ROLE OF NATTRASSIA SP. IN FRUIT ORCHARD DECLINE ...

1

ROLE OF NATTRASSIA SP. IN FRUIT ORCHARD DECLINE

AND DETERMINATION OF KEY FACTORS FOR ITS

MANAGEMENT

FAISAL SOHAIL FATEH

94-arid-224

Department of Plant Pathology

Faculty of Crop and Food Sciences

Pir Mehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2018

2

ROLE OF NATTRASSIA SP. IN FRUIT ORCHARD DECLINE

AND DETERMINATION OF KEY FACTORS FOR ITS

MANAGEMENT

by

FAISAL SOHAIL FATEH

(94-arid-224)

A thesis submitted in partial fulfillment of

the requirements for the degree of

Doctor of Philosophy

in

Plant Pathology

Department of Plant Pathology

Faculty of Crop and Food Sciences

PirMehr Ali Shah

Arid Agriculture University Rawalpindi

Pakistan

2018

7

"In the Name of Allah, the most

Beneficent, the most Merciful"

8

DEDICATION

To

MyAffectionate, Loving Parents

&

My Sweet Family

9

CONTENTS

Page

List of Tables xii

List of Figures xvii

List of Acronyms xx

Acknowledgements xxi

ABSTRACT xxii

1 GENERAL INTRODUCTION 1

1.1 EXPORT OF TARGET FRUITS 1

1.2 AREA AND PRODUCTION OF TARGET

ORCHARDS IN PAKISTAN

2

1.3 FRUIT FACING DECLINE 2

1.3.1 Citrus Decline 2

1.3.2 Mango Decline 4

1.3.2.1 Progressive or slow decline or dieback 4

1.3.2.2 Quick dieback 4

1.3.2.3 Mango sudden death (MSD) 5

1.3.2 Guava Decline 5

1.4 PATHOGENS INVOLVED IN DECLINE 6

1.4.1 Nattrassia mangiferae 6

1.4.2 Synonyms 6

1.4.3 Taxonomy 6

1.4.4 Host Range 7

1.4.5 Symptoms 7

1.4.6 Biology and Management 7

2 OCCURRENCE AND SYMPTOMATOLOGY OF CITRUS,

MANGO AND GUAVA DECLINE IN PUNJAB, PAKISTAN

9

10

2.1 INTRODUCTION 9

2.2 REVIEW OF LITERATURE 12

2.2.1 Decline of Fruit Orchards 12

2.2.1.1 Citrus decline 12

2.2.1.2 Mango decline 14

2.2.1.3 Guava decline 15

2.3 MATERIALS AND METHODS 17

2.3.1 Survey of Target Fruit Orchards for Disease Assessment 17

2.3.1.1 Profile of areas surveyed 18

2.3.1.1.1 Sargodha 18

2.3.1.1.2 Multan 18

2.3.1.1.3 Northern Irrigated Plan (Zone Iva) 19

2.3.2 Disease Incidence 35

2.3.3 Disease Severity 35

2.3.4 Disease Index 35

2.3.5 Symptomatology 36

2.4 RESULTS 36

2.4.1 Disease Assessment 36

2.4.1.1 Citrus decline assessment 36

2.4.1.2 Mango decline assessment 38

2.4.1.3 Guava decline assessment 47

2.4.2 Symptomatology 51

2.5 DISCUSSION 62

3 PREVALENCE OFNATTRASSIASP. FROM CITRUS,

MANGOAND GUAVA ORCHARDS ALONG WITH OTHER

DECLINE CAUSING FUNGI

64

3.1 INTRODUCTION 64


11


3.3.1 Sampling Methodology 68

3.3.2 Isolation and Identification of Pathogens 69

3.3.2.1 Potato dextrose agar medium (PDA) 69

3.3.2.2 Malt extract agar (MEA) 69

3.3.2.3 Stem decoction glucose agar (SDGA) 69

3.3.2.4 Sterilization 70

3.3.2.5 Isolation from infected tissues 70

3.3.3 Microscopy 70

3.3.4 Determination of Fungal Frequency Percantage 71

3.3.5 Purification of Fungal Cultures 71

3.4 RESULTS 71

3.4.1 Plant Parts Wise Fungal Frequency Percentage 71

3.4.1.1 Mycoflora from citrus decline affected trees 71

3.4.1.2 Mycoflora from mango decline affected trees 72

3.4.1.3 Mycoflora from guava decline affected trees 72

3.5 DISCUSSION 93

4 ROLE OFNATTRASSIA MANGIFERAEAND

OTHERDECLINE CAUSING FUNGI IN SYMPTOMS

DEVELOPMENT

97

4.1 INTRODUCTION 97



4.3.1 Pathogenicity Tests 101

4.3.1.1 Flap method 101

4.3.1.2 Root injury method 101

12

4.4 RESULTS 103

4.5 DISCUSSION 111

5 STUDIES ON FACTORS AFFECTING DEVELOPMENT

OFFUNGI WITH SPECIAL REFERENCE TO NATTRASSIA

SP.

114

5.1 INTRODUCTION 114



5.3.1 Effect of Temperature, Light, pH and Culture Media

onFungal Growth

117

5.3.1.1 Effect of culture media 117

5.3.1.2 Effect of temperature 118

5.3.1.3 Effect of light 118

5.3.1.4 Effect of Ph 118

5.4 RESULTS 119

5.4.1 Effect of Culture Media on Radial Mycelial Growth 119

5.4.2 Effect of Temperature on Radial Mycelial Growth 120

5.4.3 Effect of Light on Radial Mycelial Growth 122

5.4.4 Effect of pH on Radial Mycelial Growth 122

5.5 DISCUSSION 129

6 MORPHOLOGICAL VARIABILITY AMONG ISOLATES

OFCOMMONLY FOUND FUNGUS (NATTRASSIA

MANGIFERAE)FROM DECLINE AFFECTED CITRUS,

MANGO AND GUAVA TREES

132




6.3.1 Fungal Isolates 136

6.3.2 Morphological Characterization 136

6.4 RESULTS 136

6.5 DISCUSSION 145

7 FACTORS FAVOURING THE DECLINE DISEASE 147

13

ANDDEVELOPMENT OF STRATEGIES FOR ITS

MANAGEMENT




7.3.1 Surveys for Factor Favoring Decline in Fruit Orchards 150

7.3.2 Statistical Analysis 151

7.4 RESULTS 151

7.5 DISCUSSION 191

8 GENERAL DISCUSSION 198

8.1 ASSESSMENT OF DECLINE IN CITRUS, MANGO

AND GUAVA

198

8.2 COMMON SYMPTOMS OF DECLINE IN CITRUS,

MANGO AND GUAVA

201

8.3 MYCOFLORA ISOLATED FROM DECLINE

AFFECTED TREES

202

8.4

PATHOGENICITY OF THE IMPORTANT DECLINE

CAUSING FUNGI

203

8.5 FACTORS AFFECTING THE GROWTH AND

DEVELOPMENT OF NATTRASSIA SP.

203

8.6 MORPHOLOGICAL CHARACTERISTICS OF

NATTRASSIA SP. FROM CITRUS, MANGO AND

GUAVA

204

SUMMARY 206

LITERATURE CITED 210

14

List of Tables

Table No. Page

2.1 Areas surveyed for citrus decline assessment in district Sargodha 22

2.2 Areas surveyed for mango decline assessment in various districts

of Punjab

25

2.3 Areas surveyed for guava decline assessment in various districts of

Punjab

31

2.4 Mean citrus decline incidence, severity and disease index

indifferent tehsils of district Sargodha

39


indifferent location of tehsil Bhalwal of district Sargodha

39


indifferent location of tehsil KotMomin of district Sargodha

40


indifferent location of tehsil Sargodha of district Sargodha

41


indifferent location of tehsil Sahiwal of district Sargodha

42


indifferent location of tehsil Shahpur of district Sargodha

43


indifferent location of tehsil Sillanwali of district Sargodha

43

2.11 Mean mango decline incidence, severity and disease index

indifferent districts of Punjab province

46


indifferent location of district Khanewal

46


indifferent location of district Multan

48


indifferent location of district Muzaffar Garh

49


indifferent location of district Rahim Yar Khan

49

15


indifferent location of district Bahawalpur

50

2.17 Mean guava decline incidence, severity and disease index

indifferent districts of Punjab province

53


indifferent location of district Kasur

53


indifferent location of district Sheikhupura

54

2.20 Mean guava decline, severity and disease index in

differentlocation of district Nankana Sahib

55

2.21 Decline symptoms development percentages in citrus, mangoand

guava trees

61

4.1 Response of citrus plants against artificial inoculation of N.

mangiferaeandB. theobromaeusing flap method

105

4.2 Response of citrus plants against artificial inoculation of N.

mangiferaeandB. theobromaeusing root injury method

105

4.3 Response of mango plants against artificial inoculation

ofCeratotocystissp.,N. mangiferae,B. theobromaeandFusariumsp.

using flap method

106

4.4 Response of mango plants against artificial inoculation

ofCeratotocystissp.,N. mangiferae,B. theobromaeandFusarium sp.

using root injury method

107

4.5 Pathogenicity of fungi associated with guava decline by usingflap

method inoculation

108

4.6 Pathogenicity of fungi associated with guava decline by usingroot

injury method of inoculation

109

5.1 Effect of culture media on radial mycelial growth of the

fungiisolated from Citrus on PDA medium

121

5.2 Effect of culture media on radial mycelial growth of the

fungiisolated from mango on PDA medium

121

5.3 Effect of temperature on radial mycelial growth of the fungi

isolated from guava on PDA medium

123

16

5.4 Effect of temperature on radial mycelial growth of the fungi

isolated from citrus on PDA medium

123

5.5 Effect of temperature on radial mycelial growth of the

fungiisolated from mango on PDA medium

124

5.6 Effect of temperature on radial mycelial growth of the

fungiisolated from guava on PDA medium

124

5.7 Effect of light on radial mycelial growth of the fungi isolated from

citrus on PDA medium

125

5.8 Effect of light on radial mycelial growth of the fungi isolatedfrom

mango on PDA medium

125

5.9 Effect of light on radial mycelial growth of the fungi isolatedfrom

guava on PDA medium

127

5.10 Effect of pH on radial mycelial growth of the fungi isolated

fromcitrus on PDA medium

127


frommango on PDA medium

128


fromguava on PDA medium

128

6.1 Morphological characteristics of N. mangiferae isolates fromcitrus

in tehsil and district Sargodha

138

6.2 Morphological characteristics of N. mangiferae isolates fromcitrus

in tehsil Bhalwal, district Sargodha

139

6.3 Morphological characteristics of N. mangiferae isolates

frommango in district Multan

140

6.4 Morphological characteristics of N. mangiferae isolates

frommango in district Rahimyar Khan

141

6.5 Morphological characteristics of N. mangiferae isolates fromguava

in tehsil Pattoki of district Kasur

142

6.6 Morphological characteristics of N. mangiferae isolates fromguava

in tehsil Sharaqpur of district Sheikhupura

143

7.1 Factor affecting disease index (%) in selected orchards of citrusin

district Sargodha

152

17

7.2 Correlations of factors causing citrus decline disease index (%) 162

7.3 Correlation between citrus tree age groups with disease index(%) 164

7.4 Correlation between citrus tree age groups with disease index(%) 164

7.5 Correlation between mode of irrigation in citrus orchards

withdisease index (%)

165

7.6 Correlation between nutrition in citrus orchards with diseaseindex

(%)

165

7.7 Correlation between pruning in citrus orchards with diseaseindex

(%)

166

7.8 Correlation between ploughing under tree canopy in citrusorchards

with disease index (%)

166

7.9 Correlation between plant protection in citrus orchards withdisease

index (%)

167

7.10 Factors affecting decline disease index in selected orchards

ofmango growing districts in Punjab

168

7.11 Correlations of factors causing mango decline disease index (%) 174

7.12 Correlation between age group in mango orchards with

diseaseindex (%)

176

7.13 Correlation between intercropping in mango orchards withdisease

index (%)

176

7.14 Correlation between irrigation mode in mango orchards


177

7.15 Correlation between ploughing under tree canopy in citrusorchards

with disease index

177

7.16 Correlation between pruning of trees in mango orchards


178

7.17 Correlation between ploughing under tree canopy in

mangoorchards with disease index (%)

178

7.18 Correlation between plant protection in mango orchards


179

7.19 Factors affecting decline disease index in selected orchards

ofguava growing districts in Punjab

181

18

7.20 Correlations of factors causing guava decline disease index (%) 185

7.21 Correlations of factors causing guava decline disease index (%) 187

7.22 Correlation between intercropping in guava orchards withdisease

index (%)

187

7.23 Correlation between irrigation mode in guava orchards withdisease

index (%)

188

7.24 Correlation between nutrition application in guava orchards


188

7.25 Correlation between pruning of trees in guava orchards


189

7.26 Correlation between ploughing under tree canopy in

guavaorchards with disease index (%)

189

7.27 Correlation between plant protection in guava orchards


190

19

List of Figures

Figure No. Page

2.1 Agro-ecological zones 21

2.2 Areas surveyed for citrus decline in district Sargodha, Punjab,

Pakistan

24

2.3 Areas surveyed for mango decline in district Khanewal, Punjab,

Pakistan

27

2.4 Areas surveyed for mango decline in district Multan, Punjab,

Pakistan

28

2.5 Areas surveyed for mango decline in district Muzaffar Garh, Punjab,

Pakistan

29

2.6 Areas surveyed for mango decline in district R. Y. Khan, Punjab,

Pakistan

30

2.7 Areas surveyed for guava decline in district Kasur, Punjab, Pakistan 32

2.8 Areas surveyed for guava decline in district Sheikhupura, Punjab,

Pakistan

33

2.9 Areas surveyed for guava decline in district Nankana Sahib, Punjab,

Pakistan

34

2.10 Twig dieback in citrus 56

2.11 Bark Splitting and gummosis in citrus 56

2.12 Canker development in citrus 57

2.13 Citrus tree mortality 57

2.14 Bark splitting and oozing of thick darkish brown liquid in Mango 58

2.15 Gummosis and stem canker in mango decline affected tree 58

2.16 Mango Sudden Death affected dead tree (MSD) 59

2.17 Bark splitting and gummosis in decline affected guava tree 59

2.18 Tree canker in decline affected guava tree 60

2.19 Decline affected dead guava tree 60

3.1 Plant parts wise fungal frequency percentage from declineaffected

citrus trees at Chak No. 26/NB tehsil Bhalwal, Sargodha

74

3.2 Plant parts wise fungal frequency percentage from declineaffected 75

20

citrus trees at Chak No. 8 NB, tehsil Bhalwal of districtSargodha


citrus trees at Chak No. 10 N/B, tehsil Bhalwal of districtSargodha

76

3.4 Plant Parts wise fungal frequency Percentage from declineaffected

citrus trees at Chak No. 56/NB of tehsil and districtSargodha

77


citrus trees at Chak No. 112/NB of tehsil and districtSargodha

78


citrus trees at Chak No. 27 SB of tehsil and district,Sargodha

79


mango trees in BastiNandla, Multan

80

3.8 Plant Parts wise fungal frequency Percentage from declineaffected

mango trees in ChahNizamWala, Multan

81


mango trees in Basti Band Bosan, Multan

82


mango trees at Chak 22A, tehsil Liaqatpur of districtRahim Yar

Khan

83


mango trees at Chak 2P, tehsil Khanpur of district RahimYar Khan

84


mango trees at MianwaliQureshian, tehsil and districtRahimYar

Khan

85

3.13 Plant parts wise fungal frequency Percentage from declineaffected

guava trees at Sehjowal, tehsil Pattoki of district Kasur

86


guava trees at Faizpur, tehsil Ferozewala of districtSharaqpur

87


guava trees at AddaPulTorian of tehsil and districtNankana Sahib

88

3.16 Culture and macrospores of Fusarium sp. 89

3.17 Culture and conidia of Ceratocystis sp. 90

3.18 Cultural and conidia of Lasiodiplodia theobromae 91

21

3.19 Culture and spores of Nattrassia mangiferae 92

4.1 Flap method of inoculation. 110

4.2 Root injury method of inoculation. 110

6.1 N. mangiferaearthrospores from citrus. 144

6.2 N. mangiferae arthrospores from mango. 144

22

LIST OF ACRONYMS A Absent Av. Average BE Bhalwal East BW Bhalwal West CJA Carrot Juice Agar D.I. Khan DeraIsmial Khan Eds. Editors GOP Government of Pakistan J. Journal MEA Malt Extrat Agar MSDS Mango Sudden Death Syndrome N Total number of plants NARC National Agricultural Research Centre P Present Pak. Pakistan PARC Pakistan Agricultural Research Council PDA Potato Dextrose Agar PI Pattoki R.Y. Kan Rahimyar Khan Sar Sargodha SDGA Stem Decoction Glucose Agar SP Sharqpur

SW Silanwali

ACKNOWLEDGEMENTS

23

I would like to acknowledge my supervisor Prof. Dr. Tariq Mukhtar for his

constant support, patience, sympathetic attitude, inspiring guidance, constructive

criticism, timely advice and enlightened supervision during my research work and

in the accomplishment of this manuscript.

Thanks to my respected teachers Prof. Dr. Muhammad Irfan Ul Haque and Prof.

Dr. Abdul Rauf Chauhdary for their helpful advice,inputs and encouragement

during the course of my research studies and presentation of this manuscript.

I am deeply thankful to Dr. Munawar Raza Kazmi for his support and

brilliant ideas to meet the objectives of my research.

I would specially like to extend my deepest gratitude to my parents, my

beloved wife and my children for their prayers always remain with me.

Appreciation for my friends Dr. Muhammad Zakria, Dr. Atif Jamal, Mr.

Usman Raja, Dr. ShahzadAsad, Dr. Hussain Shah Hayatullah Tareen and my

officers Dr. Iftikhar Ahmad, Dr. Anjum Munir and Dr. Shahid Hameed who

always encouraged me for thecompletion of my Ph. D.

Finally, I would like to express my deepest gratitude to Ms.

ShumailaIqbal, Dr. TahiraYasmin and Dr. IshaqueMastoi who always backed

me up to complete my research in time.

(FAISAL SOHAIL FATEH)

24

ABSTRACT

Decline of citrus mango and guava is a serious threat in Pakistan. This

study was undertaken to know the role of Nattrassia sp. in fruit orchard decline, its

biology and listing down the management strategies. Its role was studies through

recording the disease incidence and severity from citrus, mango and guava

orchards in Punjab from Sargodha, Bhalwal, Multan, Rahimyar Khan, Pattoki,

Sharqpur and Ahmedpur. Disease prevalence was found in all of the above areas of

citrus mango and guava with less or more intensities. The sampling from target tree

crops resulted in almost similar type of mycoflora except Ceratocystis sp. was

isolated from only decline affected mango trees. Other fungi include Nattrassia

mangiferae, Lasiodiplodia theobromae, Botryosphaeria sp., Fusarium sp.,

Cladosporium sp., Aspergillus sp., Penecillium sp. andCurvularia sp. with different

intensities from affected twigs, branches, stem at collar region and roots. However,

the fungus Nattrassia sp. and Lasiodiplodia sp. were the most common fungi.

Pathogenicity was conducted to know the four decline causing fungi including

Nattrassia sp. It was found that Nattrassia sp. alone cannot cause tree mortality but

contributes towards the mortality in combination with other fungi. The fungus

Nattrassia isolated from citrus is morphologically and physiologically different

from isolates of mango and guava. However, there is resemblance in isolates of

mango and guava. Most of the factors that favored the disease include no concept

of pruning; faulty intercropping; flooding of the orchards; application of partially

decomposed farm yard manure; poor nutrition; inadequate plant protection

measures and less interest of the growers. The orchards can be managed by

25

adopting pruning, following recommended plant protection plans, nutrition based

on leaf, soil and water sampling; avoiding plowing under tree canopies; if

necessary to intercrop choose friendly crops. In short the decline of citrus, mango

and guava have almost common, pathogens, common symptoms, common biology

and hence the common management strategies may be adopted to avoid them in

future.

26

Chapter 1

GENERAL INTRODUCTION

Fruit orchards e.g. apple, pear, peach, grapes, citrus, mango and guava around the

world are facing decline. Symptoms of decline vary with the kind of fruit orchards

as well as the nature of causal organisms involved. The causal agents of decline

reported from around the world include fungi, bacteria, nematode, phytoplasma,

viruses and some nutrient deficiencies (Agrios, 2005).

In Pakistan important fruit trees such as citrus, mango, guava etc are facing

decline. Among these fruits, citrus and mango decline have been addressed

strongly as they are important foreign exchange earnings and export items. Mango

is being given attention in all aspects but citrus needs special attention. Due to poor

quality citrus in Pakistan we have been restricted by export markets. According to

Pakistan Horticulture Export Board, 20% of the citrus fruit is gone waste either due

to diseases or post harvest mishandling (Humayun et al., 2005; Khanzada et al.,

2004). Other fruit trees like guava and loquat are also very famous fruit of Pakistan

and are liked by all generations (Misra and Pandey, 2000).

1.1. EXPORT OF TARGET FRUITS

Pakistan is famous for citrus production especially for its kinnow and

oranges. It ranks sixth in the world with 2.3 million tons production. Pakistan

produces 95% of the world's Kinnow. According to an estimate, the country

exported kinnows amounting to Rs. 15 million thousands in year 2012. By the year

2013 the citrus export target per anum was set a 500,000 tones within the next

fiveyears, and expecting an export income of $300 million but due to some

1

27

keyconstraints that needed to be addressed these ambitious targets were not

achieved (Anonymous, 2013).

Mango ranks second after citrus in Pakistan. It is making its way to

the foreign market by its taste and shapes. The main foreign markets include

United Kingdom, France and Germany which consumes 15 % of mangoes

imported by the EU. Imports of Pakistani mango has increased since 2002 by EU

countries up to 50% i.e. 199,000 tons worth 228 million euros in 2006. The reason

for this growth is that mangoes are not grown in Europe. The major export of

mango is done by India, Mexico, Brazil and Peru. Pakistani mango has a

substantial export potential in foreign markets which has not been realized so far.

Mango exports from Pakistan reached up to Rs. 3.27 million in 2012 (Anonymous,

2013).

1.2.AREA AND PRODUCTION OF TARGET FRUIT ORCHARDS

INPAKISTAN

Citrus, mango and guava are grown on an area of 0.193; 0.175 and

0.064million hectares respectively with the production of 2.40; 1.720 and 0.490

million tons respectively (Anonymous, 2016).Citrus orchards vary from less than

10 heactares to 400 hectares (Ashraf et al., 2015), mango orchards range in size

from less than 2 hectares to 200 hectares (Khan, 2010) while guava orchards range

from less than hectare to 96 hectare (Khushk et al., 2009).

1.3. FRUITS FACING DECLINE

1.3.1. Citrus Decline

28

Citrus decline sometimes also referred to as “Citrus Dieback” which is not a

specific disease but is symptomatic expression of many disorders in the plant. The

malady is also termed as „frenching‟ or „chlorosis (Anonymous, 2000).

Citrus species which are being cultivated in Pakistan include grape-

fruit (Citrus paradisi Macfad.), mandarin (Citrus reticulata Blanco), sweet

Orange (Citrus sinensis (L.) Osbeck), bitter Orange (Citrus aurantium L.),

Lime (Citrus aurantifolia (Christm.) Swingle), Rough Lemon (Citrus

jambhiri Lush.) and Kinnow (it is a Hybrid of Citrus nobilis and Citrus deliciosa).

The kinnow is grown largely in citrus orchards of Pakistan but not a single variety

has shown resistance against decline (Anonymous, 2013).

Scientists during 1970s and 1980s reported that relatively large number of

trees were declining and showed dieback in all the citrus areas visited both on

mandarin and on sweet orange trees. Thus it appears that the decline affecting

citrus is not new. Young decline affected citrus trees grow well initially, and then

begin to show symptoms only after three or four years. (Chapot, 1975; Boveet al.,

1983).

Symptoms of decline included overall pale green-yellowish colour of

foliage; mature leaf drop and replacement of normal-sized leaves by small ones; an

upright position of small leaves along shoots; zinc-deficiency symptoms on small

leaves but without green islands on an overall yellow blade; mild leaf mottle on old

leaves; dieback and stunting (Chapot, 1975).

29

Citrus decline is known to be caused by biotic (fungi, nematodes, viruses,

phytoplasma etc.) and abiotic factors (soil, nutritional and rootstock factors).

Similarlysingle variety cultivation has further aggravated disease situation in

existing citrus gowing areas. It has been observed that decline is much more

prevalent in high rainfall receiving areas. Besides the local diseases, diseases of

international importance like citrus tristeza virus (CTV) and citrus greening have

been reported from the existing citrus growing areas and contribute to citrus

decline. (Naqvi, 2004).

1.3.2. Mango Decline

In case of mango, decline has different forms, generally known as

progressive, slow decline or die back; quick die back and sudden death (Fateh et

al., 2006). The details are given as below.

1.3.2.1. Progressive or slow decline or dieback

In progressive decline, twig and branch dieback is seen on mango trees. It

starts from top of the canopy and progresses towards the lower braches.This affect

the normal flush of trees which are supressed as well as cause leaf disoloration.

Due to weak flushes the canopy seems barren and dead twigs are generally high in

number. This kind of situation is mostly prevalent in poorly managed orchards

where proper nutrition, plant protection and irrigation practices are neglected. Such

kind of decline can be easily managed if cultivating orchards according to best

available production technology (Fateh et al., 2009).

1.3.2.2. Quick dieback

30

In this kind of dieback, trees start losing their shape quickly. Suck kind of

dieback is the indication of either deficiency or toxicity of some elements. Leaves

change colour from green to yellow and drop; the moisture does not reach the

higher branches and trees' tops look dead. This kind of drought situation allows the

opportunistic fungi to invade the plants and in response the plants produce gum of

various colors. The appropriate nutrition, irrigation at required timing as well as

foliar sprays of sustemic fungicides help in managing the quick dieback of mango

trees (Kazmi et al., 2007).

1.3.2.3. Mango sudden death (MSD)

This kind of decline is alarming and is a real threat for the mango growers.

The name "sudden death" given to this disease is tree death visible inshortest time

may be in few days or a month. Once the decline causing pathogens infect trees the

internal break down of the tissues is initiated. However, the symptoms remain

masked. As the environment becomes favourable for the rapid growth of pathogen

and trees lose vigor, healthy looking trees show severe symptoms resulting in

sudden mortality. The symptoms include leaf drying, drooping and sometimes

become leathery. The cracks appear on the stem at collar region, bark splits and

trees ooze dark brown gum. Sometimes thick dark brown liquid is released from

the colar region. In the infected trees when bark is removed from the stem, mouse

grey streaks appear which show that pathogens have fully invaded the trees and its

recovery is unlikely (Fateh et al., 2006).

1.3.3. Guava Decline

Guava Decline has been observed in the areas of sub continent in 1935

before partition. However, it was reported in 1947 for the first time (Naqvi, 2004).

31

The symptoms of guava decline include leaf discoloration, twigs necrosis, branch

splitting, drying of branches mostly at one side of the trees and irregular tree

symmetry. On removal of bark, the stem shows canker. Several fungi have been

reported from guava decline like Lasiodiplodia theobromae, Fusarium sp.,

Botryosphaeria sp. Phytophthora sp. etc. (Bukhari, 2009).

1.4.PATHOGENS INVOLVED IN DECLINE

Decline in different forest and fruit trees involve different pathogens e.g.

fungi such as Ceratocystis fimbriata, Armillaria sp., Nattrassia magniferae,

Lasiodiplodia theobromae, Phoma sp., Phomopsis sp., Fusarium sp., Viruses such

as citrus tristeza virus (CTV), Nematodes especially in case of citrus decline

(Tylenchulus semipenetrans) and in mango (Hemicriconemoides, Longidorus sp.),

bacteria such as Pseudomonas sp. and Phytoplasma. Among these pathogens, the

fungus Nattrassia magniferae has been reported from almost all of the forest and

fruit trees facing decline (Fateh et al., 2006; Bukhari, 2009).

