Tomato Leaf Curl Bangalore Research Articles

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

Identification of whitefly (Bemisia tabaci) proteins interacting with Tomato leaf curl Bangalore virus coat protein gene using Y2H system

Tomato leaf curl Bangalore virus (ToLCBV) (Geminiviridae) causes the economically important tomato leaf curl virus disease (ToLCVD), and is transmitted by the whitefly, Bemisia tabaci (Gennadius) (Aleyrodidae: Hemiptera). Successful transmission of the virus by the insect requires safe translocation of the virus through different barriers inside the vector. During the translocation, the viral coat protein will interact with many whitefly proteins, while a few of them are beneficial to virus (e.g. heat shock proteins) but some may be harmful (e.g. whitefly immune proteins). We carried out the yeast two hybrid (Y2H) assays to identify the proteins of the B.tabaci Asia 1 genetic group interacting with ToLCBV coat protein (CP). The Y2H assay initially identified a total of 425 putative interacting whitefly proteins on a low stringent selection media, and they were reduced to 324 when the yeast colonies were grown on a high stringent media, and of which about 274 colonies produced single bands in colony PCR experiments while the remaining colonies produced multiple bands. Further, high selection pressure assays confirmed a total of 102 whitefly proteins interacting with ToLCBV CP and these included the heat shock proteins (HSPs) 70 kDa, GroEL, nucleoproteins, vitellogenins, apolipophorins, 40 s ribosomal proteins, sorbitol dehydrogenase, dipeptidyl peptidase, E3 ubiquitin, annexin, GTP cyclohydrolase, tropomyosin, salivary secreted proteins, succinate dehydrogenase, lachesins, enolase and others. The identified proteins could be potential targets for novel whitefly control strategies such as using RNAi or insecticide target sites for developing future disease and whitefly control strategies.

Understanding the interactions among the crop plants, a virus, insect vector whiteflies and their endosymbionts

Interactions among the four biotic agents - the host plants, a virus, insect vector whiteflies and their bacterial endosymbionts was investigated in this study. The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an important crop pest that is infected by several endosymbiotic bacteria that can play varying roles on their hosts. Both whiteflies and endosymbionts are affected by the host plants they feed on, which in turn affect the fourth agent – the viruses, which are transmitted by the whiteflies. We investigated these interactions on five different host plants – eggplant, tomato, beans, okra and cotton - using a single whitefly species Asia 1 infected with three different bacteria Portiera, Wolbachia and Arsenophonus. B. tabaci Asia 1 transmits the Tomato leaf curl Bangalore virus (ToLCBV) effectively, which was the virus used in the study. We found host plants having a significant impact on whitefly growth and development. Eggplant was most favourable, while okra and tomato were least preferred. The endosymbiont Wolbachia was significantly affected by feeding of B. tabaci on different host plants while Portiera and Arsenophonus were unaffected. When whiteflies fed on ToLCBV-infected tomato plants, the concentration of Arsenophonus increased significantly while other endosymbionts remain unchanged. Understanding these interactions will help in managing both whiteflies and viral diseases.

Assessment of the effectiveness of Ty genes in tomato against tomato leaf curl Bangalore virus

AbstractTomato leaf curl Bangalore virus (ToLCBaV), a monopartite begomovirus transmitted by the whitefly Bemisia tabaci, has become a major constraint in tomato production in the Indian subcontinent. Earlier breeding efforts in India led to the adoption of tomato cultivars carrying the resistance gene Ty‐2. However, it has been observed recently that such cultivars/hybrids are susceptible to begomoviruses. This requires the identification of additional or new sources of resistance against ToLCBaV. The present study was undertaken to assess the effectiveness of several Ty genes in providing resistance against infection by ToLCBaV. The kinetics of virus multiplication in different Ty resistance gene stocks of tomato were estimated and compared using quantitative PCR data. Accumulation of viral genomic units and symptom severity were lower in tomato lines carrying Ty‐3 and Ty‐2 + Ty‐3 compared with those carrying Ty‐2 alone. All tested lines carrying Ty‐2 showed typical tomato leaf curl disease symptoms. Tomato lines carrying Ty‐2 + ty‐5 and ty‐5 + Ty‐6 combinations had a significantly higher disease severity index and viral genomic units compared to those carrying Ty‐3 and Ty‐2 + Ty‐3 at 30 days postinoculation. The accumulation level of ToLCBaV genomic units serves as a good indicator for resistance selection along with other parameters of disease. We present here a comprehensive overview of the effectiveness of Ty‐2, Ty‐3, and ty‐5 alone as well as the combinations Ty‐2 + Ty‐3 and Ty‐2 + ty‐5 against ToLCBaV.

