First Report of Cucumber 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. Recently, we reported an isolate of Tobacco mosaic virus (TMV) infecting P. brachycarpa. Afterward, we surveyed further for viral diseases on P. brachycarpa plants. During June 2016, virus-like symptoms, including curling, green-yellow mosaic, and deformation of leaves were observed on P. brachycarpa in a greenhouse located in Busan, South Korea. To identify a causal virus, five symptomatic leaf samples were analyzed by transmission electron microscopy using leaf dip preparations. Icosahedral virus particles (approximately 30 nm in diameter) were observed from all test leaf samples. The symptomatic leaf samples were also analyzed by DAS-ELISA using four different polyclonal antibodies specific to Tobacco mild green mosaic virus (TMGMV), TMV, Tomato mosaic virus (ToMV), and Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Germany). All samples were negative for TMGMV, TMV, ToMV, and TSWV. Cucumber mosaic virus (CMV) was identified from all five symptomatic P. brachycarpa plants by serological testing for the presence of CMV coat protein (CP) with CMV-ImmunoStrip kit (Agdia, Elkhart, U.S.A.). The presence of CMV was also confirmed by serological detection with a DAS-ELISA kit (Agdia). The virus (named CMV-Charm) caused necrotic local lesions on Chenopodium amaranticolor at 5 days post inoculation (dpi), while severe systemic mosaic symptoms were observed in Capsicum annuum and Nicotiana glutinosa 10 to 14 dpi. To fulfill Koch’s postulates, virus-free P. brachycarpa were inoculated mechanically by sap from local lesions on C. amaranticolor. The P. brachycarpa plants displayed identical symptoms 21 dpi to those observed in the Busan greenhouse. CMV-charm was detected from these inoculated P. brachycarpa plants. Meanwhile, mock-inoculated P. brachycarpa remained symptomless and virus-free, suggesting that CMV is the causal virus of the symptomatic P. brachycarpa plants. The presence of CMV-Charm in all naturally infected and mechanically inoculated P. brachycarpa plants was further verified by reverse transcription (RT)-PCR (Han et al. 2012). A 657-bp RT-PCR product from the CMV CP gene was amplified from all samples. There was no amplification from asymptomatic P. brachycarpa. RT-PCR products were cloned into pCR4-TOPO vector and selected cDNA clones were sequenced. Multiple alignment of the CMV-Charm CP sequence (accession no. LC177113) with CP sequences of other CMV isolates using MEGA6.0 software revealed identities of 96.8 to 99.2% and 70.6 to 73% to CMV isolates from subgroup I and subgroup II, respectively. CMV may pose a major threat for production of P. brachycarpa, because CMV is easily transmitted by various agents (such as aphids) (Palukaitis and Garcia-Árenal 2003) and P. brachycarpa is continually farmed in Korea