番茄细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌的四重PCR检测方法
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摘要
针对引起番茄细菌性病害的细菌性斑点病菌(Pseudomonassyringae pv. tomato,Pst)、溃疡病菌(Clavibacter michiganensis subsp. michiganensis,Cmm)、青枯病菌(Ralstonia solanacearum,Rs)以及疮痂病菌(Xanthomonas campestris pv. vesicatoria,Xcv),建立了四重PCR检测技术,为病害的快速、准确诊断提供技术支持。根据gap1基因设计Pst特异性引物BW-F/BW-R,经PCR条件优化,扩增出了375bp的特异性片段。将设计的引物与已报道的3种细菌特异性引物组合,通过设定不同的退火温度、引物浓度、循环次数以及延伸时间,探索影响四重PCR扩增的因素,优化了其反应体系。四重PCR反应体系中的引物对BW-F/BW-R、Fan1/Fan2、RS-1-F/RS-3-R和XCVF/XCVR可分别扩增出细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌长度为375、146、716和517bp的特异性目的片段,反应体系退火温度为57.1℃,4对引物的终浓度分别为0.24、0.16、0.16和0.08μmol·L~(-1),延伸时间45 s,35个循环。该四重PCR反应体系可快速检测田间番茄发病植株中的细菌性斑点病菌、溃疡病菌、青枯病菌和疮痂病菌,灵敏度达到10-1 ng·μL~(-1)。
In order to establish a rapid and specific detection method for tomato diseases,a quadruple PCR assay was designed for identifing the pathogens including Pseudomonas syringae pv. tomato(Pst),Clavibacter michiganensis subsp. michiganensis(Cmm),Ralstonia solanacearum(Rs)and Xanthomonas campestris pv. vesicatoria(Xcv). The specific primers(BW-F/BW-R)of Pst were designed based on the gap1 gene sequence,which can amplify 375 bp specific fragment by optimizing the PCR conditions. Besides,three pairs of specific primers of Cmm(Fan1/Fan2),Rs(RS-1-F/RS-3-R),and Xcv(XCVF/XCVR)used in this study were referenced previous studies. The primer concentration,the annealing temperature,amplification cycles,and the extension time were optimized to obtain the best ratio of primers and amplification conditions. Then,the rapid quadruple PCR detection of pathogens in tomato was established. The annealing temperature was 57.1 ℃,the extension time was 45 s and the number of cycles was 35. The final concentrations of BW-F/BW-R primers,Fan1/Fan2 primers,RS-1-F/RS-3-R primers and XCVF/XCVR primers were 0.24,0.16,0.16 and 0.08 μmol · L-1,respectively. The expected sizes of amplification bands were 375,146,716 and 517 bp for BW-F/BW-R,Fan1/Fan2,RS-1-F/RS-3-R and XCVF/XCVR,respectively. The quadruple PCR can simultaneously detect Pst,Cmm,Rs and Xcv in infected plants tissue,with the detection limits of 0.1 ng · μL~(-1) gDNA. This study indicated that quadruple PCR might be a useful tool for rapid and sensitive diagnosis and provided an effective technical support for pathogenic identification
In order to establish a rapid and specific detection method for tomato diseases,a quadruple PCR assay was designed for identifing the pathogens including Pseudomonas syringae pv. tomato(Pst),Clavibacter michiganensis subsp. michiganensis(Cmm),Ralstonia solanacearum(Rs)and Xanthomonas campestris pv. vesicatoria(Xcv). The specific primers(BW-F/BW-R)of Pst were designed based on the gap1 gene sequence,which can amplify 375 bp specific fragment by optimizing the PCR conditions. Besides,three pairs of specific primers of Cmm(Fan1/Fan2),Rs(RS-1-F/RS-3-R),and Xcv(XCVF/XCVR)used in this study were referenced previous studies. The primer concentration,the annealing temperature,amplification cycles,and the extension time were optimized to obtain the best ratio of primers and amplification conditions. Then,the rapid quadruple PCR detection of pathogens in tomato was established. The annealing temperature was 57.1 ℃,the extension time was 45 s and the number of cycles was 35. The final concentrations of BW-F/BW-R primers,Fan1/Fan2 primers,RS-1-F/RS-3-R primers and XCVF/XCVR primers were 0.24,0.16,0.16 and 0.08 μmol · L-1,respectively. The expected sizes of amplification bands were 375,146,716 and 517 bp for BW-F/BW-R,Fan1/Fan2,RS-1-F/RS-3-R and XCVF/XCVR,respectively. The quadruple PCR can simultaneously detect Pst,Cmm,Rs and Xcv in infected plants tissue,with the detection limits of 0.1 ng · μL~(-1) gDNA. This study indicated that quadruple PCR might be a useful tool for rapid and sensitive diagnosis and provided an effective technical support for pathogenic identification
引文
Chamberlain J S,Gibbs R A,Rainer J E,Nguyen P N,Caskey C T.1988.Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification.Nucleic Acids Research,16(23):11141-11156.
