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Chilli leaf curl virus (ChiLCV) is a monopartite begomovirus (family Geminiviridae) that spreads exclusively through an insect vector, whitefly (Bemisia tabaci). The virus causes leaf curl disease in chilli and has emerged as a serious threat to chilli production in the Indian subcontinent. Due to the lack of resistant cultivars, the management of leaf curl disease in chilli is difficult. In the present study, a novel approach is attempted to prevent ChiLCV infection by triggering RNA interference transiently in the host cell through a spray application of cocktail of dsRNAs. Three dsRNA molecules were prepared in vivo by using the E. coli strain HT115 targeting the three suppressor genes, C2, V2, and C4 of ChiLCV, separately. The cocktail of dsRNAs (dsCK) was prepared by mixing equal concentrations of the three dsRNAs (dsC2, dsV2, and dsC4) with 0.1% Celite. A single spray application of the dsCK was found to reduce disease incidence up to 66.7% in Nicotiana benthamiana plants till two weeks. Semi-quantitative PCR showed the negligible presence of the virus in the dsCK-treated plants as compared to the control. Further, the real-time analysis revealed that the plants treated with dsCK reduced the viral load up to 395-fold as compared to the control plants. This is the first report of inhibition of a chilli leaf curl virus by spray application of cocktail of dsRNAs to multiple suppressor genes of a begomovirus.

Temperature is one of the key factors influencing the viral disease development in plants. In this study effect of temperature on SYMMV accumulation and systemic movement was studied in French bean, mungbean and soybean cultivars by keeping them at temperatures range 22-25 °C, 26-29 °C and 30-33 °C. The proliferation of the virus under these conditions was confirmed through symptomatology, DAC-ELISA, RT-PCR with coat protein specific primers and RT-qPCR. French bean plants displayed symptoms by 12dpi at low temperature (22-25 °C) and by 3dpi at medium temperatures (26-29 °C) with continuous increase in viral accumulation from 0 to 24dpi. At high temperatures (30-33 °C), French bean did not show symptoms up to 12dpi but the virus accumulated and moved systemically. However, there was a decline in viral copy number after 12dpi. Mungbean plants showed mottling, mosaic and puckering symptoms at 22-25 °C and 26-29 °C and necrotic symptoms at 30-33 °C. Irrespective of temperature there was an increase in viral accumulation in mungbean from 0 days to 24dpi. Soybean plants showed veinal mild mottling at low and medium temperatures where as no peculiar symptom expression observed at high temperatures. Soybean also showed the reduction of viral copy number from 12 days to 24dpi at low and medium temperatures and from 6 days to 24dpi at high temperatures. These results indicate that temperature played an important role in SYMMV accumulation, movement and symptom expression in these three leguminous plant species. This study also demonstrates that SYMMV can replicate and spread in mungbean plants irrespective of the temperature.

Croton yellow vein mosaic virus (CYVMV), a species in the genus Begomovirus, is a prolific monopartite begomovirus in the Indian sub-continent. CYVMV infects multiple crop plants to cause leaf curl disease. Plants have developed host RNA silencing mechanisms to defend the threat of viruses, including CYVMV. We characterized four RNA silencing suppressors, namely, V2, C2, and C4 encoded by CYVMV and betasatellite-encoded C1 protein (βC1) encoded by the cognate betasatellite, croton yellow vein betasatellite (CroYVMB). Their silencing suppressor functions were verified by the ability of restoring the β-glucuronidase (GUS) activity suppressed by RNA silencing. We showed here for the first time that V2 was capable of self-interacting, as well as interacting with the V1 protein, and could be translocalized to the plasmodesmata in the presence of CYVMV. The knockout of either V2 or V1 impaired the intercellular mobility of CYVMV, indicating their novel coordinated roles in the cell-to-cell movement of the virus. As pathogenicity determinants, each of V2, C2, and C4 could induce typical leaf curl symptoms in Nicotiana benthamiana plants even under transient expression. Interestingly, the transcripts and proteins of all four suppressors could be detected in the systemically infected leaves with no correlation to symptom induction. Overall, our work identifies four silencing suppressors encoded by CYVMV and its cognate betasatellite and reveals their subcellular localizations, interaction behavior, and roles in symptom induction and intercellular virus movement.

Groundnut bud necrosis virus (GBNV) is the most significant member of the genus Orthotospovirus occurring in the Indian subcontinent. There is hardly any effective measure to prevent GBNV in crop plants. In order to develop GBNV infection prevention procedure, we examined the effect of the direct foliar application of double-stranded RNA (dsRNA) derived from the full-length NSs gene (1,320 nucleotides) of GBNV. The bacterially expressed dsRNA to the non-structural (dsNSs) gene of GBNV was purified and delivered to plants as an aqueous suspension containing 0.01% Celite for evaluating its efficacy in preventing GBNV infection in systemic host, Nicotiana benthamiana as well as in local lesion and systemic host, cowpea cv. Pusa Komal (Vigna unguiculata). The dsNSs application and challenge-inoculation were conducted in three different combinations, where plants were challenge-inoculated with GBNV a day after, immediately, and a day before the application of dsNSs. N. benthamiana plants, which were not treated with dsRNA showed severe systemic wilting and death by 9–16 days post-inoculation (dpi). The non-treated cowpea plants exhibited many chlorotic and necrotic lesions on the cotyledonary leaves followed by systemic necrosis and death of the plants by 14–16 dpi. The dsNSs treated plants in all the combinations showed significant reduction of disease severity index in both N. benthamiana and cowpea. The treatment combination where the GBNV inoculation was conducted immediately after the dsNSs treatment was found to be the most effective treatment in preventing symptom expression. The viral RNA analysis by real time PCR also showed 20 and 12.5 fold reduction of GBNV in cowpea and N. benthamiana, respectively. Our results suggest that the foliar application of dsRNA derived from the full-length NSs gene of GBNV through Celite is successful in delivering long dsRNA leading to effective prevention of GBNV infection.

