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Author(s): Y. Sivaprasad, B.V. Bhaskara Reddy, A. Sujitha and D.V.R. Sai Gopal

Article title: Genetic variability of tobacco streak virus in South India based on the analysis of coat protein

gene

Article no: GAPP_A_735883

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AQ1: Kindly provide the received and accepted dates of the manuscript.AQ2: Kindly check the sentence ‘The amplicon of c.700 bp. . .’ for sense.AQ3: Kindly check the sentence ‘The genome is positive sense. . .’ for sense.AQ4: Kindly provide the city/state of the manufacturer.AQ5: Kindly check if the sentence ‘Two micro litres. . .’ is Ok as edited.AQ6: Kindly check if ‘Achranthus aspera’ is spelt right.AQ7: Kindly note that the citations ‘Prasad Rao et al. 2003’ have been changed to ‘Prasada Rao et

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Genetic variability of tobacco streak virus in South India based on the

analysis of coat protein gene

Y. Sivaprasada,b, B.V. Bhaskara Reddya*, A. Sujithaa and D.V.R. Sai Gopalb

aDepartment of Plant Pathology, Regional Agricultural Research Station, Acharya N. G. RangaAgricultural University, Tirupati-517502, Andhra Pradesh, India; bDepartment of Virology,SVU College of Sciences, Sri Venkateswara University, Tirupati-517502, Andhra Pradesh, India

(Received &&&&; final version received &&&&)AQ1

Tobacco streak virus (TSV), a member of the genus Ilarvirus, familyBromoviridae is an important viral pathogen in peanut and other crops in SouthIndia. Fifteen TSV isolates naturally infecting groundnut, sunflower, onion, blackgram, green gram, jute, tagetes, calotropis, pumpkin, watermelon and kenafplants were collected from fields in different regions of Andhra Pradesh, TamilNadu and Karnataka. Virus was identified as TSV by direct antigen coatingenzyme linked immunosorbent assay using TSV antiserum. The CP gene fromeach isolate was amplified using TSV coat protein specific primers.AQ2 The ampliconof c.700 bp and sequenced, consisting nucleotides of 717 nucleotides, with thepotential of coding a polypeptide of 239 amino acid residues. The sequenceanalysis revealed that the CP gene shared 91–100% and 91–99% sequenceidentity with TSV at nucleotide and amino acid level, respectively. Thephylogenetic relationship based on the nucleotide sequence of these isolatesfrom different geographical regions was also analysed in this study.

Keywords: coat protein; tobacco streak virus; Ilarvirus; RT-PCR; sequenceidentities; phylogenetic relationships

Introduction

Tobacco streak virus (TSV) was first described by Johnson (1936) and is emerging asone of the most important viral diseases in several economically important crops. Ithas a wide range infecting more than 200 plant species belonging to 30dicotyledonous and monocotyledonous plant families (Fulton 1985; EPPO 2005)and it also reported from more than 26 countries worldwide (EPPO 2005). In India,TSV was first identified in sunflower in Karnataka in the year 1997 (Annual ProgressReport 1997; Singh et al. 1997) and peanut in Andhra Pradesh during 1999–2000(Prasada Rao et al. 2000; Reddy et al. 2002). Since then, this virus was found to beresponsible for serious damage to peanut, sunflower, cotton, tomato, chilli, blackgram, green gram, okra and several other annual and horticultural crops. RecentlyTSV has been observed in onion and kenaf (Sivaprasad et al. 2010; Bhaskara Reddyet al. 2012).

