Indian Tomato Leaf Curl Virus Research Articles

Evolutionary and molecular aspects of Indian tomato leaf curl virus coat protein.

Tomato leaf curl disease (ToLCD) is manifested by yellowing of leaf lamina with upward leaf curl, leaf distortion, shrinking of the leaf surface, and stunted plant growth caused by tomato leaf curl virus (ToLCV). In the present study, using computational methods we explored the evolutionary and molecular prospects of viral coat protein derived from an isolate of Vadodara district, Gujarat (ToLCGV-[Vad]), India. We found that the amino acids in coat protein required for systemic infection, viral particle formation, and insect transmission to host cells were conserved amongst Indian strains. Phylogenetic studies on Indian ToLCV coat proteins showed evolutionary compatibility with other viral taxa. Modeling of coat protein revealed a topology similar to characteristic Geminate viral particle consisting of antiparallel β-barrel motif with N-terminus α-helix. The molecular interaction of coat protein with the viral DNA required for encapsidation and nuclear shuttling was investigated through sequence- and structure-based approaches. We further emphasized the role of loops in coat protein structure as molecular recognition interface.

English

Sequence alignment of the 897 bp amplicon obtained from a diseased sunn hemp (Crotalaria juncea L.) plant DNA revealed a complete 771 bp coat protein (CP) gene flanked by 3’ regions of the AV2 and AC3 genes. Southern hybridization using (α-32P) dCTP labeled (CP) gene probe of Indian tomato leaf curl virus (IToLCV) demonstrated the association of begomovirus with the leaf curl disease of sunn hemp. Phylogenetic data suggested that, the AV2, CP and AC3 genes have closest genetic relationship with begomovirus isolates from India, China and Bangladesh, respectively. In silico recombination analysis elucidated a 297 nucleotides hot spot (346 to 643 nucleotides) within AV2 overlapping region of CP gene, amenable to genetic rearrangements, with lineage from tomato leaf curl virus Bangalore (ToLCuVB) and Indian cassava mosaic virus-Ind (ICMV) as major and minor parents, respectively. Thus, it is concluded that the recombinant CP genes related to begomoviruses are evolved from the Indian isolates, causing broad host specificity and molecular diversity among the related begomoviruses across the geographical limits of Southeast Asia. Key words: Begomovirus, sunn hemp, coat protein, recombination, phylogenetic analysis, in silico analysis.

Association of a Geminivirus with a Leaf Curl Disease of Sunn Hemp (Crotalaria juncea) in India

Natural occurrence of a geminivirus causing severe leaf curl disease on sunn hemp (Crotalaria juncea) was recorded in India. The association of a geminivirus with the disease was demonstrated by whitefly transmission tests and polymerase chain reaction (PCR) amplification of DNA fragments of expected sizes with three pairs of degenerate geminivirus primers. The PCR-amplified viral DNA fragments were further characterized by Southern hybridization with a geminivirus probe consisting of the cloned coat protein (CP) gene of Indian tomato leaf curl virus (ITLCV). Restriction fragment length polymorphism analysis of a PCR-amplified CP fragment revealed that the geminivirus from sunn hemp was different than ITLCV.

The natural occurrence of a begomovirus in sunn hemp (Crotalaria juncea) in India

Sunn hemp (Crotalaria juncea) is cultivated in India and other tropical countries as a fibre or green manure crop. During July/August 2001, a severe virus-like disease affected 80–90% of sunn hemp plants in experimental plots at the National Botanical Research Institute, Lucknow. The symptoms of the disease consisted of interveinal chlorosis, yellowing, curling and shortening of leaves, and stunting of plants. Whiteflies (Bemisia tabaci) transmitted the associated virus from diseased to healthy sunn hemp plants which developed symptoms similar to those occurring in naturally infected plants. Total DNA was isolated from the infected-leaf tissues and subjected to polymerase chain reaction (PCR) using begomovirus specific universal primers (AV494/AC1048) which were designed from the conserved sequences of the core region of the coat protein (CP) genes of several begomoviruses (Wyatt & Brown, 1996). As expected, a fragment of c. 500 bp was amplified from the DNA extracted from diseased, but not healthy, leaves. The authenticity of the PCR-amplified DNA fragment was confirmed by Southern hybridization in which DNA probes, prepared from the CP gene of Indian tomato leaf curl virus (IToLCV; Srivastava et al., 1995), cross hybridized with the PCR fragment under high stringency conditions. Although Sunn-hemp mosaic virus and some strains of Tobacco mosaic virus infect sunn hemp (Brunt et al., 1996), a begomovirus has not hitherto been reported to naturally infect this important crop species. Transmission of the sunn hemp virus by whiteflies, PCR amplification of its DNA with begomovirus specific primers and its strong hybridization with a DNA-A probe of IToLCV strongly indicate that it is a begomovirus. The extent of its relationship to IToLCV (Family Geminiviridae; Genus Begomovirus) and other begomoviruses is now being further investigated.