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, andtrans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant–virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

The present investigation was undertaken to study the expression of eight defense related genes in leaf curl resistant line DLS-Sel-10 and susceptible line Phule Mukta after different days post inoculation (dpi) with viruliferous white flies to understand their role in resistance to chilli leaf curl virus (ChiLCV). The expression level of Ca PPO, Ca AsPer, Ca ATP/ADP and CaTopoII was observed to be higher in resistant genotype DLS-Sel-10 than the susceptible Phule Mukta at all the time points studied. Expression of CaNBS-LRR increased up to 12 dpi while that of Ca Thionin, and Ca SKP1 increased up to 24 dpi in the resistant line, thereafter it started declining. The CaSPI expression did not show any specific pattern in both the test plants. The heat map clustered all the genes under study into two major clusters based on their expression profiles, one comprising CaAsPer, Ca-Thionin, CaATP/ADP transporter, CaPPO and CaTopoII while other group comprised CaSKPI, Ca NBS and CaSPI. The challenge inoculation of the test genotypes also revealed that viral titre increased at a much slower rate in DLS-Sel-10 than Phule Mukta, suggesting thereby that DLS-Sel-10 is resisting the accumulation of ChiLCV and has a more active defense machinery than Phule Mukta.

Betasatellite is mostly associated with monopartite begomovirus and is known to play a key role in expression of severe form of disease of infected dicotyledonous plants. Begomoviruses are transmitted by whitefly, causing most severe diseases in a wide range of crops. Diagnosis of the virus and the associated betasatellites is very important while breeding for resistant varieties. The current study reports an improved set of universal primers viz., CLB36F and CLB37R, with an ability to detect vast range of begomoviruses across the crops. About 31 samples of diverse plant species showing distinct symptoms of begomovirus collected from various geographical regions of India were analyzed to validate the improved universal primer pair. The results showed that the improved universal primer pair could detect the betasatellites of all the samples, whereas, the presently available primer pair viz., beta01/beta02 could detect only five out of fifteen samples. This data can be considered as a clear indication that the improved primer pair reported in the current study is a superior substitution for the detection of diverse betasatellites.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

An Indian isolate of Potato virus Y (PVY-Del-66) associated with the mild mottling symptoms in potato was characterized as a N:O type strain, which induced necrosis in tobacco and reacted strongly with the monoclonal antibody to PVY O strain. The complete genome of Del-66 sequence (9674 nucleotides) shared 96.7–99.3% similarities with the N:O strain occurring in USA and Germany. The lack of success in amplification and cloning of the full-length genome of PVY in E. coli was the major limiting factor for demonstrating infectivity of the PVY genome. The overlapping-extension PCR (OE-PCR) was successfully employed in this study for the rapid demonstration of infectivity of PVY. The Del-66 genome was amplified as 4.5 kb and 5.1 kb fragments with 30 nucleotide complementary ends. The mixture of these two fragments in the successive PCR steps with the terminal genome primers containing T7 promoter and poly-A tail resulted in a full-length construct. Rub inoculation of in vitro transcripts from this construct resulted in expression of systematic necrosis as well as the formation of typical filamentous virions in tobacco. This study for the first time characterized a hybrid strain (N:O type) of PVY occurring in India and showed a simple and rapid method of successful production of infectious in vitro transcripts of PVY by OE-PCR.

Whitefly-transmitted begomoviruses cause serious damage to many economically important food, feed, and fiber crops. Numerous vegetable crops are severely affected and chilli leaf curl virus (ChiLCV) is the most dominant and widely distributed begomovirus in chilli (Capsicum annuum) throughout the Indian subcontinent. Recently, CRISPR-Cas9 technology was used as a means to reduce geminivirus replication in infected plants. However, this approach was shown to have certain limitations such as the evolution of escape mutants. In this study, we used a novel, multiplexed guide RNA (gRNA) based CRISPR-Cas9 approach that targets the viral genome at two or more sites simultaneously. This tactic was effective in eliminating the ChiLCV genome without recurrence of functional escape mutants. Six individual gRNA spacer sequences were designed from the ChiLCV genome and in vitro assays confirmed the cleavage behaviour of these spacer sequences. Multiplexed gRNA expression clones, based on combinations of the above-mentioned spacer sequences, were developed. A total of nine-duplex and two-triplex CRISPR-Cas9 constructs were made. The efficacy of these constructs was tested for inhibition of ChiLCV infection in Nicotiana benthamiana. Results indicated that all the constructs caused a significant reduction in viral DNA accumulation. In particular, three constructs (gRNA5+4, gRNA5+2 and gRNA1+2) were most effective in reducing the viral titer and symptoms. T7E1 assay and sequencing of the targeted viral genome did not detect any escape mutants. The multiplexed genome-editing technique could be an effective way to trigger a high level of resistance against begemoviruses. To our knowledge, this is the first report of demonstrating the effectiveness of a multiplexed gRNA-based plant virus genome editing to minimize and eliminate escape mutant formation.

Evaluation of peanut germ plasm for Tomato spotted wilt virus (TSWV) resistance has been slowed by the difficulty in achieving a high rate of mechanical transmission of the virus to peanut. In this study, improvements were made and a highly efficient mechanical transmission protocol was developed. Several factors that affect the transmission efficiency were identified. Use of two antioxidants (sodium sulfite and mercaptoethanol) and two abrasives (Celite and Carborundum) and application of the inoculum by rubbing with a cotton swab dipped in the inoculum as well as pricking with an inoculation needle resulted in a significantly higher transmission rate. The most susceptible growth stage of peanut to TSWV inoculation was 2 to 3 days after germination (6 to 7 days after planting). The inoculation protocol consistently resulted in a higher percentage of infected plants from different sources of inoculum such as infected peanut, tobacco, and tomato.

