重庆辣椒上番茄斑萎病毒的血清学检测及分子鉴定
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摘要
为明确重庆地区辣椒上番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的发生情况及其外壳蛋白基因(cp)序列变异特征,采用斑点免疫杂交法(Dot-ELISA)和反转录PCR(RT-PCR)技术对采自重庆的298份辣椒病毒病样品进行了检测和分子鉴定。Dot-ELISA检测结果表明,有40份辣椒样品检测到TSWV,检出率为13.42%。通过电子显微镜在TSWV检测呈阳性的样品中观察到具包膜的球状病毒粒体。设计扩增TSWV cp的引物,选取6个TSWV阳性样品进行RT-PCR扩增,均扩增到大小约750 bp的目的片段。序列分析表明,重庆辣椒分离物TSWV-CQ(KX611497)与已报道的22个TSWV分离物cp基因的核苷酸相似性为96.1%~99.4%,与已报道的TSWV中国分离物亲缘关系较近,表现出明显的地理相关性。
To identify the occurrence and variation of the coat protein gene(cp)of Tomato spotted wilt virus(TSWV)in pepper in Chongqing,a total of 298 diseased pepper samples were collected and subjected to the detection of Dot-ELISA with specific antiserum of TSWV and RT-PCR with specific primers.Results of Dot-ELISA detection revealed that 40 samples were positive with a detection ratio of 13.42%.Typical enveloped spherical particles were observed in the TSWV-positive samples through electron microscopy.Six positive samples were randomly selected and amplified by RT-PCR with primers specific for the cp gene of TSWV,and results revealed that an approximate 750 bp fragment was obtained in each sample.Sequencing showed that cp gene of the isolate TSWV-CQ(KX611497)shared 96.1% to 99.4% identities with those of the other 22 previously reported TSWV isolates.Phylogenetic analysis showed that the isolate TSWV-CQ was most closely related to TSWV isolates of China and suggested that geographical selection force played a role in virus evolution.
To identify the occurrence and variation of the coat protein gene(cp)of Tomato spotted wilt virus(TSWV)in pepper in Chongqing,a total of 298 diseased pepper samples were collected and subjected to the detection of Dot-ELISA with specific antiserum of TSWV and RT-PCR with specific primers.Results of Dot-ELISA detection revealed that 40 samples were positive with a detection ratio of 13.42%.Typical enveloped spherical particles were observed in the TSWV-positive samples through electron microscopy.Six positive samples were randomly selected and amplified by RT-PCR with primers specific for the cp gene of TSWV,and results revealed that an approximate 750 bp fragment was obtained in each sample.Sequencing showed that cp gene of the isolate TSWV-CQ(KX611497)shared 96.1% to 99.4% identities with those of the other 22 previously reported TSWV isolates.Phylogenetic analysis showed that the isolate TSWV-CQ was most closely related to TSWV isolates of China and suggested that geographical selection force played a role in virus evolution.
引文
Adkins S.2000.Tomato spotted wilt virus-positive steps towards negative success.Molecular Plant Pathology,1(3):151-157.
Burland T G 2000.DNASTAR's Lasergene sequence analysis software.Methods in Molecular Biology,132:71-91.
Cao Jin-qiang,Xie Xue-wen,Chai A-li,Li Bao-ju.2016.Identification and control of Tomato spotted wilt virus in Ningxia.China Vegetables,(4):87-89.(in Chinese)曹金强,谢学文,柴阿丽,李宝聚.2016.番茄斑萎病毒病在宁夏的发现与防治.中国蔬菜,(4):87-89.
Ding Ming,Zhang Li-zhen,Fang Qi,Li Ting-ting,Su Xiao-xia,Li Zhan.2004.Identification,purification and antiserum preparation of Tospovirus isolate affecting potato.Southwest China Journal of Agricultural Sciences,17(Supplement):160-162.(in Chinese)丁铭,张丽珍,方琦,李婷婷,苏晓霞,李展.2004.侵染马铃薯的一个Tospovirus混合分离物的鉴定、纯化及多抗血清制备.西南农业学报,17(增刊):160-162.
