCG quinone (EGCGQ), the oxidation products of EC and EGCGand the accumulation

of the flavan-3-ols 136 . Formation of ECQ and EGCGQstrongly and negatively correlated

with the extent of lipid peroxidation in leaves, thussupporting the protective role of these

compounds in drought-stressed plants.

Polyphenol levels, PAL, PPO and peroxidase activitiesincrease with the onset of drought,

but decline with prolonged drought 137 . Chlorophyllcontent decreased, while proline levels

increased with an increase in drought intensity of teaplants 137 . Water stress reduces PAL

activity 76 . At lower PAL activity, synthesis of flavanolssuch as EGCG and ECG reduced 76 .

Levels of gallic acid and caffeine declined due to bothdrought and waterlogging stress 76 .

Drought stress increases the level of some aromaticcomponents in the fresh leaves. Most

of the terpenoid compound contents increased, while levelsgreen leaf volatiles decreased

with an increase in soil moisture deficit 138,139 .

6 Agronomic inputs and tea quality

6.1 Shade

Tea was discovered in Southeast Asia under shade. Thus,production of tea under shade

was adopted as an imitation of the original habitat 39 .However, the microclimate under

shade increased the incidence of blister blight in Indiaand Sri Lanka. Shade trees were

therefore reduced in tea plantations. Where there wasnitrogenous fertilizer application,

the shade reduction was followed by an increase in yields.In East Africa the use of

shade trees was discontinued when it was realized that theshade reduced yields 140–142

and nutrient uptake of the tea bushes 140 but shadeimproved tea quality 143 . Both quality

improvement 143 and yield decline 140 depend on theintensity of the shade 140 . Thus use of

shade in tea plantations could improve the quality but atthe expense of yields. A situation

is developing among smallholders where some trees mighthave been planted in the tea

farms in order to supplement the farmers’ energyrequirements. Such trees would provide

some amount of shade to the tea bushes. Therefore, acomprehensive study on the effect

of the limited shade may be necessary.

6.2 Fertilizers and black tea quality

In tea plantations, nutrients are lost through leaching,fixation, soil erosion and crop

harvests. Economic production therefore requires nutrientsupplementation through

the addition of inorganic and/or organic fertilizers. Theseare usually applied through

ground application or as foliar feeds. The major nutrientsfor tea production are nitrogen,

phosphorous and potassium. However, the plant also benefitsfrom the application of

other macro- and micronutrients.

6.2.1 Nitrogen

Several studies have shown that climatic conditions andagronomic practices, which

promote high green leaf production, have an adverse effecton black tea quality 11,13,87,144,145 .

Similarly, studies have recorded beneficial yield responsesfrom the application of

nitrogenous fertilizers 146–148 . Nitrogenous fertilizerapplication may therefore influence

black tea quality.

The changes in the chemical composition of black tea due tonitrogenous fertilizers

have been the subject of several studies. These studiesdemonstrated that excessive use

of nitrogen fertilizers lowers black tea quality12,48,118,119,125,149–153 . In terms of plain black tea

parameters, rates of nitrogen up to about 150 kg N/ha/yearreduce black tea TF levels.

There was no pattern in TR levels due to different rates ofnitrogen application but the TFs

followed an inverse quadratic pattern with minimum levelsbetween 150 and 300 kg N/

ha/year 85 . Caffeine levels increased with higher rates ofnitrogen application. With regard

to black tea aroma, levels of both Group I VFCs and GroupII VFCs increased with an

increase in the rates of nitrogen 85,92,150,154 . Theincrease in Group I VFCs was much larger

than that in Group II VFCs leading to a reduction in FIwith increasing nitrogen rates. Thus,

the increasing levels of nitrogen reduced black tea qualityas measured by both TF levels

and FI. In deciding the correct rates of nitrogen to beapplied, quality should also be taken

into account. Whereas nitrogen application must continuefor the economic production

of tea, excessive amounts do not give economic yieldreturns and adversely affect black

tea quality. In most tea-producing countries, the rates offertilizer application 148 have not

reached a stage where it can threaten tea quality. Caution,however, should be exercised

in order to avoid future problems. Continuous applicationof high rates of nitrogenous

fertilizer increases soil acidity 3 , which could make thesoils moribund. Only recommended

rates as adjusted from time to time, depending on researchfindings, should be applied.

In studies to establish the influence of splitting theannual nitrogenous fertilizer

application on black tea quality, in Kenya splitting had noinfluence on the black tea

quality 94,152 . Indeed the tea yields also did not varydue to the spread of the annual nitrogen

fertilizer application. This implies that the splitting ofannual nitrogen fertilizer application

should be done for other reasons, not quality or yields.Such reasons include, but not are

limited to, cash flow control, storage considerations andavailability of funds.

The caffeine levels increased 85,122 and the level offlavan-3-ols 122 changed with an increase

in nitrogenous fertilizer rates. The flavan-3-ols respondedin an inverse quadratic manner,

except EGCG whose levels linearly increased with rise innitrogenous fertilizer rates. In a

single cultivar, similar changes in pattern occurred atvarious locations within East Africa,

although the magnitude of the changes varied with thelocation of production 122 . However,

the minimum levels of total flavan-3-ols were observedbetween 150 and 225 kg N/ha/year.

The TFs in black tea had been previously observed torespond in a similar pattern 85 . Thus

the levels of these precursor chemicals, especiallyflavan-3-ol levels, directly influence the

plain black tea quality parameters. Increasing rates of Nincreased the levels of green leaf

amino acids, total polyphenols and catechins 155 . Overallblack tea quality was impaired by

the use of high rates of nitrogen 156 .

However, some black teas are classified as flavoury, makingaroma an important black

tea quality parameter. While Group II VFCs responsible forsweet flowery aroma arise

from diverse plant metabolites including terpenes, proteinsand amino acids, the Group I

VFCs are mainly the products of unsaturated fatty aciddegradation 26 . These degradation

products are collectively referred to as green leafvolatiles 157 . The influence of nitrogenous

fertilizer rates on the fatty acid composition and levelshas been a subject of various

studies. The unsaturated fatty acid levels increased withincreasing levels of nitrogen

fertilizer rates 123,125,158 . The observed patterns ofchanges were similar to the changes in

the Group I VFCs in black tea and explain the black teaaroma quality decline with rise in

nitrogenous fertilizer application rates. However, responseof the precursors of Group II

VFCs to rates of nitrogenous fertilizer application rateshas not been reported.

6.2.2 Phosphorus

In Kenya 159 , there was a variation in black tea qualityas a result of phosphatic fertilizer

application, although in Sri Lanka and China 160 , yieldand quality benefits due to the

application of phosphatic fertilizers have been recorded.In Eastern Africa, phosphatic

fertilizer application is performed mainly as an insuranceagainst possible deficiency 161 . As

part of this insurance, in East Africa, nitrogenousfertilizer application is based on various

NPK formulations 148,161 .

6.2.3 Potash

Black tea quality improvement resulting from potashfertilizer application has been

recorded in some tea-producing countries 148,162 . Increase

in potassium improved black tea

quality 163 . Potash fertilizers had no influence on thechlorophyll content of tea shoots 163 .

Potassium as muriate of potash (MOP) improved the levels ofTFs, TRs and TF/TR ratio 164 .

Quality declined when MOP was changed to sulphate of potash(SOP) 164 . SOP treatment

increased caffeine levels 164 . The leaf K content wasnegatively correlated with the crude

fibre content of made tea 164 . An antagonism was observedbetween leaf K and Mg 164 .

Overall quality of tea was impaired by the use of highrates of potassium 156 . However, in

East Africa, both yields and black tea quality variationsdue to the application of potash

fertilizer applications have been insignificant 151,159 .Thus, inclusion of the nutrient in the

fertilizer regime is purely as an insurance against diseasein East Africa 161 .

K application increased the polyphenol levels in tea leaves155,165 . K fertilizer application

increased the contents of free amino acids and caffeine ofthe various tea types 155,165,166 .

However, KCl depressed nitrate reductase activity and henceled to the accumulation of

free amino acids 166 .

6.2.4 Magnesium

Magnesium is an essential component of chlorophyll. Itplays a significant role in the

movement of phosphorus and in the production of proteins,fats, carbohydrates and other

compounds. Mg application decreased the polyphenol levelsin tea leaves 165 . The black

tea TF and TR levels were increased by Mg application 165 .The free amino acid contents in

tea leaves increased following the application of magnesiumfertilizer 166 .

6.2.5 Organic fertilizers

Enriched organic manures with inorganic fertilizersimproved tea yield but reduced black

tea quality 167 . TFs and TRs decreased with increase inthe fertilizer rate irrespective of

fertilizer type.

6.2.6 Foliar feeds and plant inoculants

Inoculating plants with arbuscular mycorrhizal fungi (AMF)improves growth 168 of tea plant.

Inoculation with AMF increased the levels of totalproteins, amino acids, caffeine and

sugar 169 . Similarly, biologically active amino acidsimproved black tea quality 170 . Foliar

applied selenium induced antioxidant activity in tea, thusinhibiting lipid oxidation 171 .

6.2.7 Bioregulators

Bioregulators, namely jibika, IAA, cycocel, thiourea,methanol, succinic acid and sucrose,

improved quality parameters such as PPO activity, caffeine,crude protein, starch, nitrogen,

carotenoid and ascorbic acid (vitamin C) of tea 172 .Maximum PPO activity was observed

with cycocel followed by succinic acid, jibika, thioureaand sucrose during the first phase

of spray, while in the second phase, PPO activity wasmaximal in sucrose-treated bushes

followed by cycocel, jibika, thiourea and succinic acid.The caffeine content was found

to be maximal in methanol followed by cycocel, IAA,thiourea and jibika as compared to

the control during the first phase of spray. Similarly,nitrogen content increased due to

methanol application at both the phases. Starch andcarotenoid contents were significantly

influenced by jibika treatment. Likewise, ascorbic acidcontent was highest in sucrose

treated bushes as compared to other treatments 172 .

6.3 Plucking/Harvesting

Various plant husbandry (tea source, plucking standards andfrequency, pruning frequency,

etc.) factors also affect the quality of tea 86 . Theobjective of plucking (harvesting) in tea

production is to obtain economic yields, producehigh-quality tea and maintain the tea

bushes in good health 40 . Plucking is therefore one of theagronomic aspects that influences

black tea quality 173 . Good leaf quality is a product ofplucking severity and frequency 12,174 .

Although emphasis is always put on plucking standards,other plucking attributes such

as plucking intervals and mode of plucking influencechemical composition and hence

quality of black tea.

6.4 Plucking standards

The size of harvested shoots is referred to as the pluckingstandard. A fine plucking standard

refers to the selection of shoots consisting mainly of leafranging from one leaf and a bud to

mostly two leaves and a bud. Coarse plucking standardconsists mainly of three or more leaves

and a bud. The chemical composition of the leaf varies withthe plucking standard 112,174,175 .

The quality potential of the leaf decreases from the buddownwards to older leaves 11 . Thus

the bud has high concentration of chemical compounds whichleads to good tea quality 38,112 .

Green leaf comprising only a bud, 1 leaf and a bud, 2leaves and a bud and up to 5 leaves and

a bud was processed and chemically analysed 175 . Caffeinelevels decreased as the plucking

standard became coarser. TF levels increased up to twoleaves and a bud and subsequently

decreased. TR levels increased up to four leaves and a budand then decreased. The Group

I VFC levels linearly increased, while Group II VFC levelslinearly decreased with the coarser

plucking standard, leading to a linear decrease in FI asthe plucking standard became coarser.

Black tea quality, total TFs, TR levels and total colourdecreased with coarse plucking 104 .

Based on the lengths of shoot growth, shorter lengths gavebetter black tea quality than

those based on the standard conventional plucking 100 .Thus, 5-cm shoot length produced

better black tea quality followed by 10-cm and 15-cmlengths, respectively. Correct plucking

standard is critical for the production of black tea ofhigh quality. Very fine plucking reduces

fresh leaf yield per plucking round and the total outputfor the grower 175 . It is important to

strike a balance between economic yield and quality so thatfarmers produce teas of high

quality at the most economic level 12,174,175 . Experience

from many tea-producing countries

shows that plucking two/three leaves and a bud improvesboth good yields and quality.

The reduction in black tea quality with coarse pluckingstandards has been observed

in many tea cultivars. However, the rate of reductionvaries with clones. There are clones

whose black tea quality is very sensitive to changes inplucking standards and some that

can withstand coarse plucking standards of up to threeleaves and a bud without much

decline in quality 54,176,177 . Thus the implementation ofa plucking standard needs to take

into consideration the clones in production.

Quality decline of black tea with coarse plucking standardshas been attributed in part to

the increase in chlorophyll content 176 . Higherchlorophyll levels increase the green appearance

in black tea, thus reducing the colour. Also the levels ofunsaturated fatty acids that produce

Group I VFCs responsible for green grassy aroma increasewith coarse plucking standards 178 .

This increase leads to an increase in the sum of Group IVFC and a reduction in the FI 175,176,179 .

Black tea quality decline with coarse plucking standardscannot be corrected neither by

adjusting fermentation duration 179 nor by alteringharvesting intervals 150 . Due to differential

growth rates and yield potentials, areas with very highyield potentials but low quality could

resort to very fine plucking 40 . This would produce leafwith a high proportion of one leaf and

a bud in the pluck analysis and thus have a higher quality

potential 40,112,174 . The yield losses

because of very fine plucking could be compensated by gainfrom improved tea quality. But

this argument may not hold sometimes depending on theprevailing market conditions 180 .

In terms of green leaf chemical components that influenceblack tea quality, the

polyphenol levels in young leaves (apical bud and the twoyoungest leaves) were higher

than in old leaves (from the tenth to the fifth leaf) 99 .The old tea leaves contain less caffeine

than young ones 181 . With the maturation of tea leaves,lipoxygenase activity and lipid

content increase 78 . The proportions of neutral andstructural lipids such as phospho-

and glycolipids increase throughout maturation. The amountsof unsaturated fatty acids

increase with shoot maturity 78 .

Fluorine is not essential to plants, but it is essentialfor bone and tooth health of

mammals. However, over exposure to F could be detrimentalto human health. Levels of

Al in tea and its implication on human health have been atopic of concern 46,182 , since tea is

an Al accumulator 183 , and may contain high Al levels inold leaves. Excessive intake of Al is

harmful to health, possibly weakening the kidney and beinglinked to Alzheimer’s disease.

The mature old leaves contain higher levels of F and Al49,50,184 . To eliminate the hazard of

over exposure to F and Al derived from tea, younger shootsshould be used for making

tea products and mature leaves should be avoided. The

fluoride and aluminium levels in

black tea, however, are not associated with considerablerisks of fluorosis and Alzheimer’s

disease, respectively 185 .

6.4.1 Plucking intervals

The economics of tea production require that all the readyand harvestable shoots be

removed. Non-removal of all crops leads to yield reductionand faster rise in bush height

or necessitating a lot of break bark. Therefore, formaximum yield benefit, all crops must

be removed at the right time (plucking interval). If theplucking standard is the same,

then quality of black tea may be consistent, provided thegrowing conditions are similar.

Correct plucking interval facilitates the removal of readycrop to produce leaf of the same

standard without sacrificing yield and quality. Theinterval between successive harvests

varies between 5 and 21 days depending on the prevailingenvironmental conditions

and the plucking policy 147 . In changing plucking roundswhile at the same time removing

the entire available crop, that is, having unselectiveplucking standards, 117,119,127,131,150,154,186

there was no change in TR levels due to long pluckingintervals. Levels of TF, caffeine and

Group II VFC and FI decreased with long plucking intervalsunder such conditions. The

sum of Group I VFC increased as longer plucking intervalswere observed. Thus, long

plucking intervals lead to lowering of black tea quality.Such decline in black tea quality

with long harvesting intervals occurs in all tea varieties186 , irrespective of nitrogenous

fertilizer rates 119,131,150,154 and location ofplantation 117,119 .

There was a decline in black tea quality with long pluckingintervals arising from

the changes in the composition of the green leaf chemicalcomponents. For example,

the unsaturated fatty acids in green leaf increase withlong plucking intervals in all

locations 124,125 . Field operations including pluckingrounds and pruning also influence PPO

and PAL enzyme activities 75 . The lipoxygenase activitydecreases with increase in plucking

intervals 187 . This decline in black tea quality with longplucking intervals is due to the

increased accumulation of coarse leaves as the pluckingintervals are lengthened. Thus the

plucking interval should be decided based on the quality ofblack tea desired, which also

compromises the production of of best commercial yields.Indeed, even the micronutrient

levels in black tea and green leaf vary due to changes inplucking intervals 188 .

6.4.2 Mode of plucking and leaf handling

Plucking is the most expensive agronomic activity in teaproduction 189,190 . Some producers

have recorded yield losses due to the inability to harvestthe entire ready crop during peak

periods. To improve leaf removal and reduce the cost of theoperation, several mechanical

harvesters have been introduced. These range from simpleshears to large combine harvester

types of mechanical plucking machines. In comparison toshear-plucked tea leaves, hand

plucked tea leaves had higher levels of TFs and caffeine,brightness, FI, Group II VFC and

sensory evaluations, but lower Group I VFC levels,irrespective of variety 186 . The quality of

machine-plucked tea leaves was inferior 191 to that ofhand-plucked tea leaves. The longer

the plucking interval, the worse the quality withmechanical harvesting. This was attributed

to the accumulation of higher quantities of coarse leafwith long plucking intervals.

Increasing the height of the plucking table by 2 cmproduced black teas with better

quality than when it was increased by 1 cm. This is due toharvesting more younger leaves.

However, after the bushes are regularly machine-plucked atoptimal plucking rounds, the

quality of resultant black teas is improved. In India,hand-plucked teas were superior to

shear-harvested teas 192 . The quality deterioration wasmainly due to mechanical injury and

non-selective plucking with shear harvesting. However, teaobtained by shear harvesting

from a continuously sheared field over a prolonged periodwas found to be superior 192 .

Handling of harvested green leaf affects the black teaquality. If the plucked leaf is not

well aerated, it develops heat that destroys cell wallmembrane to trigger early uncontrolled

fermentation. Processing such leaf leads to the low qualityof black tea 193 . Compaction

and/or bruising plucked leaf must be avoided as such

activities lead to the formation of

red leaf which makes low-quality black tea 194 . The extentof quality deterioration in black

tea is directly proportional to the amount of red leaf inthe mixture.

6.5 Pruning

Following pruning, tea bushes grow at different ratesdepending on when pruning was

done. The bushes recovering from pruning display a veryfast growth rate and produce

very heavy and large shoots. Changes in the growth ratescan be expected to cause

some changes in the chemical composition and hence in thequality of black tea. Tea

plants nearest from last pruning produce black teas withlowest quality and the quality

improves as time to next prune gets closer 195 . TF,caffeine and Group II VFC levels FI and

tasters’ evaluation increased and TR levels decreased withtime from previous prune. It is

important to mix green leaf from fields of different agesfrom previous prune to ensure that

reasonable quality is maintained. The variations in blacktea quality due to period from

pruning are partly due to the decrease in the levels ofunsaturated fatty acid levels as the

next pruning time gets closer 196 .

Several pruning technologies have been developed. Lungpruning in which part of the

plant is pruned and some parts are left growing till thepruned section has developed leaf

has proved particularly useful as it ensures that the plantis continuously photosynthesizing.

Thus even if pruning is done into drought, there is no lossof plants. From lung-pruned

tea plants, the lungs (branches that were not pruned) makemuch superior black teas

compared to the flush growing from the pruned sectionsirrespective of the nitrogen

fertilizer rates 197 . The result demonstrates that it ispossible to make black teas of different

qualities from the same plant.

The precursors responsible for tea quality, such aspolyphenols 44,187,198 , increased in the

first year and thereafter declined with time from pruning.Lipoxygenase activity declined

with time from pruning 198 . Carotenoid levels increased inthe first three years after pruning

and then declined 198 . TF content, total liquor colour(TLC) and water extract increased

with time from pruning 198 . The levels of polyunsaturatedfatty acids, total fatty acids and

various lipid fractions declined with time from pruning 198. Sum of VFC Group II showed

an enhancement, while Group I declined with time frompruning, consequently increasing

the FI and sensory evaluation value with time from pruning198 . In another study, the TC

content increased up to three years followed by a decreasein the fourth year of the

pruning cycle in all tea cultivars and the caffeine contentof tea leaves increased steadily

up to the fourth year 44 .

6.6 Environmental pollution

Phenanthrene pollution around the roots of tea plants

promotes PPO and superoxide

dismutase activities of the tea leaves leading to anincrease in the contents of water

extract, amino acid and caffeine levels while reducingprotein content 199 . In the long run,

the levels of polyphenols and total sugar declined,indicating that phenanthrene may

reduce tea quality 199 .

Proximity of tea plantations to a highway, surface dustcontamination and high levels of

lead in the soils cause elevated lead concentrations in tealeaves 200 .

6.7 Diseases

There is quality deterioration with increasing blisterblight severity 201 . Total phenols,

catechin(s), total nitrogen, amino acids, chlorophylls andPPO activity declined in tea

shoots with increasing disease severity. Levels of TFs,TRs, caffeine, highly polymerized

substances and TLC, brightness and briskness declined withincreasing disease severity.

Aroma components, particularly hexen-1-ol, phenylacetaldehyde, linalool, methyl

salicylate, geraniol, indole, b-ionone and nerolidol, andseveral unassigned components

were also lower in disease-affected teas 201 .

7 Conclusion

The main cause of variations in made tea quality isprobably due to environmental factors.

Little can be done to alter the environmental patterns inorder to achieve uniform quality.

However, manipulation of agronomic practices such as

plucking standards and nutrient use

might help to minimize the quality variations. Anyenvironment conducive to yield potential

favours low-quality potential. Compromising on total yieldby resorting to fine plucking

and applying low rates of nitrogen might minimize thequality differences. However, the

feasibility of such practices requires to be studied beforethey can be recommended for

the tea farmers.


Further research is necessary to understand the exactmechanisms of the variations in

cultivars in their responses to agronomic inputs andenvironment. Such studies should

include biotechnological approaches and in particularefforts should be directed at

identifying genes responsible for the responses.Comparisons of the responses of

genotypes to widely varying environments have beenstagnated by lack of extensive

exchange of planting materials across borders. At least,for research purposes, exchange

of planting/genetic materials should be encouraged. Mostresearchers working on tea also

need extensive collaborations across borders to strengthenresearch on the plant.

9 Acknowledgement

Extensive collaborations and consultations with formertechnical staff at the Tea Research

Foundation of Kenya, especially Prof. Calleb O. Othieno,Prof. Martin Obanda, Prof. Francis

N. Wachira, Dr David M. Kamau, and Prof. Wilson K. Ng’etichduring the preparations

of this work are gratefully acknowledged. The author thanksMaseno University for

encouraging him to write this chapter.

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6 Chapter 6 The role of arbuscularmycorrhizal fungi in tea cultivation

1 Introduction

Current soil management strategies mainly depend onsynthetic fertilizers, and are likely to

cause environmental deterioration and human health hazards.Prolonged use of chemical

fertilizers severely damages soil as trace elements are notreplenished. The crops are

harvested without doing anything for the sustainability ofthe soil. Moreover, the problem

of over-nutrition caused by inappropriate use of syntheticnutrients is deleterious to natural

microflora and disturbs soil pH. Highly soluble chemicalfertilizers dissolve into soil quite

rapidly. Since plants can only absorb a certain amount offertilizer, most of the chemicals

are leached away to groundwater, resulting in pollution. Asthese problems increase, the

importance of maintaining soil fertility for crop securityis being realized.

Tea is grown mainly in tropical and subtropical (temperate)areas. It is known to

be native to South-east Asia and has been grown sinceancient times (Harbowy and

Balentine, 1997). China, India, Sri Lanka and Kenya areleading tea producers. Tea is also

grown on a smaller scale in other countries, includingBangladesh, Japan, Indonesia,

Vietnam, Malawi, Tanzania, and Turkey. Health benefits oftea have been mentioned

since the period of ‘Ch’a Ching’ in China and ‘KissaYojoki’ in Japan (Yu, 1976; Eisai,

1211). It has been suggested that tea is an effectiveremedy for a diverse range of health

disorders. When people began to drink tea in Europe, anEnglishman, Chamberlain,

said, ‘Tea is a supreme divine treasure given for humanhealth’ (cited from Nakagawa,

2015). In one of the earliest reports, an army surgeonrecommended the use of tea in

soldiers’ water bottles as a prophylactic against typhoid(McNaught, 1906; Anonymous,

1923). Some benefits of black tea include antioxidant,anti-inflammatory, and anti

mutagenic properties. Its role in tackling high bloodpressure, oxidative damage, and

a range of cardiovascular diseases such as atherosclerosishas recently been reviewed

(Sharangi et al., 2014). Moreover, the importance ofantimicrobials from various kinds of

tea is also beginning to be recognized (Haghjoo et al.,2013; Reygaert, 2014).

Mycorrhizae have a crucial role in the conversion of aridland into fertile soil. The

significance of arbuscular mycorrhizal fungi (AMF) in cropcultivation is becoming

increasingly apparent. The increase in surface area forabsorption provided by AMF, is

primarily responsible for mycorrhizal plants being able totake up more soil nutrients,

especially P, N and S (Berrutti et al., 2015). Since AMFincrease the efficacy of fertilizer use,

they are referred to as ‘biofertilizers’ and can besubstituted for substantial amounts of

chemical fertilizers (Varma and Hock, 1999).

Methods used for measuring the function and efficacy of AMFcommunities have

involved the analysis of growth and P uptake by test plantsusing inoculum obtained from

field soils (Porter et al., 1978). Natural soil harbours aconsortium of indigenous AMF and

is often used as a source of inoculum. This was exemplifiedby the soil from established

tea gardens that harboured a wide range of promisingbio-inoculants including bacteria,

fungi and actinomycetes (Pandey and Palni, 1999, 2004,2008; Pandey et al., 1997, 2001,

2013; Trivedi et al., 2012; Rahi and Vyas, 2015). However,the use of AMF from the tea

rhizosphere as bio-inoculants became apparent a littlelater.

Although improved culture practices have resulted inincreased tea production, the

increment is not adequate to meet the increasing demand,and therefore, a faster rate of tea

production is essential. This can be achieved throughimplementation of new technologies

and better utilization of existing natural resources.Various methods to genetically improve

tea plants exist. These include cell, tissue and organculture, molecular breeding, selection

breeding, and omics. These have recently been reviewed(Mukopadhyaya et al., 2015). Due

to land limitations, expansion of the tea industry relieson yield and quality enhancement.

Important research areas include the development ofbiopesticides, biocontrol agents and

biofertilizers. However, this must take into considerationthe increasing interest in ‘organic

tea’ production. According to the Tea Board of India, thecountry’s tea production has

been expanding in recent years, increasing from 1.1 milliontonnes in 2011 to 1.2 million

tonnes in 2013 (Chang and Brattlof, 2015). The majority ofthis tea, (about 75%) is sold

in France, Germany, Japan, the United Kingdom and theUnited States. Due to sustained

efforts, production of certified organic tea in India isnow around 966 million kg, with the

majority of this being exported. The global market isexpected to reach 10.57 million tons

in volume (Majumder et al., 2010).

It is now firmly established that the influence of AMF onplant growth is generally positive.

Focus should now be on understanding the physiology andecology of this association,

the selection of appropriate fungal endophytes, and onresearching better inoculation

techniques. These measures may help to improve the uptakeof nutrients, (in particular

phosphorus) and thereby reduce the need for fertilizerapplication. Considering the

increasing interest in exploitation of soil microflora forimproving plant nutrient status from

existing soil conditions, AMF associations are receivinggreater attention. It is well known

that plantation crops are benefited through AMFassociations (Habte and aziz, 1985; Rabie

and Almadini, 2005), but information is limited in the caseof tea, even with its commercial

importance. AMF have great potential to help establish newtea crops in hilly states, (such

as Uttarakhand, India) and in areas where nutrients arelimited.

Important findings of a detailed and long-term case study,carried out on tea plantations

of the Indian Himalayan Region (IHR), are discussed below.AMF’s potential as a bio

inoculant for improving growth and tea quality is alsoexamined. The study analyzed AMF

associated with natural and cultivated tea rhizospheres inthis region. The findings are

compared with the other studies on the subject carried outsimultaneously.

2 AMF, tea and the tea rhizosphere

2.1 AMF associations in tea

Early reports of mycorrhizal association with tea appearedin the journal Tea in 1901 (cited

by Webster, 1953). The fungal species was identified asProtomyces thea which was later

classified as Rhizopus thea (Buttler, 1939). Tunstall(1926, 1930) also reported the presence

of AMF in the roots of tea. In 1989, Morita and Konishireported a low level of AMF

colonization (17%) in tea roots. Poor colonization of AMFwas correlated with the nature

of tea plants, the specific environment of tea fields, highapplication of chemicals, and

the time of sampling. However, in another investigation,high level of AMF colonization

ranging from 77 to 99% was reported in roots of tea plantsgrowing in IHR (Singh et al.,

2008a). In this study, a typical pattern of colonization intea roots by AMF was observed

to include the following: attachment of spore to the rootsurface, germination of spores

outside roots, formation of appressorium at root surface,penetration of roots, inter- and

intracellular growth of hyphae, formation of arbuscules,formation of vesicles and, in later

stages, formation of intra-radical spores (Fig. 1). Afairly high density of AMF spores was

also recorded in the tea rhizosphere soil samples. Thishigh percentage of colonization

suggested that the earlier discussed reasons for poorcolonization may not be solely

responsible for restricting the AMF colonization in tearoots. Later, more studies came

forward showing a high degree of AMF colonization in tearoots. Approximately, 70% AMF

colonization was reported by Toman and Jha (2011) in teaplantations of Upper Assam,

India. Tea plants of the Doon Valley, Uttarakhand (India),were observed with almost 100%

of root colonization by AMF (Sharma et al., 2013). Aspecific report citing a lack of AMF

colonization in tea roots also came from tea plantations inIran (Aliasgharzad et al., 2011).

2.2 The diversity of AMF associated with the tea rhizosphere

The tea rhizosphere represents a specialized ecologicalniche for various groups of

microorganisms. Characteristic features exhibited by thetea rhizosphere include the ‘negative

rhizosphere effect.’ This is defined as an inhibition ofmicrobial communities with increasing

age in established tea bushes (Pandey and Palni, 1996,2004), relatively higher antagonistic

activity, (i.e., the presence of antimicrobials producingmicroorganisms around the roots,)

(Pandey et al., 1997, 2001), and lowering of therhizosphere soil pH with as the tea bushes

age (Pandey and Palni, 1997). Edaphic and climatic factorsas well as continued interaction

between the tea roots and the surrounding soil,collectively result in the development of a

specific root and/or rhizosphere microbiome including AMF(Singh et al., 2008a).

The tea rhizosphere has been shown to be well colonized byAMF. It was dominated by

Glomus spp. with frequent occurrence of four genera ofAcaulospora, Gigaspora, Glomus

and Scutellospora. A total of 51 AMF morphospecies could bedetected from two sites

investigated and some of these are shown in Fig. 2. Highvalues of the Shannon–Weaver

index (2.05 ± 0.1) and lower values of Simpson’s index (0.2± 0.05) further represented fairly

good AMF diversity in the tea rhizosphere (Singh et al.,2008a). Similar observations have

also been reported from the tea plantations of north-eastregions of India. While overall

20 morphotypes were recovered from tea plantations in UpperAssam, the genus Glomus

along with its 15 morphotypes dominated the tea plantationsin the Upper Brahmaputra

Valley. Acaulospora was the second moderately distributedgenus, along with its five

morphotypes (Barthakur et al., 2005; Toman and Jha, 2011).However, the number of

AMF species in these studies was reduced in comparison tothe upcoming tea plantations

of Uttarakhand (Singh et al., 2008a). Later, Sharma et al.(2013) reported colonization of

Glomus fasciculatum, G. mosseae, Acaulospora scrobiculataand Gigaspora margarita

from tea plantations of the Doon Valley in Uttarakhand. Allof these studies suggested the

dominance of Glomus species in the tea rhizsophere.

2.3 AMF in natural and cultivated tea plantations

AMF species form an effective link between plants and theecosystem through symbiotic

interaction. They are responsible for environmental qualityand show diversity/dominance

Figure 1 Colonization pattern of AMF in tea roots: (a)attachment of spore on the root surface, (b)

germination of spore outside the root, (c) appressoriumformation at the root surface just before

entry, (d) intercellular growth of hypha, (e) intracellulargrowth of hypha forming coil, (f) formation

of arbuscules inside cortical cells, (g) formation ofvesicles and (h) intraradicle spore formation.

Colonization cycle restarts with extra-radical spores.

in relation to type of ecosystem, edapho-climaticconditions, seasonal variation, host

genotype and plant cover.

In cultivated ecosystems, agricultural practices includetillage, crop sequence, crop

variety /cultivar selection, fertilizer and pesticideapplications. These practices may alter

AMF populations, species composition, and rootcolonization. Soil disturbance diminishes

the number of AMF propagules since they are usuallyconcentrated in the few uppermost

centimetres of the soil profile (Schwab and Reeves, 1981).Conventional tillage disturbs the

soil, which reduces the function of AMF symbiosis throughthe breakdown of their hyphal

network (Jasper et al., 1989). Moreover, monoculture andtillage negatively influence the

diversity of AMF (Menendez et al., 2001). Land useintensity (Oehl et al., 2003) and high

input (Mader et al., 2002) also cause a reduction indiversity and colonization by AMF. In

general, there appears to be a strong correlation betweenagricultural practices and the

abundance and effectiveness of AMF root colonizastion.

The IHR has a wide geographical area of approximately 419873 km 2 comprising different

climatic zones of temperate, sub-alpine and alpine regions.The range of these zones

supports growth of diverse plant species including severalmedicinally important plants.

Tea, one of the most important cash crops of India thatalso grows well in the IHR, is a

priority crop for plantation in hilly states. Bothcultivated and abandoned tea plantations

are found in the hilly state of Uttarakhand. AMF diversityin both natural and cultivated tea

plantations of this region has been studied and compared indetail (Singh et al., 2008a).

Ranidhara (District Almora, 1760 m above mean sea level(amsl), 29º 35’ 24” N, 79º 40’

24” E) with an average annual rainfall of 828 mm andtemperature range of 4.4–29.4ºC

Figure 2 (a) Sessile spore and wall ornamentation ofAcaulospora scorbiculata, (b) wall ornamentation

of A. foveata, (c) wall ornamentation of A. rehmii, (d)loose sporocarp of Glomus aggregatum, (e) spore

of G. aggregatum, (f) part of a sporocarp of G.clavisporum, (g) part of a sporocarp of G. coremoides,

(h) loose sporocarp of G. microaggregatum, and (i) spore ofG. multicaule. Bar = 20 μm.

represented an opportunity to analyze a natural,uncultivated ecosite. Tea plants at this

site were abandoned, old (>100 years) and very likely to beof seed origin. Fertilizer

application and common agricultural practices (e.g.pruning) had not been done at this

site for at least 40 years, and the presence of otherplants including conifers was recorded

nearby. On the other hand, Kausani (District Bageshwar,1840 m amsl, 29º 51’ 4” N, 79º

35’ 62” E) represented a cultivated site. This areareceived an average annual rainfall of

1400–1600 mm and its temperature ranged from 0 to 30ºC. Ithad well-maintained and

young (6 years old) tea bushes which were raised from stemcuttings (clone AV-2) and

grown as monoculture. Plants at this site were subjected tocommon agricultural practices

including regular pruning cycles. These plants had receivedprescribed chemical fertilizers

and pesticides. Cultivated and abandoned tea bushes areshown in Fig. 3 (a1 and b1,

respectively). In this study, the AMF colonization in tearoots was higher in the cultivated

site in comparison to the natural site. It is indicative ofthe negative impact of the intensive

farming practices on AMF colonization (Helgason et al.,

1998; Douds and Millner, 1999;

Madar et al., 2000, 2002; Lekberg et al., 2008). P contentin soil is also an important

factor responsible for colonization of AMF (Sieverding andHoweler, 1985). Furthermore,

the spore density was greater in the cultivated tearhizosphere than in the natural tea

rhizosphere, contrary to the general assumption that thenumber of AMF spores is greater

in the native area than in the cultivated one (Oehl et al.,2003; Moreira et al., 2007).

Site-specific variation in AMF communities also depends onthe local environmental

conditions and management history. Changes in AMFcommunities may be observed with

both plant community succession (Janos, 1980a; Johnson etal., 1991) and with changing

land use intensity (Oehl et al., 2003). A more diverseassortment of host plants may support

Figure 3 (a1) Cultivated tea bushes; (a2) The dominant AMFmorphospecies of cultivated tea

rhizosphere – Glomus multiforum (with wall ornamentationshown in insert); (b1) Natural tea bushes;

(b2) The dominant AMF morphospheres of natural tearhizosphere – Glomus sp.7. Bar = 20 µ.

a more diverse community of AMF (Johnson et al., 2003).Analysis of natural and cultivated

tea sites found that a higher number of AMF morphospecies(35) was recorded in the

natural site when compared to the cultivated area (27). Ahigher diversity was also seen at

the natural site. The most obvious difference between thetwo AMF communities lies in the

single dominant AMF morphospecies in the cultivated site,and the six AMF morphospecies

in the natural site, being either less frequently detectedor absent in the cultivated site. The

cultivated tea rhizosphere was dominated by Glomusmultiforum in the periods of active

growth and dormancy (Fig. 3: a2). However, the natural tearhizosphere was found to be

dominated by Glomus sp. 7, the most frequently occurringmorphotype (100%) in both

the periods (Fig. 3: b2). Oehl et al. (2003) reported thatsome of the AMF species present

in the natural ecosystems get strongly suppressed underconventional high-input farming

practices, indicating loss of at least some of theecosystem functions in the latter period.

Natural tea plants harbouring a greater number of AMFmorphospecies in comparison

to the cultivated tea bushes showed a higher value ofShannon–Weaver index of diversity

during the period of active growth and dormancy (Singh etal., 2008a). These findings

were explained by the better chemical properties of soil atthe uncultivated, natural site.

These properties included organic C and pH (Wardle, 2002;Kernaghan, 2005), absence

of anthropogenic disturbance (Kernaghan, 2005) and greaterfloral diversity (Johnson

et al., 2003). Fertilizer management has a direct effect onthe performance of AMF in

any ecosystem. Generally, P fertilizer reduces AMFcolonization and effectiveness of the

symbiosis, though different species of AMF respondvariously to P additions (Schubert

and Hayman, 1986). Fields, not previously fertilized withP, show a greater decrease in

AMF colonization in response to P fertilization than fieldswith a history of P amendments

(Jasper et al., 1979). This suggests that managementpractices impose selective pressure

on AMF populations in soil.

Predominance of the genus Glomus was observed in thecultivated as well as the natural tea

bush rhizosphere. Occurrence of almost the same number ofspecies of Glomus and Acaulospora

in natural and cultivated tea plants, and the absence ofGigaspora and Scutellospora species

was thought to be related to selection pressure (Singh etal., 2008a). Agricultural practices

might have induced the selection pressure in such a waythat specific groups of organisms could

establish themselves better than others. Species of thegenus Glomus could be considered

more tolerant to various practices followed in teacultivation. Glomus is the predominant AMF

genus among the soil species described so far (Jansa etal., 2003).

2.4 Soil pH and AMF in the tea rhizosphere

Since AMF form a symbiotic relationship with plants, thefungal symbiont becomes a

major interface or connection between soil and plants.Therefore, studies on mycorrhizal

systems need to consider soil as well as plant and fungi.Soil properties can influence spore

germination, colonization of host roots, and ability of theAMF to influence the growth

and physiology of the host plant. The AMF population leveland species composition

are highly variable and are influenced by plantcharacteristics. Other influencing factors

include temperature, soil pH, soil moisture, phosphorus andnitrogen levels, heavy metal

concentrations, the presence of other microorganisms,application of fertilizers/ pesticides,

and soil salinity (Smith and Read, 1997). Species andstrains of AMF differ in their ability to

tolerate physical and chemical properties of soil (Abottand Robson, 1991).

The most important edaphic factor for the tea rhizosphereis pH since tea plants are

known to prefer acidic soils and also seem to further lowerthe soil pH (Pandey and Palni,

1997). Distribution of various AMF species may also beinfluenced by soil pH. Acidic to

neutral soils are known to harbour large numbers of AMF.Some Glomus species are very

common in neutral or alkaline soils but few are found inacidic soils, whereas species of

Acaulospora are usually found in acidic soils. Low soil pHmay have a positive, indifferent

or negative response on mycorrhiza formation. In ourstudies, the soil pH was recorded to

be 5.11 at Ranidhara and 4.20 at Kausani tea plantations.Whereas acidic pH up to 5 (at

Ranidhara) seems to favour the growth of AMF, furtherlowering of pH (at Kausani) may

have resulted in the suppression of AMF at the genus level.Occurrence of almost the

same number of Glomus species clearly indicated the hightolerance of the genus to the

harsh conditions (Singh et al., 2008a).

3 Development of AMF-based bioformulation for teaplantations

In the past 25 years, the use of chemical fertilizers foragriculture has significantly increased

throughout the world. As a result, crop yields have alsoincreased dramatically. However,

the cost of fertilizers, both in terms of currency andenergy, has risen tremendously. Costs

are expected to continue rising due to a shortage offertilizer supplies and the current cost

of energy consumption to produce the fertilizer. Economistsindicate that as energy costs

increase, fertilizer use is likely to decrease. Chemicalfertilizers are said to account for more

than half of the agricultural output or production. This isnot a sustainable trend, unless

the fertilizer efficiency can be improved or somealternative fertilizer source can be found.

The problem of producing quality AMF-based inoculum isexacerbated on account of

AMF being ‘obligate symbionts’, which require the presenceof actively growing roots during

propagation. Their obligate biotrophic nature has been alimiting factor for understanding the

physiological, biochemical and genetic characteristics ofthe organism, and its commercial

uses. Culturing AMF-based inoculum under in vitroconditions has proved challenging,

meaning that production is more efficient in the presenceof a living host root system. The

potential utilization of AMF is highly dependent on asuitable inoculum that can be easily

produced and spread on agricultural land using traditionalequipment. However, the success

of commercial AMF inoculants needs pre-evaluation on aselected crop using regional soil.

Variable results were obtained when analyzing the sporeproduction of 12 commercial AMF

inoculants, and their effect on maize growth (Faye et al.,2013).

In today’s global market, demand for organic tea issteadily increasing. Most organic

tea is grown using bio-compost as biofertilizer. AMF-basedbio-inoculants were prepared

and tested for their suitability for use as biofertilizerin tea plantations (Singh et al., 2008b).

The strategy to develop AMF-based bio-inoculant initiallyinvolved thorough testing of

the bio-inoculant using post-inoculum issues and bioassays.This was followed by mass

propagation of AMF using a ‘trap culture’ technique.

3.1 Mass propagation of AMF: trap culture

Due to the obligate symbiotic nature of AMF, theirlarge-scale production requires control

and optimization of both the host growth and the fungaldevelopment. Several inoculum

production techniques have been developed. Common methodsinclude soil-root culture,

inoculum-rich soil pellets, soil-free inoculum, nutrientfilm culture and aeroponic culture

systems. Traditionally, AMF are propagated through apot-culture technique. The initial

fungal inoculum, usually made of spores and colonized rootsegments, is incorporated

into a growing substrate for seedling production. Bothcolonized substrate and roots then

can serve as AMF inoculum.

Inoculum propagation requires isolation of AMF culture,selection of suitable host

plants, and optimum growing conditions. Pure cultures canbe obtained originally from a

single spore that germinates and colonizes roots of thehost plant. AMF inoculum can also

be generated from colonized root segments or rhizospheresoil collected directly from

field plants. AMF inoculum is then obtained throughsubsequent pot-culture generations,

using isolated spores, fine root fragments, or field soilas the starting inoculum.

A vital characteristic of the host plant is its highmycorrhizal potential, (i.e. its capacity

to be colonized by the AMF, and its ability to promote AMFgrowth and sporulation).

Other important host plant criteria include the toleranceto grow under growth chamber/

greenhouse conditions, and an extensive root system made upof solid (but non-lignified)

roots. Leek (Allium porrum L.), sudan grass (Sorghumbicolor (L.) Moench), corn (Zea mays

L.) and bahia grass (Paspalum notatum Flugge) are the mostfrequently used plant host for

inoculum propagation.

Pasteurized, steamed or irradiated growth substrates arerequired in order to avoid

culture contamination which could affect the quality of theinoculum. A well-aerated

substrate is recommended, such as coarse-textured sandy

soil, sometimes mixed with

inorganic inert materials. Inadequate mineral nutrientcomposition may affect fungal

development. Optimum P levels vary with the host plant andpropagated fungal strains. An

excess of available P can inhibit AMF propagation.Potassium, nitrogen, magnesium and

other element ratios may also affect inoculum development,especially when inert growth

substrates are used, and plant fertilization is performedartificially. Other edaphic factors

such as pH, soil temperature, light intensity, relativehumidity and environment aeration

must also be controlled to optimize AMF propagation(Ferguson and Woodhead, 1982).

Different plant species have been tested for massmultiplication of specific AMF. Two

host plants (maize and finger millet) were used for the‘trap culture’ establishment of AMF

from the tea rhizosphere (Singh et al., 2008b). These werechosen because both plants

have fast growth cycles and an extensive non-lignified rootsystem. The substrate used

for mass propagation consisted of autoclaved soil:sand(1:1) mixture, where sand was

mixed with the soil to increase aeration. Tea rhizospheresoil and root fragments served as

inoculums for mass propagation of AMF.

AMF colonization and subsequent spore formation andproduction, all depend upon

the type of host, as well as upon the duration of infectionof these symbiotic organisms.

Studies using both maize and finger millet host plants,

reported a positive effect of AMF

colonization on increasing AMF population. Soil inoculumresulted in better colonization

and spore formation during mass propagation of AMF withboth host plants. This was

explained by a number of factors such as a greater numberof propagules in soil inoculum,

(including spores, colonized root fragments andextraradical hyphae) when compared to

root inoculum (where only colonized root fragments serve aspropagules). All the root

fragments used as inoculum may not be colonized by AMF, anda definitive proportion

of colonized root fragments were not known in theinoculated root fragments. Also,

rhizosphere soil which was used as inoculum, may containmycorrhizal helper-bacteria

which could have a positive effect on the germination andcolonization stages of AMF

(Durgapal et al., 2002; Tamta et al., 2008).

Results of experiments for mass production of AMF inoculumusing finger millet and

maize as host plants indicated a gradual increase in rootcolonization and spore number

with the period of growth. Significantly higher sporeformation was recorded in finger millet

after 45 days, but not after 90 days, except for plantsinoculated with soil inoculum (Fig. 4).

This suggests that finger millet could be considered moreappropriate for producing AMF

inoculum. Therefore, soil used for growing finger milletplants was used as inoculum, and

this host was used for mass propagation of AMF. Results

also envisaged that the host plant

species favoured association with a particular AMF speciesor conversely, that AMF may

show some preference for the host species.

A total of 28 AMF morphotypes could be recovered from thetrap cultures. The AMF

consortium from natural tea rhizosphere contained 16morphotypes of Glomus and three

of Acaulospora, whereas the consortium from cultivated tearhizosphere contained 14

species of Glomus, two of Acaulospora and one ofScutellospora. A list of AMF species

recovered from trap cultures of natural tea rhizosphere andcultivated tea rhizosphere is

presented in Table 1. Spore densities in both consortiawere found to be in the range of

50–70 spores (25 g) −1 of soil. No pathogenic forms of anyfungi were detected during the

colonization in both the host plants (Singh et al., 2008b).

3.2 Use of a perennial host for mass propagation of AMF

The host plants tested so far include sorghum, barley andclover, among others. To mass

propagate AMF in a short time period, these hosts mustalso have short life cycles. This

almost invariably means that the host plant’s aerial parts,often of commercial value, are

discarded. Furthermore, a second host plant, sometimesanother species, is needed to Maize Finger millet (a) a aa (c) a a (d) (b) 0 20 40 60 80 100 120 140 160 S p o re d e n s i t y 0 20 40 60 80 100 120 140 160 45 days 90days S p o r e d e n s i t y 0 20 40 60 80 100 120 140 160180 S p o r e d e n s i t y 0 20 40 60 80 100 120 140 160180 200 45 days 90 days 45 days 90 days 45 days 90 days S po r e d e n s i t y

Figure 4 Spore density of maize and finger millet duringtrap culture establishment from root and soil

inoculum of NTR (a) and (b), respectively and CTR (c) and(d), respectively. Bars show standard error

and ‘a’ letter indicates significance as analyzed byone-way ANOVA at P ≤ 0.5 between maize and

finger millet.

continue propagation over longer periods. To overcome theseproblems, Dendrocalamus

strictus [(Roxb. Nees; common name: lathi baans-bamboo)]was tested as a perennial host

plant, for the propagation of AMF from tea rhizosphere ofcolder regions (Singh et al.,

2012). D. strictus is an evergreen, fast growing,multi-utility bamboo, which performs well

under tropical, subtropical and temperate conditions. Ithas been used to propagate a Table 1 AMF morphotypesrecovered from trap cultures prepared for NTR and CTR S.no. AMF morphospecies NTR CTR 1 Acaulospora foveata + − 2Acaulospora scrobiculata + − 3 Acaulospora spinosa + + 4Acaulospora sp. 1 + + 5 Glomus aggregatum + + 6 Glomusambisporum + + 7 Glomus ambisporum/heterosporum + − 8Glomus clarum + − 9 Glomus clavisporum + + 10 Glomusfasciculatum + − 11 Glomus geosporum + + 12 Glomusheterosporum + − 13 Glomus intraradices + − 14 Glomusmosseae + + 15 Glomus multicaule − + 16 Glomus pustulatum ++ 17 Glomus sp. 2 − + 18 Glomus sp. 6 + − 19 Glomus sp. 7 ++ 20 Glomus sp. 8 + − 21 Glomus sp. 9 + − 22 Glomus sp. 10+ − 23 Glomus sp. 12 − + 24 Glomus sp. 14 − + 25 Glomus sp.15 − + 26 Glomus sp. 17 − + 27 Glomus sp. 20 − + 28Scutellospora sp. 1 − + A ‘+’ denotes presence and a ‘−’denotes absence of the particular AMF species.

variety of AMF, such as those associated with tea plantsgrowing in mountain regions.

Comparisons were also made with two annual plants, Zea maysL. (maize) and Eleusine

coracana L. (finger millet,) by analyzing colonizationefficiency and sporulation.

Significantly higher spore formation was recorded in D.strictus after 45 and 90 days

growth, compared to the two other host plants (maize andfinger millet). This clearly

indicated D. strictus as an appealing host option.Therefore, soil from around the base of

D. strictus plants was appropriate for use as an inoculum.The plant was also considered to

be a suitable host for mass propagation of the AMForiginally isolated from the rhizosphere

of tea plants.

3.3 Post-inoculum production issues: viability andinfectivity of AMF consortia

The important features determining the potential of abio-inoculant are its infectivity

and effectiveness. Alongside this, the inoculum mustmaintain high viability even after

prolonged storage (Trivedi and Pandey, 2008). Thetemperature, humidity level, and

duration of storage are all known to influence theinfectivity of the AMF inoculum.

Generally, it is accepted that infectivity of an AMFinoculant decreases as the storage time

and temperature increase (Daft et al., 1987; Mugnier andMosse, 1987). In comparison,

storage under wet or moist conditions results in a higherinfectivity of the inoculant (Daft

et al., 1987).

Viability of AMF spores in the prepared consortia wasdetermined after storage at

room temperature, and at 4°C, up to a period of 12 months.The formulated consortia

were found to retain their viability at both room

temperature and 4°C during the storage

period. Fresh preparations of AMF consortium from thenatural tea rhizosphere possessed

62.33 ± 0.88% viable spores. Viability decreased slowly,and after 12 months of storage

at room temperature 50.66 ± 0.88% viable spores weredetected. Higher viability was

obtained with spores stored at 4°C. Very similarobservations were recorded for the

viability of spores in the AMF consortium developed fromthe cultivated tea rhizosphere.

Storage at 4°C could significantly reduce the loss of sporeviability in the AMF consortium

sourced from the cultivated tea rhizosphere, compared tostorage at room temperature,

as measured after 12 months. Analysis of AMF consortiumfrom the natural tea rhizosphere

showed viability after storage at 4ºC, which was notsignificantly higher when compared to

storage at room temperature (Singh et al., 2008b).

Similar experiments were performed to analyze infectivityof AMF inocula using a

test plant (wheat). Colonization (a measure ofinfectivity,) by AMF consortia which had

been stored either at room temperature or at 4°C, followedthe same pattern. The fresh

preparations of AMF consortia from both the natural tearhizosphere and the cultivated

tea rhizosphere could colonize up to 47.33 ± 1.20 and 37.00± 1.15% respectively of

wheat’s root length. The comparative values for AMFconsortium from the natural tea

rhizosphere were 28.00 ± 0.58 and 33.00 ± 1.15% (of root

length) after 12 months of

storage at room temperature and 4°C, respectively.Similarly, AMF consortium from the

cultivated tea rhizosphere could colonize 22.00 ± 0.58 and30.00 ± 1.00% of root length

after 12 months of storage at room temperature and 4°C,respectively. Colonization by

AMF consortium from the natural tea rhizosphere afterstorage at 4°C was not significantly

different from the AMF consortium developed from thecultivated tea rhizosphere. In this

study (Singh et al., 2008b), the consortia were storedunder semi-dried conditions. Results

from pot studies have shown that AMF viability remains highfor long periods under dry

conditions (Sylvia and Jarstfer, 1992; Pattison and McGee,1997). This could be due to

both lower fungal respiration and reduced activity ofhyphal and spore grazing soil biota

(Bakhtiyar et al., 2001). The results indicated that coldand semi-dry environments were

conductive for maintaining the inoculum’s infectivity.Storage at room temperature in semi

dried condition did not result in drastic loss in sporeviability. However, significantly higher

viability and infectivity following storage at coldtemperature (4°C) probably resulted from

improved spore germination after cold treatment.

4 Plant growth promotion following inoculation with AMFconsortia

4.1 Effects on plant growth: overview

Inoculated AMF consortia colonized the roots and promotedthe growth of both test plants

(maize and wheat). In wheat, colonization rates up to 40%and 35% were observed for

AMF consortia developed from the natural tea rhizosphereand cultivated tea rhizosphere,

respectively. In the case of maize, relatively lowercolonization was observed. Inoculation with

AMF consortia had a profound effect on the growth of boththe host plants tested. Values

for all the parameters in treated plants were greater thanthe same for respective controls.

In maize, inoculation with crude consortium developed fromthe cultivated tea rhizosphere

resulted in significantly increased root length (14%),shoot length (10%), shoot fresh weight

(19%) and root:shoot ratio (23%) when compared to thenatural tea rhizosphere. Crude

consortium developed from the natural tea rhizosphereresulted in a significant increase

in root length (15%), root dry weight (85%), root:shootratio (88%) and leaf number (7%).

In wheat, all the parameters were found to have increasedsignificantly by inoculation with

crude consortium from the natural tea rhizosphere.Increases were reported as root length

18%, root fresh 33%, root dry weight 100%, shoot length14%, shoot fresh weight 46%,

shoot dry weight 16%, root:shoot 88% and leaf number by20%. Growth was also enhanced

in wheat plants inoculated with crude consortium from thecultivated tea rhizosphere, but

this was not always statistically significant. However, asignificant increase in root dry weight

(100%), shoot fresh weight (63%), shoot dry weight (50%)

and leaves number (14%) was

observed. An increase in all the studied growth parameterswas recorded following AMF

inoculation. Wheat plants benefited more compared to maizefollowing AMF inoculation.

Mycorrhizal dependency, an important feature determiningthe benefits obtained by a host

plant, was related to a higher increase in growthparameters in wheat plants, compared to

that seen in maize. A direct and positive correlationbetween AMF colonization and growth

promotion was also observed (Singh et al., 2010).

The effectiveness of indigenous AMF populations in terms ofpromoting the growth

of maize and wheat plants was demonstrated followinginoculation with the soil-based

consortium developed from the tea rhizosphere. Generally,use of a single strain of AMF

to inoculate crops is practised (Khaliq and Sanders, 2000).Instead, a mixed consortium of

Acaulospora and Glomus was multiplied, allowed toacclimatize to the conditions at the

experimental site, and then this was used for bioassays.Inoculation with multiple AMF is

considered to provide more consistent plants benefitscompared to single mycosymbiont

based studies (Koomen et al., 1987). Examples of theeffectiveness of mixed indigenous

consortial inocula on other crops are also available (Gaurand Adholeya, 1999).

4.2 Phosphorus uptake in AMF-colonized plants

In general, P concentration in the roots and shoots ofmycorrhizal plants was higher

compared to the concentration found in control plants. Anincrease in the mineral uptake by

mycorrhizal plants has been reported by severalresearchers. However, the increased uptake

of these elements varied depending on the plant species andexperimental conditions

(Crush, 1974; Manjunath et al., 1984). In maize,inoculation by AMF consortium from the

cultivated tea rhizosphere resulted in a higher P uptakecompared to AMF consortium

from the natural tea rhizosphere. This trend was reversedin wheat plants. Similar trends

were observed for the activity levels of two enzymes–acidicphosphatase (ACP) and alkaline

phosphatase (ALP). This may be related to the enhanced Pacquisition by both maize and

wheat plants. ACP and ALP are involved in P uptake by theplants, and AMF plants are

characterized by enhanced activities of these enzymes(Thiagarajan and Ahmad, 1994).

In maize roots, inoculation with the AMF consortiumdeveloped from the natural tea

rhizosphere or cultivated tea rhizosphere resulted insignificantly higher P levels (0.04% and

0.05%, respectively) as compared to the control. Relativelyhigher P content was recorded

in shoots of maize inoculated with AMF consortia from thenatural tea rhizosphere (0.05%)

and cultivated tea rhizosphere (0.06%), as well. Similarly,significantly high P content

was recorded in wheat plants inoculated with AMF consortiafrom both the natural tea

rhizosphere and cultivated tea rhizosphere. While the root

P content in wheat plants

treated with AMF consortium from the natural tearhizosphere was 0.032%, root P content

was found to be 0.024% in plants treated with AMF consortiafrom the cultivated tea

rhizosphere. The shoot P content was also found to bestatistically higher in wheat plants

inoculated with both consortia. The P content of maizeshoots was found to be higher than

the P content in the shoots of wheat plants. ACP and ALPactivities were also enhanced

by AMF inoculation. The consortium developed from thenatural tea rhizosphere resulted

in higher ACP and ALP activities as compared to the AMFconsortium developed from the

cultivated tea rhizosphere in both the test plant species.

5 AMF inoculation, tea growth and tea quality

5.1 Effect of AMF inoculation on tea growth

A small number of studies have analyzed the effectivenessof AMF on plants grown in

acidic soil (Clark, 1997; Clark et al., 1999), but theeffect of AMF inoculation on tea plant

growth has not been widely studied. However, bacterialinoculations were shown to

positively influence the growth of tea plants raised fromeither seed or tissue culture

(Pandey et al., 2000).

Bioassays to assess the growth-promoting ability of AMFconsortia in tea plants were

performed for a one year period. Consistent results ofgrowth promotion in tea plants were

obtained, as had been observed in wheat and maize during

preliminary screening for growth

promotion (Singh et al., 2010). Since tea plants are knownto grow well in acidic soil, the

experiment was done in non-sterilized acidic soil. The useof non-sterilized soil is important to

allow tea plants to benefit from the introduction of AMF.Gazey et al. (2004) have also shown

that the introduction of inoculated AMF consortia improvesthe growth of tea plants, in the

presence of indigenous AMF. Inoculated AMF consortiacolonized the tea roots of both seed

and clonal plants to a high degree. A significantpercentage of colonization was also recorded

in the field samples. This suggests a compatibility betweentea plants and AMF.

A stimulatory effect of AMF on tea plant growth wasobserved in both seed and clonal

plants. Figure 5 shows the microscopic analysis of theroots from control and AMF

inoculated tea plants, and the effect of AMF inoculationson tea plant growth. For the

majority of growth parameters studied, a significantincrease in growth was recorded

in both seed and clonal tea plants. Data related to growthand other parameters was

recorded after one year of inoculation. In an earlier studyby Janos et al. (2001), the positive

response to AMF inoculation on lychee growth took severalmonths to become apparent.

After comparison with other woody plant species, theyconcluded that analysis of the

effects of mycorrhizae on plants grown from propagules withsubstantial mineral nutrient

reserves, must be carried out over a long time period.These nutrient reserves included

air layers, large stem cuttings (Cooperband et al., 1994;Habte and Byappanahalli, 1994)

or very large seeds (Janos, 1980b). Cooperband et al.(1994) found that AMF inoculation

of 0.4m-long cuttings from the legume tree Erythrinaberteroana Urban, had a negative

effect on shoot growth, 6 months after planting. Theyattributed this to an adequate

mineral nutrient reserve in the woody tissue meaning thatmycorrhizae imposed a carbon

cost without a noticeable benefit to the plant. Habte andByappanahalli (1994) observed

that 0.18 × 18 mm cuttings of cassava, which on averagecontained 20.2 mg P, responded

mildly to mycorrhizal inoculation, but that 21 × 9.2 mmcontaining 2.0 mg P responded

well.

Figure 5 Roots of control (a) and AMF-inoculated (b) teaplants; (c) cutting raised tea plants; control

and plants inoculated with consortia developed fromcultivated tea rhizosphere (CTR) and natural tea

rhizosphere (NTR) after one year.

5.2 Benefits of AMF inoculation on tea quality

Tea quality is determined by the presence or absence ofcertain biochemical compounds

which impart its liquor, flavour and aroma characteristics(Yaminishi, 1999). Some of these

compounds include sugars, amino acids, proteins, totalpolyphenols and caffeine. These

were studied to examine the effect of AMF inoculation ontea quality in both seed and

clonal tea plants. The results are summarized in Table 2(Singh et al., 2010). Quality-related

components come to a consistent level after two years;therefore, these analyses were

performed over a two year time frame. Most of the studiedparameters were found to

increase significantly during this period. Concentration ofcaffeine was recorded to be

lower in later flushes as compared to just after thebreakage of dormancy. Gulati and

Ranivdranath (1996) also reported that the concentration ofcaffeine is lowered in later

flushes compared to earlier ones. Non-reducing sugarcomponents were found to decrease

in most cases since total soluble sugars were not found toincrease at a comparable rate

with the reducing sugar component of total soluble sugars.

Hilton et al. (1973) reported that the quality of tea isinversely related to the growth of

tea plants. This research group also found that theaddition of nitrogen fertilizers, (which

are abundantly applied during tea cultivation) had anegative impact on the quality of

black tea. This effect was attributed to the increasedgrowth rate of tea plants after fertilizer

application. In most instances, a significant increase inthe quality parameters mentioned

above was recorded in our study. We believe this reflectsthe positive effect of AMF

inoculation on tea quality, as well as on the growth of teaplants. Since AMF association

not only improves the growth of host plants, but alsoperformance and metabolic activity,

it may have resulted in the improved quality of tea alongwith its growth.

Although most of the research relating to AMF’s effect ontea quality was undertaken

by our group, other complementary studies were performed byTomanr et al. (2012). They

also applied a small input of NPK along with AMFinoculations and compared these results

with those obtained when AMF were inoculated alone. Lowerdoses of NPK produced a

better response in terms of caffeine and flavanolsenhancement. These studies showed

the potential of AMF for growing organic tea in asustainable system, rather than the use

of high doses of agrochemicals.

6 Conclusion and future perspectives

India is regarded as one of the world’s leading areas ofbiodiversity. However, little

attention has been given to AMF in the IHR, whichcontributes silently in various ways

to the ecosystem management processes. These associationshave great economic and

agricultural significance. They can generate ecologicalsuccess by allowing both fungus

and plant to exploit habitats in which neither may growsuccessfully without the other.

Thus, the successful establishment and vigorous growth ofmany major groups of plants,

including crop species, is favoured by AMF formation.Mycorrhizal technology has

allowed the production of inoculants to significantlyimprove the survival, growth and

establishment of trees and crops. Available inoculants areeffective for upland crops

such as rice, corn, pineapple, sugarcane, peanut andtomato. Other recipients that can

benefit include orchard crops such as citrus, guava andjackfruit, and tree species used

for reforestation. In this chapter, the developments inpreparing AMF consortia for tea

plantations are summarized. However, AMF-based inoculantsare not available in large T a b l e 2 E f f e c t s o f AM F i n o c u l a t i o n o n q u a l i t y r e l a t e d pa r a m e t e r s o f s e e d a n d c u t t i n g r a i s ed t e a p l a n t s a f t e r t w o y e a r s o f i n o c ul a t i o n P a r a m e t e r s S e e d r a i s e d t e a pl a n t s C u t t i n g r a i s e d t e a p l a n t s C o nt r o l N T R C T R C o n t r o l N T R C T R S o l u b l es u g a r s ( m g g − 1 ) 2 7 4 . 3 3 ± 0 . 8 8 3 1 4 . 0 0± 6 . 6 5 b 3 2 9 . 0 0 ± 5 . 5 0 c 3 2 6 . 0 0 ± 1 . 1 5 34 5 . 3 3 ± 2 . 3 3 b 3 3 8 . 0 0 ± 1 . 1 5 b R e d u c i ng s u g a r s ( m g g − 1 ) 1 7 4 . 0 0 ± 3 . 0 5 2 5 3 . 33 ± 6 . 6 6 c 1 9 6 . 0 0 ± 2 . 3 1 b 2 1 6 . 0 0 ± 3 . 0 52 3 1 . 6 6 ± 4 . 9 1 2 0 4 . 3 3 ± 6 . 1 7 N o n r e d u ci n g s u g a r s ( m g g − 1 ) 1 0 0 . 0 0 ± 2 . 0 8 6 2 .6 6 ± 2 . 0 2 c 1 3 3 . 0 0 ± 7 . 0 9 a 1 1 0 . 0 0 ± 2 . 30 1 1 3 . 6 6 ± 7 . 2 1 1 3 3 . 6 6 ± 6 . 2 2 a T o t a l pr o t e i n s ( m g m L − 1 ) 2 . 0 3 ± 0 . 0 9 3 . 2 3 ± 0. 1 2 b 2 . 4 6 ± 0 . 0 3 b 2 . 5 0 ± 0 . 1 5 2 . 9 0 ± 0 .0 5 b 2 . 9 3 ± 0 . 0 8 a A m i n o a c i d s ( m g g − 1 )4 . 0 3 ± 0 . 0 8 4 . 7 6 ± 0 . 0 8 b 4 . 1 3 ± 0 . 0 8 4 .4 6 ± 0 . 1 4 5 . 1 6 ± 0 . 1 4 a 4 . 6 0 ± 0 . 1 1 T o t al p o l y p h e n o l s ( m g g − 1 ) 8 4 . 3 3 ± 1 . 2 0 96 . 0 0 ± 1 . 1 5 b 9 0 . 3 3 ± 0 . 8 8 a 7 3 . 8 ± 1 . 6 68 2 . 6 6 ± 1 . 7 6 a 6 9 . 3 3 ± 2 . 0 2 a C a f f e i n e( m g g − 1 ) 1 9 . 5 ± 0 . 2 5 2 2 . 1 6 ± 1 . 0 1 2 1 . 90 ± 0 . 9 5 1 9 . 4 1 ± 0 . 2 2 2 1 . 5 8 ± 0 . 6 8 a 2 2 .7 5 ± 0 . 1 4 c a = s i g n i fi c a n t a t P ≤ 0 . 0 5 ,b = s i g n i fi c a n t a t P ≤ 0 . 0 1 a n d c = s i g ni fi c a n t a t P ≤ 0 . 0 0 1 a s a n a l y z e d b y o ne w a y a n a l y s i s o f v a r i a n c e .

quantities due to their obligate symbiotic nature.Therefore, a comprehensive and

detailed study of the AMF is required to identify, assessand utilize the same for future

generations. A well-organized web-enabled database,including physical and genetic

characterization, backed by an active user-friendlyretrieval system may be useful to

evaluate nature’s unique bioresources for mankind. Theoverall concept inferred from this

chapter is that the bio-inoculants developed with thenative AMF from the tea rhizosphere

would enhance growth and yield if applied to the plantingpit. These studies highlight the

importance of supplementing fertilizer needs in tea gardenswith efficient AMF-based bio

inoculants. This could greatly increase the tea plant’snutrient uptake, and reduce costs by

lowering fertilizer requirements. There is still enoughscope to exploit AMF to its maximum

advantage in the improvement of growth, yield and qualityof tea cultivars.


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8 Acknowledgements

The Director, GBPNIHESD, Almora, and Head/Co-ordinator,Department of Bio tech

nology and Bioinformatics Centre, Barkatullah University,Bhopal, are thanked for

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7 Chapter 7 The role of microbes in teacultivation

1 Introduction

Tea is a long-duration crop and is prone to attack byseveral pests and pathogens that

ultimately result in extensive annual crop loss(Mareeswaran et al., 2015), and inorganic

chemical-based fertilizers have been applied over the lastfew decades to remedy this

situation (Adesmoyee and Kloepper, 2009). Muraleedharan andChen (1997) suggested the

application of fungicides to control tea diseases such asblister blight, grey blight, brown

blight and red rust, as well as diverse tea pests. However,the extensive use of chemical

fertilizers has a harmful effect on soil health as thechemicals can destabilize soil fertility and

thereby directly affect the native microbial populationspresent in soil (Kalia and Gosal, 2011).

The application of chemicals onto tea plantations isprohibited for several reasons,

including: deterioration of soil quality (Gurusubramanianet al., 2005), air and groundwater

pollution, undesirable residues in made tea (Muraleedharanet al., 1988; Kodomari, 1993;

Chaudhuri, 1999), escalating costs (Pimental et al., 1992),resurgence of primary pests (Das,

1959; Hazarika et al., 2009), followed by an outbreak ofsecondary pests (Cranham, 1966) and

resistance development (Kawai, 1997; Gurusubramanian etal., 2008; Roy et al., 2010; Saha

and Mukhopadhyay, 2013), variation in susceptibility,impedance of natural regulatory agents

and lethal effects in warm-blooded animals, includinghumans (Moses, 1989; Mobed et al.,

1992). To overcome these constraints, considerable efforthas been made to complement

nutritional requirement through the use of alternativebiological approaches employing

different agricultural practices and microbes, inparticular, not only to increase crop production

and plant growth, but to also maintain soil health andproductivity (Fernando et al., 2005).

Soil is a dynamic natural environment of intenseplant–microbe interactions

(Bhattacharyya and Jha, 2012), known to initiate theproduction of plant growth hormones

which are antagonists of plant pests and pathogens, inaddition to harnessing essential

micro- and macronutrients that affect plant growth (Benizriet al., 2001). Beneficial

microorganisms include a diverse array of the soilmicrobiota, such as rhizobia, mycorrhizal

fungi, actinomycetes, diazotrophic bacteria, which createopportunities to promote

nutrient mineralization, allocation and availability viatheir symbiotic associations with

plant roots. According to Saharan and Nehra (2011), soilmicroorganisms influence the

physical, chemical and biological properties of soil eitherdirectly or indirectly.

The rhizosphere is the narrow, versatile and dynamicecological environment of soil that

is naturally rich in diverse microbial communities (Benizriet al., 2001) with the ability to

influence plant development as well as protect plant rootsagainst phytopathogens. In

1978, Kloepper and Schroth (1978) introduced the term‘rhizobacteria’ to denote the soil

microbial community, bacteria, that competitively colonizedplant roots and stimulated

growth and thereby assisted in disease reduction. A diverserange of soil microorganisms,

including Arthrobacter, Azospirillum, Azotobacter,Bacillus, Burkholderia, Caulobacter,

Chromobacterium, Enterobacter, Erwinia, Flavobacterium,Klebsiella, Micrococcus,

Pseudomonas, Serratia, Xanthomonas, etc., have beendocumented for their potential to

promote plant growth and protect plants from disease(Bhattacharyya and Jha, 2012; Bal et

al., 2013; Dutta et al., 2015). According to Philippot etal. (2013), soil microorganisms have

the ability to affect the health of plants, by affectingplant growth and nutrition, and thereby

positively influencing the development of eco-friendlypractices for sustainable agriculture.

Although tea soil microbiology was initially explored in1901, knowledge of the role of

tea soil microflora in enhancing tea cultivation is stillpoorly understood (Bezbaruah and

Baruah, 1985; Baruah, 1987; Dutta et al., 2015). Thenatural abundance of microorganisms

within the tea agro-ecosystem is expected to play asignificant role in maintaining a

sustainable environment (Singh et al., 2011). Nepolean etal. (2012) investigated the

inoculation potential of soil microbes such as Azotobacter,Azospirillum and Pseudomonas

in the tea plantations of Northeast India. A similar study

using plant growth-promoting

rhizobacteria (PGPR), such as Bacillus amyloliquefaciens,Serratia marcescens and Bacillus

pumilus, for the overall improvement of growth andproductivity of tea was carried out by

Chakraborty et al. (2013). Positive plant growth–promoting(PGP) traits, such as phosphate

solubilization, siderophore production, antagonism topathogens and indole acetic acid

(IAA) production, were exhibited by these beneficialmicroorganisms, which successfully

enhanced the seedling growth of tea varieties in thenursery as well as in the field.

Due to the abundant inoculation of native antagonisticmicrobes, such as

entomopathogenic viruses, bacteria, actinomycetes andfungi, they have also been

recognized as alternative biocontrol agents against a widevariety of tea pests and

pathogens, which has led to their introduction as anattractive component in integrated

pest and disease management programmes employed in teaplantations (Kodomari,

1993). The exploitation of diverse microbials such as PGPR,arbuscular mycorrhizal fungi

(AMF) and many other useful microorganisms might lead toimproved nutrient uptake,

plant growth and plant tolerance to biotic and abioticstresses.

The present review is intended to reveal the importance ofcertain known and

putative microorganisms native to tea soil. Scientificknowledge of microbial ecology

and developing strategies, followed by mass production,commercialization and field

evaluation is needed for suitable implementation of thismicrobial-based technology in

tea plantations. Furthermore, there is also a need toexplore, utilize and determine the

efficacy of indigenous microflora associated with the soilrhizosphere of tea, not only to

reduce the load of chemical fertilizer in tea plantationsbut also to enhance plant and soil

health for the larger benefit of tea growers.

2 Soil microbial inoculants as biofertilzers: an overview

Soil is a rich natural environment (Bhattacharyya, 2012)whose health is directly influenced

by various parameters such as microbiological componentswith multifaceted metabolic

activities, root exudates, and several other biotic andabiotic factors. The microbial

diversity and interactions in soil are extremely complex(Pang et al., 2008; Bhattacharyya

et al., 2014), and determine the nature of crop health inan agro-ecosystem. The fact

that microorganisms are essential to the ecosystem due totheir vital role in accelerating

activities such as maintenance of biogeochemical cycles,degradation of xenobiotic

compounds, organic matter decomposition, nutrientacquisition, water absorption,

weed control and disease suppression (acting as biocontrolagents) has spurred scientific

investigations exploring the vast resource of microbialdiversity in soil.

Microbial biofertilizers and biopesticides are the most

effective tools for pest management

in modern agriculture (Bhardwaj et al., 2014). Studies onmicrobial biofertilizers and

biopesticides that confer many beneficial properties, suchas nitrogen fixation, phosphate

solubilization, production of plant growth regulators andother mineral nutrients, and

biocontrol mechanisms, to increase crop productivity andplant protection (Miransari,

2011; Dodd and Ruiz-Lozano, 2012) are believed to play avital role in the adoption of

innovative, effective and affordable non-chemicalapproaches to ensure sustainable crop

production in an energy-efficient and eco-friendly manner.

Microbial biofertilizers contain live microorganisms.According to Bhardwaj et al.

(2014), the application of biofertilizers onto the seed,plant surface or soil can result in

colonization of the rhizosphere and promotion of plantgrowth by improving soil fertility

and plant tolerance and by increasing the supply of primarynutrients (Vessey, 2003). These

are environmentally safe substances that eventually help tomitigate the onset of global

warming as well as reduce chemical input costs, preventdepletion of organic matter and

increase crop yields (Mia et al., 2010). Microbialbiofertilizers include all kinds of natural

soil microflora, constituting PGPRs, mycorrhizal fungi(Mishra et al., 2013), actinomycetes

and mycorrhizal helper bacteria (Garbaye, 1994). Thisbeneficial microbiota encompasses

a significant role in mobilizing nutrients from their inert

elemental and complex organic

and inorganic forms into simple available forms.

There is an increase in phosphorus (P) and nitrogen (N)uptake after the application

of PGP microbials such as Azospirillum, Phosphobacteriumand Frankia, which indicates

the potential benefit of soil microbial inoculants innutrient management (Rajendran

and Devaraj, 2004). Figure 1 illustrates a general schemefor the potential use of soil

microorganisms in sustainable agriculture. Biofertilizersare, thus, helpful in solving the

problems related to plant nutrition and diverseenvironmental stresses. It is, moreover,

important to realize the beneficial aspects ofbiofertilizer application and to integrate it

into modern agricultural practices under changing scenarios.

The Indian tea industry (ITA) has achieved significantsuccess in biofertilizer exploitation

over the last few decades, as most states in India haveincreased their production by

including biofertilizers as an essential component oforganic farming (Bhardwaj et al., 2014),

a globally recognized farming practice for the productionof safe and healthy food and

beverages. The potential of indigenous PGPR strains such asBacillus pseudomycoides,

Burkholderia sp., Enterobacter lignolyticus and Pseudomonasaeruginosa for use as

microbial biofertilizers to promote growth in teaplantation has been evaluated (Dutta et

al., 2015). A similar investigation onrhizobacteria-mediated improvement of tea plants has

been made by Chakraborty et al. (2013), who also observedthe PGP potential of certain

soil bacteria such as Bacillus amyloliquefaciens, Bacilluspumilus and Serratia marcescens.

Acetobacter, Allorhizobium, Aspergillus, Azorhizobium,Azospirillum, Azotobacter,

Bacillus, Bradyrhizobium, Mesorhizobium, Penicillium,Pseudomonas, Rhizobium etc.,

have shown high potential for use as biofertilizers insustainable agriculture (Vessey, 2003).

Similarly, the inoculation effect of beneficial microbialbiofertilizers, such as Azotobacter

chroococcum, Bacillus subtilis and Pseudomonas corrugate,in enhancing production in

Figure 1 Potential use of beneficial soil microorganisms insustainable agriculture. Adapted from

Bhardwaj et al. (2014).

tea has also been established (Mondal et al., 2004;Nepolean et al., 2012; Pandey et al.,

2013).

Tea bushes treated with beneficial microorganisms exhibitan increase in bush girth,

productivity of young shoots as well enhanced root length.Nitrogen-fixing bacteria

(NFB), phosphate-solubilizing microorganisms (PSMs),potash-solubilizing microorganisms

(KSMs), plant disease–suppressive beneficialmicroorganisms, stress-tolerant endophytes,

biodegrading microbes, etc. have potential as a functionalmicrobiota in tea plantations

whose inoculation into the tea ecosystem significantlyadvances the soil physico-chemical

properties, microbial biodiversity, soil health, plantgrowth and development, and crop

productivity (Subha Rao, 1982; Baby, 2002; Aggani, 2013).