Detection of a Whitefly-Transmitted Geminivirus in Launaea spp. in India.

Launaea acaulis (Roxb.) Babc. Aerr. and L. procumbeus (Roxb.) Ramayya & Rajgopal are weeds that grow near agricultural fields in India during the rainy season. Yellow net symptoms were observed in both species, but not in crops, in 1998 and 1999. Nicotiana tabacum cv. White Burley developed leaf curl symptoms after virus transmission from L. acaulis by whiteflies (Bemisia tabaci). Thus, the involvement of a geminivirus was suspected. Total DNA from infected and uninfected tobacco and Launaea plants was extracted by the cetyl trimethyl ammonium bromide method and precipitated by ethanol. Polymerase chain reaction (PCR), using DNA from infected and uninfected plants as a template and degenerate oli-gonucleotide primers designed from DNA-A of whitefly-transmitted African cassava mosaic virus (ACMV) (1) was performed. An expected ≈500-bp fragment was observed after agarose gel electrophoresis of the PCR product. Total DNA from infected plants gave positive signals and was homologous with the probe from cloned DNA-A of Indian tomato leaf curl virus (2) in high-stringency Southern hybridization tests. No PCR amplification and positive signals were obtained from DNA from uninfected plants. PCR amplification of the 500-bp fragment with primers from DNA-A of whitefly-transmitted ACMV resulted in positive signals, and homology with the probe indicated that L. acaulis and L. procumbens harbor whitefly-transmitted geminivirus infections. This is the first report of geminivirus infection in these Launaea spp. in India.

The contribution of tomato and alternative host plants to tomato leaf curl virus inoculum pressure in different areas of South India

Summary.Indian tomato leaf curl virus (ToLCV) (Geminiviridae: Sub‐group III) was detected both in field‐collected and laboratory‐reared B. tabaci using a triple‐antibody sandwich enzyme‐linked immunosorbent assay (TAS‐ELISA). ToLCV was detected in six of the 10 group samples of field collected B. tabaci. ToLCV was also identified in 13 weed species commonly found in Karnataka, both by symptom expression and TAS‐ELISA. ToLCV from c. 61% of infected plants was transmitted successfully to tomato by B. tabaci.Tomato plots were planted at three locations on the University of Agricultural Sciences Campus, Bangalore. Indian tomato leaf curl virus disease (ToLCVD) incidence increased most rapidly when the tomato plot was situated adjacent to an older ToLCVD‐infected tomato field. When the plots were positioned in a dryland or a wetland area, at least 500 m away from any infected tomato fields, the ToLCVD incidence increased less rapidly, although in all sites it was 100% by 11 wk after transplanting. The numbers of B. tabaci caught on yellow traps in all sites increased during weeks 1–3 after transplanting and thereafter remained at between 10–15 adults trap‐1 24 h_1. Adult numbers recorded on tomato plants by direct counts remained approximately constant at 2–4 adults plant“”1.Tomato fields were planted in three taluks (administrative areas) of Karnataka, that had different current and previous histories of tomato production. ToLCVD incidence increased most and least rapidly, respectively, in Kolar taluk where tomato is grown continuously and Doddaballapur tuluk where tomato was grown in the area for the first time. In Malur tuluk, where tomato was grown discontinuously (once a year), the incidence of ToLCVD increased at an intermediate rate.Weed host‐plant species growing near the experimental sites had averages of between 1.5–10.0 B. tabaci nymphs per plant, whereas the tomato plants had only 0.3 nymphs per plant. The percentage parasitism of B. tabaci nymphs on tomato and weed species, respectively, was 0.7% and 2–6%. Nymphs and pupae were parasitised by an Encarsia sp. and Eretmocerus mundus Mercet.The relevance and implications of these findings for the epidemiology and management of ToLCVD in Karnataka State, South India is discussed.

Yellow Net of Triumffeta Is Caused by a Geminivirus: A First Report.

