First Report of Broad bean wilt virus 2 in Dioscorea opposita Thunb. in Korea

Broad bean wilt virus 2 (BBWV2), a member of the genus Fabavirus, infects a wide range of plants, most of which are important agricultural and horticultural crops. In May 2015, to examine BBWV2 incidence in Chinese yam fields in Korea, two open fields were randomly selected in Andong City where Chinese yam has been cultivated intensively. Chinese yam plants showing virus-like symptoms, such as mosaic, stunting, mottle, yellowing, and leaf distortion, were easily found at a relatively high incidence of 10 to 15% in the surveyed fields. A total of 40 leaf samples were collected and evaluated for the presence of BBWV2 using a commercial double-antibody sandwich (DAS)-ELISA kit that detect both Broad bean wilt virus 1 (BBWV1) and BBWV2 (Agdia, Elkhardt, IN, USA). Interestingly, all the collected samples were positive by DAS-ELISA. To confirm the result from the DAS-ELISA assay and to specify whether the samples were infected with either BBWV1 or BBWV2, reverse transcription-polymerase chain reaction (RT-PCR) was performed using two sets of the primers that amplify specifically BBWV1 (5'-TGACACATATGTGGCCATGG-3' and 5'-CAGATTCTCAAGTTGGTGACAGG-3') and BBWV2 (5'-CAGAGTTCAGTAGTTCCTGCTTATG-3' and 5'-GGCATTTCAACCCTGCATAATAC-3') as described previously (Panno et al. 2014). The RT-PCR result showed that all the samples were infected with BBWV2 and no amplification was detected when using the BBWV1-specific primer set. One of the samples infected with BBWV2 was selected to determine complete genome sequence of BBWV2 isolated from Chinese yam in Korea. Total RNA extracted from the sample infected with BBWV2 was subjected to RT reactions using the Superscript III reverse transcription (Invitrogen, Carlsbad, CA, USA) and either the primer BBWVR1-3E-Rv (5'-CCCTCACTACTGAAATTTACTT-3', designed to hybridize to the 3' terminus of BBWV2 RNA1), or the primer BBWVR2-3E-Rv (5'-AAAATACTACTAAAGCTTATACATATA-3', designed to hybridize to the 3' terminus of BBWV2 RNA2). Full-length cDNAs of BBWV RNA1 and RNA2 were amplified by PCR using Phusion high-fidelity DNA polymerase (NEB, Beverly, MA, USA) with the primer BBWV-5E-Fw (5'-GTTTTAATATTTTATTAAAACAAACAGCT-3', designed to hybridize to the 5' terminus of BBWV2 RNA1 and RNA2) and either the primer BBWVR1-3E-Rv or BBWVR2-3E-Rv, respectively. Sequencing of the BBWV genome and the 5' and 3' rapid amplification of cDNA ends (RACE) of BBWV RNAs were performed as described previously (Kwon et al. 2014). The assembled full-length sequences of RNA