During a survey conducted in the grapevine orchards of Himachal Pradesh, variety of symptoms ranging from leaf yellowing, vein greening, reduced leaf size, downward rolling/cup shaped leaves to reduced fruit bearing were observed. Symptomatic leaf samples were collected and analyzed by serological (DAS-ELISA) and molecular methods (RT-PCR, PCR) for viruses and phytoplasma known worldwide on grapevine. DAS-ELISA was used for detection of Grapevine leafroll associated virus 1, 2 and 3 (GLRaV-1, 2 & 3), Grapevine virus A (GVA), Grapevine fan leaf virus (GFLV), Grapevine fleck virus (GFkV) and successfully detected GLRaV-1 & 3 and GFkV. All these samples were complemented with RT- PCR along with GVb and phytoplasma (additional to ELISA) using specific primers. Specific amplification in RT-PCR for GLRaV-1 (~232 bp), GLRaV-3 (~300 bp), GFkV (~179 bp) and GVB (~440 bp) confirmed the presence of these pathogens. Overall, ELISA and RT-PCR results confirmed the presence GLRaV-3 (66.7 %), GLRaV-1& GFkV (50 %), and Grapevine virus B (GVB) (12.5 %) in symptomatic plants. None of the samples were found positive for GFLV, GLRaV-2 and phytoplasma. Mixed infection was common and none of the plants were found virus free. To the best of our knowledge this is the first report of detection of GFkV and GVB in India.
Keywords: DAS-ELISA; GFkV; GLRaV; GVA; GVB; RT-PCR.