番茄细菌性斑点病菌实时荧光定量PCR检测方法的建立及应用
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摘要
以番茄细菌性斑点病病原菌丁香假单胞菌番茄致病变种(Pseudomonas syringae pv. tomato,Pst)的致病相关基因HrpZ为靶序列,设计了特异性引物Pst3F/Pst3R,能从Pst基因组DNA中特异性扩增出大小为161 bp的目的片段。建立的Pst实时荧光定量PCR检测技术体系的检测灵敏度比普通PCR高1 000倍。利用实时荧光定量PCR检测体系,检测模拟带菌种子中Pst的带菌量,检测下限为4.21 cfu·g~(-1);检测人工接种叶片组织中Pst的带菌量,检测到1级发病叶片带菌量为4.15×102 cfu·g~(-1)。对田间采集的63个番茄细菌性斑点病明显症状和疑似症状样本,分别进行了实时荧光定量PCR、普通PCR和病原菌分离检测,检测到54个样本中含有Pst,3种方法检测结果一致。结果表明,建立的Pst实时荧光定量PCR检测体系具有特异性强、灵敏度高的特点,可以快速准确地定量检测番茄种子和发病组织中Pst的含量,为番茄细菌性斑点病的早期预防和流行监测提供了有效的技术手段。
In the present work,a specific primer pair Pst3F/Pst3R was designed based on the pathogenicity-related gene HrpZ of Pseudomonas syringae pv. tomato(Pst),and a target fragment of 161 bp was specifically amplified from the genomic DNA of Pst. A real-time fluorescent quantitative PCR(qPCR)assay was developed for quantifying Pst in contaminated tomato seeds and infected tissues. The detection sensitivity of qPCR established in this study was 1 000 times higher than that of conventional PCR. The detection threshold was 4.21 cfu per gram of artificially contaminated tomato seeds. The amount of Pst was detected to be 4.15 × 10~2 cfu per gram of inoculated leaves with disease-grade level 1. For naturally infected tomato tissue samples,PCR,qPCR and classical culture methods were used for determination of Pst. Quantifiable levels of Pst were detected in 54 out of the 63 samples,and the results of the three methods were consistent. These studies showed that qPCR assay is a highly rapid and reliable method to quantify Pst in tomato seeds and infected tissues of leaves,stems and fruits. Application of the assay may potentially improve pathogen identification and disease management.
In the present work,a specific primer pair Pst3F/Pst3R was designed based on the pathogenicity-related gene HrpZ of Pseudomonas syringae pv. tomato(Pst),and a target fragment of 161 bp was specifically amplified from the genomic DNA of Pst. A real-time fluorescent quantitative PCR(qPCR)assay was developed for quantifying Pst in contaminated tomato seeds and infected tissues. The detection sensitivity of qPCR established in this study was 1 000 times higher than that of conventional PCR. The detection threshold was 4.21 cfu per gram of artificially contaminated tomato seeds. The amount of Pst was detected to be 4.15 × 10~2 cfu per gram of inoculated leaves with disease-grade level 1. For naturally infected tomato tissue samples,PCR,qPCR and classical culture methods were used for determination of Pst. Quantifiable levels of Pst were detected in 54 out of the 63 samples,and the results of the three methods were consistent. These studies showed that qPCR assay is a highly rapid and reliable method to quantify Pst in tomato seeds and infected tissues of leaves,stems and fruits. Application of the assay may potentially improve pathogen identification and disease management.
引文
Bao Qi,Cao Meng-qi,Zhou Yu,Zheng Yu,Li Chang-long,Wang Hai-yan,Wang Jun,Sheng Cheng,Wu Fu-an.2016.Detection of Pseudomonas syringae pv.mori on real-time quantitative PCR technology.Sericulture Science,(2):210-218.(in Chinese)包奇,曹梦琪,周雨,郑煜,李长龙,王海燕,王俊,盛晟,吴福安.2016.基于实时荧光定量PCR技术检测桑丁香假单胞菌.蚕业科学,(2):210-218.
Bashan Y,Okon Y,Henis Y.1985.Detection of cutinases and pectic enzymes during infection of tomato by Pseudomonas syringae pv.tomato.Phytopathology,75(8):940-945.
Bryan M K.1933.Bacterial speck of tomatoes.Phytopathology,23(11):897-904.
Charkowski A O,Huang H C,Collmer A.1997.Altered localization of HrpZ in Pseudomonas syringae pv.syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria.Journal of Bacteriology,179:3866-3874.
