四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定

doi:10.16420/j.issn.0513-353x.2021-0592

园艺学报 ›› 2022, Vol. 49 ›› Issue (9): 2007-2016.doi: 10.16420/j.issn.0513-353x.2021-0592

四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定

韩帅¹^,², 吴婕¹^,², 张河庆¹^,², 席亚东¹^,²^,^*()

¹四川省农业科学院植物保护研究所,农业农村部西南作物有害生物综合治理重点实验室,成都 610066
²四川省农业科学院园艺研究所,蔬菜种质与品种创新四川省重点实验室,成都 610066

收稿日期:2022-06-22 修回日期:2022-08-27 出版日期:2022-09-25 发布日期:2022-10-08
通讯作者: 席亚东 E-mail:Xiyadong2002@126.com
基金资助:
四川省科技计划项目(2021YFYZ0021);四川省农业科学院现代农业学科建设推进工程项目(2021XKJS087);四川省农业科学院科技成果中试熟化与示范转化工程项目(2021ZSSF021)

Identification and Sequence Analysis of Tomato Spotted Wilt Orthotospovirus Infecting Lettuce in Sichuan

HAN Shuai¹^,², WU Jie¹^,², ZHANG Heqing¹^,², XI Yadong¹^,²^,^*()

¹Key Laboratory of Integrated Pest Management on Crops in Southwest,Ministry of Agriculture and Rural Affairs, Institute of Plant Protection,Sichuan Academy of Agriculture Sciences,Chengdu 610066,China
²Vegetable Germplasm Innovation and Variety Improvement Key Laboratory of Sichuan Province,Horticulture of Research Institute,Sichuan Academy of Agriculture Sciences,Chengdu 610066,China

Received:2022-06-22 Revised:2022-08-27 Online:2022-09-25 Published:2022-10-08
Contact: XI Yadong E-mail:Xiyadong2002@126.com

摘要/Abstract

摘要：

为明确引起四川彭州莴笋病毒病的病毒种类,通过生物接种和电子显微镜方法,结合小RNA测序技术和反转录PCR对采集病样进行鉴定和分析。摩擦接种试验表明回接莴笋的发病症状与田间相似,矮牵牛叶片出现局部枯斑;利用电镜可观察到叶肉细胞中有大量病毒粒体聚集于内质网池,其形态特征与正番茄斑萎病毒属（Orthotospovirus）病毒相似;通过小RNA测序技术获得了番茄斑萎病毒（tomato spotted wilt orthotospovirus,TSWV）接近全长的序列,表明侵染该批次莴笋样品的病原物可能是TSWV。利用RT-PCR方法确定田间和回接莴笋及枯斑寄主中均存在TSWV,进一步证明四川彭州莴笋病毒病是由TSWV侵染引起的,这是四川首次发现TSWV为害莴笋。对拼接序列和N基因序列分别构建系统发育树可知,彭州分离物TSWV-SCPZ分别与韩国分离物（YY和K）和美国分离物（MR-01）亲缘关系最近。

关键词: 莴笋, 番茄斑萎病毒, 小RNA测序, 电子显微技术

Abstract:

Biological inoculation,electron microscopy and small RNA sequencing were employed to identify the virus infecting lettuce in Pengzhou,Sichuan Province,followed by reverse transcription（RT）-PCR for verification. Mechanical inoculation showed similar symptom on back inoculated lettuce to field samples,and necrosis was observed on Petunia hybrida leaves. Numerous virions which had similar morphology characteristics to members of the genus Orthotospovirus were observed,and they clustered in the cisternae of endoplasmic reticulum in the mesophyll cells. Nearly complete sequence of tomato spotted wilt orthotospovirus（TSWV）was obtained by small RNA sequencing,revealing that the pathogen in infected samples might be TSWV. RT-PCR results indicated the presence of TSWV in both field and inoculated lettuce plants and indicator plant,further conformed that TSWV was the pathogen causing lettuce virus disease in Pengzhou. This was the first report on TSWV infecting lettuce in Sichuan. The phylogenetic tree constructed based on assemble sequences showed that Pengzhou isolate（TSWV-SCPZ）had close relationship with Korea isolate （YY and K）,whereas more close related to America isolate（MR-01）based on N gene. These isolates shared a similar evolutionary pattern with other isolates present in Korea and America.

