Shallot virus X in Indian shallot, a new virus report for India

Shallot, Allium cepa var aggregatum, is cultivated in Tamil Nadu and other southern states of India. Leaves from five plants with mild mosaic & chlorotic symptoms were collected from the field and fixed in 2.5 % glutaraldehyde. Flexuous rod shaped particles of two different lengths measuring ~ 650 and ~800 nm were observed in negatively stained leaf dip preparations under the electron microscope. Immuno-sorbent electron microscopy was performed using antisera to the potyvirus Onion yellow dwarf virus (OYDV) available at our centre, and the carlavirus Garlic latent virus (GarLV), obtained from Dr. D.E. Lesemann, Braunschweig, Germany. OYDV and GarLV are commonly reported viruses infecting garlic and onion in India (Ghosh and Ahlawat 1997; Arya et al., 2006). The smaller flexuous particles were decorated by antisera to GarLV (Fig. 1) but neither OYDV or GarLV antisera decorated the larger flexuous virus particle (Fig. 2).

Based on the length and morphology of the larger particles RT-PCR was performed to determine whether the virus was a member of the Flexivirus genus. Total RNA was extracted from 100 mg of leaves using an RNeasy Plant Minikit (Qiagen) according to the manufacturers protocol and RT-PCR was performed using the primers 5'-CYGCTAAGCTATATGCTGAARGG-3' and 5'-TGTTRCAARGTAAGTTTAGYAATATCAACA-3' previously designed from conserved regions at the 3'-end of ORF6 and 3'-end of the non coding region of Allexivirus sequences (Dovas et al., 2001). An expected amplicon of ~ 200 base pairs was obtained (Fig. 3) which was cloned in pGEM-T Easy vector (Promega) and sequenced. Sequence analysis of the 191 nucleotide product using BioEdit (version 7.0.5.3.) showed a 90% sequence identity with the Allexivirus, Shallot virus X (Accession no M97264). To the best of our knowledge this is the first report of Shallot virus X in India and the first published report of Garlic latent virus in field samples of shallot from India.