Association of a recombinant Cotton leaf curl Bangalore virus with yellow vein and leaf curl disease of okra in India.

A begomovirus isolate (OY136A) collected from okra plants showing upward leaf curling, vein clearing, vein thickening and yellowing symptoms from Bangalore rural district, Karnataka, India was characterized. The sequence comparisons revealed that, this virus isolate share highest nucleotide identity with isolates of Cotton leaf curl Bangalore virus (CLCuBV) (AY705380) (92.8 %) and Okra enation leaf curl virus (81.1-86.2 %). This is well supported by phylogentic analysis showing, close clustering of the virus isolate with CLCuBV. With this data, based on the current taxonomic criteria for the genus Begomovirus, the present virus isolate is classified as a new strain of CLCuBV, for which CLCuBV-[India: Bangalore: okra: 2006] additional descriptor is proposed. The betasatellite (KC608158) associated with the virus is having more than 95 % sequence similarity with the cotton leaf curl betasatellites (CLCuB) available in the GenBank.The recombination analysis suggested, emergence of this new strain of okra infecting begomovirus might have been from the exchange of genetic material between BYVMV and CLCuMuV. The virus was successfully transmitted by whitefly and grafting. The host range of the virus was shown to be very narrow and limited to two species in the family Malvaceae, okra (Abelmoschus esculentus) and hollyhock (Althaea rosea), and four in the family Solanaceae.

Molecular characterization of distinct bipartite begomovirus infecting bhendi (Abelmoschus esculentus L.) in India.

Yellow vein mosaic disease of okra is a whitefly transmitted begomovirus causing heavy economic loss in different parts of India. The okra isolate (OY131) of this virus from a bhendi plant [(Abelmoschus esculentus L.) Moench] showing yellow vein mosaic, vein twisting, reduced leaves, and a bushy appearance in the Palem region, New Delhi, India, was characterized in the present study. The complete DNA-A and DNA-B sequences have been determined and are comprised of 2,746 and 2,703 nucleotides, respectively. The betasatellite (DNA-ß) component was absent in the sample. The genome organization was typically of biparite begomoviruses, which were characterized earlier. Comparison of DNA-A component with other known begomoviruses suggest that this virus, being only distantly related (<85.9% similarity with its nearest relative, BYVMV) to other known begomoviruses, is a new species. We have tentatively assigned the genome to a novel geminivirus species Bhendi yellow vein mosaic Delhi virus [BYVDV-IN (India: Delhi: okra)]. DNA-B showed highest sequence identity (87.8% identical) to that of a ToLCNDV (AY158080). The phylogenetic analysis of the present isolate is distinct from all other viruses; however clusters with ToLCNDV group infect different crops. The recombination analysis revealed that this isolate has sequences originated from ToLCNDV. This is the first known bhendi yellow vein mosaic disease associated bipartite begomovirus from India.

Diversity and phylogeography of Begomovirus-associated beta satellites of Okra in India.

BACKGROUND: Okra (Abelmoschus esculentus; family Malvaceae) is grown in temperate as well as subtropical regions of the world, both for human consumption as a vegetable and for industrial uses. Okra yields are affected by the diseases caused by phyopathogenic viruses. India is the largest producer of okra and in this region a major biotic constraint to production are viruses of the genus Begomovirus. Begomoviruses affecting okra across the Old World are associated with specific, symptom modulating satellites (beta satellites). We describe a comprehensive analysis of the diversity of beta satellites associated with okra in India. RESULTS: The full-length sequences of 36 beta satellites, isolated from okra exhibiting typical begomovirus symptoms (leaf curl and yellow vein), were determined. The sequences segregated in to four groups. Two groups correspond to the beta satellites Okra leaf curl beta satellite (OLCuB) and Bhendi yellow vein beta satellite (BYVB) that have previously been identified in okra from the sub-continent. One sequence was distinct from all other, previously isolated beta satellites and represents a new species for which we propose the name Bhendi yellow vein India beta satellite (BYVIB). This new beta satellite was nevertheless closely related to BYVB and OLCuB. Most surprising was the identification of Croton yellow vein mosaic beta satellite (CroYVMB) in okra; a beta satellite not previously identified in a malvaceous plant species. The okra beta satellites were shown to have distinct geographic host ranges with BYVB occurring across India whereas OLCuB was only identified in northwestern India. Okra infections with CroYVMB were only identified across the northern and eastern central regions of India. A more detailed analysis of the sequences showed that OLCuB, BYVB and BYVIB share highest identity with respect ßC1 gene. ßC1 is the only gene encoded by beta satellites, the product of which is the major pathogenicity determinant of begomovirus-beta satellite complexes and is involved in overcoming host defenses based on RNAi. CONCLUSION: The diversity of beta satellites in okra across the sub-continent is higher than previously realized and is higher than for any other malvaceous plant species so far analyzed. The beta satellites identified in okra show geographic segregation, which has implications for the development and introduction of resistant okra varieties. However, the finding that the ßC1 gene of the major okra beta satellites (OLCuB, BYVB and BYVIB) share high sequence identity and provides a possible avenue to achieve a broad spectrum resistance.

Fruit Distortion Mosaic Disease of Okra in India.