Cabbage and collard greens were inflicted with a previously undescribed virus-like disease during the fall 2000. Symptoms on leaves were yellow spots, vein clearing, mosaic, curling, and puckering. Symptomatic plants were widespread in Brooks, Colquitt, Grady, and Pierce counties in Georgia. Disease incidence ranged from 10 to 20% in the majority of the fields surveyed but some fields had 100% incidence. Fields were heavily infested by Bemisia argentifolii and the symptoms were suggestive of a whitefly-transmitted geminivirus infection. A polymerase chain reaction (PCR)-based diagnostic test for geminivirus was conducted. Total DNA was extracted from symptomatic cabbage and collard green plants collected from commercial fields. The two primers, 5'-GCCCACATYGTCTTYCCNGT-3' and 5'- GGCTTYCTRTACATRGG-3' (2,3), are “universal” for genus Begomovirus of family Geminiviridae. The primer pair could amplify a part of the replicase-associated protein and coat protein and the complete common region of DNA-A. The PCR gave a DNA band of expected size (1.1 kb) from both symptomatic cabbage and collard green samples, whereas no such product was obtained from healthy samples, suggesting that the causal agent could be a geminivirus. To establish the identity of the virus, the 1.1 kb PCR product was cloned into pGEM-T Easy (Promega) and sequenced. GenBank search showed that the geminivirus isolated in Georgia was most closely related (98% sequence identity) to Cabbage leaf curl virus (accession number U65529) reported from Florida (1). The virus was mechanically transmitted to healthy cabbage and collard green plants under experimental conditions. To our knowledge, this is the first report of Cabbage leaf curl virus from Georgia. References: (1) A. M. Abouzid et al. Phytopathology 82:1070, 1992. (2) S. S. Pappu et al. Plant Dis. 84:370, 2000. (3) M. R. Rojas et al. Plant Dis. 77:340–347, 1993.

Tomato spotted wilt virus (TSWV) of the genus Tospovirus, family Bunyaviridae (1), causes an economically important virus disease in tomato in several parts of the world. The virus has a wide host range that includes numerous crops and weeds and is transmitted by at least seven species of thrips. Tomato crops in the Subukia, Bahati, and Kabazi areas of the Nakuru District in Kenya were affected by a disease suggestive of TSWV infection during the November 1999 to March 2000 tomato-growing season. Farmers reported up to 80% losses of their potential yields. Characteristic symptoms were noticed on fruits, especially when they were green. Distinct concentric rings on fruits, which later turned into brown, uneven ripening, were the most visible symptoms. Foliage did not develop pronounced symptoms, but mild bronzing was observed in a few cultivars. However, foliage senesced prematurely, starting with older leaves. Foliar symptoms were mistaken for blight infection, and as a result, excessive fungicides were applied that failed to manage the disease. To test for TSWV infection, tomato leaf samples collected from the fields were tested initially with a TSWV test kit (HortiTech, Horticulture Research International, Wellesbourne, UK), and the results were confirmed by double-antibody sandwich-enzyme-linked immunosorbent assay with antibodies from Agdia Inc. (Elkhart, IN). Further molecular characterization was done using reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from symptomatic leaves of tomato cv. Money Maker using the RNeasy mini kit (Qiagen Inc., Valencia, CA). Using primers 5′ TTAAGC AAGTTCTGTGAG 3′ and 5′ ATGTCTAAGGTTAAGCTC 3′ specific to the nucleoprotein (N) gene of TSWV, the N gene was amplified by RT-PCR (2). A 777-bp product of the expected size was obtained from symptomatic plants, whereas no amplification was obtained from noninfected tomato. The PCR product was cloned into pGEM-T Easy (Promega, Madison, WI) and sequenced. A search of GenBank revealed a sequence identity of 95 to 99% with the N genes of known TSWV isolates. To our knowledge, this is the first report TSWV infection of tomato in Kenya. Considering its wide host range, future surveys should be directed toward estimating its incidence in tomato and other TSWV-susceptible crops, such as Irish potatoes, pepper, peanut (groundnut), beans, and a wide variety of ornamental cut flowers in Kenya. References: (1) J. W. Moyer. Tospoviruses (Bunyaviridae). Pages 1803–1807 in: Encyclopedia of Virology. A. Granoff and R. G. Webster, eds. Academic Press, San Diego, CA, 1999. (2) Jain et al. Plant Dis. 82:900, 1998.

Spotted wilt, caused by Tomato spotted wilt virus (TSWV), is a devastating disease of many crops including peanut (Arachis hypogaea). Because the virus has a broad host range and is spread by ubiquitous thrips, disease management by traditional means is difficult. Developing new peanut cultivars with resistance to TSWV presents a significant challenge since existing genetic resistance in peanut germ plasm is limited. A genetic engineering approach appears to have great potential for resistance enhancement to TSWV. Transgenic peanut progenies that expressed the nucleocapsid protein of TSWV were subjected to natural infection of the virus under field conditions during the growing seasons of 1999 and 2000 in Tifton, GA, and in three locations (Tifton, GA, Marianna, FL, and Headland, AL) in 2001. Significantly lower incidence of spotted wilt was observed for the transgenic progeny in comparison to the nontransgenic checks in the field (in multiple years and locations) as well as during challenge inoculation under controlled environmental conditions. This transgenic event could potentially be used in a traditional breeding program to enhance host resistance.

Large cardamom (Amomum subulatum Roxb.) is affected by the serious disease ‘foorkey’, which is of unknown etiology (1). Excessive sprouting and formation of bushy dwarf clumps at the base of mother plants that gradually die, characterize the disease. In surveys in the Sikkim-Darjeeling Hill area during 2002 and 2003, as much as 27% of plants were found to be affected. Electron microscopy of negatively stained, partially purified preparations from field-infected large cardamom plants showed the presence of isometric particles measuring 17 to 20 nm. The causal agent was successfully transmitted by the aphid Micromyzus kalimpongensis Basu, in a persistent manner. Ten randomly selected field samples gave A405 0.1 to 0.23 compared with 0.57 with positive control with enzyme-linked immunosorbent assay using antiserum to an Indian isolate of Banana bunchy top virus (BBTV). A fragment of approximately 850 bp was amplified from the diseased plants using polymerase chain reaction (PCR) with primers 5′-ATGGCGCGATATGTGGTATGC-3′ and 5′-TCAGCAAGAAAACCAACTTTATTC-3′, designed to amplify the putative Rep gene based on the sequences of DNA1 of BBTV. The PCR product was cloned and sequenced (GenBank Accession No. AY 485960) and showed 80 to 82% identity with the corresponding region of DNA1 of BBTV and 47.6 to 48.5% with other recognized nanoviruses. These findings indicate that a nanovirus is associated with ‘foorkey’ disease of large cardamom, which is tentatively named as Cardamom bushy dwarf virus. Reference: (1) P. M. Varma and S. P. Capoor. Indian J. Agric. Sci. 34:56, 1964.