French J M,Goldberg N P,Randall J J,Hanson S F.2016.New Mexico and the southwestern US are affected by a unique population of Tomato spotted wilt virus(TSWV)strains.Archives of Virology,161:993-998.
Gao Wei,Wang Yong,Zhang Chun-xiang,Zhang An-sheng,Zhu Xiao-ping.2016.Investigation and pathogen preliminary identification of pepper virus disease in Tianjin.Shandong Agricultural Sciences,48(3):91-94.(in Chinese)高苇,王勇,张春祥,张安盛,竺晓平.2016.天津地区辣椒病毒病调查及毒源种类初步鉴定.山东农业科学,48(3):91-94.
Guo Si-yao,Tong Yan,Huang Ya,Luo Xin-fu,Qing Ling.2015.Preliminary identification and analyses of viruses causing pepper virus disease in Chongqing,China.Acta Horticulturae Sinica,42(2):263-270.(in Chinese)郭思瑶,童艳,黄娅,罗信福,青玲.2015.重庆辣椒病毒病病原初步鉴定和分析.园艺学报,42(2):263-270.
Huang Ren-zhong,Ge Wen-dong,He Ye,Huang Qi-zhong,Zhe Wen-jun,Liu Xiao-bo,Wu Zhao-long.2015.The effectiveness of planting disaster insurance in Chongqing Shizhu.China Vegetables,1(10):10-12.(in Chinese)黄任中,葛文东,何叶,黄启中,柘文君,刘晓波,吴朝龙.2015.重庆石柱辣椒种植灾害保险的实施效果.中国蔬菜,1(10):10-12.
Lei Lei,Lin Qing,L(u|")Zhong-hua,Huang Ren-zhong,Huang Qi-zhong,Shi Si-ru.2001.The species identification and strain ascertainment of pepper virus in Chongqing.Southwest Gardening,29(2):28-29.(in Chinese)雷蕾,林清,吕中华,黄任中,黄启中,史思茹.2001.重庆市辣椒病毒病毒原种类鉴定及株系确认.西南园艺,29(2):28-29.
Li Fei,Wu Qing-jun,Xu Bao-yun,Xie Wen,Wang Shao-li,Zhang You-jun.2012.Tomato spotted wilt virus was identified in Beijing.Plant Protection,38(6):186-188.(in Chinese)李飞,吴青君,徐宝云,谢文,王少丽,张友军.2012.北京地区发现番茄斑萎病毒.植物保护,38(6):186-188.
Lian S,Lee J S,Cho W K,Yu J,Kim M K,Choi H S,Kim K H.2013.Phylogenetic and recombination analysis of Tomato spotted wilt virus.PLoS ONE,8(5):e63380.
Lin Qing,Lu Zhong-hua,Huang Ren-zhong,Huang Qi-zhong,Lei Lei,Shi Si-ru.2001.The identification of TMV and CMV in pepper of Chongqing.China Vegetables,(3):13-15.(in Chinese)林清,吕中华,黄任中,黄启中,雷蕾,史思茹.2001,重庆市辣椒TMV和CMV的鉴定.中国蔬菜,(3):13-15.
Liu Chen-chen,Sun Miao,Yu Guan-hua,Qing Ling.2013.The ELISA detection of Tomato spotted wilt virus(TSWV)infecting tobacco in Chongqing/Proceedings of the Eleventh National Congress and the 2013 Academic Annual Meeting of China Society of Plant Protection.Beijing:China Agricultural Science and Technology Publishing House:401.(in Chinese)刘陈晨,孙淼,余冠华,青玲.2013.侵染重庆烟草的番茄斑萎病毒的ELISA检测//中国植物保护学会第十一次全国会员代表大会暨2013年学术年会论文集,北京:中国农业科学技术出版社:401.
Liu Yong,Li Yuan-yuan,Wu Zhu-yan,Rao Xue-qin.2010.Identification of Tospovirus on pepper in Guangzhou.Acta Phytophylacica Sinica,37(4):383-384.(in Chinese)刘勇,李媛媛,吴竹妍,饶雪琴.2010.广州辣椒上番茄斑萎病毒属病毒的鉴定.植物保护学报,37(4):383-384.
