河南省小麦全蚀病菌的鉴定、进化分析及标志基因筛选
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摘要
为了解河南省郑州市中牟市小麦全蚀病菌的变种类型及进化情况,筛选全蚀病菌侵染小麦的分子标记,采用形态学观察、科赫氏法则验证、ITS序列分析和系统进化树构建等方法对分离的小麦全蚀病菌进行鉴定,并对其侵染后小麦病原相关蛋白(pathogen-related protein,PR)基因的表达进行实时定量PCR分析。结果表明,显微形态观察初步确定分离的小麦全蚀病菌为禾顶囊壳Gaeumannomyces graminis(Sacc.)v. Arx.&Olivier,经科赫氏法则验证、ITS序列比对及系统进化树分析进一步证明该病菌均为禾顶囊壳。本研究分离的小麦全蚀病菌与地域相距较远的英国、美国的小麦全蚀病菌间的同源性比与来自中国陕西省的小麦全蚀病菌的同源性更高。采用禾顶囊壳4个变种的特异性引物进行PCR扩增,所有分离病菌均扩增出了小麦变种的特异性条带,证实分离菌株为禾顶囊壳小麦变种G. graminis var. tritici。实时定量PCR分析发现,病原相关蛋白基因PR4a、PR4b、PR2、PR10受到小麦全蚀病菌侵染后表达量均显著上调,在侵染后第5～6天达到最高峰,表明这些基因可作为小麦全蚀病菌侵染小麦的标志性基因。
In order to completely identify the variant types and evolutionary relationship of wheat take-all disease in Zhongmou of Zhengzhou City of Henan Province, and screening for the molecular marker genes of wheat, a series of experiments were carried out in this research. The isolated pathogens were identified with morphological characters, the ribosomal DNA internal transcribed spacer(ITS) sequence, the expression patterns of pathogenesis-related(PR) proteins were characterized after pathogen inoculation, for the screening of the marker genes of take-all fungus infection. Microscopic morphological observation results showed that the pathogen was Gaeumannomyces graminis(Sacc.) Arx & Olivier,and molecular identification and phylogenetic tree results further confirmed it. The phylogenetic tree results had also verified that the isolated fungus had closer homology with the isolates from UK and USA than that from Shaanxi Province of China. All the isolated pathogens in the four pairs of variety-specific primers used to identify the fungus turned out to be G. graminis var. tritici, according to the results of PCR amplification. The expression level of PR4 a, PR4 b, PR2 and PR10 post infected with G. graminis,analyzed by quantitative real-time PCR(q RT-PCR), had increased significantly and reached a peak on the fifth and sixth days after inoculated with the pathogen. In conclusion, all the above PR genes could be served as marker genes of G. graminis.
In order to completely identify the variant types and evolutionary relationship of wheat take-all disease in Zhongmou of Zhengzhou City of Henan Province, and screening for the molecular marker genes of wheat, a series of experiments were carried out in this research. The isolated pathogens were identified with morphological characters, the ribosomal DNA internal transcribed spacer(ITS) sequence, the expression patterns of pathogenesis-related(PR) proteins were characterized after pathogen inoculation, for the screening of the marker genes of take-all fungus infection. Microscopic morphological observation results showed that the pathogen was Gaeumannomyces graminis(Sacc.) Arx & Olivier,and molecular identification and phylogenetic tree results further confirmed it. The phylogenetic tree results had also verified that the isolated fungus had closer homology with the isolates from UK and USA than that from Shaanxi Province of China. All the isolated pathogens in the four pairs of variety-specific primers used to identify the fungus turned out to be G. graminis var. tritici, according to the results of PCR amplification. The expression level of PR4 a, PR4 b, PR2 and PR10 post infected with G. graminis,analyzed by quantitative real-time PCR(q RT-PCR), had increased significantly and reached a peak on the fifth and sixth days after inoculated with the pathogen. In conclusion, all the above PR genes could be served as marker genes of G. graminis.