Chen S,Cao Y Y,Li T Y,Wu X X.2015.Simultaneous detection of three wheat pathogenic fungal species by multiplex PCR.Phytoparasitica,43(4):449-460.
Gao Shi-gang,Zeng Rong,Xu Li-hui,Luo Jin-yan,Chen Lei,Dai Fu-ming.2016.Triplex PCR Detection for Corynespora cassiicola,Colletotrichum orbiculare and Pseudomonas syringae pv.lachrymans.Scientia Agricultura Sinica,49(16):3119-3129.(in Chinese)高士刚,曾蓉,徐丽慧,罗金燕,陈磊,戴富明.2016.三重PCR检测黄瓜靶斑病菌、炭疽病菌和细菌性角斑病菌.中国农业科学,49(16):3119-3129.
Gao Yong-yang,Wang Nan,Gao Guan-peng,Wang Wei.2010.Triplex PCR detection of Cladosporium cucumerinum,Fusarium oxysporum fsp.niveum and Mycosphaerella melonis in infected plant tissues.Acta Phytopathologica Sinica,40(4):343-350.(in Chinese)高永洋,王楠,高观朋,王伟.2010.瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测.植物病理学报,40(4):343-350.
Huang Wen,Xu Jin,Zhang Hao,Xu Jing-sheng,Ding Wei,Feng Jie.2016.Development of a LAMP Approach for Detection of Ralstonia solanacearum.Scientia Agricultura Sinica,49(11):2093-2102.(in Chinese)黄雯,徐进,张昊,许景升,丁伟,冯洁.2016.植物青枯菌LAMP检测方法的建立.中国农业科学,49(11):2093-2102.
Jones J B,Lacy G H,Bouzar H,Stall R E,Schaad N W.2004.Reclassification of the Xanthomonads associated with bacterial spot disease of tomato and pepper.Systematic&Applied Microbiology,27(6):755-762.
Luo Wen-bin,Li Hua-wei,Tang Hao,Qiu Si-xin,Ji Rong-chang,Xu Yong-qing,Liu Zhong-hua,Qiu Yong-xiang.2015.Establishment and application of multiplex PCR rapid Detection of potato viruses.Acta Horticulturae Sinica,42(2):280-288.(in Chinese)罗文彬,李华伟,汤浩,邱思鑫,纪荣昌,许泳清,刘中华,邱永祥.2015.马铃薯5种病毒多重PCR检测技术的建立及应用.园艺学报,42(2):280-288.
Markoulatos P,Siafakas N,Moncany M.2002.Multiplex polymerase chain reaction:a practical approach.Journal of clinical laboratory analysis,16(1):47-51.
Ozdemir Z.2005.Development of a Multiplex PCR Assay for Concurrent Detection of Clavibacter michiganensis ssp.michiganensis and Xanthomonas axonopodis pv.vesicatoria.Plant Pathology Journal,4(2):133-137.
Park D S,Shim J K,Kim J S,Lim R S,Hahn J H,Kim H G.2009.Sensitive and specific detection of Xanthomonas campestris pv.vesicatoria by PCR using pathovar-specific primers based on rhs family gene sequences.Microbiological Research,164(1):36-42.
Pastrik K H,Elphinstone J G,Pukall R.2002.Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of16S-23S ribosomal intergenic spacer region with internal positive control.European Journal of Plant Pathology,108(9):831-842.
Pizano J F,García G A,Sánchez-Rico K F,González-Chavira M M,Guevara-González R J,Torres-Pacheco I,Guevara-Olvera L.2016.Detection of Clavibacter michiganensis ssp.michiganensis by PCR in in tomato plants(Lycopersicon esculentum Mill.).Revista Mexicana de Ciencias Agrícolas,7(6):1347-1357.