Triumffeta rhomboidiaceae Jacq. (Tiliaceae family) is an annual rainy season weed that is commonly found throughout India. For the last 3 years, during the rainy season, several plants of T. rhomboidiaceae in and around the gardens of the National Botanical Research Institute have been found with vein yellowing symptoms. The initial symptoms were vein clearing but in later stages the veins became yellow and thickened. In severe cases, the chlorosis extends into interveinal areas, resulting in complete yellowing of the leaves. In a few cases, green leafy or thorny enations could be seen on the dorsal side of the leaf. The disease was investigated to identify the causal agent. Vector transmission studies showed that the causal agent is transmitted by the whitefly, Bemisia tabaci, from infected to healthy seedlings of T. rhomdoidiaceae. Since whitefly transmission of the disease is consistent with a geminivirus as the causal agent, the role of such a virus was investigated. DNA isolated from Triumffeta plants (both from the infected plants in the field as well as from those inoculated experimentally in the greenhouse) showing above mentioned symptoms was amplified with two sets of degenerate primers, PAL1v1978/PAR1c496 (set 1) and PAL1v1978/PCRc1 (set 2), that have been shown to be specific for DNA-A of whitefly transmitted geminiviruses (WTGs), in polymerase chain reaction (1). We could amplify DNA-A fragments of approximately 1.2 kb from set 1 and 0.7 kb from set 2, as expected (1). DNA isolated from healthy seedlings gave no amplification of such fragments. Identification of the amplified DNA fragments (from infected samples) to be of geminiviral in nature was confirmed by Southern blot hybridization carried out under high stringency conditions. DNA-A of Indian tomato leaf curl virus (2) was used as a general probe for WTGs for the above hybridization experiment. Therefore, Triumffeta yellow net disease is caused by a geminivirus. A review of literature revealed that there is no record of a viral disease affecting this weed and, therefore, this is the first report of a viral disease affecting this plant.

Leaf Curl Disease of Carica papaya from India May Be Caused by a Bipartite Geminivirus.

Papaya has considerable economic importance to agriculture in India. Papaya leaf curl disease was first reported in 1939 by Thomas and Krishnaswamy (3). This disease is of moderate incidence and widely distributed in India. Recent observations of papaya fields in India indicated that there has been a continued increase in the incidence of papaya leaf curl disease (as shown by symptoms), resulting in severe economic losses. The disease is characterized by downward curling and cupping of leaves followed by vein clearing and thickening. Enations develop in the form of frills on green veins. The affected leaves become leathery and brittle and the petioles become twisted in a zig-zag manner. Diseased plants may bear a few small fruits, which are distorted in shape and tend to fall prematurely. The disease could be transmitted by the whitefly Bemisia tabaci Genn. Therefore, possible involvement of a geminivirus was suspected. Three different cloned geminiviral DNAs, Indian tomato leaf curl virus (ITLCV) (2), tomato yellow leaf curl virus from Sardinia (TYLCV Sar), and tomato golden mosaic virus (TGMV), were used as probes (with radioactive labeling) to detect the presence of geminiviral DNA from infected papaya tissue in both slot-blot and Southern blot hybridization studies with high stringency washes. These DNA probes gave strong signals with DNA isolated from infected papaya tissue whereas they did not give any signals with DNA from healthy tissue. Further, successful polymerase chain reaction (PCR)-based amplification of fragments from both DNA-A and DNA-B components with geminivirus degenerate primers (1) was accomplished only from the DNA of infected papaya plants. The PCR-amplified DNA fragments gave positive signals in Southern blot hybridization with the three geminiviral DNA probes. These results suggest that the causal agent of papaya leaf curl disease is a bipartite geminivirus that may be provisionally called papaya leaf curl virus (PLCV).

Ageratum, Croton and Malvastrum harbour geminiviruses : evidence through PCR amplification

Ageratum conyzoides, Croton bonpladianum and Malvastrum coromandelianum are common weeds found around agricultural fields. In several cases these were found to exhibit vein yellowing and yellow mosaic symptoms. Using degenerate primers specific for whitefly-transmitted geminiviruses (WTGs), and total DNA isolated from such infected plants (exhibiting the above symptoms) as a template, 1.2kbp fragments were amplified and were shown to have homology to DNA-A of Indian tomato leaf curl virus (ITLCV) by Southern hybridization. In control experiments the same primers failed to amplify any DNA fragments from the total DNA isolated from healthy plants (no symptoms as above). These results show that Ageratum, Croton and Malvastrum harbour geminivirus(es).