Seed treatment, soil treatment, foliar applications, etc.are the methods usually followed

when using microbial biofertilizers in tea plantation. Theprocedure of treatment application

may vary and is dependent upon the implementation ofvarious cultural practices and type

of planting material in use. Although most chemicals intheir recommended doses do not

have a significant inhibitory effect on microbial products,biofertilizers or treated seeds

should not be mixed with chemical fertilizers, insecticidesor pesticides (Mondal et al.,

2015) until the compatibility assessment of biologicalsubstances have been confirmed

with tested agrochemicals (Dutta et al., 2016). Organicmatter has an immensely important

influence on the growth and proliferation of differentkinds of microbial bioinoculants,

and soil rich in organic matter normally supports theluxuriant growth of beneficial

microorganisms. In soils poor in organic matter, the growthand metabolism rates of such

organisms are drastically reduced and restricted only tothe rhizospheric microenvironment.

3 Nitrogen-fixing microbial biofertilizers

Nitrogen (N) is an essential macronutrient in plantmetabolism (Leghari et al., 2016). It

is the key elemental component in chlorophyll, thebiomolecule that enables plants to

photosynthesize and facilitate growth. N is also an

essential constituent of protein, nucleic

acids and growth hormones. There are reports of enhanceduptake and utilization of

certain other nutrients, such as potassium and phosphorus,due to N influence and it

thereby indirectly controls the major nutrient supplynecessary for normal plant growth

and development (Bloom, 2015). The age of the growing plantand its characteristics, soil

and climatic variations, etc. are considered to be the keyfactors which influence the N use

efficiency of plants. In conventional agriculturalpractices, fertilizers with high N content

such as urea or ammonium nitrate are usually applied toincrease N levels in soil.

Being a foliage crop, the requirement of N in tea soil isquite high, as N plays an

important role in increasing tea productivity. In teaplantation soil, the requirement for

N is usually met by chemical fertilizers such as urea, andsulphate of ammonia. However,

high doses of chemical fertilizers have an adverse effecton crop sustainability (Saha et al.

2000), as well on soil health and productivity. Theexploitation of N fertilizer requires fossil

fuel as an energy source, which is in short supply andexpensive, and contributes to global

warming, eventually affecting environmental quality andcreating health hazards to human

beings (Altman and Hasegawa, 2012; Gepts, 2012). Excessnitrogenous fertilizer run-off

via leaching leads to the eutrophication of terrestrial andaquatic systems, natural decline

of soil microflora and acidification of water, ozone layerdepletion, etc. (Byrnes, 1990). This

necessitates the search for alternative strategies that canprovide significant N to growing

plants in an efficient and sustainable manner.

The application of NFB that have the ability to reduce thechemical fertilizer load

would potentially contribute to a substantial reduction inproduction cost, and positively

influence the world’s economy, thereby reducing healthrisks to environments (Altman and

Hasegawa, 2012; Dodd and Ruiz-Lozano, 2012; Shridhar, 2012;Wang et al., 2012). NFB

are microorganisms that have the ability to transformatmospheric nitrogen into a fixed

form that is available for utilization by plants. Thegeneral scheme of biological nitrogen

fixation can be represented by the following equation: N 8H8e ------------2NH H 2 3 2 + + + + −

This is an energy-intensive process requiring 16equivalents of ATP and produces two

molecules of NH 3 and one molecule of H 2 . Cavalcante andDobereiner (1988) recovered the

first nitrogen-fixing acetic acid bacterium, Acetobacterdiazotrophicus (presently known as

Gluconacetobacter diazotrophicus) from tissues of thesugarcane plant, and Muthukumarasamy

et al. (2002) isolated the bacterium from tea roots.Besides, Gluconacetobacter diazotrophicus,

many diazotrophic bacteria such as Azotobacter,Azospirillum, Herbaspirillum and Clostridium

have the ability to fix atmospheric N 2 and thereby makeit available to plants. Among these

genera, free-living N 2 -fixers such as Azotobacter andAzospirillum have established their

identity as potential NFB to be used in tea soil (Wani etal., 2013).

Azotobacter is an aerobic, free-living soil microbe (Kumar,2014) that plays an important

role in maintaining the N 2 cycle in nature, as it derivesits nutrients from organic matter

present in soil and root exudates and thereby fixesatmospheric nitrogen which is finally

absorbed by adjoining roots. Besides their immensepotential for nitrogen fixation,

Azotobacter are also known to secrete certaingrowth-promoting substances like IAA,

gibberellic acid and cytokinins as well as some antibioticsand vitamins such as thiamine

and riboflavin (Revillas et al., 2000) that can reduce theincidence of soilborne plant

pathogens. When applied as soil treatment, they canmultiply rapidly and develop a thick

microbial load around the rhizosphere, which ultimatelyenvelops the entire root system.

Azotobacter, being aerobic in nature, gives better resultsin well-aerated, light- or medium

textured soil. Azotobacter chroococcum can improve plantgrowth by enhancing seed

germination and advancing the root architecture viaphytopathogen inhibition (Gholami et

al., 2009) around the root systems of crop plants (Mali andBodhankar, 2009).

Likewise, an Azospirillum biofertilizer may also be used inany non-legume crop (Saikia et al.,

2013) such as tea to increase the nitrogen supply andpromote nutrient management. Being

microaerophilic in nature, Azospirillum can thrive even inwaterlogged conditions (Sahoo

et al., 2014), thereby aiding the nutrient profile for theadvancement of plant growth and

development (Ilyas et al., 2012). The ability ofAzospirillum strains to withstand waterlogged

conditions has been demonstrated by Kadouri et al. (2003).For tea productivity, Azospirillum

is one of the most important microbes as it is known toenhance the nutrient level in tea

seedlings or cuttings (Phukan et al., 2012). Efficacy wasachieved for Azospirillum-treated

rootings of vegetatively propagated (VP) cuttings and forthe growth of VP plants as well as

seedlings.

Azospirillum, upon direct inoculation to plant or soil, canmultiply rapidly and move

towards available root systems or intercellular spaces, andcan thereby establish a symbiotic

relationship with the host (Santi et al., 2013). Accordingto Bashan et al. (2004) Azospirillum

acts as an effective root colonizer and its use is notlimited by host specificity. It has been

reported that Azospirillum inoculation can change rootmorphology via the production

of plant growth hormones (Bashan et al., 2004) andsiderophores (Sahoo et al., 2014).

Azospirillum are also known to stimulate the density andlength of root hairs as well as

increasing biomass through the production of plantgrowth-regulating substances (de

Souza et al., 2015) and atmospheric nitrogen fixation. Theproduction of plant growth

hormones such as IAA and gibberellic acid,exopolysaccharides, siderophores and poly

β-hydroxybutyrate by Azospirillum has been reported (Tienet al., 1979; Etesami et al., 2015;

Cecagno et al., 2015). An Azospirillum biofertilizer maybenefit the target crop by promoting

grain yield and vegetative growth successively. Bacterialbiofertilizers like Azotobacter and

Azospirillum are also known to secrete antibiotics (Ahemadand Kibret, 2014; Beneduzi et al.,

2012; Bhattacharyya and Jha, 2012) that protect the targetplant from pathogenic attack and

environmental stress. Field applications with a microbialconsortium of these biofertilizers

might result in better yield performances than microbialtreatments with a single application

(Gharib et al., 2008).

4 Phosphate-solubilizing, potash-solubilizing andcellulose-degrading microbial biofertilizers

4.1 Phosphate-solubilizing microbial biofertilizers

Phosphorus (P) is a vital nutrient requirement in all typesof plants, including tea. It plays an

indispensable role in photosynthesis, respiration, energystorage and transfer, cell division

and enlargement, and several other biochemical reactions inthe living plant (Patel and

Parmar, 2013). It is also vital for seed formation andcontributes significantly towards

disease resistance. It has been estimated that more than40% of the crop yields on the

world’s agricultural land are limited by P availability.The requirement of P is usually met

upon the addition of phosphate-rich chemicals in the formof aluminium or iron phosphates.

However, these fertilizers are quite expensive (Tilak etal., 2005; Richardson, 2007) and

injudicious application adversely affects the environment,lowers soil fertility and reduces

microbial diversity and function. According to Sharpley andTunney (2000), extensive P

run-off and leaching activities lead to the eutrophicationof aquatic bodies which indirectly

creates serious health hazards to living organisms,including human beings.

PSMs play a key role in P-cycling in soil (Richardson,2007). Morgan et al. (2005) reported

that there is an increase in rhizosphere microfloralpopulations followed by lateral root

formation, and the production of extensive root hairsystems in plants that are P-deficient

in order to seek a greater volume of soil. PSMs are knownto convert the insoluble form of

soil phosphorus to the soluble form through mechanisms suchas acidification, chelation

of cations bound to P, secretion of various types oforganic acids such as citric, fumaric,

glutamic, lactic, maleic, succinic and α-ketoglutric acidprotons, and the release of P from

phosphates and phytates via the corresponding hydrolases(Siddiqui, 2006; Richardson

et al., 2009; Hayat et al., 2010).

PSMs include different groups of microorganisms which notonly assimilate P from

insoluble forms of phosphates, but also cause a largeportion of soluble phosphates to be

released in quantities in excess of their requirements.Among the bacteria, Agrobacterium

sp., Arthrobacter sp., Azospirillum sp., Azotobacter sp.,Bacillus sp., Burkholderia sp.,

Enterobacter sp., Flavobacterium sp., Pseudomonas sp.,Rhizobium sp. and Serratia sp.,

and among the fungi, Aspergillus sp., Penicillium sp. andSchwanniomyces occidentalis are

considered to be the predominant members which convertinsoluble inorganic phosphate

in soil into soluble plant-usable forms (Rodriguez andFraga, 1999; Tilak et al., 2005; Avis et

al., 2008; Hayat et al., 2010). It has been reported thatphosphate solubilizers of bacterial

origin normally survive and work well in soils of pHranging from 6.0 to 7.5, while for acidic

soils, fungal strains are more effective. The effect ofPSMs on soil available phosphate

and growth of young tea has been observed by Phukan et al.(2016). Bacillus subtilis and

Aspergillus niger were recorded as the predominantphosphate solubilizers resulting in

considerable P fertilizer savings, particularly inP-deficient acidic soils. In addition to this,

considerable amounts of phytate (inositol phosphate) alsooccur in soils complexed with

minerals such as iron, calcium and magnesium (Gerke, 2015).Soil bacteria like Bacillus sp.

have the ability to produce an enzyme, commonly known asphytase, which hydrolyses

phytate and releases phosphate that can be utilized by theplant. The presence of phytate

solubilizing microorganisms in tea soils of Northeast Indiaand their efficacy to solubilize

bound P in soil have been determined by Pramanik et al.(2014). There was an increased

available P content in the soil due to inoculation ofphytate-solubilizing microbes. The

ability of PSMs is of immense interest to agriculturalmicrobiologists in order to enhance

the availability of P for effective plant growth andsubsequently enhance plant immunity

against pathogenic attack.

4.2 Potash-solubilizing microbial biofertilizers

Potassium (K) is a vital component of plant nutrition andperforms a multitude of important

biological functions in order to maintain quality plantgrowth and development (Bahadur

et al., 2014), and additionally helps crops to developresistance to stress as well as insect

pests and diseases. According to Meena et al. (2014), mostglobal agricultural land is

deficient in K, which includes three-quarters of the paddysoils of China and two-thirds of

the wheat belt of Southern Australia.

K is usually present in soil but mainly in an unavailableform or as K-bearing minerals,

and its availability to plants is highly variable mainlydue to complex soil dynamics, which

are strongly influenced by plant root and soil interactions(Basak and Biswas, 2010).

For instance, in developing countries like India, Sri Lankaand Kenya there is practically

no reserve of K-bearing minerals for the manufacture of Kfertilizers. As a result, the

consumption of all K fertilizers in those soils is

imported, which leads to a huge amount

of expenditure. Thus, in most developing countries, thebioactivation of soil K reserves

as well as K-bearing minerals is an urgent demand in orderto alleviate potash fertilizer

limitation.

Certain soil microorganisms such as Acidithiobacillusferrooxidans, Arthrobacter sp.,

Azotobacter sp., Azospirillum sp., Bacillus mucilaginosus,Bacillus edaphicus, Burkholderia

sp., Frateuria sp., Klebsiella sp., Paenibacillus sp.,Pseudomonas sp., Rhizobium sp. and

Serratia sp. (Sheng, 2005; Lian et al., 2008; Liu et al.,2012) have the ability to solubilize

the unavailable form of K-bearing minerals to release K inan available form (Setiawati and

Mutmainnah, 2016). The production of organic acids secretedduring K transformation

and nutrient cycling (Parmar and Sindhu, 2013; Sessitsch etal., 2013), biofilm formation

and enhanced mobilization is considered to play a key rolehere. Similar research on the

use of a biological potassium biofertilizer to investigatethe activation of minerals so as

to alleviate the shortage of K fertilizer, particularly inChina and South Korea, has been

reported (Lin et al., 2002; Han et al., 2006; Liu et al.,2006). With the existing economic crisis

for tea growers, it is necessary to choose microbialfertilizers that can improve the mineral

status of tea soil (Bhattacharyya et al., 2016). KSM wouldserve as an effective biofertilizer

if integrated with a reduced level of K fertilizers, which

could create opportunities for

maximum crop yield with low-cost technology.

The isolation and characterization of potash mobilizersfrom native tea soil has been

made by Bhattacharyya et al. (2016). The efficiency of theisolated strains was initially

tested on nursery grown bean (Phaseolus vulgaris L.) plants(Tocklai Ann. Sc. Report

2014–15). The isolates were able to enhance the available Klevel in the soil after in vitro

incubation. Total microbial population numbers; levels ofavailable K and P in the soil;

and various plant growth parameters like plant dry weight,number of fresh leaves and

branches, pods, legumes, leaf moisture and root length weresignificantly increased after

the soil was treated with potash mobilizers compared withan untreated control (Fig. 2).

Four strains of the Bacillus genera (KMB03, KMB06 and KMB08identified as Bacillus

pumilus and KMB11 as Bacillus subtilis based on theirbiochemical as well as molecular

characteristics using 16S rDNA homology) were found to beeffective at increasing the

available potash in tea soil. Exploitation and screening ofthese indigenous and native

beneficial soil microbial strains, thus, creates thepossibility of utilizing them as possible

bioinoculants in tea alone or in combination with low dosesof potash fertilizers to reduce

cost and support eco-friendly quality tea production.

Figure 2 Inoculation effect of potent KMB strains isolatedfrom tea field on nursery-grown bean

(Phaseolus vulgaris L.) plants. Adapted from Bhattacharyyaet al. (2016).

4.3 Cellulose-degrading microbial biofertilizers

Cellulose is the most abundant carbohydrate present inplant residues or organic matter

(Hopkins et al., 1990). Cellulose decomposition usuallyoccurs in two stages. In the first

stage, the long chain of cellulase is broken down intocellobiase and then into glucose

via hydrolysis in the presence of enzymes, that is,cellulase and cellobiase. This reaction

is followed by the oxidation of glucose which is ultimatelyconverted into CO 2 and water.

The entire reaction of cellulose decomposition isrepresented below, where cellulolytic

microorganisms like bacteria, fungi and actinomycetes playa significant role in cellulose

degradation through the excretion of enzymes such ascellulase or cellobiase. 1 Cellulase Cellobiase CelluloseHydrolysis Cellobiose Hydr � →�� olysis Oxidatio n OxidaGlucose 2 Glucose Organic acids � →�� →�� tion 2 2 CO+H O� →��

A cellulose-degrading bacterial (CDB) inoculant is abeneficial microbiological tool to aid

the recovery of energy from degraded ecosystems during thepruning of tea plantations,

as it may lead to the efficient use of renewableconventional resources via microbiological

processes rather than non-renewable conventional ones.Exploitation and implementation

of cellulose-degrading bacteria in tea cultivation isimportant not only to recycle the

vast amount of cellulosic biomass deposited on the floor ofthe tea plantations during

the pruning stage, but it is also essential to minimizebiomass pollution as well as control

fungal diseases and improve the organic matter content ofsoil (Balamurugan et al., 2011).

Consequently, this study is also helpful in opening novelavenues of progressive thoughts

and ideas, where tea growers might depend either on reduceddoses of synthetic fertilizers

or use both cellulose-degrading bacteria along withsynthetic fertilizers (in minimum dosage)

under an integrated nutrient management programme toincrease tea productivity.

5 Microbial management of pests and diseases in tea

5.1 Microbial pesticides

Microbial pesticides are substances that promote plantgrowth by controlling the activity of

diverse plant pests including disease-causingphytopathogens and other tea pests. Bacterial

genera like Bacillus spp. and Pseudomonas spp.; fungalisolates such as Aspergillus sp.,

Beauveria bassiana, Gliocladium sp., Metarhiziumanisopliae, Paecilomyces sp., Penicillium

sp., Trichoderma spp. and Verticillium lecanii;actinomycetes like Streptomyces sp.; and

mycorrhizal fungi have raised agricultural productivity dueto their potential as biopesticides

in tea plantations. According to Ahmed and Holmstrom(2014), iron-chelating compounds

present in antagonistic microbes make them a bettercompetitor than phytopathogens.

An array of mechanisms such as mycoparasitism, productionof antibiotics, siderophores,

hydrogen cyanide (HCN), hydrolytic enzymes, and thedevelopment of acquired and

induced systemic resistance (ISR) (Somers et al., 2004;Chandler et al., 2008) might work

in a collaborative way to promote microbial pesticides fortheir antagonistic action against

diverse tea pests. Microbial pesticides may use more thanone of these mechanisms for

pest suppression and to enhance plant growth, asexperimental evidence suggests that

plant growth stimulation and pest control are the netresult of multiple interactions that

may be activated simultaneously. The pesticide-degradingability of microorganisms such

as R. leguminosarum, R. trifolii, Penicillium citrinum,Penicillium oxalicum, Staphylococcus

sp. and Bacillus circulans has been recently established(Huidrom and Sharma, 2014).

The biodegradation of pesticide molecules, such as dicofoland fenpropathrin, by

Pseudomonas spp., has been demonstrated by Sarkar et al.(2009). The phenomenon

of quorum regulation can affect the expression of microbialbehaviour, as beneficial

microorganisms have been reported to interact with theresident microbial community in

the rhizosphere (Lugtenberg and Kamilova, 2009).

5.2 Microbial management of diseases in tea: an overview

Tea diseases are considered to be an important bioticconstraint (Sarmah, 1960a) that

leads to significant tea crop losses worldwide. Theincidence and intensity of pathogenic

attack, however, varies with change in climate, elevation

and the use of planting material.

Prolonged drought leads to death of the tea bushes, whileexcessive rainfall may cause

overall loss of the crop. As tea plantations are mostlyrain-fed and the cropping season

needs a stable microclimate with alternating wet and dryperiods (Anita et al., 2012; Ali et

al., 2014), tea is naturally susceptible to theestablishment of several tea pathogens.

Diseases of tea bushes may be caused by one or more factorssuch as presence of fungi,

algae and bacteria, and animal parasites; adverse soilconditions; unfavourable climate;

and mechanical damage or viral attack. Based on the natureof pathogenesis, tea diseases

fall into two basic categories, primary and secondary teadiseases. Primary tea diseases

arise spontaneously on direct occurrence and areresponsible for causing death of healthy

tissues or tea bushes even under the best environmentalconditions. It is not associated

with previous disease, injury or event, but may lead tosecondary disease development.

Secondary tea diseases may be harmful if the health of thetea bushes is impaired due to

injury or any other cause (Sarmah, 1960b).

Out of all the biotic agents, fungi alone are responsiblefor causing a large number

of recognized tea diseases. In addition to fungal diseases,an alga, Cephaleuros spp.

(Cephaleuros parasiticus and Cephaleuros mycoidea) is alsoresponsible for causing

disease in tea, popularly known as red rust. Although red

rust is secondary in occurrence,

it is common in tea bushes, both on plains as well as onhills and is known for damaging

young tea plants by penetrating deep into the tissues ofthe host cells resulting in die-back

of the stem. In addition, tea seeds, damaged by the teaseed bug (Poecilocoris latus), are

often invaded by bacteria. More recently, Dutta et al.(2015) reported leaf blight in tea

plantations of Darjeeling in the district of West Bengal,caused by Nigrospora sphaerica.

It has been suggested that introducing a decade of organic,non-toxic tea plantations

throughout the world, as an alternative to the use ofsynthetic chemicals to control various pests

and pathogens (Pandey et al., 2013), is important mainlydue to the non-availability of effective

chemicals as well as dispensing with the use of hazardoustoxic chemicals. The microbial

tea rhizosphere has important PGP abilities that may beexploited for crop enhancement

and pest and disease suppression in tea (Phukan et al.,2005; Phukan et al., 2007, 2012).

Aspergillus, Azotobacter, Azospirillum, Fusarium,Gliocladium Penicillium, Trichoderma and

different phosphate solubilizers like Bacillus andPseudomonas are known for their significant

contributions to tea cultivation (Bezbaruah and Baruah,1985; Baruah, 1987). Considerable

success has also been achieved in utilizing antagonisticmicrobes to control tea pathogens

(Barthakur, 1995; Barthakur et al., 1998, 2002;Saravanakumar et al., 2007; Pallavi et al., 2012;

Kanimozhi and Ponmurugan, 2013; Pandey et al., 2013;Mokhtar and El-Mougy, 2014).

According to Harman et al. (2004) biological control viaantagonistic microorganisms is a

promising, non-chemical, eco-friendly approach for managingthe incidence and severity

of plant diseases. Microbial biocontrol agents have theability to infect a given host

through diverse modes of actions such as mycoparasitism,hyperparasitism, production

of siderophores, volatile and non-volatile compounds,antibiotics and hydrolytic

enzymes. (Chandramouli and Baby, 2002; Benitez et al.,2004; Ponmurugan and Baby,

2007; Gnanamangai and Ponmurugan, 2012). Bacterial strainssuch as Bacillus spp. and

Pseudomonas spp.; fungal isolates like Aspergillus sp.,Gliocladium sp., Penicillium sp. and

Trichoderma spp; and mycorrhizal fungi act as potentialbioinoculants in tea plantations

(Barthakur et al., 2001, 2004; Anita et al., 2012; Moranget al., 2012).

The efficacy of Bacillus subtilis to control foliar teadiseases such as black rot and blister

blight, and T. viride in preventing Poria branch canker,thorny stem blight and primary root

diseases have been established (Barthakur, 2011). T. virideexhibited an inhibition of up

to 65.77% of Phomopsis theae, the causal agent of Phomopsiscanker in tea (Anita and

Ponmurugan, 2011). Sowndhararajan et al. (2013) developedstrategies for the integrated

management of blister blight disease in tea using thebiocontrol agent Ochrobactrum

anthropi along with chemical fungicides. The resultsundoubtedly indicated the potential

of Ochrobactrum anthropi as a biopesticide for theeffective management of blister blight

disease in tea. Evaluations of the biocontrol action ofPseudomonas aeruginosa, isolated

from the tea soil of Barak Valley, Assam, against the brownroot rot pathogen, Fomes

lomaoensis has been established (Morang et al., 2012). Theantagonist showed significant

inhibition of disease development (reduced by up to33.33%). The significance of

secondary metabolites and enzymes produced by T. atrovirideisolates against Phomopsis

canker has been examined by Anita et al. (2012). Thesyntheses of extracellular enzymes

such as amylase, cellulase, chitinase, polygalacturonaseand protease are associated with

the antagonist activity of the biocontrol agent. The invitro antagonistic activity of Bacillus

spp. and Pseudomonas against grey blight disease in tea hasalso been established

(Pallavi et al., 2012). The results indicated theproduction of cell wall lytic enzymes as the

key components responsible for the antagonistic activity ofthe biocontrol agents.

PGPR-induced defence responses in tea plants againstblister blight disease was

evaluated by Saravanakumar et al. (2007), who tested theefficacy of PGPR bioformulations

such as Pseudomonas and Bacillus spp., against Exobasidiumvexans, the causal agent

of blister blight in tea under field conditions.

Saravanakumar et al. (2007) reported that

foliar application of Pseudomonas fluorescens at seven-dayintervals is most effective

in reducing the severity of blister blight disease. Defenceenzymes such as chitinase,

β-1,3-glucanase, peroxidase (PO), phenolics, phenylalanineammonia lyase (PAL) and

polyphenol oxidase (PPO) in Pseudomonas fluorescens-treatedplants might be involved.

An integrated disease management approach to control greyblight in tea was undertaken

by Premkumar et al. (2012). Bacillus sp., Pseudomonas sp.and Trichoderma sp. were found

to be effective microbial agents against the attack offungal pathogens. The bioefficacy of

microbial biocontrol agents such as Pseudomonasfluorescens, Streptomyces sannanensis

and T. atroviride against bird’s eye spot disease of teaplants has been evaluated by

Gnanamangai and Ponmurugan (2012). The results indicatedmaximum potential by

Streptomyces sannanensis in terms of disease protection.

The prospect of actinomycetes as a biocontrol agent tocontrol tea diseases in

Northeast India has also been investigated (Bhattacharyyaet al., 2015). Antagonistic

actinomycetes such as Streptomyces nojiriensis,Streptomyces griseoluteus, Streptomyces

somaliensis and Streptomyces sannanensis were found to beeffective in controlling major

tea pathogens, namely Exobasidium vexans, Corticiuminvisum, Ustulina zonata, Fomes

lamaeonsis, Pestalozzia theae and Poria hypobrunnea, to

varying degrees (Sarmah et al.,

2005; Barthakur, 2011), and thereby may lead to bioprimingof the plants. Thus, reduced

dependence on synthetic chemicals and applying soilmicrobial inoculants to maintain an

intact ecosystem (pesticide and contaminant-free) is theemergent strategy for tea growers,

as persistent use of chemical fertilizers in soil graduallydeteriorates the soil quality as well

as causing the stagnation of quality tea production.

The application of microbial biofertilizers andbiopesticides to acidic soils appears

to be crucial, as this type of soil has the ability topromote plant growth and suppress

pathogenic attack. However, under field conditions, thesuccess of microbe-based

‘fertilizer’ technology depends on the availability ofsuitable bioinoculants in appropriate

formulations. The microbial source, shelf life andcontamination of the microbial

formulations should be verified before their applicationunder field conditions to control

diverse tea diseases. Scientific investigation of theantagonistic properties of microbial

biocontrol agents involved in the tea ecosystem, with asolid indication of the interaction

between the tea plants, pests, other natural enemies andmicrobial inoculants, as well as

the linkage between the entire microbial consortium (Wardleet al., 2004; Hazarika et al.,

2009) needs to be worked out in order to maintain a moresustainable and productive

ecosystem in the future.

6 Important interactions and mechanisms of action in themicrobial management of disease

6.1 Microbial interactions contributing to biologicalcontrol

Biological control is an important component of anintegrated pest management strategy

(Chidawanyika et al., 2012; Roy and Muraleedharan, 2014).It may be defined as the

method of reducing pest populations, such as insects,mites, weeds and pathogens, using

other organisms. It basically relies on predation,parasitism, herbivory or other natural

mechanisms, but typically also involves an active humanrole. Odum (1953) observed

microbial interactions such as mutualism, protocooperation,commensalism, neutralism,

antagonism, competition, parasitism and predation aseffective strategies in biological

control.

Mutualism is an association between two or more specieswhere all the species may

derive benefit while being together. Sometimes, it may bean obligatory interaction

involving close physical or biochemical contact (e.g.plants and mycorrhizal fungi);

mutualism may be facultative or opportunistic.Protocooperation is a type of mutualism

where the organisms do not depend exclusively on each otherfor their survival. Most

microorganisms involved in protocooperation behave as afacultative mutualist as their

survival rarely depends on any specific host.

Commensalism is a type of symbiotic interaction between twoliving organisms, where

one organism benefits and the other is neither harmed norbenefits. Most plant-associated

microbes are assumed to be commensals with regard to thehost, as their presence

individually or in total rarely results in positive ornegative consequences to the host.

Neutralism describes unbiased biological interactions whenthe population density of one

living species has absolutely no effect on the other.Related to biological control, it can be

defined as the inability of a pathogen to associate withanother organism.

Antagonism is result of the production of some antagonisticmolecules (e.g. antibiotics),

originating from antimicrobial screening activities. Theability to produce multiple

antibiotics by biocontrol agents is helpful in suppressingthe activity of a diverse array of

harmful pathogenic competitors. An efficient biocontrolagent is one that can produce

adequate quantities of antibiotics in the vicinity of theplant pathogen (Vargas Gil et al.,

2009). Competition within or between species results indecreased growth, activity or

fecundity of the interacting organisms. Biocontrol occurswhen non-pathogens compete

with pathogens for nutrients in and around the host plant.The process of competition is

considered to be an indirect interaction between thepathogen and the biocontrol agent.

Here, the pathogens are excluded from the association withthe host plants by depleting

their natural food, as well as physical occupation of theinfected site.

Parasitism is a symbiosis in which two phylogeneticallyunrelated organisms co-exist over

a prolonged period of time. In this type of association,one organism (usually the parasite)

benefits and the other (the host) is harmed. Interestingly,host infection and parasitism by

relatively avirulent pathogens can lead to the biocontrolof more virulent pathogens via

stimulation of the host’s defence system. Predation refersto the hunting or killing of one

organism by another for consumption or sustenance.Biocontrol is successful to varying

degrees in different microbial interactions (Haas andDefago, 2005) and depends on the

environmental framework within which they usually occur. Asthe bioagent represents a

living system, it needs to be mass produced and formulatedso that it remains viable for

a considerable period.

6.2 Mechanism of action

Tea diseases are the result of tripartite interactionsamong the host, pathogen and

environment. Biocontrol agents are the living organismsthat interact with the components

of this disease triangle to manage the disease. Biocontrolagents generally possess a

diverse array of mechanisms to manipulate the soilenvironment and thereby create a

healthy situation for disease management (Junaid et al.,2013). Direct and indirect

antagonism are the two basic mechanisms usually involvedduring the biocontrol of plant

diseases. Direct antagonism results from the physicalcontact or high degree of pathogen

selection by the tested biocontrol agent.

Hyperparasitism is considered to be the direct form ofantagonism (Pal and Gardener,

2006). It involves the tropic growth of the biocontrolagent towards the target pathogen

followed by coiling, final attack and dissolution of thepathogenic cell wall via enzymatic

activities. T. harzianum exhibits antagonism againstRhizoctonia solani (Altomare et al.,

1999) and is a classic example of hyperparasitism.Mycoparasitism and competition are

two other direct antagonism mechanisms of biocontrol agentsrecorded, so far, against

the target pathogen. Mycoparasitism is directly underenzymatic control. Harman (2000)

reported the involvement of chitinase and β-1,3-glucanasein Trichoderma-mediated

biological control. A pathogen must effectively compete foravailable nutrients while

colonizing the host, and competition for micronutrientsexists among the biocontrol agents

and the target pathogens since the former have a moreefficient nutrient uptake system.

This property of microbial antagonists can be attributed tothe production of iron-binding

ligands, called siderophores, as in Erwinia carotovora(Kloepper et al., 1980), resulting in

iron becoming unavailable to the pathogen, leading to lesspathogen infection.

Biocontrol agents compete with the pathogen for physicaloccupation in the soil

habitat as well as for nutrient acquisition, and therebymay reduce the colonizing

ability of a particular pathogen. Applications of abeneficial microbial consortia to the

seeds or seedlings of certain plants have resulted inincreased efficiency of ISR against

diverse pathogens (Ramamoorthy et al., 2001). Signallingmechanisms such as salicylic

acid (SA)-independent jasmonic acid (JA), ethylene(ET)-dependent signalling and NPR

(non-expressor pathogenesis-related genes) 1-dependentsignalling might be involved

in enhancing the expression of JA-responsive genes andET-responsive genes in

Pseudomonas fluorescens-inoculated plants, resulting ininduction and development of

ISR. The JA and ET response in plant growth–promotingbacteria-inoculated plants might

activate npr-1 gene expression that encodes the NPR-1protein followed by activation

of the defence-related gene (Jain et al., 2014). NPR-1proteins are known as master

regulators in defence pathways as after receiving theproceeding signal, this protein

activates expression of either the PR gene or adefence-related gene for the establishment

of salicylic acid resistance (SAR) and ISR, respectively.Different defence-related genes

activated by JA and ET are shown in Fig. 3. Expression ofpathogen-inducible genes Hel

(encoding a hevein-like protein), ChiB (encoding a basicchitinase) and pdf 1.2 (encoding

a plant defensin) and the proteins encoded by them areshown, which have significant

antifungal activity via the ET signalling pathway(Penninckx et al., 1996).

Likewise, methyl salicylate and methyl jasmonate may alsowork as a volatile signal for

the distal part of the plant. According to Bharathi (2004),microbial inoculants may induce

Figure 3 Gene activation during the development of ISR.Adapted from Jain et al. (2014).

systemic resistance through activation of variousdefence-related enzymes such as PO, PPO,

PAL, chitinase and ß-1, 3-glucanase and antifungalmetabolites (AFMs) like phenazines,

pyrrolnitrin, 2, 4-diacetylphloroglucinol (DAPG),pyoluteorin, viscosinamide and tensin

(Bloemberg and Lugtenberg, 2001). Kidarsa et al. (2011)generated information on

pyoluteorin biosynthesis in Pseudomonas fluorescens Pf-5and 2, 4-diacetylphloroglucinol

in Pseudomonas fluorescens Q2-87. Microbial inoculants areknown to release lytic

enzymes and several other biocidal volatiles that mighthave significantly contribute to

suppressing plant pathogens through hydrolysing a widevariety of polymeric compounds

like chitin, proteins, cellulose, hemicellulose and DNA.Production of HCN by certain

fluorescent pseudomonads is believed to be involved in thesuppression of root diseases

through blockage of the cytochrome oxidase pathway ofphytopathogens.

Detoxification of pathogen virulence factors is another

mechanism of biological control

by microbial inoculants. Antagonistic microbials such asBacillus sp., and Pseudomonas

sp., are able to detoxify albicidin, a toxin produced by X.albilineans (Walker et al., 1988).

The production of a specific protein is usually involvedhere, which reversibly binds

with the pathogen toxin and establishes growth inhibitionof the microbial competitor

(Zhang and Birch, 1997). The genetic modifications ofbiocontrol agents expressing

1-aminocyclopropane-1-carboxylate (ACC) deaminase genesalso appear to be useful

in the management of plant diseases. Biocontrol agentscontaining ACC deaminase

can boost plant growth, particularly under stressedconditions such as salinity, drought,

waterlogging, excess temperature, pathogenicity andcontaminants that usually arise in

response to a multitude of abiotic and biotic stresses(Saleem et al., 2007). Biocontrol

agents induce abiotic stress by enhancing plant toleranceagainst adverse situations.

Microbial-induced plant tolerance includes both physicaland chemical changes.

Antioxidants to remove reactive oxygen species, cytokininsto reduce ABA accumulation,

IAA for better root growth and nutrient uptake,participation of ACC deaminase in ET

reduction, extracellular polymeric substance in biofilmformation and better soil aggregation,

etc. are considered to be potent mechanisms usuallyinvolved in plant-induced systemic

tolerance. Table 1 represents some of the generalmechanisms or molecules that might

be associated with microbial bioagents which are effectiveagainst diverse tea pathogens.

Recently, it has been reported that certain microbialinoculants quench pathogen quorum

sensing capacity by degrading autoinducer signals andthereby might block expression of

numerous virulence genes (Molina et al., 2003; Dong et al.,2004; Morello et al., 2004). Since

most plant pathogens rely upon autoinducer-mediatedquorum-sensing to turn on gene

cascades for their key virulence factors (von Bodman etal., 2003), this approach provides

tremendous potential to reduce the onset of pathogeninfection. However, for effective

action to reduce disease incidence, the biocontrol agentsmust be able to aggressively

colonize the plants and should have the potential todominate the ecological niche.

7 Tea pest management: microbiological approach

Sustainable tea cultivation increasingly relies onalternatives to conventional chemical

pesticides for pest management that are eco-friendly andreduce the amount of pesticide

residues in made tea (Roy and Muraleedharan, 2014). Theexploitation of microbial

biocontrol agents is a positive outcome from severaldecades of extensive investigations

(Leggett and Gleddie, 1995). In order to produce pesticidecontaminant-free tea,

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microbial control measures have been gradually recognizedin different tea-growing

areas under integrated pest management programmes (Hazarikaet al. 2009; Ye et al.

2014). The approach necessitates the isolation,identification and exploitation of native

entomopathogens in tea ecosystems.

Entomopathogens can provide effective control measures aswell as conserve biodiversity

and serve as alternatives to broad-spectrum chemicalinsecticides (Lacey et al., 2001; Roy

and Muraleedharan, 2014). Due to their specificity forinsects, entomopathogens (fungi,

bacteria and viruses) are believed to be ideal candidatesfor incorporation into integrated

pest management programmes where their effects on othernatural enemies would be

minimal. However, initial effort to use entomopathogenicfungi for the control of a tea

pest (tea leaf roller) commenced in 1914 (Hotta, 1914) inJapan. More than 40 species

of entomogenous fungi, which infect tea pests in China,have been reported, such as

Beauveria bassiana (Bals.) Vuill., Metarhizium anisopliae(Metschn.) Sorokin, Paecilomyces

spp., Aschersonia aleyrodis Webb. and Aegerita webberFawcett (Yu and Lin, 2008). The

potential of Beauveria bassiana, a native entomopathogen tocontrol Helopeltis theivora

(by up to 50%), a major sucking pest of tea leaves inNortheast India, has been reported

(Barthakur et al., 2004). The field performance of a nativeentomopathogen, Metarhizium

anisopliae, against a live wood-eating termite of tea inCachar has been examined (Debnath

et al., 2012). There was a significant reduction in termiteinfestation due to the application

of Metarhizium anisopliae over a conventional termiticide.The fungus Paecilomyces

tenuipes (Peck) Samson was isolated from a psychid pest oftea from Darjeeling, India

(Debnath, 1986), which was also found to be infectious toflushworm Cydia leucostoma

Meyr (Debnath, 1997). Similarly, the efficacy of Beauveriabassiana against Helopeltis

and other insect pests of tea has been documented byHazarika and Puzari (2001). The

antagonistic activity of V. lecanii against tea thrips hasalso been evaluated (Babu et al.,

2008). There are reports illustrating the potential ofentomopathogenic fungi, such as

Acremonium sp., Aspergillus flavus, Aspergillus niger,Cladosporium sp., Curvularia sp.,

Fusarium sp., and Trichoderma sp., which caused seriousinfection in Helopeltis theivora

in tea plantations of Assam, N.E. India (Bordoloi et al.,2012).

Most of the reviews on bacterial pathogens of tea pests(Barbora, 1995; Agnihothrudu,

1999; Barthakur et al., 2003; Barthakur, 2011; Hazarika etal., 2001, 2005, 2009; Ye et al.,

2014) document the potential of Bacillus thuringiensis asthe entomopathogenic bacteria.

In China, Bacillus thuringiensis is primarily used tocontrol tea pests and its efficacy against

lepidopterous larvae was recorded to be more than 95% (Yeet al., 2014). Two kinds of

Bacillus thuringiensis preparations are registered for usein tea plantations in Japan. One

of them is the spore-dead Bacillus thuringiensis

preparation (named TOAROW-CTR) and

another is the living spore crystal mixture preparation(named BACILEXR) (Kodomari, 1993).

The efficacy of Bacillus thuringiensis var. kurstaki ineliminating Buzura suppressaria, Adalia

bipunctata and Scirtothrips dorsalis (up to 45–95% foreach) in Northeast India has been

reported (Gurusubramanian et al., 2008). However, theutilization of Bacillus thuringiensis

based insecticides for pest control is prohibited incountries like Japan and India owing

to its harmful effect on the silkworm industry (Kodomari,1993; Muraleedharan, 2006).

The efficacy of Pseudomonas fluorescens against Oligonychuscoffeae, which infests

tea, has been reported (Roobakkumar et al., 2013). Insectpathogenic properties of the

entomopathogenic bacterium, Serratia marcescens, have alsobeen established (Flyg et

al., 1980; Ye et al., 2014).

The study of insect viruses associated with tea pests hasbeen underway since late

1970s. Insect-specific viruses (ISV) are highly effectivein the natural control of caterpillar

pests of tea. According to Tsai et al. (1978) ISV may bepromising candidates as biocontrol

agents. To date, in China, 82 species of viruses associatedwith tea insects have been

reported (Zheng et al., 1985; Hong, 1998; Zhang and Tan,2004; Ye et al., 2014). In

China, insect viruses, such as Buzura suppressaria NPV(BusuNPV), Eucalyptus obliqua

NPV (EcobNPV), Euproctis pesudoconspersa NPV (EupeNPV),

Andraca bipunctata GV

(AnbiGV) and Adoxophyes orana GV (AdorGV), have been usedas large-scale biocontrol

agents in tea cultivation. The application of BusuNPV hasresulted in more than 90%

mortality of the 1st and 2nd generations of Buzurasuppressaria within ten days of spraying

polyhedral suspensions at 3 × 10 12 PIB/ha (Peng et al.,1998). In Japan, granuloviruses

(GVs), entomopoxviruses (EPVs) and the nuclear polyhedrosisvirus (NPV) have been

successfully employed to control tea pests, particularlyAdoxophyes honmai and Homona

magnanima (Tortricidae: Lepidoptera) (Kodomari, 1993;Nakai, 2009). Symptoms of

NPV infection on Buzura suppressaria have also beeninvestigated by Narayanan et al.

(2006) and Mukhopadhyay et al. (2007). Virulence of NPVagainst Buzura suppressaria

was established in vitro (De et al., 2008). Thus, thediscovery of NPVs, GVs and EPVs

in different lepidopteran pests of diverse tea-growingareas, and their killing efficacy

with special reference to cross infectivity, opens up newavenues of developing this viral

pathogen into a biopesticide.

8 Selection and characterization of microbial productsfor commercialization

Microbial inoculants are strain and crop specific (Trabelsiand Mhamdi, 2013).

Product type, nature of active ingredients, crop type andapplication strategies, and

topographical variations usually determine the market

demands of microbial biofertilizers

and biopesticides. However, the main aim with regard to theuse of biofertilizers and

biopesticides is to obtain higher revenues and increaseproductivity in order to enable

sustainable agricultural practice. The currentbiofertilizer market represents about 5% of

the total chemical fertilizer market (BCC Research, 2014).It is even predicted that the

biofertilizer market share might reach up to 1.66 billionUnited States Dollar (USD) by

2022 and will rise at a compound annual growth rate of13.2% between 2015 and 2022

(Timmusk et al., 2017). According to Yaish et al. (2016)the global market for biopesticides

in terms of revenue was estimated to be worth about 5billion USD in 2011, which is

about 2.5% of the global market for chemical exploitationin the crop field. The isolation,

characterization and identification of microorganisms andselection of beneficial microbial

strains for desirable characters through rigorousscreening, followed by field evaluation

of bioinoculants are essential in introducing microbe-basedtechnology into agriculture.

The isolation of an effective strain is a prime criterionfor improved agricultural practices,

which is typically achieved using pathogen-suppressivesoils either by dilution plate

technique or by baiting the soil with fungal structuressuch as sclerotia (Nakkeeran et al.,

2005). The selectivity of plant growth-promotingmicroorganism (PGPM) strains is one of

the limiting factors for their commercialization. Theselection of an efficient biocontrol

agent requires a thorough understanding of the dynamics andcomposition of the microbial

community colonizing the rhizosphere and itscharacterization with special reference to plant

growth promotion and disease suppression (Trivedi et al.,2012). The selection of carrier

material that may aid the stabilization and protection ofmicrobial cells during storage and

transformation, and standardization of nutrient content tomaintain the population load of

inoculated microbial culture are considered to be essentialwhile developing a microbial

formulation. Clay mineral, diatomaceous soil and whitecarbon as a mineral, and rice, wheat

bran and discarded feed as organic matter, etc. are knownto be carriers in the solid type of

microbial products. It is important to seriously considermechanisms of control while using

microbial products. In most cases, agriculturalpractitioners (farmers/planters) occasionally

misunderstand this carrier effect as microbial action.

Mass production is usually achieved through liquid,semi-solid and solid fermentation

techniques (Manjula and Podile, 2001). According to Compantet al. (2005) efficacious

microbial strains may be selected on the basis of a massscreening technique. Gas

chromatographic analysis of cellular fatty acids is stilluseful to identify efficacious bacterial

strains (Sasser, 1990). A developed inoculant should besafe, cost-effective and easy

to switch under different agro-climatic conditions. DNA andRNA homology tests are

effective tools for the characterization of biocontrolagents (Hershkovitz et al., 2013), and a

protein profile analysis technique has proved to be usefulfor patenting purposes (Maiti et

al., 2009). Terminal restriction fragment lengthpolymorphism (Osborn et al., 2000) using

a probe-target sequence and restriction endonucleasedigestion pattern are of use for the

identification of potent strains.

The successful commercialization of microbialbiofertilizers and biopesticides depends on

the linkages between scientific organizations andstakeholders. According to Nandakumar

et al. (2001), the commercial success of microbialinoculants depends on the isolation and

screening of an antagonist followed by pot and field trialsto determine the efficacy and

possible impact on other beneficial microorganisms andpredators. The selection of an

effective microorganism, on the other hand, tends to behighly targeted in comparison to

conventional agrochemicals as the latter may also work asbroad-spectrum products that

eventually impact many different kinds of organisms. Thisselective property of microbial

biocides may result in variable quality and efficacy undera complex field environment

where diverse players act simultaneously. Mass productionfollowed by formulation

development along with viability testing and fermentationactivities to optimize the quality

parameters for developing industrial links are essentialsteps towards commercialization.

However, commercialization of biocontrol products is amulti-step process that involves a

wide range of research links to different activities, suchas isolation of microorganisms from

a natural environment, evaluation of antagonisticproperties of isolated strains against test

plants both in vitro and under field conditions, selectionof the best microbial inoculants for

mass production, microbial formulation, compatibilitytesting, identification, registration

and release.

The quality of microbial biocides is an important factorand results in their success or

failure and acceptance or rejection by farmers. Qualitymanagement is essential and must

be performed continually to control the microbial productsin favour of the customers.

Counting live microbial cells through spread plate or dropplate methodologies for a viable

count is another essential criterion to be followed beforerecommending the microbial

products for commercialization. Economical and viablemarket demand, consistent and

broad-spectrum action, safety and stability, longer shelflife, low capital costs, easy

availability of carrier materials, etc. are the key pointsto remember when recommending

particular microbes for field evaluation. Entrepreneurshiprequires patent application

of the identified strain. Quality control is crucial toretain the confidence of farmers to

determine the efficacy of the target strain.

Formulation is the process of blending active ingredientssuch as fungal spores with

inert materials such as diluents and surfactants in orderto alter the physical characteristics

of microbial inoculants into a more desirable form. Theability to survive and replicate

might increase the formulation requirements, depending onthe target and the stage of

the biocontrol agent. There are also challenges with themicrobial registration process.

Registration of the biocontrol agent mainly depends ontoxicity and environmental fate.

According to the pesticide act, India (1968), several dataare required for the registration

of any kind of biocontrol agent such as the systemic nameand common name of the

biocontrol agent along with its natural source of origin,general morphology, details of

the manufacturing process, mammalian toxicity,environmental toxicity, residual analysis,

etc. According to the report of Timmusk et al. (2017),regulations in the European Union

(EU), with respect to the lack of quality restrictionsconcerning biofertilizer application,

have left us in a situation where national or regionalrules are being applied, which tend

to vary. However, it is a two-phased process. The activeingredient within a biofertilizer

must be authorized by the EU Commission DG SANCO(Directorate General for Health

and Consumer Affairs) and, subsequently, the formulatedproduct must be of national

authorization. Thus, the procedure for registeringbiocontrol agents within the EU is

long and complicated; while deadlines are not met andnumerous steps are difficult to

even comprehend. The new legislation gives specific statusto non-chemical and natural

alternatives to conventional chemical pesticides andrequires them to be given priority

wherever possible. Biopesticides generally qualify aslow-risk active substances under

the legislation. Low-risk substances are granted initialapproval for 15 years rather than

the standard 10. Professionals and specialized personnelare involved in solving many

issues around the globe relating to mass production,optimization of the bio-product,

commercialization, field applications and associativeenvironment-related issues.

9 Conclusions, future prospects and challenges

The adoption of biological techniques for sustainable teacultivation is a pertinent strategy for

an efficient and ideal agricultural growth with minimalgeneration of adverse environmental

impacts that may affect water resources, ecosystems or thequality of human life. Modern

agriculture offers immense opportunity to exploitbeneficial soil microbial resources such

as nitrogen fixers, phosphate solubilizers, potashmobilizers, cellulose degraders, AM

fungi, etc., as well as biopesticides (microbial biocontrolagents and entomopathogens),

to optimize crop benefit and maintain soil quality.According to Raja (2013) there is a rising

trend towards organic agriculture using biological-basedorganics as an alternative to

agrochemicals.

Organic farming ensures food safety as well as aidingbiodiversity conservation and

functioning in the soil. It is highly likely to bedependent on the native soil microflora

which constitutes all kinds of useful bacteria and fungiincluding the AMF and PGPR. A

synergistic interaction of PGPR and AMF is recorded ashighly suitable to fertilizer use

patterns in agricultural soil. An enhanced plantnutrient-use efficiency with PGPR and AMF

in an integrated nutrient management system has beenachieved by Adesemoye et al.

(2008), and thereby suggested the application of microbeswith reduced doses of fertilizer

in agricultural soil.

Microbial inoculants are of paramount significance inintegrated nutrient and pest

management schedules and thereby assist in the generationof healthy agricultural practices

(Adesemoye and Kloepper, 2009). Microbial biofertilizersand biopesticides fulfil diverse

beneficial interactions in tea soil and thereby lead topromising solutions for sustainability.

As the rhizosphere is an ideal habitat for the isolation ofbeneficial microorganisms (Mendes

et al., 2013), considerable effort has been made to explorethe microbial diversity of the

surface and sub-surface soil of tea plants undercultivation in diverse topographical regions.

Different species of microbial inoculants are now known to

antagonize tea pathogens via

mycoparasitism, producing volatile and non-volatileantibiotics, competing for nutrients

and space, as well as their diverse beneficial activitiescontributing towards enhanced

crop production. The use of microbials such as viruses(NPV/GV), spore-forming bacteria

(Bacillus strains) and fungi (Beauveria, Metarhizium,Verticillium, Paecilomyces), holds great

promise in this regard. Most of the tea estates, however,refrain from risking crop loss due

to pest attack and liberally use synthetic pesticides under‘no threshold category of pest

management’ (Pedigo, 2002), and as microbial insecticidesare pest specific, the potential

market for these products is rather limited.

The concept of exploiting native microbial resources in teais still a novel concept.

For instance, the application of certain NFB, such asRhizobium sp., in tea soil is still

under experimentation to evaluate their formulation in thetea ecosystem, which may be

due to poor acid tolerance of the bacterial strains(Samelis et al., 2001). The optimum

temperature requirement for the normal growth of bacterialinoculants ranges from 28 to

32°C. However, fungal strains like Aspergillus niger cantolerate a temperature up to 39°C.

Low temperatures, that is, below 20°C, can drasticallyreduce the growth and solubilization

potential of microbial inoculants. Soil pH also plays avital role in influencing the growth

and metabolism of microbial biofertilizers (Khan et al.,

2009). The activity of microbial

biofertilizers and biopesticides, thus, depends on a largenumber of factors. Since the

effectiveness of microbial inoculants is affected by theprevailing environmental conditions,

crop protection and productivity-related research have toenvisage the challenge of

finding appropriate screening procedures to select suitablemicroorganisms able to

work efficiently under the current changing scenarios. Theimpact of the environment on

the performance of the beneficial microbiota might help, inthis regard, to predict the

resulting output and to develop an effective pest anddisease management schedule.

Extensive knowledge of the structure and diversity ofmicrobial inoculants is essential to

identify the microbial mechanism of action, optimuminoculum size and field performance

to effectively understand its exploitation in tea soil as apotent biofertilizer.

The recent exploitation of tailor-made core microbiometransfer therapy (Gopal et al.,

2013) in agriculture might be helpful in managing diverseplant diseases of economic

importance. Detailed evaluations of microbial pathogenicitybased on physiological,

biological and genetic studies, mass production, fieldapplication, aspects of microbial

interactions with other natural enemies and bio-safetyissues must be carried out for

developing effective microbial biopesticides. Reliable dataas well as confirmed protocols

are needed to develop, isolate and prepare standardmicrobial inoculums which would

ultimately serve the tea industry. It is also imperative toknow the status of the market,

the registration process, and related microbial researchand development in order to

determine the suitability of the microbial biofertilizersand biopesticides as an alternative

to chemicals to enhance the quality of tea in terms of itsproduction and disease control, as

well as to overcome the challenges of placing biocontrolagents as a reliable component

for tea growers in the changing scenario.

The integration of beneficial plant–microbe symbiosis andmicrobiome interactions may

represent a promising solution to improve sustainable teacultivation. According to Timmusk

et al. (2017), studies on microscale information technologyfor microbiome metabolic

reconstruction have the potential to advance thereproducible application of microbes under

natural conditions. A major focus in the coming decades isthe development of safe and

eco-friendly methods through exploitation of beneficialmicroorganisms in sustainable crop

production. Current research indicates that integratedpractices require less chemical inputs

on the soil and facilitate the incorporation of residuesthat would otherwise go to dumping

sites and landfills, which represent relevant reductions onthe environmental impact associated

with agricultural activities.

The additional advantages of exploiting microbial

inoculants as biofertilizers and

biopesticides include longer shelf life with no adverseeffect to the ecosystem.

Biofertilizers and organic manure in suitable combinationwith chemical fertilizers may

add optimal crop productivity and thereby could mitigatedifferent issues related to

environmental and health hazards (Gurusubramanian et al.,2005). Engineering the

rhizosphere to encourage beneficial plant–microbeinteractions is a great challenge for

the future. Undoubtedly, the management of rhizosphereopens up novel opportunities

for future agricultural developments as it has alreadysealed its importance in exploiting

beneficial microbial resources to reduce the input ofagrochemicals and thereby assist

in achieving sustainable environmental and economic goals.More focus and proper

understanding about the behaviour of indigenous andregion-specific microbial strains

of tea-growing areas is needed to be carried out in thecoming decades to exploit

their potential in efficient nutrient allocation anddisease management.

10 Acknowledgements

The corresponding author is thankful to the Director,Tocklai Tea Research Institute

(TTRI), Tea Research Association (TRA), Jorhat, Assam,India, for providing the necessary

facilities to undertake the study. The authors are indebtedto the Departmental faculty

members for their copious encouragement. The corresponding

author is grateful to

Dr S. K. Pathak, Former Dy. Director (ASA)/Chief Scientist& Head, Mycology and

Microbiology Dept.; Dr B. K. Barthakur, Ex-Head, Mycologyand Microbiology Dept.;

and Dr T. S. Barman, Ex-Head, Plant Physiology and BreedingDept., TTRI, TRA, Jorhat,

Assam, India, for their noteworthy suggestions during thepreparation of the chapter

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8 Chapter 8 Diseases affecting tea plants

1 Introduction

Tea is usually grown as a monoculture crop over large landareas. It can be cultivated

under different climatic conditions and using variousagricultural practices. The tea plant

is maintained in its vegetative phase throughout itseconomic lifespan. The plant is

susceptible to diseases at various stages of its lifecycle, which affect the growth of tea

plants, reduce their economically sound lifespan and reducecrop yields. Such diseases

also pose the biggest challenge to pesticide-freeproduction of tea; yet many of today’s

tea consumers demand cups of high-quality, pesticide-freetea.

Fungi, bacteria and viruses cause diseases in tea, withfungal diseases being the most

problematic. The leaves, stems and roots of tea bushes aretargeted by pathogens. Leaf

diseases have a direct bearing on the quantity of theharvest and the quality of the final

product. Stem diseases are persistent and cannot be easilycured, while root diseases

pose a severe threat because they are often only diagnosedat a later stage of the disease

when symptoms appear on parts of the tea plant above theground. Crop loss due to

disease varies between tea growing countries, depending onclimatic conditions. Tea

crop loss due to pests and diseases has been reported at10–20% in Southern India

(Sathyanarayana and Barua, 1983).

Management of diseases to reduce crop loss and the cost ofproduction, while

maintaining the quality of made tea and protecting theenvironment and humans

through the rational and safe use of fungicides, is one ofthe biggest challenges faced

by tea producing countries. With global climate change, theemergence of new diseases,

the spread of existing diseases beyond their currentboundaries and the evolution of what

are currently minor diseases are all serious threats.

There are various chapters (Hainsworth, 1952; Balasuriya,2008; Rattan, 2012) and

review papers (Chen and Chen, 1982; Lehmann-Danzinger,2000) which focus on tea

diseases and their management. This chapter will focus onmajor diseases which pose a

threat to tea production, recent advances in diseasediagnosis, and disease management

strategies. It will also aim to pinpoint for readers thegaps in our knowledge and describe

future research needs.

2 Foliar diseases affecting tea

Tea plants are primarily grown in order to harvest theiryoung shoots (‘two leaves and a

bud’). Foliar diseases have a direct effect on harvest,bush health and the quality of the

final product. Blister blight, grey blight, brown blight,bacterial shoot blight, black blight

and red rust are the key foliar diseases in major teagrowing countries.

2.1 Blister blight (Exobasidium vexans Massee)

Occurrence and importance

Blister blight has been known to occur in North east Indiasince 1855 (Venkata Ram, 1971).

It gradually spread to Japan by 1912 and Vietnam by 1930(De Weille, 1959). Tea in South

India remained free of blister blight until 1946, when thedisease was also introduced to

Sri Lanka (Tubbs, 1946). Later, blister blight was reportedin Sumatra and Java (Indonesia)

(De Weille, 1959). Currently, the disease is a menace totea production in almost every tea

growing country in Asia. Tea cultivation in Africa andAmerica so far remains free of blister

blight.

Pathogen

Blister blight is caused by the fungus Exobasidium vexansMassee. This is an obligate

fungus or biotroph belonging to the Phylum Basidiomycota.So far, tea is the only known

host of this pathogen.

Factors affecting the disease

Tea plants are susceptible to this disease at the nurserystage as well as at mature stages.

Only young, succulent leaves and stems are invaded byblister blight (Gadd and Loos,

1949), when mature, leaves and stems become resistant tothe disease. In addition to

direct crop loss, the costs of managing the disease anddelayed recovery of the pruned

bushes after attack make blister blight a serious economicconcern.

Relative humidity of more than 80%, a temperature of15–25°C and long leaf wetness

periods (11–13 h) are crucial for basidiospore germination(Gadd and Loos, 1949).

The disease is therefore prevalent during wet, cold weather(during monsoons), and when

tea is grown at higher altitudes.

Infection and symptoms

Infection is initiated by germination of airbornebasidiospores, and this germination

is highly influenced by environmental conditions. Agerminating spore forms a germ

tube and appressorium, which aids direct penetration of thecuticle of young leaf and

stem tissues. Following infection, the fungus growsintercellularly and forms lemon

green translucent spots (Loos, 1951) on the first or secondleaf of a harvestable tea

shoot.

The fungus continues to grow inside the tissues, obtainingnutrients through haustoria.

Expansion of cells usually results in the characteristicblister with an indentation on the

upper surface of the leaf and bulging on the lower surface(Fig. 1), although occasionally the

reverse occurs. A developed hymenium below the epidermis onthe lower surface exerts

pressure, pushing the hymenium out and rupturing the lowerepidermis. Basidiospores

are produced on the tips of basidia outside the leaftissues, giving the blisters on the

lower surface a powdery white coating (Gadd and Loos,1949). Soon after the release of

basidiospores, blisters start to die off from the centre.The fungus takes up to 21 days to

complete its life cycle (Loos, 1951).

How E. vexans survives during dry, unfavourable periodsremains obscure.

The basidospores are shortlived and killed by directsunlight (De Weille, 1957; Gunasekera

et al., 1997) and high temperature (Gadd and Loos, 1949).No special overwintering

structures or alternative host/s have been identified.Occasionally, thick-walled spores have

been seen (Gadd and Loos, 1949; Ajay et al., 2009), buttheir role remains uninvestigated.

There is evidence that E. vexans survives in the dry seasonwith the infected leaves under

the tea canopy (Ajay et al., 2009). In support of this,basidiospores have been trapped

throughout the year in Sri Lanka (Kerr and Shanmuganathan,1966) and in India (Ajay et al.,

2009) in the atmosphere.

Figure 1 Blister blight with characteristic white powderyblisters on the underside of the leaf and

corresponding shiny indented areas on the upper surface.

Crop loss

Blister blight caused enormous crop losses soon after itsintroduction to South India

(Venkata Ram, 1971), Sri Lanka (Portsmouth, 1951) andIndonesia (De Weille, 1959). Studies

consistently showed that a crop loss of about 20–40%occurred in unprotected areas,

compared to fields that were sprayed with fungicides(Schweizer, 1950; Portsmouth, 1951;

Loos, 1952; Ordish, 1952).

Management

Thinning of shade trees with the onset of the monsoon isimportant in blister blight

management, because shade favours disease development(Venkataramani, 1973;

Balasooriya, 2008). Removal of infected leaves before thefungus sporulates on them,

which can be achieved by frequent harvesting (shorterplucking rounds) and/or fish

leaf (hard) plucking, helps to maintain blister blight atlower levels (Portsmouth and

Loos, 1949).

Chemical control has been found to be effective in themanagement of blister blight.

Copper formulations such as copper hydroxide, copper oxideand copper oxychloride

have been identified as cheap, promising fungicides inblister blight management

(Portsmouth and Loos, 1949). Several systemic fungicideshave been incorporated into

tea plant spraying (Arulpragasm et al., 1987). Currently,hexaconazole, propiconazole,

tebuconazole and bitertanole are used in blister blightmanagement (Arulpragasam et al.,

1987; Chandra Mouli, 1992; Chandra Mouli and Premkumar,1995, 1997).

2.2 Grey blight (Pestalotia theae and Pestalotiopsislongiseta)


This disease has been reported in Japan, India, Sri Lanka,Korea (Park et al., 1996a,b) and

Kenya. It appears to be a serious problem in Japan (Takeda,2003), Kenya and India (Joshi

et al., 2009a). The total crop loss due to grey blight isestimated to be 17% in Southern

India (Joshi et al., 2009a; Premkumar et al., 2012) and10–20% in Japan (Horikawa, 1986).


Pestalotia theae is generally considered to be a weakpathogen, and therefore less

problematic than other diseases. Stresses and wounds to teaplants create favourable

conditions for disease infection and development. Matureleaves, young shoots and

bare stalks are affected by this pathogen. Infection onyoung shoots results in dieback of

shoots. The incidence of grey blight is higher withcontinuous mechanical harvesting, and

lower with hand plucking (Sanjay et al., 2003).

Symptoms

The initial symptoms of grey blight are brown spots onleaves. These spots later turn grey

in colour, with brown margins and concentric rings (Fig.2). The enlarged lesions can cover

up to half of the leaf area. The centre of the lesion isdotted with minute, black acervuli.

The pathogen generally infects mature foliage, bare stalksand young shoots.

Management

Grey blight can be effectively controlled by copperfungicides (Balasuriya, 2008;

Anonymous, 2014), thiophanate-methyl, benomyl orchlorothalonil (Hirokiwa, 1986), or

carbendazim and/or mancozeb(http://www.upasitearesearch.org).

2.3 Brown blight and Anthracnose (Colletotrichum spp.)


Brown blight has been reported in Sri Lanka, India, China,Indonesia, Bangladesh,

Taiwan, Hawaii and Japan. The damage caused by the diseasein most of these

countries is negligible, and so the disease is generallyconsidered of minor

importance. However, brown blight is important in Japan,especially on ‘Yabukita’,

the most popular tea cultivar (Yoshida et al., 2010) andChina (Wang et al., 2015;

Wang et al., 2016a).

Pathogen

The pathogen which causes brown blight in Sri Lanka(Willis, 1899) and China (Wang

et al., 2015) has been identified as C. camelliae. Thedisease is also referred to as

tea anthracnose. (Wang et al., 2015; Liu et al., 2015).Colletotrichum theae-sinensis

is reported to be the pathogen causing tea anthracnose inJapan (Yoshida and

Takeda, 2006). Colletotrichum gloeosporioides (Guo et al.,2014), C. acutatum (Chen

et al., 2016), C. cocodes and Gloeosporium theae-sinensis(lately Disculata Discula

theae-sinensis) (Moriwaki and Sato, 2009) were alsoreported to be involved in tea

anthracnose.

Figure 2 Tea leaves infected with grey blight (left) andbrown blight (right).


Brown blight/anthracnose is severe during periods of highhumidity and high temperature

(Chen and Chen, 1982). Leaf damage caused by sun scorch andhail predisposes foliage

to brown blight infection (Kuberan et al., 2012).

Symptoms

The initial symptom of this disease is the appearance ofsmall, yellowish green, diffused

spots on leaves. These spots later become larger, darkbrown, necrotic lesions with a

yellowish green margin with characteristic concentricrings. Minute black fructifications

generally appear on the lesion. Blight spots develop fromthe margin and spread inwards

(Fig. 2). The spots spread to a large area of the leaf orsometimes to the whole leaf.

The disease can affect young leaves, green twigs andshoots, and can develop into shoot

blight and dieback.

Management

In countries where brown blight and anthracnose areproblematic, they can be efficiently

controlled by the use of copper oxychloride (Anonymous,2014) and systemic fungicides

such as thiophanate-methyl and chlorothalonil.

2.4 Red rust (Cephaleuros parasiticus and C. mycoidea)


Red rust is an important disease affecting tea in North

India and Bangladesh (Sana, 1989).

In Bangladesh, about 8–25% of tea estates are affected bythis disease (Sana, 1989). Tea

yields are linearly and inversely correlated with theseverity of red rust disease (Huq et al.,

2010).


Red rust can attack both young and old tea plants underadverse soil and climatic conditions.

Sarmah et al. (2016) observed that succulent young leavesand shoots are generally more

susceptible to red rust. The predisposing factors aremainly poor soil fertility, alkalinity and

lack of aeration of the soil, hard pan, inadequate orcomplete absence of shade, drought

and waterlogging (Gadd, 1949; Balasuriya, 2008).

Symptoms

As the algae grow epiphytically on tea leaves, the affectedleaves generally bear thin red or

white superficial discs with lobed margins, composed offilaments arranged in rows which

radiate from a centre (Fig. 3). When the discs mature, theyproduce red, erect filaments

and some of them bear sporangia with spores. The discsoften turn white when they

are lichenized. The leaves become variegated (yellow orwhite) and the cells underneath

the alga become generally discoloured. Stem colonizationhardens the leaves and causes

longitudinal cracks. In extreme cases, red rust causessevere damage to young tea plants

by killing patches of stem tissue, resulting dieback of the

stem.

Management

Nutrient balance, especially using fertilizers whichprovide supplementary potassium, is

known to reduce the severity of red rust (Huq et al.,2010). Copper fungicides also give

satisfactory control of red rust (Balasuriya, 2008). It isreported that some tea cultivars are

more susceptible to red rust than others in India (Sarmahet al., 2016).

2.5 Bacterial shoot blight (Pseudomonas syringae pv. theae)


Bacterial shoot blight is one of the major diseasesaffecting tea cultivation in Japan

(Horikawa, 1985). The disease occurs in both young andmature tea plants. However,

young plants are more seriously affected by bacterial shootblight.


The disease is favoured by low temperatures, and severeoutbreaks are associated with

frost and cold damage (Ando, 1988). The disease is mostproblematic from late autumn

to spring.

Symptoms

The first symptom of the disease is the appearance ofsmall, water-soaked, dark greenish

brown spots on the leaf blades and leaf petioles of maturedleaves. These spots gradually

Figure 3 Red rust symptoms on a tea leaf.

enlarge and often coalesce with each other. Where veins or

midribs are attacked, the spots

enlarged along them, forming elongated lesions.Water-soaked, dark green and narrow

areas form along the edges of the lesions. Shoots areusually infected through expansion

of the leaf lesions, but not through direct infection. Thediseased leaves defoliate easily,

but the new shoots of first flush are scarcely infected(Horikawa, 1985).

Management

Bacterial shoot blight can be managed by timely applicationof copper bactericides

(Tomihama, 2006).

2.6 The effects of foliar disease on the quality of made tea

The commercial value of the tea plant arises from the useof its tender shoots to produce

the non-alcoholic beverage of tea, which has various healthbenefits. The quality of black

tea is determined by its colour, brightness, appearance,liquoring properties, strength

and aroma. The quality of the harvested leaves and shootshas a significant impact on the

quality of the final product (Owuor et al., 1990). For thisreason, leaf diseases are of major

concern.

There are contradictory claims from authors regarding theeffect of foliar pathogens on

made tea quality. Some authors have argued that thebiochemical changes which take

place in foliage during pathogenesis improve tea quality(Rajalakshmi and Ramarethinam,

2000; Charkraborty et al., 2002). However, others have

showed that foliar infections

impair physiological functions and reduce the quality ofthe final product. Blister blight,

brown blight and grey blight result in a considerablereduction of the photosynthesis and

transpiration rates of the tea cultivar UPASI-9 (Ponmuruganet al., 2016). These diseases

caused a significant decrease in the polyphenol, catechin,amino acid and dry matter

content of green leaves. The changes in the biochemicalconstituents in green leaves

resulted in lower levels of theaflavin, thearubigin andcaffeine when compared to made

tea produced from healthy shoots. There was a drop in theproduction of volatile flavour

compounds (compounds of terpenoids) which give tea asuperior flavour. In contrast,

factors which negatively affect tea quality, such as fibrecontent and highly polymerized

substances, were found to be higher in made tea producedfrom infected leaves. Volatile

compounds which impart inferior flavour to tea are alsoproduced in significant quantities

in infected shoots.

Among the foliar diseases of tea, blister blight caused byE. vexans affected the overall

tea quality more extensively than the other foliarpathogens (Gulati et al., 1993, 1999;

Baby et al., 1998; Chakraborty et al., 2002; Mur et al.,2015; Ponmurugan et al., 2016).

3 Stem diseases affecting tea

3.1 Cankers


Canker is the most widely prevalent stem disease of tea inalmost in all tea growing

countries (Ponmurugan et al., 2006). Cankers arepersistent. They attack twigs and stems,

kill or griddle branches, and ultimately travel downwardskilling the entire bush, thus

shortening the lifespan of attacked bushes. Canker causedby Macrophoma theicola is

one of the major diseases of tea in Sri Lanka (Balasuriya,2008). This disease has also been

reported from India (Sarmah, 1960) and Bangladesh.

Phomopsis theae has been identified as the major pathogencausing stem canker disease

in India (Venkata Ram, 1979), China (Chen and Chen, 1982),Kenya (Onsando, 1988), Japan

(Kasai et al., 1965), Bangladesh (Ahmad et al., 2013) andSri Lanka (Shanmuganathan,

1965). The disease causes the death of 2- to 8-year-old teabushes, after complete girdling

of the collar region. It therefore substantially reducestea plantation establishment and

production (Khare et al., 2009).

Pathogens

Several fungal species have been reported to cause cankersin tea (Petch, 1923; Gadd,

1949). Stem and branch cankers caused by Botryosphaeriaceaespp. (formally M. theicola)

and collar canker caused by P. theae have been reported tocause considerable damage

in tea worldwide.

Symptoms

Macrophoma theicola enters the stem when it is stillpencil-thick and kills the bark, forming

a small, elongated, red patch. At this stage, the activityof the fungus appears to be

arrested and a callus begins to grow from the edge. Thecanker enlarges slowly and forms

the typical symptoms on the branches, giving the appearanceof slightly sunken lesions

surrounded by a ring of callus growth (Fig. 4).

Phomopsis cankers are generally found on the branches andcollar, and at the base of

twigs, leaf scars and prune cuts. They are associated withwounds or injuries caused by

mechanical damage. The attacked bushes show yellow or brownleaves on a killed branch

or an entire bush. A close examination of such a bush willshow a canker or lesion located

at the collar of a dead bush or at the base of a killedbranch.

Cankers are developed by gradual killing of the bark at oraround the site of infection.

Old cankers have the raised margin due to development of acallus; a thick ridge of callus

forms at the upper margin of the canker. The diseased areamay be regular or irregular in

shape, often sunken and grey to black in colour.


The incidence and severity of canker diseases areassociated with environmental conditions

and cultural practices (Arulpragasam, 1989). Poor soilconditions, drought and moisture

stress to plants are the predisposing environmental factors

for canker diseases (Balasuriya,

2008).

Management

Successful management requires integrated measures. Plantsshould be protected from

moisture stress during dry periods by adequate thatchingand establishing adequate shade.

Soil health should be improved, and disease-resistantcultivars should be planted (Sarmah,

1960; Arulpragasam, 1989; Balauriya, 2008). The applicationof systemic fungicides can

also provide adequate control (Arulpragasam, 1989;Ponmurugan et al., 2006; Balasuriya,

2008). The planting of healthy, vigorous plants and theremoval of damaged branches

during pruning can help to minimize the damage caused bycankers.

3.2 Wood rot

General wood rot

Rotting of the main and side branches of tea bushes is aserious issue in almost all tea

growing countries in Asia and Africa. Tea bushes are prunedperiodically to stimulate

vigorous vegetative growth (Kulasegaram andKathiravetpillai, 1981). After pruning, the

exposed bush frame is liable to sun scorch. Furthermore,the area between the cut surface

and the newly developing bud dies naturally. Severalsaprophytic and pathogenic fungi

enter through these openings and initiate rotting of wood(Fig. 5). The resulting wood rot

is considered to be general wood rot, caused by multiple

factors. A DNA sequencing

based investigation revealed that Aspergillus aculeatus, A.tamari, Bionectria ochroleuca,

Fusarium solani, F. oxysporum and other Fusaria,Purpureocillium lilacinum, Rhizomucor

variabilis, Trametes corrugata, Trichoderma harzianum andLasiodiplodia sp. were all

associated with decayed tea stumps of the TRI 4042 cultivar(Senanayake et al., 2016).

These fungi produce polygalactouronase and/or pectatelyase,or laccase to digest the

wood. The rotting fungi can also enter into stem tissuesthrough wounds caused by

mechanical devices and stem cankers. Despite the fact thatwood rot gradually reduces

the vigour of the plant and lowers yield, it neverthelessattracts stem pests, especially live

wood termites (Glyptotermes dilatatus). Bioactive compoundsin the decaying stems play

an important role in attracting the termites (Senanayake etal., 2015).

Figure 4 Cankers on a (a) stem and (b) collar region of atea bush.

Hypoxylon wood rot (Nemania diffusa)

Wood rot in tea bushes caused by the fungus Hypoxylonserpens (N. diffusa) has been

reported in several countries, including India, Kenya,Malawi, Zimbabwe and Sri Lanka

at high elevations at or above 1500 m. In Kenya, thedisease is second in importance,

after Armillaria root rot (Onsando, 1985). Thecharacteristic symptoms normally appear

after the bushes have attained an age of 15–20 years.

Initial symptoms are wilting of the

foliage of a branch, followed by scorching. Infectedbranches snap off easily near the base.

Figure 5 Wood rot in the branches of a tea bush (top);characteristic black encrustation of Nemania

diffusa on a rotting bush (bottom).

Characteristic black encrustations (stroma) develop at thebases of infected branches

(Fig. 5). The disease appears to be disseminated mainlythrough pruning knives (Balasuriya

and Adikaram, 2009) and the infection progresses frompruning cuts during wet weather.

Under natural field conditions, infection at 60–90% andover 90% in a susceptible cultivar

could bring about 27 and 36% yield reductions,respectively, showing that the disease can

cause significant damage to tea (Balasuriya and Adikaram,2002).

Management of wood rots

The lifespan of affected bushes can be prolonged bymaintaining sanitary conditions in the

field, sanitary pruning to remove rotted branches as far aspossible and painting pruned

cuts with fungicidal wound dressings. A long-term solutionwould be to replace badly

rotted tea bushes.

4 Root diseases affecting tea

Several fungi are known to attack tea roots, and so to killtea bushes when their supply of

water and nutrients is disrupted due to infection. Wheninfected with these fungi, plants

show wilting of leaves followed by sudden yellowing or

browning. A characteristic feature

of root diseases is the sudden onset of symptoms(Lehmann-Danzinger, 2000). An infected

bush suddenly dries up, as though it had been scorched, anddies with all its leaves still

attached (Fig. 6a). In some instances, the leaves may falloff gradually until the branches

thin down and die off (dieback) (Arulpragasam, 1988).

The major pathogens causing root disease in tea arefacultative soil inhabitants,

belonging to the basidiomycetes and ascomycetes groups.Progression of root diseases

is very slow, and it may take several years from initialinfection to the death of bushes

(Sarmah, 1960). Red root rot, brown root rot, black rootrot, charcoal stump rot and

Armillaria root rot are the primary root diseases of tea.Primary root diseases spread by

means of airborne spores, by direct contact of roots withdiseased material or by freely

growing mycelium in the soil. Secondary root diseases areassociated with adverse soil

conditions or impaired health and vigour of tea bushes dueto other causes. Violet root

rot, Ganaderma rot and Diplodia root disease are secondaryroot diseases in tea. For

example, violet root rot always follows poor soil aerationor waterlogging of the soil

(Sarmah, 1960).

In this section, a brief account is given of the primaryroot diseases which are widespread

among tea growing countries, or which cause considerableeconomic damage.

4.1 Red root rot (Poria hypolateritia, current name –Ceriporiopsis hypolateritius)

Occurrence, importance and pathogen

This disease is of major economic importance in Sri Lanka,South India and Indonesia.

Red root disease usually occurs in patches. Although thedisease can attack both young

and mature plants, its occurrence is more common in youngplants. The fungus can

spread through wind-borne spores or cords of mycelium(rhizomorphs) growing freely

through soil.

Symptoms

At the early stage of infection, white cords of myceliumappear on the root surface. Later,

these cords turn red, fuse with one another to form anetwork spreading over the root

(Fig. 6b) and later expand laterally to form continuoussheets. Soil is usually found to be

attached to the roots. Infected roots have a mottled, redand white appearance. There is

often a very thin film of white mycelium between the woodand the bark (Petch, 1923). At the

advanced stages of disease development, the infected rootdisintegrates into a soft pulp.

4.2 Brown root rot (Phellinus noxius)


This disease is prevalent in all tea growing areas in India(Morang, 2013), Sri Lanka and

Indonesia.

Symptoms

Root infection occurs by infected material coming intocontact with healthy plants

(Satyanarana and Venkataramanan, 1979). Once attacked bythe pathogen, the roots of

Figure 6 (a) A tea bush showing scorching of leaves due toroot disease, (b) red root rot, (c) black root

rot, (d) charcoal root rot and (e) brown root rot.

the tea bushes are encrusted with a mass of earth and smallstones, difficult to remove by

washing or rubbing. The mycelium of the fungus is alsocemented to the root. These mycelia

initially appear as white to brown woolly masses on thesurface of roots, and later turn black/

dark brown among the earth and stones encrusted to theroots (Fig. 6e). Between the bark

and the wood, there is usually a thin layer of white orbrownish mycelium. At the initial stages

of infection, the roots do not exhibit much evidence ofdecay. However, in the later stages,

roots are permeated with yellowish brown sheets, which givea honeycomb structure.

4.3 Black root rot (Rosellinia arcuata and R. bunoides)


This is an important disease in Sri Lanka, Indonesia andIndia. It is common in both young

and mature tea plants. The infection spreads throughwind-borne spores, direct contact

with diseased material or mycelial cords growing freely inthe soil.

Symptoms

The fungus forms irregular, black, cobwebby cords ofmycelium on the root surface.

Woolly grey to black mycelium can also be found on the mainstem a few inches above

the soil surface. Numerous small, white, star-shapedmycelium develop on the surface of

the wood, under the bark (Fig. 6c). Small black points ornarrow black strands can be seen

on the surface of and embedded in wood infected with R.bunoides.