Quinterovásquez G A,Bazántejeda M L,Martínezpe?afiel E,Martínezpe?afiel E,Kameyamakawabe L,Bermúdez-Cruz R M.2013.Multiplex PCRto detect four different tomato-infecting pathogens.Folia Microbiologica,58(4):269-276.
Singh D,Sinha S,Yadav D K,Chaudhary G.2014.Detection of Ralstonia solanacearum from asymptomatic tomato plants,irrigation water and soil through non-selective enrichment medium with hrp gene-based bio-PCR.Current Microbiology,69(2):127-134.
Sint D,Raso L,Traugott M.2012.Advances in multiplex PCR:balancing primer efficiencies and improving detection success.Methods in Ecology&Evolution,3(5):898-905.
Umesha S,Avinash P.2015.Multiplex PCR for simultaneous identification of Ralstonia solanacearum and Xanthomonas perforans.Biotech,5(3):245-252.
Wu Xing-hai,Shao Xiu-ling,Deng Ming-jun,Chen Chang-fa,Li Yan,Liang Cheng-zhu.2007.Study on rapid Detection of Clavibacter michiganensis subsp.michiganensis by Real-time Fluorescent PCR.Acta Agriculturae Jiangxi,19(3):34-36.(in Chinese)吴兴海,邵秀玲,邓明俊,陈长法,厉艳,梁成珠.2007.番茄溃疡病菌实时荧光PCR快速检测方法研究.江西农业学报,19(3):34-36.
Xia Ming-xing,Zhao Wen-jun,Ma Qing,Zhu Shui-fang.2006.Rapid detection of Tomato bacterial canker(Clavibacter michiganensis subsp.michiganensis)with real-tmie fluorescent PCR.Acta Phytopathologica Sinica,36(2):152-157.(in Chinese)夏明星,赵文军,马青,朱水芳.2006.番茄细菌性溃疡病菌的实时荧光PCR检测.植物病理学报,36(2):152-157.
Yin Yan-ni,Huang Yan-xia,Ge Yun-ying,Guo Jian-hua.2006.Molecular diagnostic techniques for several commonly used phytobacteria.Plant Protection,32(6):1-4.(in Chinese)尹燕妮,黄艳霞,葛芸英,郭坚华.2006.几种常用植物病原细菌分子检测方法.植物保护,32(6):1-4.
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(1):85-90.
Chen S,Cao Y Y,Li T Y,Wu X X.2015.Simultaneous detection of three wheat pathogenic fungal species by multiplex PCR.Phytoparasitica,43(4):449-460.
Gao Shi-gang,Zeng Rong,Xu Li-hui,Luo Jin-yan,Chen Lei,Dai Fu-ming.2016.Triplex PCR Detection for Corynespora cassiicola,Colletotrichum orbiculare and Pseudomonas syringae pv.lachrymans.Scientia Agricultura Sinica,49(16):3119-3129.(in Chinese)高士刚,曾蓉,徐丽慧,罗金燕,陈磊,戴富明.2016.三重PCR检测黄瓜靶斑病菌、炭疽病菌和细菌性角斑病菌.中国农业科学,49(16):3119-3129.
Gao Yong-yang,Wang Nan,Gao Guan-peng,Wang Wei.2010.Triplex PCR detection of Cladosporium cucumerinum,Fusarium oxysporum fsp.niveum and Mycosphaerella melonis in infected plant tissues.Acta Phytopathologica Sinica,40(4):343-350.(in Chinese)高永洋,王楠,高观朋,王伟.2010.瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测.植物病理学报,40(4):343-350.
Huang Wen,Xu Jin,Zhang Hao,Xu Jing-sheng,Ding Wei,Feng Jie.2016.Development of a LAMP Approach for Detection of Ralstonia solanacearum.Scientia Agricultura Sinica,49(11):2093-2102.(in Chinese)黄雯,徐进,张昊,许景升,丁伟,冯洁.2016.植物青枯菌LAMP检测方法的建立.中国农业科学,49(11):2093-2102.
Jones J B,Lacy G H,Bouzar H,Stall R E,Schaad N W.2004.Reclassification of the Xanthomonads associated with bacterial spot disease of tomato and pepper.Systematic&Applied Microbiology,27(6):755-762.
