First report of konjac mosaic virus infecting Codonopsis pilosula in China.

Radix Codonopsis pilosulae is a perennial herb of the genus Codonopsis, family Campanulaceae, and its dry root is frequently used in traditional Chinese medicine for hundreds of years for replenishing qi deficiency, strengthening the immune system, improving poor gastrointestinal function, decreasing blood pressure, etc. (He et al. 2015). However, there have been no previous reports of virus infection in C. pilosula. In July 2021 and July 2022, we collected 61 C. pilosula samples exhibiting virus symptoms of yellowing, mottling, and crinkling from fields in Gansu Province. A composite of six leaf samples was submitted to Biotech Bioengineering (Shangai) Co. Ltd. for small RNA sequencing. Total RNA of C. pilosula was extracted according to the manufacturer's directions using the total RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). The library was constructed using the TruSeq™ Small RNA Sample Prep Kits (Illumina, San Diego, USA), and was sequenced using the Illumina Hiseq 2000/2500 with a single-end read length of 1X50bp. Samples were sequenced to obtain 14349505 raw reads and 10081026 clean reads by removing low quality reads. Quality-controlled qualified reads were assembled using SPAdes (Bankevich et al., 2012) with a k-mer value of 17 and the obtained results were compared with NCBI's non-redundant nucleotide database. A contig of 8195 bp in length, with an 85% query coverage of the reference genome was annotated as homologous to konjac mosaic virus (KoMV, AB219545.1) with 80.60% nucleotide similarity. The virus-specific primers F 5`- ATAGCGGAAACGGCATT-3` and R 5`- GGCACGGCAGATAAACAC -3` were designed based on the contig to validate the sequencing results in individual samples. One of the original composite samples was KoMV positive. Polymerase chain reaction (PCR) products were resolved in 1.5% agarose gel and an ∼954 bp fragment was obtained (Fig. 1A). The PCR amplicons were submitted to Beijing Tsingke Biotech Co. Ltd. for Sanger sequencing. The obtained sequence (PP790593) was searched against the NCBI nucleotide database using the BLASTn algorithm. Results showed that it shared 98.85% nucleotide sequence identity with the genome of KoMV (MK770338). This is the first time that KoMV was found to naturally infect C. pilosula that was first identified in Amorphophallus Konjac in Japan(Shimoyama et al., 1992). KoMV belongs to the genus Potyvirus, family Potyviridae. To analyze the phylogenetic relationships of KoMV, partial coat protein (cp) genes of genus Potyvirus were downloaded from NCBI and a phylogenetic tree was constructed using the Neighbor-Joining method implemented in MEGA 11.0 software (Tamura et al. 2021) with default parameters. The KoMV isolate obtained from Gansu C. pilosula in this experiment clustered with KoMV sequences isolated from Angelica sinensis collected in China, which again proving that the virus is KoMV (Fig.1B). Additionally, a total 61 C. pilosula samples showing similar virus disease symptoms and suspected to be infected were collected from 'fields' and tested by RT-PCR with primers KoMV-F and KoMV-R. Among these samples, 29 were positive for KoMV. The infection rates of the KoMV in C. pilosula suspected of being infected were 47.54%. The detection rates of Weiyuan, Zhangxian, Lintao, Minxian and Yuzhong were 55.55%, 50.00%, 50.00%, 75.00% and 35.00%, respectively. C. pilosula infested with KoMV may produce symptoms of chlorotic (Fig.1D). Accordingly, larger scale C. pilosula investigations must be conducted to determine the distribution and prevalence of KoMV in China. This study will promote the research on virus diseases of C. pilosula and will help growers make informed disease management decisions.