In August 2002, ridge gourd (Luffa acutangula) plant samples exhibiting yellowing of leaves were collected from the experimental farm of Tamil Nadu Agricultural University, Coimbatore, India. Mechanical inoculations of ridge gourd seedlings using the above samples resulted in chlorotic spots on inoculated leaves and vein clearing followed by chlorotic rings and yellow netting along veins of newly developed leaves. The virus was reisolated from the chlorotic spots of the seedlings. Extracts from the field samples as well as mechanically inoculated seedlings reacted with antiserum to Watermelon silver mottle virus (WSMoV) in direct antigen coating enzyme-linked immunosorbent assay, suggesting the association of a tospovirus belonging to WSMoV serogroup (2). Symptomatic leaves from the second mechanical passage exhibiting yellow netting along the veins were subjected to reverse transcription-polymerase chain reaction (1) to identify the genus Tospovirus. Using the primer pair (5′TCTGTCCT(C/T) TTGAA (G/T) GTCCA3′ and 5′AGAGCAATCGAGGCGCT3′) derived from the conserved sequences of Groundnut bud necrosis virus (GBNV) and WSMoV, part of the nucleocapsid (N) protein gene and the complete noncoding region upstream of the N gene's coding sequence were cloned and sequenced. Comparative sequence analysis of 291-bp region of the N gene revealed that the genus Tospovirus infecting ridge gourd shared maximum identity both at nucleotide (94%) and amino acid (97%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), which is a distinct species of WSMoV serogroup infecting watermelon in India (1). In contrast, only 76 to 81% and 82% identity at nucleotide and amino acid levels, respectively, was observed with the corresponding region of the N genes of GBNV and WSMoV. Natural infection of WBNV in cucurbits except watermelon in India is not known. To our knowledge, this is the first report of natural infection of ridge gourd by WBNV. References: (1) R. K. Jain et al. Arch. Virol. 143:1637, 1998. (2) J. W. Moyer. Tospoviruses (Bunyaviridae). Pages 1803–1807 in: Encyclopedia of Virology. A. Granoff and R. G. Webster, eds. Academic Press, New York, 1999.

An unusual disease of tomato characterized by leaf mottling and necrotic streaks on veins, shortened internodes, necrosis of terminal buds, and concentric rings on fruits was observed during 2010 to 2011 surveys in tomato growing regions of Godagari Upzila, Rajshahi district, Bangladesh. Disease incidence in popularly grown F1 hybrid cultivars, which include Sobal, Abhiruchi, Salamat, Bangobir, and BARI hybrid tomato-5 and -6 in about 40 commercial fields, ranged from 40 to 90%. Extracts from the field samples (n = 10) reacted with polyclonal antiserum to Groundnut bud necrosis virus (GBNV) in direct antigen coated ELISA, suggesting the association of a tospovirus antigenically related to serogroup IV topsovirus (1). To identify whether the tospovirus was a distinct virus species, ELISA-positive samples were subjected to total RNA extraction with an RNeasy Plant Mini Kit (Qiagen, Chatsworth, CA) followed by reverse transcription (RT)-PCR with tospovirus-specific primers (5′-ATGGTTGAAAAGAGCAAGAATGATGC-3′) and degenerate primer (5′-CTTCTTATGAAGTGTACTCACCATAAGTCATCC-3′) derived from the conserved sequences of GBNV, Watermelon bud necrosis virus (WBNV), and Capsicum chlorosis virus (CaCV) (2). The RT-PCR product was cloned into pGEM-T Easy vector (Promega, Madison, WI) and sequenced at Department of Biochemistry, University of Delhi, South Campus, Delhi, India (GenBank Accession No. JQ692083). The sequences of cloned fragments were assembled. Analysis of the 477-bp region of the nucleocapsid protein (N) gene revealed that the tomato tospovirus shared maximum identity both at the nucleotide (96%) and amino acid (97%) levels with the corresponding region of GBNV. In contrast, only 78 to 81% and 85 to 87% identity at nucleotide and amino acid levels, respectively, was observed with the corresponding region of the N genes of CaCV, WBNV, and Watermelon silver mottle virus. These results suggested the association of GBNV with the diseased tomato samples. To our knowledge, this is the first report of GBNV infecting tomato in Bangladesh and regular surveys are necessary to ascertain the prevalence and incidence of GBNV in other crops. References: (1) R. K. Jain et al. J. Virol. Methods 130:162, 2005. (2) M. Tsompana and J. W. Moyer. Tospovirus. Page 157 in: Encyclopedia of Virology. Academic Press, New York, 2009.

The present investigation was undertaken to screen Solanum pseudocapsicum, against chilli leaf curl disease (ChiLCD) which is the most destructive virus disease in terms of incidence and yield loss in chilli peppers. The screening was done both under natural epiphytotic condition and through challenge inoculation using viruliferous whiteflies. The genus, which is perennial in nature and has a life span of about 10 years, was found free of infection under field screening even after four years of continuous exposure to high disease pressure. Under challenge inoculation, S. pseudocapsicum was found asymptomatic upon infection with ChiLCV and ToLCNDV, the two pre-dominant begomovirus prevalent in the study area. S. pseudocapsicum plants exposed to leaf curl disease under field condition was analysed for presence of ChiLCV through PCR and was found to show presence of virus indicating it to be a symptomless carrier of the disease. S. pseudocapsicum has proven to be a source of resistance against ChiLCD.