Margaria P,Bosco L,Vallino M,Ciuffo M,Mautino G C,Tavella L,Turina M.2014.The NSs protein of Tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis.Journal of Virology,88(10):5788-5802.
Massumi H,Samei A,Pour AH,Shaabanian M,Rahimian H.2007.Occurrence,distribution and relative incidence of seven viruses infecting greenhouse-grown cucurbits in Iran.Plant Disease,91(2):159-163.
Meng Jiao-rong,Zhao Jia-xin,Cai Qi,Zou Cheng-wu,Li Zhan-biao,Liao Yong-mei,Chen Bao-shan.2012.The investigation of virus affecting occurrence and pathogen identification in tobacco in Guangxi.Guangxi Plant Protection,25(4):1-2.(in Chinese)蒙姣荣,赵家新,蔡琦,邹承武,李战彪,廖咏梅,陈保善.2012.广西烟草病毒病发生情况调查和病原病毒的鉴定.广西植保,25(4):1-2.
Parrella G,Marchoux G.2003.An update of the host range of Tomato spotted wilt virus.Journal of Plant Pathology,85(4):227-264.
Ren Xi-yi,Liu Yong-xiang,Tan Yu-mei,Sun Cheng-long,Huang Yong-hui,Liu Zuo-yi.2014.Distribution characteristics of main tobacco virus disease in Guizhou,Guizhou Agricultural Sciences,42(5):117-120.(in Chinese)任锡毅,刘永翔,谭玉梅,孙成龙,黄永会,刘作易.2014.贵州烟草病毒病的主要种类及分布特点.贵州农业科学,42(5):117-120.
Riley D G,Joseph S V,Srinivasan R,Diffie S.2011.Thrips vectors of Tospoviruses.Journal of Integrated Pest Management,1(2):1-10.
Scholthof K G,Adkins S,Czosnek H,Palukaitis P,Jacquot E,Hohn T,Hohn B,Saunders K,Candresse T,Ahlquist P,Hemenway C,Foster GD.2011.Top 10 plant viruses in molecular plant pathology.Molecular Plant Pathology,12(9):938-954.
Sivparsad B,Gubba A.2011.Evaluation of tomato(Lycopersicon esculentum Mill.)plants with natural and transgenic resistance against Tomato spotted wilt virus(TSWV)isolates occurring in the Republic of South Africa(RSA).African Journal of Agricultural Research,6(13):3013-3020.
Soler S,Cebolla-Cornejo J,Nuez F.2003.Control of diseases induced by Tospoviruses in tomato:an update of the genetic approach.Phytopathologia Mediterranea,42(3):207-219.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,28(10):2731-2739.
Timmerman-Vaughan G M,Lister R,Cooper R,Tang J.2014.Phylogenetic analysis of New Zealand Tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution.Archives of Virology,159(5):993-1003.
Ullman D E,Whitfield A E,German T L.2005.Thrips and Tospoviruses come of age:mapping determinants of insect transmission.Proceedings of the National Academy of Sciences,102(14):4931-4932.
Xu Ze-yong,Zhang Zong-yi,Chen Jin-xiang.1989.Biological properties of a Guangdong isolate of Tomato spotted wilt virus(TSWV).Acta Phytopathologica Sinica,(4):198-198.(in Chinese)许泽永,张宗义,陈金香.1989.番茄斑萎病毒(TSWV)广州分离物生物学特性研究.植物病理学报,(4):198-198.
Yao Ge.1992.Tomato spotted wilt virus was identified in tobacco in Sichuan.Tobacco Science&Technology,(6):2-4.(in Chinese)姚革.1992.四川晒烟上发现番茄斑萎病毒.烟草科技,(6):2-4.
Yu Mao-de,Zhao Ai-chun,2016.Control of pests and diseases of mulberry in summer in Chongqing.Newsletter of Sericultural Science,36(2):18.(in Chinese)余茂德,赵爱春.2016.重庆市夏季桑树主要病虫害防治技术要点.蚕学通讯,36(2):18.
