Development of Antibodies, Infectious clones and Infectivity studies of Cotton leaf curl Burewala virus (CLCuBuV)

Abstract

Cotton leaf curl disease (CLCuD) is a major hindrance to cotton production across Pakistan. The disease is caused by a complex of begomoviruses with a single known betasatellite, Cotton leaf curl Multan betasatellite. Leaf samples from cotton plants, both symptomatic (leaf curling, vein thickening, enation) and non-symptomatic, were collected from major areas in Punjab, Pakistan during 2010-2013. Total DNA was isolated from these samples using a CTAB-method, followed by PCR. From the symptomatic samples, 30 full-length begomovirus components (10 DNA-A, 10 betasatellite and 10 alphasatellite) were cloned and completely sequenced. The isolates C28 and C49 with their associated cognates alpha-and beta-satellites were used to produce partial tendem repeat constructs for agroinoculation. Cotton leaf curl Burewala virus (recently re-named as Cotton leaf curl Kokhran virus-Burewala) with Cotton leaf curl Multan alphasatellite and betasatellite was the most frequent virus identified in most of the cotton belt of Punjab. One new strain, a recombinant alphasatellite was identified. For this new strain, the name “Cotton leaf curl Burewala virus-Layyah” was proposed. The partial tandem repeat constructs were infectious to Nicotiana benthamiana, N. tabacum, Solanum lycopersicum, Cucurbita maxima and cucumber. For immunological studies, polyclonal antisera were raised to the coat protein of CLCuKoV-Bu. Antiserum was tested as serologically positive in arrays of assays like DAS-ELISA and western blot for detection of viral coat protein of the begomovirus. The present study is significant for the development of antibodies for rapid detection of cotton infecting begomoviruses on a large scale and production of infectious clones for screening material for resistance studies.