Key words: lettuce, tomato spotted wilt orthotospovirus, small RNA sequencing, electron microscopy

中图分类号:

S636.2

韩帅, 吴婕, 张河庆, 席亚东. 四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定[J]. 园艺学报, 2022, 49(9): 2007-2016.

HAN Shuai, WU Jie, ZHANG Heqing, XI Yadong. Identification and Sequence Analysis of Tomato Spotted Wilt Orthotospovirus Infecting Lettuce in Sichuan[J]. Acta Horticulturae Sinica, 2022, 49(9): 2007-2016.

图/表 6

参考文献 28

[1]	Chen Dao, Zhang Jie,Wu Zujian,Ding Xinlun. 2021. Genome sequence of Strawberry Pallidosis-associated virus isolated in China. Acta Horticulturae Sinica, 48 (1):146-152. (in Chinese)
	陈道, 张洁, 吴祖建, 丁新伦. 2021. 草莓白化相关病毒中国分离物全基因组分析. 园艺学报, 48 (1):146-152.
[2]	Chen Dongliang, Li Mingyuan, Li Xuemei, Wang Lingling, Huang Conglin. 2018. Identification of Tomato spotted wilt virus from lettuce in Beijing. China Vegetables,(1):65-69. (in Chinese)
	陈东亮, 李明远, 李雪梅, 王玲玲, 黄丛林. 2018. 北京地区莴苣中番茄斑萎病毒的鉴定. 中国蔬菜,(1):65-69.
[3]	Ciuffo M, Mammella M, Vallino M, Caciagli P, Turina M. 2016. Molecular identification and biological characterization of a new potyvirus in lettuce. Archives of Virology, 161 (9):2549-2554. doi: 10.1007/s00705-016-2920-x pmid: 27287434
[4]	Fan Xudong, Dong Yafeng, Zhang Zunping, Ren Fang, Hu Guojun, Zhang Mengyan, Li Chen. 2021. Detection and sequence analyses of grapevine leafroll-associated virus 7 isolates in China. Acta Horticulturae Sinica, 48 (2):347-354. (in Chinese)
	范旭东, 董雅凤, 张尊平, 任芳, 胡国君, 张梦妍, 李晨. 2021. 中国葡萄卷叶相关病毒7检测及基因变异分析. 园艺学报, 48 (2):347-354.
[5]	Fan Xudong, Dong Yafeng, Zhang Zunping, Zhang Mengyan, Ren Fang, Hu Guojun. 2019. Small RNA sequencing and RT-PCR detection of viruses infecting‘Shine Muscat’grapevines. Acta Phytopathologica Sinica, 49 (6):749-755. (in Chinese)
	范旭东, 董雅凤, 张尊平, 张梦妍, 任芳, 胡国君. 2019. ‘阳光玫瑰’葡萄病毒小RNA测序鉴定及RT-PCR检测. 植物病理学报, 49 (6):749-755.
[6]	Fang Zhongda. 1998. Plant pathology and entomology research methods. 3 rd ed.ed. Beijing:China Agriculture Press: 280-288. (in Chinese)
	方中达. 1998. 植病研究方法. 3版. 北京:中国农业出版社:280-288.
[7]	Fontes M G, Cabral C S, Lima M F, Fonseca M E N, Boiteux L S. 2019. Phenotypic expression and species-specific reaction of Lactuca sativa‘PI 342444’to groundnut ringspot virus(GRSV)and tomato spotted wilt virus(TSWV)isolates. European Journal of Plant Pathology, 155:231-238. doi: 10.1007/s10658-019-01766-3
[8]	Gong Mingxia, Zhao Hu, Wang Meng, Wu Xing, Zhao Zengjing, He Zhi, Huang Jinmei, Meng Shengde, Wang Risheng. 2022. Identification of viruses infecting peppers in Guangxi by small RNA deep sequencing and RT-PCR. Acta Horticulturae Sinica, 49 (5):1060-1072. (in Chinese)
	龚明霞, 赵虎, 王萌, 吴星, 赵曾菁, 何志, 黄金梅, 孟生德, 王日升. 2022. 广西辣椒病毒的sRNA深度测序和RT-PCR鉴定. 园艺学报, 49 (5):1060-1072.
[9]	Han Shuai, Zhang Heqing, Wu Jie, Li Honghao, Xi Yadong. 2020. Identification and detection of Tomato spotted wilt virus and Pepper mild mottle virus infecting peppers in Sichuan using small RNA sequencing. Acta Phytopathologica sinica, 50 (2):21-28. (in Chinese)
	韩帅, 张河庆, 吴婕, 李洪浩, 席亚东. 2020. 侵染四川辣椒的番茄斑萎病毒和辣椒轻斑驳病毒的小RNA测序检测及鉴定. 植物病理学报, 50 (2):21-28.
[10]	Kikkertm M, van Lent J, Storms M, Rodegom P, Kormelink R, Goldbach R. 1999. Tomato spotted wilt virus particle morphogenesis in plant cells. Journal of Virology, 73 (3):2288-2297. pmid: 9971812
[11]	King A M Q, Adam M J, Carstens E B, Lefkowitz E. 2012. Virus taxonomy: ninth report of the international committee on taxonomy of viruses. New York:Elsevier:737-739.
[12]	Martínez R T, Almeida M D, Rodriguez R, Oliveira A D, Melo F L, Resende R O. 2018. Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing. Virology Journal, 15 (24):1-9. doi: 10.1186/s12985-017-0913-3 URL
[13]	Moreno A, Fereres A. 2012. Virus diseases in lettuce in the mediterranean basin. Advances in Virus Research, 84:247-288. doi: 10.1016/B978-0-12-394314-9.00007-5 pmid: 22682170
[14]	O’Malley P J, Hartmann R W. 1989. Resistance to Tomato spotted wilt virus in lettuce. HortScience, 24:360-362. doi: 10.21273/HORTSCI.24.2.360 URL
