刺盘孢属真菌PCR检测方法的建立
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摘要
【目的】刺盘孢属(Colletotrichum)真菌是重要的植物病原菌,引起植物炭疽病。建立刺盘孢属真菌PCR检测方法。【方法】比对分析刺盘孢属及其近似属的ITS序列,设计刺盘孢属特异性引物,建立PCR扩增体系,验证引物的特异性和灵敏度。【结果】设计出刺盘孢属特异性引物ITS-c3/c4,建立了刺盘孢属常规PCR检测体系。建立的PCR体系能从刺盘孢属菌株中扩增出449 bp的特异性条带,刺盘孢属的近似属菌株未能扩增到条带。灵敏度试验可检测到DNA的浓度为2.2 pg/μL。【结论】用建立的PCR体系对炭疽病梨果实样品进行检测,可从发病组织中检测到刺盘孢属真菌。建立的PCR方法可靠、灵敏度高,能够快速、准确检测出刺盘孢属真菌。
【Objective】 Colletotrichum fungi are important plant pathogens that cause plant anthracnose. This project aims to establish a PCR detection method for the Colletotrichum.【Method】The specific primers of the Colletotrichum were designed by comparing and analyzing the internal transcribed spacers(ITS) sequences of the Colletotrichum and allied genera, and the PCR amplification system was established to verify the specificity and sensitivity of the primers.【Result】A pair of species-specific primer ITS-c3/c4 was designed for Colletotrichum and a conventional PCR detection system was established. The established PCR system could amplify a specific band of 449 bp from the Colletotrichum, but the allied genera could not do so. The concentration of DNA was 2.2 pg/L by sensitivity test. The established PCR system was used to detect the anthracnose pear fruit samples and the fungi of the Colletotrichum could be detected from the pathogenic tissues.【Conclusion】The established PCR method is reliable and sensitive, which can be used to quickly and accurately detect the Colletotrichum.
【Objective】 Colletotrichum fungi are important plant pathogens that cause plant anthracnose. This project aims to establish a PCR detection method for the Colletotrichum.【Method】The specific primers of the Colletotrichum were designed by comparing and analyzing the internal transcribed spacers(ITS) sequences of the Colletotrichum and allied genera, and the PCR amplification system was established to verify the specificity and sensitivity of the primers.【Result】A pair of species-specific primer ITS-c3/c4 was designed for Colletotrichum and a conventional PCR detection system was established. The established PCR system could amplify a specific band of 449 bp from the Colletotrichum, but the allied genera could not do so. The concentration of DNA was 2.2 pg/L by sensitivity test. The established PCR system was used to detect the anthracnose pear fruit samples and the fungi of the Colletotrichum could be detected from the pathogenic tissues.【Conclusion】The established PCR method is reliable and sensitive, which can be used to quickly and accurately detect the Colletotrichum.
引文
[1]向梅梅,张云霞,刘霄.炭疽菌属真菌分类的研究进展[J].仲恺农业工程学院学报,2016,29(4):1-8.XIANG Mei-mei,ZHANG Yun-xia,LIU Xiao.(2016).Research progress on the taxonomy of Colletotrichun [J].Journal of Zhongkai University of Agriculture and Engineering,29(4):1-8.(in Chinese)
[2] 韩长志.炭疽菌属真菌分类研究现状及发展趋势[J].中国植保导刊,2015,35(6):24-30.HAN Chang-zhi.(2015).Research review on taxonomy of genus Colletotrichum and its development trend [J].China Plant Protection,35(6):24-30.(in Chinese)
[3] Tao,G.,Hyde,K.D.,& Cai,L.(2012).Species-specific real-time pcr detection of Colletotrichum kahawae.Journal of Applied Microbiology,114:829-835.
[4] 杨友联,刘永翔,刘作易.炭疽菌属真菌分类学研究进展[J].贵州农业科学,2011,39(1):152-157.YANG You-lian,LIU Yong-xiang,LIU Zuo-yi.(2011).Research Progress on Fungus Taxonomy of Colletotrichum [J].Guizhou Agricultural Sciences,39(1):152-157.(in Chinese)
[5] 索相敏,王献革,李学营,等.rDNA ITS 序列在果树真菌病害研究中的应用[J].江西农业学报,2015,27( 5) :22-24.SUO Xiang-min,WANG Xian-ge,LI Xue-ying,et al.(2015).Application of rDNA ITS Sequence in Studies of Fungal Diseases of Fruit Tree [J].Acta Agriculturae Jiangxi,27(5) :22-24.(in Chinese)
[6] 陈汝,王金政,薛晓敏,等.红富士苹果果实炭疽病病菌的分离与鉴定[J].江苏农业科学,2017,45( 11) :79-81.CHEN Ru,WANG Jin-zheng,XUE Xiao-min,et al.(2017).Isolation and Identification of Anthracnose Pathogen in Red Fuji Apple Fruit[J].Jiangsu Agricultural Sciences,45(11):79-81.(in Chinese)
[7] 李河,周国英,徐建平.一种新油茶炭疽病原多基因序列鉴定[J].植物保护,2015,41(2):92-96.LI He,ZHOU Guo-ying,XU Jian-ping.(2015).Pathogen identification of a new anthracnose of Camellia oleiferain China based on multiple -gene sequences [J].Plant Protection,41(2):92-96.(in Chinese)
[8] 邓维萍,杨敏,杜飞,等.云南葡萄产区葡萄炭疽病病原鉴定及致病力分析[J].植物保护学报,2013,40(1):61-67.DENG Wei-ping,YANG Min,DU Fei,et al.(2013).Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity [J].Journal of Plant Protection,40(1):61-67.(in Chinese)
[9] 李海云,靳帅,张学勤,等.大豆炭疽病菌Colletotrichum chlorophyti的鉴定[J].植物保护,2017,43(2):163-166.LI Hai-yun,JIN Shuai,ZHANG Xue-qin,et al.(2017).Identification of Colletotrichum chlorophyti causing soyben anthracnose [J].Plant Protection,43(2):163-166.(in Chinese)
[10] Cannon,P.F.,Damm,U.,Johnston,P.R.,& Weir,B.S.(2012).Colletotrichum - current status and future directions.Studies in Mycology,73:181-213.(Complete)
[11] Weir,B.S.,Johnston,P.R.,& Damm,U.(2012).The colletotrichum gloeosporioides species complex.Studies in Mycology,73(1):115-180.
