毛白杨叶片疮痂病原菌分离与鉴定
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摘要
在江苏南京市毛白杨叶片上发现一种新型叶斑病害。本研究,使用组织分离法,先后获得5个形态相似的真菌分离株,进而分别单孢分离纯化,编号E1到E5并提交中国林业微生物菌种保藏管理中心保存。通过克隆测序获得5个菌株的ITS、LSU、RBP2和TEF1-α基因片段,序列比对发现所有菌株的ITS序列完全一致,但LSU、RBP2和TEF1-α序列存在着不同程度的差异。结合形态学观察和公共数据库比对的分类鉴定,表明该病原菌属于痂囊腔菌属(Elsino觕),是首次报道的新种,我们将之命名为Elsino觕tomentosae。接种杨树组培苗叶片的致病性检测发现5个菌株均能侵染毛白杨和山新杨,但不能侵染杂种山杨和欧美杨,表明E. tomentosae可能在白杨派树种中具有一定的专化性。
A new leaf spot disease was found on Populus tomentosa in Nanjing, Jiangsu Province. Five isolates of fungi with similar morphologies were obtained by tissue isolation method and purified by single conidia isolation.Five pure cultures from each isolates were referred as E1 to E5 and submitted to China Forestry Culture Collection Center. The ITS, LSU, RBP2 and TEF1-α gene sequences of these five strains were obtained by cloning and sequencing. Sequence alignment revealed that the ITS sequences of all isolates were identical, but there were some differences in LSU, RBP2 and TEF1-α sequences. Combined morphological observation and BLAST in public database, the results showed that these fungi were classified as a novel species in genus Elsino觕, for which the name Elsino觕 tomentosae was proposed. Pathogenicity analysis of the seedling leaf in the group of poplar with inoculation revealed that all five strains could infect P. tomentosa and P. davidiana×P. bolleana, but could not infect P.tremula×P. tremuloides and P. deltoides×P. euramericana. That result implied that E. tomentosae might have host specialization within the section Leuce of Populus genus.
A new leaf spot disease was found on Populus tomentosa in Nanjing, Jiangsu Province. Five isolates of fungi with similar morphologies were obtained by tissue isolation method and purified by single conidia isolation.Five pure cultures from each isolates were referred as E1 to E5 and submitted to China Forestry Culture Collection Center. The ITS, LSU, RBP2 and TEF1-α gene sequences of these five strains were obtained by cloning and sequencing. Sequence alignment revealed that the ITS sequences of all isolates were identical, but there were some differences in LSU, RBP2 and TEF1-α sequences. Combined morphological observation and BLAST in public database, the results showed that these fungi were classified as a novel species in genus Elsino觕, for which the name Elsino觕 tomentosae was proposed. Pathogenicity analysis of the seedling leaf in the group of poplar with inoculation revealed that all five strains could infect P. tomentosa and P. davidiana×P. bolleana, but could not infect P.tremula×P. tremuloides and P. deltoides×P. euramericana. That result implied that E. tomentosae might have host specialization within the section Leuce of Populus genus.