Viral diseases: a threat for tomato cultivation in Indo-Gangetic eastern plains of India

Tomato, worldwide, is infected by several viral diseases which cause stunting, leaf curl, yellowing, mosaic, mottling, necrosis and shoe-string symptoms on plants, leaves or fruits. Among them, bud necrosis disease caused by an orthotospovirus is emerging as a major constraint to the cultivation of tomato for resource-poor farmers. In the Indo-Gangetic eastern plains, bud necrosis disease incidence on tomato ranged from 0 to 45% under field conditions during 2015 and 2016, along with other diseases such as leaf curl and mosaic caused by begomoviruses (0–35%) and tobamoviruses (0–18%) respectively. Thirty four viral infected samples collected from 11 different villages were screened for different viruses using serological and PCR-based methods. The result revealed that most samples were positive for RNA (peanut bud necrosis virus, tomato mosaic virus) and DNA (tomato leaf curl New Delhi virus, tomato leaf curl Gujarat virus, tomato leaf curl Palampur virus, tomato leaf curl Bangalore virus, tomato leaf curl Karnataka virus) viruses along with their satellites (DNA-α and DNA-β) respectively. Further, the incidence of bud necrosis correlates to the weather parameters, which demonstrate that a long dry spell leads to higher incidence of viral disease, whereas lower incidence was observed during the rainy period considering a reduced vector population.

Rapid detection of tomato leaf curl Bengaluru virus through loop mediated isothermal amplification assay.

A loop-mediated isothermal amplification (LAMP) technique was employed to develop a simple and rapid method for the detection of tomato leaf curl Bangalore virus (ToLCBaV) in diseased plants of tomato (Solanum lycopersicum). Six sets of primers were designed for LAMP technique targeting the conserved AC1 region and successfully detected ToLCBaV. No reaction was detected in the tissues of healthy plants by either the LAMP or the polymerase chain reaction (PCR). The LAMP products can be visualized by presence or absence of turbidity and staining (0.2μL for 25μL LAMP product) directly in the tube with nucleic acid stain dye which allowed easy detection. Sensitivity of LAMP assay is 100 times of conventional PCR technique. Although, both the LAMP and the PCR methods were capable of detecting ToLCBaV in infected tissues of tomato, the LAMP method would be more useful than the PCR method for detection of ToLCBaV infection in tomato plants because it is more rapid, simple and accurate method.

Suppressors of RNA silencing encoded by tomato leaf curl betasatellites

Virus encoded RNA-silencing suppressors (RSSs) are the key components evolved by the viruses to counter RNA-silencing defense of plants. Whitefly-transmitted begomoviruses infecting tomato crop code for five different proteins, ORF AC4, ORF AC2 and ORF AV2 in DNA-A component, ORF BV1 in DNA-B and ORF beta C1 in satellite DNA beta which are predicted to function as silencing suppressors. In the present study suppressor function of ORF beta C1 of three betasatellites Tomato leaf curl Bangalore betasatellite ToLCBB-[IN:Hess:08], Cotton leaf curl Multan betasatellite CLCuMB-[IN:Sri:02] and Luffa leaf distortion betasatellite LuLDB-[IN:Lu:04] were examined. Agroinfiltration of GFP-silenced Nicotiana tabaccum cv. Xanthi with the cells expressing betaC1 protein resulted in reversal of silenced GFP expression. GFP-siRNA level was more than 50-fold lower compared to silenced plants in plants infiltrated with betaC1 gene from ToLCBB. However, in the case of 35S-beta C1 CLCuMB and 35S- beta C1 LuLDB construct, although GFP was expressed, siRNA level was not reduced, indicating that the step at which beta C1 interfere in RNA-silencing pathway is different.

