Molecular characterization of bhendi yellow vein mosaic virus

Abstract

Bhendi/Okra (Abelmoschus esculents (L.) Moench) is one of the important vegetable crops grown extensively throughout the tropical, subtropical and warm regions of the temperate zones of the world. Bhendi yellow vein mosaic virus (BYVMV) is one of the most sever disease which take a heavy toll in India. A roving survey revealed that the occurrence of BYVMV disease incidence ranged from to be 23.0 to 67.67% in Kamataka, 45.89 to 56.78% in Andhra Pradesh, 23 to 75.64% in Tamil Nadu, 42.45 to 75.64% in Kerala, 23 to 85.64% in Maharashtra, 24.85 to 65.78% in Haryana, 35.76 to 57% in Uttar Pradesh, 45.45% in Delhi, 67.78% in Chimdigarh and 45.89 to 66.78% in Rajasthan and The highest disease incidence was recorded in the districts of Gulbarga (75.0%) and least is in Bangalore rural (44.01%). Single whitefly of B biotype and two whiteflies indigenous B. tabaci could able to transmit BYVMV with 30% and 20% efficiency. Nine B biotype and 10 indigenous viruliferous whiteflies required for 100% BYVMV transmission. Minimum AAP and lAP found to be 15 mints, in B biotype and 20 mint. in indigenous whiteflies. 100% transmission obtained in 24 hrs. AAP and 16 hrs. lAP given to B- biotype compared to indigenous whitefly which required 24 hrs. AAP and lAP. Females of B-biotype and indigenous B. tabaci were more efficient in transmitting BYVMV compared to males. Age group of 7-15 days old Bhendi plants were found more susceptible for infection. BYVMV was successfully purified from the systemically infected okra leaves using the modified procedure with addition of sucrose gradient. The UV spectrum observations of 260/280 ratios showed 1.20-1.27 OD with the yield of 12-15 mg/Kg of infected leaves. SDS-PAGE analysis and western blotting of the purified preparation resulted in the presence of band with an approximate molecular weight of 28000 Daltones, which is expected monomeric size of BYVMV coat protein. The direct antigen coating ELISA (DAC-ELISA) was standardized and successfully used to detect virus using polyclonal antibody raised against ACMV and goat anti-mouse IgG-alkaline phosphatase conjugate. The ELISA results showed that BYVMV detect in infected leaf extracts up to 1:10000 antigen dilution. However, optimum reaction has been obtained at 1: 1000 antigen concentrations. Further the BYVMV also detect in infected flowers, pollen, petals, calyx and fruits but not in seed. Dot immuno binding Assay (DIBA) was standardized and successfully used to detect virus using polyclonal antibody raised against ACMV. The result showed that BYVMV detected in non purified sample up to 1: 1000 antigen dilution and 1: 10000 dilution of purified virus with the antibody dilution of 1: 500 from the infected leaf tissues. Dot blot hybridization was standardized using diagoxigenin labeled probes. The methods were found to be equally sensitive in detecting viral nucleic acid up to a concentration of O.OO1 ng from purified BYVMV. Further, the BYVMV isolates were detected using DNA was extracted from the infected leaf tissues. This method could also be used as diagnostic tool for identification of BYVMV with more precision. The complete DNA-A sequence of 113 isolates nucleotide sequence of the DNA-A genome of different isolates size ranged from 27722 to 2793nt. The complete nucleotide sequence of 113 isolates has 84 to 100% similarity among themselves. Ten groups are formed in phylogenetic grouping that have sequence identity of less than 89% with other reported begomoviruses. Ten species were identified as distinct from already known begomovirus species in India. These are Bhendi yellow vein Haryana virus (BYVHV), Bhendi yellow vein Guntur virus (BYVGV), Bhendi yellow vein India virus (BYVIV), Bhendi yellow vein Kamataka Virus (BYVKV), Okra leaf curl India virus (OKLCIV). Bhendi Yellow Vein Kerala Virus (BYVKV), Bhendi Yellow Vein Delhi Virus (BYVDV), Bhendi Yellow Vein Trichy Virus (BYVTV), Bhendi Yellow Vein Bhubaneswar Virus (BYVBV), Okra Enation Leaf Curl Virus (OELCV). Most of isolates of BYVMV showed high percentage of nucleotide sequence and amino acid identity with in ORFs AVl, AV2, ACI AC2 AC3, AC4, AC5 and intergenic region with BYVMV.NOL751, BYVMV-Madurai, OYVMV, BYVMV-Pak301, CLCuVA and (CLCuMV-(Okra] and some isolates showing the nucleotide sequence and amino acid identity ToLCNDV-A VT. The complete DNA-B sequence of 7 isolates nucleotide sequence genome of different isolates size ranged from 2695nt (OY77, OY81, OY174) to 2707nt (OYI64). The complete nucleotide sequence of 7 isolates has 84 to 100% similarity among themselves lead to form four groups which are associated with okra yellow vein mosaic disease namely the isolates OY77, OY142, OY81 and OYT closely with Tomato leaf curl New Delhi luffa isolates (EF620535) and Tomato leaf curl New Delhi AVTl isolate (AY438563 ToLCNDV [Lucknow] and ToLCNDV-sever [Jessor] isolates. the isolate OY164 form separate group with ToLCNDV-AVTl and ToLCNDVluffa. Pk. and one isolate OY131 which shows the nucleotide identity of less than 85% with other okra DNA-B we considered as distinct species of okra yellow vein virus. The full length of 36 isolates Beta DNA molecules ranging in size from 1324 to l403nt in length the sequence of these presumed full length DNA ~ molecules have these conserved 207 features an A-rich region, a satellite conserved region (SCR), and a single open reading frame (putative coding region of gene C 1). The complete nucleotide sequence of 36 Beta DNA molecule revealed the occurrence of four types of Beta DNA molecules were present among the isolates viz., Bhendi yellow vein beta satellite (BYVB), okra leaf curl betasatellite (OLCuB), Ludwigia leaf distortion betsatellite (LuLDB), Croton yellow vein mosaic beta satellite (CroYVMB) and these may are associated with BYVMV. 57 different genotypes were screened for BYVMV under glasshouse and 29 different genotypes under natural conditions. None of genotypes tested was highly resistant or immune. Only 3 genotypes showing resistant reaction, 3 genotypes showing moderately resistant.