引文
Beckman P.M.,and Payne G.A.,1982,External growth,penetration and development of Cercospora zeae-maydis in corn leaves Phytopathology,72(72):810-815
Bitancourt A.A.,and Jenkins A.E.,1936,Elsinofawcettii,the perfect stage of the Citrus scab fungus,Phytopathology,26(4)393-395
Brook P.J.,1973,Epidemiology of grapevine anthracnose,caused by Elsinoe ampelina,New.Zeal.J.Agr.Res.,16(3):333-342
Dong J.G.,Fan Y.S.,Gui X.M.,An X.L.,Ma J.F.,and Dong Z.P2008,Geographic distribution and genetic analysis of physiological racesof setosphaeria turcica in Northern China,Am.J.Agr.Biol.Sci.,3(1):389-398
Fan X.L.,Barreto R.W.,Groenewald J.Z.,Bezerra J.D.,Pereira O.L.,Cheewangkoon R.,Mostert L.,Tian C.M.,and Crous P.W.,2017,Phylogeny and taxonomy of the scab and spot anthracnose fungus Elsino觕(Myriangiales,Dothideomycetes),Stud.Mycol.,87(C):1-41
Feng H.M.,Chen X.T.,Chen Y.,Chen J.X.,and Wu Y.T.,2015,Genealogy analysis of banana germplasm based on r DNAITS sequences,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):830-835(冯慧敏,陈雪婷,陈友,陈家献,武耀廷,2015,香蕉种质资源基于r DNA ITS序列的谱系分析,基因组学与应用生物学,34(4):830-835)
Gonzalez E.,and Sutton T.B.,1999,First report of Glomerella leaf spot(Glomerella cingulata)of apple in the United States,Plant Disease,83(11):1074
Han Z.M.,Yin T.M.,Li C.D.,Huang M.R.,and Wu R.L.,2000,Host effect on genetic variation of Marssonina brunnea pathogenic to poplars,Theor.Appl.Genet.,100(3-4):614-620
Harada Y.,Sawamura K.,and Konno K.,1974,Diplocarpon mali sp.nov.,the perfect state of apple blotch fungus Marssonina coronaria,Japanese Journal of Phytopathology,40(5):412-418
Huang Z.H.,1992,The study on the regionalization of the distributional region of Populus tomentosa,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),14(S3):26-32(黄智慧,1992,毛白杨分布区气候区划的研究,北京林业大学学报,14(S3):26-32)
Hyun J.W.,Yi S.H.,Mackenzie S.J.,Timmer L.W.,Kim K.S.,Kang S.K.,Kwon H.M.,and Lim H.C.,2009,Pathotypes and genetic relationship of worldwide collections of Elsinospp.causing scab diseases of citrus,Phytopathology,99(6):721-728
Jenkins A.E.,1931,Lima-bean scab caused by Elsino,Journal of Agricultural Research,42(1):13-23
Jenkins A.E.,1934,Sphaceloma perseae,the cause of avocado scab,Journal of Agricultural Research,49(10):859-869
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870-1874
Leonard K.J.,1977,Virulence,Temperature optima,and competitive abilities of isolines of races T and O of bipolaris maydis,Phytopathology,67(10):1273-1279
Leonard K.J.,1978,Polymorphisms for lesion type,fungicide tolerance,and mating capacit in Cochliobolus carbonum isolates pathogenic to corn,Can.J.Bot.,56(15):1809-1815
Liu H.X.,Jia X.Z.,Lu Q.,and Liang J.,2005,Occurrence and control of poplar canker disease in China,Shijie Linye Yanjiu(World Forestry Research),18(4):60-63(刘会香,贾秀贞,吕全,梁军,2005,中国杨树溃疡病的发生与防治,世界林业研究,18(4):60-63)
Liu Y.J.,Whelen S.,and Hall B.D.,1999,Phylogenetic relationships among ascomycetes:evidence from an RNA polymerseⅡsubunit,Mol.Biol.Evol.,16(12):1799-1808
Mccubbin W.A.,1933,The lima bean scab situation,J.Econ.Entomol.,26(3):625-630
Park P.,Nishimura S.,Kohmoto K.,Otani H.,and Tsujimoto K.1981,Two action sites of AM-toxin I produced by apple pathotype of Alternar alternata in host cells:an ultrastructural study,Can.J.Bot.,59(3):301-310
