Significant Acheivements and Current Status : Virology

Narayan Rishi* Department of Plant Pathology

CCS Haryana Agricultural University Hisar 125 004

ONE HUNDRED YEARS OF PLANT PATHOLOGY IN INDIA: AN

OVERVIEW

Editors

S. S. Chahal, R. K. Khetrapal and T. S. Thind

Scientif Publishers (India), Jodhpur;

on behalf of the Indian Socioety of Mycology and Plant Pathology, Udaipur

2006 pp. 143-205

_____________________________________________________________ *President, Indian Virological Society, B-6, Old Campus, CCS Haryana Agricultural University, Hisar 125 004, India. E. Mail: narayan.rishi@gmail.com

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Significant Acheivements and Current Status : Virology

Narayan Rishi* Department of Plant Pathology

CCS Haryana Agricultural University Hisar 125 004

Adolf Eduard Mayer in 1880s for the first time thought that mosaic symptoms in tobacco termed by the Dutch growers as “bunt”, “rust” or “smut” is distinct therefore; to prevent confusion he termed it “mosaic disease of tobacco”. Thus the first ever name of a viral disease was inadvertently coined. ‘Virus’ (venom/poison) a Latin word was first used for the causal agent of this disease when Martinus Beijerinck gave the classical ‘contagium vivum fluidum’ theory in 1898. Since then several landmark discoveries viz. insect, nematode, fungus, dodder and seed transmission of viruses; local lesion host used for quantitative assay of tobacco mosaic and other viruses; antigenicity and immunogenicity; nucleoprotein nature; isolation, purification and electron microscopy; inclusion bodies; infectivity by nucleic acid; viroids; satellite virus; nanovirus; DNA 1 and DNA β etc. coupled with the recent advancements in molecular studies lead to the development of strong system of classification of viruses. Today Virology is a separate dynamic science. In the recent years, developments were so fast that now there are as many as 15 journals exclusively devoted to the science of Virology. In India root (wilt) disease of coconut (Cocos nucifera L.) is known to be present since 1882 (Table 1) in south Kerala (Butler, 1908, Varghese, 1934, Nampoothiri and Koshy, 1998). Thereafter, almost simultaneous to ‘contagium vivum fluidum’ theory (1898), spike disease of sandal (Santalum album L.), was observed in Karnataka in 1899 (McCarthy, 1903). However the long understood viral etiology of these diseases proved to be elusive when Solomon et al. (1983) and Varma et al. (1969) respectively reported association of mycoplasma like organisms (MLOs) later termed as phytoplasma with these diseases. Thus the first virus disease studied in India became mosaic of sugarcane reported by Dastur in 1923. There after several other virus diseases (Table-1) were recorded in the country. Upto mid fifties about 70 plant virus diseases were recorded infecting 59 plant species in19 families (John. 1957). When Dastur in 1923 noticed sugarcane mosaic in Bihar, it was very much dreaded because then recently in 1920 it caused epiphytotics in Louisiana, USA resulting into near collapse of the sugar industry there (Abbot, 1961). In this furore, for the first time in India detailed studies were undertaken on any plant virus disease (McRae, 1926, McRae, 1932, McRae and Subramaniam, 1928, McRae and Subramaniam, 1933, McRae and Subramaniam, 1934, Rafay, 1935, Chona and Rafay, 1950). Some important findings of these studies were; that sugarcane mosaic disease was wide spread in India, it induced 10% reduction in yield, it did not affect the quality of the juice and sugarcane varieties in India were tolerant to this disease. It was later reported that losses due to this disease were much higher (Rishi et al., 1975) and also there was deterioration in the quality of juice (Bhargava, 1971). For the first time in India internationally accepted sugarcane differentials were used and several strains of this virus were identified (Rishi, 1969, Bhargava, et al., 1972, Bhargava, 1971, 1975, Rao et al., 2002).

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________________________________________________________________________________________________ Table 1: Earliest reported viral diseases in India Disease Reference

Root wilt of coconut, known to be present Butler, 1908 since 1882 in south Kerala (Later phytoplasma, Solomon et al., 1983) Spike of sandle, observed in 1899 McCarthy, 1903 (Later phytoplasma, Varma et al., 1969) Yellow leaf disease of arecanut palm observed in 1914 Varghese, 1934

(Later phytoplasma, Nayar and Selsikar, 1978) Cotton stenosis Kottur and Patel, 1920, Likhite, 1939 (Later phytoplasma Capoor et al., 1972) Tristeza disease of citrus Browm, 1922 Mosaic of sugarcane Dastur, 1923 Yellow vein mosaic of Okra Kulkarni, 1924 Clump disease of groundnut Sundararaman, 1927 Pigeonpea sterility mosaic disease Mitra, 1931 Leaf curl of Zinnia elegans Mathur, 1933 Sesamum phyllody Kashiram, 1930, Pal and Pushkarnath, 1935, (Later phytoplasma, Cousin et al., 1970) Tobacco leaf curl Pal and Tandon, 1937, Pruthi and Samuel, 1937, 1939 Pansukh of rice Dastur, 1937

(Later rice tungro, Varma et al., 1999) Little leaf of brinjal Thomas and Krishnaswami, 1939

(Later phytoplasma, Varma et al., 1969) Leaf curl of chillies Uppal, 1940 Mosaic disease of cowpea Vasudeva, 1942 Potato virus Y and potato leaf roll Pal, 1943 Mosaic disease of bottlegourd Vasudeva and Lal, 1943 Bigbud disease of tomato Vasudeva and Lal, 1944 Degeneration of potatoes Vasudeva and Lal, 1944, 1945 Melon mosaic (also seed transmission) Vasudeva and Pavgi, 1945 Mosaic of cardamom Uppal et al., 1945 Tomato leaf curl Vasudeva and Samraj, 1948 Yellow mosaic of Phaseolus lunatus, mosaic of Capoor and Varma, 1948a, b, c respectively Lagenaria vulgaris and enation mosaic of Dolichos lablab Linn.

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In the first half of 20th century no basically trained manpower in plant virology was available in the country, nevertheless, some important information on the problems of plant virus diseases were generated (Dastur, 1923, Uppal, 1934, Pal, 1943, Vasudeva and Lal, 1945, 1945) that necessitated the establishment of exclusive plant virology laboratories in India (Table 2). In 1940s Vasudeva at IARI, New Delhi inducted a few pathologists to initiate work on the problems of plant virus diseases and made some very important contributions like information on tomato leaf curl disease and degeneration of potato tubers infected with viruses. In 1950s three basically trained virologists viz. S. P. Capoor, K. S. Bhargava and S. P. Raychaudhuri trained under the then two most leading plant virologists in world viz. Sir Frederick C. Bawden, Rothamsted Experimental Station, Harpenden, Herts, UK and Prof. L. O. Kunkel, Boyce Thomson Institute (now University), New York, USA. These plant virologists developed strong schools of Plant Virology at Pune, Gorakhpur, Kalimpong and New Delhi. Simultaneously T. Sadasivan at the University of Madras and G. S. Verma at the University of Lucknow developed work on the basic aspects of plant viruses. These centers also came up as strong schools of Plant Virology. ________________________________________________________________________ Table 2: Establishment of plant virology laboratories in India – a chronicle Place Year___ Division of Plant Pathology, Indian Agricultural Research Institute (IARI), Pusa, Bihar, 1905 shifted to New Delhi in 1934 after the great earth quake in Pusa, Bihar Sub station, Shimla (originally for wheat rust (1828), later also a center on fruit viruses) 1940s Sub station, Pune, became IARI, Regional Station, Pune 1939, 1952 Central Potato Research Institute, Patna, shifted to Simla in 1956 1949 Dept. of Botany, University of Madras, Madras 1950s Dept. Botany, DSB College, Nainital, shifted to Dept. Botany, Univ. of Gorakhpur, Gorakhpur 1952, 1958 Regional Station, Kalimpong 1956 Dept. of Botany, University of Lucknow, Lucknow 1960s Dept. of Plant Pathology, GB Pant University of Agriculture & Tech., Pantnagar 1960 Dept. of Plant Pathology, Punjab Agricultural University, Ludhiana 1960 Central Tuber Crops Research Institute, Thiruvananthpuram 1963 Directorate of Rice Research, Hyderabad 1965 Dept. of Plant Pathology, Tamil Nadu Agricultural University, Coimbatore 1965 Plant Virology Laboratory, National Botanical Research Institute, Lucknow 1966 Central Rice Research Institute, Cuttack 1966 BC Krishi Vishwavidyalaya, Kalyani 1966 Indian Institute of Horticultural Research, Bangalore 1967 Dept. of Plant Pathology, CCS Haryana Agricultural University, Hisar 1970 Dept. of Plant Pathology, Dr. YSP University of Agriculture & Forestry, Solan 1970 SK University of Agriculture & Technology, Srinagar 1970 Central Plantation Crops Research Institute, Kasargod 1971 Marthwada Agricultural University, Parbhani 1972 Div. Microbiol. & Pl. Pathology, Central Institute of Medicinal and Aromatic Plants, Lucknow 1980s Institute of Himalayan Bioresource Technology, Palampur 1986 Department of Plant Pathology, H P Krishi Vishwavidyalaya, Palampur 1986 Plant Virology Unit, Division of Plant Quarantine, NBPGR, New Delhi 1989 Dept. of Plant Biotechnology, Madurai Kamaraj University, Madurai 1992 Dept. of Plant Molecular Biology, University of Delhi South Campus, New Delhi 1997

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In the ancient period, symptoms resembling virus diseases and their management have been recorded in the ancient Indian and Chinese civilizations. A lesson on ‘Vrikshaaurved’ in the 6th century ‘Brihatsanghita’ by Varahmihir gives strong indication that ‘Vrikshaaurved’ was quite advanced even before 6th century. In the ‘Vrikshaaurved’ by Surpal, which was compiled more than one thousand years ago, diseases of plants and their remedial measures using plant and animal products are given in ‘shloka’ Nos. 165-222. Diseases resembling symptoms of viruses were controlled by ‘panchmool’ (root of five plant species), cow milk and ghee etc. The Asian Agri-history Foundation and it Chairman Dr. Y. L. Nene have done a great service to the present day Indian Agriculture by procuring a copy of this great Indian compilation from Oxford and getting it translated in English and Hindi (Anon., 1996). Rich flora in India, with diverse temperate climate is very suitable for the colonization of insect vectors and perpetuation of large number of viral diseases (Table 3). Since 1960s onwards, introduction of newer technologies in agriculture, frequent movement of seed and plant material in different areas, unwarranted use of insecticides, ecological changes and appearance of newer strains of viruses led to exponential increase in the viral disease problems. Geminiviruses and potyviruses are today economically most important. Wide prevalence of geminiviruses is due to the development of natural recombinants and increased population of whitefly. Potyviruses are very successful pathogens inciting a large number of diseases incurring substantial quantitative and qualitative losses. They have large host range and their large numbers of efficient vectors are present in nature. Some of the potyviruses are seed borne specially in leguminous crops and are major limiting factor in crop production (Rishi, 2000). Cucumoviruses and Tospoviruses are the next important groups. By conservative estimates, viral diseases of plants incur annual losses of more than one thousand crore rupees in India (Varma and Ramachandran, 1994). In the first half of the 20th century fungal pathologists who did not have training in virology worked on virus diseases in India. Reports of appearance of sugarcane mosaic virus in all the areas (Dastur, 1923) and degeneration of potatoes due to viruses (Vasudeva and Lal, 1944, 1945) served as alarm to the rising problems of plant virus diseases in the country. In the 50s and 60s a few well-trained virologists in England and USA initiated systematic work in virology and developed strong schools of virology at New Delhi, Poona, Kalimpong, Madras, Lucknow, Gorakhpur, Bangalore, Shimla and Cuttack (Table 2). Establishment of Advanced Center for Plant Virology (ACPV) at IARI New Delhi in 1988 under the leadership of Prof. Anupam Varma and formation of Indian Virological Society (IVS) in 1984 at CCS Haryana Agricultural University, Hisar with Dr. S. P. Raychaudhuri as founder President were the milestones in strengthening work in Plant Virology in India. For suitable human resource development; ACPV as a part of National Agricultural Technology Project (NATP) organized group training on modern techniques in Plant Virology during 1999-2003. In addition hand-on-training was also organized for trained manpower in Plant Virology to take up work on molecular characterization of Plant Viruses. In mid 80s work on molecular virology was initiated at New Delhi, Lucknow and Bangalore. Later this was also initiated at Madurai, Palampur, Shimla, Hisar, Hyderabad and Varanasi. A number of viruses have been characterized at

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these centers and genetically modified plants of a few species developed. Since 1985 IVS regularly organizes annual meeting and National Symposium on current burning topics. ______________________________________________________________________________ Table 3: Natural infections of viruses on crops and weeds in Eastern India*_________________ Virus Host Plant_________________ Bean yellow mosaic potyvirus Cowpea (Vigna sinensis Savi) Cucumber mosaic virus Pointed gourd (Trichosanthes dioca Roxb.), cucumber (Cucumis sativus L.), Cleome viscosa L. Cucumber green mottle mosaic virus Coccinia grandis (L.) Vogt Watermelon mosaic potyvirus 1 Pumpkin (Cucurbita moschata Poir)

Ridgedgourd ( Luffa acutangula Roxb.) C. grandis (L.) Vogt

Watermelon mosaic potyvirus 2 Pumpkin (C. moschata Poir), Solanum nigrum L. Zucchini yellow mosaic potyvirus Pumpkin (C. moschata Poir), Ridgedgourd ( L. acutangula Roxb.),

Bittergourd (Momordica charantia L.), C. grandis (L.) Vogt Potato virus Y Potato (S. tuberosum L.), Cucumber (C.

sativus L.), brinjal (S. melongena L.), chilli (Capsicum Annuum L.), Teramnus labilis SW., Datura metel L.

Potato virus X Potato (S. tuberosum L.), Glycosmis arborea Corr. Papaya ringspot potyvirus Papaya (Carica papaya L.) Dasheen mosaic virus Elephant foot (Amorphophallus paeonifolius Dennst.) Nicol.) Amaranthus mosaic potyvirus Amaranthus viridis L. Tobacco mosaic virus Ridgedgourd ( L. acutangula Roxb.),

Pointed gourd (T. dioca Roxb.), brinjal (S. melongena L.), C. grandis (L.) voigt, Colocasia esculenta (L.) Schott.

Southern bean mosaic sobemovirus Cowpea (V. sinensis Savi) Furovirus-like particles Sweet potato (Ipomoea batatas L.) Badnavirus-like particle Okra (Abelmoschus esculentus) Tobamovirus-like particles Okra (A. esculentus L.) Tobravirus-like particles Jute (Corchorus olitorious L.) Potyvirus-like particles Chilli (C. annuum L.), Peperomia

pellucida (Kunth), S. indicum L. Potexvirus-like particles Alternanthera sessilis (L.) R. Br. ExDC. Isometric particles Pumpkin (C. moschata Poir), T. labilis

SW. Filamentous particles Ageratum conyzoides L., Calotropis gigantia (L.) R. Br. Ex Ait, Colocasia esculenta (L.) Schott Short rod-like particle Clerodendrum viscosum Vent. ______________________________________________________________________________ * Based on Plump et al., 2000

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One International Conference on Virology in the Tropics was also organized in 1991 at Lucknow and the next is scheduled at New Delhi in February 2008. Biannual issue of Indian Journal of Virology is the official publication of IVS. International Working Group on Tropical Virology (ViroTrop) under IVS occasionally brings out publication in the form of edited book entitled Topics in Tropical Virology containing invited chapters on important topics. ViroTrop so far has organized three International Training Programs in India, Vietnam and Thailand for virologists from developing countries. Caulimovirus Carnation etched ring virus (CERV) isolates have been detected using virus specific primers and expected products of CERV (1350 bp) were obtained. Of these the fragments of CERV have been cloned in suitable vector. Cloned fragment of CERV has been partially sequenced and it showed 90-95 % homology with the sequences available in the database for CERV. Its sequence has been submitted to EMBL Data Base as Carnation etched ring virus partial gene for polyprotein and coat protein (Acc. No. AJ549330). The primers were designed for coat protein, movement protein, aphid transmission protein, polyprotein and inclusion bodies protein. These genes were amplified, cloned, sequenced and sequences were submitted to EMBL Database with Acc. Nos. AJ619715, AJ619716 and AJ619974. Complete genome of CERV has been amplified using designed primers that gave an amplification of approximately 8 kb. The authencity of the amplicon has been confirmed by initial sequencing. CERV has also been detected in Carnations collected from Solan (HP). The CP, MP, Aphid transmission (At) (Acc. No. AJ830017) and DNA binding genes (Acc. No. AJ830018) have been amplified and cloned for sequencing. CERV genome was sequenced by amplifying complete genome of CERV using primers from 5’ and 3’ end and with internal primers that amplify individual genes and parts of the genes. The genome was found to comprise of 7924bp with GC content of 37%. The different genes encoded by CERV were compared with the other known CERV isolate and caulimoviruses (Accession No. AJ853858). They were found most conserved with respect to poly protein region (37-65% amino acid homology) while least to inclusion body matrix protein (5-37% amino acid homology). Duplex and multiplex PCR protocols were standardized to amplify different genes of CERV genome in one reaction. Phylogenic analysis based on different genes showed that CERV has independently evolved among caulimoviruses but is closely related to Cauliflower mosaic virus. Although CERV has been previously characterized from Holland but it is an effort to completely characterize the Asian isolate of CERV (Raikhy et al., 2003). Symptoms of CERV varied seasonally. Sometimes it is symptomless on Dianthus caryophyllus or exhibited necrotic fleck on leaves. Badnavirus Citrus yellow mosaic disease (CYMD) is widely distributed in India and is economically important in sweet orange (Citrus sinensis (L) Osbeck) and pummelo (C. grandis (L) Osbeck). It is graft transmissible to 13 citrus cultivars and in nature mealybug (Planococcus citri) is the vector. Symptoms of CYMD are severe mosaic with significantly lesser fruit setting with deteriorated juice quality reduced productive life of the citrus trees. The virus particles are non-enveloped bacilliform and measured 130X 30nm (Ahlawat et al., 1996). The causal virus is designated as Citrus yellow mosaic virus

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(CYMV) of genus Badnavirus and family Caulimoviridae with genome as dsDNA. It is weak immunogen thus serodiagnosis is not preffered. Baranwal et al., (2003) reported a highly reliable PCR based method of detection and diagnosis. In total DNA isolation from CYMV infected sweet orange leaves using DNeasy Kit (Qiagen), high concentration of polyphenolics and tannins were the interfering factors. They were removed with the addition of sodium sulphite to the DNA extraction protocol. DNA thus extracted survived at various temperatures for much longer time than without sodium sulphite. The amplification was also better and DNA yield was also higher. The sequenced amplicons (638 base pair) had 89% identity with the earlier sequence (AF347695) of an Indian isolate of CYMV in RT and Rnase H domain of ORF III polyprotein. This shows possible variability in the Indian isolates of CYMV. Symptoms of banana streak disease (BSD) caused by Banana streak virus (BSV) of genus Badnavirus and family Caulimoviridae is confusing with those of CMV on banana. Like CYMV, BSV is also weakly immunogenic hense PCR is preferred for detection and diagnosis. BSV particles are bacilliform measuring 30X130-150 nm with circular dsDNA genome of ~7.4 kb. The virus is transmissible only to plant species in family Musaceae with mealybug. For PCR test using BSV-K1 and BSV-K3 isolates from Kannara, Kerala, primers were designed based on the available sequence data (AJ002234) from the conserved domain of RT and RNase H. The sequence alignment of amino acids showed a very high degree of identity (98%) amongst BSV-K1 and BSV-K3 isolates and they clustered with Nigerian isolate of BSV-Onne (Cherian et al., 2004). Sugarcane bacilliform virus (SCBV) has been first reported in the world sugarcane germplasm collection of the Sugarcane Breeding Institute, Coimbatore. The symptoms were mild mottling, chlorotic stripes, no tillers developed from the main shoot, and canes had reduced number of internodes which were shorter. This gave a characteristic stunted growth. It was serologically related with BSV in DAC-ELISA and ISEM tests (Viswanathan et al., 1996). Mealybug species Sacchricocon sacchari, Desmicoccus boninsis and Planococcus citri could transmit this virus. The natural hosts are Sorghum halepense, Brachiaria spp., Panicum maximum and Rottboellia exaltata. SCBV has also been recorded in Maharastra, Uttar Pradesh and Haryana (Rao et al., 2002). A mosaic disease on black pepper has been recently observed in Kerala. The symptoms are vein clearing, chlorotic flecks, mottling, interveinal chlorosis and leaf curling. The virus was transmitted to healthy black pepper plants by grafting and with mealybug (Ferrisia virgata (Cockerell)). Sap transmission could be done with difficulty. The virus particles were bacilliform measuring 30X120 nm. In DAC-ELISA test the virus was found serologically related to Banana streak virus (BSV) and Sugarcane bacilliform virus (ScBV) but not to Rice tungro bacilliform virus and Commelina yellow mottle virus. The serological affinity was stronger with BSV (Bhat et al., 2003). For Rice tungro bacilliform virus, see the portion on Rice tungro virus.