引文
Barret M,Frey-Klett P,Guillerm-Erckelboudt AY,Boutin M,Guernec G,Sarniguet A.2009.Effect of wheat roots infected with the pathogenic fungus Gaeumannomyces graminis var.tritici on gene expression of the biocontrol bacterium Pseudomonas fluorescens Pf29Arp.Molecular Plant-Microbe Interactions,22(12):1611-1623
Bertini L,Leonardi L,Caporale C,Tucci M,Cascone N,di Berarsino D,Buonocore V,Caruso C.2003.Pathogen-responsive wheat PR4 genes are induced by activators of systemic acquired resistance and wounding.Plant Science,164(6):1067-1078
Caruso C,Chilosi G,Caporale C,Leonardi L,Bertini L,Magro P,Buonocore V.1999.Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum.Plant Science,140(1):87-97
Cook RJ.2003.Take-all of wheat.Physiological&Molecular Plant Pathology,62(2):73-86
Enjalbert J,Duan X,Leconte M,Hovm?ller MS,de Vallavieille-Pope C.2005.Genetic evidence of local adaptation of wheat yellow rust(Puccinia striiformis f.sp.tritici)within France.Molecular Ecology,14(7):2065-2073
Guilleroux M,Osbourn A.2004.Gene expression during infection of wheat roots by the‘take-all’fungus Gaeumannomyces graminis.Molecular Plant Pathology,5(3):203-216
Gutteridge RJ,Bateman GL,Todd AD.2003.Variation in the effects of take-all disease on grain yield and quality of winter cereals in field experiments.Pest Management Science,59(2):215-224
Lu S.2014.Zn2+blocks annealing of complementary single-stranded DNA in a sequence-selective manner.Scientific Reports,4:5464
Liu X,Yang LH,Zhou XY,Zhou MP,Lu Y,Ma LJ,Ma HX,Zhang ZY.2013.Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.Journal of Experimental Botany,64(8):2243-2253
Liu ZP,Wang LX,Li RX,Du W,Suo FL.2001.Preliminary identification on the variety of wheat take-all pathogens in Bayannol,Inner Mongolia.Acta Agriculturae Boreali-Sinica,16(4):132-135(in Chinese)[刘正坪,王立新,李荣禧,杜微,索凤兰.2001.内蒙古巴彦淖尔盟小麦全蚀病菌变种类型初步鉴定.华北农学报,16(4):132-135]
Mohammadi M,Srivastava-Jocelyn S,Hall JC,Kav NNV,Deyholos MK.2012.Two wheat(Triticum aestivum)pathogenesis-related10(PR-10)transcripts with distinct patterns of abundance in different organs.Molecular Biotechnology,51(2):103-108
Rachadwong S,Cramer CL,Grabau EA,Stromberg VK,Lacy GH,Stromberg EL.2002.Gaeumannomyces graminis var.avenae,graminis,and tritici identified using PCR amplification of avenacinase-like genes.Plant Disease,86(6):652-660
Rong W,Qi L,Wang JF,Du LP,Xu HJ,Wang AY,Zhang ZY.2013.Expression of a potato antimicrobial peptide SN1 increases resistance to take-all pathogen Gaeumannomyces graminis var.tritici in transgenic wheat.Functional&Integrative Genomics,13(3):403-409
Sari E,Etebarian HR,Aminian H.2008.Effects of Pseudomonas fluorescens CHA0 on the resistance of wheat seedling roots to the take-all fungus Gaeumannomyces graminis var.tritici.Plant Production Science,11(3):298-306
Walker J.1972.Type studies on Gaeumannomyces graminis,and related fungi.Transactions of the British Mycological Society,58(3):427-457