Bemisia tabaci Phylogenetic Groups in India and the Relative Transmission Efficacy of Tomato leaf curl Bangalore virus by an Indigenous and an Exotic Population

Bemisia tabaci adults from various host-plant species were collected from 31 regions across India. 266 B. tabaci samples were first screened by RAPD-PCR to examine molecular variability and to select individuals with different fingerprints. Host-plant and region of collection were then used to select 25 individuals for PCR amplification and sequencing of their partial mitochondrial cytochrome oxidase subunit one (mtCOI) genes. Pairwise comparisons with mtCOI consensus sequences showed that the majority of these samples had <3.5% sequence divergence from groups currently termed Asia I, Asia II-5, Asia II-7, and Asia II-8. The biotype-B B. tabaci from India clustered into the Middle East-Asia Minor 1 group. A new group of B. tabaci from Coimbatore, collected from pumpkin, was related most closely to the Asia I group (6.2% sequence divergence from the consensus Asia I sequence). To increase our understanding of the epidemiology of tomato leaf curl disease (ToLCD) and the key B. tabaci genetic groups involved in virus spread, the indigenous Asia I and the exotic biotype-B population from South India were used to carry out transmission experiments using Tomato leaf curl Bangalore virus (ToLCBV). The acquisition access periods (AAP), inoculation access periods (IAP), latent periods (LP), and ToLCBV transmission efficiencies of the two populations were compared and the biotype-B had the more efficient transmission characteristics. These results are discussed in relation to recent changes in the epidemiology of tomato leaf curl disease in South India.

Tomato leaf curl Bangalore virus (ToLCBV): infectivity and enhanced pathogenicity with diverse betasatellites

Genomic components of a begomovirus isolated from tomato plants showing leaf curl and stunting symptoms in farmer's fields at Hessarghatta village near Bangalore, India, were cloned by rolling-circle amplification. The virus was identified as a variant of strain C of the species Tomato leaf curl Bangalore virus and designated as Tomato leaf curl Bangalore virus-C[India:Hessarghatta:2008], ToLCBV-C[IN:Hess:08]. The betasatellite isolated from these samples belongs to the betasatellite species Tomato leaf curl Bangalore betasatellite. ToLCBV-C[IN:Hess:08] induced severe symptoms in Nicotiana benthamiana and Solanum lycopersicum plants when co-inoculated with the cognate betasatellite, Tomato leaf curl Bangalore betasatellite-[India:Hessarghatta:2008], ToLCBB-[IN:Hess:08] and with two other non-cognate betasatellites, Cotton leaf curl Multan betasatellite-[India:SriGanganagar:2002] and Luffa leaf distortion betasatellite-[India:Luffa:2004].

Molecular diversity of the DNA-β satellites associated with tomato leaf curl disease in India

DNA-beta satellites, referred to here as betasatellites, were found associated with tomato leaf curl disease (ToLCD) in India. The size of eight betasatellites isolated from different geographical locations in India varied from 1353 to 1424 nt; these molecules had an ORF beta C1, an adenine-rich region, and a satellite conserved region. Their nucleotide sequence identity varied from 45 to 93%. In phylogenetic analysis, these betasatellites grouped according to their geographic locations rather than the host species. Two new betasatellites, tomato leaf curl Bangalore betasatellite and tomato leaf curl Maharashtra betasatellite, were identified.

Production of monoclonal antibodies to Tomato leaf curl Bangalore virus

SummaryPurified Tomato leaf curl Bangalore virus (ToLCBV) was injected into mice and the splenocytes were used for establishing hybridoma lines. Initial screening of culture supernatants showed that 13 lines produced antibody, and after further screening four produced functional monoclonal antibodies. Upon characterisation, these were found to be of low affinity, probably due to host protein contamination and poor yield of native virus in the original preparations. In order to circumvent these problems, the coat protein of ToLCBV was over‐expressed in Escherichia coli. Fusion experiments using recombinant coat protein as antigen yielded two primary hybridoma clones G11 and E4 that exhibited good affinity of binding to the antigen. Sub‐cloning yielded four monoclonal antibodies G11E7E7, G11E7G12, E4E2 and E4G6. G11E7E7 and G11E7G12 successfully detected ToLCBV in infected leaf extracts of tomato and Nicotiana benthamiana, viruliferous whiteflies and weed samples. These monoclonal antibodies could also detect other type III geminiviruses such as Pumpkin yellow vein mosaic virus and Bhendi yellow vein mosaic virus. Thus these monoclonal antibodies can be used for testing field‐collected samples.