[15]	Pappu H R, Jones R A C, Jain R K. 2009. Global status of tospovirus epidemics in diverse cropping systems:successes achieved and challenges ahead. Virus Research, 141 (2):219-236. doi: 10.1016/j.virusres.2009.01.009 pmid: 19189852
[16]	Shang Weina, Zheng Kuanyu, Dong Jiahong, Zhang Zhongkai, Hong Jian, Zhou Xueping. 2016. Comparison of cytopathological changes of leaves infected with Tomato spotted wilt virus and Tomato zonate spot virus. Journal of Chinese Electron Microscopy Society, 35 (3):269-274. (in Chinese)
	尚卫娜, 郑宽瑜, 董家红, 张仲凯, 洪健, 周雪平. 2016. 感染番茄斑萎病毒和番茄环纹斑点病毒的植物叶片细胞病理变化观察. 电子显微学报, 35 (3):269-274.
[17]	Tang Yafei, Pei Fan, Li Zhenggang, She Xiaoman, Yu Lin, Lan Guobing, Deng Mingguang, He Zifu. 2019. Identification of viruses infecting peppers in Guangdong by small RNA deep sequencing. Scientia Agricultura Sinica, 52 (13):2256-2267. (in Chinese)
	汤亚飞, 裴凡, 李正刚, 佘小漫, 于琳, 蓝国兵, 邓铭光, 何自福. 2019. 基于小RNA深度测序技术鉴定侵染广东辣椒的病毒种类. 中国农业科学, 52 (13):2256-2267.
[18]	Wang Kaina, Zhan Binhui, Zhou Xueping. 2019. Detection and partial biological characterization of Tomato spotted wilt tospovirus in Heilongjiang Province. Plant Protection, 45 (1):37-43. (in Chinese)
	王凯娜, 战斌慧, 周雪平. 2019. 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. 植物保护, 45 (1):37-43.
[19]	Wei Ningsheng, Jia Changsheng. 1987. Isolation and identification of three viruses infecting lettuce. Chinese Journal of Virology, 3 (1):102-103. (in Chinese)
	魏宁生, 贾长盛. 1987. 莴苣病毒病病毒的分离及鉴定. 病毒学报, 3 (1):102-103.
[20]	Yan Jiawen, Yuan Qifeng, Xie Pu, Wang Lijuan, Ma Yuhua, Wang Zhengyuan. 2019. Identification of viruses infecting Passiflora edulis in Guizhou Province by small RNA sequencing. Chinese Journal of Tropical Crops, 40 (8):1577-1584. (in Chinese)
	严佳文, 袁启凤, 解璞, 王立娟, 马玉华, 王正媛. 2019. 利用小RNA测序技术检测贵州西番莲病毒. 热带作物学报, 40 (8):1577-1584.
[21]	Yao Ge. 1992. Tomato spotted wild virus(TSWV)on cigar tobacco in Sichuan. Tobacco Science & Technology,(4):2-4. (in Chinese)
	姚革. 1992. 四川晒烟上发现番茄斑萎病毒. 烟草科技,(4):2-4.
[22]	Zhang Mengyan, Zhang Zunping, Ren Fang, Hu Guojun, Fan Xudong, Dong Yafeng. 2020. Establishment and application of a real-time fluorescent quantitative RT-PCR for detection of Grapevine fabavirus. Acta Horticulturae Sinica, 47 (1):187-194. (in Chinese)
	张梦妍, 张尊平, 任芳, 胡国君, 范旭东, 董雅凤. 2020. 葡萄蚕豆萎蔫病毒实时荧光定量RT-PCR检测方法及应用. 园艺学报, 47 (1):187-194.
[23]	Zhang Y L, Yu N T, Huang Q X, Yin G H, Guo A P, Wang X F, Xiong Z G, Liu Z X. 2014. Complete genome of Hainan papaya ring spot virus using small RNA deep sequencing. Virus Genes, 48:502-508. doi: 10.1007/s11262-014-1042-3 URL
[24]	Zhang Z J, Wang D Y, Yu C M, Wang Z H, Dong J H, Shi K R, Yuan X F. 2016a. Identification of three new isolates of Tomato spotted wilt virus from different hosts in China:molecular diversity,phylogenetic and recombination analyses. Virology Journal, 13 (8):1-12. doi: 10.1186/s12985-015-0456-4 URL
[25]	Zhang Z K, Zheng K Y, Dong J H, Fang Q, Hong J, Wang X F. 2016b. Clustering and cellular distribution characteristics of virus particles of tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts. Virology Journal, 13 (11):1-9. doi: 10.1186/s12985-015-0456-4 URL
[26]	Zhang Zhongkai, He Yuefeng, Fang Qi, Cheng Xiaofei, Dong Jiahong, Yin Yueyan, Ding Ming. 2010. Cytopathological characterization of three distinct Tospovirus species in infected host plants. Southwest China Journal of Agricultural Sciences, 23 (5):1522-1524. (in Chinese)
	张仲凯, 何跃锋, 方琦, 程晓非, 董家红, 尹跃艳, 丁铭. 2010. 种番茄斑萎病毒属病毒侵染寄主植物的细胞病理特征. 西南农业学报, 23 (5):1522-1524.
[27]	Zhang Zhongkai, Li Yi. 2001. Plant virus in Yunan. Beijing:Science Press:49-57. (in Chinese)
	张仲凯, 李毅. 2001. 云南植物病毒. 北京:科学出版社:49-57.
[28]	Zheng Kuanyu, Wu Kuo, Dong Jiahong, Fang Qi, Zhang Zhongkai. 2015. Infection and damage of Tomato spotted wilt virus on lettuce vegetables in Yunnan. Plant Protection, 41 (5):174-178. (in Chinese)
	郑宽瑜, 吴阔, 董家红, 方琦, 张仲凯. 2015. 番茄斑萎病毒对云南莴苣类蔬菜的侵染危害. 植物保护, 41 (5):174-178.