Wang AY,Wei XN,Rong W,Dang L,Du LP,Qi L,Xu HJ,Shao YJ,Zhang ZY.2015.GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat.Functional&Integrative Genomics,15(3):375-381
Wang MN.2002.Genetic difference of Gaeumannomyces graminis(Sacc.)v.Arx&Oliver.Ph.D Thesis.Yangling:Northwest A&FUniversity(in Chinese)[王美南.2002.禾顶囊壳[Gaeumannomyces graminis(Sacc.)v.Arx&Oliver]遗传差异研究.博士学位论文.杨凌:西北农林科技大学]
Wang MN,Shang HS.2000.Variety type and biological characteristics of wheat take-all fungus in Shaanxi.Acta Agriculturae BorealiOccidentalis Sinica,9(4):78-82(in Chinese)[王美南,商鸿生.2000.陕西小麦全蚀病菌变种类型及其主要特性.西北农业学报,9(4):78-82]
Wang MN,Shang HS.2005.The molecular techniques of diagnosis used in take-all disease.Journal of Northwest A&F University(Natural Science Edition),33(4):69-73(in Chinese)[王美南,商鸿生.2005.分子生物学技术在全蚀病诊断中的应用.西北农林科技大学学报(自然科学版),33(4):69-73]
Wang MZ,Han QY,Fu YP,Xiao YN.2000.Identification of the takeall fungus occurring in Qianjiang of Hubei Province.Journal of Huazhong Agricultural University,19(2):112-114(in Chinese)[王明祖,韩群营,付艳平,肖炎农.2000.湖北省潜江市发生的小麦全蚀病病原鉴定.华中农业大学学报,19(2):112-114]
Xie YR,Chen ZY,Brown RL,Bhatnagar D.2010.Expression and functional characterization of two pathogenesis-related protein10 genes from Zea mays.Journal of Plant Physiology,167(2):121-130
Xu F,Yang GQ,He WL,Song YL,Wang JM,Li YH.2013.Evaluation of resistance to take-all disease in different wheat cultivars or lines.Plant Protection,39(2):143-146(in Chinese)[徐飞,杨共强,何文兰,宋玉立,王俊美,李亚红.2013.不同小麦品种(系)对全蚀病的抗性鉴定与评价.植物保护,39(2):143-146]
Xu F,Yang GQ,He WL,Wang JM,Song YL,Li YH.2014.Composition and virulence diversity in Gaeumannomyces graminis var.tritici populations infecting wheat in Henan Province.Journal of Plant Protection,41(2):255-256(in Chinese)[徐飞,杨共强,何文兰,王俊美,宋玉立,李亚红.2014.河南省小麦全蚀病菌群体组成及致病力分化.植物保护学报,41(2):255-256]
Yao JM,Wang YC,Zhu YG.1992.A new variety of Gaeumannomyces graminis on maize.Mycosystema,11(2):99-104(in Chinese)[姚健民,王永成,朱有釭.1992.全蚀病菌在玉米上的新变种.菌物学报,11(2):99-104]
Zhang QE,Cao KQ,Hu TL,Wang ST,Yang JY,Wang PP.2008.Identification of the varieties of Gaeumannomyces graminis causing wheat take-all in Hebei.Plant Protection,34(4):18-21(in Chinese)[张秋娥,曹克强,胡同乐,王树桐,杨军玉,王培培.2008.河北省小麦全蚀病菌变种类型鉴定.植物保护,34(4):18-21]
Zhang Y,Zhang PP,Ma XM,Fu LL,Qiao YE,Chen PP,Li CW.2012.Identification and phylogenic analysis of powdery mildew on Triticum aestivum in two cities of Henan.Acta Agriculturae Boreali-Sinica,27(1):189-192(in Chinese)[张怡,张佩佩,马晓萌,傅露露,乔月娥,陈佩佩,李成伟.2012.河南两地市小麦白粉病菌的分子鉴定和进化分析.华北农学报,27(1):189-192]
Zhu TH,Xing XP,Sun SD.2004.The antagonism mechanisms and diseases control trials of Trichoderma strain T97against several plant fungal pathogens in greenhouse.Journal of Plant Protection,31(2):139-144(in Chinese)[朱廷恒,邢小平,孙顺娣.2004.木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验.植物保护学报,31(2):139-144]