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Begomovirus Tomato (Lycopersicon esculentum), an important vegetable and raw material for several edible products are widely infected by tomato leaf curl virus disease (ToLCD). Sinha et al., (2004) collected ToLCVD samples from tomato in four distinct zones in the country viz New Delhi (ToLCNDV-IARI), Jabalpur (ToLCV-Jb), Raipur (ToLCV-Rp) and Dharwad (ToLCV-Dh). Replication initiator protein (Rep) gene of these isolates was amplified using gene specific primers. The amplicons obtained had 1086bp in each case. Sequence analysis of these amplicons revealed two distinct subgroups – ToLCV-Nde with bipartite genome having 94-95% homology and tomato leaf curl from Bangalore (ToLCBV) with monopartite genome that showed only 73-75% homology. Phylogenetic analysis showed that all the four isolates collected from distinct zones of the country belonged to subgroup I i.e. ToLCV-Nde. The Rep gene nucleotide sequence of Dharwad isolate (ToLCV-Dh) had more similarity with ToLCV-Nde as compared to only 75% similarity with subgroup Ii i.e. ToLCBV from Bangalore (Sinha et al., 2004). This supports independent origin of these virus isolates and that the geographical divergence in the country had no influence (Malathi and Varma, 2003). Alignment of ToLCNDV-IARI Rep protein governing Rep oligomerization, DNA binding and DNA cleavage with other viruses inducing ToLCVD showed that sequences were conserved in α-helix involved in DNA cleavage, three motifs and P-loop for ATP binding inspite of high degree of variability at extreme N- and C-terminal (Dasgupta et al., 2004). Leaf curl disease on chilli is commonly seen in India. The symptoms are leaf curling, stunting and shortening of internodes and petiole resulting into crowding of leaves. The disease is transmissible on chilli and tomato using Bemisia tabaci producing typical symptoms on chilli as observed in field and leaf curl symptoms on tomato. CP region was amplified using total DNA isolated from infected chilli plants. The amplification product was of ~800 bp, it was cloned and sequenced and Blast search analysis of nt showed 89-93% identity with Tomato leaf curl New Delhi virus (ToLCNDV), 86% identity with Pepper leaf curl Bangladesh virus and 81% with Chilli leaf curl virus –[Multan] (Khan et al., 2005). It is the first report of natural infection of ToLCNDV on chilli in India. A similar report on chilli in Pakistan (Hussain et al., 2004) indicates that chilli is an important alternate host of ToLCNDV in the Indian subcontinent. Jacob et al. (2003) reported an improved method of agroinoculation of bipartite bigomoviruses using single Agrobacterium strain over the routine method of using two strains of Agrobacterium independently harboring tandem repeats of DNA-A and DNA-B. Co-delivery of Mungbean yellow mosaic virus DNA-A and DNA-B from pGV2260∷pGV1.3A (a co-integrate vector) and pGA1.9B (a binary vector) in one Agrobacterium strain enhanced agro-infection from 33 to 78% and 24 to 61%. When co delivery was done using pGA1.9A and pPZP1.9B (both binary vector), agroinoculation increased to 100% from 63 and 74%. Clones of bipartite Mungbean yellow mosaic virus (MYMV) from field infected V. mungo (MYMV-Vig) were obtained. They were DNA-A clone KA301 and five DNA-B clones viz. KA21, KA22, KA27, KA28 and KA34. The sequence identity in 150-nt common region (CR) of DNA-A and DNA-B was highest (95%) with KA22 DNA-B and lowest (85.6%) with KA27 DNA-B. Agroinoculation of DNA-A and KA22 DNA-B

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induced more severe symptoms in V. mungo with high virus titer. Similarly agroinoculation of and DNA-A and KA27 DNA-B in V. radiata showed more severe symptoms and high virus titer (Balaji et al., 2004). Nucleotide sequencing of YMV infected soybean in central India identified it as a strain of Mungbean yellow mosaic India virus and from southern India a strain of Mungbean yellow mosaic virus. Sequence similarity between DNA-A components of these two species was higher i.e. 82% whereas between their DNA-B components it was lower i.e. 71% (Girish and Usha, 2005). Mungbean yellow mosaic India virus (MYMIV) blackgram isolate, cowpea isolate MYMIV- [Cp] and soybean isolate MYMIV- [Sb] though reveal >90% nucleotide sequence identity yet they show distinct hostrange (Varma et al., 1992). The specific host barrier in a bipartite bigomovirus may be either due to inefficient replication governed by DNA-A or impaired movement governed by DNA-B. In a study host barrier of MYMIV in infecting cowpea was investigated by using hybrid constructs by exchanging DNA-A and DNA-B components of MYMIV, MYMIV- [Cp] and MYMIV- [Sb]. It was observed that reassortment of genetic components amongst the isolates occurred in all the leguminous hosts but not in cowpea. Very low recovery of viral DNA and atypical leaf curl symptoms produced suggested barriers both in viral replication and systemic movement (Surendranath et al., 2005). Yellow mosaic disease of legumes in India is caused by MYMV and MYMIV. Host range studies of MYMIV- [Sb] isolate were worked out using whitefly and agroinoculation. It was observed that like MYMIV- [Cp], MYMIV- [Sb] also infected cowpea. It therefore differed from MYMIV blackgram isolate and MYMIV- [Mg] mungbean isolate which do not infect cowpea. Analyses of DNA-B nucleotide sequence of these MYMIV isolates differed, that explains the host range variation (Usharani et al., 2005). Bhendi (Abelmoschus esculentus) is widely infected by bhendi yellow vein mosaic virus (BYVMV) that induces drastic reduction in fruit yield and seed quality. Jose and Usha (2000) gave a protocol that successfully eliminates the problem of polyphenols and mucilage having polysaccharides that inhibits Taq polymerase and interferes in isolating viral DNA. BYVMV in India is monopartite containing DNA-A component which when agroinoculated systemically infects bhendi but produces only mild leaf curling. When DNA-A component is co-agroinocolated with a small satellite DNAβ component it produces typical symptoms of BYVMV (Jose and Usha, 2003). Cotton leaf curl virus (CLCuV) disease is a serious problem in cotton in the northern Indian states. PCR based technique can be of wide application in diagnosis of field samples. Complete nucleotide sequence of a CLCuV isolate collected from Haryana and maintained on cotton cv. HS-6 was tried (Sharma et al., 2005). The specific primers from CLCuV used were forward (CPF) 5’-AATTATGTCGAAGCGAGCTGC-3’ and reverse (CPR) 5’-TAATATCAATTCGTTACAGAG-3’ and the amplification reaction was carried in 50 μl containing 2.5 mM MgCl2, 150 μM each of the four dNTPs, 25 pmoles of each primer and 1 unit of Taq Plymerase and the leaf extract/plasmid DNA (1 μg). Comparison of CP gene sequence with some other mono and bipartite showed maximum

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identity of 97.3% to Pakistan isolate CLCuV-62 and the amino acid sequence homology was 99%. In another study, hybridization with [α-32P] dCTP radiolabelled CLCuV-DNA-A probe was used for detection of CLCuV infection in cotton cvs. HS-6, H-1098, F-846, H-777, H-182, LH-1556 and RST-9 raised in glasshouse and field samples collected from Hisar, Sirsa and Dabawali and in six weed and other hosts (Sharma et al., 2004). Additional details on CLCuV are summarized in a recent review paper (Rishi, 2004). Several complete viral clones were obtained for the first time from cassava plants from field locations in Andhra Pradesh, Tamilnadu and Kerala. The work drew a detailed picture of the biodiversity of the virus in the country, which has important implications for disease prediction in crops. DNA sequence analysis and infectivity studies also showed that the cloned viruses were functional and can be modified further for various future uses. The work shows that certain viral proteins, related to movement have very strong signals to target specific locations within plant cells. A new virus, Sri Lankan cassava mosaic virus was shown to be responsible for cassava mosaic disease in India. Two movement-related proteins, the Movement protein and the Nuclear Shuttle protein were functionally analyzed for the domains representing intracellular targeting by fusing them with GFP and using fluorescence microscopy on biolistically-bombarded tobacco leaves (Patil et al., 2005). RNA-interference (RNAi) is one technology, which has immense potential in biology in the coming years. Work on using this technology for virus resistance was initiated about two years ago. Experiments using the cassava mosaic virus replication as a marker have indicated that the RNAi-based targeting of the replicase gene can decrease the replication of the viral DNA in transfected leaves of tobacco. This technology is still under development (Dasgupta, personal communication, 2005). DNA 1 and DNA β: A single stranded circular 1376 nucleotides long satellite-like DNA molecule is found in CLCuV. This was named as DNA 1, which encodes a nanovirus rolling circle replication initiator protein. This indicates that DNA 1 can replicate itself in the host plant but needs a helper virus CLCuV for movement and insect transmission. Recently diversity in DNA 1 has been reported (Briddon et al., 2004). Other single stranded DNA molecules of 1350 nt named DNA β has been found in nine host species existing with bigomoviruses. Symptom modulating role of these DNAs in monopartite bigomoviruses have been demonstrated. DNA βs have highly conserved single ORF. Phylogenetic analyses of 26 DNA βs revealed two groups, one originates from hosts in family Malvaceae and the other from more diverse group of plants of families Solanaceae and Compositae (Briddon et al., 2003). Further highlights of the work done on bigomoviruses in India have been covered in earlier reviews (Rishi, 2004, Singh et al., 2004, Varma and Malathi, 2003, Nene, 1972). Nanovirus Banana bunchy top virus (BBTV) disease is the most devastating virus disease of banana causing yellowing of leaves followed by debilitation and death of plants. Symptoms of naturally infected plants persist. Primary transmission is through infected suckers and

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secondary transmission through banana aphid Pentalonia nigronervosa Coq. The virus is persistently transmitted and aphids carry the virus throughout their life but the virus is not passed transovarially to the next generation. BBTV is not sap transmissible. Host range is confined only to Musa spp. In M. sapientum cv. Cavandish it induces yellowing of leaves and stunting. BBTV particles are isometric, non-enveloped of 18-20 nm diameters, and belong to genus Nanovirus and family Circoviridae. The genome consists of multiple ssDNA molecules of ~1kb size. The Australian isolate has 6 DNA components which appear to be structurally similar in being (+) sense that transcribe in one direction and contain a conserved stem loop structure and other domains in non-coding region (Hull, 2002). The disease was first seen in Fiji in 1879 (Brunt et al., 1990). In India BBTV showing similar symptoms as described above was characterized and efficient serological detection technique standardized (Manickan et al., 2001). To manage this dreaded disease, a number of companies are now supplying BBTV free banana saplings generated through tissue culture techniques, but due to presence of efficient banana aphid vector and inoculum in field it is essential to monitor the presence of BBTV in P. nigronervosa in field for the success of tissue culture technique to manage the disease (Thiribhuvanmala et al., 2005). Viruliferous IInd instar nymphs were fed for 24 hrs on BBTV infected banana leaf and serially transferred to healthy banana plants at 24 hrs interval till the aphids survived. Presence of BBTV in these aphids was checked by DAC-ELISA. It was noticed that BBTV was present in IInd instar nymphs and adults but not in Ist instar. This may possibly be due to feeding behaviors of nymphs and adults than the amount of virus ingested. Potyvirus Strains of sugarcane mosaic virus (SCMV) using sugarcane differentials were reported in northern India (Rishi, 1969, Bhargava, 1971, 1975, Bhargava et al., 1972). So far ten strains have been identified using sugarcane and sorghum differentials (Rao et al., 2002). Incidence of SCMV goes upto 100% resulting into serious losses (Jain et al., 1998). Three years data on assessment of losses due to SCMV in popular sugarcane varieties revealed that incidence of 50% since early stage of crop results into 20-25% cane yield loss (Rishi and Ram, unpublished). Recently a virus inciting mosaic disease of sugarcane in Andhra Pradesh has been identified as a strain different than SCMV group. The molecular studies revealed it as a new virus named sugarcane streak mosaic virus (SCSMV) a member of genus Tritimovirus in family Potyviridae. The virus particles were flexuous of ca 890X15 nm, CP sequencing indicated it a possible Potyvirus but different than many potyviruses reported. Sequence analysis of 3’-terminal 1084 nt showed 93.6% identity in the CP coding region with sugarcane streak mosaic virus (Pakistan isolate). Glycosylation of CP resulted into its higher molecular weight i.e. 40 kDa as against 34 kDa deduced from amino acid sequence (Hema et al., 1999). Later based on cDNA generated from purified RNA preparations of SCSMV-AP and comparison of partial ORF, C’ 1420 amino acids sequence with nine other members of Potyviridae and SCSMV-PAK revealed 100% identity with Indian isolates, 92% with SCSMV-PAK and only 20-30% identity with other members. The most variable genes among potyviruses are P1 and P3 and the most conserved one NIb, the RNA dependent RNA polymerase. A detailed analysis of this region and comparison with members of each genus in Potyviridae revealed maximum identity was with SCSMV-PAK. It was

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concluded that SCSMV is not a member of Tritimovirus and may represent a new genus in family Potyviridae (Hema et al., 2002). While studying antigenic diversity of sugarcane mosaic virus isolates from different northern and western India states, presence of SCSMV was confirmed from western U. P. (Rao et al., 2004). Widespread occurrence of an apparently unrecorded mosaic disease of sorghum (Sorghum bicolor cv. M.P. Cherry) was observed in Uttar Pradesh. The incidence ranged from 7 to 32%. The symptoms on naturally infected plants consisted of severe mosaic mottling and stunting of entire plant having few reduced ears. The dilution end point of the virus was 10-5, thermal inactivation point 55oC and longevity in vitro 7 days at room temperature (25+_3oC). The virus was readily transmitted by sap but could not be transmitted through seeds of infected sorghum. It was systemic in graminaceous hosts only viz. sorghum, Johnshongrass, Sudangrass and maize. The virus particles were long flexuous having an average length of 715 nm and it showed positive serological relationship with MDMV-antisera in tube precipitin, DAC-Elisa and ISEM test. The virus was identified as an isolate of maize dwarf mosaic virus (MDMV). Occurrence of MDMV on Sudangrass in India was also reported from Eastern Uttar Pradesh (Rao et al., 1996) Papaya ring spot virus (PRSV) a member of genus Potyvirus of family Potyviridae induces severe losses in papaya and cucurbits all over world. In India this virus has been recorded all over the country and is the major limiting factor in papaya cultivation (Varma, 1988). Variability in coat protein (CP) gene of PRSV isolates from various locations in India and implications on the development of transgenics were reported (Jain, et al., 2004). CP sequences of eleven isolates were studied in comparison with available sequences from other parts of world. Nuclear inclusion b (NIb) and CP regions from six isolates were amplified in two steps using two different sets of primers. Partial fragments of NIb and CP that overlapped were amplified using HRP 50 and HRP 83 primers, where as CP region alone were amplified using HRP 52 and RKJ 3 primers. CP region of the remaining five isolates of PRSV were amplified with primers HRP 52 and RKJ 3. Nucleotide and amino acid sequences of these eleven isolates were compared with the available sequences of fourteen isolates of PRSV from India, Asia, North and South America and Australia. At amino acid level there was considerable heterogeneity in CP sequences of Indian isolates. Seven of the eleven isolates had divergence of 0-11% where as remaining four had divergence of 0-7%. The variation in CP-coding region was 840-858 nt encoding protein of 280-286 amino acids. The Indian isolates were differentiated in two clusters but the sequence variation had no correlation with geographic origin. Small cardamom (Eletteria cardamomum Maton.) is said to be the queen of spices. It is cultivated in 1000 kms stretch of Western Ghats of south India. This crop is widely infected by cardamom mosaic virus disease (CdMVD) that incurs yield depression of 70-100% in one to three years. The characteristic symptoms are mosaic and light green stripes on leaf, marked reduction in the size of capsules and severe stunting of the diseased plant. Aphid Pentalonia nigronervosa transmits it. Centuries of cultivation of cardamom in the Western Ghats lead to considerable variation in CDMVD and steep fall in production and annual export of 2,383 tons in the earlier years to 270 tons in 1986 (Jacob and Usha, 2001). Molecular studies on the virus isolate revealed viral RNA of 8.5

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kb and coat protein (CP) mol. wt 38 kDa. Partial nuclear inclusion body gene, CP gene and 3' untranslated region (3' UTR) of viral RNA were amplified by reverse transcription and PCR and cloned and sequenced. The viral origin of these clones was confirmed by Northern hybridization with viral RNA. It was concluded that the virus isolate is Cardamom mosaic virus (CdMV) of the genus Macluravirus of the family Potyviridae (Jacob and Usha, 2001). ELISA and Western blotting analyses of the symptom variant isolates of CdMV coupled with sequence comparisons of CP and 3' UTR confirmed high degree of genetic variability in CdMV (Jacob et al., 2003). Banana bract mosaic virus (BBrMV) was the only known Potyvirus that naturally infected banana in Philippines (Thomas et al., 1997). BBrMV induced prominent dark streaks on the inflorescence bracts. On petioles and pseudostem purple colored streaks are seen and occasionally interveinal chlorotic streaks on leaves. In India, similar symptoms were reported in parts of Andhra Pradesh, Maharashtra and Tamil Nadu and were locally known as ‘kokkan disease’. Thomas et al., (1997) reported that ‘kokkan disease’ from India and Sri Lanka positively reacted with the antiserum of BBrMV in ELISA test. Banana samples in Maharashtra and Tamil Nadu showing similar symptoms contained Potyvirus-like particles (760X12 nm). Kiranmai et al., (2005) collected BBrMV disease samples of banana plants cv. Kovvur Bontha from West Godavari district in Andhra Pradesh that reacted positively with antiserum of datura leaf distortion virus (DLDV) a potyvirus in ELISA test. The purified BBrMV preparations had A260/280 and Amax/min ratios of 1.25 and 1.10 respectively. Virus coat protein showed one major polypeptide of 38kDa and three minor polypeptides in 12% SDS-PAGE. The virus isolate reacted positively with antisera of Bean yellow mosaic virus, Blackeye cowpea mosaic virus, lettuce mosaic virus, Pepper mild mottle virus, Pepper mottle virus, Peanut stripe virus, Sugarcane mosaic virus, Pea seed-borne mosaic virus, Potato virus Y and Datura leaf distortion virus using DAC-ELISA test. Out of 55 suspected banana plants tested 20 were positive to DLDV antiserum and 14 to CMV-B antiserum in DAC-ELISA tests. BBrMV is not transmissible mechanically. The transmission is vertical through vegetative propagation. Host range is confined to Musa spp. only. The Indian isolate of BBrMV has been thus identified as a potyvirus similar to Banana bract mosaic virus a member of genus Potyvirus reported from Philippines. In a survey Aglaonema sp. grown in Kangra Valley (H.P.), showed mosaic on leaves. Sap inoculations from these plants evoked chlorotic lesions on C. amaranticolor, C. quinoa, N. benthamiana and Saponaria vaccaria and mosaic on Philodendron sp. using epidermal strips from infected plants as sources. Yellowing of veins in some inoculated plants of C. quinoa and N. benthamiana was also observed. Amaranthus caudatus, Capsicum annuum, Cucumis sativus, Datura stramonium, Gomphrena globosa, N. clevelandii, N. glutinosa, N. rustica, N. tabacum cvs. White Burley and Samsun NN, Petunia hybrida, Phaseolus vulgaris and Zinnia elegans remained symptomless. The aphids Myzus persicae, Aphis craccivora and A. gossypii transmitted the virus non- persistently. From these diseased plants, virus was purified. In electron microscopy, filamentous particles (c. 750 x 11 nm) were present in the leaf epidermis of infected plants and in purified preparations. In ultrathin sections of the leaves cylindrical inclusions characteristic of potyvirus were observed. In double diffusion tests and DAS-

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ELISA the purified preparations reacted with specific antibodies to Dasheen mosaic potyvirus (from Agdia, USA). These observations lead to the conclusions that the virus infecting Aglaonema is Dasheen mosaic virus (Ram et al., 2003). BYMV was the major virus (100%) found to infect gladiolus crop. Diagnostics, such as RT-PCR, were standardized to diagnose BYMV (amplification of 750bp), The polyprotein gene of BYMV was amplified and sequenced which was submitted to the EMBL Nucleotide Database (Acc. No. AJ579917) (Katoch et al., 2003). ELISA, EM, IEM and RT-PCR could detect Iris severe mosaic virus (ISMV) in Iris x hollandica cv. Bluemagic by. ELISA and results showed strong positive reaction in the cultivars tested (Kulshrestha et al., 2004). Electron microscopy of negatively stained preparations from clarified virus concentrate and clarified virus extract revealed the presence of flexuous virus particle ca. 760nm x 12nm indicating it to be a potyvirus. IEM studies employing trapping, clumping and decoration using the ISMV specific antibodies indicated the virus to be an isolate of ISMV. About 850bp amplification product obtained in RT-PCR was cloned and sequenced. The sequence of PCR amplified product of ISMV has been submitted to EMBL Data base (Acc. No. AJ 549755). Iris (Iris x hollandica Hort. cv. Bluemagic and Cassablanca) plants showing mosaic symptoms were tested for the presence of Iris mild mosaic potyvirus (IMMV) by host range, ELISA (using antibodies specific for IMMV), RT-PCR, IC-RT-PCR using potyvirus group specific primers and antibodies specific to IMMV, IEM and cytopathology. Sap inoculation did not result in any symptoms in any of the inoculated test plants, only local lesions were observed on C. amaranticolor and C. quinoa. ELISA revealed the presence of IMMV in Iris leaves, bulb and bulblet, RT-PCR resulted in the amplification of ~335bp long fragment in leaves and bulblet while nothing could be detected in bulbs. IC-RT-PCR resulted in the amplification of~335 bp in leaf tissue. Enhanced trapping and clumping of virions with IgG to IMMV and occurrence of cylindrical inclusions in the cytoplasm, typical of a potyvirus, further confirmed the identity of the virus as IMMV (Kulshrestha, et al., 2005). The amplified fragment has been sequenced, analyzed and found to have 50-62% nucleotide sequence homology with the other established potyviruses from the database (Acc. No. AJ507397). Tewari and Shukla (1984) reported the effect of three strains of watermelon mosaic virus (WMV) on peroxidase activity in infected Cucurbita maxima Dusch. at different intervals after inoculation. There was general increase in in peroxidase activity in infected leaves with increase in the post inoculation period. The highest increase was on 5th day in case of all the three strains of WMV. For problems of potyviruses on potato readers may see an excellent bulletin published by CPRI, Shimla (Khurana, 1999). Closterovirus In India Brown (1922) recorded presence of tristeza disease causing failure of malta sweet orange on sour orange rootstock. This disease is caused by Citrus tristeza virus