4.4 Charcoal root rot (Ustulina zonata and U. deusta)


Charcoal root rot is the commonest root disease in India,Sri Lanka, Indonesia and Malaysia.

In Malaysia it poses a very significant problem. Thedisease spreads by direct contact of

roots with infected material or wind-borne spores.

Symptoms

Roots attacked by this fungus have no visible mycelium ontheir surface. However, when

the bark is removed, large white or brownish whitefan-shaped patches of mycelium are

seen on the wood surface. The fans are often fused togetherto form a very thin, continuous

sheet (Gadd, 1949). Wood is permeated by irregular singleor double black bands or lines

(Fig. 6d). The fungus produces a characteristiccharcoal-like, black, brittle fructification at

the collars or main stems of diseased bushes.

4.5 Armillaria root rot (Armillaria spp.)


Armillaria root rot is a major economic concern in certaintea growing countries in Africa.

In Kenya the disease is common if tea is grown afterdeforestation. The disease is not

identified as a constraint on large plantations. However,about 50% yield loss has been

recorded on smallholder farms in Kenya (Onsando et al.,1997).

Pathogen

Armillaria root rot in tea has been attributed to thepathogen Armillaria mellea. Recent

studies of Otieno et al. (2003) and Mwenje et al. (2006)characterized isolates of Armillaria

based on both morphology and DNA sequencing data.Accordingly, they confirmed at

least three distinct Armillaria groups associated with rootrot of tea in Kenya, viz. Armillaria

fuscipes, A. mellea ssp. nipponica and A. hinnulea.

Symptoms

The first symptom of infection by Armillaria is chloroticfoliage: wilting and defoliating

leading to death. Plants may appear somewhat stunted. Thesebushes also tend to flower

prematurely (Onsando, 1986). Longitudinal cracks areusually present on the collar above

the soil level as well as on the taproot and lateral roots.Under the bark, sheets of creamy

white, fan-shaped mycelia can be seen (Fig. 7). The woodhas a strong mushroom-like

smell. Rhizomorphs (slender, root-like, dark brown to black‘shoestrings’ with a white

interior) develop beneath the bark. Affected bushes tend tooccur in patches, usually

around old tree stumps, but sometimes isolated bushes are

affected.

4.6 Management of root diseases

Unlike foliar and stem diseases, root diseases cannot beidentified at the early stage of

infection. The symptoms of root diseases are mostly visibleafter the death of bushes,

and hence curing a diseased plant seems to be impossible.Removal of symptomatic and

Figure 7 Longitudinal cracking of the stem at the collarregion (left) and a white mycelial growth of

the fungus overlying the wood (right) of tea bushesinfected by Armillaria spp. (Photo courtesy – Tea

Research Institute, Kenya).

dead bushes is the only available option. As root pathogensare soil-borne in nature,

rehabilitation of soil before replanting is highlyrecommended.

Soil fumigation with methyl bromide was an effectivecontrol for root diseases (Venkata

Ram and Joseph, 1974). The phasing out of methyl bromide,due to its detrimental effects,

has made the management of root disease in tea a morechallenging task. A prophylactic

treatment of fungicide spray is recommended under fieldconditions in Sri Lanka

(Balasuriya, 2008). Several biocontrol agents aresuccessfully used for the management of

root diseases (see Section 6.1).

5 Development of resistance: resistance of fungi tofungicides and tea plants to diseases

5.1 Fungicide resistance and the emergence of new biotypes

The introduction of synthetic fungicides almost 50 years

ago revolutionized chemical

protection of tea diseases, providing highly efficientdisease management. However, it

has been discovered that pathogenic fungi adapt tofungicide treatments by mutations

(rapid) or gradual mechanisms, leading to resistance andloss of fungicide efficacy.

Among tea pathogens, grey blight pathogens P. longiseta andP. theae (Oniki et al., 1986;

Omatsu et al., 2012), and anthracnose fungus G.theae-sinensis (Oniki et al., 1985) have

shown resistance to benzimidazole. P. longiseta has alsoshown resistance to strobilurin

(Omatsu et al., 2012; Yamada et al., 2016). Pradeepa et al.(2013) have shown that there is

a clear trend towards reduction of sensitivity tohexaconazole (DMI) in M. theicola isolates

which are exposed to the fungicide in Sri Lanka. Thoughfungicides are extensively used

to control blister blight in tea worldwide, resistantstrains or new biotypes have not yet

been found in E. vexans. Judicial use of fungicides andsafe alternatives are necessary to

manage the emergence of resistance in tea pathogens.

5.2 Resistance of tea plants to diseases

Tea plants show variation in their level of resistance totea diseases. The resistance or

susceptibility of a tea cultivar to pathogens depends onits genetic makeup and the

structural and/or biochemical defences operating in varyingdegrees among tea cultivars.

These defences are also influenced by environmentalconditions and agronomic practices.

Punyasiri et al. (2005) showed a relationship between thechemical constituents of tea

leaves and blister blight disease resistance. Levels of(-)-epicatechin were significantly

higher in tea cultivars resistant to blister blight leafdisease than those in susceptible

cultivars, while the reverse was true for(-)-epigallocatechin gallate. The high resistance

of a purple green leafed cultivar, TRI 2043, was attributedto the higher catechin content.

Changes in levels of pathogenesis-related (PR) proteins,chitinase (CHT), β-1,3-glucanase

(bGLU), peroxidase (POX) and total protein have beenreported during blister blight

infection (Charkraborty et al., 2002, 2005; Sinniah et al.,2014).

Some morphological and anatomical characters also showedcorrelation with resistance.

The higher resistance to blister bight in resistant teacultivars has been attributed to higher

trichome density, leaf L/W ratio and cuticle thickness(Sinniah et al., 2015; Jeyaramaraja

et al., 2005). Germination of basidiospores of E. vexanswas higher on the leaf surface

and in leaf wax extract of the susceptible cultivars thanfor those of the resistant cultivars.

Generation of H 2 O 2 and O 2 − at the site of infectionin the resistant tea cultivar, leading to

hypersensitive reaction (HR) and failure in generation of H2 O 2 and O 2 − at the early stages

of infection cycle in susceptible cultivars, has beenreported (Sinniah et al., 2014).

A relationship between polyphenol content and the

resistance of tea plants to P. theae

was reported by Langat et al. (1998). Higher sugar contentleads to greater susceptibility

of some tea cultivars to collar canker caused by P. theae(Ponmurugan and Baby, 2007).

The molecular basis of disease resistance in tea plants isdiscussed in Section 7.2.

6 Recent advances in the management of tea diseases

An integrated approach to the management of tea diseasesencompasses 1) modifying

agricultural practices, 2) protecting susceptible plantmaterials and 3) establishing resistant

plant materials. Chemical protection of susceptiblematerials has proven effective in

controlling foliar diseases, and so comes under strategy 2.However, growing concerns

about pesticide residues in made tea, non-target effects tothe environment and human

health, and the escalating cost of disease management haveled researchers to explore

alternative, safer plant protection products.

6.1 Biological control of tea diseases

Biological control agents are becoming increasinglyimportant as a sustainable alternative

to chemical protection or as an important component inintegrated disease management.

Several bacterial strains have been identified as potentialbiocontrol agents for management

of foliar diseases. Pseudomonas fluorescens Pf1(Saravanakumar et al., 2007), Ochrobactrum

anthropi BMO (Sowndhararajan et al., 2013), Bacillussubtilis, actinomycetes strains (MM

AC/02 & 05) (Barthakur et al., 2002; Sarmah et al., 2005)and Rhodotorula rubra (Rayati, 2011)

have been found to be effective against blister blight.Bacillus sp. (Premkumar et al., 2005;

Hong et al., 2005; Oh et al., 2005) and Pseudomonas spp.(Sanjay, 2005) have also been

reported to act against the grey blight pathogen P. theae.The bacterium Micrococcus luteus

and an anti-fungal compound extracted from M. luteus showedantagonism to G. cingulata

(brown blight pathogen) (Chakraborty et al., 1998).

Fungal antagonists have been used to effectively controlstem and root pathogens.

Trichoderma harzianum effectively protected stems from H.serpens (Premkumar, 2005),

collar canker (Ponmurugan et al., 2002), horse hair blight(Balasuriya and Pradeepa, 2005),

red root rot (Balasuriya and Pradeepa, 2005), brown rootrot and charcoal stump rot

(Barthakur, 2002; Sarmah et al., 2005; Elango et al.,2015). Elango et al. (2015) reported

that a combination of Streptomyces griseus and T. harzianumincreased green leaf yield as

well as improved made tea quality.

It has been noted that the effect of these biocontrolagents is not consistent. The

degree of control against blister blight achieved variedfrom 22 to 50% (Barthakur, 2002;

Sarmah et al., 2005; Rayati, 2011). The level of protectionachieved under field conditions

is influenced by the type of formulation, method ofapplication (Hazarika et al., 2000),

carrier materials and organic matter status in the soil

(Premkumar et al., 2009). A mixture of

biocontrol agents or consortium has been found to be moreeffective than the individual

agents used alone (Nepolean et al., 2015).

6.2 Botanicals in tea disease management

Botanical antimicrobial extracts from plants are used asgreen pesticides in agriculture.

A number of plant extracts have been tested on tea bushesas protection against various

diseases. Treatment of tea plantlets with formulations madefrom purified compounds

of Xanthium strumarium and Clausena excavata reducedincidences of blister blight

significantly (Ramashish, 2015). The purified preparationcompared favourably with

the synthetic fungicide bavistin, whereas the crude extractwas slightly less effective

(Ramashish, 2015). Aqueous extracts of 5–10% of Cassiaalata/Polygonum hamiltoni/

Acorus calamus/Adhatoda vasica/Equisetum arvense/Polygonumhydropiper/Tagetes

patula, applied at 15-day intervals, have been recommendedfor use in India (Anonymous,

2014).

6.3 Induced resistance in tea disease management

One promising strategy is the induction of host resistanceusing plant defence elicitors. This

offers the prospect of broad-spectrum, long-lasting diseasecontrol using the plant’s own

resistance mechanisms (Hammerschmidt and Yang-Cashman,1995). Various biosynthetic

pathways are activated in treated plants, depending on the

compound used. A wide

range of natural (organic/inorganic) and syntheticchemicals are used as natural defence

activators in plants. Some biological and chemicalelicitors have been tried successfully

against tea pathogens.

Chemical elicitors

Treatments with the elicitors acibenzolar-S-methyl andsalicylic acid resulted in reduced

severity of blister blight disease in nursery plants underfield conditions. Resistance to the

pathogen increased because of the significant increase inthe activities of phenylalanine

ammonia lyase, peroxidase and β-1,3-glucanase in tea leavestreated with the elicitor

treatments, compared to untreated leaves (Ajay and Baby,2010). A mixture of salicylic

acid or 1,2,3-benzothiadiazole with 0.05% copper hydroxideresulted in a reduction of

disease severity comparable to that achieved with therecommended fungicide treatment

(1%) (Sinniah et al., 2014).

Yoshida et al. (2010) showed that grey blight andanthracnose lesion development

was suppressed by treatment with the elicitors probenazole,prohydrojasmon and

tiadinil. All the tested elicitors induced diseaseresistance in tea plants systemically, and

the induced resistance continued for at least 30 days aftertreatment. The growth and

chemical composition of tea shoots were not changed by theelicitor treatments (Yoshida

et al., 2010).

Biological elicitors

Plant growth-promoting rhizobacteria (PGPR) have beenreported to induce systemic

resistance against blister blight (Saravanakumar et al.,2007), brown root rot and charcoal

stump rot (Mishra et al., 2014). PGPR strains (Pseudomonassp.) reduced the viscosity loss

of cellulose and pectin caused by the pathogens for bothbrown root rot and charcoal

stump rot, and increased the activity of defence-relatedenzymes l-phenylalanine ammonia

lyase, peroxidase and polyphenol oxidase (Mishra et al.,2014).

7 Advances in the molecular biology of tea diseases

Molecular biology provides several techniques to accuratelyidentify plant pathogens,

reveal plant-pathogen interactions, demonstrate theexpression of genes involved in

pathogenesis and resistance, and develop resistant plantmaterials. The development of

molecular biology and biotechnology techniques forapplication to tea diseases is still

in its infancy. At present, disease diagnosis andidentification of pathogens still mainly

rely on conventional techniques. However, attempts havebeen made to develop DNA

based methods to identify the pathogens of tea, and a fewstudies are available which

give insight into the molecular mechanisms behind tea plantresistance to diseases.

The following section summarizes the advancement ofmolecular biological techniques

used to study tea diseases.

7.1 Identification and characterization of tea pathogens

Recent advances in DNA sequencing technologies andbioinformatics enable accurate

identification of plant pathogens to species level and evento strain level. Accurate and

rapid identification of pathogens helps in devising diseasemanagement strategies and

surveillance. Recent advances have enabledreclassification, renaming and recognition of

new species of some tea pathogens.

Maharachcikumbura and co-workers (Maharachchikumbura etal., 2013, 2014, 2016) have

done considerable work to resolve confusion in phylogeny ofthe grey blight pathogen

in tea. The grey blight pathogen P. theae has beendifferentiated from Pestalotia and

renamed as Pseudopestalotiopsis theae, and it has beenclassified under a new genera

Pseudopestalotiopsis, based on morphological and multi-locisequence data from the

internal transcribed spacer (ITS), partial β-tubulin andpartial translation elongation factor

1-alpha gene regions (Maharachchikumbura et al., 2014). Theassociation of the new

species, Pestalotiopsis furcata (Maharachchikumbura et al.,2013) and Pseudopestalotiopsis

ignota (Maharachchikumbura et al., 2016), has also beenidentified. Based on their results,

Maharachchikumbura et al. (2016) suggested that grey blightmay be caused by a species

complex of Pseudoestolotiopsis.

Phylogenetic studies based on RAPD indicated the existenceof genetic diversity in the

blister blight pathogen E. vexans in Southern India (Joshiet al., 2009b) and Sri Lanka (Kumari

et al., 2010). rDNA sequence analysis (ITS+LSU) revealed noor low genetic diversity from

samples collected from different tea growing regions in SriLanka (unpublished data). This

may be due to the lower resolution of rDNA sequences inrevealing intraspecific variation

within a species, or due to lower genetic difference in thepathogen in this region. The

genetic diversity of the important pathogen E. vexanswarrants further investigation using

multi-loci sequencing or other advanced techniques.

M. theicola was believed to be the canker causing agent intea in Sri Lanka. However,

based on the ITS sequences and morphologicalcharacteristics of isolates, three anamorphic

species, Botryosphaeria dothidea, B. mamane andLasiodiplodia theobromae, have

recently been identified as responsible for canker diseasein Sri Lanka (Pradeepa et al.,

2014). C. acutatum has been identified as causing brownblight in tea in China (Chen et al.,

2016). So far, the molecular characterization oridentification of major root pathogens of

tea remains an unexplored area.

7.2 Molecular mechanisms of resistance to disease in teaplants

Plants possess two types of resistance. Vertical resistance(gene-for-gene) is governed

by a single gene and is effective only against the pathogenrace/strain which has a

corresponding gene. Horizontal resistance involves manygenes (polygenic) and protects

plants against a number of pathogens or strains. Certaintea cultivars exhibit horizontal

resistance to some pathogens.

Data has recently been published on global gene expressionin young leaf tissues

during the different stages of blister blight, for bothresistant and susceptible genotypes

(Jayaswall et al., 2016). Infection with E. vexansactivates various R genes, along with

defence-related enzymes, to prevent the spread of thedisease after infection. This is

followed by the activation of transporters in resistantgenotypes, which are involved in

overcoming the virulence of the pathogen (Jayaswall et al.,2016). In severe cases of the

disease, retrotransposons are activated and, by genomereshuffling, it is possible that

these produce a new type of R gene(s), so that the plant isable to recognize the new

kind of virulence produced by the pathogen (Fig. 8).Transcription factors are activated

in the plant to further enhance the activity of R and otherdefence-associated genes (see

Jayaswall et al., 2016 for further details). Expressionstudies indicate that salicylic acid

and jasmonic acid may induce synthesis of antimicrobialcompounds, which are required

to overcome the virulence of E. vexans. Twenty-fiveresistant genes (25 R genes), 1337

defence-related enzymes, 9 multidrug-resistant transporters(MDR), 66 transcription

factors, 9 retrotransposons, metacaspases and chaperons areinvolved in defence against

E. vexans. Differentially expressed, 149 defence-relatedtranscripts/genes were also

identified in resistant genotype (SA6). Among the R genes,the four R genes RIN4, RPM1,

RPS2 and RPP13 were prominent, irrespective of the diseasedevelopment stages. This

study further suggests the putative defence mechanismagainst BB in tea is comparable

with model plant Arabidopsis.

Figure 8 Diagrammatic representation of putative defencemechanism in tea against blister

blight based on different categories of immune-relatedcandidates identified in RNA-seq data.

(Source: Jayaswall et al., 2016 ).

Bhorali et al. (2012) reported that PR and defence-responsetranscripts differentially

expressed in the resistant cultivar include chitinase,endo-glucanase, beta-glucosidase,

wound-induced protein, protease inhibitor, thaumatin-likeprotein, cystatin, a blight

associated protein p12 and aspartic proteinase. Thetranscripts encoding proteins which

have a function in defence-related signal transductionpathways (serine/threonine

protein kinase, leucine-rich repeat transmembrane proteinkinase, oxo-phytodienoic acid

responsive protein, energy processes, ATP-binding, calciumion-binding or calmodulin

related protein, salicylic acid-binding protein,

chitin-inducible gibberellin-responsive

protein and calreticulin) were also found to be highlyinduced during infection. Expression

profiling further demonstrated that HIR, WIN2 GST, CBL-CIPKand CIGR are involved in

contributing towards the development of most resistanceresponses. Some of the genes

were responsive early and upregulated within 72 h afterinoculation, while others were

responsive later and only highly induced 240 h afterinoculation. The authors also found

that genes related to metabolism, energy and transcriptionfactors participated in the

development of resistance responses against blister blightinfection.

A study using a transgenic plant with the introducedSolanum tuberosum class I chitinase

gene (AF153195) showed resistance to E. vexans by thecreation of a hypersensitivity

reaction. This result suggests that constitutive expressionof the potato class I chitinase

gene can be exploited to improve resistance of tea plantsto E. vexans (Singh et al., 2015).

Wang and co-workers (2016b) used genome-wide analysis toelucidate the defence

mechanism of tea plants against brown blight pathogen C.camelliae. Different levels

of genes were involved in plant hormone biosynthesis andsignalling, plant-pathogen

interaction and secondary metabolic pathways (includingphenylpropanoid biosynthesis)

in the resistant genotype ZC108, as compared to thesusceptible genotype. Several genes,

including the MADS-box transcription factor and genesinvolved in phenylpropanoid

metabolism (CAD, CCR, POD, beta-glucosidase, ALDH and PAL),contributed to

anthracnose disease tolerance in ZC108. The resistance oftea plants to grey blight

caused by Pestalotiopsis longi was found to be controlledby two independent, dominant

resistance genes Pl 1 and Pl 2 , based on parent-offspringgenetic analysis (Takeda, 2002).

7.3 Resistance breeding and molecular markers

Breeding disease-resistant cultivars is an importantobjective for all tea breeding

programmes. Conventionally, resistant plants are developedby crossing resistant parents,

followed by phenotypic selection of plants with resistanttraits. This approach has been

time-consuming, laborious, expensive and can be influencedby environmental factors

and knowledge and experience of the diseases. Molecularmarkers have proved to be a

powerful tool in facilitating the introgression ofresistance genes into breeding material

(Gunasekera, 2007). Molecular markers are PCR-based,sequence-based or hybridization

based. Attempts have been made to develop molecular markersfor blister blight

resistance using F1 progeny of TRI2023 (blister blightsusceptible) and X TRI2043 (blister

blight resistant). A primer, EST-SSR073, was found togenerate a distinct band specific to

BB-resistant individuals (Karunarathna et al., 2013).

RAPD and SCAR markers have been developed for early

identification of blister blight

resistance or susceptibility. These DNA markers will enablethe selection of blister blight

resistant/susceptible plants(http://www.upasitearesearch.org/research/auto-draft-13/).The

above studies clearly reflect the potential of molecularmarkers in differentiating blister blight

resistant individuals from susceptible ones, and hencecould be useful in early selection of the

trait by a simple PCR assay, independent of growth stageand field assessments. This would

enable marker-assisted selection or marker-assisted teabreeding for disease resistance.

8 Disease forecasting for tea

Since the introduction of blister blight into South Indiaand Sri Lanka, attempts have

been made to reduce the need to manage the disease usingfungicides by use of

disease forecasting. In Sri Lanka, postponement of coppercontact fungicide spray was

suggested, based on sunshine hours since postponement bysuccessive 5-day periods

until the average sunshine for the previous five daysexceeded 3 h and 45 minutes (Visser

et al., 1959, 1962). It was found that 20 h of sunshine perprevious five days, or 10 h of

sunshine per previous four days, was sufficient to delay aspraying round (Mulder and de

Silva, 1960). However, Venkata Ram (1971) showed that thisscheme of delayed spraying

did not give satisfactory control under South Indianconditions. Disease incidence was

predicted three weeks in advance by combining sunshinehours and number of spores in

the environment (Kerr and de Silva, 1969).

In Java (Indonesia), the blister blight infection rate andseverity were predicted based

on microclimatic variables such as leaf wetness duration,leaf temperature and relative

humidity. This involved assuming that only weather factorswere simulative and also

assuming that plant, pathogen and other cultural practiceswere favourable but not an

influence on the data to be predicted. The model was foundto be useful, except for when

the assumptions failed (Gunadi, 2001). A computer-basedforecasting model has been

developed to predict blister blight incidence in India(Premkumar et al., 2002).

In addition to weather parameters, inoculum levels and theavailability of a susceptible

host also influence the accuracy of predictions. It istherefore difficult to put forecasting

models into general use. Further, there are severallogistical barriers to grower adoption

of disease forecasting systems. A fungicide spray warningis of little use if it is inconvenient

to adopt, adds additional costs and labour, and is notalways accurate. Grower adoption of

warning systems is unlikely to increase unless thesebarriers are overcome.

9 Conclusion

This chapter has focused mainly on tea diseases of majoreconomic importance, and diseases

which are prevalent among tea growing countries. Research

carried out in Sri Lanka and

India has been highlighted, and research from othercountries has been included when the

information is available. Emphasis has been placed onrecent developments in the field of plant

pathology, in order to identify the knowledge gaps andfuture needs of the global tea industry.

10 Future trends

Tea pathogens have been identified through conventionaltechniques. It is necessary to

confirm these identifications using more advancedtechniques and to discover if different

strains of particular pathogens exist among the differenttea growing countries. This will

allow better understanding of the epidemiology of teadiseases and will help us to devise

suitable management strategies.

The diseases of tea have not been thoroughly studied inview of the changing global

climate. It is imperative to look at how major diseasessuch as blister blight and stem

canker will react to climate change, and tea breeding mustfocus on meeting the resulting

future challenges. Disease forecasting techniques for majordiseases need to be given

serious consideration and developed to the stage where theycan be incorporated into

disease management strategies.

Technical innovations have given birth to modern sprayersand continue to improve chemical

treatments for pests and diseases, making them increasinglyeffective in most crops. However,

this technological innovation has not yet reached teacultivation. Spraying technology should

be developed to suit the tea industry’s needs. Thisrequires recognition that tea is cultivated

over varying topology and elevations. Technologicalinnovation for tea cultivation should

target higher efficiency, minimization of chemical usageand the safety of spray personnel.

Although every breeding programme targets diseaseresistance, there are still gaps in our

knowledge of disease-resistant mechanisms and the genesresponsible for resistance. There is also

progress to be made in marker-assisted breeding forresistance to most of the major tea diseases.

Safe chemicals and biocontrol agents for widely variableenvironments should also be

explored further to ensure consumer and environmentalsafety. One good approach to

addressing both plant protection and consumer safety is theformulation of new generation

chemicals that can extend the time intervals of applicationwhile reducing the pre-harvest

interval. New innovations may provide even moreuser-friendly products for the grower

and offer new disease control possibilities (e.g. controlof new species or better control of

difficult-to-control diseases).


For information on tea diseases in Sri Lanka andrecommended practices for management •http://www.tri.lk/technology-dissemination/recommendations• Advisory circulars DM1 to DM6 and PU2

For information on tea diseases in India •

For policy on usage of plant protection formulations in teaplantations of India •www.teaboard.gov.in/pdf/notice/Plant_Protection_Code.pdf

For information on tea disease in Vietnam •

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9 Chapter 9 Insect pests of tea: shothole borers, termites and nematodes

1 Introduction

Tea (Camellia sinensis (L) O Kuntze) is a perennial treecrop that has been grown for

hundreds of years and is therefore exposed to the ravagesof a wide variety of pests.

Depending on the climatic conditions and localenvironmental niche, every part of the plant

is vulnerable to attack by one or more herbivorous pests.Currently there are records of

just over 1000 arthropod pests (Chen and Chen, 1989) andseveral pathogenic nematode

species attacking the tea crop (Gnanapragasam, 2014).However, amongst these, only a

few are known to cause economic damage to tea (Table 1).

Pest incidence in tea is particularly severe in the Asiancountries, including Bangladesh,

China, India, Indonesia, Japan and Sri Lanka, compared tothe African countries and other

newer tea-growing areas of the world. It is interesting tonote that pests can be very

damaging and widespread in one country, but non-existent,or less important, in others. As

an example, the major pests of tea in Sri Lanka are thelive wood tea termite (Glyptotermes

dilatatus Bugnion and Popoff, 1910) and Postelectrotermesmilitaris Desneux, 1904) and

pathogenic nematodes (Pratylenchus loosi Loof, 1960 andRadopholus similis Cobb,

1893; Thorne, 1949), whilst in India, P. loosi, R. similisand species of live wood termites

are considered as only localized problems. Widespread

infestation by the tea mosquito

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bug (Helopeltis theivora Water House, 1886) and jassids(Empoasca flavescens, Gillete,

1898) are considered to be of economic importance inBangladesh, north India, China

and Vietnam, whilst jassids and the tea mosquito bug areonly occasionally encountered

in localized areas of Sri Lanka and are only considered a

minor pest.

This and an accompanying chapter provide a complete surveyof known important pests

affecting tea crops. The pests of tea can be divided intofour categories:

• Perennial pests: Perennial pests are ideally adapted tothe particular environment and have found a permanentenclave. They remain in the tea bush and reach injuriouspopulation levels every year. They cause damage to the teabush over varying periods with gradual crop losses leadingto death of the tea bushes. Pests include the shot holeborer (SHB) (Euwallacea fornicatus Eichhoff, 1868), livewood tea termite (Glyptotermes dilatatus Bugnion andPopoff, 1910; Postelectrotermis militaris Desneux, 1904;Neotermes greeni Desneux, 1904) and pathogenic nematodes(Pratylenchus loosi Loof, 1960; Radopholus similis Cobb,1893, Thorne, 1949; Meloidogyne brevicauda Loos, 1953).

• Seasonal pests: Seasonal pests attack the crop onlyduring specific seasons of the year, causing temporarydeclines in the crop. These are mostly kept under controlby natural predators and parasites, and pest outbreaksonly occur when the environment becomes conducive for thepopulation to build up above damaging threshold levels.These pests are widespread globally, for example,leaf-eating caterpillars or different species ofphytophagous mites.

• Occasional pests: These cause only occasional economicdamage, usually due to environmental influences or humanactivities, for example, beetle grubs (Holotrichiadisparalis, Arrow, 1916) or the red borer (Zeuzera coffeae,Nietner, 1861).

• Minor pests: These usually do not cause economic damage,for example, tea leaf miner (Melanagromyza theae Mejere),aphids (Toxoptera aurantii, Boyer de Fonscolombe, 1841),scale insects (Coccus viridis Green, 1900), Saissetiacoffeae (Walker, 1852), the faggot worm or bag worm(Clania cramerii Westwood), red slug (Eterusia aedeacingala Moore, 1877) or ants (Oecophylla smaragdinaFabricius, 1775).

The prevalence and occurrence of these pests in tea-growingareas are primarily determined

by the specific agro-climatic condition, the type of

cultivar (susceptible, tolerant and

resistant varieties) and the cultural practices adoptedwithin a given specific location.

This chapter reviews SHBs, termites and nematodes(perennial pests). An accompanying

chapter discusses caterpillars and other seasonal,occasional and minor pests.

2 Shot hole borers

SHB beetles belong to the class of Insecta, orderColeoptera and family Scolytidae.

There are about eight to nine species of scolytids known toattack tea (Banerjee, 1983;

Muraleedharan, 1992), of which the SHB (Euwallaceafornicatus) is the most serious

pest. In Japan, two other ambrosia beetles, Xylosandrusgermanus (Blandford, 1894)

and Xylosandrus (Xyleborus) compactus (Eichhoff, 1875), arereported to cause serious

damage to tea (Kaneko et al., 1965).

2.1 Shot hole borer (Euwallacea fornicatus Eichhoff, 1868)

Euwallaceae fornicatus is a tiny wood boring beetlebelonging to the family Scolytidae,

often referred to as the ambrosia beetle because of itssymbiotic activity with the ambrosia

fungus. The fungus carried in the buccal cavity of theparent female beetle (Fernando,

1960) is cultivated on the walls of the galleries in thetea plant stem (Gadd and Loos,

1947). The beetle was first described by Eichhoff (1868)from a specimen collected in

Ceylon (Sri Lanka) (Speyer, 1917) and was referred to asXyleborus fornicatus. Later Wood

(1989) transferred X. fornicatus to the genus Euwallacea,and since then, it is generally

referred to as Euwallaceae fornicatus.

2.1.1 Geographical distribution

Euwallacea fornicatus is a pest of tropical countries andis one of the few ambrosia beetles

known to infest both healthy and stressed plants. It wasfirst noted as a pest of tea in 1892

in Sri Lanka at an elevation range of 600 m above mean sealevel (Gadd, 1946a; Austin,

1956). Although the pest has been reported in Bangladesh,Indonesia, Malaysia, China,

Korea, Philippines, New Guinea, Taiwan, Fiji and Hawaii(CABI, 2015; James, 2007), it is a

major pest of tea in Sri Lanka and in areas of mid- andlow-elevation in some parts of south

India (Muraleedharan, 1986). Waterhouse (1993) has reportedit to be an important pest

of tea in Malaysia as well.

This SHB is widely distributed in the warmer tea-growingareas of Sri Lanka in the

150–1400 m elevational range, although occasionalinfestation occurs at sea level as

well. A survey carried out by Walgama and Pallemulla (2005)indicated consistently high

infestation levels at the elevational range of 600–1200 m,with variations in infestation up

to a limit of 1400 m. Beyond 1400 m, due to the coolerclimate, the borer is unable to

breed and colonize. This corresponds with observations madeearlier by Gadd (1949) and

Sivapalan and Shivandarajah (1977).

2.1.2 Alternative hosts

Euwallacea fornicatus has a very wide host range (Browne,1961; Danthanaryana, 1968;

Amarasinghe and Devi, 2003). Although SHBs are attracted tomany hosts, the fungi carried

by the SHB are reported to reproduce successfully only oncertain trees (Faber, 2015). The

main alternative hosts reported include avocado (Perseaamericana, Mill), citrus (Citrus

spp.), derris (Derris scandens Roxb.), rubber (Heveabrasiliensis Mull. Arg), castor (Ricinus

communis L.), cocoa (Theobroma cacao L), white teak(Gmelina arborea Roxb), nutmeg

(Myristica fragrans Houtt), cinchona (Cinchona pubescensVahl), litchi (Litchi sinensis Sonn),

pomegranate (Punica granatum L.), loquat (Eriobotryajaponica (Thunb) Lindl), guava

(Psidium guajava L), kitul palm (Caryota urens L), Mimosabracaatinga Hoehne, Albizia

moluccana Riq, Erythrina lithosperma Miq. (Vitarana, 2003;Cabi Compendium, 2015) and

neem Azadirachta indica A. Juss (Amarasinghe and Devi,2003).

2.1.3 Symptoms of damage

A yellowing of foliage and broken branches (Fig. 1a and b)give a scorched appearance

(Fig. 1c). There is sometimes defoliation as well. Thedensity of infested bushes varies in

a linear fashion with denser infestations near theboundaries of fields. Walking through

infested tea fields causes snapping of branches. Detailedinspection of broken branches

reveals galleries that have been bored into by the pest.

The galleries occupied by the

pest show the different stages of this pest. Prune cuts ofinfested plants reveal tiny holes

of not more than 1.0 mm in diameter in the younger branchesand larger holes in older

Figure 1 (a) A young tea plant showing branch breakage dueto SHB infestation. (b) A mature tea

branch snapped due to SHB damage showing entrance togalleries formed by SHB (Courtesy Tea Res.

Inst. Sri Lanka). (c) Mature tea bush with branch breakageand scorched leaves due to SHB damage

(Courtesy Tea Res. Inst. Sri Lanka). (d) Long-term damageby SHB showing advanced wood-rotted tea

branches (Courtesy Tea Res. Inst. Sri Lanka). (e) A splittea stem showing SHB galleries and SHB brood

(large-adult females, small-adult males) (Courtesy Tea Res.Inst. Sri Lanka).

branches. Long-term attack by this pest causes secondarydamage due to wood rot and

decay, which are easily visible by the gnarled appearanceof the damaged shoots (Fig. 1d).

2.1.4 Biology and ecology

The biology and ecology of this pest have been studied indetail by several workers in Sri

Lanka (Speyer, 1917; Jepson, 1920, 1921; Gadd, 1941a,b,1947a,b, 1949; Gadd and Loos,

1947; Judenko, 1958a,b; Cranham, 1961, 1963; Calnaido,1964, 1965; Danthanarayana,

1967, 1968; Danthanarayana et al., 1968; Sivapalan, 1975,1976); in India by Muraleedharan

(1986) and Radhakrishna (2016) and in Indonesia byKalshoven (1958). Browne (1961) and

Walgama (2012) have reviewed the biology and ecology of

Euwallacea fornicatus in South

east Asia and Sri Lanka, respectively. Infestation occurswhen, after mating, young adult

females bore into the wood to initiate new galleries forthe development of the brood. The

adult females are dark brownish to black with a sclerotizedbody; they are 2–2.8 mm in length

(breadth half of the length) and have well-developed wings(Fig. 1e). The males are smaller

(1.5–1.67 mm long), devoid of wings and spend their entirelife within the mother gallery.

Mating occurs within the gallery. After mating, adultfemale beetles carrying developing

eggs (gravid females) move out of the entrance hole of thegallery and search for new sites

to initiate a fresh gallery. According to Green (1903), thepoint of entry is usually leaf scars.

Initiation of galleries can take place at any time of theyear if there are suitable branches.

Galleries are formed only on the younger branches at aparticular stage of maturity (with

new reddish brown striped bark) and not on mature brancheswith brown to grey bark

(Sivapalan, 1976). The number of borers in pruned framesdeclines, presumably due to

reduction in sap flow after pruning (Cranham, 1966). Stemstypically become attractive

for gallery construction and brood development when theyare about 8–14 months from

pruning (depending on the prevailing environment andcultivar). Most of the galleries in

Sri Lanka are vertical along the pith, but galleries havebeen found to traverse around the

xylem cylinder, forming circular or spiral rings as well.Mixed galleries are also observed.

In thin twigs, larvae were found mostly in straightgalleries and in larger braches in circular

galleries (Gadd, 1947a). Radhakrishnan (2016) also reportedvertical, horizontal or mixed

galleries.

Increase in sap flow and growth of the bushes are conduciveto beetle development

and provide optimum conditions for gallery formation(Sivapalan, 1975). Galleries are

constructed close to the outside woody cylinder of thestem, often separated from the

cambium by only a thin layer of young wood (Cranham, 1966).According to Danthanarayana

(1970), about 70% of the galleries are constructed at thenodes. Investigations of the

pattern of brood galleries within the plant frames haveshown that distribution depends

on the type of branch (primary, secondary or tertiary)rather than the total available branch

length or thickness (Sivapalan and Delucchi, 1973). Certainplant volatiles present in tea

bark attract the adult female beetle (Sivapalan, 1974;Karunaratne et al., 2009).

Egg laying begins as soon as the entrance tunnel iscompleted. The parent female

beetle is known to die within the gallery after the broodhas developed and emerged.

Only one brood is developed by the parent female and thegallery made by a female is not

occupied again by another female. The larvae are white,legless and do not feed on the

tea plant but on ambrosia fungus. This is introduced intothe galleries by the adult female

and the success of brood development depends on the growthof the fungus. The newly

formed galleries have smaller cavities and the fungus isobserved to colonize without

causing any damage to the tissues of tea. Followingabandonment of the galleries by

the brood, there is reduced growth of ambrosia fungus andsaprophytic fungi have been

observed to take over (Kumar et al., 1998).

Gadd and Loos (1947) identified the fungus asMonacrosporium ambrosium. In south

India, Mouli and Kumar (1988) reported the fungus Fusariumtumidum in the galleries, whilst

Parthiban and Muraleedharan (1992, 1996) later identifiedthis as Fusarium bugnicourtii

(Bray ford). There are three instars and pupation takesplace within the galleries. Following

emergence, the young females remain in the galleries forseveral days, mate within the

parent gallery with the smaller flightless males and emergethrough the original gallery

entrance. This is reported to be an ‘inbreeding polygynous’mating system where one or

few males inseminate their many sisters (Kirkendall, 1993).

Detailed laboratory studies on semi-artificial mediaindicated a distinct rhythm and more

than 70% of the total young adults emerged between 11 amand 1 pm (11–13 h), with peak

emergence at 12 noon. No emergence was observed after 4 pmuntil 7 am the following

morning, These studies resembled field observations

(Sivapalan and Shivanandarajah, 1977).

Males have a shorter lifespan and are produced continuouslyand develop more rapidly

than females. The ratio of females to males is low andalthough the wingless males do not

normally leave the parental gallery, they have beensometimes observed to emerge and

crawl on the surface of the bark, occasionally entering agallery made by another female

and mating with the females in that gallery. Such behaviourcauses a small amount of

cross-breeding (CABI, 2015).

Life history is dependent on temperature and, as perstudies made by Walgama and

Zalucki (2007), about 373 DD was reported to be necessaryfor the development of one

generation, at 15°C.

In southern India, the life cycle is reported to be 39–48days (Cabi, 2015); in Malaysia,

the life cycle is reported to be 29–33 days and in SriLanka, the life cycle varies from 34 to

60 days (Speyer, 1917; King 1940, Gadd, 1947a). Thediscrepancy in the observations was

due to the method of estimation, the elevation and theclimate (Walgama, 2012).

Simulation models developed by Walgama (2008) agreed withthe earlier field studies

from Sri Lanka that report that the SHB evolves throughmultiple generations within a year

in the pruning cycle and that colonization takes placecontinuously in a pruning cycle.

Based on the thermal requirement and accumulation ofdaytime temperature, the author

predicted 2, 6, 9 and 12 generations per year for theelevation ranges of (a) above 1200 m,

(b) 600–1200 m in the mid-country dry zone (eastern slopesof Sri Lanka), (c) 600–1200 m in

the mid-country wet zone (western slopes of Sri Lanka) and(d) below 600 m (low-country

districts of Sri Lanka).

2.1.5 Means of dispersal

Since males cannot fly, dispersion is mainly by the wingedfemales through flight, aided

by the wind and convection currents. Calnaido (1964, 1965)showed that the beetles fly

only during the day, between 11am and 1 pm, with flightceasing at 4 pm. Transport of a

pruned litter could also serve as a source of infestation.

Once the population attains its peak level within a pruningcycle, there is mass migration

of young fertilized females dispersed further by windcurrents. Attacks are also brought

about by immigrants from neighbouring infested fieldsduring dispersal flights (Sivapalan,

1976). In rare instances, wingless males have been foundcrawling on the surface of bark and

entering another gallery (CABI, 2015). Following pruning ofthe tea, the surviving residual

broods initiate new attacks on the newly forming branchesfollowing successive prunes.

2.1.6 Damage to tea

Damage to tea by the SHB is twofold: direct mechanicaldamage is a result of gallery

formation on both primary and secondary branches andsecondary long-term protracted

debilitation is caused by wood rot on both primary branchesand on the exposed galleries

on larger prune cuts (Austin, 1956), with greater damagecaused to circular galleries

(Radhakrishnan, 2016). If the fractured branches are leftunattended, wood rot that sets in

through the fractured wounds at successive prunings isattacked by scavenging termites.

The result is progressive frame debilitation, leading toweakening of frames and poor

recovery from pruning and ultimately the death of thebushes, a process that becomes

accelerated during successive droughts. The loss of crop ismore pronounced at this stage

(Sivapalan, 1985).

2.2 Xylosandrus (Xyleborus) compactus

Xylosandrus (Xyleborus) compactus was described by Eichhoff(1875) to be in the genus

Xyleborus, although it was thereafter transferred toXylosandrus by Browne (1963).

Xylosandrus compactus is considered one of the mostimportant SHB beetles infesting tea

in Japan, causing economic damage. It cultivates theambrosia fungi in its galleries as food

for the larvae. It is a subtropical species native tosoutheast Asia.


This species has a wide host range. Major crops to which itis reported to cause serious

damage are coffee (Coffea L), avocado (Persea americana,Mill), cocoa (Theobroma cacao

L), mahogany (Swietenia macrophylla, King), Erythrina

(Desr. A.Juss), Melia azedarach and

Annona muricata L.


The symptoms of attack are necrosis of the leaves andbranches as well as die back, wilting

and branch breakage. Small entrance holes to the galleriescan also be observed on the

twigs with off-white piles of dust that can be seen in thebored holes.


Biology and ecology of this pest have been studied ondifferent hosts by several workers.

The example of tea in Japan was studied by Kaneko (1965)and Kaneko et al. (1965).

X. compactus (Eichhoff.) attacks mainly twigs of about 5–8mm diameter and only the adult

females attack. Eggs are white shiny ovoids laid in thegallery, and pupation and mating

occur within the galleries (Hoffman, 1941; Hara andBeardsley, 1979). The mature larva is

creamy white with a pale brown head and is legless. Thebody of the pupa is creamy white

and the same size as the adult. The adult female is darkbrown to black in colour and is

larger than the males. The males are wingless.

As in the case of Euwallacea fornicatus, a single male ineach gallery mates with his

sisters. The adults emerge through the entrance holes madeby the parent beetle. The ratio

of females to males varies, but is usually 9:1 (Hara andBeardsley, 1979). Two generations

per year are reported (late July to late August and from

late August to September) and this

species is reported to hibernate within the branches.


Dispersal to hitherto uninfected areas is mainly by flight.Entwistle (1972) found that

adult females dispersed at least 200 m and it is likelythat dispersal over several

kilometres is possible, especially if aided by wind. Theyare also dispersed by the

transport of infested plants and infested branches. As inthe case of Euwalaceae

fornicatus, sometimes a male moves out of its own galleryand enters a neighbouring

gallery (Peer and Taborsky, 2004).

2.2.5 Damage to tea

Primary damage to tea is caused by the adult females whichexcavate galleries in the

branches of tea plant. As in the case of Euwalaceaefornicates, compactus is also one of

those few species of ambrosia beetle which attacks healthytea branches. Within a few

weeks of infestation, the branches show signs of wilting.Thereafter, secondary damage is

caused by the invasion of pathogens, causing wood rot.

2.3 SHB beetle, Xylosandrus (Xyleborus) germanus

The female of this species of Xylandrous was described inJapan by Blandford (1894)

and was in the genus Xyleborus Eichhoff. Thereafter,Hoffmann (1941) transferred it to

Xylosandrus. However, it is still often referred to in theliterature as Xyleborus germanus.

The male of this species was described by Eggers (1926).Xylosandrus (Xyleborus)

germanus (Bldf.) is considered as one of the most importantSHB beetles infesting tea in

Japan and causing economic damage. It cultivates ambrosiafungi in its galleries as food

for the larvae. It is a subtropical species native tosoutheast Asia. This species is often

referred to as the ‘black timber bark beetle’.


This species has a wide host range.


Wilting and yellowing of foliage, rotting of the root, thefeeding site is inside the roots and

early senescence occurs.


The biology and ecology of this pest in tea were studied inJapan by Kaneko (1965),

Kaneko et al. (1965) and Kaneko and Takagi (1966). Thefemales are larger than the males.

The type of infestation caused by the species is reportedto be different on different

host trees. Although in other hosts they were observed toattack the branches, in Japan,

X. germanus was found to attack mainly the roots of the teaabout 1.5 cm in diameter, at depths

of 1 ft (Kaneko and Takagi, 1966). Usually, they attackstressed trees, although occasionally

attacks have been reported on apparently healthy trees onother hosts (Hoffmann, 1941).

Infestation occurs through mated females. Eggs are laidloosely in the gallery over

a period of time and the larvae feed on the ambrosia fungusgrowing on the walls of

the gallery. The size of the brood has been reported tovary significantly, from 1 to 54

individuals, with an average of 16 (Kaneko and Takagi,1966; Weber and McPherson, 1983).

Pupation and mating take place within the gallery, andusually a single male in each gallery

mates with his sisters. The new generation of femalesemerges through the entrance hole

made by the parent. The males are smaller than the femalesand are flightless. Although

males stay mostly within the gallery, Peer and Taborsky(2004) have reported that males of

X. germanus to occasionally move out of the entrance holein search of females.

The optimum temperature range for development was reportedto be 21–23°C and

25–27°C (Kaneko et al., 1965; Kaneko and Takaji, 1966). Twogenerations per year have

been reported (from June to early July and from late Augustto September). X. germanus

hibernates in the roots, and the death rate of hibernatingbeetles is reported to be high.

Adults overwinter in the host plants, often clustering ingalleries (Hoffmann, 1941; Kaneko

and Takagi, 1966; Weber and McPherson, 1983).

X. germanus is often reported to be found together withXylosandrus compactus on tea

(Kaneko and Takagi, 1966).


Dispersal is by flight and the adult females are known to

come out of the ground for

dispersion and have been observed to disperse over 200 km,mostly aided by wind. The

flight period of the adults is usually between April andAugust, but may extend to March

and September (Kaneko and Takagi, 1966; Weber andMcPherson, 1991).

2.3.5 Damage to tea

As in the case of other SHBs, they do not feed on tea, butthe damage occurs during

excavation of the roots to form galleries and secondaryattack by other pathogenic fungi.

3 Termites of tea: general comments

Termites belong to the class Insecta. They are socialinsects belonging to the primitive

order Isoptera. They are often known as white ants or woodants due to the similarity of

the social organization of the latter, but their structureand development differ from the

true ants, wasps and bees belonging to the highly developedorder Hymenoptera. They

are supposed to be closely related to wood-eatingcockroaches.

There are two types of termites damaging tea plants:

• Live wood termites causing direct damage to tea;

• Scavenging termites causing indirect damage.

4 Live wood termites

This section starts by discussing live wood termites(Sections 4.1 to 4.3). The termites

that cause primary damage to tea belong to the familyKalotermitidae, which are dry

wood termites that feed above the ground on damp and rottedwood and do not have

any connection with the soil. Since they are directlyinjurious to the tea plants, shade

trees and form galleries within living tissues, they arealso referred to as ‘live wood

tea termite’. Unlike the case of the highly organizedcolony of the family Termitidae

(mound-building termites), which have thousands ofindividuals with distinct castes, in

the primitive family Kalotermitidae, the colony size isusually small and the castes few

and simple. In a typical termite colony, the castes consistof larvae, workers, soldiers

and reproductive forms. In Kalotermitidae, there are noworkers and the work of the

colony is carried out by nymphs (pseudogates) whosedevelopment into the winged

adult is controlled depending on the needs of the colony.Food gathering, gallery

construction and feeding of the other castes are all doneby the nymphs. In a well

developed colony, there are also supplementary reproductivetermites (neotonics)

which are sexually mature but unable to fly, may have shortwing pads and serve as

substitute or complementary kings and queens (Cranham,1966; Danthanarayana and

Fernando, 1970).

Severe damage to tea by live wood tea termites has so farbeen reported only from

Sri Lanka (Ranaweera, 1962; Sivapalan and Senaratne, 1977;Sivapalan et al., 1977) and

Indonesia (Damiri, 2014). Although there are reports oflive wood termite infestation

in the tea areas in India (Borthakur, 1984; Das and Kakoty,1991), Bangladesh (Ahmed,

1996; Mamun and Ahmed, 2011), Kenya, Malawi, Tanzania,Uganda (Rattan, 2000), China,

Indonesia (Damiri, 2014), Nepal (Shrestha and Thapa, 2015),Malaysia (Danzinger, 2000)

and localized areas of Tanzania, they are mostlyMicrotermes sp., Odontotermes sp. and/

or Coptotermes sp. These are scavenging termites which haveoccasionally been found to

cause damage to the tender roots of tea and are thussometimes grouped with live wood

termites (Cranham, 1966).

There are three species of live wood termites which causedirect damage to tea,

viz. Postelectrotermis militaris, Desneux, 1904; Neotermisgreeni, Desneux, 1904 and

Glyptotermes dilatatus, Bugnion and Popoff, 1910.

4.1 Up-country live wood termite, Postelectrotermismilitaris


This species of live wood termite is reported to be foundonly in Sri Lanka and Indonesia.

Postelectrotermis militaris Desneux was first recorded inSri Lanka at an elevation of

1000 m (Green, 1890). It is yet restricted to only a fewlocations above 800 m in Sri Lanka,

and since it is located at higher elevations it is commonlyreferred to in Sri Lanka as the

‘up-country live wood tea termite’.

4.1.2 Alternative host

Although tea is the preferred host, it also attacks Acaciadecurrens Willd, Albizia falcata

(L.) Backer, Casuarina sp. (Casuarina equisetifolia L),Cedrela toona Oken, Crotalaria sp. L,

Erythrina lithosperma Miq, Eucalyptus sp., Grevillearobusta A.Cunn. ex R.Br and Tephrosia

vogelii Hook.F (Cranham, 1966).


Infestation goes unnoticed until galleries are formed inthe branches, which are observed

only at pruning (Fig. 2a). However, especially duringdrought, it has been observed that the

affected plants appear weak and wilting. At prune, theaffected branches expose hollow

stems with earth-like faecal matter filling up the hollow.The galleries extend beyond the

collar into the distal ends of the roots and often whitesoft-bodied organisms may be

found in the galleries. Infested bushes are mostly found inpatches.


Biology and life cycle have been studied by Jepson (1930)and Dantharayana and Fernando

(1970). Development of colonies is very slow and unlike inthe case of Glyptotermes

dilatatus, winged adults are rarely found. Infestationoriginates in a healthy lateral root,

supposed to be by root contact, and the colony extends itsgallery upwards to the collar,

to the other adjacent roots and then the branches (Fig.2a). The galleries formed by

this species are wider than those of G. dilatatus and N.greeni and more regular and

Figure 2 (a) A trunk of a tea bush hollowed out byPostelectrotermis militaris (Courtesy Tea Res. Inst.

Sri Lanka). (b) A dead tea bush severely damaged byGlyptotermes dilatatus (Courtesy Tea Res. Inst.

Sri Lanka). (c) Adult Glyptotermes dilatatus on rotted endsof a tea bush (Courtesy Tea Res. Inst. Sri

Lanka). (d) Galleries formed by Glyptotermes dilatatus(Courtesy Tea Res. Inst. Sri Lanka). (e) Workers

of Glyptotermes dilatatus (Courtesy Tea Res. Inst. SriLanka). (f) Severe damage by Glyptotermes

dilatatus, extended up to the trunk (Courtesy Tea Res.Inst. Sri Lanka). (g) Alates of Glyptoermes

dilatatus (Courtesy Tea Res. Inst. Sri Lanka). (h) Damageby Odontotermes sp. (Courtesy Tea Res. Inst.

Sri Lanka). (i) Damage by Microtermes sp. (Courtesy TeaRes. Inst. Sri Lanka).

continuous. The colony of this species contains only around3000–5000 individuals. Fully

winged adults are rare. In some instances, a few alate(winged) reproductive forms have

been found inside tea bushes which have been subjected tosevere termite damage,

although their emergence from these bushes was not observed(Ranaweera, 1962). When

unfavourable conditions prevail inside a bush or when thehost bush is reduced to a mere

shell, there is a possibility of a few individuals beingoccasionally released to form new

colonies (Ranaweera, 1962).

The queen is brown and slightly larger than the othercastes, and she freely moves

within the galleries. Egg laying is not confined to aparticular region. In well-established

colonies, numbers of wingless and short-wingedsupplementary reproductive termites are

present along with eggs, larvae, nymphs and soldiers. Thenymphs (pseudogates) feed on

the live heart wood of the roots.

4.1.5 Dispersal

This species does not exist in winged form and the spreadof the colony to the adjacent

healthy bushes is only via root contact. No entry holeshave ever been reported on

branches. Groups of supplementary reproductive termites areknown to migrate towards

the adjacent bushes to form new colonies (Green, 1907;Cranham, 1966).

4.1.6 Damage to tea

Damage is formed by the nymphs (pseudogates) during feedingand gallery formation.

Since they consume only the heart wood, leaving the sapwood untouched, damage is

mostly mechanical. Infestation goes unnoticed until theentire heart wood is excavated. In

prolonged attack, only the shell of the plant remains(King, 1937).

4.2 Neotermes greeni (= Calotermes greeni)

This species was reported by Green (1890) from materialcollected in Sri Lankan (Ceylon)

tea and the pest was named after him.


So far this species is reported to be found only in SriLanka and occurs sporadically at

elevations ranging from sea level up to 1500 m. At lowerelevation tea areas in Sri Lanka, this

species occurs concurrently with G. dilatatus (Vitarana,2003, Vitarana, 1986/Mohotti, 2008).


Grevillea robusta A.Cunn. ex R.Br is the preferred host.Occasionally it attacks Albizia

falcata (L) Backer and Erythrina lithosperma Miq.


During drought, the branches wilt and dry branches appear.An inspection of affected

branches shows honeycomb-like network of galleries. Thesegalleries are not continuous

and do not extend beyond the collar region. Inside thegalleries, soft-bodied stages of the

termites could be seen.


As in the case of G. dilatatus, the winged adult pairenters the host at a weak point in the

branch or the trunk to initiate colony formation. Duringthe process of development, the

heart wood of the stems and branches is eaten in a regularhoneycomb that looks like a

network of galleries which are narrower than those made byP. militaris. The size of the

colony is the same as P. militaris, but the number ofsupplementary reproductive termites

is low.

4.2.5 Dispersal

Dispersal is made by the flight of the adult winged pair.

4.2.6 Damage to tea

Attack on tea is usually confined to few isolated bushes.Damage occurs during feeding

and gallery formation by the nymphs. It does not completelyhollow out the heart wood

and in the advanced stage of attack, the sap wood and barkare also eaten, thus interfering

with the uptake of water causing wilting and drying ofbranches. The galleries rarely enter

below the soil level (Cranham, 1966).

4.3 Glyptotermes dilatatus

Glyptotermes dilatatus, Bugnion and Popoff, is the mostimportant live wood termite pest

of the tea plant and was first recognized as a pest of teain 1908. It was observed in the

lower elevation tea-growing areas of Sri Lanka, ataltitudes below 600 m. This pest was

earlier referred to as Kalotermes dilatatus (Bugnion andPopoff, 1910; Krishna 1961) and

had been first detected in southern Sri Lanka, near thecoastal area.

It was earlier considered as an occasional and innocuouspest (Jepson, 1941). However,

damage and loss of crops became serious from the mid-1960s,with the planting of sappier

clonal tea in Sri Lanka, and the spread of infestationbecame exponential, especially

amongst the newly replanted clonal tea fields(Danthanarayana and Fernando, 1970;

Sivapalan et al., 1977, 1980).


The alternative hosts of this species are reported to be

Albizia (Albizia moluccana Riq.),

Artocarpus integrifolia. R. Forster and G. Forster, Coffearobusta L, Caryophyllus aromaticus

(L.) Merrill and Perry, Casuarina equisetifolia L, Erthrynalithosperma Miq, Grevillea robusta,

Hevea brasiliensis Mull Arg, Moringa oleifera Lam andTheobroma cacao L.


The presence of G. dilatatus is often detected at pruning,when the prune cuts reveal the

tell-tale signs of typical galleries. In advanced stages ofattack, the galleries extend into the

conducting tissues, interfering with uptake of water,resulting in early wilting of affected

branches during dry weather and the mature leaves having acoppery texture. During

prolonged dry weather, the affected branches die, and thedead branches are readily

noticed in the field (Fig. 2b). When the galleries reachthe sap wood, they cause damage to

the bark and the wounds callous over, giving rise to thecharacteristic pitted appearance on

the base of mature branches and the collar region(Vitarana, 1986/Mohotti, 2008).


The biology of this pest was studied by Jepson (1926,1929), Pinto (1941), Cranham (1966),

Danthanaryana and Fernando (1970), Sivapalan and Seneratne(1977) and Sivapalan et al.

(1977).

A colony is initiated by the winged swarming adult termites(Fig. 2c), which enter the

aerial part of the host plant (trunk or branch) onlythrough the pruning snags that have

died back and suffered wood rot and not through clean-cutsurfaces nor through branch

tips that have died back but not formed wood rot (Sivapalanand Seneratne, 1977).

Volatile and non-volatile compounds present in these rottedstumps are known to attract

the swarmers (Samarasinghe et al., 1999; Senenayake et al.,2015). There could be more

than one pair of colony founders exploring these weakspots. Having located such weak

points, these swarmers mate after de-alation and beginburrowing the initial gallery cell to

deposit the eggs. The galleries formed are small, likethose of N. greeni, but less regular in

size and pattern and have a ‘honey-combed’ appearance (Fig.2d). Two or more colonies

can exist within a branch. The hatched nymphs transforminto different castes, including

soldiers and supplementary reproductive termites (neotenicnymphs), and the rest remain

as immature nymphs, performing the duties of ‘workers’(pseudogates). The nymphs

(pseudogates) (Fig. 2e) continue excavating galleries deepinto the branch and progress

towards the main trunk and collar but rarely extend intothe roots (Fig. 2f) (Jepson, 1941;

Ranaweera, 1962; Sivapalan et al., 1977; Sivapalan andSenaratne, 1977).

A few of the young nymphs themselves become reproductive(neotenic nymphs) and

commence egg laying. Within a relatively short period ofone or two years, the entire bush

frame is excavated by galleries and once these reach thebase of the trunk (collar), further

tunnelling ceases and excavation into the roots is avoided.

When the galleries reach the base of the trunk (collar),due to overcrowding and the

shortage of available food, a large proportion of thenymphs metamorphose into mature

swarming adults (Fig. 2g).

4.3.4 Dispersal

Dispersal is by swarming of winged adults. Althoughswarming is staggered, the

transformation to the winged adult is reported to beinfluenced by the weather. The

winged adults swarm, mainly at dawn and increasingly duringhumid weather, especially

prior to the onset of the long monsoonal months. Too muchwet weather decreases

swarming. Apart from the weather, swarming is influenced bythe number of tea bushes in

advanced stages of attack.

4.3.5 Damage to tea

As in the other two cases of live wood termites attackingtea, damage occurs primarily

during feeding and gallery formation. Initially, thepseudogates eat into the heart wood.

Later, with expansion of the galleries and an increase incolony size, it eats into the sap

wood and bark, causing death of branches and even entirebushes. Damaged bushes are

usually scattered and not in groups within a field.

5 Scavenging termites

The following sections discuss scavenging termites.Scavenging termites are those which

cause indirect damage to tea. They form nests in the soiloutside the plants, in mounds,

underground or in ‘carton nests’ in hollow tree trunks andfeed on dead and decayed wood

and rotted bark and snags, thus serving as secondary pests.On a few occasions direct damage

has also been observed (Cranham, 1966). During the processof such feeding on dead and

moribund tissues of young plants caused by collar rot, deepplanting, damage by chafer

beetle larvae, mechanical damage and adverse physiologicalconditions due to drought or

waterlogging, a certain amount of healthy tissues are alsoconsumed (Ranaweera, 1962).

Scavenging termites, however, do have some social benefits.They break down woody

plant materials and return nutrients to the soil. Moundsand other structures built by

termites are usually enriched in soil organic matter andfine particles (Cranham, 1966; Holt

and Coventry, 1990).

These termites belong to the higher termite familiesRhinotermitidae and Termitidae

which form highly specialized colonies.

Rhinotermitidae are moist wood termites and have a highlyorganized social organization,

with thousands of individuals in a colony which includesworkers, soldiers and reproductive

termites. They do not build mounds but have nests in thesoil or old tree roots below

ground (Cranham, 1966).

Termites belonging to Termitidae have also highly organizedcolonies in the ground, or

in mounds above ground or in ‘carton nests’ in hollow treetrunks (Cranham, 1966).

The scavenging termites indirectly reported to be injuriousto tea include Coptotermes

ceylonicus Holmgren, Hospitalitermes monoceros König,Odontotermes ceylonicus

Wasmann, Odontotermes horni Wasmann and Odontotermesredemanni Wasmann.,

Microcerotermis, Ancistrotermes sp. and Pseudacanthotermessp.

Dispersal is by flight. Older workers also move in searchof food and form colonies aided

by secretion of pheromones.

5.1 Coptotermes ceylonicus

Coptotermes sp. belongs to the family Rhinotermitidae.Unlike Kalotermitidae, termites

belonging to the family Rhinotermitidae have well-developedsocial organization and

consist of thousands of individuals in a colony withdistinct castes of workers, soldiers and

reproductive forms.


Coptotermes ceylonicus Holmgren, 1911 is native to SriLanka (Cranham, 1966) and south

India (Ananda-Rao, 1939). In Sri Lanka it is mostly foundat elevations below 900 m and

occasionally up to 1400 m. Coptotermes sp. is also reportedin the tea areas of Bangladesh

and Indonesia (Damiri, 2014). The species reported in

Bangladesh is Coptotermes heimi

Wasmann (Ahmed, 2014).


The presence of termites may be indicated by earth-coveredrunways and the Coptotermes

runways are often soil lined and flattened in appearance(CABI, 2017).


This is a subterranean scavenging termite and does notbuild mounds. It forms nests in the

soil or in old tea roots below the ground (Jepson, 1930;Vitarana, 2003). It is also known

to attack buildings. When disturbed, the soldier ejects amilky white fluid from a gland in

the head (Cranham, 1966).

5.1.4 Damage to tea

Although it is a scavenging termite, it is known tooccasionally damage healthy roots and

branches and therefore is usually included along with livewood termites (Cranham, 1966).

Ananda Rau (1939) reported this termite to be a pest ofliving tea bushes in south India.

Such attacks on live tissues perhaps occur in substandardor pest attacked nursery plants

where the roots are damaged and/or with rotted ends, or inmature bushes weakened due

to continuous environmental climatic stress.

5.1.5 Dispersal

Dispersal is by flight of adults as well as by human-aidedtransport.

5.2 Hospitalitermems monoceros

Hospitalitermems monoceros König 1779 belongs to the familyTermitidae which form

highly socialized colonies. This species is commonly knownas the black termite of Ceylon

(Sri Lanka).

5.2.1 Geographic distribution

This species is native to Sri Lanka. Although recently thepresence of this species has been

documented in Western India, there are no reports of itsinvasion into tea gardens (Poovoli

et al., 2014).


It forms carton nests in the hollow of trees or sometimesattaches to branches. They

forage on the open ground, unprotected by earthengalleries, particularly at night

(Cranham, 1966). During day time they are mostly confinedto their nests (Poovoli et al.,

2014).

5.2.3 Damage to tea

These are secondary pests of tea plants. They have beenobserved to frequent sick tea

bushes, but do not cause damage to living tissues(Ranaweera, 1962).

5.3 Odontotermes sp.

This is a subterranean termite and belongs to the familyTermitidae.


This species is native to Sri Lanka and India. Odontotermessp. are found in Sri Lanka

(Cranham, 1966), south India (Anon, 2015), northeast India,(Barthukar, 2011), Bangladesh

(Ahmed, 2014) and Africa (Rattan, 2000).


The species O. redemanni Wasmann 1893 is reported to buildmounds, whilst

Odontotermes ceylonicus Wasmann 1902 and O. horni Wasmann,1902 nest in the soil

without building mounds (Cranham, 1966). O. redemanni isoften found along with other

ground-inhabiting termites.

In addition to being a scavenger, O. redemanni also attacksbuildings (Vitarana, 1986/

Mohotti, 2008). In Sri Lanka they are mostly found inlow-country tea estates.

5.3.3 Damage to tea

O. horni is a subterranean termite and attacks the fallenbark of living trees but does

not damage living tissue. It is known to make earthensheet-like covers on the wood of

trees underneath which it feeds (Vitarana,1986/Mohotti,2008). Although it is a scavenging

termite, it was reported to attack the tender roots ofnursery plants and new clearings

during water stress. They have been observed to kill youngnursery plants by chewing

young roots and stems (Ranaweera, 1962; Cranham, 1966)(Fig. 2h).

5.4 Microcerotermis sp.


Microcerotermis species belongs to the family Termitidaeand was originally reported in Sri

Lanka, northeast India, Africa, Indonesia, Bangladesh andMalaysia. The species reported

in Sri Lanka is Microcerotermis greeni Holmgren. and thatin Bangladesh Microcerotermes

championi Snyder.


Microcerotermes greeni Holmgren, 1912, is a subterraneanspecies which builds nests

in soil and does not build mounds, whilst some otherspecies of this genus are known to

build carton nests (Ranaweera, 1962; Cranham, 1966).

5.4.3 Damage to tea

A species of Microcerotermis has been reported to attackthe live wood of tea in north

India (Das, 1958) but there is no evidence of this speciesattacking tea in Sri Lanka. They

attack the tea bushes on wood-rotted ends as well as smallcracks or holes on branches

formed by SHB. Often, infested plants in the field may becovered with soil runways (Fig.

2i) under which termites may be found. If infestationoccurs at the nursery stage, it causes

sudden wilting and death of bushes (Nada et al., 2013).

5.5 Ancistrotermes sp.


Ancistrotermes sp. including A. latinotus Holmgren, 1912,has been mostly reported in

the tea areas in Africa. Ancistrotermes pakistanicus Ahmedwas reported in Bengal and

northeast India.

5.5.2 Damage to tea

This species mostly feed on woody litter and plant debris.However, in Malawi and Africa,

widespread damage to tea has been reported. It is known toenter roots or the collar

region of bushes below ground and penetrate into the branch(Rattan, 2000).

5.6 Pseudacanthotermes sp.

This species (including P. militaris Hagen) belongs to thefamily Termitidae (sub-family

Macrotermitinae). It is a subterranean fungus growingtermite and its distribution is patchy

in tea areas. It is widely spread in sugar cane plantationsin Africa. It is a litter feeder, but

has occasionally been reported to cause damage to tea inMalawi, Kenya, Tanzania and

Uganda (Benjamin, 1968a,b; Rattan, 2000).

6 Nematodes

Nematodes belong to Phylum Nematoda. These aremulticellular translucent worm

like organisms with a tubular digestive system open at bothends. They exist in a wide

range of habitats. Those that are parasitic on plants aremostly microscopic and feed

on different parts of the plants. The nematodes that areparasitic to tea are all root

feeders.

There are several species of plant-parasitic nematodesidentified from different tea

growing regions of the world. Amongst these, the ones whichhave been confirmed to

cause economic damage to tea include Meloidogyne spp,

Goeldi, 1892; Pratylenchus

loosi Loof, 1960; Pratylenchus brachyurus Godfrey, 1929,Filipjev and Schuurmans

Stekhoven 1941; Radopholus similis Cobb, 1893 andHemicriconemoides kanayaensis

(Nakasono and Ichinohe 1961; Sivapalan, 1972; Gnanapragasamand Mohotti, 2005;

Gnanapragasam, 2014).

6.1 General symptoms of root-feeding nematodes

The above-ground symptoms of all root-feeding nematodes arevery similar. The affected

plants appear unthrifty with poor foliage and weakenedframes and sometimes defoliation.

The leaves are dull and pale, mainly due to the nutritionalimbalance resulting from poor

intake of essential nutrients by the damaged roots. Theheavily infested plants are stunted

with premature flowering and fruiting (Fig. 3a).

The most widely distributed species of nematodes in thetea-growing areas of the world

are the root-knot nematodes, Meloidogyne sp. This isfollowed by the Pratylenchus sp.

6.2 Root-knot nematode, Meloidogyne sp.

Meloidogyne sp. belong to the order Tylenchida, familyMeloidogynidae.

Prior to 1949, the root-knot nematodes were all known asHeterodera marioni, Cornu,

1879, Goodey. In 1949, Chitwood published his revision andgave the generic name of

Meloidogyne, which was the name given by Goeldi in 1887 tothe root-knot nematode of

coffee roots in Brazil (Chitwood, 1949, Moens et al., 2009).

Figure 3 (a) A heavily nematode-infested mature tea plantshowing stunted growth, defoliation and

premature flowering. (b) Meloidogyne brevicauda infestedtea roots with large galls (Courtesy Tea Res.

Inst. Sri Lanka). (c) Advanced stage Meloidogyne brevicaudainfestation on tea roots, showing root

galling and death of roots (Courtesy Tea Res. Inst. SriLanka). (d) A young tea plant heavily infested

with Pratylenchus loosi showing stunted growth, pale leavesand premature flowering and seeding.

(e) Roots of a tea plant heavily infested with Pratylenchusloosi, showing sparse root growth with brown

dried-up roots. (f) Brownish lesions formed on storageroots of tea by Pratylenhus loosi. (g) A tea field

showing vacancies due to death of nematode infested plants.

The only species of Meloidogyne which attacks mature tea isMeloidogyne brevicauda

Loss (1953a). All other species of Meloidogyne sp. attackonly the young tea plants, mainly

in nurseries, whilst the mature tea plant developsresistance by about 12–14 months of

age (Sivapalan, 1972; Gnanapragasam and Mohoti, 2005;Gnanapragasam, 2014).

6.3 Root-knot nematode of mature tea, Meloidogynebrevicauda

This species was identified by C. A. Loos in 1953 in SriLanka (Loos, 1953a). Amongst the

different species of root-knot nematodes, M. brevicauda isthe only one which feeds on

both young and mature tea. As such, during earlier years,it was suspected of belonging to

a specialized race of M. javanica (Gadd and Loss, 1946).However, later it was confirmed as

a separate species and referred to as Meloidogynebrevicauda, Loss, 1953.


Sri Lanka (Loos, 1953a; Sivapalan, 1972); Coonoor, southIndia (Venkata Ram, 1963; Mehta

and Somasekhar, 1998; Muraleedharan and Selvasundaram,2001); West Bengal (Mukerjea

and Dasgupta, 1982) and Fujian, China (Changshang, 1984).


Apart from tea, it has been reported so far only in twohosts, viz. Saffron (Crocus sativus),

only from Azerbaijan (Kasimova and Atakishieva, 1980) andMorinda officinalis How

(medicinal Indian mulberry) from Fujian, China (Changshang,1984).


Infestation with M. brevicauda is recognizable by theformation of typical tumour-like galls

on the roots which are larger than those formed by otherMeloidogyne sp. and many of

the infected roots display pinhole pits (Fig. 3b and 3c).Though rare, the pear-shaped

females can be occasionally seen protruding from the eggmasses. As in the case of

other Meloidogyne sp., it has a distinct perineal pattern(a unique structure located at the

posterior end of the female near the anal-vulva region)which helps in identification under

the microscope. The above-ground symptoms of damage arevery similar to those induced

by other pathogenic nematodes. The effect of infestation inbushes is most evident during

the period of recovery from pruning (Loos, 1953a;Sivapalan, 1972).


M. brevicauda was first described by Loos in 1953 (Loos,1953a) and thereafter additional

morphological features of this species were described byEisenbach and Gnanapragasam

(1986, 1992).

Early investigations in Sri Lanka revealed the presence ofthis species of nematode only

in mature seedling tea fields (Loss, 1953a). Subsequently,some clonal tea was observed

to be infested (Gnanapragasam et al., 1985).

This is a sedentary endoparasite. The life cycle of M.brevicauda is very similar to that

of other Meloidogyne sp. The female lies embedded in thehost tissue and lays eggs in

a gelatinous matrix. The first-stage juvenile is inside theegg case and it moults into a

second-stage juvenile which is motile and this is the onlyinfective stage.

These second-stage infective juveniles move into the soiland penetrate the root behind

the root cap and commence feeding. Like all plant-parasiticnematodes, this species also

possesses a stylet for injecting secretions into the hostas well as ingesting nutrients from

host plant cells. The secretion of salivary juices excretedvia the stylet induces the formation

of giant cells which serve as feeding sites. Continuousfeeding enlarges the giant cells and

the proliferation of neighbouring cortical cells, leading

to the formation of tumour-like galls.

As the potential female larvae mature within the roottissue, they change shape from the

typical vermiform shape to a sausage-shaped form and thenfinally to a sexually mature

pear-shaped form and continue to remain sedentary. When thefemale deposits her eggs,

she ceases to feed and the galled tissues gradually dry up(Sivapalan, 1967).

The pear-shaped females of M. brevicauda are very large andabout 5–6 times the size

of the female M. incognita. Mean hatch per egg mass is onlyaround ten, whilst in other

common species of root-knot nematode, this is of the orderof 200–600 juveniles/egg

mass (Gnanapragassam and Manuelpilai, 1981). Males arevermiform but extremely rare.

M. brevicauda is sensitive to warmer temperatures and isencountered only in very cold

climates. In Sri Lanka it has been restricted to only threeestates, at altitudes of 1500–

2000 m with very low soil temperatures, that is, 12–14°C.

Detailed studies carried out in controlled soil temperaturetanks showed rapid build-up

and successful parasitism at 12°C, and at highertemperatures there was a decline in the

population, which coincided with field observations(Gnanapragasam, 1988). With the

change in climate in the cooler regions of Sri Lanka, thespread of this species is reported

to be decreasing (Mohotti, 2009).

6.3.5 Dispersal

The spread of infestation is very slow and infection ismainly by the second-stage juveniles

which are free-living. They either re-invade the originalhost or move to adjacent plants.

Other means of dispersal are movement of plant material andsoil debris.

6.3.6 Damage to tea

Gall formation on the roots and feeding are by females.Damage to attacked roots can

be due to mechanical damage during feeding or invasion, anddue to withdrawal of

nutrients and/or physiological and biochemical damage dueto secretion of salivary juices.

Damaged roots reduce the rate of uptake of nutrients andwater. Secondary pathogens

aggravate the damage further.

6.4 The root-knot nematodes of young tea


The first report of root-knot nematode infestation in youngtea was from south India,

where it was found to infest large numbers of tea seedlings(Barber, 1901). In Sri Lanka,

it was reported in seedling tea in 1928 (Stuart Light,1928) and since the 1960s, with the

propagation of only clonal cultivars, it is rarelyencountered in tea in this country. The

common species that attack tea are M. incognita, Kofoid andWhite, 1919, Chitwood, 1949,

M. javanica, Treub, 1885, Chitwood, 1949 and M. arenaria,Neal, 1889, Chitwood, 1949.

On the other hand, M. hapla Chitwood, 1949 was rarely foundto infest tea (Banerjee, 1967;

Gnanapragasam, 1985). The distribution of different speciesin the different tea-growing

countries is reported in Gnanapragasam and Mohotti (2005)and Gnanapragasam (2014).

6.4. 2 Symptoms of damage

The root-knot nematode of young tea forms galls on both thetap root and the feeder

roots of young tea plants (Gadd and Loos, 1946). Thesegalls are very much smaller than

that of M. brevicauda. On closer inspection of the affectedroots, very often the pearl

shaped minute females can be seen protruding from theepidermis. This species can also

be identified under the microscope by checking the perinealpatterns of the females.

Above-ground symptoms are similar to those formed by otherplant-parasitic root

feeders.


The biology and ecology of root-knot nematodes have beenstudied by several scientists

on different hosts. Although the host−parasiterelationships of root-knot nematodes

species may vary in the different host plants, their lifecycle is almost the same.

These species have four larval stages and an adult stage.The larvae and the adult males

are vermiform, whilst the adult females are rounded orpear-shaped. The first larval moult

is within the egg. The second-stage larvae has a shortfree-living stage in the soil in the

rhizosphere area. This second-stage juvenile represents theinfectious stage and it can

move and invade fresh roots or re-invade the roots of theoriginal host, usually at the root

tips, causing some of the root cells to enlarge forminggiant cells on which the nematodes

feed and develop. The free-living second-stage larva doesnot feed until it invades the

root. The male nematodes remain vermiform and eventuallyleave the roots, but the

females undergo morphological stages and become rounded andremain embedded in

the roots of the host, laying their eggs into a gelatinousmass (egg sac). The number of

eggs laid in the egg sacs may vary according to the species.

Most root-knot nematodes (other than species such as M.brevicauda) are well adapted

to different climatic and soil conditions and the length ofthe life cycle is temperature

dependent. In China, the optimum soil temperature for pestincidence in tea has been

reported to be 20–30°C, in soils with 20% moisture (Rong etal., 1984).

6.4.4 Dispersal

Same as M. brevicauda.

6.4.5 Damage to tea

Damage is only to young tea up to the age of 12–14 monthsand is less severe and only

small galls are formed. The devitalized root cap and gallformation on the roots affect

absorption of nutrients and water, thus weakening thebushes.

6.5 Root lesion nematodes, Pratylenchus sp. Filipjev 1936Pratylenchus loosi Loof 1960

Pratylenchus loosi belongs to the order Tylenchida, familyPratylenchidae. P. loosi was

earlier referred to as Tylenchus pratensis by de Man in1884, as T. gulosus by Kuhn in

1889 and thereafter as Anguillulina pratensis de Man (Gadd,1939). Later, the name was

changed to Pratylenchus pratensis de Man (Gadd, 1947c) andthen to Pratylenchus coffeae

Zimmermann (Loos, 1953b). It is now referred to asPratylenchus loosi Loof (Hutchinson,

1961). The last name, Pratylenchus loosi, was given inrecognition of the fact that as a

species, it differs from P. coffeae and as a tribute to Mr.Clive Loos from Sri Lanka who

carried out much of the earlier work on this nematodespecies.


Pratylenchus loosi is endoparasitic on tea, widelydistributed around the world and one

of the most serious pests of tea in Sri Lanka (Gadd, 1939;Gadd and Loos, 1946); Loos,

1953b; Sivapalan, 1972). It is also reported to causedamage in China (Chen and Chen,

1982; Li, 1985), Bangladesh (Khan et al., 2003, 2006),Japan (Kaneko and Ichinohe, 1963;

Takagi, 1967, 1969; Ohta et al., 2014), Iran (Maafi, 1992;Hajieghrari et al., 2005), north

India (Mukherjea and Dasgupta, 1982) and Korea (Park etal., 2002).

In Sri Lanka, although it could be found in tea fields atall altitudes, damage to tea

is mostly confined to elevations of 900–1800 m (Hutchinsonand Vythilingam, 1963;

Sivapalan, 1972). In contrast, in Japan, being located inthe cooler temperate zone, it can

be encountered in all tea areas (Takagi, 1969; Gotoh, 1976).

6.5.2 Alternative host range

There are several alternative hosts of this species(Gnanapragasam and Mohotti, 2005).


The above-ground symptoms of damage are very similar tothose caused by all other

endoparasitic nematodes, which have been described above(Fig. 3d).

Roots of infested plants are stunted, sparse and brownishand dried-up (Fig. 3e). Dark

brown necrotic patches or lesions of varying sizes aredisplayed on peeling the bark of the

larger storage roots, hence its name root lesion nematode(Fig. 3f) (Gadd, 1939; Visser,

1959; Sivapalan, 1967, 1972; Gnanapragasam, 1986/Mohotti,2008; Gnanapragasam

and Mohotti, 2005). Since they are microscopic, detailedidentification can be made only

under the microscope.