Watermelon bud necrosis virus (WBNV) and Groundnut bud necrosis virus (GBNV) are two closely related tospovirus species infecting several crops in India. In the present study, specific diagnostic assays for these two most predominant tospoviruses were developed by comparing host reactions and genome sequence information. Bioassay was developed by sap transmission of both the viruses to a set of host plants, cowpea, groundnut and watermelon. WBNV induced only local symptoms including chlorotic spots on cowpea cv. Pusa Komal, typical chlorotic ring spots on cv. C-152, bud necrosis on watermelon and no symptoms on groundnut. Whereas, GBNV induced both local and systemic symptoms including chlorotic/necrotic spots and veinal necrosis on both the cowpea cultivars, systemic yellowing and bud necrosis on groundnut and no symptoms on watermelon. Single and duplex RT-PCR was developed for diagnosis of WBNV and GBNV by designing primers based on differential sequence from Gn/Gc genes located on M RNA and from nucleocapsid protein (NP) gene located on S RNA genome. The duplex RT-PCR using NP gene specific primers was successfully utilised for the diagnosis of natural infection of GBNV in dahlia and muskmelon and mixed infection of GBNV and WBNV in chrysanthemum. The amplified products of duplex RT-PCR were sequenced and phylogenetic analysis confirmed the authenticity of the RT-PCR assay as well as confirmed infection of WBNV and GBNV in the new natural hosts. The bioassay and RT-PCR developed in this study will be useful in detecting single and mixed infection of GBNV and WBNV strains in different crops.

Polyclonal antibodies were raised against the bacterial expressed fused coat proteins (CPs) of Potato virus Y (PVY) and Potato virus X (PVX). Truncated CP sequences of PVY (~246 bp) and PVX (~243 bp) were amplified by PCR, cloned into T&A cloning vector and subsequently mobilized in a protein expression vector pET-28b (+). The recombinant CP was expressed as a fusion protein (~20 kDa) with His-tag and purified from E. coli BL21 (DE3) using His-Bind resin. The specificity of the recombinant protein was confirmed by Western blot using previously made polyclonal antibodies against each virus. Polyclonal antibodies developed against the fused CPs in rabbit detected natural infection of PVY and PVX in potato leaf samples collected from IARI experimental farm, by direct antigen-coated enzyme-linked immunosorbent assay (DAC-ELISA).

Potato (Solanum tuberosum) is one of the important vegetable crops in the world and its production is seriously affected by apical leaf curl disease in northern India. This paper reveals the role of cucurbits in maintaining Tomato leaf curl New Delhi virus (ToLCNDV) and Potato apical leaf curl (PALCD) disease in that region. The affected plants showed severe leaf curling and stunted growth. The begomovirus causing leaf curling and mosaic disease in cucurbits could be easily transmitted by the whitefly to potato crops and develop apical leaf curl disease in northern India. The movement of the virus by whiteflies from cucurbits to potato and tomato is possible because of overlapping of planting and harvesting dates of these crops. The causal virus was identified as a begomovirus on the basis of whitefly transmission, PCR, dot blot hybridization, cloning and sequencing of the coat protein gene. The comparison of full length coat protein gene sequence homology revealed that 90% identity with the coat protein gene of ToLCNDV- [Luffa] isolate and the phylogenetic tree derived from these sequences with other selected begomoviruses formed a close cluster with ToLCNDV isolates. The findings proved that the virus causing disease in cucurbits could easily move to tomato and potato and cause leaf curl disease naturally. This is the first observation on the role of sponge gourd for maintenance of ToLCNDV and serving as a host for PALCD in northern India. The findings indicate that the causal organism is a strain of ToLCNDV.

Evaluation of 130 accessions of rapeseed-mustard germplasm grown at the National Bureau of Plant Genetic Resources, New Delhi, India during the winter season (2011–2012) revealed the occurrence of a leaf curl disease in seven accessions. The occurrence of the disease was observed in another 62 of 525 accessions evaluated during 2012–2013. The association of a monopartite begomovirus and betasatellite was established with the symptomatic plants by whitefly transmission and PCR amplification. The complete nucleotide sequences of the begomovirus (JX270684, 2745 nucleotides), obtained by rolling circle amplification, showed the highest sequence identity (98.1%) with the weed-infecting begomovirus, Croton yellow vein mosaic virus. Analysis of recombination indicated the probable occurrence of many overlapping inter- and intraspecific recombination events. The sequence of betasatellite (JX270685, 1355 nucleotides) showed the highest sequence identity (95.7%) with Croton yellow vein mosaic betasatellite. Begomoviruses were not previously known to naturally infect rapeseed-mustard. This is the first report of the emergence of a weed-infecting begomovirus–betasatellite complex in rapeseed-mustard germplasm in India and raises the concern on utilization of such susceptible germplasm in crop improvement programmes.

An epidemic of chilli leaf curl disease was recorded in 2004 in Jodhpur, a major chilli-growing area in Rajasthan, India. Several isolates were efficiently transmitted by the whitefly (Bemisia tabaci), all of which induced severe leaf curl symptoms in chilli. A single whitefly was capable of transmitting the virus, and eight or more whiteflies per plant resulted in 100% transmission. The minimum acquisition access period (AAP) and inoculation access period (IAP) were 180 and 60 min, respectively. The virus persisted in whiteflies for up to 5 days postacquisition. Of 25 species tested, the virus infected only five (Capsicum annuum, Carica papaya, Solanum lycopersicum, Nicotiana tabacum and N. benthamiana). The virus was identified as Chilli leaf curl virus (ChiLCV), which shared the closest sequence identity (96.1%) with an isolate of ChiLCV from potato in Pakistan and showed sequence diversity up to 12.3% among the ChiLCV isolates reported from India and Pakistan. A betasatellite was identified, which resembled most closely (97.3%) that of Tomato leaf curl Bangladesh betasatellite previously reported from chilli and tomato leaf curl in India. The betasatellite was very different from that reported from chilli leaf curl in Pakistan, indicating that different betasatellites are associated with chilli leaf curl in India and Pakistan. We describe here for the first time the virus–vector relationships and host range of ChiLCV.

The complete genome of a Potato virus X (PVX) isolate from India (ptDel-9), which occurred symptomlessly in potato but induced ringspots on Nicotiana tabacum cv. Xanthi and necrotic mosaic on Nicotiana benthamiana, was sequenced. The genome was 6435 nucleotides long (JF430080) and contained five open reading frames. The isolate was closely related to those reported from the Eurasian region (95.1–97.1% sequence similarity) and distantly related to those reported from South America (77.2–77.9%). The CP gene was expressed in Escherichia coli as a 76-kDa fusion protein with maltose-binding protein and used to generate polyclonal antibodies, which successfully detected PVX in field samples of potato by ELISA. In 20% of field samples, for which ELISA failed, the virus was successfully detected by RT-PCR. This is the first report of molecular characterization of PVX occurring in India.

