中国番茄黄斑驳相关病毒感染龙葵的首例报告李正钢,汤亚飞,小满她,GuobingLan,林宇,何子福*广东省植物保护高技术重点实验室,植物保护研究所,广东省农业科学院,广州,510640,中华人民共和国。番茄黄斑驳相关病毒(TYMaV)是一种新发现的与小叶叶片分泌相关的细胞病毒,黄色斑点,Puckering,和中国番茄植株中的斑驳症状(Xu等人。,2017)。2020年5月,在汕头市发现了表现出叶片褶皱和马赛克症状的龙葵植物(eXtraS1),广东,中国。为了确定病原体,收集三个有症状的植物的叶子,并用TRIzol试剂(Takara,草松,日本)。约100μgRNA混合物,由从三个样本中提取的等量的总RNA组成,进行小RNA深度测序和组装(sRSA)(Kreuze等人。,2009).用先前描述的方法构建小RNAcDNA文库(Mi等人。,2008).用IlluminaHiSeqXTen平台进行小RNA深度测序。VirusDetect(Zheng等人,2017)用于分析序列数据。结果显示,序列数据包括约1100万个读段,并且在去除宿主来源的重叠群后产生194个独特重叠群。随后,使用BLASTn搜索病毒数据库筛选独特的重叠群.将105个独特的重叠群映射到TYMaV基因组(参考序列,KY075646),将21个独特的重叠群映射到番茄萎黄病病毒(ToCV)基因组的RNA1片段(参考序列,KY618796),将67个独特的重叠群映射到ToCV基因组的RNA2片段(参考序列,KY618797),并将一个独特的重叠群映射到辣椒脉斑驳病毒(PVMV)基因组(参考序列,FJ617225)(eXtraS1)。要验证TYMaV检测的sRSA结果,用两个引物对TYMaV-F1/R1(5'-TCATTAGACTCAGGCCTAATCCTCAAAGT-3\'/5'-GATATGGAGACGTCAAAGTCAAAGTCAAGTGGA-3\')进行RT-PCR,和TYMaV-F2/R2(5\'-TATGCGGCAGCTTTCATCTGTCATCTATAGACCCT-3\'/5\'-ATGACCTAGCTTCAATAACAGTCGCG-3\'),根据sRSA结果进行设计。所有有症状的样本检测为TYMaV(eXtraS2)阳性。用TYMaVN蛋白特异性抗体的Western印迹进一步验证了结果(eXtraS2)。为了获得在汕头鉴定的TYMaV的近全长序列,设计13个引物对以扩增病毒片段。然后将扩增的PCR产物引入pMD19T(Takara,草松,日本),并由SangonBiotechCo.(上海,中国)。从13个重叠序列(参考序列,MW527091)。TYMaV-ST基因组包含13401nt并且与参考基因组(KY075646)共享84.93%的核苷酸序列同一性。此外,从广东省不同地点采集了37份黑曲霉样品和20份附近有病毒性疾病症状的番茄样品,中国。通过RT-PCR检测出六份黑曲霉样品和五份番茄植物样品对TYMaV呈阳性,表明该病毒在被调查地区广泛传播。这些结果与sRSA测定的结果一起也表明,在原始黑曲霉植物中显示的疾病症状可能不一定是由TYMaV或由TYMaV单独引起的。据我们所知,这是中国首次报道TYMaV感染黑血病。黑草是一种常见的杂草,属于茄科,可以在田间用作TYMaV的水库。需要进一步的研究来验证是否确实如此,并了解这种病毒的特征,包括其传播,致病性,和经济意义。作者声明没有利益冲突。资助这项工作得到了广东省重点研究发展计划(2018B020202006)的支持,广东省农业科学院农业优势产业学科团队建设项目(202103TD和202105TD),广州市科技计划(202102020504),和科学创新战略专项资金-高水平农业科学院建设(R2019PY-QF003)。参考资料:Kreuze,J.F.,etal.2009.病毒学.388:1.Mi,S、etal.2008.细胞。133:116.徐,C.,etal.2017.JVirol.91:11.郑,Y.,etal.2017.病毒学.500:130.