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(CTV) of family Closteroviridae, which has been recorded in all the citrus growing regions in India (Ahlawat and Pant, 2004). The decline symptoms is due to phloem necrosis in sour orange rootstock. The virus particles are 2000 nm in length and 10-12 nm in widthhaving +sense ssRNA of about 19,256 nt that encode 12 ORF that code 17 proteins (Manjunath et al., 1993, Ahlawat and Pant, 2004). National Research Centre of Citrus, Nagpur has developed integrated management program of this disease (Ghosh, 2002). Potexvirus Potato virus X is of worldwide importance in potato. It is dependent on potato for survival. The symptoms on potato are mild mosaic that is barely perceptible to latent in many varieties. In potato var. Craigs Defiance it induces top necrosis and local lesion hosts are Gomphrena globosa, Chenopodium amaranticolor, C. quinoa and Potato clone A6. In the northwestern and northeastern plains of India, higher virus incidence is noticed because of the favourable weather conditions and ecological factors. This result into corresponding higher yields losses in this area (Nagaich et al., 1969). Higher virus incidence since the early stage of the crop leads to higher tuber yield depressions. Continued use of virus contaminated potato tubers lead to tuber degeneration and very high yield losses (Garg, 1987). The virus particles are flexous rods with helical symmetry, measuring 470-580 X 18 nm. Genome consists of single species of ssRNA. It is highly contagious, readily sap transmissible and transmits in nature by contact. For further details readers may see bulletin published by CPRI, Shimla (Khurana, 1999). Cymbidium mosaic virus (CymMV), infecting orchids has been established on various herbaceous plants like Datura stramonium and Nicotiana benthamiana (Sherpa et al., 2003). Coat protein (672 bp) and movement protein 1 (MP1) (690bp), MP2 and MP3 (276 bp) genes of CymMV have been amplified using designed specific primers (Accession nos. AJ566130 & AJ566131) and sequenced (Accession nos. AJ564562, AJ581997, AJ581998, AJ585202, AJ585203, AJ585204, AJ620244, AJ619959, and AJ623307). The variability in coat protein (CP) gene sequences of CymMV isolates naturally infecting orchids was investigated. Samples were collected from different regions of India including Pakyong, East Sikkim. Some plants were identified that showed virus like symptoms such as chlorotic mosaic, severe to mild chlorosis and/necrosis, colour breaking, deformation, reduction in size and yield of flowers characteristics of CymMV. Using DAS-ELISA Vanda sp., Dendrobium hybrid White, Pompora, Sakura, Cymbidium sp., Cymbidium grandiflorum, Dendrobium sp., D. aciniciformae and Straupsis undulata gave a positive reaction with the polyclonal antibodies specific to CymMV. The samples were tested for CymMV by RT-PCR using CymMV primers that gave a product of expected size (672 bp). The amplified products were cloned, sequenced. The nucleotide sequences and the amino acid sequences were found to be 87-97 % identical at nucleotide level and 67-98 % at amino acid level with other CymMV isolates. Following confirmation of CymMV infection an extensive screening of hundred different orchids were conducted by Northern blotting using CymMV coat protein gene labeled with 32p as a probe. In total around sixteen infected samples were identified as being CymMV positive among 100 different orchids. Out of twenty-four genera, CymMV was observed in seven orchid genera viz Cymbidium, Dendrobium, Oncidium, Paphiopedilum, Phaius, Straupsis and Vanda. These orchids can

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act as a potential source of CymMV infection for adjoining orchid plants. Coat protein gene of about 669bp was amplified from different orchids collected from Orissa, Sikkim, Kerala and Bengalore (Acc. No: AJ564562, AJ581998, AJ581997, AJ585202, AJ585204, AJ585203, AJ620244, AJ698947 and AJ871374). The nucleotide sequences and the amino acid sequences were found to be 87-97 % identical at nucleotide level and 67-98 % at amino acid level. Such high sequence conservation suggests that CymMV coat protein gene is highly conserved and is a suitable candidate for development of diagnostic and to provide resistance (through transgenic) to orchids cultivated in different geographical locations. RdRp gene (4.2 kb) was amplified by RT-PCR. Different primers sets are designed to amplify the different region of RdRp gene. A potexvirus has been recently recorded on Asparagus using ELISA (Zaidi personal communication). Cucumovirus A mosaic disease on Egyptian henbane (Hyoscyamus muticus L.) of family Solanaceae was noticed at the experimental farm of CIMAP, Lucknow which, is widely spreading. In severe farm it produces pronounced mosaic and crinkling on leaf, stunting, reduction in fresh weight and total alkaloids content. This plant is an important source for hyoscyanine and hyoscine alkaloids that are used in different modern drugs. The virus is sap transmissible and by aphids (Myzus persicae Sulz and Aphis gossypii Glover) in non-persistent manner on a number of hosts. Virus particles were isometric measuring 28 nm in diameter with a central core and capsid protein was 26 K. Viral nucleic acid was infective when treated with DNase but completely lost infectivity when treated with RNase and S1 nuclease thus showing ssRNA. The viral genome was tripartite (RNA1, RNA2, RNA3) with a subgenomic RNA (RNA4). Similar findings were reported on CMV that infected Banana in India (Kiranmai et al., 1996). Serologically the virus isolate was closely related to CMV-S and CMV-A (Raj et al., 1997) and distantly to CMV-T, CMV-CD, CMV-P and tomato aspermy virus. Based on these findings the virus isolate on Egyptian henbane was identified as a strain of CMV (Samad et al., 2000). Looking to the economic importance of CMV in banana in India, Kiranmai et al. (1996) compared three different ELISA tests to evaluate relative efficacy for routine detection and diagnosis of CMV in banana. It was concluded that double antibody sandwich (DAS)-ELISA is ideal for detection of virus in pseudostem sap exudates by pin pricking in large-scale testing of banana plants that could detect virus up to 1μl/well. For dot-blot hybridization test cDNA to RNA genome of CMV was cloned and a clone with 1521 and 334 bp inserts were selected and used for probe preparation. Non-radioactive digoxigenin (DIG) probe appeared better than 32P probe (1521 bp) and detected virus up to 5pg in pseudostem sap exudates and leaf extract (Kiranmai et al., 1998). Virus isolates that showed severe mosaic and leaf deformation on brinjal, mosaic and leaf puckering on chilli and mosaic and fern leaf and stunting on tomato in Chitoor district in Andhra Pradesh were respectively identified as CMV-Br, CMV-Ch and CMV-To (Kiranmai et al., 1997). The differential hosts identified are Dolichos lablab for CMV-To, Cucumis sativus for CMV-Br and Datura metel for CMV-Ch. CMV-Br and CMV-Ch were serologically distinct but related to CMV-To. Stunt disease of black pepper (Piper nigrum L.) has become important in Kerala and Karnataka. Leaves of infected plants are crinkled, brittle, leathery and small with chlorotic patches and streaks on the internodal

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areas and general stunting of the vine. The disease is sap transmissible with chlorotic/necrotic local lesions on C. amaranticolor, C. quinoa, V. unguiculata, V. radiata and V. mungo and systemic mosaic on Cucumis sativus and five solaneous plant species. It is serologically related with CMV isolates on banana, brinjal, chilli, tomato in India and CMV-L (USA) and CMV-A (China). The genome is dsRNA with four RNA species (Sarma et al., 2001). CP gene of CMV isolate associated with this disease was sequenced and was 657 nucleotides coding for protein of 218 amino acids. It was found most closely associated with CMV isolate infecting Egyptian henbane in India a member of subgroup I. It showed 94-95% nucleotide identity with CMV subgroup I isolates recorded in India and 96-99% identity at amino acid level. In general it showed 92-99% amino acid identity with members of subgroup I, and 77-79% with members of subgroup II. It was concluded that stunt disease of black pepper in India is caused by a strain of CMV of subgroup I (Bhat et al., 2005). A virus disease showing severe mosaic, leaf deformation, crinkling and curling, stunting, delayed flowering and reduced seed setting on Amaranthus tricolor L. and A. hypochondriacus L. (grain species) was studied (Raj et al., 1997). The virus was sap transmissible and non-persistently aphid (Myzus persicae (Sulze) and Aphis gossypi (Glove)) transmissible on ten plant species. It gave necrotic local lesion (NLL) on Chenopodium amaranticolor Coste & Reyn, C. murale L., Spinacea oleracea L., and NLL on inoculated leaves of Nicotiana rustica L., N. tabacum cvs. Samsun NN and White Burley followed by severe mosaic. Seed transmission was 16.5% in A. hypochondriacus. The virus was serologically related to CMV-C and CMV-D but not to CMV-L, S, T and Pet strains. On the basis of non-persistent aphid transmission, presence of 28 nm isometric particles, 26 kD mol. wt. of coat protein, serological relation to CMV-C strain, ssRNA (infectious), this virus isolate was identified as CMV. Chrysanthemum plants showed diffused chlorosis on leaves, chlorotic dots near veins and stunting at the experimental farm of NBRI, Lucknow. These symptoms differed from those of chrysanthemum aspermy virus and tomato aspermy virus (TAV). It was therefore, worked out to identify the causal virus (Srivastava et al., 1992). On the basis of host reaction, physical properties, isometric virus particles of 29nm diameter, 24.5 kd coat protein, ssRNA the virus isolate was identified as chrysanthemum strain of CMV. Natural infection of CMV on chrysanthemum has not been reported earlier. It was occasionally used as diagnostic host to differentiate CMV and TAV. CMV infected chrysanthemum may serve as a potential reservoir for CMV dissemination to a number of economically important plants in field and kitchen garden. Raj et al. (1995) reported coat protein (CP) sequence homology of two Indian isolates of CMV on Dianthus barbatus (CMV-CR) and Physalis minima (CMV-P). RNA4 of CMV-CR and CMV-P were used as template for cDNAs synthesis using random primers. They were cloned in Bluescript II KS (+) phagemid at Sma I site and sequenced. Comparison of 249 nt sequences of 5’ coding regions of CP gene and their putative products indicate that these strains may be in CMV subgroup I. This is the first report of the natural occurrence of CMV on Datura innoxia. CMV infection on Gladiolus psittacinus was identified on the basis of RT-PCR using CP gene specific primers and Southern hybridization with probe derived from cDNA (Raj et al., 1999, 2002). These tests were found most sensitive for reliable detection of CMV in gladiolus leaf and corm tissues.

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Widespread occurrence of an apparently unrecorded mosaic disease of pea (Pisum satvium L. Var. Arkel) was observed in and around Gorakhpur (Eastern, U.P.). The symptoms in naturally infected plants consisted of severe mosaic mottling and puckering of leaves followed by stunting of the entire plant and few small pods. The DEP of the virus was between 10-6 to 10-7, TIP 75oC to 80oC and longevity in vitro 27 days at room temperature (20-25oC); 32 days at 4oC and 46 days on dessication over calcium chloride at room temperature. The virus was readily transmitted by sap, aphids (non-persistent type) and seed but not by any beetle tested. It was systemic in leguminous hosts only viz. Arachis hypogaea, Cassia tora, Crotolaria juncea, C. sericea, Cyamopsis tetragonoloba, Dolichos biflorus, D. labalab, Pisum sativum, Vigna mungo, V. radiata, V. sinensis and Vicia faba and it was localized in Chenopodium amaranticolor and C. ambrisoides. The symptoms in different leguminous hosts varied from mild mosaic mottling, chlorotic spots, vein clearing and vein banding to reduction of leaves, pods and entire plant. It gave a positive precipitin test with antiserum of cucumber mosaic virus. The shape of virus particles was isometric having a diameter of 32-2 nm. Phosphate buffer (pH 7.0; 0.1 M) was found to be the best extraction medium for the virus. The standard inoculum could be clarified by chloroform. The maximum precipitation of virus was obtained with a mixture of 4% polyethylene glycol (PEG; 6000 MW) and 1% sodium chloride followed by 1% Triton-x. The purified sample had absorption maximum at 260 nm and minimum at 242 nm. The nucleic acid content was approximately 9.8%. On the basis of above characteristics, the virus under study appeared as a member of cucumovirus group. It might be a new member/strain of this group, which remains to be established. However, the said virus has been referred to as cucumovirus (Rao, 1986). A similar type of disease showing identical symptoms on pea was observed in Western U.P. and Haryana. On the basis of symptoms, host range and serological relationship it was identifies as a strain of CMV (Rishi et al., 1992). CMV has been found to infect alstroemeria plants by ELISA and RT-PCR. The amplified product has been cloned and sequenced (Acc. No. AJ635301) (Verma et al., 2005). Nine cultivars of Alstroemeria hybrids viz. Alladin, Amor, Capri, Cindrella, Pluto, Rosita, Serena, Tiara and Variety No.14 plants were found to be infected with Cucumber mosaic virus (CMV). The virus was identified on the basis of host range, insect transmission, ELISA, electron microscopy and RT-PCR (using virus-specific primers). In RT-PCR the expected size of amplicon (540 bp) was observed in virus-infected plants. The eluted DNA on sequencing was found to be of 533 bp containing 215 nt of intercistronic region and 318 nt of coat protein gene of RNA3 (Accession no. AJ635301). In BLAST search the sequence showed 96-98% homology with the available sequences (D28488, AJ564331, D28486, AB004780, D42080, D00462 and AJ276481) of CMV subgroup I. In northern hybridization the positive signals for the presence of CMV were obtained in 22 out of 36 plants (61.1%) of these nine cultivars, showing wide spread CMV in alstroemeria. Plants of chrysanthemum cv. Fish Tail were found to be infected with Tomato aspermy virus (TAV), of genus Cucumovirus and family Cucumoviridae (Verma et al., 2005b). In RT-PCR an expected product of 660 bp (CP gene) was obtained using primers specific for this virus. The DNA has been cloned and sequenced (Acc. No. AJ550020). Different

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TAV Indian strains were amplified and sequenced. These sequences were submitted to the EMBL Nucleotide Database with accession Nos. AJ586134, AJ582718 and AJ580841. The complete genome of TAV was amplified using single tube RT-PCR that amplifies all the three RNAs (RNA 1, RNA 2 and RNA 3). Chrysanthemum cultivars were screened by DAS-ELISA using virus/group specific antibodies for the presence of TAV. Nineteen cultivars viz. Shyamal, Regol Time, Chandrama, fish Tail, Maghi Pink, Shanti, Sharadhar, Punjab Gold, Flood, Punjab Joy, Kirti, Chaman, Jubilee, Kaka Sonu, Vasantika, Atlantus, Discovery, Yellow Spider and Snow White were found to be infected with this virus. Gerbera plants were found to exhibit colour break symptoms on the petals, asymmetrical ray florets and deformed flowers. The virus evoked chlorotic local lesions on C. album, C. amaranticolor and C. quinoa, while systemic mosaic on C. sativus, N. benthamiana, N. clevelandii, N. glutinosa and N. tabacum cv. Samsun. This was found to be transmitted nonpersistently by M. persicae and A. gossypii and was identified as CMV by ELISA using CMV specific antibodies. In electron microscopy of gerbera leaves, polyhedral particles c. 29 nm were observed. Total RNA was isolated from the infected plants of gerbera and N. glutinosa using RNAqueousTM (Ambion, USA). CMV specific primers (2) were used to detect the virus by RT-PCR that gave an amplicon of 540 bp in virus-infected plants. PCR products on sequencing were found to be 533 bp long containing partial intercistronic region and partial coat protein gene (Acc. No. AJ634532) of CMV RNA3. In Blast search the sequence shows 91-99% homology with that of CMV subgroup I. This is the first definitive report of CMV on gerbera from India (Verma et al., 2004). Screening of 17 cultivars of Asiatic and Oriental Hybrid lilies and L. longiflorum was done by ELISA. CMV was found only in three cultivars of lily in combination with Lily symptomless virus (LSV). It was found that ELISA could detect these viruses in leaves at flower emergence stage in comparison to bulb scales. RT-PCR carried out for virus detection also showed that there were one or more viruses that cause disease in lily. All the cultivars taken for study except two were infected with LSV, whereas seven cultivars were infected by Lily mottle potyvirus (LmoV) in combination with LSV. Only two cultivars had mixed infection of LSV, LMoV and CMV as detected by RT-PCR. C. sativus was used as propagation host for CMV. M. persicae transmitted the virus in non-persistent manner. Electron microscopy showed isometric particles of 29nm in purified preparation. The molecular weight of viral coat protein was found to be 29KDa. The virus reacted with CMV specific antibodies both in ELISA and immunosorbent electron microscopy. Antiserum produced against the virus has dilution of 1:64 in double diffusion test. In RT- PCR 500bp fragment was obtained with CMV specific primers and sequenced (Acc. No. AJ564331). The complete CMV (infecting Asiatic lily cv. Romano) coat protein gene was sequenced (Acc. No. AJ585086).

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By RT-PCR various genes namely 1a (2.9 kb), 2a (2.5 kb), 2b (335 bp) and 3a (840 bp)) RNA2 (3.0 kb) and RNA3 (2.2kb) of CMV have been amplified using the designed specific primers. The amplified products have been cloned and sequencing is under progress for confirmation of the results. RNA2, RNA3, 1a and 2a genes have been amplified from L. longiflorum while CP, 2b and 3a genes of CMV have been amplified from Asiatic Hybrid, Oriental Hybrids and Tiger lily (Ram et al., 1999). Tobamovirus An Indian strain of Odontoglossum ringspot virus (ORSV) of genus Tobamovirus was detected (Sherpa et al., 2004) by DAS-ELISA and verified by RT-PCR using primers, which amplifies ORSV. The amplified product was cloned and sequenced. The nucleotide sequence and the amino acid sequence were compared with other ORSV isolates and found to be 96-100% identical at both nucleotide and amino acid level with other ORSV isolates. Multiple sequence alignment of deduced amino acid sequences revealed considerable homology to other ORSV. This is the first report of ORSV coat protein sequence from an Indian strain. The primers were also designed for coat protein gene of Odontoglossum ring spot virus infecting orchids (Acc. Nos. AJ566612 & AJ566632). The complete coat protein gene has been amplified (477 bp) by RT-PCR, cloned and sequenced (Acc. No. AJ564563). In ELISA it was found that most of the orchids collected from Sikkim, Chail and Palampur regions were infected with ORSV indicating its widespread presence. In RT-PCR using the primers U1 and L1 the orchid samples found to be symptomless gave an amplification of approximately 290 bp. The samples that showed negative result in ELISA were found to be positive in PCR. The pair of primer (osu and osd) amplified the complete coat protein gene of ORSV (477 bp). On sequence alignment it was found that Indian isolate of ORSV shares 96-100% sequence homology with the other available sequences both at nucleotide level and amino acid level. This is the first report of ORSV coat protein sequence from an Indian strain. A method of detection of ORSV by tissue slot blotting technique, which is very much sensitive, has been developed. Cloned ORSV CP gene in pUC 18 vector was used as a radiolabelled and used as a probe. Orchid samples (100 in no.) collected from different parts of Sikkim were analyzed. More than 40% of the orchid samples tested, were infected with the virus. This assay can be used for the certification of virus-free orchid materials because of its high sensitivity. Out of 15 cvs. of Rose indexed for TMV by ELISA, nine cvs. Anvil Spark, Montezuma, Sonia, Raktagandha, Pink Panther, Virgo, Queen Elizabeth, First Prize and IceBerg were found to be positive for TMV (Zaidi, unpublished). A white mosaic disease on Egyptian henbane not reported earlier was seen on the experimental farm of CIMAP, Lucknow during 1996 (Samad et al., 1999). The symptoms initiated as small white patches that gradually covered 80% of leaf area. Later these leaves turned yellow and shed from the plants. The virus was readily sap transmissible but not by aphids or through seeds of infected plants. It showed necrotic local lesions on Nicotiana glutinosa, Chenopodium amaranticolor and C. murale. The virus particles measured 300 nm in length and 16 nm in diameter. Based on biological and physical properties the virus isolate was identified as a strain of TMV.