1.4.1. Nattrassia magniferae

1.4.1.1. Synonyms

This fungus has different synonyms e.g. Dothiorella mangiferae,

Exosporina fawcettii, Fusicoccum dimidiatum (Penz.), F. eucalypti, Hendersonula

agathidis, H. cypria Nattrass, H. toruloidea Nattrass, Neofusicoccum mangiferae,

Neoscytalidium dimidiatum, Scytalidium dimidiatum, S. lignicola and Torula

dimidiate (Farr et al., 2005).

1.4.1.2. Taxonomy

32

The Nattrassia mangiferae belongs to Phylum Ascomycota; Class

Ascomycetes; Sub ClassDothidiomycetes; FamilyBotryosphaeriacea; Genus

Nattrassia and Species: N. mangiferae(Farr et al., 2005).

1.4.1.3. Host range

In fruit trees mango, guava, citrus, apple, plum, mulberry, fig and walnut

are the hosts. In forest trees the recorded hosts are Arbutus sp., Euclyptus sp.,

Madrone sp.Besides causing different plant diseases, this fungus is equally harmful

to human beings causing skin diseases (Atta and Aref, 2013).

1.4.1.4. Symptoms

The fungus Nattrassia magniferae causes different symptoms in different

trees e.g. dieback, cankers, blossom blight, wilt, necrosis, decline and death. As

mentioned above the symptoms of slow or quick die back can result from infection

by the fungus N. mangiferae. The symptoms are confused with those produced by

other pathogens such as Botryodiplodia theobromae and Botryosphaeria

sp.Brownish streaks appear when the bark is removed from branches and stem as

well.The trees are apparently healthy, with masked symptoms and produce rotten

fruits (Farr et al., 2005).

1.4.1.5. Biology and management

A very few scientists made efforts to study the biology of this potential

pathogen which is involved in many of the decline diseases in fruits and forest

trees. To devise effective strategies for its management,biology of N.

mangiferaewas studied in South Africa. In this regard many chemicals were

applied in preharvest stage of the trees to see their effect on management of pre and

33

post harvest diseases of citrus fruit. Among all thetested fungicides, pre-harvest

applications of copper-oxychloride on monthly basis during fruit set till harvest

significantly managed the disease (Saimaan, 1997).

Keeping in view importance of the fungus associated with decline of fruit trees,

lack of information on its biology and management, the present studies were

designed with the following objectives:

Determination of prevalence of Nattrassia sp. in target orchards (mango, citrus

and guava) of Punjab.

Studies on the biology and epidemiology of Nattrassia sp. causing decline in

fruit orchards and

Determination of factors causing tree decline for the management of Nattrassia

sp.

34

Chapter 2

OCCURRENCE AND SYMPTOMATOLOGY OF CITRUS,

MANGO AND GUAVA DECLINE IN PUNJAB, PAKISTAN

2.1. INTRODUCTION

Pakistan is an agricultural country. The Gross Domestic Product (GDP) of

Pakistan was 20.9 % in 2014-15. Livelihood of 43.5 % of rural population exists in

agriculture.Enhance productivity and less vulnerability are the main objectives of

food security. To increase economic growth and reducing poverty in Pakistan

through agriculture, a policy framework coupled with best socio political climate,

good governance is fundamental. The government of Pakistan is focusing on

horticulture, livestock and fisheries. Along with developed nations efforts have

been made to improve production technologies and linking farmers directly with

markets and industry (Anonymous, 2015).

Horticulture is the main hub of agricultural economy. Approximately12

million tons of fruits, vegetables and spices are produced on yearly basis. Among

the important fruits citrus contributes2,000,000, mangoes 1,000,000, dates63000

and apples 400,000 tons annually. There are few minor fruits which are harvested

round the year in different geographical zones of Pakistan.

Citrus, mango and guava are grown on an area of 190,000,170,000 and

63000 hectares respectively with the productionsof 240,000, 1,720,000and 490,000

tons respectively (Anonymous, 2016).

Citrus is leading the fruits industry in Pakistan and occupies first position.

Pakistan produces approximately 3 to 4 % of the world citrus and the exports are

9

35

limited to only about 0.8 % of harvest. It is a known fact thatup to 95 % of the

world's kinnow is produced in Pakistan. In Punjab 85% area under citrus is in tehsil

Sargodha and Bhalwal. The rest of 15% is shared by Sillanwali, Sahiwal and

Sahpur tehsils. However, Bhalwal is leading in terms of citrus cultivation. Kinnow

is the famous variety in these areas (Sharif and Waqar, 2005).

In Pakistan, citrus has been planted in the new areas but the average

economic life of citrus is still not comparable with other citrus producing countries.

Fifty years is the average economic life of citrus all around the world. However,

the average life of citrus in Pakistan is about 25 years and trees start to decline after

7-10 years of production (Chaudhry, 2003).

Citrus decline in Pakistan has been thought to be found since 1960s from

the Khyber Pakhtoonkhwah province. However, it was reported in 1970 in the form

of dieback and the causes were attributed to deficiency symptoms and drainage

problems in the orchard (Chapot, 1970).

In citrus, particular decline symptoms are wilt and chlorosis of leaves

followed by leaf and fruit drop. Affected branches show die back starting from top

to the lower branches, a healthy tree suddenly wilts, turns yellow and dies rapidly

(Safdar et al., 2010).

Mango decline in Pakistan was reported from the Punjab province in one of

the orchards at Muzaffar Garh in 1995. The symptoms like leaf drooping, bark

splitting, gummosis, stem canker and stem bleeding appeared. This type of decline

was called quick decline. Twig dieback and the branches dieback are also observed

but that might be due to many reasons (Fateh et al., 2006; Naqvi, 2004).

36

The association of pathogenic fungi and role of bark beetle in mango

decline has been studied in Pakistan as well as few other countries where the

disease has become a dilemma. The most common symptoms produced were leaf

drying, discoloration gum oozing, cankers development under the bark and

vascular blockage. The symptoms intensify when Hypocryphalus

mangiferae,commonly known as bark beetle bores in the stem. Some of local

mango varieties of Pakistan like Malda, Langra and Anwar Ratol show a little bit

tolerance to decline.The mycoflora from decline affected treesand the bark beetle

(H. mangiferae) includedLasiodiplodia theobromae, Ceratocystis fimbriata and

Phomopsis sp. (Masood et al., 2011).

Guava decline have been found existing in India since 1935 but it

wasreported for the first time in 1947. Severely decline affected trees show leaf

drooping and discoloration, dead twigs and rotted, discolored stems. The branches

die on one side of the trees initially and later cover the entire canopy. The tree

mortality occurs with complete defoliation and dry wood.

During a survey of guava growing areas in Sheikhupura district of Punjab,

Pakistan maximum disease incidence was recorded in Tehsil Sharaqpur followed

by Pattoki of district Kasur (Safdar et al., 2015).

As orchards in the Punjab province are declining, trees are becoming

debilitated and less productive, therefore, the first step was planned to conduct

surveys in major areas to determine the incidence and prevalence so that proper

management measures might be suggested to the farmers.

The objectives of this study were as under:

37

1) To assess the disease occurence and severity in the orchards of

citrus, guava and mango from some selected locations famous for the

production of the commodity in the province of Punjab.

2) To find out the similarity of symptoms of citrus, mango and guava

decline affected trees.

2.2. REVIEW OF LITERATURE

2.2.1. Decline of Fruit Orchards

Many fruit orchards in Pakistan have prevalence of decline disease

including citrus, mango, guava, loquat and pomegranate. However, special

importance is given to citrus, mango and guava decline being the most important

fruit crops of Pakistan.

2.2.1.1. Citrus decline

Citrus decline is a global problem in citrus growing areas. Mostly the

mature trees become victim of decline. The recovery of an infected tree is quite

difficult. Twig blight of citrus is mostly confined to tree canopies but its symptoms

resemble with other internationally important diseases like greening, CTV and slow

decline (caused by nematode Radophilus citrophilus) in Florida (EPPO/CABI,

1996a, b and c). The decline affected citrus trees show zinc deficiency. The

significance of high levels of zinc is not known but its analysis may be helpful for

diagnosis of decline (Young et al., 1980a and b).

Other than apparent symptoms, there is also change in tree physiology.

Studies have shown that in decline affected citrus trees water conductivity in the

xylem is impaired. The trees failed to intake water and food because the xylem was

38

blocked by light-yellow dark-brown filamentous plugs known as tylosis. The

dieback symptoms appear due to lack of water transport to the canopy (Brlansky et

al., 1985).

Most of the scientists thought that it was citrus tristeza virus which caused

citrus decline and they did research with this vision. A survey was conducted in

citrus growing areas of Khyber Pakhtunkhwah province of Pakistan to assess

various citrus diseases. Most common diseases in citrus orchards and nurseries

included citrus tristeza, citrus variegation, citrus exocortis, citrus cachexia

(xyloprosis) and citrus greening. High incidence of these devastating pathogens has

caused the severe citrus decline, drastic yield and quality losses in citrus fruits in

the region (Arif et al., 2005).

Another school of thought said that phytoplasma and bacteria are involved

in citrus decline. Burney et al. (2007) during a survey of citrus growing areas in

Punjab found that citrus decline was a result of combination of bacteria and

phytoplasma. The leaf sample showed that Xanthomonas compestris is the most

prevalent bacterium with 50% incidence in Jhang, 17.24% in Sahiwal and no

incidence was found in Faisalabad, Toba Tek singh and Kasur. Phytoplasmal

disease incidence was found to be 27.58% in Sahiwal, 15.62% in Toba Tek Singh

while it was not encountered in Jhang.

The fungal involvement in citrus decline was found in Oman as well.

During survey conducted in various citrus growing districts, it was observed that

citrus varieties i.e. acid limes and sweet limes exhibited gummosis at collar region.

The isolation from bark and roots resulted in 19 fungal species. Among these the

39

most common were Lasiodiplodia hormozganensis, L. theobromae and Fusarium

solani. On artificial inoculation of these common fungi, acid lime and sweet lime

seedlings reproduced decline and gummosis symptoms. The nursery studies on

sweet lime showed association of 12 fungal species, which is evidence that

nurseries act as a main source for some citrus pathogens (Al Sadi et al., 2010).

During a survey in Pakistan from citrus growing districts especially

Sargodha and Faisalabad, decline incidence was found higher in Sargodha except

Sillanwali and Kot Momin tehsils. The isolated fungi from decline affected tree

tissue and root samples were Fusarium semitectum, F.solani, Helminthosporium

sp. and Phytophthora sp., with varying frequencies. (Safdar etal., 2010).

Nematologists said that these are nematodes causing citrus decline.

Khanzada et al. (2007) conducted surveys in different districts of Punjab and

collected roots and soil samples from the orchards showing decline symptoms. In

all areas the incidence of Tylenchulus semipenetrans, the cause of slow decline was

the maximum (56%) in variety Feutrell‟s Early followed by 40% in lemon and

orange, 35% in Kinnow and Musambi. The minimum incidence (20%) was

observed in grape fruit. A survey in Punjab showed that the nematode (T.

semipenetrans) population was recorded above economic threshold level in all

citrus growing tehsils of Sargodha districts and this might also contributing towards

citrus decline (Safdar etal., 2010).

2.2.1.2. Mango decline

Mango decline is prevalent in the world especially in Brazil, Oman,

Pakistan and India. A fungus Diplodia theobromae associated with sudden decline

40

of mango for the first time was reported in Oman. During 1999 up to 60% of trees

in parts of the Al Batinah region had died of strange wilt disease. Trees showed

gummosis, wilting, browning of leaves and died in 5-6 months after appearance of

first symptoms. Dead wood was dark brown. It was thought that a bark beetle

(Cryphalus scabrecollis), is responsible for spread of the disease (Al-Adawi et al.,

2002).

The etiology of mango sudden death in Pakistan was first time reported in

Pakistan during 2005. Incidence of the disease was high in mango growing areas of

province Sindh as compared to the Punjab province. The mycoflora isolated

included Botryodiplodia theobromae, Phytophthora sp., and Fusarium sp. In the

mean time a new fungus was reported in Oman during 2006, known as

Ceratocystis fimbriata which attacked mango in combination with L. theobromae.

Later it was reported new species Ceratocystis omanensis (Al- Adawi et al., 2006).

In Pakistan the fungus C. fimbriata for the first time was reported in Sindh

from declining mango trees. It's identification was confirmed by morphological

characteristics of perithecia (brown to black with globose base, necks almost 800-

900 µm long with ostiolar hyphae), ascospores (elliptical 4-8 × 2-5 µm, hat shaped)

conidiophores (hyaline, septate up to 150 µm long and conidia that were

cylindrical, sometimes in chains and truncated at the ends (Fateh et al., 2006).

2.2.1.3. Guava decline

Guava decline is also termed as guava wilt; Rosemary (2001) reported that

in guava decline leaves start wilting from the top of the tree. Sometimes, it is a

rapid decline and sometimes it is a slow decline. There is general wilting of all the

41

leaves. The leaves become yellow or purple and drop. During the initial stages, the

leaves dry out and often remain on the tree. If there are fruit on the tree, fruit

development ceases immediately, and you get mummified fruit on the tree. Guava

wilt disease killed off orchard trees randomly, and at the end, there would be no

orchard left. The original research concentrated on trying to control the disease

with chemicals, but was totally ineffective. There are no chemicals available to

control the disease.

Guava decline is distributed in many countries of the world. Most of the

guava is cultivated in India (where it is known as poor men‟s apple) and Pakistan.

Guava decline in India is considered a soil borne disease associated with

association of several pathogens. Among most common pathogens, Fusarium

oxysporum f. sp. psidii and F. solani were reported. A new potential fungus

Gliocladium roseum was also identified. Heavy losses in guava production in India

have been recorded due to this disease (Misra, 2007).

In South Africa guava decline has been reported to cause by a fungus

identified as Penicillium vermoesenii. Earlier, the fungus has been associated with

blight of ornamental palms in the United States and Belgium. It was later reported

from guava in the above mentioned countries. The Koch's postulates confirmed the

pathogenicity of the fungus in the glasshouse and field conditions by producing

symptoms like wilting, chlorosis and defoliation in nursery seedling as well as

branches of mature trees (Schoeman et al., 2008).

Avelar-Mejia et al. (2003) described the effect of guava tree decline on the

anatomy of branches and roots of guava. Guava tree decline in Calvillo,

42

Aguascalientes and Apozol, Zacatecas, Mexico did not affect the anatomy of the

branches of the tree. The disease was associated with root histological alterations

caused by Meloidogyne sp. infestation. The bark, phloem and vascular cambium

tissues showed hyperplasia and hypertrophy, as well as an increase in polyphenol

content and necrosis. The decline was related with an increase in lignin and

polyphenol content, and with the reduction in root starch. Fungi were observed in

the xylem and cambium of severely affected trees, although the genus and species

were not determined. The partial plugging of xylem vessels was not related with

the decline.

During a survey in one of the guava growing districts of the Punjab

province of Pakistan i.e. Sheikhupura, decline disease incidence was reported as

high as 36% inTehsil Sharaqpur. The isolated fungi included B. theobromae,

Fusarium oxysporum f.sp. psidii, Phytophthora parasitica, F. solani,

Helminthosporium sp. and Curvularia lunata. The pathogenicity of most

dominating fungus (B. theobromae) was confirmed on green house potted guava

plants of 2 years (Safdar et al., 2015).

2.3. MATERIALS AND METHODS

2.3.1.Survey of Fruit Orchards for Disease Assessment

According to world map Pakistan is located between 24o to 37

o latitudes in

north and 61o to 75

o longitudes in east. It is spread over 1600 kilometres from north

to south and from east to west 885 kilometres. The total area of the country is 796,

095 square kilometres. Pakistan has best climate for agriculture ranging from

subtropical to semi-arid. The per anum rainfall ranges from 125 mm (extreme

southern plains) to 900 mm in the mountainous and northern plains. Monsoon

43

season is known for heavy rainfall and receives 70 % of the total rainfall in

generarlly from month of July to September. The remaining 30 % rainfall is in

winter. In plains summers is extreme with maximum temperature more than 40 °C,

while the temperature in winter is a few degrees above the freezing point.

2.3.1.1. Profile of areas surveyed

A brief description of the areas surveyed for incidence and prevalence of decline is

given below.

2.3.1.1.1. Sargodha

Sargodha is known as Pakistan's best citrus-producing area. It is situated

220 kilometers from Islamabad, the capital. Summer season in the district is hot

and temperature may reach to 50 oC (122

oF) while, in winter temperature can be as

low as 2-3 oC. The climate of the district is quite suitable for agriculture;

wheat, rice, and sugarcane are its main field crops. However, Sargodha district is

famous for citrus (Kinnow) production. The district Sargodha comprises of six

tehsils i.e. Sargodha, Bhalwal, Sillanwali, Shahpur, Sahiwal and Kot Momin.

2.3.1.1.2 Multan

Multan is famous for saints and mango. There are many shrines of saints in

the district. Many crops are grown in Multan but the only crop linked with its name

is mango. Flat and alluvial plains are best combination for cultivation of fruit crops.

The canal system running in Multan brings soil from distant places and makes the

land more fertile.

Multan has very harsh climate in summer and the temperature exceeds even

52 oC (126

oF) whereas, in extreme winter, the minimum recorded temperature is

44

approximately −1 oC (30

oF). Average rainfall is about 186 mm. Multan is also

popular for its dust storms.

Basically, Pakistan is divided into ten agro-ecological zones depending

upon climate, land utilization and availability of water (Fig.2.1). Maximum fruits

and other field crops in Pakistan are cultivated in northern irrigated plan (Zone

IVa) among other agro-ecological zones. Following are the characteristics of Zone

IVa.

2.3.1.1.3 Northern irrigated plain (Zone IVa)

Northern Irrigated Plainis mostly characterized as flood plain with semi-

arid to arid climate. The eastern side receives more average per annum rainfall (300

to 500 mm) than southwest (200 to 300 mm). The soils range from sandy, loam-

clay to loam. The canal system in the northern part helps in cultivation of irrigated

crops like wheat, rice, sugar cane and millets. The south is suitable for cotton,

sugar cane, maize, citrus and mangoes (PARC, 1998).

Survey for the current study was planned in such a way that the areas which

were best known for the target host plants must be visited. The survey was

conducted in the citrus, mango and guava orchards for disease estimation as well as

sampling. For citrus the survey was conducted in tehsils of district Sargodha. In

Sargodha district a total of 88 locations with three orchards from each location

were surveyed (Table 2.1). In each orchard randomly 5 out of 40 trees were

observed. Hence a total of 264 citrus orchards were surveyed and a total of 1320

trees were observed for disease estimation and sampling. The orchards were

surveyed using double diagonal method. Randomly five trees from an orchard were

observed for recording the data.

45

For mango, the surveys were conducted in 5 districts of Punjab viz.

Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur. In total 50

locations were visited, in each location 3 mango orchards were surveyed and in

each orchard 5 out of 35 trees were observed. A total of 150 mango orchards were

surveyedand 750 mango trees were observed (Table 2.2).For disease assessment in

guava, surveys were done in disttrict Kasur,Sheikhupura and Nankana Sahib. In

total 26 locations and in each locationthree orchards were surveyed. Similarly from

each orchard, 5 trees out of 40 were observed for the disease assessment and

sample collection. Thus a total of 90 orchards and 390 trees wereobserved (Table

2.3).

46

I: Indus Delta

II: Southern Irrigated

III (a & b) Sandy Desert

IV (a & b) Nothern Irrigated Plains

V Barani Lands

VI Wet Mountains

VII Northern Dry Mountains

VIII Western Dry Mountains

IX Dry Western Plateau

X Suleman Piedmont

Fig. 2.1: Agro-ecological zones of Pakistan

47

Table 2.1: Areas surveyed for citrus decline assessment in district Sargodha.

S. No. Tehsils Localities

1 Bhalwal Chak No. 4 SB, 7 ML, 7SB, 7 ASB 8NB, 9

NB, Chak 9 Lokri, 10 NB, 13 NB, 18 NB, 22

NB, 23 NB, 26 NB

2 Sargodha Chak No. 24 SB, 27 SB, 28 SB, 30 NB, 48

NB, 53 SB, 56 NB, 90 NB, 91 NB, 93 SB, 94

BN, 95 NB, 95SB, 101 SB, 112 NB, 115 SB,

and 122 SB.

3 Sillanwanli Chak No. 118 NB, 119 SB, 120SB, 122 SB,

4123 SB, 124 SB, 127 SB, 127 NB,137 SB,

147 NB and 148 NB.

4 Shahpur Malakwal, Wadhi, kot Maghrib, Shahpur

Saddar, Noor Kallu, Kandaan Kalan,

Jhavarian, Hussain Shah, Kudyana, Allahdad

Wala, Chachar Sharf, Chak 128 NB

5 Sahiwal Kot Pehalwan, Biral Sharif, Nawabpur, Vijh,

Nehang, Chohal, Tirkhanwala, Haveli

Majuka, Pindi Wala, Sial Sharif, Sangoraka,

Farooka, Sial Dholka, Muhammad Wala,

Jahane Wala, Dherowal, Chatror and Radhan

48

S. No. Tehsils Localities

6 Kot Momin Chak No. 9 SB, 19 SB, 20 SB, 21 SB, 65 SB,

66 SB, Rawan, Dera Thoye Wala, Ghulapur

Bangla, Jalla Makhdum, Takht Hazara,

Naseerpur Kalan, Mateela, Dodha and Midh

Road

49

Fig. 2. 2: Areas surveyed for citrus decline in district Sargodha, Punjab, Pakistan

50

Table 2.2: Areas surveyed for mango decline assessment in various districts of

Punjab.

S. No. District Tehsils Localities

1 Khanewal Kabirwala Qadirpur Rawan, 5 Kassi,

Solgi, Matti Tal, Abbas Pur,

8 Kassi, Maula Pur, Bilawal

Pur, Basti Toheed Nagar and

Hassan Pur

2 Multan Multan Qasim Bela, Nandla, Chah

Nizam Wala, and Basti Band

Bosan.

Shujabad Basti Khokhran, Shahpur

Ubbha, Abbas Pura

Jalalpur Pirwala Ghazipur, Basti Malkani,

Manik Wali

3 Muzaffar Garh Muzaffar Garh Makhan Bela, Rohillanwali,

Shah Jamali

Kot Addu Ali Wala, Musay Wala,

Shuhrat Wala, Basti Drigh

Alipur Muradpur Pull, Basti

51


Nukray, Basti Jat Lashari

4 Rahim Yar Khan Rahim Yar Khan Taranda Muhammad Panah,

Wahi Shah Muhammad,

Mianwali Qureshian

Sadiqabad Ahmad Pur Lumma

Khan Pur Chak 2P, Chak 3 P, Bagh o

Bahar,

Liaqat Pur Chak 22 A, Chak 23 A,

Islam Nagar

5 Bahawalpur Bahawalpur Khanqah Sharif, Chak 13

BC, Chak 23 BC, Nowshera,

Munshi Wala

52

Fig. 2. 3: Areas surveyed for mango decline in district Khanewal, Punjab, Pakistan

53

Fig. 2. 4: Areas surveyed for mango decline in district Multan, Punjab, Pakistan

54

Fig. 2. 5: Areas surveyed for mango decline in district Muzaffar Garh, Punjab,

Pakistan

55

Fig. 2. 6: Areas surveyed for mango decline in district R. Y. Khan, Punjab,

Pakistan

56

Table 2.3: Areas surveyed for guava decline assessment in various districts of

Punjab.


1 Kasur Kasur Qaisar Garh, Dolaywala,

Noor Shah Wali, New City

Kasur, Sadar Diwan

Chuniyan Khara, Kot Gurdas Wala

Pattoki Gehlan Pathak, Rukan Pura,

Sehjowal

3 Sheikhupura Ferozewala Faizpur interchange, Adda

Thabal, Noor Shah, Burj

Attari, Saggian Khurd,

Thikriwala

Sharaqpur Sharaqpur, Ghareebabad,

Sukhanwala, Kot Mahmood

4 Nankana Sahib Nankana Sahib Jalal Nou, Giller Wala,

Magtan Wala, Adda Pul

Torian, Chachkay Gill,

Mirza Pur, Mandi Faziabad

and Mor Khunda

57

Fig. 2. 7: Areas surveyed for guava decline in district Kasur, Punjab, Pakistan

58

Fig. 2. 8: Areas surveyed for guava decline in district Sheikhupura, Punjab,

Pakistan

59

Fig. 2. 9: Areas surveyed for guava decline in district Nankana Sahib, Punjab,

Pakistan

60

Prevalence, incidence and severity data were recorded to calculate the disease

index % age. Others parameters were also recorded which included tree age, tree

height, variety, damage, symptom of the decline or sudden death, irrigation and

fertilizer regimes etc.

2.3.2. Disease Incidence

The incidence of decline in citrus, mango and guava was calculated by the

formula (Rehman et al., 2011 a).

Disease Incidence (%)= Number of declined plants× 100

Total number of plants observed

2.3.3. Disease Severity

Disease Severity of mango, citrus and guava decline was recorded using 0-5

visual rating scale(Kazmi et al., 2005). Where:

0=Healthy Plants; 1=1-10%decline; 2=11-20%; 3=21-30%, 4=31-50% 5=More

than 50%.

2.3.4. Disease Index

Disease Index gives the exact status of the disease combining the disease

incidence and severity (Kazmi et al., 2005).

Disease Index (%) = 0(n1)+1(n2)+2(n3)+3(n4)+4(n5)+5(n6)× 100

N 5

Where

n1: No. of trees in 0 rating



61




N: Total Number of Trees

2.3.5. Symptomatology

Symptoms were carefully observed from each decline affected citrus,

mango and guava trees. For current study, 50 trees each of citrus, mango and guava

were selected and the %age of trees showing decline symptoms such as gummosis,

bark splitting, die back, canker formation, stem bleeding etc was calculated.

2.4. RESULTS

2.4.1. Disease Assessment

As a result of survey for the disease assessment and observation taken, the

disease incidence, severity and disease index was calculated as follows:

2.4.1.1. Citrus decline assessment

Disease incidence, severity and disease index were found to be variable in

district Sargodha. Maximum mean disease incidence was recorded in tehsil

Sargodha (94.06%) followed by tehsil Shahpur (93.33%) and Sahiwal (73.33%),

while it was the minimum in tehsil Sillanwali (35.73%). Similarly, mean disease

severity was the maximum in tehsil Sargodha (1.47) and the minimum (0.64)

wasobserved in Sillanwali. The rest of the tehsils has intermediate disease situation.

Likewise, maximum disease index was observed in tehsil Sargodha (29.41%)

followed by tehsil Shahpur (26.75%) and Bhalwal (23.77%) On the other hand

62

minimum disease index was found in tehsil Sillanwali (10.36%) as shown in table

2.4.

In tehsil Bhalwal of Sargodha almost 54% of the locations had 100%

disease incidence. Similarly, 61.5% locations fell into disease severity rating of "2"

which was the maximum rating observed in any location of tehsil Bhalwal (Table

2.5).

In tehsil Kot Momin 27% of the locations showed 100% disease incidence

while 80% locations had disease severity rating of "1", maximum rating observed

in any location of tehsil Kot Momin. Disease index ranged from 12-28% as shown

in Table 2.6.

In tehsil Sargodha, 71% of the locations observed had 100% disease

incidence and in 47% locations maximum disease severity rating "2" was recorded.

Disease index ranged from 20-40% (Table 2.7). In tehsil Sahiwal, 61% locations

observed had 100% disease incidence; 11% locations showed disease severity

rating "2" (maximum). Disease index ranged from 12-32% as shown in Table 2.8.

In Shahpur tehsil 58% locations had 100% disease incidence and 25%

locations showed maximum disease severity rating "2" while disease index ranged

from 19-35% (Table 2.9). Similarly, in Sillanwali only one location had 100%

disease incidence maximum disease severity rating was "2". Disease index

however, ranged from 4-32% (Table 2.10)where growers are following integrated

orchard management approach (Fateh et al., 2017).

Generally disease index of more than 15% of any disease at any location or

any host is considered alarming. However, in district Sargodha overall disease

index ranged from minimum 10.36 to maximum 29.41% which is a real panic for

63

the growers as well as entire citrus industry and requires the immediate attention of

the scientists and other stakeholders. There is lack of value chain and supply chain

studies and training of the growers‟ at large scale based on the best available

integrated approaches. Nominal quarantine measures in the country has also

aggravated the disease situation which renders the growers to either change their

crops or they sell their properties to land mafia for housing societies. It is

concluded that the situation of citrus decline is alarming in district Sargodha and

almost every orchard faced the problem. The situation is alarming and warrants

strict control strategies for its management. It is also emphasized that the farming

community be trained in this regard.