Chen Chang-jun,Li Jun,Zhao Wei,Wang Jian-xin,Zhou Ming-guo.2011.Detection of Sclerotinia sclerotiorum by a quantitative real-time PCR.Acta Phytopathologica Sinica,41(5):516-525.(in Chinese)陈长军,李俊,赵伟,王建新,周明国.2011.利用实时荧光定量PCR技术检测油菜菌核病菌.植物病理学报,41(5):516-525.
Chen Hong-ming,Wang Xue-feng,Zhou Yan,Zhou Chang-yong,Guo Jun,Li Zhong-an.2015.Biological characterization and RT-PCRdetection of a new disease in Eureka lemon.Journal of Plant Protection,43(4):557-563.(in Chinese)陈洪明,王雪峰,周彦,周常勇,郭俊,李中安.2015.尤力克柠檬上一种新病害的生物学特性及RT-PCR检测技术.植物保护学报,43(4):557-563.
Chen Si,Huang Kun,Xu Wen-tao,Li Yuan,Luo Yun-bo.2006.Real-time fluorescence quantitative PCR method for detection of E.coli o157:h7.Journal of Agricultural Biotechnology,14(5):779-782.(in Chinese)陈思,黄昆,许文涛,李媛,罗云波.2006.实时荧光定量PCR方法检测大肠杆菌o157:h7.农业生物技术学报,14(5):779-782.
Cheng Ying-chao,Kang Hua-jun,Shi Yan-xia,Chai A-li,Zhang Hong-jie,Xie Xue-wen,Li Bao-ju.2018.Development and application of real-time fluorescent quantitative PCR for detection of Phytophthora capsici.Acta Horticulturae Sinica,45(5):997-1006.(in Chinese)程颖超,康华军,石延霞,柴阿丽,张红杰,谢学文,李宝聚.2018.辣椒疫霉菌RT-PCR检测技术的建立及应用.园艺学报,45(5):997-1006.
Cottyn B,Baeyen S,Pauwelyn E,Verbaendert I,Vos P D,Bleyaert P,H?fte M,Maes M.2011.Development of a real-time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse-grown lettuce,and its detection in irrigating water.Plant Pathology,60(3):453-461.
Dieter K.2002.Quantification using real-time PCR technology:applications and limitations.Trends in Molecular Medicine,8(6):257-260.
Dou Ya-ya,Ruan Xiao-lei,Yuan Yue,Liu Qiong-guang,Li Hua-ping.2013.Utilization of a real time PCR based approach for rapid quantitative detection of bacterial soft rot of banana.Acta Horticulturae Sinica,40(2):380-388.(in Chinese)豆亚亚,阮小蕾,袁月,刘琼光,李华平.2013.利用实时荧光PCR方法检测香蕉软腐细菌.园艺学报,40(2):380-388.
Fanelli V,Cariddi C,Finetti-Sialer M.2007.Selective detection of Pseudomonas syringae pv.tomato using dot blot hybridization and real-time PCR.Plant Pathology,56:683-691.
Jones J B,Gitaitis R D,Mccarter S M.1986.Fluorescence on single-carbon sources for separation of Pseudomonas syringae pv.syringae,Pseudomonas syringae pv.tomato,and Pseudomonas viridiflava on tomato transplants.Plant Disease,70(2):151-153.
Paul D.2010.Development of a real-time PCR assay for detection and quantitation of group B Streptococci with special reference to infection of vascular endothelium[M.D.Dissertation].Manchester:University of Manchester.
Ross A,Somssich I E.2016.A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana.Plant Methods,12:48.
Wang Dao-ze,Zhang Li-li,Tao Zhong-yun,Xie Guan-lin.2016.Development of a real time quantitative PCR assay for the specific detection of Pseudomonas syringae pv.maculicola in crucifers.Journal of Plant Protection,43(4):559-566.(in Chinese)王道泽,张莉丽,陶中云,谢关林.2016.实时荧光定量PCR法检测十字花科细菌性黑斑病菌.植物保护学报,43(4):559-566.
Wang Ying-ying,Chai A-li,Li Bao-ju.2015.Symptoms and integrated control of tomato bacterial speck.China Vegetables,(11):82-84.(in Chinese)王莹莹,柴阿丽,李宝聚.2015.番茄细菌性斑点病症状多样性与综合防治.中国蔬菜,(11):82-84.
Wen Chao-hui,Wang Jun-ping,He Su-qin.2013.Identification and detection of tomato bacterial spot pathogens in Gansu.Chinese Vegetables(6):68-73.(in Chinese)文朝慧,王军平,何苏琴.2013.甘肃地区番茄细菌性斑点病菌的鉴定与检测.中国蔬菜,(6):68-73.