Zou BH,Ding Y,Liu H,Hua J.2018.Silencing of copine genes confers common wheat enhanced resistance to powdery mildew.Molecular Plant Pathology,19(6):1343-1352
Bertini L,Leonardi L,Caporale C,Tucci M,Cascone N,di Berarsino D,Buonocore V,Caruso C.2003.Pathogen-responsive wheat PR4 genes are induced by activators of systemic acquired resistance and wounding.Plant Science,164(6):1067-1078
Caruso C,Chilosi G,Caporale C,Leonardi L,Bertini L,Magro P,Buonocore V.1999.Induction of pathogenesis-related proteins in germinating wheat seeds infected with Fusarium culmorum.Plant Science,140(1):87-97
Cook RJ.2003.Take-all of wheat.Physiological&Molecular Plant Pathology,62(2):73-86
Enjalbert J,Duan X,Leconte M,Hovm?ller MS,de Vallavieille-Pope C.2005.Genetic evidence of local adaptation of wheat yellow rust(Puccinia striiformis f.sp.tritici)within France.Molecular Ecology,14(7):2065-2073
Guilleroux M,Osbourn A.2004.Gene expression during infection of wheat roots by the‘take-all’fungus Gaeumannomyces graminis.Molecular Plant Pathology,5(3):203-216
Gutteridge RJ,Bateman GL,Todd AD.2003.Variation in the effects of take-all disease on grain yield and quality of winter cereals in field experiments.Pest Management Science,59(2):215-224
Lu S.2014.Zn2+blocks annealing of complementary single-stranded DNA in a sequence-selective manner.Scientific Reports,4:5464
Liu X,Yang LH,Zhou XY,Zhou MP,Lu Y,Ma LJ,Ma HX,Zhang ZY.2013.Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.Journal of Experimental Botany,64(8):2243-2253
Liu ZP,Wang LX,Li RX,Du W,Suo FL.2001.Preliminary identification on the variety of wheat take-all pathogens in Bayannol,Inner Mongolia.Acta Agriculturae Boreali-Sinica,16(4):132-135(in Chinese)[刘正坪,王立新,李荣禧,杜微,索凤兰.2001.内蒙古巴彦淖尔盟小麦全蚀病菌变种类型初步鉴定.华北农学报,16(4):132-135]
Mohammadi M,Srivastava-Jocelyn S,Hall JC,Kav NNV,Deyholos MK.2012.Two wheat(Triticum aestivum)pathogenesis-related10(PR-10)transcripts with distinct patterns of abundance in different organs.Molecular Biotechnology,51(2):103-108
Rachadwong S,Cramer CL,Grabau EA,Stromberg VK,Lacy GH,Stromberg EL.2002.Gaeumannomyces graminis var.avenae,graminis,and tritici identified using PCR amplification of avenacinase-like genes.Plant Disease,86(6):652-660
Rong W,Qi L,Wang JF,Du LP,Xu HJ,Wang AY,Zhang ZY.2013.Expression of a potato antimicrobial peptide SN1 increases resistance to take-all pathogen Gaeumannomyces graminis var.tritici in transgenic wheat.Functional&Integrative Genomics,13(3):403-409
Sari E,Etebarian HR,Aminian H.2008.Effects of Pseudomonas fluorescens CHA0 on the resistance of wheat seedling roots to the take-all fungus Gaeumannomyces graminis var.tritici.Plant Production Science,11(3):298-306
Walker J.1972.Type studies on Gaeumannomyces graminis,and related fungi.Transactions of the British Mycological Society,58(3):427-457