TSV is the type species of the genus Ilarvirus, family Bromoviridae.AQ3 The genomeis positive sense, linear, tripartite ssRNA with 50 terminal cap structures. The

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*Corresponding author. Email: bvbreddy68@gmail.com

CE: PU QA: EP COL: PG: kothandamk 11/10/12 19:40 2008 – Style 1 (B5)GAPP_A_735883 (XML) RefSty-(C

CSE name-year)

Archives of Phytopathology and Plant Protection

Vol. 00, No. 0, Month 2012, 1–8

ISSN 0323-5408 print/ISSN 1477-2906 online

� 2012 Taylor & Francis

http://dx.doi.org/10.1080/03235408.2012.735883

http://www.tandfonline.com

30 terminus is not polyadenylated, sometimes forming strong tRNA-like structures.The RNA-1 (3.5 Kb) and RNA 2 (3.0 Kb) encode proteins involved in viral RNAreplication. RNA-3 (2.3 Kb) encodes protein that is required for cell to cellmovement. Only RNA 4 ‘‘encodes the coat protein of c.28 kDa (Xin et al. 1998;Scott 2001). RNA-1–3 are genomic and encode proteins 1a (119 kDa), 2a (91 kDa)and 3a (32 kDa), respectively, whereas RNA-4a and RNA-4 are subgenomicexpressed from RNA-2, and RNA-3 encodes 2b (22 kDa) and coat protein (28 kDa),respectively. It is infectious only in presence of its coat protein or RNA-4 (Fulton1985; Sanchez-Navarro and Pallas 1994; Ansel-Mckinney et al. 1996). The virus isreadily sap transmissible and is naturally transmitted through pollen from infectedplants with the aid of thrips, such as Scirtothrips dorsalis, Frankliniella schultzeii, F.fusca, Thrips palmi and Megalurothrips usitatus (Reddy et al. 2002; Prasada Raoet al. 2003).

Materials and methods

Disease survey and sample collection

A survey on the incidence and severity of TSV on different plants/crops in differentstates of South India (Andhra Pradesh, Tamil Nadu and Karnataka) was carried outduring the last four years (2007–2011) so as to understand the distribution of disease.Naturally affected groundnut samples showing necrotic spots on leaves and necrosisof stem and bud in groundnut (Groundnut-TPT, Groundnut-NLR, Groundnut-ATP, Groundnut-Tamil Nadu and Groundnut-Bangalore) were observed. Mosaicand chlorotic or necrotic spots on leaves and necrosis on stems in Black gram-TPT,Green gram-TPT, Tagetes-TPT, Sunflower-TPT, Onion-KNL, Jute-TPT, Calotro-pis-NLR, Kenaf-TPT, Pumpkin-TPT and Watermelon-KDP were also observed.Association of Ilarvirus with the samples was first established by direct antigen-coated enzyme linked immunosorbent assay (DAC-ELISA) (Clark and Joseph 1984)using polyclonal antiserum directed against the CP of TSV.

Isolation of total RNA and RT-PCR

The total RNA from 100 mg of healthy and TSV infected leaf tissues of Groundnut-TPT, Groundnut-ATP, Groundnut-NLR, Groundnut-Bangalore, Groundnut-TamilNadu, Black gram, Green gram, Sunflower, Jute, Pumpkin, Onion, Calotropis,Kenaf-TPT, Tegetes-TPT and Watermelon-KDP was isolated using RNase plantminikit according to the manufacture’s instructions (Qiagen, &&, USA).AQ4 Theresulting total RNA was incubated with TSV-CP gene specific reverse primer at 658Cfor 5 min and snap-chilled on ice for 2 min. cDNA was synthesised using M-MuLVreverse transcriptase (Fermentas, USA) at 428C for 1 h. The genome sense primer,TSV-CP-F-50-ATGAATACTTTGATCCAAGG-30 and antisense primer, TSV-CP-R-50-TCAGTCTTGATTCACCAG-30 were used to amplify the coat protein gene ofTSV (Bhat et al. 2002).AQ5 Two micro litres of cDNA were amplified in a 25 ml reactionvolume containing 2.5U of Taq DNA polymerase (Fermentas, &&, USA)AQ4 , 10 pmolof forward (TSV-CP-F) and reverse primer (TSV-CP-R), 2.5 mMMgCl2 and dNTPsof 10 mM each. PCR amplification conditions included an initial denaturation cycleof 2 min at 948C, followed by 35 cycles of denaturation for 30 s at 948C, annealingfor 1 min at 488C and extension for 1 min at 728C with a final extension 60 min at728C. The amplified products were resolved following electrophoresis through a 1%

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2 Y. Sivaprasad et al.

agarose gel and visualised in a UV gel documentation system (Alpha Innotech, &&,USA)AQ4 after staining with ethidium bromide (10 mg/ml).