2.4.1.2. Mango decline assessment

According to results shown in Table 2.11, maximum mean disease severity

2.70 was observed in district Bahawalpur followed by Khanewal (2.30), Multan

(2.11), Muzaffar Garh (2.00). Minimum disease severity (1.90) was recorded in

Rahim Yar Khan District.

Similarly maximum mean disease incidence (100%) was found in

Khanewal followed by Bahawalpur (99.30%), Muzaffar Garh (96.00%), Multan

(96.00%) and minimum disease incidence (90.00%) was observed in Rahim Yar

Khan.Disease index which gives the actual picture of the disease was found to be

the maximum in Bahawalpur (53.30%) followed by Khanewal (46.50%), Rahim

Yar Khan (39.10%), Muzaffar Garh (37.30%) while minimum disease index 36.89

was found in Multan district (Table 2.11).

According to table 2.12, in tehsil Kabirwala of district Khanewal mean

disease severity raged from 2-3 based on 0-5 rating scale. Maximum mean disease

64

Table 2.4: Mean citrus decline incidence, severity and disease index in different

tehsils of district Sargodha.

Sr.

No.

Tehsil Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Bhalwal 1.38 72.85 23.77

2 Kot Momin 0.80 64.87 15.80

3 Sargodha 1.47 94.06 29.41

4 Sahiwal 0.89 73.33 19.28

5 Shahpur 1.25 93.33 26.75

6 Sillanwali 0.64 35.73 10.36

Data are means of five replicates


locations of tehsil Bhalwal of district Sargodha.

Sr.

No.

Location Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

L Chak No. 4 SB 2 100 31

2 Chak No. 7 ML 2 100 32

3 Chak No. 7SB 2 100 40

4 Chak No. 7 ASB 2 100 37

5 Chak No. 8NB 2 100 33

6 Chak No. 9 NB 1 67 15

7 Chak No. Chak 9 Lokri 2 100 36

8 Chak No. 10 NB 0 0 0

9 Chak No. 13 NB 2 93 31

10 Chak No. 18 NB 2 100 37

11 Chak No. 22 NB 0 0 0

12 Chak No. 23 NB 1 87 17

13 Chak No. 26 NB 0 0 0


65


locations of tehsil Kot Momin of district Sargodha.

Sr.

No.

Location Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Chak No. 9 SB 0 0 0 2 19 SB 0 0 0

3 20 SB 1 60 12

4 21 SB 1 80 19

5 65 SB 1 73 16

6 66 SB 1 87 23

7 Rawan 0 0 0

8 Dera Thoye Wala 1 100 24

9 Ghulapur Bangla 1 100 29

10 Jalla Makhdum 1 80 16

11 Takht Hazara 1 60 12

12 Naseerpur Kalan 1 73 17

13 Mateela 1 100 25

14 Dodha 1 100 28

15 Midh Road 1 60 16


66


locations of tehsil Sargodha of district Sargodha.

Sr.

No.

Location Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Chak No. 24 SB 1 80 20

2 Chak No 27 SB 2 100 37

3 Chak No 28 SB 1 73 20

4 Chak No 30 NB 1 100 27

5 Chak No 48 NB 1 100 29

6 Chak No 53 SB 2 100 31

7 Chak No 56 NB 1 100 27

8 Chak No 90 NB 2 100 32

9 Chak No 91 NB 1 73 24

10 Chak No 93 SB 2 100 40

11 Chak No 94 NB 2 100 37

12 Chak No 95 NB 2 100 33

13 Chak No 95SB 2 100 32

14 Chak No 101 SB 1 73 24

15 Chak No 112 NB 1 100 29

16 Chak No 115 SB 2 100 31

17 Chak No 122 SB 1 100 27


67


locations of tehsil Sahiwal of district Sargodha.

Sr.

No.

Location Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Kot Pehalwan 1 100 28

2 Biral Sharif 1 93 25

3 Nawabpur 1 100 24

4 Vijh 1 100 28

5 Nehang 0 0 0

6 Chohal 1 100 23

7 Tirkhanwala 0 0 0

8 Haveli Majuka 2 100 32

9 Pindi Wala 1 100 24

10 Sial Sharif 2 100 31

11 Sangoraka 1 60 12

12 Farooka 1 67 16

13 Sial Dholka 1 100 23

14 Muhammad Wala 1 100 24

15 Jahane Wala 0 0 0

16 Dherowal 1 100 28

17 Chatror 1 100 29

18 Radhan 0 0 0


68


locations of tehsil Shahpur of district Sargodha.

Sr.

No.

Location Mean

Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Malakwal 1 67 19 2 Wadhi 2 87 35

3 Kot Maghrib 1 93 20

4 Shahpur Saddar 1 100 27

5 Noor Kallu 1 100 27

6 Kandaan Kalan 2 100 31

7 Jhavarian 1 100 28

8 Hussain Shah 1 80 25

9 Kudyana 1 93 23

10 Allahdad Wala 1 100 25

11 Chachar Sharf 1 100 28

12 Chak 128 NB 2 100 33



locations of tehsil Sillanwali of district Sargodha.

Sr. No. Location Mean Disease

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Chak No. 118 NB 1 33 15 2 Chak No. 119 SB 1 73 24 3 Chak No. 120SB 0 0 0 4 Chak No. 122 NB 1 53 12 5 Chak No. 123 SB 2 100 32 6 Chak No. 124 SB 0 0 0 7 Chak No. 127 SB 0 20 4 8 Chak No. 127 NB 1 67 15 9 Chak No. 137 SB 0 0 0 10 Chak No. 147 NB 0 0 0 11 Chak No. 148 NB 1 47 12


69

severity "3"was recorded in Matti Tal, Bilawal Pur and Basti Toheed Nagar

followed by disease severity "2" in Qadirpur Rawan, 5 Kassi, Solgi, Abbas Pur, 8

Kassi, Maula Pur and Hassan Pur. Mean disease incidence observed was 100% in

all orchards observed in all locations. However, maximum disease index 65% was

observed in Basti Toheed Nagar followed by 57% in Bilawal Pur, 51% in Matti

Tal, 49% in Maula Pur, 47% in 8 Kassi, 41% in Solgi, 40% in Abbas Pur, 39% in

Qadirpur Rawan and 5 Kassi and minimum 37% in Hassan Pur.

Survey condcuted in 3 tehsils of Multan showed mean disease severity

ranging from 1-3. Maximum mean disease severity "3"was observed in Chah

Nizam Wala (Tehsil & district Multan) and Ghazi Pur (Tehsil Jalalpur Pirwala,

district Multan) followed by disease severity "2" in Nandla, Basti Band Bosan

(Tehsil Multan); Basti Khokhran, Shahpur Ubbha, Abbas Pura (Tehsil Shujabad)

and Manik Wali ( Tehsil Jalal Pur Pirwala). Minimum mean disease severity "1"

was observed in Qasim Bela of Multan. Mean disease incidence was 100% in all

orchards except Qasim Bela and Nandla in tehsil Multan where it was 67%.

Disease index was maximum in 52% in Chah Nizam Wala (Multan) and Ghazipur

(Jalalpur Pirwala) followed by 40% in Shahpur Ubbha and Abbas Pura (Shujabad),

39% in Manik Wali (Jalalpur Pirwala), 33% Bast Band Bosan (Multan), 32% Basti

Khokhran ( Shujabad), 31% Nandla (Multan) and minimum 13% in Qasim Bela of

tehsil Multan (Table 2.13).

In Muzaffar Garh district, 3 tehsils were covered i.e. Muzaffar Garh, Kot

Addu and Ali Pur. Mean disease severity ranged from 1-3. Maximum mean disease

severity "3" was recorded in Basti Drigh of Muzaffar Garh tehsil and Basti Nukray

of Alipur tehsil followed by disease severity "2" in Makhan Bela, Rohillanwali,

70

Shah Jamali (Tehsil Muzaffar Garh), Shuhrat Wala (Kot Addu), Muradpur Pull and

Basti Jat Lashri (Ali Pur). Minimum mean disease severity "1" was observed in Ali

Wala and Musay Wala of tehsil Kot Addu, District Muzaffar Garh. Mean disease

incidence 100% was observed in Rohillanwali, Shah Jamali (Muzaffar Garh); Ali

Wala and Shuhrat Wala, Basti Drigh (Kot Addu) and Murad Pur Pull, Basti Nukray

and Basti Jatt Lashri of tehsil Ali Pur. Maximum disease index (52%) was

calculated in Basti Drigh of Kot Addu tehsil and Basti Nukray of Ali Pur tehsil

followed by 44% in Makhan Bela and Shah Jamali (Muzaffar Garh); 41% Basti Jat

Lashari (Ali Pur), 40% Shuhratt Wala (Kot Addu); 31% Muradpur Pull (Ali Pur);

29% Ali Wala (Kot Addu), 27 % Rohillanwali (Muzaffar Garh) and minimum 13%

Musay walla (Kot Addu) (Table 2.14).

In district Rahim Yar Khan 4 tehsils i.e. Rahim Yar Khan, Sadiqabad, Khan

Pur and Liqat Pur were surveyed. Maximum mean disease severity "3" was

observed in Wahi Shah Muhammad of tehsil Rahim Yar Khan only followed by

disease severity "2" in all locations. Disease incidence was 100% in all

locationssurveyed. However, maximum disease index (52%) was found in Wahi

Shah Muhammad (Rahim Yar Khan) followed by 49% in Chak 23 A (Liaqat Pur);

48% Chak 2P (Khan Pur). and Islam Nagar (Liaqat Pur); minimum 37 % in

Taranda Muhammad Panah & Mianwali Qureshian of tehsil Rahim Yar Khan

(Table 2.15).

Tehsil Bahawalur and Ahmedpur East were surveyed in the district of

Bahawalpur. Mean disease severity ranged from 2-4 based on 0-5 rating scale.

Maximum mean disease severity "4" was recorded in Chak 23 BC of tehsil

Bahawalpur followed by "3" in Khanqah Sharif, Chak 13 BC, Munshi Wala (Baha-

71

Table 2.11: Mean mango decline incidence, severity and disease index in different

districts of Punjab province.

Sr. No. District Mean Disease

Severity

(0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Khanewal 2.30 100 46.50 2 Multan 2.11 92.67 36.89 3 Muzaffar Garh 2.00 96.00 37.30 4 Rahim Yar Khan 1.90 90.00 39.10

5 Bahawalpur 2.70 99.30 53.30 Data are means of fivereplicates


location of district Khanewal.

Sr. No. Tehsil Location Mean Disease

Severity (0-5)

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index (%)

1 Kabirwala

Qadirpur

Rawan

2 100 39 5 Kassi 2 100 39 Solgi 2 100 41 Matti Tal 3 100 51 Abbas Pur 2 100 40 8 Kassi 2 100 47 Maula Pur 2 100 49 Bilawal Pur 3 100 57 Basti

Toheed

Nagar and

3 100 65 Hassan Pur 2 100 37

Data are means of fivereplicates

72

Walpur) and Mehrab Wala and Basti Johnan (Ahmedpur East). Minimum mean

disease intensity "2" was recorded in Nowshera (Bahawalpur) and Channi Goth,

Muhabbat Pur and Basti Khokhran of tehsil Ahmedpur East. Mean disease

incidence was 100% in all locations except in Khanqah Sharif (Bahawalpur) which

was 93%. Maximum disease index 76% was observed in Chak 23 BC

(Bahawalpur) followed by 61% in Basti Johnan and it was minimum in Channi

Goth as shown in Table 2.16.

2.4.1.3 Guava decline assessment

During the decline assessment in guava, results showed that among three

guava growing districts, the maximum severity was recorded in district

Sheikhupura (0.90) followed by district Kasur (0.78), while, the minimum

severitywas observed in district Nankana Sahib (0.57).

Maximum guava decline incidence was found in district Kasur (62.89%)

followed by Sheikhpura (61.30%) whereas it was minimum in district Nankana

Sahib (56.29%).

However, a maximum disease index of 18% was recorded in district Kasur

followed by Sheikhpura but the minimum disease index of 14% was observed in

district Nankana Sahib (Table 2.17).

73


locations of district Multan.


Severity (0-5)

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index

(%) 1 Multan Qasim Bela 1 67 13

Nandla 2 67 31

Chah

Nizam

Wala

3 100 52

Basti Band

Bosan

2 100 33

2 Shujabad Basti

Khokhran

2 100 32

Shahpur

Ubbha

2 100 40

Abbas Pura 2 100 40

3 Jalalpur

Pirwala

Ghazipur 3 100 52

Manik Wali 2 100 39


74


locations of district Muzaffar Garh.


Severity (0-5)

Severity (0-5)

Mean Disease

Incidence (%)

Disease

Index

(%) 1 Muzaffar

Garh

Makhan

Bela

2 93 44

Rohillanwal

i

2 100 27

Shah Jamali 2 100 44

2 Kot Addu Ali Wala 1 100 29

Musay

Wala

1 67 13

Shuhrat

Wala

2 100 40

Basti Drigh 3 100 52

3 Alipur Muradpur

Pull

2 100 31

Basti

Nukray

3 100 52

Basti Jat

Lashari

2 100 41



locations of district Rahim Yar Khan.

Sr.

No.

Tehsil Location Mean Disease

Severity (0-5)

Mean

Disease

Incidence

(%)

Disease

Index (%)

1 Rahim Yar

Khan

Taranda

Muhammad

Panah

2 100 32

Wahi Shah

Muhammad

3 100 52

Mianwali

Qureshian

2 100 32

2 Sadiqabad Ahmad Pur

Lumma

2 100 41

3 Khan Pur Chak 2P 2 100 48

Chak 3 P 2 100 44

4 Liaqat Pur Chak 22 A 2 100 45

Chak 23 A 2 100 49

Islam Nagar 2 100 48


75


locations of district Bahawalpur.


Severity (0-5)

Mean

Disease

Incidence

(%)

Disease

Index (%)

1 Bahawalpur Khanqah

Sharif

3 93 53 Chak 13 BC 3 100 52

Chak 23 BC 4 100 76 Nowshera 2 100 49

Munshi

Wala

3 100 57

2 Ahmed Pur

East

Channi

Goth

2 100 32

Mehrab

Wala

3 100 60

Muhabbat

Pur

2 100 48

Basti

Khokhran,

2 100 45

Basti

Johnan

3 100 61

Data are mean of five replicates.

76

In District Kasur mean disease severity of guava decline ranged from 0-1

based on 0-5 rating scale. Maximum mean disease severity "1" was recorded in all

the villages except Saddar Diwan (Kasur) and Kot Gurdas Wala (Chuniyan).

Maximum mean disease incidence of 93% was recorded in Khara of tehsil

Chuniyan followed by Gehlan Phatak and Sehjowal of Pattoki tehsil (73%), while,

the minimum was observed in Dolaywala (53%). Maximum disease index was

observed in Sehjowal (24%) followed by Khara and Gehlan Phatak (23%). On the

other hand minimum disease index (17%) was found in Dolaywala (Kasur) (Table

2.18).

In Sheikhpura district 2 tehsils were surveyed i.e. Ferozewala and

Sharaqpur. Maximum mean disease severity "2" was observed only in Faizpur

interchange. Disease severity " 1" was in all the locations except in Sharaqpur and

Sukhanwala (Tehsil Sharaqpur). Maximum mean disease incidence of 93% was

observed in Faizpur interchange followed by 80% in Noor Shah, while, the

minimum incidence was recorded in both Ghareebabad and Kot Mahmood.

Maximum disease index 33% was recorded in Faizpur Interchange followed by

28% in Burj Attariand it was minimum (12%) in Ghareebabad (Table 2.19).

In tehsil and district Nankana mean disease severity of "1" was observed in

more than 50% locations. Similarly,disease incidence 80% in Adda Pull Torian

followed by 67% in Jalal Nou. Maximum disease index in district Nankana was

observed 27% in Adda Pull Torian followed by Chackay Gill (19%), Jalal Nou

(15%) and minimum 12 % in Mirzapur (Table 2.20).

2.4.2. Symptomatology

77

The symptoms development percentageswere calculated by randomly

selecting 50 trees of each citrus, mango and guava. According to Table 2.21,

recorded data showed that dieback was maximum (42%) in guava followed by 38%

in citrus and minimum (28%) in mango trees. Bark splitting was shown to be

maximum by mango (38%), while, in citrus and guava bark splitting trees were

30% and 16% respectively. Maximum gummosis was recorded in 48%mango trees,

followed by 42% in citrus and minimum 32% in guava. Cankers were observed in

36% citrus trees (maximum), while in mango and guava it was 20% and 10%

respectively. Thirty percent (30%) mango trees showed stem bleeding followed by

28% in citrus and 8% in guava trees. Maximum mortality (8%) was observed in

mango, followed by (4%) guava and 3% citrus trees (Table 4.5). It was noted that

the similar symptoms observed in citrus, mango and guava included twigs dieback

(Fig. 2.10), bark splitting and gummosis (Fig.2.11, 2.14and 2.17), canker

development (Fig. 2.12, 2.2.15 (b) and 2.18. Finally, the mortality of decline

affected trees occur (Fig. 2.13, 2.16 and 2.19).

The results show that all citrus, mango and guava tree showed somewhat

similar symptoms with greater or lesser extent. These symptoms may appear either

due to similar predisposing factors or due to the common pathogens. However,

bark splitting in citrus, mango and guava trees happen differently, this is may be

due to their wood structure, physiology or the varietal character. Moreover, sudden

death is found less in citrus and guava as compared to mango. Dieback symptoms

have been most common among target commodities. Similarly canker development

can also be seen very commonly in all cases. Leaf drooping and drying but

remaining attached to trees was a common symptom in sudden death.

78

Table 2.17:Mean guava decline incidence, severity and disease index in different

districts of Punjab province.

Sr.

No.

District Mean

Disease

Severity

(0-5)

Mean

Disease

Incidence

(%)

Disease Index

(%)

1 Kasur 0.78 62.89 18.00

2 Sheikhupura 0.90 61.30 17.40

3 Nankana Sahib 0.57 56.29 14.00

Data are mean of five replicates

Table 2.18: Mean guava decline incidence, severity and disease index in different

locations of district Kasur.

Sr.

No.

Tehsil Location Mean

Disease

Severity

(0-5)

Severity (0-

5)

Mean

Disease

Incidence

(%)

Disease

Index (%)

1 Kasur

Dolaywala 1 53 17

Noor Shah

Wali

1 67 20

New City

Kasur

1 67 19

Sadar Diwan 0 47 9

2 Chuniyan

Khara 1 93 23

Kot Gurdas

Wala

0 33 8

3 Pattoki

Gehlan

Pathak

1 73 23

Rukan Pura 1 60 19

Sehjowal 1 73 24


79

Table 2.19: Mean guava decline incidence, severity and disease index in different

location of district Sheikhupura.

Sr.

No.


Disease

Severity

(0-5)

Severity (0-

5)

Mean

Disease

Incidence

(%)

Disease

Index (%)

1 Ferozewala Faizpur

interchange,

2 93 33

Adda Thabal 1 67 16

Noor Shah 1 80 17

Burj Attari 1 67 28

Saggian

Khurd

1 67 19

Thikriwala 1 60 17

2 Sharaqpur Sharaqpur 0 33 8

Ghareebabad 1 53 12

Sukhanwala 0 40 9

Kot

Mahmood

1 53 15


80

Table 2.20: Mean guava decline, severity and disease index in different location of

district Nankana Sahib.

Sr.

No.


Disease

Severity

(0-5)

Severity (0-

5)

Mean

Disease

Incidence

(%)

Disease

Index (%)

1 Nankana

Sahib

Jalal Nou

1 67 15

Giller Wala 0 47 9

Magtan Wala 0 40 8

Adda Pul

Torian

1 80 27

Chachkay

Gill

1 60 19

Mirza Pur 1 60 12

Mandi

Faziabad

0 40 8


81

Fig.2.10: Twig dieback in citrus.

Fig.2.11: Bark Splitting and gummosis in citrus.

82

Fig.2.12: Canker development in citrus.

Fig.2.13:Citrus tree mortality.

83

Fig.2.14: Bark splitting and oozing of thick dark brown liquid in Mango.

Fig. 2.15 (a) Fig. 2.15 (b)

Fig.2.15 (a) and (b): Gummosis and stem canker in mango decline affected tree.

84

Fig.2.16: Mango Sudden death affected dead tree (MSD).

Fig. 2.17: Bark splitting and gummosis in decline affected guava tree.

85

Fig. 2.18: Tree canker in decline affected guava tree.

Fig. 2.19: Decline affected dead guava tree.

86

Table 2.21: Percentage of citrus, mango and guva trees showing various decline

symptoms.

Data are mean of 50 replicates

Orchards Die-

back

Bark

splitting

(% trees)

Gummosis

(% trees)

Canker

formation

(% trees)

Stem

bleeding

(% trees)

Complete

Mortality

(% trees)

Citrus

19/50

(38%)

15/50

(30%)

21/50

(42%)

18/50

(36%)

14/50

(28%)

3/50

(6%)

Mango

14/50

(28%)

19/50

(38%)

24/50

(48%)

10/50

(20%)

15/50

(30%)

4/50

(8%)

Guava

21/50

(42%)

8/50

(16%)

16/50

(32%)

5/50

(10%)

4/50

(8%)

2/50

(4%)

87

2.5. DISCUSSION

The overall results indicate disease havoc in citrus growing areas of

district Sargodha which is due to a number of biotic and abiotic factors. Most of the

scientists thought that it was CTV causing et al., 2009 and Arif et al., 2015). Others

thought that phytoplasma and bacteria are involved in citrus decline (Burney et al.,

2007). Some considered bacteria e.g. Xanthomonas compestris as the cause of

citrus decline. Fungal involvement in citrus decline was studied in Oman, Iran and

Pakistan. A list of fungi has been reported to cause decline symptoms. e.g.

Lasiodiplodia hormozganensis, L. theobromae, Fusarium solani, Phytophthora sp.,

Neoscytalidium dimidiatum and Nattrassia mangiferaebeing the most common

(Safdar etal., 2010).

Among abiotic stresses there are two categories which are related to soil i.e.

salinity, nutrient toxicity/ deficiency, compaction/ hardpan in subsurface and other

is irrigation problem including water logging and water stress (Ali et al., 2014).

The human manipulations regarding orchard management as well as intercropping

are aggravating decline problem in existing citrus growing areas. Most of the

orchards in Sargodha, Bhalwal and Shahpur had old citrus plantations with

intercropping of wheat, berseem and barley. Removal of deadwood and pruning are

not commonly practicedin these orchards which haveresulted in profused branching

of trees. However, better situation was found in terms of orchard management in

Kot Momin and Sillanwali tehsils.

In mango areas, maximum decline disease index of 53.30% was recorded in

district Bahawalpur. During survey the large overlapping trees have been observed

in this district. Most of the areas surveyed had more than 100 years old plantations.

88

The age of trees have been reported to have significant relationship with decline

symptoms (Khaskheli et al., 2011). The growers were not even aware of the

disease but they thought that senescens had been resulting in such symptoms of the

trees. Secondly mango orchards were not given importance by the growers rather

they were taking care of intercropped plants. The large farms were owned by the

absentee landlords who stayed abroad and left orchard management on their

attendants. These attendants strongly believed in traditional mango cultivation and

showed harshness towards the adoption of innovative technologies.

In case of guava, maximum disease index was found in Kasur (18%)

followed by Sheikhupura (17.40%) and minimum 14% in Tehsil Nankana. Kasur

being the nursery industry area is looking for guava trees in high density for sale

rather than better management to earn from the fruit. Most of the trees were not

grown in proper rows, rather overlapping with each other in such a way that all the

operations like sprays, pruning and machine operations were difficult. Improper

cuttings and non treatment of woundsresulted in guva decline. However, proper

guava cultivation has been observed in Sheikhupura with intercropping of wheat

and berseem. Ploughing inside the orchards increased tree infection by making

injuries. Nankana is comparatively low in disease as most of the platations were

with proper planning and better management strategy. The main reason for low

severity in Nankana Sahib District may bedue to a new cultivaing area for guava;

the trees are young and are planted in a systematic way.

89

Chapter 3

PREVALENCE OF NATTRASSIA SP. FROM CITRUS, MANGO

AND GUAVA ORCHARDS ALONG WITH OTHER DECLINE

CAUSING FUNGI

3.1. INTRODUCTION

Tree decline in citrus, mango and guava has been a hot issue in Pakistan

and some of the countries around the globe. Different researchers around the world

have reported different causes of these declining trees. Mostly described terms for

the decline factors are predisposing, inciting and contributing factors (Malik et al.,

2004; Safdar et al., 2010 and Safdar et al., 2015).

Predisposing factors are long term factors that change slowly such as soil,

site and climate. These affect tree's ability to respond to any kind of injury or

disease inducing agents. In case of citrus, mango and guava decline all of these

factors have a key role. The changing climate especially prevailing drought

conditions have tremendously affetced citrus, mango and guava cultivation as well

as other fruit, forest and ornamental trees. Similarly, sites also play important role

as the plantation near the roads or canals are mostly decline affected either because

of hard soil pans, road or canal widening and other human interventions which

cause the root injury of the trees and result in the decline symptoms. Saline and

saturated soils are other predisposing factors which create hindrance in the orchard

management. Salinization of soil and water is an important factor for increased

desertification in arid and semi-arid regions of the world (Szabolcs, 1992). About

6.3 million hectares of arable land in Pakistan is affected to varying degrees of

salinity/sodicity.

64

90

Inciting factors are of short duration and appear in biological or

physiological form. They generally produce dieback symptoms or we can say they

are just starter of the disease. These might be twig dieback causing fungi, insects,

phytoplasma, late spring frost, drought and salt spray (Burney et al., 2007).

The role of contributing factors starts after inciting factors which further

weaken the plants and ultimately moratlity occurs. These include pathogenic fungi,

vectors (in case of decline the bark beetle), canker causing fungi, bacteria, viruses

and phytoplasma etc (Kazmi et al., 2005).

The most important pathogens worked out among other contributing biotic

and abiotic factors causing tree decline are the fungi. Maximum fungi have been

isolated from the roots of decline affected citrus trees. The isolated fungi were

Nattrassia mangiferae, Fusarium sp., Alternaria sp. and Aspergillus sp. from the

area of Sargodha, Bhalwal, Kot Momin, Sillanwali and Toba Tek Singh areas of

the Punjab province (Khan et al., 2012; Ali et al., 2014). Several fungi isolated

from decline affected mango trees include Ceratocystis fimbriata, Lasiodiplodia

theobromae, Fusarium sp., Nattrassia mangiferae, Phomopsis sp. (Kazmi et al.,

2005; Fateh et al., 2006 and Masood et al., 2010). Fungi associated with guava

decline include Fusarium sp., Nattrassia mangiferae, Macrophomina sp.,

Cephalosporium sp., Alternaria sp., Pythium sp., Verticillium sp., Trichthecium sp.

(Mirzae, 2003; Miskita et al., 2005).

It is hypothesized that sometimes these fungi individually or sometimes

with the interaction of more than one fungi result in the appearance of decline

symptoms. These different fungi are found in different profiles or parts of the

decline affected trees. The fungus N. mangiferae have a wide host arrange around

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the globe and has covered field crops, ornamental, forest and fruit trees. However,

in Pakistan, the fungus was not studied thoroughly on systematic basis. Hence, the

present study was conducted to determine the frequency of Nattrassia

mangiferaealong with other mycoflora associated with decline of citrus, mango and

guava and to find out common mycoflora in different plant parts of the affected

trees so that a common management may be devised for decline of all fruit crops.


Lonsdale (1992) reported that Nattrassia mangiferae was found to be an

important pathogen of mango in South Africa. It was found to be the cause of

blossom blight. Initial symptoms of the disease were sudden wilt followed by rapid

dying and drying out of inflorescences. In the later stages of the disease, side axes

and individual flowers dropped, fruitlets shriveling up and turned black and also

dropped. Other fungi such as Alternaria alternata and Colletotrichum

gloeosporioides did not cause blight, but instead, induced small, dark necrotic spots

(lesions) on the inflorescences.