P. loosi is a migratory endoparasite and attacks both youngand mature tea which is found

in the root cortex. However, it is very common to encounterlarge populations in the soil

in the rhizosphere of tea, where feeder roots are present.

The existence of pathotypes of P. loosi and ‘P. loosispecies complex’ was reported by

Pourjam et al. (1997, 1999), Mohotti (1998) and Mohotti et

al. (2002).

The life cycle is similar to all other Pratylenchus sp. Alljuvenile and adult life stages

of lesion nematodes are worm-shaped and motile, and alllife stages (except the

egg and J1) can cause infection. The nematodes are mostlyattracted to the growing

parts of the roots where they penetrate and enter near theroot tips and during their

movement form tunnels in the cortex (Gadd, 1947c). Theyhave prominent stylets

which puncture the root tissues and feed by secretingenzymes during which process

lesions are formed.

When the parasitized roots are severely damaged or becomeover-parasitized, they

move into the soil in search of fresh roots. The femalesdeposit the eggs within the tunnel,

at the rate of one or two eggs per day, and the life cycleis reported to be around 40–50

days (Seinhorst, 1977; Takagi, 1969). Egg laying was foundto be delayed in the absence

of males (Gadd and Loos, 1941).

Soil population of this species varies at different timesof the year and this is directly

correlated to soil moisture and rainfall (Sivapalan, 1972).

Soil temperature plays a significant role in thedistribution. The highest population

occurs in the temperature range 18–24°C. Severepathogenicity is also observed, mostly

within this temperature range (Sivapalan and Gnanapragasam,1975). Greatest damage to

tea plants is observed in clayey soils, whilst the damageis less in gravelly and least in sandy

soils (Sivapalan, 1971). Under the controlled conditionsmaintained in the greenhouse, the

damage threshold of P. loosi was estimated to be 40nematodes per 100 g soil at 24°C

(Gnanapragasam and Manuelpillai, 1984).

6.5.5 Dispersal

All larval stages (other than J1) of males and females movein the soil in search of

fresh feeder roots. Such movement, however, is only over ashort distance. Important

means of dispersal to hitherto uninfested areas are by thedissemination of infested

plants from contaminated nurseries, movement of infestedsoil and water, poor soil

conservation measures adopted in infested areas and use ofcontaminated irrigation

water in nurseries.

6.5.6 Damage to tea

Damage to tea is caused during root penetration, movementthrough the cortex and

feeding activities. The roots of both young and mature teaare damaged by this pest,

and once infected, they remain in the roots during thehost’s life time (Sivapalan, 1972;

Gnanapragasam, 1986/Mohotti, 2008). Secondary damage iscaused by invasion of fungi,

causing disease complex (Arulpragasam and Adaikkan, 1983;Hoseini et al., 2010). In Sri

Lanka, disease complexes with SHB beetle are oftenencountered in the warmer regions,

accentuating the damage further (Gnanapragasam, 2002, 2014).

The infestation of P. loosi in mature tea is normally inpatches (Fig 3g) and decline in

crops brought about by this pest ranges between 4 and 35%depending on the population

density and the environmental factors. The extent of damageis, however, greater in

nurseries and young clearings, where the casualties canrange between 60 and 100%

(Gnanapragasam, 1988).

Under adverse conditions, significant populations ofnematodes found in such storage

roots undergo a stage of inactive hibernation whereby theycan remain viable for as long

as 3–4 years within the storage roots.

6.6 Root lesion nematode, Pratylenchus brachyurus


This species is restricted to only a few tea-growingcountries. It is reported in Assam, India

(Basu, 1968; Mukherjee and Dasgupta, 1982), Malawi(Corbett, 1967) and Vietnam (Ryss

and Fam Tkhan’ Bin’, 1989).


This species has a very wide host range and some of theimportant hosts are reported to be

coffee (Coffea L), peanuts (Arachis hypogaea, L), pineapple(Ananas comosus (L.) Merr), potato

(Solanum tuberosum L), soybeans (Glycine max (L.) Merr),sugar cane (Saccharum officinarum

(L.)), tomatoes (Solanum lycopersicum L), citrus (Citruslimon (L.)) and cotton (Gossypium

barbadense L), tobacco (Nicotiana tabacum L) and peaches(Prunus persica (L.)).


Above-ground symptoms are similar to those formed by allother root-feeding termites.

Dark red necrotic patches are observed on the infestedroots.

6.6.4 Biology and life cycle

This is also a migratory endoparasite. P. brachyurus causesdamage to only young tea,

about 1- to 3-year-old plants. Life cycle is very similarto that of all the other Pratylenchus

sp. The first-stage juveniles hatch within the egg.Infestation is by all juvenile stages (other

than J1) and they enter the root and move throughout,laying eggs singly in the root

cortex and a few in the soil. Males are rare.

This species is reported to survive long periods ofdrought, during which they remain

quiescent (Basu, 1968).

6.6.5 Dispersal

Similar to that of P. loosi.

6.6.6 Damage to tea

As in the case of other Pratlylenchus sp., damage is causedduring movement into the

deep tissues and also during feeding. During feeding, itmoves deep into the root tissues,

and induces the formation of lesions on the epidermallayer. But since they are found in

restricted areas, damage is not widespread. Secondarydamage can be caused by invasion

of other pathogens.

6.7 Burrowing nematode, Radopholus similis

This is a migratory endoparasitic nematode of tea and feedson both young and mature

tea.


Radopholus similis is reported on tea from only a fewcountries: Zimbabwe, South Africa

(Keetch and Buckley, 1984) and Sri Lanka (Sivapalan, 1968;Gnanapragasam, 1983, 1990).


This species of nematode is distributed worldwide in bananaMusa Carl Linnaeus, 1753 (other

than in west and central Africa) and in many other cropssuch as citrus (Citrus L), avocado

(Persea americana Mill), coconut (Cocos nucifera L), pepper(Piper nigrum L), arecanut (Areca

catechu L), sugar cane (Saccharum officinarum L) andhorticultural crops such as Anthurium,

Anthurium acaule (Jacq.) Schott. Holdeman (1986) hasreported 365 hosts.


Above-ground symptoms are similar to those caused by otherroot-feeding nematodes

(Sivapalan, 1968). The roots of infested plants are sparseand dry. Lesions are observed on

roots and are smaller than those formed by P. loosi on tea(Gnanapragasam, 1983).


The biology of this pest is very similar to that of P.loosi. Although fertilization takes place,

parthenogenesis also occurs. Unlike Pratylenchus sp.,infection of the host is only by

the motile juveniles and females. Males have weak styletand thus do not feed and are

therefore not infective. It is an obligate parasite andneeds living tissues to survive and

cannot remain in the soil for long periods. However, as inthe case of Pratylenchus sp., it

can move in and out of the host and soil.

This species is quite sensitive to low temperatures.Uniformly distributed, high

rainfall favours build-up of the population, whilst verywet or dry soils cause decline in

the population (Gnanapragasam, 1993). Damage to tea wasfound to be significantly

worse in gravely, sandy and loamy soils, while populationbuild-up is poor in clayey soil

(Gnanapragasam, 1994).

Results of pot experiments carried out at 25 ± 1°C revealedsevere damage to tea

brought about by a low initial population level of 28nemas/100 g of soil. When exposed to

additional environmental stress, such as other pest attack,drought and poor soil condition,

the damage threshold level could be even lower(Gnanapragasam and Hearth, 1989).

6.7.5 Pathotypes

Field observations and differential host trials indicatedthe existence of different biological

races of R. similis in tea areas (Gnanapragasam et al.,1991; Gnanapragasam, 1994), which

were later confirmed by molecular analysis of the differentraces (Hahn et al., 1994).

6.7.6 Dispersal

Similar to that by P. loosi.

6.7.7 Damage to tea

Damage to tea is very similar to that caused by P. loosiand females attack both young

and mature tea. However, infestation is not widespread andR. similis are found only in

the warmer tea-growing areas. As in the case of P. loosi,several casualties have been

observed in nurseries and new clearings, whilst in maturetea areas, infestation is only in

patches. The lesions formed are usually smaller than thoseof P. loosi.

6.8 Hemicriconemoides kanayaensis

Hemicriconemoides kanayaensis belongs to the familyCriconematidae and was reported

as one of the important nematode pests of tea in Japan(Nakasono and Ichinohe, 1961;

Takagi, 1969) and Taiwan (Sivapalan, 1972; Chen et al.,2007). In the tea areas of Taiwan,

in addition to H. kanayaensis, H. californianus has alsobeen reported (Chen et al., 2007).

Hemicriconemoides mangiferae are reported from the teaareas of northeast India

(Bhattacharya et al., 2012) and Bangladesh (Khan et al.,2003).


Tea is the only reported host of H. kanayaensis (Takagi,1969).


This is an ectoparasitic nematode. Root damage to tea is in

the form of sloughing off of

the root cortex, revealing a brownish discoloured stele(Takagi, 1969). The above-ground

symptoms are stunting and yellowing of foliage.


These are obligate migratory ectoparasites and attackmostly the rootlets of the tea plant.

The major portion of a population is female, with only 0.6%males. Males and females

have a cylindrical body tapering at either end. Femaleshave a cuticular sheath attached

at the anterior terminus and vulva region. Males are moreslender than the female and are

devoid of sheaths.

Oviposition takes place over a period of 15–20 days duringJune/July. The entire life cycle

is reported to be completed in 100 days (Kaneko andIchinohe, 1963; Takagi, 1969). There

are four juvenile stages without a sheath. The second-stagejuvenile (J2) is the infective

stage in which the termites hatch from the eggs, feed onthe root and thereafter moult into

J3 and J4. The J4 develops into either a female with asheath or a male without a sheath.

Males do not feed, whereas the juveniles J2–J4 feed onroots. Males and juveniles, along

with females, can be found in soil. The females arepartially embedded, with the anterior

portion of their bodies inside the root and they feed oncortical cell tissue.

Maximum populations are encountered at a depth of 30 cm.

6.8.4 Damage to tea

Damage to tea occurs mainly during feeding activity andinvasion by other pathogens. Large

populations of this nematode have brought about severe croploss. An increase in nitrogenous

fertilizer is reported to reduce populations of thisspecies of nematode (Kaneko and Ichinohe,

1963; Takagi, 1969). Not much work on this pest has beendone over the last few decades

and thus the current status of this pest and the extent ofthe damage it causes are not known.


The currently available detailed knowledge on the lifehistory and host plant and natural

enemy interaction could be further strengthened by carryingout in-depth studies on any

changes in behaviour of pest–host interaction. Some aspectsof this work are already in

progress for tea pest as well as other pests. Properliterature survey and interaction with

other scientists working on these lines of studies would behelpful.

8 Acknowledgements

I extend my sincere thanks to Professor Chen Zong Mao,Academician, Tea Research

Institute, Chinese Academy of Agricultural Scinces,Hanzhou, China, Professor S.

Kodomari, Senior Consultant of World Tea Union (in China),Dr. Baruah, Director of the

Tocklai Tea Research Institute and Dr. Somnath Roy(entomologist), Tocklai Research

Institute, Jorhat, Assam, India; Dr. Muraleedharan, pastDirector of the Tocklai Research

Institute; Dr. Radhakrishnan, Director, UPASI Tea ResearchFoundation, South India; Dr.

Keerthi Mohotti, Deputy Director Research and NematologistTea Research Institute, Sri

Lanka; Mr. Dharmampriya Samansiri, Head Advisory andExtension Division Tea Research

Institutr, Sri Lanka; and Mr. Ajith Prematunge, ResearchOfficer, Entoology Division of

the Tea Research Institute of Sri Lanka for providing mewith photographs of insects and

publications in their respective institutes. Last but notleast I thank my brother Lakshman

Gnanapragasam for helping me to organize the photographs.

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10 Chapter 10 Insect pests of tea:caterpillars and other seasonal,occasional and minor pests

1 Introduction

This chapter is one of the two chapters providing acomprehensive review of insect pests

of tea. The pests of tea can be divided into fourcategories:

• Perennial pests: Perennial pests are ideally adapted to aparticular environment where they find a permanentenclave. They remain present in the tea bush and reachinjurious population levels every year. They cause damageto the tea bush over varying periods with gradual croplosses leading to death of the tea bushes. Pests includethe shot hole borer (Euwallacea fornicatus Eichhoff, 1868),live wood tea termite (Glyptotermes dilatatus Bugnion andPopoff, 1910, Postelectrotermis militaris Desneux, 1904,Neotermes greeni Desneux, 1904) and pathogenic nematodes(Pratylenchus loosi Loof, 1960, Radopholus similis Cobb,1893, Thorne 1949, Meloidogyne brevicauda Loos, 1953).

• Seasonal pests: Seasonal pests attack the crop onlyduring specific seasons of the year, causing temporarydeclines in the crop. These are mostly kept under controlby natural predators and parasites, and pest outbreaksonly occur when the environment becomes conducive for thepopulation to build up above damaging threshold levels.These pests, for example, leaf-eating caterpillars, ordifferent species of phytophagous mites, are widespreadglobally.

• Occasional pests: These cause only occasional economicdamage, usually due to environmental influences or humanactivities, for example, beetle grubs (Holotrichiadisparalis Arrow) or the red borer (Zeuzera coffeaeNietner, 1861).

• Minor pests: These usually do not cause economic damage,for example, tea leaf miner (Melanagromyza theae Mejere),aphids (Toxoptera aurantii, Boyer de Fonscolombe, 1841),scale insects (Coccus viridis Green 1900, Saissetia coffeaeWalker, 1852), the faggot worm or bag worm (Clania crameriWestwood), red slug (Eterusia aedea cingala Moore, 1877)or ants (Oecophylla smaragdina Fabricius, 1775).

This chapter discusses caterpillars and other seasonal

pests, and occasional and minor

pests. The companion chapter provides an introduction tothe range of insect pests before

focusing specifically on shot hole borers, termites andnematodes.

2 Caterpillars and other seasonal pests

The tea tortrix belongs to the order Lepidoptera, familyTortricidae. Amongst the tea

tortricids, the species which cause economic damage to teainclude Homona coffearia,

Adoxophyes honmai and Homona magnanima.

2.1 The tea tortrix, Homona coffearia Nietner

The tea tortrix, Homona coffearia, was first described as apest of coffee by Nietner (1861)

and subsequently, when coffee was replaced by tea, thispest transferred its activity to tea.

The first outbreak in tea in Sri Lanka was recorded byGreen (1890).


Sri Lanka (Green, 1903; King, 1933), Indonesia (Dharmadi,1983; Zeiss and Braber, 2001),

India (Hazarika et al., 2009), China (Chen and Kunshan,1987), Malaysia, Formosa (Taiwan)

(King, 1933; Baptist, 1956) and Nepal (Shrestha and Thapa,2015).


Homona coffearia is mainly regarded as a pest of tea(Camellia sinensis (L.) O Kuntze) and

coffee (Coffea L.), but being highly polyphagous, is foundin many other crops as well

(King, 1933; Van der Geest and Evenhuis, 1991).


Damage is indicated by the appearance of larval nests madeby rolling one leaf over

another with the sides spun together or by webbing togethertwo or more leaves (Fig. 1a).

In this, Homona coffearia can be distinguished fromCaloptilia theivora Wlsm., which rolls

the leaf from the tip downwards. Inside the nest, thecaterpillar or the pupal stage of the

pest may be found. The infested leaf nest appears withseveral holes, which later become

scorched and blackened. The appearance of flat yellow toorange egg masses on the

leaves also indicates infestation.


This is mostly a dry weather pest and, in Sri Lanka, ismainly confined in the elevation

range of 900–1800 m. It occurs during the period ofDecember to April, and August/

September in the southwest region and in May to June/Julyin the northeast monsoonal

areas. Tortrix outbreaks are commonly found to fluctuatethrough the years and several

years of high incidence may be followed by years of lowincidence.

The biology and life history of this pest were studied byJardine (1918), Hutson (1927),

King (1933), Gnanapragasam (1979, 1983) and Gnanapragasamand Sivapalan (1980),

and the life cycle is similar to that of any typical moth.Although earlier studies had

indicated oviposition to begin within 24 h of emergence(King, 1933), laboratory studies

consistently indicated oviposition taking place only afterthree days (Gnanapragasam,

1979), which agrees with the field studies wherein theemission of sex pheromones of

virgin females significantly increased from the second dayfrom eclosion (Sivapalan and

Vitarana, 1975).

Flat, compact pale yellow egg masses (Fig. 1b) are laid bythe female moths on the

upper surfaces of mature leaves and the colour of the eggmasses deepens with age

(Fig. 1c). The first instar larvae are tiny (1.5 mm), paleyellow in colour and very agile.

Following hatching, the majority of larvae move to thegrowing points of the bush and

begin feeding on the flush. From the second to the fifth(final) instar, they make leaf nests

with silken threads. Younger larvae are mostly found in theflush and older in both flush

and maintenance foliage. The older caterpillars aregreenish in colour with a shiny black

head and have a black horny patch on the back of the firstsegment. They are about 2 cm

in length when fully grown (Fig. 1d).

Several nests are usually constructed by one larva in thecourse of its development

and the larvae feed and grow inside the nest with pupationtaking place inside the final

nest. The adult female is small, pale brown and bell-shapedin outline and the male is

smaller and darker with more intense pigmentation (Fig.1e). The development of the

males is always faster than that of the females and themales have a shorter lifespan

(4.74 ± 1.1 days) than the females (8.1 ± 1.9 days). Adefinite rhythm for pupation and

adult eclosion was reported by Gnanapragasam and Sivapalan(1980). The sex ratio of the

emerging adults is 1:1. The development istemperature-dependent, and, in Sri Lanka,

one generation lasts about seven to nine weeks in thecooler, higher elevations, and five

to six weeks or less at warmer, lower elevations. InIndonesia, one generation was reported

to last seven to eight weeks (Simanjuntak, 2002).

2.1.5 Dispersal

Dispersal is mainly by the winged adult male and femalemoths. The newly hatched larvae

move to the other parts of the bush suspended on silkenthreads and move up to the flush

to make nests. (i) (j)

(k) (l) (m)

(a) (b) (c)

(d) (e) (f)

(g) (h)

2.1.6 Damage to tea

Damage to tea is caused by the larvae during the formationof leaf nests and feeding

activity. The adult moths do not feed. The larvae nibblethe leaf at random, moving from

one spot to another. At first, the leaf nest completelycovers the caterpillar, but later,

several holes are formed and the nest appears scorched

(Fig. 1f) and the leaves are no

longer fit for harvesting. They do not completely defoliatethe bushes as in the case of

nettle grub attack.

2.2 The smaller tea tortrix, Adoxophyes honmai


Adoxophyes honmai Yasuda and Homona magnanima Diakonoff arereported from Japan

(Akira, 1997), China (Chen and Kunshan, 1987), Taiwan andVietnam (Razowski, 2008).

Adoxophyes honmai is reported as the major pest of tea incentral and southern Japan,

whereas Homona magnanima is more prevalent in southernJapan (Hazarika et al., 2009).

All three species have the same type of lifestyle andfeeding habits.


This is mainly a pest of tea, but it is also known to occuron many other trees and shrubs

(Yasuda, 1998).


Symptoms are similar to those of Homona coffearia. The typeof leaf nest with the larvae

inside can be easily identified. The leaves are notentirely stripped but have holes due to

feeding and a scorched appearance.


Adoxophyes honmai is often referred to as the smaller teatortrix. The life histories of

Homona coffearia, Adoxophyes honmai and Homona magnanimaare very similar.

The female lays pale yellow oval-shaped egg masses on theundersurface of young tea

leaves. The newly hatched larva is pale yellowish whitewith a dark brownish head and

has a body length of about 1.5 mm. Subsequently, the bodycolour changes to green

or yellowish green, and mature larvae grow to about 20 mmin length and have a brown

to yellowish-brown head (Yasuda, 1998) (Fig. 1g). Adultmales have ochreous-yellowish

forewings marked with dark brown, with paler hind wings(Fig. 1h). Females have tawny

ochreous forewings with fewer markings than the males (Fig.1i). Hind wings are yellowish.

Figure 1 (a) Leaf nest of Homona coffearia (Courtesy TeaRes. Inst. Sri Lanka). (b) Egg mass of Homona

coffearia – (early stage). (c) Mature egg mass of Homonacoffearia. (d) Larvae of Homona coffearia

(Courtesy Tea Res. Inst. Sri Lanka). (e) Adult Homonacoffearia (female – left; male – right) (Courtesy Tea

R es. Inst. Sri Lanka). (f) Severe damage to tea by Homonacoffearia (Courtesy Tea Res. Inst. Sri Lanka).

(g) Larvae of Adoxophyes honmai – Courtesy Prof. ChenZongmao. (h) Adult male of Adoxophyes

honmai. (i) Adult female of Adoxophyes honmai. (j) Larvaeof Homona magnanima – (Courtesy Prof.

Madoka Nakai). (k) Webbing of leaves by Homona magnanima.(l) Adult female of Homona magnanima

(Courtesy Prof. Kodomari). (m) Adult male moth of Homonamagnanima (Courtesy Prof Chen Zongmao).

As in the case of Homona coffearia, Adoxophyes honmai makeleaf nests by binding and

rolling several leaves together, often enclosing the buds

and feed from within these leaf

nests. Pupation occurs inside the leaf nest and the pupaeare dark brown in colour. The

temperature also seems to have an effect on the lifehistory of this species. No development is

reported to occur below 10.3˚C and 417 DD days are requiredto complete the development

from egg to adult emergence (Nabeta et al., 2005). Thepupal period is reported to be 5–6

days in summer and about 10 days in spring and autumn.There are generally five to six

larval instars, and in the overwintering population,sometimes an additional larval stage has

been reported. No diapause has been observed in winter.This species generally has four

generations per year and sometimes five in the southernpart of Japan.

2.2.5 Dispersal

As in the case of Homona coffearia, dispersal is mainly byflight of both male and female

moths and the flight activity of Adoxophyes honmai isreported to be influenced by age

as well as by temperature. Both sexes, as well as the matedand unmated females, do not

show any difference in the flight activity when they are1–4 days old, and the flight activity

peaks in 2-day-old adults. However, in 5- to 8-day-oldadults, females fly a longer distance

than the males. Both sexes are reported to fly actively at23°C and their continuous long

flights are reduced at temperatures below 18°C (Shirai andKosugi, 2000).

2.2.6 Damage to tea

Damage to tea occurs at all stages of caterpillar larvae.Adult moths do not feed. The

manner of folding of leaves and their voracious feedingactivity on the tea leaves causes

damage to the leaves as well as defoliation leading to croploss. It also affects the quality

of the leaf. Unlike in the case of Homona magnanima, thedamage caused to tea by

the larvae of Adoxophyes honmai is uniformly distributed onthe tea bush. Although

Adoxophyes honmai usually attacks the first harvest of theyear, it may cause considerable

damage in the second and third harvests as well. The larvaeare known to pass winter

inside the rolled leaf nest (Gireesh Nadda et al., 2013).

2.3 The oriental tea tortrix, Homona magnanima

Homona magnanima is often commonly referred to as orientaltea tortrix or tea leaf roller.


Homona magnanima has been reported from China, Japan andVietnam (Razowski, 2008).

2.3.2 Alternative host plants

Larvae are polyphagous and have a wide host range.


Symptoms of damage are similar to those of Homona coffeariaand Adoxophyes honmai. The

morphology of the caterpillars can however help in theidentification of the specific species.


The adult female of Homona magnanima lays pale greenishyellow oval-shaped egg

masses on the upper surfaces of leaves which become paleyellow within a few days.

The development is temperature-dependent and mature larvaehave a yellowish to dark

green body and brown head with black markings (Fig. 1j).The prothoracic plate is brown

with a dark border posteriorly. The newly hatched larvadisperses from the egg mass and

makes a nest by webbing two mature leaves together. Themature larva makes a large nest

by webbing several mature leaves together and pupatesinside the nest (Fig. 1k). These

larvae do not make any hibernaculum and occasionally feedduring the warmer winter

days. First-generation larvae enter diapause in the fourthor fifth instar (Tamaki, 1991).

Females are larger than the males (Fig. 1l) and the malesare pale yellowish brown

with a greyish head, antennae and thorax (Fig. 1m). Theforewing is brown-coloured with

dark-orangish-brown markings and the hind wing is greyish.The adult females have an

elongated rectangular forewing which is pale yellowishochreous-coloured and sprinkled

with brown markings. The hind wings are half-greyish andhalf-yellow (Diakonoff, 1948).

This species has four generations per year. An additionalgeneration is often observed in

the southern part of Japan.

Studies carried out in the laboratory at 25°C and L:D 18:8indicated the mating activity

of Homona magnanima Diakonoff to be the highest at 2–4 daysfrom emergence in both

sexes, with activity of the female being at a peak when sheis three days old and that of the

male at two days. Males were most active 3–5 h afterlight-off, irrespective of age. There

were no differences between single and multiple mating withrespect to the total number

of eggs deposited, egg viability or the lifespan of theadults (Kanoh et al., 1983).

2.3.5 Dispersal

Dispersal is caused by the flight of the male and femalemoths.

2.3.6 Damage to tea

As in the case of Homona coffearia and Adoxophyes honmai,damage is caused by the

larvae during the folding of leaves to form nests andfeeding activity on the leaves.

2.4 The looper caterpillar, Biston suppressaria

The looper caterpillar belongs to the order Lepidoptera,family Geometridae. Unlike the

other Lepidopterans, they have only one pair of legs andlack the first two or three pairs

of ventral prolegs. During movement, they pull their bodiesinto loops and hence their

name, looper caterpillars. Because of this characteristicmovement, they are also called

‘earth measurer’ or ‘inch worms’.


The earliest known species of looper caterpillar is Buzurasuppressaria. This species

occurs in northeast India (Das, 1963, 1965; Rau, 1973),south India (Muraleedharan,

1991), Bangladesh (Mamun and Ahamed, 2011), China (Chen andKunshan, 1987), Japan

(Kodomari et al., 1987), Nepal (Shrestha and Thapa, 2015),Indonesia (Dharmadi, 1983),

Malaysia (Nair et al., 2008) and Sri Lanka (Cranham, 1966a).

Large-scale use of the pesticide Dieldrin to control smallhive beetle (SHB) resulted

in severe outbreak of this pest in Sri Lanka during themid-1960s (Danthanarayana and

Kathiravetpillai 1969a). However, with minimal use ofpesticides since the 1970s in this

country, this pest has continued to be kept under naturalcontrol and is rarely encountered

at present (Seneratne, 1986/Mohotti, 2008). The parasitoidApanteles sp. seems to be one

of the main means for keeping this pest under control(Danthanarayana and Kathiravetpillai,

1969b). Similar control by Apanteles sp. was reported inChina by Chen and Kunshan (1987).

According to Muraleedharan (1991), this pest is consideredto be a minor pest in south India

as well, although it is considered to be a destructive pestof tea in northeast India. Of late,

four looper species have been recorded in northeast India,viz. Buzura bengaliaria Guenee

1858, Buzura suppressaria Guen, Hyposidra talaca Walk 1860and Hyposidra infixaria

Walk 1860 with Buzura suppressaria reported to have beenoutnumbered by two hitherto

unrecorded geometrid species in this country, viz.Hyposidra talaca and Hyposidra infixaria

(Das et al., 2010; Antony et al., 2012). Hyposidra talacahas been reported as a pest of tea in

Java and West Malaysia. Hyposidra infixaria (Walk.), whichis a rare pest globally, has been

reported for the first time in tea recently in northeastIndia (Nair et al., 2008). Besides the

aforementioned looper species, the giant loopers Ascotisselenaria Schiffermüller 1775 and

Cleora scriptaria Walker 1860 were also recorded recentlyfrom the Terai tea plantations

in northeast India, but was reported not to be considered amajor pest (Das et al., 2010)

(Fig. 2a–e). Ascotis selenaria has been reported to causesevere defoliation to tea in Japan

(Akira, 1997), Taiwan and the USSR (Izhar and Wysoki, 1995).


Buzura sp. (looper caterpillar) is a polyphagous pest andhas a wide host range.


The symptoms of damage are similar for all the species oflooper caterpillars mentioned

earlier. The typical holes on the leaves formed during thefeeding activity of larvae of this

pest are similar to the ones caused by twig caterpillars.However, the larvae and moths

of these two species differ in size and colouration and canbe easily distinguished. When

disturbed, the mature larvae stretch out on the branch andlie motionless.


The life cycle of looper caterpillars, Buzura suppressaria,is very similar to that of twig

caterpillars and has been described in northeast India byDas (1965), Rau (1973) and

Chutia et al. (2011); in Sri Lanka by Danthanaryana andKathiravetpillai (1969a,b) and in

China by Yu (1985) and Zhu et al. (1986). Five to sixgenerations of looper caterpillars per

year have been reported in Sri Lanka and India, whilst inChina, there are reports of only

two to three generations (Peng et al., 1991). The completelife cycle is reported to last

from about seven and a half to nine weeks (Danthanaryanaand Kathiravepillai 1969a,b;

Muraleedharan, 1991) and the development period is reportedto vary in different species

(Das et al., 2010). The occurrence of this pest is reportedto be around February to May

and July to October in northeast India (Anon., 2012).

The eggs of looper caterpillars look like the eggs of twigcaterpillars. They are turquoise

blue in colour and darken before hatching. They are laid2–3 m above the ground level on

crevices of the bark of shady trees in several batches of200–600 eggs each, mostly during

the night. The larvae undergo five distinct stages. In SriLanka and India, the larval duration

is reported to be around 4–5 weeks in tea fields, whilst inthe tea fields of Bangladesh, it

is three weeks (Mamun and Ahmed, 2011) and in China about4–7 weeks, lengthened to

5–9 weeks under in vitro conditions (Yu, 1985).

Infestation on tea is caused by the larvae. The first-stagelarvae of the looper caterpillar

are similar in size to that of the twig caterpillar butdiffer in colouration. The younger larvae

are greenish brown with greenish white lines along the back

and side, and as the larvae

mature, they become mottled grey or brownish and thecaterpillar appears wrinkled and

resembles the bark of a tea bush. They are easilycamouflaged whilst resting on the twigs

and branches during the day. The fully grown larvae areabout 6–7 cm in length and are

much thicker than the twig caterpillars (Cranham, 1966a).

The pupae are laid in the soil under tea bushes and shadytrees. These are brownish

black, with serrated ridges, one on either side of theanterior end of the body and a short

process at the posterior end. They do not spin silkencocoons. The pupal period lasts

for about 3–4 weeks and the moths emerge about two and ahalf to three weeks later.

Copulation takes place soon after emergence, usually within24 h. Males die within three

Figure 2 (a) Larva of Buzura suppressaria (CourtesyTocklai, TRA). (b) Larva of Hyposidra talca (Courtesy

Tocklai, TRA). (c) Larva of Hyposidra infixaria (CourtesyTocklai, TRA). (d) Ascotis sp. (Courtesy Tocklai,

TRA). (e) Larva of Cleora sp. (Courtesy Tocklai, TRA).

(a) (b)

(c) (d) (e)

days and the females die as soon as the last batch of eggsare laid, which is about three

to five days after emergence. The fecundity of the femaleis reported to be 482 ± 205.

The moths are similar to those of twig caterpillars buthave a larger wing span (45–

50 mm) and are greyish in colour with wavy yellow and black

bands and a series of spots

along the margin. The females are generally larger andmales can be easily identified by

the feathered antennae. The heads of the moths are palebrownish yellow and the thorax

and abdomen have orange–yellow stripes (Danthanarayana,1966; Seneratne, 1986/

Mohotti, 2008). The different morphological characteristicsof the looper caterpillars are

described in Anon., (2011).

2.4.5 Movement and dispersal

The first instar larvae suspended by silk threads getdispersed mostly by the wind. Some

larvae also crawl in a loop-like movement down the trunk ofthe trees and find their way

to other plants growing beneath these trees. The moths aregenerally sluggish, weak

fliers and rest during the day, flattened against the treetrunks or rocks with their wings

expanded.

2.4.6 Damage to tea

As in the case of twig caterpillars, looper caterpillarsfeed on the tender leaves of tea

(Camellia sinensis) and nibble irregular holes along themargins. Mature larvae eat the

whole leaf and cause defoliation. Attack by loopers issevere in a field recovering from

prune, new clearing and nursery plants and can result inthe death of the bushes. The

damage occurs mostly during the night and early mornings.In northeast India, Hyposidra

sp., which was earlier mostly found in forest and fruit

trees, is reported to be the main

defoliator (Das et al., 2010). The damage threshold levelof this pest determined in China

on tea bushes was found to be about five larvae per plant(Chen and Kunshan, 1987). In

Sri Lanka and south India, it is considered to be a minorpest.

2.5 Twig caterpillar, Ectropis bhurmitra

Twig caterpillars, Ectropis bhurmitra Walker, belong to theorder Lepidoptera, family

Geometridae. It is commonly referred to as the twigcaterpillar as it very much resembles

dead twigs whilst resting. As in the case of loopercaterpillars, these are also known as

‘inch worms’ because they move 1 in at a time (Cranham,1966a).


The twig caterpillar was first recorded in Sri Lanka(Ceylon) in 1884 from an unknown

host plant (Moore, 1884–87) and later as a pest of tea byGreen in 1900. As in the case

of the looper caterpillar, this pest is now onlyoccasionally encountered in Sri Lanka

and is being kept under natural control. Although Rau(1936) reported this pest to

be damaging to tea in south India, according toMuraleedharan (1991), this had not

been a serious pest of tea in south India. This pest hasbeen reported only recently in

West Bengal (Majumdar, 2008) and Darjeeling, north India(Prasad and Mukhopadhyay,

2013). It has been earlier reported from China (Chen andKunnshen, 1987), Indonesia

(Cranham, 1966a; Dharmadi, 1983), Taiwan (Hutachen andTublin, 1995) and Papua New

Guinea (APPPC, 1987).

2.5.2 Host range

This pest has a wide host range (Cranham, 1966a; Beeson,1941).


The damage caused to leaves by this pest is similar to thatcaused by looper caterpillars

but the size and colouration of the larvae and moths aredifferent. The small black pellets

of frass may also be noticed on the ground below theaffected bushes. Disturbance of

the suspect bushes with a stick will often cause some ofthe younger larvae to fall down

on silken threads. The mature larvae, when disturbed, standup erect at an angle to the

branch on which they get a foothold or hang down fromleaves (Vitarana, 1989).


The life history has been described by Cranham (1966a),Seneratne/Mohotti (1986/2008)

and Prasad and Mukhopadhyay (2013), with slight variations.The moths of twig caterpillars,

although active at night, are not strong fliers. Duringdaytime, they are very sluggish

and rest flattened against tree trunks or rocks with wingsfully expanded and become

camouflaged against the tree trunk. Mating takes placeusually on the day of emergence

from the pupae and egg laying commences on the second andthird days of copulation

and continues for five days. The males usually die soonafter mating and the females die

after depositing the last batch of eggs. Eggs are small,rounded or oval and bluish green.

They are laid in several batches of around 64 eggs each inthe crevices of the bark of shady

trees and the incubation period is 7–10 days. The completelife cycle from egg to egg lasts

from 8 to 9 weeks and there are about six generations peryear.

The first-stage larvae are tiny, blackish in colour with arow of white triangular markings

on either side of the body. The colour becomes brownishwith succeeding moults and

the mature larvae measure around 4–5 cm in length and aredark brownish in colour. It

resembles the red wood of twigs and is easily camouflaged(Fig. 3a). Pupation occurs on

the soil surface and the pupa is smooth, glossy and reddishbrown and measures about

1.2–2.0 mm in length (Cranham, 1966a; Danthanaryana, 1966;Seneratne, 1986/Mohotti,

2008). The adult moths are pale brown or greyish in colourand spotted, with darker

markings forming wavy lines across the wings. They haveblurry dark blotches near the

middle of each forewing (Fig. 3b). They are smaller thanthe moth of the looper caterpillar

and have a wing expanse of about 4–4.5 cm (Cranham, 1966a).

Figure 3 (a) Larvae of Ectropis bhurmitra. (b) Adult mothof Ectropis sp. (top – male, and bottom – female

(Courtesy Prof. Chen Zong Mao). (c) Tea damaged by Ectropissp. (Courtesy Prof. Chen Zong Mao).

(a) (b) (c)

2.5.5 Movement and dispersal

Adult moths are weak fliers and dispersal is mostly by thenewly hatched larvae. Heavy

mortality (89.6–98.2%) of larvae is reported during thisprocess (Danthanarayana and

Kathiravetpillai, 1969b). The larvae move along the bark inloop-like movements.

2.5.6 Damage to tea

The damage caused to tea by twig caterpillars (Ectropisbhurmitra) is very similar to that

caused by looper caterpillars (Buzura suppressaria) (Fig.3c) and occurs mostly during the

night and early mornings (Danthananryana, 1966; Cranham,1966a). The young larvae

feed on the epidermis of the tender leaves and when mature,they eat out holes or feed

on the margins of leaves on the maintenance foliage.

2.6 Tea leaf roller, Caloptilia theivora (= Gracillariatheivora)

Caloptilia theivora belongs to order Lepidoptera, familyGracillariidae. This species was

first recorded as a pest of tea in Japan by Hotta (1918),under the name Gracillaria theivora.

Thereafter, Issiki (1950) transferred it to the genusCaloptilia.


The tea leaf roller is reported as an important pest innortheast India (Green, 1890; Watt

and Mann, 1903), Bangladesh (Mamun and Ahmed, 2011),Indonesia (Kalshowen, 1950),

Nepal (Shrestha and Thapa, 2015), China (Chen and Kunshan,1987), Japan (Akira, 1997),

Korea (Lee et al., 1995) and Taiwan (Muraleedharan, 2002).It is considered to be only

a minor occasional pest of tea in Sri Lanka (Cranham,1966a; Seneratne, 1986/Mohotti,

2008) and south India. It is sometimes referred to as the‘tea triangle leaf roller’ in Japan,

because of the shape of the rolled leaf.


The caterpillar rolls the leaf from the tip downwards,forming a sort of cone, and the nest

formed contains wet blackish frass within (Fig. 4).

Figure 4 Leaf nest formed by rolling leaf from tipdownwards by Caloptilia theivora.


This pest is often encountered on tea recovering frompruning. In northeast India, the

occurrence of this pest is reported during the period ofJune to November, with peak

occurrence in November (Devi et al., 2016). Infestationbegins with the laying of eggs on

the underside of the leaf (an average of 55–71 throughoutthe season).

The newly hatched larvae are tiny and during the first twoinstars mine into the leaf and

live protected under the lower epidermis. The third andlater instars emerge outside and

roll the leaf tip downwards, thus differing from the nestformed by the tea tortrix. Fully

grown larvae are about 2.5 cm in length, slender andoff-white to pale green in colour with

pale heads. They eat the epidermis of the leaf within thenest and, unlike the tea tortrix,

the wet frass is left within the leaf roll. The maturelarvae leave the roll and spin silken

cocoons in the depression of the leaf or nearer the midrib.

The adult moth is small, narrow-winged with fringed marginsand dark in colour

and iridescent. Studies on the life history of this pest byLee et al. (1995) indicated six

generations per year in Korea. The average longevities ofadults were reported to be

8.4–14.5 days in spring and autumn and 6.3–8.6 days insummer. During winter, the larvae

live within the rolled leaves.

2.6.4 Dispersal

Dispersal is caused by the flight of the moths.

2.6.5 Damage to tea

Damage occurs mainly during feeding, which is confined tothe surface of the leaf within

the roll and does not feed through. Severe infestationcould cause defoliation and drying

of the plant. They also damage the buds whilst forming anest (Cranham, 1966a; Gireesh

Nadda et al., 2013).

2.7 Bunch caterpillar, Andraca bipunctata

Andraca bipunctata belongs to the order Thysonoptera.Although the bunch caterpillar

earlier belonged to the family Bombycidae, it has recentlybeen transferred to the family

Endromidae (Zwick et al., 2011).


It is considered a serious pest in northeast India (Nathand Rahman, 2012), China (Chen

and Kunchan, 1987) and is also found in Indonesia, Nepal(Shrestha and Thapa, 2015),

Taiwan and Indo-China. It is not found to date in Sri Lanka.

2.7.2 Hosts

The occurrence of Andraca bipunctata was reported oncultivated tea shrubs: Camellia

sinensis var. sinensis (Chinese variety) and Camelliasinensis var. assamica (Assamese

variety). Camellia japonica and Camellia drupifera.


The type of damage to tea leaves (scraped leaves and cutmargins), clustered leaves, the

morphology of the caterpillars and the manner in which theyare bunched together on the

branches help to identify the presence of this caterpillar.


The caterpillars generally bunch together on the branchesof the host plant, hence its name,

‘Bunch caterpillar’ (Fig. 5a). The adult moth is brown incolour. Females are larger than

males and there are dark wavy lines on the forewings withtwo white spots near the outer

margin. Hind wings are brown. The occurrence of this pestis reported to be usually during

March to April, May to June, July to August and October toNovember (Anon., 2012).

Temperature has a great influence on the life cycle andstudies on biology carried out

in the laboratory by Nath and Rahman (2012) indicated five

larval instars with a total life

cycle of 61.18 ± 0.88 days. Studies carried out by Ghoraiet al. (2010), however, indicated a

longer period. Infestation begins with the laying of eggsin clusters (100–120 eggs) on the

underside of the leaves. Eggs are small and yellowish. Themature larvae measure 65 mm

and have a tawny yellow colour with a reddish tinge andblackish brown transverse stripes

(Fig. 5b). The larvae have five instars with pupationoccurring on the ground amongst

dried leaf litter. Pupae are reddish brown in colour (Wanget al., 2011) (Fig. 5c). Soon after

emergence, the caterpillars feed on the egg shells andthereafter initially feed on the

tissues of the leaf surface. Later, they consume wholeleaves.

Figure 5 (a) Larvae of Andraca bipunctata bunched together(Courtesy Prof. Chen Zong Mao). (b) Larvae

of Andraca bipunctata (Courtesy Prof. Chen Zong Mao). (c)Pupae of Andraca bipunctata (Courtesy

Prof. Chen Zong Mao). (d) Damage to tea by Andracabipunctata (Courtesy Prof. Chen Zong Mao). (a) (b) (c) (d)

2.7.5 Movements and dispersal

Dispersal is caused by the flight of adult moths and by thecaterpillars moving from bush

to bush in groups.

2.7.6 Damage to tea

Damage to tea is caused by the gregarious feeding of thelarvae excepting the first instar.

The young larvae eat on the epidermis of young leavescommencing from the margin,

whilst mature larvae feed on the entire leaves, leavingonly the veins and venules, and

cause defoliation (Fig. 5d). Prior to pupation, a bunch ofcaterpillars are known to destroy

a large number of tea bushes during the feeding activityand crop loss due to feeding has

been reported to increase from the second instar onwards(Panigrahi, 1997), whilst at the

fifth instar, the larvae stop feeding for 40–48 h beforecommencing to pupate (Panigrahi,

1996).

2.8 Nettle grub

The nettle grubs are larvae of the family Limacodiidae,coming under the order Lepidoptera.

This pest obtained its name due to the presence of stingingspines on the body. The

species which do not have spines are called gelatine grubs.Nettle grubs are tropical dry

weather pests. The earliest record of nettle grubs as apest of economic importance was

made by Neitner (1872) when he identified Parasa lepidaCramer 1799, the blue-striped

nettle grub, in Sri Lanka, which was then known asLimacodes graciosa Westwood 1848,

a pest of coffee. This attained pest status in tea in 1890(Green, 1900; Austin, 1931–32).

Following this, the nettle grub Natada nararia Mo.(Macroplectra nararia Moore 1859) was

reported in Sri Lanka in 1897 (Green, 1900). The othercommon nettle grubs in tea areas

are Aphendala recta (=Thosea recta, Hampson 1893),saddle-backed nettle grub, Thosea

cervina (Moore 1877). In Indonesia, the species Setora

nitens Walker (coconut nettle grub)

is also recorded (Dharmadii, 1983). These pests haveseveral natural enemies and thus

are mostly under natural control and not considered to bemajor pests, although with a

sudden outbreak, they could cause serious loss of crop.

The damage caused by all the different species are similarand are in the form of small

irregular patches on the undersurface of the leaf which areinitially yellowish and later become

brown. The older grubs (third instar) form large holes onthe leaves and in severe cases, they

eat the entire foliage, leaving only the midribs. Theappearances of the typical larvae and

cocoons serve as a means for the identification of thedifferent species (Cranham, 1966a).

2.9 Fringed nettle grub (Macroplectra nararia, Moore 1859)


The first record of the nettle grub Macroplectra narariaMoore 1859, then referred to as

Natada nararia Moore (= Darna nararia), but commonlyreferred to now as the small nettle

grub or fringed nettle grub, was reported in tea in SriLanka (Ceylon) in 1897 (Green, 1900).

However, currently only sporadic outbreaks occur in SriLanka during the dry season. It is

considered as an occasional pest in northeast India andsoutheast India as well (Cranham,

1966a; Rao, 1973).


They have several hosts which have been reported by Austin(1931–32) and Browne

(1968). The main Alternative hosts include coffee (CoffeaL.), coconut (Cocos nucifera

L.), African oil palm (Elaeis guineensis Jacq.), dadaps(Erythrina lithosperma Miq.) and

banana (Musa L.).


The biology and ecology of this pest have been described byseveral authors (Austin,

1931–32; Hutson, 1932; Gadd et al., 1946; Ananda Rau, 1934;Cranham, 1966a; Cranham

and Fernando, 1960). The females are larger than the malesand the adult moths are pale

brown in colour and become active only at dusk. Egg layingtakes place within three days

from adult emergence, between 6.30 pm and midnight. Asingle female can lay about

500 eggs and they are laid singly on the upper surface ofthe leaf. The eggs hatch after

about one week and are shiny and initially appear paleyellow or greenish, but become

opaque or pale brown prior to hatching. Following hatching,the young larvae migrate to

the undersurface of the leaves and begin feeding.

The larvae (grubs) are short, fleshy, stout and oval inshape with a retractile head and

minute feet. The newly hatched larvae are about 0.1 mmlong, whilst the older mature

larvae are about 1.3 cm in length. The colour of the maturelarvae ranges from yellowish

green to apple green and the larvae have a dark mediandorsal stripe, with pale yellow

borders on each side. The body is fringed with white

stinging hairs (Fig. 6a) and there

are also two pairs of anterior tubercles bearing muchshorter hairs. The larval stage is

reported to last about 5–6 weeks.

Pupation takes place on the ground under the bush, amongstthe dead leaves or

in crevices amongst stones, and sometimes on the lowerbranches of the bush or on

mature leaves. The cocoon is globular and one-sixth of aninch in diameter, consisting

of a hard papery shell covered with thin webbing of darkbrown or purplish silk and very

much resembles a large seed. Austin (1931–32) reported thelife cycle from egg to adult

to take about ten weeks. This coincides with the frequencyof generations observed by

Gadd et al. (1946).

Figure 6 (a) Fringed nettle grub, Macroplectra nararia(Courtesy Tea Res. Inst. Sri Lanka). (b) Damage

to tea leaf by nettle grub (Courtesy Tea Res. Inst. SriLanka).

(a) (b)

2.9.4 Movements and dispersal

Moths migrate in the night time. However, mass migrationhas never been observed and

movement is also not induced by wind (Austin, 1931–32).They are also known to be

attracted to light and move towards it. Another method ofdispersal is by the transport of

plants from one district to another.

2.9.5 Damage to tea

Damage occurs during feeding. The younger larvae nibble andscrape away patches of the

lower portion of the leaf, leaving the upper epidermisintact. When many larvae feed on

the same leaf, small brownish patches appear. The oldergrubs (third instar larvae) make

larger holes and can eat whole portions of the leaf,including the epidermis (Fig. 6b).

When infestation is heavy, the entire foliage is eaten,leaving only the midribs, and there is

severe defoliation (Cranham and Fernando, 1960; Cranham,1966a; Rao, 1973; Seneratne,

1986/Mohotti, 2008; Muraleedharan, 1991). The presence ofthese caterpillars can cause

indirect disruption of work in the plantation, as theirritation caused by the grubs on the

skin hinders the movement of the workers into infestedareas.

2.10 Blue-striped nettle grub, Parasa lepida


This species is common in many countries, for example,Bangladesh (Viqar et al., 2008),

India, Sri Lanka (Austin, 1931–32), Vietnam, Malaysia andIndonesia (Dharmadi, 1983).


It is reported to have a wide host range; some of theseinclude coffee (Coffeae L.), rubber

(Hevea brasiliensis, Mull. Arg.), Citrus (L.) Osbeck, oilpalm (Elaeis guineensis Jacq.), cocoa

(Theobroma cacao L.), cassava (Manihot esculenta Crantz),ebony (Diospyros ebenum Koenig),

coconut (Cocos nucifera L.), Gliricidia (Gliricidia sepium(Jacq.) Kunth ex Walp), banana (Musa

L.), winged bean (Psophocarpus tetragonolobus (D) D .C),mango (Mangifera indica, L.),

poplar (Populus deltoides, W. Bartram ex Humphry Marshall)and Litchi chinensis Sonn.


The fully grown larvae are fleshy and about 3–4 cm long andgreenish yellow in colour and

have three pale blue longitudinal stripes edged with darkblue or black, one prominent

in the middle dorsally and the other two lateral to it.They also have three pairs of ruddy

brown tufts and four pairs of tubercles with stingingspines which are green with black tips.

Eggs are oval, flat and overlap each other and are laid inclusters of 10–30. A single female

lays more than 500 eggs which hatch in 6–8 days. Larvaldevelopment is reported to be

slower than that of Macroplectra nararia and pupaldevelopment is around 1–1.5 months.

The morphologies of male and female moths have beendescribed by Austin (1931–32).

2.10.4 Dispersal

Dispersal is caused by the flight of the moths.

2.10.5 Damage to tea

These are gregarious feeders and feeding is limited todefinite areas on the maintenance

foliage (Cranham, 1966a). This species mostly occurs alongwith Thosea sp. and

Macroplectrus sp. and damage is mostly due to the combinedattack (Austin, 1931–32).

2.11 Saddle-backed nettle grub, Thosea cervina


This pest was first reported in tea in Assam, India, in1894 and is thus often referred to as

the Assam nettle grub. It is also referred to as the teaseed nettle grub in northeast India

because the cocoon resembles a tea seed (Cranham, 1966a).In Sri Lanka, south India and

Indonesia, it was reported as a pest of tea in 1906(Austin, 1931–32 (Damiri, 2014).


The eggs are yellowish and elliptical in shape, flat andshiny and are usually located along the

margin of the undersurface of the leaves. The mature larvaeare about 2.5–4 cm long, greenish

in colour and with three prominent white to brown markingsmid-dorsally. The central marking

is saddle-shaped, hence its name, saddle-backed nettlegrub. The larvae pupate about 1-in

deep in the soil or on leaf litter and the life cycle isslow with the larvae pupating after 45–66

days. Moths emerge from the cocoon at dusk and are activeduring the early part of the night.


Larvae feed on the leaf tissue on the undersurface,beginning at the tips where the eggs

were laid and later devour large portions of lamina. Wheninfestation is severe, the entire

leaf can be stripped off, leaving only the midrib.

2.12 Slug caterpillar moths, Aphendala recta (Thosea rectaHampson, 1893)


It is native to Sri Lanka (Cranham, 1966a) and is also

reported from south India, Vietnam

(Robinson et al., 2009) and Indonesia (Bernard, 1917).


The fully grown larvae are about 1.5 cm and green in colourwith narrow, silvery white

dorsal band markings and short spines. The cocoons are seenattached to leaves or twigs

and are about 1 cm long, oval in shape and dark brown incolour, with lighter brown

patches. The adult moths are greyish brown and measureabout 2.5 cm across the wings.


Young larvae scrape off the undersurface of the leaf andmature larvae eat large portions

of the leaves (Cranham, 1966a).

2.13 Gelatine grub, Narosa conspersa


The gelatine grub Narosa conspersa can be found in the teaareas where the other nettle

grubs are found but it is not a serious pest.


The larvae are yellowish to green in colour with whitesubdorsal lines and a series of white

sublateral spots. It measures about 0.7–1.0 cm in length.The body does not have any

spines but does have a prominent dorsal ridge which istransversally segmented. The

legs are retractile. The cocoon is oval, measures 0.2 in,is white with an operculum at one

end and is closely attached to leaves or twigs (Cranham,1966a; Austin, 1931–32). Mating

takes place during the day as well as at night. Larvae aredifficult to detect as they are

camouflaged in the leaves, but the cocoons are white andcan be easily seen.


Damage is caused by the larvae which feed on tea leaves,but it is not of economic

importance.

2.14 Large gelatine grub, Belippa laleana Mo.


This pest is found in the same areas where other nettlegrubs occur but only in small

numbers, and is of minor importance.


The larvae are squat, rounded, about 1.5 cm in length,bluish green in colour, gelatinous

and slug-like in appearance and movement. Cocoons areoblong and light brown in colour.

2.15 Tea flush worm, Cydia leucostoma

This pest belongs to the order Lepidoptera, familyTortricidae.


This tortricid species is reported to damage tea inBangladesh (Mamun and Ahmed, 2011),

India (Meyrick, 1912; Muraleedharan and Selvasundaram,1986), Taiwan and Indonesia

(Java and Brunei) (Dharmadi, 1983; Shrestha and Thapa,2015). It is considered only as a

minor pest in south India and is not reported yet in thetea areas of Sri Lanka.


The appearance of the tight rolls of the buds and topleaves of young shoots made by the

larvae indicates infestation with this pest. Larvae can befound inside the rolled leaves. The

affected leaves become rough, crinkled and leathery.


Tea seems to be the main host.


This is a dry weather pest (Van Der Gest and Evenhuis,1991), and the habits resemble

those of Homona coffearia (Fletcher, 1920). The eggs arepale yellow or brown and are

laid singly on the undersurface of mature leaves and hatchafter 5–10 days (Simanjuntak,

2002). There are five instars. Larvae are about 1 cm long,with the body being green in

colour and the head being dirty yellowish with a darkmarginal spot. The adult moth is

small, less than 1 cm long and blackish brown in colourwith a wing span of 11–15 mm.

Moths are active during morning and evening hours and aremostly on the wing from May

to October (Meyrick, 1912), whilst Devi et al. (2016)reported this to occur from March to

April and August to October, with peak occurrence in April.

Soon after emergence, the larvae make tight rolls of theterminal buds and top leaves,

within which they feed. In the initial stages, the larvaescrape the upper epidermal layer

of young leaves. Feeding thereafter extends to palisadeparenchyma, but they rarely feed

on the spongy mesophyll. As the caterpillar grows, thegrowing leaves of the bud are

continuously webbed, preventing the unfolding of the leaves(Van Der Gest and Evenhuis,

1991; Simanjuntak, 2002).

2.15.5 Dispersal

Movement from place to place is caused by caterpillars andmoths.


The larval stage causes damage to the tea during rolling ofthe leaves to form the nest as well

as during the feeding activity, and infestation affectsboth the yield and the quality of the tea

(Murthy and Chandrasekaran, 1979; Tamaki, 1991; Van DerGest and Evenhuis, 1991).

3 Sucking pests

Insects with specialized mouth parts which pierce the hosttissues and suck the plant sap

are called sucking insect pests.

Sucking pests of tea include the Tea Mosquitoe Bug, LygusBug, Tea Jassid, Tea Thrips

and Mites which, other than mites, belong to the classInsecta, whilst mites belong to the

order Acari, class Arachnida and are thus not insects.

3.1 Tea mosquito bug Helopeltis theivora

Helopeltis belongs to the order Hemiptera, family Miridae,genus Helopeltis. Amongst

41 species of mirids in the genus Helopeltis (Hazarika etal., 2009), about 18 are reported

to be associated with tea (Saha and Mukhopadhyay, 2012). Ofthese, the most important

damaging species are Helopeltis theivora Water House,Helopeltis antonii Sign. and

Helopeltis schoutedeni Reuter. Helopeltis theivora iswidely distributed in the tea areas in

Asia (India, Bangladesh, Indonesia, Java, Malaysia, SriLanka, Sumatra, Laos and Vietnam).

Helopeltis antonii is reported in India, Sri Lanka,Indonesia and Vietnam (Watt and Man,

1903; Hazarika et al., 2009; Saha and Mukhopadhyay, 2013,Ahmed and Mamun, 2014),

whilst the main mosquito bug in Africa is Helopeltisschoutedeni, which is reported to

cause severe loss of crop (Rattan, 1992).

Recently, two new species Helopeltis bradyi Waterhouse andPachypeltis maesarum

Kirkaldy have been recorded in south India on tea (Srikumaret al., 2015). It had earlier

been reported in India on cashew and black pepper byStonedahl (1991).


The tea mosquito bug, Helopeltis theivora Waterhouse, wasfirst recorded on tea in Java

in 1847 (Rao, 1970) and in India in 1868 in the Cacharregion (Watt and Mann, 1903). This

polyphagous-sucking pest is widely distributed in almostall the tea-growing countries, other

than Japan and Korea (Saha and Mukhopadhyay, 2013).Helopeltis sp. is considered to be

an important pest of tea in northeast India including Assamand West Bengal (Das, 1965;

Sundararaju and Sundarababu, 1999; Sudhakaran andMuraleedharan, 2006; Debnath and

Rudrapal, 2011), Indonesia (Koningsberger, 1908; Leefmans,1916), Bangladesh, (Ahmed

et al., 1992, 2013), Africa (Rattan, 1992) and Vietnam(Pasquier, 1932) and it is also reported

from Laos (Pasquier, 1932), West Malaysia (Lever, 1949;Corbett, 1930), Taiwan (Hsiao,

1983; Mulien, 2015), Nepal (Shrestha and Thapa, 2015),Papua New Guinea (Smith et al.,

1985), Uganda (Hargreaves, 1936), China (Yong-ming andQi-An, 1985) and Sri Lanka

(Cranham, 1966a). In Sri Lanka, it is considered to be aminor pest confined to only a few

districts in the low country (Cranham, 1966a; Seneratne,1986/Mohotti, 2008).

3.1.2 Host range

Tea is reported to be the main host but being polyphagous,it attacks many economic

crops (Mukhopadhyay and Roy, 2009; Sundararaju andSundarababu, 1999). Some of these

include Mikania Wild 1803, Cinchona (Cinchona officinalis(L.) Ruiz), cashew (Anacardium

occidentale L.), Cinnamomum Schaeff, camphor laurel(Cinnamomum camphora (L.) J.

Presl), cocoa (Theobroma cacao, L.), tephrosia (Tephrosiapurpurea (L.) Pers.), guava

(Psidium guajava L.), coffee (Coffea arabica, L.), jackfruit (Artocarpus heterophyllus, Lam.),

mango (Mangifera indica L.) and kapok (Ceiba pentandra (L.)Gaertn).


Tea plants infested with tea mosquito bugs have leaves withnecrotic patches with holes

in the middle (larger than those formed by the Lygus bug)

resembling water-soaked

areas at the point of feeding, which blacken with age asthe cells die and stripes of corky

tissues develop around the lesion. The affected leavesoften curl and become deformed,

sometimes dying from the tip or edges. The bushes alsobecome stunted and the entire

flush gives a scorched appearance from afar.


Biology and ecology of this pest have been extensivelystudied by several entomologists

(Das, 1965; Mann, 1902; Gope and Handique, 1991; Zeiss andBrarber, 2001; Atmadja,

2003; Ahmed and Mamun, 2014; Sudhakaran and Muraleedharan,2006; Roy et al., 2009),

and Roy et al. have carried out an extensive review of thework done by various scientists

on the biology and ecology of this pest (Roy et al., 2015).Temperature and rainfall have

an influence on the incidence of this pest. Damage to teaplants occurs throughout the

year, but this pest prefers warm moist conditions and istherefore predominant in moist

and shaded areas, especially after monsoon showers andmisty weather (Cranham, 1966a;

Zeiss and Braber, 2001; Chakraborty and Chakraborty, 2005).This species is reported to

prefer low jat tea compared to the Assam variety (Cranham,1966a). Although it attacks

throughout the year, the damage is reported to be severefrom May to September in

northeast India (Das, 1957; Debnath and Rudrapal, 2011). Insouthern India, the incidence

of the TMB was reported to be high from July to Decemberand low from January to June

(Muraleedharan, 1992a,b).

The behaviour and life cycle resemble those of Lygus bugs.Both males and females

resemble mosquitoes, hence the name (Fig. 7a). Females arelarger (5–10 mm) than males

(around 4 mm). Adults are longer and narrower than Lygusand are black in colour, with a

red thorax and a black-and-white abdomen. Wings aregreenish brown in colour and at

the time of resting or moving on the leaves, the wings arefolded close to the abdomen.

Adults live for about 8–13 days. There exists colourvariability in the populations collected

from Vietnam, south India, Assam and the Dooars, withspecial reference to the head

and pronotum, as has been reported by Stonedahal (1991),Mann (1907), Bora and

Gurusubramanian (2007), Sarmah and Bandyopadhyay (2009) andRoy et al. (2009).

Infection occurs through the mated female which lays eggswith the aid of an ovipositor

into the tender green stems and occasionally in thepetioles and midribs of the leaves. A

female lays 4–10 eggs per day, laying around 220 eggsduring its lifetime. The egg is oval,

white in colour with two filamentous threads of unevenlength. There are five nymphal

instars and the nymphs have an ant-like appearance withelongated legs and range in

colour from copper or orange to green. The duration of thelife cycle has been reported

to vary depending on the climatic conditions (Cranham,1966a; Das, 1984). Zeiss and

Braber (2001) reported eight generations. The carbohydratecontent and the C/N ratio

are reported to have an influence on the development of theHelopeltis (De Jong, 1938

in Kalshoven, 1981).

Figure 7 (a) Adult tea mosquito bug (Courtesy Tocklai,TRA). (b) Adult tea mosquito bug and damage

on tea leaves (Courtesy UPASI, TRI).

(a) (b)

Adults are more visible in the early and late hours of theday and take shelter under tea

leaves, especially in the lower frame, during daytime orwhen disturbed (Cranham, 1966a;

Hazarika et al., 2009). On a cloudy day, this pest is foundmostly on the top branches of

the bushes and when disturbed, the nymphs and adultsquickly move down the stems or

fall to the ground.

3.1.5 Dispersal

Although the adults have the ability to fly up to 4 km(Cheramgoi, 2010), the spread is

mainly by wind (Das, 1965).

3.1.6 Damage to tea

Damage by all species of Helopeltis is similar. Injury isinduced by young (nymphs) and

adult mosquitoes, both of which have sucking mouthpartswith which they suck the sap of

young leaves, buds and tender stems. Nymphs are reported tocause more severe damage

than adults because the nymphs move less than the adults.Damage is also caused during

the injection of saliva containing toxic enzymes, whichcauses the tissues to break down

around the puncture, causing brown necrotic lesions (Fig.7b). Mechanical damage is

caused during the insertion of eggs into plant tissues, andcracks are formed on stems

inducing overcallusing and leading to stunted growth anddieback (Das, 1965; Roy, 2008).

The hypersensitive reaction and anatomical changes to youngtea leaves during feeding

are described by Ahemed et al. (2013). Das (1984) reportedabout 80 feeding lesions by

a single late-instar nymph in 24 h, whilst Hainsworth(1952) reported 150 feeding spots

in a day by an adult. Studies carried out by Sana and Haq(1974) indicated the damage

to be greater at night time, whilst Muraleedharan (2002)indicated greater damage in

the morning and evening. Fully grown fifth instar nymphswere observed to be the most

voracious feeders and produced the largest number oflesions (Bhuyan and Bhattacharyya,

2006). Helopeltis schoutedeni is reported to form diebackand stem cankers on tea (Rattan,

1992). Apart from causing crop loss, this pest also affectsthe quality of tea (Sudhakaran

and Muraleedharan, 2006). In a severe attack, bushesvirtually cease to form new shoots

and the affected area may not crop for weeks.

3.2 Lygus bug, Lygus sp.

The Lygus bug belongs to the order Hemiptera, familyMiridae.


It has been reported from India, Sri Lanka and Japan, butit may be present in many other

tea-growing countries as well. In Sri Lanka, it is a minor,occasional pest and occurs only

in localized areas at very high elevations of 1300–2000 min tea areas bordering jungles

(Calnaido, 1959; Seneratne, 1986/Mohotti, 2008).


It feeds on a wide variety of hosts including many weedsand cover crops. Tea is reported

to be a less favoured host (Cranham, 1966a).


Small punctures can be found in the middle of necroticpatches (smaller than those formed

by the tea mosquito bug), which become small holes onmature leaves. The leaves become

ragged and deformed, giving a corroded appearance.


This species has been described by Cranham (1966a) and Duan(1994). The adult bugs

are delicate-winged insects, about 5 mm long and greenishyellow in colour. Infestation

is caused by the adults when they embed eggs in stems,stalks and midribs of leaves.

They have five larval instars. The young nymphs resemblethe adults but are wingless.

From the third instar onwards, the rudiments of wingsappear which progressively increase

in size with each stage. The adults are known to live forseveral weeks. The bug prefers

shady moist conditions and is known to favour low jat tea.They are active from dusk to

dawn when the feeding activity takes place. During the day,they are hardly seen and live

amongst the bushes (Cranham, 1966a). The attack can occurthroughout the year, but it is

more common during April, May and June (Anon., 2012).

3.2.5 Dispersal

Adults are mobile and dispersal is caused by flight.

3.2.6 Damage to tea

Symptoms of damage are similar to those caused byHelopeltis. Damage is caused by adults

and nymphs which feed during night time on the buds andyoung flush by inserting the needle

like proboscis through which saliva containing toxin isinjected. The cells around the punctures

die and small brown necrotic spots are formed. As the leafenlarges, the small punctures

become holes and the leaves become ragged and deformed. Thedamage is noticeable on

the upper maintenance foliage at the end of attack(Cranham, 1966a). Mechanical damage

also occurs whilst embedding the eggs in the midrib of theleaves and stalks and stems.

3.3 Tea jassid, Empoasca sp.

The tea jassid, Empoasca flavescens Gillette, 1898(=Empoasca vitis Gothe 1871), belongs

to the order Hemiptera, family Cicadellidae laterille 1802.It is commonly referred to as the

green fly, or leaf hopper, and is a major sucking pest of

tea in northeast India (Das, 1965).


This pest attacks tea in Bangladesh (Mamun and Ahmed,2011), China (Chen and Kunshan,

1987; Xu et al., 2005), Japan (Akira, 1997), Indonesia(Zeis and Braber, 2001), Taiwan

(Hsiao, 1983) and Vietnam (Zhao et al., 2000) but is onlyoccasionally encountered on tea

in localized areas in south India and Sri Lanka. In northIndia, this pest was considered to

be a minor pest until recently. It has now taken the statusof a major one (Saha et al., 2012).

There are different species of jassids attacking tea invarious tea-growing countries

(Saha and Mukhopadhyay, 2013). The leaf hopper pest inChina was hitherto reported as

Empoasca vitis Gothe 1875. However, recent studies carriedout in China have identified

this species to be Empoasca Matsumurascaonukii Matsuda,1952 (XiaoFei et al., 2015;

DaoZheng et al., 2015; Long-Quing et al., 2015).


Apart from tea, it is also known to attack castor (Ricinuscommunis L.), cotton (Gossypium

barbadense L.), lady’s finger (Abelmoschus esculentus (L.)Moench.), brinjal (Solanum

melongena L.) and potato (Solanum tuberosum L).


Symptoms of infection appear as yellow or brownish patchesalong the margin of the

leaves, followed by the distortion of leaf veins andcurling of leaves downwards (Fig. 8a).

The leaves thereafter dry up. This characteristic symptomis known as ‘rim blight’ or

‘hopper burn’ (Das, 1965).


This pest does not like direct light and is found commonlyon the underside of the leaves

(Simanjuntak, 2002). When disturbed, they jump and moveaway, hence the name leaf

hopper. The adults are small, yellowish green and about2.5–2.75 mm in length. The hind

legs have two parallel rows of spines which extend allalong the hind tibiae. The wings are

also green and transparent (Fig. 8b). Eggs are laid insidethe soft tissues of the tea buds,

veins and midribs of the leaves. A single female canproduce about 100 eggs during her

lifetime. Eggs hatch into nymphs within 6–13 days. Thehatching period is longer during

the warmer months (Zeiss and Braber, 2001).

The nymphs are like the adults but wingless (Fig. 8c). Fournymphal stages are reported

and the total duration of nymphal stages is reported to bearound 7–16 days. The newly

hatched nymphs are white in colour and with each instar,the size of nymphs increases

and they develop a greenish tinge. The final instar isabout 2 mm long. The anatomical

character, the caffeine and the soluble protein content ofthe tea shoots were reported to

have an effect on population density of this pest (Zhang etal., 1994). The development

is temperature-related and is faster in warmer

temperatures. The entire life cycle is 12–30

days. The lifespan of the adult varies between 14 and 21days and females live longer than

males (Zeiss and Braber, 2001). Too much rain or extremelydry weather, however, is not

favourable for the development of the insect (Zhu et al.,1993; Widayat and Winasa, 2006).

Figure 8 (a) Damage to tea leaves by Empoasca. (b) AdultEmpoasca sp. (Courtesy Prof. Chen Zong

Mao). (c) Nymph of Empoaska sp. (Courtesy Prof. Chen ZongMao).

(a) (b) (c)

This pest is reported to be found in India mostly duringFebruary–July (Anon., 2012) and

in Sri Lanka during April, May and June (Cranham, 1966a).

3.3.5 Damage to tea

Damage to tea is caused during the feeding activity of theadults and nymphs. Insertion of

needle-shaped mouth parts during sucking causes mechanicalinjury. Further injury is caused

by the secretion of saliva, containing enzymes, into thevascular tissue whilst feeding, which

interferes in the uptake of water and nutrients (Zhu etal., 1993; Zeiss and Braber, 2001;

DeLong, 1971). Feeding is mostly confined to theundersurface of young leaves and mainly

in the middle area of the leaves, but the effect of damageis reflected at the periphery.

Damaged leaves develop yellow or brown spots. Withcontinued feeding, the leaves become

stunted and dry. Occasionally, the leaf may curl, due tofeeding on the upper surface (Saha

and Mukhopadhyay, 2013). In newly planted young tea,infestation causes the drying of new

shoots and the plant becomes stunted and may even die(Zeiss and Braber, 2001). Severe

attacks on a pruned bush affect the recovery from prune andformation of new shoots and

leaves dry up and defoliate. They also hamper the growth ofnewly planted tea, causing

drying of shoots and stunting (Zeiss and Braber, 2001; Sahaand Mukhopadhyay, 2013).

3.4 Tea thrips, Scirtothrips dorsalis sp.

Tea thrips belong to the order Thysonoptera, familyThripidae.


The species of thrips reported in the tea areas of theworld include Scirtothrips dorsalis

Hood 1919, Scirtothrips bispinosus Bagnall 1924,Heliothrips haemorrhoidalis Bouche

and Physothrips (= Taeniothrips) setiventris Bagnall(Cranham, 1966a; Kodomari, 1978;

Muraleedharan and Kandasamy, 1980; Chen and Kunshan, 1987;Mamun and Ahmmed,

2011; Mirab-balou et al., 2014). Amongst these, the mostwidespread species in tea areas

causing damage to tea is Scirtothrips dorsalis Hood,commonly referred to as chilli thrips

(India, Sri Lanka, Japan, China and Taiwan). In southIndia, the species Scirtothrips bispinosus

(Bagnall) is reported to be a major pest of tea(Ananthakrishnan, 1963). In Darjeeling district,

the species Taeniothrips setiventris (Bagnall) is reportedto cause severe damage, whilst the

common species in the plains of north India is Scirtothrips

dorsalis (Das, 1965). In Malawi

and Zimbabwe, another species, viz. Scirtothrips aurantii,has been reported. In Sri Lanka,

thrip is considered to be only an occasional minor pest(Seneratne, 1986/Mohotti, 2008).


Being polyphagous, it is found in several plant speciesincluding vegetable crops, cotton

(Gossypium barbadense L.), citrus (Citrus limon (L.)) andfruit and ornamental crops in

southern Asia (Ananthakrishnan, 1993; CABI/EPPO, 1997;CABI, 2003).


Two or three corky lines of scarred tissues parallel to themidrib on the underside and

pressed out on the upper surface of the leaves occur, butthe leaves do not curl up as in an

attack caused by yellow mites. Leaves are puckered,hardened and deformed.


Thrips are tiny sucking insects which prefer to feed ontender tissues such as young leaves

and buds of plants. They are mostly found on young plantsand fields recovering from

prune (Dev, 1964). In northeast India, the occurrence isreported to be generally during

January–July (Anon., 2012), whilst in south India, thepopulation buildup is reported to

be during November/December and reaches a peak inFebruary/March or April/May

(Anon., 2015). Eggs are laid on tender leaves. The larvaeare tiny (0.1 mm in length) and

are found on the undersurface of leaves. It has both bitingand sucking mouth parts.

There are four nymphal stages. The first two nymphal stagesare active and are the ones

which feed on the leaves. They are creamy white in colourand resemble the adult, but

are wingless. The last two nymphal stages (pre-pupa andpupa) do not feed and passed

inside a cocoon on the surface of the soil or leaf litterfrom which the adult emerges.

Sometimes, they pupate in the lower base of the plant orcrevices of bark. Adults are

small slender insects, around 2 mm, and have acharacteristic brown-coloured abdomen

(Fig. 9a). Wings are long and narrow fringed with longhairs and are kept close to the

abdomen when not in flight.

3.4.5 Dispersal

Dispersal is caused by adults. They are weak fliers andaided by wind.

3.4.6 Damage to tea

Damage to tea is caused during the feeding activity of theadults and the first two active

nymphal stages. They have both sucking and biting mouthparts and they penetrate the

leaf surface and suck the plant sap. They commence feedingon folded and unfolded

buds and when the leaves unfold, two parallel corky linesparallel to the midrib can be

observed on the undersurface and pressed on the uppersurface (Fig. 9b). This very much

resembles damage caused by yellow mite, but in the latter,the corky area completely

covers the midrib and lower surface of the leaf and theleaf curls up. The leaves damaged

by thrips appear puckered, hardened, deformed and dullerand dark green in colour. They

do not cause serious damage and damages last only for a fewweeks (Das, 1965; Cranham,

1966a).

Figure 9 (a) Adult thrips. (b) Thrips damage on tea leaf.(a) (b)

3.5 Mites

Mites are a small inconspicuous species widely distributedaround the world in varying

habitats. There are more than 15 species of phytophagousmites reported to attack tea

(Roy et al., 2014). The common and most widely distributedspecies causing damage

include tea red spider mite Oligonychus coffeae Nietner,1861 (Tetranychidae),

scarlet mite (Brevipalpus californicus Banks, 1904(Tenuipalpidae)), yellow/broad mite

Polyphagotarsonemus latus Banks, 1904 (= Hemitarsononemuslatus Banks, 1904)

(Tarsonemidae) and purple mite (Calacarus carinatus Green,1890) (Eriyophida). The other

less common but also damaging species include Brevipalpusphoenicis Geijskes, 1936

(Tenuipalpidae), pink mite, Acaphylla theae Watt, 1898(Eriyophida) and Tetranychus

kanzawai Kishida, 1927 (Tetranychidae).

The four common species, Oligonychus coffeae, Brevipalpuscalifornicus,

Polyphagotarsonemus latus Banks and Calacarus carinatus,

were reported from the earliest

days of tea planting in Sri Lanka (Green, 1890), India(Watt and Mann, 1903) and Indonesia

(Bernard, 1909). These occur in almost all the tea-growingareas of the world.

The mite species Tetranychus kanzawai Kishida is reportedto be an important pest of

tea in Japan, China, Taiwan and the Philippines (Ho, 2000).In China, an additional species,

Daidatarsonemus De Lean (Tarsonemidae), has been reported(Lin and Liu, 1995). The

pink mite, Acaphylla theae (Watt), has also been reportedto be found in the tea-growing

areas of Bangladesh, India, Malaysia, Indonesia, Taiwan andChina (Danzinger, 2000). Pink

mite was reported in Sri Lanka for the first time in 1999(Vitarana, 2000). Mites, with the

exception of yellow mites, are known to be more abundant inareas having low shades.

3.6 Red spider mite, Oligonychus coffeae

The red spider mites belong to the family Tetranychidae.


The red spider mite, Oligonychus coffeae Nietner, 1861, isthe most widely distributed mite

species in Southeast Asia and is considered to be a seriouspest of tea in northeast India. It is

also widely distributed in Bangladesh, Burundi, Florida,the United States, India, Indonesia,

China, Japan, Kenya, Malawi, Pakistan, QueenslandAustralia, Sri Lanka, Taiwan, Uganda,

Zimbabwe and Vietnam (Pasquier, 1904; Kalshoven, 1951,1981; Pritchard and Baker, 1955;

Hu and Wang, 1965; Cranham, 1966a; Danthanarayana andRanaweera, 1972; Rattan, 1992,

1994; Gotoh and Nagata, 2001; Senaratne, 1986/Mohotti,2008; Roy et al., 2014).


Although it has a wide host range, its main host is tea.


The bronzing of the upper surface of the leaf induced bythe damage caused by the adults

and nymphs of this pest is visible from a long distance(Fig. 10a). Closer examination

shows the tiny mite species running around on the leafsurface. The tiny white cast skins

are also visible to the naked eye. When the thumb ispressed on the leaf containing mites,

the blood of the mites will appear as pin dots.

Figure 10 (a) Infestation with red spider mite (CourtesyTea Res. Inst. Sri Lanka). (b) Adult red spider

(Courtesy Tocklai, TRA). (c) Advanced stage of infestationwith red spider mite (Courtesy Tea Res. Inst.

Sri Lanka). (d) Tea leaves infested with scarlet mites(Courtesy Tea Res. Inst. Sri Lanka). (e) Tea leaves

infested with purple mites (Courtesy Tea Res. Inst. SriLanka). (f) Young tea leaves infested with pink

mites. (g) Young tea leaves infested with yellow mites(Courtesy Tea Res. Inst. Sri Lanka). (h) A tea leaf

infested with yellow mite (Courtesy Tea Res. Inst. SriLanka). (i) Adult kanzawa mite (Courtesy Prof.

Kodomari, Japan). (j) Adult kanzawa mites (Courtesy Prof.Kodomari, Japan). (a) (b) (c) (e) (d) (f) (g) (h) (j)(i)


This mite is the largest amongst the other common species

and can be seen with the

naked eye. It spins a fine silken web on the upper leafsurface and is hence referred to

as the spider mite. Its biology and life history have beendescribed by several authors in

different tea-growing countries, including north and southIndia (Das, 1959a; Rau, 1965;

Selvasundaram and Muraleedharan, 2003; Roy et al., 2014),Bangladesh (Ahmed and

Sana, 1990), China and Taiwan (Hu and Wang, 1965), SriLanka (Cranham, 1966a), Central

Africa (Rattan, 1994; Sudoi, 1997) and Japan (Gotoh andNagata, 2001). The adult female

is about 0.32–0.44 mm in length and elliptical in shape,broadly rounded at the posterior

end, crimson in colour at the anterior end and withpurplish markings on the posterior

(Fig. 10b). The male is small and slimmer and the abdomentapers to a point posteriorly.

Infestation begins with the laying of eggs on the uppersurface of the leaves. The eggs

are spherical, smooth, bright red in colour and, prior tohatching, become light orangish.

There are three development stages, the six-legged larvae,protonymph and deutro

nymph. The different stages overlap and a quiescent periodprecedes each moult. Unlike

the case of Tetranychus kanzawai Kishida, red spider mitedoes not enter dormancy to

survive adverse periods.

These mites aggregate along the midrib and vein on theupper surface of the leaves.