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The first ever report of tobacco mosaic virus infection on scotch spearmint (Mentha gracillis Sole) was noticed in 1992 (Samad et al., 2000). The plant is cultivated for mint oil, menthol and carvone widely used in pharmaceutical industry and some of the editives. Symptoms on M. gracillis appear as mosaic, vein banding, deformation and stunting. The virus is readily sap transmissible but not by aphids and seeds from infected plants. The virus isolate gave local lesions on inoculated leaves of N. glutinosa, C. album, C. amaranticolor and C. murale. The purified virus preparation had A260/A280 ratio of ca 1.19 and mol. Wt. of capsid protein 17,000±500 Ddaltons by SDS-PAGE. The virus was serologically related to TMV-U1 and closely related to Brinjal necrotic mosaic virus (BNMV). No serological relationship was found with TMV-A1, TMV-D and cucumber green mottle mosaic tobamovirus. On the basis of these studies the virus was identified as a strain of TMV (Samad et al., 2000). Tospovirus Of late Tospoviruses of family Bunyaviridae have emerged as important pathogens inducing serious losses in several crops in India viz. cowpea, mungbean, soybean, potato and tomato (Varma et al., 2002, Jain, et al., 2002, 2004a). Tospoviruses identified in India are Groundnut bud necrosis virus (GBNV), Groundnut yellow spot virus (GYSV) and Watermelon bud necrosis virus (WBNV). It appears that GBNV is endemic in India on groundnut; it has a wide host range (Ghanekar et al., 1979, Reddy et al., 1992) and in course of time spread to other crops in nature (Jain et al., 2004a). Potato stem necrosis disease (PSND) caused by a strain of GBNV and named GBNV-Po is seen since 1982. It has widely spread in Northwest and central parts of India. In Madhya Pradesh and Rajasthan upto 90% disease incidence has been recorded. The virus is sap, and graft transmissible on cowpea causing local lesions. In nature it is transmitted by Thrips palmi. The characteristic symptoms on potato are necrotic spots on stem and petiole, spots on leaves accompanied with deformation and stunting. The virus was serologically related to tospoviruses GBNV from India and Watermelon silver mottle virus (WSMV) from Taiwan. The associated virus particles were quasi-spherical and measured 70-110 nm (Khurana et al., 2001). Nucleocapsid protein (N) gene sequences from multilocational (Gujarat, Madhya Pradesh and Rajasthan) PSND samples on potato were studied to confirm the association of GBNV with PSND (Jain, et al., 2004a). The virus isolates were maintained on diagnostic host Vigna unguiculata cv. Pusa Komal. Total RNA was extracted and N gene was amplified using RT-PCR and primer pair representing the 1st and the last 21 bases of the coding region of N gene respectively as available in literature. This successfully amplified N gene. The amplicons thus obtained were transformed, sequenced and analyzed. It had an ORF of 831 bases coding for a protein of 276 amino acids that was 1 and 30 amino acids longer than that of the corresponding gene of WBNV and GYSV. However it had 97-99% identity with GBNV both at amino acid nucleotide sequences. This confirmed that PSND in India is caused by GBNV. Within potato GBNV collected from different locations had 98-99% sequence identity with high degree of conserved sequence. On the other hand GBNV-Po N gene sequence had 41-81% nucleotide and 16-84% amino acid sequence identity with other tospoviruses. In another study nucleotide and amino acid sequences of movement protein (NSm) gene located on M RNA of the GBNV isolates collected from different hosts and locations in

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India were compared (Akram et al., 2004). Diseased cowpea and tomato samples from Kerala showing chlorotic and brown necrotic spots, groundnut leaves showing chlorotic and necrotic spots from Tamil Nadu, and potato showing severe stem and leaf necrosis from Rajasthan and Madhya Pradesh were collected. Presence of Tospovirus in these samples were tested with ELISA using polyclonal antisera against WSMV and identified as GBNV on the basis of nucleoprotein gene sequences. Amplification of NSm gene was done as per reported method (Jain, et al., 2004a) and sequenced. Analyses of the sequences showed one ORF of 924 bases coding for a protein of 307 amino acids. GBNV isolates from different hosts and locations under study had highly conserved NSm with 93-100% identity there by suggesting common origin of these isolates. They also showed 98-100% identity at amino acid level with GBNV-type isolate (GBNV-GNAP) but 82-83% with WSMV and 34-65% with other Tospoviruses. Singh and Krishnareddy (1996) reported a tospovirus disease on watermelon in Karnataka on the basis of transmission, host range and serological relationship with watermelon strain of tomato spotted wilt virus (TSWV-W) from Taiwan and GBNV from India, though in host range it differed from GBNV. Symptoms on watermelon were mottling, crinkling, yellowing of leaves, necrotic streaks on wines, shortened internodes and necrosis and dieback of buds. Molecular studies revealed that nucleocapsid protein gene amino acid sequence was most closely related with that of watermelon silver mottle tospovirus from Taiwan (84%) and GBNV from India (82%). On the basis of sequence divergence and variation in host range this virus isolate was designated as watermelon bud necrosis tospovirus and considered as a distinct species in serogroup IV (Jain et al., 1998). Carmovirus Examination of virus like symptoms on twenty-nine cultivars of carnations showed different types and degrees of symptoms on the leaves. Plants were tested by ELISA for Carnation mottle virus (CarMV). On the basis of ELISA results CarMV was found to be present in twenty-seven carnation cultivars. Culture of CarMV is maintained on Saponaria vaccaria and C. ambrosioides. The virus has also been detected by using specific primers for CarMV and its full-length CP gene has been amplified and cloned in suitable vector for further analysis. Carnation mottle virus gene for coat protein, genomic RNA has been submitted to EMBL Data Base (Acc. No. AJ549954). Also movement protein genes i.e p7 and p9 were also amplified, cloned and sequenced and have been submitted to the EMBL Database with Acc. No. AJ584843, AJ584842. Complete genome (4005 bp) of CarMV has been amplified using designed primers. The genome has been sequenced completely (Acc. No. AJ811998). Coat protein and movement protein genes of four Indian isolates were compared with the available sequences in the database. CP gene of annual and perennial carnations showed high homology (95–100%) compared to isolates reported from different parts of the world. MP genes (p7 and p9) showed 95–98% nucleotide homology while at the amino acid level homology of 86–98% for p7 and 92–98% for p9 respectively was obtained. The virus isolate therefore, is identified as Carnation mottle virus a species of genus Carmovirus and family Tombusviridae (Singh et al., 2005). CarMV has also been detected in the carantions

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collected from Solan and Palampur. The CP, movement protein genes (p7 and p9) have been cloned for further confirmation. Illarvirus Recently Tobacco streak virus (TSV) of genus Illarvirus (Subgroup I), family Bromoviridae has been observed causing serious necrosis diseases in sunflower (Helianthus annuus) and groundnut in Andhra Pradesh, Tamil Nadu and Maharashtra. TSV particles are icosahedral measuring 27-35 nm in diameter. It has tripartite genome of positive sense, ssRNA. The RNA-1 is 2.9 kb, RNA-2 is 2.7 and RNA-3 is 2.2 kb. The symptoms on sunflower are necrosis of leaf, petiole, stem and floral calyx and floral malformation and on groundnut necrosis of stem and terminal leaflets leading to death of the plants. TSV has also appeared on cotton, mungbean and sunn-hemp. One sample each of virus infected sunflower from Andhra Pradesh, Karnataka, Maharashtra and Tamil Nadu, one each of mungbean, cotton and sunn-hemp from Tamil Nadu, Maharashtra and Karnataka were collected and presence of TSV in these samples was tested using DAC-ELISA. The primer pair used was derived from the beginning of the first 20 bases of the coding region of the reported CP gene sequence of TSV on sunflower in India. Amplification was done using RT-PCR. Sequence analyses of the CP gene and dendrogram illustrating phylogenetic relationship revealed highly conserved CP gene (99-100%) suggesting common origin. This also explains close serological affinity in these isolates. As compared to USA isolates upto 12% sequence divergence were observed (Bhat et al., 2002). During a survey of begonia (Begonia semperflorens), plants were observed showing characteristic ring symptoms on leaves. On mechanical inoculation from infected leaves to healthy begonia plants, it produced characteristic rings on begonia and dark colored lesions on Cyamopsis tetragonoloba, typical of Prunus necrotic ring spot virus. Total RNA was isolated from infected leaf tissue using QIAGEN RNeasy Plant mini kit. RT-PCR was performed using PNRSV specific primers and an amplification of 785bp fragment was obtained as expected indicating the presence of PNRSV in begonia (Verma et al., 2002). Carlavirus Necrosis disease of cardamom that was observed in 1988 in the Nilgiri Hills and pockets of Kerala show chlorotic or necrotic patches on leaves. The only known method of transmission is through infected rhizomes. Based on partial characterization, i.e. Mol. Wt. of CP gene (37 kDa) , virus purification using two cycles of differential centrifugation followed by 10-40% sucrose linear density gradient centrifugation ,indirect ELISA test giving positive response with the antisera of potato carlavirus S and carnation latent carlavirus it is identified as a carlavirus. Mixed infection of cardamom necrosis virus with CdMv is also observed even in symptomless plants of cardamom. (Saravanakumar et al., 1998). In survey of chrysanthemum cvs. grown in H.P. different types of symptoms were observed. Out of 36 cvs. examined 7 were found to be negative for Chrysanthemum B virus (CBV) in DAS-ELISA. The virus has a very narrow host range infecting only N.

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clevelandii, N. glutinosa, N. rustica, Petunia hybrida and Vicia faba. In cytopathology, infected cells showed absence of specific inclusion body (ies), presence of virus particles in large numbers along the outer envelope of chloroplast with abnormalities like electron dense matrix, loss of chloroplast envelope and extensive invagination of cytoplasm. In immune electron microscopy there was heavy decoration and trapping of virus particle with the antiserum specific to CBV. The particle length was ca. 680 x 12 nm. The causal virus was found to be Chrysanthemum B carlavirus on the basis of host range, DAS-ELISA, immune electron microscopy and cytopathology (Verma et al., 2003). Screening of 17 cultivars of Asiatic and Oriental Hybrid lilies and Lilium longiflorum was done by ELISA. Out of 17, fourteen cultivars of A.H. and O.H. lilies and L. longiflorum were found to be infected with Lily symptomless virus (LSV). It was found that ELISA could detect the virus in leaves at flower emergence stage in comparison to bulb scales. Healthy lily plants were used as propagation host for LSV. Myzus persicae transmitted this virus non-persistently. Molecular weight of coat protein was found to be 31 KD. In EM, particles of 640nm were observed which showed clumping with LSV specific antisera in immunosorbent electron microscopy and in ELISA. Cytopathological studies showed cytoplasmic invasion in chloroplast and cluster of viruses along nucleolar membrane. Antiserum produced against the virus has dilution of 1:32 in double diffusion test. 900bp fragment was obtained in RT-PCR with LSV specific primers. Lily symptomless virus CP gene was sequenced and submitted to EMBL Database with Acc. No. AJ585052. LSV isolates infecting L. longiflorum, L. tigrinum, Asiatic and Oriental Hybrid (Acc. No. AJ748277) lilies have been characterized at the level of coat protein sequence. The Indian isolates among them show 78-96 % homology. With other LSV isolates (from the world) the Indian isolates show 83-98% homology. LSV-L (L. longiflorum), (Acc. No. AJ748320) and LSV-A (Asiatic hybrid), (Acc. No. AJ585052) isolates have unique stretches in the middle portion of the protein as compared to other LSV isolates and even Indian isolates. The isolate infecting tiger lily (Acc. No. AJ781318) has been found to be different from the isolates that have been characterized from the world. It shows 78-84% homology at the protein level. At the same time LSV-T (L. tigrinum) shows lot of variability in the C-terminal of the protein. A stretch of 41 amino acids in the C-terminal is unique to this isolate. LSV-T has been proposed to be a distinct isolate of LSV infecting L. tigrinum indigenous to India (Singh et al., 2005). Luteovirus Yellow leaf syndrome (YLS) has been recently recognized as new disease of sugarcane (Comstock et al., 1994). Symptoms consist of yellowing of leaves with a diagnostic deep or bright yellow midrib, often when the rest of the lamina is still green. In India, occurrence of sugarcane yellow leaf virus (SCYLV) was reported in five states (Uttar Pradesh, Bihar, Uttaranchal and Haryana and Tamil Nadu) on the basis of symptomatology, serology and particle morphology (Rao et al., 2000, 2001). Electron microscopy of the virus infected samples in leaf and stalk juice revealed the presence of icosahedral particles of 25 nm in diameter. The virus was further characterized by ELISA (Gaur et al., 2003) and by RT- PCR (Gaur, 2003).

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Immuno based PCR was performed using both healthy and infected leaf samples using Harper et al. (1999) methods. The primers 5’-CGXATCGYTAZZATNNG-3’and GCTATGGGCAGATGCCC-3’ (X=G; Y=T; Z=C and N=any nucleotide, Gibco BRL) were used to prime the amplification. The genome sense universal primer 5’- CGXATCGYTAZZATNNG- 3’ was derived from the beginning of the first bases of the coding region. The genome antisense primer, 5’-GCTATGGCAGATGCCC-3’represented the last 17 bases of coding region of the coat protein gene. Amplification was performed in an automated thermal cycle performed for 1 cycle of 37º C for 30 min and 94ºC for 5 min.; 30 cycles of 94ºC for 1 min, 58ºC for 1 min. and 72ºC for 1 min. followed by 72ºC for 5 min. for each tubes. In IC RT-PCR, a single band of expected size (ca. 352 bp) corresponding to coat protein was observed. The identity of ca 352 bp product was confirmed by cloning and sequencing. The complete genome of sugarcane yellow leaf luteovirus-Indian isolate was 5899 nucleotides long (Gaur, 2003). However, the coat protein gene of SCYLV-India isolate was 590 nt long (3648-4238). The coat protein gene could potentially code for a protein of 196, long amino acid. There were no detectable similarities between 5’ and 3’ untranslated region (UTR) and any sequences in the database. The 5’ UTR starts with the sequence TATA, which is consistent with the 5’ terminal motif of many Luteoviruses. The 3’ UTR is 220, nucleotide long. The genome of SCYLV-India was compared with corresponding genomes from known SCYLV isolates at the nucleotide and amino acid sequence levels. Comparative alignment study showed that there is 100% homology between Indian isolate of SCYLV with Texas and Australian isolates. However, there were changes only in 5 amino acids compared to the sequence from Texas, but no such differences were found when compared to an isolate from Australia (CP cv. 65-357). Unrooted phylogenetic tree analysis of different SCYLV revealed that among the different isolates used for comparison, SCYLV-India was most closely related to CP92-1654, Florid 1999, LHo83-153, SP71-6163B, Q136 Argentina and CP65-357AUS isolates forming one cluster. Comparative sequence analysis showed that SCYLV India shared 100% sequence identity with SCYLV Texas, Florida and CP 92-1654 of Australia at nucleotide (97-100%) as well as amino acid (92-100%) levels. In contrast 46% nucleotide sequence identity was observed with coat protein genes of other isolates used in comparative study.

The 5899 nucleotide long single-stranded RNA genome of sugarcane yellow leaf virus India isolate (SCYLV-IND) includes six major ORFs. SCYLV-IND ORFs 1 and 2 are most closely related to their Polerovirus counterparts, whereas SCYLV-IND ORFs 3 and 4 most closely related to counterparts in Luteovirus genome, and SCYLV-IND ORF 5 is most closely related to the read through protein gene of the only known Enamovirus. These differences in affinity result from inter-species recombination. The phylogeny of the sequence also suggests that Indian isolate of SCYLV has more close affinity CP92-1654, Florid 1999, LHo83-153, SP71-6163B, Q136 Argentina and CP65-357AUS rather than CC85-964, CC84-75 and SP71-6163C. Based on the sequence data presented here SCYLV-India should be included in the Luteovirus genus. The molecular characterization of the SCYLV revealed that the RNA is linear, unipartite having 5899 nucleotide with 196 amino acid capsid protein. The sequence data and ORFs presented here are consistent with previously reported findings that include SCYLV, the causative agent of the yellow leaf disease in India as a possible member of the family Luteoviridae

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(Smith et al., 2000). The comparison of the coat protein sequence revealed that SCYLV is also closely related to the viruses in the genus Luteovirus. This is the first confirmation of occurrence of SCYLV on sugarcane in India on the basis of molecular characterization. Earlier occurrence of this virus was reported in Tamil Nadu, Haryana, Maharasthra and Uttar Pradesh on the basis of symptomatology, particle morphology and serology (Rao et al., 2000, 2001; Singh, 2001). Studies on more isolates of SCYLV from different geographical regions of India would be further necessary to assess biological and molecular diversity among SCYLV strains in India and to ascertain their taxonomic status. Potato leaf roll virus (PLRV) disease is a serious problem in potato in India and other parts of world. Symptoms are reddening of top leaves that become erect and upward rolling of leaves from margins. Plants grown from infected tubers are stunted with upward rolling of lower leaves that lead to degeneration of tubers (Garg, 1987). Virus particles are confined to phloem and concentration is low that create problem in purification of PLRV with higher/appreciable yield. Diagnostic hosts are Physalis floridana and Datura metel that produce systemic symptoms. The virus is persistently transmitted by aphids Myzus persicae, which is most efficient vector. Detection and diagnosis of PLRV was earlier dependent on visual symptoms on leaves and cut potato tubers. Dhawan and Rishi (1990) for the first time in India successfully purified the virus and raised high titer antiserum, which was used in ELISA test. Pecluvirus During 1998-2001, symptoms resembling those of red-leaf mottle disease caused by peanut clump virus was observed on sugarcane cv. CoSe 93232, CoS 767, 96-137, Co 97017 and Co 9916 in Uttar Pradesh and Haryana. Affected plants showed various degrees of mottling (mild to severe), which later turned into red streaks of varying intensities. CoSe 93232 showed white streaks or bands parallel to mid vein, which was converted to red streaks. The symptoms were not associated with marked growth disorders but slight stunting in many diseased plants was observed. Chlorotic lesions were observed on Chenopodium amaranthicolor Coste et Reyn by inoculated infected sugarcane leaf extracts. Leaf dip preparations under electron microscopy revealed the presence of rigid rods averaging 250 x 20 nm in extracts of mechanically inoculated plants of C. amaranthicolor (chlorotic lesions) and Saccharum hybrids (mottling). Sugarcane leaf extracts were highly reactive in DAC-ELISA and western blotting test against polyclonal antiserum of peanut clump virus prepared from sugarcane isolate at CIRAD-CA, Montpellier, France. The virus isolate was identified as peanut clump virus –sugarcane isolate, the causal agent of red leaf mottle disease and is also the first record of PCV infecting sugarcane in India (Rao et al., 2002). In the seventh report of ICTV peanut clump virus is placed as a member of genus Pecluvirus and as yet not assigned to any family (Regenmortel et al., 2000). Mandarivirus Citrus ring spot virus disease was first described in India as a strain of psorosis-A disease (Ahlawat, 1989). It was later identified as citrus ringspot virus (Pant et al., 1997, Shelly et al., 1999). ICTV has recognized citrus ringspot virus as a new and only species Indian

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citrus ringspot virus (ICRSV) of a new genus Mandarivirus (Adams et al., 2004) under new family Flexiviridae. This family is named as it includes flexous virions and includes existing genera viz. Allexivirus, Capillovirus, Carlavirus, Foveavirus, Trichovirus, Vitivirus and the new genus Mandarivirus. ICRSV particles are flexous measuring 650X13 nm. The genome is ssRNA of 7.6 kb with six ORFs. The natural host is only citrus where it propagates through grafting. Mechanically it is transmissible to Chenopodium spp. and a few leguminous plants. For further details on this and other virus and virus like problems on citrus, readers may see a recent review (Ahlawat and Pant, 2003). Nepovirus Mosaic virus on roses when transferred on N. megalosiphon which is the specific host of Strawberry latent ringspot virus (SLRSV) of family Comoviridae showed symptoms. Presence of SLRSV in N. megalosiphon was confirmed by RT-PCR. 181bp portion of CP gene of SLRSV was amplified by using virus specific primers and the amplified fragment was cloned in pGEM-Teasy vector system for further confirmation. The virus isolate was identified as SLRSV (Kulshrestha et al., 2004). Rice tungro virus disease (mixed infection of Machlovirus + Badnavirus) The characteristic symptoms of rice tungro virus disease (RTD) are stunting, yellow- orange discoloration of leaves and reduced tillering. Earlier only spherical virus particles were found associated with RTD. Later reports confirmed association of both bacilliform and spherical particles with RTD (Saito et al., 1976, Jones et al., 1991). The bacilliform spherical particles (30-35 nm diameter and 110-400 nm in length) were identified as Rice tungro bacilliform virus of genus Badnavirus of family Caulimoviridae containing dsDNA while spherical particles measuring 30 nm in diameter were identified as Rice tungro spherical virus of genus Machlovirus of family Sequiviridae (Hull, 1996). Rice tungro bacilliform virus is dependent on Rice tungro spherical virus for its transmission by leafhopper Nephotettix virescens. First report on the complete sequencing and analysis of Rice tungro bacilliform virus (RTBV) DNA from two Indian isolates was made by the virus group working at the Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi. This work showed very clearly that the virus, in India, exists as a different strain from the previously known strains from Southeast Asia. Subsequently, investigations were carried out on the diversity of this virus in India and it was found consisting of many different molecular species at the field level (Joshi and Dasgupta, 2001, Nath et al., 2002, Joshi et al., 2003). The promoter region of a RTBV clone, obtained from field locations in West Bengal was then intensively investigated by functional dissection, which has important implications for transgenic gene expression in rice. This work has shown the existence of positive and negative regulatory elements in the viral promoter, inclusion or deletion of which gives rise to stage-specific and tissue-specific transgene expression in diverse plants such as rice, tobacco and even bacteria. To protect any intellectual property associated with this work, to the University, a provisional application for an Indian Patent