In North America, eucalyptus dieback for the first time was reported in

1994 caused by the fungus N. magniferae. Dieback symptoms were observed in

branches; rifting between the phloem and the xylem. During summer cankers and

exuding gum were also observed on Eucalyptus camaldulensis Dehnh. trees in

southwestern Arizona. N. mangiferae was the dominant fungus which has been

consistently isolated from the affected branches (Michael, 1994).

In south-eastern Iran, the fungus N. mangiferae was reported from guava

and Ficus religiosa, causing branch die-back. The pathogenicity was confirmed

through Koch's postulates. This pathogen repported for the first time in 2000 in

92

Iran and has been a threat to Irani citrus and guava industry since then (Mirzaee et

al., 2003).

Decline of Pacific Madrone has been a popular disease caused by N.

mangiferae inducing cankers on stem and blight of shoots. Due to attack by N.

mangiferae trees were weakened and opportunistic fungi like Fusicoccum aesculii

nvaded stressed trees. These two pathogens were also reported on declining

Madrone trees in Washington, USA but N. mangiferae was the most prominent and

primary pathogen (Elliott and Robert, 2002).

Further studies on the fungus N. mangiferae causing canker of Pacific

madrone were based on morphological and molecular methods. The asexual spores

confirm the pathogen, however, sequencing of the ITS region of the ribosomal

rDNA of sexual state had linkages with genus Botryosphaeria. The fungus showed

resemblance to Fusicoccum anamorphs, closely related Botryosphaeria species and

has a similar pathology (Elliott and Robert, 2003).

Msikita (2005) isolated the fungus N. mangiferae from cassava causing root

rot in Bénin and West Africa. The pathogenicity was confirmed on different

cultivars of cassava, showing fungal lesions, while control plants remained

symptom free. Other pathogens causing root rot included Macrophomina

phaseolina, Fusarium sp. B. theobromae and Pythium sp.

David et al. (2005) reported that pacific Madrone had been facing decline

for thirty years. It was reported that fungus causing decline has been present in

western North America since 1968. Prior to this research, plant pathologists had

misidentified the causal fungus, which is now known to be N. mangiferae.

93

Jayasinghe and Silva (2007) for the first time reported N. mangiferae

causing foot canker of Hevea seedlings. One of the predisposing factors for the foot

canker was sun-scorch and hence it must be considered in other declining trees as

well where N. mangiferae is isolated.

3.3.MATERIALS AND METHODS

3.3.1. Sampling Methodology

During survey of citrus mango and guava orchards, samples were taken

from the twigs, branches, stem at collar region, bark and the roots of decline

affected trees. The samples were taken from randomly selected villages keeping in

mind the maxiumum orchards present in the areas. For citrus samples were

collected from samples 26 NB, 8NB and 10NB from tehsil Bhalwal of district

Sargodha. Similarly in tehsil and district Sargodha, samples were taken from 56

NB, 112 NB and 27 SB villages.

In mango growing areas the samples for isolation were taken from Nandla,

Chah Nizam Wala and Basti Band Bosan in district Multan while, in district Rahim

Yar Khan samples were taken from villages of Chak 22A (tehsil Liaqatpur), Chak

2P (tehsil Khan Pur) and Mianwali Qureshian.

To know the fungal frequencies in guava decline orchards, the samples

were taken from Sehjowal, tehsil Pattoki of district Kasur, Faizpur (tehsil

Ferozewala of district Sheikhupura) and Adda Pul Torian(tehsil and district

Nankana Sahib).

The samples were used for the isolation, identification and further

pathogenicity studies. The sample pieces were cut with sterilized sharp tool and

kept in paper bags which after proper labeling were placed in the shopping bags to

94

avoid moisture loss.

3.3.2. Isolation and Identification of Pathogens

All the diseased samples of mango, citrus and guava were brought to

Mango Pathological Lab. at National Agricultural Research Centre, Islamabad and

the isolations were made aseptically in the laminar flow chamber placed in

isolation room. All glassware and media were sterilized by autoclaving at 121oC at

15 Psi for 20 min. For the isolation of fungi Potato Dextrose Agar (PDA), Malt

Extract Agar (MEA) and Stem Decoction Glucose Agar (SDGA) media were used.

Recipe of the media used is given below.

3.3.2.1.Potato dextrose agar medium (PDA)

For making Potato Dextrose Agar (PDA) medium a readymade mixture of

the chemical company BDH was used

Potato Dextrose Agar = 39 g

Sterilized Distilled water = to make volume up to 1000 ml

Medium was made homogeneous by heating in a microwave oven for 3

minutes in half filled 1000 ml flasks, air tightened with cotton plug and later

sterilized by autoclaving at 121oC at 15 Psi for 20 minutes.

3.3.2.2.Malt extract agar (MEA)

This medium was prepared by mixing following ingredients:

Malt Extract= 20 g

Agar= 20 g

Distilled water= to make volume up to 1000 ml

3.3.2.3. Stem decoction glucose agar (SDGA)

Citrus/mango/guava stem pieces = 200 g

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Agar= 20 g

Glucose=20 g

Distilled water= to make volume up to 1000 ml

The respective stem pieces of citrus, mango and guava were boiled in 1000

ml water for 10 min. cooled down and then again boiled so that half of the volume

is left. Concentrated water sieved through muslin cloth in beaker and more water

was added to make the volume up to 1 L before adding other ingredients.

3.3.2.4. Sterilization

The media were sterilized in autoclave at 121oC and 15 PSi. All glassware

including flasks, petriplates and other inoculation equipment was also sterilized in

the autoclave.

3.3.2.5. Isolation from infected tissues

All the infected tissue samples were cut into small pieces and disinfested by

rinsing into 1 % sodium hypochlorite for 1 min. followed by washing thrice with

sterilized distilled water. The washed pieces were dried on filter papers to remove

excessive moisture and aseptically placed in the 90 mm petridishes containing

either PDA or MEA medium. The plates were wrapped with parafilm to avoid

moisture loss and contamination and incubated 25oC ±.

3.3.3. Microscopy

Slides were prepared by taking a minute portion of fungal colony from the

growing margin with the help of inoculating needle and identified by viewing

under the Labomed Florescent Microscope made in USA microscope,using the

magnifications of X100, X200, X400 and X1000. Isolated fungi were identified on

the basis of their colony characteristics, conidial morphology, ascospores, pycnidia,

96

pericthecia etc. The identification was based on key used by Fateh et al., 2010 and

Domsch et al., 1980.

3.3.4. Determination of Fungal Frequency %age

The fungal frequency was determined using the following formula for

tissue inoculation:

Fungal frequency (%) =Number of colonies of a particular fungus × 100

Total no. of colonies of all fungi in 90 mm petridish

3.3.5. Purification of Fungal Cultures

Cultures of individual fungi were purified using single spore technique. The

spores were observed through Olympus stereoscope and were picked with the help

of very thin wire inoculating needle and placed in 90 mm petridish containing

PDA. The pure cultures were preserved in mineral oil for further studies.

3.4. RESULTS

3.4.1. Plant Parts Wise Fungal Frequency %age

The plant parts wise % frequency of different fungi associated with

declining trees is given below.

3.4.1.1. Mycoflora from citrus decline affected trees

Six fungi (Nattrassia mangiferae, Fusarium sp., Botryodiplodia

theobromae, Alternaria sp., and Phytophthora sp.) were isolated from different

parts (twigs, branches, bark, stem at collar region and roots) of affected samples of

citrus trees from randomly selected villages. In Chak No. 26 NB of tehsil Bhalwal

of district SargodhaN. mangiferae was found at maximum frequency (40%) in the

bark of tree followed by 22% in stem at collar region, 7% on branches and only 3%

frequency was observed from the root samples. Fusarium sp. frequency was

maximum (12%) from both branches as well as stem at collar region followed by

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11% from both twigs and roots. However no Fusarium sp. colony appeared from

bark samples. Botryosphaeria sp. was isolated in maximum frequency (39%) twigs,

27% from branches and minimum of 17% from bark. But it was absent in other

plant parts. Lasiodiplodia theobromae frequency was maximum from branch

samples (34%), from stem at collar region 27 %, from twigs 24%, 12 % from bark

and only 2% from roots. Alternaria was mostly found in twigs(14%), 4% from

branch samples, 12% from bark and of minimum 6% from the roots. However, the

fungus Phytophthora sp. (12%) was only recovered from roots (Fig.3.1). Almost

similar trends were observed from other randomly selected villages i.e. Chak 8 NB,

Chak 10 NB, Chak 56 NB, Chak 112 NB and Chak 27 SB as shown in Fig.3.2, 3.3,

3.4, 3.5 and 3.6 respectively.

3.4.1.2. Mycoflora from mango decline affected trees

From mango decline affected trees, samples were taken from six randomly

selected villages (Basti Nandla, Chah Nizam Wala, and Basti Ban Bosan of district

Multan and Chak 22 A, Chak 2 P and Mianwali Qureshian of district Rahim Yar

Khan). From the infected trees, six fungi were found associated with different plant

parts in varying frequencies. These fungi associted with declining mango trees

were N. mangiferae, Fusarium sp., Ceratocystis fimbriata, Lasiodiplodia

theobromae, Alternaria alternata and Phytophthora species. The plant wise

percentage frequencies of these fungi are given in Fig.3.7, 3.8, 3.9, 3.10, 3.11 and

3.12.

3.4.1.3 Mycoflora from guava decline affectedtrees

Similarly from decline affected guava trees sampling was done in in three

randomly selected locations situated in districts of Kasur, Sheikhpura and Nankana

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Sahib. Six fungi viz. N. mangiferae, Fusarium sp, Botryosphaeria sp, L.

theobromae, Alternaria alternata and Phytophthora sp. were isolated.

The percentage frequencies of different fungi isolated from different plant

parts of decline affected guava trees from Sehjowal (Tehsil Pattoki of district

Kasur) are given in Fig.3.13. N.mangiferae frequencywas maximum (14%) from

bark samples followed by branches (8%) and minimum7% from twig samples.

Fusarium sp. frequency was maximum from roots (13%)followed by 9 %

(branches) and minimum 5% from stem at collar region. The % frequency of

Botryosphaeria sp. was maximum (16%) from branches followed by 6% from twig

samples. Lasiodiplodia theobromae frequency was maximum (27%) from stem at

collar region followed by branches (15%), twigs (12%) and minimum 3% from

bark samples. Likewise, Alternaria sp. frequency maximum (12%) was found from

all branches, barks and stem at collar region and minimum 11% from root samples.

The fungus Phytophthora sp. was only recovered from roots (4%).

The percentage frequencies of these six fungi from other two selected

locations more or less followed the similar trends and are given in Fig.3.14 and

3.15. The culture and conidia of some of these fungi are given in Fig. 3.16 (a and

b), 3.17 (a and b), 3.18 (a and b) and 3.19 (a and b).

Another important point was that in case of citrus and guava there was not

any record of Ceratocystis sp. which was solely isolated from mango. It does not

mean that it may not be found in citrus or guava but it may be present in some

stage of the same genera. The results conclude that the most common fungus found

in all types of decline is Nattrassia mangiferae.

99

Fig.3.1: Plant parts wise fungal frequency %age from decline affected citrus trees

at Chak No. 26/NB tehsil Bhalwal, Sargodha.

0

5

10

15

20

25

30

35

40

45

Twig

branches

Bark

Stem at collar region

Roots

100


at Chak No. 8 NB, tehsil Bhalwal of district Sargodha.

0

5

10

15

20

25 Twig

branches

Bark


Roots

101


at Chak No. 10 N/B, tehsil Bhalwal of district Sargodha.

05

1015202530354045 Twig

branches

Bark


Roots

102

Fig.3.4: Plant Parts wise fungal frequency %age from decline affected citrus trees

at Chak No. 56/NB of tehsil and district Sargodha.

0

5

10

15

20

25

30

35

Twig

branches

Bark


Roots

103

Fig. 3.5: Plant parts wise fungal frequency %age from decline affected citrus trees

at Chak No. 112/NB of tehsil and district Sargodha.

0

5

10

15

20

25

30

35

40

45

Twig

branches

Bark


Roots

104

Fig.3.6. Plant parts wise fungal frequency %age from decline affected citrus trees

at Chak No. 27 SB of tehsil and district, Sargodha.

0

5

10

15

20

25

30

35Twig

branches

Bark


Roots

105

Fig.3.7: Plant parts wise fungal frequency %age from decline affected mango trees

in Basti Nandla, Multan.

0

5

10

15

20

25Twig

branches

Bark


Roots

106

Fig.3.8: Plant Parts wise fungal frequency %age from decline affected mango trees

in Chah Nizam Wala, Multan.

0

5

10

15

20

25

30 Twig

branches

Bark


Roots

107

Fig.3.9: Plant parts wise fungal frequency %age from decline affected mango trees

in Basti Band Bosan, Multan.

0

5

10

15

20

25 Twig

branches

Bark


Roots

108

Fig.3.10:Plant parts wise fungal frequency %age from decline affected mango trees

at Chak 22A, tehsil Liaqatpur of district Rahim Yar Khan.

0

5

10

15

20

25

30Twig

branches

Bark

Stem at collar

regionRoots

109

Fig.3.11: Plant parts wise fungal frequency %age from decline affected mango

trees at Chak 2P, tehsil Khanpur of district Rahim Yar Khan.

0

5

10

15

20

25

30

Twig

branches

Bark


Roots

110

Fig.3.12: Plant parts wise fungal frequency %age from decline affected mango

trees at Mianwali Qureshian, tehsil and district Rahim Yar Khan.

0

5

10

15

20

25

30

35 Twig

branches

Bark


Roots

111

Fig. 3.13: Plant parts wise fungal frequency %age from decline affected guava

trees at Sehjowal, tehsil Pattoki of district Kasur.

0

5

10

15

20

25

30 Twig

branches

Bark


Roots

112

Fig.3.14: Plant parts wise fungal frequency %age from decline affected guava trees

at Faizpur, tehsil Ferozewala of district Sharaqpur.

0

5

10

15

20

25

30

35Twig

branches

Bark

Stem at collar

region

113

Fig.3.15: Plant parts wise fungal frequency %age from decline affected guava trees

at Adda Pul Torian of tehsil and district Nankana Sahib.

0

5

10

15

20

25

30

35

40 Twig

branches

Bark

Stem at collarregion

114

Fig.3.16 (a)

Fig.3.16 (b)

Fig.3.16: (a) and (b) Culture and macrospores of Fusarium sp.

115

Fig.3.17 (a)

Fig.3.17 (b)

Fig.3.17: (a) and (b): Culture and conidia of Ceratocystis sp.

116

Fig.3.18 (a)

Fig.3.18 (b)

Fig.3.18: (a) and (b): Cultural and conidia of Lasiodiplodia theobromae.

117

Fig.3.19 (a)

Fig.3.19 (b)

Fig. 3.19: (a) and (b): Culture and spores of Nattrassia mangiferae.

118

3.5. DISCUSSION

A number of fungi have been isolated from different decline affected trees

and different plant parts. These include Botryodiplodia, Botryosphaeria, Nattrassia

mangiferae, Fusarium, Alternaria, Pythium, Phytophthora and Ceratocystis species

(Fateh et al., 2006; Kazmi et al., 2007; Fateh et al., 2016, 2017). These fungi have

been found infecting the trees singly or in combination (Anwar et al., 2012). It has

been observed that Botryodiplodia, Botryosphaeria and Nattrassia mangiferae

were mostly found in the twigs and branches of all affected trees. They may be the

cause of twig blight and branch die back in the affected trees (Safdar et al., 2015;

Mirzae et al., 2003; Atta and Aref, 2013). Another probable reason for their co-

existance is their common lineage (Crous and Palm, 2004). Nattrassia and

Botryodiplodia were also observed on the bark as well as on the inner side of the

bark where bark splitting occurred. Therefore, they might have an additional role in

bark splitting. Fusarium, Ceratocystis have been found in the vascular tissues of

the plants to stop the nutrients inflow and cause the wilting symptoms. However, it

is impotant to note that Ceratocystis was only found from the vascular tissues of

mango but it was not isolated from citrus and guava (Mirzaee et al., 2003; Miskita

et al., 2005; Fateh et al., 2006 and Masood et al., 2010;).

Maximum frequencies of N. mangiferae and B. theobromae recorded in the

present study agreed with those described by Giha, (1975); Farr et al. (2005) and

Fateh et al.(2016). It seems that infection by these two fungi has reached an

epidemic stage. This is much evident from the host range of these fungi.

Especially, the fungus N. mangiferae caused branch wilt of Banyan trees (Ficus

beneghalensis) Khartoum, Sudan (Giha, 1975); top dying and mortality of Gmelina

119

arborea in India (Harsh and Tiwari, 1992). The fungus had an endemic attack on

Eucalyptus camaldulensis and on citrus trees in Arizona (Sigler et al., 1997);

caused butt rot of Zanthoxylum bungeanum in China (Cao and Wang, 1989). N.

mangiferae invaded number of fruit trees including almond, peach, plum, guava

and forest trees like Eucalyptus sp., Populus sp. and Pinus sp. in Iraq (Al Zarrari et

al., 1979;Shawkat et al., 1979). The continuing victims of the fungus include

lemon trees (gummosis) in Lebanon (Hartmann and Niehaus, 1974) and bananas

(Tip rot) in Jamaica (Meredith, 1963; Jones and Stover, 2000). In USA, the canker

of Pacific madrone (Arbutus menziesii) (Davis and DeVay. 1975 and Far et al.,

2005) and foliar disease of strawberry trees (Arbutus unedo) in Europe

(Tsahouridou and Thanassoulopoulos, 2000) was also reported to be caused by the

fungus. In Africa it attacked cassava and white yam (Msikita et al., 1997) and the

most importantly the fungus has been reported to cause skin and nail diseases and

fungal keratitis in humans (Kindo et al., 2010). Being ubiquitous in distribution, it

is not sparing trees in Pakistan as well.Although the fungus has not been studied

frequently, yet few scientists have reported it on citrus, mango and guava (Fateh et

al., 2010; Anwar et al., 2012; Fateh et al., 2016, 2017)

The high frequencies of N. mangiferae in the tropical fruits such as mango

and guava are also due to the wide range of temperature these fungi can withstand.

For example Namsi et al.(2010) reported that N. mangiferae grew well within the

range of 20 to 40oC. Sehlar et al. (1997) and Mahmood et al. (2002) showed

similar results for the growth of B. theobomae.

Another important reason for high frequencies of these fungi is that in the

sampling area there were large overlapping trees. This affects the frequencies in

120

two ways: firstly, the large trees with overlapping branches are not the well

managed trees i.e. they do not receive proper pruing or protective spray regimes.

Thus the opportunistic fungi found their way through possible wounds due to

ploughing or other such cultural practices and invaded the trees without any danger

to their own survival (Mirzae et al., 2003). Secondly, the large trees are mostly old

age or senescence is itself a contributing factor in tree decline (Khaskheli et al.,

2011; Anwar et al., 2012).

In case of mango, Ceratocystis sp. is unique, as it was not found in both

citrus and guava decline affected orchards even though the sampling parts were

same for all target fruit trees. One reason can be the resistance of these plants

towards this fungus which is yet to be studied or they still did not come in touch

with the fungus and are safe. However, across the globe, the fungus Ceratocystis is

known to cause mango and many forest tree decline as well as decline in field

crops. The fungus for the first time was reported in Pakistan by Fateh et al. (2006)

during sampling in mango orchards of Sindh province. Later, the fungus has been

repeatedly reported by the relevant scientists in mango as well as shisham (Masood

et al., 2010; Safdar et al., 2012; Fatehet al., 2016). Like N. mangiferae and B.

theobromae, Ceratocystis genera also penetrate the plants through wounds and

establish itself in the xylem vessel, it clogs the xylem and the sap is collected in the

small pockets. These pockets later rupture and a thick dark color liquid oozes out

which is the characteristic of Ceratocystis sp. (Fateh et al., 2006;Al-Adawi et

al.,2006). Unlike other fungi, the fungus Ceratocystis likes low temperatures. Its

infection starts in the mid of October to the end of February which are mostly cool

121

months in Pakistan. Hence, its management is possible as October to January is

mostly the dormant period of mangoes in the country.

The present study was very significant to report the similar pathogens in

citrus, mango and guava decline as this will encourage, similar management pattern

for similar fungi in all trees. However, special care will be required in case of

mango in future that the deadly fungus Ceratocystis sp. may not enter other fruit

orchards from mango ones. This also provides a complete profile that which part of

the plant is occupied by which fungi and to devise management accordingly.

122

Chapter 4

ROLE OF NATTRASSIA MANGIFERAE AND OTHER

DECLINE CAUSING FUNGI IN SYMPTOMS DEVELOPMENT

4.1. INTRODUCTION

There are four major fungi, which have been consistently reported to cause

the tree decline in fruit trees, forest and other ornamental plants. These fungi

include Nattrassia mangiferae,Ceratocystis sp., Lasiodiplodia theobromae, and

Fusarium sp. The symptoms produced by the inoculation of these fungi alone or in

combination are dieback, gummosis, bark splitting, canker formation and mortality

of the tree (Kazmi et al., 2007; Fateh et al., 2006).

Different scientists used different methods for the confirmation of

pathogenicity of these fungi associated with decline (Khanzada et al., 2004; Urbez-

Torres et al., 2008; Masood et al., 2010). Some of the scientists used green house

potted plants and others used branches of the mature trees. In green house potted

plants the plugs from fresh cultures were inoculated through flap method, stem

injury method and root injury method. In case of mature trees, branches have been

artificially inoculated through flap methods (Mirzaee et al., 2003).

In Oman, citrus decline study was conducted by artificial inocultion of acid

lime and sweet lime seedlings of age six month to one year. The seedlings were

apparently healthy before inoculation. The procedure was done by making injury at

the base line of the seedlings and insertion of 5 mm mycelial plug in it that has

been taken from 5-6 day-old culture of the fungi grown on potato dextrose agar.

The plugs were wraped by moist cotton to avoid drying of the mycelium or culture.

Seedlings were placed in completely randomized design in a glasshouse under

25oC. The test was replicated 5 times and the symptoms were observed on

97

123

seedlings on weekly basis for 3-4 months for the development of gum, dieback or

death. Re-isolations were made to confirm the association Gof the inoculated fungi

with the observed symptoms. The isolations were done as explained previously

(Al-Sadi et al., 2010).

Similar studies have also been conducted on mango for C. fimbriata and L.

theobromaecausing mango decline. Individual as well as combine inoculation to

healthy mango seedling was done in five replications for each treatment under

completely randomized design (CRD) in a green house. For inoculation 5mm plug

of fungi growing on PDA was placed in slanting cuts under the bark with sterilized

scalpel and then covered with parafilm (Mullen et al., 1991). In case of control,

only agar slant without any fungal growth was used for insertion. The

pathogenicity was confirmed by re isolation of the inoculated fungi (Masood et al.,

2011).

To conduct the pathogenicity for guava decline, detached guava twigs were

used for inoculation of the fungi. A moist chamber was developed by placing a

blotter sheet on a plastic tray (50 × 40 × 7.5 cm). Twigs of 8‒10 cm length and

2‒3 cm thickness were cut from healthy branches, thouroughly washed twice with

distilled water, dried on sterilized blotting paper and re-sterilized with 2% sodium

hypochlorite. Twigs were injured with the help of a cork borer. Fungal disks from

the respective culture were placed in the holes made as well as uninjured sites of

twigs. The uninoculatedtwigs served as control. There were three replications with

five twigs per replication. The twigs in trays were incubated in a growth chamber at

28 ± 2°C. The observations were recorded regularly for symptoms or fungal lesion

development (Shah et al., 2010).

124

The evaluation of symptoms during pathogenicity has been reported in

many different ways. Some of the scientists evaluated by using different rating

scales, while others just described the symptoms produced. However, the

pathogenicity was confirmed after re-isolation of pathogens from artificially

inoculated symptomatic plants.

The aim of present study was to determine the role of Nattrassia sp. in

causing decline along with other fungi. Therefore, the pathogenicity of four

selected fungi was done individually and in combination which have been

associated with decline symptoms directly or indirectly. These fungi included

Nattrassia sp., Fusarium sp., Ceratocystis sp. and Botryodiplodia theobromae.


The fungal involvement in citrus decline was found in Oman. Among these

the most common were Lasiodiplodia hormozganensis, L. theobromae and

Fusarium solani. On artificial inoculation of these common fungi, acid lime and

sweet lime seedlings reproduced decline and gummosis symptoms. The nursery

studies on sweet lime showed association of 12 fungal species, which is an

evidence that nurseries act as a main source for some citrus pathogens (Al Sadi et

al., 2010).

Abbasher et al. (2013) reported that the fungus Nattrassia mangiferae

(Nattrass) is considered to be the pathogen of branch wilt of citrus, one of the most

hazardous diseases that spread in Wad Medani province, Gezira state, Sudan. The

survey for the prevalence of fungi isolated from the branch wilt disease samples in

Wad Medani area revealed that that there were 2 different isolates of N.

125

mangiferae. The 1st one was isolated from ficus (Ficus nitida) and the 2

nd was

isolated from lime (Citrus aurantifolia Swingle.).

A study was conducted to observe the synergistic effect of the fungus

Fusarium semitectum Berk & Rev. and a nematode Tylenchulus semipenetrans

Cobb. was determined singly and in combination on citrus. Maximum reduction in

leaves, root length, shoot length, fresh shoot weight, fresh root weight, stem

diameter and number of feeder roots were observed in the plants having combined

treatment of F. semitectum and T. semipenetrans. Thus, existence of synergistic

relationship between F. semitectum and T. semipenetrans in citrus decline was

confirmed (Safdar et al., 2013).

Masood et al., 2011 conducted a study to identify the association of

pathogenic fungi with mango quick decline tree and the bark beetle. From diseased

tree as well as from H. mangiferae, the most frequently isolated fungi were

Lasiodiplodia theobromae, Ceratocystis fimbriata and Phomopsis sp. The fungi

were re-isolated from artificially inoculated and symptomatic mango plants. After

six months of inoculations, disease symptoms i.e., wilting, oozing and black streaks

were developed which showed significant differences among all treatments.

Similar research was done in Oman as well on mango sudden decline.

Affected mango trees showed wilting that began on one side and later spread to

involve the entire tree. Trees exude amber-coloured gum from the bark of their

trunks or branches and vascular tissues are discoloured. It was found that vascular

wilt pathogen Ceratocystis fimbriata caused decline disease in combination

with Lasiodiplodia theobromae. Isolates of these fungi from affected trees, caused

126

infection on artificially inoculated seedlings and later recovered from these (Al

Adawi et al., 2006).

Arif (2013) reported the fungi causing mango and guava decline in

Pakistan. These includedCeratocystis fimbriata, Botryodiplodia theobromae,

Fusarium solani, Nattrassia mangiferae and Fusarium oxysporum f.sp psidii. All

of these fungi proved to be pathogenic when inoculated on mango and guava

plants.


4.3.1. Pathogenicity Tests

To test the reponse of various fungi isolated from their respective hosts i.e.

citrus, mango and guava artificial inoculation was done individually as well as in

combination to potted plants of 1 and half to 2 years old, visually healthy and taken

from reliable nurseries. The pathogenicty tests were done under green shade of

NARC, Islamabad. The inoculation was done through flap method and root injury

method.

4.3.1.1.Flap method

In the flap method a T shape cut was given in the bark of plants and 2 plugs

of 5 mm from freshly growing cultures of test fungi were placed by opening T-

shape cut and then wrapped with the help of parafilm. As a control of this method

plugs from PDA without cultures were inserted.

4.3.1.2. Root injury method

In this method the plants' roots were injured with the help of sterilized knife

and then covered with soil. Spore suspension (5 plugs of 5mm from fresh cultures

of fungi were mixed in 200 ml sterilized distilled water) was applied as soil drench.

127

In this method the spore suspension of each fungus was mixed and stirred well

before applying as soil drench.

The treatments for citrus in both flap and root injury methods were as

follows:

T1= N. mangiferae

T2= B. theobromae

T3= Nattrassia + B. theobromae

T4= 5 mm plug from PDA (Control)

The treatments for mango in both flap and root injury methods were as

follows:

T1= Ceratocystis sp.