Okra (Abelmoscus esculentus (L.) Moench) is an important vegetable crop of India and other subropical and tropical countries. In 2000 and 2001, in the states of Karnataka and Tamil Nadu, okra was severely affected by a new disease. Since that time, the disease has spread to other states: Andhra Pradesh, Madhya Pradesh, Haryana, and Maharashtra. Chlorotic spots, chlorotic leaf blotches, distortion of leaves, chlorotic streaking, distortion of fruits, and severe yield losses as much as 63% characterize the disease. The causal virus induces local and systemic chlorotic and necrotic lesions on Vigna unguiculata (L.) Walp. cv. C-152 and Chenopodium amaranticolor Coste & Reyne., chlorotic local lesions and mosaic on Cucumis sativus L., necrotic local lesions on Gossypium hirsutum L. and black gram (Vigna mungo L.), and chlorotic local lesions and systemic necrosis on sunflower (Helianthus annuus L.). Host reactions on these species are similar to those described for the ilarvirus Tobacco streak virus (TSV) (3). Electron microscopic observation of leafdip preparations from field samples and partially purified virus preparations revealed the presence of isometric virus particles measuring 25 to 30 nm in diameter. The virus was purified from mechanically inoculated okra by differential and sucrose density gradient centrifugation, and disease symptoms were reproduced in okra mechanically inoculated with the purified virus. In direct antigen coated enzyme-linked immunosorbent assay and immunosorbent electron microscopy tests, the purified virus and sap extracts reacted positively with polyclonal antibodies to TSV, the ilarvirus associated with sunflower necrosis and peanut stem necrosis diseases (1,2), but did not react positively to Turnip mosaic virus and Okra mosaic virus that are previously reported to infect okra. In reverse transcription-polymerase chain reaction (RT-PCR), using oligonucleotide primers designed to amplify the entire coat protein region of TSV, an approximately 800-bp DNA fragment was obtained from purified virus and okra displaying fruit distortion mosaic disease (OFDM) but not from healthy okra. On the basis of host range, serological relationship, electron microscopy, and RTPCR amplification, the virus causing OFDM is an ilarvirus closely related to TSV. To our knowledge, this is the first report of the occurrence of an ilarvirus in okra, and is the third and most recent report of an ilarvirus related to TSV causing disease in crops on the Indian subcontinent (1,2). References:(1). A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2). A. S. Reddy et al. Plant Dis. 86:173, 2002. (3). S. W. Scott. Tobacco streak virus. No 381 in: Descriptions of Plant Viruses. CMI/AAB, Surrey, U.K., 2001.

Outbreak of Tobacco streak virus Causing Necrosis of Cucumber (Cucumis sativus) and Gherkin (Cucumis anguria) in India.

Cucumber (Cucumis sativus L.) and Gherkin (Cucumis anguria L.) are important cucurbitaceous vegetables grown in India for slicing and pickling. During the 2000 to 2002 rainy season and summer, a new virus disease, causing yield losses of 31 to 75% in Bangalore, Bellary, Davanagiree, and Tumkur districts of Karnataka State, infected cucumber and gherkin. Symptoms were tip necrosis characterized by necrotic lesions on leaves, and a general leaf and stem necrosis extending to mid veins, petioles, flower buds and tip, eventually resulting in dieback of vines. Tissue extracts from symptomatic leaves of cucumber and gherkin were mechanically inoculated on several herbaceous indicator plants (cowpea, cucumber, pepper, Zinnia, watermelon, Chenopodium amaranticolor, sunflower, Nicotiana glutinosa, N. tabacum, and Gomphrena globosa). On most hosts, symptoms of chlorotic or necrotic lesions followed by mottle or systemic necrosis were observed. Back-inoculation from the symptomatic indicator plants onto cucumber and gherkin resulted in symptoms typical of those observed in the field. Electron microscopic examination of leaf-dip preparation and ultra thin sections of virus infected plant samples showed the presence of isometric particles 25 to 28 nm in diameter. Similar types of particles were observed when infected samples were trapped in immunosorbent electron microscopy with polyclonal antibodies specific to Tobacco Streak virus (TSV) but not to Watermelon silver mottle virus (WSMV). Enzymelinked immunosorbent assay tests using leaf extracts of field-collected samples and sap-inoculated plants showed positive reaction to antibodies of TSV (1) but not to antibodies of Cucumber mosaic virus, WSMV, Watermelon bud necrosis virus, Papaya ring spot virus W strain, and Zucchini yellow mosaic virus. Reverse transcription-polymerase chain reaction (RT-PCR) of RNA extracts of infected samples of field and inoculated symptomatic plants was done by using primers derived from TSV RNA3 specific for the coat protein (CP) region of TSV (2). A 800-bp specific DNA fragment was amplified from infected cucumber and gherkin but not from healthy control plants. Sequence analysis of cloned PCR fragments revealed nucleotide identities of 99% with TSV isolates from cotton, mungbean, sunnhemp, and sunflower (GenBank Accessions Nos. AF515824, AF515823, AF515825, and AY061929) and 88% with TSV-WC (GenBank Accession No. X00435). On the basis of host range, serological relationship, electron microscopy, and sequence analysis of the CP region, the virus was identified as a strain of TSV. To our knowledge, this is the first report of natural occurrence of TSV on cucumber and gherkin in India. References: (1). A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2). B. J. C. Cornelissen et al. Nucleic Acids Res.12:2427, 1984.