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has been filed at the Patent Office, New Delhi, in July 2004 (Dasgupta, personal communication, 2005). For a detailed account on RTD readers may consult a recent review paper by Muralidharan et al., (2004). Virus like particles (unidentified viruses) inducing important diseases Urdbean leaf crinkle virus: Urdbean leaf crinkle virus (UBLCV) disease was first seen at Pantnagar on Vigna mungo. There after this disease has been observed in all the urdbean and mungbean growing areas in India. Important symptoms are leaf crinkling, puckering and distortion. The natural hosts are V. radiata, V. unguiculata, V. aconitifolia and Cajanus cajan. UBLCV is sap transmissible and in nature by Coleopteran insect vector Henosepilachna dodecastigma. Seed transmission up to 18% has been reported. The virus particles are non-enveloped isometric measuring 25-30 nm in diameter. In ultrathin sections, particles were found in cytoplasm, nucleus and chloroplasts. There was hypertrophy of infected cells, mitochondria became filiform and no inclusion bodies were seen. Serological relationship is unknown. Sharma et al., (2000) studied implication of initial UBLCV contamination level in urdbean seeds on the epidemiology of leaf crinkle disease. For further details please see Nene (1972) and Albrechtsen and Rishi (1999). Pigeonpea sterility mosaic virus disease: Sterility mosaic disease (SMD), first described in 1931 from Pusa, Bihar State, India (Mitra, 1931), is restricted to pigeonpea growing countries in Asia. SMD is the major constraint on pigeonpea production in India. The disease is sometimes referred to as the “Green Plague” because at flowering time affected plants are green with excessive vegetative growth and have no flowers or seedpods. Under congenial condition, SMD spreads rapidly like a plague leading to severe epidemics. SMD infection at an early stage results in a 95-100% loss in yield, whilst losses from late infection (>45 day-old plants) depend on the level of infection and range from 25 to 95%. Seeds from partially affected plants are discolored and shriveled with about 20% reduction in dry weight (Jones et al., 2004). SMD causes greater yield losses than any other disease affecting pigeonpea in India: in 1984, losses due to SMD were estimated at 205,000 tons of grain valued at US$76 million and, in India and Nepal in 1993, losses were US$280 million. More recent studies on the economic impact of SMD are lacking, but the losses are expected to exceed US$300 million. The causal agent of such an important disease was not known despite several attempts over several decades. Graft transmission experiments showed that it was an infectious agent, transmitted under natural conditions by the eriophyid mite, Aceria cajani Channabasavanna (Acari: Arthropoda) (Nene, 1995). The extensive studies for the SMD causal agent have ruled out the involvement of fungus, a bacterium or a phytoplasma-like agent. The invariable association of vector mites with diseased plants led to a speculation that SMD may be the result of mite toxemia. However, this was excluded by critical experiments using SMD agent-free mite colonies on SMD-susceptible pigeonpea cultivars. Based on symptoms and transmission by mites, the SMD agent was assumed to

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be a virus. However, efforts to isolate a putative virus or virus-like agent using various techniques were not successful (Nene, 1995). A new purification procedure was developed which resulted in isolation of aggregates of highly flexuous, apparently irregularly branched, filamentous virus-like particles (VLPs) of 8 to 11 nm diameter and of undetermined length, resembling particles of tenuiviruses (Kumar et al., 2003). Comparable preparations from healthy pigeonpea leaves were free from such particles. The purified virus preparations contained a major protein of 32 kDa and up to 6 segmented RNA species of size 6.8-1.1 kb. Such particles were isolated consistently from all SMD-affected plant samples collected from different locations of peninsular India and from SMD-affected pigeonpea samples infected by graft inoculation, and by infective mites (A. cajani). Because of this very close association, the virus was named, Pigeonpea sterility mosaic virus (PPSMV) and was the first evidence of a causal agent for SMD (Kumar et al., 2003). Purified PPSMV VLP preparations were not infective to plants, but virus was transmitted experimentally, but with difficulty, by mechanical inoculation of fresh leaf sap extracts of SMD-affected pigeonpea to Nicotiana benthamiana and N. clevelandii, but not to pigeonpea. However, it was not possible to transmit the agent from infected Nicotiana species to pigeonpea by mechanical inoculation of sap (Kumar et al., 2002a). Polyclonal antibodies to PPSMV VLP preparations were produced which were very effective in detecting PPSMV in plant tissues by the double antibody sandwich-ELISA (Kumar et al., 2003). ELISA test detected PPSMV in all SMD-affected pigeonpea plants infected either experimentally by A. cajani, or by grafting, or naturally in the field at several different locations in India and Nepal, and in infected accessions of wild pigeonpea. Leaves from hundreds of healthy or uninoculated pigeonpea plants were negative in ELISA. This demonstrated the complete and specific association of PPSMV with SMD, and provides very strong evidence that PPSMV is the causal agent of the disease, ending the search for one of the most elusive plant pathogens. Unequivocal evidence that PPSMV is the causal agent depends on fulfilling Koch’s postulates but several technical difficulties prevent this, including the unstable nature of the virus and the difficulty of infecting pigeonpea by mechanical inoculation. The properties of PPSMV indicate that it is a previously undescribed virus with an unusual combination of properties. In the size and appearance of its VLPs and the number and sizes of its protein and RNA components, it is similar to viruses in the genus Tenuivirus. However, all tenuiviruses are phloem limited, transmitted by Delphacid plant-hoppers and infect plant species in the Poeaceae. Ultrastructural studies of PPSMV-infected pigeonpea and N. benthamiana plants identified 100-150 nm quasi-spherical membrane bound-bodies (MBBs) and fibrous inclusions (FIs) (Kumar et al., 2002b). The MBBs were labeled in situ specifically with antiserum to PPSMV, indicating that they contain the PPSMV-specific 32 kDa antigen. The FIs found in PPSMV-infected cells are possibly a non-structural inclusion protein of PPSMV (Kumar et al., 2002b). PPSMV also resembles tospoviruses that share many properties with tenuiviruses. Thus, the filamentous VLPs of PPSMV resemble the nucleoprotein particles of Tomato spotted

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wilt virus (TSWV) and the MBBs of PPSMV are similar to, though larger than, those of TSWV. Despite these similarities, serological tests failed to detect any relationship of PPSMV to Maize stripe virus and Peanut bud necrosis virus a tenuivirus and tospovirus respectively that are endemic in the Indian subcontinent. Furthermore, whereas tospoviruses, tenuiviruses and several other membrane-associated plant viruses are transmitted in a persistent and propagative manner by their invertebrate vector species, an eriophyid mite transmits PPSMV in a semi-persistent manner. Moreover, the nucleotide sequence of c. 2 kb of PPSMV-RNA and the monoisotopic masses of the 32 kDa nucleoprotein, show no similarity with these viruses, or with any other organisms in databases. The VLPs of PPSMV show some morphological similarity to species in the genus Ophiovirus, but members of this genus differ from PPSMV in the number and sizes of their protein and RNA components and there is no serological relationship detected between PPSMV and three members of this genus. PPSMV shows most similarity with High Plains virus (HPV) as eriophyid mites transmit each virus, has 4-7 RNA species, a virus-specific 32 kD protein, MBBs of similar size and morphology, and is mechanically transmitted with difficulty in sap extracts but not in purified preparations. However, no serological relationship was detected between these two viruses. MBBs similar to those detected in PPSMV- and HPV-infected plants are also detected in plants affected with other eriophyid mite-transmitted agents that cause fig mosaic, wheat spot mosaic, thistle mosaic and rose rosette diseases. These agents, together with PPSMV and HPV, probably represent species in a new genus of plant viruses (Kumar et al., 2003; Jones et al., 2004).

These very recent advances in understanding of the SMD etiology, detection and transmission of PPSMV and of resistance to it in wild Cajanus species, has been a major step towards sustainable management of SMD. PPSMV occur as various geographic isolates. Three isolates occurring in peninsular India [From Patencheru (Andhra Pradesh), Bangalore (Karnataka) and Coimbatore (Tamil Nadu)] were characterized. All these viruses have distint bio-chemical properties and differ in their virulence. It is likely that several PPSMV isolates occur in India with divergent properties. Characterization of these isolates is essential for precise selection of resistant varieties for effective disease management (Jones et al., 2004).

This work program, involving partnerships between ICRISAT, Scottish Crop Research Institute (UK) and national centers in India to address strategic and applied research, coupled with technology development and transfer, has demonstrated the ‘power of partnership’ in comprehending an what was believed to be an incomprehensible problem of the 20th Century. Virus Structures and Capsid Assembly Proteins play vital role as catalyst and building blocks of macromolecular assemblies in living systems. Identical protein subunits organize in highly symmetrical way for certain key cellular functions and structure. The most complex structures are those of isometric viruses. Advances in experimental X-ray crystallography coupled with computer technology have led to understanding of three-dimensional structure of a number of isometric viruses. Capsid/protein coat encapsidate and protect the genome (nucleic acid) of viruses. Harrison et al., (1978) first reported such studies on three-dimensional

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structure of tomato bushy stunt virus followed by a number of other viruses at different centers (Murthy and Savithri, 1998). These studies generate information on the diversity in viral architecture and viral evolution. In India Prof. M. R. N. Murthy of Molecular Biophysics Unit, Indian Institute of Science, Bangalore has developed a strong group on such studies. At this center extensive work has been done on assembly and structure of physalis mottle tymovirus (Munshi et al., 1987, Kekuda et al., 1993, Sastri et al., 1999, Umashankar et al., 2003), and sesbania mosaic sobemovirus (Subramanya et al., 1993, Lokesh et al., 2002, Sangita et al., 2004). Satellite RNA The ssRNA positive sense genome of CMV consists of three RNA species and a fourth subgenomic RNA that acts as messenger RNA for CP of 24 kDa. In addition to these RNA species some CMV isolates support replication and encapsidation of 330-391 nt ssRNA species, which was designated as satellite RNA (sat-RNA). Kaper and Waterworth (1977) were the first to demonstrate the symptom modulating property of satRNA while studying tomato necrosis disease caused by CMV and the associated satRNA identified as CARNA 5. In India Raj et al. (2000) demonstrated systemic stem necrosis and death of N. benthamiana, severe form of leaf deformation in N. tabacum cv. White Burley and blisters on N. tabacum cv. Samsun NN when CMV RNA+satRNA were used for inoculations. Inoculations of CMV RNA alone did not induce severe symptoms. Amongst Indian isolates CMV-C, CMV-T and CMV-A showed presence of satRNA. For separation of RNA species nucleic acid was extracted from purified virus particles by disrupting them using sodium dodecyl sulphate (10 g/l) and equal volume of phenol/chloroform followed by ethanol precipation and resuspension in diethyl pyrocarbonate. Electrophoresis of extracted nucleic acid on agarose gel 15 g/l showed bands of RNA1, RNA2, RNA3, RNA4 and RNA5 (sat-RNA). RNA1 and RNA2 were very close and did not separate as distinct bands. The satRNA was of 350 bp. Satellite RNAs depend on their helper viruses for replication and encapsidation. Readers may consult review paper by Kaper (1994) on exploitation of satRNA in biological control of viral diseases. Viroid Viroid induced diseases were recorded in the country much before their etiology got established. The first reports of viroid association came with diseases like potato spindle tuber and exocortis of citrus from the United States of America. (Diener, 1971; Semancik and Weathers, 1972). Although Saraswati and Mishra (1989) were the first in India to report the association of a viroid with tomato bunchy top disease, that was reported to be a virus disease by Pandey and Summanwar (1982). Later this viroid isolate was sequenced and characterized as a distinct strain of citrus exocortis viroid (Mishra et al., 1991). Similarly, Patil and Warke (1968) first reported the exocortis-like disease on Mosambi sweet orange (Citrus sinensis) trees on Rangpur lime (C. limonia) rootstock from Maharashtra. The symptoms observed were scaling and splitting of bark starting from bud union and stunting of diseased trees. More reports came from other parts of the country viz. Delhi, (Nariani et al, 1968), Punjab (Kapur et al, 1974, Cheema et al., 1984). However, the viroid nature of the disease with these symptoms became established with the report of Ramachandran et al., (1993). Later, extensive surveys undertaken in citrus

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orchards throughout the country have now established that exocortis is widely prevalent in the states of Maharashtra, Punjab, Karnataka, Andhra Pradesh and Delhi (Rustem et al., 1997). In a recent survey of citrus orchards in certain parts of Central India, symptoms of bark scaling, bark splitting and leaf yellowing were observed on Nagpur mandarin (C. reticulata) (7 plants) and mosambi (C. sinensis) (10 plants) grafted on rough lemon and Rangpur lime rootstocks. Molecular analysis of the samples revealed the presence of Hop stunt viroid (HSVd), suggesting that HSVd is also a component in producing bark-scaling symptoms in rootstocks, which were previously considered to be due to CEVd-infection alone. This isolate of HSVd has also been sequenced and the isolate designated as HSVd-RL (Ramachandran et al., 2005). Yellow corky vein disease of sweet orange (Citrus sinensis cv. Sathgudi) was reported by Reddy et al., (1974). Association of at least two viroid components viz., Citrus exocortis viroid and Hop stunt viroid with this disease was reported by Rustem et al., (2000). The viroid group at the Advanced Center for Plant Virology, IARI, New Delhi has recently reported characterization of one of the components associated with the disease and shown it to a Hop stunt viroid variant and named it Hop Stunt viroid -yellow corky vein variant (HSVd-ycv) (Roy and Ramachandran, 2003). Viroids were detected in asymptomatic plants of Coleus and seed lots collected in Delhi and that obtained from Indo-American Hybrids, Bangalore. This viroid appeared to be different from potato spindle tuber viroid (PSTVd) that was used as reference (Ramachandran et al. 1992). Singh et al., (1978) reported chlorotic mottle of Chrysanthemum morifolium (Ram) Hemsl. without confirming its etiology but only recently it was shown to be caused by Chrysanthemum stunt viroid (Mathur et al., 2002). Chrysanthemum plants in experimental fields of IARI, New Delhi and in the local commercial nurseries were examined, where 50% of the plants showed stunting, mild chlorosis of young leaves, delayed blooming and high percentage of sap and seed transmission. Presence of low molecular weight RNA similar in electrophoretic mobility to Potato spindle tuber viroid (PSTVd) was observed in symptomatic plants Stability of this RNA to high temperature, sensitivity to RNase and insensitivity to DNase further confirmed its viroid nature. This led the authors to tentatively designate this viroid to be Chrysanthemum stunt viroid (Mathur et al., 2002) of family Pospiviroidae. The technique of R-PAGE (Return Poly Acrylamide Gel Electrophoresis) has been standardized as a potent tool for viroid detection in India by Ramachandran et al. (1991). Using this tool association of viroid-like RNA has been observed in symptomatic palnts of tomato, tobacco, citrus and Vinca and in asymptomatic plants of coleus and grapes. Natural occurrence of a viroid in apple trees from Himachal Pradesh was also reported using electrophoretic technique (Thakur et al. 1995). The priced apple cv. Starking (Royal) Delicious showed dapple symptoms on the fruits i.e. blemished fruits at maturity. Symptoms appear in mid-July as small circular spots, which are prominent on immature fruits. The spotted area surface is somewhat flattened. In cvs. Golden Delicious and Yellow Newton, pigmented areas appear on fruits that turn brown, russetted and scarred with several tiny fissures and some of them turn into deep craks. Such fruits become unsaleable in the market and incur heavy loss. Results of two-dimensional PAGE clearly showed a band of RNA at the same position as that of ASSVd-Japan isolate. It was therefore concluded that the isolate under study is viroid and a strain of ASSVd. Dapple

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apple viroid is closely related to ASSVd. These are reported to be seed borne therefore; an infected tree is a potential source of inoculum. Today, diagnostics for quick detection of viroids and molecular probes are available for citrus (Ramachandran et al, 2003), which can be easily applied to other cases and viroid detection, made feasible for researchers, orchardists and quarantine laboratories. Phytoplasma diseases Coconut root (wilt) disease: Coconut root (wilt) disease is known to occur in south Kerala since 1882 (Butler, 1908, Varghese, 1934) with the great floods that occurred in the area (Menon and Pandalai, 1958). The first authentic report of the disease is from Kottayam district of Kerala (Butler, 1908, Pillai, 1911). Originally this disease was known as coconut root disease but considering the associated foliar symptoms Nagaraj and Menon (1955) thought that it should be more appropriately known as ‘wilt’. Subsequently the disease was known as coconut root (wilt) disease. Initial symptoms of the disease are flaccidity and characteristic bending or ribbing along the entire length of leaflets of the central and outer whorls. The bending of the leaflets may be due to impaired stomatal regulation resulting into excessive water loss. Subsequent symptoms are wilting and drooping of the leaves, paling/yellowing and necrosis of older leaflets of outer whorl. Inflorescence necrosis, lesser number of female flowers and pollen sterility render the diseased trees unproductive. Many consider rotting of roots as one of the symptoms of the disease. The root decay varied from 12-94.4% depending on intensity of the disease. Rotting of roots, and rootlets start from tips backward. The proportion of smaller roots rotting was much higher. Yield depression varied from 43-80% depending on the stage of the disease. Since the yield decline is gradual Swaminathan (1983) called this malady as ‘coconut decline’. Other symptoms are shedding of nuts, poor quality copra, thinner and lesser firm husk, shell does not harden properly and turn black and kernel is of uneven thickness, does not dry normally and remain flexible. Earlier from time-to-time fungi, bacteria, nematodes and viruses were considered as the causal agent of this disease (Nampoothri and Koshy, 1998). Solomon et al., (1983) reported association of mycoplasma like organisms MLOs) later termed as phytoplasma. They observed MLOs in the sieve tube cells of roots, tender stem, petiole and root. Dine’s stain and DAPI that bind with the nucleic acid of phytoplasma and show as fluorescence staining under light microscope is used as the diagnostic tool to evaluate phytoplasma infection (Nienhaus et al., 1982). Agar gel diffusion test (Solomon et al., 1983a) and indirect ELISA using the enzyme Horse radish peroxidase and the substrate tetra methyl benzidine gave very satisfactory results in detection and diagnosis of coconut root (wilt) disease (Sasikala et al., 1998). In field this disease is transmitted by lace bug Stephanitis typical (Distant) (Shanta et al., 1964, Mathen, et al., 1990). This has been confirmed using Dine’s stain and DAPI. Association of phytoplasma with this disease has been further confirmed by tetracycline sensitivity (Pillai et al., 1991). Butler (1908) and Pillai (1911) reported that coconut root disease affected about 24,000 hactares of coconut plantations in Kerala. The disease steadily increased and affected

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more than 30% coconut plantations in 7, 50,000 hactares in Kerala. The yield dipression ranged 43-80% depending on the stage of disease development. For further details readers may consult the book Coconut Root (wilt) Disease edited by Nampoothiri and Koshy (1998). Grassy shoot disease of sugarcane: This disease is endemic in the country since 1955. Rishi et al., (1973) first reported association of mycoplasma like bodies with this disease. The characteristic symptoms are excessive tillering with narrow leaves. In severe cases it gives crowded grass like appearance with almost total loss of cane formation. For further details two review chapters may be seen (Rishi and Chen, 1989, Rao and Dhumal, 2002) Little leaf disease of brinjal: Little leaf disease of brinjal (Solanum melongena) was first reported in 1939 by Thomas and Krishnaswami. The diseased plants show progressively smaller leaves with soft laminae, pale and glabrous, plants are stunted with poor flower and fruit setting. Varma et al., (1969) first reported association of mycoplasma like organisms with this disease. It is transmitted through Jassid Hishimonus phycitis. Further details on this disease may be seen in a chapter by Mitra (1988). X disease of peach: The initial symptoms of this disease appear in the first week of May i.e. 7-8 weeks after bud break in the form of leaf chlorosis with longitudinal upward rolling and occasional red spotting. These leaves later become pale to yellow red in color and there is early defoliation. However a tuft apparently healthy at the top of the diseased branch remains attached. In severe form of disease fruits are shriveled, small in size and prematurely drop. In Himachal Pradesh the disease incidence went up to 70%, the commercial cvs. infected are July Elberta and Sunhaven. The disease is transmissible to the healthy seedling by budwood grafting, T-budding and bark chipping. In February there was 60% transmission by budwood grafting but in June there was 100% transmissin through T-budding and bark chipping. This disease showing similar symptoms was earlier reported from Darjeeling hills in 1970s (Ahlawat and Chenulu, 1979). Based on symptomatology, transmissibility, presence of phytoplasma in the sieve tube elements of the infected leaves and symptom remission by oxytetracycline treatment it was concluded that the peach maldy in Himachal Pradesh is related to X disease of peach reported in Darjeeling and California, USA (Thakur et al., 1998). Though the disease was present in the northeastern Himalayas since 1970s but in Himachal it disseminated recently perhaps through infected planting material. Such high incidence (70%) also reached through grafting of infected bud/bark. Witches’ broom disease of acid lime: Acid lime (Citrus aurantifolia (L.) Swingle) is one of the most important citrus fruits in India, which covers 20% of the total citrus area in the country. Witches’ broom disease in acid lime was first observed in 1995 in Maharashtra and increased to 5% incidence in 1998 in the states of Maharashtra, Karnataka, Tamil Nadu and Andhra Pradesh. The characteristic symptoms are small chlorotic leaves, excessively proliferating shoots and shortened internodes. These lead to