Resistance of tomato and sweet-pepper genotypes to Tomato leaf curl Bangalore virus and its vector Bemisia tabaci

The primary objective of this study was to identify sources of resistance to Tomato leaf curl Bangalore virus—Bangalore strain 4 (ToLCBV-[Ban4]) and its whitefly vector Bemisia tabaci (Gennadius) in tomato and sweet-pepper genotypes, because resistant cultivars did not exist. A total of 25 wild and cultivated tomato and nine sweet-pepper genotypes obtained from the Asian Vegetable Research and Development Centre, Taiwan and local cultivars were screened at the University of Agricultural Sciences, Bangalore, India, both by whitefly-mediated inoculation of virus in the glasshouse and natural infection in the field. Resistance to virus was assessed by disease incidence, symptom severity and DNA hybridisation tests. Resistance to B. tabaci was measured by counting the number of eggs, nymphs and pupal cases on the tomato genotypes. The wild species Lycopersicon peruvianum INRA sel. and L. chilense LA 1969 were resistant to ToLCBV-[Ban4] but highly susceptible to whiteflies, whereas L. hirsutum LA 1777 was resistant to both the virus and the vector. Among the L. esculentum genotypes, H-24, FL 744-6-9, FL 699 and FL 699 sp + were tolerant to ToLCBV-[Ban4], but were susceptible to whiteflies. Of the nine pepper genotypes inoculated with the virus, only five became infected, at low incidence levels and none were suitable for the development of B. tabaci. Use of tomato lines with resistance to both the virus and the vector offer the best way of controlling these related problems, and the prospects for tomato leaf curl virus disease management in South India are discussed.

A conserved zinc finger motif in the coat protein of Tomato leaf curl Bangalore virus is responsible for binding to ssDNA.

Tomato leaf curl Bangalore virus (ToLCBV-[Ban5]) from south India belongs to the genus Begomovirus of the family Geminiviridae. So far, only the DNA A component has been identified in this virus. In this paper we report the overexpression of ToLCBV-[Ban5] coat protein (CP) in E. coli and its characterization. The purified recombinant CP bound preferentially to ssDNA in a sequence non-specific manner. Deletion of 50 amino acids from the N-terminus, including a putative N-terminal alpha helix, did not result in the loss of binding to DNA. A search for motifs responsible for DNA binding indicated a conserved putative zinc finger motif in the CPs (corresponding to residues 65-85 in ToLCBV-[Ban 5] CP) of begomoviruses. The role of this motif in DNA binding was evaluated through site-directed mutagenesis of the conserved cysteines (C68, C72) and histidines (H81, H85) present within this motif to alanine. The results indicate that the zinc finger motif is indeed involved in binding to zinc and DNA.

Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India

Cotton leaf curl disease (CLCuD) causing viruses belong to the Begomovirus genus of the family Geminiviridae. Most begomoviruses are bipartite with two molecules of circular single stranded DNA (A and B) encapsidated in icosahedral geminate particles. However, the begomoviruses associated with CLCuD have DNA-beta instead of DNA-B. In this communication we report the complete genomic sequence of DNA-A component of two CLCuD-causing begomoviruses, cotton leaf curl Kokhran virus-Dabawali (CLCuKV-Dab), tomato leaf curl Bangalore virus-Cotton [Fatehabad] (ToLCBV-Cotton [Fat]) and partial sequences of two other isolates cotton leaf curl Rajasthan virus-Bangalore (CLCuRV-Ban) and cotton leaf curl Kokhran virus-Ganganagar (CLCuKV-Gang). A phylogenetic analysis of these isolates along with other related begomoviruses showed that ToLCBV-Cotton [Fat] isolate was closest to the tomato leaf curl Bangalore virus-5 (ToLCBV-Ban5) where as CLCuKV-Dab isolate was close to the cotton leaf curl Kokhran virus-Faisalabad1 (CLCuKV-Fai1), cotton leaf curl Kokhran virus-72b (CLCuKV-72b) and cotton leaf curl Kokhran virus-806b (CLCuKV-806b) isolates from Pakistan. The phylogenetic analysis further showed that the ToLCBV-Cotton [Fat] and CLCuKV-Dab isolates along with CLCuKV-Fai1, CLCuKV-72b and CLCuKV-806b are closer to the ToLCBV, tomato leaf curl Gujarat virus (ToLCGV), tomato leaf curl Gujarat virus-Varanasi (ToLCGV-Var) and tomato leaf curl Sri Lanka virus (ToLCSLV) isolates, where as cotton leaf curl Alabad virus-804a (CLCuAV-804a), cotton leaf curl Multhan virus (CLCuMV) cluster with the isolates from cotton leaf curl Rajasthan virus (CLCuRV) and okra yellow vein mosaic virus (OYVMV). These results demonstrate the extensive variability observed in this group of viruses. The AC4 ORF is the least conserved among these viruses. In order to further asses the variability in the CLCuD-causing begomoviruses, the region showing minimum similarity in the DNA-A sequence was first determined by a comparison of segments of different lengths of the aligned sequences. The results indicated that region 2411-424 (771 nt) was the least conserved. A phylogenetic tree constructed using the sequences of all the CLCuD causing begomoviruses, corresponding to the least conserved region showed that they form two distinct clusters.