Cloning and sequencing of PCR products

The PCR product (*700 bp) was eluted by QIAquick gel extraction kit (Qiagen,&&, USA)AQ4 and cloned into pTZ57R/T vector (Fermentas, &&, USA) according toAQ4the manufacture’s instructions. The resulting recombinant plasmids were trans-formed into Escherichia coli strain DH5a cells. Recombinant clones were confirmedthrough colony PCR and restriction digestion using enzymes BamHI and XbaI. Theresulting clones with expected size DNA inserts were sequenced using M13 universalprimers at Eurofins Genomics India Pvt. Ltd, Bangalore.

Sequence analysis and phylogenetic relationship

The sequences were assembled and a sequence identity matrix was generated usingBioedit sequence alignment editor (version 5.09) Hall (1999). Multiple alignmentwere performed, a phylogenetic tree was constructed, and a bootstrapped consensusdendrogram was generated with 1000 replications using Neighbour Joiningalgorithm of MEGA 4 (version 4.02) (Tamura et al. 2007). The coat protein genesof our isolates were compared with other known TSV isolates (Table 1) fromGenBank (Benson et al. 1999).

Results

Distribution of TSV

The samples were collected from different regions of South India (Andhra Pradesh,Tamil Nadu and Karnataka). The symptoms of TSV showed in the form ofchlorosis, necrotic lesions on leaves and necrosis of veins, midrib, petioles and stemand finally causing death of the plant. The presence of the virus in symptomaticleaves was confirmed by DAC-ELISA using polyclonal antibodies.

In RT-PCR analysis, a c.700 bp fragment corresponding to the coat protein ofTSV was amplified from total RNA isolated from the symptomatic leaf tissue(Figure 1). No such amplification was observed with total RNA extracted fromhealthy leaf tissue. The identity of the 700 bp product was confirmed by cloning andsequencing. The selected clones (Groundnut-TPT, Groundnut-NLR, Groundnut-ATP, Groundnut-TN, Groundnut-Bangalore, Black gram-TPT, Green gram-TPT,Tagetes-TPT, Onion-KNL, Jute-TPT, Calotropis-NLR, Kenaf-TPT, Watermelon-KDP, Pumpkin-TPT and Sunflower-TPT) were sequenced and deposited in NCBIGenBank (FJ447357, GQ401240, HM131488, HM770023, HM622157, HQ324117,HM131487, FJ447358, HM131490, HQ324116, HQ199846, JN254782, JN254783,JF968417 and GU355899) respectively. The CP gene of TSV was 717 nucleotideslong, coding for a protein of 239 amino acids. The CP gene of isolates from thepresent study was compared with the corresponding genes from known Ilarviruses(Table 1) at the nucleotide and amino acid levels.

The nucleotide sequence homology studies revealed that our isolates have 98–99% identity with other TSV isolates (Table 1). On the contrary, 91–100%nucleotide and 91–99% amino acid identity were observed with the coat protein geneof other members of the genus Ilarvirus.

100

105

110

115

120

125

130

135

140

145

Archives of Phytopathology and Plant Protection 3

150

155

160

165

170

175

180

185

190

195 Table

1.

Sequence

identity

ofpresentisolatesatnucleotide(below

thediagonal)andaminoacid(abovethediagonal)levels.