Serological studies on the accumulation and localisation of three tomato leaf curl geminiviruses in resistant and susceptible Lycopersicon species and tomato cultivars

SummaryAccessions of wild Lycopersicon spp. and selected Fl hybrid tomato cultivars were compared for their resistance to three whitefly‐transmissible geminiviruses: Indian tomato leaf curl virus (ITmLCV) and tomato yellow leaf curl viruses from Sardinia (TYLCV‐Sar) and Senegal (TYLCV‐Sen). The resistance of different plant lines was expressed in different ways but in most instances a given line reacted similarly to graft inoculation with the three viruses. L. pimpinellifolium LA1478 produced as much virus antigen, assessed by triple antibody sandwich‐ELISA, as the susceptible cv. Moneymaker but developed only very mild symptoms and is therefore tolerant of infection. In L. hirsutum LA1777 and L. peruvianum CMV‐INRA, very mild or no symptoms developed but antigen concentrations were substantially less than in Moneymaker. L. chilense LA1969 remained symptomless and its antigen concentration was < 1% of that in Moneymaker. Symptoms were mild or barely evident in the Fl hybrid cultivars. Cultivars Tyking and Fiona had antigen concentrations about 5–10% of those of Moneymaker, whereas TY20, Top 21 and Tyger had intermediate antigen concentrations. In a few instances, the extent to which virus accumulation was restricted depended on the challenge virus. Accumulation of TYLCV‐Sen in TY20, Top 21 and Tyger was less affected than that of the other two viruses, and accumulation of TYLCV‐Sar in accessions LA1777 and CMV‐INRA was less affected than that of TYLCV‐Sen or ITmLCV.Tissue‐printing tests showed that ITmLCV and TYLCV‐Sen antigens were confined to phloem tissue. In Tyking, the number of virus antigen‐containing phloem traces and the antigen content of individual traces were less than in Moneymaker but the partitioning of antigen between internal and external phloem was unaffected.

Host range, vector relations and serological relationships of cotton leaf curl virus from southern India

SummaryIn 1989 to 1991, leaf curl disease was observed in cotton (Gossypium bar‐badense cv. Local) grown in kitchen gardens in five districts in Karnataka State, India, and in 1994 it was recorded in G. hirsutum cv. Sharada in two districts. Symptoms consist of leaf curling, vein thickening, leaf enations, and stunting and distortion of plants. The disease is caused by cotton leaf curl virus (CLCuV‐K), which was transmitted by the whitefly Bemisia tabaci to 24 plant species in six families. Hosts include bean (Phaseolus vulgaris), pepper, tobacco, tomato and several weeds, almost all of which developed leaf curl, with or without vein thickening. CLCuV‐K was transmitted from cotton to cotton by adult B. tabaci after an acquisition access period of 1 h, could be inoculated in 5 min, had a minimum latent period of 8 h and was retained by viruliferous insects for up to 9 days. Female B. tabaci transmitted more frequently than males. CLCuV‐K is a whitefly‐transmitted geminivirus. It reacted with two out of 17 monoclonal antibodies (MAbs) raised to African cassava mosaic virus and five out of 10 MAbs raised to Indian cassava mosaic virus. CLCuV‐K isolates from different locations in Karnataka had similar epitope profiles. As judged by these profiles, CLCuV‐K is closely related to Indian tomato leaf curl virus from Karnataka, is distinguishable from several other whitefly‐transmitted geminiviruses found in India and is still more distantly related to those, including cotton leaf crumple virus from the USA, found in other continents.CLCuV‐K infected all cultivars tested of G. barbadense and one of six cultivars of G. hirsutum but none of G. arboreum or G. herbaceum.

Transmission of tomato leaf curl geminiviruses by Bemisia tabaci: effects of virus isolate and vector biotype

SUMMARYCultures of Bemisia tabaci from Ivory Coast (IC), Pakistan (PK) and USA (US B‐type) were compared for the frequency with which they transmitted three tomato geminivirus isolates: Indian tomato leaf curl virus from Bangalore (ITmLCV), and tomato yellow leaf curl viruses from Nigeria (TYLCV‐Nig) and Senegal (TYLCV‐Sen). Frequency of transmission from tomato to tomato depended both on the whitefly culture and the virus isolate. US B‐type and IC whiteflies transmitted TYLCV‐Sen more frequently than ITmLCV whereas PK whiteflies transmitted ITmLCV more frequently than TYLCV‐Sen. US B‐type whiteflies transmitted both viruses four to nine times more frequently than IC whiteflies. TYLCV‐Nig was transmitted rarely by US B‐type and not at all by IC whiteflies.Previous work indicates that the geminivirus coat protein controls vector transmissibility. The differential adaptation of TYLCV‐Sen to transmission by US B‐type whiteflies and of ITmLCV to PK whiteflies was associated with a large difference in epitope profile of the coat proteins of the two viruses. Also, the readily transmissible TYLCV‐Sen differed appreciably in epitope profile from the poorly transmissible TYLCV‐Nig, which reached a consistently greater concentration in source tissues but lacked epitope 18. However, the lack of epitope 18 in ITmLCV did not prevent its transmission by US B‐type whiteflies. Differences in frequency and specificity of geminivirus transmission by whitefly cultures from different countries therefore were associated with differences among epitope profiles of the coat proteins of the viruses, but the structural features of the proteins that control transmission remain to be determined.