T2= Nattrassia sp.

T3= Botryodiplodia theobromae

T4= Fusarium sp.

T5= Nattrassia + Ceratocystis

T6= Ceratocystis + B. theobromae

T7= Ceratocystis+ Fusarium

T8= Nattrassia+ B. theobromae

T9= Nattrassia + Fusarium

T10= B. theobromae+ Fusarium

T11= Nattrassia + Ceratocystis + B. Theobromae

T12= Nattrassia + Fusarium + B. Theobromae

T13= Nattrassia + Fusarium + Ceratocystis

T14= B. Theobromae + Fusarium + Ceratocystis

128

T15= Nattrassia + Ceratocystis + B. Theobromae + Fusarium


The treatments for guava in both flap and root injury methods were as

follows:

T1= N. mangiferae

T2= B. theobromae

T3= Fusarium sp.

T4= Nattrassia + B. theobromae

T5= Nattrassia + Fusarium

T6= Fusarium + B. Theobromae

T7= Nattrassia +B. Theobromae + Fusarium


Each of the above treatment was tested in triplicate and a range of

symptoms was observed on citrus, mango and guava seedlings over a period of two

months.

4.4.RESULTS

The inoculation results of different fungi by flap method (Fig.4.1) on 1 to 2

years citrus potted plants have been shown in the Table 4.1. The results indicate

that when Nattrassia sp. was inoculated alone (T1), bark splitting, stem canker and

gummosis appeared but the plants survived. On inoculation of Botryodiplodia

theobromae alone (T2), stem canker and gummosis was shown without mortality

of the plants. The combination of Nattrassia sp + Botryodiplodia theobromae (T3)

showed similar symptoms as N. mangiferae alone. The control plants (T4) without

inoculation of any fungi only produced minor gummosis.

129

The inoculation results of different fungi by root injury method (Fig.4.2)

through soil drenching the fungal spore suspension on citrus plants have been

shown in the Table 4.2.

It was observed when Nattrassia sp. was inoculated alone (T1), bark

splitting and gummosis were observed but plants survived. On inoculation of B.

theobromae alone (T2), showed only gummosis. The combination of N.

mangiferae+ B. theobromae (T3) also showed same results as N. mangiferae alone.

The control plants (T4) without inoculation of any fungi only produced gummosis.

Pathogenicity of different fungi on potted mango plants by flap method is

given in Table 4.3. The results indicated that T1, T5, T6, T7, T13, T14 and T15

produced all symptoms of decline i.e. leaf drooping, bark splitting, stem canker,

stem gummosis and the final mortality. In all of these cases Ceratocystis sp. was

either inoculated alone or in combination with other fungi. In case of other

treatments some of the decline symptoms were observed but no mortality was

recorded. However, in case of control plants only stem gummosis was seen that

may be due to the injury done for inoculation.

Mango plants in case of root injury inoculation method almost showed

similar results to that of flap method except, in case of Fusarium sp. inoculated

alone also caused mortality of seedling (Table 4.4).

Guava plants response to different fungal inoculations was same as that of

mango. Plants of T7 in both flap and root injury method showed all decline

symptoms and eventually died. Other treatments similar to mango showed some of

the decline symptoms but no mortility occured.

130

Table 4.1: Response of citrus plants against artificial inoculation of N. mangiferae

and B. theobromae using flap method.

Treatments Decline symptoms in citrus(flap method)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

N. mangiferae (Nm)

- + + + -

T2

B. theobromae (Bt)

- - + + -

T3

Nm + Bt

- + + + -

T4

Control

- - - + -

+ = Symptoms present ; - = Absent

Table 4.2: Response of citrus plants against artificial inoculation of N. mangiferae

and B. theobromae using root injury method.

Treatments Decline symptoms in citrus(Root injury)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

N. mangiferae (Nm)

- + - + -

T2

B. theobromae (Bt)

- - - + -

T3

Nm + Bt

- + - + -

T4

Control

+ - - + -


131

Table 4.3: Response of mango plants against artificial inoculation of Caratocystis

sp., N. mangiferae, B. theobromae and Fusarium sp. using flap

method.

Treatments Decline symptoms in mango(flap method)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

Ceratocystis sp.(Cs) + + + + +

T2

N. mangiferae (Nm) - + - + -

T3

B. theobromae (Bt) - + + + -

T4

Fusarium sp. (Fu) + - + + -

T5

Nm + Cs + + + + +

T6

Cs + Bt + + + + +

T7

Cs + Fu + + + + +

T8

Nm + Bt - + + + -

T9

Nm + Fu - + + + -

T10

Bt + Fu - - + + +

T11

Nm + Cs + Bt - - - + -

T12

Nm + Fu + Bt + + + + -

T13

Nm + Fu + Cs + + + + +

T14

Bt + Fu + Cs + + + + +

T15

Nm + Bt + Cs + Fu + + + + +

T16

Control - - - + -


132

Table 4.4: Response of mango plants against artificial inoculation of Caratotocystis

sp., N. mangiferae, B. theobromae and Fusarium sp. using root injury

method.

Treatments Decline symptoms in mango(root injury)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

Ceratocystis sp.(Cs) + + + + +

T2

N. mangiferae (Nm) - - - + -

T3

B. theobromae (Bt) - + - + -

T4

Fusarium sp. (Fu) + + + + +

T5

Nm + Cs + + + + +

T6

Cs + Bt + + + + +

T7

Cs + Fu + + + + +

T8

Nm + Bt - + + + -

T9

Nm + Fu + + + + -

T10

Bt + Fu + + + - +

T11

Nm + Cs + Bt + + + + +

T12

Nm + Fu + Bt - + + + -

T13

Nm + Fu + Cs + + + + +

T14

Bt + Fu + Cs + + + + +

T15

Nm + Bt + Cs + Fu + + + + +

T16

Control - - - + -


133

Table 4.5: Pathogenicity of fungi associated with guava decline by using flap

method inoculation.

Treatments Decline symptoms in guava

(Flap method)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

N. mangiferae (Nm)

- + - + -

T2

B. theobromae (Bt)

- + + + -

T3

Fusarium sp. (Fu)

+ - + + -

T4

Nm + Bt

- + + + -

T5

Nm + Fu

+ + - - -

T6

Fu+ Bt

+ + + + -

T7

Nm + Bt + Fu

+ + + + +

T8

Control

- - - - -


134

Table 4.6:Pathogenicity of fungi associated with guava decline by using root injury

method of inoculation.

Treatments Decline symptoms in guava(root injury)

Leaf

drooping

Bark

splitting

Stem

canker

Stem

gummosis

Mortality

T1

N. mangiferae (Nm)

- + - + -

T2

B. theobromae (Bt)

- + - + -

T3

Fusarium sp. (Fu)

+ - - + -

T4

Nm + Bt

- + + + -

T5

Nm + Fu

+ + - + -

T6

Fu+ Bt

+ + + + -

T7

Nm + Bt + Fu

+ + + + +

T8

Control

+ - - + -


135

Fig.4.1: Flap method of inoculation.

Fig.4.2: Root injury method of inoculation.

136

The response of plants in all cases showed that Nattrassia alone cannot

cause the mortality of plants. However, in combination with other fungi it can

produce all the symptoms we observe in the decline affected trees of citrus mango

and guava. Moreover, the fungus Ceratocystis sp. has the ability to cause the

mortality of plants which is more common in case of mango.

4.5. DISCUSSION

The present study was done to evaluate the role of N. mangiferae incausing

decline among other popular fungi that has been repeatedly reported from various

declining trees. It was necessary to include those fungi in pathogenicity experiment

as various scientists reported the cause of decline differently, for example

Mahmood et al.(2002) reported that the cause of decline is B. theobromae. Similar

results were shown by Khanzada et al.(2005). However, Fateh et al. (2006); Al

Adawi et al. (2006) and Masood et al. (2010) reported the cause of the disease was

Ceratocystis sp. Similar results were also reported for citrus and guava decline,

where the role of B. theobromae and Fusarium sp. were given more importance

than other fungi (Safdar et al., 2015; Bokhari et al., 2008). There was another

school of thought who has reported that in case of guava decline, there is an

association of Fusarium sp. and Meloidogyne sp. (Gomes et al., 2012).

The similar behavior of N. magiferae and B. theobromae upon individual

inoculation for producing symptoms irrespective of the hosts once again confirms

that they inherit a common lineage as discussed in Chapter 3. It is important to

discuss here that most of these fungi are endophytic. Endophytic fungi live

symbiotically with the majority of plants by entering their cells (Hirsch and Braun,

137

1992) and are utilized as an indirect defence against herbivores. This means that

probably when the fungus is alone within a plant, it plays its symbiotic part, but

when combined with other fungi it becomes aggressive and damages plants. In the

present study, individual fungi except Ceratocystis were not able to produce

mortality at all. However, the combinations of these fungi caused mortality in the

green house potted plants; these results are in agreement with those of Fateh et

al.(2006); Al Adawi et al.,

(2006); Masoodet al. (2010) and Fateh et al. (2016).

Among the two methods used for pathogenicity, root injury was the most

effective for infection development and causing the mortality of plants. This also

confirms the findings that the decline causing fungi and in particular the

Ceratocystis sp. when enter the plants through wounds during cultural practices

such as plowing, hoeing and weeding, invade roots, move to xylem part where they

block the intake of water and nutrient inflow. Thus, cause permanent wilting or

mortality of the tree (Fateh et al., 2006; Al Adawi et al., 2006; Kazmi et al, 2007;

Masood et al., 2010 and Fateh et al., 2016). This also suggests that when some

screening studies are planned especially for varietal resistance against Ceratocystis

sp. or other decline causing fungi, the root injury method should be adopted.

The fungus Fusarium sp. in case of mango has been able to cause mortality

but in only one case when it was inoculated through root injury method. This once

again strengthens the statement that the root injury method of inoculation was the

most effective to cause disease which enabled Fusarium as well to cause mortality.

In case of guava the death caused by Fusarium was already reported by Pandey and

Dwivedi (1985).

138

The fungus N. mangiferae in field conditions enters the plants through

wounds not only from the branches through bark cracks but also through stem and

injured roots. This fungus in the presence of other fungi such as Fusarium sp., B.

theobromae, Ceratocystis sp. plays an equal role for creating a situation where the

plants normal functioning is disturbed in target fruits i.e. mango, citrus and guava

as reported by Safdar et al.(2015) and Fateh et al. (2016).

139

Chapter 5

SOME PHYSIOLOGICAL STUDIES OF FUNGI ASSOCIATED

WITH DECLINE

5.1. INTRODUCTION

Basic knowledge about the pathogen, its survival, spread and other related

aspects are very important for the better management of any disease. Physiological

studies include the study of factors that contribute towards the physiological

development of the fungi. These factors can be different sources of carbon and

nitrogen by using different culture media to observe the best growth on a particular

medium (Walker and White, 2005).

It is a known fact that physiological development offungi is largely affected

by environmental factors such as temperature, light and pH. Most of the time these

studies are the part of epidemiology and are conducted in vitro (Charlie and

Watkinson, 1995).

The factors that influence physiological development of fungi include the

optimum ranges of temperature, light, pH and carbon sources that can either favour

or inhibit the growth of fungi. Therefore, these studies lead to develop the

management strategies for different diseases (Hubbali et al., 2010).

As the effects of these physiological factors have not been studied on

decline inciting fungi in Pakistan. Therefore, the present studies were conducted to

assess the effects of different physiological factors on the growth of the fungi

isolated from citrus, mango and guava with special emphasis on N. mangiferae.


Physiological studies have been done on decline causing fungi on several

114

140

different hosts. For example Lasiodiplodia theobromae, causing decline of

common tea (Camellia sinensis) does not sporulate or poorly sporulates on potato

dextrose agar (PDA). Therefore, a study was planned to determine the effects of

culture media, carbon source, nitrogen source, temperature, pH and light on

mycelial growth and sporulation. Among several carbon sources tested, glucose

and sucrose were found superior for growth. The addition of tea root extract to

potato dextrose agar medium was found to be the most suitable for mycelial growth

and sporulation of L. theobromae. It was found that the fungus grew at

temperatures ranging from 20 to 36 oC, with optimum growth at 28

oC and no

growth was noted at 40 oC. There was no significant effect of different light periods

on growth of L. theobromae, however, light enhanced sporulation. The fungus

grew at pH 3.0-8.0 and optimum growth was observed at pH 6.0. Tea root extract

supplemented potato dextrose agar medium with pH 6.0 was the most suitable for

production of conidia of L. theobromae at 28 oC (Saha et al., 2008).

However, Kausar et al. (2009) reported that Optimum temperature for both

Fusarium solani and Lasiodiplodia theobromae fungi was 25 ºC. They found that

continuous light supported growth more as compared to darkness (for 24 hours)

and 12 hours light plus 12 hours darkness for the growth of fungi. Among two

media tested for both fungi, grew maximum on Poatao Dextrose Agar (PDA)

medium but least grew on the water agar (WA) medium.

According to Alam et al. (2001) Botryodiplodia theobromae grew and

sporulated well at 15-40°C, but optimum between 25-30oC. Among culture media

PDA showed maximum mycelial growth and sporulation. However, no growth was

observed at 10 and 45°C.

141

Similarly,Ceratocystis fimbriata is a decline cauing pomegranate decline,

grew well in all most all pH ranging from 2.0 to 11.0. It indicates that the fungus

can survive in all soil types and locations. Maximum growth has been recorded at a

pH of 7.5 with no significant difference with that on pH 7 and 8. The fungal growth

was inhibited below pH 5.5 and above pH 9.0. Lowest growth was observed at pH

2.0. The maximum growth of the fungus was observed at a temperature of 30oC

and failed to grow at 45oC (Sonyal el al., 2015). However, the best growth from

other hosts has been obtained at 25oC. This shows that the fungus when isolated

from different hosts can behave differently.

Fusarium oxysporum f. sp. psidii and F. solani, cause wilt in guava and are

highly variable pathogens. Physiological studies of the fungi revealed that

maximum mycelial growth was obtained on PDA followed by ME broth as liquid

medium. Maximum sporulation was recorded in oatmeal agar (OMA). The

optimum temperature and pH for growth of Fusarium sp. was 28oC and 5.5

respectively. The isolates had differences in their colony growth, mycelial mass,

macro-conidia, and micro-conidia produced. These variations were characters of

each of the isolates with respect to cultural and physiological characters (Gupta et

al., 2010).

Dring a field study on cashew diseases three fungal generaLasiodiplodia,

Fusarium and Pestalotiafound were cultured on Potato Dextrose Agar medium.

The optimum temperature for the growth of Fusarium was 250C while all the

temperature treatments 20oC, 25

oC and 30

oC were found suitable for the growth of

Lasiodiplodia and Pestalotia. Moreover, alternating 12 hours light and12 hours

darkness was more suitable for the growth of Fusarium than continuous darkness.

142

The growth of both Lasiodiplodia and Pestalotia were not affected by the light

condition.


5.3.1.Effect of Temperature, Light, pH and Culture Media on Fungal Growth

The effect of culture media, temperature, light and pH was observed on

mycelial growth of fungi including Nattrassia mangiferae, Ceratocystis sp.,

Botryodiplodia theobromae and Fusarium sp. in 90 mm petridish. The study was

done to have the basic information of suitable temperature, growth medium, pH

and sensitivity to alternate light and dark periods. For this purpose the temperature

ranges used were, 20oC, 22

oC, 25

oC and 28

oC, the pH ranges 5.5, 6.0 and 6.5.

Light and dark periods of 12 hrs respectively and complete 24 hours of continuous

light and continuous dark. The culture media tested were MEA, PDA and SDGAM.

In all cases other than target variable tested all conditions were kept constant. The

radial growth was measured by the ruler from the central point of inoculation at the

back of 90 mm Petri dish containing culture. Four observations were taken from

around and the average was calculated.

5.3.1.1. Effect of culture media.

Four culture media i.e. Potato Dextrose Agar (PDA), Malt Extract Agar

(MEA), Stem Decoction Glucose Agar (SDGA) and Carrot Juice Agar (CJA) were

evaluated to find out the most suitable one for the growth of the fungi. Each culture

medium was prepared in 1 L of water and autoclaved at 121 oC at 15 psi for 20

min. These were cooled to 45 oC and then poured in 90 mm Petri dishes for

solidification. A plug of 5 mm was taken from freshly growing culture of each of

the fungi was taken and inoculated to plates containing above mentioned media.

143

The plates were observed for mycelial growth everyday and on 7th day the final

reading was taken. The experiment was done in triplicate.

5.3.1.2. Effect of temperature

As mentioned above 5 mm culture plugs were taken by sterilized cork borer

from advancing margin of freshly growing target fungal cultures and inoculated on

PDA containing 90 mm petridishes. The incubation was done on PDA at 20oC, 22

oC, 25

oC and 28

oC for 7 days till the final reading was taken for radial mycelial

growth measurement. The experiment was done in triplicate.

5.3.1.3. Effect of light

For the effect of light and darkness on mycelial growth of isolated fungi, 5

mm culture discs were cut with sterilized cork borer from advancing margins of the

colonies of isolated fungi and inoculated on PDA plates separately for seven days.

To create condition of darkness carbon paper was used to wrap the petridishes.

Another set of petridishes of fungal culture was without wrap. All petridishes were

incubated at 25+1oC in triplicates and alternating light and darkness (12 hours light

plus 12 hours darkness) and complete darkness (24 hours) was provided to record

the effects in terms of mycelial growth. The mycelial growth in mm was recorded

on 7th day of inoculation.

5.3.1.4. Effect of pH

Target fungi were inoculated as bove in petridishes containing PDA and

incubated at optimum temperature 25+ 1 oC. The pH of PDA medium was adjusted

5.5, 6.0 and 6.5 with 0.1 N NaOH/HCl with pH meter. Optimum pH was

determined by calculating colony diameter on daily bases till final reading on 7th

day.

144

5.4.RESULTS

5.4.1. Effect of Culture Media

The culture media had significant effects on the growth of decline causing

fungi. Among isolates from citrus, the fungusN. mangiferaeshowed maximum

radial growth on MEA followed by PDA which was not significantly different

from that of MEA. Minimum growth was recorded on SDGA and CJA. Similarly,

there was significant difference in growth of Fusarium sp. on PDA and MEA. The

fungi Botryosphaeria sp. and B. theobromae both showed no significant difference

in growth on PDA and MEA. The growth on SDGA and CJA was significantly less

as compared to PDA and MEA (Table 5.1).

The fungi isolated from declining mango trees had significant differences in

growth on different media.N. mangiferae gave maximum growth on MEA medium

PDA. The growth of the fungus was significantly less on SDGA and CJA media

(Table 5.2).

Unlike N. mangiferae isolated from citrus, which showed no significant

difference in growth on MEA and PDA media, the isolate from mango showed

significant difference in radial mycelial growth on the media. The growth on MEA

was significantly higher than on PDA medium. This shows that isolates of same

fungus from different hosts can behave differently when growth is tested on same

culture media. This is because that fungus can have varying virulence levels on

different hosts (Makun et al., 2010)

The isolates of fungi from guava showed that N. mangiferae has significant

difference in growth on MEA from rest of the culture media giving the maximum

145

(75 mm) radial growth. Similarly, B. theobromae from guava showed significant

difference in growth on PDA and MEA (Table 5.3).

The results in table 5.3 showed that N. mangiferae and Fusarium sp. gave

best growth on PDA and MEA. The fungus Ceratocystis sp. grew well on SDGA

and CJA. The Botrysphaeria sp. and B. theobromae showed variable responses in

growth on different culture media. This also showed that the fungi isolated from

mango and guava had resemblance regarding radial mycelial growth on tested

culture media. However, the fungal isolates from citrus species behaved differently.

5.4.2. Effect of Temperature on Radial Mycelial Growth

The decline causing fungi behaved differently when grown on different

temperatures.

The statistical analysis showed highly significant results regarding the

effect of temperature on radial growth. The growth of Nattrassia mangiferae

increased from 20oC to 25

oC and then declined. Maximum radial growth of all the

fungi was recorded at 25oC, below and above this temperature their growth

declined (Table 5.4).

All the fungi isolated from mango grew to the maximum at a temperature of

25oC except Ceratocystis sp. which showed maximum growth at 22

oC as shown in

Table 5.5.

Similarly, the fungi isolated from guava followed the same growth patterns

at different temperature regimes as shown by the isolates from citrus and mango

(Table 5.6).

146

Table 5.1: Effect of culture media on radial mycelial growth of the fungi isolated

from Citrus.

Fungi Culture Media

(Average growth in mm)

PDA MEA SDGA CJA

Nattrassia mangiferae 80 e 81 e 74 d 67 c

Botryosphaeria sp. 85 e 84 e 71 d 70 cd

Botryodiplodia theobromae 80 e 79 e 75 d 70 cd

Fusarium sp. 65 c 61 a 57 b 58 b

Data are mean of threereplicates


from mango.

Fungi

Culture Media


PDA MEA SDGA CJA

Nattrassia mangiferae 69 ef 75 fg 68 e 65e

Ceratocystis sp. 43 a 51 b 55 c 54 c

Botryodiplodia theobromae 84 ef 72 f 71 f 67 e

Fusarium sp. 70 f 65 e 58 cd 58 cd


147

5.4.3. Effect of Light on Radial Mycelial Growth

Light significantly affected the growth of fungi isolated from different

hosts. It was found that continuous light or continuous dark did not have any

significant effect on the growth of fungi isolated from citrus. However, alternate

light and dark periods were found significant for the growth of fungi as shown in

Table 5.7. Similar findings were reported by Alam et al. (2001) and Sharma et al.

(2005).

The fungi isolated from declining mango trees followed the same pattern as

shown in Table 5.8. B. theobromae and Fusarium sp. had no significant differences

in any case. The results are in line with Rehman et al. (2011).

In case of the fungal isolates from guava the growth of B. theobromae when

kept in continuous dark was the minimum. However, it showed maximum growth

in alternate light and dark (80 mm) followed by continuous light conditions (75

mm). Thus, it did not show the significant difference in growth when placed in

either complete dark or under alternate light and dark hours. From the results it

seemed that the fungus had photophobic behavior. The rest of the fungi has no

clear significant difference in all cases (Table 5.9).

5.4.4. Effect of pH on Radial Mycelial Growth

The pH had significant effect on the growth of Botryosphaeria sp. it

showedmaximum growth at pH 5.5. Similarly,Fusarium sp. had significant

difference in growth at pH 5.5 from other pH ranges. The rest of the fungi have no

clear significant difference (Table 5.10).

148

N. mangiferae isolated from mango showed significant difference in growth

at different pH regimes. At pH 5.5, it gave maximum growth followed by that at

pH 6.0 and 6.5 respectively.


from guava.

Fungi

Culture Media


PDA MEA SDGA CJA

Nattrassia mangiferae 70 def 75 fg 67 de 65 d

Botryosphaeria sp. 75 fg 78 g 54 ab 51 a

Botryodiplodia theobromae 82h 73 f 70 def 68 de

Fusarium sp. 71 def 64 d 57 bc 58 bc

Data are mean of three replicates

Table 5.4: Effect of temperature on radial mycelial growth of the fungi isolated

from citrus on PDA medium.

Fungi Temperature (oC)


20 22 25 28

Nattrassia mangiferae 80 def 81 def 84 ef 78 cdef

Botryosphaeria sp. 74 cd 78 def 85 f 80 def

Botryodiplodia theobromae 76 cde 80 def 85 f 81 def

Fusarium sp. 43 a 54 b 71 c 68 c


149


from mango on PDA medium.

Fungi Temperature (oC)


20 22 25 28

Nattrassia mangiferae 68c 75d 83e 80e

Ceratocytis sp. 55b 57b 55b 41a

Botryodiplodia theobromae 67c 72cd 84e 82e

Fusarium sp. 41a 55b 74d 69c



from guava on PDA medium.

Fungi

Temperature (oC)


20 22 25 28

Nattrassia mangiferae 64 b 68 bc 81f 75 def

Botryosphaeria sp. 67 bc 70 bcd 79 ef 78 ef

Botryodiplodia theobromae 70 bcd 73 cdf 80 f 75 ef

Fusarium sp. 48 a 53 a 79 ef 65 b


150

Table 5.7: Effect of light on radial mycelial growth of the fungi isolated from citrus

on PDA medium

Fungi

Light Conditions


12 hrs light+

12 hrs dark

Continuous

light

Continuous

dark

Nattrassia mangiferae 80 d 65 ab 68 ab

Botryosphaeria sp. 77 d 69 bc 75 cd

Botryodiplodia theobromae 80 d 75 cd 70 bc

Fusarium sp. 62 a 68 a 65 ab


Table 5.8:Effect of light on radial mycelial growth of the fungi isolated from

mango on PDA medium.

Fungi

Light Conditions


12 hrs light+

12 hrs dark

Continuous

light

Continuous

dark

Nattrassia mangiferae 76 ef 74 ef 71 e

Ceratocystis sp. 56 cd 48 ab 43 a

Botryodiplodia theobromae 79 f 74 ef 74 ef

Fusarium sp. 59d 55 cd 52 bc


151

While, Ceratocystis sp. and Fusarium sp. showed significant difference in

growth at pH 6.5.Ceratocystis sp. gave maximum growth at pH 6.5 and Fusarium

sp. gave minimum growth at pH 6.5. The fungus B. theobromae followed same

pattern as shown in Table 5.11. The results coincide with those of Lathal et

al.(2012) and Rehman et al. (2011).

The isolates of N. mangiferae, Botryosphaeria sp. and Fusarium sp. from

guava had significant difference in growth at 5.5 pH. All fungi showed maximum

growth at pH 5.5 (69 mm, 65 mm and 62 mm respectively).

The fungus B. theobromae had no clear significant difference in growth in

any case and grew maximum at pH 5.5 (75 mm) followed by 72 mm and 68 mm

respectively at pH 6.0 and 6.5 (Table 5.12).

Results showed that the alternate light and dark conditions have

sigfunginificant effect on growth of N. mangiferae, Botryosphaeria sp., B.

theobromae and Fusarium sp. However, the fungus Ceratocystis sp. has shown

significant growth in contiuous dark condition.

The results regarding pH have shown that all of the fungi like low pH level.

As pH increases the mycelial growth is reduced. The results are somehow similar

in citrus and guava. However, in case of mango, Ceratocystis sp. has ascending

mycelial growth trend. The lowest pH range 5.5 and 6.0 has significantly reduced

growth as compared to pH level 6.5.

Similarities were also found in growth pattern of fungi associated with

Botryosphaeriacae family. This was very astonishing that within the same mango

trees the isolated mycoflora have different choices of environmental conditions,

especially the fungi belong to Botryospaeriace and Ceratocystis sp.

152

Table 5.9: Effect of light on radial mycelial growth of the fungi isolated from

guava on PDA medium.

Fungi

Light Conditions


12 hrs light+

12 hrs dark

Continuous

light

Continuous

dark

Nattrassia mangiferae 69 abcd 65 abc 63 ab

Botryosphaeria sp. 70 bcd 64 abc 61 a

Botryodiplodia theobromae 80 e 75 de 72 cd

Fusarium sp. 64 abc 61 a 65 abc

Data are mean ofthree replicates

Table 5.10: Effect of pH on radial mycelial growth of the fungi isolated from citrus

on PDA medium.

Fungi

pH of medium


5.5 6.0 6.5

Nattrassia mangiferae 71 ef 68 def 63 cd

Botryosphaeria sp. 74 f 62 cd 59 bc

Botryodiplodia theobromae 70 ef 66 de 65 cde

Fusarium sp. 59 bc 54 b 47 a


153

Table 5.11: Effect of pH on radial mycelial growth of the fungi isolated from

mango on PDA medium.

Fungi

pH of medium


5.5 6.0 6.5

Nattrassia mangiferae 79 gh 71ef 68 ef

Ceratocytis sp. 49 a 51a 57 bc

Botryodiplodia theobromae 81h 78gh 74fg

Fusarium sp. 65de 59cd 51ab

Data are mean ofthree replicates

Table 5.12: Effect of pH on radial mycelial growth of the fungi isolated from guava

on PDA medium.