Isolates

FJ447357

GQ401240

FJ447358

HM131487

HM131488

HM131490

HM770023

HM622157

HQ324116

HQ324117

HQ199846

JN254782

JN254783

JF968417

GU355899

AF515823

AY940158

AY590139

DQ864452

AY940155

DQ058079

EF159702

AY940151

AY501483

DQ864458

DQ864456

AY940157

DQ864459

AF515825

AY510129

AM933669

DQ225172

AY501484

EF159703

FJ447357

100

99.1

98.7

99.1

99.5

99.5

99.5

99.5

99.5

98.3

99.5

99.1

99.5

98.7

99.5

99.1

99.1

98.3

98.3

98.7

99.5

98.7

97.4

98.7

98.7

99.1

99.1

97.8

99.5

99.1

91.5

99.1

99.1

98.7

GQ401240

99.5

100

98.7

99.1

99.5

99.5

99.5

99.5

99.5

98.3

99.5

99.1

99.5

98.7

99.5

99.1

99.1

98.3

98.3

98.7

99.5

98.7

97.4

98.7

98.7

99.1

99.1

97.8

99.5

99.1

91.5

99.1

99.1

98.7

FJ447358

99.3

99.4

100

98.7

99.1

99.1

99.1

99.1

99.1

98.3

99.1

98.7

99.1

98.3

99.1

98.7

98.7

97.8

97.8

98.3

99.1

98.3

97.8

98.3

98.3

98.7

98.7

97.4

99.1

98.7

91.5

98.7

98.7

98.3

HM13148799.4

99.3

99

100

99.5

99.5

99.5

99.5

99.5

98.3

99.5

99.1

99.5

98.7

99.5

99.1

99.1

98.3

98.3

98.7

99.5

98.7

97.4

98.7

98.7

99.1

99.1

97.8

99.5

99.1

91.5

99.1

99.1

98.7

HM13148899.7

99.5

99.3

99.4

100

100

100

100

100

98.7

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

HM13149099.5

99.4

99.1

99.5

99.5

100

100

100

100

98.7

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

HM77002399.7

99.5

99.3

99.7

99.7

99.8

100

100

100

98.7

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

HM62215799.7

99.5

99.3

99.4

99.7

99.5

99.7

100

100

98.7

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

HQ324116

99.7

99.5

99.3

99.4

99.7

99.5

99.7

99.7

100

98.7

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

HQ324117

99.1

99

98.7

98.8

99.1

99

99.1

99.1

99.4

100

98.7

98.3

98.7

97.8

98.7

98.3

98.3

97.4

97.4

97.8

98.7

97.8

97

97.8

97.8

98.3

98.3

97

98.7

98.3

91.5

98.3

98.3

97.8

HQ199846

99.4

99.3

99

99.4

99.4

99.5

99.7

99.4

99.4

98.8

100

99.5

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

JN254782

99.5

99.4

99.1

99.5

99.5

99.7

99.8

99.5

99.5

99

99.5

100

99.5

98.7

99.5

99.1

99.1

98.3

98.3

98.7

99.5

98.7

97.4

98.7

98.7

99.1

99.1

97.8

99.5

99.1

91.5

99.1

99.1

98.7

JN254783

99.7

99.5

99.3

99.7

99.7

99.8

100

99.7

99.7

99.1

99.7

99.8

100

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

JF968417

99

98.8

98.6

99

99

99.1

99.3

99

99

98.7

99

99.1

99.3

100

99.1

98.7

98.7

97.8

97.8

98.3

99.1

98.3

97

98.3

98.3

98.7

98.7

97.4

99.1

98.7

91.5

98.7

98.7

98.3

GU355899

99.8

99.7

99.4

99.5

99.8

99.7

99.8

99.8

99.8

99.3

99.5

99.7

99.8

99.1

100

99.5

99.5

98.7

98.7

99.1

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