Soybean (Glycine max) is one of the most important crops as a source of high-quality protein and oil. In India, soybean area has expanded with the introduction of high-yielding varieties with wide adaptability, which also favored the development of new diseases with increased incidence. During 2016, 38 samples from initial varietal trials of soybean cultivars from India exhibiting symptoms such as mosaic, mottling, necrosis, and puckering were collected from the fields of the Indian Agricultural Research Institute, New Delhi, India, and tested for the presence of viruses, namely, Cowpea mild mottle virus, Groundnut bud necrosis virus, Soybean mosaic virus, Soybean vein necrosis virus, Soybean yellow mottle mosaic virus (SYMMV), and Tobacco streak virus. Crude sap from 3 of the 38 samples strongly reacted with the polyclonal antibodies raised against SYMMV in direct antibody coating ELISA (A₄₀₅ₙₘ= 1.2 to 1.8) (Sandra et al. 2015). To confirm the SYMMV infection, total RNA was extracted from the ELISA-positive soybean leaf samples showing mosaic and mottling symptoms using the SV Total RNA isolation system (Promega, Madison, WI) followed by reverse-transcription polymerase chain reaction (RT-PCR) with coat protein (CP) gene specific primers (forward, 5′-ATGAATTCATGAATGGAACAATGCTCACCGT-3′; reverse, 5′-CAAAGCTTAGTGTGTGGGTTTTAACTGGCGTTGTTA-3′) (Sandra et al. 2015). The RT-PCR product of 1,065 base pairs was cloned into TA cloning vector (Real Biotech Corporation), and the clones were sequenced in both orientations and deposited in GenBank (accession no. MG768974). Sequence analysis of the CP gene showed 98 to 100% identity with Indian isolates (KR260903, KR260902, and KP259608) followed by 82 and 92% similarity with Korean isolates of SYMMV at nucleotide and amino acid levels, respectively. Identity at the nucleotide and amino acid level of 29 to 42% and 16 to 25% was observed with the corresponding CP region of other carmoviruses. For further confirmation, French bean seedlings (cv. Pusa Parvati) were mechanically sap inoculated with an extract from an ELISA-positive soybean leaf sample, and these seedlings showed systemic chlorotic blotches after 10 to 12 days postinoculation, which were then confirmed by ELISA and RT-PCR as above. Previously, SYMMV was reported on soybean from South Korea and North America (Li et al. 2009; Nam et al. 2009). More recently, SYMMV was reported on mungbean and urdbean from India (Sandra et al. 2015). To our knowledge, this is the first report of natural infection of soybean by SYMMV in India and suggests that this newly identified SYMMV may be adapting to other leguminous host plant species.

Sponge gourd is a popular vegetable grown throughout India. Tomato leaf curl New Delhi virus, the causal virus of tomato leaf curl disease, has recently been reported to be associated with sponge gourd, causing up to 100% crop loss under epidemic conditions. We have collected 30 genotypically diverse genotypes of sponge gourd from different parts of India, screened these for resistance under natural epiphytotic conditions, and then confirmed the results through challenge inoculation with a purified strain of the virus under insect-proof greenhouse conditions. The minimum vulnerability index was recorded in genotype DSG-6 (3.33), followed by DSG 7 (6.0) under the challenge (whitefly-populated) inoculation conditions. Two susceptible genotypes (‘Pusa Sneha’ and NSG-1-11), both possessing desirable fruit characters, were crossed with the two most promising resistant lines (DSG-6 and DSG-7) and the disease reaction of segregating and backcross generations studied through challenge inoculation with a purified strain of virus under insect-proof greenhouse conditions. A chi-square (χ2) test of frequency distribution based on the vulnerability index of the F2 progenies of the two resistant × susceptible crosses revealed monogenic dominant Mendelian ratio 3(R):1(S) to be the best fit in all crosses. This monogenic dominant model was further confirmed by the 1(R):1(S) ratio found to be best fit for the test cross with the susceptible parent. These results reveal that resistance to Tomato leaf curl New Delhi virus associated with yellow mosaic disease of sponge gourd is controlled by a single dominant gene in the genetic background of the resistant parents (DSG-6 and DSG-7) and that these two lines can be effectively utilized for the development of high-yielding and yellow mosaic disease-resistant varieties/hybrids of sponge gourd. This is the first conclusive identification of a resistant source and the inheritance of resistance against Tomato leaf curl New Delhi virus in sponge gourd.

The genetics of resistance to Cucumber mosaic virus (CMV) in Cucumis sativus var. hardwickii R. Alef, the wild progenitor of cultivated cucumber was assessed by challenge inoculation and by natural infection of CMV. Among the 31 genotypes of C. sativus var. hardwickii collected from 21 locations in India the lowest mean percent disease intensity (PDI) was recorded in IC-277048 (6.33%) while the highest PDI was observed in IC-331631 (75.33%). All the four cultivated varieties (DC-1, DC-2, CHC-1 and CHC-2) showed very high PDI and susceptible disease reaction. Based on mean PDI, 8 genotypes were categorized as resistant, 13 as moderately resistant, 9 as moderately susceptible and one as susceptible. A chi-square test of frequency distribution based on mean PDI in F2 progenies of six resistant × susceptible crosses revealed monogenic recessive Mendelian ratio 1(R):3(S) to be the best fit. This monogenic recessive model was further confirmed by 1(R):1(S) ratio as the best fit for back cross with resistant parent and no fit for either 3:1 or 1:1 in the back cross with the susceptible parent. The results revealed that CMV resistance in C. sativus var. hardwickii was controlled by a single recessive gene. Considering the cross compatibility between C. sativus var. hardwickii and cultivated cucumber, the resistance trait can be easily transferred to cultivated species through simple backcross breeding.

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.

