黑龙江地区番茄斑萎病毒的鉴定及其部分生物学特征分析
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摘要
近年来,番茄斑萎病毒Tomato spotted wilt tospovirus (TSWV)在我国多个地区发生严重危害。本研究采用小RNA深度测序和RT-PCR相结合的方法对采自黑龙江的番茄病毒病样品中的病毒进行了鉴定。提取番茄样品的RNA并构建小RNA文库进行测序,数据分析发现比对至TSWV基因组的reads数占比对到病毒核酸总reads数的92.64%,表明侵染此番茄样品的病原物可能是TSWV。利用RT-PCR方法进一步确定侵染此番茄样品的病毒为TSWV,将其命名为TSWV-HLJ1。对TSWV-HLJ1的核苷酸序列进行分析并构建系统进化树,结果表明TSWV-HLJ1与TSWV云南甜椒分离物(TSWV-YNgp)的亲缘关系最近。介体传毒试验表明,西花蓟马可将TSWV-HLJ1传播感染健康寄主植物。摩擦接种试验表明,TSWV黑龙江分离物能够侵染本氏烟、辣椒和番茄。这是我国首次报道在黑龙江地区发现TSWV的危害。
Recently, Tomato spotted wilt tospovirus(TSWV) has been reported in several areas of China causing serious damage. To identify the viruses in tomato samples exhibiting virus-like symptoms from Heilongjiang Province, small RNA deep sequencing and RT-PCR were adopted. Total RNA of tomato samples was extracted and the siRNA library was constructed and sequenced. Data analysis showed that the siRNA reads aligned to TSWV accounted for 92.64 % of the total siRNA reads to the virus sequences deposited in GenBank, indicating that the pathogen infecting the tomato samples might be TSWV. The RT-PCR further confirmed the presence of TSWV, which was named TSWV Heilongjiang isolate(TSWV-HLJ1). The phylogenetic tree based on the whole nucleotide sequence of TSWV isolates indicated that the TSWV-HLJ1 was more closely related to TSWV Yunnan isolate(TSWV-YNgp). Vector transmission experiment showed that Frankliniella occidentalis could acquire TSWV-HLJ1 and transmit it to the healthy plants successfully. Mechanical inoculation experiment showed that TSWV-HLJ1 could infect Nicotiana benthamiana, Capsicum annuum L. and Lycopersicon esculentum Mill. This was the first report of TSWV infecting tomato in Heilongjiang Province.
Recently, Tomato spotted wilt tospovirus(TSWV) has been reported in several areas of China causing serious damage. To identify the viruses in tomato samples exhibiting virus-like symptoms from Heilongjiang Province, small RNA deep sequencing and RT-PCR were adopted. Total RNA of tomato samples was extracted and the siRNA library was constructed and sequenced. Data analysis showed that the siRNA reads aligned to TSWV accounted for 92.64 % of the total siRNA reads to the virus sequences deposited in GenBank, indicating that the pathogen infecting the tomato samples might be TSWV. The RT-PCR further confirmed the presence of TSWV, which was named TSWV Heilongjiang isolate(TSWV-HLJ1). The phylogenetic tree based on the whole nucleotide sequence of TSWV isolates indicated that the TSWV-HLJ1 was more closely related to TSWV Yunnan isolate(TSWV-YNgp). Vector transmission experiment showed that Frankliniella occidentalis could acquire TSWV-HLJ1 and transmit it to the healthy plants successfully. Mechanical inoculation experiment showed that TSWV-HLJ1 could infect Nicotiana benthamiana, Capsicum annuum L. and Lycopersicon esculentum Mill. This was the first report of TSWV infecting tomato in Heilongjiang Province.
引文
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[3] 董家红, 尹跃艳, 徐兴阳, 等. 番茄斑萎病毒在云南的发生为害[C]//吴孔明:公共植保与绿色防控——中国植物保护学会2010年学术年会.北京: 中国农业科学技术出版社,2010: 784.
[4] 佟爱仔, 赵兴能, 孔宝华, 等. 侵染云南烟草的番茄斑萎病毒(TSWV)的RT-PCR检测[J]. 云南农业大学学报(自然科学版), 2012, 27(6): 809-814.
[5] SCHOLTHOF K B, ADKINS S, CZOSNEK H, et al. Top 10 plant viruses in molecular plant pathology [J]. Molecular Plant Pathology, 2011, 12(9): 938.
[6] WHITFIELD A E, ULLMAN D E, GERMAN T L. Tospovirus-thrips interactions [J]. Annual Review of Phytopathology, 2005, 43(43): 459.
