Molecular Detection, Cloning and in-silica Analysis of Genomic Components of Tomato Leaf Curl Virus Associated With Leaf Curl Disease in Chilli (Capsicum Annum L.)

Abstract

Chilli (Capsicum annum L.) is a high value spice crop cultivated in tropical and sub-tropical countries (Berke et a/., 2~01). Chilli Leaf Curl Disease (ChLCD) is an important limiting factor affecting chilli production on Indian subcontinent and is caused by begomoviruses transmitted by whitefly Bemisia tabaci (Green and Kali, 1994). During November' 2011, very high disease incidence (up to 100% of plants) was observed in chilli fields of Juranpur, Jahidpiur, Gosipur and Nareda villages of Meerut district. Symptoms similar to tomato leaf curl, such as upward leaf curling,·crowding and yellowing of leaves and stunting of whole plants were observed in all the chilli fields (Hussain et a/., 2004). To confirm the association of begomovirus with the disease, polymerase chain reaction (PCR) was performed using total DNA extracted from infected chilli leaves and five different sets of primers designed to amplify coat protein region, replication protein region, movement protein gene, complete DNA-B and betasatellite DNA. Amplifications of expected size i.e. (-750bp) for coat protein region, (-1200 bp) for replication protein gene and ( -1300 bp) for betasatellite component were produced. No amplification was observed for movement protein gene and DNA-B. These amplicons were cloned and sequenced. The bands corresponding to coat protein gene and betasatellite DNA were eluted and cloned in pTZ57Rff cloning vector which was 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. A BLAST search of GenBank revealed close similarity of the sequences with coat protein gene (A VI) and betasatellite components of other chilli begomoviruses reported from Panipat, New Delhi, Lucknow, Bangladesh and Pakistan (HM 143901, HM143911, GQ284842, AJ875157 and AM279668), which are all tentative strains of Tomato leaf curl l'irus (ToLCV). Based on these findings the virus infecting chilli has been tentatively identified as an isolate of ToLCV with monopartite genome and betasatellite. Wide spread distribution and diversity of begomoviruses pose a threat to world agricultural system (Mansoor et al., 2003). 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.