编号 No.	株系名称 Isolate	来源 Source	寄主 Host
1 2 3	YNgp	中国云南Yunnan,China	辣椒 Capsicum annuum
	TSWV-YN	中国云南Yunnan,China	番茄 Solanum lycopersicum
	TSWV-QLD1	澳大利亚Australia	辣椒 Capsicum annuum
4	PVR	西班牙Spain	辣椒 Capsicum annuum
5	Pepper1 CY-CN	韩国Korea	辣椒 Capsicum annuum
6	Pepper2 CY-CN	韩国Korea	辣椒 Capsicum annuum
7	CG-1	中国云南Yunnan,China	莴苣 Lactuca sativa
8	NJ-JN	韩国Korea	番茄 Solanum lycopersicum
9	YN5575	中国云南Yunnan,China	大丽花Dahlia
10	WA7	澳大利亚Australia	番茄 Solanum lycopersicum
11	LS3	韩国Korea	细叶益母草Leonurus sibiricus
12	TSWV-5	韩国Korea	牛繁缕Stellaria aquatica
13	TSWV-8	韩国Korea	山莴苣Lactuca indica
14	TSWV-HJ	韩国Korea	葎草Humulus yunnanensis
15	YN5573	中国云南Yunnan,China	豌豆Pisum sativum
16	YY	韩国Korea	辣椒 Capsicum annuum
17	K	韩国Korea	辣椒 Capsicum annuum
18	HS	韩国Korea	辣椒 Capsicum annuum
19	LK-1	南非South Africa	番茄Solanum lycopersicum
20	TSWV-QLD2	澳大利亚Australia	辣椒 Capsicum annuum
21	Mum-A5	津巴布韦Zimbabwe	菊花Chrysanthemum
22		中国云南Yunnan,China	鬼针草Bidens pilosa
23	MR-01	美国America	菊苣Cichorium intybus
24	YNHS	中国云南Yunnan,China	花生Arachis hypogaea
25	TSWV-BJFC-Ze	中国北京Beijing,China	百日菊Zinnia elegans
26	HLJ-1	中国黑龙江Heilongjiang,China	番茄 Solanum lycopersicum
27	Tarquinia	意大利Italy	辣椒 Capsicum annuum
28	JJ	韩国Korea	辣椒 Capsicum annuum