[7] ZHANG Z J, WANG D Y, YU C M, et al. Identification of three new isolates of tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses [J]. Virology Journal, 2016, 13(1): 1-12.
[8] 赫尔 R.马修斯植物病毒学[M].北京:科学出版社,2007:377.
[9] 郑雪, 刘春明, 李宏光, 等. 云南省红河地区传播番茄斑萎病毒属病毒的蓟马及其寄主植物种类调查[J]. 中国植保导刊, 2013, 33(3): 41-44.
[10] ZHAO W W, WAN Y R, XIE W, et al. Effect of spinosad resistance on transmission of tomato spotted wilt virus by the western flower thrips (Thysanoptera: Thripidae)[J]. Journal of Economic Entomology, 2016, 109(1): 62-69.
[11] AHMED M S, ADEL A R, KHALID A A. Cucurbit yellow stunting disorder virus and watermelon chlorotic stunt virus induced gene silencing in tobacco plants [J]. African Journal of Biotechnology, 2018, 17(7): 189-197.
[12] KREUZE J F, PEREZ A, UNTIVEROS M, et al. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses [J]. Virology, 2009, 388(1): 1-7.
[13] 宋静静,蒙姣荣,邹承武,等.用小RNA深度测序鉴定广西冬种马铃薯病毒[J].中国农业科学,2013,46(19):4075-4081.
[14] 仇心钰, 郝丹丹, 韩成贵, 等. 利用小RNA深度测序对我国北方地区甜菜病毒病的调查[C]//吴学宏.植物病理学研究进展——中国植物病理学会青年学术研讨会论文选编.北京: 中国农业大学出版社, 2015: 126.
[15] 苏秀, 徐毅, 陈莎, 等. 利用小RNA深度测序和组装技术鉴定紫藤花叶病病原[J]. 植物病理学报, 2015, 45(1): 88-92.
[16] 王芳, 高正良, 周本国, 等. 利用小RNA高通量测序技术检测玉米病毒[J]. 植物病理学报, 2017, 47(3): 422-427.
[17] 廖倩, 张治军, 张玉秀,等. 西花蓟马数量与番茄斑萎病毒发生流行的关系[J]. 浙江农业学报, 2015, 27(9): 1601-1605.
[18] 姚革. 四川晒烟上发现番茄斑萎病毒(TSWV)[J]. 中国烟草科学, 1992(4): 2-4.
[19] 李飞, 吴青君, 徐宝云, 等. 北京地区发现番茄斑萎病毒[J]. 植物保护, 2012, 38(6): 186-188.
[20] 李洁, 迟胜起, 杨勤民, 等. 山东烟台地区发生番茄斑萎病毒病危害[J]. 植物保护, 2017, 43(1): 228-232.
[21] 曹金强, 谢学文, 柴阿丽, 等. 番茄斑萎病毒病在宁夏的发现与防治[J]. 中国蔬菜, 2016(4): 87-89.
[22] 饶雪琴, 刘勇, 李媛媛, 等. 广东番茄上检测到Tospovirus病毒[J]. 植物病理学报, 2010, 40(4): 430-432.
[23] 张璐璐. 防治西花蓟马的球孢白僵菌液固双相发酵条件优化[D]. 北京:中国农业科学院, 2016.
[24] 吴鑫本.番茄斑萎病毒的防治与检疫[J]. 现代农业科学, 2008, 15(11): 95-96.
[25] 张洁,陈永对,吴阔,等.6种杀虫剂对烟田番茄斑萎病毒属病毒传毒蓟马的药效试验[J].江苏农业科学,2014,42(4):95-97.
[26] 吕印谱, 丁征宇, 宋宝安, 等. 抗植物病毒剂作用机制研究进展[J]. 中国植保导刊, 2007, 27(10): 14-16.
[27] FUNDERBURK J, REITZ S, OLSON S, et al. Managing thrips and tospoviruses in tomato [J]. Entomology & Nematology, 2015, 18(6): 785-788.
[2] BRITTLEBANK C C. Tomato diseases [J]. Journal of the Department of Agriculture in Victoria, 1919, 17: 1348-1352.
[3] 董家红, 尹跃艳, 徐兴阳, 等. 番茄斑萎病毒在云南的发生为害[C]//吴孔明:公共植保与绿色防控——中国植物保护学会2010年学术年会.北京: 中国农业科学技术出版社,2010: 784.