First report of tomato yellow mottle-associated virus infecting Solanum nigrum in China Zhenggang Li, Yafei Tang, Xiaoman She, Guobing Lan, Lin Yu, and Zifu He† Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, 510640, P. R. China. Tomato yellow mottle-associated virus (TYMaV) is a newly found cytorhabdovirus associated with epinasty of leaflet blades, yellow spots, puckering, and mottling symptoms in tomato plants in China (Xu et al., 2017). In May 2020, Solanum nigrum plants exhibiting leaf crinkling and mosaic symptoms (eXtra S1) were found in Shantou city, Guangdong, China. To identify the causal pathogens, the leaves of three symptomatic plants were collected and subjected to total RNA extraction with TRIzol Reagent (Takara, Kusatsu, Japan). About 100 μg RNA mixture, which consisted of an equal amount of total RNA extracted from the three samples, was subjected to small RNA deep sequencing and assembly (sRSA) (Kreuze et al., 2009). Small RNA cDNA library was constructed with the method described previously (Mi et al., 2008). Small RNA deep sequencing was performed with Illumina HiSeq X Ten platform. VirusDetect (Zheng et al., 2017) was used to analyze the sequence data. The result showed that the sequence data includes about 11 million reads and generated 194 unique contigs after removal of host-derived contigs. Subsequently, the unique contigs were screened using BLASTn search against the virus database. One hundred and five unique contigs were mapped to TYMaV genome (reference sequence, KY075646), 21 unique contigs were mapped to RNA1 segment of tomato chlorosis virus (ToCV) genome (reference sequence, KY618796), 67 unique contigs were mapped to RNA2 segment of ToCV genome (reference sequence, KY618797), and one unique contig was mapped to pepper veinal mottle virus (PVMV) genome (reference sequence, FJ617225) (eXtra S1). To verify the sRSA result for TYMaV detection, RT-PCR was performed with two primer pairs TYMaV-F1/R1 (5\'-TCATTAGACTCAGGCCTAATCCTCA AAGT-3\'/5\'-GATATGGAGACGTCCAAGTTCAAAGGGATGGA-3\'), and TYMaV-F2/R2 (5\'-TATGCGGCAGCTTTCATGTCTATAGACCCT-3\'/5\'-ATGACCTAGCTTCAATAACAGTCGCG-3\'), which are designed according to the sRSA result. All the symptomatic samples tested positive for TYMaV (eXtra S2). Western blot with TYMaV N protein-specific antibody further verified the result (eXtra S2). To obtain the nearly full-length sequence of TYMaV identified in Shantou, 13 primer pairs were designed to amplify the viral fragments. The amplified PCR products were then introduced into pMD19T (Takara, Kusatsu, Japan) and sequenced by Sangon Biotech Co. (Shanghai, China). The nearly full-length sequence of TYMaV Shantou isolate (TYMaV-ST) was assembled from the 13 overlapping sequences (reference sequence, MW527091). TYMaV-ST genome comprises of 13401 nt and shares 84.93% nucleotide sequence identity with the reference genome (KY075646). In addition, 37 S. nigrum samples and 20 tomato samples nearby with viral disease symptoms were collected from different sites of Guangdong province, China. Six S. nigrum samples and five tomato plant samples tested positive for TYMaV by RT-PCR, suggesting a wide spread of the virus in the surveyed region. These results together with those of the sRSA assay also suggest that the disease symptoms shown in the original S. nigrum plants may not necessarily be caused by TYMaV or by TYMaV alone. To our knowledge, this is the first report of TYMaV infecting S. nigrum in China. S. nigrum is a common weed which belongs to the family Solanaceae and may serve as a reservoir for TYMaV in the fields. Further research is needed to verify whether this is indeed the case, and to understand the characteristics of this virus including its transmission, pathogenicity, and economic significance. The authors declare no conflict of interest. Funding This work was supported by the Key Research and Development Program of Guangdong Province (2018B020202006), the Agricultural Competitive Industry Discipline Team Building Project of Guangdong Academy of Agricultural Sciences (202103TD and 202105TD), the Science and Technology Program of Guangzhou (202102020504), and Special Fund for Scientific Innovation Strategy-Construction of High-Level Academy of Agriculture Science (R2019PY-QF003). References: Kreuze, J. F., et al. 2009. Virology. 388: 1. Mi, S., et al. 2008. Cell. 133: 116. Xu, C., et al. 2017. J Virol. 91: 11. Zheng, Y., et al. 2017. Virology. 500: 130.