Leaf temperature and light penetration within the tea

bushes influence mite distribution

(Gireesh Nadda et al., 2013). They breed mainly on theyoung maintenance foliage but

under severe pest attack, they will feed on the youngshoots as well (Cranham, 1966a). The

Rhodoxanthin content of tea leaves has been shown to act asa feeding and reproductive

stimulae for Oligonychus coffeae (Fernando, 1967).According to Radhakrishnan (2004),

severe infestation with this species of mite brings about acrop loss of 18%.

High temperature, dry conditions and absence of shade areconducive for the outbreak

of this pest (Seneratne, 1986/Mohotti, 2008; Muraleedharan,1999). The optimal

temperature for growth and development is reported to be30°C and the lower threshold

for the development is 10°C with 232.6° days (Gotoh andNagata, 2001). In northeast

India, this species is reported to be active throughout theyear, but populations increase

from early March to early April and injury is severe duringMay and June (Das, 1959a;

Choudhury et al., 2006; Mukhopadhyay and Roy, 2009). Insouth India, the incidence

of red spider mite is high during January–May and lowduring June–December, with a

peak population in April/May (Selvasundaram andMuraleedharan, 2003). In Sri Lanka,

outbreaks occur during dry weather, that is, May toSeptember in the northeast monsoon

zone and January to April in the southwest monsoon zone(Amarasena et al., 2011).

3.6.5 Dispersal

Mites can move to only a short distance by walking.However, they disperse over long

distances by the transportation of infested plant parts,carried by pluckers, cattles, weed

host and also by wind.

3.6.6 Damage to tea

Oligonychus coffeae favours low jat tea and the attack ispronounced in water-logged

areas, drains and where there is poor cover of tea(Cranham, 1966a). The bushes along the

boundary of roads are very often found to be severelyattacked by this species more than

inside (Das, 1959a) (Fig. 10c). The mites have piercing(Chelicerae) and sucking mouth

parts and feeding is caused by the active stages (adultsand active stages of nymphs). They

cause injury to the leaf surface by piercing the plantcells and sucking out the sap contents.

The mite normally attacks the upper surface of the matureleaves and the affected leaves

turn brown, then bronze, and under heavy attack, they canmove to the lower surface as

well. Severe infestation brings about defoliation.

3.7 Scarlet mite, Brevipalpus californicus

The scarlet mites belong to the family Tenuipalpidae.


There are about three species of scarlet mites reportedfrom tea-growing areas of which

the common one attacking tea is Brevipalpus californicusBanks1904 (=Brevipalpus

australis Tucker) (Pritchard and Baker, 1958), which is acommon pest of tea reported in Sri

Lanka (Cranham, 1966a) and south India and Africa (Rattann,1992). On the other hand,

Brevipalpus phoenicis, Geijskes, 1936 is the main mitespecies reported from Bangladesh

(Ali and Haq, 1973), north India (Das, 1963), Indonesia(Dharmaadi, 1983) and Hawaii

(Randall et al., 2008). Morphological characteristics ofboth these species are very similar.

All these Brevipalpus species of mites belong to the familyTeniplapidae and, unlike

spiders, mites do not spin webs.

Brevipalpus phoenicis has several common names such as thered-and-black flat mite in

Hawaii (Haramoto, 1969), the scarlet mite in India and SriLanka (Das, 1961; Banerjee, 1969,

1971; Baptist and Ranaweera, 1955; Cranham, 1966a;Danthanarayana and Ranaweera,

1970, 1974) and the red-crevice mite in East Africa(Banerjee, 1976).


This species of mite attacks a wide variety of plantspecies including citrus plants and

ornamental plants (Pritchard and Baker, 1958; Oomen, 1982).


Feeding of scarlet mites induces dark brown necrosis alongthe midrib and and petiole on

the lower surface of leaves and necrotic patches alongmargins of leaves leading to heavy

defoliation (Fig. 10d). The leaves are sometimes curved.Unlike the case of spider mites,

the mites are not visible to the naked eye but can be seenwith a hand lens. White dust-like

specks are noticeable to the naked eye at the base of leaf,near the midrib and along the

margin. The leaves appear bronze from afar.


The scarlet mite was first recognized as an important pestof tea in Java (Koningsberger,

1903; Bernard, 1909). Since then, its life history has beenstudied in detail by several

workers (Baptist and Ranaweera, 1955; Banerjee, 1965, 1971,1976; Haramoto, 1969;

Zaher et al., 1970; Oomen, 1982). Infestation begins withthe laying of eggs singly on the

undersurface of the leaves, especially near the midrib andon the petiole, at the base of

leaf (Cranham, 1966a; Anon., 1994; Zeiss and Braber, 2001).Although they are sedentary,

occasionally a small percentage of mites are found on theupper surface, usually following

a long drought. Deterioration in leaf quality makes themmove to younger leaves. Eggs are

bright red and elliptical in shape. The adult mite issmaller and flatter than the red spider

mite, ovate and scarlet in colour with some black marks onthe body and hence its other

name, the black mite (Oomen, 1982). The life cycle isreported to be very similar to that

of red spider mites, although male mites are rarely foundand unfertilized eggs produce

the females. Males are few and rare in the population ofBrevipalpus phoenicis. Razoux

Schultz, 1961 estimated the proportion of males on tea as

about 1.5%. Development

and fecundity are reported to be lower than the other mitespecies and the buildup of

the population is slow. Application of copper fungicides isreported to increase the mite

population (Oomen, 1982).

Baptist and Ranaweera (1955) reported a five-week periodfor the entire life cycle in the

laboratory at about 4500 ft elevation, whilst Zeiss andBraber (2001) reported 2–47 days,

depending on the season. The populations are known toincrease during the dry season,

and peaks at the end of the dry season (Oomen, 1982). Innortheast India, it is reported to

occur mostly during February–November (Anon., 2012). In SriLanka, they occur during dry

weather from December to May in southwest monsoon areas andJune to September in

the northeast monsoon zone and decline during wet weather.Scarlet mites are supposed

to favour high jat tea and are also suspected to attackstressed bushes. They are more

common in the latter years of the pruning cycle and areseldom reported until the second

year after prune (Cranham, 1966a).

3.7.5 Dispersal

Similar to red spider mites.

3.7.6 Damage to tea

As in the case of other mites, damage to the tea leaves iscaused by the piercing and

sucking mouth parts of the adults and nymphs. This speciesof mite generally does not

attack the flush, but attacks the maintenance foliage,especially near the midrib and

petiole. However, during dry weather, if there is heavyinfestation, they move to attack the

young leaves as well. Damage induces dark brown necrosis ofthe midrib and petiole on

the undersurface of leaves and also causes marginalnecrotic patches on the leaves and

sometimes the leaves are curved. Severe defoliation canoccur even, at low mite density

per leaf, causing the death of petiole. Defoliation isoften seen with the occurrence of

the first showers of rain by which time the population hasdwindled (Danthanraayana and

Ranaweera, 1972). The loss of crop due to damage by thisspecies is reported to be 13%

in Indonesia, 8–17% in south India and 12% in Kenya (Wilsonand Clifford, 1992; Kumar

et al., 2004; Rattan, 1992).

3.8 Purple mites, Calacarus carinatus

Purple mites, Calacarus carinatus Green, 1890, belong tothe family Eriophydae, which

includes other pests such as blister mites, rust mites, budmites and gall mites. These are

minute and worm-like and have only two pairs of legs.


North and south India, Sri Lanka, Indonesia, China(Cranham, 1966a; Muraleedharan et al.,

1988; Anon., 2012) and Bangladesh (Mamun and Ahmed, 2011).


The damaged leaves are dull with purplish brown

discolouration giving a bronzed

appearance (Fig. 10e). Heavy infestation gives a dustyappearance due to the leaves

becoming covered, especially along the midveins, withthousands of white cast skins of

the mites. Examination with a hand lens will show thousandsof white caste skins and also

the mites themselves.


The biology and ecology of this species were described byKing (1937) and Das and

Sengupta (1962). They are very small and cannot be seenwith the naked eye. The adult

female is dark purple in colour with five longitudinalwhite waxy ridges on the dorsal side.

The body is elongated and spindle-shaped and is broaderanteriorly. The two pairs of legs

are directed forward and move slowly. The abdomen of themale is shorter than that of the

female. Infestation begins with the laying of the eggs bythe adults on the undersurface of

the leaves. The eggs develop into larvae, protonymphs andadults and the total life stages

take about 6–11 days. About 22–23 generations per year werereported in China and this

species does not go into diapause (Haiyuan and Jian, 1988).

This species of mite attacks mainly the older foliage butduring dry weather, they can

be seen in the younger leaves up to the second and thirdleaves from the bud. Although

they can be seen both on the upper and lower surfaces ofthe leaves, they are reported to

prefer the upper surface, especially along the midrib andmargin (Light, 1927). In Sri Lanka,

it is commonly reported in upcountry districts on high jatteas attacked by scarlet mites. It

occurs mostly in teas which are in the first year afterpruning and is reported to predispose

to attack by scarlet and red spider mites or occurs alongwith pink mites (Cranham, 1966a).

In northeast India, it is reported to occur mostly duringFebruary–November (Anon., 2012).

In south India, peak numbers of mites were reported inJanuary, April, May and November

(Muraleedharan and Chandrasekharan, 1981). In Japan, thepopulation increase is reported

from June to October (Shiao, 1976). High temperature andhigh relative humidity, as well

as heavy rainfall, were reported to significantly reducemite numbers (Muraleedharan et al.

(1994). Low temperatures were also reported to have anadverse effect on mite populations

(Muraleedharan and Chandrasekharan, 1981). Danthanarayanaand Ranaweera (1972), in

addition to pointing out the effect of rainfall on the mitepopulation, also suggested that

mite numbers could be influenced by certain biochemicalprocesses in tea leaves such

as the presence of Rhodoxanthin acting as phagostimulants.Calacarus carinatus was

reported to prefer shaded tea, except in August, inTripura, India (Pande and Nandi, 1983).

3.8.4 Dispersal

Similar to red spider and scarlet mites.

3.8.5 Damage to tea

It is reported to be the least injurious amongst the mitespecies attacking tea (Cranham,

1966a). As in the other plant-feeding mites, the damage totea is due to piercing and

sucking plant sap by the adults and nymphs. They generallyattack the mature leaves, but

under severe infestation, especially during dry weather,can occur in younger leaves as well.

They do not cause much defoliation. According to studiesconducted in south India, the

loss due to eriophyid mites was only about 5%(Muraleedharan and Radhakrishnan, 1990).

3.9 Pink rust mite (or) orange Acaphylla theae


Pink mite, Acaphylla theae Watt, 1898, sometimes referredto as pink rust mite, belongs

to the same family as the purple mite and is a serious pestin northeast India, south India,

Bangladesh, China, Taiwan, Nepal, Malaysia and the formerUSSR (Yeh and Huang, 1975;

Danzinger, 2000). It was reported from Sri Lanka only sincelate 1999, and is present only

in localized areas (Vitarana, 2000).


Damage symptoms are in the form of yellowish discolourationof leaves, which appear

pale, dry and leathery and the veins and margins of theleaf become pinkish and affected

leaves curve upwards (Fig. 10f). White cast skins are alsofound on the leaf surfaces.

Severely damaged leaves appear crinkled.


Adults are minute and cannot be observed, even under a handlens. They are orange

coloured and carrot-shaped. Eggs are shiny and globular andlaid singly on the undersurface

of the leaves. Eggs hatch in 2–3 days. There are twonymphal stages and they are white in

colour and are found on the undersurface of young leaves.

During the dry season, they multiply fast, causing severedamage. Studies carried out in

Taiwan by Yeh and Huang (1975) indicated 30 generations peryear and the development

of one generation took 9.5 days at 23.1°C and 18.2 days at15.9°C. Studies carried out

in China, on the other hand, by Kuang and Zhao (1988),indicated 22–23 generations per

year. According to the latter, it took 11 days for thedevelopment at 21°C; egg hatch was

affected beyond 33°C and at 35°C hatching ceased. Seneratneand Mohotti (Seneratne,

1986/Mohotti 2008) reported 6–7 days for one generation.Kunshan et al. (2003) observed

the mite population to reduce below 18°C and above 27°C.The most favourable relative

humidity for the development was reported to be between 75and 90%. Increase in

population was inhibited when relative humidity was lowerthan 50% or higher than 95%.

Population density was reported to be the highest onpubescent varieties of tea with

round-shaped leaves (Yeh and Huang, 1975) and high jat tea(Assamica variety). The

removal of shade trees increased the population density.

They are known to be present

throughout the year but heavy rains control them. Innortheast India, the occurrence of this

pest was reported to be mostly during February–November(Anon., 2012). In south India,

the population buildup initiates in November/December andattains a peak in February/

March, declining during May/June. Life cycle is completedin 6–9 days (Anon., 2015). This

mite is often found with purple mites on the same leaf.

3.9.4 Dispersal

Similar to other tea mites.

3.9.5 Damage to tea

As in the case of other tea mites, the feeding activity ofadults and nymphs causes damage

to tea leaves. Damage is on both surfaces of the leaves andis mostly restricted to tender

leaves. It also affects the tender stems and petioles. Itis reported to favour high jat tea as

opposed to low jat (Seneratne, 1986/Mohotti, 2008).Affected leaves turn pale and show

upward curling. In severe infestations, leaves becomeleathery and brownish. The affected

plants appear sickly and are stunted in growth.

3.10 Yellow mite/Broad mite, Polyphagotarsonemus latus

The yellow mite, Polyphagotarsonemus latus, Banks, belongsto the order Tarsonemidae.

It was first described by Banks (1904), as Tarsonemuslatus, from the terminal buds of

mango in Washington, D.C., USA (Denmark, 1980). Thisspecies is distributed worldwide

in many crops and has many names. In Sri Lanka and India,it is referred to as yellow

mite, in Bangladesh as yellow jute mite, in Romania asbroad spider mite and in Africa

as broad rust mite. This species belongs to the familyTarsonemidae and as with other

Tarsinomids has a very simple life cycle (Gadd, 1946). Itwas first detected in tea in south

India in 1955 (Cranham, 1966a) and was considered as aminor pest. It is reported from

China (Chen and Kunshan, 1987), Indonesia (Damiri, 2014),Bangladesh (Cabi, 1986) and

Sri Lanka (Cranham, 1966a).


The infested young leaves are stunted, unthrifty and appeardistorted, with two light corky

brown-striped areas parallel to the midrib on the undersideof the leaves (Fig. 10g). The

corky area usually covers the whole midrib and may coverthe entire lower surface. The

upper surface has a wrinkled appearance. The affectedleaves also become coppery brown.


The biology of yellow mite in tea was described by Gadd(1946). The yellow mite has

four stages in its life cycle: egg, larva, nymph and adult.The entire life cycle takes about

five days and therefore the buildup of population can bevery rapid. The young larvae

hatch from the eggs in about 2–3 days. Larvae areslow-moving and pass into a quiescent

nymphal stage. The adults are minute, whitish and paleyellow in colour and can be seen

as minute white dust-like specs on the undersurface of theleaves. Males are smaller and

have suckers on the posterior end which help to carry thequiescent female.

Yellow mites are active and fast-moving. Adults and nymphsof this mite are seen on

younger leaves, especially the top two to three leaves andthe bud. The males move around

rapidly in search of the quiescent females which theyremove from the leaf and carry with

the aid of the suckers and move to the younger leaves. Assoon as the female emerges

from the quiescent stage and becomes an adult, copulationtakes place. According to

Gadd (1946), unfertilized females produce only maleoffspring, whilst fertilized females

produce both sexes. The mites prefer areas of high humidityand low temperature and

shady areas. Yellow mite is common in tea recovering frompruning and in young tea. They

occur mostly during the post-monsoonal periods ofJanuary–March and August–October.

In prolonged dry weather, the population declines.

3.10.3 Dispersal

Short-distance movement may be accomplished by walking.Apart from other common

modes of dispersal of all phytophagous mites, such astransportation by plant parts and

wind, this particular mite species has a special mode ofdispersal by the males. The adult

male gets attracted to female pupae and lifts them up withtheir specialized hind legs and

carries them to new feeding sites. The movement of malescan be seen with a hand lens.


Yellow mite attacks the flush and therefore causes directloss to crop. Feeding is caused

by adults and nymphs. Damage is caused during piercing andsucking of the leaf sap

and affected leaves become rough and brittle; thus, thequality is also affected. In severe

infestations, affected leaves curl downward (Fig. 10h).Although the period of attack often

declines fast, the bush takes a longer period to recover.

3.11 Kanzawa spider mite Tetranychus kanzawai Kishida

The Kanzawa spider mite belongs to the order Tetranychidae.


This is an important mite species known to damage tea inJapan, China, Taiwan and the

Philippines (Ho, 2000).


It is a polyphagous species. Some of the important hostsinclude peanut (Arachis hypogaea

L.), papaw (Carica papaya L.), Citrus sp. strawberry(Fragaria ananassa Duchesne), soybean

(Glycine max (L.) Merr.), apple (Malus pumila Miller 1768),mora (Morus alba L1753),

sweet cherry (Prunus avium L 1755), peach (Prunus persicaL. Batch, 1801), aubergine

(Solanum melongena L.) and grape vine (Vitis vinifera L.).


Damaged leaves appear with yellowish spots. In heavilyinfested bushes, the leaves

become completely yellow and dry. Webbing appears on theundersurface of the leaf.


Life history of this species on tea has been reported byTsai et al. (1989) and Ho (2000).

The female (Fig 10i and 10j) lays about 2–3 eggs per day onthe undersurface of the

leaves. The development of the mite is reported to betemperature-dependent with

the optimal temperature being 12–37˚C; development ceasesat 12˚C (Ho, 2000) and

the entire life cycle is completed in about 40 days. InJapan, this species is reported to

undergo diapause at 18°C or below (Takafuji et al., 2000).Tsai et al. (1989) reported the

development to be slow at lower temperatures and thehighest around 30°C. The mean

generation time ranged from 12.4 days at 30°C to 53.9 daysat 15°C. Studies carried out

on host plant preference indicated the presence ofdifferent races and tea populations

take only tea as a host, although intrapopulation crosseswere successful amongst some

populations (Gomi and Gotoh, 1996).

3.11.5 Dispersal

Similar to other phytophagous mites.


Damage is caused during the feeding of adults and nymphswhen they suck the cell

contents and induce yellow necrotic spots on the affectedleaves. Heavily infected leaves

become very dry.

4 Occasional and minor pests

4.1 White grub (Cockchafer larvae)

The larvae of scarabaeid beetles (cockchafers), arecommonly called ‘white grubs’. The

species belongs to the order Coleoptera, familyScarabaeidae. Amongst the several

species of white grubs occurring in tea soils, Holotrichiadisparalis Arrow Hadton, 1916,

is the only one which causes severe damage to the roots ofyoung tea and it belongs

to the subfamily Melolonthinae Samouelle, 1819. The othercommon species that are

encountered in tea areas are Microtrichia costata Walker(family: Melonthinae) and Anomla

walkerie Arrow (subfamily Ruteline), which usually feed ondead organic matter, although

occasionally they have been observed to cause damage to tea(Fernando, 1971).


Bangladesh (Mamun and Ahmed, 2011), Sri Lanka (Cranham,1966a), India, (Das, 1965;

Sharma and Bisht, 2012) and China (GuangZao, 2006). In SriLanka, it is mostly found at

higher elevations, above 1200 m, and in areas underrehabilitation grasses.


Wattle (Acacia sp. Martius 1829), Albizzia Durazz 1771,Erythrina ligthosperma Miq,

Guetemala grass (Tripsacum laxum Scrib and Merr, Mana grass(Cymbopogon confertiflorus

(Steud.) Stapf), lawns, patnas.


Above-ground symptoms are in the form of yellowing offoliage, defoliation and dieback.

Inspection of the tea root system indicates that the rootshave been chewed off, leaving

behind only the callused stumps. Ring-barking of the stemat the soil level is also noticed.

This should not be confused with ‘collar rot’ formed due todeep planting, or fungus attack

as collar rot due to deep planting often attracts the otherspecies such as Mitotrichia

sp. (and not Holotrichia disparilis), which feed only onthe dead and moribund bark of

the collar, inducing secondary damage. Inspection of thesoil indicates the presence of

‘C’-shaped larvae (grubs) in the soil. The grubs ofHolotrichia could be easily distinguished

from the other white grubs by the black vomit formed fromthe mouth when handled. The

arrangement of a double row of spines (‘rasters’) and hairson the ventral side of the last

abdominal segment also help to identify this species(Cranham, 1966a).


The biology of this pest has been described by Cranham(1966a). The eggs are soft,

ellipsoid, off-white in colour and are laid in the soil.The incubation period takes around

12–18 days. There are four larval instars in Holotrichiadisparalis, whilst the other chafers

have three. The larvae are fat, whitish or cream-colouredgrubs, generally 1.5-in long

(when fully grown), with the body curved in the form of theletter ‘C’ and they live in soils

at a depth of 5–20 cm. When the soil is dry, they movedeeper (Fig. 11a). The larvae are

reported to mature during November–December.

Prior to pupation, the larvae stop feeding and the colourof the larvae becomes opaque

or off-white or yellowish. When it stops feeding, the bodybecomes less ‘C’-shaped. It

hollows out an earthen cell several inches below the groundand pupates. Pupation is

generally from December to January. Pupa is at first softand yellowish brown, but later

hardens and becomes dark in colour, although not encastenedwithin a pupal case.

The adults are about 0.75-in long and are mostly brown incolour (Fig. 11b). They fly

during the night time and are attracted in large numbers tolight, along with other chafer

species. During daytime, they are found resting amongstleaf litter. There is only one

main generation per year. Adult emergence is usually at thebeginning of rains. In Sri

Lanka, the peak adult emergence is between March and Mayand smaller numbers of

adult emergence are in the later months, with lessoverlapping of generations (Cranham,

1966a).

4.1.5 Dispersal

Adults fly nocturnally and a large number of them areattracted to light.

4.1.6 Damage to tea

Damage to tea is caused by the feeding activity of thelarvae, mostly on tender tea

roots. They chew off the roots, leaving a callused stump,or ring bark the stem at the

Figure 11 (a) Larvae of white grub, Holotrichia disparalis(Courtesy Tea Res. Inst. Sri Lanka). (b) Adult

white grub Holotrichia disparalis (Courtesy Tea Res. Inst.Sri Lanka). (a) (b)

soil level. Damage is serious only in the first and secondyears from planting and in the

nurseries and is more pronounced during dry weather and insoils with high organic

matter. According to Cranham (1966a), dense populations ofabout 1–2 grubs per

sq. ft of soil surface could cause severe losses of about40–50%, or sometimes less.

Adult beetles feed on the foliage of neighbouring trees andcause defoliation. There

are instances where large patches of very young tea plantshave been wiped out by

these grubs (Sonan, 1939; Cranham, 1966a; Fernando, 1971;Gireesh Nadda et al., 2013;

Arulpragasam and Adaikkan, 1983).

4.2 The red borer, Zeuzera coffeae Nietner

Red borers belong to the order Lepidoptera, family Cossidae.


This pest is reported from India, Sri Lanka, China,Malaysia, Indonesia (Cranham, 1966a;

Danzinger, 2000) and Vietnam.


It feeds on a wide range of hosts, some of which includeCasuarina L., Erythroxylum P.

Browne, Acalypha L., Phyllanthus L., Hydnocarpus Gaertner,Annona L., Cinnamomum

Schaeff, Persea Mill, Amherstia nobilis wall, Cassia,Pericopsis elata, (Harms) van Meeuwen,

Hypericum perforatum L., cotton (Gossypium L.) Hibiscus L.,Cedrela odorata L., Melia

azederach L., Swietenia Jacq., Psidium L., Grevillearobusta A. cunn ex R. Br, Coffea

L., Citrus sp., Cestrum L., cocoa (Theobroma cacao L.,Clerodendrum infortunatum L.),

Lantana L. and Tectona grandis L.


The appearance of reddish pink frass, similar to largepellets of sawdust, on the ground

and under the bush is indicative of the presence of thispest. Holes about 5 mm in diameter

would be visible a few centimetres above the collar. Splitstems of affected plants show

galleries, with dark reddish caterpillars or chestnut brownpupae.


Moths are large, with a wingspan of 4.5 cm, whitish incolour and spotted with black and

dark blue spots. They are strong fliers. The development isslow and takes about 4–5

months. Yang et al. (1997) reported that there was only onegeneration per year in China

and that the pest overwintered at the fifth instar stage.The lower threshold temperature

for the development in China was reported to be 15.0°C forpupae and 17.6°C for eggs.

The development of these stages required 174 ± 12 and 124 ±

7 day °C, respectively.

Infestation begins when eggs are deposited into thecrevices of the stem and the eggs

resemble strings of amber-coloured beads. The mature larvais about 3.5-cm long, and is

reddish brown in colour with a small head and largemandibles. The pupae are 1-in long

and are chestnut brown in colour. Pupation is within aspecially constructed chamber,

which has an exit hole through which it wriggles out andemerges into a moth. This pest

mainly attacks young plants and is thus found in newclearings.

4.2.5 Dispersal

Moths are strong fliers and can move long distances. Newlyhatched caterpillars are

dispersed mainly by the wind.

4.2.6 Damage to tea

Damage is caused when larvae bore into young stems with theaid of powerful mandibles

and tunnel downwards, devouring the woody parts, especiallythe pith (Fig. 12a and b).

Holes are made at intervals to eject the excreta and woodparticles. The leaves of affected

plants wither and turn brown, but remain attached to theplant. With the growth of the

larva, the tunnel is also extended and may extend intothicker branches and even up to the

root. The affected stem may be completely hollowed out,leaving only a thin shell of bark

and wood and eventually the plant dies (Cranham, 1966b;Gireesh Nadda et al., 2013).

4.3 Tea aphids, Toxoptera aurantii Boyer de Fonscolombe

The tea aphids belong to the order Hemiptera, familyAphididae. The species that occurs

on tea is called Toxoptera aurantii, Boyer de Fonscolombe,which is also often referred

to as the black citrus aphid. Although countries likeChina, Bangladesh and northeast

India have very often listed it under major pests, this islisted here under minor pests as

economic damage is, on a comparative basis, less.


It is widely distributed in all the tea-growing countries,namely Bangladesh (Mamun and

Ahmed, 2011), India (Das, 1965), Japan (Akira, 1997), China(Han et al., 2014), Vietnam

(Kha Zui Nga, 1982), Nepal (Shrestha and Thapa, 2015),Indonesia (Dharmaadi, 1983),

Sri Lanka 1966 (Cranham, 1966a) and Tanzania (Msomba,2015). In countries like China,

it is considered as a major pest, whilst in Sri Lanka andsouth India, it is considered as an

occasional minor pest.

Figure 12 (a) Split tea stem showing red borer, Zeuzeracoffeae, inside the bored tunnel (Courtesy

Tea Res. Inst. Sri Lanka). (b) Tea stem damaged by redborer, Zeuzera coffeae (Courtesy Tea Res. Inst.

Sri Lanka).

(a) (b)


It is very common in citrus (Citrus medica L.), cocoa(Theobroma cacao L.), coffee (Coffea

L.), fig (Ficus benjamina L. 1767), Rambutan Nepheliumlappaceum L., mango (Mangifera

indica L.), sour sop (Annona muricata L.) and severalornamental plants.


Appearance of distorted leaves and presence of sooty mouldson mature leaves. The

insects can be seen clustered on the underside of tenderleaves and shoots.


Infestation starts with the winged females alighting on thehost and forming large numbers

of colonies. These are small soft-bodied, sucking insects,dark brown in colour and found

in clusters on the tender leaves and buds or the undersideof young leaves (Fig. 13).

Winged and wingless females are present. The first-stagenymphs have legs which help

them to crawl. The latter stage nymphal instars and adultsare stationary and attached to

the underside of the leaves with their sucking mouth parts.

The wingless females are about 1–2 mm in length, dark brownin colour with a flask

shaped body. On the sixth segment of the abdomendorsolaterally, they have a pair of

tubular protuberances (cornicles) projecting backwards,which is characteristic of aphids,

and through which waxy substance is secreted; this helps todeter predators (Cranham,

1966a). They are viviparous and reproduction is caused byparthenogenesis. The offspring

develop as tiny wingless females, although a few wingedfemales have also been reported

Figure 13 Tender tea leaf and stem encrusted with teaaphid, Toxoptera aurantii (Courtesy Tea Res.

Inst. Sri Lanka).

to form colonies themselves (Cranham, 1966a). This pestexcretes sticky honey dew via

the anus which falls on the leaves below and encourages thegrowth of sooty mould on

the plants. Ants have a symbiotic relationship with theaphids. They protect the aphid and

at the same time carry them to other shoots, therebyhelping to develop fresh colonies

(Cranham, 1966a; Gupta and Shanker, 2007; Devi et al.,2010).

4.3.5 Dispersal

Winged females are dispersed long distance by wind andconvection currents. On

alighting on shoots, they start colonies viviparously. Antsalso help in the dispersal as they

carry them from one bush to another. When disturbed, theydo not move fast.

4.3.6 Damage to tea

Damage to tea occurs during the feeding activity by theadults and nymphs when they suck

the sap of the host plant with their piercing and suckingmouth parts. The affected leaves are

distorted and curled. They attack mainly the buds and youngleaves. Since flush is removed

during harvesting, they are not a problem in mature tea andare usually encountered in

nurseries, new clearings and tea recovering from prune.When infestation is heavy, recovery

from pruning is reported to be delayed (Cranham, 1966a). In

south India, the population is

higher from January to April and lower during June/July(Anon., 2012, 2015).

4.4 Scale insects

The scale insects belong to the families Coccidae andDiaspididae, order Hymenoptera.

Those with soft scales belong to family Coccidae and thosewith armoured scales to family

Diaspididae; mealy bugs belong to family Pseudococcidae.There are several species of

scale insect observed in tea gardens and the common onesare the soft-scaled (a) brown

bug (Saissetia coffeae Walker) and (b) green bug (Cocusviridis Green). Both of these were

earlier pests of coffee (Coffea L.) in Sri Lanka (thenCeylon) during the years 1882–86 and

were studied by Nietner (1861).

1 Brown bug (Saissetia coffeae, Walker): These are alsoreferred to as the brown scale, helmet scale, coffee brownscale or hemispherical scale bug. This species was earlierrecorded by Green as Lecanium hemisphaericum Targ. Themature scales of the brown bug are hemispherical in shapewith a marginal rim. Initially, they are pinkish and laterbecome deep brown in colour, and 2–3 mm in length and1.25–2 mm wide (Cranham, 1966a).

2 Green bug (Cocus viridis Green): This is also referred toas the green coffee scale bug. This species was earlierrecorded by Green as Lecanium vididae (Cranham, 1966b).Green bugs, as the name suggests, are yellowish green incolour and flat and oval in shape. Although less convexthan the brown bug, they are about 2.5–3.25 mm in lengthand 1.5–2 mm in breadth. The anterior end is pointed, withtwo tiny black eyes (Cranham, 1966a).


Cinchona officinalis (L.), guava (Psidium guajava),Adiantum sp., Asparagus officinalis L.

1753, Gardenia sp. and Loranthus sp.


As in the case of aphids, the appearance of sooty mouldindicates their presence.


Biology and ecology of this scale insect are similar tothose of the aphids. They occur in

clusters and are observed adhering to tender stems and theundersurface of young leaves

and in heavy attacks, the stems are encrusted with maturescales (Fig. 14). Their behaviour

is very similar to the aphids. However, scale insectsusually do not occur in healthy vigorous

plants and their presence is an indication of an imbalanceof water and nutrients in the

host (Cranham, 1966b). As in the case of the aphids, thescale insect also excretes honey

dew and is attended by ants (Das, 1959b). Infestationbegins with the laying of eggs. With

the green bug, the eggs are hatched almost immediatelyafter exclusion under the adult

female, and a succession of larvae are produced with thegeneration overlap. A mass of

empty egg shells will be found under the adult female.

With the brown bug, a mass of eggs is laid under the maturefemale from which larvae

hatch later, all almost at the same time (Cranham, 1966a).The emerging crawlers (first

instar nymph) in both species disperse and attachthemselves to tender plant parts. There

are three nymphal stages in all scale insects. Crawlersrepresents the only mobile stage.

The remaining two nymphal stages and adults are stationaryand only under adverse

conditions will females move small distances (Hill, 1983).In green bugs, males are not

reported. Although winged males have been reported in thebrown bug, this is rare. In

Figure 14 Tea stem infested with scale insect (Courtesy TeaRes. Inst. Sri Lanka).

both species, reproduction is parthenogenetic. Wheneveradult male brown bugs are

present, they do not feed and die within a few days.

4.4.4 Dispersal

Females are sedentary and remain attached to the feedingsite. Crawlers move short

distances on the leaf area in search of a suitable feedingsite until one is found (Hill, 1983).

Their dispersal from one bush to another is aided by wind.They are also dispersed by

animals, ants, etc. In warmer temperatures, crawlers arereported to settle at a feeding site

in about two days, whilst in cooler temperatures, it takesabout a week (Hill, 1983).

4.4.5 Damage to tea

Damage to tea is caused when the adult female and nymphalstages of the scale insects

feed on the leaf sap with their piercing and sucking mouthparts.

4.5 Root mealy bug, Dysmicoccus sp. (Pseudococcidae:Homoptera: Hemiptera)


Appearance of filamentous masses adhering to the bases ofleaves and sometimes on the

axils of leaves and twigs. Similar observations can be madeon rooted cuttings and young

plants in nurseries (Vitarana, 1989).


Adult females are wingless but have functional wings.Although winged males are present,

reproduction is parthenogenetic. These attack the roots ofyoung tea plants in nurseries

(Fig. 15a and b). The presence of this bug is indicative ofpoor drainage (Cranham, 1966a).

4.5.3 Damage to tea

Damage is not noticeable. It is an occasional pest

4.6 Lobster caterpillar, Neostauropus alternus(Notodontidae: Lepidoptera)

This caterpillar belongs to the family Notodontidae and isa minor pest in tea areas around

the world and is rarely found in large numbers.


India (Watt and Mann, 1903), Sri Lanka (Green, 1890),Indonesia (Dharmadi, 1983) and the

Philippines.


They are considered a pest on pulses, tea, coffee, rambutan(Nephelium lappaceum L.)

and mango. (Mangifera indica L.), Cassia sp., Albizzia sp.,Durazz 1772, Grevillea robusta

A cunn ex R.Br, Crotalaria L.


The presence of grotesquely shaped larvae which stillresemble crumpled leaves. When

disturbed, they rear up, raising the head and thorax, andcurve the back and tail in a

threatening pose. The damaged leaves appear with holes orparts of the leaves eaten,

leaving behind the midrib.


This pest is often found in the nurseries and young teaplantations. Their eggs are whitish,

finely sculptured and laid in small clusters. Thecaterpillars are 1.5–2.0 in long, have a double

series of prominent humps on the back and the last segmentis expanded into lateral flanges

and is grotesquely shaped. The terminal segment has a pairof slender claspers. The colour

of the larvae varies from dark brown to greyish-blacktinged with green and resembles dried

leaves. When disturbed, they rear up their long legs in athreatening stance, with the head

and thorax raised and with a curved back and tail raisedand with claspers held erect. This

gives them the name ‘lobster caterpillar’, althoughsometimes this pest is called the crab

caterpillar. The larval period is 3–4 weeks. Pupation takesplace within a woolly cocoon

formed by two or three leaves spun together and adultsemerge after 10–14 days. Moths

are pale greyish brown with a few reddish brown spots(Cranham, 1966a; Beesan, 1941).

Figure 15 (a–c) Examples of tea roots damaged by mealy bug,Dactylopius sp. (Courtesy Tea Res. Inst.

Sri Lanka).

(a) (b) (c)

4.6.5 Damage to tea

Damage to tea leaves occurs during the feeding activity ofthe caterpillars when they

devour all leaves from young plants. In the cases of severedamage, entire branches are

stripped of leaves.

4.7 Red slug (Eterusia aedea cingala Moore)

Although the larvae of this species resemble nettle grubsin their slug-like appearance,

they lack stinging hairs and belong to the familyZygaenidae. It was noted as a localized

pest of tea from a long time ago (Green, 1890). Thesubspecies recorded in north India is

E. a. magnifica Butler and in south India and Indonesia E.a. virescens (Cranham, 1966a).

In Sri Lanka, it is considered a minor pest, whilst severedefoliation to tea by this pest has

been reported in Darjeeling, north India (Das et al., 2006).


It is polyphagous and has several hosts.


Brick-red-coloured larvae resemble nettle grubs but withoutstings. Pupae are present in

closely woven pinkish cocoons in the fold of leaves.


Eggs are pale white in colour and oval in shape. The fullygrown larvae are 2.5–3.0 cm in

length, broad and are reddish-brown or brick-red-colouredand pupae are creamy white.

Larvae have six rows of longitudinal tubercles on the bodyand eject a viscous, non-irritant

fluid through them as a defence mechanism againstpredators. At pupation, larvae spin a

closely woven straw-coloured cocoon. Adult moths arebrightly coloured in hues of black

and metallic blue, with a yellow abdomen. In northeastIndia, it is known to occur during

Mar–Apr/May–Jun/Jul–Aug,/Sep–Oct (Anon., 2012).

4.7.4 Dispersal

Dispersal is caused by the flight of moths as well asmigration of caterpillars through the

soil.

4.7.5 Damage to tea

Larvae feed on the maintenance foliage on both upper andlower surfaces and occasionally

cause severe defoliation.

4.8 The leaf miner (Melanagromyza theae de Mejere)

This tiny fly belongs to the order Diptera and familyAgromyzidae and is mostly a pest

on young tea plants, in new clearings and nurseries. Inmature tea, infestation occurs

following pruning, when new flush is formed (Gireesh Naddaet al., 2013).


Signs of injury appear as whitish, winding thick bandsunderneath the cuticle on the upper

surface of the leaf.


The adult is winged and active. The life cycle begins withthe female laying the eggs

singly on the upper surface of the leaves, inserting them

below the upper epidermis.

The hatched larvae/maggots mine below the upper epidermis(and not on lower surface)

and the mine widens as the larvae grow. Whilst feeding,this pest leaves characteristic

winding white lines. The fully grown larva pupates at theend of the tunnel (Cranham,

1966b).

4.8.3 Dispersal

Dispersal is caused by the active winged adult.

4.8.4 Damage to tea

Damage occurs during the excavation of mines and feeding onthe leaf sap. In severe

cases, affected leaves bear extensive mines and leaves turnbrown, dry and drop down.

Severely mined leaves become unfit for harvesting (GireeshNadda et al., 2013). Damage

is mostly in new clearing and nurseries.

4.9 Red ant, Oecophylla smaragdina Fabricius


The ants build nests by spinning together a number ofleaves with silk (Fig. 16a). Worker

ants hold the leaves together, whilst others carry thelarvae, which secrete the silk to

bind the leaves (Fig. 16b). The leaf nests appear undamagedand stay green for several

days.

Figure 16 (a) Ant pest Oecophylla smaragdina (Courtesy TeaRes. Inst. Sri Lanka). (b) Ant nest (Courtesy

Tea Res. Inst. Sri Lanka).

(a) (b)

4.9.2 Damage to tea

The webs cause dieback but the injury is negligible. Thepresence of ants causes discomfort

to the workers as they bite severely. They also causeindirect damage to tea by tending

scale insects, preventing them being attacked by otherparasitoids and predators. They

also carry the nymphs of aphids, mealy bugs and scaleinsects from tea bush to tea bush,

thereby spreading infection. They also, however, havebeneficial functions as they are

predacious on other insects (Cranham, 1966b).

4.10 Cut worm (Noctuidae)

There are several species of cut worms. They are notserious pests of tea and cause

occasional damage to tea plants in nurseries and newclearings. According to Gadd

(1947), the common cut worm in tea areas was Agrotis(Euxoa) segetum Schiff. According

to Green (1890) the cut worm was Agrotis suffuse Hubner.


Appearance of stem girdling at the base associated withdamage to the lowest leaves

(Vitarana, 1989).


These are occasional pests of tea nurseries and newclearings. The eggs are laid on leaves

and twigs. The fully grown larvae are dark brown with palestrips and about 3.5 cm long,

with three pairs of short thoracic legs and four pairs of

abdominal feet. During the day,

they are hidden in debris and soil and come out at night tofeed. Pupation occurs in

the soil in an earthen cell. The moths have dull brownforewings, mottled and with dark

markings. They are nocturnal and attracted by light. Duringdaytime, they rest, with wings

folded under leaves and debris (Cranham, 1966a).


Damage is caused by caterpillars during feeding, when theyoften girdle the base of the

stems or roots of young plants in the nurseries, leading tothe death of entire plants and

hence their name ‘cut worms’. They also attack themaintenance foliage near the base of

the plant and make irregular holes.

4.11 Army worm (Spodoptera litura)


This is also a noctuid moth and is similar to the cut worm.Larvae are olive green in

colour with pale lateral stripes. They do not hide in thesoil like the cut worms and

pupate on the surface of soil. The moth has greyish brownforewings streaked with

silvery lines and the hind wings are white. The larvae movein large numbers and when

food becomes scarce, they move gregariously in search offood supply, hence its name

‘army worm’.


They feed on ground cover and form irregular holes on

mature leaves. They do not occur

in new clearings and nurseries and damage is confined tolower maintenance foliage of the

mature tea. It causes only occasional damage and becomesserious only in large numbers

(Cranham, 1966a). According to Beesan (1941), it has fivegenerations in south India.

4.12 Bag worm or faggot (Psychidae)


The presence of the cone-shaped nest made of plant debrisin which the larvae and the

females reside. These hang from a branch suspended by athread from a leaf or twig.


There are many species recorded on tea. The large faggotworm recorded in Sri Lanka is

Eumeta crameri, which is also known as C. crameri Westwood.This species is common

in India and Indonesia. The other species recorded in teaare Mantha alipes Moore and

Acanthopsyche subteralbata Hampson. The former occurs ontea and shady trees, whilst

Acanthopsyche subteralbata is found only on shady trees.Another species occasionally

reported in tea causing damage is Chalioides vitrea Hampson(Cranham, 1966a). This

species constructs a portable case with silk, covered withthe fragments of leaf, bark or

twigs. When feeding, the head and thorax project from thecase, but at the slightest alarm,

withdraw into the case. At maturity, the larva reversesitself and pupates within the case.

Prior to pupation, the bag is suspended from the bush. Thefemale moths are degenerated,

without wings and legless, and emerge from the pupal casebut remain inside the larval

case. The adult females resemble a worm-like egg sac. Theysometimes lack even antenna,

legs and mouth parts. The male pupa pushes itself outsidefrom the bottom of the case and

a male moth emerges. Male moths are reddish brown withwings and are efficient fliers.

During copulation, the male alights on a case having afemale within and the female lays

500 eggs inside the case; the incubation period is 10–15days (Cranham, 1966a; Anon.,

2015).


Psychids are reported to feed by eating holes right throughthe leaves. They also cause

damage to bark and to young plants by cutting the twigs.Although they are considered

pests of tea, damage is minimal. However, they form severedefoliation of shady trees

(Light, 1928).

5 Conclusion

A vast amount of work has been carried out in the past onthe biology and ecology of the

major insect, mite and nematode pests in the differenttea-growing countries. However, the

majority of this work was carried out in the twentiethcentury. Over the last few decades, there

has been a progressive rise in global temperatureaccompanied by changes in weather and

climate and the tea-growing areas have been exposed toconsecutive droughts, intense rains

and unpredictable weather patterns. Insects, mites andnematodes, being poikilothermic, are

greatly influenced by such changes in the weather pattern.Individual responses to climatic

changes will, however, depend on their geographic range,trophic level and natural history

(Strange and Ayres, 2010). A change in climate brings aboutchanges in their behaviour,

changes in geographical distribution, increases ordecreases in population density, increase

in the number of generations, their survival period,fecundity and dispersal, changes in

interspecific interactions and the evolution of pestbiotypes. The climate also has an effect on

the host and predators/parasites and their interactions.Although work is being carried out on

the effect of climate on pests, more in-depth studies arenecessary to understand the impact

such change in climate has on the physiology and behaviourof pests, their predators and

parasites and host plants and their interactions with oneanother. It would also be useful to

carry out studies on the biology and ecology of variouspredators and parasites attacking major

insect pests of tea and to study their behavioural changeswith the change in environment.

Early detection of pest infestation will facilitate correcttiming of treatment. Using the

currently available knowledge on biology and ecology,population models should be

developed to predict population buildup in the differentlocations and integrate this with

the change in the weather pattern. Such models arecurrently present only for few pests of

tea, for example, SHB. Early detection would help preventthe escalation of pests beyond

economic injury levels. In any location where tea plantsare found to lack vigour and there

are potential signs of pest infestation, the first step inpest management is to identify the

particular pest/pests causing damage. This should be doneby locating the feeding site

on the host, checking for the type of damage induced(chewing, sucking, boring and root

damage) and signs of damage (discolouration, necrosis,twisting of leaves, curling, leaf

nests, holes in leaves and root rot). Microscopic pestscould be identified by inspection

under a microscope. In some cases, symptoms of damage areaccentuated or masked

when the disease complex is formed in which case theexamination of the entire plant

part would be necessary. Such an identification warrants aproper knowledge of the pest

morphology and biology and is time-consuming. Currently,computer-based recognition

systems have been developed to identify insect speciesautomatically. This could be

applied to tea pests in the future for a rapididentification.

Molecular and biochemical studies should also be activelypursued to help to identify

different races/pathotypes and study their interactions andpathogenicity on different hosts

as well as their interactions under different environmental

conditions. These studies would

further strengthen integrated management. Most plants havecues to attract predators

and parasites of pests and/or deter harmful organisms.Biochemical studies should be

carried out to identify these volatiles which will alsohelp in the better understanding of

pest/host/predator to parasite interactions.

6 Acknowledgements

The author wishes to extend her sincere thanks to Prof.Chen Zong Mao Academician,

Tea Research Institute, Chinese Academy of AgriculturalSciences, Hanzhou, China; Prof.

S. Kodomari, Senior Consultant of World Tea Union (inChina); Dr Baruah, Director of

the Tocklai Tea Research Institute; Dr Somnath Roy(entomologist), Tocklai Research

Institute, Jorhat, Assam, India; Dr Muraleedharan, pastDirector of the Tocklai Research

Institute; Dr Radhakrishnan, Director, UPASI Tea ResearchFoundation, South India; Dr

Keerthi Mohotti, Deputy Director Research and Nematologist,Tea Research Institute, Sri

Lanka; Mr Dharmampriya Samansiri, Head Advisory andExtension Division Tea Research

Institute, Sri Lanka; and Mr Ajith Prematunge, ResearchOfficer, Entomology Division of

the Tea Research Institute of Sri Lanka for providing herwith photographs of insects and

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11 Chapter 11 Integrated pest managementof insect, nematode and mite pests of tea

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h e r D i t h i o c a r b o m a t e M e t a m s o d i u m 0. 0 2 0 . 1 0 S r i L a n k a P h e n y l p y r a z o l e Fi p r o n i l 0 . 0 0 5 – 0 . 0 0 2 – – B a n g l a d e s h, S r i L a n k a U n c l a s s i fi e d D a z o m e t B an g l a d e s h R y n a x a p y r B a n g l a d e s h M i ti c i d e O r g a n o p h o s p h a t e D i m e t h o a t e0 . 0 5 – 1 – – B a n g l a d e s h E t h i o n 3 – 0 . 3 5– N o r t h e a s t I n d i a T r i a z o p h o s 0 . 0 2 –– – – B a n g l a d e s h O r g a n o t h i o p h o s p h at e Q u i n a l p h o s 0 . 0 5 – 0 . 1 0 . 0 1 – B a n g la d e s h P y r e t h r o i d B i f e n t h r i n 5 3 0 3 0– 3 0 B a n g l a d e s h , C h i n a F e n p r o p a t h ri n 2 2 2 5 1 2 B a n g l a d e s h F e n v a l e r a t e 0. 0 5 – 1 – – B a n g l a d e s h F l u c y t h r i n a t eC h i n a A v e r m e c t i n A b a m e c t i n 0 . 0 2 – 1– – B a n g l a d e s h C h l o r f e n a p y r B a n g l ad e s h C y fl u m e t o f e n N o r t h e a s t I n d i aC y h e x a t i n B a n g l a d e s h E m a m e c t i n b en z o a t e 0 . 0 2 – 0 . 5 – – B a n g l a d e s h E t o xa z o l e 1 5 1 5 1 5 – 1 5 S o u t h I n d i a , n o r t he a s t I n d i a F e n a z a q u i n 1 0 – – 3 – B a n g la d e s h , s o u t h I n d i a , n o r t h e a s t I n d ia , C h i n a F e n p y r o x i m a t e 0 . 1 2 0 4 0 0 . 2– B a n g l a d e s h , s o u t h I n d i a , n o r t h e as t I n d i a F l u f e n z i n e 0 . 1 – – 0 . 0 5 – N o rt h e a s t I n d i a H e x y t h i a z o x 4 – 3 5 0 . 0 11 5 B a n g l a d e s h , s o u t h I n d i a , n o r t h ea s t I n d i a P r o p a r g i t e 0 . 0 5 1 0 5 1 0 5 B an g l a d e s h , s o u t h I n d i a , n o r t h e a s t In d i a , I n d o n e s i a P y r i d a b e n C h i n a , In d o n e s i a S p i r o m e s i f e n 5 0 4 0 3 0 1 – S ou t h I n d i a , n o r t h e a s t I n d i a O r g a n o ch l o r i n e D i c o f o l 2 0 5 0 3 5 4 0 N o r t h e a st I n d i a , C h i n a I n o r g a n i c S u l p h u r E xe m p t e d B a n g l a d e s h , S o u t h I n d i a , n or t h e a s t I n d i a , S r i L a n k a T a b l e 1 ( C on t i n u e d ) I n s e c t i c i d e T e c h n i c a l n am e

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12 Chapter 12 Pesticide residues in tea:challenges in detection and control

1 Introduction

Tea (Camellia sinensis (L.) O Kuntze) is an important cropthat is grown in a number of

countries. The beverage produced from its processed leavesis consumed across the

world because of its special flavour and stimulating effecton the human body. Global

tea production has increased by 4.2% annually over the lastdecade and was around

5.13 billion tons in 2014 (Chang, 2015). This popular drinkis prepared by extracting the

mostly non-fermented (green tea), fermented (black tea) andsemi-fermented (oolong tea)

leaves of the tea plant by adding hot water. A cup of teatastes good when the soluble

substances present in the dry tea leaves come into theliquor in the right proportions.

For this reason, many scientists across the world areinterested in working more intensely

with tea quality and, consequently, the factors thatenhance quality are well known.

Fresh tea shoots are extremely rich in polyphenoliccompounds, the largest group being

the catechins (flavan-3-ols), which constitute 30–35% ofthe dry weight of the material

(Engelhardt, 2010; Harbowy and Balentine, 1997; Robertson,1992). They are present as

‘catechins’ in fresh tea leaves or in green tea. In blacktea, catechins undergo fermentation

to yield oxidation products such as theaflavins (TF) andthearubigins (TR). These oxidation

products of polyphenols are responsible for the colour,flavour, taste, briskness and overall

strength of tea liquor. TF have several beneficial healtheffects, including antioxidant

activities (Almajano et al., 2008; Dufresne and Farnworth,2001; Ganguly, 1995; Gardner

et al., 2007; O’Coinceanaima et al., 2004; Shibasaki andKitakawa, 2003). Apart from

caffeine, tea also contains a unique amino acid, theanine,which is found only in tea plants.

Theanine makes up to 50% of all amino acids and is reportedto have health benefits as

well (Ekborgott et al., 1997; Engelhardt, 2010; Haskell etal., 2008).

Tea is mostly cultivated in tropical and subtropicalclimates as the plant needs a hot,

moist climate and well-drained acidic soils of adequatedepth (Baruah, 1989). These

climatic requirements and the tea ecosystem, whichundergoes continuous monocropping,

are also congenial for the spread of a large number ofpests and diseases (Banerjee,

1983; Das, 1965) which needs to be managed, often with theuse of pesticides, to protect

the crop. However, pesticides invariably leave residues inthe treated crop (Barooah and

Borthakur, 1994, 2008; Barooah et al., 1994; Chen andHaibin, 1988; Chen and Liu, 2003;

Hou et al., 2013). It is therefore vital that the plantprotection schedules are properly

evaluated to ensure food safety, which is why settingmaximum residue limits (MRLs) for

all pesticides is so crucial. The MRL is a legal limit. Itrepresents the highest level of a

pesticide residue that is permitted in or on food or feedfollowing the use of pesticides in

accordance with good agricultural practices (GAPs).

The use of pesticides in tea has become increasinglydifficult because of growing

concerns for food safety, and this is reflected in thestringent MRLs set by national and

international regulatory bodies. Since tea is a highlytraded commodity throughout the

world, the quality requirement of tea has undergoneconsiderable changes over the

years. The factors that undermine quality, such as thepresence of pesticide residues and

other contaminants, therefore have a great influence on thetea trade globally. This very

important and sensitive subject is discussed in thischapter under the following headings:

• Measuring pesticide residues in tea

• Review of recent research on the extent of pesticideresidues in tea

• Conventional methods for detecting residues in tea

• Food safety standards for tea and the challenges ofmaintaining maximum residue limits (MRLs)

• Strategies for reducing pesticide residues in tea.

2 Measuring pesticide residues in tea

The necessity for measuring pesticide residue levels in teaarises primarily from two

reasons. One is to generate data on pesticide residues inorder to assess risk, which is

required for fixing MRLs. Another reason is to check forconformance with the regulatory

standards (MRLs). The generation of residue data fromsupervised field trials is essential

and is constantly being carried out in many tea-producingcountries where the target

pesticides are known and single residue methods maysuffice. However, conformance

testing involves simultaneously screening for the residuesof a large number of pesticides

in tea samples with an unknown history. Therefore, itessentially requires multi-residue

methods that satisfactorily cover the entire range ofpesticides which may be present in a

sample in trace quantities.

The natural constituents of tea may vary with climate,soil, cultural practices, cultivars,

growing season and processing methods (Bhuyan et al., 1991;Gulati et al., 2009;

Jeyaramraja et al., 2003; Mahanta et al., 1988; Sabhapanditet al., 2005; Tamuly et al.,

1993). Residues of the pesticides applied to the tea plantsoften end up in the tea beverage

in spite of the fact that the treated shoots undergowithering (loss of moisture), maceration

or rolling, oxidation (mostly referred to as fermentation)and high-temperature drying

(around 100ᵒC) during tea processing. The intricatecomposition (or matrix) of the tea plant

makes trace-level, multi-residue analysis in tea morechallenging as the co-extractives from

tea severely interfere with the target analytes (Cajka etal., 2012; Steiniger et al., 2010).

As tea has a difficult matrix, many establishedlaboratories fail performance testing on the

tea matrix, for example, the Food Analysis PerformanceAccreditation System (FAPAS)

Proficiency Testing. An AOAC collaborative study on a ‘Highthroughput analytical

method for determination of multi-classes and multi-kindsof 668 residues of pesticides

and chemical pollutants in Tea by GC–MS, GC–MS/MS andLC–MS/MS’, by Guo-Fang

Pang (AOAC study director), is currently in progress forvalidation. If an AOAC method can

be established as an ‘Official Method of Analysis’, itwould certainly help all stakeholders

to comply with the regulations. Another pertinent point toconsider in selecting a test

method is that the default MRLs for a number of pesticidesin tea have been set at very low

levels or at the limit of determination. In addition, newermolecules have replaced many of

the older pesticides and these are not amenable toconventional methods of analysis. The

different analytical techniques that have been used formeasuring pesticide residue levels

in tea will be discussed in detail later in this chapter.

3 Review of recent research on the extent of pesticide

residues in tea

In a review of research carried out in China since the1960s on 17 pesticides in tea,

Chen and Haibin (1988) argued that photolysis and dilutiondue to growth were

the most important factors affecting the persistence ofpesticide residues in/on

the growing tea plant, while vapour pressure played asignificant role in the loss of

pesticide residues during tea processing. The amount ofpesticide extracted during

the tea infusion process was highly related to the watersolubility of the compounds.

The authors suggested that all four factors should beconsidered as integral elements

in studies on pesticide residues in tea. The authors havealso reported that about

25.2–67% of the pesticides applied to tea foliage could bedegraded during the tea

manufacturing process.

Nimura et al. (1989) reported a high incidence of HCH intea produced in Japan. In

another Japanese study, Nagayama et al. (1996) observedresidues of 30 different

organophosphorous pesticides in Japanese tea samples,including residues of fenitrothion,

phosalone and chlorpyriphos. de Silva and Thiemann (1991)reported significant levels of

organochlorine pesticide residues in Sri Lankan teas.Studies reported by Buchholtz (1988)

and Fernandez et al. (1993) in Germany also showed residuesof organochlorine pesticides

in tea, although these fell within the European Union MRLs.

In a survey carried out in India, Muraleedharan (1994)monitored residues of ethion

and dicofol in teas from the Nilgiri hills. The studyshowed that most of the teas (about

93% of the samples) were found to contain residues ofethion, a widely used acaricide

in India, at levels below 1 mg/kg, which were within theIndian and EU MRLs, and that

none of the samples tested exceeded the MRLs for dicofol.In another survey carried

out in various tea-growing states in southern India,residues of ethion, dicofol and

endosulfan were monitored. Residues in all the samples werewithin the EU MRLs.

Barooah (2005) reported on the monitoring of tea samplescollected from tea

gardens in Northeast India. The residues that were detectedwere mostly confined

to ethion, dicofol, endosulfan, chlorpyriphos andquinalphos. A series of pesticide

residue awareness campaigns were organized by the TocklaiTea Research Institute,

Jorhat (Assam), to make the tea growers of the region moreaware of the issues of

pesticide residues. Since 1994, Tocklai has conducted AreaScientific Committee

seminars and special workshops. As a result of theseconcerted efforts, there has been

a downward trend in the occurrence of residues in thesurveys conducted since then

for about 2000 tea samples collected from Northeast Indiaand monitored at Tocklai

(Barooah et al., 2013).

A large-scale survey of the tea produced in southern Indiawas carried out during

2006–08. It monitored residues of dicofol, ethion,quinalphos, hexaconazole,

fenpropathrin, fenvalerate and propargite (Seenivasan andMuraleedharan, 2011). The

results showed that less than 0.5% of 912 samples hadresidues of the target pesticides

and no sample exceeded EU MRLs. In a recent nationalpesticide residue monitoring

programme, carried out in India during 2014–15, only fourout of 174 tea samples were

found to contain residues above the MRL (Anon., 2015). Thestakeholders of these

samples were cautioned to take corrective steps.

Radhakhrishnana and Shanmugaselvan (2015) recentlyconducted an extensive

basket survey of market samples of tea in southern India tomonitor residues of the

target pesticides, that is, hexaconazole, quinalphos,deltamethrin, glyphosate, paraquat

dichloride, chlorpyriphos, fenpyroximate, lambdacyhalothrin, ethion, dicofol, propargite,

benomyl, carbendazim, mancozeb, tridemorph, fenazaquin,fenvalerate, DDT and

bifenthrin. No residues were detected in the majority ofthe samples (90%), while in the

positive samples, the residues were below the respective EUand Indian (FSSAI) MRLs. The

results showed that none of the banned pesticides –monocrotophos, DDT and BHC –

were detected in the surveyed tea samples and the residuesthat were detected were only

of the approved pesticides.

A report from the Tea Research Institute at the ChineseAcademy of Agricultural

Sciences in Hangzhou indicated that the most widely usedpesticides in tea plants in China

in the recent past were endosulfan, imidacloprid,bifenthrin, cypermethrin, deltamethrin,

acetamiprid and propagite (in that order). In the same

report, on the basis of around

50 000 tea samples tested, it was observed that residues offenvalerate, fenpropathrin,

imidacloprid and acetamiprid exceeded the respective EUMRLs. However, in recent

years, the proportion of tea samples containing residueshigher than the EU MRLs has

decreased significantly (Chen, 2013).

The decreasing trend of residues detected in tea producedin the two major tea-growing

countries – India and China – in recent years is a goodindication of the concerted efforts of the

tea growers, research institutes and governments. This isencouraging news to all tea lovers

globally.

In the period 2004–07, concerns were raised about S-241(Octochlorodiphenyl ether)

in tea samples exported to Europe. S-241 is not a pesticidebut a synergist for synthetic

pyrethroids. Studies carried out by the Tea ResearchInstitute at Hangzhou, China, showed

that tea samples can pick up residues of S-241 frommosquito coils or aerosols used inside

tea houses (Chen, 2013).

A monitoring study was carried out in local markets inTehran (Iran) by Amirahmadi

et al. (2013), where samples consisting of bothdomestically grown and imported teas

were screened for residues of 25 pesticides belonging todifferent chemical groups

using a validated GC–SQ–MS method. Results showed that tenout of 56 samples were

contaminated with residues of endosulfan sulphate(0.005–0.02 mg/kg) and five samples

were contaminated with bifenthrin (<0.01–0.035 mg/kg).These levels were below the EU

MRLs. The highest levels of residues were detected in thedomestically grown samples,

for both endosulfan sulphate and bifenthrin. There is noMRL for these two pesticides in

tea in Iran that can be used for comparison, and none ofthe positive samples contained

more than one pesticide.

About 21 million kilos of tea are produced in Nepal, in 19000 ha. Among the different

crops grown, tea accounts for the highest pesticide useafter cotton. To avoid crop loss,

most farmers, including private companies and small teagrowers, use more than 20

pesticides in their gardens (Shrestha and Thapa, 2015).There is no detailed study on

the monitoring of residues in tea in Nepal. Similarly, verylittle information is published

on recent monitoring studies from Sri Lanka, Kenya,Indonesia and other tea-producing

countries, although in tea-consuming countries, a number ofreports are available on

regular monitoring programmes.

Different food safety regulatory agencies carry outmonitoring programmes to test for

pesticide residues in tea. In Canada, pesticide residues intea have been monitored under

the Canadian National Chemical Residues Monitoring Programand/or the Food Safety

Action Plan. The Canadian Food Inspection Agency carried

out surveys and tested coffee,

fruit juice and tea for the presence of pesticide residues.The recent surveys indicated

no health risks to consumers. A 2010–11 study found that75% of the tea samples tested

met Health Canada standards for pesticide residues. Thehighest number of violations

was with bifenthrin (11), cypermethrin (10), imidacloprid(22) and thiamethoxam (18). In a

similar survey carried out in 2009–10 involving 100samples, 59% were compliant, while

the higher residues detected were of bifenthrin (8),cypermethrin (5) and imidacloprid (11)

(Canadian Food Inspection Agency, 2010–11).

In the United States, the Total Diet Survey, carried out bythe Food and Drug Administration

(FDA) in 2008, showed low levels of residues in 42 teasamples imported in the United States

and 71.4% of the samples were without residues (FDA Survey,2008). Tea was also one of

the imported commodities that were monitored in 2011 by theFDA. The results, which were

based on 15 samples, showed a violation percentage of 26.7%(FDA Survey, 2011).

Hayward et al. (2015) surveyed tea samples from 18 vendorsin the United States for

pesticide residues. The results showed that out of 62black, green, white and oolong tea

samples, 31 had residues (up to 3.2 mg/kg) ofanthraquinone, azoxystrobin, bifenthrin,

buprofesin, chlorpyrifos, cyhalothrin, cypermethrin,p,p-DDE, p,p- and o,p-DDT,

deltamethrin, endosulfan, fenvalerate, heptachlor,

hexachlorocyclohexanes (α-, β-, γ- and

δ-HCH), phenylphenol, pyridaben, tebuconazole, tebufenpyradand triazophos.

Feng et al. (2015) monitored residues of 32 pesticides in223 samples of green tea,

oolong tea, black tea and pu-erh tea from three teadistricts of China during 2010–12.

Results showed that 189 samples were tested positive, outof which 39 exceeded the EU

MRLs. Oolong tea samples were found to be the mostcontaminated, and bifenthin was

the pesticide that was most detected in excess of the MRL.However, the potential health

risk to tea consumers was below the safe limits.

In 2012 and 2013, the Tea and Herbal Infusion, Europe(THIE) (formerly known as the

European Tea Committee, ETC) established a database tocompile the results obtained

from monitoring of more than 14 000 analyses, correspondingto more than three million

individual results pesticide residues in teas from 29different origins worldwide. More

than 500 different pesticides were monitored. Only 1.02% ofthe total number of samples

tested positive and 0.13% of the samples had individualpesticide traces above the EU

MRLs. This clearly confirms that tea is a safe and healthybeverage (Hann, 2015).

4 Conventional methods for detecting residues in tea

In pesticide residue analysis, only validated methods ofsample extraction, clean-up and

estimation are used as instruments and, together with thematrix effects, they significantly

contribute to the accuracy of results. For the analyticalmethod to be demonstrated as

valid, it needs to be capable of providing mean recoveryvalues at each fortification level

within the range of 70–120%, with a repeatability relativestandard deviation (RSD) of less

than 20%. In a multi-residue screening method, the numberof target pesticides that can

be analysed in a single run is large, but the recoveriesfor a few pesticides may be slightly

outside this range. For enforcement or conformance testing,the method used should be

sensitive, selective and robust.

The conventional methods of detecting pesticide residues intea invariably involve

(1) extraction of the samples, mostly with organicsolvents, (2) clean-up by liquid–liquid

partitioning (often between aqueous sodium chloridesolution and dichloromethane/n

hexane/ethyl acetate) and/or using silica gel, alumina orflorisil column chromatography

and (3) estimation of the residues in the cleaned extracteither by using GLC (gas–liquid

chromatography) with ECD (electron capturedetector)/nitrogen phosphorus detector/

FPD (flame photometric detector) or by using HPLC(high-pressure liquid chromatography)

with a ultraviolet/photodiode array detector directly or byusing an HPLC–fluorescence

detector, once the residues in the clean extract have beenidentified (Banerjee and

Banerjee, 2012; Barooah and Borthakur, 1994; Barooah etal., 1994; Barooah et al., 2011;

Bhattacharya et al., 1995; Chen et al., 1992; Chen and Liu2003; Gajbhieye et al., 1989;

Gopal et al., 1987; Gupta and Shanker, 2008, 2009; Jaggi etal., 2000; Kumar et al., 2005;

Moinfar and Hosseini, 2009; Oh, 2007; Pramanik et al.,2005; Sanyal et al., 2006; Sharma

et al., 2008; Wu et al., 2009; Xia et al., 2008). Many ofthese studies have emphasized the

need for the rehydration of the dry tea leaf samples priorto solvent extraction to improve

recovery.

Barooah and Borthakur (1994) extracted residues ofalphamethrin in tea shoots and

black tea samples by blending the samples with a mixture ofacetonitrile and water (65:35,

v/v). Clean-up was undertaken first by liquid–liquidpartitioning (into n-hexane) and was

followed by acidic alumina column chromatography (elutingwith n-hexane-acetone, 95:5

v/v), and estimation was done by GLC–ECD using a glasscolumn (2 × 2 mm i.d.) packed

with 1.95% OV210 + 1.5% OV17 on 100/120 mesh ChromosorbWHP. Residue levels were

confirmed by a reanalysis on another similar column coatedwith a different phase (3%

OV-10 on 100/120 mesh Chromosorb WHP). The method was alsofound to be suitable

for the estimation of a number of other syntheticpyrethroid insecticide residues in tea in

the authors’ laboratory.

Residues of ethion and dicofol in both CTC and orthodoxblack tea could conveniently

be extracted by pre-soaking the dry tea leaves in water(for 30 minutes), followed by

blending the brew (tea liquor) with a mixture of acetoneand n-hexane (1:4, v/v). Clean-up

of an aliquot of the extract was by acidic alumina columnchromatography, and estimation

of the residues was by GLC–ECD, using a wide-boreopen-tubular capillary column coated

with 5% phenyl substituted methylsiloxane (Barooah et al.,1994). The residues in the tea

brew were also analysed by partitioning an aliquot of thebrew, after cooling to room

temperature, with n-hexane, and estimation of the residueswas undertaken directly by

GLC–ECD.

Cho et al. (2008) reported a simultaneous multi-residuemethod to determine 14

different pesticides (flufenoxuron, fenitrothion,chlorfluazuron, chlorpyrifos, hexythiazox,

methidathion, chlorfenapyr, tebuconazole, EPN, bifenthrin,cyhalothrin, spirodiclofen,

difenoconazole and azoxystrobin) in green tea usingpressurized liquid extraction (PLE)

and liquid–liquid extraction (LLE). In the PLE method, thetea samples were extracted with

acetone/n-hexane (20:80, v/v) in a pressurized liquidextractor and analysed by GLC–ECD.

The residues were confirmed by GC–MS in a selectedion-monitoring mode. In the LLE

method, the green tea samples were extracted with acetoneby blending. The extracts,

after dilution with aqueous sodium chloride solution, werepartitioned into n-hexane and

dichloromethane. The two organic phases were combined,concentrated and cleaned up

using a florisil column, eluting with acetone/n-hexane (2:8v/v). The eluate was dried and

the residues were re-dissolved in acetone and injected intoGLC–ECD. Both methods gave

limits of quantification (LOQs) that were lower than theMRLs established by the Korea

Food and Drug Administration (KFDA). They were precise andhave been successfully

applied to the analysis of real samples.

Zhang et al. (2012) reported a method for the simultaneousdetermination of residues

of three pesticides (procymidone, pyridaben andbeta-cypermethrin) in tea solutions.

The method involved extraction of residues using apetroleum ether–ethyl acetate

(3:1, v/v) mixture, clean-up using a florisil solid-phaseextraction (SPE) cartridge and

subsequent determination by GLC–ECD. The overall averagerecoveries (at spiked

levels of 0.05–0.5 mg/kg) from green and black tea samplesranged from about 86 to

106% with an RSD of 1.29–4.97% and LOQs at 0.025–0.038mg/kg. The method could

be applied for conformance testing with respect to EU MRLsfor these compounds.

An HPLC-based analytical method was employed by Gupta andShankar (2008) for

measuring residues of acetamiprid in black tea and teashoots. Samples were extracted by

shaking with acetonitrile and cleaned up by liquid–liquidpartitioning (after dilution with

sodium chloride solution), first with petroleum ether(fraction discarded) and again with

dichloromethane, followed by column chromatography usingactivated silica gel mixed

with activated carbon (6:0.1, m/m) and elution withacetone–hexane mixture (1:1, v/v). The

residues were finally reconstituted in the mobile phaseacetonitrile–water (70:30, v/v) and

estimated by HPLC–DAD (…).

A number of research works have been published on residuesin dry tea leaves (black

tea) and tea liquor, mostly employing the conventionalmethods, as described earlier

(Bhattacharya et al., 1995; Gupta et al., 2008; Jaggi etal., 2000; Manikandan et al., 2009;

Pramanik et al., 2005; Saha et al., 2000). There arepublished multi-residue methods available

in the literature (Germany DFG method S 19, 1999;Analytical method, Department of

Food Safety Ministry of Health, Labour and Welfare, Japan,2006). In addition to column

chromatographic clean-up, other methods, such as gelpermeation chromatography

(GPC) and SPE, have also been applied for removing the teaco-extractives (Huang et

al., 2007). Cai et al. (2003) appliedpolyphenylmethylsiloxane as a coating for solid-phase

microextraction (SPME), combined with microwave-assistedextraction, to determine the

concentrations of organochlorine pesticides in Chinese tea.The extracts were analysed

by GLC–ECD.

5 Advanced methods for detecting residues in tea

There have been rapid changes in the regulatory standardsof many international standard

bodies, and the most stringent have been the EU MRLs. Thelatter have affected the tea

trade because many default MRLs are set at an LOQ of 0.01or 0.02 mg/kg. Many of the

conventional methods have detection limits that are muchhigher than these levels. In

addition, the use of new pesticides, such asneonicotinoids, which have been introduced

in tea in recent years, has been on the rise and requiresclose monitoring for compliance

to the standards, using sophisticated techniques likeLC–MS/MS. In many developed

countries today, the speed, sensitivity and precision ofanalysis make the triple quadrupole

GC–MS/MS and LC–MS/MS techniques the methods of choice formeasuring pesticide

residues in tea.

Huang et al. (2007) reported a GC–MS method for thesimultaneous determination

of 102 pesticide residues in teas. The method involvedextraction with acetone–ethyl

acetate–hexane, clean-up by GPC and SPE, and identificationand quantification of the

selected pesticides by GC–MS under retention time-lockedconditions for a runtime of 120

minutes. Pesticide residues could be determined in lowsub-ppb, with average recoveries

of 60–121% (a mean of 88%) and RSDs of 3.0–20.8% (mean13.7%) for all analytes and the

LODs were much lower than the EU MRLs. The authorsdemonstrated that the traditional

methods could not provide an effective solution to minimizethe matrix effects of tea.

Tan et al. (2014) have described a pre-treatment techniqueof matrix solid-phase

dispersion coupled with pressurized column chromatography,combining the extraction

and clean-up steps in the analysis of residues of 14organochlorines from fresh tea

leaves. The method involves mixing 1-g portions ofmacerated fresh tea leaves with 4 g

of alumina and transferring the mixture to a glass columnfilled with 0.2 g of activated

carbon and eluted with 20 mL of ethyl acetate-n-hexane. Theresidue levels of the HCH

isomers (alpha-, beta-, gamma- and delta-), heptachlor,aldrin, PCB 101, endosulfan-I,

endosulfan-II, endrin, dieldrin, p,p-DDD, p,p-DDT and mirexin the cleaned extract were

estimated by the conventional technique of GLC–ECD andconfirmed using the triple

quadrupole GC–MS/MS in the multiple reaction monitoring(MRM) mode. The method

offered a recovery of 70–75% and a detection limit at theppb level.

Pan et al. (2014) monitored the residues of acephate anddimethoate at different stages

during the manufacturing of black and green tea. Residuelevels were measured by

extracting 2-g portions of tea samples with 10 mL ofacetonitrile, followed by a separation

of phases achieved by the addition of sodium chloride,centrifugation and clean-up

of the supernatant by dispersive-SPE (d-SPE) using PSA,

C18, GCB and magnesium

sulphate. The residues in the supernatant obtained aftercentrifugation of the cleaned

extract were estimated using the conventional GLC–FPDmethod. Results showed that

the black tea manufacturing process degraded more residues(86–94%) of both acephate

and dimethoate than that of green tea (49–77%) and that themaximum loss of residues

(33–63%) was during drying, while the fixing (in the caseof green tea processing), rolling

and fermentation (oxidation) stages had a little influenceon the residues.

In addition to polyphenols and chlorophylls, theco-extracted caffeine creates a strong

matrix effect in the analysis of tea samples by bothGC–MS/MS and LC–MS/MS methods.

A new clean-up method, demonstrated by Oellig and Schwack(2012), was the use of

planar SPE, which has been proven to be highly efficient inproducing almost matrix-free

extracts. This avoided the much-reported tea matrix effectsand permitted the use of pure

solvent standards. By applying this technique to initialextracts of green and black tea,

colourless extracts that were almost free of interferenceswere obtained for acetamiprid,

penconazole, azoxystrobin, chlorpyrifos, pirimicarb,fenarimol and mepanipyrim for

analysis by LC–MS/MS. The method offered recoveries of72–114%, at spiking levels of

0.01 and 0.1 mg/kg, with very good precision.

A rapid method for the determination of 135 pesticide

residues in green and black tea

leaves, employing gas chromatography (GC) in tandem withmass spectrometry (GC–MS/

MS), was developed and validated by Cajka et al. (2012).Prior to processing, the dry tea

leaves were hydrated by adding water to a homogenizedsample, and extracted with

acetonitrile in the presence of inorganic salts. Most ofthe co-extracts in the crude extract

were subsequently removed by liquid–liquid partitioning,using hexane and a 20% (w/w)

aqueous sodium chloride solution. The importance of thehydration of dry tea samples

prior to extraction for achieving good recoveries wasdemonstrated with the subsequent

pesticide residues. Recoveries for most of the analyteswere in the acceptable range of

70–120% and the RSDs were less than 20% for the two teamatrices and offered an LOQ

of 0.01 mg/kg for most pesticides. The method wassuccessfully used by the authors to

test real tea samples for MRL conformance.

A method for determining residue levels of cyhalothrin,flufenoxuron, fenitrothion, EPN,

bifenthrin, difenoconazole, triflumizole and azoxystrobinresidues in dry green tea leaves

and in tea liquor was developed by Cho et al. (2014) usingGC and a micro-electron

capture detector (μECD). The method involved the hydrationof the dry tea samples prior

to extraction with acetonitrile, clean-up, estimation byμECD and confirmation by the triple

quadrupole GC–MS/MS. Recoveries were within the range of

77–116% with an RSD of 14%

and an LOQ of 0.015–0.03 mg/kg. The method was adequate forconformance testing in

Korea as the LOQs were lower than the MRLs set by the KFDAfor all the pesticides (listed

earlier) in tea (0.05–10 mg/kg).

Mukherjee et al. (2015) reported a liquid–liquid partitionmethod for extracting residues

of dinotefuran from tea samples (green leaf and a tea brew)by taking 2 g of previously

homogenized samples and 100 mL of 5% sodium chloridesolution in a 500-mL separating

funnel and partitioning the residues into dichloromethane(100-, 50- and 50-mL portions).

The dichloromethane extracts were dried over anhydroussodium sulphate, concentrated

and cleaned up on an Accu BOND C18 cartridge (3 mL, 500 mg)and d-SPE using 25 g

each of PSA and GCB along with anhydrous sodium sulphate(300 mg). It was analysed

using a triple quad LC–MS/MS method.

The use of hyphenated techniques in measuring residues intea has been reported by

Schurek et al. (2008). A head-space (HS) SPME technique,coupled with a comprehensive,

two-dimensional GC–time-of-flight (TOF) MS, was employed todevelop a method for

determining the presence of 38 pesticides in black andgreen tea matrices. This hyphenation

of SPME in the HS mode, along with a comprehensive,two-dimensional GC with a high-speed

TOF MS (HS-SPME–GC × GC–TOF MS), proved to be a quickalternative to a conventional

ethyl acetate-based extraction and GPC clean-up GC–MSmethod. Using a matrix-matched

calibration, the authors could achieve an LOQ of0.001–0.028 mg/kg with a fair degree of

repeatability (RSD < 24%) and employed this method toanalyse market tea samples.

There is now a trend to shift from the labour-intensivetraditional methods to fast and

simple approaches, such as QuEChERS (quick, easy, cheap,effective, rugged and safe)

(Anastassiades et al., 2003), which represents a newmilestone for pesticide residue

analysis. Many laboratories have also used this techniquein the tea matrix with some

modifications (Cajka et al., 2012; Kanrar et al., 2010;Steiniger et al., 2010).

A multi-residue method was developed for the rapiddetermination of pesticide residues

in Chinese tea using ultra-performance liquidchromatography–electrospray in tandem

with mass spectrometry (UPLC/MS/MS) by Chen et al. (2011).The QuEChERS method

was used to prepare the sample. In order to minimize thematrix effects from the tea, an

SPE cartridge layered with graphite carbon/aminopropylsilanized silica gel was applied

to complement the QuEChERS method. Matrix-matchedcalibration curves were applied

to compensate for the matrix effects. The method offered anLOQ at 0.01–0.02 mg/kg

and recoveries ranged from 70% to 120%. The RSDs met the EUquality control guideline.

Wang et al. (2014) carried out a study on pesticide residue

transfer rates (%) from dried

tea leaves to tea liquor or brew. Pesticide residues wereextracted from tea liquor using a

modified QuEChERS method and were analysed with the help ofan UHPLC/ESI–MS/MS

method. The method was validated to identify and quantifyup to 172 pesticides in both

tea leaves and tea liquor using a six-point matrix-matchedcalibration with isotopically

labelled standards or a chemical analogue as the internalstandard. The method offered

81–110% recoveries for about 95% of the pesticides,precision (RSD ≤ 20%) and a

measurement uncertainty of less than 40%. Results from 44incurred tea samples showed

different transfer rates up to 92.4%, with the differencesrelated to the type of tea.

