DETECTION AND CHARACTERISATION OF TOBACCO LEAF CURL VIRUS ISOLATES INFECTING FCV TOBACCO IN INDIA

Abstract

Tobacco leaf curl disease is widespread in several states in India causing economic losses to farmers. In the present study an attempt has been made to identify and characterize 11 tobacco leaf curl virus isolates collected from FCV tobacco growing region of Andhra Pradesh and Karnataka based on virus coat protein gene (cp). PCR primers specific to coat protein gene (cp) of tobacco leaf curl virus were designed based on the sequences of tobacco leaf curl virus-Karnataka1 (TbLCV-Kar1) and tobacco leaf curl virus-Karnataka2 (TbLCV-Kar2) genes available in the NCBI data base and used to amplify the DNA isolated from the leaf curl infected plants. In all the samples, amplicon of 725bp was observed confirming the presence of leaf curl virus in all the collected samples. Sequencing of the resultant amplicons from selected isolates and analysis using NCBI-BLAST for their similarities in the existing database revealed more than 70% similarity with Tomato leaf curl Karnataka virus, Sunflower leaf curl virus isolate ToLCKV, Croton yellow vein mosaic virus and Papaya leaf curl virus from Carica papaya indicating that the presence of leaf curl virus. The study also identified the existence of sequence differences in leafcurl isolates. In order to facilitate for further studies, ~725 bp purified PCR amplicon was ligated in to the pDRIVE commercial cloning vector in E. Coli. The presence of the insert in the vector was confirmed through the plasmid isolation and restriction analysis with ECoRI enzyme. Thus, the designed primers in the study can be used in identification and confirmation of leaf curl in the tobacco samples.