AF515823

99.5

99.4

99.1

99.3

99.5

99.4

99.5

99.5

99.5

99

99.3

99.4

99.5

98.8

99.7

100

99.1

98.3

98.3

98.7

99.5

98.7

97.4

98.7

98.7

99.1

99.1

97.8

99.5

99.1

91.5

99.1

99.1

98.7

AY940158

99.5

99.4

99.1

99.3

99.5

99.4

99.5

99.5

99.5

99

99.3

99.4

99.5

98.8

99.7

99.4

100

98.3

99.1

99.5

99.5

99.5

98.3

99.5

99.5

100

100

98.7

99.5

100

91.5

99.1

99.1

98.7

AY590139

98.8

98.7

98.4

98.6

98.8

98.7

98.8

98.8

98.8

98.3

98.6

98.7

98.8

98.1

99

98.7

98.7

100

98.3

97.8

98.7

97.8

96.6

97.8

97.8

98.3

98.3

97

98.7

98.3

90.7

98.3

98.3

97.8

DQ864452

99

98.8

98.6

98.7

99

98.8

99

99

99

98.4

98.7

98.8

99

98.3

99.1

99.1

99.1

98.7

100

98.7

98.7

98.7

97.4

98.7

98.7

99.1

99.1

97.8

98.7

99.1

91.5

98.3

98.3

97.8

AY940155

99.4

99.3

99

99.1

99.4

99.3

99.4

99.4

99.4

98.8

99.1

99.3

99.4

98.7

99.5

99.3

99.5

98.6

99

100

99.1

99.1

97.8

99.1

99.1

99.5

99.5

98.3

99.1

99.5

91.1

98.7

98.7

98.3

DQ058079

99.7

99.5

99.3

99.4

99.7

99.5

99.7

99.7

99.7

99.1

99.4

99.5

99.7

99

99.8

99.5

99.5

98.8

99

99.4

100

99.1

97.8

99.1

99.1

99.5

99.5

98.3

100

99.5

92

99.5

99.5

99.1

EF159702

99

98.8

98.6

98.7

99

98.8

99

99

99

98.4

98.7

98.8

99

98.6

99.1

98.8

99.1

98.1

98.6

99.3

99

100

97.8

99.1

99.1

99.5

99.5

98.3

99.1

99.5

91.1

98.7

98.7

99.1

AY940151

99.1

99

99

98.8

99.1

99

99.1

99.1

99.1

98.6

98.8

99

99.1

98.4

99.3

99

99.3

98.3

98.7

99.1

99.1

98.7

100

97.8

97.8

98.3

98.3

97

97.8

98.3

90.3

97.4

97.4

97

AY501483

99.4

99.3

99

99.1

99.4

99.3

99.4

99.4

99.4

98.8

99.1

99.3

99.4

98.7

99.5

99.3

99.5

98.6

99

99.7

99.4

99.3

99.1

100

99.1

99.5

99.5

99.1

99.1

99.5

91.1

98.7

98.7

98.3

DQ864458

99.4

99.3

99

99.4

99.4

99.5

99.7

99.4

99.4

98.8

99.4

99.5

99.7

99

99.5

99.3

99.5

98.6

99

99.4

99.4

99

99.1

99.4

100

99.5

99.5

98.3

99.1

99.5

91.1

98.7

98.7

98.3

DQ864456

99.7

99.5

99.3

99.4

99.7

99.5

99.7

99.7

99.7

99.1

99.4

99.5

99.7

99

99.8

99.5

99.8

98.8

99.3

99.7

99.7

99.3

99.4

99.7

99.7

100

100

98.7

99.5

100

91.5

99.1

99.1

98.7

AY940157

99.3

99.1

98.8

99

99.3

99.1

99.3

99.3

99.3

98.7

99

99.1

99.3

98.6

99.4

99.1

99.4

98.4

98.8

99.3

99.3

98.8

99

99.3

99.3

99.5

100

98.7

99.5

100

91.5

99.1

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DQ864459

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AF515825

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DQ225172

99.5

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AY501484

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EF159703

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Figure 1. &&&.AQ13

Figure 2. &&&.

