First Report of Cucumber Mosaic Virus in Atractylodes macrocephala in Korea

Abstract

Atractylodes macrocephala Koidz (family Asteraceae) is a perennial herb that is widely grown in many Asian countries. Its roots are used in traditional medicine to improve the immune system and to treat gastrointestinal disease, inflammation, and cancer because it contains valuable pharmacological ingredients such as atractylon, atractylenoid, eudesmol, and hinesol (Lee et al. 2007). In May 2018, virus-like symptoms including leaf curl, mosaic, and yellow spot were observed on the leaves of the A. macrocephala plants grown in a commercial field in Jaecheon city, Korea. Because virus diseases in A. macrocephala remain rather unstudied, total RNA extracted from the collected symptomatic leaves was subjected to Illumina RNA sequencing to identify the causal agent of the disease as described previously (Seo et al. 2017). In brief, a transcriptome library generated using the TruSeq Stranded Total RNA with Ribo-Zero Plant kit (Illumina, San Diego, CA) was analyzed by an Illumina HiSeq2000 sequencer. Raw reads were de novo assembled to contigs by the Trinity pipeline and analyzed against the viral reference genome database in GenBank by BLAST searches (Schelhorn et al. 2013). The entire RNA sequencing procedure was performed by Macrogen (Seoul, South Korea). Among the analyzed contigs, only three large contigs (3,333, 2,778, and 2,188 nt, respectively) corresponding to near-complete sequences of cucumber mosaic virus (CMV, genus Cucumovirus) genomic RNAs were identified. To confirm the presence of CMV, eight symptomatic leaf samples and three asymptomatic samples collected from the field were subjected to reverse transcription polymerase chain reaction (RT-PCR) using specific CMV primers (CMV-R3-Fw, 5′-ATGGACAAATCTGAATCAACCAGTGCTGGT-3′, position RNA3 1,257 to 1,284; and CMV-R3-Rv, 5′-TCAGACTGGGAGCACTCCAGATGTGGG-3′, position RNA3 1,887 to 1,913). The RT-PCR resulted in amplification of specific PCR fragments with an estimated size of 657 bp from all symptomatic samples, but not from the asymptomatic samples. CMV infection of the symptomatic samples was confirmed by serological testing with a CMV-specific ImmunoStrip kit (Agdia, Elkhart, IN). An asymptomatic sample was used as a negative control in serological testing. To determine the complete genome sequence of the identified CMV (named CMV-JC), three RNA segments were analyzed by the 5′ and 3′ rapid amplification of cDNA ends method (Kwon et al. 2014). In addition, the entire contig sequences obtained by RNA sequencing were confirmed by de novo sequencing. The entire sequences of CMV RNA 1, RNA2, and RNA3 were determined to be 3,359 nt (GenBank accession no. MH594044), 3,047 nt (MH594045), and 2,212 nt (MH594046), respectively. The highest nucleotide sequence identities were 96% with RNA1 of the CMV isolate DS (GenBank accession no. KU255787), 99% with RNA2 of the CMV isolate AM (JX993910), and 96% with RNA3 of the CMV isolate FT (KU255786). Sap inoculation of CMV-JC induced typical CMV symptoms of severe mosaic and stunting in Nicotiana tabacum, N. glutinosa, and N. benthamiana and mosaic and yellow spot symptoms in A. macrocephala. Phylogenetic analysis based on full-length RNA3 sequences using the maximum likelihood method implemented in MEGA7 software indicated that CMV-JC belonged to subgroup IB (Kim et al. 2014). CMV has a wide host range and is easily transmitted by various aphids in nature. In particular, CMV is still one of the most prevalent viruses in various crops including pepper and tomato worldwide, despite continuous breeding and introduction of various CMV-resistant cultivars. In this work, we first identified CMV from A. macrocephala in Korea and report its complete genome sequence.