牛磺酸(ArctiumlappaL.,属于菊科),是韩国的食用植物和东方药草(汉和古,1993).2023年7月,在大邱的一个温室中,在大约4,000种植物中的9种植物中观察到了斑叶和叶子发黄的牛场,韩国。为了确定致病病毒种类,从每个植物中收集九个有症状的叶子,并使用市售的免疫试纸条进行测试(Agdia,埃尔克哈特,美国)用于黄瓜花叶病毒(CMV)和番茄斑萎病毒(TSWV)。九个样品中的七个仅对TSWV测试为阳性。韩国的TSWV于2004年首次报道了来自Yesan的甜椒(Kim等人。,2004年),此后已传播到各种农作物。1995年,夏威夷首次报道了TSWV感染牛磺酸的植物(Bautista等人。,1995),但在韩国尚未报道感染TSWV的牛磺酸。为了进一步确认TSWV的存在,使用RNeasyPlantMini试剂盒(Qiagen,希尔登,德国),并用于逆转录聚合酶链反应(RT-PCR)测定,使用特定的引物组扩增了TSWV的777bp的核衣壳基因(N基因)(Yoon等人。,2014).为了获得牛蛙植物中TSWV的完整基因组序列,名为TSWV-DG,L的碎片,M,并对S段进行扩增和测序。L的完整基因组序列(8914nt),M(4773nt),通过重叠RT-PCR扩增子获得S(2946nt)片段。将RT-PCR产物克隆到pGEM-TEasy载体中,并使用Sanger方法(Bioneer,韩国)。将完整的基因组序列保藏至GenBank(分别为LC790665、LC790666和LC790667)。BLASTn分析显示每个TSWV-DG片段的序列具有99.5%的最大核苷酸同一性,99.5%,和99.5%与TSWV-L,TSWV-M,和TSWV-S(分别为OM154971、OM154970和OM154969),从中国的水滴草(Oenanthecrocata)中分离出来(Qiu等人。,2023年)。为了评估TSWV-DG的生物活性,在25℃下,在3周时,用感染的牛磺酸叶的汁液机械接种拉帕和烟草,并保持目视检查病毒症状。TSWV-DG在A.lappa的全身叶片上产生症状,包括褪绿斑点和变黄,在N.benthamiana的叶子上,其中包括接种后14天的褪绿斑点和马赛克图案。同时,模拟接种的A.lappa和N.benthamiana仍然没有症状。随后通过免疫试纸条和RT-PCR分析确认接种叶片上TSWV的存在。TSWV可能对拉帕的生产构成重大威胁,在韩国被种植为绿叶蔬菜和根茎类蔬菜。此外,A.lappa可能不仅有TSWV感染造成损害的风险,而且还可能是TSWV感染的潜在来源,从而构成向韩国其他主要作物传播的风险,如胡椒或土豆(Yoon等人。,2014).这是TSWV在韩国感染牛磺酸的第一份报告。
Burdock (Arctium lappa L., belongs to the family Asteraceae), is an edible plant and an oriental medicinal herb in Korea (Han and Koo, 1993). In July 2023, burdocks showing chlorotic ringspots and yellowing on the leaves were observed in nine of approximately 4,000 plants in a greenhouse in Daegu, South Korea. To determine the causal virus species, nine symptomatic leaves from each individual plant were collected and tested using commercially available immunostrips (Agdia, Elkhart, USA) for cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV). Seven out of nine samples tested positive for TSWV only. TSWV in South Korea was first reported on sweet pepper from Yesan in 2004 (Kim et al., 2004) and has since spread to various crops. The first report of TSWV infecting burdock plants in the world was from Hawaii in 1995 (Bautista et al., 1995), but TSWV-infected burdock has not been reported in Korea. To further confirm the presence of TSWV, total RNA was extracted from TSWV-positive burdock leaves using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and used in reverse transcription-polymerase chain reaction (RT-PCR) assays with a specific primer set that amplifies 777 bp of nucleocapsid gene (N gene) of TSWV (Yoon et al., 2014). To obtain the complete genome sequence of this TSWV in the burdock plant, named TSWV-DG, fragments of L, M, and S segments were amplified and sequenced. The complete genome sequences of the L (8914 nt), M (4773 nt), and S (2946 nt) segments were obtained by overlapping RT-PCR amplicons. RT-PCR products were cloned into the pGEM-T Easy vector, and selected DNA clones were sequenced using Sanger method (Bioneer, Korea). The complete genome sequences were deposited to GenBank (LC790665, LC790666, and LC790667, respectively). BLASTn analysis showed that sequences of each TSWV-DG segment had maximum nucleotide identities of 99.5%, 99.5%, and 99.5% with TSWV-L, TSWV-M, and TSWV-S (OM154971, OM154970, and OM154969, respectively), which were isolated from water dropwort (Oenanthe crocata) in China (Qiu et al., 2023). To assess the biological activity of TSWV-DG, A. lappa and Nicotiana benthamiana were inoculated mechanically with sap from infected burdock leaves and maintained for visual inspection of virus symptoms at 25 ℃ at 3 weeks. TSWV-DG produced symptoms on the systemic leaves of A. lappa, that included chlorotic spots and yellowing, and on the leaves of N. benthamiana, that included chlorotic spots and mosaic patterns from 14 days-post-inoculation. Meanwhile, mock-inoculated A.lappa and N.benthamiana remained symptomless. The presence of TSWV on the inoculated leaves was subsequently confirmed through Immunostrip and RT-PCR analyses. TSWV may pose a significant threat to the production of A. lappa, which is cultivated as both leafy greens and root vegetables in Korea. Furthermore, A. lappa may not only be at risk of damage from TSWV infection but also act as a potential source of TSWV infection, thereby posing a risk of transmission to other key crops in Korea, such as pepper or potato (Yoon et al., 2014). This is the first report TSWV infecting burdock in South Korea.