Archives of Phytopathology and Plant Protection 5

The present study isolates (Groundnut-TPT, Groundnut-ATP, Groundnut-Bangalore, Groundnut-NLR, Tagetes-TPT, Sunflower-TPT, Jute-TPT and Blackgram-TPT) grouped together along with sunn-hemp, cowpea, urdbean, mungbean,tridax, chilli and groundnut formed a separate clade (Clade I). Niger, AchranthusasperaAQ6 , soybean, okra, parthenium, gherkin, pumpkin, tagetes, cotton, jute andsunflower formed a separate clade (Clade II). Groundnut-TN, Watermelon-KDP,Kenaf-TPT, Onion-KNL and Calotropis-NLR formed a separate clade (Clade III).Green gram-TPT formed a separate clade (Clade-IV). Pumpkin-TPT formed asepate clade (Clade V) (Figure 2).

Discussion

TSV is easily sap transmissible to the members of Leguminosae, Cucurbitaceae,Solanaceae, Asteraceae, Compositae, Malvaceae, Chenopodiaceae and was observedin localised and systemic infections. Various symptoms exhibited by different hostsincluded chlorosis, necrosis on leaves followed by veinal necrosis and streaks onstems and finally resulted in plant death. Peanut and sunflower are the mostimportant oil seed crops that are affected; they are grown in many countries of theworld. Black gram, green gram, pumpkin, kenaf and onion are mostly cultivatedvegetable crops in India. Jute is one of the most important fiber crops and it is grownin different countries of the world. Tagetes is one of the flowering ornamental cropsin India. Calotropis is a weed and also an important medicinal plant.

Earlier reports indicated that TSV infects several economically important cropssuch as groundnut, sunflower, cotton, tobacco, cowpea, mungbean, sunn-hemp,green gram, black gram and bhendi (Okra) (Bhat et al. 2002; Babu et al. 2003;Prasada Rao et al. 2003). Flowering ornamental plant such as marigold and weedssuch as Acalypha indica, Abutilon indicum, Achranthus aspera, Calotropis gigantean,Cleome viscosa, Commenlina benghalensis, Croton sparsiflorus, Digera arvensis,Euphorbia hirta, Euphorbic geniculata, Legasca mallis, Lecus aspera, Partheniumhysterophorus and Tridax procumbens are also infected (Prasada Rao et al. 2003)AQ7 .TSV is naturally transmitted through pollen from infected plants with the aid ofthrips, such as S. dorsalis, F. schultzeii, F. fusca, T. palmi and M. usitatus (Reddyet al. 2002; Prasada Rao et al. 2003). The finding by Sdoodee and Teakle (1987) andGreber et al. (1991) confirmed that thrips free of pollen did not transmit the virus.Seed transmission of TSV was reported in several crops species in the world (Kaiseret al. 1982; 1991; Fulton 1985; Sdoodee and Teakle 1987, 1993)AQ8 . However, seedtransmission was not found in naturally or experimental infected groundnut,sunflower, parthenium or several other annual crops infected with TSV in India(Prasada Rao et al. 2003; Reddy et al. 2007).AQ9 Krishnareddy et al. (2003) reportedsuggest seed transmission of TSV to 2.7-65.7% in cucumber and gherkin in SouthernKarnataka. Recently, mosaic and chlorotic spotting on leaves was observed inonions in Kurnool district of Andhra Pradesh, India (Sivaprasad et al. 2010).

The variability studies of TSV not only is useful in establishing differences amongstrains that infect different crops, but also aids in evolving transgenic plants withresistance to TSV. Studies on host–vector–virus relationship pertaining to TSVinfection in various crop species are also one area of study that needs attention. Sincethe TSV in all the hosts is transmitted by thrips (Reddy et al. 2002; Prasada Raoet al. 2003), detailed investigation on management of vector population and theirrole in transmission of disease at different time intervals is necessitated.

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Incorporating host plant resistance through transgenics in varieties that have innateresistance to thrips incidence is also an emerging concept in virus disease control.

Acknowledgements

The authors are thankful to the Acharaya N.G Ranga Agricultural University, Hyderabad,India for financial assistance.

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