Tomato leaf curl Karnataka virus from Bangalore, India, Appears to be a Recombinant Begomovirus

ABSTRACT The genome of Tomato leaf curl virus (ToLCV) from Bangalore, India, a whitefly-transmitted geminivirus, was cloned (pIND9) and sequenced. The circular DNA of 2,759 nucleotides (U38239) is organized similarly to that of other begomoviruses with monopartite genomes. Comparison of the nucleotide sequence of pIND9 with other tomato-associated begomoviruses from India (Tomato leaf curl Bangalore virus [ToLCBV, Z48182]) and Tomato leaf curl New Delhi virus-Severe (ToLCNdV-Svr, U15015) showed moderate DNA sequence identities (82 to 87%) between capsid protein (CP) genes but low identities (66 to 67%) for the intergenic regions and the replication-associated protein (Rep) genes (75 to 81% identity). Phylogenetic trees generated with nucleotide sequences of the Rep and CP genes of 26 begomoviruses indicated that this ToLCV is distinct from other begomoviruses and that it may be a recombinant virus derived from at least three different viral lineages. Tomatoes (Lycopersicon esculentum) inoculated with the cloned DNA monomer of ToLCV (pIND9) via particle bombardment developed leaf curling and yellowing symptoms. The virus was transmitted by Bemisia tabaci biotype B from tomatoes infected via particle bombardment to healthy tomatoes and by sap inoculation from infected tomatoes to tomato, Nicotiana benthamiana and N. tabacum. This ToLCV is a distinct member of the genus Begomovirus from India that differs from the previously characterized Tomato leaf curl Sadasivanagar virus isolate Bangalore 1 (L12739), ToLCBV (Z48182), ToLCBV isolate Bangalore 4 (AF165098), and the bipartite ToLCNdV (U15015, U15016). Thus, this ToLCV is named Tomato leaf curl Karnataka virus (ToLCKV).

Virus transmission studies and diagnostic detection of four begomovirus isolates in selected crop hosts and in Bemisia tabaci biotype B*

Virus transmission studies were conducted under glasshouse conditions using the vector Bemisia tabaci biotype B to determine how effectively isolates of the begomoviruses Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl Bangalore virus (ToLCBV) could be transmitted to phaseolus bean, capsicum and tomato test plants, the latter host used as a positive control for transmission. Diagnostic detection of viruses in these host crops and vector was also evaluated. Polymerase chain reaction (PCR) detection of TYLCV in bean cv. Wade and capsicum cv. Bellboy was achieved 4 weeks after fumigation in asymptomatic plants. Detection of TYLCV in tomato controls was achieved 2 weeks after fumigation with improved frequency of detection at 4 weeks. PCR was found to be a more sensitive method than triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for the detection of TYLCV isolates in all hosts. ToLCBV was detected by PCR and TAS-ELISA in bean. TYLCV was also detected by PCR in the vector, with a novel internal positive control. This work was carried out to facilitate the development of a diagnostic protocol for the begomoviruses causing tomato yellow leaf curl under the EU SMT programme project –‘Diagnostic protocols for organisms harmful to plants’ (DIAGPRO).