Natural infection of tospoviruses on three cucurbitaceous (Cucumis sativus, cucumber; Luffa acutangula, ridge gourd; Citrullus lanatus, watermelon) and three fabaceous (Vigna unguiculata, cowpea; Phaseolus vulgaris, French bean; Dolichos lablab, sem) vegetable crops in India was identified on the basis of nucleocapsid protein (NP) gene characteristics. The complete NP gene of the cowpea isolate from Kerala and the sem isolate from Tamil Nadu was 831 nucleotides long, encoding a protein of 276 amino acids. For other Tospovirus isolates from cucumber, French bean, ridge gourd and watermelon, the partial NP gene (291 nt) was sequenced. Comparative NP gene sequence analyses revealed that fabaceous isolates shared maximum identity both at the nucleotide (92–97%) and amino acid (93–97%) levels with the corresponding region of Groundnut bud necrosis virus (GBNV), whereas cucurbitaceous isolates shared maximum identity both at nucleotide (93–99%) and amino acid (95–98%) levels with the corresponding region of Watermelon bud necrosis virus (WBNV), results suggesting that the Tospovirus isolates infecting fabaceous hosts should be regarded as strain of GBNV, whereas those infecting cucurbitaceous hosts as a strain of WBNV. Nucleocapsid protein gene was conserved both in GBNV and WBNV isolates originating from different hosts and locations.

Spotted wilt disease, caused by Tomato spotted wilt virus (TSWV), is an economically important disease in peanut, pepper, tobacco, and tomato in the southeastern United States. However, very little is known about the biological variability existent in the virus population. Fourteen isolates of TSWV collected in Georgia were evaluated for symptom severity. The majority of the isolates produced severe systemic necrosis. One mild (GATb-1) and one severe (GAL) isolate were further examined because of the distinct differences in their virulence and symptomatology on tobacco. GATb-1 caused a few chlorotic spots and mild systemic symptoms, whereas GAL produced a large number of local lesions and severe systemic necrosis. Distinct differences in the response of selected commercial cultivars of peanut, tobacco, and tomato to GATb-1 and GAL infection were observed. GAL was lethal to a widely grown tobacco cultivar, K326. Georgia Green, a field resistant peanut cultivar, and C11-2-39, a breeding line with the highest level of known resistance to TSWV, were more susceptible to GAL than to GATb-1. BHN 444, a newly released TSWV-resistant tomato cultivar, showed a resistant reaction, whereas Stiletto, a newly released TSWV-resistant pepper cultivar, was susceptible to both GATb-1 and GAL isolates. Information on the biological diversity of TSWV may be useful in developing more durable TSWV-resistant crops.

Screening of peanut germ plasm for resistance to Tomato spotted wilt virus (TSWV) has been largely inefficient due to the lack of a screening technique based on mechanical transmission of the virus under controlled environmental conditions. We have studied the reaction of three peanut cultivars (Georgia Green, Georgia Runner, C-99R) and one breeding line (C11-2-39) using a highly efficient mechanical inoculation procedure. The disease response was studied at two temperature regimes, 25 to 30°C (low temperature) and 30 to 37°C (high temperature). Based on percent transmission, symptomatology, distribution of TSWV, and relative levels of TSWV nucleocapsid (N) protein, Georgia Runner and Georgia Green were found to be susceptible, whereas C-99R and C11-2-39 were resistant. Of the four genotypes tested, C11-2-39 had the highest level of resistance to TSWV. The results correlated with the field performance of the genotypes except in the case of Georgia Green, which could not be distinguished from TSWV-susceptible Georgia Runner. Exposure of the inoculated plants to higher temperature (30 to 37°C) resulted in a better resistant response as reflected by reduced systemic infection, localized symptom expression, restricted viral movement, and reduced levels of TSWV antigen. To our knowledge, this is the first report of differential response of peanut genotypes to TSWV using mechanical inoculation. The four peanut genotypes should be useful as reference standards for the initial screening and identification of sources of TSWV resistance in peanut germ plasm.

Reciprocal cross hybridization between DNA-A and ONA-B molecules showed that MYMV genome contains certain regions common to each other. “Common region” of MYMV belongs to the 0.55 kb Kpnl-Clal fragment of DNA-A and 0.25 kb Xbal-Hind III fragment of DNA-B. Hybridization of DNA-A restriction fragments with the full length coat protein region probe to Indian cassava mosaic virus (ICMV) showed the location of MYMV coat protein coding region in DNA-A. The greater extent of homology of ICMV coat protein region falls within the 400 base pair region of Clal-Clal fragment in DNA-A of MYMV-Bg. The homology between ICMV and MYMV-Bg coat protein gene appeared to be less than 50%. Cross hybridization between DNA-A and DNA-B molecules helped to trim out the homologous region and the DNA-A and DNA-B specific probes could be tailored.

Agroinoculation of blackgram (Vigna mungo) by the cloned genomic components (DNA-A and DNA-B) of a non-sap transmissible isolate of mungbean yellow mosaic geminivirus (MYMV) has been demonstrated. It has been shown that MYMV has a bipartite genome. Tandem dimeric constructs of DNA-A and DNA-B were infectious when inoculated together and induced systemic yellow mosaic symptoms. Agroinoculation of germinated seeds of blackgram was superior to inoculation of stems of seedlings. Infection of blackgram plants was confirmed by ELISA, dot blot and Southern hybridization. Agroinfected plants contained different forms of DNA characteristic of whitefly transmitted geminiviruses. The virus from agroinfected plants was transmissible by the whitefly Bemisia tabaci.

Sponge gourd (Luffa cylindrica) is an economically important vegetable crop cultivated throughout India. The crops were severely infected with begomovirus disease, showing yellow mosaic and leaf distortion symptoms, with 100% incidence at the end of season. The association of Tomato leaf curl New Delhi virus (ToLCNDV) with this disease has been reported earlier (Varma & Giri 1998; Sohrab et al. 2003; Tiwari et al. 2012).The virus was easily transmitted by sap to ridge gourd (Luffa acutangula) , sponge gourd and Nicot iana benthamiana. Inoculated plants produced typical yellow mosaic symptoms (Fig. 1). The sap transmission was confirmed by coat protein gene amplification, cloning and sequencing. The coat protein sequence (AY309957) showed highest identity (95.1%) with a severe strain of ToLCV-NDe (U15015). Several factors affecting the efficient sap transmission of begomovirus were identified, viz., buffer combinations, source and age of inoculum, genotypes and species of plants, temperature, seasons. Use of an antioxidant (sodium sulfite and β-mercaptoethanol) and abrasive (celite and corborundum) resulted in significant transmission. The optimal buffer combination (0.1M sodium phosphate buffer pH 7.2, 1:4 w/v, 0.15% sodium sulfite with a pinch of celite and corborundum powder) resulted in 100% infection on 7-day-old seedlings at room temperature during the summer season. The occurrence of sap transmission was observed to be 90% under field conditions. This is Phytoparasitica (2013) 41:591–592 DOI 10.1007/s12600-013-0319-8