Zhang Ying,Mo Rang-yu,Li Long-yun,Qu Xian-you.2011.Occurrence and control of new pest of Polygonum multiflorum-Scirtothrips dorsalis.Plant Doctor,24(2):28-29.(in Chinese)张应,莫让瑜,李隆云,瞿显友.2011.何首乌新害虫——茶黄蓟马发生规律及防治方法.植物医生,24(2):28-29.
Zhang Zhen-jia,Wang De-ya,Yu Cheng-ming,Wang Zeng-hui,Dong Jia-hong,Shi Ke-rong,Yuan Xue-feng.2016.Identification of three new isolates of Tomato spotted wilt virus from different hosts in China:molecular diversity,phylogenetic and recombination analyses.Virology Journal,13(1):1-12.
Zhang Zhong-kai,Ding Ming,Fang Qi,Zhang Li-zhen,Li Ting-ting,Peng Lu-bo,Su Xiao-xia,Li Zhan.2004.The preliminary study of the occurrence and distribution of Tospovirus in Yunnan.Southwest China Journal of Agricultural Sciences,17(Supplement):163-168.(in Chinese)张仲凯,丁铭,方琦,张丽珍,李婷婷,彭潞波,苏晓霞,李展.2004.番茄斑萎病毒属(Tospovirus)病毒在云南的发生分布研究初报.西南农业学报,17(增刊):163-168.
Zhang Zhong-kai,Fang Qi,Ding Ming,Peng Lu-bo,Zhang Li-zhen.2000.The electron microscope identification of Tomato spotted wilt virus in tobacco.Journal of Chinese Electron Microscopy Society,19(3):339-340.(in Chinese)张仲凯,方琦,丁铭,彭潞波,张丽珍.2000.侵染烟草的番茄斑萎病毒(TSWV)电镜诊断鉴定.电子显微学报,19(3):339-340.
Zheng Kuan-yu,Wu Kuo,Dong Jia-hong,Fang Qi,Zhang Zhong-kai.2015.Identification 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.
Burland T G 2000.DNASTAR's Lasergene sequence analysis software.Methods in Molecular Biology,132:71-91.
Cao Jin-qiang,Xie Xue-wen,Chai A-li,Li Bao-ju.2016.Identification and control of Tomato spotted wilt virus in Ningxia.China Vegetables,(4):87-89.(in Chinese)曹金强,谢学文,柴阿丽,李宝聚.2016.番茄斑萎病毒病在宁夏的发现与防治.中国蔬菜,(4):87-89.
Ding Ming,Zhang Li-zhen,Fang Qi,Li Ting-ting,Su Xiao-xia,Li Zhan.2004.Identification,purification and antiserum preparation of Tospovirus isolate affecting potato.Southwest China Journal of Agricultural Sciences,17(Supplement):160-162.(in Chinese)丁铭,张丽珍,方琦,李婷婷,苏晓霞,李展.2004.侵染马铃薯的一个Tospovirus混合分离物的鉴定、纯化及多抗血清制备.西南农业学报,17(增刊):160-162.
French J M,Goldberg N P,Randall J J,Hanson S F.2016.New Mexico and the southwestern US are affected by a unique population of Tomato spotted wilt virus(TSWV)strains.Archives of Virology,161:993-998.
Gao Wei,Wang Yong,Zhang Chun-xiang,Zhang An-sheng,Zhu Xiao-ping.2016.Investigation and pathogen preliminary identification of pepper virus disease in Tianjin.Shandong Agricultural Sciences,48(3):91-94.(in Chinese)高苇,王勇,张春祥,张安盛,竺晓平.2016.天津地区辣椒病毒病调查及毒源种类初步鉴定.山东农业科学,48(3):91-94.
Guo Si-yao,Tong Yan,Huang Ya,Luo Xin-fu,Qing Ling.2015.Preliminary identification and analyses of viruses causing pepper virus disease in Chongqing,China.Acta Horticulturae Sinica,42(2):263-270.(in Chinese)郭思瑶,童艳,黄娅,罗信福,青玲.2015.重庆辣椒病毒病病原初步鉴定和分析.园艺学报,42(2):263-270.
