First Report of Grapevine Yellow Speckle Viroid-2 Infecting Grapevine (Vitis vinifera) in India

Abstract

In the grape vineyards of India, symptoms of viral and sub-viral pathogen have been observed on a wide scale. Three, out of five viroids reported worldwide, have been recorded from grape vineyards in India viz., Hop stunt viroid (HSVd), Grapevine yellow speckle viroid-1 (GYSVd-1) (1) and Australian grapevine viroid (AGV) (2) while there are no reports of Grapevine yellow speckle viroid-2 (GYSVd-2) and Citrus exocortis viroid (CEVd) infection from India. To determine the presence of GYSVd-2, grapevine samples exhibiting symptoms like yellowing, vein banding, yellow speckle, downward curling and mixed infections maintained in the glasshouse, were subjected to reverse transcription (RT)-PCR with primer pair GYSVd-2-Pl (5’-ACTAGTACTTTCTTCTATCTCCGAAGC-3’) and GYSVd-2-Pl (5’-ACTAGTCCGAGGACCTTTTCTAGCGCTC-3’) (3) for the amplification of its complete genome. In agarose gel electrophoresis, an amplicon of expected size (375 bp) was obtained in the three tested grapevine varieties (Thomson seedless, sharad seedless, flame seedless). Amplicon from one of the samples was cloned into pGEM-T Easy vector (Promega, Madison, WI) and two clones were sequenced. Sequences of both the clones were 100% identical (375 bp). Sequences were submitted to GenBank (Accession numbers MG780425; MG780426). BLAST analysis of the clones revealed 98% nt. sequence identity with Grapevine yellow speckle viroid-2 (GYSVd-2) (GenBank accession number HM211853.1.). Following this, symptomatic as well as non-symptomatic samples from 14 grapevine varieties were collected from grapevine fields of IARI, New Delhi and tested for the presence of GYSVd-2. Out of fourteen varieties, four were found positive for GYSVd-2. GYSVd-2 belongs to the genus Apscaviroid and has been reported from China, Australia (3) and Thailand (4) and known to produce yellow speckle symptoms in plants. To the best of our knowledge this study represents the first report of GYSVd-2 from India and will help in viroid indexing for grapevine certification programme in India.