The kaolin-based particle film Surround has been shown to suppress various insect pests and foliar diseases while reducing canopy temperature and improving water use efficiency in certain agricultural production systems. The usefulness of Surround was examined against important production constraints in peanut, including tomato spotted wilt, leaf spots, and aflatoxin contamination. Field experiments were conducted during 2001 using multi-varietal trials with or without spray treatment of Surround (75 lbs/acre) + NuFilm-17 (8 oz/acre). The effects of Surround application were evaluated for control of thrips and tomato spotted wilt on genotypes Georgia Green, C11-2-39, C34-24, and Sunoleic 97R; for control of leaf spots on AgraTech 201, GK 7 High Oleic, and C-99R; and for control of aflatoxin contamination on Aspergillus-inoculated plots of genotypes Georgia Green, AgraTech 201, and GK 7 High Oleic. In these experiments, Surround applications had little or no effect on thrips populations, tomato spotted wilt severity and incidence, leaf spot severity, drought stress, aflatoxin contamination, chlorophyll content, specific leaf area, and pod yield. In all experiments, host genotype effects were more effective in reducing disease and increasing yield than was Surround protection. Although foliar applications of particle films may be useful for producing certain vegetables and fruits, its benefits for addressing peanut production constraints are limited. Accepted for publication 7 April 2004. Published 19 April 2004.

) is an important spice crop cultivated through-out India. Leaf curl disease of Chilli has emerged as a serious problem inthe Jodhpur district, the major chilli growing area of Rajasthan state. Dur-ing December 2004, very high disease incidence (up to 100% of plants)was observed in farmers’ fields in Narwa and Tinwari villages. The char-acteristic field symptoms were upward curling, puckering and reduced sizeof leaves. Severely affected plants were stunted and produced no fruit. Thevirus from field samples from Narwa village was transmitted by whitefly(Bemisia tabaci) to 50–100% of chilli test plants, which produced veinclearing, curling and stunting symptoms. Electron microscopic examina-tion of field samples revealed few, typical geminate particles. The presenceof a begomovirus was confirmed by PCR using the degenerate primersAVF28 5′-GCCCACATYGTC TTYCCNGT-3 ′ and AV29R 5′-GGCTTYCTRT ACATRGG-3 ′, which gave a ca.1·0 kb product. Cloning and sequencingof the PCR product yielded a 995 bp sequence (Acc. No. DQ445255). ABLAST search of GenBank revealed close similarity of the sequence withthe intergenic region and part of the replication initiator protein, AV1 andAV2 genes of Chilli leaf curl virus-[Pakistan: Multan] (ChiLCuV-[Pk:Mul];AF336806) reported from Pakistan (Shih et al., 2003).In India, Tomato leaf curl New Delhi virus (ToLCNDV) was recentlyshown to be associated with chilli leaf curl disease occurring in Lucknow(Khan et al., 2006). Two more Begomovirus species, Cotton leaf curlMultan virus (CLCuMV) (Hussain et al ., 2004) and Pepper yellow leaf curlIndonesia virus (PepYLCIDV) (Tsai et al ., 2006), have been reported to beassociated with chilli leaf curl in Pakistan and Indonesia respectively.Sequence analysis showed that the virus isolated from Jodhpur wasdistantly related (59·1-67·9 % identity) to CLCuMV, PepYLCIDV andToLCNDV. However, it shared 96·5% identity with ChiLCuV-[Pak:Mul].Given the close sequence identity with ChiLCuV-[Pk:Mul], the virusisolated from Jodhpur is considered to be ChiLCuV. This is the firstreport of ChiLCuV affecting chilli in India and the recent report of Khanet al. (2006) show that chilli leaf curl in India is caused by more than onebegomovirus.ReferencesHussain M, Mansoor S, Iram S, Zafar Y, Briddon RW, 2004. First report of

Sponge gourd (Luffa cylindrica), an important cucurbitaceous vegetable in India, is affected by a disease (2) causing yellow spots on newly emerged leaves, mosaic, mild leaf curling and distortion, small leaves, and misshapen fruits. Nearly 100% of sponge gourd plants were symptomatic in Delhi. Geminivirus-like particles were observed with electron microscopy of uranyl acetate-stained leaf-dip preparations of the diseased plants collected from experimental fields at the Indian Agricultural Research Institute in New Delhi during May and June of 2002. The virus was transmitted by the whitefly (Bemisia tabaci) to sponge and ridge gourd (L. acutangula) after an acquisition and inoculation access period of 24 h each. Whitefly-inoculated plants produced typical yellow mosaic symptoms and contained geminate particles. Nucleic acid extracted from the field-infected and experimentally infected plants hybridized with 32P-labeled probe to DNA-A of Indian cassava mosaic virus, suggesting association of a begomovirus. The viral DNA, isolated by the alkali denaturation method (1) from the experimentally infected sponge gourd plants, was cloned in pBS SK+ at the EcoRI site. A clone with an insert of 2,658 bp was sequenced (GenBank Accession Nos. AJ557219, AJ555488, and AY309957) which shared 89.6 to 95.1% identity with the DNA-A of different strains of Tomato leaf curl virus-New Delhi (ToLCV-NDe). The highest sequence identity (95.1%) was with the severe strain of ToLCV-NDe (GenBank Accession No. U15015). The data suggest that the begomovirus associated with the yellow mosaic disease of L. cylindrica in India is a putative strain of ToLCV-NDe. Reference: (1) K. M. Srivastava et al. J. Virol. Methods 51:297, 1995. (2) A. Varma and B. K. Giri. Virus diseases. Pages 225–245 in: Cucurbits. N. M. Nayar and T. A More, eds. Oxford and IBH Publishing House Private Ltd., New Delhi, India, 1998.