First report of Tobacco mosaic virus infecting Pimpinella brachycarpa in Korea

Pimpinella brachycarpa (family Apiaceae) is an edible green, leafy vegetable frequently consumed in Korea. The plant species is commonly used to prepare ‘Namul,’ a seasoned raw vegetable dish. During February 2016, virus-like symptoms, including curling, green-yellow mosaic, deformation, and necrosis of leaves were observed on P. brachycarpa in a greenhouse located in Busan, South Korea. Ten symptomatic plants of leaf samples were analyzed by transmission electron microscopy and leaf dip-preparations. Typical tobamovirus-like particles of rigid rod shape and ∼300 × 18 nm length/width were observed in the leaf samples of P. brachycarpa plants. The symptomatic leaf samples were also analyzed by DAS-ELISA using five different polyclonal antibodies specific to Cucumber mosaic virus, Tobacco mild green mosaic virus, Tobacco mosaic virus (TMV), Tomato mosaic virus, and Tomato spotted wilt virus (Loewe Biochemica, Germany). All 10 symptomatic P. brachycarpa plants tested positive for TMV only. Diagnosis was further confirmed through lateral flow immunoassay (LFI), and were tested with TMV-specific LFI developed in our laboratory. All samples were positive for TMV, except for a nonsymptomatic leaf of P. brachycarpa used as negative control. To confirm that TMV (named TMV-Charm) was the cause of the disease observed in P. brachycarpa, pathogenicity test was carried out using two Nicotiana species containing the N-gene, resulting in necrotic local lesions 3 days postinoculation (dpi). To fulfill Koch’s postulates, virus-free P. brachycarpa inoculated mechanically by sap from local lesions on N. glutinosa, showing identical symptoms observed on the symptomatic P. brachycarpa 21 dpi and TMV-Charm was reisolated from the inoculated P. brachycarpa. Meanwhile, mock-inoculated P. brachycarpa remained symptomless and virus-free, suggesting that TMV is the causal virus of the symptomatic P. brachycarpa plants. To further confirm the obtained serological and biological results, total RNA was extracted from 10 symptomatic P. brachycarpa and analyzed by RT-PCR using primers specific to TMV (Choi et al. 2009). An 807-bp RT-PCR product amplifying a fragment of the capsid protein (CP) gene was amplified from all samples. There was no amplification from nonsymptomatic P. brachycarpa. RT-PCR products were purified and cloned into pCR4-TOPO vector (Yoon et al. 2014). Selected cDNA clones were sequenced and analyzed using BLAST, showing the determined sequences of the amplified RT-PCR products matched with TMV CP gene. A multiple alignment of the obtained TMV CP sequences with other TMV isolates using MEGA6.0 showed 99.3% identity to TMV strain described previously (accession no. AAM64216). All these results demonstrate that TMV is the cause of the disease in P. brachycarpa. TMV may pose a major threat for production of P. brachycarpa, because farming of P. brachycarpa plants is continuously performed in Korea. To our knowledge, this is the first report of TMV infecting P. brachycarpa in Korea.