Wreikat B I,Al-Banna L S,Khlaif H M.2006.Detection of identification of bacterial speck of tomato(Pseudomonas syringae pv.tomato)by polymerase chain reaction.Jordan Journal of Agricultural Sciences,2:45-56.
Xu Yan-yan,Chen Lu,Li Jin-ping,Xie Xue-wen,Shi Yan-xia,Li Bao-ju.2013.Development and application of real-time fluorescent quantitative PCR for detection of Pseudomonas tolaasii.Acta Horticulturae Sinica,40(1):169-178.(in Chinese)徐岩岩,陈璐,李金萍,谢学文,石延霞,李宝聚.2013.平菇细菌性褐斑病病原菌RT-PCR检测方法的建立及其应用.园艺学报,40(1):169-178.
Yunis H,Bashan Y,Okon Y,Henis Y.1980.Weather dependence,yield losses and control of bacterial speck of tomato caused by Pseudomonas tomato.Plant Disease,64:937-939.
Zaccardelli M,Spasiano A,Bazzi C,Merighi M.2005.Identification and in planta detection of Pseudomonas syringae pv.tomato using PCRamplification of hrpZPst.European Journal of Plant Pathology,111:85-90.
Zhang Hui.2003.IMS-PCR for the detection of bacterial carriers of tomato bacterial spot disease[M.D.Dissertation].Harbin:Northeast Agricultural University.(in Chinese)张卉.2003.IMS-PCR检测番茄细菌性斑点病种子带菌研究[硕士论文].哈尔滨:东北农业大学.
Zhao Ting-chang,Sun Fu-zai,Song Wen-sheng.2001.Identification of pathogenic bacteria of bacterial spot disease in tomato.Journal of Plant Pathology,31(1):37-42.(in Chinese)赵廷昌,孙福在.宋文生.2001.番茄细菌性斑点病病原菌鉴定.植物病理学报,31(1):37-42.
Bashan Y,Okon Y,Henis Y.1985.Detection of cutinases and pectic enzymes during infection of tomato by Pseudomonas syringae pv.tomato.Phytopathology,75(8):940-945.
Bryan M K.1933.Bacterial speck of tomatoes.Phytopathology,23(11):897-904.
Charkowski A O,Huang H C,Collmer A.1997.Altered localization of HrpZ in Pseudomonas syringae pv.syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria.Journal of Bacteriology,179:3866-3874.
Chen Chang-jun,Li Jun,Zhao Wei,Wang Jian-xin,Zhou Ming-guo.2011.Detection of Sclerotinia sclerotiorum by a quantitative real-time PCR.Acta Phytopathologica Sinica,41(5):516-525.(in Chinese)陈长军,李俊,赵伟,王建新,周明国.2011.利用实时荧光定量PCR技术检测油菜菌核病菌.植物病理学报,41(5):516-525.
Chen Hong-ming,Wang Xue-feng,Zhou Yan,Zhou Chang-yong,Guo Jun,Li Zhong-an.2015.Biological characterization and RT-PCRdetection of a new disease in Eureka lemon.Journal of Plant Protection,43(4):557-563.(in Chinese)陈洪明,王雪峰,周彦,周常勇,郭俊,李中安.2015.尤力克柠檬上一种新病害的生物学特性及RT-PCR检测技术.植物保护学报,43(4):557-563.
Chen Si,Huang Kun,Xu Wen-tao,Li Yuan,Luo Yun-bo.2006.Real-time fluorescence quantitative PCR method for detection of E.coli o157:h7.Journal of Agricultural Biotechnology,14(5):779-782.(in Chinese)陈思,黄昆,许文涛,李媛,罗云波.2006.实时荧光定量PCR方法检测大肠杆菌o157:h7.农业生物技术学报,14(5):779-782.
Cheng Ying-chao,Kang Hua-jun,Shi Yan-xia,Chai A-li,Zhang Hong-jie,Xie Xue-wen,Li Bao-ju.2018.Development and application of real-time fluorescent quantitative PCR for detection of Phytophthora capsici.Acta Horticulturae Sinica,45(5):997-1006.(in Chinese)程颖超,康华军,石延霞,柴阿丽,张红杰,谢学文,李宝聚.2018.辣椒疫霉菌RT-PCR检测技术的建立及应用.园艺学报,45(5):997-1006.
Cottyn B,Baeyen S,Pauwelyn E,Verbaendert I,Vos P D,Bleyaert P,H?fte M,Maes M.2011.Development of a real-time PCR assay for Pseudomonas cichorii,the causal agent of midrib rot in greenhouse-grown lettuce,and its detection in irrigating water.Plant Pathology,60(3):453-461.