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premature defoliation and distorted twigs and in advanced stage infected branches show die back symptoms. Not all the branches of the infected tree show witches’ broom symptoms and there was no flower and fruit formation. Phytoplasmas were seen under electron microscope in the sieve tube cells of phloem in the infected leaves. The disease was transmissible through dodder from infected citrus to periwinkle and back. The infected sieve elements showed yellowish green fluorescence when stained with a DNA binding flurochrome DAPI (4’, 6-diamidino-2-phenyllindole). No such fluorescence was observed in the sieve elements of healthy plants (Ghosh et al., 1999). Diagnostic tools of phytoplasmas: Recently molecular tools are available for the reliable detection and diagnosis of phytoplasmas (Davis et al., 1990, Kirkpatrick et al., 1994, Smart et al., 1996 and Lee et al., 1998, Seemuller et al., 1998). Siddiqui et al., (2001) applied molecular characterization methods based on sequence homology of 16S ribosomal DNA (rDNA) and 16S-23S intergenic spacer region (ISR) to conclude that little leaf disease of brinjal and little leaf disease of periwinkle (Catharanthus roseus) phytoplasmas in Bangladesh are closely related. Further these strains are identical to brinjal little leaf phytoplasma in India and belong to clover proliferation group (Lee et al., 1998, Seemuller et al., 1998). Virus fungus interaction Several microorganisms when present in community on plants may have antagonistic and/or synergistic interaction or may react independent of each other. Yarwood (1951) studied interaction of several viruses and uredinial stages of rusts of bean, sunflower, snap dragon and beet. He found that rust infected bean plants became more susceptible to some of plant viruses. The increased susceptibility to viruses could be due to quantitative and qualitative increase of amino acids in rusted bean leaves. In India mild mosaic infection on potato had synergistic effect on the natural susceptibility of Phytophthora infestans (Mukhopadhyay and Sen Gupta 1967). Similarly potato plants were more susceptible to Alternaria solani if preinfected with potato virus Y (PVY) (Nagaich and Prasad, 1970). Interaction between P. infestans and potato virus X (PVX) strain PVX-0 and PVY-3 were studied in potato cv. Kufri Chandramukhi (Kalra et al., 1989). It was routine observation during regular field visits that potato plants infected with mosaic disease showed lesser severity to late blight. This field observation was quantified experimentally. It was found that prior infection of PVX and PVY on potato reduced the susceptibility of these plants to P. infestans resulting into lesser severity of late blight symptoms. The result was best obtained when potato leaflets were inoculated with P. infestans 72 hrs after inoculating PVY. Lesions caused by fungus on the leaflets appeared 3 day later and were much smaller than on leaflets inoculated with alone. P. infestans infection was 25% lesser than on leaflets inoculated with the fungus alone. Virus titer was assayed to see the influence of P. infestans infection. Extract from second leaf from top was inoculated on local lesion host C. amaranticolor for PVY and Gomphrena globosa for PVX. It was observed that number of local lesions was lesser in the plants infected with virus and fungus as compared to leaves infected with virus alone. Inoculation of virus after P. infestans did not influence development of fungus symptoms. Similar studies were conducted on whole plants of potato where time period between virus and fungus inoculation ranged between 0-30 days. The appearance of P. infestans

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symptoms was delayed by 2 days when PVY was inoculated 20 or 30 days earlier. The numbers of leaves showing late blight symptoms were lesser. All the leaves of potato plants inoculated with the fungus alone collapsed but in the plants preinoculated with PVY 30 days prior, the numbers of collapsed leaves were much lesser. These findings open new avenue for search of milder or attenuated strains of PVY inducing resistance to P. infestans without themselves inducing any or negligible loss in yield. Possibility of using hyphal components of P. infestans coupled with milder or attenuated strains of virus should also be explored. Effect of extracts, lechates and washings from healthy and PVY infected leaves were studied on the disease severity of P. infestans (Kalra et al., 1990). Seedlings of potato cv. Kufri Chandramukhi were raised in earthen pots (20 cm ø) containing soil and farmyard manure (2:1). Extracts of PVY-3 infected and virus free leaves were prepared. For lechates leaf laminae were cut into 1 cm2 pieces and suspended in sterile distilled water (3g/50ml) and for washings leaves were immersed in sterile distilled water (3g/50ml) and flask placed on rotary shaker at 26 0 C and ~ 160 rpm for 8 h. Dilutions (1:1, 1:2, 1:3) of above suspensions were used with corresponding dilutions of healthy control. Equal volume of suspensions of sporangia (400/ml) and zoospores (1000/ml) were mixed with equal volume of suspensions of virus infected leaves. Extracts, lechates and washings from infected leaves inhibited the liberation and germination of zoospores. Late blight lesions formed on inoculated leaves were smaller and with lower mean disease severity values (Kalra et al., 1990). For more details on virus fungus interactions readers may see reviews covering various aspects published earlier (Rishi and Kalra, 1998, 2003, Kalra and Rishi 2000). Epidemiology Knowledge of intricacies of mode and course of dissemination of virus diseases is very important in developing effective management strategies. For this identification of the virus, information on sources of primary inoculum (contaminated seeds/vegetative propagules in vegetatively propagated crops, alternate hosts, collateral hosts, and volunteer plants), host susceptibility, efficacy of vector, virus vector relationship and correlation of weather parameters and vector population and spatial factors are the important aspects for understanding the epidemiology of a virus disease. Scanty informations on epidemiology of virus diseases in the country are available. In potato seed certification program avoidance of the period of aphid populations above the critical level opened new opportunities to take up seed certification in the plains of northwestern and northeastern India (Pushkernath, 1967). It was recommended that in these plains potato seed crop should be grown in the aphid free period of October-December and dehaulming should be done in the last week of December when aphid population crosses the critical level. In Jalandhar conditions when dehaulming was done on December 25, the virus incidence remained within the permissible limit of 1%, but if haulms were cut on January 5, the aphid population was three times higher than the critical level thereby enhancing the virus incidence exponentially (Verma and Vashisth, 1985). Trivedi et al., (1998) gave a model for predicting aphid population, which is helpful in deciding the date of dehaulming.

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Quite interesting information on epidemiology of rice tungro virus disease has been summarized by Muralidharan et al., (2003). This covers inter-species hybridization, the vector leafhoppers in nature and their role in sudden outbreak of tungro disease. It was experimentally proved using the blue and green types of Nephotettix virescens and two ecotypes of N. nigropictus that were allowed for mating and hybrids produced were tested for relative efficacy of tungro disease transmission. Tobacco leaf curl virus (TbLCV) disease is one of the most destructive diseases of tobacco in India (Pal and Tandon, 1937). Leaves of diseased plants develop vein thickening, curling, veinal depressions, enations and plants are stunted. TbLCV is transmitted by whitefly (WF) Bemisia tabaci (Pruthi and Samuel, 1939). Status of TbLCV disease in tobacco in five major tobacco growing states (Andhra Pradesh, Karnataka, Gujarat, Bihar and West Bengal), WF population, host range and management were studied (Valand and Muniyappa, 1992). Incidence of Disease varied between 5.4-77% that was highest in Andhra Pradesh and lowest in West Bengal. This was in proportion to the WF count/plant, which was 32, the highest in Andhra Pradesh and 5 the lowest in West Bengal. In an experiment in which, sequential sowing of tobacco done from February to June at Bangalore (Karnataka) all the plants contracted disease within 90 days. The number of WF count had a positive correlation with the final incidence of disease. TbLCV could be transmitted to 35 plant species using WF. The host range includes Beta vulgaris, Capsicum annuum, Carica papaya, Cymopsis tetragonoloba, Lycopersicon esculentum, Sesamum indicum, Phaseolus vulgaris and Petunia hybrida. TbLCv isolates (45) collected from different parts in India when transmitted produced four distinct types of symptoms on tobacco cvs. Samsun, and Anand 119. Conservation of virus free germplasm - National Bureau of Plant Genetic Resources (NBPGR), New Delhi National Bureau of Plant Genetic Resources (NBPGR) established in 1976, is the nodal agency for management of plant genetic resources including germplasm exchange and quarantine processing. In the beginning the plant quarantine processing involved detection of fungi, nematodes and insect pests and salvaging of infected material. The plant virology activity was initiated with the establishment of a Plant Virology Unit in the Division of Plant Quarantine in 1989 to ensure that the imported germplasm and research material especially legumes, are also screened for seed-transmitted viruses before release. Over the years the Plant Quarantine Division developed facilities through international and national donor agencies. Now it possesses a series of greenhouses at its headquarters at New Delhi and at its Regional Station at Hyderabad, which were built under an INDO-US-AID Project and recently established a Containment facility of Containment Level - 4 under a DBT-ICAR project for processing the imported transgenic germplasm. It has developed a well-equipped laboratory comprising all the facilities for serological and molecular detection of seed-transmitted viruses. The Regional Station of NBPGR located at Hyderabad established in 1985 undertakes quarantine processing for germplasm and other research material of ICRISAT and other institutes of South India, also has a well-equipped Virology Unit with highly trained man power. All groundnut imports into India are channeled through Regional Station. The testing groundnut germplasm includes a non-destructive seed ELISA procedure to obtain a part of cotyledonary tissue to detect

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the seed borne viruses and growing virus free seeds in the greenhouse for 4 weeks under observation and releasing healthy seedlings to grow in post entry quarantine isolation field. The harvest from healthy plants will be released to the indenter. For testing the imported genetic resources for viruses they are grown in the post-entry quarantine greenhouses and the seedlings showing viral symptoms are indexed by deploying a combination of techniques comprising infectivity test, electron microscopy and variants of ELISA. The harvests only from virus-free plants are released to the indenters. To meet these objectives following six projects are at hand and projects at 7 and 8 have been completed (Personal communication Dr. Ravi K. Khetarpal, Head, Division of Plant Quarantine, NBPGR, New Delhi).

1. Post-entry Quarantine Processing of Exotic Germplasm

2. Detection and Identification of Viruses in Quarantine and Supportive Research

3. Analysis of Risk of Viruses Associated with Exchange of Germplasm

4. Detection of Viruses in in vitro Cultures of Germplasm Meant for Conservation

5. Diagnostics of Emerging Plant Viruses

6. National Facility for Plant Quarantine and Transgenic Planting Material

7. NATP Mission Mode project on Diagnostics and Development of Seed Certification Protocols for Management of Seed-transmitted Viral Diseases of Grain Legumes (Financed by World Bank-aided National Agricultural Technology Project).

8. Investigation and Exploitation of Natural and Engineered Resistance to Pea seed borne mosaic virus in Pea (Financed by European Union, Collaborative Project of France, UK and Denmark).

Some of the important findings of these projects are enumerated below:

• Intercepted a number of destructive seed-transmitted viruses which are either not known to occur in India or are known to possess virulent strains. (Chalam et al., 2005a, 2005b; Khetarpal et al., 1992, 1994, 2001; Parakh et al., 1994, 2005a, 2005b, 2005c, 2005d). SMV though known in India on soybean was intercepted in imported transgenic soybean germplasm (Singh et al., 2003). The details are given in Table 4.

• Demonstrated the occurrence of Barley yellow dwarf virus isolate MAV on wheat and of Zucchini yellow mosaic virus on summer squash in India for the first time (Khetarpal et al., 1993a; Chalam et al., 2003).

• Identified sources of tolerance in garlic, to garlic mosaic disease (Khetarpal et al., 1991).

• Demonstrated the effect of PSbMV on seed yield and the higher seed transmission rate of the virus by smaller seeds of pea. Also demonstrated the hitherto

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unreported phenomenon of latency in seed transmission of PSbMV in pea (Khetarpal and Maury, 1990; Khetarpal et al., 1988, 1990).

• Produced antiserum to PSbMV, which detects specifically the seed-transmitted virus in pea seed embryos thus eliminating the tedious step of decortication of testas, the infection of which is not correlated to the seed transmission. This is of great importance for seed certification in routine (Khetarpal and Maury, 1987; Masmoudi et al., 1994).

• Developed and standardized the techniques of Immuno-capture Reverse Transcription PCR for detection of PSbMV in pea seeds and evaluated its efficacy with ELISA in-group testing of seeds (Phan et al., 1997).

• Isolated a virulent pathotype of PSbMV overcoming the cluster of recessive resistance genes in pea (Khetarpal et al., 1990, 1997a, 1997b). This discovery has led to the formulation of a European Union Collaborative Project entitled “Investigation and exploitation of natural and engineered resistance to Pea seed borne mosaic virus in pea”.

• Carried out in-depth studies on variability of PSbMV isolates, isolation of a virulent pathotype of the virus, molecular characterization of the virulence determinant and testing of transformed pea plants for resistance after the incorporation of both natural and engineered resistance (Khetarpal et al., 1993b).

• Blackgram mottle virus was successfully detected in seedlings of four varieties of urdbean. The extent of seed transmission ranged between 5 and 10% and virus was successfully transmitted to healthy urdbean seedlings by sap inoculation and the time of appearance of symptoms varied in varieties. The identity of the virus was further confirmed by immunosorbent electron microscopy (Dinesh Chand et al., 2004a).

• In the project on seed certification for management of seed-transmitted viral diseases of grain legumes with cooperating centers in Gujarat and Karnataka, extensive surveys carried out for three years in nine major legume-growing states of the country revealed varied incidence of BCMV and urdbean leaf crinkle disease of urdbean and mungbean, Black-eye cowpea mosaic virus (BlCMV) and CABMV of cowpea, SMV of soybean and PSbMV of pea depending on location and the crop variety. Based on the results of field surveys complemented by seed testing, a national map on prevalence of seed-transmitted viruses of grain legumes was prepared.

Epidemiological studies carried out in cases of BlCMV/ cowpea, CABMV/ cowpea, PSbMV/ pea and SMV/ soybean revealed a correlation in viral disease incidence with aphid vector population, and appreciable losses in seed yield in these cases were demonstrated. Based on virus spread using a known level of initial seed/ seedling infection, the seed standards for certification against both BlCMV of cowpea and SMV of soybean were fixed as 0.5%. Antisera to BlCMV and SMV were produced in bulk and immunodiagnostic kits were prepared. Group testing of embryos using ELISA was standardized for quality control of seeds. It is expected that the results would be optimally utilized at national level

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for seed certification of grain legumes (Chalam et al., 2002, 2004a; Dinesh Chand et al., 2002, 2004; Khetarpal et al., 2003a).

• Standardized Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Dot Immunobinding Assay techniques for detection of BCMV, Bean common mosaic necrosis virus, PSbMV and SMV in leaf and seed samples. Also standardized Real-time RT-PCR protocols to detect BCMV and PSbMV in leaf and seed samples (Chalam et al., 2004).

• Assessed the prevalence of seed-transmitted viruses in different crops in fields and in seed production centers of Mauritius and prepared a national map of distribution of the viruses under an FAO/ TCP project (Khetarpal, 2003).

• Undertook gap analysis on seed health certification and transboundary movement of seeds including that of viruses (Khetarpal, 2004; Khetarpal et al., 2004c; Maury et al., 1998; Maury and Khetarpal, 1997)

• Provides regular technical input on policy issues related to plant protection and plant quarantine with special reference to the Agreement on Sanitary and Phytosanitary Measures of the WTO to the government and to professional organizations and societies. Also represents the Bureau in all national apex bodies on policy matters related to WTO Agreements and biosafety concerns with respect to imported transgenic planting material and in reviewing the National Plant Quarantine System (Khetarpal and Gupta, 2002; Khetarpal et al., 2005).

• As part of human resource development/strengthening, imparted short-term trainings on seed-transmitted viruses related to epidemiology, detection and certification of seeds. Also imparted training to researchers/ students from Brazil, Fiji, France, Madagascar, Mauritius, Nepal and Vietnam on the use of serological and molecular virus detection techniques.

• Prepared a checklist of seed-transmitted viruses for quarantine purposes and developed a strategy for post-entry quarantine processing for exotic germplasm material for seed-transmitted viruses (Chalam et al., 2005c; Dev et al., 2005; Kumar et al., 1994).

NBPGR-Regional Station, Hyderabad • A total of 5378 groundnut germplasm accessions imported from 39 countries

were tested by a non-destructive seed ELISA test followed by grow-out test in greenhouse for the detection of seed-borne viruses of quarantine importance. Both peanut mottle virus (PeMoV) and peanut stripe virus (PStV) were detected in seeds from Philippines and USA, only PStV in seeds from Myanmar and China and only PeMoV in seed from Malawi and Uganda (Prasada Rao et al., 2004, Demski et al., 1993).

• First reprot of peanut stripe virus (PStV) occurrence in India: During monitoring surveys in 1987-raining season, PStV a seedborne virus of quarantine importance was observed on a multilocational groundnut varietal trail (IET-SB) of AICRPO at 6 out of 12 experimental stations surveyed. The virus was identified based on host range, transmission (sap, aphid and seed), physical properties, serological affinities, purification and particle morphology (Prasada Rao et al., 2004).

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• Containment of PStV: Peanut stripe, a seedborne virus of quarantine importance to India, inadvertently introduced into Junagadh and subsequently was shown up on experimental material of AICORPO grown at several stations was systematically eliminated from these stations by regular monitoring surveys and timely actions in collaboration with Directorate of Plant Protection, Quarantine and Storage, ICRISAT and NRCG (Prasada Rao et al., 1993, 1995).

• Identification of resistance to peanut stripe, peanut mottle and tomato spotted wilt viruses: Genotypes of section Arachis, Erectorides and Rhizomatosae were screened against PStV, PeMoV and TSWV, by inoculation with sap, and graft. The viruses did not infect twelve accessions, five in the section Arachis, one in section Erectordes and six in the section Rhizomatosae despite repeated sap inoculations. In addition, by single graft inoculation PI 262817, 421707 and 468363 were not infection with any of the three viruses, when a single plant of these 3 accessions was simultaneously graft inoculated with the viruses PI 262817 was resistant to all three viruses (Prasada Rao et al., 1993).

• Elimination of PStV from the virus infected groundnut seed: To salvage all imported groundnut seed infected with PStV, an in vitro method to eliminate PStV from groundnut seed was developed for use in quarantines. Growing PStV infected groundnut seed on Murashige and Skoog (MS) medium supplemented with 40 mg/L ribavirin for 16 weeks eliminated PStV from the growing plants in vitro. The procedure will help in releasing virus free plants in quarantine (Prasada Rao et al., 1995).

• Identification of tobacco streak virus as causal agent of sunflower necrosis disease: The sunflower necrosis disease, first recorded in parts of Karnataka during 1997, which has spread to Andhra Pradesh, Tamilnadu and Maharastra has been identified due to tobacco streak virus, disease of quarantine importance to India (Prasada Rao et al., 2000).

• Identification of tobacco streak virus as causal virus of peanut stem necrosis disease: Peanut stem necrosis disease occurred in an epidemic form in Anantapur district in Kharif 2000, affecting an area of 2.25lakh out of 7 lakh hectares grown. The disease has been identified due to tobacco streak virus (TSV), based on host range, transmission, physico-chemical properties, serological affinities and particle morphology (Reddy et al., 2002).

• Studies on the epidemiology of tobacco streak virus on groundnut under Anantapur conditions: The virus infects economically important crops such as soybean, mungbean, urdbean, cotton, okra and marigold in addition to sunflower and groundnut. The disease spreads by thrips in the presence of pollen from TSV infected plants. Thrip transmission occurs through wounding of leaf tissue as well as infected pollen and their proximity during thrips feeding, rather than a specific virus-vector interaction. Parthenium, a symptomless carrier of TSV growing in fallow lands, roadsides and on field bunds produces several flushes during its life cycle, thus ensuring continuous supply of pollen, plays an important role in the perpetuation and spread of the disease. Seed transmission tests conducted on groundnut, sunflower, soybean, urdbean, mungbean, marigold and Parthenium indicated that the virus is not seed transmitted in these crops. The favorable factors for PSND incidence under Anantapur conditions are: early rains either in

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late May or early June that encourages germination and growth of Parthenium, groundnut sowing in July by which time Parthenium is in full bloom, having normal rains during the crop season with one or two dry spells for the thrip movement and disease spread. The above congenial conditions occurred during kharif 2000 and as well in kharif 2004. The disease incidence ranges from 15-100% depending upon the proximity of Parthenium (Prasada Rao et al., 2003).

Interception of Exotic Plant Viruses - Department of Plant Protection and Quarantine Plant Quarantine plays a major role through legal regulations to facilitate safe movement of plants/seeds/plant materials in international trade. The Destructive Insects and Pests Act (DIP Act) was formulated in 1914 and various notifications issued under the DIP Act, 1914 to prevent the introduction and spread of exotic pests by way of regulating the import of plants and plant material. The import of agricultural commodities is currently regulated under the “Plant Quarantine Order 2003” issued under the DIP Act, 1914 and amendments issued there under. The import of Germplasm/Genetically modified plants/plant materials for research by Private or Public Institutes is regulated through the Director, National Bureau of Plant Genetic Resources (NBPGR), New Delhi. In the past, there are instances of inadvertent introduction and establishment of several exotic viruses into our country in spite of best efforts in implementing the Plant Quarantine Regulations (Table 4). To cite a few, banana bunchy top virus, soybean mosaic virus, sunflower necrosis virus and peanut stripe virus, are some of the important viruses, which got introduced into India.

