First report of Youcai mosaic virus in Raphanus raphanistrum subsp. Sativus in Korea

Radish (Raphanus raphanistrum subsp. sativus), a species of the family Brassicaceae, is widely consumed as Kimchi and various other foods in South Korea. During October 2016, virus-like symptoms, including stunting and mild mosaic of leaves, were observed on a radish plant (cv. Seoho) on a farm located in Wanju, South Korea. The symptomatic leaf sample was also analyzed by DAS-ELISA using four different polyclonal antibodies specific to Cucumber mosaic virus, Turnip mosaic virus, Turnip yellow mosaic virus, and Turnip yellows virus (Loewe Biochemica, Germany). None of the four viruses was detected in the radish leaf sample. The symptomatic leaf sample was also analyzed by transmission electron microscopy (TEM) in leaf dip preparations. Typical tobamovirus-like particles of rigid rod shape and about 300 × 18 nm length/width were observed in the leaf sample of radish plant. To further confirm the obtained TEM result, total RNA was extracted from the symptomatic radish plant and analyzed by reverse transcription (RT)-PCR using degenerate primers specific to genus Tobamovirus (Agdia, U.S.A.). An approximate 400-bp RT-PCR product was amplified from the radish sample and the amplified RT-PCR product was purified and cloned into pCR4-TOPO vector (ThermoFisher Scientific, U.S.A.). Selected cDNA clones were sequenced, and the BLASTN search showed that determined sequences of the amplified RT-PCR product had the highest identity with isolate Br of Youcai mosaic virus (accession no. JN634066). To confirm that YoMV (named YoMV-Rad) was the cause of the disease observed in radish, pathogenicity test was carried out using two Nicotiana species containing the N-gene, resulting in necrotic local lesions 4 days postinoculation (dpi). To fulfill Koch’s postulates, virus-free radish plants inoculated mechanically by sap from local lesions on N. glutinosa, showing identical symptoms observed on the symptomatic radish 30 dpi and YoMV-Rad was reisolated from the inoculated radish plants. Mock-inoculated radish plants remained symptomless and virus-free, suggesting that YoMV is the causal virus of the symptomatic radish plant. To determine the complete coat protein (CP) gene sequence of YoMV-Rad, primers YoMV-CP-For (5′-ATGTCTTACAACATCACGAGCTCGAAT-3′) and YoMV-CP-Rev (5′-CTATGTAGCTGGCGCAGTAGTCCAAGG-3′) were synthesized according to the sequence of YoMV-Br. A 474-bp RT-PCR product amplifying the CP gene was amplified from the symptomatic radish. Sequencing result of selected cDNA clones for YoMV-Rad showed the determined sequences of the amplified RT-PCR products matched with YoMV CP gene. A multiple alignment of the obtained CP sequence of YoMV-Rad (accession no. LC270132) with other YoMV isolates using MEGA6.0 showed 92.0 to 98.0% identity to YoMV strains described previously. All these results demonstrate that YoMV is the cause of the disease in radish. During fall and winter 2016, 300 Korean radish plants showing virus-like symptoms on farms were collected in Wanju and Kochang and subjected to RT-PCR using the above YoMV-CP primers. Incidence of YoMV in radish plants was 3%. To our knowledge, this is the first report of YoMV infecting radish in Korea.