[12] Damm,U.,Cannon,P.F.,Woudenberg,J.H.C.,& Crous,P.W.(2012).The colletotrichum acutatum species complex.Studies in Mycology,73(73):37-113.
[13] U.Damm,P.F.Cannon,J.H.C.Woudenberg,et al.(2012).The Colletotrichum boninense species complex[J].Studies in Mycology ,(73):1-36.
[14] Damm,U.,O"Connell,R.J.,Groenewald,J.Z.,& Crous,P.W.(2014).The colletotrichum destructivum species complex - hemibiotrophic pathogens of forage and field crops.Studies in Mycology,79(79):49-84.
[2] 韩长志.炭疽菌属真菌分类研究现状及发展趋势[J].中国植保导刊,2015,35(6):24-30.HAN Chang-zhi.(2015).Research review on taxonomy of genus Colletotrichum and its development trend [J].China Plant Protection,35(6):24-30.(in Chinese)
[3] Tao,G.,Hyde,K.D.,& Cai,L.(2012).Species-specific real-time pcr detection of Colletotrichum kahawae.Journal of Applied Microbiology,114:829-835.
[4] 杨友联,刘永翔,刘作易.炭疽菌属真菌分类学研究进展[J].贵州农业科学,2011,39(1):152-157.YANG You-lian,LIU Yong-xiang,LIU Zuo-yi.(2011).Research Progress on Fungus Taxonomy of Colletotrichum [J].Guizhou Agricultural Sciences,39(1):152-157.(in Chinese)
[5] 索相敏,王献革,李学营,等.rDNA ITS 序列在果树真菌病害研究中的应用[J].江西农业学报,2015,27( 5) :22-24.SUO Xiang-min,WANG Xian-ge,LI Xue-ying,et al.(2015).Application of rDNA ITS Sequence in Studies of Fungal Diseases of Fruit Tree [J].Acta Agriculturae Jiangxi,27(5) :22-24.(in Chinese)
[6] 陈汝,王金政,薛晓敏,等.红富士苹果果实炭疽病病菌的分离与鉴定[J].江苏农业科学,2017,45( 11) :79-81.CHEN Ru,WANG Jin-zheng,XUE Xiao-min,et al.(2017).Isolation and Identification of Anthracnose Pathogen in Red Fuji Apple Fruit[J].Jiangsu Agricultural Sciences,45(11):79-81.(in Chinese)
[7] 李河,周国英,徐建平.一种新油茶炭疽病原多基因序列鉴定[J].植物保护,2015,41(2):92-96.LI He,ZHOU Guo-ying,XU Jian-ping.(2015).Pathogen identification of a new anthracnose of Camellia oleiferain China based on multiple -gene sequences [J].Plant Protection,41(2):92-96.(in Chinese)
[8] 邓维萍,杨敏,杜飞,等.云南葡萄产区葡萄炭疽病病原鉴定及致病力分析[J].植物保护学报,2013,40(1):61-67.DENG Wei-ping,YANG Min,DU Fei,et al.(2013).Identification of the pathogen causing grape anthracnose in Yunnan and its pathogenicity [J].Journal of Plant Protection,40(1):61-67.(in Chinese)
[9] 李海云,靳帅,张学勤,等.大豆炭疽病菌Colletotrichum chlorophyti的鉴定[J].植物保护,2017,43(2):163-166.LI Hai-yun,JIN Shuai,ZHANG Xue-qin,et al.(2017).Identification of Colletotrichum chlorophyti causing soyben anthracnose [J].Plant Protection,43(2):163-166.(in Chinese)
[10] Cannon,P.F.,Damm,U.,Johnston,P.R.,& Weir,B.S.(2012).Colletotrichum - current status and future directions.Studies in Mycology,73:181-213.(Complete)
[11] Weir,B.S.,Johnston,P.R.,& Damm,U.(2012).The colletotrichum gloeosporioides species complex.Studies in Mycology,73(1):115-180.
[12] Damm,U.,Cannon,P.F.,Woudenberg,J.H.C.,& Crous,P.W.(2012).The colletotrichum acutatum species complex.Studies in Mycology,73(73):37-113.
[13] U.Damm,P.F.Cannon,J.H.C.Woudenberg,et al.(2012).The Colletotrichum boninense species complex[J].Studies in Mycology ,(73):1-36.
[14] Damm,U.,O"Connell,R.J.,Groenewald,J.Z.,& Crous,P.W.(2014).The colletotrichum destructivum species complex - hemibiotrophic pathogens of forage and field crops.Studies in Mycology,79(79):49-84.