Fungi

pH of medium


5.5 6.0 6.5

Nattrassia mangiferae 69 bcd 51a 49 a

Botryosphaeria sp. 65 bc 52 a 51a

Botryodiplodia theobromae 75 d 72 cd 68 bcd

Fusarium sp. 62 b 54 a 51a


154

5.5. DISCUSSION

It is clear from the results that when the growth of fungal isolates from

citrus was tested against various media i.e. PDA, MEA, SDGA and CJA, three

fungi among 4 have shown no significant difference in the growth. These fungi are

Nattrassia mangifera, Botryodiplodia theobromae and Botryosphaeria sp. As

discussed in chapter 3 that these fungi share a common lineage and hence they

showed no significant difference on each of the culture media used for their

growth. However, Fusarium sp. growth was significantly different from the rest of

the fungi in the study. The significantly shown growth may be due to its slow

growing nature already reported in the past (Schroeder et al. 2006; Kang et al.,

2014). The other reason can be its growth response to a selective medium rather

than a range of media (Vanwyk et al., 2006; Kang et al., 2014).

It was astonishing to know that N. mangiferae isolates from mango showed

completely different behaviour in term of growth within stipulated time i.e.

significantly less growth was observed in all of the tested culture media as

compared to its isolates from citrus. It is due to the fact that the growth of N.

mangiferae in mango is hampered by plant defence mechanisms (Elliott and

Edmonds, 2008) and it is reasonably slow growing in nature. However, further

research is needed in this regard. Among other fungi, Ceratocystis sp., showed

significantly slow growth on all of the media tested. The difference was also

observed in its growth on potato dextrose agar (PDA) and malt extract agar (MEA)

but no significant difference was found onstem decoction glucose agar (SDGA)

and carrot juice agar (CJA). Ceratocystis sp. is known for its complexity in growth

155

and very choosy in physiological conditions especially temperature (Fateh et al.,

2010; Rehman et al., 2011; Masood et al., 2011). Secondly, culture media like

SDGA and CJA media were particularly developed for the fungus Ceratocystis sp.

based on the experience of scientists associated with this fungus (Moller and De

Vay, 1968; Barnes et al., 2003). During the study it was observed that on

inoculation of infected tree samples on SDGA and CJA, only the fungus

Ceratocystis sp. was isolated but its growth was observed in bits and patches

spread throughout the 90 mm petridish but once re-culturing was done the normal

growth was observed in the petri plates and this is the new finding of the study.

There was not much difference in growth among the fungal isolates from guava

when compared with mango. This may be due to the fact that guava in Pakistan is

grown with similar climate as mangoes. Sometimes, guava is intercropped with

mango in orchards (Khushk et al., 2009).

Unlike N. mangiferae isolated from citrus which showed no significant

difference in growth on MEA and PDA media, the isolate from mango showed

significant difference in radial mycelial growth on the media. The growth on MEA

was significantly higher than that on PDA medium. This shows that isolates of

same fungus from different hosts can behave differently when growth is tested on

same culture media. This is because that fungus can have varying virulence levels

on different hosts (Makun et al., 2010).

The response of growth of N. mangiferae isolates from citrus was much

interesting. It showed the aggressive growth from 20oC to 25

oC and started

declining after 28oC. The results are in agreement with those of Jamali and

Banihashemi(2010) and Rehman et al.(2011). It followed similar pattern in growth

156

when isolated from mango as well as guava. However, relatively less growth

among the isolates from mango and guava was observed as compared to citrus

isolates. The reason can again be the similar climatic areas occupied by mango and

guava. It is important to note that the fungus Ceratocystis sp. again preferred a low

temperature range as compared to other fungi, in case of mango (Fateh et al., 2006

and Rehman et al., 2011). The results also suggest that fluctuation in temperature

regimes in field condition affect both the growth of trees as well as the growth of

Ceratocystis sp. This fact has been supported by Reynolds(1992).

Mycelial growth was seen more in the isolates of Nattrassia when provided

alternate light and dark periods as compared to continuous light or continuous dark

periods. However, again the isolates from mango and guava did not show

significant differences in any condition of light and dark (Elliott and Edmonds,

2003). Nattrassia isolates from citrus were the least affected by the pH range of 5.5

to 6.5. However, in mango and guava the isolates performed well at pH up to 6.00.

The results are contradictory to the actual climatic conditions especially in the

mango orchards where soil pH is more than 7 andN. mangiferae keeps growing but

the results are in agreement with the findings of Namsi et al. (2010) andEl Atta and

Aref (2013).

157

Chapter 6

MORPHOLOGICAL VARIABILITY AMONG ISOLATES OF

COMMONLY FOUND FUNGUS (NATTRASSIA

MANGIFERAE)FROM DECLINE AFFECTED CITRUS,

MANGO AND GUAVA TREES

6.1. INTRODUCTION

The most common fungi found from decline affected trees are Nattrassia

mangiferae and Botryodiplodiatheobromae. These fungi are found in different

parts of the plants causing different symptoms. However, the same specie might

have some different characteristics when invading different hosts. Mostly these

fungi invade twigs and branches of the trees (Al- Adawi, 2002, Asif et al., 2011).

The fungus N. mangiferae belongs to class Coelomycetes. The fungus is

polymorphic and has two spore stages, the pycnidial and the arthroconidial stage.

Some anamorphs of Botryosphaeria are similar to Nattrassia both in their behavior

on the host plant and morphology. They attack stressed plants especially due to

drought or physical wounding. The fungus can cause canker, branch die back, twig

die back and blossom blight. Mostly these fungi are endophytic and become

aggressive to cause symptoms when the host is under stress conditions (Denman et

al., 2003).

N. mangiferae has been reported to cause a number of diseases in different

host plants including: branch wilt of walnut, citrus, figs,branch canker of

eucalyptus, madrone, acasia and mango, dieback and trunk canker of guava (El-

Atta and Aref, 2013).

The fungus N. mangiferae is basically a wound invading pathogen of forest

and fruit trees and has been reported to be the cause of foot canker of Hevea

132

158

brasiliensis seedlings in Sri Lanka. The cultural characteristics and the morphology

of the reproductive structures such as thallospores, pycnidia, pycnospores of Hevea

isolate from Sri Lanka resembles with the isolate of N. mangiferae obtained from

other hosts in different parts of the world (Jayasinghe et al., 1997).

In the year 2000, in the Jiroft region, South-Eastern Iran, N. mangiferae was

isolated from both Ficus religiosa, which showed branch die-back and elongated

cankers in trunks, and Psidium guajava (guava) trees, which showed branch die-

back. The pathogenicity of the fungus was demonstrated by fulfilling Koch's

postulates. These diseases are a potential threat to the citrus industry and P.

guajava production in the region. This is the first report of N. mangiferae inciting

die-back and trunk cankers on F. religiosaand die-back on P. guajava (Mirazeeet

al., 2003).

A survey of branch wilt disease was conducted in 2008, 2009 and 2010 in

South Kordofan State. The survey was done on different hosts inclunding citrus,

mango and other fruit trees. The tree showed symptoms of dieback, bark cracking

and sloughing off, gum exudates on pockets at the base of infected branches.

N. mangiferae was dominant pathogen in the samples collected from branch

wilt affected trees. Other opportunistic fungi found were such as Penicillium sp.,

Aspergillus niger and Fusarium oxysporum.

Since N. mangferae has been reported from a variety of hosts causing

variety of symptoms, the present study was aimed to see the morphological

variations among different isolates of these fungi based on their location, tree and

plant parts from which they were isolated with a hypothesis that there may be some

morphological variations among the isolates.

159

6.2.REVIEW OF LITERATURE

In Pakistan the fungus C. fimbriata for the first time was reported in Sindh

from declining mango trees. It's identification was confirmed by morphological

characteristics of perithecia (brown to black with globose base, necks almost 800-

900 µm long with ostiolar hyphae), ascospores (elliptical 4-8 × 2-5 µm, hat shaped)

conidiophores (hyaline, septate up to 150 µm long and conidia that were

cylindrical, sometimes in chains and truncated at the ends (Fateh et al., 2006).

Lasiodiplodia theobromaewhich is the anamorph of Botryosphaeria

rhodina is an opportunistic fungus attacking on more than 500 tree species in the

tropics and subtropics. During surveys of different plant diseases in Australia and

Venezuela various isolates were some what different from L. theobromae based

upon morphology and subsequently characterized and ITS and EF1-α nucleotide

sequences. These were described as three new speciesi.e. L. venezuelensis sp.

nov., L. crassispora sp. nov. and L. rubropurpurea sp. nov. Theywere easily

distinguished from each other (Burgess et al., 2006).

Nattrassia mangiferaeanother opportunistic wound invading fungus of

many forest and fruit trees was reported to cause of Hevea brasiliensis foot canker

in Sri Lanka. The morphology of the reproductive structure: thallospores, pycnidia,

pycnospores of Hevea isolate from Sri Lanka resembled with the isolate of N.

mangiferae obtained from other hosts in different parts of the world (Jayasingheet

al., 1997).

Elliott and Edmonds in 2003 identified N. mangiferae, causing a canker

disease of Pacific madrone, using morphological and molecular methods. Only

asexual spores were observed, but sequencing of the ITS region of the ribosomal

160

rDNA places the sexual state in the genus Botryosphaeria. Resembles of the fungus

was found with Fusicoccumwhich is anamorphs of closely related Botryosphaeria

species and had a similar pathology.

Several pecies of Botryosphaeria affect mango trees and fruit. These are

mainly identified on the morphology of the anamorphs, include,Dothiorella

dominicana, D. mangiferae (Natrassia mangiferae), D. aromatica and an

unidentified species, Dothiorella „long‟. The genus name Dothiorella, however, is

acknowledged as a synonym of Diplodia. To achieve isolates representing all four

Dothiorella spp. the anamorphs of known Botryosphaeria sp., were based on

conidial morphology and DNA sequence data. The morphological and molecular

results confirmed that the fungi previously identified from mango as species of

Dothiorella belong to Fusicoccum. This study provided basis for the identification

of Botryosphaeria species from mango, which is important for disease control and

to uphold quarantine regulations (Slippers et al., 2005).

In, Iran, a study was conducted using morphological and molecular

methods to identify the causal agent of black locust treesdecline. The isolates of the

fungus produced arthroconidia, and the fungus identified wasNeofusicoccum

mangiferae.Pathogenicity test under the bark produced similar symptoms as in

natural infection. Species classification was confirmed by analysis of the internal

transcribed spacer (ITS) sequencing. The funus was mostly similar to N.

mangiferae(Nazerianet al., 2015).

A fungus resembling the asexual morphs in the family Botryosphaeriaceae

was isolated from a fallen leaf of an orchid collected in Thailand. After

morphological and phylogenetic analyses it was placed as Neoscytalidium sp.

161

(Huang et al., 2016).


6.3.1. Fungal Isolates

To study the morphological characterization, the samples were collected

from decline affected citrus trees from representative orchards of tehsil and district

Sargodha and tehsil Bhalwal of Sargodha. For mango, the orchards of districts

Multan & Rahim Yar Khan and for guava tehsil Pattoki of district Kasur and tehsil

Sharaqpur of Sheikhupura. The areas were selected as representatives, based on

their commodity based popularity as well as number of orchards. The samples were

taken from twigs, branches and stem at collar region. The cultures were purified by

sub culturing and finally 30 isolates were selected out of 300 samples based on

their hosts used in the present study. The methodology for isolation and

identification has already been mentioned in chapter 3.

6.3.2. Morphological Characterization

For morphological characterization, each isolate was maintained on Malt

Extract Agaer (MEA) medium. A 5 mm mycelial plug was taken from fresh

growing culture of the fungi N. mangiferae and B. theobromae and aseptically

placed in the center of petridish (90 mm). After 5 days at 25oC, morphological

characters like colony color and patterns were studied. However, the conidial

characters and measurements were recorded on conidial appearance.

The measurement of conidia, perithecia and other target structure was done

using method reported by Fateh et al., 2006.

6.4.RESULTS

162

The morphological variability of the most common decline causing fungi N.

mangiferae was studied based on hosts i.e. citrus mango and guava, plant parts i.e.

branch and twig and location. The results are shown in the tables 6.1 to 6.6.

The isolates for decline affected citrus trees collected from tehsil and

district Sargodha showedmorphologically similar characteristics irrespective of the

plant part and location. The isolates on malt extract agar medium showed dark grey

mycelial and produced appressed colonies five days after inoculation. However,

there was a slight difference in the measurement of chain of arthrospores which

were somewhat elongated (Fig.6.1), hyaline aseptate, and fusoid becoming 2-3

septate with central cell dark brown (Table 6.1).

In tehsil Bhalwal of Sargodha, isolates had exactly similar characteristics

with one another. They even did not show slight change in the measurement of

arthrospores. They also showed resemblance to the isolates taken from tehsil and

district Sargodha (Table 6.2).

In case of mango, the isolates of N. mangiferae showed major difference in

the mycelial pattern in the 90 mm petri plates. The mycelia started to rise instead of

running in the appressed form. Mycelial color in case of mango after 5 days of

inoculation turned creamy grey which was completely different fromN. mangiferae

isolates taken from decline affected citrus trees. The arthrospores were also not

much elongated continuously and mostly they were round shaped in the chain

(Table 6.3). The isolates from district Rahim Yar Khan were also similar to the

isolates taken from district Multan (Table 6.4).

The isolates taken from decline affected guava trees show more closeness

towards the mango isolates. There has been mixture of elongated and roundish

163

Table 6.1: Morphological characteristics of N. mangiferae isolates from citrus in

tehsil and district Sargodha.

Isolate Tree/

Plant

Part

Mycelial

color

(after 5

days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

SW-6 Citrus/

Branch

Dark grey Appressed 5-12.5x2.5-10 Somewhat elongated

hyaline aseptate,

fusoid becoming 2-3

septate with central

cell dark brown

SW-7 -ditto- -ditto- -do- 5-12.0x2.5-10 -ditto-

SW-8 -ditto- -ditto- -do- 4.5-12.5x2.5-9 -ditto-



Sar-6 Citrus/

Twigs

-ditto- -do- 4.5-12.0x3.0-

10

-ditto-

Sar-7 -do- -ditto- -do- 5-12.0x2.5-10 -ditto-



Sar-10 -do- -ditto- -do- 4.5-12.0x3.0-

10

-ditto-

Table 6.2: Morphological characteristics of N. mangiferae isolates from citrus in

tehsil Bhalwal, district Sargodha.

Isolate Tree/

Plant

Part

Mycelial

color

(after 5

days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

BE-10 -ditto- Dark grey Appressed 5-12.0x2.5-10.5 elongated hyaline

aseptate, fusoid

becoming 2-3 septate

with central cell dark

brown

BW-6 Citrus/

Twigs

-ditto- -ditto- 5-12.0x2.5-10 -ditto-

BW-7 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -ditto-



BW-

10

-ditto- -ditto- -ditto- 5-12.0x2.5-10.5 -ditto-

164

Table 6.3: Morphological characteristics of N. mangiferae isolates from mango in

district Multan.

Isolate Tree/

Plant

Part

Mycelial

color

(after 5

days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

BBR-6 Twigs Creamy

grey

Raised 5-12.0x2.5-10 Hyaline aseptate,

fusoid becoming 2-3


cell dark brown

BBR-7 -ditto- -ditto- -ditto- 4.9-12.0x2.5-10 -ditto-

BBR-8 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto-

MKR-

8

-ditto- -ditto- -ditto- 5-12.0x3.0-10 -ditto-

MKR-

9

Branch -ditto- -ditto- 4.4-12.5x2.5-10 -ditto-

MKR-

10

-ditto- -ditto- -ditto- 5.2-12.0x2.5-10 -ditto-

MSR-

6


MSR-

7


MSR-

8


MSR-

9

-ditto- -ditto- -ditto- 5-12.0x2.5-10 -ditto-

165

Table 6.4: Morphological characteristics of N. mangiferae isolates from mango in

district Rahim Yar Khan.

Isolate Tree/

Plant

Part

Mycelial

color (after

5 days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

LP-7 Twigs Creamy

grey

Raised 4.2-12.5x2.1-10 Hyaline aseptate,

fusoid becoming 2-3


cell dark brown

LP-8 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto-

LP-9 -ditto- -ditto- -ditto- 4.2-12.5x2.5-10 -ditto-

KP-6 Branch -ditto- -ditto- 5.1-12.5x2.5-10 -ditto-

KP-7 -ditto- -ditto- -ditto- 5-12.0x2.5-10 -ditto-

KP-8 -ditto- -ditto- -ditto- 5.2-12.0x2.7-10 -ditto-

MQ-6 -ditto- -ditto- -ditto- 4.5-12.5x2.5-10 -ditto-



Table 6.5: Morphological characteristics of N. mangiferae isolates from guava in

tehsil Pattoki of district Kasur.

Isolate Tree/

Plant

Part

Mycelial

color (after

5 days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

PI-12 Twigs Creamy

grey

Raised 4.5-12.5x2.0-10 Hyaline aseptate,

fusoid becoming 2-3


cell dark brown

PI-13 -ditto- -ditto- -ditto- 4.0-12.0x2.0-10 -ditto-



PI-16 Branch -ditto- -ditto- 4.5-12.0x2.5-10 -ditto-

PI-17 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -ditto-



PI-20 -ditto- -ditto- -ditto- 5-12.5x2.5-10.5 -ditto-

166

Table 6.6:Morphological characteristics of N. mangiferae isolates from guava in

tehsil Sharaqpur of district Sheikhupura.

Isolate Tree/

Plant

Part

Mycelial

color (after

5 days)

Mycelial

pattern

Arthrospores

measurement

(LxW) µm

Description of

arthrospores

SP-11 Twigs Creamy

grey

Raised 5-12.5x2.5-10 Hyaline aseptate,

fusoid becoming 2-3


cell dark brown

SP-12 -ditto- -ditto- -ditto- 4.5-12.0x2.5-9 -do-

SP-13 -ditto- -ditto- -ditto- 5-12.5x2.5-10 -do-



SP-16 Branch -ditto- -ditto- 4.5-12.0x2.5-10 -do-

SP-17 -ditto- -ditto- -ditto- 4.0-12.5x2.5-

10.2

-do-


SP-19 -ditto- -ditto- -ditto- 5-12.0x2.5-10.2 -do-


167

Fig.6.1:N. mangiferae arthrospores from citrus

Fig.6.2:N. mangiferae arthrospores from mango

168

arthrospores (Fig. 6.2). However, there has been no difference in mycelial colours

and pattern when compared with mango isolates.

6.5. DISCUSSION

The morphological characteristics of the fungus described in current study

were in agreement with those reported by Wilson (1949); Calavan and Wallace

(1954); Sutton and Dyko (1989) and El Atta and Nori (2014). The similarity

between isolates of mango and guava shows that there has been a shift of attack of

N. mangiferae either from mango to guava or otherwise. The chances are that these

are the same fungal isolates which attack both trees as both are grown in tropical

climate and as discussed earlier that sometimes guava is intercropped in mango

orchards and hence there has been a transfer of the fungus from mango to guava.

It has been found during the pathogenicity tests that the symptoms produced

by N. mangiferae in both trees were almost similar, which is again a confirmation

of their common origin (Fateh et al., 2016). In case of citrus, the morphology of N.

mangiferae was a little bit different. One of the reasons might be the difference in

climate where citrus is grown and secondly, it can be due to the fact that in citrus

the fungus has been reported to produce branch wilt with cracks in a particular

check pattern, while in mango and guava they either cause branch dieback, branch

canker or twig die back (Mirzae et al., 2002; Saaiman, 1997).

However, the morphology of N. mangiferae isolates from all citrus, mango

and guava had close resemblance with madrone canker isolates in USA. Madrone

isolates of N. mangiferae are characterized as hyaline, aseptate, guttulate conidia

becoming 1- to 3-septate, verisicolored or brown. Conidia had thin-walls, mostly

fusoid but occasionally oblong or clavate with truncate bases. The isolates differed

169

in conidial length, width as well as in arthrospore size. Although there were

statistically significant differences, there does not appear to be a geographical

pattern in spore size or shape. The researchers in US conclude that N. mangiferae

isolated from Pacific madrone belonged in the teleomorph genus Botryosphaeria

and hence, diseases caused by this fungus can be managed using methods

developed for other Botryosphaeria pathogens, such as B. dothidea(Elliott and

Edmonds, 2003).

170

Chapter 7

FACTORS FAVORING THE DECLINE DISEASE AND

DEVELOPMENT OF STRATEGIES FOR ITS MANAGEMENT

7.1. INTRODUCTION

Tree decline have been a universal problem and none of the forest, fruit and

ornamental trees are safe from the disease. Mostly decline in trees has very

complex etiology and is attributed to pathogens, various native organisms, climatic

factors and agricultural or urban pollution. Where particular biotic or abiotic

factors could not be singled out, they have been regarded as predisposing, inciting

or contributing factors in „diseases of complex etiology‟. For example ongoing

monitoring of eucalypt decline during recent droughts in eastern Australia, together

with extensive one-time observations across temperate Australia, provided

opportunities to further examine some hypotheses of decline and dieback that were

largely based on retrospective investigations (Jurskis, 2005). Dieback in intervals

can be distinguished from the process of chronic decline. Repeated dieback was

associated with natural climatic extremes whereas chronic decline was associated

with human management. Decline of forests in nature was associated with

exclusion of fire and grazing, while decline of rural trees was mostly associated

with pastures development (Fensham, 1998). Trees growing low in the landscape

on soils with poor drainage and aeration were especially predisposed to decline. It

appears that chronic abiotic stress causes tree decline when the function of roots is

impaired by changes in soils. Climatic extremes can accelerate severe declines

associated with human management. A variety of pests, „pathogens‟ and parasites

can take advantage of trees that are stressed by environmental changes, especially

eutrophication. Similarities between diebacks and declines in the Atlantic and

147

171

Pacific regions suggest a simple unifying concept of tree decline and dieback

(Karnosky, 2003).

A gradual decline in tree health is a common problem in urban and rural

areas of Pakistan. Decline is not limited to certain specific trees but it is galloping

towards almost all woody vegetation of forest, ornaments and fruits. Trees live

long and over period of years they are invaded by number of insects, diseases and

environmental effects. These trees are called stressed trees and often end up in

decline and mortality (Bigler et al., 2004).


Pakistan has tropical and sub tropical climate in most of the fruit growing

areas. Much information has been generated in general for the perennial crops in

the tropics and sub tropics in the world. Diseases in the tropics may be complicated

by interactions between different pathogens, or between pathogens and insect pests

(Holliday, 1980; Ploetz, 2006; Vandermeer et al., 2010; Anonymous, 2010).

Disease complexes involving a number of fungal pathogens or fungi and

nematodes are common in tropical situations. Interactions between pathogens and

environmental stress may also occur. Crops can become more susceptible to

pathogen infections when weakened by environmental stresses such as drought,

temperature extremes, and exposure to sunlight or wind (Agrios, 2005). Stressed

plants, or plants sustaining damage caused by insects or other pathogens, may also

be susceptible to attack by secondary pathogens or pathogens that infect through

wounds. Nutrient deficiencies may increase the susceptibility of crops to disease. In

tropical perennial crops, poor plant nutrition is likely to be a particularly important

contributing factor to production losses (Palti, 1981). In addition to lower

172

production due to nutrient deficiency, low nutrition may predispose plants to

diseases, increasing losses further. Nutrient deficiency causes the plant to become

weakened and generally more susceptible to infection. Under such conditions,

infection by weakly pathogenic species that would normally cause few problems

may become more serious. The incidence and severity of particular diseases may

also be linked to deficiencies of particular nutrients (Schroth et al., 2000).

Scientists agreed that the insect pests and diseases are not the only cause of

tree decline but the tree age, environmental factors, tree nutrition, soil chemistry

and above all the neglecting right production practices play a crucial role for

decline of the trees. For example citrus decline in Pakistan is a complex or

syndrome. As the phytoplasma, viruses, nematodes and fungi are involved in citrus

decline but at the same time the production practices like tree density in the

orchard, orchard sanitation, orchard irrigation and nutrition etc. are not accurate

(Arif et al. 2005; Burney et al., 2007). All these circumstances invite decline not

only for citrus but also in other forest, ornamental and fruit trees (Bigler et al.,

2006).

Mango decline has been a problem for Pakistan since it was first time

reported in 1995 at Muzaffar Garh. Lot of work has been done on mango decline in

the past few years and the production technology is being altered to manage the

issues (Fateh et al., 2006). Mango decline is a general term and it is used for

different disorders in the mango trees which either slowly or rapidly decline the

mango tree or eventually cause the death of tree

Guava decline is another big threat in Pakistan. As we have already limited

area under cultivation and the stressed condition someday will let vanish this

173

precious fruit from lands of Pakistan. It is a national problem of Pakistan because

since last decade the guava production has been declining. The problem of guava

decline has been prevailing in Lahore, Sheikhupura, Faisalabad, Jhang and

Sargodha (Khushk et al., 2009).

Therefore, the present study was done to know the common factors

favouring decline disorders especially in mango citrus and guava and on the basis

of these factors listing the management strategies that can be adopted to avoid tree

decline in future.


7.3.1.Surveys for Factor Favoring Decline in Fruit Orchards

A proforma was designed to have survey in the citrus, mango and guava

orchards randomly in their respective production areas as mentioned in table 7.1,

7.2 and 7.3 to record parameters such as tree crop, age of trese, variety,

intercropping, method of irrigation, nutrition applied, pruning of trees, ploughing in

the orchards and plant protection measures adopted to co relate this with disease

index (%) found. The detail of the parameters was as under:

Tree Crop: Mango, citrus, guava

Age of the tree: Age of the tree was taken in the form of range. The trees were

divided into different age groups i.e. less than 10 years, 10-15, 16-25, 26-50 and

more than 50 years were represented by 3.

Variety: individual tree variety was taken into records

Intercropping: The field crop, other trees grown or otherwise no intercropping

was observed

Irrigation: Source method and time of irrigation was observed

174

Nutrition: Kind of nutrients applied or not applied

Ploughing under tree canopy: Ploughing in the orchard especially under tree

canopy was present or absent.

Plant protection measures: If any plant protection measures are adopted?

Disease Incidence in the Orchard and average severity: Incidence of the disease

was recorded as the % age of affected plants in an observed population. While, the

severity was recorded based on the area of infected individual tree.Factors were

correlated with disease index (%).

7.3.2. Statistical Analysis

For the statistical analysis, SPSS software was used wherever needed. First

of all the data was coded in binary form. Such kind of coding is needed where in

data we have "Yes" or "No"entries. The data was imported from excel spread sheet

to SPSS software. Finally the correlation was chosen for the analysis to see effect

of each factor on decline disease index of citrus mango and guava.

7.4.RESULTS

According to table 7.1 survey conducted in 6 tehsils of district

Sargodha for observing the factors that can favor the decline disease in these areas

has shown interesting results. In Tehsil Bhalwal 8citrus orchards out of 13

locations fell in the age group of 16-25 while the rest 5 are in the age group of 10-

15. The sole variety of citrus cultivated in these orchards was "Kinnow". In all

young orchards disease index either was low or zero. This is because the young

175

Table 7.1: Factor affecting disease index (%) in selected orchards of citrus in district Sargodha.

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Bhalwal

Chak No.

4 SB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 31

Chak No.

7 ML 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 32

Chak No.

7SB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 40

Chak No.

7 ASB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 37

Chak No.

8NB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 33

Chak No.

9 NB 10-15 Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 15

Chak No.

Chak 9

Lokri 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 36

Chak No.

10 NB 10-15 Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste)

0

176

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Chak No.


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 31

Chak No.

18 NB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 37

Chak No.


Canal/F

lood

P

*(NPK) P A

P (Lime

paste) 0

Chak No.


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 17

Chak No.


Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Sargodha Chak No.

24 SB 16-25 Kinnow A

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 20

Chak No

27 SB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 37

Chak No


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 20

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 27

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 29

177

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 31

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 27

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 32

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 24

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 40

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 37

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 33

Chak No

95SB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 32

Chak No


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 24

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 29

178

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 32

Chak No


Canal/F

lood

P

(FYM) A P

P (Lime

paste) 25

Sillanwali Chak No.


Canal/F

lood

P

(NPK) P A

P (Lime

paste) 15

Chak No.


Canal/F

lood

P

(NPK) P P

P (Lime

paste) 24

Chak No.

120SB 10-15 Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Chak No.


