First Report of Broad Bean Wilt Virus 2 in Achyranthes bidentata in Korea

Achyranthes bidentata Blume (family Amaranthaceae) is a traditional medical herb with a range of pharmaceutical properties (Zhou et al. 2009). Although A. bidentata has been grown commercially in many geographic areas of Korea, little information is available on viral diseases of this crop. In August 2017, during a survey conducted in a commercial field in Naju city, Korea, approximately 5% of A. bidentata plants showed virus-like symptoms of mosaic and yellowing. Leaf samples were collected from 10 symptomatic plants and tested to identify the causal agent(s). Total RNA isolated from a collected symptomatic leaf was analyzed by Illumina RNA sequencing (Seo et al. 2017). The entire RNA-sequencing procedure was performed by Macrogen (Seoul, South Korea). The assembled contigs (84,264 contigs) were analyzed against the viral reference genome database in GenBank by BLASTn and BLASTx searches. Among the contigs, only two large contigs were clearly of viral origin. Nucleotide blast analysis showed that the first and second contigs (5,918 and 3,544 bp, respectively) have maximum identities of 98 and 88% to RNA1 of the isolate RP2 (GenBank accession no. JX183223) and RNA2 of the isolate Ty (AB746939) of broad bean wilt virus 2 (BBWV2), respectively. The 10 collected samples were tested by reverse transcription polymerase chain reaction (RT-PCR) using BBWV2-specific primers designed based on the assembled contig sequence (BBWV2-R1-Fw, 5′-TCGCCTAGATCGTGGTCCTG-3′, corresponding to RNA1 nucleotide position 4,182 to 4,201; and BBWV2-R1-Rv, 5′-CAGTGAGCGGAAAGCCAGAA-3′, corresponding to RNA1 nucleotide position 4,758 to 4,777). All samples yielded specific PCR fragments with an estimated size of 596 bp, and sequences of the amplified products were confirmed to correspond to that of the contig by de novo sequencing. To confirm the RT-PCR result, a double-antibody sandwich ELISA assay was performed using a commercial kit (Agdia, Elkhart, IN), and all tested samples were positive for BBWV2. To obtain the complete genome sequence of the BBWV2 A. bidentata isolate, terminal sequences of both RNA segments were analyzed by the 5′ and 3′ rapid amplification of cDNA ends method as described previously (Kwon et al. 2014). The assembled full-length sequences of BBWV2 RNA1 and RNA2 were 5,950 and 3,597 nucleotides in length, respectively, and deposited in GenBank under the accession numbers MH447988 and MH447989, respectively. Sap inoculation of the BBWV2 isolate induced mild mosaic symptoms in Nicotiana benthamiana and pepper (Capsicum annuum L.). BBWV2, which belongs to the genus Fabavirus in the family Secoviridae, has a wide host range and is easily transmitted by various aphids. In particular, BBWV2 is an emerging virus in various crops including pepper and legume plants in Korea (Kwak et al. 2013). To control the virus, a large-scale survey may be required for in-depth epidemiological investigation of BBWV2 in Korea. To the best of our knowledge, this is the first report of BBWV2 in A. bidentata.