Cnidium officinale is a perennial plant in the family Apiaceae. It is native to China and cultivated in China, Japan, and Korea for its roots for medicinal purposes. In August 2019, 63 C. officinale plants showing symptoms of vein chlorosis, yellowing and chlorotic spots (Supplementary Fig. 1) were collected from commercial farms in Bonghwa and Youngyang, Gyeongsangbuk-do, South Korea. Reverse transcription and polymerase chain reaction (RT-PCR) was performed to confirm the presence of apple stem grooving virus (ASGV), cnidium vein yellowing virus 1, cnidium vein yellowing virus 2, lychnis mottle virus, and Cnidium virus X with specific primers (Supplementary Table 1). Forty-one out of the sixty-three samples were positive for ASGV in mixed infection with one or more of the other four viruses. Nicotiana benthamiana plants mechanically inoculated with the crude sap of one of the ASGV-infected C. officinale plants showed mosaic symptom on upper leaves 10 days post inoculation (dpi). Infection was confirmed by RT-PCR and Sanger sequencing. N. benthamiana plants systemically infected with ASGV-CO-kr1 isolate alone were used for subsequent sequencing and host range test. Twenty-day old seedlings of 23 species of plants (two to 14 species for each family) from the families Solanaceae, Chenopodiaceae, Cucurbitaceae, Fabaceae and Amaranthaceae (Supplementary Table 2) were mechanically inoculated with sap of ASGV-CO-kr1-infected N. benthamiana plants. ASGV-CO-kr1 infected all tested 23 species as confirmed by symptomology, RT-PCR, and Sanger sequencing at 10 to 20 dpi. The MP and CP genes of ASGV-CO-kr1 were amplified by RT-PCR with specific primers 4300-4325F/5642-5666R and 5592-5612F/6475-6499R, respectively (Supplementary Table 1). The amplicons were cloned and sequenced (GenBank accession numbers: MP = MW889883 and CP = MW889884). Multiple sequence alignment using the MegAlign program in DNASTAR showed that the complete CP and MP genes of ASGV-CO-kr1 shared 89.9%-99.7% and 83.1%-99.5% identities, respectively at the nucleotide (nt) level and they shared 92.4%-99.6% and 93.8%-99.4% identities, respectively at amino acid (aa) level with corresponding sequences of 34 other ASGV isolates from various host plants and countries. Phylogenetic analysis with the Maximum Likelihood method using the MEGA X program (Kumar et al., 2018) showed that ASGV-CO-kr1 grouped with isolates Cuiguan (KR185346), BH (LC480456), and YY (LC480457) based on the CP aa sequences, while it grouped with isolates SG (LC475148) and TL101 (MH108976) based on the MP aa sequences. ASGV is known to naturally infect apples, European pear, Asian pear, citrus, apricot, cherry, kiwifruit, loquat, lily, and lotus (Clover et al., 2003; He et al., 2019; Hu et al., 2017; Liu et al., 2017; Yanase et al., 1975). To the best of our knowledge, this is the first report of the natural infection of ASGV in C. officinale. C. officinale plants are propagated by root division, so they are susceptible to infection with viruses. The result of this study is important for generating virus-free seedlings to produce C. officinale.
Vitrification Cryo-Foil for Apple Cryopreservation and the Seesaw Effect Between Shoot Recovery and Virus Eradication