Canal/F

lood

P

(NPK) P A

P (Lime

paste) 12

Chak No.


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 32

Chak No.

124 SB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Chak No.

127 SB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 4

Chak No.

127 NB

10-15

Kinnow P

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 15

179

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Chak No.

137 SB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Chak No.

147 NB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Chak No.

148 NB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 12

Shahpur

Malakwal 16-25 Kinnow P

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 19

Wadhi 16-25 Kinnow P

Canal/F

lood

P

(FYM) P P

P (Lime

paste) 35

Kot

Maghrib 16-25 Kinnow P

Canal/F

lood

P

(FYM) P P

P (Lime

paste) 20

Shahpur

Saddar 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 27

Noor

Kallu 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 27

Kandaan

Kalan 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 31

Jhavarian 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

180

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Hussain

Shah 16-25 Kinnow P

Canal/F

lood

P

(FYM) P P

P (Lime

paste) 25

Kudyana 16-25 Kinnow P

Canal/F

lood

P

(FYM) P P

P (Lime

paste) 23

Allahdad

Wala 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 25

Chachar

Sharf 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

Chak 128

NB 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 33

Sahiwal Kot

Pehalwan 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

Biral

Sharif 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 25

Nawabpur 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 24

Vijh 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

Nehang 10-15 Kinnow A

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 0

181

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Chohal 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 23

Tirkhanwa

la 10-15 Kinnow A

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 0

Haveli

Majuka 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 32

Pindi

Wala 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 24

Sial Sharif 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 31

Sangoraka

10-15

Kinnow P

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 12

Farooka

10-15

Kinnow P

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 16

Sial

Dholka 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 23

Muhamma

d Wala 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 24

Jahane

Wala 10-15 Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

182

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Dherowal 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

Chatror 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 29

Radhan

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

Kot Momin Chak No.

9 SB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

19 SB

10-15

Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0


Canal/F

lood

P

(NPK) P A

P (Lime

paste) 12


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 19


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 16


Canal/F

lood

P

(FYM) P P

P (Lime

paste) 23

Rawan 10-15 Kinnow A

Canal/F

lood

P

(NPK) P A

P (Lime

paste) 0

183

FYM: Farmyard Manure; N: Nitrogen; P: Phosphorus; K: Potassium

Tehsil Location Age

Group

Variety Intercr

op

(P/A)

Irrigati

on

(T/C),

(HE/

Fl)

Nutriti

on

(P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index (%)

Dera

Thoye

Wala 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 24

Ghulapur

Bangla 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 29

Jalla

Makhdum

10-15

Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 16

Takht

Hazara

10-15

Kinnow A

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 12

Naseerpur

Kalan

10-15

Kinnow A

Canal/F

lood

P

(FYM) P P

P (Lime

paste) 17

Mateela 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 25

Dodha 16-25 Kinnow P

Canal/F

lood

P

(FYM) A P

P (Lime

paste) 28

Midh

Road 16-25 Kinnow P

Canal/F

lood

P

(FYM) P A

P (Lime

paste) 16

184

orchards 10-15 years group following pruning, fertilizer regimes, without

ploughing under tree canopy and plant protection spray regimes while from

orchards of 16-25 age reciprocal observations were done. Almost similar trend was

seen in other tehsils i.e. Sargodha, Shahpur and Sahiwal. However, best

management of the orchards among all tehsils was observed in tehsil Kot Momin

and Sillanwali. (Table 7.1).

The overall correlation of various factors with the citrus decline

disease index (%) was analysed using SPSS softare. The results are shown in Table

7.2. A perfect correlation was observed regarding age group, intercropping and

nutrition of the orchards with disease index percentage. One to one correlation gave

the accurate picture. The results were significant regarding the correlation between

the age groups and disease index %. It was observed that in young orchards disease

was minimum or nil, while in other higher age groups it was vice versa. Therefore,

at alpha 0.02 a perfect correlation was found using two tailed correlation test

(Table 7.3).

The results are significant when we observe correlation of intercropping

(Table 7.4), nutrition application (Table 7.6), pruning of trees (Table 7.7) and

ploughing under the tree canopy (Table 7.8) at the 0.01 level (2-tailed). The mode

of irrigation (Table 7.5) and plant protection measures adopted (Table 7.9) were

almost constant, hence correlation could not develop.

It is obvious from the results that orchards need care and management to

avoid decline, especially the snescence affects the physiology of trees and they

cannot resist degradation of cells.On the other hand if the growers adopt best

management practices like pruning of the trees for deadwood removal, aeration and

185

Table 7.2:Correlations of factors causing citrus decline disease index (%).

Age Group

Intercrop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protectio

n (P/A) If

P (which)

Disease

Index(%)

Age Group Pearson

Correlation 1 .687

** .

b .687

** .621

** -.835

** .

b .843

**

Sig. (2-

tailed) .000 . .000 .000 .000 . .000

N 86 86 86 86 86 86 86 86

Intercrop (P/A) Pearson

Correlation .687

** 1 .

b .663

** .607

** -.634

** .

b .714

**

Sig. (2-

tailed) .000 . .000 .000 .000 . .000

N 86 86 86 86 86 86 86 86

Irrigation (T/C),

(HE/Fl)

Pearson

Correlation .b .

b .

b .

b .

b .

b .

b .

b

Sig. (2-

tailed) . . . . . . .

N 86 86 86 86 86 86 86 86

Nutrition (P/A) Pearson

Correlation .687

** .663

** .

b 1 .624

** -.805

** .

b .736

**

Sig. (2-

tailed) .000 .000 . .000 .000 . .000

N 86 86 86 86 86 86 86 86

`Pruning (P/A) Pearson

Correlation .621

** .607

** .

b .624

** 1 -.723

** .

b .718

**

186

Age

Group

Intercrop

(P/A)

Irrigatio

n (T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Sig. (2-

tailed) .000 .000 . .000 .000 . .000

N 86 86 86 86 86 86 86 86

Ploughing under

tree canopy

(P/A)

Pearson

Correlation -.835

** -.634

** .

b -.805

** -.723

** 1 .

b -.849

**

Sig. (2-

tailed) .000 .000 . .000 .000 . .000

N 86 86 86 86 86 86 86 86

Plant Protection

(P/A) If P

(which)

Pearson

Correlation .b .

b .

b .

b .

b .

b .

b .

b

Sig. (2-

tailed) . . . . . . .

N 86 86 86 86 86 86 86 86

Disease

Index(%)

Pearson

Correlation .843

** .714

** .

b .736

** .718

** -.849

** .

b 1

Sig. (2-

tailed) .000 .000 . .000 .000 .000 .

N 86 86 86 86 86 86 86 86

**. Correlation is significant at the 0.01 level (2-tailed).

b. Cannot be computed because at least one of the variables is constant.

187

Table 7.3: Correlation between citrus tree age groups with disease index (%)

Disease

Index (%)

Age Group

Disease Index (%) Pearson Correlation

Sig. (2-tailed)

N

1

86

.843**

0.000

86

Age Group Pearson Correlation

Sig. (2-tailed)

N

0.116

0.000

86

1

86


Table 7.4:Correlation between intercrop in citrus orchards with disease index

(%)

Disease

Index (%)

Intercrop

(P/A)


Sig. (2-tailed)

N

1

86

0.714**

0.000

86

Intercrop (P/A)Pearson Correlation

Sig. (2-tailed)

N

0.714**

0.000

86

1

86


188

Table 7.5: Correlation between modes of irrigation in citrus orchards with disese

index (%)

Disease Index

(%)

Irrigation

(T/C), (HE/FI)


Sig. (2-tailed)

N

1

86

.a

.

86

Irrigation(T/C),(HE/FI) Pearson Correlation

Sig. (2-tailed)

N

.a

.

86

.a

86

a. Cannot be computed because at least one of the variables is constant

Table7.6: Correlation between nutrition in citrus orchards with disese index (%).

Disease Index

(%)

Nutrition

(P/A)


Sig. (2-tailed)

N

1

86

0.736**

0.000

86

Nutrition (P/A)

Sig. (2-tailed)

N

0.736**

0.000

86

1

86

a. Cannot be computed because at least one of the variable is constant

189

Table 7.7: Correlation between prunings in citrus orchards with disease index (%).

Disease index

(%)

Pruning

(P/A)


Sig. (2-tailed)

N

1

86

0.718**

0.000

86

Pruning (P/ A) Pearson Correlation

Sig. (2-tailed)

N

0.718**

0.000

86

1

86


Table 7.8: Correlation between ploughing under tree canopy in citrus orchards with

disease index (%).

Disease Index

(%)

Ploughing

under Tree

Canopy (P/A)


Sig. (2-tailed)

N

1

86

-0.849**

0.000

86

Plouging under tree canopy (P/A) Pearson

Correlation

Sig. (2-tailed)

N

-0.849**

0.000

86

1

86


190

Table 7.9: Correlation between plant protection in citrus orchards with disease

index (%).

Disease Index

(%)

Plant

Protection

(P/A)

Disease index (%) Pearson Correlation

Sig. (2-tailed)

N

1

86

.a

.

86

Plant Protection (P/A) Pearson Correlation

Sig. (2-tailed)

N

.a

.

86

.a

86

a. Cannot be computed because at least one of the variables is constant

191

Table 7.10:Factors affecting decline disease index in selected orchards of mango growing districts in Punjab.

District Tehsil Location Age

Group

Variety Interc

rop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index

(%)

Khane

wal

Kabir

wala

Qadirpur

Rawan

26-50 Chaunsa P Canal/

Flood

P (FYM) A P A 39

5 Kassi 26-50 Chaunsa P

Canal/

Flood P (FYM) A P A 39

Solgi 26-50 Chaunsa P

Canal/


Matti Tal 26-50 Chaunsa P

Canal/


Abbas

Pur 26-50 Chaunsa P

Canal/


8 Kassi 26-50 Chaunsa P

Canal/


Maula

Pur 26-50 Chaunsa P

Canal/


Bilawal

Pur 26-50 Dosehri P

Canal/


Basti

Toheed

Nagar 26-50 Chaunsa P

Canal/


Hassan

Pur 26-50 Desi P

Canal/

Flood P (FYM) P P P (Imida) 37

192


Group

Variety Inter

crop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index

(%)

Multan

Multa

n

Qasim

Bela 16-25 Desi P

Canal/

Flood P (NPK) P A

P

(Lambda,

CoCl) 13

Nandla 16-25 Desi P

Canal/

Flood P (FYM) P P

P

(Boadeau

x paste) 31

Chah

Nizam

Wala, 26-50 Desi P

Canal/


Basti

Band

Bosan 26-50 Dosehri P

Canal/

Flood P (FYM) P P

P

(Boadeau

x paste) 33

Shuja

bad

Basti

Khokhra

n 26-50 Chaunsa P

Canal/

Flood P (FYM) P P

P

(Boadeau

x paste) 32

Shahpur

Ubbha 26-50 Chaunsa P

Canal/

Flood P (FYM) P P A 40

Abbas

Pura 26-50 Chaunsa P

Canal/


Jalalp

ur

Pirwal

a Ghazipur 26-50 Chaunsa P

Canal/


193


Group

Variety Interc

rop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index

(%)

Manik

Wali 26-50 Desi P

Canal/


Muzaff

ar Garh

Muzaf

far

Garh

Makhan

Bela

26-50 Desi P

Canal/


Rohillan

wali 26-50 Desi P

Canal/

Flood P (NPK) P P P (Imida) 27

Shah

Jamali 26-50 Desi P

Canal/


Kot

Addu

Ali Wala

16-25 Chaunsa P

Canal/


Musay

Wala

16-25

Tota

Pari P

Canal/

Flood P (NPK) P A

P

(Lambda,

CoCl,

Nativo) 13

Shuhrat

Wala 26-50 Desi P

Canal/

Flood P (FYM) A P P 40

Basti

Drigh 26-50 Desi P

Canal/

Flood P (FYM) A P P 52

Alipur Murad

pur Pull

26-50 Chaunsa P

Canal/

Flood P (FYM) P P

P

(Boadeau

x paste) 31

Basti

kray 26-50 Chaunsa P

Canal/Floo

d P (FYM) A P A 52

194


Group

Variety Interc

rop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index

(%)

Basti Jat

Lashari 26-50 Desi P

Canal/


Rahim

Yar

Khan

Rahim

Yar

Khan

Taranda

Muham

mad

Panah 26-50 Chaunsa P

Canal/


Wahi

Shah

Muham

mad 26-50 Chaunsa P

Canal/


Mianwali

Qureshia

n 26-50 Chaunsa P

Canal

/Flood P (FYM) A P

P

(Boadeau

x paste) 32

Sadiq

abad

Ahmad

Pur

Lumma 26-50 Desi P

Canal/


Khan

Pur

Chak 2P

26-50 Desi P

Canal/


Chak 3 P

26-50 Desi P

Canal/


Liaqat

Pur

Chak 22

A 26-50 Desi P

Canal/


Chak 23

26-50 Desi P

Canal/Floo

d P (FYM) A P A 49

195


Group

Variety Interc

rop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutrition

(P/A)

Pruning

(P/A)

Ploughing

under tree

canopy

(P/A)

Plant

Protecti

on (P/A)

If P

(which)

Disease

Index

(%)

Islam

Nagar 26-50 Desi P

Canal/


Bahawa

lpur

Baha

walpu

r

Khanqah

Sharif More

than 50 Dosehri P

Canal/


Chak 13

BC

More

than 50 Chaunsa P

Canal/


Chak 23

BC

More

than 50 Chaunsa P

Canal/


Nowsher

a 26-50 Chaunsa P

Canal/


Munshi

Wala 26-50 Chaunsa P

Canal/


Ahme

Pur

East

Channi

Goth

26-50 Chaunsa P

Canal/


Mehrab

Wala, 26-50 Dosehri P

Canal/


Muhabba

t Pur, 26-50 Dosehri P

Canal/


Basti

Khokhra

n, 26-50 Langra P

Canal/


196

Basti

Johnan 26-50 Langra P

Canal/



197

reducing height, they can keep trees young and energeting for a long time. This can

also be supplemented by using required nutrition after soil and water testing.

The results also showed that there is potential if we bring change in the

methods of irrigation which is currently flooding, in which most water is wasted. If

the growers adopt high efficiency irrigation such as drip, furrow or sprinkler

irrigation this might reduce decline of trees further. Similarly the crop protection

measures are almost uniform in all tehsils. If the growers start measures after

proper identification of pests and diseases, they can add in health of trees and

orchards.

To study factors favouring mango decline, a survey was done in the district

of Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur. In

Kabirwala tehsil of Khanewal almost at all locations mango orchard trees fell into

age group of 26-50 years. At8locations chaunsa variety while in others locations

desi and dosehri variety of mango was cultivated. Intercropping was common in all

locations with wheat and cotton crop. At all of the locations and even the entire

districts observed canal water was used as irrigation through flooding in mango

orchards. All growers used farm yard manure as nutrition in the areas surveyed at

Kabirwala. Pruning was seen only at one location and ploughing under tree canopy

was a common practice. No plant protection measures adopted except in one

location where growers mostly spray Imidacloprid without knowing the need of

chemical. Amost similar situation was found in district Multan except at 2 locations

i.e. Qasim Bela and Nandla where orchards were relatively young and growers

followed good agricultural practices and disease index (%) was found low.

198

Table 7.11:Correlations of factors causing mango decline disease index (%).

Age

Gro

up

Inte

rcro

p

Irri

_m

od

e

Nu

trit

ion

Pru

nin

g

Plo

ugh

ing

un

der

tre

e

can

op

y

Pla

nt

Pro

tect

ion

Dis

ease

Ind

ex (

%)

Age Group Pearson Correlation 1 .a .

a .

a -.089 .030 -.068 .116

Sig. (2-tailed) . . . .548 .837 .644 .433

N 48 48 48 48 48 48 48 48

Intercrop Pearson Correlation .a .

a .

a .

a .

a .

a .

a .

a

Sig. (2-tailed) . . . . . . .

N 48 48 48 48 48 48 48 48

Irri_mode Pearson Correlation .a .

a .

a .

a .

a .

a .

a .

a

Sig. (2-tailed) . . . . . . .

N 48 48 48 48 48 48 48 48

Nutrition Pearson Correlation .a .

a .

a .

a .

a .

a .

a .

a

Sig. (2-tailed) . . . . . . .

N 48 48 48 48 48 48 48 48

Pruning Pearson Correlation -.089 .a .

a .

a 1 -.342

* .633

** -.676

**

Sig. (2-tailed) .548 . . . .017 .000 .000

N 48 48 48 48 48 48 48 48

Ploughing under tree

canopy

Pearson Correlation .030 .a .

a .

a -.342

* 1 -.311

* .542

**

Sig. (2-tailed) .837 . . . .017 .032 .000

N 48 48 48 48 48 48 48 48

199

Age

Gro

up

Inte

r

crop

Irri

_m

od

e

Nu

trit

ion

Pru

nin

g

Plo

ugh

ing

un

der

tre

e

can

op

y

Pla

nt

Pro

tect

ion

Dis

ease

Ind

ex (

%)

Plant Protection Pearson Correlation -.068 .a .

a .

a .633

** -.311

* 1 -.600

**

Sig. (2-tailed) .644 . . . .000 .032 .000

N 48 48 48 48 48 48 48 48

Disease Index (%) Pearson Correlation .116 .a .

a .

a -.676

** .542

** -.600

** 1

Sig. (2-tailed) .433 . . . .000 .000 .000

N 48 48 48 48 48 48 48 48

*. Correlation is significant at the 0.05 level (2-tailed).


a. Cannot be computed because at least one of the variables is constant.

200

Table 7.12: Correlation between age agroup in mango orchards with disease index

(%).

Diseas index

(%)

Age Group


Sig. (2-tailed)

N

1

48

0.116

0.433

48


Sig. (2-tailed)

N

0.116

0.433

48

1

48

Correlation is not significant at the 0.01 level (2-tailed).

Table 7.13: Correlation between itercropping in mango orchards with disease index

(%).

Diseas index

(%)

Intercrop


Sig. (2-tailed)

N

1

48

.a

.

48

Intercropping Pearson Correlation

Sig. (2-tailed)

N

.a

.

48

.a

48


201

Table 7.14: Correlation between irrigation modes in mango orchards with disease

index (%).

Diseas index

(%)

Irrigation

mode


Sig. (2-tailed)

N

1

48

.a

.

48

Irrigation mode Pearson Correlation

Sig. (2-tailed)

N

.a

.

48

.a

48


Table 7.15: Correlation between nutrition in mango orchards with disease index

(%).

Diseas index

(%)

Nutrition


Sig. (2-tailed)

N

1

48

.a

.

48

Nutrition Pearson Correlation

Sig. (2-tailed)

N

.a

.

48

.a

48


202

Table 7.16: Correlation between pruning of trees in mango orchards with disease

index (%).

Diseas index

(%)

Pruning


Sig. (2-tailed)

N

1

48

-0.676**

0.000

48

Pruning Pearson Correlation

Sig. (2-tailed)

N

-0.676**

0.000

48

1

48

**. Correlation is significant at the 0.01 level (2-tailed)

Table 7.17: Correlation between ploughing under tree canopy in mango orchards

with disease index (%).

Diseas index

(%)

Ploughing

under tree

canopy


Sig. (2-tailed)

N

1

48

0.542**

0.000

48

Plowing under tree canopy Pearson Correlation

Sig. (2-tailed)

N

0.542**

0.000

48

1

48


203

Table 7.18: Correlation between pant protection in mango orchards with disease

index (%).

Diseas index

(%)

Plant

Protection


Sig. (2-tailed)

N

1

48

-0.600**

0.000

48

Plant Protection Pearson Correlation

Sig. (2-tailed)

N

-0.600**

0.000

48

1

48


204

Locations surveyed in Muzaffar Garh, Rahim Yar Khan and Bahawalpur

had shown results on same pattern as that of Khanewal and Multan (Table 7.10).

The correlation through statistical analysis showed the significance of various

factors with disease index (%) of mango decline (Table 7.11). The results were

significant against the factors like pruning (Table 7.16), ploughing under the

canopy of mango trees (Table 7.17) and the plant protection measures adopted

(Table 7.18) at the 0.01 level (2-tailed). Other factors like age group had shown no

significance, as tree of all ages were victim of decline. The correlation of

intercropping, irrigation and orchards nutritions was not found due to similarity in

the practices.

According to table 7.19 for guava districts Kasur, Sheikhupura and

Nankana were surveyed. In district Kasur, trees age group of 10-15 and 16-25 with

variety mix of Safeda, Allahabad and Karela. Intercropping of either wheat or

berseem was observed. In all of the orchards irrigation source was canal and

method was flooding. Proper nutrition was followed only at few places. Similar

situations were found in district Sheikhupura and district Nankana.

The statistical correlation showed that the guava decline is significantly

affected by age group, nutrition, pruning, ploughing under tree canopy and plant

protection measures adopted or neglected in the guava orchards. (Table 7.21, 7.23,

7.25. 7.26 and 7.27). However, decline in guava is not as severe as it has been

observed in mango.

The above results indicate that decline diseases can appear in any age group

of the disease but with different disease index % ages. However, it is very clear that

the young orchards are relatively healthy in all commodities and in all locations.

205

Table 7.19:Factors affecting decline disease index in selected orchards of guava growing districts in Punjab.


Group

Variety Inte

rcrop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutritio

n (P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protectio

n (P/A) If

P (which)

Disease

Index(%

)

Kasur Kasur Qaisar

Garh

16-25 Allahaba

d

P Canal/Flood A A P A 17

Dolaywa

la

10-15 Allahaba

d

P Canal/Flood A A P P (Lime

paste)

20

Noor

Shah

Wali

16-25 Safeda P Canal/Flood A A P A 19

New City

Kasur


Sadar

Diwan

10-15 Safeda P Canal/Flood P (FYM) P A P (Lime

paste)

23

Chuniy

an

Khara 16-25 Karela P Canal/Flood A A P A 8

Kot

Gurdas

Wala

10-15 Karela P Canal/Flood P (FYM) A A P

Bordeaux

Paste

23

Pattoki Gehlan

Pathak


206


Group

Variety Inte

rcrop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutritio

n (P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protectio

n (P/A) If

P (which)

Disease

Index(%

)

Rukan

Pura

16-25 Allahaba

d


paste)

24

Sehjowal 26-50 Allahaba

d


Sheikhupu

ra

Feroze

wala

Faizpur

interchan

ge,

26-50 Karela P Canal/Flood A A P A 16

Adda

Thabal

10-15 Safeda P Canal/Flood A A A P (Lime

paste)

17

Noor

Shah

10-15 Allahaba

d


paste)

28

Burj

Attari

16-25 Allahaba

d


Saggian

Khurd

16-25 Safeda P Canal/Flood A A P P (Lime

paste)

17

Thikriwa

la

16-25 Safeda P Canal/Flood A A P P (Lime

paste)

8

Sharaq

pur

Sharaqpu

r

16-25 Safeda P Canal/Flood P (NPK) A A P

(Bordeaux

paste)

12

207


Group

Variety Inte

rcrop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutritio

n (P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protectio

n (P/A) If

P (which)

Disease

Index(%

)

Ghareeba

bad

10-15 Karela P Canal/Flood A A P P (Lime

paste)

9

Sukhanw

ala

10-15 Karela P Canal/Flood P (NPK) P A P

(Bordeaux

paste)

15

Kot

Mahmoo

d

10-15 Safeda P Canal/Flood A P P A 15

Nankana

Sahib

Nankan

a Sahib

Jalal

Nou


Giller

Wala

10-15 Safeda P Canal/Flood P (FYM) P A P (Lime

paste)

8

Magtan

Wala

10-15 Safeda P Canal/Flood P (NPK) P P P

(Bordeaux

paste)

27

Adda Pul

Torian


Chachka

y Gill


208


Group

Variety Inte

rcrop

(P/A)

Irrigation

(T/C),

(HE/Fl)

Nutritio

n (P/A)

Prunin

g (P/A)

Plough

ing

under

tree

canopy

(P/A)

Plant

Protectio

n (P/A) If

P (which)

Disease

Index(%

)

Mirza

Pur

10-15 Safeda P Canal/Flood A P A A 8

Mandi

Faziabad

10-15 Safeda P Canal/Flood P (NPK) P P P (Lime

paste)

17


209

Table 7.20: Correlation of factors causing guava decline with disease index

Age

Gro

up

Inte

rcro

p

Irri

gatt

io

n m

od

e

Nu

trit

ion

Pru

nin

g

Plo

ugh

in

g u

nd

er

tree

can

op

y

Pla

nt

Pro

tect

io n

Dis

ease

Ind

ex

(%)

Age Group Pearson Correlation 1 .a .

a -.332 -.415

* .318 -.383

* .608

**

Sig. (2-tailed) . . .091 .031 .106 .049 .001

N 27 27 27 27 27 27 27 27

Intercrop Pearson Correlation .a .

a .

a .

a .

a .

a .

a .

a

Sig. (2-tailed) . . . . . . .

N 27 27 27 27 27 27 27 27

Irri_mode Pearson Correlation .a .

a .

a .

a .

a .

a .

a .

a

Sig. (2-tailed) . . . . . . .

N 27 27 27 27 27 27 27 27

Nutrition Pearson Correlation -.332 .a .

a 1 .427

* -.677

** .350 -.684

**

Sig. (2-tailed) .091 . . .026 .000 .074 .000

N 27 27 27 27 27 27 27 27

Pruning Pearson Correlation -.415* .

a .

a .427

* 1 -.299 .105 -.562

**

Sig. (2-tailed) .031 . . .026 .130 .603 .002

N 27 27 27 27 27 27 27 27

Ploughing

under tree can.

Pearson Correlation .318 .a .

a -.677

** -.299 1 -.276 .594

**

Sig. (2-tailed) .106 . . .000 .130 . .164 0.001

N 27 27 27 27 27 27 27 27

210

Age

Gro

up

Inte

rcro

p

Irri

gati

on

mod

e

Nu

trit

ion

Pru

nin

g

Plo

ugh

ing

un

der

tre

e

can

op

y

Pla

nt

Pro

tect

ion

Dis

ease

Ind

ex (

%)

Plant Protection Pearson Correlation -.383* .

a .

a .350 .105 -.276 1 -.571

**

Sig. (2-tailed) .049 . . .074 .603 .164 .002

N 27 27 27 27 27 27 27 27

Disease Index

(%)

Pearson Correlation .608**

.a .

a -.684

** -.562

** .594

** -.571

** 1

Sig. (2-tailed) .001 . . .000 .002 .001 .002

N 27 27 27 27 27 27 27 27

*. Correlation is significant at the 0.05 level (2-tailed).


a. Cannot be computed because at least one of the variables is constant.

Table 7.21: Correlation between age groups in guava orchards with disease index (%).

Diseas index

(%)

Age Group


Sig. (2-tailed)

N

1

27

0.608**

0.001

27


Sig. (2-tailed)

N

-0.608**

0.001

27

1

27


Table 7.22: Correlation between intercropping of trees in guava orchards with disease

index (%).

Diseas index

(%)

Intercrop


Sig. (2-tailed)

N

1

27

.a

.

27

Intercrop Pearson Correlation

Sig. (2-tailed)

N

.a

.

27

1

27

a. Cannot be computed because of the variables is constan

ii

ii

Table 7.23: Correlation between irrigation modes in guava orchards with disease index

(%).

Diseas index

(%)

Irrigation

mode


Sig. (2-tailed)

N

1

27

.a

.

27

Irrigation mode Pearson Correlation

Sig. (2-tailed)

N

.a

.

27

.a

27

a. Cannot be computed because of the variables is constan

Table 7.24: Correlation between nutrition applications in guava orchards with disease

index (%).

Diseas index

(%)

Nutrition


Sig. (2-tailed)

N

1

27

-0.684**

0.000

27

Nutrition Pearson Correlation

Sig. (2-tailed)

N

-0.684**

0.000

27

1

27


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Table 7.25: Correlation between pruning of trees in guava orchards with disease index

(%).

Diseas index

(%)

Pruning


Sig. (2-tailed)

N

1

27

-0.562**

0.002

27

Pruning Pearson Correlation

Sig. (2-tailed)

N

-0.562**

0.002

27

1

27


Table 7.26: Correlation between ploughing under tree canopy in guava orchards with

disease index (%).

