First Report of Tomato Spotted Wilt Orthotospovirus Infecting <i>Erigeron breviscapus</i> in Yunnan, China

The Asteraceae family plant Erigeron breviscapus (Vant.) Hand.-Mazz. (EBHM), commonly known as Dengzhan Asarum, was first recorded in the South Yunnan Materia Medica. E. breviscapus is widely utilized for treating stroke hemiplegia due to its ability to enhance blood circulation, clear collaterals, and alleviate pain (Liu et al., 2024). This species is recognized as a natural medicinal resource in China and is increasingly recognized as the primary source of such materials in Yunnan Province (Dong et al., 2022). In July 2023, virus-like symptoms, including leaf chlorosis and necrosis, were observed in 20 E. breviscapus plants in Luxi Province, Yunnan, China, with an incidence rate of approximately 10% among the examined specimens (Figure 1a-1c). Electron microscopy revealed the presence of spherical virions, measuring between 80 and 120 nm, which exhibited morphological characteristics consistent with members of the Orthotospovirus genus. These virions were localized within the cytoplasm of the mesophyll cells (Figure 1d). Total RNA was extracted from the infected E. breviscapus plants using the TRNzol Universal Reagent (Tiangen Biotech, Beijing, China), and cDNA synthesis was performed using the PrimeScript™ IV 1st strand cDNA Synthesis Mix (TaKaRa, Beijing, China). A 280 bp PCR product amplified using the dTospo-F2/dTospo-R2 primer pair (Zheng et al., 2020) was cloned and sequenced. A total of 10 samples were subjected to RT-PCR and sequencing. A 20 μL reaction system was prepared in PCR tubes, consisting of cDNA (100 ng/μL), forward primer (10 μM), reverse primer (10 μM), PrimeSTAR Max DNA Polymerase (1x) (TaKaRa, Beijing, China), and ddH2O. The PCR reaction procedure included the following steps: 98 ◦C for 10 s; followed by 35 cycles of 98 ◦C for 20 s, 56 ◦C for 1 min, and 72 ◦C for 1 min; concluding with an extension at 72 ◦C for 10 min. A BLASTn search of the sequenced amplicons against the NCBI GenBank viral database revealed a 97.83% nucleotide identity with the corresponding sequences of the TSWV-BJFC-Ze strain (MN493846.1), which was isolated from Zinnia elegans in China. This finding confirmed the presence of the tomato spotted wilt orthotospovirus in the E. breviscapus samples. The primers were specifically designed to target the partial genome of each segment reported for TSWV-BJFC-Ze (MN493846, MN493847, MN493848) to amplify the S, M, and L segments obtained via RT-PCR. The expected PCR product sizes were S (3 kb), M (4.8 kb), and L (8.9 kb). The segments were amplified using KOD One™ PCR Master Mix (Toyobo), followed by cloning, sequencing, and assembly. The assembled S, M, and L segments of TSWV-EBHM-1 were found to be 2,956 nt (PP596540), 4,773 nt (PP596541), and 8,913 nt (PP596542), respectively. A BLASTn analysis confirmed that the S, M, and L segments exhibited 99.23%, 99.43%, and 99.56% identity, respectively, to the TSWV-HLJ-1 (MG878875, MG878874, MG878873) sequences. We achieved 98% coverage of the virus genome through cloning and sequencing, which was sufficient to confidently establish its identity. Our team also conducted a disease incidence survey in three fields of E. breviscapus in Luxi province. Prior to the identification of TSWV, a symptom survey was performed, and samples were collected from symptomatic plants. We recorded a symptom incidence of 30% across all three fields. To our knowledge, this is the first report of an E. breviscapus infection by TSWV. The experiment provides valuable insight into the potential host range and transmission of TSWV, which could inform future disease management strategies.