RT-PCR based detection and molecular characterization of Prunus necrotic ring spot virus (PNRSV) in peach

Abstract

Prunus necrotic ring spot virus (PNRSV) is an important virus of worldwide occurrence in stone fruits. It is a complex Ilarvirus consisting of five RNA components viz., RNA1, RNA2, RNA3, RNA4 and RNA5. Surveys conducted in four major peach growing districts of Himachal Pradesh revealed its occurrence on the basis of visual symptoms. Since PNRSV is a latent virus DAS-ELISA was further used to confirm its presence in most of the symptomatic trees. Subjecting the test samples to RT-PCR revealed the exact identity and resulted in molecular characterization of PNRSV in three trees of peach cv. July Elberta with highest concentration of PNRSV based on OD values obtained in DAS-ELISA. RNA3 component of 665bp size of PNRSV in coat protein (CP) was identified on 1.0 percent agarose gel. Cloning and transformation further confirmed the same RNA3 component in PNRSV in E. coli DH5α cells using PCR.