First report of tomato spotted wilt virus infecting water dropwort in China.

Water dropwort (Oenanthe javanica) is an aquatic perennial plant that has been cultivated in many regions in Asia for thousands of years. In China, it is an economically important vegetable that has been consumed as food, while also being used as a folk remedy to alleviate diseases (Liu et al., 2021). In 2021, during a disease survey of a greenhouse in Beijing, China, chlorotic spots were detected on many water dropwort plants (Fig. S1A). Twenty-seven water dropwort samples were collected for the extraction of total RNA using the TRIzol reagent (Invitrogen, USA). High-quality RNA samples from three water dropwort plants were combined and used as the template for constructing a single small RNA library (BGI-Shenzhen Company, China). The Velvet 1.0.5 software was used to assemble the clean reads (18 to 28 nt) into larger contigs, which were then compared with the nucleotide sequences in the National Center database using the BLASTn algorithm. Thirty-eight contigs matched sequences in the tomato spotted wilt virus (TSWV) genome. No other viruses were detected. Twenty-seven leaf samples were analyzed in an enzyme-linked immunosorbent assay (ELISA) with anti-TSWV antibody (Agdia, USA), which revealed 17 positive reaction. Two sets of primer pairs targeting different parts of the S RNA (Table S1) was used to verify the TSWV infection on water dropwort by reverse transcription (RT)-PCR followed by Sanger sequencing (BGI-Shenzhen, China). The TSWV target sequences were amplified from 17 samples, which was consistent with the ELISA results. The sequenced 861-bp PCR product shared 99.8% nucleotide sequence identity with TSWV isolate MR-01 (MG593199), while the 441-bp amplicon shared a 99.2% nucleotide sequence identity with MR-01 (MG593199). To obtain the whole genome sequence of TSWV (S, M, and L RNA sequences), specific RT-PCR primers were designed (Table S1) and used to amplify their respective fragments from one representative sample (TSWV-water dropwort). The amplified products were inserted into PCE2TA/Blunt-Zero vector (Vazyme Biotech Co., Ltd, China) and then sequenced (BGI-Shenzhen, China). The S, M, and L RNA sequences were determined to be 2,952 nt (accession no. OM154969), 4,776 nt (accession no. OM154970), and 8,914 nt (accession no. OM154971), respectively. BLASTn analysis demonstrated that the whole genome sequence was highly conserved. The nucleotide identities between this isolate and other TSWV isolates ranged from 98.6% to 99.6% (S RNA), 98.9% to 99.2% (M RNA), and 97.3% to 98.7% (L RNA). Using MEGA 7.0, the phylogenetic relationships of TSWV were determined on the basis of the S, M, and L RNA full-length sequences (Kumar et al., 2016). In the S RNA derived phylogenetic tree, the water dropwort isolate was closely related to the MR-01 isolate from the USA (MG593199). In the M RNA and L RNA derived phylogenetic trees, the water dropwort isolate formed a branch with only a TSWV isolate from eggplant. Additionally, the M and L RNA sequences were most similar to sequences in TSWV isolates from China and Korea, respectively (Fig. S1B). To the best of our knowledge, this is the first report of water dropwort as a natural host for TSWV in China and the second report worldwide since the first finding in the Korea (Kil et al. 2020). TSWV has caused serious problems on many crops in the world, and the infection of TSWV on water dropwort in a greenhouse should not be looked lightly. Firstly, the virus can be passed on from generation to generation in infected water dropwort due to the vegetative propagation mode of the plant in production, thus threaten the production of this vegetable crop. In addition, infected water dropwort may serve as a reservoir for the virus, thus potentially posing a threat for causing TSWV spread in the affected greenhouses. The author(s) declare no conflict of interest. Funding: This research was supported by the Beijing Academy of Agriculture and Forestry Foundation, China (QNJJ202131, KJCX20200212, and KJCX20200113). References: Kil et al. 2020. Plant Pathol. J. 36: 67-75 Kumar et al. 2016. Mol Biol Evol, 33: 1870-1874 Liu et al. 2021. Horticulture Research. 8:1-17.