In a recent study, a method for analysing 101 pesticides ingreen tea leaves was

developed and validated by Hou et al. (2016). Green teasamples (5 g), which were taken on

50-mL polypropylene tubes, were extracted with acetonitrile(20 mL) by vortexing them for

one minute in a QuEChERS extraction bag (magnesium sulphate+ sodium chloride + tri

sodium citrate didydrate + disodium hydrogen citratesesquihydrate), followed by a

centrifugation clean-up of an aliquot of the supernatantwith SPE cartridges (PSA/GCB).

Finally, the residues were reconstituted inn-hexane-acetone (9:1, v/v) and analysed using

the triple quad GC–MS/MS. Most of the pesticides showed70–120% recoveries at 0.05

and 0.1 mg/kg spiked levels with good precision (<20% RSD).The method offered an

LOQ of 0.001–0.025 mg/kg.

Concerns about residues in tea are mostly confined toinsecticides, acaricides and

fungicides, as they are directly applied to the teafoliage. Herbicides, which are also

widely used in tea plantations, are not usually the targetfor residue analysis as these are

never applied directly to the tea plants. These are mostlyapplied on the soil or on the

weeds covering the ground between the tea bushes and hencethe scope of herbicides

leaving any residues in tea seems to be remote.Consequently, the analytical methods for

examining herbicide residues are scanty. However, somereports indicate the detection of

low levels of herbicide residues in tea as well (see thestudies on residues of paraquat in

tea by Barooah et al. (1995), unpublished work).

Glyphosate is a herbicide that is widely used in teaplantations for broadleaf weed

control. In view of the current restrictions on the use ofparaquat in Europe and of 2,4-D

in Japan, glyphosate has been the single most popularherbicide of recent times in India

and other tea-producing countries. Most of the teaproducers rely heavily on glyphosate.

Although it has a simple structure (N-(phosphonomethyl)glycine), its residue analysis

has been very difficult to accomplish because it ispractically insoluble in any organic

solvents and lacks any chromophores. This makes the

detection of pesticides by any of

the conventional techniques difficult. Goon et al. (2015)have recently developed and

validated a method based on the extraction of tea samples(2 g) with 0.1 N sodium

hydroxide (10 mL) along with dichloromethane (10 mL) in anultrasonic bath, followed by

centrifugation, adjusting the supernatant to pH 7 andderivatization using FMOC-Cl. The

derivatized extracts were analysed using LC–ESI–MS/MS underthe MRM scanning mode.

Using this method, the authors have observed no detectableresidues of glyphosate in

tea following the application of the herbicide on thelow-lying weeds covering the soil

between the tea bushes. The residues of the herbicide werebelow detectable levels

(<0.06 mg/kg) in the soil after 60 days.

Both copper oxychloride (COC) and copper hydroxides (CuOH)are foliar fungicides that

are widely used for the control of blister blight, blackrot and rust diseases in tea plantations

in India and Sri Lanka. Due to the prevailing growingconditions, the incidence of these

diseases is quite widespread, requiring the frequentapplication of these fungicides. The

residues of these two compounds in the treated crop aremeasured as the total amount

of copper following extraction of dry tea samples byashing/acid digestion and estimation

usually by Flame–AAS (atomic absorption spectrophotometry).Since copper is also

absorbed from the soil by the plants’ roots and is an

essential micronutrient, the copper

content detected in tea at any given time cannot beattributed to residues arising from the

application of copper fungicides alone. In the field trialscarried out at Tocklai in 2007 and

2008, the residues of copper (measured as the differencewhen compared to an untreated

control crop) in tea seven days after foliar sprays of COCand CuOH were within the

current Indian maximum limit of 150 mg/kg, but many timeshigher than the EU MRL of

40 mg/kg (unpublished work at the authors’ laboratory).This is a concern.

2,4-D is a widely used herbicide in the tea plantations ofIndia, and in fact was introduced

in India as a tea herbicide to control mikania (Mikaniamicrantha Kunth), a dreaded weed

at that time. The analytical method employed for measuringresidue levels of 2,4-D in tea

involved extraction of the tea samples with boiling waterand, after cooling the extracts,

blending with 2-propanol. An aliquot of 2-propanol extractwas shaken with hexane

and 0.03 (N) HCL. The hexane layer was concentrated,residues were reconstituted in

methanol, converted into a derivative withtrimethylsilyldiazomethane and then estimated

by capillary column GC–ECD (Bhattacharya, 2011).

6 Food safety standards for tea and the challenges of

maintaining maximum residue limits (MRLs)

Different food safety regulatory agencies have set MRLs andestablished monitoring

programmes to test for pesticide residues in tea. The EUstandards are by far the most

stringent. However, there are some aspects of this standardthat need careful review. The

key points in the EU regulations are that the amounts ofresidues found in food must be

safe for consumers and must be as low as possible. It fixesMRLs for all food and animal

feed, which can be found in the MRL database on theEuropean Commission website. For

tea, the reader may use the following link and click on theExport to Excel button: http://

ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=product.resul

tat&language=EN&selectedID=243 (accessed on 9 April 2017).

6.1 EU standards

Dr Thomas Henn of the ETC has summarised some of the keyissues affecting the situation

in the EU as follows: ‘continuous change of residueregulations and the loss of authorization

for chemicals in Europe; the need for the industry to lookahead, replace old chemicals

and ensure enough new chemicals are available; that to thepesticide companies tea is not

an important crop; that consumers are sensitive topesticide residues; climate change and

sustainability; safe evaluation of pesticides in the futuremay be based on multiple residue

analysis; the analytical issues surrounding low LODs’(Report of the Working Group on

MRLs on Tea. Intersessional Meeting of theIntergovernmental Group on Tea, Mombasa,

Kenya, held on 18–19 July 2011, p. 8).

The EU regulation 149/2008/EC of the Commission (Anon.,2005), establishing annexes

II–IV to the EU pesticide residue regulation 396/2005/EC(Anon., 2008), came into effect

on 1 September 2008 and several revisions have been madesince.

There are three issues that need attention:

• MRLs have been fixed for some pesticides in tea which canbe complied with.

• MRLs for a large number of pesticides have been fixed ata lower limit of analytical determination, creating muchdifficulty in the tea trade.

• MRLs for elements that are naturally present in tea needto be revoked.

These issues may be viewed against the challenges beingfaced in tea-producing countries

(Barooah et al., 2010). These challenges can be summarizedas follows:

• The choice of pesticides has become very limited.

• A number of pesticides approved for use in tea in sometea-producing countries have been withdrawn for use inEurope.

• A number of approved pesticides in some tea-producingcountries are not accepted in Japan.

• Recurring severe pest and disease attacks have resultedin huge crop losses.

• Climate change has led to the emergence of new and minorpests as major challenges.

• Very low default MRLs are not practically achievable forreliable pest control.

These challenges indicate the need for fixing realisticMRLs as well as ensuring compliance

through the regular testing of teas in competentlaboratories. Therefore, it is essential to

have access to the right contacts within the European Unionand to have the scientific

data to support a review of the standard. The erstwhileETC, now renamed THIE, has been

working with the Tea Research Institutes in readily sharinginformation. Another route is

to approach the manufacturers, whose support will becrucial in revising/fixing EU MRLs

in order to ensure a smooth tea trade. Since 2005, theseapproaches have been initiated

by Tea Research Institutes in India, China, Sri Lanka andKenya, through the Working

Group (WG) on MRLs in Tea of the Intergovernmental Group onTea. That has yielded

results. Table 1 compares current EU MRLs with those of theCodex for tea, along with

our assessment of the scope of compliance based onsupervised field trials (Barooah and

Borthakur, 1994, 2008; Barooah et al., 1994, 2011) thatwere carried out in accordance

with GAP in India.

The Codex has MRLs for 17 chemicals in tea. Only 12 ofthese chemicals are currently

approved for use in tea in India, out of which ninechemicals comply with both EU T a b l e 1 C o m p a r i so n o f c o d e x a n d E U M R L s o f p e s t i c i d e si n t e a P e s t i c i d e s C O D E X M R L ( m g / k g )E U M R L ( m g / k g ) P e s t i c i d e s C O D E X M R L( m g / k g ) E U M R L ( m g / k g ) P a r a q u a t 0 . 20 . 0 5 * E t h i o n – 3 M e t h i d a t h i o n 0 . 5 0 .1 * P r o f e n o p h o s – 0 . 1 C l o t h i a n i d i n 0. 7 0 . 7 P r o p i c o n a z o l e – 0 . 1 F e n p r o p at h r i n 2 2 2 , 4 D – 0 . 1 C h l o r p y r i f o s 2 0 .1 * P h o s a l o n e – 0 . 1 D e l t a m e t h r i n 5 5 He x a c o n a z o l e – 0 . 0 5 P r o p a r g i t e 5 0 . 05 * , # L c y h a l o t h r i n – 1 E n d o s u l f a n 1 03 0 F e n a z a q u i n – 1 0 E t o x a z o l e 1 5 0 . 0 5

T h i a c l o p r i d – 1 0 P e r m e t h r i n 2 0 0 . 1 *F l u f e n o x u r o n – 1 5 T h i a m e t h o x a m 2 0 20 H e x y t h i a z o x 1 5 4 C y p e r m e t h r i n 1 5 0. 5 A c e t a m i p r i d – 0 . 1 ( 0 . 0 5 ) * B i f e n th r i n 3 0 5 Q u i n a l p h o s – 0 . 1 F l u b e n d i am i d e 1 5 0 . 0 2 * G l y p h o s a t e – 2 . 0 D i c o fo l 5 0 2 0 O x y fl u o r f e n – 0 . 0 5 I n d o x a c ar b 5 5 h t t p : / / w w w . c o d e x a l i m e n t a r iu s . n e t / p e s t r e s / d a t a / c o m m o d i t i es / d e t a i l s . h t m l ? i d = 1 0 1 * L o w e r l i mi t o f a n a l y t i c a l d e t e r m i n a t i o n . # Ef f e c t i v e 3 O c t o b e r 2 0 1 5 .

and Codex MRLs (paraquat, clothianidin, fenpropathrin,deltamethrin, endosulfan,

thiamethoxam, bifenthrin, hexythiazox and dicofol),although currently endosulfan is

withdrawn due to a Supreme Court of India order. Propargiterecently lost authorization in

the EU, reducing its MRL from 5 to 0.05 mg/kg. The EU MRLscan be complied with for five

chemicals (L-cyhalothrin, fenazaquin, thiacloprid,flufenoxuron and glyphosate); the Codex

MRLs are yet to be fixed. For another six chemicals(phosalone, profenophos, quinalphos,

propiconazole, hexaconazole and copper), compliance withthe EU MRL is difficult following

their use as blanket sprays. There is no Codex MRL forthese chemicals yet.

6.2 Japan’s positive list system

Japan introduced the positive list system w.e.f. May 2006covering more than 400

agrochemicals (see

introduction.html (accessed on 12 June 2016)). While MRLsfor a number of compounds

used in tea have been fixed at realistic levels, a uniformlimit (UL) of 0.01 mg/kg has been

established for agricultural chemicals without MRLs, usinga toxicological threshold of

1.5 µg/day as the basis on which to determine the UL (seeTable 2).

6.3 Challenges posed by Japan’s ULs

2,4-D, fenazaquin, oxyflurofen, simazine and a few othergrowth regulators are not

accepted in tea in Japan, and hence ULs were assigned atthe 0.01 mg/kg level. 2,4-D is a

most serious case as it is one of the few herbicides thatis used widely in the tea fields. The

EU standard is ten times higher than that of Japan, andensuring conformance to the EU

standard is much easier when using the multi-residueLC–MS/MS method. The complex

matrix of black tea poses a major challenge duringanalysis, even when using the triple

quadrupole LC–MS/MS at concentrations below 0.05 mg/kg for2,4-D when multi-residue

methods are used. The UL is far below the LOQ of some ofthe laboratories, leading to

difficulties in assuring conformance to Japanese standardsas well (Barooah et al., 2013).

The non-availability of an alternative to 2,4-D is a majorconcern.

6.4 MRL setting initiatives

However, some very good progress is being made in settingMRLs in many tea-consuming as

well as tea-producing countries. China has recently fixedan MRL for 35 pesticides in tea, and

Kenya has adopted Codex MRLs for 14 pesticides in teas astheir national MRLs. The Food

Safety Authority of India has notified seven MRLs in tea

and notifications for other chemicals

are due. The United States has set up realistic MRLs, andCanada and Australia have also set

up MRLs based on the data provided by both manufacturersand Tea Research Institutes.

India and Sri Lanka have jointly submitted residue data oncopper to the THIE for submission

to the EU. The Tocklai Tea Research Institute has activelycollaborated with manufacturers

of propargite to ensure that it meets the threshold forimportation into the EU (since 2014).

The WG on MRLs in Tea was constituted at the Bali Meetingof the Food and Agriculture

Organization (FAO)-Intergovernmental (IGG) on Tea in 2005to assist in generating and

submitting the data required for fixing the MRLs ofpesticides in tea. Since the 1990s,

pesticide residues in tea have been a major non-tarifftrade barrier affecting the global

tea trade. The problem was due mostly to certain defaultMRLs, which had been set at

analytical detection limits. The only way to tackle thisproblem was to help fix realistic

MRLs, which would be acceptable to all stakeholders, inorder to ensure food safety as

well as the smooth operation of the tea trade globally. Thedata generated in the tea

producing countries under the initiatives of the WG havealso led to the establishment of

realistic MRLs in many tea-importing countries, such asAustralia, Canada and the United

States, which was in line with the decision taken at the20th session of the IGG on Tea

(Colombo, 30 January–1 February 2012) (Ref. Document: CCP12/14 E February 2012) to

change the nomenclature from ‘harmonization of tea MRLs’ to‘Achieve global cooperation

in obtaining MRLs in tea’. The setting of realistic MRLs inthe EU in the recent years has had

a positive impact on the export of tea to the EU. HavingMRLs in the importing countries

(Table 3) has opened up new markets for the tea-producingcountries because they can

now trade in products which would otherwise have had toface very low default MRLs. The

default MRLs are a major trade barrier, and the collectivework of this WG is expected to

replace them with realistic MRLs that will satisfy allstakeholders (Ref. Document: CCP:TE

16/CRS15, Report of The Working Group on MRLs and MRLs inTea Brew, FAO-IGG on

Tea, Naivasha, Kenya, 25–27 May 2016).

The National Food Safety Standard-Maximum Residue Limitsfor Pesticides in Food was

issued by the Ministry of Agriculture of the People’sRepublic of China in July 2015. The

draft document GB 2763-2015 indicated more than 35 MRLs forpesticide in tea, as listed

in Table 4.

Kenya introduced a Draft Kenya Standard KS 2128: 2015 inAugust 2015, which included

a code of practice in tea. In the Annex C of this standard,MRLs have been proposed for

14 compounds based on Codex MRLs, as shown in Table 5.

Research findings at different tea research institutes haveshown that all the residues

detected in tea brews are not present in the cup of tea onedrinks. This is an important

issue that needs to be considered when fixing MRLs. The WGson MRL and Tea Brew

of the FAO-IGG on Tea, on behalf of China and India,presented a policy document

entitled ‘Assessment of MRLs for Pesticides in Tea’ at the44th CCPR session at Shanghai,

China, on 23–28 April 2012, under Agenda item 12 (b). Thepresentation was supported

by two reference documents (Ref. Document: PR 44 CRD 10(China) and PR 44 CRD 29

(India)). The presentation highlighted the fact thatalthough dry tea leaves are the traded

commodity, it is the brew or liquor which is actuallyconsumed. Hence all standard-setting

bodies, including JMPR, the Codex and the nationalregulatory agencies, should consider

the residue in tea brew, or in both the brew and the tealeaves, when setting MRLs. The

concept of using the brew factor (Barooah et al., 2011) andcompilations of the available

data on the extent of the possible transfer of pesticideresidues into the tea liquor from dry

tea (Chen, 2011) has been the basis of this argument.

Table 2 Some unified limits of agrochemicals in tea in Japan

Sl no. Chemicals Type of use UL (mg/kg) Remarks

1 2,4-D Herbicide 0.01 w.e.f. May 2006

2 Oxyfluorfen Herbicide 0.01 w.e.f. May 2006

3 Simazine Insecticide 0.01 w.e.f. May 2006

4 Fenazaquin Acaricide 0.01 w.e.f. May 2006

5 Gibberellic acid Plant growth regulator 0.01 w.e.f. May2006

6 Triacontanol Plant growth regulator 0.01 w.e.f. May 2006

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6.5 Brew factor and risk assessment using brew factor

The brew factor, described by Barooah et al. (2011), can bedetermined from residue data,

as follows: Brew factor BF Residues in tea brew Residues indry tea ( ) = ÷ leaves

The residues in tea brew are the residues in the tea liquorexpressed in mg per kg of the

dry tea leaves (black or green tea) used for preparing thebrew. It is obtained by dividing

the amount of residues in the brew (mg) by the amount ofblack or green tea used (kg) for

preparing the brew. The residues in the dry tea leaves(black or green tea) are expressed

in mg/kg. The brew factor, therefore, has no units.

The actual intake of residues through tea consumption canbe determined by

incorporating this new parameter, termed ‘brew factor’,into the conventional estimates

of the theoretical maximum daily intake. Thecompound-specific brew factor improves

risk assessment and will enable the setting of realistic

MRLs for pesticides in tea. A policy

document entitled ‘Guidance Document on Risk AssessmentUsing Brew Factor for Fixation

Table 4 MRLs for pesticides in tea in China

Pesticides China MRL mg/kg Pesticides China MRL mg/kg

Acephate 0.1 Fenitrothion 0.5

Bifenthrin 5 Fenvalerate 0.1

Buprofezin 10 Flucythrinate 20

Carbendazim 5 Glufosinate-ammonium 0.5

Carbofuran 0.05 Glyphosate 1

Cartap 20 Hexachlorocyclohexane 0.2

L-cyhalothrin 15 Hexythiazox 15

Cyfluthrin 1 Imidaclothiz 3 *

Cypermethrin 20 Indoxacarb 5

DDT 0.2 Isazofos 0.01

Demeton 0.05 Isocarbophos 0.05

Deltamethrin 10 Methomyl 0.2

Diafenthiuron 5 * Omethoate 0.05

Dicofol 0.2 Permethrin 20

Difenoconazole 10 Phosfolan 0.03

Diflubenzuron 20 Phoxim 0.2

Endosulfan 10 Pyridaben 5

Ethoprophos 0.05 Fenpropathrin 5

Fenazaquin 15

* Not regulated.

of MRLs of Pesticides in Tea’ was presented in the 48th

session of the CCPR held in China

on 25–30 April 2016. Residue data in both dry tea leavesand tea brew will henceforth be

submitted for MRL fixation(http://www.fao.org/fao-who-codexalimentarius/sh-proxy/en/?

lnk=1&url=https%253A%252F%252Fworkspace.fao.org%252Fsites%252Fcodex%252FM

eetings%252FCX-718-48%252FCRD%252Fpr48_crd21x.pdf (accessedon 6 April 2017)).

7 Strategies for reducing pesticide residues in tea

Tea production is geographically limited to a few areasaround the world and it is highly

sensitive to changes in growing conditions. The fact thatits ideal growing conditions are

at a high risk from climate change, and are expected tochange significantly as a result of

climate change (Chen, 2012), has thrown up many challengesfor the global tea industry.

One serious concern that needs to be addressed is thepressure arising from increasing

numbers of pests. It is now generally agreed that thereliance on pesticides should be

reduced and replaced by different pest managementstrategies. Such strategies can

integrate various methods, such as host plant resistance,cultural interventions, biological

control, the use of behaviour modifying chemicals, colourtraps, light traps and physical

control.

Some of the approaches that are being recommended by theTea Research Institutes in

India (Barooah at al., 2010) for adoption by the teaproducers are the regular monitoring

of pests at the grassroots level for early detection,integrated pest management (IPM)

schemes that place a greater emphasis on culturalpractices, the use of botanicals and

Table 5 List of pesticides with MRLs in Kenya (adapted fromCodex)

Sl no. Pesticides Kenya MRL (mg/kg) Codex MRL (mg/kg)

1 Paraquat 0.2 0.2

2 Methidathion 0.5 0.5

3 Clothianidin 0.7 0.7

4 Fenpropathrin 2 2

5 Chlorpyrifos 2 2

6 Deltamethrin 5 5

7 Propargite 5 5

8 Endosulfan 10 10

9 Etoxazole 15 15

10 Hexythiazox 15 15

11 Cypermethrins (including alpha- & zeta- cypermethrin)15 15

12 Permethrin 20 20

13 Thiamethoxam 20 20

14 Bifenthrin 30 30

microbial biocides, changing application methods, usingpesticides only as a component

part of IPM schemes, the restricted use of foliar sprays ofnutrients and plant growth

regulators, as well as monitoring the inputs used in teaplantations for contaminants and

monitoring the teas for MRL conformance.

On a larger canvas, the reclassification of tea as minorcrop in JMPR, in order to speed

up the setting of MRLs for tea and to obtain MRLs for thechemicals that are used in tea

producing and tea-consuming countries, will cut down thetime and cost of generating

data. Other recommendations include the convergence ofmethodologies for MRL

setting, the agreement of a common risk assessment processfor tea and the transparency

of risk assessment processes in different countries, inaddition to enhancing the speed of

replacing old chemicals with promising new ones with fewerresidue problems.

India, China, Sri Lanka, Kenya and Indonesia togetherproduced almost 80% of the

world’s tea (Chang, 2015) and steps taken to aidconformance at the source will go a

long way in ensuring food safety. Tea is plucked inseven-day cycles in India, Sri Lanka,

Indonesia and Kenya, and may be a little longer in thecolder regions of China. However,

a number of pesticides have been found to linger in teaplants with half-lives between 1.2

and 3.9 days (Barooah and Borthakur, 1994; Barooah et al.,1994; Chen and Haibin 1988;

Gajbhiye et al., 1989; Gopal et al., 1987; Xia et al.,2008). The rapid growth of tea shoots

may help lower the residues but a shorter plucking cycletends to leave higher amounts

of residues in the plants. Hence monitoring for pests atthe grassroot level should form an

integral management practice and, during the plucking

season, only spot spraying should

be followed as far as possible.

The effect of washing the tea shoots to reduce residues ofthree pesticides was studied

at the Tocklai Tea Research Institute. Most of the residuesof quinalphos, dicofol and

endosulfan were present on the surface of the tea shootseven 3–7 days after spraying.

Washing could remove 22–49% of the residues from thefield-treated samples of tea shoots

(Barooah and Borthakur, 2008). The introduction of awashing step before processing

the crop therefore has the potential to decontaminateresidues in tea. This is an area of

research which needs to be explored.

8 Conclusion and future trends

Tea is the most popular beverage globally; it is the drinkof the common man. As more

people become aware of the health benefits of tea, it isonly natural that greater numbers

of people will turn to tea. Tea is produced in more than 45countries and involves intensive

cultivation practices. Tender tea shoots are plucked andprocessed in factories to dry the

tea leaves, which are then traded. However, unlike otheragricultural and horticultural

crops, tea is never consumed directly. Consumers mostlydrink the infusion of tea leaves in

hot water. As a result, in terms of assessing food safetyfrom the point of view of pesticide

residues, it is necessary to consider the residues in boththe marketed, but not eaten,

forms of tea and in the form in which tea is consumed. Anumber of studies have indicated

that only a proportion of the residues detected in the drytea leaves are in fact transferred

into the liquor. This has led to the proposal that a riskassessment should be based on the

brew factor rather than the dry leaves, and that the brewfactor should be used for fixing

realistic MRLs of pesticides in tea that will satisfy allstakeholders. Realistic MRLs will assist

in the smooth operation of the tea trade globally as wellas ensure food safety for millions

of tea consumers.

From the foregoing discussion, it is clear that in order toaccurately measure the trace

levels of residues of a wide range of pesticides in thecomplex tea matrix, the use of modified

QuEChERS extraction and clean-up methods, followed byestimation using GC–MS/MS and

LC–MS/MS techniques will be an absolute requirement – inview of the speed, selectivity and

sensitivity offered by these techniques. Such a method willensure the food safety of the tea

that is traded globally in the current context and infuture. Laboratories engaged in the residue

analysis in tea need to make use of matrix match standards,adequate acceptance criteria

and the proper training and care that is required toidentify and avoid false positives as well.

Regular participation in FAPAS and other such InternationalProficiency Testing programmes

will enhance the confidence of the testing laboratories,especially in the tea-producing

countries. The continuous generation of residue data on newand existing pesticides from

multi-locational supervised field trials in alltea-producing countries, and the sharing of such

data and common submissions to the Codex, EU and otheragencies will accelerate the

upward revision of many default MRLs, which are a majorobstacle to non-tariff trade today.

This could be a good roadmap for the journey ahead.

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13 Chapter 13 Instrumentation andmethodology for the quantification ofphytochemicals in tea

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14 Chapter 14 The potential role for teain combating chronic diseases

1 Introduction

Tea, made from the leaves of the plant Camellia sinensis,has been used for medicinal

purposes since ancient days and is now a popular beverage.During the past 30 years,

tea, especially green tea polyphenols, has been studied forits potential beneficial health

effects. These include the prevention of cancer, diabetes,cardiovascular diseases (CVDs)

and neurodegenerative diseases; reduction of body weightand alleviation of metabolic

syndome (MetS) (reviewed in Yang and Hong, 2013; Yang etal. 2009, 2011a, 2016). Most

of these beneficial effects are believed to be due to thepresence of polyphenols in green

tea, although caffeine also contributes to some of theeffects. A unique amino acid, theanine

(γ-ethylaminol-glutamic acid), has been shown to haveneuroprotective effects.

Cancer and CVDs are the two most common diseases and thetop two leading

causes of death in many countries. Overweight, obesity anddiabetes are emerging as

major health issues, and the closely related MetS alsopredisposes individuals to CVDs.

Neurodegenerative diseases are becoming major socialconcerns because of the ageing

of the world’s population. If tea could prevent or delaythe development of these diseases,

the public health implications would be substantial.Because of this, there is immense

scientific and public interest on this topic. A literaturesearch on PubMed using the key

words ‘Tea and Health’ yielded 3831 publications, ‘Tea andcancer’ – 3657 publications,

‘Tea and diabetes’ – 640 publications, ‘Tea and heartdisease’ – 142 publications and ‘Tea

and weight control’ – 290 publications. Unfortunately, someof the beneficial effects found

in the laboratory may have been over-extrapolated to humanhealth and propagated in the

news media, popular magazines and even review articles.

This chapter reviews these topics, including possible humanrelevance of the published

results. Selected examples are used to illustrate thebeneficial health effects and possible

mechanisms involved. Information from recently publishedmeta-analyses and systematic

reviews are used to help assess the relative strengths ofthe existing data. Suggestions for

future research are made. The author believes this articlewill enhance our understanding

of the health effects of tea consumption.

2 Chemical properties, bioavailability andbiotransformation of tea constituents

2.1 Tea constituents and their properties

Depending on the post-harvest processing of the tea leaves,the common types of tea

consumed are green tea, black tea and oolong tea, withblack tea accounting for more

than 78% of the world tea production. In the manufacturingof green tea, the tea leaves are

heated or steamed, rolled and dried, which inactivates theenzymes with minimum oxidation

of the constituents. The drying of the tea leaves helps tostabilize the tea constituents

upon storage. Green tea possesses characteristicpolyphenolic compounds known as

catechins, which include (–)-epigallocatechin-3-gallate(EGCG), (–)-epigallocatechin (EGC),

(–)-epicatechin-3-gallate (ECG) and (–)-epicatechin (EC).The structures of catechins together

with theaflavins from black tea are shown in Fig. 1. Tealeaves also contain lower quantities

of other polyphenols such as quercetin, kaempferol andmyricetin, as well as alkaloids such

as caffeine and theobromine. A typical brewed green teabeverage (e.g. 2.5 g tea leaves

in 250 ml of hot water) contains 240–320 mg of catechins,of which 60–65% is EGCG, with

20–50 mg of caffeine (Balentine et al., 1997; Sang et al.,2011).

In the manufacturing of black tea, the tea leaves arewithered, crushed and allowed to

undergo enzyme-mediated oxidation in a process commonlyreferred to as ‘fermentation’.

During this process, most of the catechins are oxidized,dimerized and polymerized to

form theaflavins and thearubigins (Balentine et al., 1997;Sang et al., 2011). Theaflavins

are produced from the dimerization of two catechinmolecules. Theaflavins exist in four

forms (theaflavin, theaflavin-3 gallate,theaflavin-3‘-gallate and theaflavin-3,3‘-digallate)

and contribute to the orange colour and characteristictaste of black tea. Thearubigins

are heterogeneous polymers of tea catechins with reddish

brown colour, but the

structures are poorly understood. In brewed black tea,catechins, caffeine, theaflavins

and thearubigins each account for 3–10%, 3–6%, 2–6% andgreater than 20% of the dry

weight, respectively. Oolong tea is manufactured bycrushing only the rims of the leaves

and limiting fermentation to a short period to producespecific flavour and taste of the tea.

Oolong tea contains catechins, theaflavins and thearubiginsas well as some characteristic

components: epigallocatechin esters, dimeric catechins(such as theasinensins) and

dimeric proanthocyanidins (Sang et al., 2011).

Tea catechins are strong antioxidants, scavenging freeradicals and also preventing the

formation of reactive oxygen species (ROS) by chelatingmetal ions (reviewed in Sang

et al., (2011)). In vivo, EGCG and other catechins canserve as antioxidants, but they

may also cause the formation of ROS in the mitochondriaunder certain conditions (Li

et al., (2010), Tao et al. (2014)). The ROS may activatenuclear factor erythroid 2-related

factor 2 (Nrf2)-mediated antioxidant and othercytoprotective enzymes (Shen et al., 2005;

Wang et al., 2015; James et al., 2015). EGCG is also knownto undergo superoxide

catalysed auto-oxidation in vitro to produce ROS that caninduce cell death (Hou et al.,

2005). Nevertheless, such auto-oxidation of EGCG isunlikely to occur in internal organs,

because of the lower oxygen partial pressure (than in

solution in vitro) and the presence

of antioxidant enzymes in animal tissues (Hou et al.,2005). Therefore, results on EGCG

obtained from cell culture studies need to be interpretedwith caution.

An important biochemical property of catechins is theirH-bonding, via their phenolic

groups, to proteins, lipids and nucleic acids. The multipleH-bond formation provides

high-affinity binding to these biomolecules. As will bediscussed subsequently, the binding

of EGCG to many proteins has been proposed to be a keymechanism for its anti-cancer

activities. Black tea polyphenols, with more phenolicgroups, may bind to biomolecules

with even higher affinity than EGCG.

2.2 Bioavailability and biotransformation

According to Lipinski’s Rule of 5 (Lipinski et al., 2001),compounds that have 5 or more

hydrogen bond donors, 10 or more hydrogen bond acceptors, amolecular weight

greater than 500 and Log P greater than 5 are usuallypoorly absorbed following oral

administration. This is due to their large actual size(high molecular weight), large apparent

Figure 1 Structures of catechins and theaflavins.

size (due to the formation of a large hydration shell) andhigh polarity (Lipinski et al.,

2001). The bioavailabilities of tea polyphenols follow thisprediction (reviewed in Yang

et al. (2008a), Chow and Hakim (2011)). Both human andanimal studies have shown that

the bioavailability of EC (molecular weight 290 and 5phenolic groups) is higher than that

of EGCG (molecular weight 458 and 8 phenolic groups). Inrats, following intragastric

(i.g.) administration of decaffeinated green tea (200mg/kg), the absolute plasma

bioavailabilities of EGCG, EGC and EC were 0.1, 14 and 31%,respectively. EGCG, EGC

and EC in plasma had elimination half-lives of 165, 66 and67 min, respectively (Yang

et al., 2008b). However, the absolute plasmabioavailability of EGCG in mice following

i.g. administration of EGCG (75 mg/kg) was much higher at26.5%, with more than 50%

of EGCG present as glucuronide conjugates. Levels of EGCGin the small intestine and

colon were 20.6 and 3.6 ng/g, respectively (Yang et al.,2008b). In humans, following oral

administration of the equivalent of two or three cups ofgreen tea, the peak plasma levels

of tea catechins (including the conjugated forms) wereusually 0.2–0.3 µM. With high

pharmacological oral doses of EGCG, peak plasmaconcentrations of 2–9 µM and 7.5 µM

were reported in mice and humans, respectively (Yang etal., 2008a). Conversely, theaflavin

and theaflavin-3,3‘-digallate (molecular weights of 564 and868 and containing 9 and 14

phenolic groups, respectively) were reported to haveextremely low bioavailability when

administered orally (Mulder et al., 2001). However, morestudies are needed in this area.

EGCG and other tea catechins undergo extensivebiotransformation (reviewed in Yang

et al. (2008a)). Because of the catechol structure, EGCGand other catechins are readily

methylated by catechol-O-methyltransferase. EGC is alsoreadily methylated to form

4‘-O-methyl-EGC. This metabolite as well as4“-O-methyl-EGCG and 4‘,4“-dimethyl

EGCG have been detected in human and animal plasma andurine samples after the

ingestion of tea. In addition to methylation, catechins arealso glucuronidated by UDP

glucuronosyltransferases and sulphated bysulphotransferases. Multiple methylation and

conjugation reactions can occur on the same molecule. Forexample, we have observed

methyl-EGCG-glucuronide, EGCG-glucuronide-sulphate,dimethyl-EGCG-diglucuronide

and methyl-EGCG-glucuronide-sulphate as urinary metabolitesin mice (Sang et al., 2011).

Active efflux has been shown to limit the bioavailabilityof many polyphenolic

compounds. The multidrug resistance-associated protein 2(MRP2), located on the

apical surface of the intestine and liver, mediates thetransport of some polyphenolic

compounds to the lumen and bile, respectively (Jemnitz etal., 2010). Therefore, EGCG

and its metabolites are predominantly effluxed from theenterocytes into the intestinal

lumen, or effluxed from the liver to the bile and excretedin the faeces, with little or none

of these compounds in the urine of humans and rats.However, low levels of urinary EGCG

metabolites (in the conjugated forms) can be detected in

mice (Sang et al., 2011). Tea

catechins can be degraded in the intestinal tract bymicroorganisms. We have observed

the formation of ring fission metabolites5-(3‘,4‘,5‘-trihydroxyphenyl)-γ-valerolactone

(M4), 5-(3‘,4‘-dihydroxyphenyl)-γ-valerolactone (M6) and5-(3‘,5‘-dihydroxyphenyl)-γ

valerolactone (M6‘) in human urine and plasma samplesseveral hours after the ingestion

of tea (Li et al., 2000; Lee et al., 2002). These compoundscan undergo further degradation

to phenylacetic and phenylpropionic acids.

Several investigators have reported the pharmacokinetics oftea polyphenols in human

volunteers. For example, we found that in the oraladministration of 20 mg green tea solids

per kg body weight, it took 1.4–1.6 h for the catechins toreach peak plasma concentrations

(Lee et al., 2002). This resulted in maximal plasmaconcentrations of 77.9, 223 and 124 ng/

mL, respectively, for EGCG, EGC and EC. EGCG, EGC and EChad terminal half-lives of

3.4, 1.7 and 2 h, respectively. Plasma EGC and EC werepresent mainly in the conjugated

forms, whereas 77% of the EGCG was in the free form.Methylated EGCG and EGC were

also present in human plasma (Yang et al., 2008a; Lee etal., 2002). Chow et al. (2003)

demonstrated that, following four weeks of oraladministration of EGCG (800 mg, once

daily), there was an increase in the systemicbioavailability, but the molecular basis for this

observation remains to be investigated.

In contrast to the limited bioavailabilities of catechins,Caffeine has bioavailability close

to 100% and is mainly metabolized by cytochromes P450 1A2to dimethylxanthines and

theophylline (Arnaud, 2011). Theanine is also almost 100%bioavailable and is metabolized

to ethylamine and glutamic acid (Scheid et al., 2012; vander Pijl et al., 2010). Both caffeine

and theanine readily cross the blood–brain barrier and areneurologically active.

3 Tea and cancer prevention

3.1 Inhibition of carcinogenesis in animal models

Tea and its major constituents have been demonstrated toinhibit tumorigenesis in many

animal models for different organ sites, including thelung, oral cavity, oesophagus,

stomach, small intestine, colon, skin, liver, pancreas,bladder, prostate and mammary

glands (reviewed in Yang et al. (2009, 2011a)). Most of thestudies were conducted with

green tea, green tea polyphenol preparations or pure EGCG,administered through the

drinking water or diet. Some of the examples are discussedas follows.

At least 20 studies have demonstrated the inhibitory effectof tea or tea preparations

on lung tumorigenesis (reviewed in Yang et al. (2011a)).Most of the experiments were

conducted in tobacco carcinogen-treated or transgenic mousemodels and a few studies

in rat and hamster models. Inhibitory activities have beendemonstrated when green tea

preparations were administered during the initiation,promotion or progression stages

of carcinogenesis. Treatment with green or black teaextracts for 60 weeks also inhibited

the spontaneous formation of lung tumours as well asrhabdomyosarcomas in A/J mice.

These results demonstrate the broad activities of teapreparations in the inhibition of lung

neoplasia at different stages of carcinogenesis.

Inhibitory effects of tea against tumorigenesis in thedigestive tract, including the oral

cavity, oesophagus, stomach, small intestine and colon,have been shown in more than 30

studies (Yang et al., 2011a). For example, tea preparationswere shown to inhibit chemically

induced oral carcinogenesis in a hamster model andoesophageal carcinogenesis in a rat

model. EGCG also inhibited tumorigenesis in rat stomach andforestomach induced by

N-methyl-N-nitro-N-nitrosoguanidine. The inhibitory effectsof tea and tea polyphenols on

intestinal tumorigenesis in mice have been consistentlyobserved in different laboratories.

The effects of tea preparations on colon tumorigenesis inrats, however, have not been

consistent (Yang et al., 2011a). Our recent animal studiesshowed that in AOM-treated

rats, administration of Polyphenon E (PPE; a standardizedtea polyphenol preparation

that contains 65% EGCG, 25% other catechins and 0.6%caffeine), at a dose of 0.24%

in the diet, decreased ACF formation, adenocarcinomaincidence and adenocarcinoma

multiplicity (Yang et al., 2011a).

As reviewed previously (Yang et al., 2009, 2011a), most ofthe reported studies have

demonstrated the cancer-preventive activities of teacatechins against carcinogenesis at

different organ sites and the evidence is strong. However,there are also some studies

that did not observe cancer-preventive effects. The reasonsfor the discrepancies may be

complex. One possible reason is the dose andbioavailability of the tea catechins used. For

example, in studying the prevention of mammarycarcinogenesis, the concentrations of

EGCG in the mammary tissues may be too low to be effectivein some of the experiments

(Yang et al., 2011a). Although EGCG and other catechins arethought to be the major

cancer-preventive agents in tea, effective inhibition ofcarcinogenesis by caffeine in the

lung and skin, but not in the colon, has been demonstrated(Yang et al., 2011a).

3.2 Tea consumption and cancer risk in humans

3.2.1 Observational epidemiological studies

In contrast to the strong evidence for thecancer-preventive activities of tea constituents

in animal models, results from epidemiological studies havenot been consistent

in demonstrating the cancer-preventive effect of teaconsumption in humans.

A comprehensive review by Yuan et al. (2011) concluded thatconsumption of green

tea was frequently associated with a reduced risk of uppergastrointestinal tract cancer,

after adjusting for confounding factors and limited datasupported its protective effect

of lung and hepatocellular carcinogenesis. However, intakeof black tea in general was

not associated with a lower risk of cancer (Yuan et al.,2011). We agree with this general

conclusion, and would like to add the following discussions.

In a meta-analysis of 25 epidemiological studies on teaconsumption and colorectal

cancer published in 2006 (Sun et al., 2006), no associationwas found between black

tea and colorectal cancer, and the results on green teawere mixed. Subsequently, a

prospective study on women in Shanghai found a reduced riskof colorectal cancer in

green tea drinkers (Yang et al., 2007). On the other hand,a cohort study in Singapore

found that green tea consumption had a statisticallynon-significant increased risk for

advanced stage colon cancer only in men (Sun et al., 2007).The observed adverse effect

in men may be related to smoking or the pro-inflammatoryactivities of tea catechins as

were observed in animal models (Guan et al., 2012).

In a meta-analysis on the relation between green teaconsumption and breast cancer

published in 2010, an inverse association was found in thefour case–control studies but not

in the three cohort studies (Ogunleye et al., 2010). Twoadditional cohort studies in Japan

and China also did not find an association. These resultsare consistent with the rather weak

evidence from animal studies on the prevention of mammarycancer by tea. Similarly, an

inverse association between green tea consumption andprostate cancer was found in two

case–control studies, but not in four prospective cohortstudies (Yuan et al., 2011).

Smoking appears to be a strong interfering factor instudies on digestive tract cancers. For

example, in a case–control study on the effect of green teaconsumption on oesophageal

cancer in Shanghai, a protective effect was only observedin women, who were mostly non

smokers (Gao et al., 1994). Similarly, in the ShanghaiMen’s Health Study, green tea drinking

was found to reduce the risk of colorectal cancer amongnon-smoking men, but not for men

in general (Yang et al., 2011b). In a recent large-scale,population-based case–control study

in urban Shanghai, regular green tea drinking wasassociated with a significant reduction

of pancreatic cancer risk in women – who were mostlynon-smokers, but not in men – who

were mostly smokers and former smokers (Wang et al., 2012).A recent systematic review of

epidemiological studies in Japan on green tea consumptionand gastric cancer indicated

no overall preventive effect of green tea in cohortstudies. However, a small consistent

risk reduction was found in women (mostly non-smokers), andthe result was statistically

significant in the pooled data of six cohort studies(Sasazuki et al., 2012).

3.2.2 Intervention studies

Many intervention trials have been conducted with greentea, and some have found a

beneficial effect of green tea polyphenols in preventingthe development or progression

of cancer. For example, in a double-blinded, Phase II trialin Italy, 30 men with high-grade

prostate intraepithelial neoplasia (PIN) were given 300 mgof green tea catechins twice

daily for 12 months (Bettuzzi et al., 2006). Only onepatient developed prostate cancer,

whereas nine of the 30 patients with high-grade PIN in theplacebo group developed

prostate cancer (highly statistically significant).However, in a recent trial in Florida with a

similar design using PPE (containing 400 mg of EGCG) in 97men with high-grade PIN and/

or atypical small acinar proliferation (ASAP),supplementation for 6–12 months showed no

differences in the number of observed prostate cancer casesbetween the treatment group

(n = 49) and the placebo group (n = 48) (Kumar et al.,2015). Nevertheless, there was a

decrease in the cumulative rate of prostate cancer plusASAP in the treatment group, in

subjects without ASAP at baseline. A decrease in serumprostate–specific antigen was also

observed in the PPE-supplemented group (Kumar et al., 2015).

An earlier randomized controlled trial (RCT) on oral cancerprevention in China, with a

mixed tea product (3 g/day administered orally ortopically) in patients with oral mucosa

leukoplakia for six months, showed significant decrease inthe number and total volume

of proliferation index and silver-stained nucleoliorganizer regions (Li et al., 1999).

Nevertheless, a later Phase II RCT in the United Stateswith green tea extract (GTE; 500,

750 or 1000 mg/m 2 , two times daily) for 12 weeks, topatients with oral pre-malignant

lesions (n = 28), showed possible beneficial effects inlessening oral pre-malignant lesions,

in part through reducing angiogenic stimulus (stromavascular endothelial growth factor),

but it was not statistically significant (Tsao et al.,2009). Some recent intervention studies

on breast cancer and oesophageal adenocarcinoma werelimited to bioavailability and

some biomarker studies (Crew et al., 2012; Joe et al.,2015). At present, the earlier

optimistic expectation of cancer-preventive activity by teapolyphenols, based on

laboratory results, has not materialized in RCTs. More than20 human trials with green tea

polyphenol preparations are ongoing in the United States,China and Japan (NIH clinical

trials website 1 ). Some of these studies may yield clearconclusions concerning cancer

preventive activities of green tea polyphenols.

3.3 Mechanistic considerations

Many mechanisms have been proposed for cancer prevention bytea constituents, and

this subject has been reviewed (Yang et al., 2009, 2011a).ROS have been shown to play

key roles in carcinogenesis; the antioxidant actions of teacatechins could be an important

mechanism for cancer prevention. Another possible mechanism

is through the binding

of EGCG to target proteins, leading to the inhibition ofmetabolic or signal transduction

pathways. As reviewed previously, the 67-kDa lamininreceptor, Bcl-2 proteins, vimentin,

peptidyl prolyl cis/trans isomerase (Pin 1) and otherproteins have been proposed as

targets for EGCG (Yang et al., 2009, 2011a). It isreasonable to assume that the high-affinity

1 http://clinicaltrials.gov search on 30 August 2016.

binding proteins reported in the literature could serve asinitial targets, but this point

remains to be substantiated in animal models. Some of theproposed mechanisms based

on studies in cell lines, however, may not be relevant tocancer prevention.

Apparently, mechanisms derived from cancer preventionstudies in animal models are

likely to be more relevant. These include the induction ofapoptosis in different animal

models, inhibition of the phosphorylation of c-Jun andErk1/2 in lung tumorigenesis

model, suppression of phospho-Akt and nuclear β-cateninlevels in colon cancer

models, inhibition of the IGF/IGF-1R axis in colon,prostate and other cancer models

and suppression of vascular endothelial growthfactor–dependent angiogenesis in

lung and prostate cancer models (Yang et al., 2011a). It isstill unclear whether these

molecules are direct targets for EGCG or downstream eventsof the primary action.

Based on the limited human data, actions of tea polyphenols

in reducing oxidative

stress and enhancing the elimination of carcinogens may beconsidered as important

mechanisms.

4 Reduction of body weight, alleviation of metabolicsyndrome and prevention of diabetes

Overweight, obesity and type 2 diabetes are emerging asmajor health issues in many

countries. MetS is a complex of symptoms that includeenlarged waist circumference

and two or more of the following: elevated serumtriglyceride, dysglycemia, high blood

pressure and reduced high-density lipoprotein-associatedcholesterol (Ford, 2005). The

possible beneficial effects of tea consumption on bodyweight reduction and MetS

alleviation could have huge public health implications.

4.1 Studies in animal models

The effects of tea, tea polyphenols and EGCG on body weightand MetS have been

studied extensively in animal models (reviewed in Yang andHong (2013), Huang et al.

(2014), Yang et al. (2016)). Most of the studies showedthat consumption of GTE or EGCG

significantly reduced the gaining of body weight and/oradipose tissue weight, lowered

blood glucose or insulin levels and increased insulinsensitivity or glucose tolerance. Most

of these studies used high-fat diets or genetically inducedobese/diabetic rodent models.

For example, in mice fed with a high-fat (60% of thecalories) diet, we found that dietary

EGCG treatment (0.32% in diet) for 16 weeks significantlyreduced body weight gain,

body fat and visceral fat weight (Bose et al., 2008). Theseresults were also reproduced in a

second study using a high-fat/Western-style diet (Chen etal., 2011). EGCG treatment also

attenuated insulin resistance, plasma cholesterol andmonocyte chemoattractant protein

levels in mice on high-fat and high-fat/Western diets (Boseet al., 2008; Chen et al., 2011).

Similar results were also observed in several recentstudies (Okuda et al., 2014; Byun et al.,

2014; Ortsater et al., 2012; Lee et al., 2015). Green teapolyphenols also alleviate MetS

in other animal models. For example, in insulin-resistantrats, treatment with green tea

polyphenols significantly decreased blood glucose, insulin,triglycerides, total cholesterol,

low-density lipoprotein (LDL) cholesterol and free fattyacids (Qin et al., 2010). In insulin

resistant beagle dogs, oral administration of GTE (80 mg/kgdaily, before the daily meal)

for 12 weeks also markedly increased insulin sensitivityindex (Serisier et al., 2008).

Diet-induced liver steatosis, which predisposes to livercancer, is becoming a common

disease, and its possible prevention by tea consumptionwarrants more investigation.

We have shown that EGCG treatment reduces the incidence ofhepatic steatosis, liver

size (48% decrease), liver triglycerides (52% decrease),plasma alanine aminotransferase

concentration (67% decrease) and liver pathology in micefed with a high-fat diet (Bose

et al., 2008, Chen et al., 2011). Tea catechins have beenreported to also reduce hepatic

steatosis and liver toxicity in rodents treated withethanol, tamoxifen or endotoxins, or

rodents with liver ischaemia/reperfusion injury (reviewedin Sae-tan et al. (2011)). These

findings have potential for practical applications.

4.2 Studies in humans

4.2.1 Randomized controlled trials

The effects of tea consumption on body weight andbiomarkers of MetS have been studied

in many short-term RCTs during the past decade. Systematicreviews and meta-analysis

covering more than 26 earlier RCTs indicated the beneficialeffects of tea consumption

in reducing body weight and alleviating MetS (Hursel etal., 2009; Phung et al., 2010).

Most of these studies used green tea or GTE with caffeine,in studies for 8–12 weeks,

on normal weight or overweight subjects. Some of the morerecent RCTs also showed

that daily consumption of 458–886 mg of green tea catechinsby moderately overweight

Chinese subjects for 90 days reduced body fat (Wang et al.,2010), and that daily intake

of PPE capsules (containing 400 or 800 mg EGCG and loweramounts of other catechins,

but small amounts of caffeine) for two months bypostmenopausal women in the United

States decreased blood levels of LDL cholesterol, glucoseand insulin (Wu et al., 2012). In

another study, GTE supplementation (379 mg per day) to

obese patients for three months

decreased body weight and waist circumference (Suliburskaet al., 2012). Improvements

in lipid profiles, including the decrease in levels oftotal cholesterol, LDL cholesterol and

triglycerides, were also observed (Suliburska et al.,2012). Similarly, an RCT in patients with

obesity-related hypertension showed that consumption of GTE(379 mg daily) for three

months reduced fasting serum glucose and insulin levels(Bogdanski et al., 2012).

A recent metabolomics study with healthy male subjectsdemonstrated that GTE

supplementation (1200 mg catechins and 240 mg caffeinedaily) for seven days increased

lipolysis, fat oxidation and citric acid cycle activityunder resting conditions without

enhancing adrenergic stimulation (Hodgson et al., 2013).The role of caffeine in these

studies was inconsistent among the different studies. Ameta-analysis of metabolic studies

showed that both a catechin–caffeine mixture and caffeinealone dose-dependently

stimulated daily energy expenditure, but only thecatechin-caffeine combination

significantly increased fat oxidation (Hursel et al., 2011).

In contrast to the above-described beneficial effects, tworecent studies in English adults

did not show such beneficial effects (Hursel et al., 2011;Mielgo-Ayuso et al., 2014). The

first study was in obese Caucasian women, after anenergy-restricted diet intervention used

supplementation with EGCG (200 mg daily) for 12 weeks

(Mielgo-Ayuso et al., 2014). The

second study used daily supplementation with green tea(>560 mg EGCG) plus caffeine

(0.28–0.45 mg) for 12 weeks (Janssens et al., 2015). Thereasons for these contradictory

results are not known. The relatively low dose of EGCG usedin the first study and the

different populations, with different physiological anddietary conditions, could also be

contributing factors. An intriguing issue is the timefactor, that is, the increase in energy

expenditure with catechins may be observed in short-term(7–10 days) studies, but not in

long-term studies (Janssens et al., 2015). This may berelated to our previous observations

in mice that the plasma levels of EGCG increased in thefirst several days of daily catechin

administration, but the EGCG levels decreased gradually to~13% of the maximal levels

upon prolonging treatment (Kim et al., 2000). More researchis needed concerning the

time-dependent changes in bioavailability and healthbeneficial effects of catechins.

4.2.2 Epidemiological studies

Two epidemiological studies suggested the beneficialeffects of green tea consumption on

MetS (Chang et al., 2012; Vernarelli and Lambert, 2013).One study on elderly Taiwanese

males in a rural community indicated that tea drinking,especially for individuals who drank

240 ml or more tea daily, was inversely associated withincidence of MetS (Chang et al.,

2012). The second, a cross-sectional study of US adults,

showed that intake of hot (brewed)

tea, but not iced tea, was inversely associated withobesity and biomarkers of MetS and

CVDs (Vernarelli and Lambert, 2013). These results areexciting and need confirmation

from additional studies. On the other hand, two recentcross-sectional studies in Japan did

not find a preventive effect of green tea consumptionagainst MetS (Takami et al., 2013;

Pham et al., 2014).

The lowering of body weight and alleviation of MetS by teashould lead to the reduction

of type 2 diabetes. Such an association was found in some,but not all, human studies

(reviewed in Yang and Hong (2013), Huang et al. (2014),Wang et al. (2014) and Sae-tan et

al. (2011)). For example, a prospective cross-sectionalstudy with US women aged 45 years

and older showed that consumption of more than four cups oftea per day was associated

with a 30% lower risk of developing type 2 diabetes,whereas the consumption of total

flavonoids or flavonoid-rich foods was not connected toreduced risk (Song et al., 2005).

A retrospective cohort study of 17 413 Japanese adults aged40–65 years indicated that

daily drinking of more than six cups of green tea (but notoolong or black tea) lowered

the morbidity of diabetes by 33% (Iso et al., 2006). Theeffects of caffeine in these

epidemiological studies are unclear. A meta-analysis basedon seven studies (286 701

total participants) showed that individuals who drank three

to four or more cups of tea per

day had a lower risk of type 2 diabetes than thoseconsuming no tea (Huxley et al., 2009).

4.3 Mechanistic considerations

There are many proposed mechanisms for the above-describedactions of tea polyphenols,

and they can be summarized into two major types of actions.One is the action of tea

polyphenols in the gastrointestinal tract and the other isthe action produced by tea

polyphenols after systemic absorption in different organs.The combined effects would

reduce body weight, alleviate MetS and reduce the risk ofdiabetes and CVDs.

4.3.1 Actions in the gastrointestinal tract

Ingestion of green tea polyphenols has been shown toincrease faecal lipid and total

nitrogen contents, suggesting that polyphenols can decreasedigestion and absorption

of lipids and proteins (reviewed in Yang et al. (2016)).For example, in mice fed a high-fat

diet, EGCG dose-dependently decreased food digestibilityand increased the faecal mass;

with a 13 C-triglycerides-enriched diet, EGCGsupplementation increased 13 C levels in the

faeces (Friedrich et al., 2012).

The possibility that tea may affect gut microbiome has beenstudied in mice. For

example, green tea powder feeding affected gut microbiotaand reduced the levels of

body fat, hepatic triglyceride and hepatic cholesterol; thereduction was correlated with the

amount of Akkermansia and/or the total amount of bacteriain the small intestine (Axling

et al., 2012). The abundance of Akkermansia muciniphila hasbeen shown previously to

be increased in prebiotic-treated ob/ob mice, which hadlower fat mass compared to

the control ob/ob mice (Everard et al., 2011). Changing gutmicrobiota, for example, by

the administration of Saccharomyces boulardii, has alsobeen shown to reduce hepatic

steatosis, low-grade inflammation and fat mass in obese andtype 2 diabetic db/db mice

(Everard et al., 2014). In humans, green tea consumptionhas been reported to increase

the proportion of the Bifidobacterium species in faecalsamples (Jin et al., 2012). Increase

in intestinal Bifidobacteria by a prebiotic (oligofructose)has been shown to decrease

biomarkers for diabetes in mice (Cani et al., 2007). Theseresults suggest the possibility

that tea may alleviate MetS by enriching the probioticpopulation in the intestine. However,

a recent study in humans indicated that long-term green teaconsumption did not change

the gut microbiota (Janssens et al., 2016). More studies inthis area with green and black

tea preparations are needed. The possibility that the EGCGactivates the gut–brain–liver

axis, intestinal endocrine systems or intestinal receptorscould be important and warrants

investigation.

4.3.2 Actions in internal organs

Many studies showed that ingestion of tea catechins

suppressed gluconeogenesis

and lipogenesis and enhanced lipolysis in a coordinatedmanner (reviewed in Yang

et al. (2016)). These results suggest that the actions oftea catechins are mediated by

energy-sensing molecules, possibly AMP (adenosinemonophosphate)-activated protein

kinase (AMPK). In response to falling energy status, AMPKis activated to inhibit energy

consuming processes and promote catabolism to produceadenosine triphosphate (ATP)

(Long and Zierath, 2006; Hardie et al., 2012; Hardie,2015). In addition to maintaining

cellular energy homeostasis, AMPK also responds todifferent hormone signals to maintain

whole body energy balance (Hardie, 2015). We propose thatthe activation of AMPK is

the main mechanism for EGCG and other catechins toinfluence energy metabolism and

to alleviate MetS. The activation of AMPK by EGCG and greentea polyphenols has been

demonstrated in vivo and in vitro (Murase et al., 2009;Banerjee et al., 2012; Zhou et al.,

2014; Collins et al., 2007; Serrano et al., 2013). Thereare also reports indicating that

AMPK was activated in adipose tissues and skeletal muscleby black tea, oolong tea and

Pu-erh teas (Yamashita et al., 2014; Yamashita et al.,2012). The detailed mechanism by

which EGCG activates AMPK is still unclear, although theinvolvement of ROS has been

suggested based on studies in vitro (Collins et al., 2007).The situation in vivo could be

different due to the lack of auto-oxidation of EGCG. EGCGhas been reported to inhibit

mitochondrial oxidative phosphorylation to decrease ATPlevels (Valenti et al., 2013).

Another possibility is that EGCG may serve as an uncouplerof oxidative phosphorylation.

Either action could increase the AMP (ADP) to ATP ratiosand activate AMPK.

The downregulation of the two key enzymes ingluconeogenesis, PEPCK and G-6-Pase,

and associated decrease in glucose production in the liverby EGCG has been shown

to be mediated by AMPK activation (Collins et al., 2007).The activated form, p-AMPK,

is also known to phosphorylate and inactivate ACC, therate-limiting enzyme of fatty

acid synthesis. The resulting lowered levels of malonyl-CoAcan activate CPT-1, which

facilitates long-chain fatty acyl CoA transport intomitochondria for β-oxidation (Long and

Zierath, 2006). The possible role of AMPK in mediating theactions of tea constituents in

affecting other genes and proteins to increase catabolismand decrease anabolism has

been reviewed (Yang et al., 2016).

4.3.3 Overall mechanistic considerations

We proposed the hypothesis that most of these beneficialeffects can be explained by the

decreased absorption of macronutrients and the systemiceffects of tea catechins after

absorption in metabolic regulation, which are mediatedmostly by the activation of AMPK.

We further hypothesize that the relative importance of

these two types of action depends

on the types and amounts of tea consumed as well as thedietary conditions. For example,

with black tea, the decrease in nutrient absorption,especially with a high-fat diet, may

play a more important role than its systemic effects,because of the low bioavailability of

theaflavins and thearubigins. Even though our ‘AMPKhypothesis’ proposes that AMPK

plays a major role in mediating the actions of EGCG ongluconeogenesis, fatty acid

synthesis and catabolism of fat and glucose, actions thatare independent of AMPK could

also be involved. Some of these actions are described inthis chapter, and some have been

discussed in reviews by Wang et al. (2014) and Kim et al.(2014).

5 Lowering of blood cholesterol, blood pressure andincidence of cardiovascular diseases

5.1 Studies in humans

The alleviation of MetS by tea logically leads to thereduction of the risks for CVDs (reviewed

in Deka and Vita (2011), Di Castelnuovo et al. (2012) andMunir et al., (2013)). The lowering

of plasma cholesterol levels and blood pressure as well asimprovement of insulin sensitivity

and endothelial function by green tea has been verified inmany studies (reviewed in Munir

et al. (2013)). In a review of 11 RCTs, both green andblack teas decreased LDL cholesterol

and blood pressure (Hartley et al., 2013). The strongestevidence for the reduction of CVD

risk by the consumption of green tea was provided by large

cohort studies in Japan. In the

Ohsaki National Health Insurance Cohort Study (n = 40 530),deaths due to CVDs were

decreased dose-dependently by tea consumption at quantitiesof one to more than five

cups of tea per day (Kuriyama et al., 2006b). In anotherstudy with 76 979 Japanese adults,

the consumption of green tea was also associated withdecreased CVD mortality, but daily

consumption of more than six cups of tea was needed tomanifest the effect (Mineharu et

al., 2011). Correlation between the consumption of tea andthe decreased risk of stroke

was reported by two studies from China and Japan (Liang etal., 2009; Kokubo et al.,

2013). A meta-analysis of 14 prospective studies, covering513 804 participants with a

median follow-up of 11.5 years, found an inverseassociation between tea consumption

and risk of stroke, and the protective effect of green teaappeared to be stronger than that

of black tea (Shen et al., 2012). Many, but not all,studies in the United States and Europe

demonstrated an inverse association between black teaconsumption and CVD risk (Arab

et al., 2009; de Koning Gans et al., 2010; Mukamal et al.,2006; Sesso et al., 2003; Deka and

Vita, 2011). A meta-analysis, including 6 case–control and12 cohort studies (5 measured

green tea and 13 measured black tea as the exposure), founda reduced risk of coronary

artery disease by 28% via green tea consumption (10%decrease in risk with an increment

in consumption of one cup per day). However, there was nosignificant protective effect

from black tea (Wang et al., 2011).

5.2 Possible mechanisms

As discussed above, reduction of body weight andalleviation of MetS by tea consumption

would decrease the stress to the cardiovascular system.Beneficial effects of tea catechins

in lowering plasma cholesterol levels, preventinghypertension and improving endothelial

function contribute to the prevention of CVDs. Thecholesterol-lowering effect is likely

due to the decrease in cholesterol absorption orreabsorption by catechins as well as the

decrease in cholesterol synthesis via the inhibition of3-hydroxy-3-methylglutaryl-coenzyme

A reductase (mediated by the activation of AMPK). Enhancednitric oxide signalling has

been suggested as a common mechanism for catechins todecrease blood pressure and

the severity of myocardial infarction (Munir et al., 2013).Several studies showed that green

tea or black tea polyphenols increased endothelial nitricoxide synthase (eNOS) activity

in bovine aortic endothelial cells and rat aortic rings(Jochmann et al., 2008; Lorenz et al.,

2004; Aggio et al., 2013), possibly mediated by AMPK andother signal pathways. Tea

catechins may suppress the expression of caveolin-1, anegative regulator of eNOS (Li et

al., 2009). Similarly, EGCG has also been shown to lowerthe expression of endothelin-1,

possibly through the activation of AMPK, and this reduces

vasoconstrictor tone and may

directly increase bioavailability of nitric oxide toimprove endothelial function (Akiyama et

al., 2009). AMPK has also been suggested to regulate theantioxidant status (Colombo

and Moncada, 2009) and to mediate the anti-inflammatoryactivity of EGCG in endothelial

cells (Wu et al., 2014). EGCG has also been shown to inducethe expression of haeme

oxygenase 1 in aortic endothelial cells (Pullikotil et al.,2012), and this may increase anti

inflammatory activity to benefit the cardiovascular system.While moderate doses of EGCG

have yielded beneficial effects, a very high dose (1% indiet) has been shown to promote,

rather than to attenuate, vascular inflammation inhyperglycemic mice (Pae et al., 2012).

6 Neuroprotective effects of tea

Several epidemiological studies have suggested that teadrinking was associated with the

improvement of cognitive function. For example, green teaconsumption was associated

with a lower prevalence of cognitive impairment amongelderly Japanese (Kuriyama et

al., 2006a; Tomata et al., 2012) and with better cognitiveperformance in community

living elderly Chinese (Feng et al., 2010). Other studieshave described a moderate risk

reduction of Parkinson’s disease (PD) in tea drinkers(Barranco Quintana et al., 2009; Li et

al., 2012). A recent meta-analysis of 13 articles involving901 764 participants showed a

linear dose relationship for decreased PD risk with tea,

coffee and caffeine consumption

(Qi and Li, 2014). Another meta-analysis of 20observational epidemiological studies

involving 31 479 participants, however, found that caffeineintake from tea or coffee was

not associated with the risk of cognitive disorders(including dementia, Alzheimer’s disease

and cognitive impairment/decline) (Kim et al., 2015). Arecent review article suggested

that tea, coffee and caffeine may be protective againstlate-life cognitive impairment/

decline, but the association was not found in all cognitivedomains and lacked a distinct

dose–response association (Panza et al., 2015). Teadrinking was also associated with

lowering risks of depressive symptoms (Hintikka et al.,2005) and psychological distress

(Hozawa et al., 2009).

Laboratory studies also showed that EGCG protected againstneurodegenerative

diseases such as Parkinson’s and Alzheimer diseases inrodent models (Levites et al.,

2001, Rezai-Zadeh et al., 2005). More recent studies havealso demonstrated the

neuroprotective effects of tea catechins against okadaicacid-induced acute learning and

memory impairment in rate (Li et al., 2014) and against6-hydroxydopamine-induced

behavioural and depressive changes in a rat model of PD(Bitu Pinto et al., 2015).

Interestingly, a recent study in anN-methyl-4-phenyl-1,2,3,6-terahydropyridine-induced

Parkinsonian monkey model demonstrated that oral

administration of tea polyphenols

alleviates motor impairment, dopaminergic neuronal injuryand cerebral α-synuclein

aggregation (Chen et al., 2015). There are also studiessuggesting beneficial effects of

dietary EGCG in combination with exercise on brain healthand slows the progression of

Alzheimer’s disease in TgCRNDS mice (Walker et al., 2015).Similarly, EC plus exercise are

also neuroprotective against cognitive deficiencies andprogression of moderate or mid

stage Alzheimer’s disease in APP/PS1 mice (Zhang et al.,2016).

Based on animal and cell culture studies, catechins andtheanine are also considered

to be responsible for the neuroprotective action of greentea. The proposed mechanisms

of action of EGCG and catechins include their antioxidantand iron-chelating activities

as well as anti-inflammatory and signal-modulatingactivities related to neuronal cell

growth (reviewed in Weinreb et al. (2009) and Mandel et al.(2008)). Since neuronal cells

are sensitive to oxidative damage, antioxidant actions ofcatechins are important for the

protective effect. Iron accumulation in brain is a commonfeature of neurodegeneration,

and iron is also involved in the production of amyloidprecursor protein and β-amyloid

formation (Rogers et al., 2002). EGCG is known to reversethe iron-dependent events in

many in vitro models. Although several reports indicatedthat EGCG can pass the blood–

brain barrier and exert a neuroprotective action, it isunclear whether EGCG can reach the

brain at levels high enough for neuroprotection in humans.

The characteristic amino acid theanine, which can cross theblood–brain barrier, is

considered to be an important compound for theneuroprotective actions of tea. Several

studies indicated that theanine relieved anxiety symptomsin patients with schizophrenic

and schizoaffective disorders (Ritsner et al., 2011), and acombination of GTE and theanine

improved memory and attention in subjects with mildcognitive impairments (Park et al.,

2011). Theanine was also effective in improving sleepquality in boys diagnosed with

attention-deficit hyperactivity disorder (Lyon et al.,2011). As an analogue of glutamate, a

neuroexcitatory transmitter, theanine may act as anantagonist against glutamate receptors

and prevent glutamate-induced excitatory neuronal toxicity.Since the affinity of theanine

to the receptors is low, interference in glutaminetransporter and modulation of levels

of other neurotransmitters, such as dopamine andγ-aminobutyric acid, have also been

suggested to be neuroprotective mechanisms of theanine(Kakuda, 2011).

Overall, there are many human and animal studies suggestingthe protective effects of

tea consumption against several types of cognitivedysfunctions. Longer follow-up studies

on a large number of subjects are needed to fully confirmthese effects.

7 Conclusion

As discussed above, many laboratory and epidemiologicalstudies strongly suggest the

beneficial health effects of tea consumption in theprevention of chronic diseases. Evidence

from human intervention studies, however, is lackingconcerning some of the beneficial

effects, such as the prevention of cancer. In animalstudies, conditions are usually optimized

to demonstrate the hypothesized effects, and the doses oftea preparations used are

usually higher than the levels of human consumption.Effects of interfering factors, such

as smoking, physical activity and dietary intake of coffee,calories, fat and fibre, make it

difficult to interpret human data. After taking thesefactors into consideration, a clearer

pattern may emerge.

The possible molecular mechanisms by which tea constituentslower body weight,

alleviate MetS and prevent diabetes, CVDs,neurodegenerative diseases and cancer

are summarized in Fig. 2. The antioxidant activities of teapolyphenols are believed

to contribute all these beneficial effects. These involvethe direct antioxidant effect of

polyphenols, their chelation of metal ions and theiractivation of the Nrf2-mediated

cellular defence system. The binding of tea polyphenols tolipids and proteins (including

inhibitions of digestive enzymes) and decreasingmacronutrient absorption in the intestine

appear to be a major mechanism for body weight reduction in

animals and humans who

Figure 2 Possible mechanisms for the prevention of chronicdisease by tea constituents. Abbreviation:

NDDs = neurodegenerative diseases.

ingest excessive amount of calories. The bodyweight-lowering effect provides beneficial

effects to alleviate or prevent MetS, diabetes and CVDs andperhaps also cancer. The

activation of AMPK, possibly a consequence of polyphenolsbinding to mitochondrial

election transport proteins, is also expected to alleviateMetS and diabetes, which

reduces the risk of CVDs. For the prevention ofneurodegenerative diseases, although

tea polyphenols have been showing activity in laboratorystudies, epidemiology studies

suggest the important contribution of caffeine, becausecoffee has similar effects. Theanine

could play a major role in preventing neurodegeneration andimproving mental health.

Multiple mechanisms have been proposed for thecancer-preventive activities of tea

polyphenols and caffeine. The binding of catechins, such asEGCG, to specific enzymes,

receptors and signalling molecules provides excitinginsights for further investigations on

the mechanisms of prevention of many of these diseases. Howa molecule such as EGCG

could specifically affect the functions of many proteinsand the expression of many genes

as discussed in this chapter is intriguing and requiresfurther investigation.

Bioavailability is an important issue in determining the

biological effects of tea polyphenols

in internal organs. This factor could explain the resultsthat many of the beneficial effects

were observed with green tea but not with black tea. Teapolyphenols that are not absorbed

into the blood, however, may exert their effects in thegastrointestinal tract, for example,

in decreasing lipid absorption. This is probably why blacktea is also effective in lowering

body weight, body fat and cholesterol levels. Theintestinal microbiota may degrade tea

polyphenols, as has been shown for catechins andtheaflavins (Li et al., 2000; Chen et al.,

2012). Some of the metabolites may have interestingbiological activities. The microbial

degradation of black tea polyphenols has not beensufficiently characterized and more

research is needed. The effects of tea consumption onintestinal microorganisms have been

studied (Axling et al., 2012; Jin et al., 2012; Janssens etal., 2016), and more comprehensive

research on the microbiota using newer approaches wouldprovide more information.

In studies dealing with MetS and diabetes, beneficialeffects have been observed in

individuals consuming three or four cups of tea (600–900 mgcatechins) daily. However,

such cause–effect and dose–response relationship in theprevention of other diseases is

not clear. Intervention studies on the prevention ofcancer, CVDs and neurodegenerative

diseases are difficult to conduct. Because of budgetconsiderations, most intervention

studies are limited to a length of a few or several years,usually with high-risk populations.

A drawback of using a high-risk population is that thelatent disease may have progressed

to a stage that is reflective to the intervention agents.If a general population is used,

the intervention period may not be long enough for themanifestation of the disease

preventive effects.

In epidemiological studies, the lack of beneficial effectsof green tea consumption

observed in some studies could be due to the relatively lowquantities of tea consumed.

For protection against certain diseases, the effects oflower levels of tea consumption (one

to three cups per day) may be subtle. However, cautionshould be applied in the use of

high doses of tea extracts for disease prevention. Manycases of hepatotoxicity due to the

consumption of GTE-based dietary supplements have beenreported (reviewed in Mazzanti

et al. (2009) and Koyama et al. (2010)). Because of thehepatotoxicity concern, French and

Spanish regulatory agencies suspended market authorizationof a weight reduction product

containing GTEs (Sarma et al., 2008). In addition, becauseof the strong binding activities of

tea polyphenols to minerals and biomolecules, ingestion oflarge quantities of tea extracts

may cause nutritional and other problems, even though suchproblems are not expected to

occur due to regular tea beverage consumption (Yang andHong, 2013).

In order to gain a better understanding of the healtheffects of tea consumption, more

research is needed. Some suggestions are as follows:

1 More laboratory studies to elucidate the biochemicalbasis for the reported health effects of different typesof tea. The relevance of these results to human healthshould be evaluated.

2 More large and long-term prospective studies with specialattention paid to the quantity and types of teaconsumption, smoking status, diet, physical activities,genetic polymorphism and other possible interferingfactors.

3 More well-designed intervention studies, based on stronglaboratory data and with adequate duration.


For general information about tea, look at other chaptersof this book. For the effects of

2015).

9 Acknowledgements

This work was supported by NIH grants CA120915, CA122474and CA133021. We thank

Ms. Vi P. Dan for her assistance in the preparation of thismanuscript.

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15 Chapter 15 Tea cultivation underchanging climatic conditions

1 Introduction

Intense human activity over the last two centuries hasresulted in a rapid global climate change.

The Intergovernmental Panel on Climate Change (IPCC)reported that the concentrations of

the main greenhouse gases CO 2 , CH 4 and N 2 O havereached 393.1 ppm, 1819 ppb and

325.1 ppb in 2012, increasing by 41%, 160% and 20%,respectively, compared to the pre

industrial era (IPCC, 2013). As a result, the speed ofglobal warming has increased and the

global mean surface temperature has increased by 0.85°Cover the period 1880–2012. The

world meteorological organisation confirmed thecontinuation of this warming trend over

the past few decades, with the five-year period from 2011to 2015 being the warmest on

record globally, and 2015 being the warmest year on recordto date (WMO, 2016).

Despite the increase in the mean temperature, many extremeweather events, including

the breaking of temperature records, heavy precipitationand prolonged intense hot,

dry seasons, occur. As a result, droughts, floods, storms,landslides, outbreaks of pests

and diseases and a loss of diversity are now frequentlyexperienced. For example, the

probability of extreme high temperatures increased by afactor of 10 or more in some

locations during the period 2011–15 (WMO, 2016).

Tea is one of the most important cash crops worldwide,

playing a significant role in rural

development, poverty reduction and food security indeveloping countries. It is planted

in 58 countries in the five continents, the majority beingin Asia and Africa. The total area

of land under tea cultivation is 4.37 million ha, with anannual production of 5.30 million

tons in 2015 (ITC, 2016). Smallholders are the main forcein tea production, especially

in the mountainous regions. It was estimated that China andIndia have 20 million and

three million rural labourers, respectively, involved intea production and processing.

Smallholders constitute 73%, 60% and 47% of the total teaproduction in Sri Lanka, Kenya

and Indonesia, respectively. Tea also plays a vital role inrelation to economic development.

For example, in Sri Lanka, it generates 1.3 billion USdollars in exports, comprising 14.84%

of the total export earnings or 59.72% of the agriculturalexport earnings. Tea exports

contributed 20% to the total national foreign exchangeearnings in Kenya (Azapagic et

al., 2016).

As it is a rain-fed mono-cropping system, tea cultivationdepends on weather conditions

for optimal growth. Global climate change, therefore, has agreat impact on tea growth

and development. Ochieng et al. (2016) found that tea wouldbe one of the crops most

adversely affected by climate change. Increasingtemperatures and extreme weather

events are posing a significant threat to the resilience of

tea production systems. There is,

therefore, an urgent need to develop mitigation andadaptation strategies to cope with

climate change. We have attempted to review recent researchmodelling and the potential

impact of climate change on tea cultivation. An attempt isalso made to suggest strategies

for mitigating its adverse influence on abiotic stress andtemperature fluctuation in order

to reduce the risks presented by climate change and topromote sustainable development

of tea industry.

2 Climate change and climatic variability

2.1 The extent of climate change

It has been established that the global mean surfacetemperature has increased since the

late nineteenth century. Each of the past three decades hasbeen successively warmer at

the Earth’s surface than any of the previous decades in theinstrumental record. Globally

averaged combined land and ocean temperature data ascalculated by a linear trend show

a warming of 0.85°C over the period 1880–2012 (IPCC, 2013).

As with many other regions of the world, the majortea-producing countries such

as China, India, Sri Lanka and Kenya have witnessed asignificant change in climate in

the last few decades. Han et al. (2016) evaluated climatechange in representative tea

producing cities at various latitudes in China, includingHaikou, Kunming, Hangzhou

and Jinan. The results showed that the annual mean

temperature and extreme lowest

temperature have increased by 1.0–1.6°C and 2.1–3.8°C,respectively, over the last 50

years. The highest temperature was not significantlydifferent, but the number of hot days

having a temperature >35°C had significantly increased,particularly in Hangzhou city.

The annual precipitation did not show a discerniblefluctuation. However, the number of

rainy days (>0.1 mm) and the atmospheric relative humidityhave fallen by 6.7–13.2 d and

3.3–9.4%, respectively, over the same period. Similarly,the annual hours of sunshine and

its percentage decreased from 207.1 h and 56.3% in 1950 to158.0 h and 42.9% in 2010,

respectively.

The main tea-growing areas of northeast India, Assam andWest Bengal, showed a

steady increase in mean temperature with the averageminimum temperature having

increased by about 1.3°C over the last 100 years. However,the annual precipitation has

steadily declined. For example, annual precipitation in theSouth bank region at the TTRI

of Assam has declined by more than 200 mm in the last 96years (Bhagat et al., 2016).

Similarly, Kenya, a tropical tea-producing country, alsoshowed an increase in temperature

and a decline in rainfall over the last few decades, withmean temperature increase of

0.1°C and a decrease in rainfall of 153 mm in Kericho overthe last 54 years (Bore and

Nyabundi, 2016). In Sri Lanka, temperatures have increased

markedly over the last one

and a half centuries and the warming rate has acceleratedin recent years. For example,

the mean temperature increased at a rate of 0.016°C peryear during the period 1961–90

and 0.025°C per year during the period 1987–96 (Esham andGarforth, 2013).

In terms of future climate, global surface temperaturechange for the end of the

twenty-first century is likely to exceed 1.5°C relative to1850–1900, and warming will

continue beyond 2100 (IPCC, 2013). In terms oftea-producing regions, Dutta (2014)

applied predicted models derived by WorldClim and IPCC4 andfound that the average

temperature may increase by 2°C in Northeast India in 2050,while there will be a little

variation in the rainfall pattern as compared to today. Themean temperature in Sri Lanka

is predicted to increase by 2.4°C by the end of the century(Basnayake, 2011), and the

minimum and maximum mean temperatures are predicted toincrease between the ranges

of 0.5–0.7°C and 0.6–0.9°C, respectively, by 2050 (Eshamand Garforth, 2013). The mean

air temperature in East Africa is predicted to increase byabout 2.5°C by 2025 and 3.4°C

by 2075, while rainfall is predicted to increase by about2% by 2025 and 11% by 2075

(Bore and Nyabundi, 2016).

A gradual increase in global-scale precipitation isprojected in the twenty-first

century, but the changes in response to warming will not be

uniform, with some regions

experiencing increases, and others experiencing decreasesor little change. High-latitude

land masses are likely to experience greater amounts ofprecipitation due to the additional

water carrying capacity of the warmer troposphere. Manymidlatitude and subtropical arid

and semi-arid regions are likely to experience lessprecipitation (IPCC, 2013).

