Molecular detection and Characterization of a begomovirus causing leaf curl disease of Tomato (Lycopersicon esculentum)

Abstract

Tomato (Lycopersicon escu/entum) is a high value crop cultivated in tropical and sub-tropical countries. Tomato Leaf Curl Disease (ToLCD) is an important limiting factor affecting tomato production on Indian subcontinent and is caused by begomovirus transmitted by whitefly Bemisia tabaci. During November' 2013, very high disease incidence (up to 5% of plants ) was observed in tomato fields of Surani, Bafawat, Lawar, Pavli khas and Pawarsa villages of Meerut district. Symptoms such as upward leaf curling, crowding and yellowing of leaves and stunting of whole plants were observed in all the tomato fields. To confirm the association ofbegomovirus with the disease, polymerase chain reaction (PCR) was performed using total DNA extracted from infected toma.to leaves and four different sets of primers designed to amplify coat protein region, replication protein region, complete DNABand betasatellite DNA. Amplification of 774 bp for coat protein region and 1086 bp for replication protein gene were produced. No amplification was observed for betasatellite, movement protein gene and DNA-B. These amplicons were cloned and sequenced. The bands corresponding to coat protein gene and rep protein gene were eluted and cloned in pTZ57RIT and pGEM -T Easy cloning vector which were followed by confirmation using plasmid DNA isolation, colony PCR and Restriction digestion approaches. The positive results were subjected to sequencing and were further subjected to in-silico analysis. aBI..,AST search of GenBank revealed close similarity of the sequences with coat protein gene (A VI) and rep protein components of other tomato begomoviruses reported from Haryana, New Delhi, Lucknow, Bangladesh and Pakistan (FN645905, HM989845, DQI19573, AJ875157, DQ I I 6885), Which are all tentative strains of Tomato Leaf curl virus (ToLCV). Based on these findings the virus infecting tomato has been tentatively identified as an isolate of ToLCV with monopartite genome . Wide spread distribution and diversity of begomoviruses pose a threat to world agriculture system. The cloned components can be used to determine pathogenicity of the individual component and can further be used to develop resistant plant varieties using biotechnological approaches .