Huang Ren-zhong,Ge Wen-dong,He Ye,Huang Qi-zhong,Zhe Wen-jun,Liu Xiao-bo,Wu Zhao-long.2015.The effectiveness of planting disaster insurance in Chongqing Shizhu.China Vegetables,1(10):10-12.(in Chinese)黄任中,葛文东,何叶,黄启中,柘文君,刘晓波,吴朝龙.2015.重庆石柱辣椒种植灾害保险的实施效果.中国蔬菜,1(10):10-12.
Lei Lei,Lin Qing,L(u|")Zhong-hua,Huang Ren-zhong,Huang Qi-zhong,Shi Si-ru.2001.The species identification and strain ascertainment of pepper virus in Chongqing.Southwest Gardening,29(2):28-29.(in Chinese)雷蕾,林清,吕中华,黄任中,黄启中,史思茹.2001.重庆市辣椒病毒病毒原种类鉴定及株系确认.西南园艺,29(2):28-29.
Li Fei,Wu Qing-jun,Xu Bao-yun,Xie Wen,Wang Shao-li,Zhang You-jun.2012.Tomato spotted wilt virus was identified in Beijing.Plant Protection,38(6):186-188.(in Chinese)李飞,吴青君,徐宝云,谢文,王少丽,张友军.2012.北京地区发现番茄斑萎病毒.植物保护,38(6):186-188.
Lian S,Lee J S,Cho W K,Yu J,Kim M K,Choi H S,Kim K H.2013.Phylogenetic and recombination analysis of Tomato spotted wilt virus.PLoS ONE,8(5):e63380.
Lin Qing,Lu Zhong-hua,Huang Ren-zhong,Huang Qi-zhong,Lei Lei,Shi Si-ru.2001.The identification of TMV and CMV in pepper of Chongqing.China Vegetables,(3):13-15.(in Chinese)林清,吕中华,黄任中,黄启中,雷蕾,史思茹.2001,重庆市辣椒TMV和CMV的鉴定.中国蔬菜,(3):13-15.
Liu Chen-chen,Sun Miao,Yu Guan-hua,Qing Ling.2013.The ELISA detection of Tomato spotted wilt virus(TSWV)infecting tobacco in Chongqing/Proceedings of the Eleventh National Congress and the 2013 Academic Annual Meeting of China Society of Plant Protection.Beijing:China Agricultural Science and Technology Publishing House:401.(in Chinese)刘陈晨,孙淼,余冠华,青玲.2013.侵染重庆烟草的番茄斑萎病毒的ELISA检测//中国植物保护学会第十一次全国会员代表大会暨2013年学术年会论文集,北京:中国农业科学技术出版社:401.
Liu Yong,Li Yuan-yuan,Wu Zhu-yan,Rao Xue-qin.2010.Identification of Tospovirus on pepper in Guangzhou.Acta Phytophylacica Sinica,37(4):383-384.(in Chinese)刘勇,李媛媛,吴竹妍,饶雪琴.2010.广州辣椒上番茄斑萎病毒属病毒的鉴定.植物保护学报,37(4):383-384.
Margaria P,Bosco L,Vallino M,Ciuffo M,Mautino G C,Tavella L,Turina M.2014.The NSs protein of Tomato spotted wilt virus is required for persistent infection and transmission by Frankliniella occidentalis.Journal of Virology,88(10):5788-5802.
Massumi H,Samei A,Pour AH,Shaabanian M,Rahimian H.2007.Occurrence,distribution and relative incidence of seven viruses infecting greenhouse-grown cucurbits in Iran.Plant Disease,91(2):159-163.
Meng Jiao-rong,Zhao Jia-xin,Cai Qi,Zou Cheng-wu,Li Zhan-biao,Liao Yong-mei,Chen Bao-shan.2012.The investigation of virus affecting occurrence and pathogen identification in tobacco in Guangxi.Guangxi Plant Protection,25(4):1-2.(in Chinese)蒙姣荣,赵家新,蔡琦,邹承武,李战彪,廖咏梅,陈保善.2012.广西烟草病毒病发生情况调查和病原病毒的鉴定.广西植保,25(4):1-2.