Table 4. Seed-transmitted Viruses Intercepted in Germplasm Imported During 1989-2005

S. No.

Virus Intercepted

Crop Source of Import Technique Used

Glycine max AVRDC (Taiwan), IITA (Nigeria), Brazil, Myanmar, USA

Phaseolus vulgaris♣ CIAT (Colombia), Canada, Kenya, USA

Vigna radiata♣ Japan

1. Alfalfa mosaic virus (AMV)

V. unguiculata IITA (Nigeria)

Grow-out, DAS-ELISA

Hordeum vulgare ICARDA (Syria) 2. Barley stripe mosaic virus (BSMV)*

Triticum spp. USA Grow-out, Infectivity test, EM, DIBA

G. max♣ AVRDC (Taiwan), IITA (Nigeria), USA

P. vulgaris CIAT (Colombia), CIS, Hungary, Kenya, USA

V. radiata AVRDC (Taiwan), Japan, USA

3. Bean common mosaic virus (BCMV)

V. unguiculata IITA (Nigeria), Guyana

Grow-out, Infectivity test, EM, DAC-ELISA, DIBA

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4. Bean common mosaic necrosis virus (BCMNV)

P. vulgaris Kenya Grow-out, DAS-ELISA,

G. max IITA (Nigeria), Myanmar, USA P. vulgaris CIAT (Colombia)

5. Bean yellow mosaic virus (BYMV)

V. faba ICARDA (Syria), Bulgaria, Spain

Grow-out, EM, DAC-ELISA, DAS-ELISA

6. Broad bean stain virus (BBSV)*

Vicia faba ICARDA (Syria), Bulgaria Grow-out, EM, DAC-ELISA, DAS-ELISA

7. Cherry leaf roll virus (CLRV)*

P. vulgaris CIAT (Colombia) Grow-out, DAC-ELISA

G. max♣ AVRDC (Taiwan), IITA (Nigeria), Myanmar, USA

V. radiata♣ AVRDC (Taiwan)

8. Cowpea aphid-borne mosaic virus (CABMV)

V. unguiculata IITA (Nigeria), Eritrea, Guyana, The Philippines, USA

Grow-out, EM, DAC-ELISA

V. radiata♣ USA 9. Cowpea mosaic virus (CPMV) V. unguiculata IITA (Nigeria)

Grow-out, DAS-ELISA

10. Cowpea mottle virus (CPMoV)*

V. unguiculata The Philippines Grow-out, Double diffusion test

G. max AVRDC (Taiwan), IITA (Nigeria), Brazil, Myanmar, USA

P. vulgaris CIAT (Colombia)

11. Cucumber mosaic virus (CMV)

V. unguiculata IITA (Nigeria)

Grow-out, DAS-ELISA

Pisum sativum AVRDC (Taiwan), Australia, Bulgaria, Colombia, Eritrea, Germany, Holland, Nepal, Russia, Syria, USA

12. Pea seed-borne mosaic virus (PSbMV)

V. faba♣ ICARDA (Syria), Bulgaria, Spain

Grow-out, Infectivity test, EM, DAC-ELISA, DAS-ELISA, DIBA

G. max♣ IITA (Nigeria), USA 13. Southern bean mosaic virus (SBMV)

P. vulgaris♣ CIAT (Colombia) Grow-out, DAS-ELISA

G. max AVRDC (Taiwan), IITA (Nigeria), Australia, Brazil, Hungary, Thailand, USA

14. Soybean mosaic virus (SMV)#

P. vulgaris♣ CIAT (Colombia)

Grow-out, Infectivity test, EM, DAS-ELISA, DIBA

15. Tobacco ring spot virus (TRSV)

G. max IITA (Nigeria), Myanmar Grow-out, DAS-ELISA

16. Tomato black ring virus

P. vulgaris♣ CIAT (Colombia), Brazil, Canada, Kenya, USA

Grow-out, DAS-ELISA

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(TBRV) V. unguiculata♣ IITA (Nigeria) 17. Carlavirus

particles Lilium spp. Israel Grow-out, EM,

Infectivity test 18. Unidentified

flexuous Potyvirus

Helianthus annuus France, USA Grow-out, EM, Infectivity test

P. acutifolius USA V. angularis Germany V. catjang France

19. Unidentified filamentous virus

V. unguiculata France, USA

Grow-out, EM

20. Unidentified isometric virus

V. radiata AVRDC (Taiwan), Germany, Indonesia

Grow-out, EM, Infectivity test

* Virus not reported from India ♣ Viruses present in India but not recorded on the host on which intercepted # Virus intercepted also in transgenic soybean imported from USA DAC-ELISA = Direct Antigen Coating – indirect Enzyme-linked Immunosorbent Assay DAS-ELISA = Double Antibody Sandwich – Enzyme-linked Immunosorbent Assay DIBA = Dot Immunobinding Assay EM = Electron Microscopy The Government of India by way of “New Policy on Seed Development” in 1988 liberalized import of seed/ planting material for improving agriculture production and thereby improving agriculture economy of the country. To facilitate safe import, the import of seeds/plants of flowers and vegetables were permitted only through the ports of 5 major Stations viz. New Delhi, Mumbai, Chennai, Kolkata and Amritsar. The five major stations were strengthened under the FAO UNDP Project on ‘Strengthening of Plant Quarantine Facilities in India’. The Specialists in the fields of Plant Virology, Plant Bacteriology, and Plant Nematology were positioned for effective monitoring and screening of imported plants and plant materials. The major stations, especially New Delhi and Chennai are provided with Electron microscope, Ultra and high speed Centrifuge, Gel documentation system, ELISA detection kit and PCR thermal cyclers for molecular diagnosis of viruses. The technical officers/staff have been trained in molecular detection of viruses at Advanced Center for Virus Research, Division of Plant Pathology, Indian Agriculture Research Institute, New Delhi 110012. Major interceptions of plant viruses: Cymbidium mosaic (potex virus) on Dendrobium/ Aranda plants imported from Thailand, Singapore; Chilli Leaf crinkle virus in seeds imported from Germany; Pea seed-borne mosaic virus on Pea seeds imported from Australia; Leaf streak (poty virus) in Oilpalm seed sprouts imported from Costa Rica; Rose mosaic virus on rose plants imported from Holland; Soybean mosaic virus on soybean seeds imported from USA; Chicory yellow mosaic virus on Chicory seeds imported from Holland; Odontoglossum ring spot (tobamo virus) on Dendrobium plants

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imported form Thailand (Dr. O. R. Reddy, Jt. Director, Dept. Pl. Prot. & Quarantine, Personal Communication, 2005). Induced resistance Once a plant in field is infected with any virus, the general practice to prevent further dissemination is to uproot and burn. But this practice is not acceptable to the farmers if the virus disease incidence is high and thus they are tempted to use hazardous pesticides to control insect vectors. This practice is more common in vegetables that are short duration and high remunerative crops; thus 13% of the total pesticide consumption in India is in vegetables. Application of pesticides sometimes may be helpful in case of persistent viruses but are erroneously applied to control non-persistent viruses leading to environmental pollution and health hazards. Both these viruses can be efficiently managed through ecofriendly approaches if epidemiological studies are conducted to precisely understand their mode of perpetuation, alternate/collateral hosts including weeds, relationship of macro and micro climatic conditions, vector population and disease development. Some of the botanicals have given promising results in field trials. Change in agronomical practices, use of non-host barrier crops and mulches have also given encouraging results in virus disease management. Virus resistant transgenics have also been developed in a few cases. Allard (1918) and Duggar and Armstrong (1925) for the first time reported the presence of inhibitory substances in plants when they could not transmit sap transmissible mosaic virus infecting Phytolacca decandra L. (pokeweed) and tobacco mosaic virus. In India a very strong school has come up on applications of botanicals in management of virus diseases under the leadership of Prof. H. N. Verma at the University of Lucknow. Virus inhibitors are mostly present in leaves. They have also been reported in flowers, fruits, seeds, bark, roots and rhizomes. When virus inoculum is incubated with extracts of certain healthy plants and inoculated it shows decreased or totally suppressed symptoms as compared to full-blown symptoms in control. However, uniform consistency have not been found in the response of these inhibitors sometimes even in the same experiment. A number of factors such as plant ecology, climatic conditions, pH of extract, proteases and nucleases are responsible for such variability. Verma et al., (1995, 1995a) have compiled a list of 157 plants belonging to families Amranthaceae, Caryophyllaceae, Chenopodiaceae, Nyctaginaceae, Phytolaccaceae, Solanaceae and Verbenaceae showing antiviral properties; eleven plants where nature of inhibitors have been identified and nine plants, of which extracts/compound isolated from induced systemic resistance against virus infection. All the plants neither contain similar inhibitors nor their antiviral properties\mechanism is similar. For consistent results with the antiviral phytoproteins isolated from non-host healthy plants callus culture and organogenesis in Boerhaavia diffusa and micropropagation of Clerodendrum aculeatum through adventitious shoot were reported (Gupta et al., 2004, Srivastava et al., 2004). In B. diffusa callus induction was best in stem explants and these calli had the best shoot regeneration capability, and the best virus inhibitory property. The in vitro regenerated plants also showed virus inhibitory activity and were devoid of the effect of seasonal variation in the production of antiviral protein known as B. diffusa systemic resistance inducing protein (BD-SRIP). Similarly in micropropagated C. aculeatum the amount of resistance inducing protein was

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consistent in all the seasons. The C. aculeatum-systemic resistance inducing protein (CA-SRIP) had the molecular weight of 34kDA. Cloning and characterization of CA-SRIP encoding gene revealed 1218 nt with ORF of 906bp (Kumar et al., 1997). These findings may be very helpful in mass production of these antiviral phytoproteins and developing transgenics (Baulcombe, 1994). Antiviral factor (AVF) systemically present in virus-infected plant when added to virus inoculum decreases virus infectivity. In tobacco a single dominant N gene governs AVF activity. Some possible analogies have been discussed between AVF and interferon (Gianinazzi, 1982). Further, a substance inhibiting virus replication (IVR) was found in the TMV infected tobacco protoplast of N gene bearing plant. This IVR consisted of two biologically active components of 26,000 and 57,000 mol wt (Lobenstein and Gera, 1981). In susceptible host virus interfering agent (VIA) is present both at the virus inoculum treated site and non-treated site. Plant extract containing VIA when mixed with virus inoculum it greatly reduces virus infectivity. VIA is low mol wt protein that is neither host nor virus specific and its maximum titre is between 12-24 hrs after inoculation. It seems that AVF, IVR and VIA do not compete amongst them but are the general defense responses of plant virus interface. Gupta et al., (1974) reported the first antiviral agent, which acted systemically from a fungus Trichothecium roseum. Raychaudhuri and Prasad (1965) reported effect of microbial growth products in the infectivity of radish mosaic virus. Prospects of application of some of these inhibitors in field inducing systemic resistance and symptom reversal have been reported (Verma and Awasthi, 1979, Verma and Awasthi, 1980, Verma and Dwivedi, 1984, Awasthi et al., 1985, Prasad et al., 1995, Verma et al., 1996, Verma and Baranwal, 1999, Singh et al., 2004). For more details readers may consult some excellent reviews (Verma et al., 1995, 1995a, Verma et al., 1998, Verma and Baranwal, 1999, Baranwal and Verma, 2000). Non-host barrier crops, mulches, and insecticides and cultural practices Use of non-host barrier/trap crop appreciably reduced the incidence of leaf curl of chilli and enhanced chilli fruit yield (Dhawan and Rishi, 1999, Dhawan et al., 2002). Use of reflective aluminum polythene mulches reduced the incidence incidence of watermelon mosaic virus in cucurbits and increased yield in Australia in comparison to use of insecticides and oil sprays that failed to give positive results (McLean et al., 1982). Reflected UV light from shining surface seems to act as repellent to the landing vector. In India Khan and Mukhopadhyay (1985) using yellow polythene mulches delayed the appearance of yellow vein mosaic disease on Okra. Yellow color attracts the insect vector as a result instead of host plants, they land on the mulches. Pun et al., (2005) studied the effect of leaf extracts of Bougainvillea spectabilis, Prosopis chilensis, Sorghum vulgare and neem oil, neem seed kernel extract, Thuja 30, acetyl salicylic acid, Endosulfan, Monocrotophos and water as control on the management of whitefly and Okra yellow mosaic virus (OYMV) disease. Out of these neem oil (3%, v/v) neem seed kernel extract (5%, w/v) and leaf extract of Bougainvillea (10%, w/v) and Prosopis (10%, w/v) were most effective in decreasing the whitefly count and the OYMV incidence. Neem oil, neem seed extract and Monocrotophos reduced the whitefly count (plant-1) to 0.92, 1.01 and 1.05 as compared to 1.74 in control. Neem oil sprayin was most effective in reducing the OYMV incidence by 59.8% compared to control. This was followed by neem seed kernel extract and leaf extracts of Bougainvillea and Prosopis. Surprisingly virus disease

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incudence in Monocrotophos sprayed plots were high dispite it reduced the whitefly count. The maximum fruit yield (5.99 t ha-1) was from the plots treated with neem oil as against (2.26 t ha-1). Prasad and Kudada (2005) achieved significant increase in fruit yield using barrier and inter crops to manage papaya ring spot virus (PRSV) disease. Intercropping with pigeonpea (both annual and perennial type), sorghum, tree castor and barrier/border (two rows) of sorghum and tree castor were tried for two years. It was found that perennial pigeonpea both as barrier/border and in intercropping with papaya reduced the PRSV incidence significantly compared to control plot. Analyses of data revealed that out of 16 yields attributing characters including disease incidence (6, 12 and 18 months), four viz. fruit weight, fruit length and fruit circumference accounted for 97.45% (R2 =0.9745) variation in fruit yield. Strong positive correlation of fruit yield (r=0.81) and the highest direct effect was of fruit length. Best results were obtained when papaya crops were intercropped with perennial type pigeonpea for long duration followed by tree castor barrier/inter cropping. No significant difference in disease incidence and fruit yield were seen with sorghum as barrier/inter crop. Valand and Muniyappa (1992) studied management of tobacco leaf curl disease in nursery bed until transplanting after 45 days of sowing using nylon nets of 40 mesh. In addition effects of barrier crops sunflower (Helianthus annus) and castor (Ricinus communis) were also studied. In nylon covered nursery no virus incidence was recorded. In uncovered nursery the virus incidence was 5.4% at 45 days after sowing and Wf counts were 31, 49 and 73 per 100 seedlings at 15, 30 and 45 days after sowing. Barrier crops reduced the virus incidence by 50% as compared to control. There were fewer WFs on tobacco but on sunflower the count was 133 adults and on castor 381 adults. In case of F8 ToLCV resistant lines TLB 111, TLB 130 and TLB 182 use of physical barrier (polythene sheet) effectively reduced WF immigration and increased emigration and adult mortality of WF. Disease incidence in these plots was significantly lower and yields were significantly higher. Turnip mosaic potyvirus is a serious problem on radish in Himachal Pradesh. Several efficient vectors of this virus are present in nature. Application of insecticides in case of non-persistently transmitted viruses is not helpful. Wheat as barrier/trap crop along with spraying of Rogor reduced the disease incidence by 29.44-34.79% as compared to control. Yield obtained was also 183.18-187.93 q/h as against 129.25-139.43 q/h in control. Rogor alone did not give such encouraging results (Sharma et al., 2003). Heavy population of whitefly was controlled at IHBT, Palampur, using yellow sticky traps in the field of Pickrorhiza kurroa a medicinal plant (Zaidi, personal communication). Applications of barrier crops and mulches, though effective and ecofriendly could not get wide applicability in most of the cases. Smallholdings in India discourage farmers to adopt these practices. In addition plastic mulches have disposal problems especially when burning of these materials is now prohibited.

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Effect of inter-row spacing on incidence of bean common mosaic potyvirus (BCMV) was studied in Frenchbean/rajma (Phaseolus vulgaris L.) crops (Dhar and Gurha, 1999). There is considerable increase in the area of rajma in the northern and mideastern plains of India. With this increase in acreage disease problems such as BCMV, which is, seed borne has increased many fold. This disease induces yield loss of 55%. The primary transmission is through contaminated seeds and secondary transmission by aphids. Field trials in split plot design with inter-row spacings of 30, 45 and 60 cms were conducted. In case of 30 cms spacing in one set rouging of infected plants was done and no such rouging was done in another set. It was found that disease incidence was more than 50% lesser in 30 cms spacing with much higher yields than the disease incidence and yield in 60 cms spacing. Lower healthy (apparently) plant population in the plots with rouging did not compensate the yield. This was therefore, recommended for seed crops. Genetics of resistance and breeding program In a classical work Kaloo and Banerjee (1990) developed tomato leafcurl virus (ToLCV) disease resistant tomato inbred line H24, which is used by AVRDC, Taiwan in breeding program. The resistance-governing gene in Lycopersicon hirsutum f. glabratum (B6013) (Banerjee and Kaloo, 1987) was mapped at AVRDC and found an introgression located on lower end of chromosome 11 (Hanson et al., 2000). Information on genetics of resistance to yellow mosaic diseases of cowpea and mungbean has been discussed earlier (Rishi, 2004). Three f8 resistant lines of tomato TLB 111, TLB 130 and TLB 182 derived from Lycopersicon hirsutum f. glabratum (B6013) were tested successfully at farmers fields in southern India and approved for release. No virus could be detected in TLB seedlings inoculated with ToLCV-Ban-4 when PCR tested three months after transplanting (Muniyappa et al., 2002). Seed certification Seed certification is rather most effective way in reducing the potential inoculum load in field. In case of vegetatively propagated crops the vegetative propagules are the sequential source of inoculum leading to degeneration of the crop. In India CPRI, Shimla has developed a very successful seed certification program (Pushkarnath, 1967, Nagaich et al., 1969, Khurana, 1999). The certified seeds have performed well in field in drastically containing the virus disease incidence and enhancing yield manyfold. Similarly in case of citrus National Research Center for Citrus (NRC), Nagpur has done a very commendable job in developing citrus budwood certification program. An ambitious program of producing disease-free planting material of Nagpur mandarin, acid lime and sweet orange is going on at NRC, Nagpur using the most advanced internationally accepted techniques of nursery management. This technology is giving divend in quality and quantity of fruit yield to the citrus industry that is growing at an average rate of 9.3% per annum. In the last four years more than one lakh virus free planting material of citrus was developed at this center and given to farmers (Ghosh, 2002, Anon., 2004).

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Tissue culture techniques, thermotherapy and chemotherapy to eliminate viruses Effect of virazole or ribavirin (1-β-D-ribofuranosyl-1, 2, 4-triazole-3-carboxamide) and some of the dye viz. malachite green, acridine orange, ethidium bromide and eosin Y were tried on eggplant mottled crinkle virus-Indian isolate (EMCV-1) infected shoot tips of eggplant (Solanum melongena) under aseptic conditions (Raj et al., 1991). The shoot tips were growm on Murashige and Skoog (MS) medium supplemented with 6-benzyl-aminopurine (BAP) 2mg/l and 3-indole acetic acid (IAA) 0.5 mg/l. To this different treatments (50-150mg/l) of viralzole and dyes were added. Corresponding suitable control were maintained for comparison. Virus assay in the differentiated shoots was done by sap inoculation on local lesion indicator plant C. amaranticolor using extract taken out by macerating the shoots in 1:2 phosphate buffer (0.1 M, pH 7.0) containing 0.1% sodium sulphite and Ouchterlony double diffusion test. The results revealed that virus inhibition of 44-100% was achieved with acridine orange, ethidium bromide and viralzole. Malachite green and eosin Y were not effective. It was concluded that viralzole treatment in in vitro explant culture was effective in production of virus-free plantlets of S. melongena. There are earlier reports of inhibiting viruses by this technique using viralzole and dyes (Aminuddin et al., 1986, 1988). As per need for plant quarantine in international floriculture trade, techniques for the production of virus tested carnation plants were developed and standardized and virus free carnation plants developed (Zaidi et al., 1991). To strengthen this program on national basis ELISA based diagnostic kits for Carnation mottle virus (CarMV) and Carnation etched ring virus (CERV) have been developed (Zaidi personal communication). Similar exercises have been successfully done in case of other ornamentals at IHBT, Palampur. Alstroemeria hybrids cv. Serena plants found to be infected with CMV were produced CMV-free in vitro using meristem tip culture and thermotherapy. Meristem tips (0.3-0.4 mm size) from virus-infected plants were grown in MS medium supplemented with 3% sucrose, BAP (2 mg/l) and NAA (1 mg/l) with pH 5.7. The plants grown from meristem tips were transferred in MS medium supplemented with BAP (1 mg/l) and NAA (0.1 mg/l) and sucrose 1.5% for rooting and were kept in BOD incubator for 35-40 days at 37-38ºC. Plants developed with roots (72%) were found to be CMV-free by DAS-ELISA using CMV-specific antibodies (Verma et al., 2005). Prunus necrotic ringspot virus-free Begonia semperflorens plants were raised using in vitro techniques (Verma et al., 2005a). Petioles of infected plants were used to raise virus-free begonia. The petioles were grown in MS medium amended with 0.2 mg/l NAA and 0.2 mg/l BAP (pH 5.8). For rooting, simple MS medium (half strength) without any hormone was used. In rooting medium, shoots were given chemotherapy (virazole, 2-thiouracil and 6-azauracil) and thermotherapy (38°C for 16h light period and 22°C for 8h dark period) separately and in combination. Virazole at the concentration of 20 mg/l was found to be more effective (22.5 & 12.5% virus-free plants as indexed by ELISA and RT-PCR, respectively) in comparison to other chemicals. Thermotherapy for 25 days gave 35 and 25 % virus-free plants as indexed by DAS-ELISA and RT-PCR. A combination of both treatments gave 68 and 57.5 % virus-free plants as indexed by DAS-ELISA and RT-

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PCR, respectively. At higher concentrations all three chemicals were found to be phytotoxic. Chrysanthemum cv. Regol Time plants found to be infected with Tomato aspermy virus (TAV) were produced TAV-free in vitro using meristem tip culture, chemotherapy and thermotherapy. Meristem tips (0.3-0.4 mm size) from TAV infected plants were grown in MS medium supplemented with 3% sucrose, BAP (2 mg/l) and IBA (0.05 mg/l) at pH 5.8. None of the plants were found to be virus-free as indexed by RT-PCR. Virazole at concentration of 5-10 mg/l, 2-thiouracil, Amantadine hdrochloride and Acyclovir were used in chemotherapy in different concentration (10-30 mg/l). None of these chemicals were found effective to eradicate the virus. The plantlets were transferred in fresh MS medium and were kept in BOD incubator for thermotherapy for 40 days at 37-38ºC. After that the meristem tips were taken from these treated plants and transferred in MS medium for thermotherapy. By RT-PCR 62% plants and by ELISA 77% plants grown from these meristem were found to be TAV-free. The virus-tested plants have been transferred for hardening (Verma et al., 2004a,b). Strawberry mottle virus (SMoV) is a serious problem in cultivation of strawberry. Meristem tip culture has been successfully used to raise virus free saplings of strawberry (Kaur et al., 2000). The procedure involves selection of newly formed shoots of strawberry plants showing prominent symptoms and virus testing, meristem tip culture, shoot differentiation, rooting and plantlet formation, plantlet hardening and acclimatization and test for SMoV using cvs. Chandler and Fern. Shoot tips comprising the apical dome and primordial excised under aseptic conditions (0.3-0.7 mm) and explants cultured on MS medium supplemented with BA (0.5 mg/l) + Kn (o.1 mg/l) for cv. Fern and 0.5 mg/l each of BA and Kn for cv. Chandler. Meristem sprouted in 10-15 days after culture and shoots differentiated in 5-6 weeks. Most of the shoots rooted within a week of the transfer on MS + IBA. Presence of SMoV was evaluated by inoculation on the indicator plant Cucumis sativus. It was observed that plants derived from tips upto 3mm long were free from virus. There was inverse correlation between the size of meristem and rate of virus elimination. Inhibition of the infectivity of two strains of watermelon mosaic virus was studied using latex from ten angiosperms from five different families (Tewari and Shukla, 1982). Virus inoculum was mixed with latex (1:1 v/v) and incubated for five minutes at 20 0 C and inoculated on the cotyledonary leaves of Cucurbita pepo cv. Caserta. It was seen that latex of Jatropha curcas decreased the transmission by 92.6%, Calotropis procera by 86.6%, Argemone maxicana 85.5%, and Euphorbia hirta 78.5 %. The incubation period also increased in these cases. Zaidi et al., (1988) reported inhibition of spinach mosaic virus using extracts of some medicinal plants. Bavistin (50% w/w carbendazim) and Benomyl (50% w/w benlate) are the potential chemotherapeutants of certain virus diseases and improved the growth and yield of virus-infected plants (Tomlinson, 1982). The benzimidazole derivatives have cytokinin like activity. Cytokinins decrease the breakdown of chlorophyll thereby delaying senescence and they also increase nucleic acid and protein syntheses (Fletcher 1969, Waygood,