2.2 Extreme weather events

As temperatures increase, many extreme weather and climateevents have been observed

since around 1950, and future increases in long-termdroughts and precipitation extremes

are very likely in Africa, East Asia, South Asia andSoutheast Asia (IPCC, 2013) – the main

tea-producing regions of the world. For example, on theglobal scale, there has been a

decrease in the number of cold days and nights and anincrease in the number of warm

days and nights, while the number of heavy precipitationevents over land has increased

in more regions than it has decreased. The worldmeteorological organisation not only

recorded the warmest year in 2015, but found large numbersof extreme weather and

climate events, including heat and cold waves, tropicalcyclones, flooding, droughts and

severe storms during the period 2011–15 (WMO, 2016).

In tea-growing areas, Han et al. (2016) reported that thefrequency of climate

variations and extreme weather events, such as drought,high daily precipitation and

late spring cold spells, had increased significantly inChina in recent years. In Kenya,

extreme weather events were characterised by a rising trendof hail and an increasing

incidence of frost and heavy rainfall over a short period,followed by prolonged

dry periods (Bore and Nyabundi, 2016). Tea farmers rankedrecurrent droughts and

changes in rainfall patterns as the most importantindicators of climate change in

Kenya (Mwendwa and Giliba, 2012).

3 Effects of climate change on the suitability of teaplanting areas and plucking duration

3.1 Basic environmental requirements for tea growth anddevelopment

Tea originated in southwestern China, at the centre of theYunnan and Guizhou plateaus,

a junction of tropical and subtropical areas. This regionis characterised by a warm climate,

abundant rainfall, high humidity and sufficiently diffusedlight. Under such conditions, tea

plants have gradually evolved specific characteristics ofadaptation to warm, moist weather,

diffused light and acidic soils. The basic environmentalrequirements for tea growth and

development are listed in Table 1. Pronounced changes intemperature, precipitation,

relative humidity, rainy days and annual sunlight hourswill not only directly affect tea

yield and quality, but will also change other basicparameters necessary for its growth

and development, such as soil pH, water content, organicmatter, nutrient availability,

pest and disease management, ecological systems around teagardens and eventually

tea processing. Tea is one of the tree species mostaffected by changes in bio-climatic

suitability in Yunnan province, China (Ranjitkar et al.,2016).

3.2 Shift of tea production areas

With increasing temperatures, especially a rise in theminimum temperature, the tea

growing area would be extended to higher latitudes andhigher altitude ecosystems. This

might be beneficial for tea production in relatively coldclimate regions such as highland and

subtropical areas, but would have a negative effect inlowland and tropical areas. Therefore,

those growing regions which are currently well known maybecome unsuitable for tea

cultivation in the future. This could trigger a shift insuitable locations for the cultivation of

Table 1 Basic environmental requirements for tea growth anddevelopment

Climate parameter Extreme lowest Normal range Optimum

Temperature (°C) −20 (var. sinensis), −8 (var. assamica)13–26 18–23

Annual accumulated

temperature (≥10°C) 3000 4000–8000 6000–7000

Annual precipitation (mm) 500 800–2500 1500–2000

Annual relative humidity (%) 60 70–90 80–85

Soil moisture (% of water

holding capacity) 50 60–95 70–90

Soil pH (in water suspension) 3.0 3.5–6.5 4.5–5.5

some varieties if high-quality tea is to be obtained. Forexample, in the Uji area of Kyoto,

one of the oldest and most famous green-tea-producing areasin Japan, tea quality has been

gradually affected by climate change (Ashardiono andCassim, 2014). In China, the main tea

producing area could gradually shift from south to north.The area under tea cultivation in

the South Tea District (Hainan, Guangdong, Guangxi, Fujianand Taiwan provinces) totalled

371.8 thousand ha in 2014, a 59.8% increase compared tothat of 1983. Meanwhile, the

tea area in the North Tea District in China (Shandong,Shanxi, Gansu, Henan and Hubei

provinces) was 618.6 thousand ha in 2014, a seven-foldincrease over 1983. However, the

northern boundary of tea-producing areas in China isunlikely to move further north beyond

Qing Ridge and the Wai River due to limited precipitationand higher soil pH in the northern

part of this region. In Kenya, the optimum tea-producingzone is expected to shift to a

higher altitude of between 2000 and 2300 m by 2050,compared with the current altitude of

between 1500 and 2100 m. By 2050, the tea areas ataltitudes between 1400 and 2000 m

will suffer the greatest decrease in suitability and theareas around 2300 m will have the

highest increase in suitability when compared to thepresent day.

Overall, the suitability of tea-growing areas is expectedto decline by 22.5% by 2075

(Bore and Nyabundi, 2016). Suitable areas will shift up thealtitudinal gradient: those

retaining some suitability will see decreases of between 20and 40%, compared with

today’s suitability of 60–80% in Uganda (Eitzinger et al.,2011). In Eastern Africa, up to

40% yield loss is expected due to the reduction in suitableareas caused by temperature

increase (Adhikari et al., 2015). Due to differingsensitivity to temperature increases, the

reduced suitable area for Camellia sinensis var sinensiswas greater than that for Camellia

sinensis var assamica in the Yunnan province of China(Ranjitkar et al., 2016).

3.3 Tea plucking duration

The temperature increase in subtropical areas with distinctseasons will extend the duration

of growing and plucking. It is reported that the number ofdays warmer than 10°C, which is

regarded as the starting temperature for tea sprouting, mayincrease by 15 d if the annual

mean temperature increases by 1°C (Yang, 2006). With anincrease in spring temperatures, the

tea budding time will be advanced and the harvest can beginearlier. Lou et al. (2015) found

that the plucking date of the Longjing-43 tea cultivar inChina was significantly advanced

by 0.88–1.28 d/decade, based on phenological and economicoutput models established

using meteorological data from 1972 to 2013. In addition,Lou et al. found that the amount

of high-quality tea decreased significantly. However,economic output depended on the

scale of the farm. In tea plantations, the economic outputcould be reduced due to difficulty

in employing sufficient pluckers for high-quality tea,while smallholders experienced no

significant decline in profits (Lou et al., 2015). Earlyplucking of spring tea would increase the

yield, but would probably pose a risk of more extremeweather events, such as later spring

coldness. In Sri Lanka, the delayed onset of the monsoonnot only reduced tea production,

but also shortened the cultivation season (Esham andGarforth, 2013).

4 Effects of climate change on tea production

Climate change brings both advantages and disadvantages forthe growth and development

of tea and will ultimately have a considerable impact onproduction. The beneficial changes

include increases in temperature and CO 2 . The adverseimpacts include a decrease in rainy

days and in relative humidity, and an increase in climateextremes and variations, such as

drought, flood and extremely cold and hot weather. Theseadverse climate changes will

cause serious problems for tea production and sustainabledevelopment. The impact of

the reduction of sunny days will depend on the degree ofchange and the location.

4.1 Modelling and temperature

Econometric modelling is normally used to assess the effectof climate change on crop

yields. Hsiang (2016) provides overviews of the methodsused. Normally, regression models

are estimated using panel data sets of crop yield. Duncan

et al. (2016) used a garden

level panel data set and estimated statistical models inAssam, India, to identify the causal

effect of monthly temperature, monthly precipitation,drought intensity and precipitation

variability on tea yield and found that monthlytemperatures above 26.6°C had a negative

effect on yield. An extra one degree of warming at anaverage monthly temperature of 28°C

would result in a 3.8% reduction in yield. Wijeratne (1996)and Challinor et al. (2007) also

found that yield tended to decline with increasingtemperature in higher mean temperature

regions (>25–26°C). But in a region or season with lowtemperatures, an increase in

temperature would increase yield. For example, the meantemperature in Lushan Mountain

at an altitude of 1020 m in April was 13.8°C and the yieldwas only 74.8% of that at an

altitude of 212 m and a mean temperature of 17.6°C (Han etal., 2017).

4.2 Rainfall or monsoon

Boehm et al. (2016) analysed the effect of monsoon dynamicsand weather on tea

production by using provincial-level data of tea productionin China and found yield to

be more sensitive to precipitation than to temperature. Anincrease in the retreat date of

the monsoon and an increase in monsoon precipitation areassociated with a decrease in

tea yield. IPCC (2013) predicts that global measures ofmonsoon by the area and summer

precipitation are likely to increase in the twenty-first

century, as the monsoon circulation

weakens. The monsoon season is predicted to be longer withan earlier arrival and a

similar or later retreat date. The increase in seasonalmean precipitation is likely to be most

pronounced in the East and South Asian summer monsoons,while the change in other

monsoon regions is subject to greater uncertainty (IPCC,2013).

Changes in the monsoon season will affect tea productionbecause the quantity and

variability of rainfall are crucial. The effects ofprecipitation relative to temperature are

even more important in tropical countries such as Kenya andSri Lanka (Bore et al., 2013;

Esham and Garforth, 2013). Diminishing rainfall reduces teayields, but this depends on

its distribution over time. During long rains, teaproduction is lower when compared with

short rains. This is due to long rainy periods reducingsunshine and the photosynthesis of

tea leaves. Extreme rainfall events such as floods anddroughts will also negatively affect

tea yield (Esham and Garforth, 2013; Duncan et al., 2016).Wijeratne et al. (2007) found

that a reduction of monthly rainfall by 100 mm could reduceproductivity of made tea by

30–80 kg/ha/month in Sri Lanka.

The reduction in annual rainy days (even with the samequantity of precipitation)

and relative humidity, which are closely correlated, willadversely affect tea production.

For example, in Jinan city in China, the mean annual rainy

days, precipitation and

relative humidity were only 75 d, 689 mm and 58%,respectively, over the last 60 years.

A further reduction in these parameters will certainlyreduce tea growth and production.

Furthermore, the rise in temperature will increase soilevaporation and plant transpiration,

causing water shortage or seasonal drought in areas withlow precipitation. Soil water

deficits showed a negative correlation with tea yields(Bore et al., 2013), and higher water

availability increased the growth of new leaves (Ahmed etal., 2014). The highest tea

leaf production per hectare depends on 4000–4600 mm annualrainfall, according to an

analysis of field experiment results with weather data inBangladesh (Ali et al., 2014).

Tea generally exhibits a positive interaction betweenrainfall and temperature because its

production depends on stable temperatures and consistentrainfall patterns (Ochieng et

al., 2016). Any significant change in temperature andprecipitation will affect production.

4.3 CO 2 concentration

The increase in CO 2 concentration could improvephotosynthesis in tea plants. Jiang et al.

(2005) found that CO 2 concentrations of 550 or 750µmol/mol increased the net photosynthesis

of tea shoots by 17.9% and 25.8%, respectively, compared toambient air CO 2 concentration.

An increase in CO 2 concentration also resulted in thereduction or disappearance of midday

depression. Our research showed that plant height, fresh

shoot weight and root weight of tea

seedlings were 63.2 cm, 9.6 g and 9.9 g at 800 µmol/mol CO2 , which increased by 13.5%, 24.7%

and 67.8%, respectively, compared to that under the ambientCO 2 concentration of 380 µmol/

mol (Table 2) (Li et al., 2017). CO 2 enrichment alsopromoted photosynthesis and respiration

in tea plants. However, the photosynthesis wouldacclimatise to high CO 2 concentration, so

that the increase in yield would not be as great asexpected since CO 2 concentration increases

gradually with long-term climatic change. In addition, asthe concentration of CO 2 increases,

other plant growth limiting factors such as a shortage ofnitrogen and microelements will

appear, further reducing the benefit of the CO 2concentration increment to tea yield. However,

this could be overcome by proper nutrient management.

4.4 Pests and diseases

The incidence and proliferation of pests, diseases andweeds will increase with climate

change (Lal, 2005). Warmer weather helps insects andpathogens to survive in winter,

which is a critical time for their reduction, and thushelps to shorten the damaging period

by increasing the number of annual generations andreproduction rates in some pests. For

example, the tea geometrid (Ectropis obliqua) has sixgenerations per year in Hangzhou

under normal weather conditions. However, this willincrease to seven generations if the

mean temperature rises above 20°C during October (Wang andJin, 2010). The numbers

of over-wintering tea green leafhoppers and theirreproductive rate are significantly

correlated with the number of days with a daily meantemperature above 0°C in Hangzhou

Table 2 Effect of CO 2 enrichment on the growth of the teaplant

Treatment Height (cm) Fresh shoot weight (g) Fresh rootweight (g) Root/shoot ratio

Ambient CO 2 55.7 ± 3.73 b 7.7 ± 0.98 b 5.9 ± 0.74 b 0.47 ±0.031 a

Elevated CO 2 63.2 ± 4.65 a 9.6 ± 1.47 a 9.9 ± 1.32 a 0.60± 0.064 b

Different letters in the same column denote significantstatistical difference (P < 0.05).

district (Yang, 2005). The higher temperature, togetherwith lower relative humidity, also

favours some pests. The incidence of diseases is positivelycorrelated to temperature and

negatively correlated to sunlight hours (Li et al., 2010).For example, the incidence of

Phomopsis canker disease in southern Indian tea plantationswas significantly related to

local climate (Ponmurugan et al., 2013). Thrips(Scirtothrips dorsalis Hood) and the tea

green leafhopper (Empoasca flavescens Fabricius), whichwere previously considered as

minor or occasionally serious pests in localised areas oftea plantations, are now established

as regular, and at times major pests in the tea plantationsof North Bengal, spread over the

sub-Himalayan slopes and the adjoining plains of Terai andthe Dooars (Saha et al., 2012).

Our recent investigation also showed that the leaf disease

‘blister blight’ (Exobasidium

vexans Massee), which normally occurs in tropical Yunnanand Hainan Provinces, has

become a problem in Songyang and Pujian Counties ofZhejiang Province, the typical

subtropical zone. The incidence of anthracnose disease(Colletotrichum gloeosporioides)

also increased significantly with elevated CO 2concentrations which reduces the

endogenous caffeine content of tea leaves (Li et al.,2016). Pest activities, tea plant and

pest interactions could be changed due to altered plantphysiology and/or morphology

under increased CO 2 concentration (Chakraborty et al.,2000). Due to the increasing

incidence and activities of pests and diseases, the averageyield could be reduced by up

to 40% if no proper measures were taken (Oerke et al.,1996).

4.5 Soil quality

An increase in temperature speeds up the microbe depletionof soil organic matter while

reducing the time needed to release nutrients from chemicalfertilisers.

Intense daily precipitation may cause severe flooding orlandslides, which remove fertile

top soils. The enrichment of CO 2 and other air pollutantssuch as SO 2 and NO 2 will cause

strong acid precipitation that further increases soilacidity. It should be noted that tea soils

in China are already too acidic, one-third being below pH4.5 (Han et al., 2002). Further

acidification will therefore degrade soil quality and tea

production.

4.6 Solar radiation

A reduction in sunlight hours resulting from fewer rainydays and more cloudy days

with diffused light would be beneficial for tea growth anddevelopment in tropical or

subtropical areas currently receiving intense solarradiation. However, it would be harmful

for cultivation in high mountains or ecosystems with lowsolar radiation. Boehm et al.

(2016) found that a 1% decrease in solar radiation in theprevious growing season was

associated with 0.554–0.864% decrease in tea yields.

4.7 Extreme climate events

Climate extremes and variations, such as drought, flood andvery cold or hot weather, will

cause serious problems for tea production and sustainabledevelopment. Because of its

unpredictable nature, it would be the most significantfactor influencing year-to-year tea

production. Ahmed et al. (2014) found that the tea growthduring the spring drought in

Yunnan Province is 50% lower than that compared to themonsoon period. The previous

year’s weather factors had a significant impact on teayields (Boehm et al., 2016), heat

stress causing obvious alterations in the leaf phenotypeand significantly decreasing the

net photosynthetic rate and tea yield (Li et al., 2015).

5 Effects of climate change on tea quality

The most commonly used tea quality parameters include freeamino acids, polyphenols or

catechins, caffeine and water-extracted substances. With acertain polyphenol concentration,

higher levels of free amino acids give better green teaquality. Polyphenols or catechins are

precursors to the theaflavins and thearubigins developed inblack tea processing, so higher

levels of polyphenols, to some extent, deliver better blacktea quality. The amino acids and

polyphenols in tea shoots are mainly a result of nitrogenand carbon metabolisms and their

balance in tea plants, which are significantly affected byclimate change.

5.1 Temperature and rainfall

Relatively low temperatures are beneficial to nitrogenmetabolism, especially the bio-synthesis

of amino acids. High temperatures have a positive impact oncarbon metabolism and the bio

synthesis of polyphenols. The mean temperature 15 d beforetea plucking has a significant

negative correlation with amino acids, although it ispositively correlated with polyphenols

(Huang, 1985). For example, epigallocatechin (EGC),epicatechin (EC), epicatechin gallate

(ECG) and epigallocatechin gallate (EGCG) increased with arise in the daily average

temperature. Long periods of precipitation led to thedecline in EGC, EC, ECG, EGCG and

their total catechins content but increased C (catechin)content (Wang et al., 2011).

The relative humidity of a tea-growing location is decidedby water availability and

temperature. The amount of sunlight hours is related to theintensity of solar radiation and

affects temperature. Amino acid concentrations in teashoots are positively correlated with

relative humidity and negatively correlated with sunlighthours. However, tea polyphenols

show just the opposite trend, which is a negativecorrelation with relative humidity and a

positive correlation with sunlight hours (Huang, 1985; Weiet al., 2011).

The majority of famous Chinese teas are typically producedin mountainous regions

and near rivers or lakes. Mountainous tea-growing regionsare characterised by altitudes

between 300 and 1000 m, higher precipitation and higherrelative humidity, more diffused

light, lower temperature and higher diurnal temperaturevariation compared to low lands

(Wang and Jin, 2010). These are favourable weatherconditions under which tea plants

synthesise and accumulate amino acids and othernitrogen-bearing quality components.

Our research also showed that with the increasing altitudeof cultivation, amino acids

increased and polyphenols decreased, resulting in a lowerratio of tea polyphenols to

amino acids (Table 3) (Han et al., 2017).

Further studies show that among the amino acids, theconcentration of theanine,

glutamic acid, arginine, serine, γ-aminobutyric acid andaspartic acid increased significantly

with increasing elevational gradients. However,epigallocatechin-3-gallate (EGCG) and

ECG significantly decreased under these conditions,indicating that catechin galloylation

was inhibited at lower temperatures (Han et al., 2017).Tea-growing regions near lakes or

rivers also have a high relative humidity, more cloudy daysand diffused light and relatively

stable temperatures. Such weather conditions also help innitrogen metabolism and the

bio-synthesis of amino acids.

Water availability will affect tea quality through a changeof individual secondary

metabolites. In a greenhouse experiment, higher wateravailability significantly

increased total methylxanthine and phenolic concentrationsin tea leaves, but decreased

concentrations of EGCG (Ahmed et al., 2013). However, afield study on the effects of

monsoon rains on tea showed that too much water brings theopposite result. Ahmed et

al. (2014) compared quality of tea sampled from the springdrought and after the onset

of the East Asian Monsoon in the Yunnan Province of China.The results showed that

concentrations of catechin and methylxanthine secondarymetabolites, major compounds

which determine tea functional quality, were up to 50%lower during the monsoon, while

total phenolic concentrations and antioxidant activityincreased.

5.2 CO 2 concentration

An elevated CO 2 level exerts a significant impact on teaquality. A growth chamber study

showed that elevated CO 2 at 500 and 700 µmol/moldecreased tea amino acids by

1.7–4.5% and 6.7–12.2%, respectively, when compared to thecurrent ambient air CO 2

concentration. At the same time, the caffeine content wasalso reduced by 3.1–4.6% and

5.1–10.7%, respectively. However, polyphenol concentrationsincreased by 3.8–6.0% and

6.9–11.3%, respectively. The soluble saccharide contentincreased by 8.4–14.4% and

18.1–28.2%, respectively (Jiang et al., 2006).

From the above evaluation, it may be seen that globalwarming will probably reduce green

tea quality, while to some extent increasing that of blacktea. Of course, an inappropriate

ratio of polyphenols to free amino acids in shoots willalso cause a deterioration in black

tea quality.

Other climate variations and extremes such as late springcold spells, frost, hail, flooding

and drought will not only damage the growth and developmentof tea plants, but will

also lead to deterioration in tea quality. Climatevariations and extremes also resulted in

biodiversity loss in tea-growing areas (Mwendwa and Giliba,2012), which could cause

pest and disease outbreaks and increase pesticide residuein tea products.

6 Adaptation and mitigation strategies

Planning for climate change adaptation and mitigationinitiatives is essential, not only

for dealing with the negative impacts of climate change,but also in order to create cost

effective opportunities and benefits for sustainabledevelopment of the tea industry. These

strategies should have at least three levels: governmentpolicy, technology and technical

development and community involvement for the extension ofadaptation and mitigation

measures. These should be integrated for the best outcomes.

Table 3 Tea polyphenols, free amino acid contents and theirratios in green tea as influenced by

changes in cultivation altitude

Sampling site Elevation (m) Total polyphenols (TP, mg/g)Free amino acids (AA, mg/g) TP/AA ratio

Cumaling 212 245.2 ± 4.7 a 26.1 ± 2.7 d 9.5 ± 1.2 a

Mazu temple 420 242.0 ± 7.2 a 29.2 ± 2.0 cd 8.3 ± 0.8 a

Songshuling 574 200.8 ± 4.8 b 35.2 ± 2.0 b 5.7 ± 0.4 b

Xiaotianchi 828 199.0 ± 5.2 b 33.3 ± 3.1 bc 6.0 ± 0.7 b

Jingzuo

station 1020 167.3 ± 18.4 c 43.1 ± 2.3 a 3.9 ± 0.3 c

Means denoted by different letters in the same column aresignificantly different (P < 0.05).

6.1 Governmental policies and strategies

Climate change is often referred to as a global problem,requiring top-down international

and national strategies to achieve substantial climatechange adaptation and mitigation.

For example, individuals are unlikely to takeresponsibility for the global accumulation

of atmospheric greenhouse gases. Therefore, effectivelyintegrated and coordinated

government policies or strategies are required forcost-effectiveness and consistency of

implementation.

Establishing international and national networks on climate

change

International and national networks involving internationaland interdisciplinary research and

communication units can be initiated based on coordinatedpolicies or/and strategies to

advance research collaboration on impact assessments ofclimate change. Such networks will

help to set up policies through integrating the natural,social and cultural aspects of climate

change. The Climate Change working group in the FAOIntergovernmental Group on Tea

should be strengthened to collect and collate the availableresearch data to determine the

impact of climate change on the tea economy, toidentify/suggest mitigation and adaptation

strategies and to develop appropriate long-termtechnologies. Collaborative research

networks with a focus on climate change should beestablished at international and national

levels. For example, a national legal framework capable ofmanaging water resources in

accordance with anticipated climate change impacts shouldbe built for adjusting water

allocations and improving usage efficiency. Extensionnetworks to implement mitigation and

adaptation measures at different governmental levels shouldbe initiated.

Strengthening investment in field infrastructure

Infrastructure construction and improvement, such asdrainage and irrigation systems, road

construction, ecosystem diversity and rebalancing should be

strengthened. Infrastructure

is considered a very good investment from the cost–benefitanalysis point of view, even

in the absence of climate change. Taking into account thehigh cost across the board to

individual’ tea plantations, governments should play a keyrole in this area.

Promoting organic, good agricultural practices (GAP) andclimate

smart tea production

Organic farming is seen as a system capable of contributingto climate mitigation and

sustainable agriculture. Recent research shows thatorganically managed tea agro

ecosystems can enhance soil carbon sequestration throughincreasing soil organic matter

(carbon) levels (Han et al., 2013a; Subramanian et al.,2013). Organic tea soils also have

statistically significant higher levels of soil pH, totalnitrogen content and soil microbial

biomass, carbon (C), nitrogen (N) and phosphorus (P) andtheir ratios to total organic C,

N and P, respectively (Han et al., 2013a). This translatesinto better plant nutrient content,

increased water retention capacity and better soilstructure and thus to higher yields and

greater soil resilience (FAO, 2009).

Organically managed tea systems are further associated withhigher levels of biodiversity,

including natural predators for pest control (Li et al.,2014; Saikia et al., 2014; Liu et al.,

2015). Pest and disease damages and the imbalance ofnutrients, especially shortages of

nitrogen, will decrease tea yields by 20–30%. However,organic tea has a 20–50% higher

price compared to conventional tea, compensating for theloss in yield. Promoting organic

tea production is therefore not only beneficial to farmers’incomes, but offers significant

scope for climate change adaptation.

GAP and/or green farming consists of a collection ofprinciples which take into account

economic, social and environmental sustainability. Theseapply to on-farm production and

post-production processes for safe and healthy food andnon-food agricultural products.

They also apply to integrated pest management, integratedplant nutrient management

and conservation agriculture, which are beneficialtechniques for mitigation and adaption

to climate change.

Climate-smart agriculture is an approach which helps toguide the transformation

and reorientation of agricultural systems to effectivelysupport development and ensure

food security in a changing climate. It aims to tacklethree main objectives: a sustainable

increase in agricultural productivity and incomes,adaptation and the building of resilience

to climate change and the seeking of opportunities toreduce greenhouse gas emissions

and increase carbon sequestration.

Precision agriculture is a high-tech farming system. Ituses global positioning systems,

geographical information systems and remote sensing to

collect the spatial variability

of parameters related to crop yield and quality, such asplant-growing situations, terrain

features, soil organic matter, moisture and nitrogencontents and pH. It utilises agricultural

inputs by machines and equipment controlled by the ExpertTechnology System. Precision

agriculture is an effective and efficient way of combatingclimate change, offering

considerable savings in natural resources and optimisingcrop yield and quality.

6.2 Research and development priorities for new andimproved technologies

Traditional coping mechanisms will not always be adequatefor dealing with the expected

medium- and long-term impacts of climate change. Innovativeagricultural technologies

and practices can play a significant role in mitigation ofand adaptation to climate

change, especially in developing countries whereagricultural productivity is low, while

poverty, vulnerability and food insecurity remain high.Shaping research priorities and

developing and disseminating innovative technologies arecentral to dealing with climate

change, reducing negative environmental impacts andimproving the sustainability of tea

production.

Developing new models for predicting the impact of climate

change on tea cultivation

There are numerous models for predicting change in theclimate itself, such as the extent

of average temperature changes and shifts in precipitationpatterns. Many models also

predict the effects of climate change on crop production.However, few focus specifically

on tea cultivation. It is therefore necessary to developspecific models for predicting future

climate change in tea-growing regions and their impact ontea cultivation. Parameters

in the simulation models should include not only meantemperature, precipitation and

CO 2 concentrations, but also the climate variability andextremes. The impact of climate

change on tea production should focus on tea quality andthe suitability of tea-growing

areas, as well as on yield. Quantitative analysis should beincluded in the prediction

models. Early warning and monitoring systems should beestablished according to the

results of predictive models.

Reducing emissions of greenhouse gases

The continued emission of greenhouse gases will causefurther warming and changes in

all components of the climate system. Substantial andsustained reductions of greenhouse

gas emissions, such as the use of clean developmentmechanisms in tea growing, could

limit climate change while not compromising production. N 2O is a key greenhouse gas

produced in tea soils. Tea, as a leaf harvest crop, is fedwith large amounts of fertiliser,

especially those which are nitrogen-based. The annualnitrogen application rates in typical

Chinese tea fields ranged from 0 to 2600 kg/ha with an

average of 553 kg/ha (Han and

Li, 2002). When 300, 600 and 900 kg N/ha are applied, N 2O-N emissions accounted

for 1.43%, 1.96% and 3.44% of the nitrogen applied,respectively (Han et al., 2013b).

Reducing nitrogen application, by using slow-releasenitrogen fertilisers and balanced

fertilisation, can significantly increase the efficiency ofnitrogen use (Han et al., 2008).

Additional technologies and practices should be developedto increase nitrogen use

efficiency and reduce N 2 O emissions. The reduction of CO2 emissions could also improve

soil quality through increasing soil organic carbon.

Enhancing carbon sequestration

Increasing soil organic matter not only enhances carbonsequestration, but also improves

water retention capacity, soil structure and thebiodiversity which increases yield and

improves soil resilience to flooding, erosion, drought andheavy rainfall. Increasing soil

organic matter also improves nitrogen use efficiency. Inthe case of China, the average

organic carbon in the top 0–40 cm of topsoil is only 1.08%(Han et al., 2002). An increase

in topsoil organic carbon of 0.1% could increasesequestration by 5.2 ton/ha, or 13.78

million tons, equivalent to 50.52 million tons of CO 2 inthe Chinese tea sector. There

is great potential in carbon sequestration and newtechnologies should be developed

to increase soil organic matter along with organic farming,GAP, no or low tillage and

mulching.

Developing new resistant cultivars

There is an urgent need to develop new tea cultivars todeal with climate change. These

will need high tolerance to heat, cold and drought stress,high resistance to pests and

diseases, high nitrogen nutrient use efficiency and highnet photosynthesis, especially

in response to higher CO 2 concentrations. For example,tea cultivars with characteristics

of deep-growing roots and high metabolite content (e.g.amino acids and sugars) have

proved highly resistant to drought (Thiep et al., 2015;Nyarukowa et al., 2016).

Improving soil and water conservation capacity

Climate change has a negative effect on the basic elementsof food production, such

as soil, water and biodiversity. Most tea fields arelocated on the rain-fed slopes of

mountainous areas in which tea yields depend not only onthe amount of rainfall, but also

on its utilisation. With increasing temperatures and highevaporation and transpiration,

drought will probably be a normal phenomenon in the comingyears. Therefore, to

develop soil and water conservation measures, increasingsoil water holding capacity will

be very important in reducing the impact of drought andmaintaining tea production.

Besides establishing contour terraces, mulching, plantingcover crops and installing

contour-staggered trenches, ecosystem diversity and water

conservation agents should

be considered for further research and development.Adopting small-scale irrigation in tea

fields will also increase resilience to drought.

Improving integrated tea production ecosystems

Tea is a perennial, mono-cultured crop and tea farmecosystems are generally simple.

Creating more diverse tea agro-ecosystems could maximiseadaptation to hot weather,

flooding or drought and increased CO 2 . Proper shademanagement and eco-agricultural

models, such as pig raising – biogas slurry – tea, and tea– green manure – animal husbandry

– biogas slurry – tea should be further studied andpromoted. Conservation agriculture,

precision agriculture, organic agriculture and othersustainable farming systems should all

be integrated in adaptation to climate change.

6.3 Community involvement and technology extension

Smallholder tea farms account for 60% of tea productionglobally and are more vulnerable

to the impacts of climate change than larger farms. Inaddition to the development of

new technologies and techniques, awareness of climatechange and its impact, public

education, information exchange, indigenous andcommunity-based adaptation strategies

and the extension of new technology, crop insurance shouldbe promoted to deal with

climate change and extremes.

Creating awareness of climate change and its impact

Public awareness of climate change and its impact on thetea industry is one of the key

strategies in implementing effective participatory climateadaptation on a large scale.

Public access to information on climate change and itseffects, especially those specific to

the region of target audiences, should be made available inlocal communities.

Extending mitigation and adaptation measures

Climate change mitigation and adaptation measures should beextended immediately,

especially in the most vulnerable areas and in keytea-producing areas. Indigenous and

community-based measures should be encouraged. New andintegrated adaptation

measures should be tested or evaluated locally beforeextension on a large scale.

Community and individual participations in national orregional adaptation planning

processes should be encouraged. Technical and financialsupport, scientific and managerial

personnel training and other capacity-building programmesshould be strengthened in

local communities.

Common mitigation and adaptation measures comprise theplanting of drought

tolerant cultivars, increased application of organicfertilisers, adoption of irrigation and

water harvesting techniques, mulching, inter-cropping forhigher diversity and balanced

ecosystems, better infrastructure and early warning andmonitoring systems. Farmers

located in areas which are likely to become unsuitable for

tea cultivation in the future

need to identify alternative crops. The use of local orindigenous/traditional knowledge

is also an important aspect which needs to be revived andpromoted with appropriate

scientifically based evidence.

Developing crop insurance to minimise the risks of

meteorological disasters

Tea producers, especially small holders and low-incomefarmers, should be encouraged

to purchase meteorological disaster insurance to reduce therisks of climate impacts.

Insurance for late spring cold spells is currently sold bythe leading insurance companies in

China and is supported by the government of ZhejiangProvince. The increasing frequency

of extreme weather events, such as drought, and relatedchange in ecosystems such as

pest outbreaks will seriously affect tea production. Moreinsurance schemes should be

developed and promoted.

7 Conclusion

The world has witnessed a significant upsurge in climatechange and it will continue even

in greater pace in the coming decades. Climate change doesnot only mean the rising

temperature and CO 2 elevation; the extreme weatherevents, such as drought, heavy

precipitation and frost, will also be more and morefrequent, which has a huge negative

impact on tea production. Therefore, it is necessary todevelop management strategies

to cope with climate change in order to reduce the risksand promote sustainable

development of the tea industry.

With the increase of temperature, the currently well-knowntea-growing regions

might not be suitable for cultivation and would shift tohigher latitudes and higher

altitude ecosystems, and the tea-growing period can belengthened in subtropical

areas. Tea production would be benefited by temperatureincrease and CO 2 elevation,

but significantly affected by drought, heavy rains, frostsand proliferation of pests

and diseases, and soil degradation. Tea quality would bedeteriorated if the ratio

of free amino acids to polyphenols is unbalanced. Anappropriate plan for climate

change adaptation and mitigation should be developed andextended for sustainable

development of tea industry. The adaption and mitigationstrategies should have three

levels: government policy, R&D for new technologies andtechniques and community

involvement and technology extension, which should beintegrated and implemented

immediately.


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9 Acknowledgements

This work was supported by the Science and TechnologyInnovation Project of the Chinese

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16 Chapter 16 Assessing and reducing theenvironmental impact of tea cultivation

1 Introduction

Environmental issues like climate change affect thesustainability of agricultural crop

production all over the world and the plantation sector isno exception. Currently,

there is a growing demand for environmental awareness andprecautionary measures

to mitigate or reduce the rate of environmentaldegradation, or stop it altogether.

Therefore, it is becoming vital to assess the environmentalimpact of different agricultural

cropping systems. In particular, following the UnitedNations Conference on Sustainable

Development (Rio+20) in 2012, the global community wasurged to follow a sustainable

consumption and production (SCP) strategy. Considering itsimportance, it was named as

the 12th of the sustainable development goals (SDGs) (UNEP,2015).

Tea being the most popular, widely consumed beverage in theworld has become

an important plantation crop in many countries. As aperennial crop occupying a large

proportion of arable land, assessing its environmentalimpact would benefit the economy

of tea growing countries immensely.

The environmental impact of tea can be measured byperforming a life cycle analysis

(Brentrup et al., 2004). The carbon footprint calculationis one step in the life cycle analysis

in tea production systems. The total CO 2 (or its

equivalent) emissions from the ‘flush shoot’

to teacup is quantified with the objective of assessing theenvironmental impact over the

entire production cycle. This should include all the inputsinvolved in the production of tea

shoots including emissions at different stages ofcultivation, manufacturing, packaging,

transportation and consumption. Therefore, theenvironmental impact has to be considered

at different stages of the life cycle. While consideringthe environmental impact of tea,

besides greenhouse gas (GHG) emissions there are severalother issues to be considered

as well. Most important amongst them are depletion ofabiotic resources, land use, global

warming, stratospheric ozone depletion, human toxicity,ecotoxicity, eutrophication and

acidification (Brentrup et al., 2004). This chapterattempts to address the impact on the

environment at different stages of the life cycle of tea,namely nursery, land preparation,

crop establishment, immature tea, mature tea,manufacturing, transportation (including

both the inputs as well as the product), consumption andalso waste disposal. Further,

possible ways and means of reducing such impacts are alsodiscussed with the objective

of reducing the carbon footprint in tea.

2 The environmental impact of tea cultivation

Tea plantations are raised by planting cuttings, seeds orboth. The plants are raised in

a nursery for a period of about one year. In order to starta tea nursery, soil fumigation

is carried out with chemicals to eradicate soil-borne pestsand diseases, mainly the

parasitic nematodes. Methyl bromide was until recently usedto fumigate nursery soil.

However, due to its ozone depleting nature andphyto-toxicity (producing bromide

residues, a groundwater pollutant), its use was bannedfollowing the Montreal Protocol

in 1997. As a result, the search for alternate methods ofsoil fumigation began (Vitarana

et al., 2002).

Tea plantations are established at the expense of naturalforests, resulting in the

destruction of biodiversity and soil (Van der Wal, 2008).The destruction of plant species

leads in turn to the loss of many other species too.Consequently, it has been reported

that the number of both the lion-tailed macaque in Indiaand the Horton Plains slender

loris in Sri Lanka, both of which are on the IUCN’s RedList of endangered species,

declined (Smith, 2010; William, 2011). When establishing atea crop, all the other

vegetation is uprooted and the soil is rehabilitated toprevent soil-borne pest and

disease infestations. During this uprooting of vegetation,the soil is loosened resulting

in significant soil erosion. Also during the cropestablishment period, that is, the first

two to three years, there is a high possibility the landwill be exposed and become

eroded (Van der Wal, 2008). It has been reported that thetea lands in many places had

lost topsoil in the order of 300–450 mm during the past 100years which is equivalent

to 3–4.5 million kg ha −1 of soil loss (Zoysa et al.,2008). Yan et al. (2003) reported

that the direct impact of soil erosion on the environmentcan be on-site as well as

off-site. On-site impacts are a thinning soil layer,deterioration of soil structure and

decreased soil nutrients whereas, the off-site impacts arethe pollution of water bodies.

These eroded topsoils accumulate in water bodies reducingtheir capacities due to

sedimentation. Ultimately, it will lead to flooding and itsconsequential environmental

hazards. The eroded fertile topsoil also causes algal bloomand eutrophication of

water bodies which disturb the aquatic biota. Further, itwill be necessary to apply

more and more fertilizers ultimately increasing energyconsumption and changes in

adaptability of land use (Yan et al., 2003). According toShcherbak et al. (2014) the

emission response to increasing nitrogen fertilizer inputis exponential. Therefore,

global warming will also be accelerated causing more harmto the environment than

expected.

Unlike most other perennials, tea is harvested at seven- toten-day intervals throughout

its lifespan. Therefore, it is necessary to replenish thedepleting nutrients continuously

to avoid any economic loss. These intensive cultivationpractices necessitate the use of

synthetic fertilizers and chemicals. The monoculture natureof tea plantations aggravates

this issue as they are usually lacking natural enemies andheavily dependent on chemicals

to protect the tea bushes and to achieve higherproductivity (Van der Wal, 2008). The use

of such chemicals may cause other environmental hazardssuch as global warming, soil

erosion and eutrophication. However, studies conducted onthose lines are rare. Instead,

many studies have been conducted on the positive impacts oftea cultivation such as

carbon sequestration in different countries and regions(Kamau et al., 2008; Wijeratne

et al., 2014, 2015).

In the tea manufacturing process, old machines are commonlyused which consume a lot

of energy. The use of such machines increases the carbonfootprint of tea immensely. The

conventional dryers used in Darjeeling tea (Doublet andJungbluth, 2010) are one such

example. It has been reported that withering, drying,grading and packing tea requires

4–18 kWh per kg of processed tea, in comparison to the needfor 6.3 kWh per kg of steel.

Also, it has been reported that in some parts of EastAfrica, where power is expensive and

unreliable, many tea factories use standby dieselgenerators which have high polluting

potential (Van der Wal, 2008).

If fuel wood is transported from distant places to teafactories, the carbon footprint

will be increased due to the use of fossil fuel fortransportation. Furthermore, if the fuel

wood is not dried properly before using them in furnaces,the carbon footprint would

again be increased as the burning becomes inefficient.Similarly, if the processed tea is

transported far away for marketing, the use of fossil fuelincreases again causing a greater

environmental impact. In countries where the totalproduction is consumed locally within

the country itself the environmental impact will be lowercompared to countries which

export their products due to long-distance freightscreating a greater carbon footprint.

However, as explained by Doublet and Jungbluth (2010) thecontribution of transportation

activities to the carbon footprint compared to thecultivation and preparation is much less

and will not significantly affect the carbon footprint oftea.

A calculation of the carbon footprint has been developedfor tea plantations in India and

Kenya and these were used in tea promotion campaigns.Several companies are currently

practising this strategy, trying to use these values as amarketing tool. But the availability

of research papers on the same in peer-reviewed journals isstill scarce. According to

a study done by Nigel Melican as mentioned by Sauer (2009)the carbon footprint of

tea varies from 200 g CO 2 per cup to −6 g CO 2 per cupdepending on how it is grown,

processed, shipped, packed, brewed and discarded. Also, itwas mentioned that loose

leaf tea would have an average of 20 g CO 2 per cupwhereas, that of a teabag would be

ten times higher due to the use of carbon-intensivepackaging.

According to Munasinghe et al. (2013), boiling water toprepare tea is one of the highest

energy consuming activities affecting the carbon footprintof tea which is in agreement

with the findings of Azapagic (2013) and Doublet andJungbluth (2010). In fact, around

70% of the total impact results from the use of electricityfor boiling water to prepare tea.

According to the results of Nigel Melican as mentioned bySauer (2009) when electricity

is used for boiling water, energy is wasted at manydifferent stages such as at the point of

electricity generation, grid losses along the wires,transformer losses as voltage is stepped

up and down and finally through heating water in thekettles. All these wastages account

for the increase in carbon footprints and the consequentialenvironmental impact of tea.

Finally, if the remaining consumed tea is discardedproperly it provides a good opportunity

to reduce the carbon footprint through recycling.

3 Making tea cultivation more sustainable

As a result of an increased desire to minimizeenvironmental impacts, many environmentally

friendly alternatives are being tested for methyl bromideincluding soil solarization, the use

of metham sodium, dazomet and different organicformulations in different concentrations.

Sri Lanka has been recorded as the first tea growingcountry which has totally phased out the

use of methyl bromide and thereby earned the ozone-friendlylabel (Gunawardena, 2011).

Soil rehabilitation is a common practice before replantingtea in all tea growing

countries. This is done to recondition the soil. Due to themonoculture and perennial

nature of the crop, the tea lands are depleted of nutrientsand there is a high possibility of

building up soil-borne pests and disease causing organisms.Use of mana (Cymbopogon

confertiflorum) prior to replanting is a good way ofcontrolling nematode infestation

without using chemicals. It also increases the organiccontent of the soil. The addition of

compost, cover crops or mulching will also help to increasethe organic matter in the soil

immensely.

High doses of synthetic fertilizers are conventionallyapplied to obtain better yields and

tea growth. However, it has been reported that the sameresults could be obtained using

low levels of nitrogen (N) and phosphorus (P) together withmicrobial inoculants compared

to the use of a chemical fertilizer alone or the inoculantalone (Nepolean et al., 2012; Saikia

et al., 2011). According to recent studies on biofilmbiofertilizers (BFBF), it is possible to

reduce the use of recommended chemical fertilizer by 50% atthe nursery stage when it

is applied with BFBF (De Silva et al., 2014). Also, it hasbeen found that the use of site

specific phosphorus-solubilizing bacteria allows the growerto reduce the use of fertilizer

by one-third which will ultimately reduce the use ofsynthetic fertilizers (Thennakoon

et al., 2016). Site-specific fertilizer recommendationsprevent the use of chemical

fertilizers unnecessarily without compromising the cropyield. It replenishes the soils with

only the depleted nutrients, thereby reducing fertilizerwastage which would ultimately

reduce eutrophication and algal bloom. Similarly,slow-release fertilizers release nutrients

according to the requirements of the plants, thus reducingenvironmental pollution.

Improving the organic content of the soil will also improvethe fertilizer use efficiency

while reducing wastage. Organic cultivation also increasesthe soil carbon pool, thereby

reducing the atmospheric CO 2 concentration and mitigatingclimate change (Cracknell

and Njoroge, 2014). Furthermore, the addition of organicinputs increases the efficient use

of fertilizers and reduces indirect emissions associatedwith fertilizer production. Also, it

leads to reduced soil erosion, another GHG emission source,by binding the soil particles

together.

Well-managed tea produces good canopy cover whichultimately protects the land by

reducing splash erosion. However, during the cropestablishment period it is necessary to

apply thatching materials to cover the soil which willreduce the evaporation of water and

splash erosion. Development of contour drains with lock andspills and leader drains will

drain excess water efficiently from the tea lands withouteroding the soil.

Where tea is cultivated with shade trees, a lot ofenvironmentally positive impacts can

be achieved while achieving economic benefits throughimproved yields. They help to

improve the micro-climate surrounding the tea bushes whichwill ultimately improve the

physiology of tea and help to utilize the inputsefficiently resulting in lower wastage. They

prevent direct contact of rainwater on the ground andreduce the speed of rainwater

resulting in less soil erosion. The addition of shade treelopping into the tea plantations

will increase the organic carbon content (OC) of the soilwhich will ultimately reduce

the quantity of applied fertilizer as well as its wastage.It will also help to improve the

microbial activity in the soil. Shade trees provide nestingand resting places for birds and

other fauna which will act as natural enemies for the pestand disease causing organisms.

Furthermore, it has been observed that tea plantationswhich comprise tea and shade

trees have comparable carbon sequestration potentials tothe reported values of agro

forestry systems (Wijeratne, 2015; Wijeratne et al.,2014a,b,c). This carbon sequestration

potential of tea plantations will be useful in reducing thecarbon footprint of tea. Also,

the addition of different types of shade trees and windbreaks will enhance biodiversity

in the tea plantations and together with their lopping willprovide a better source of

renewable energy. It is necessary to utilize the landswhich are unsuitable for growing tea

to grow fast-growing tree species which can act more as CO2 scrubbers as well as generate

renewable sources of fuel wood for the tea manufacturingprocess. This will reduce energy

wastage through transportation too. Tea plantations whichare accredited by the Rain

Forest Alliance have less environmental impact compared tonon-accredited ones as they

avoid the use of synthetic inputs for at least a 5-mdistance along the roadsides.

If hydropower is used to generate electricity, theenvironmental impact could be

reduced compared to electricity generated using coal orfossil oils. Electricity wastage in

the tea manufacturing process can be addressed by usingon-site electricity generation

techniques such as mini hydropower stations. In that waythe energy wastage from electricity

generation to consumption as described earlier could bereduced enormously. However,

the electricity wastage reduction and its environmentalimpacts using mini hydropower

needs to be balanced against the resultant environmentalimpact on the spray zone and

downstream vegetation. It has been reported that the uniqueforest vegetation present in

the spray zone of waterfalls are badly affected and facethe threat of extinction due to the

construction of mini hydropower stations (Silva and Silva,

2016).

New energy efficient technologies should be adopted ratherthan using the old and

inefficient methods of tea manufacture. The use ofenergy-saving stoves, energy-efficient

boilers and the replacement of factory lights with LEDbulbs are some examples. Fluidized

bed drying where hot water is blown directly into the dryerand the process of fluidization

moves the tea leaves can be adopted instead of conventionaltea drying as it saves a lot

of energy, in fact almost half the amount (Doublet andJungbluth, 2010). Obtaining fuel

wood for factory production from sustainably managedforests that do not reduce the area

or density of natural forest cover and drying them well toabout 20% moisture content

ensures optimum calorific value (Cracknell and Njoroge,2014).

Also, production of tea in bulk should be promoted as faras possible as it has a much

lower environmental impact compared to teabags. When it isnecessary to make teabags,

recyclable materials should be used. Bulk tea can also bepacked using reusable containers

or recyclable paper.

As the highest energy use in the life cycle of tea is atthe consumption stage, alternate

tea which uses cold water instead of boiling water willhelp to reduce the carbon footprint

immensely. The production of cold-water-soluble instantblack tea (Perera et al., 2016) is

a good alternative. Also reducing the amount of boiling

water and using energy efficient

kettles are the other options to reduce the carbonfootprint of tea. Further, using gas

instead of electricity to boil the water would help toreduce the carbon footprint of tea as

then there will only be one step of energy loss that is,from burning fossil fuel to increasing

the water temperature (Sauer, 2009). The carbon footprintof tea can be further reduced

by heating water in a closed vessel as then the evaporativeloss of water is prevented

(Munasinghe et al., 2013).

At the time of disposal of used tea, it would be better ifit was composted rather than just

thrown away. Also, waste tea can be used to remove badodours from the household or

in beauty cultural activities. The cardboard and plasticwrappings should be recycled and/

or reused without being dumped in landfills. In addition,the tea factory wastes should be

disposed of properly by separating the solids fromwastewater and composting, reducing

the pathogens from the wastewater and the dissolvedchemical/nutrients to acceptable

levels, and aerating the wastewater to reduce CH 4emission. Constructed wetlands and

gravel bed hydroponics can be used to treat the wastewater(Cracknell and Njoroge,

2014).

4 Case studies: carbon sequestration and production

As it has been identified that the CO 2 emissions at thecultivation stage are one of the major

contributors to the increase in the carbon footprint oftea, the following two case studies

were conducted to discover the potential of carbonsequestration and CO 2 emissions in

major tea growing regions of Sri Lanka.

4.1 Measurement of carbon sequestration potential of teaplantations in Sri Lanka

Carbon sequestration is a concept which has gainedattention as a feasible and cost

effective option in mitigating climate change. Theprincipal objective of the present work

was to estimate the carbon sequestration potential of SriLankan tea plantations using

experimental fieldwork. There, the actual carbonsequestration of tea in different tea

growing regions of Sri Lanka, namely Low-country (LC),Mid-country (MC), Up-country

(UC) and Uva, was estimated using the stock differencemethod of the Inter-governmental

Panel of Climate Change. Standing biomass and carbonconcentrations of different

parts of seedling and VP (TRI2025) tea plants were measuredby destructive sampling in

selected tea plantations with similar management using theoven dry method and Walkley

and Black method, respectively. Sampling on two occasionswith a considerable interval

between the two sampling enabled estimation of carbonsequestration rates based on the

rate of average net carbon storage in biomass. Biomass andcarbon gain of selected high

and medium shade tree species were also estimated usingallometric equations.

Estimations from the experimental fieldwork showed thatseedling tea plants are superior

to VP tea plants in sequestering carbon. The differences inpercentage distribution of total

biomass within the tea bush in seedling and VP tea wereidentified as the major reasons

for this observation.

The addition of shade trees in tea plantations increasedits carbon sequestration potential

substantially. Tea plantations with high and medium shadetrees in LC, MC, UC and Uva

had carbon sequestration potentials of 6.7, 3.5, 2.3 and5.1 Mg of C ha −1 yr −1 , respectively

(Fig. 1). These values were comparable with the reportedcarbon sequestration values of

smallholder agro-forestry systems and mesic savannas butlower than those for tropical

rainforests. This study further emphasized the need toestablish and manage shade trees

in tea plantations not only to enhance yields, but also toensure better environmental

resilience. Therefore, based on management, extent ofcultivation and perennial nature,

the carbon sequestration potential of tea plantations inSri Lanka is significant in mitigating

climate change. Also, possibilities should be explored forsecuring payments for carbon

sequestration by tea plantations to compensate for economiclosses due to possible yield

reductions as a result of climate change, especially inwarmer regions.

4.2 Determination of soil respiration in UC and LC teagrowing regions of Sri Lanka

Soil respiration (SR) is defined as total CO 2 productionin soils resulting from the respiration

of soil organisms and roots. SR is an important function interrestrial ecosystems as it

contributes to global carbon cycling and climate change.Determining SR values or

the CO 2 efflux is important in this context. The totalglobal emission of CO 2 from soil is

recognized as one of the largest fluxes in the globalcarbon cycle and small changes in

the magnitude of SR could have a significant effect on theconcentration of CO 2 in the

atmosphere. As tea (Camellia sinensis (L) O. Kuntz) is oneof the most important plantation

crops grown in a large proportion of Sri Lanka, it is ofthe utmost importance to get an

idea about CO 2 emissions from tea lands to remain as aprofitable industry. Therefore,

this study was carried out to discover the CO 2 emissionsin UC and LC tea lands, the two

extremes of the tea growing regions in Sri Lanka duringboth wet and dry seasons. The soil

samples were collected along the active root zone depth ofthe tea plants, that is, up to a

depth of 0–45 cm from experimental sites using a stratifiedrandom sampling technique

taking the tea growing region as the main strata. Soilmoisture content was measured

gravimetrically and OC was measured using the Walkley–Blackmethod in parallel with 0 1000 2000 3000 4000 5000 60007000 LC MC UC UVA C a r b o n s e q u e s t r a t i o n p ot e n t i a l ( k g o f C h a – 1 y r – 1 ) Tea GrowingRegion 100%tea+100%HS+100%MS 100%tea+50%HS+50%MS100%tea+0%HS+0%MS

Figure 1 Carbon sequestration potential of tea plantations

of Sri Lanka with different densities of high

shade (HS) and medium shade (MS) trees in different teagrowing regions of Sri Lanka (Low-country

(LC), Mid-country (MC), Up-country (UC) and Uva).

SR measurements using the Anderson method. Twelvereplicates were taken for each

treatment. The relationships of SR with the soil moisturecontent and OC were developed

using regression analysis. Data was analysed using SASsoftware package version 9.1 and

the results are given in Fig. 2.

Accordingly, SR was 4.07 mg C m −2 h −1 and 2.16 mg C m−2 h −1 in UC and LC tea lands,

respectively. SR was significantly higher in wet conditionsthan dry conditions too. The

relationship between SR and soil moisture was highlysignificant and positive compared to

the OC which was complex and negligible.

5 Summary and future trends

Table 1 presents a brief summary of the major environmentalimpacts of tea that have

been discussed earlier in this chapter and possiblemitigation measures at different stages

of tea production for easy reference.

Environment is a priority area today where climate changeoccurs as a result of global

warming. Most of the time anthropogenic activities are themain cause of global warming.

The natural environment is altered for the well-being ofhumans and the consequences are

suffered by all living beings. As a result, the value ofthe environment has been recognized

by the global community and seventeen SDGs have beenidentified (United Nations

Department of Public Information, 2016).

Due to growing awareness of the impact of global climatechange, it is necessary to

consider the environmental impact of each and every productbeing traded. This becomes

mandatory, especially with the responsible or SCP conceptwhich is the 12th SDG (United

Nations Department of Public Information, 2016). Munasingheet al. (2013) explained

that businesses are carefully inspected for theenvironmental impact of their products

and there is a growing trend for customers to purchase moreenvironmentally friendly

products. Therefore, unless these trends are addressed bythe tea industry there will be

no sustainable future for it.

Insufficient research has been conducted into reformingpolicies which address the

global environmental impact of tea. It is high time tocommence such research and

Figure 2 Variation of soil respiration in Up-country (UC)and Low-country (LC) tea growing regions

(means with the same letter are not significantly differentat α = 0.05).

reduce its environmental impact while unveiling thepotential to benefit those who follow

environmentally sustainable tea production.


As this is a comparatively new trend in the world, theavailability of peer-reviewed

articles with high scientific reputation is rare. However,most of the basic details can be

found on the internet. Brentrup et al. (2004) clearlyexplained the theoretical concept of

environmental impact assessment methodology foragricultural systems. Therefore, it has

become possible to conduct new research to assess theenvironmental impact of different

products on the market and make informed decisions inpurchasing the correct product.

Also, the websites of the Inter-governmental Panel onClimate Change, United Nations

Framework Convention on Climate Change, United NationsEnvironment Programme,

Rainforest Alliance and the Ethical Tea Partnership willprovide a fair amount of detail.

Azapagic, A. (2013). Life cycle assessment of tea producedin Kenya. In National Multi-Stakeholder Workshop held on29–30 April 2013 at Naivasha. Retrieved fromwww.fao.org/fileadmin/.../FAO_Uni_Manchester_AZAPAGIC_LCA_of_tea.pdf (Last accessed15 November 2016).

Brentrup, F., Kusters, J., Kuhlmann, H. and Lammel, J.(2004). Environmental impact assessment of agriculturalproduction systems using the life cycle assessmentmethodology I. Theoritical

Table 1 Summary of environmental impacts of tea andpossible mitigation measures at different

stages of tea industry

Stage Environmental impact Possible mitigation measures

Cultivation O 3 layer depletion Use of alternative methodsfor soil fumigation GHG emission Rational use offertilizers in combination with organic, BFBF, inoculantsand SSFR Destruction of biodiversity Addition of shadetrees, wind breaks and green manure crops Soil erosionDevelopment of well-planned drains system, increase of

organic matter content, cover crops, live mulches etc.

Manufacturing GHG emission Use of energy-efficientmachinery Combustion of fuel wood Use of sustainablymanaged well-dried fuel wood, energy-efficient machineryetc. Environmental pollution due to wastewater Separatingand treating wastewater before releasing into theenvironment, constructed wetlands and gravel bedhydroponics

Consumption GHG emission Use of energy-efficient kettles,boiling only the required amount of water, use of coldwater soluble tea concept of a LCA method tailored to cropproduction. European Journal of Agronomy, 20:247–64.

Cracknell, R. and Njoroge, B. (2014). Mitigating ClimateChange in the Tea Sector. International Trade Centre,Geneva, p. 102.

De Silva, M. S. D. L., Jayasekera, A. P. D. A.,Seneviratne, G., Abeysekera, U. P., Premathunge, E. W. T.P. and Wijesekera, S. N. (2014). Soil fertility improvementthrough Biofilmed Biofertilizers: Potential for fieldapplication in tea cultivation. In A. P. Keerthipala (Ed.)Proceedings of the fifth symposium on Plantation CropResearch – ‘Towards a Green Plantation Economy’. SugarcaneResearch Institute, Uda Walawe, 70190, Sri Lanka, pp.229–36.

Doublet, G. and Jungbluth, N. (2010). Life cycle assessmentof drinking Darjeeling tea, conventional and organicDarjeeling tea. Practical training report. ESU-servicesLtd., Uster. Retrieved from

Gunawardena, N. (2011). Ozone friendly pure Ceylon tea.Retrieved from http://www.businesstoday.lk/article.php?article=3453 (Last accessed 11 November2016).

Munasinghe, M., Deraniyagala, Y. and Dasanayake, N. (2013).Economic, social and environmental impacts and overallsustainability of the tea manufacturing industry in SriLanka. SCI Research Study Report. Munasinghe Institute fordevelopment, Colombo, Sri Lanka.

Nepolean, P., Jayanthi, R., Pallavi, R. V., Balamurugan,A., Kuberan, T., Beulah, T. and Premakumar, R. (2012).Role of biofertilizers in increasing tea productivity.Asian Pacific Journal of Tropical Biomedicine, S1443–5.

Perera, G. A. A. R., Amarakoon, A. M. T., Illeperuma, D. C.K. and Edirisinghe, E. N. U. (2016). Applying membranefiltration technique in manufacturing cold water solubleinstant black tea. In V. R. M. Vidhanaarachchi, H. M. I.K. Herath, M. K. Meegahakumbura, A. D. N. T. Kumara and M.K. F. Nadheesha (Eds) Proceedings of sixth symposium onPlantation Crop Research – ‘Plantation Agriculture towardsNational Prosperity’. Coconut Research Institute, Lunuwila,Sri Lanka, 2, pp. 27–41.

Saikia, D. N., Sarma, J., Dutta, P. K. and Baruah, D. K.(2011). Sustainable productivity of tea throughconservation of bio-mass, addition of bio-fertilizers andreduction of inorganic fertilizer. Two and a Bud, 58:109–17.

Sauer, J. L. (2009). Tea’s carbon footprint. Retrieved fromhttp://www.samovartea.com/teas-carbonfootprint/(Last citedon 16 November 2016).

Shcherbak, I., Millar, N. and Robertson, G. P. (2014).Global metaanalysis of the nonlinear response of soilnitrous oxide (N2O) emissions to fertilizer nitrogen.Proceedings of the National Academy of Sciences of theUnited States of America (PNAS), 111(25): 9199–204.

Silva, E. I. I. and Silva, E. N. S. (2016). Mini-hydro, aninjurious novel threat to highland forest ecosystems ofSri Lanka. In Proceedings of the International ResearchSymposium 2016. Ministry of Mahaweli Development andEnvironment, Sri Lanka, p. 47.

Smith, L. (2010). Found: Sri Lankan primate thought to beextinct for 60 years. The Guardian. Retrieved from

Tennakoon, P. L. K., Rajapaksha, R. M. C. P. andHettiarachchi, L. S. K. (2016). Potentials of plant growthpromoting rhizobacteria based microbial inoculants fornutrient management in tea (Camellia sinensis (L.) O.Kuntze). In V. R. M. Vidhanaarachchi, H. M. I. K. Herath,M. K. Meegahakumbura, A. D. N. T. Kumara and M. K. F.Nadheesha (Eds) Proceedings of sixth symposium onPlantation Cro Research – ‘Plantation Agriculture towardsNational Prosperity’. Coconut Research Institute,Lunuwila, Sri Lanka, 1, pp. 149–61.

United Nations Department of Public Information (2016).Sustainable Development Goals; Guidelines for the use ofthe SDG logo, including the colour wheel and seventeenicons. Retrieved from

United Nations Environment Programme (2015). Sustainableconsumption and production indicators for the future SDGs.UNEP Discussion paper, March 2015.

Van der Wal, S. (2008). Sustainability issues in the teasector; a comprehensive analysis of six leading producingcountries. Centre for Research on MultinationalCorporations (SOMO), Amsterdam, The Netherlands. Retrievedfrom https://papers.ssrn.com/sol3/papers.cfm?abstract_id=1660434 (Last cited on 15 March 2017).

Vitarana, S. I., Liyanage, D. D., Herath, U. B., Navaratne,N., Jayaratne, U. P., Udamulla, G. P., Prematunga, A. K.and Jayawickrama, A. K. (2002). Chemical and non-chemicaldisinfestation of tea soils. The Tea Research Institute ofSri Lanka, Talawakelle, Sri Lanka.

Wijeratne, T. L., De Costa, W. A. J. M. and Wijeratne, M.A. (2014a). Carbon sequestration potential of teaplantations of Sri Lanka. Proceedings of the 228thExperiment and Extension Forum of the Tea ResearchInstitute of Sri Lanka, Colombo, Sri Lanka. 31 January2014.

Wijeratne, T. L., De Costa, W. A. J. M. and Wijeratne, M.A. (2014b). Carbon sequestration potential of teaplantations in Sri Lanka as an option for mitigatingclimate change; a Step towards a greener economy. In A. P.Keerthipala (Ed.) Proceedings of the 5th Symposium onPlantation Crop Research – ‘Towards a green PlantationEconomy’. Sugarcane Research Institute, Uda Walawe, 70190,Sri Lanka, pp. 205–12.

Wijeratne, T. L., De Costa, W. A. J. M. and Wijeratne, M.A. (2014c). Carbon Sequestration: an underexploitedenvironmental benefit of tea plantations in Sri Lanka. TeaBulletin, 23(1&2): 1–5.

Wijeratne, T. L. (2015). Estimation of carbon sequestrationby Sri Lankan tea plantations and its variation withfuture climate change. PhD thesis. Postgraduate Instituteof Agriculture, University of Peradeniya, Sri Lanka.

Wijeratne, T., De Costa, J. and Wijeratne, M. (2015).Carbon sequestration of tea plantations as an adaptationfor climate change. Abstract presented at the 4thInternational Conference on Climate Change Adaptation 2015held in Colombo Sri Lanka from 22–23 November 2015, p. 41.

William, M. (2011). Special report on environmental damageand human rights abuses blight global tea sector.Ecologist. Retrieved fromhttp://www.theecologist.org/News/news_analysis/847970/

Yan, Z., Hong, A., Bu-zhuo, P and Hao, Y. (2003). Soilerosion and its impacts on environment in Yixing teaplantations of Jiangsu province. Chinese GeographicalScience, 13 (2): 142–8.

Zoysa, A. K. N., Anandacoomaraswamy, A. and De Silva, M. S.D. L. (2008). Management of soil fertility in tea lands.In A. K. N. Zoysa (Ed.) Handbook on Tea, Tea ResearchInstitute of Sri Lanka, Talawakelle, Sri Lanka, pp. 16–33.

17 Chapter 17 Cultivation, production andmarketing of organic tea

1 Introduction

Tea qualifies as organic only when it achieves an overallproduction system that promotes

environmentally and socially sound, economically viablemethods that are based on

recycling natural resources, maintaining biodiversitywithout any synthetic inputs. The

number of organic tea producers and volume of organic teatraded in the world market

have increased substantially over the last few years. Thisdevelopment can be explained

by a number of factors. In the first place, tea growershave become more aware of

environmental problems such as erosion and contamination ofsoil and water sources, as

well as severe health hazards connected with an intensivesystem of tea production. Tea is

generally grown in ecologically fragile hilly tracts.Adopting organic farming would protect

the environment and prevent contamination of rivers andstreams originating from these

hills. A further reason for the rise in organic tea can beexplained by the fact that demand

for organic tea has grown steadily due to increasedconsumer awareness of pesticide

residues and presence of heavy metals in conventional teas.

Organic tea cultivation sustains the health of soil,ecosystems and people. It is a system

that relies on enhancing the soil’s natural fertility usingorganic manures (composts)

biofertilizers, green manures, mulching and bio-pesticides.

In the organic field, tea is

produced in the absence of synthesized chemicals likepesticides, fungicides, herbicides

and growth regulators; sewage sludge; genetically modifiedorganisms; ionizing radiation;

and concentrated fertilizers. Non-synthetic/naturallyoccurring (manufactured and/or mined)

products, bulky organic manures (farmyard manure, compost,green manures etc.) and

concentrated manures (oil cakes, blood meal, fish manureetc.) are used. Pests and diseases

are controlled by regulation of the micro-climate,introduction of biological control agents

and/or the use of biological products naturally extractedwithout the use of inorganic solvents.

1.1 Organic and inorganic tea – biochemical comparison

Many comparison studies reveal that the contents ofminerals, vitamins, proteins and

carbohydrates in organic food are similar to those inconventional food, but the amount

of defence-related secondary metabolites may be greater inorganic food (Brand and

Mølgaard, 2001). Tea is a product where secondarymetabolites such as polyphenols are

quality parameters. Also, there is a belief thatorganically grown teas are better quality

due to the lack of synthetic inputs. Is organic teahealthier? Such information is important

in promoting the production and consumption of organic tea.Han and Yan (2014) while

conducting an experiment in six gardens, having bothorganic and conventional tea

located in the Zhejiang and Fujian provinces, eastern

China, reported that organic tea had

significantly higher polyphenols, includingepigallocatechin gallate (EGCG) epicatechin

gallate (ECG) epigallocatechin(EGC) and water extracts,than those found in the

conventional tea. The concentrations of prolineу-aminobutyric acid were also significantly

higher in organic tea. It was further concluded thatorganic tea has greater health benefits

and environmental adaptability than conventional tea. InDarjeeling, India, Kumar et al.

(2012) observed that biosynthesis of polyphenols, prolineand lipids was greatly influenced

by organic cultivation and their total content wasrelatively higher in organic than in

inorganic fields located at different locations andaltitudes. Bagchi et al. (2015) reported

that the content of secondary phenolic compounds like totalphenolics, gallocatechin

gallate (GCG), EGCG and ECG was higher in organic plotsthan in conventional plots.

An increase in the polyphenol content of organically grownfield crops has also been

confirmed in previous studies (Daniel et al., 1999; Nicolaset al., 1994; Johansson et al.,

2014). A discussion on cultivation practices for theproduction of organic tea, producing

countries, yield trends, market development and trade inorganic tea follows.

2 Establishing and maintaining a new organic tea

plantation

2.1 Selection of site

When establishing a new organic tea plantation the designof the farm is crucial for optimum

utilization of resources within the plantation itself. Thefield needs to be sufficiently isolated

to ensure there is no possibility of any pollutants orcontaminants flowing or drifting into

the field from adjacent areas. The minimum depth of soilprofile should be 1.5–2.0 metres

and organic matter status should be medium to high leveldepending on the elevation

and rainfall of the area. A perennial source of water freefrom pollutants is essential in the

field for large-scale compost preparation when cultivatingorganic tea. A detailed, fully

documented history over a period of about ten years, togive details of external inputs

during the pre-conversion period, should be maintained tofacilitate inspection of organic

tea cultivation.

2.2 Conversion from conventional to organic cultivation

The transition to organic tea production will have economicand environmental impacts on

tea growers in particular and on society as a whole (Tran,2009). For an existing plantation,

the minimum conversion period should be three years fromthe last usage of synthetic

agrochemicals in the field.

In the case of cultivation on virgin land and where recordsare available that no chemicals

were applied previously, the conversion period can berelaxed by the accredited certifying

agencies.

An analysis of the experiences of tea planters who haveconverted their conventional

tea estates to organic systems indicates that the mainproblems encountered during the

conversion process include a reduction in yield,infestation of pests, unexpectedly high

workloads in peak periods and financial difficulties (GhoshHajra, 2006). Planning the

conversion entails being aware of the problems which canoccur and attempting to avoid

them by advance preparation.

The first stage is an assessment of the motives behind theconversion and whether they

form a suitable basis for a successful conversion, togetherwith an analysis of the current

situation on the estate, including size and layout of theestate, soil texture and nutrient

status, manure handling systems, livestock availability,rainfall, workers and capital. During

conversion additional labourers are required for trenching,compost preparation, weed

control, application of bulky and concentrated organicmanures and shade regulation.

Other costs like planting of trees, grasses, Citronella,etc., application of manure, organic

pesticides and biofertilizers depend upon the extent towhich integrated farming can be

practised. For tea, where chemical fertilizers are appliedat a few hundred kg per hectare,

compost and cakes are required in tons per hectare,elevating the labour costs. When

the labour cost can be as high as 60% of the totalproduction cost, it can pose a serious

financial risk.

The second part involves the development of a targetorganic system and a plan for

the transition between the current conventional system andthe organic endpoint (Lamkin,

2002). Technical questions, such as a measure for soilimprovement, direct weed, insect

pests and disease control techniques, appropriatecultivation practices and suitable

manure management systems are not very important at thisstage of the planning process,

although they do need to be considered before theconversion process gets underway.

Capital investment towards the purchase of livestock (ifnot sufficiently available in

the estate) improved livestock housing, feeding and manurehandling systems as well

as facilities for processing, packaging and marketing ofprocessed tea might also be

necessary.

Tea can be marketed as ‘organic tea’ only after thecompletion of three years’ post

conversion. Sometimes during conversion the accreditedcertifying body allows tea to

be labelled and sold as ‘organic in conversion’ tea, it canat best fetch prices only slightly

higher than conventionally produced tea.

3 Maintenance of new and converted organic

plantations

3.1 Boundaries and buffers

Organic fields must have distinct, defined boundaries andbuffer zones (100 metres or

more) between organic and conventional fields on all sidesof the field to prevent contact

with prohibited substances applied to adjoining land notunder organic management.

3.2 Terraces

The construction of terraces in sloping land is essentialto minimize the rapid loss of surface

soil. Terraces should follow the contour of the slope andthe upper surface of the terrace

may slope slightly towards the hillside to prevent soilwash.

3.3 Drains

A high water table resulted in frequent waterlogging insome tea fields. Waterlogged

plants exhibit certain symptoms such as scanty shootproduction, yellowing of leaves and

premature defoliation, dieback in plucking points, poorrecovery after pruning, infection

by violet root rot, red rust diseases and loss of vigour.To cope with this situation it has

become necessary to provide artificial drainage facilitiesto drain excess water below the

root zone. Drainage is one of the most effective means ofincreasing tea productivity.

Provision of an efficient drainage system to maintain awater table at a minimum depth of

100 cm is considered adequate for growing tea. Diggingshould be started from outfall

and go upward. In addition to boundary drains, contourdrains must be dug at regular

intervals.

3.4 Soil reaction

The soil pH should be maintained at 4.5–5.5 by applyingagricultural lime or dolomite

once in a pruning cycle and the quantity of liming materialshould be determined on the

basis of soil pH, rainfall and yield of tea. Dolomite alsoserves as a source of magnesium. It

must be mentioned that agricultural lime should not beapplied to land ear-marked for tea

planting; if done, planting should be taken up only afterone year. The rate of application

of dolomite is as follows (see Table 1). Table 1 Rate ofapplication of dolomite to raise the soil pH level pH Rateof dolomite lime application (tons/ha) 4.7 to 4.9 1.0 4.5to 4.6 1.5 4.3 to 4.4 2.0 4.0 to 4.2 2.5 <4.0 3.0

In the case of extremely low pH values (risk of Altoxicity) the use of gypsum (CaSO 4 ) is

also permitted. However, it is advisable to get priorapproval from the accredited certifying

body before application of these materials. In the organictea field, constant soil testing

is mandated and even when the land becomes free ofcontaminants and new planting is

carried out, the soil must be constantly monitored toachieve the level for which organic

certification can be granted.

3.5 Soil organic carbon (SOC)

In the organic field, it is important to maintain anyimprovement in the SOC content to

sustain productivity and support the biological activityand diversity that contribute to

nutrient cycling. SOC is one of the most importantconstituents of the soil due to its

capacity to affect plant growth as both a source of energy

and a trigger for nutrient

availability through mineralization (Saeed et al., 2014).Assessment of soil quality in tea

fields is important to determine the extent of degradationand introduce sustainable

land management practices. In Sri Lanka, Wanniarachchi etal. (2003) observed that land

management has a significant impact on the quantity of soilcarbon in tea plantations.

He further reported that the rehabilitation processes haveincreased the soil carbon

content, nearly 15% of the added carbon is lost during theinitial years after planting tea.

Forest soils had an organic carbon (in the O-15cm soillayer) range of 3.39 to 4.42 kg m 2

compared to 3.02 to 3.18 kg m −2 seen in rehabilitated tealands. In the second year, the tea

field lost over 40% of its SOC after being rehabilitatedand planted with tea. SOC can be

considered a strong indicator of soil quality(Wanniarachchi et al., 2003; Kalita et al., 2016).

3.6 Livestock and animal husbandry

Livestock play a major role in organic agriculture as theintermediary between the

utilization of crop residues or fodder produced at theestate and return of nutrients as

manure. To produce more organic fertilizers, an animal farmunder organic or GAP (Good

Agricultural Practices) management may be established in alarge organic tea estate

or by its neighbours. The animal waste could be processedas an organic fertilizer. For

small organic gardens, an ecological agricultural system

‘pig raising - biogas slurry - tea

garden’ is recommended (Anon., 2014a; Han et al., 2011).Biogas slurry supplies not only

the nutrients, but also water for tea plants. It has beenreported that the biogas slurry

produced from 45 pigs for one hectare garden increasesyield by 18% compared to the

no-manure control plots (Han et al., 2011). The number oflivestock kept on a particular

estate is therefore dependent upon the amount of fodderavailable, or the size of crop

area used to grow fodder on the site itself. Plantingfodder crops (especially legumes) is

particularly helpful for improving the fertility of thesoil. Hedges can be useful not only as

windbreaks and as protection against erosion but also as aconstant source of forage for

cattle. The workers in the organic tea estates areencouraged to raise animals, primarily

cows and poultry.

3.7 Planting materials

For preference, low input responsive clones or seed stockswith broad tolerance to

different stresses should be used as planting materials.Use of genetically modified seeds

or planting materials is prohibited in the organic field.After planting, the field should be

mulched (6 to 8 cm thick) in the absence of a cover crop,but the mulch must not touch

the stem of the young plants. The organic techniques duringand after planting need to

be critically observed to ensure no external chemicals areapplied to make the plants grow

faster or make them stress-free following their planting.

3.8 Manuring

Manuring ensures the availability of those essentialnutrients normally deficient in the soil

at optimum quantities and replacement of nutrients removedby using organic manure

demanded by the tea bushes for sustainable productivity. Itis, therefore, vital for the

organic tea producers to establish a manuring programmeright at the beginning. The

nutrient content of the soil – particularly potassium,phosphorous, magnesium and also

the trace elements – should be analysed regularly.

In the organic tea estates, nutrients are beingsupplemented using well-composted

farmyard manures, biogas slurry, neem cakes, oil cakes,lopping of green manures, shade

tree droppings, etc. Bone meal, fish meal and other organicmanure available from the

unpolluted environment can also be applied. The estimatednutritional values of some

organic waste materials are presented in Table 2. Wood ashmay be applied only in mature

tea fields in the pruned year, preferably in the dryperiods by broadcasting 15 cm away

from the collar. In general, most of the workers in theorganic tea estate are supplied with

a cow or goat per household. The droppings of sheep andgoats contain higher nutrients

than farmyard manure and compost. It is applied to thefield in two ways. The sweepings

from sheep or goat sheds are placed in pits for

decomposition and later applied to the

field; however the nutrients present in the urine arewasted using this method. In Sri

Lanka, organic tea growers are now encouraged to use moregoat dung as it gives the

plants enough copper and zinc(http://www.ipsnews.net/2000/07/development-sri-lanka

organic-farming-revives-barren-land/). The workers areencouraged to grow fodder for

their cows in areas that are unsuitable for tea. Theplantation buys the compost or the

organic manure from the workers.

Han et al. (2013) conducted a systematic comparative studywith conventional tea fields

in the Zhejiang province of eastern China and reported thatsoil pH, organic carbon and

total nitrogen contents were higher in the organic fieldsmainly due to use of higher inputs

of organic fertilizer and in the absence of chemicalfertilizers. The organic carbon content

in organic fields was 7.2% higher on average than theconventional ones. In addition,

the microbial biomass carbon; ninhydrin-nitrogen andbiomass P; the ratios of C mic :C org ,

Nnin mic :N tot , P mic :P tot ; and the net Nmineralization and nitrification rates were significantly

higher in the organic fields in most of the comparativepairs. It has further been reported

that the longer the period of organic management, the moreorganic C and microbial

biomass C were present. However, inorganic N, available Pand K concentrations were

generally lower in organic fields. No significant

differences were found in available Ca,

Mg, Na, Fe, Mn, Cu and Zn concentrations between the twofarming systems. These

findings suggest that organic farming could promote soil Csequestration and microbial

biomass size and activity in tea fields without croprotation, but more N-rich organic

manures, natural P and K fertilizers will be required forsustainable organic tea production

in the long term.

Since processed tea is obtained from harvested tea leaf, nochemical fertilizers are

permitted in the organic tea gardens and application ofhigh nitrogen organic manures is

recommended. A commercial bio-fertilizer with Trichodermaharzianum and other effective

Table 2 Nutrient status of some organic waste materials

Category Source Nutrient content (%) wt/wt Nitrogen (N)Phosphoric acid (P 2 O 5 ) Potash (K 2 O)

Animal waste Cattle dung 0.3–0.4 0.10–0.15 0.15–0.20 Cattleurine 0.80 0.01–0.02 0.5–0.70 Sheep and goat dung (mixed)3.0 1.0 2.0 Night soil 1.2–1.5 0.8 0.5 Human urine 1.0–1.20.1–0.2 0.2–0.3 Leather waste 7.0 0.1 0.2

Compost Farmyard manure 0.5–1.0 0.15–0.20 0.5–0.6 Poultrymanure 2.87 2.90 2.35 Town compost 1.5–2.0 1.0 1.5 Ruralcompost 0.5–1.0 0.2 0.5 Water hyacinth compost 2.0 1.0 2.3Tea waste compost** 3.60 0.60 2.30

Oil cakes Castor 5.5–5.8 1.8 1.0 Coconut 3.0–3.2 1.8 1.7Cotton seed 3.9(6.5)* 1.8(2.8)* 1.6(2.1)* Ground nut4.5(7.8)* 1.7(1.7)* 1.5(1.4)* Karanj (Pongamia pinnata)3.9–4.0 0.9–1.0 1.3 Neem 5.2 1.0 1.4 Niger 4.8 1.8 1.3Mahua (Bassia latifolia) 2.5–2.6 0.8 1.8 Rapeseed 5.1 1.81.0 Linseed 5.5 1.4 1.2 Safflower 4.8(7.8)* 1.4(2.2)*1.2(2.0)* Sesame 6.2 2.0 1.2

Animal meals Blood 10–12 1.2 1.0 Meat 10.5 2.5 0.5 Horn andhoof 13.0 0.3–1.5 – Raw bone 3–4 20–25 − Steamed bone 1–2

25–30 − Fish 4–10 3–9 1.8 (Continued)

Category Source Nutrient content (%) wt/wt Nitrogen (N)Phosphoric acid (P 2 O 5 ) Potash (K 2 O)

Miscellaneous

materials Paddy husk** 0.15 0.13 0.22 Paddy straw** 0.500.07 0.60 Maize straw*** 0.40 1.50 1.60 Thatch grass** 0.500.10 0.50 Guatemala grass*** 1.6 0.60 1.70 Napier grass***0.8 0.40 1.30 Mimosa*** 2.6 0.60 1.00 Tea pruning litter**2.0 0.50 1.50 Shade tree droppings** 2.5 0.70 0.80

Wood ash Ash, coal 0.73 0.45 0.53 Ash, household 0.5–1.91.6–4.2 2.3–12.0

Figures in parentheses are for decorticated (after removalof husk of the seeds) material; *Gaur (1994); **Chakravartee

and Sinha (1994); *** Banerjee (1993).

