Identification and diagnosis of chilli veinal mottle potyvirus (CVMV) infecting hot pepper (Capsicum annuum L.)

Abstract

Chilli Veinal Mottle Potyvirus (ChiVMV) is an important virus disease of chilli pepper in India. The virus produced initial symptoms of mosaic mottling on young leaves 10 days after inoculation followed by characteristic vein banding, reduction in leaf size, cupping in leaves and leaf distortion. Host range studies of ChiVMV revealed that only Plant species belonging to family Solanaceae (9 species out of 16 species of Solanaceae) were susceptible. Hosts, which took systemic infection, are Capsicum annuum L. cvs. Arka Lohit and California wonder, Datura metel, Nicotiana glutinosa L. Nicotiana tabaccum L. cvs. Bhavya and Gold line. One plant that found to be local lesion host is Nicotiana tabaccum L. cv. Hartensis. The ultra thin section of infected tissues revealed the presence of cytoplasmic cylindrical inclusions as pinwheels and short curved inclusion bodies typical of potyvirus group. ChiVMV was successfully purified from systemically infected Datura metel L. leaves 3-4 weeks after inoculation. First the virus was extracted using 0.02 M HEPES buffer (pH 7.5) containing mercaptoethanol, sodium diethyl dithiocarbamate, triton X-100 and urea. The virus was finally concentrated by sucrose density and differential centrifugation in HEPES resuspension buffer (pH 7.5) containing sodium sulfite and urea. The UV absorption spectrum of purified preparations of ChiVMV showed typical nucleoprotein pattern with a maximum and minimum absorbance at 260 nm and 240 nm respectively. The A260/2s0 ratios measured from several purified preparations were in the range of 1. 110 1 to 1.2101 and the virus yield estimated was 1.0 to 1.2 mg/100 g of leaf tissue. Electron microscopy of the purified preparation of ChiVMV after negative staining with 2 per cent uranyl acetate revealed the presence of numerous virus particles which were flexuous rods measuring about 640 to 700 nm long, 12 nm in diameter indicating the relationship of virus under study belonging to potyvirus group. SDS Polyacrylamide gel electrophoresis of the purified preparation of ChiVMV coat protein on 12 per cent SDS gel revealed the presence of a band with an approximate molecular weight of 35000 daltons in freshly purified virus. A direct antigen coating ELISA (DAC-ELISA) and Dot Immuno Binding Assay (OIBA) were standardized and successfully used to detect virus and identify resistant and susceptible lines.