Wang AY,Wei XN,Rong W,Dang L,Du LP,Qi L,Xu HJ,Shao YJ,Zhang ZY.2015.GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat.Functional&Integrative Genomics,15(3):375-381
Wang MN.2002.Genetic difference of Gaeumannomyces graminis(Sacc.)v.Arx&Oliver.Ph.D Thesis.Yangling:Northwest A&FUniversity(in Chinese)[王美南.2002.禾顶囊壳[Gaeumannomyces graminis(Sacc.)v.Arx&Oliver]遗传差异研究.博士学位论文.杨凌:西北农林科技大学]
Wang MN,Shang HS.2000.Variety type and biological characteristics of wheat take-all fungus in Shaanxi.Acta Agriculturae BorealiOccidentalis Sinica,9(4):78-82(in Chinese)[王美南,商鸿生.2000.陕西小麦全蚀病菌变种类型及其主要特性.西北农业学报,9(4):78-82]
Wang MN,Shang HS.2005.The molecular techniques of diagnosis used in take-all disease.Journal of Northwest A&F University(Natural Science Edition),33(4):69-73(in Chinese)[王美南,商鸿生.2005.分子生物学技术在全蚀病诊断中的应用.西北农林科技大学学报(自然科学版),33(4):69-73]
Wang MZ,Han QY,Fu YP,Xiao YN.2000.Identification of the takeall fungus occurring in Qianjiang of Hubei Province.Journal of Huazhong Agricultural University,19(2):112-114(in Chinese)[王明祖,韩群营,付艳平,肖炎农.2000.湖北省潜江市发生的小麦全蚀病病原鉴定.华中农业大学学报,19(2):112-114]
Xie YR,Chen ZY,Brown RL,Bhatnagar D.2010.Expression and functional characterization of two pathogenesis-related protein10 genes from Zea mays.Journal of Plant Physiology,167(2):121-130
Xu F,Yang GQ,He WL,Song YL,Wang JM,Li YH.2013.Evaluation of resistance to take-all disease in different wheat cultivars or lines.Plant Protection,39(2):143-146(in Chinese)[徐飞,杨共强,何文兰,宋玉立,王俊美,李亚红.2013.不同小麦品种(系)对全蚀病的抗性鉴定与评价.植物保护,39(2):143-146]
Xu F,Yang GQ,He WL,Wang JM,Song YL,Li YH.2014.Composition and virulence diversity in Gaeumannomyces graminis var.tritici populations infecting wheat in Henan Province.Journal of Plant Protection,41(2):255-256(in Chinese)[徐飞,杨共强,何文兰,王俊美,宋玉立,李亚红.2014.河南省小麦全蚀病菌群体组成及致病力分化.植物保护学报,41(2):255-256]
Yao JM,Wang YC,Zhu YG.1992.A new variety of Gaeumannomyces graminis on maize.Mycosystema,11(2):99-104(in Chinese)[姚健民,王永成,朱有釭.1992.全蚀病菌在玉米上的新变种.菌物学报,11(2):99-104]
Zhang QE,Cao KQ,Hu TL,Wang ST,Yang JY,Wang PP.2008.Identification of the varieties of Gaeumannomyces graminis causing wheat take-all in Hebei.Plant Protection,34(4):18-21(in Chinese)[张秋娥,曹克强,胡同乐,王树桐,杨军玉,王培培.2008.河北省小麦全蚀病菌变种类型鉴定.植物保护,34(4):18-21]
Zhang Y,Zhang PP,Ma XM,Fu LL,Qiao YE,Chen PP,Li CW.2012.Identification and phylogenic analysis of powdery mildew on Triticum aestivum in two cities of Henan.Acta Agriculturae Boreali-Sinica,27(1):189-192(in Chinese)[张怡,张佩佩,马晓萌,傅露露,乔月娥,陈佩佩,李成伟.2012.河南两地市小麦白粉病菌的分子鉴定和进化分析.华北农学报,27(1):189-192]
Zhu TH,Xing XP,Sun SD.2004.The antagonism mechanisms and diseases control trials of Trichoderma strain T97against several plant fungal pathogens in greenhouse.Journal of Plant Protection,31(2):139-144(in Chinese)[朱廷恒,邢小平,孙顺娣.2004.木霉T97菌株对几种植物病原真菌的拮抗作用机制和温室防治试验.植物保护学报,31(2):139-144]
Zou BH,Ding Y,Liu H,Hua J.2018.Silencing of copine genes confers common wheat enhanced resistance to powdery mildew.Molecular Plant Pathology,19(6):1343-1352