Some nutrients and their organic source:

Nitrogen: Farm yard manure (FYM) compost, organicfertilizers, cow urine, etc.

Phosphorus: FYM, compost, organic fertilizers, etc.

Potassium: FYM, compost, organic fertilizers, etc.

Calcium: Lime and dolomite.

Magnesium: Dolomite

Table 2 (Continued)

microbes and relatively high nitrogen content was developedand used extensively in

organic tea fields in China. Slow-release organicfertilizers are applied in Japan. The

organic fertilizer is applied biannually, in China and anincrease in yield of at least 10%

could be achieved compared to an annual application inSeptember (Anon., 2014a).

Literature on composting, green manure, preparation of

liquid manures (viz. mix manure,

jivamrut, sanjivak, amritpani, panchagavya, vermiwash,etc.) biofertilizers, vermicomposting

for application into the organic tea field exists (GhoshHajra, 2006; Ghosh Hajra, 2014).

Organic manure (see Table 3) should not be broadcast in thetea fields. Preferably it

should be applied in staggered trenches. The dimensions ofthe trench should be 2 × 0.3

× 0.4 m with 2 m between the trenches. Trenches may betaken across the slope once in

every two to three rows depending on the gradient.

In a tea field, nutrients are removed in various ways. Theloss was greater where soil

husbandry was poor. The nutrients are continuously removedat each plucking round,

consequently in a year a considerable amount is removedfrom the soil. Wood is taken as

fuel when pruning, resulting in nutrients retained in thewood also being removed from

the soil. There are some who believe that a ‘balancedmanuring program’ is one where

the amount of any nutrient removed by the crop is replacedin the manuring programme

for the following year. A balanced manuring programme isone which provides adequate,

but not excessive, supplies of all plant nutrients to thesoil system. When soil supplies of

nutrients are low (as indicated by soil tests) adequateamounts must be supplied in the

fertilizer programme. Soil testing then is key to arrivingat a balanced fertilizer programme.

3.9 Biodynamic agriculture and preparations

Biodynamic (BD) agriculture, as one of the organicagricultural farming methods, was

proposed by Steiner (1974). The BD farming method strivesfor diversified, resilient

and ever-evolving farms which could provide ecological,economical and physical long

term sustainability for humankind (Turinek et al., 2009).It incorporates the practices of

composting and mixed farming systems with the use of animalmanures, crop rotations,

animal welfare, regarding the farm as an organism/entityand local distribution system

(Reganold, 1995). All of which contribute towards theprotection of the environment,

safeguarding biodiversity and improving the livelihoods offarmers. Nowadays, there are

more than 5000 BD farms in 50 countries, the area of which,over 150,000 ha, is certified

according to Demeter Standards (Anon., 2015a). Demeter’s‘biodynamic’ farm standard

is a comprehensive organic farming method that requires thecreation and management

of a closed system minimally dependent on importedmaterials, and instead meets its

Table 3 Manuring for organic tea 1

Manure Quantity Nutrients (Kg/ha) N P K

Manuring of organic tea pruned year (for 2500–4000 kg madetea/ha/annum)

1 Compost * 10 t/ha 199 14 72

2 Rock phosphate (20% P 2 O 5 ) 400 kg/ha − 35 − (or)

1 Neem cake ** (1 split) 2.5 t/ha 121 9 26

2 Castor cake *** (1 split) 2.5 t/ha 121 9 28

3 Rock phosphate (20% P 2 O 5 ) 400 kg/ha − 35 −

Manuring of organic tea other years (for 2500–4000 kg madetea/ha)

1 Compost 15 t/ha 298 21 108

2 Woodash 5.5% K (1 split/1 application) 0.5 t/ha − − 28

3 Rock phosphate (20% P 2 O 5 ) 400 kg/ha − 35 − (or)

1 Neem cake ** (2 splits) 5 t/ha 242 18 52

2 Castor cake *** (2 splits) 5 t/ha 242 18 56

3 Woodash 5.5% K (1 split) 0.5 t/ha − − 28

4 Rock phosphate (20% P 2 O 5 ) 400 kg/ha − 35 −

* Compost (1.99% N, 0.14% P and 0.72% K, moisture less than10.0%).

** Neem cake (4.85% N, 0.36% P, 1.03%K).

*** Castor cake (4.82% N, 0.35 P, 1.11% K).

1 IFOAM guidelines (www.ifoam.bio);www.upasitearesearch.org (accessed 24 Sept., 2016).

needs from the living dynamics of the farm itself. Next toorganic standards of agriculture,

Demeter Standards demand the use of BD preparations, thekeeping of farm animals, use

of animal manures, and the encouragement of localproduction and distribution systems

using local breeds and varieties. The BD method can also becalled a ‘Holistic Farming

System’ and became the subject of research efforts duringthe past decades.

BD preparations (see Table 4) are one of the main featuresof BD agriculture.

The details of BD preparations are as follows:

Cow horn manure (BD 500): Uncovered cow horns were filledwith straw-free dung from

a lactating cow (see Fig. 1a) with the lactating rings atthe base (see Fig. 1b) and buried

in the ground during the autumn and winter months. Thehorns were then dug out (see

Fig. 1c) in the spring. The dung is totally transformedinto humus. Soon after removing the

manure from the horn, it must be protected from drying outby storing in a suitable glass,

ceramic vessel or mud pot covered with wet peat or moss(see Fig. 1d). 75g of preparation

BD 500 in 40 litres of water for one ha is the standardrecommendation.

Horn silica (BD 501): This silica spray is based on veryfinely ground quartz crystals. The

quartz is powdered by crushing it with a pounding stone, apestle and mortar and finally

ground between glass plates. The powder is made into slurryby adding water, packed

into the horns (see Fig. 1e) and buried during the springand summer months. The horns

are dug out of the ground in the winter and stored intransparent glass containers near

a window sill. One set (one gram) of BD preparation 501 in13.5 litres of water for 0.4 ha

of soil can be applied as a fine mist over the canopy oftea bushes early in the morning

during the ascending period of the month.

Table 4 Numbers of BD preparations, their main ingredients,mode of use and predicted influence I

Preparation

code Main ingredient 2 Use Mentioned in connection with

BD 500 Cow manure Field spray Soil biological activity

BD 501 Silica Field spray Plant resilience

BD 502 Yarrow flowers (Achillea millefolium L.) Compostpreparation/inoculant K and S processes

BD 503 Chamomile flowers (Matricaria recutita L.) Compostpreparation/inoculant Ca and K processes

BD 504 Stinging nettle shoots (Urtica dioica L.) Compostpreparation/inoculant N management

BD 505 Oak bark (Quercus robur L.) Compostpreparation/inoculant Ca processes

BD 506 Dandelion flowers (Taraxacum officinale Web.)Compost preparation/inoculant Si management

BD 507 Valerian extract (Valeriana officinalis L.) Fieldspray, compost preparation/inoculant P and warmthprocesses

BD 508 Horsetail plants (Equisetum arvense L.) Field sprayPlant resilience

1 Turinek et al. (2009).

2 The procedure of preparation and fermentation isdescribed in detail by Steiner (1974). BD preparations are

designed to be used together on a farm/farming system.

Revitalizing preparations made from yarrow flowers (BD 502)(see Fig. 1f) chamomile

flowers (BD 503) stinging nettle shoots (BD 504) (see Fig.1g) and oak bark (BD 505) are

used in small doses providing healing qualities and as anaid in combating plant diseases

(Rosenberg and Linders, 2004).

BD 506: Dandelion flowers (see Fig. 1h) are sewn in cowmesentery and sun-dried. The

fresh valerian extract (see Fig. 1i) is pressed and the

juice put into bottles. The bottles are left

uncorked for six weeks to allow fermentation to finish,then corked and stored in a dark cellar.

Figure 1 Common biodynamic preparations applied in teafields. (a) Cow horns filled with straw-free

manure which are buried in the ground; (b) Horns havecharacteristic lactating rings at the base;

(c) Manure after removal from horn; (d) Ceramic containersare surrounded on all sides; (e) Ground

quartz is put into cows’ horn and buried in the soil; (f)Yarrow plant with flowers; (g) Nettle plant; (h)

Dandelion plant with flower; (i) Valerian plant withflowers.

BD 508: Dried shoots of horsetail herb (Equisetum arvense)– tea is made in a vat,

covered and filled with rain water.

The short- and long-term trials carried out on crops otherthan tea (Pfiffner and Mäder

1997; Mäder et al., 2002; Fließbach et al., 2007; Zallerand Köpke, 2004; Berner et al., 2008;

Bougnom et al., 2012) with the inclusion of BD farmingmethods where BD preparations

as given in Table 4 were used. The results show the effectof BD preparations on yield

and on some on-going processes in compost piles and longterm in the soil. However,

the exact mode of action of BD preparations which presentthe greatest difference from

organic production, remains unexplained. Published researchdata will be analysed and

future development will be brought into focus to betterunderstand and explain the BD

farming method.

3.10 Weed management

Complete eradication of weeds does not generally occur inorganic farms. A balanced

weed population can provide a favourable micro-climate andthe activities of the plant

roots help to improve soil biological activity andstructure. Weeds in general are controlled

manually by hand pulling during dry periods and slashingduring monsoons. Uprooted and

slashed weeds may be retained in the field. Weeds can alsobe managed by different eco

friendly practices. Thermic weed control is permitted. Theuse of any kind of herbicides is

prohibited.

3.11 Insect pest and disease management

The most common pests for tea are mites, thrips, teamosquito bug, leaf-eating caterpillars,

mirids, scolytid beetles and termites. Pest and diseasecontrol in organic farming is

primarily preventative rather than curative. Preventativecultural techniques viz. (1) green

manure (2) a balanced manuring programme (3) mulching andmechanical control can be

adopted.

Field scouting at regular intervals is vital to monitor theinitial development of pests and

diseases in each section of the tea estate. Based on thefindings of field monitoring if pest

infestation reaches the economic threshold level (ETL) thenappropriate plant protection

measures should be taken immediately.

A considerable number of natural enemies of these pestshave been identified (see

Table 5) and some of them have proved capable, under theright conditions, of keeping

the pest population below the ETL (Ghosh Hajra, 2002,2006). International regulations

stipulate that pests and diseases should be tackledprimarily by planting tolerant or

resistant cultivars. Even low levels of resistance areimportant as it minimizes the need for

other control methods. It has been observed that China –varieties are more susceptible

to attack by red spiders and scarlet mites while Assamcultivars are favoured by eriophyid

mites. The incidence of Helopeltis is greater on China jatsin Assam; the clone TV-1 is

highly susceptible to this pest. Flushworm incidence isgreater on the clone UPASI-17

considered ‘cambod’ in nature. Soft wooded tea plants areknown to be easily attacked

by termites. Clones with a high content of alphaspinasterol are susceptible to damage by

the shot hole borer. The use of certain Sri Lankanselections viz. TRI 2024, TRI 2025 which

are popular in South India should be avoided in shot holeborer-prone areas. In Taiwan,

Table 5 Important natural enemies of some tea pests*

Pest Natural enemy

Red spider mite

Oligonychus coffeae

(Nietner) Verania vincta Garh., Stethorus gilvifronsMulsant, Scymnus sp., Verticillium lecanii (fungus),Agistemus hystrix., Chrysopa sp., Phytoseiulus persimilis

(Exotic sp.), Chrysoperla carnea Stephens, Exothoriscaudata, Pronematus sp., Cunaxa sp., Amblyseius herbicolus(Chant), A. ovalis, A. mcmurtry, Staphilinid beetle.

Pink and purple

mites,

Acaphylla theae (Watt),

Calacarus carinatus Amblyseius herbicolus (Chant),Lestodiplosis sp., Scolothrips rhagebianus Priesner

Bunch caterpillar

Andraca bipunctata

Walker Cylindromya sp.(Techinid fly), Cantheconideafurcellata (Wolff), Pencillium and Rhizopus (Fungi),bacteria, dipterous fly

Red slug caterpillar

Eturesia magnifica

Butler Apanteles sp., Exorista heterusiae, bacteria

Looper caterpillar

Buzura suppressaria

Guenee Sarcophagus sp., Apanteles taprobanae Cameron,Aphanogmus monilae, Bacteria

Flush worm

Cydia leucostoma

(Meyrrick) Apanteles aristaeus Nixon, Calleida nilgirensisStraneo, Asympiesella indis

Leaf roller

Caloptilia theivora

(Walsingham) Sympiesis dolichogaster Ashmead, MestocharellaJavensis

Tea tortrix

Homona coffearia

Nietner Phytodietus spinipes (Cameron), Palexoristasolennis (Walker)

Cut worm

Spodoptera litura

(Fabricius) Peribaea orbata (Wiedemann)

Aphid

Toxopteraaurantii,

(Boyer de Fons.) Syrphus balteatus, Asarcina aegrota,Scymnus pyrocheilus (Mulsant), Verania vincta, Cryptogonusbimaculata, Cryptoonus or Cryptogonus biculus (Gyllenhal),Coccinella repanda,Coccinella septempunctata, Lemniabissellata (Mulsant), Pseudaspidimerus circumflexus(Mots.), Aphelinus sp., Menochilus sexmaculatus (Fab.),Jaurevia pubescens (Fab.), Aphidius colemani Vierick,Lipolexis scutellaris Mackauer, Episyrphus balteatus DeGeer, Paragustibialis (Fallen), Leis dimidiata varquindecimmaculata, Trioxysindicus, Chrysoperla carneaStephens

Jassid

Empoasca flavescens

Fabr. Cephalosporium sp. (Fungus), Drynid wasp, (Continued)

Pest Natural enemy

Helopeltis

Helopeltis theivora

Waterhouse Oxyopes sp., Melamphus sp., praying mantis,Chrysoperla carnea Stephens, Reduviid bug, Spider,Mermethid nematode, Ichneumonoids

Scale insects

Eriochiton theae Green Coccinella septempunctata,Spalgisepius, Blastothrix sp.

Chrysomphalus

aonidium Ashmead Chilocorus circumdatus, C. nigritus,Pharoscymnus horni, Comperiella bifasciata, Prospeltellasp., Aphytis chrysomphali, Aspidiotiphagus citrinus

Fiorinia theae

(Green) Jauravia quadrinotata, Scuymnus sp., Aphytis theae,Prospeltella sp., Aspidiotiphaus sp.

Aonidiella aurantii

(Maskell) Aphytis chrysomphali, C. bifasciata

Saissetia formicarii

(Green) Coccophagus acanthosceles

* Ghosh Hajra (2006).

Table 5 (Continued)

TTES No. 12 is the recommended variety for organic teaplantations due to its superior

pest resistance (Lin, 2015).

3.11.1 Botanicals

Certain products derived from plants are used for tea pestcontrol. Formulations containing

azadirachtin obtained from the neem tree (Azadirachtaindica) have been found effective

against pink and purple mites and caterpillar pests such asflushworms and leaf rollers. The

bioefficacy of neem oil (NO) and neem seed kernel powder(NSKP) has been observed in a

field trial in Darjeeling and found NO and liquid soap at1% concentration offered the best

control (60%) followed by NO at 0.5% (51%) and NSKP at 100g (42%) on some sucking

pests. The average cost benefit ratio was 1: 2.13 (Bisenand Ghosh Hajra, 1995). Extracts (1%)

of the aerial parts of some common weeds viz. Polygonum

runcinatum, Artemisia vulgaris,

Eupatorium glandulosum, Urtica sp., Lantana, etc., havebeen found effective against some

sucking pests in Darjeeling tea estates viz. aphids,Jassids (Ghosh Hajra, 2001). Products

from plants viz. Annona squamosa, Lantana camara, Bidenspilosa, Calotropis gigantea,

Ocimum basilicum and Vitex negundo have been reported tohave pesticidal properties

(Muraleedharan, 2004). The spray (2%) of lemon grass(Cymbopogon citratus, C. nardus

and C. flexous), citronella (Cymbopogon winterianus)palmarosa (C. martini) and other

vegetative oil had good aphid control, Jassid andHelopeltis antonii (Ghosh Hajra et al.,

1994; Mohapatra, 2001). However, the problem associatedwith commonly-used botanicals

and bio-pesticides is their varying degree of success inthe field.

3.11.2 Cultural control

This is probably the most economical and widely applicablemethod of pest control.

Certain routine cultural operations such as plucking,pruning, shade regulation and weed

control are manipulated to reduce the incidence of pests.The tea mosquito bug lays a

large number of eggs on the broken ends of plucked shoots.Intensive removal of stalks

during plucking is recommended to reduce the incidence ofthe tea mosquito bug. Leaf

folding caterpillars can be removed manually whileharvesting. A few rounds of black

plucking reduce the incidence of thrips and Helopeltis.

Proper shade management will

help prevent the excessive buildup of thrips, mites andalso reduce the incidence of

blister blight disease. Certain caterpillars like flushworm, leaf rollers and tea tortrix can

be controlled to an extent by manually removing theinfested shoots during plucking.

Blister blight disease can also be controlled by the use ofresistant clones and modifying

the micro-climate by thinning shade trees. In general,clones are less susceptible to blister

blight disease compared to seedling tea. Certain SouthIndian selections viz. SMP-1 and

SA-6 show some resistance to blister blight disease(Muraleedharan, 2005).

Weeds offer excellent hiding places and serve as alternatehosts to many tea pests.

Mikania micrantha, M. cordata, Bidens biternata, Melastomamalabathericum, Eugenia

jambolana, Rosa sinensi, Jasminum scandens and Polygonumchinense are the alternate

hosts of the tea mosquito bug in North East India.Effective weed control assumes greater

significance in the management of tea mosquitoes. Thepopulation of Pratylenchus loosi

decreased to a certain extent when castor oil and mahua(Madhuca longifolia) cake were

added to the soil (Gnanapragasam, 1991).

Root diseases viz., root splitting, black, red and brownroot disease may occur in patches.

In order to control black root disease (Rosellinia arcuataPetch) burial of pruned branches

in the infested field should be avoided. Preparations of

Trichoderma viride or Gliocladium

virens at 200 g/pit could be applied at the time ofplanting. The following measures are

recommended to control red, brown and root splittingdiseases (www.upasitearesearch.org)

• Isolation of infected area

• Including one circle of healthy bushes – in case of Fomesinfection

• Including two circles of healthy bushes – in case ofPoria infection

• Making trenches 1.3 m deep and 45 cm width

• Putting soil inside the infected patch

• Uprooting and burning the bushes in situ

• Rehabilitating soil with Guatemala grass

• Using of Bio- control agents (200 g per planting pit)Trichoderma harzianum Red root and Root splitting diseaseT. viride Black root disease T. harzianum T. hamatum: T.viride, T. resei and T. koningii Brown root disease G.virens Red, Brown and Root splitting diseases.

It has been observed in South India that the spread ofprimary root diseases such as Poria

hypolateritia and Fomes noxius can be prevented byisolating disease patches by making

trenches 120 cm deep and 45 cm wide surrounding thediseased plants (Muraleedharan,

2004). Further isolation of the infected area, putting soilinside the infected patch and

uprooting and burning the bushes in situ, rehabilitatingthe soil with Guatemala or Napier

or Manna grass for at least two years before replanting orrefilling and using bio-control

agents are important. The application of beneficialmicroorganisms has been attempted

to control tea root diseases. T. viride and T. harzianumshowed inhibitory activity against

branch canker (Poria hypobrunnea) (Barua et al., 1989). InNorth East India when the

propagules of Trichoderma were spread on the cut surface ofstems after pruning and

then inoculated with the pathogen, the rate of infectiondecreased from 29.2% to 3.3%

after 12 months and from 51% to 5.8% after 24 months. T.viride and T. harzianum are

also effective against Charcoal stump rot (Ustulina zonata)and brown root rot (F. noxius)

(Hazarika et al., 2000). Micrococcus luteus was found to behighly antagonistic to brown

blight (Glomerella cingulata) disease in India. In Japan,Horikawa (1988) observed that the

conidia of Pestalotia longiseta and P. theae (Grey blight)were destroyed by Streptomyces

roseosporus after three days of inoculation. Black rot(Corticium theae and C. invisum)

by strains of Bacillus subtilis was reported in North EastIndia (69 and 79%, respectively,

reduction over control) (Agnihothrudu, 1999). Theapplication of Pseudomonas strain,

isolated from tea soils of North East India, as abio-control agent against brown rot disease

of tea was recorded as satisfactory (Misra et al., 2004).

In Japan, the most damaging tea insect pest is white peachscale (Pseudaulacaspis

pentagona) and the major tea diseases are anthracnose(Discula theae-sinensis) and gray

blight (Pestalotiopsis longiseta). Pest-resistant cultivarshave been selected (see Table 6)

by assay and selection methods. These four cultivars arerecommended for organic tea

cultivation.

A list of permitted and restricted products for control ofpests and diseases in organic

production are presented in Table 7.

3.12 Manual workers

Additional workers are required for organic tea cultivationcompared to conventional tea

production. The extra workers are required for trenching,compost preparation, weed

control, application of bulky concentrated organic manures,shade regulation, etc. It has

been observed that (see Table 8) 976 workers are requiredper hectare per year for an

organic tea estate in the Darjeeling hills in comparisonwith 850 workers on a conventional

estate, but it would vary depending upon various factors.

To ensure the labour force is available for differentoperational works throughout the year

on the tea estate is one of the most important of themanagement strategies. The tea sector

is far more labour intensive compared to other plantationcrops. The land:man ratio in the

tea crop sector is about 2.5 labour units per hectare,which is higher than for natural rubber

Table 6 Disease and insect-resistant tea cultivars in Japan

Cultivar Registered year Disease Insect Anthracnose Grayblight White peach scale

Minamisayaka (late budding green tea cultivar) 1 1991 R R R

Benifuki (high quality black tea cultivar) 2 1993 R R S

Yumekaori (semi-early budding green tea

cultivar) 3 2006 S R R

Saeakari (semi-early budding, vigorous and

high yielding green tea cultivar) 4 2010 R R S

R: resistant, S: susceptible; 1 Furuno et al. (1997 and2001); 2 Takeda et al. (1994); 3 Nagatomo et al. (2007);4 Yoshida

et al. (2011).

Table 7 Products allowed for plant and disease control inorganic agriculture*

Inputs Condition for use

Substances from plant and animal origin

Azadirachta indica (neem preparations) Permitted

Neem oil Restricted

Preparation of rotenone from DerrisellipticaLonchocarpus,

Thephrosia spp. Restricted

Gelatine Permitted

Propolis Restricted

Plant based extracts – garlic, pongamia, etc. Permitted

Preparation on basis of pyrethrins extracted fromChrysanthemum

cinerariaefolium, containing possibly a synergist Pyrethrum

cineriiafolium Restricted

Preparation from Quassia amara Restricted

Release of parasite predators of insect pests Restricted

Preparation from Ryaniaspecies Restricted

Tobacco tea Prohibited

Lecithin Restricted

Casein Permitted

Sea weeds, sea weed meal, sea weed extracts, sea salt andsalty water Restricted

Extract from mushroom (Shitake fungus) Permitted

Extract from Chlorella Permitted

Fermented product from Aspergillus Restricted

Natural acids (vinegar) Restricted

Minerals

Chloride of lime/soda Restricted

Clay (e.g. bentonite, perlite, vermiculite, zeolite)Permitted

Copper salts/inorganic salts (Bordeaux mix, copperhydroxide, copper oxychloride)

used as a fungicide depending upon the crop and under thesupervision of

accredited Certification Body Restricted

Mineral powders e.g. stone meal Prohibited

Diatomaceous earth Restricted

Light mineral oils Restricted

Permanganate of potash Restricted

Lime sulphur (calcium polysulphide) Restricted (Continued)

Inputs Condition for use

Silicates, clay (Bentonite) Restricted

Sodium bicarbonate Restricted

Sulphur (as a fungicide, acaricide, repellant) Restricted

Microorganismused for biological pest control

Viral preparation (e.g. granulosis virus, nuclearpolyhedrosis virus) Permitted

Fungal preparations (Trichoderma spp.) Permitted

Bacterial preparations (Bacillus spp.) Permitted

Parasites, predators and sterilized insects Permitted

Others

Carbon dioxide and nitrogen gas Restricted

Soft soap (potassium soap) Permitted

Ethyl alcohol Prohibited

Homeopathic and ayurvedic preparations Permitted

Herbal and biodynamic preparations Permitted

Traps

Physical methods (chromatic traps, mechanical traps, stickytraps and pheromones) Permitted

*Source: www.apeda.gov.in Restricted – means that theconditions and the procedure for use shall be set by the

accredited certification agency.

Table 8 Requirement of workers (ha −1 year 1 ) fordifferent field operations in the organic and conventional

tea fields of the Darjeeling hills (altitude 1000–2000metre)*

Sl. no. Operation Workers (no.) Organic/Conventional

1 Weed management a. weeding (top of the bush) - 10 mandays/year b. sickling (pruned bush) - 84 man days/year c.sickling (unpruned bush) - 63 man days/year 204/157

2 Compost preparation 90/55

3 Manure application 45/25

4 Insect pest and disease management 78/60

5 Mulch collection and mulching 18/12

6 Pruning 75/75

7 Rehabilitation and green crops (Guatemala grass, etc.)50/50

8 Plucking average 32 round � 13 workers/round 416/416Total 976/850

*Average of three tea estates each

Table 7 (Continued)

at around 0.9 units, or for coconut sector, where the ratiois about 1 unit of labour for roughly

5 hectares

It has been reported that a crop of 11,650 kilograms perhectare requires 3.7 to 4.9

workers per hectare to pluck the tea shoots and maintainthe fields in Kerala, South India

(http://www.kerala.com/keralatea/html/growingfrm.htm).Normally it should be 10, 15,

20, or 25–30 pluckers per hectare per round dependingwhether for first, second, third,

fourth or fifth year fields, respectively(http://tourismmunnar.com/plantat/default.htm).

It has been reported that the high cost of production oforganic tea in South India (more

than 60%) is due to the man days required (see Table 9)compared to conventional tea

(Anon., 2014b).

4 Post-harvest and manufacturing practices

4.1 Manufacturing practices

For estates having both conventional and organic farmingactivities, the manufacture of

organic tea should be carried out in a separate factory to

eliminate any possibility of coming

into contact with the conventional tea. The manufacture of‘organic tea in conversion’

and ‘certified organic tea’ should preferably be done onseparate days, with the utmost

care being taken to properly clean and wash the factorywith pressurized water before

the manufacture of ‘organic tea’ and after the manufactureof organic tea in conversion.

The use of additives is not permitted during themanufacturing process of organic tea. The

processing unit of organic tea should possess ISO, HazardAnalysis and Critical Control

Point (HACCP) certification by the relevant accreditationagency.

4.2 Storage and packing

Organically produced and manufactured teas should be storedpreferably in separate

storerooms and warehouses where no fumigants, insecticidesor fungicides are used.

Vacuum, steam or high pressure water cleaning is permitted.In the case of small organic

tea producers, it would be worthwhile having smaller commonstorage facilities if they are

interested in storing their tea more profitably. This wouldalso facilitate common inspection

by the certifying body.

Table 9 Comparative cost (per kg) of organic tea overconventional tea in South India 1

Sl. no. Cost components Organic Conventional Cost per kg(INR) Cost per kg (INR)

1 Total variable cost 77.66 35.41

2 Total fixed cost 34.15 16.62

3 Manufacturing cost 24.50 24.50

4 Overhead expenses 16.67 16.00

5 Total cost 152.98 92.53

6 % increase over non-organic 60.48%

1 Anon. (2014b).

Packaging material for organic tea must be biodegradableand eco-friendly. Waste

generating package material should be avoided. Organic teamay be packed in plywood

chests or biodegradable packing materials on the same dayof production and the organic

quality grade should be clearly indicated on each chest orcontainer along with the invoice

number of dispatch. When the full standards required arefulfilled, products can be labelled

as ‘Produce of Organic Agriculture’.

4.3 Possible contamination sources

Proper care must be taken to ensure that the produce doesnot become contaminated by

foreign substances. Possible contamination sources include:

• Substances (e.g. copper and lead from abrasion) emanatingfrom processing machinery that the tea comes into directcontact with;

• wood protection preparations used to protect woodencrates (e.g. pentachlorophenol (PCP));

• glues used to make the crates (often containingformaldehyde);

• glues used in consumer packages often containcontaminants (e.g. PCP);

• ink used in crates and bags often contain contaminants.

4.4 Transportation and shipment

The chests of organic tea should be transported separatelyand there should not be

any chance of it coming into contact with the conventionaltea. Before shipment to

its destination, organic tea chests should be stored in aseparate place apart from the

conventional tea.

5 Inspection and certification of organic tea

The most important aspect in the modern era of organicfarming is the certification

programme which consists of:

• standards (rules)

• inspection (checking whether the rules are implemented)and

• certification (judgement).

Organic farming can be distinguished from other methods ofsustainable agriculture by

this certification programme. The organic standards definewhat can be labelled 'certified

organic' and sold commercially as such.

A certificate is only valid if it is awarded by anaccredited certifying agency. Certification

by any agency includes:

1 Certification attested by the certification manager

2 Inspection carried out by an inspector

3 Adoption of standards attested by a quality controlmanager.

Certification programmes vary among countries or regionsdue to differences in

environmental, climatic, social and cultural factors.

Certification acts as a trust-building system betweengrowers and customers, helping

authentication of the product, enabling transparency,strengthening the position of the

primary producer and helping in market promotion of theproduct (Ghosh Hajra, 2014).

The production chain has to be inspected by ‘certificationbodies’ that follow specific

guidelines.

The basic requirements for certification are as follows:

• Minimum of one full inspection a year

• Full implementation of the relevant standards

• Conversion period followed

• Sustainable production system

• Identification of product flow and audit procedure

• Clear management responsibilities

• Parallel production (where organic and non-production ina field are indistinguishable) is not allowed.

Representatives of the certification agency will make fieldvisits to check the organic

growing techniques, taking random samples of leaf andmarketable tea and also check the

bookkeeping system. Analysis of soil, macro and micronutrients and heavy metal should

be done regularly to reveal usage of nutrients andcontamination, if any. Tissue analysis

of mother leaf/harvested leaves and marketable teas shouldalso be carried out at certain

intervals to reveal nutrient levels, chemical residues and

quality of tea.

In India, 50% of the total cost of certification subject toa ceiling of INR 1 00 000 (US$

1612) is borne by the government which facilitates a largenumber of units securing

organic tea certification.

6 Future prospects for organic tea cultivation

• Some tea growers believe growing organic tea is no longera fad but necessary to increase sustainability andproductivity.

• Growers considering conversion to organic cultivation ata later date fear a potential decrease in yield. Theincreased requirement for field workers is a barrier toadoption. The production patterns tend to be morediversified.

• Growers do not normally obtain any price premium duringconversion to organic. Due to lower yields and higherproduction costs, the transition to organic production maylead to financial losses within this period.

• There is a scarcity of organic manure in many places andit is quite expensive especially when it involvestransportation from the production site to the plantationsite.

• Deficiency of potassium and zinc in the organic field maybecome apparent in due course.

• Difficulties in controlling pests such as Helopeltis,mites, caterpillars, blister blight, etc., in the organictea field.

• Lack of adequate marketing channels can prevent anorganic tea grower from securing a premium price for theirproduce. If there is no assured market, organic tea willhave to be sold at rates similar to conventional teas.

• Growers are not sure whether the premium will remain atleast at the current level.

• There is still a fear that an increasing supply levelmight lead to a collapse in price premiums.

• High cost of certification.

7 Organic tea yield trends

During the past few years conversion of conventional teagardens to organic tea in

India, China and Sri Lanka has continued steadily. Duringthe transition from chemical

based agriculture to natural farming a decline inproductivity has been observed. In the

Darjeeling hills, it was observed that the decline in yieldof processed tea 3 to 14% in the

first year after conversion, 10 to 17% in the second yearand this depressing effect on

yield was significant until the fourth year when thereduction was around 48% in estate C

(see Table 10). In most of the tea estates, yield was foundto stabilize later but there was

no net gain (Ghosh Hajra, 2011a). Crop loss of up to 40%after conversion to organic was

observed in six tea estates in the Darjeeling hills (Anon.,2014b). Decreased tea yield of 21

to 33% was reported in the first year after the transitionfrom conventional (240 to 300 kg

N ha −1 year −1 ) to organic cultivation (Werf, 1990). Theexperience in North India has also

been more or less similar with the exception of the fourthyear, when the decline in yield

was only 20%. Crop loss up to 44 percent after conversionhas also been reported in India

(Anon., 2014b).The sudden withdrawal of inorganicfertilizers might be the reason for this

reduction in yield, but there are a few exceptions whereyields in organic agriculture are

comparable in both systems (Ghosh Hajra, 2006). The low

yield of organic tea is probably

due to the limitation of available nutrients, especially N,P and K. Therefore, it is important

in organic tea fields that a relatively large amount ofN-rich organic fertilizers and sufficient

natural P and K fertilizers are applied to ensure asufficiently high yield in the long term

(Han et al., 2013).

Table 10 Processed tea yield trends of three organic teaestates in the different valleys of Darjeeling

hills, India along with per cent of loss in yields observedafter conversion to organic

Esta-tes Before conversion After conversion 1st year 2ndyear 3rd year 4th year 5th year 6th year Y L Y L Y L Y L YL Y L

A 470 455 3.19 390 17.02 452 3.82 388 17.4 407 13.4 368 21.7

B 597 510 14.57 500 16.24 407 31.82 402 32.66 515 13.73 49616.9

C 385 353 8.31 347 9.87 204 47.01 200 48.05 220 42.85 29024.67

Average 484 439 8.69 412 14.37 354 27.55 330 32.7 381 23.32385 21.09

Y = Yield of processed tea (kg/ha); L = Per cent of loss inyield after conversion to organic.

8 Major producing countries of organic tea

Organic tea was introduced to the world market in themid-1980s. Since then, certain

pockets in the Eastern Himalayas, the Western Ghats ofIndia, mainly in up country of

Sri Lanka and China have been converted to organicprocesses. There are also organic

tea fields in Japan, Nepal, Bangladesh, Tanzania and Turkeyas well. The organic tea

production in Taiwan, Malaysia, Vietnam, Argentina,Indonesia and Papua New Guinea

are very minor and do not have any significant influence onthe world market.

8.1 India

India and Sri Lanka are among the pioneers growing organictea for more than 25 years.

In India there are 77 certified organic gardens altogethercovering 15 726 hectares and

producing 11 million kg tea (see Table 11). Around 1% ofthe total tea produced in

India is organic tea. (Anon., 2014a). Presently, productionis also gaining momentum in

the small-holder sector. Most of the organic gardens arecertified by IMO (Institute for

Marketecology, Switzerland) SGS, Control Union (formerlyknown as SKAL International

(the Netherlands)) and the biodynamic tea gardens mostlylocated in the Darjeeling hills

are certified by Demeter. Gardens are engaged in in-houseactivities to produce farm

inputs such as composts, vermi-compost, vermi-wash, herbalbrew to work as pesticides

and weedicides (Anon., 2016a).

8.2 Sri Lanka

Organic tea was first adopted at the Haldummulla tea estatein 1983 which made the first

organic tea exports in 1987 (Anon., 2016a). Organic teaproduction is expanding and a large

number of players are being certified under planters’certification schemes as being capable

Table 11 Conventional tea production (A) as a percentage of

global production (B) and certified

organic tea production (C) as a percentage of globalorganic production (D) in some important tea

producing countries in 2013 1

Country A* B C* D

China 1924.5 38.00 35 62

India 1200.4 23.70 11 19.48

Sri Lanka 343.1 6.77 7 12.39

Japan 84.7 1.67 1.98 3.50

Nepal 18.3 0.36 0.06 0.10

Bangladesh 66.2 1.30 0.07 0.12

Tanzania 32.4 0.63 0.11 0.19

Turkey 227 4.48 0.42 0.74

Others 1169.6 23.09 0.82 1.45

Global 5063.9 56.46

*In million kg; 1 Source: Chang (2015); Anon. (2013);Anon. (2014a); Anon. (2016a);http://caykur-tea.com/en/tea-nation-turkey/organic

tea.html (accessed 21 July 2016).

of exporting organic tea. The cooperative sector andorganized small-holder tea grower

organizations engage in organic and BD tea cultivation inupper, mid and low elevations and

the main growing districts are Badulla, Kandy and NuwaraEliya. The majority of tea lands

converted to organic however, are marginal and low yieldingtea fields. A very small area is

available in rehabilitated lands for initiation with newcultivars. Tea Research Institute (TRI)

clones viz. TRI 2023 and TRI 2043 and estate selection DT1,CY9 and DN are cultivated in

most of the organic tea estates. TRI 2043 is mainly used toproduce silver tips, TRI 2023 is

resistant to Short Hole Borer pest and high yielding(Abeysinghe, 2011). The major organic

tea estates are certified by National Association ofSustainable Agriculture (NASSA), Australia

IMO, JAS Japanese Agricultural Standards (JAS) ControlUnion, Biosuisse, Naturland,

OF&OG (Organic Farmers & Growers Ltd. UK), USDA Organic,National Organic Programme

(NOP) and Demeter. As per the Institute of OrganicAgriculture (FiBL, Switzerland) statistics

in the year 2012, the total amount of certified conversionto organic and BD teas in Sri

Lanka covers around 10 000 ha. It has increased from 0.78 %in 2000 to over 5 % in 2015

(Anon., 2016a). The production is in the range of 400–800made tea kg/ha/year. Tea is not

sold by public auction but only through direct sales. Thereare 14 organic certified factories

(Abeysinghe, 2011) producing 7000 metric tons of tea and isexpected that 10,000 metric

tons of processed tea will be produced in 2016 (Anon.,2016a).

8.3 China

Organic tea is one of the leading crops in China and thegardens are generally located in

mountainous areas. Organic tea cultivation is establishedin around 45 000 hectares and

produced 35 million kg of tea (see Table 11). In the lastdecade, the area under organic

tea and its production has increased by 33 and 50 times,respectively (Han et al., 2011).

Around 79% of all tea is cultivated using traditionalorganic methods, but relatively little

is certified (Bolton, 2015). Around 700 enterprises areinvolved in organic tea production

(Qiaoi, 2014) and are certified by IMO, ECOCERT (France),BCS (Germany) JONA (Japan)

and OCIA (the United States) according to the standards ofthe destination market (e.g.

EU regulation, NOP and JAS for export of the product. Localcertification is conducted

by domestic certifiers first by OFDC and OTRDC (Qiao etal., 2015). In a comparative

study conducted in Wuyuan, China, and Kandy, Sri Lanka, itwas observed that organic

agriculture and fair trade certification could provideeconomic and social benefits where

farm income is the main source of household income (Qiao etal., 2015).

8.4 Japan

There are about 2500 hectares of organic tea fields inJapan. A total of 927 metric tons

of JAS-certified organic green tea have been produced in2001, and it increased to 1986

metric tons in 2011 (3.4% increment) (Anon., 2013).

8.5 Nepal

Total production of organic tea is 60 tons (0.06 millionkg). Guranse tea estate, one of

two organic tea estates, covers 250 hectares in Dhankutasituated in the eastern hills of

Nepal. Kanchanjangha (KTE) is another organic tea estatelocated at Ranitar eastern Nepal

certified in 1997. Both estates are certified as organic bythe NASAA. KTE is an orthodox

tea-producing garden that grows black and green tea. KTEexports its produce to Europe,

the United States and Japan. It also produces tea which isexported and supplied locally

to the renowned hotels and resorts in Nepal.

TeeGschwendner GmbH, a specialist tea retailer in Germany,has entered into a

partnership with Deutsche Gesellschaft für InternationaleZusammenarbeit (GIZ) GmbH

to provide long-term support to small growers in East Nepal– Fikkal and Ilam – and help

them meet international standards for organically producedtea. The teas were certified by

IMO. This organic tea was then purchased by TeeGschwendnerGmbH and sold all over

the world as organic Nepali tea, labelled as ‘Spirit ofSunderpani’, named after the area

Sunderpani in Fikkal where the tea plants grow(http://www.includenepal.org/images/

publications/teappp). In Nepal, shortages of organicfertilizers, a costly organic farm

certification process and lack of market access havesometimes discouraged organic tea

producers. Further, lack of logo and proper branding, hasmeant quality cannot be assured

although demand for the product is on the rise in theglobal market.

8.6 Tanzania

Two gardens viz. Luponde and Herculu, growing organic teaaccount for 0.19% of the

total world production. Luponde gardens under Mufindi TeaCo. Ltd are located at the

Livingstonia Mountains at 6800 to 7200 feet above sealevel. Luponde received organic

certification in 1988 from the Soil Association and iscurrently certified through Ecocert,

Madagascar. The Herkulu organic tea estate of 230 ha ownedby Bombay Burmah Trading

Corpn. Ltd., located in the Usambara region, began in 1994and was certified organic by

IMO. They are currently manufacturing orthodox tea inaddition to CTC (cut, tear and curl).

Luponde is also producing organic green and white tea.

8.7 South Korea

There are 18 tea fields in Boseong county. Green tea fromBoseong County was awarded

‘organic certification’ from the Control Union in theNetherlands.

8.8 Turkey

Cay Isletmeleri Genel Müdürlügü (Çaykur) a state-ownedenterprise, is the most

established tea producer in Turkey. 1000 hectare of teaplantation has organic certification

while 2877 hectares of it are still in the conversionperiod. It produced 150 tons of organic

tea in 2010. (Sakli, 2011;

Çaykur established the Hemsin Organic Tea Factory. IMO hascertified the organic tea

range by Çaykur as a 100% agricultural organic product. Thecompany owes its leading

position to its dominance within organic tea and held an89% value share within organic

black tea and a 97% value share within organic green tea in2015 (Anon., 2016b). The

company expects to have converted its entire tea productionto organic by 2023 (http://

North East region of Turkey, organic tea has beencultivated by Karali Cay Co. in the

Rize province since 2000 and recently new gardens have beenestablished in the Trabzon

province. Sometimes tea, Maize and cabbage are cultivatedtogether in some organic

fields (Ghosh Hajra, 2011a,b).

8.9 Bangladesh

Kazi & Kazi Tea Estate Ltd is the only organic tea gardenin Bangladesh and is located in

Tetulia, in the northernmost district of Panchagarh only 50km south of Darjeeling, India.

Its exports make up 60 000 kilograms, the rest being soldat auctions and local markets.

The tea garden has been certified 100% organic by the USDepartment of Agriculture and

by SGS organic production standards in accordance with EUstandards.

8.10 Malaysia

Sabah Tea Plantation (STP) (1200 acre) is the only teaplantation in the state of Sabah,

where it is also the third largest tea plantation after BOHtea and Bharat Tea in Cameron

Highland, Malaysia. STP is certified by the Control Unionfor the production of organic tea

(Chin et al., 2011). The company exported 15% of itsproduct to countries like Australia,

New Zealand, Canada, Britain and Singapore with the rest

being used for the domestic

market. This tea is available in supermarkets viz. Jusco,Carrefour, Tesco and Cold Storage.

8.11 Vietnam

Organic tea production is just at the initial stage. ThaiNguyen province is well known

for its high quality and quantity tea production area. Astochastic frontier analysis was

applied to estimate profit levels for the production oforganic tea which shows organic tea

production has a higher profit efficiency level (0.836)than conventional tea production

(0.454). If the price premium is removed, the probabilitythat organic tea farmers incur a

negative profit is about 22.5% and the probability that thefarmers receive profits below

average observed profit increases by 42.5% (Tran andYanagida, 2015).

8.12 Taiwan

Organic tea products in Taiwan have been developed quitediversely. Since 1995, there

have been a total of 101 organic tea farmers in Taiwanconsisting of approximately 170

hectares or 1% of the total Taiwanese tea gardens. NantouCounty (68 hectares) Miaoli

County (30 hectares) Taipei County (29.11 hectares) andHualian County (16.86 hectares)

have relatively large portions of Taiwanese organic teagardens. Green, oolong, black,

GABA tea, Akai (red) oolong, honey flavour tea, green teapowder and RTD tea are

marketed (Lin, 2015).

The organic tea production sectors in Malaysia, Vietnam,Argentina, Taiwan, Chile,

Papua New Guinea and Indonesia are marginal and presentlydo not have a significant

influence in the world market for organic tea.

9 Major markets for organic tea

Over the past decades, its consumption has grown globallyby more than 10% per year.

This is mainly due to health concerns of consumers in theimporting countries. Further, with

a worldwide emphasis on global connectivity and an influxof younger people drinking tea,

there is a strengthening demand for organic and sustainabletea products. As a result, the

organic market for beverage and food items labelled‘organic’, ‘Bio’, ‘all natural’, ‘additive/

preservative free’, ‘non GMO’, ‘free range’, etc., havedeveloped. Still the organic tea

sector is a very small part at 1.11% of the tea industrycompared to conventional tea, but

the number of organic tea producers and the volume oforganic tea traded in the world

market have recorded significant growth over the lastcouple of years. It is a niche market

where the product sells at a premium price. The averagedeclared value for all organic

teas, regardless of origin, was $10.18 per kilo in 2014 orabout $4.63 a pound (Bolton,

2015). The countries with major markets in organic tea donot produce tea domestically

and therefore the demand for certified organic tea isgrowing rapidly all over the world.

9.1 India

India leads the world in organic black tea output and SriLanka is also quite strong. Organic

green tea is mainly exported from China. Organic teaconstituted 2% of the total organic

food products (263687 metric tons) exported from India in2015-16 (http://apeda.gov.in/

apedawebsite/organic/Organic_Products.htm)). The entireproduction is exported to the

United Kingdom, Germany, the United States, Japan andAustralia. Organic Darjeeling Tea

is now making inroads into new markets like Russia, Japan,China and the UAE. It is also

gaining shelf-space with the US and the UK coffee retailerssuch as Starbucks, Whittards of

Chelsea and Peet’s Coffee & Tea Inc. Germany has become amajor market for Darjeeling

organic tea. Projectwerkstatt GmbH, Berlin is a leadingDarjeeling tea buyer. During 2010,

the sale of organic tea in Germany was €35.5 million ofwhich 3.6 million was for black

tea and 4.2 million for green tea (GfK household panel).The rest was herbal and fruit tea

(personal communication with Diana Schaack, AgrarmarktInformations-Gesellschaft mbH,

Germany). The market share of organic tea (black and greentea) in Germany in 2009 was

4.1% (Quelle Deutscher Teeverband). Organic Darjeeling teais sold at € 25-30 kg1 in

retail stores in the European market and at $25-30 kg1 inthe United States. The premium

first and second flush of Darjeeling tea is sold largelythrough private deals. The price

of organic tea however depends mainly on the quality. The

following observations were

reported while assessing the US consumer market for Indianorganic tea (Sen, 2010).

• Both organic tea and food markets are expanding rapidlyin the United States.

• The majority of the target market is not aware of organictea products.

• More than 55% of people know of organic tea and purchaseit.

• Consumer satisfaction with organic tea is related to itsprice.

• Most consumers (more than 65%) do not want to pay morethan 1.5 times the price of regular tea for organic tea.

• Female consumers and less educated consumers rely heavilyon word of mouth as an information source on organic tea.

• The grocery is the most effective place to sell organictea products.

• Advertising plays a big role in the promotion of organicgreen tea.

• Neither organic black tea nor organic green tea is vieweddifferently across different demographic groups.

• An awareness of organic black tea is not necessarilyassociated with an awareness of organic green tea, andvice-versa.

9.2 Sri Lanka

After the establishment of the National Organic ControlAgency (NOCA) by the Sri Lanka

Export Development Board (commonly known as EDB) organicstandards and regulations

were established pertaining to the cultivation, production,processing and trade of

organic products. The Sri Lankan organic tea industrycaters for black, green and silver

tip teas in bulk and processed teas, value-added teas withflavours and environmentally

friendly packages which fetch a minimum of 2-3 fold premiumprices in the international

markets. The UK, Australia, Germany, Japan, the Netherlandsand the United States are

the traditional buyers of Sri Lankan organic tea(Abeysinghe, 2011). Other destinations are

Italy, France, Canada, Singapore and Spain (Anon., 2016a).The local demand for organic

teas is now increasing. Once upon a time, it was a growthsector but now in Sri Lanka the

organic certification process is very costly – a factorthat makes organic tea 10–15% more

expensive than regular tea.

9.3 China

The domestic market is growing rapidly and presently Chinahas a domestic market in

Beijing, Shanghai, Guangzhou and other big cities. China isexporting organic tea to Japan,

the European Union and the United States. Domestic salesare larger than the quantity

exported. ‘Gengxiang’ has become the famous organic teabrand in China. Almost all

organic tea exported is certified according to EU and NOPregulations. Some tea gardens

have applied for JAS certification. Around 75–80% oforganic teas were exported to the

United States and Germany followed by France, Denmark, theUnited Kingdom, Japan

and Holland (Qiao et al., 2011). There are around 20countries importing 10,000 tons of

organic tea from China. (Lin, 2010).

9.4 Japan

Mitsui Ringyo is the major supplier of certified organictea (JAS Marked). The company

markets organic tea under the Nitto Koucha brand and mostlysupplies to the supermarkets.

Its major customers are Daiei, SOGO and Ito Yokado . MitsuiRingyo does not sell organic

tea to specialist retailers. The company is the largestsupplier of JAS-certified organic

green tea in Japan and it imports small volumes of blackorganic tea from India and Sri

Lanka. There are numerous other companies supplying organictea in Japan but only a few

deal with JAS-certified organic tea.

9.5 Tanzania

Cha Dô, the largest organic and fair trade speciality teablender in Germany, buys

exclusively from the Herkulu estate to mix with organic teafrom other producing regions.

(van Reenen et al., 2010). Cha Dô supplies blended teas tothe UK and North American

markets. Kirchner, Fischer & Co GmbH (K, F & Co.) own thelabel Mt. Everest Tea Company,

which is one of the oldest specialty tea companies inGermany. Specializing in orthodox

blends, K, F & Co. has only begun purchasing Luponde teasin 2010. Luponde organic

orthodox teas have a direct route to retail in the UKmarket. Luponde has a shop in the

Burlington Arcade in London which sells Luponde teaexclusively. There is also a domestic

value chain for organic tea. Chai TTB and Chai Bora, the

two leading tea packers in the

Tanzanian market, both offer organic brands for the localmarket. These are sold in the

main supermarkets in the large urban centres in thecountry, in the airport shops and in

the tourist areas.

9.6 South Korea

Presently, in the South Korean Republic, the slow-downgrowth of conventional green

tea has affected the growth of organic green tea. Althoughthe rate of growth is not

as significant, organic green tea sales are stillincreasing (see Table 12). New products

launched by major players in 2013 were confined to RTD tea,where retail prices are

relatively reasonable due to a low content of localingredients. Supported by increased

interest in health and well-being among local consumers,organic beverages will record

positive growth over the coming years. However, the rate ofgrowth is expected to slow

down gradually as economic prospects are not bright enoughto support a booming market

(Anon., 2015b). Nokchawon is the leading manufacturer oforganic green tea, accounting

for 37% of sales values in 2013. The company leads overallorganic beverage sales since

organic green tea accounted for 63% of the total value oforganic beverage sales in 2013.

Nokchawon specializes in tea products and as growth inorganic soft drinks is increasing,

the sales share of Nokchawon may be diluted in the nearfuture (Anon., 2015b).

9.7 Turkey

Since 2009, all Çaykur products made for export to Europehave carried the European

organic Biosiegel label

Çaykur organic teas are available in the market as OrganicHemsin, Organik Rize Cayi,

Organik Zumrut Yesil Cay and Organik Hemsin Cay.

The organic tea market is also emerging in Asia at a rapidrate. Among the young

consumers in Hong Kong, Singapore and all over South EastAsia, the trend for organic tea

has been noticed (Ghosh Hajra, 2011a). Raising awareness ofthe health and environmental

benefits from organic tea production, product labellingwith quality control certification

would create a higher and more competitive price fororganic tea products domestically

in the long run.

9.8 The organic tea market in Europe

The stronger European markets for organic teas have enjoyedyears of consumer

education, mandatory certification and offerings from majorbrands. Leading destinations

for organic tea are Germany and the UK. Among EU countries,Germany boasts the

highest growth within the organic beverage market. Thegrowth per cent of organic tea

during 2010–2014 was 8.7 (see Table 13). However, Germansare extremely price sensitive

and prudent spenders. During the most recent recession,overall consumer spending on

food and non-alcoholic beverage remained relatively static(Anon., 2016c). Consumption

in other European countries is far more limited. Bulkorganic tea is sold in Germany via

organic shops or health food stores and in the UK viaconventional retailers. It is also being

Table 12 Forecast sales of organic tea in the Republic ofKorea (Unit: $1,000) 2013 2014 2015 2016 2017 2018

Organic tea & green tea 2,914 2,888 2,850 2,799 2,737 2,669

Source: Euromonitor International from trade associations,trade press, company research, trade interviews, trade

sources.

marketed via special tea shops viz. De Drie Mollen, SimonLevelt and Algra Mocca d'Or

in the Netherlands. Black tea still constitutes the largestshare of EU tea consumption, but

green tea is increasing in popularity. The major organicimporters/traders/blenders in the

UK are Clipper Teas, Whist bray, Hampstead Tea, DragonflyTeas, Qi Herbal Health and in

Germany Oasis, Lebensbaum, Kloth & Köhnken. Major organictrading houses are more

often located in the Netherlands and Germany. The premiumtea producer in London is

Twinings and they have organic lines specifically for thecatering and institutional markets.

Private labels are of less importance in the organic teamarket than the organic coffee

market. For example, both Super de Boer and Albert Heijn inthe Netherlands have only

one type of organic tea which is also Fair Trade certified.

The share of organic tea in the UK market was 1.9 (onlymultiple retail sales) and organic

tea had a 12.8% share of the food and drink market during2014-2015 (Anon., 2016d). In

many EU countries the focus within the tea market is moreon Fair Trade than on organic

teas and the number of supermarkets carrying organic tea isfar more limited. Fairtrade

certified organic tea accounted for 7% of the UK tea salesin 2009 with the top 3 retailers

being Tesco (market share 31%), Asda (17%) and Sainsbury(16%). Most of the tea

packing occurs in the EU and this leaves little opportunityfor value addition in developing

countries but, on the other hand, also means thatinvestments in processing facilities are

not needed.

9.9 The organic tea market in the United States

Globally, there are likely to be more natural productretailers selling private-label organic

teas. Additionally, many big retailers already have theirorganic and private label lines (Kim,

2012). In the United States, organic tea is the fastestgrowing section of the tea industry

and demanding teas from organic and fair trade farms. Blackorganic offerings (flavoured,

unflavoured, breakfast blends, etc.) remain the mostpopular, followed by green and

oolong at a distant third (Ghosh Hajra, 2011a). The UnitedStates imported 3078 metric

tons of organic tea in 2014 but that represents only 1.5%of the total tea imported for

domestic consumption. In 2013, the US organic tea importvalue amounted to about US$

42.1 million (Anon., 2016e). Imports of USDA-certifiedorganic tea continue to climb with

brands including Adagio Tea, Republic of Tea, Mighty Leaf,Celestial Seasonings, Rishi

Organic Tea, DAVIDs TEA, Teavana and Choice Organic Teas(all offering USDA-Certified

Organic tea) reporting strong sales (Bolton, 2015). Thereare a couple of other brands and

companies such as Tazo, Stash Tea, Jones Soda, NumiOrganic, Traditional Medicines,

Sweet Leaf and Steaz. Honest Tea and Tazo Tea achievedprominence in ready-to-drink tea

with their organic products and organic teas represent lessthan one-half of 1% of the total

tea market. Much of the US growth in organic tea has beenthrough incremental additions Table 13 Market share –Historic/Forecast of organic black and green tea in GermanyRetail sales value (US$ millions) Year-on-year growth %2009 2010 2014 2009–10 2010–14 Organic tea 73.8 74.9 81.41.6 8.7 Organic black tea 12.3 12.8 15.3 4.1 19 Organicgreen tea 3.6 3.6 3.8 1.3 5 Source: Anon. (2016c).

or line extensions of organic offerings by non-organic(conventional) tea brands and not

full line organic tea companies.

Presently, China is the world’s largest supplier of organicgreen tea in the United States.

In 2016, China exported 1500 metric tons of organic greentea to the United States,

accounting for 67% of total organic green tea imports.Total imports from China were

valued at $152 million in 2016, up 7% compared to 2015.After China, Japan is shipping

16.5% of organic green tea imports. In the smaller categoryof black fermented tea, which

includes puer and dark tea, China has seen a significant

50% increase in value over 2015

to $1.38 million in 2016 (Bolton, 2017). The GlobalAgricultural Trade System (GATS)

maintained by the US Department of Agriculture (ForeignAgriculture Service) tracks three

green and fermented black tea by value and quantity. Theyindicated that the value of

all organic green tea (three categories) imported was $26million and $13.5 million for

organic black teas. The data largely ignore specialtyDarjeeling black tea which is usually

imported by air and bulk import of black tea since verylittle is certified organic.

TechSci Research in its another report ‘United StatesOrganic Tea & Coffee Market, By

Type, By End User, Competition Forecast & Opportunities,2012–2022’ states that the

organic tea and coffee market in the United States isanticipated to grow at a CAGR of over

13% during 2017–2022(https://www.techsciresearch.com/report). The report alsostates

that West region accounted for the largest share in the USorganic tea and coffee market

due to huge population base, increasing disposable incomeand the highest number of

organic farms in the region. However, the demand fororganic tea products is also growing

at a healthy pace owing to increasing number of immigrantsfrom the South East Asian

countries who prefer to opt for tea.


Development of organic farming is dependent on the

evolution of market structure and

performance. Careful selection and development of targetmarkets and distribution channels

focus on the right customers, improvement in customerservice skills and conversion of

occasional buyers into regular organic buyers through largeretail outlets are of the utmost

importance. Such marketing requires different skills toregular marketing. Further, greater

efforts should be directed at expanding demand. Forexample, there is scope for increasing

per capita consumption in producing countries as they arelow compared to traditional

exporting countries. In the global movement towardsconservation and nurturing of

the environment, respect for the ecosystem and concern forthe well-being of all living

creatures, the organic way of growing tea will certainlyachieve more popularity and organic

tea producers could seize the emerging opportunity of thisniche world market.


• Breeding of varieties with high nutrient use efficiencyespecially low nitrogen requirement, or with high pest anddisease resistance especially to major pests.

• Soil fertility improvement.

• Effective use of green and animal manures, soilamendments at times and at rates that meet tea nutrientneeds to increase nutrient use efficiency and minimizenutrient losses to the environment.

• Comparative study on the effect of BD preparations andother organic manure on soil microbial dynamics, yield andquality of organic tea.

• To study productivity, profitability, sustainability,

produce quality and input-use efficiencies of tea underorganic farming in different agro-ecological regions.

• Green manure for integrated management of nutrients,insects, disease and weeds.

• Development of pest management strategies in organic teaproduction in collaboration with farmers utilizingcomplementary biological control strategies.

• Biological control of plant pathogenic nematodes inorganic tea production.

• Weed management with a better understanding of theecology and potential use of weeds in organic tea fields.

• Potential of weed flora to increase biodiversity.

• Effective use of renewable energy (e.g. biogas) renewablematerials, renewable agricultural inputs and non-renewableenergy efficiency in the tea fields and processingfactories.

• Improvement in soil organic carbon content to sustainproductivity and to support the biological activity anddiversity that contribute to nutrient cycling.

• Does development of organic matter in the soil have asignificant value in terms of carbon sequestration?

• Quantitative and qualitative information on how muchcarbon sequestrated and greenhouse gas emissions reduced,low carbon regulation/standard development, and attendvarious activities to power the organic movement ofsustainability

• Comparative study on carbon credit between organic andconventional tea gardens.

• Development of market for organic tea

• Minimization of cost for production of organic tea

• Understanding consumer motivation to buy organic tea at apremium price.

• Marketing strategies to ensure prominence of suitabletypes of organic tea in the marketplace.

• To what extent will subsidies in the organic tea sector

promote sustainable farming practices and what factorswill influence this?

• How the organic tea sector is going to broaden its appealand role of non-economic factors in conversion to organictea from conventional tea production.

12 Acknowledgements

The author is thankful to the reviewers and the editor fortheir constructive comments and

suggestions.

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18 Chapter 18 Supporting smallholders intea cultivation

1 Introduction

The Bandung International Tea Convention (BITC) 2014 andthe 21st session of the

FAO Intergovernmental Group (IGG) on Tea were both held inBandung, Indonesia, in

November 2014. The Convention identified some majordifficulties facing stakeholders in

the tea trade in both producing and consuming countries,including problems faced by

smallholders. Both meetings then facilitated consensusbuilding on methods to improve

global tea trading, with smallholders as the vital cog inthe industry.

In general, tea smallholders require the following methodsof support: • assistance to increase the productivity oftheir tea plants and quality of the tea they produce •encouragement and advice on how to become self-supportingthrough better farmer organization • business education,leading to better incomes and profits, thereby improvedlivelihoods

The following sections explore some of these themes.

2 Smallholders and their role in tea cultivation

Tea has traditionally been considered the most importantbeverage crop in Indonesia.

There are three players in the tea industry in Indonesia:state-owned plantations, private

companies and smallholders, as shown in Table 1.

The total area under tea cultivation is shrinking due to amove towards more lucrative

cash crops. Over the last ten years, nearly 30 000 ha ofland under tea cultivation has

been lost. Nevertheless, it is estimated that the tea

industry as a whole (i.e. including

downstream) employs 1.5 million people in Indonesia everyyear, from a population of

257 million. Tea as a commodity is not only a foreigncurrency earner and provider of

employment, tea plantations also contribute to theconservation of the environment,

especially by preventing soil erosion and flooding in hillyareas.

Small-scale tea growing has been a traditional source oflivelihood in rural communities

in Indonesia, mainly in West Java and Mid Java. Thedefinition of smallholder in Indonesia

is based on holding size: smallholders are those who growtea on plots of between 0.5

and 2 hectares. They mainly sell the green leaf withoutprocessing, as they do not have

the necessary processing facilities. Only a few smallholdergroups have their own factories,

particularly for ‘pan-fired green tea’.

Of the 1.5 million people employed in the tea industry, itis estimated that more than

350 000 are smallholder farmers. In fact, the teacultivation area operated by smallholders

covers nearly 45% of the total area under tea plantation inIndonesia, 129 000 ha. It is

therefore clear that the role of smallholders in theIndonesian tea industry is very important.

The cultivation of this commodity can serve as a means ofproviding opportunities for self

employment for millions of families, and it provides workand income throughout the year

to smallholders.

3 Problems facing smallholders

There are a variety of problems that worsen the position ofsmallholders in tea producing

countries across the globe, including decreasing prices,increasing cost of production,

asymmetrical market power, non-transparent trading in termsof both tariff and non-tariff

barriers and the impacts of global warming.

In Indonesia, major risk factors for smallholderscultivating tea include price volatility,

encompassing the occasional crash in the price of greenleaf due to leaf abundance or a

drop in the price of processed tea. When prices continue toremain at non-economic levels

Table 1 Indonesia’s tea production (Ton)

Year Smallholders State-owned plantations Private-ownedplantations Total

2007 38,937 80,274 31,012 150,223

2008 38,593 78,354 37,024 153,971

2009 45,239 75,451 36,211 156,901

2010 50,947 73,524 32,133 156,604

2011 51,507 65,144 34,125 150,776

2012 51,528 65,251 34,170 150,949

2013 51,737 58,814 34,909 145,460

2014* 50,897 58.484 34,370 143,751

* Preliminary

Source: Directorate General of Estate Crops, MoA. 2015.

over long periods, the entire sector is affected and it isthe smallholder who always takes

the hardest hit. The regulations on the pricing of thegreen leaf need to be strengthened,

and the distortions in the market place due to perishablenature of their produce in the

form of green leaf be ironed out. The use of informationand communication technology

(ICT) tools, especially cell phone technology, for thedissemination of market information

may also need to be encouraged. Another market complicationis that due to competition

and trade liberalization, consumer demands are becomingincreasingly complex with

higher concerns for food safety and product traceabilityand it is not possible for poor

small growers to fully meet these demands.

Smallholders also have a poor bargaining position in thevalue chain, as the management

of the supply chain, for example, the leaf collectioncentres and transportation system, is

mostly in the hands of middleman. The power ofintermediaries in the supply chain needs

to be reduced. The number of factories buying tea fromsmallholders is currently too

small and needs to be increased. Continuous contractsbetween smallholders and tea

processing factories are scarce, and/or unavailable, butneed to become commonplace.

Productivity in most smallholdings is low, at 1000 kg madetea/ha/year to 1700 kg or

less. This compares to that of large state- andprivate-owned plantations, which produce

more than 2300 kg made tea/ha/year. The productivity ofsmallholders is thought to be

weak due to low crop yields. In some places, low productionvolumes are also considered

to be due to severely dry climates as a consequence ofglobal warming. To improve

productivity, smallholders need access to credit workingcapital and resources to purchase

fertilizers and seedlings of high-yielding tea clones touse as their planting material. Most

of the small farmers currently have limited access tocredit due to the small size of their

landholdings and the fact that they do not hold clear landcertificates.

4 Disseminating good agricultural practices and improvingmarket knowledge

Most smallholders in Indonesia have been acquainted withtea growing technology for many

years, with some having more than 30 years of experience.They are therefore well informed

about basic maintenance of the tea plants on their farms.Efforts to support smallholders

in tea cultivation aim at increasing productivity and teaquality through the dissemination

of good agricultural practices (GAP), including the correctapplication of fertilizer, effective

plucking and pruning, planting shade trees, methods toprotect tea plants from pests and

diseases, and best practice in tea harvesting. Extensionofficers also advise on technological

adaptations, for example, introducing the practice ofplanting higher yielding clones,

teaching methods to mitigate the impacts of climate changeon tea cultivation and assisting

with routes to improve energy efficiency in tea processing

factories.

A case study of a particular farm has demonstrated that awell-managed smallholder site

can reach a level of productivity of up to 4000 kg madetea/ha/year. This farm has become

a benchmark for other smallholders to reach higherproductivity levels for better incomes.

Unfortunately, the best technologies recommended by groupssupporting smallholders

are not fully applied by growers in the anticipated manner,due to problems they face, for

example, lack of access to the microcredit required to fundmaintenance of the tea plant.

Poor knowledge of business practices and trading alsocauses smallholders to

become vulnerable to leaf agents and to the factoriesbuying the green leaf. There

have therefore also been capacity-building efforts focusedon improving smallholders’

general understanding of business management, marketing,input sourcing, materials

management and sourcing of finance. Shifting the status ofsmallholders from smallholder

groups to cooperatives or companies owned and operated bysmallholders will produce

better results for smallholders in the tea supply chain, asdiscussed in Section 5. To date,

however, less attention has been devoted to helping farmersorganize themselves to

improve their position in the market.

Government support programmes initiated in Indonesia in2014 aim to increase the tea

prices received by farmers and to improve farmer income and

welfare. They mainly focus

on farm sub-systems and attempt to speed up improvements inproductivity, quality and

the supply chain. It is estimated that there is an area of57 000 ha requiring rejuvenation,

rehabilitation and intensification. Efforts includereplacing old tea bushes with superior

tea clones and training extension officers, staffinstructors and farmers to improve the

knowledge and skills, motivation and entrepreneurship ofsmallholder farmers. In 2014,

1500 ha of tea plantations were rehabilitated and 1700 hawere intensified in the West

Java province.

5 Organizing smallholders to improve their position inthe market

5.1 Self-help groups and smallholder-owned companies

The position of smallholders in the market could beimproved if they were to strengthen

their organization. In particular, smallholders could worktogether by forming self-help

groups (SHGs). The self-help model, which is built onself-respect and self-determination,

incorporates the belief that a community, no matter howbackward, has resources that can

be mobilized in order to make improvements and bring aboutsocial change, both for the

individual and the community. Each SHG is built as aneconomic institution and consists of

a group of farmers who have the determination toself-organize for mutual benefit.

One approach to setting up an SHG is as follows:

1 An SHG is organized for every 10 ha, comprising of 10–25members depending upon the size of the land holdings.Every five SHGs form a nodal group and every five nodalgroups form an apex group.

2 Each SHG has a managerial group of five elected members,one of which is also part of the nodal groups’ managerialgroup. One of the nodal group’s managers is elected to actas secretary. The secretaries of the nodal groups form themanagement of the apex group. These apex groups arefederated to a state-level federation, and one of thesecretaries of the apex groups functions as the secretaryof the federation.

3 Funds are arranged by levying a token annual membershipfee at the nodal group level.

4 The federation is the coordinating agency and has liaisedwith the state government, financial institutions, the TeaBoard and other relevant authorities.

The models can be modified according to the localconditions to make the smallholders

a viable force in the development of tea in their areas.Since each SHG will be set up and

managed by the growers themselves, its chance of success ishigh.

Under the continuous guidance of an extension officer,these SHGs could initiate

programmes to increase both productivity and the quality oftea eligible to be processed.

They should also be connected to processing factories forwin-win cooperation. They

require microcredit facilities from local banks and a fairtrade system to flourish.

SHGs can then combine to become smallholder-owned companies(SOCs). Under

this new concept, smallholder farmers progressively shiftfrom being organized into

small groups to forming a legal tea company. This systemcould be implemented

by dividing the area under cultivation by smallholdersusing a cluster system. Each

company could manage a tea cluster with an area of 300 haof smallholder tea land at

minimum. The land could be intensively improved, taking thespecific problems of that

particular location into account, to reach an averageproductivity of at least 1700 kg

made tea/ha/year.

5.2 Changing patterns of partnership between smallholdersand processors

Figure 1 shows a typical pattern of partnership betweensmallholders and tea processors.

Following this pattern, the price of the tea leaf isdetermined by the factory owner based

on negotiation and agreement between smallholder farmerswith the factory. Alternatively,

it might be agreed using a price formula or criteria basedon the percentage of tea leaf of

eligible quality accepted by the processing factory (thisis especially the case for pan-fired

green tea factories).

Rather than selling fresh green leaf to private companiesthat own processing facilities,

smallholders are beginning to demand a model encompassingsmallholder factory

ownership, so that they can operate their own teafactories. There is the opportunity for

factories to be held through a cooperative, so that thesmallholders each own a share of Farmer Group/ SHG AFarmer Group/ SHG E Farmer Group SHG C Farmer Group/ SHG DFarmer Group/ SHG B Union SHG Tea Factory Partnership,Selling Tea leaf Pattern I PARTNERSHIP BETWEEN SHG +PROCESSOR Consumer

Figure 1 Pattern I: Typical partnership between self-helpgroup (SHG) and processor.

the factory. This gives a sense of ownership to the farmersand guarantees sustainable

cooperation for mutual benefit.

The second pattern of partnership is shown in Fig. 2. Inthis pattern the smallholders are

all part of the same cooperative organization.

Whichever model is followed, it is important for SHGs toimprove communication

between farmers and tea packing companies to betterunderstand market demand. SHGs

should make efforts to produce tea meeting nationalstandards and engage actively in tea

promotion and carry out human resources capacity building,becoming micro-economic

institutions in their villages.

5.3 The role of institutional partnerships

SOCs could also be developed further through institutionalpartnerships. In this context,

partnership is considered an instrument of cooperationbased on the trust between a

partner and smallholder group to reach common goals.Possible types of partnership

include nucleus estate programmes, agribusiness operationalpartnerships, general trade

relationships, sub-contracts and agency relations. The maingoals of partnership are to

increase incomes, to sustain agribusiness and to increasethe attractiveness of smallholder

groups as business partners. To be considered as a partner,an organization should have

an appropriate tea culture technology, management skills, atea processing factory,

market access and stand-by working capital. Theorganization should also have a strong

commitment that the available opportunity is a businesspartnership opportunity (i.e. it

would not be acting as a trader). The partnership shouldbuild a management unit that

straddles the sectors of production, processing andmarketing.

Features and advantages of institutional partnershipsinclude: • Inter-dependence. The company partner needs theraw material from the smallholder farmers and the farmersneed guidance on leaf collection and an efficient leafcollection system. Tea Factory Pattern II ECONOMIC SCALEConsumer TEA AREA : 300 HA NUMBER OF FARMERS : 450 FAM.TEA LEAF PRODUCTION 5 TON/DAY PRODUCING : 1,050 KG MADETEA/DAY LEAF ELIGIBLE TO PROCESS SHG

Figure 2 Pattern II: Cooperative organization. • Mutualbenefit. The partnership would allow the smallholders andcompany partner to increase income and profit as well assustain their tea businesses. • Strengthening of businessposition. The smallholder and company partner areconducting business ethically and with equal rights toreach a sustainable partnership.

There needs to be a willingness to change mind-sets toenhance and strengthen

partnerships. Other factors that require work forsuccessful partnerships include

communication, transparency, willingness to exchange andreceive ideas, and ability to

build up the trust with one another. Company partnersshould also be expecting to actively

engage in empowering the smallholder farmers. It isenvisaged that the partnerships

model could be successful if smallholder groups couldincrease their capabilities in (a)

co-creating business planning and conducting partnershipagreements, (b) collecting and

utilizing funds and (c) increasing good relationships withother institutions to find and

utilize business opportunities.

5.4 Future trends

The empowerment of smallholders is viewed as a keycomponent of programmes to

revitalize national tea agribusinesses. The paradigm shifttowards the establishment of

SHGs, which then become SOCs, is key in this regard. Theestablishment of cooperatives

and collective ownership of processing factories bysmallholders is a feature of the tea

industry in successful tea producing countries, such as inKenya, Sri Lanka and India. In

order to support smallholders to improve yields and teaquality, consistent and continued

guidance on the application of GAP by the extensionofficers is needed, and, to be more

competitive in the tea business, SHGs and SOCs need thefull support of the government.

6 Case studies: Kenya and Sri Lanka

Kenya

One example of a country with a well-organized andsuccessful smallholder organization

is Kenya. The Kenya Tea Development Authority (KTDA) wasformed in 1964 to take over

the function of the Special Crops Development Authority andwas privatized in 2000,

becoming KTDA Ltd. The core activities of KTDA Ltd arehelping smallholder farmers to

manage their tea agronomy and processing activities,marketing and selling the black

tea they produce, and providing support services to promotesmallholder tea businesses.

KTDA Ltd is now a major producer of black tea and isprobably the single largest tea

producer in any country.

In relation to the smallholder education, established GAPis disseminated to the

smallholder tea farmers by extension services staff, basedboth at the factories and at the

leaf collection centres (one supervisory staff membercovers between 10 and 15 collection

centres using a motorbike). Education has been throughconventional extension method

of demonstration, for example through field visits andfield days. The KTDA is introducing

a new extension methodology called ‘farmers field schools’(FFS) in order to push small

scale farmers to achieve higher growth and sustainabilityin all areas, but particularly in

green leaf production. FFS are a participatory approach toextension, schools without walls,

where the field is the primary learning venue. They involveexperimentation, discussion

and decision-making by the farmers themselves; theextension staff becomes facilitators,

rather than teachers. The farmers are organized into groupsof 30 to form a school and

learn by discovering the problems that hinder them fromachieving their production goals,

and then experimenting with the best methods to solve them.The farmers themselves

become the experts by conducting their own field studiesand learning by doing.

A particular challenge facing small-scale tea farmers inKenya is the steady increase

in production costs both at the factory and farm level,with fuel, electricity, labour and

fertilizer being the major contributors. With thecontinuous increase in price of oil, wood

is being introduced as an alternative fuel, as it is acheaper energy source. All factories

are therefore also required to raise 150 000 tree seedlingsannually for use as fuel. In

addition, attempts are being made at establishingmini-hydroelectric power projects

within smallholder factory catchments.

Sri Lanka

Sri Lanka’s tea industry has two main sectors: the statesector and the smallholding sector.

About 210 621 ha is devoted to tea cultivation, of which 91667 ha (43.5%) is smallholdings

and estates and 118 954 ha (56.5%) is state-owned.

Smallholders produce 74% of the tea in Sri Lanka, and henceplay a leading role in the

country’s tea industry; however, despite this highpercentage, it is considered that their

performance could be improved. Realizing the importance ofthe smallholder sector for

the development of the tea industry, the Sri Lankangovernment established the Tea Small

Holdings Development Authority (TSHDA) (Act No. 35 of1975), to look after the interests

of private sector tea smallholdings. The principal

responsibilities of the TSHDA are to

provide extension services to smallholders and manage thetax rebate for replanting and

infilling tea. The organization also supplies fertilizerand planting materials. The TSHDA is

recognized as a successful institutional arrangement thatis supportive of smallholders and

helps them to tackle the issues they face.

The primary causes for the poor performance of thesmallholdings sector in Sri Lanka

were due to lack of technical support for implementingimproved technology, poor facilities

for leaf collection due to a lack of organized collectioncentres and difficulties with transport

to the processing factories. As a result of the operationof various intermediaries (middle

men) in the supply chain of the produce from the grower tothe processing factory, the net

income to the grower was also meagre. A survey of teasmallholders in Central and Southern

Sri Lanka on the variables fertilizer application, pest anddisease control, weed control,

plucking and pruning concluded that the rate of adoption ofinnovations in the smallholding

sector was far behind that of the estate sector, pointingto lack of knowledge as one of the

major constraints in adopting innovations in thesmallholding sector. Increasing extension

services was recommended as a solution. Following theexternally funded Smallholder

Tea Development Project, which took place in 1989,significant progress in smallholding

performance was seen. This project reformed and

strengthened extension services and

research, as well as building up tea smallholder societiesand associations.

7 Conclusions

Some countries with developed tea industries have beensuccessful in their efforts to

support smallholders, as a vital cog in the supply chain.Lessons can be learned from the

way they have assisted tea smallholders over the pastdecades. Many projects funded

by government and international donors dedicated tosmallholders, for example, have

significantly raised tea productivity and quality, and haveimproved the price paid.

Strengthening smallholder organization is the mosteffective way to alleviate the

difficulties currently faced by small-scale farmerscultivating tea. The bargaining position

of smallholders needs to be strengthened through theformation of SHGs, which may then

become SOCs, a type of small medium enterprise. If suitablebusiness support is provided,

small-scale growers can make better connections with themarket place, in particular

obtaining the right pricing structures from processingfactories purchasing their tea leaf.

With support from professional management, smallholders canraise their position in the

value chain and improve their livelihoods.

A continual process of farmer empowerment is also aprerequisite to the successful

improvement of the position of smallholders in teaagribusiness. In order to improve

farmers’ standards of living and use farmers’ potential tothe full, mind-sets in the tea

industry must change and effective, fair, trusting andsustainable partnerships be developed

between individuals, groups and farmers’ institutions withcorporate partner institutions.

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