RT-PCR and CP gene based molecular characterization of a cucumber mosaic cucumovirus from Aligarh, U.P., India

Abstract

A virus disease of garden sage ( Salvia splendens Ker-Gawl.) was observed and characterized showing symptoms of severe mosaic, mottling and distortion of leaves being remain shortened and growth retarded. The virus was transmitted to the healthy plants of Salvia spp. as well as many other hosts by mechanical inoculation, Myzus persicae Sulzer and Aphis gossypii Glover transmit the virus in non-persistent manner. Purified sample in EM showed spherical particles c.28 nm in diameter. DAC- ELISA [1] was performed with crude sap, specific polyclonal anti-serum (PVAS 242a, ATCC, USA) and alkaline phosphatase-linked secondary antibodies (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH or DSMZ, Germany). The mean absorbance at 405 nm for negative and positive controls were 0.061 ± 0.008 and 0.349 ± 0.003 respectively, while infected samples were recorded four-times more than the value of negative controls with values that ranged between 0.289 ± 0.005 and 0.325 ± 0.003. RT-PCR was performed using total RNA as templates and CMV Coat Protein (CP) gene specific reverse and forward primers, gel was electrophoresed on 1% agarose, an amplification of expected size 650 bp fragment was obtained only in the infected sample which proved that the present virus is a strain of CMV, the type member of the genus cucumovirus belonging to the family Bromoviridae.