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1965). Saigopal et al., (1988) observed chemotherapy of peanut green mosaic virus (PGMV) a potyvirus using Bavistin wp (50% carbendazim, BASF India Ltd.). PGMV naturally infecting groundnut (Arachis hypogaea L.) in India induces pod yield loss of 20-25%. Seeds of groundnut cv. ‘TMV-2’ were sown in the ex0perimental field. PGMV were sap inoculated on young seedling of groundnut 25 days after sowing. Three sprays of Bavistin at 0.5% in tap water were given respectively on the day of inoculation and 15 and 30 days after inoculation. Data recorded showed suppressed symptoms on sprayed plants, increased chlorophyll content, increased- shoot and root length, number of pegs, pods, side shoots, total leaves, dry weight of shoot, root and pods and decreased abcission as compared to unsprayed plants. It was argued that during the pertiod of pod filling (70-80 days) four top leaves that are better exposed to sunlight play pivotal role in contributing more photosynthates to pods. Bavistin increased the area of top 4 leaves. Saigopal et al., (1990) reported increased total lipid content in Bavistin sprayed PGMV infected groundnut plants as compared to unsprayed infected plants. This explains suppression of foliar symptoms and analogous biochemical changes in greening of etiolated leaves and delayed senescence. Earlier decreased chlorosis and analogously increased plant growth and grain yield was observed when rice tungro virus-infected rice plants were treated with carbendazim ((Thomas and John, 1980). Effects of Benlate (methyl-1-butyl carbamoyl-2-benzimidazole) on symptom expression, virus-acquisition by aphid vector and sap inoculation of urdbean leaf crinkle virus (ULCV) were studied (Bhardwaj et al., 1982). ULCV particles are isometric; it is seed borne and non-persistently transmitted by Aphis craccivora Koch and Acyrthosiphon pisum Harris. Seedlings raized from healthy seeds of highly susceptible urdbean cv. Kulu-1 in pots were given Benlate treatment in the form of soil drenching at 1% and higher concentration. Seedlings given Benlate treatment before aphid inoculation feeding or sap transmission did not develop disease symptoms. However at lower concentration of Benlate the % transmission was reduced. Post inoculation application of fungicide was less effective. When infected plants were given Benlate treatment at 2% or higher concentration, the aphids failed to acquire the virus. Transgenics Genetically engineered resistance in plants by introducing a suitable foreign gene using transformation techniques are referred as transgenics. Such resistance to plant viruses may be induced by pathogen derived resistance (PDR) or non-viral gene mediated resistance. PDR strategies may be aimed either at production of protein that confer resistance to broader range of virus strains and viruses or accumulation of viral nucleic acid conferring very high level of resistance to a specific virus strain. Expressions of coat protein interfere with virus disassembly process, mutant movement protein can sequester the host factors and complete or partial replicase protein can interfere with viral replication. Resistance mediated through non-viral genes are (i) Genes encoding the antibodies to viral protein, (ii) Ribosomal in activating protein, (iii) Antisense RNA, (iv) Protease inhibitors, (v) Satellite RNA, (vi) Enhansing salisylic acid (Baulcombe, 1994, 1996, Beachy, 1997).

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Only a few laboratories in India are engaged in development of transgenics. Abridged information on development of virus resistant transgenics in India are given here: Cotton: Cotton leaf curl disease (CLCuD) has assumed economic significance since early 1990s when it appeared in big way (Rishi and Chauhan, 1994) in the northern India states of Punjab and Rajasthan areas bordering Pakistan. Earlier to this CLCuD widely appeared in cotton growing areas and incurred severe yield losses. Virus species inducing CLCuD are of genus Begomovirus of family Geminiviridae. In a DBT financed network project involving this laboratory and Department of Biochemistry and Department of Microbiology and Cell Biology of Indian Institute of Science, Bangalore efforts were made to identify the cotton leaf curl virus isolates (CLCuV) causing CLCuD in northern India and exploiting the suitable viral genomic construct for developing transgenics. Cotton leaf curl Kokhran virus –Dabawali (CLCuKV-Dab), Tomato leaf curl Bangalore virus-Cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and Cotton leaf curl Kokhran virus-Ganganagar (CLCuKV-Gang). CLCuKV-Dab is most widely prevalent in Haryana, Rajasthan and Punjab (Kirthi et al., 2004). CLCuV resistant cotton transgenic was developed in cotton var F846 expressing antisense movement protein gene (AV2) through Agrobacterium-mediated transformation (Sanjaya et al., 2005). Antisense-based resistance may work better with DNA viruses that transcribe their genome into mRNA in the nuclei. A binary plasmid pPZP with antisense AV2 and nptII gene in the T-DNA driven by cauliflower mosaic virus (CaMV0 35S promoter and (NOS) terminator sequence was mobilized into Agrobacterium tumefaciens disarmed strain LBA 4404 by freeze-thaw method. Viral gene integration in the transformed plants was confirmed by PCR and Southern blot hybridization analyses. The induced character segregated in the Mendelian pattern. T3 seeds are under test for virus resistance at this laboratory and at Advanced Center of Plant Virology, IARI, New Delhi. Tomato: The most devastating viruses infecting tomato are those with the generic name Tomato leaf curl virus (ToLCV). Antisense gene strategy was adopted for development of transgenic tomato having possibility of stable resistance against ToLCV. Tomato plants transformed with antisense rep gene construct showed stable inheritance of transgene in subsequent generations and its potential for resistance to the disease. For development of ToLCV resistant tomato transgenic, transgene (replicase gene of Tomato leaf curl virus) was characterized and its conserved functional motifs were identified (Dasgupta et al., 2004). Construct was developed using full-length antisense rep gene. Six transgenic events were found to be having single to multiple integration of transgene. All the six events were characterized at T1 stage and inheritance was found to follow Mendelian pattern. Two single insertion lines were carried forward to T2 generation. Resistance evaluation in all the six lines at T1 stage and two lines having single insertion at T2 stage was carried out. All the six lines at T1 stage showed 50-77% resistance as compared to control. Two single insertion events, when selfed to produce T2 generation showed >80% resistance as compared to control. Gene silencing assays demonstrated the RNA mediated resistance through antisense suppression of the viral genome. Physalis mottle tymovirus (PhMV) infection induces serious yield depression in tomato. Agrobacterium-mediated resistance to PhMV was achieved in tobacco (Nicotiana

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tabacum cv. Havana) transgenics by expression of coat protein and 3’ noncoding region. (Ranjith Kumar et al., 1999). Subsequently Sree Vidya et al., (2000) developed PhMV resistant genetically modified tomato (Lycopersicon esculentum var. Pusa Ruby) by integrating coat protein (CP) gene. CP gene was cloned into plasmid pBI CP binary vector by deleting GUS gene and mobilized into Agrobacterium by freeze-thaw method. Tomato cotyledonary leaves were precultured for 48 h in the Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L Thidiazuron and 2% sucrose and infected with Agrobacterium suspension (0.5 OD) for 5 min. These explants were blotted dry with Whatman No 1 filter paper and co-cultivated for 48h again on the regeneration medium. They were then washed with MS liquid medium and transferred to fresh regeneration medium containing 50mg/L kanamycin to select for transformants and 400mg/L cefotaxime to inhibit Agrobacterium contamination. The explants resistant to kanamycin were sub cultured every two weeks in fresh medium. Putative transformants were separated and transferred to half strength MS medium containing 25mg/L kanamycin and 200mg/L cefotaxime for rooting. PCR and Western blotting analyses confirmed CP gene integration into these transgenic plants. The T1 progenies obtained by selfing were germinated on MS medium plate with 100mg/L kanamycin. Kanamycin resistant and sensitive seedlings were in the ratio of 3:1 thereby showing that T1 progenies segregated in 3:1 ratio. Gene silencing resulting into recovery of Tomato leaf curl virus (ToLCV) infected tomato plants was achieved using homologous replicase gene constructs that produce RNAs duplex (sense (viral origin) and antisence (transgene)). This production of dsRNA template acts to produce sequence-specific degradation of viral genome and recovery of infected plants (Praveen et al., 2005). Potato: Virus diseases of potato are one of the major constraints in growing healthy profitable potato crops in India. Efforts were made at Central Potato Research Institute, Shimla in collaboration with Bhabha Atomic Research Center, Trombay in developing virus resistant potato transgenics (P. Naik, personal communication, 2005). CP gene of an Indian PVY0 strain was used for Agrobacterium-mediated transformation of potato var. Kufri Jyoti. Glasshouse evaluation at CPRI, Simla showed total protection from PVY. In addition CP gene of PLRV, P1 proteinase gene of PVY and movement protein gene of potato stem necrosis virus are being exploited for developing genetically modified potato by posttranscriptional gene silencing. Rice: Since 1999, the group on the molecular biology of plant-virus interactions lead by Dr. Indranil Dasgupta of the Dept. of Plant Molecular Biology and Dr. M. V. Rajam of the Dept. of Genetics of the University of Delhi South Campus are involved in genetic engineering of rice for viral resistance, as a part of a Network Project funded by Department of Biotechnology, Government of India. Many transgenic lines of rice have been produced, expressing the viral transgenes. Molecular analyses of the integration patterns of five different virus-derived transgene constructs have shown that there are about 20 different transgenic lines. They contain different genes derived from RTBV as well as the other causative virus for tungro disease, Rice tungro spherical virus (RTSV). The transgenes have been integrated in the rice genome in 1-3 copies. The resistance

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strategies are both protein-mediated and RNA-mediated. When a few of the above lines were challenged with RTBV and RTSV by Leafhopper vectors, some of the transgenic lines showed only 20%-50% accumulation of the viruses, as compared to non-transgenic control plants (I. Dasgupta, personal communication, 2005). Since April 2004, work to develop virus-based gene silencing vectors for functional genomics of rice under the Network Project on rice functional genomics, funded by Department of Biotechnology, Government of India has started. In this project, RTBV DNA is being modified to replicate independently in rice plants, following introduction through Agrobacterium, a process known as agro-infection. Cloning of any rice gene in such a vector is expected to produce gene silencing of the corresponding gene in the plant. Work in this project is under progress (I. Dasgupta, personal communication, 2005). Vigna mungo: A novel method of curing yellow mosaic virus (YMV) infected Vigna mungo seedlings ten days after agroinoculation using RNA interference (RNAi), which is conserved in plants (Pooggin et al., 2003). RNAi processes dsRNA into 21-25 nt short-interfering RNAs (siRNAs) using RNase III-like enzyme. These siRNAs can cleave target RNA leading to post-transcriptional gene silencing (PTGS). YMV agroinoculated V. mungo seedlings 10 days postinoculated were bombarded with 1µm gold particles covered with interfering DNA construct resulted in complete recovery of YMV infection. This recovery lasted till to the senescence. Very little viral DNA could be recovered using PCR as compared to control. For further details on transgenics, please see reviews authored by Varma et al., (2002), Dasgupta et al., (2003) and Kirthi and Savithri (2003). Beneficial viruses Rice necrosis mosaic virus: A virus-based technology using rice necrosis mosaic virus (RNMV) has been developed at Central Research Institute for Jute and Allied Fibers, Barrackpore (1996). RNMV is a soil borne virus transmitted by a fungus Polymyxa graminis. Ghosh (1979) first recognized this virus on rice at Central Rice Research Institute, Cuttack. On rice it induces necrotic and chlorotic streaks (0.5-3.0 mm), checks the growth of plant and reduces grain yield. The virus particles were rod shaped having length peaks of 275 and 550 nm and width 13-14 nm in saps of infected rice and L. perenis plants. The Indian isolate of RNMV showed strong serological relationship with Japanese isolate of RNMV. It belongs to genus Bymovirus of family Potyviridae (Shukla et al., 1994).

While studying the host range it was observed that the RNMV inoculated plants of Ludwigia perenis (= L. parviflora) an annual weed host the growth and juvenility of the plants were increased many fold (Ghosh, 1982). Virus infection increased the biomass from 8 to 220 g and longevity from 3 to 9 months with greater vigour and enhanced juvenility. Similar results were found when RNMV was artificially inoculated on fiber crops like jute (Corchorus olitorius cv. JRO-632 and C. capsularis cv. JRC-212) and mesta (Hibiscus sabdariffa cv. HS-4288 and H. cannabinus cv. HC-583). In nature this virus does not infect these dicotyledonous plants. Field inoculation of RNMV at 10 days

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after germination (AG) without any recommended fertilizer gave best results in fiber yield as compared to inoculation at 40 and 70 days (AG). There was 38.9% increase in JRO-632, 20.6% in JRC-212, 34.7% in HS-4288 and 30.6% in HC-583. The virus infection improved the vascular system of these fiber crops (Ghosh and Mitra, 1987). The stem diameter and leaf and root sizes also increased. Correspondingly the yield determining factors like number of fiber wedges and fiber bundles increased by 322 and 52 % respectively in the RNMV treated plants. Only half dose of the normally recommended fertilizer was needed to give enhanced fiber yield in these plants. In case of JRC 212 full dose of fertilizer enhanced the yield but the increase was not economical when cost benefit ratio was compared (Ghosh, 1988). Seeds obtained from these virus-energized plants of both diploid and tetraploid types showed transmission of the growth enhancing property upto 3rd generation. Multilocational on farm trials were conducted at farmers’ field and other institutions using cv. JRO-524 with reduced dose of fertilizer, where increase in yield was 2-40% over control.

Ghosh (1982) reported that RNMV triggers synthesis of endogenous growth hormones (IAA and Cytokinin in inoculated plants. When normal seeds of cv. JRO-632 were soacked in sap extracted from RNMV infected rice leaves dilute (1:10) in sterile water for 1-24 hrs it was observed that 16-18 hrs soacking produced 50% more fiber. Similarly, spraying this at 10 days and 40 days after germination of the cv. JRO-632 raized from normal seeds showed 13% increase of dry fibers.

Beneficial applications of plant viruses in gene technology: Caulimo viruses having dsDNA and bigomoviruses having ssDNA and some of the RNA viruses (TMV, PVX, BMV etc.) have been used as vehicle for foreign gene integration with some definite advantages. In addition viruses have been used as sources of control elements for producing transgenics. Some viruses have also been used for presenting heterologous peptides for vaccine production and in functional genomics.

Further suggested readings are: -

Raychaudhuri, S. P. 1977. A Manual of Virus Diseases of Tropical Plants. The MacMillan Company of India Ltd., Delhi.

H. N. Verma 2003. Basics of Plant Virology. Oxford & IBH Publishing Co. Pvt. Ltd., New Delhi.

Lobenstein, G. and G. Thottappilly 2004. Virus and Virus-like Diseases of Major Crops in Developing Countries. Kluwer Academic Publishers, Dordrecht/Boston/London

Nayudu, M. V. 2005. Plant Viruses. Tata McGraw Hill, New Delhi, Bangalore

Epilogue Since the first report of virus disease (Dastur, 1923) and first reports of phytoplasmas (McCarthy, 1903, Butler, 1908) in the country, even with limited resources excellent work has been done on the semeiology and other biological properties of a number of viruses. Identification of viruses and strains were done on the basis of symptomatology, transmissibility, host range, physical and chemical properties, virus morphology and

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serological affinity. Some stray studies on search of the sources of resistance, genetics of resistance to virus diseases and breeding of virus resistant varieties was done. Though these informations of classical Plant Pathology are still quite relevent, but with the development in the molecular techniques during the last two decades or more there was breakthrough in the characterization of viruses, which led to developmet of strong universally acceptable system of classification of viruses. In addition now we have highly reliable and efficient molecular techniques for detection and diagnosis of virusese, which will strengthen the programs of work on virus diseases. I have tried to give here an abridged account of the representative work on viruses of several genera viz. Caulimovirus and Badnavirus (Caulimoviridae), Bigomovirus (Geminiviridae), Nanovirus (Circoviridae), Potyvirus and Carlavirus (Potyviridae), Closterovirus (Closteroviridae), Potexvirus and Nepovirus (Comoviridae), Cucumovirus (Cucumoviridae), Tobamovirus (no family assigned), Tospovirus (Bunyaviridae), Carmovirus (Tombusviridae), Illarvirus (Bromoviridae), Luteovirus (Luteoviridae), Pecluvirus, Mandarivirus (Flexiviridae), Machlovirus (Sequiviridae) and viroids (Paspiviroidae) and two of the unidentified important viruses viz. urdbean leaf crinkle and pigeonpea sterility mosaic that indicate status of research and the current trend that is developing in Plant Virology in India. In this exercise I have tried to discuss work done on detection and characterization of viruses, epidemiology and ecofriendly approaches of management, at all most all the important centers in the country. Earlier also; status of Plant Virology research and teaching in India, and constraints therein were discussed (Nene, 1986, Reddy, 1990, Bhargava, 1992, Savithri, 1995) and very useful suggestions made. Unfortunately there is little progress on the lines of suggestions given by highly respected Plant Virologists. The economy of India is primarily based on agriculture. In the current scenario of strong competetion in the internationally open market system and intellectual property rights (IPR) the country cannot afford the annual yield losses of more than Rs. 1000/- crore per annum by conservative estimates due to virus diseases alone (Varma and Ramachandran, 1994). In addition produce obtained from virus infected plants are of depleted quality and low marketability. For the purpose of export of our agricultural produce in the international market it should be certified virus free. But for a few places (Table 2), lack of trained manpower and lack of adequate facility in virology both in teaching and research work lead to weak practical training at graduate and postgraduate level. The weather parameters and ecological factors in the plains of India are ideal for perpetuation of a large number of viruses of wide biodiversity and their vectors and biotypes. The intensive and extensive agriculture, large-scale free movement of plant and seed material including exotic ones and extensive use of insecticides lead to appearance of newer strains of viruses, newer biotypes of insect vectors and introduction of diseases in new areas. In a developing country like India our strategies to handle these problems should be agriculture oriented so as to find direct field applicability. Consequently there is an urgent need to strengthen the Agricultural Universities primarily and other active centers of Plant Virology for developing competent trained manpower to cater to the needs of agriculture institutes and corporates to efficiently handle the specific problems of virus diseases in most professional way. Advantages of this exercise will directly reach

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to the end users i.e. our farmers for enhancing the quality and quantity of yields. As a supplement to it molecular characterization of important viruses, easy availability of highly sensitive detection and diagnostic kits are essential for a more successful breeding programme for viral disease resistance and epidemiological studies. In the preceding paragraphs an abridged account of the representative work being done on different virus genera in the country depict the initiation of the molecular work on viruses and their ecofriendly management in the country but on a very small scale only in premier institutions. Attention to following considerations is urgently required to address the enormous problems of virus diseases of different agroclimatic zones in a vast country like India: -

• There is an urgent necessity to open a National Centre of Plant Virology for coordination and direction in research, teaching and extension programmes in the national perspective.

• Suitable centers in the country should be identified to shoulder the responsibility of National Reference Centers and Sera Bank on specific groups of viruses. Full fiscal and manpower facilities should be provided to these centers. As of now the most important are bigomoviruses, potyviruses, tospoviruses and cucumoviruses.

• Genetically pure seeds of identified diagnostic hosts should be made readily available to all the teaching and research institutions in India.

• Minimum facilities for strengthening practicals in teaching program in Virology should be identified and provided to all the agricultural universities and other active places of teaching in Plant Virology for on hand training of the students. This should be supported by efficient network facility for easy access to data and knowledge available at global level.

• Identified centers should conduct regular bench training for updating. • Effective seed certification program should be initiated to handle all the important

seed and vegetatively transmitted viruses so as to reduce the inoculum load in fields.

• Disease maps of important diseases should be prepared so as to take up the seed production program in disease and vector free/low vector population areas.

• Plant qurantine programme should be further strengthened. Acknowlwdgement Sincere thanks to Drs. Y. S. Ahlawat, Padma Ramachandran, V. G. Malathi, A. A. Zaidi, S. K. Ghosh, P. Naik and Indranil Dasgupta for generously sharing some of the unpublished informations and for some useful discussions. Drs. R. K. Khetrapal and R.D.V.J.Prasada Rao have compiled the portion on ‘Conservation of virus free germplasm - National Bureau of Plant Genetic Resources’, Dr. O. R. Reddy on ‘Interception of exotic viruses - Department of Plant Protection and Quarantine’, Dr. P. Lava Kumar on ‘Pigeonpea sterility mosaic virus’, Dr. Padma Ramachandran on ‘Viroids’ and Dr. A. A. Zaidi has compiled portions on viruses on ornamentals and their management discussed.

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