Luo Wen-bin,Li Hua-wei,Tang Hao,Qiu Si-xin,Ji Rong-chang,Xu Yong-qing,Liu Zhong-hua,Qiu Yong-xiang.2015.Establishment and application of multiplex PCR rapid Detection of potato viruses.Acta Horticulturae Sinica,42(2):280-288.(in Chinese)罗文彬,李华伟,汤浩,邱思鑫,纪荣昌,许泳清,刘中华,邱永祥.2015.马铃薯5种病毒多重PCR检测技术的建立及应用.园艺学报,42(2):280-288.
Markoulatos P,Siafakas N,Moncany M.2002.Multiplex polymerase chain reaction:a practical approach.Journal of clinical laboratory analysis,16(1):47-51.
Ozdemir Z.2005.Development of a Multiplex PCR Assay for Concurrent Detection of Clavibacter michiganensis ssp.michiganensis and Xanthomonas axonopodis pv.vesicatoria.Plant Pathology Journal,4(2):133-137.
Park D S,Shim J K,Kim J S,Lim R S,Hahn J H,Kim H G.2009.Sensitive and specific detection of Xanthomonas campestris pv.vesicatoria by PCR using pathovar-specific primers based on rhs family gene sequences.Microbiological Research,164(1):36-42.
Pastrik K H,Elphinstone J G,Pukall R.2002.Sequence analysis and detection of Ralstonia solanacearum by multiplex PCR amplification of16S-23S ribosomal intergenic spacer region with internal positive control.European Journal of Plant Pathology,108(9):831-842.
Pizano J F,García G A,Sánchez-Rico K F,González-Chavira M M,Guevara-González R J,Torres-Pacheco I,Guevara-Olvera L.2016.Detection of Clavibacter michiganensis ssp.michiganensis by PCR in in tomato plants(Lycopersicon esculentum Mill.).Revista Mexicana de Ciencias Agrícolas,7(6):1347-1357.
Quinterovásquez G A,Bazántejeda M L,Martínezpe?afiel E,Martínezpe?afiel E,Kameyamakawabe L,Bermúdez-Cruz R M.2013.Multiplex PCRto detect four different tomato-infecting pathogens.Folia Microbiologica,58(4):269-276.
Singh D,Sinha S,Yadav D K,Chaudhary G.2014.Detection of Ralstonia solanacearum from asymptomatic tomato plants,irrigation water and soil through non-selective enrichment medium with hrp gene-based bio-PCR.Current Microbiology,69(2):127-134.
Sint D,Raso L,Traugott M.2012.Advances in multiplex PCR:balancing primer efficiencies and improving detection success.Methods in Ecology&Evolution,3(5):898-905.
Umesha S,Avinash P.2015.Multiplex PCR for simultaneous identification of Ralstonia solanacearum and Xanthomonas perforans.Biotech,5(3):245-252.
Wu Xing-hai,Shao Xiu-ling,Deng Ming-jun,Chen Chang-fa,Li Yan,Liang Cheng-zhu.2007.Study on rapid Detection of Clavibacter michiganensis subsp.michiganensis by Real-time Fluorescent PCR.Acta Agriculturae Jiangxi,19(3):34-36.(in Chinese)吴兴海,邵秀玲,邓明俊,陈长法,厉艳,梁成珠.2007.番茄溃疡病菌实时荧光PCR快速检测方法研究.江西农业学报,19(3):34-36.
Xia Ming-xing,Zhao Wen-jun,Ma Qing,Zhu Shui-fang.2006.Rapid detection of Tomato bacterial canker(Clavibacter michiganensis subsp.michiganensis)with real-tmie fluorescent PCR.Acta Phytopathologica Sinica,36(2):152-157.(in Chinese)夏明星,赵文军,马青,朱水芳.2006.番茄细菌性溃疡病菌的实时荧光PCR检测.植物病理学报,36(2):152-157.
Yin Yan-ni,Huang Yan-xia,Ge Yun-ying,Guo Jian-hua.2006.Molecular diagnostic techniques for several commonly used phytobacteria.Plant Protection,32(6):1-4.(in Chinese)尹燕妮,黄艳霞,葛芸英,郭坚华.2006.几种常用植物病原细菌分子检测方法.植物保护,32(6):1-4.
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(1):85-90.