Virus transmission studies and diagnostic detection of four begomovirus isolates in selected crop hosts and in Bemisia tabaci biotype B*

Virus transmission studies were conducted under glasshouse conditions using the vector Bemisia tabaci biotype B to determine how effectively isolates of the begomoviruses Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl Bangalore virus (ToLCBV) could be transmitted to phaseolus bean, capsicum and tomato test plants, the latter host used as a positive control for transmission. Diagnostic detection of viruses in these host crops and vector was also evaluated. Polymerase chain reaction (PCR) detection of TYLCV in bean cv. Wade and capsicum cv. Bellboy was achieved 4 weeks after fumigation in asymptomatic plants. Detection of TYLCV in tomato controls was achieved 2 weeks after fumigation with improved frequency of detection at 4 weeks. PCR was found to be a more sensitive method than triple‐antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA) for the detection of TYLCV isolates in all hosts. ToLCBV was detected by PCR and TAS‐ELISA in bean. TYLCV was also detected by PCR in the vector, with a novel internal positive control. This work was carried out to facilitate the development of a diagnostic protocol for the begomoviruses causing tomato yellow leaf curl under the EU SMT programme project –‘Diagnostic protocols for organisms harmful to plants’ (DIAGPRO).

Evidence for recombination among the tomato leaf curl virus strains/species from Bangalore, India.

Tomato leaf curl virus (ToLCV) belongs to the Begomovirus genus of the family Geminiviridae. These viruses have circular single stranded DNA molecules as their genome encapsidated in icosahedral geminate particles. Generally the Begomoviruses are bipartite with respect to their genomic composition. ToLCV from South India is unique in that only DNA A component has been isolated and sequenced thus far and there is no evidence for the presence of DNA B component. In this communication we report the genomic sequences of DNA A component of two strains of Tomato leaf curl Bangalore virus (ToLCBV), one from Bangalore, ToLCBV [Ban 5] and the other from Kolar (70 kms from Bangalore), ToLCBV [Kolar]. We have examined the possibility of recombination between strains/species that co-exist within the same geographical location. A novel method has been used to analyze the variation of ToLCV sequences reported from Bangalore and to assess the frequency and importance of recombinational events among the strains/species existing in Bangalore. The results indicate that there are potential sites of recombination in AV1, AV2, AC1 and intergenic regions of the viral genome and this accounts for the observed variability in these strains/species.

First Report of the Bemisia tabaci B Biotype in India and an Associated Tomato leaf curl virus Disease Epidemic.

In May 1999, in the Kolar district of Karnataka State, Bemisia tabaci numbers on tomato increased by approximately 1,000-fold that observed previously (3). This was associated with an epidemic of severe tomato leaf curl disease that caused complete crop failure. DNAs extracted from 35 symptomatic tomato leaf samples collected within the epidemic region all gave the expected 500 to 600 bp amplicon with begomovirus-specific primers A/B (1). These primers amplify from the conserved nonanucleotide TAATATTAC in the common region of DNA-A to the conserved amino acid sequence CEGPCKYG within the coat protein gene. AluI and TaqI restriction patterns of all 35 polymerase chain reaction (PCR) products were identical. One PCR product from an epidemic (GenBank no. AF321929) and a non-epidemic (AF321930) site (Bangalore) were cloned and sequenced. The two 531-bp inserts showed 96% nucleotide identity to each other and 94% nucleotide identity to the equivalent region of Tomato leaf curl Bangalore virus (ToLCBV-Ban-4) (AF165098), suggesting that the epidemic was caused by an indigenous ToLCBV strain. Adult B. tabaci were collected from tomato plants at nine sites within the epidemic. DNA was extracted from 9 to 13 individuals per site and analyzed by RAPD-PCR using primers OpB20 and OpB11. Eighty to 100% of individuals per site had identical patterns to those of B biotype individuals from Israel and Florida, which were different to the patterns produced by the indigenous Indian B. tabaci. Adult B. tabaci from the epidemic and nonepidemic (Bangalore) regions were cultured separately on zucchini plants (n = 20) vars. Fordhook and Ambassador. Distinct silverleaf symptoms appeared in all plants fed on by the epidemic B. tabaci, but not on those fed on by the nonepidemic whiteflies. Irregular ripening of tomatoes was also a widespread problem in the epidemic area. Cytochrome oxidase I (COI) (720 bp) gene sequences were obtained for epidemic (AF321927) and nonepidemic (AF321928) B. tabaci, which had only 80% nucleotide identity to each other. A GenBank BLAST search showed that the former were most similar to B biotype whitefly from Israel (AF164667; 97%) and Texas (AF164675; 99%). The B biotype transmits Indian ToLCBV (2) and its introduction into India is of great concern as it is already associated with a devastating plant-disease epidemic.