Parrella G,Marchoux G.2003.An update of the host range of Tomato spotted wilt virus.Journal of Plant Pathology,85(4):227-264.
Ren Xi-yi,Liu Yong-xiang,Tan Yu-mei,Sun Cheng-long,Huang Yong-hui,Liu Zuo-yi.2014.Distribution characteristics of main tobacco virus disease in Guizhou,Guizhou Agricultural Sciences,42(5):117-120.(in Chinese)任锡毅,刘永翔,谭玉梅,孙成龙,黄永会,刘作易.2014.贵州烟草病毒病的主要种类及分布特点.贵州农业科学,42(5):117-120.
Riley D G,Joseph S V,Srinivasan R,Diffie S.2011.Thrips vectors of Tospoviruses.Journal of Integrated Pest Management,1(2):1-10.
Scholthof K G,Adkins S,Czosnek H,Palukaitis P,Jacquot E,Hohn T,Hohn B,Saunders K,Candresse T,Ahlquist P,Hemenway C,Foster GD.2011.Top 10 plant viruses in molecular plant pathology.Molecular Plant Pathology,12(9):938-954.
Sivparsad B,Gubba A.2011.Evaluation of tomato(Lycopersicon esculentum Mill.)plants with natural and transgenic resistance against Tomato spotted wilt virus(TSWV)isolates occurring in the Republic of South Africa(RSA).African Journal of Agricultural Research,6(13):3013-3020.
Soler S,Cebolla-Cornejo J,Nuez F.2003.Control of diseases induced by Tospoviruses in tomato:an update of the genetic approach.Phytopathologia Mediterranea,42(3):207-219.
Tamura K,Peterson D,Peterson N,Stecher G,Nei M,Kumar S.2011.MEGA5:molecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods.Molecular Biology and Evolution,28(10):2731-2739.
Timmerman-Vaughan G M,Lister R,Cooper R,Tang J.2014.Phylogenetic analysis of New Zealand Tomato spotted wilt virus isolates suggests likely incursion history scenarios and mechanisms for population evolution.Archives of Virology,159(5):993-1003.
Ullman D E,Whitfield A E,German T L.2005.Thrips and Tospoviruses come of age:mapping determinants of insect transmission.Proceedings of the National Academy of Sciences,102(14):4931-4932.
Xu Ze-yong,Zhang Zong-yi,Chen Jin-xiang.1989.Biological properties of a Guangdong isolate of Tomato spotted wilt virus(TSWV).Acta Phytopathologica Sinica,(4):198-198.(in Chinese)许泽永,张宗义,陈金香.1989.番茄斑萎病毒(TSWV)广州分离物生物学特性研究.植物病理学报,(4):198-198.
Yao Ge.1992.Tomato spotted wilt virus was identified in tobacco in Sichuan.Tobacco Science&Technology,(6):2-4.(in Chinese)姚革.1992.四川晒烟上发现番茄斑萎病毒.烟草科技,(6):2-4.
Yu Mao-de,Zhao Ai-chun,2016.Control of pests and diseases of mulberry in summer in Chongqing.Newsletter of Sericultural Science,36(2):18.(in Chinese)余茂德,赵爱春.2016.重庆市夏季桑树主要病虫害防治技术要点.蚕学通讯,36(2):18.
Zhang Ying,Mo Rang-yu,Li Long-yun,Qu Xian-you.2011.Occurrence and control of new pest of Polygonum multiflorum-Scirtothrips dorsalis.Plant Doctor,24(2):28-29.(in Chinese)张应,莫让瑜,李隆云,瞿显友.2011.何首乌新害虫——茶黄蓟马发生规律及防治方法.植物医生,24(2):28-29.
Zhang Zhen-jia,Wang De-ya,Yu Cheng-ming,Wang Zeng-hui,Dong Jia-hong,Shi Ke-rong,Yuan Xue-feng.2016.Identification of three new isolates of Tomato spotted wilt virus from different hosts in China:molecular diversity,phylogenetic and recombination analyses.Virology Journal,13(1):1-12.
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