Molecular Detection and Characterjzatlon Of a Begomovirus Causing Leaf Curl Disease of Pot a to (Solanu1n Tuberosutn)

Abstract

Potato (Solanum tuberosum) is one of the most widely cultivated vegetables crops of India. Potato Apical Leaf Curl Disease (PALCO) is an important factor affecting potato production on Indian subcontinent and is caused by begomoviruses transmitted by whitefly Bemisia /abaci. During January'2014, very high disease incidence (up to 5--7%) was observed in potato fields ofLawar, Macchri, Dulhera, Surani and Pabli khas villages of Meerut district. Symptoms similar to tomato leaf curl, such as stunting, crinkling, vein thickening and leaf distortion were observed in the potato fields. To confirm the associatjon of begomovirus with the disease, polymerase chain reaction (PCR) was performed using total DNA extracted from infected potato leaves and four different sets of primers designed to amplify coat protein region, replication protein region, complete DNA-Band betasatellite DNA. Amplifications of expected size i.e. (-770 bp) for coat protein region, (-1086 bp) for :eplication protein region and (- 2700) for DNA-B were produced. No amplification was observed for betasatellite DNA. These amplified products were cloned and sequenced. The bands corresponding to coat protein ge~e and replication protein gene were eluted and cloned in pTZ57RIT and in pGEM-T Easy c!oning vectors which was followed by confirmation using cdlony PCR, plasmid DNA isolation and Restriction digestion approaches. Rolling Circle Amplification was also performed and the amplified product was eluted and cloned in pTZ 19R which w~s further confirmed using plasmid DNA isolation and Restriction digestion. T~e positive results were subjected to sequencing and were further subjected to in-silico analysis. The sequences obtained from coat protein gene and replication protein gene were submitted to GenBank, NCBI with accession no. AB9761 05 and AB9761 04. The sequence data was further validated by BLAST analysis. and phylogene~ic tree was constructed using MEGA5.0 software which revealed close similarity of sequences with coat protein gene (A VI) and replication protein gene (AC I) components of other potato begomoviruses, which are all tentative strains of Tomato Leaf Cur/ New Delhi Virus (ToLCNDV). Based on these findings the virus infecting potato has been tentatively identified as an isolate ofToLCNDV with bipartite genome. Widespread distribution and diversity of begomoviruses pose a threat to world agricultural system. The cloned components can be used to detem1ine pathogenicity of the individual component and can further be used to develop resistant plant varieties using biotechnological approaches.