Rehner S.A.,and Samuels G.J.,1994,Taxonomy and phylogeny of Gliocladiu analysed from nuclear large subunit ribosomal DNA sequences,Mycol.Res.,98(6):625-634
Su H.R.,He H.,Huang Q.Z.,and Lu N.H.,2014,First report of black spot of Acanthus ilicifolius caused by Fusarium solan in China,Plant Disease,98(10):1438
Sung G.H.,Sung J.M.,Hywel-Jones N.L.,and Spatafora J.W.2007,A multi-gene phylogeny of Clavicipitaceae(Ascomycota,Fungi):identification of localized incongruence using a combinational bootstrap approach,Mol.Phylogenet.Evol.44(3):1204-1223
Tan M.K.,Timmer L.W.,Broadbent P.,Priest M.,and Cain P.1996,Differentiation by molecular analysis of Elsinospp causing scab diseases of citrus and its epidemiological implications,Phytopathology,86(10):1039-1044
Tian C.M.,Shang Y.Z.,Zhuang J.Y.,Wang Q.,and Kakishima M.,2004,Morphological and molecular phylogenetic analysis of Melampsora species on poplars in China,Mycoscience,45(1):56-66
Vilgalys R.,and Hester M.,1990,Rapid genetic identification and mapping of enzymatically amplified ribosomal DNAfrom several Cryptococcus species,J.Bacteriol.,172(8):4238-4246
White T.J.,Burns T.D.,Lee S.B.,and Taylor J.W.,Innis M.A.,Gelfand D.H.,Sninsky J.J.and White T.J.,eds,1990,Amplification and sequencing of fungal ribosomal RNA genes for phylogenetics,San Diego,California,Academic Press,pp.315-322
Zhu Z.T.,Lin H.B.,and Kang X.Y.,1995,Studies on allotriploid breeding of Populus tomentosa B301 clones,Linye Kexue(Scientia Silvae Sinicae),31(6):499-505(朱之悌,林惠斌,康向阳,1995,毛白杨异源三倍体B301等无性系选育的研究,林业科学,31(6):499-505)
Bitancourt A.A.,and Jenkins A.E.,1936,Elsinofawcettii,the perfect stage of the Citrus scab fungus,Phytopathology,26(4)393-395
Brook P.J.,1973,Epidemiology of grapevine anthracnose,caused by Elsinoe ampelina,New.Zeal.J.Agr.Res.,16(3):333-342
Dong J.G.,Fan Y.S.,Gui X.M.,An X.L.,Ma J.F.,and Dong Z.P2008,Geographic distribution and genetic analysis of physiological racesof setosphaeria turcica in Northern China,Am.J.Agr.Biol.Sci.,3(1):389-398
Fan X.L.,Barreto R.W.,Groenewald J.Z.,Bezerra J.D.,Pereira O.L.,Cheewangkoon R.,Mostert L.,Tian C.M.,and Crous P.W.,2017,Phylogeny and taxonomy of the scab and spot anthracnose fungus Elsino觕(Myriangiales,Dothideomycetes),Stud.Mycol.,87(C):1-41
Feng H.M.,Chen X.T.,Chen Y.,Chen J.X.,and Wu Y.T.,2015,Genealogy analysis of banana germplasm based on r DNAITS sequences,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(4):830-835(冯慧敏,陈雪婷,陈友,陈家献,武耀廷,2015,香蕉种质资源基于r DNA ITS序列的谱系分析,基因组学与应用生物学,34(4):830-835)
Gonzalez E.,and Sutton T.B.,1999,First report of Glomerella leaf spot(Glomerella cingulata)of apple in the United States,Plant Disease,83(11):1074
Han Z.M.,Yin T.M.,Li C.D.,Huang M.R.,and Wu R.L.,2000,Host effect on genetic variation of Marssonina brunnea pathogenic to poplars,Theor.Appl.Genet.,100(3-4):614-620
Harada Y.,Sawamura K.,and Konno K.,1974,Diplocarpon mali sp.nov.,the perfect state of apple blotch fungus Marssonina coronaria,Japanese Journal of Phytopathology,40(5):412-418
Huang Z.H.,1992,The study on the regionalization of the distributional region of Populus tomentosa,Beijing Linye Daxue Xuebao(Journal of Beijing Forestry University),14(S3):26-32(黄智慧,1992,毛白杨分布区气候区划的研究,北京林业大学学报,14(S3):26-32)