Dieter K.2002.Quantification using real-time PCR technology:applications and limitations.Trends in Molecular Medicine,8(6):257-260.
Dou Ya-ya,Ruan Xiao-lei,Yuan Yue,Liu Qiong-guang,Li Hua-ping.2013.Utilization of a real time PCR based approach for rapid quantitative detection of bacterial soft rot of banana.Acta Horticulturae Sinica,40(2):380-388.(in Chinese)豆亚亚,阮小蕾,袁月,刘琼光,李华平.2013.利用实时荧光PCR方法检测香蕉软腐细菌.园艺学报,40(2):380-388.
Fanelli V,Cariddi C,Finetti-Sialer M.2007.Selective detection of Pseudomonas syringae pv.tomato using dot blot hybridization and real-time PCR.Plant Pathology,56:683-691.
Jones J B,Gitaitis R D,Mccarter S M.1986.Fluorescence on single-carbon sources for separation of Pseudomonas syringae pv.syringae,Pseudomonas syringae pv.tomato,and Pseudomonas viridiflava on tomato transplants.Plant Disease,70(2):151-153.
Paul D.2010.Development of a real-time PCR assay for detection and quantitation of group B Streptococci with special reference to infection of vascular endothelium[M.D.Dissertation].Manchester:University of Manchester.
Ross A,Somssich I E.2016.A DNA-based real-time PCR assay for robust growth quantification of the bacterial pathogen Pseudomonas syringae on Arabidopsis thaliana.Plant Methods,12:48.
Wang Dao-ze,Zhang Li-li,Tao Zhong-yun,Xie Guan-lin.2016.Development of a real time quantitative PCR assay for the specific detection of Pseudomonas syringae pv.maculicola in crucifers.Journal of Plant Protection,43(4):559-566.(in Chinese)王道泽,张莉丽,陶中云,谢关林.2016.实时荧光定量PCR法检测十字花科细菌性黑斑病菌.植物保护学报,43(4):559-566.
Wang Ying-ying,Chai A-li,Li Bao-ju.2015.Symptoms and integrated control of tomato bacterial speck.China Vegetables,(11):82-84.(in Chinese)王莹莹,柴阿丽,李宝聚.2015.番茄细菌性斑点病症状多样性与综合防治.中国蔬菜,(11):82-84.
Wen Chao-hui,Wang Jun-ping,He Su-qin.2013.Identification and detection of tomato bacterial spot pathogens in Gansu.Chinese Vegetables(6):68-73.(in Chinese)文朝慧,王军平,何苏琴.2013.甘肃地区番茄细菌性斑点病菌的鉴定与检测.中国蔬菜,(6):68-73.
Wreikat B I,Al-Banna L S,Khlaif H M.2006.Detection of identification of bacterial speck of tomato(Pseudomonas syringae pv.tomato)by polymerase chain reaction.Jordan Journal of Agricultural Sciences,2:45-56.
Xu Yan-yan,Chen Lu,Li Jin-ping,Xie Xue-wen,Shi Yan-xia,Li Bao-ju.2013.Development and application of real-time fluorescent quantitative PCR for detection of Pseudomonas tolaasii.Acta Horticulturae Sinica,40(1):169-178.(in Chinese)徐岩岩,陈璐,李金萍,谢学文,石延霞,李宝聚.2013.平菇细菌性褐斑病病原菌RT-PCR检测方法的建立及其应用.园艺学报,40(1):169-178.
Yunis H,Bashan Y,Okon Y,Henis Y.1980.Weather dependence,yield losses and control of bacterial speck of tomato caused by Pseudomonas tomato.Plant Disease,64:937-939.
Zaccardelli M,Spasiano A,Bazzi C,Merighi M.2005.Identification and in planta detection of Pseudomonas syringae pv.tomato using PCRamplification of hrpZPst.European Journal of Plant Pathology,111:85-90.
Zhang Hui.2003.IMS-PCR for the detection of bacterial carriers of tomato bacterial spot disease[M.D.Dissertation].Harbin:Northeast Agricultural University.(in Chinese)张卉.2003.IMS-PCR检测番茄细菌性斑点病种子带菌研究[硕士论文].哈尔滨:东北农业大学.
Zhao Ting-chang,Sun Fu-zai,Song Wen-sheng.2001.Identification of pathogenic bacteria of bacterial spot disease in tomato.Journal of Plant Pathology,31(1):37-42.(in Chinese)赵廷昌,孙福在.宋文生.2001.番茄细菌性斑点病病原菌鉴定.植物病理学报,31(1):37-42.