Diseas index

(%)

Ploughing

under tree

canopy


Sig. (2-tailed)

N

1

27

0.594**

0.001

27


Sig. (2-tailed)

N

0.594**

0.001

27

1

27


iv

iv

Table 7.27: Correlation between plant protection in guava orchards with disease index

(%).

Diseas index

(%)

Plant

Protection


Sig. (2-tailed)

N

1

27

0.571**

0.002

27

Plant Protection Pearson Correlation

Sig. (2-tailed)

N

0.571**

0.002

27

1

27


The senescence in tree has also greatly aggravated the problems.Especially in case of

mango where the age group is either 26-50 years or even more, the disease index has

touched 76% in Bahawalpur. However, in case of citrus and guava as the orchards

were not too old the disease index was not much high.

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7.5. DISCUSSION

A significant correlation was observed in citrus for trees age and disease index.

It means that with an increase in tree age there was a corresponding increase in disease

index.

With increasing age, the efficiency of tree for performing physiological

function lowersi.e. lower photosynthetic rates, decreased growth rates, shifting of

carbon resources to different parts of the plant and reductions in foliar efficiency, leaf

size and gas exchange rates (Kaufmann, 1996; Ryan and Yoder, 1997; Carrer and

Urbinati, 2004; Martínez-Vilalta et al., 2007).Similarly, the larger tree size and the

structural complexity associated with tree aging increase the maintenance respiration

costs and reduce the efficiency of water transport; both tend to reduce growth (Weiner

and Thomas, 2001; Carrer and Urbinati, 2004; Pennisi, 2005).

It has been reported that citrus productivity life span is only ten years, which is

the prime age of production, whereas it is not uncommon for citrus orchards to remain

in production for over 50 years in many other countries.It has also been mentioned that

the average age of citrus tree in Pakistan is about 25 years. Therefore, the significant

correlation between citrus age and disease index in the current study is supported by

the previous findings of Ahmed et al.(2006) and Niaz et al. (2004). It has also been

observed that fruit trees which are invaded by fungi in early age, react more severely

in the advanced stage (Fraser and Singh, 1966).

In Pakistan more than 75% farmers are considered small landholders and even

their holdings are shared. The small landholdings compel the growers for intensive

cropping. Same is the case with citrus orchards like other fruit trees in Pakistan.

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Therefore, intercropping of different kinds of fodders, cereals and other trees are a

common practice in the country. Intercropping in the orchards is in a way beneficial

for citrus growers, as it meets the needs of fodder for his livestock, grain for household

use, fuel for his kitchen etc. At the same time the harmful pests and pathogen of

diseases are harboured by these alternate hosts. This happens only when the

intercropping is done with competing plants and unmatching life cycles. Same

happened in trees under current study.

It was observed that rigorous and deep ploughing was done even under the

canopy of trees which resulted in increased tree decline incidence as compared to

orchards without intercropping. Intercropping also disturbs citrus orchards with

unwanted spray and irrigation regimes received by the intercropped plants (Johnson,

2006). However, from rest of the world it has been reported that the intercropping of

leguminous crops in citrus orchards help in the fertility of the orchards. Similarly if

intercropped plants receive nitrogen, phosphorus and potassium fertilizers, indirectly

these are also available to citrus trees as well as encourage the population of friendly

insects. For example Sohail et al.(2013) reported that maizeintercropped plots of each

citrus variety had low infestation of citrus miner and high population of coccinellids

and Chrysoperla carnea when maize was intercropped in citrus orchards between the

tree rows away from tree canopies.

In most of the surveyed orchards in citrus areas, nutrition was being provided

either in the form of farmyard manure or as NPK. However, it was important to know

that only about 18% of the areas surveyed were using the recommended doses of NPK

fertilizers (700:350:700 g of NPK per tree with 4-6 kg FYM). Nutrient management in

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citrus influences flowering, fruit set, fruit size, the amount of vegetative growth and

other plant characteristics. Nutrient management can help growers to have earlier,

heavier fruit set (Ibrahim et al., 2004; Abd-Allah, 2006; Alva et al., 2006). Improper

crop nutrition impairs plant genetic resistance to invasivepathogens, decreases yield

and reduces productive life of the plant. It is interesting that in the surveyed areas

where intercropping was practiced, NPK fertilizers were applied to the intercropped

crops but growers did not apply fertlizers to citrus trees (Razi et al., 2011).

In case of mango, significant correlation was found in pruning, ploughing

under the tree canopies and plant protection measures adopted. The large unpruned

trees were mostly the victim of tree decline. Zero pruning in mango orchards has been

reported in Pakistan to be a predisposing factor of mango decline (Naqvi and Perveen,

2015). Pruning is basically done to reduce tree size, to givie shape, removal of

deadwood and upright branches as they are not productive, rather they help in

hibernation of various insect pests and disease causing organisms. Pruning helps in

increasing vigor of trees (Masood et al., 2014). It was seen that in Multan, where the

pruning has been observed in quite a few orchards, the disease index percentage is

relatively low as compared to other areas where it was not practiced. It is important to

remember that only pruning without plant protection measures does not provide

suitable results. This is because decline causing fungi are opportunistic in nature and

enter through wounds. By pruning trees only they have a good chance to invade trees.

Hence, pruning coupled with plant protection measure come up with effective tree

management (Al Adawi et al., 2006; Fateh et al., 2006; Fateh et al., 2009). The

pruning and plant protection are integral for the health of mango trees (Poland et al.,

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2006). Similarly, ploughing under tree canopy which injures roots provides an easy

entry for the opportunistic fungi to invade mango trees and cause mortality. It has been

observed especially, the fungus Ceratocystis fimbriata in mango has entered through

root wounds (Saeed et al., 2007; Masoodet al., 2011).

In guava, a significant correlation was recorded for disease index with age

group, nutrition, pruning, ploughing under tree canopy and plant protection measures.

As it has been discussed in citrus and mango, the age of tree is one of the biggest

factors inviting the decline of trees, same is the case in guava as well (Khushk et al.,

2009). It has been discussed in details earlier in case of mango that how pruning and

plant protection measures are linked. Pruning reduces the disease pressure and

improves the vigor of guava trees (Tomita et al., 2016). The nutrition in guava

orchards is seldom found especially the application of NPK is not adopted. However,

nutrition is given in the form of farmyard manure. However, it has been reported that

one percent increase in these factors results in 0.21 and .016 percent increase in guava

yield (Ishtiaq et al., 2012). The major difference that has been observed in the severity

of decline disease of guava from mango can be the difference in height as well as the

density of leaves and branches. It was observed that guava trees were not as large as

mango trees, having few branches which would allow the aeration and sunlight as

well. However, tree injuries were common which were the entry point of various fungi

as reported by Abbas et al.(2014). It is also worth mentioning to discuss here that

Pakistan is among the water deficient countries and cannot afford flood irrigation for

its orchards or other crops. Secondly, the excessive standing water is lethal in two

ways that it causes suffocation of trees as well as it can act as medium for spread of

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diseases from diseased trees to healthy ones. The country has been introduced to high

efficiency irrigation systems and there is dire need to adopt them for conserving

precious water as well as protect plants (Malik et al., 2004; Khushket al., 2009; Mirjat

et al., 2011).

Based on the results of above study, the following short term management

recommendations can be given:

1. Make sure to buy healthy planting material from reliable nurseries.

2. Pruning for having vigorous trees and providing quality fruits.

3. Follow recommendation of plant protection specialists for suitable and safe

spray regimes.

4. Analysis based nutrition for producing quality fruits may be adopted under

the guidance of nutrition specialists.

5. Wounding of the tree may be avoided to check entry of pests and

pathogens.

6. Intercrop with companion crops, such as legumes or with matching life

cycles.

7. Floor management may be encouraged, using local grass species without

disturbing the root zones of trees.

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Chapter 8

GENERAL DISCUSSION

8.1. ASSESSMENT OF DECLINE IN CITRUS, MANGO AND GUAVA

The decline disease assessment in citrus, mango and guava growing areas of

Punjab presents a gloomy picture for the respective industries. In all three

commodities the decline has resulted from a number of biotic factors such as citrus

tristeza virus (Arif et al., 2005); phytoplasma, bacteriae.g. Xanthomonas compestrisare

involved in citrus decline (Burney et al., 2007). In Pakistan, the role of fungi in citrus

decline has not been studied widely by scientists. However, the fungal involvement in

citrus decline is a popular research topic in Oman, Iran and even in USA (Sutton and

Dyko, 1989; Mirzaee et al., 2003; Al Sadi et al., 2014). In Pakistan very limited

research has been done to acknowledge the fungi in decline development. A list of

fungi has been reported to cause decline symptoms. This IncludesLasiodiplodia

hormozganensis, L. theobromae, Fusarium solani, Phytophthora sp., Neoscytalidium

dimidiatum and Nattrassia mangiferaebeing the most common (Al Sadi et al., 2014;

Safdar etal., 2010; Fateh et al., 2016).

Besides biotic factors, the human manipulations regarding orchard

management, floods, poorly drained soils, water logging, droughts, and climatic

chnges push trees towards decline. It has been observed that poorly drained soils and

deficiencies lead to die back (Chauhdary, 2003). Most of the orchards in Sargodha,

Bhalwal and Shahpur had old citrus plantations with intercropping of wheat, berseem

and barley even under the canopy of trees, competing with feeder roots of trees in the

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root zone.In the surveyed orchards where dead wood removal and pruning was not

commonly practiced theorchards looked like forest(Ali et al., 2004).

The most available nutrition to the citrus trees was farmyard manure which at

many places were dumped fresh in the orchards and caused burning of leaves. This

practice can badly influence the soil pH and soil salinity. Imbalanced nutrition resulted

in deficiency and toxicity of some elements e.g. zinc deficiency in the orchards which

also promoted die back symptoms in trees. Although, it has been reported that citrus

orchards are mostly macronutient deficient (Stenico et al., 2009). Another major issue

is the quality of sub soil water which is unfit for irrigation and played an important

role to destroy the citrus orchards. Canal water is not frequently available and flooding

is done whenever it is available. It means there is no timely irrigation followed in the

orchards.

Generally, more than 15% disease index of any disease at any location or any

commodity is considered alarming. In district Sargodha overall disease index ranged

from minimum 10.36 to maximum 29.41% which requires the immediate attention of

the scientists and other stakeholders to have more research for management of the

system through trainings and education (Fateh et al., 2017).

Among the insect pests, leaf miner, citrus psylla, whitefly and lemon butterfly

were common. The gaps in the orchards were filled with poor quality planting

material. So a complex of soil nutrients, insect pests and improper irrigation was

observed which are all limiting factors for the growth and favorable for the disease

(Khan et al., 2011).

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In tehsils like Kot Momin and Sillanwali,the growers were following integrated

orchard management approaches e.g. pruning of trees; plant protection sprays for

insect pest and diseases on proper guidance from plant protection specialists; soil and

water analysis for nutrition; orchard floor management without intercropping and

unnecessary ploughing in the orchards. These good agricultural practices make

Sillanwali and Kot Momin better as compared to other citrus growing tehsils.

Moreover, nutritional study has been conducted in all tehsils of Sargodha and it was

obvious that the above mentioned 2 tehsils were comparatively much better in

nitrogen, potassium and phosphorus contents (Razi et al., 2011). Pruning in most of

the orchards resulted in proper shapes of trees and no brancheswere touching the

ground oroverlapping each other. Ploughing was not being done under the tree

canopies and hence there was no chance of damage to the roots to provide openings to

opportunistic fungi. Pruning of the trees reduces the pests and diseases and improves

the equal flow of nutrients to whole tree making it vigorous to produce more quality

food. It keeps the size of the tree in control and makes easy the field practices, sprays

as well as fruit harvesting without much damage (Van Vuuren and Graca, 1996). In

few orchards an effort was made to paste the lime and Bordeaux paste as well. This

was enough to avoid the opportunistic fungi invading the stems as well as insects

creeping into the bark cracks such as spider mite (Overmeer, 1985).

The localities surveyed in Bhalwal include Chak No. 10 /NB, Chak No. 10/1

were well managed. Moreover, the growers were literate and well aware of the orchard

management. Most of them were posted on the influential posts as well as had links to

the experts in citrus production technology. This may also be due to the international

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projects running in the area. The trees were well pruned with good shapes having ages

of 10-15years. Many orchards were having the lime pasting and some were sprayed

with suitable pesticides. There was almost an equal ratio of farmers who were

maintaining the orchards themselves as well as those whose orchards were maintained

by the attendants. These orchards were being continuously monitored by the Global

Gap Certification Project of USAid. The experts guided the growers at each stage to

perform different activities at different phenological stages (Anonymous, 2008).

Mango decline is also a problem of all traditional mango growing areas of

Pakistan. The survey to record the occurrence of mango decline was done in

Khanewal, Multan, Muzaffar Garh, Rahim Yar Khan and Bahawalpur distrcits which

are famous for mango production. Maximum disease index of 53.30% was recorded in

district Bahawalpur. During survey the large overlapping trees were observed in this

district. Most of the areas surveyed had more than 100 years old plantations (Fateh et

al., 2016). The growers were not even aware of the disease and they thought that

senescens had resulted in such symptoms of the trees. Secondly, mango orchards are

not given first importance rather the intercropped crops are considered important to the

growers. The large farms are owned by the absent landlords who stay abroad and leave

orchard management on their managers (munshies). These munshies strongly believe

in traditional mango cultivation and show harshness towards the adoption of

innovative technologies (Khushk et al., 1996).

The farmers have lost their interest in the mango orchards and taking more

interest in the housing colonies. The orchards were neglected and animal grazing and

lopping of trees were common. Most of the trees have been uprooted. The existing

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trees had many wounds. The age of the trees ranged from 26-50 years. The old age

itself a problem for the trees as they are more vulnerable to be attacked by the diseases

(Kazmi et al., 2005; Malik et al., 2005). However, mango tree decline was not limited

to only old age trees but equally found on young trees as well. The trees were large in

size, overlapping with each other giving a complete shade in the orchard and not

allowing any penetration of sunlight. The pruning of the trees was like sin for the

growers (Jiskani, 2002).

Flood irrigation was adopted in the orchards which could easily spread the

disease from infected trees to healthy ones. The water was directly touching the stems

of the trees and leaf drop was much more common. The chemical fungicides which

were not supposed to be for mango were sprayed intentionally on the trees i.e. the

chemicals left over after spraying on cotton and other crops (Iqbal et al.. 2007).

Along the Multan Khanewal and Multan Shujabad Road, many orchards were

pruned and few orchards also showed the lime pasting around tree trunk that showed

that these orchards are not as neglected as the orchards along Band Bosan Road.

Another reason may be that orchard along Shujabad road were in the area of Mango

Research Station Shujabad and had links with the expert. Therefore, the growers were

taking interest in the management of their orchards and decline occurrence in these

orchards was little bit less (Fateh et al., 2016).

Guava decline is another big threat in Pakistan. As we have already limited

area for guava cultivation in Punjab and due to the stressed conditions someday this

precious fruit will vanish from lands of Pakistan. It is a national problem of Pakistan

because since last decade the guava production has been declining. The problem of

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guava decline has been prevailing in Lahore, Sheikhupura, Faisalabad, Jhang and

Sargodha (Khushk et al., 2009). Maximum guava decline disease index was observed

in Kasur (18%) followed by Sheikhupura (17%) and minimum of 14% in Tehsil

Nankana. Kasur being the nursery industry area, the nursery growers are interested in

guava in high density for sale rather than in better management to earn from the fruit.

Most of the trees are not grown in proper rows, rather overlapping with each other in

such a way that all the operation like sprays, pruning and machine operations are

difficult. Improper cuttings and non treatment of wounds are giving rise to guava

decline situation. However, proper guava cultivation has been observed in

Sheikhupura but with intercropping of wheat and berseem. Ploughing inside the

orchards is increasing tree infection by making injuries. Nankana is comparatively low

in disease as mostly there are new plantations with proper planning and better

management strategy.

It is concluded that the situation of decline in citrus, mango and guava orchards

is alarming and almost every orchard faced the problem. The situation warrants strict

control strategies for its management. It is also emphasized that the farming

community be trained in this regard to adopt better management strategies.

8.2. COMMON SYMPTOMS OF DECLINE IN CITRUS, MANGO AND

GUAVA

The common symptoms found in the orchards included the bark splitting,

gummosis, leaf drooping and discoloration, twig blight, die back and the eventual tree

mortality. In case of citrus decline, however, there are few additional symptoms like

yellowing, dwarfing or bunching which are attributes of viruses and phytoplasma.

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Symptoms are always the expression of some disorders of the plants which result from

being invaded by the biotic factors like viruses, fungi, phytoplasma, nematodes etc and

also the result from human manipulations (Safdar et al., 2010). In mango decline the

first symptoms that appear in declining tree is leaf drooping that shows that the

nutrients flow has stopped inside the vascular tissues. Actually the decline causing

fungi enter through the wounded roots or stem and invade the vascular tissues. They

keep growing and form the tylosis in the vascular bundles, thus blocking the flow of

nutrients. Resultantly, the nutrients keep depositing in the air pockets which when

overflow are ruptured the bark and oozing out as thick dark fluid. In most of the

affected trees the stain of overflowing fluid were seen. When the stem of the affected

trees are scraped after peeling of the bark, the dark brown to grey cankers are found

(Kazmi et al., 2005; Fateh et al., 2006; Masood et al., 2010). Similar symptoms have

been observed in guava but the leaf drooping wass not as much apparent as in mango.

However, leaf drying and leaf drooping is found in many trees. The dark stains at the

base of trees are also observed in guava (Rangaswami, 1984).

8.3. MYCOFLORA ISOLATED FROM THE DECLINE AFFECTED TREES

A number of fungi have been isolated from different decline affected trees and

different plant parts. These include Botryodiplodia, Botryosphaeria, Nattrassia

mangiferae,Fusarium, Alternaria, Pythium, Phytophthora and Ceratocystis species.

These fungi have been found infecting the trees singly or in combination. For

exampleBotryodiplodia, Botryosphaeria and Nattrassia mangiferae were mostly found

in the twigs and branches of all affected trees. They may be the cause of twig blight

and branch die back in the affected trees. Nattrassia and Botryodiplodia were also

xvii

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observed on the bark as well as on the inner side of the bark where bark splitting

occurred. Therefore, they might have an additional role in bark splitting.

FusariumandCeratocystis have been found in the vascular tissues of the plants to stop

the nutrients inflow and cause the wilting symptoms (Fateh et al., 2016). However, it

is impotant to note thatCeratocystis was only found from the vascular tissues of mango

but it was not isolated from citrus and guava (Mirzaeeet al., 2003; Miskita et al., 2005;

Fateh et al., 2006;Masood et al., 2010).

8.4. PATHOGENICITY OF THE IMPORTANT DECLINE CAUSING

PATHOGENS

Two different methods were adopted to check the pathogenicity of all the four

commonly occuring fungi individually as well as in combination. These four fungi

were Ceratocystis, Nattrassia, Botryodiplodia and Fusarium sp. Individual fungi

except Ceratocystis was not able to produce mortality at all. However, the

combinations of these fungi caused mortality in the green house potted plants.

Infact,Botryosphaeria species are endophytic in nature and become aggressive only

when they are combined with the virulent fungi. The opportunistic fungi when entered

the plants' system through wound, they start producing decline symptoms in the plants.

Among the two methods, the root injury method was more appropriate as the root

injury frequently produced the symptoms. This can be related to the fact that when

ploughing is done under the tree canopies, the roots are damaged and they provide

opening to the fungi to enter and cause tree decline (Al-Adawi et al., 2006; Urbez-

Torroset al., 2008).

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8.5. FACTORS AFFECTING THE GROWTH AND DEVELOPMENT OF

NATTRASSIA SP.

In the samples taken from citrus plants, the growth ofNattrassia isolates

was least affected by the temperature range from 20-28oC. However, in case of mango

and guava, the temperature below 25 o

C reduced the growth of the fungi (Alizada et

al., 2000). This may be due to the fact thatN. mangiferae isolates have been adopted to

high temperatures in the tropical areas as compared to subtropical or temperate

regions. Mycelial growth was seen more in the isolates of Nattrassia when provided

alternate light and dark periods as compared to continuous light or continuous dark

periods. However, again the isolates from mango and guava did not show significant

differences in any condition of light and dark (Elliott and Edmonds, 2003). Nattrassia

isolates from citrus were the least affected with the pH range of 5.5 to 6.5. However, in

mango and guava the isolates performed well at pH up to 6.00. The results are

contradictory to the actual climatic conditions especially in the mango orchards where

soil pH is more than 7 but N. mangiferae keeps growing. Among four culture media

used for the growth of N. mangiferae, it only preffered to grow maximum on potato

dextrose agar and malt extract agar media (El Atta and Aref, 2013). Other two media

i.e. carrot juice agar and stem decoction agar media produced less growth of the

fungus. However, it was found that these two media give pure growth of Ceratocystis

which is also a decline causing fungi (Fateh and Kazmi, 2010).

8.6 MORPHOLOGICAL CHARACTERISTICS OF NATTRASSIA SP. FROM

CITRUS, MANGO AND GUAVA

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The similarity between isolates of mango and guava shows that there

has been a shift of attack of N. mangiferae either from mango to guava or otherwise.

The chances are that these are the same fungal isolates which attack both trees as both

are grown in tropical climate and as discussed earlier that sometimes guava is

intercropped in mango orchards and hence there has been a transfer from mango to

guava (Sutton and Dyko, 1989; El Atta and Nouri, 2014).

It has been found during the pathogenicity tests that the symptoms produced by

N. mangiferae in both trees were almost similar, which is again a confirmation of their

common origin (Fateh et al., 2016). In case of citrus, the morphology of N. mangiferae

was a little bit different. One of the reasons might be the difference in climate where

citrus is grown and secondly, it can be due to the fact that in citrus the fungus has been

reported to produce branch wilt with cracks in a particular check pattern, while in

mango and guava they either cause branch dieback, branch canker or twig die back

(Mirzae et al., 2002; Saaiman, 1997).

However, the morphology of N. mangiferae isolates from all citrus, mango and

guava had closed resemblance with madrone canker isolates in USA. Madrone isolates

of N. mangiferae are characterized as hyaline, aseptate, guttulate conidia becoming 1-

to 3-septate, verisicolored or brown. Conidia had thin-walls, mostly fusoid but

occasionally oblong or clavate with truncate bases. The isolates differed in conidial

length, width as well as in arthrospore size. Although there were statistically

significant differences, there does not appear to be a geographical pattern in spore size

or shape. The researchers in US conclude that N. mangiferae isolated from Pacific

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xx

madrone belonged in the teleomorph genus Botryosphaeria and hence, diseases cause

by this fungus can be managed using methods developed for other Botryosphaeria

pathogens, such as B. dothidea(Elliott and Edmonds, 2003).

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SUMMARY

In Pakistan important fruit trees such as citrus, mango, guava etc are facing

decline. Among these fruits, citrus and mango decline have been addressed strongly as

they are important foreign exchange earnings and export items. Mango is being given

attention in all aspects but citrus needs special attention. Due to poor quality citrus in

Pakistan we have restricted export markets. According to Pakistan Horticulture Export

Board, 20% of the citrus fruit is gone waste either due to diseases or post harvest

mishandling (Humayun et al., 2005; Khanzada et al., 2004). Other fruit trees like

Guava and Loquat are also very famous fruit of Pakistan and are liked by all

generations.

The common symptoms found in the orchards included the bark splitting

gummosis, leaf drooping and discoloration, twig blight, die back and the eventual tree

mortality.

Decline in different forest and fruit trees involve different pathogens e.g. fungi

such as Ceratocystis fimbriata, Armillaria sp., Nattrassia magniferae, Lasiodiplodia

theobromae, Phoma sp., Phomopsis sp., Fusarium sp., viruses such as Citrus Tristeza

Virus (CTV), nematodes especially in case of citrus decline (Tylenchulus

semipenetrans) and in mango (Hemicriconemoides, Longidorus sp.), bacteria such as

Pseudomonas sp. and phytoplasma. Among these pathogens the fungus Nattrassia

magniferae has been reported from almost all of the forest and fruit trees facing

decline.

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Citrus decline incidence, severity and disease index were found to be variable

in district Sargodha. Maximum mean disease incidence was recorded in tehsil

Sargodha (94.06%) followed by tehsil Shahpur (93.33%), similarly, mean disease

severity was the maximum in tehsil Sargodha (1.47), Bhalwal (1.38). Maximum

disease index was observed in tehsil Sargodha (29.41%) followed by tehsil Shahpur

(26.75%), Bhalwal (23.77%), Sahiwal (19.28%), and Kot Momin (15.80%). On the

other hand minimum disease index was found in tehsil Sillanwali (10.36%).

In mango maximum mean disease severity 2.70 was observed in district

Bahawalpur followed by Khanewal (2.30). Similarly maximum mean disease

incidence (100% ) was found in Khanewal followed by Bahawalpur (99.30%). Disease

index was found maximum in Bahawalpur (53.30%) followed by Khanewal (46.50%).

Results show that maximum guava decline severity was recorded in district

Sheikhupura (0.90) followed by district Kasur (0.78). Minimum severity was observed

in district Nankana Sahib (0.57). Maximum guava decline incidence was found in

district Kasur (62.89%) followed by Sheikhpura (61.30%) while minimum incidence

was observed in district Nankana Sahib (56.29%). However, regarding disease index

district Kasur and Sheikhpura were not too far (18.00 and 17.40 respectively but

minimum disease index (14% ) was recorded in district Nankana Sahib.

Mycofloral studies showed that in all kind of decline of citrus, mango and

guava mostly common pathogens are involved in producing range of symptoms like,

leaf drooping, bark splitting, twig or branch dieback, stem canker and eventual tree

mortality. Moreover, an important fact found was that mostly N. mangifereae, L.

theobromae and Botryosphaeria sp. were found in case of twig or branch dieback.

206

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However, Fusarium sp. and Ceratocystis sp. were found in the vascular regions.

Two different methods were adopted to check the pathogenicity of

Ceratocystis, Nattrassia, Botryodiplodia and Fusarium sp. Individual fungi except

Ceratocystis was not able to produce mortality at all. However, the combinations of

these fungi led the green house potted plant to mortality. In fact Botryosphaeria

species are endophytic in nature and become aggressive only when they are combined

with the virulent fungi.

During physiological studies it was found that Nattrassia isolates growth was

least affected by the temperature range from 20-28oC. However, in case of mango and

guava, the temperature below 25 o

C reduced the growth of the fungi (Alizada et al.,

2000). This may be the reason that the Nattrassia isolates have been adopted to high

temperature in the tropical areas as compared to subtropical or temperate regions.

Edmonds, 2003).Nattrassia isolates from citrus were least affected with the pH range

of 5.5 to 6.5. However, in mango and guava the isolates have been preferring pH up to

6.00. The results are contradictory to the actual climatic conditions especially in the

mango orchards where soil pH is more than 7. butNattrassia keeps growing. This may

be due to low pH of fluent in the twigs and branches of mango tree which favour the

growth of this fungus. Among four culture media used for the growth of Nattrassia, it

only like to grow maximum on potato dextrose agar (PDA) and malt extract agar

(MEA) media (El Atta and Aref, 2013). Other two media i.e. carrot juice agar and stem

decoction agar media produced less growth of the fungus. However, it was found that

these two media give pure growth of Ceratocystis which is also a decline causing fungi

It can be concluded from all of the above discussion that Nattrassia mangiferae

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is a common pathogen of citrus mango and guava decline. It is mostly involved in the

twig blight or die back of the trees. However, when it is combined with other

aggressive isolates it can contribute towards the mortality of trees. There are few

morphological differences among the isolates from mango and citrus, therefore it

emphasizes to have a molecular study in future to see whether, it is Nattrassia

mangiferae or other species of Nattrassia are also prevailing in Pakistan. Similarly

there is different preference of light, temperature and pH for the development of the

growth. The factors which favour the disease development other than fungi are

intercropping in the orchards; ploughing under tree canopy causing root injury or

damage by other field implements; application of fresh or partially decomposed farm

yard manure, deficiency of the nutrients because of poor nutrition plans. Ignorance to

pruning, which is a best tool to avoid pests and diseases and produce high quality fruits

without compromising on yield. Above all the interst of the growers to manage their

orchards.

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