Hyun J.W.,Yi S.H.,Mackenzie S.J.,Timmer L.W.,Kim K.S.,Kang S.K.,Kwon H.M.,and Lim H.C.,2009,Pathotypes and genetic relationship of worldwide collections of Elsinospp.causing scab diseases of citrus,Phytopathology,99(6):721-728
Jenkins A.E.,1931,Lima-bean scab caused by Elsino,Journal of Agricultural Research,42(1):13-23
Jenkins A.E.,1934,Sphaceloma perseae,the cause of avocado scab,Journal of Agricultural Research,49(10):859-869
Kumar S.,Stecher G.,and Tamura K.,2016,MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets,Mol.Biol.Evol.,33(7):1870-1874
Leonard K.J.,1977,Virulence,Temperature optima,and competitive abilities of isolines of races T and O of bipolaris maydis,Phytopathology,67(10):1273-1279
Leonard K.J.,1978,Polymorphisms for lesion type,fungicide tolerance,and mating capacit in Cochliobolus carbonum isolates pathogenic to corn,Can.J.Bot.,56(15):1809-1815
Liu H.X.,Jia X.Z.,Lu Q.,and Liang J.,2005,Occurrence and control of poplar canker disease in China,Shijie Linye Yanjiu(World Forestry Research),18(4):60-63(刘会香,贾秀贞,吕全,梁军,2005,中国杨树溃疡病的发生与防治,世界林业研究,18(4):60-63)
Liu Y.J.,Whelen S.,and Hall B.D.,1999,Phylogenetic relationships among ascomycetes:evidence from an RNA polymerseⅡsubunit,Mol.Biol.Evol.,16(12):1799-1808
Mccubbin W.A.,1933,The lima bean scab situation,J.Econ.Entomol.,26(3):625-630
Park P.,Nishimura S.,Kohmoto K.,Otani H.,and Tsujimoto K.1981,Two action sites of AM-toxin I produced by apple pathotype of Alternar alternata in host cells:an ultrastructural study,Can.J.Bot.,59(3):301-310
Rehner S.A.,and Samuels G.J.,1994,Taxonomy and phylogeny of Gliocladiu analysed from nuclear large subunit ribosomal DNA sequences,Mycol.Res.,98(6):625-634
Su H.R.,He H.,Huang Q.Z.,and Lu N.H.,2014,First report of black spot of Acanthus ilicifolius caused by Fusarium solan in China,Plant Disease,98(10):1438
Sung G.H.,Sung J.M.,Hywel-Jones N.L.,and Spatafora J.W.2007,A multi-gene phylogeny of Clavicipitaceae(Ascomycota,Fungi):identification of localized incongruence using a combinational bootstrap approach,Mol.Phylogenet.Evol.44(3):1204-1223
Tan M.K.,Timmer L.W.,Broadbent P.,Priest M.,and Cain P.1996,Differentiation by molecular analysis of Elsinospp causing scab diseases of citrus and its epidemiological implications,Phytopathology,86(10):1039-1044
Tian C.M.,Shang Y.Z.,Zhuang J.Y.,Wang Q.,and Kakishima M.,2004,Morphological and molecular phylogenetic analysis of Melampsora species on poplars in China,Mycoscience,45(1):56-66
Vilgalys R.,and Hester M.,1990,Rapid genetic identification and mapping of enzymatically amplified ribosomal DNAfrom several Cryptococcus species,J.Bacteriol.,172(8):4238-4246
White T.J.,Burns T.D.,Lee S.B.,and Taylor J.W.,Innis M.A.,Gelfand D.H.,Sninsky J.J.and White T.J.,eds,1990,Amplification and sequencing of fungal ribosomal RNA genes for phylogenetics,San Diego,California,Academic Press,pp.315-322
Zhu Z.T.,Lin H.B.,and Kang X.Y.,1995,Studies on allotriploid breeding of Populus tomentosa B301 clones,Linye Kexue(Scientia Silvae Sinicae),31(6):499-505(朱之悌,林惠斌,康向阳,1995,毛白杨异源三倍体B301等无性系选育的研究,林业科学,31(6):499-505)