First Report of Tomato Zonate Spot Virus Infecting <i>Cyclocodon lancifolius</i> (Hong Guo Ginseng) in China

Cyclocodon lancifolius (Roxb.) Merr. (Campanulaceae), commonly known as Hong Guo Ginseng (Pan et al., 2024), is a perennial herb widely distributed in tropical and subtropical East Asia, mainly cultivated in Yunnan, Sichuan, and Guizhou provinces of China, and has potential therapeutic effects on diabetes, antioxidation, and gout (Ma et al., 2023). Tomato zonate spot virus (TZSV), Orthotospovirus tomatozonae in the genus Orthotospovirus of the family Tospoviridae, was first reported infecting tomato (Solanum lycopersicum) and pepper (Capsicum annuum) in Yunnan, China, in 2008 (Dong et al., 2008). In November 2024, a total of 10 symptomatic C. lancifolius plants showing virus-like symptoms, including leaf malformation, chlorosis, and necrosis, were collected from Malipo County, Wenshan Prefecture, Yunnan Province (n=5) and the Fangxiangliqi Garden of Yunnan University of Chinese Medicine (n=5) (Fig. S1). Spherical viral particles with a diameter of 80 to 100 nm were observed in the sap of diseased leaves by transmission electron microscopy (Fig. S2). Reverse transcription-polymerase chain reaction (RT-PCR) screening was performed using degenerate primers CIFor/CIRev for potyviruses and dTospo-F2/dTospo-R2 for orthotospoviruses (Huang et al., 2018), with total RNA extracted from diseased leaves as the template. Only the latter primers amplified 312-bp fragments, while CIFor/CIRev yielded none. Sequencing and BLASTn analysis of these 312-bp fragments showed 95.53% nucleotide identity with the TZSV tomato-YN isolate (accession no. OR603945.1). The virus isolate was named TZSV-HGG. Total RNA from a sample pool (n=4) was sequenced on the Illumina NovaSeq 6000 (150-bp paired-end) by Lingke Biotechnology Co., Ltd. (Kunming, China). After quality control and filtering, 238,829,666 clean reads were obtained and assembled into contigs using SPAdes v. 3.15.3. Through online BLASTn search, six contigs were found to have nucleotide identities of 94.13%, 94.13% to 99.38%, and 95.53% to 98.77% with partial sequences of the L, M, and S segments of TZSV, respectively. To characterize the genome of the virus, specific primers were used to amplify the full-length genome sequence by fragment amplification. Following PCR product purification, cloning, and Sanger sequencing analysis, the amplified sequences were assembled and confirmed to be identical to high throughput sequencing assembled contigs by BLASTn (Fig. S3). The L, M, and S segments of the TZSV-HGG genome are 8,917 nt, 4,696 nt, and 3,212 nt, respectively, and have been deposited in GenBank (accession numbers PV892472, PV892473, and PV892474). Blastn analysis showed that these segments share 96.31%, 97.31%, and 94.24% nucleotide sequence identity with the L (NC_010491.1), M (NC_010490.1), and S (NC_010489.1) segments of the TZSV tomato-YN isolate, respectively. Compared to the TZSV tomato-YN isolate, the M RNA intergenic region contains a 252 nt deletion, and the S RNA intergenic region contains a 67 nt deletion. Mechanical inoculation and RT-PCR confirmed TZSV-HGG systemically infects N. tabacum cv. K326, causing chlorosis and necrotic spots at 14 days post-inoculation. To our knowledge, this is the first report of natural infection of C. lancifolius by TZSV, expanding the host range of this virus and providing critical bases for disease epidemic surveillance and integrated control.