编号 No.	株系名称 Isolate	来源 Source	寄主 Host
1 2 3	YNgp	中国云南Yunnan,China	辣椒 Capsicum annuum
	TSWV-YN	中国云南Yunnan,China	番茄 Solanum lycopersicum
	TSWV-QLD1	澳大利亚Australia	辣椒 Capsicum annuum
4	PVR	西班牙Spain	辣椒 Capsicum annuum
5	Pepper1 CY-CN	韩国Korea	辣椒 Capsicum annuum
6	Pepper2 CY-CN	韩国Korea	辣椒 Capsicum annuum
7	CG-1	中国云南Yunnan,China	莴苣 Lactuca sativa
8	NJ-JN	韩国Korea	番茄 Solanum lycopersicum
9	YN5575	中国云南Yunnan,China	大丽花Dahlia
10	WA7	澳大利亚Australia	番茄 Solanum lycopersicum
11	LS3	韩国Korea	细叶益母草Leonurus sibiricus
12	TSWV-5	韩国Korea	牛繁缕Stellaria aquatica
13	TSWV-8	韩国Korea	山莴苣Lactuca indica
14	TSWV-HJ	韩国Korea	葎草Humulus yunnanensis
15	YN5573	中国云南Yunnan,China	豌豆Pisum sativum
16	YY	韩国Korea	辣椒 Capsicum annuum
17	K	韩国Korea	辣椒 Capsicum annuum
18	HS	韩国Korea	辣椒 Capsicum annuum
19	LK-1	南非South Africa	番茄Solanum lycopersicum
20	TSWV-QLD2	澳大利亚Australia	辣椒 Capsicum annuum
21	Mum-A5	津巴布韦Zimbabwe	菊花Chrysanthemum
22		中国云南Yunnan,China	鬼针草Bidens pilosa
23	MR-01	美国America	菊苣Cichorium intybus
24	YNHS	中国云南Yunnan,China	花生Arachis hypogaea
25	TSWV-BJFC-Ze	中国北京Beijing,China	百日菊Zinnia elegans
26	HLJ-1	中国黑龙江Heilongjiang,China	番茄 Solanum lycopersicum
27	Tarquinia	意大利Italy	辣椒 Capsicum annuum
28	JJ	韩国Korea	辣椒 Capsicum annuum

