Isolation and characterization of Pepper leaf curl virus PepLCV the suspected causative agent of leaf curl disease LCD in bell pepper hot pepper and tomato crops in different districts of Himachal Pradesh

Abstract

newline xvi newlineABSTRACT newlineGeminiviridae is the largest and economically one of the most important plant virus families. newlineAmong total nine genera of the family, the largest genus Begomovirus consists of more than 320 newlinerecognized species. The members of the genus have a bipartite (with DNA-A and DNA-B, as newlinetwo circular ssDNA components of 2.5-2.6 kb in length) or monopartite genome (having DNAA, newlinea circular ssDNA molecule of ~2.7 kb in length). The leaf curl disease (LCD) has been a newlinemajor constraint on cultivation and production of peppers and tomato crops in India. newlineSymptomatic bell-pepper, chilli and tomato plant samples were collected from five districts of newlineHimachal Pradesh state of India. PCR-based detection was done using coat protein specific newlineprimers for Pepper leaf curl virus, which are highly conserved throughout the begomoviral newlinespecies. It was 35 samples out of total 94 symptomatic samples of bell-pepper and 11 samples newlineout of total 40 samples of chilli plants which showed positive amplification. No amplification newlinewas observed in total 40 symptomatic tomato plant samples. Different sets of primers were newlinedesigned for amplification of DNA-A component of PepLCV and associated DNA-and#946; satellites. newlineThe respective amplicons were cloned in E. coli (DH5and#945;) using pGEM-T vector and sequenced. newlineOverall 10 viral isolates (viz., BC1-R1, SPC3, SPP4, SPP7, SPP10, V-Bell, SPUR1 and SiKP7 newlineinfecting bell-pepper, while KP28 and BP32 infecting chilli) have been identified belonging to newlinegenus Begomovirus on basis of their CP gene sequences. Among all these identified begomoviral newlineisolates, the whole genome sequencing was able to be achieved only for 4 isolates (viz., BC1-R1, newlineSPC3, SPP4 and SiKP7) which were confirmed as variants of single strain of ChiLCV-India newline(MK161454) with 98% to 99% identity showing close phylogenetic relationship. Five DNA-and#946; newlinesatellite sequences associated with the identified isolates (viz., SPC3, SPP4, SiKP7, SPP10 and newlineKP28) were found to be new variants of Chilli leaf curl betasatellites (ChiLCBs) with gt78% newlinenucleotide identity. While, one DNA-and#946; satellite associated with SPUR1 isolate was found to be newlinenew variant of Tomato leaf curl Bangladesh betasatellite (ToLCBB) with 93% nt identity. The newlinestatus of association of helper DNA-and#946; satellites with SPP7 and V-Bell isolates stayed newlineunconfirmed. But, BC1-R1 and BP32 isolates were confirmed not having any association with newlinehelper DNA-and#946; satellites, whereby they produced only moderate to mild form of disease newlinesymptoms in their infected host plants. Variations at specific amino acid positions were also newlinetraced CP and and#946;C1 protein sequences using MultAlin. newlineKeywords: Geminiviridae, Begomovirus, Leaf curl disease (LCD), Pepper leaf curl virus, Chilli newlineleaf curl virus, DNA-A, DNA-and#946; satellites.