[4] 佟爱仔, 赵兴能, 孔宝华, 等. 侵染云南烟草的番茄斑萎病毒(TSWV)的RT-PCR检测[J]. 云南农业大学学报(自然科学版), 2012, 27(6): 809-814.
[5] SCHOLTHOF K B, ADKINS S, CZOSNEK H, et al. Top 10 plant viruses in molecular plant pathology [J]. Molecular Plant Pathology, 2011, 12(9): 938.
[6] WHITFIELD A E, ULLMAN D E, GERMAN T L. Tospovirus-thrips interactions [J]. Annual Review of Phytopathology, 2005, 43(43): 459.
[7] ZHANG Z J, WANG D Y, YU C M, et al. Identification of three new isolates of tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses [J]. Virology Journal, 2016, 13(1): 1-12.
[8] 赫尔 R.马修斯植物病毒学[M].北京:科学出版社,2007:377.
[9] 郑雪, 刘春明, 李宏光, 等. 云南省红河地区传播番茄斑萎病毒属病毒的蓟马及其寄主植物种类调查[J]. 中国植保导刊, 2013, 33(3): 41-44.
[10] ZHAO W W, WAN Y R, XIE W, et al. Effect of spinosad resistance on transmission of tomato spotted wilt virus by the western flower thrips (Thysanoptera: Thripidae)[J]. Journal of Economic Entomology, 2016, 109(1): 62-69.
[11] AHMED M S, ADEL A R, KHALID A A. Cucurbit yellow stunting disorder virus and watermelon chlorotic stunt virus induced gene silencing in tobacco plants [J]. African Journal of Biotechnology, 2018, 17(7): 189-197.
[12] KREUZE J F, PEREZ A, UNTIVEROS M, et al. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses [J]. Virology, 2009, 388(1): 1-7.
[13] 宋静静,蒙姣荣,邹承武,等.用小RNA深度测序鉴定广西冬种马铃薯病毒[J].中国农业科学,2013,46(19):4075-4081.
[14] 仇心钰, 郝丹丹, 韩成贵, 等. 利用小RNA深度测序对我国北方地区甜菜病毒病的调查[C]//吴学宏.植物病理学研究进展——中国植物病理学会青年学术研讨会论文选编.北京: 中国农业大学出版社, 2015: 126.
[15] 苏秀, 徐毅, 陈莎, 等. 利用小RNA深度测序和组装技术鉴定紫藤花叶病病原[J]. 植物病理学报, 2015, 45(1): 88-92.
[16] 王芳, 高正良, 周本国, 等. 利用小RNA高通量测序技术检测玉米病毒[J]. 植物病理学报, 2017, 47(3): 422-427.
[17] 廖倩, 张治军, 张玉秀,等. 西花蓟马数量与番茄斑萎病毒发生流行的关系[J]. 浙江农业学报, 2015, 27(9): 1601-1605.
[18] 姚革. 四川晒烟上发现番茄斑萎病毒(TSWV)[J]. 中国烟草科学, 1992(4): 2-4.
[19] 李飞, 吴青君, 徐宝云, 等. 北京地区发现番茄斑萎病毒[J]. 植物保护, 2012, 38(6): 186-188.
[20] 李洁, 迟胜起, 杨勤民, 等. 山东烟台地区发生番茄斑萎病毒病危害[J]. 植物保护, 2017, 43(1): 228-232.
[21] 曹金强, 谢学文, 柴阿丽, 等. 番茄斑萎病毒病在宁夏的发现与防治[J]. 中国蔬菜, 2016(4): 87-89.
[22] 饶雪琴, 刘勇, 李媛媛, 等. 广东番茄上检测到Tospovirus病毒[J]. 植物病理学报, 2010, 40(4): 430-432.
[23] 张璐璐. 防治西花蓟马的球孢白僵菌液固双相发酵条件优化[D]. 北京:中国农业科学院, 2016.
[24] 吴鑫本.番茄斑萎病毒的防治与检疫[J]. 现代农业科学, 2008, 15(11): 95-96.
[25] 张洁,陈永对,吴阔,等.6种杀虫剂对烟田番茄斑萎病毒属病毒传毒蓟马的药效试验[J].江苏农业科学,2014,42(4):95-97.
[26] 吕印谱, 丁征宇, 宋宝安, 等. 抗植物病毒剂作用机制研究进展[J]. 中国植保导刊, 2007, 27(10): 14-16.
[27] FUNDERBURK J, REITZ S, OLSON S, et al. Managing thrips and tospoviruses in tomato [J]. Entomology & Nematology, 2015, 18(6): 785-788.
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