病毒种类 Virus	Contigs数量 Contigs count	长度/bp Length
Tomato spotted wilt orthotospovirus	34	67 ~ 2 004
Cucumber mosaic virus	3	80 ~ 488
Garlic virus A	1	495
Trichoderma hypovirus	1	273
Papaya ringspot virus	1	194
Grapevine leafroll-associated virus 1	1	134

病毒种类 Virus	Contigs数量 Contigs count	长度/bp Length
Tomato spotted wilt orthotospovirus	34	67 ~ 2 004
Cucumber mosaic virus	3	80 ~ 488
Garlic virus A	1	495
Trichoderma hypovirus	1	273
Papaya ringspot virus	1	194
Grapevine leafroll-associated virus 1	1	134

四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定

Identification and Sequence Analysis of Tomato Spotted Wilt Orthotospovirus Infecting Lettuce in Sichuan

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 28

相关文章 10

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	忽靖宇, 阙开娟, 缪田丽, 吴少政, 王田田, 张磊, 董鲜, 季鹏章, 董家红. 侵染鸢尾的番茄斑萎病毒鉴定[J]. 园艺学报, 2023, 50(1): 170-176.
[2]	杨菲, 张爱红, 王秀芝, 李希望, 邸垫平, 苗洪芹, 张尚卿, 孙祥瑞, 孙茜. 内蒙古赤峰地区番茄斑萎病毒的鉴定[J]. 园艺学报, 2021, 48(6): 1208-1216.
[3]	吴淑华1,，涂丽琴1,，咸文荣2，闫佳会2，高丹娜1，吉颖2，陶小荣3，周益军1，郭青云2,，季英华1,. 青海辣椒上番茄斑萎病毒检测及鉴定[J]. 园艺学报, 2020, 47(7): 1391-1400.
[4]	邱孝军, 谭群运, 肖清明, 梅时勇, 张冀芳. 长沙地区萝卜病毒病病原鉴定与萝卜种质抗病性评价[J]. 园艺学报, 2020, 47(10): 1947-1955.
[5]	李云洲1，默宁2，闫见敏1，曹贺贺3，庞胜群4，梁燕2,*. 番茄斑萎病毒病研究进展[J]. 园艺学报, 2018, 45(9): 1750-1760.
[6]	孙淼1，荆陈沉1，楚成茹1，吴根土1，孙现超1，谢艳2，刘勇3，青玲1,*. 重庆辣椒上番茄斑萎病毒的血清学检测及分子鉴定[J]. 园艺学报, 2017, 44(3): 487-494.
[7]	方琦, 丁铭, 董家红, 尹跃艳, 张磊, 苏晓霞, 李婷婷. 云南澳洲坚果苗木感染番茄斑萎病毒属病毒初报[J]. 园艺学报, 2013, 40(2): 350-354.
[8]	邱树亮, 王孝宣, 杜永臣, 高建昌, 国艳梅, 朱德蔚, 胡鸿, 李宝聚, 石延霞. 番茄斑萎病毒TSWV 的鉴定及抗病种质的筛选[J]. 园艺学报, 2012, 39(6): 1107-1114.
[9]	方琦;董家红;丁铭;尹跃艳;张丽珍;李婷婷;苏晓霞;李展;张仲凯;. 侵染水鬼蕉和花朱顶红的番茄斑萎病毒属病毒的电镜和DAS-ELISA诊断 [J]. 园艺学报, 2011, 38(10): 2005-2009.
[10]	刘雅婷;郑元仙;李永忠;李正跃. 云南省建水县番茄上Tospovirus属病毒的初步鉴定 [J]. 园艺学报, 2009, 36(3): 437-440.

四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定

Identification and Sequence Analysis of Tomato Spotted Wilt Orthotospovirus Infecting Lettuce in Sichuan

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 28

相关文章 10

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: