玉屏油茶叶枯病病原菌分子鉴定及防治药剂筛选
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摘要
为了明确贵州玉屏油茶叶枯病病原菌的种类,评价不同药剂对致病菌的毒力,筛选出高效防治药剂,本研究对玉屏油茶典型叶枯病病叶进行了病原菌的分离与纯化,通过病原菌回接验证与形态学鉴定初步确定分离所得的4株致病菌为拟盘多毛孢属真菌。再采用ITS-TUB2基因序列进一步将4株致病菌鉴定为拟盘多毛孢属的小孢拟盘多毛孢菌(Pestalotiopsis microspora)。结合菌丝生长速率和孢子萌发对5种杀菌剂对叶枯病病原菌的抑制效果的研究结果表明,5种杀菌剂中,咪鲜胺和戊唑醇对菌丝生长毒力的EC50分别为0.013μg/mL、0.055μg/m L,对孢子萌发毒力的EC50分别为0.093μg/m L、0.082μg/m L,均优于其余3种试剂,可作为高效药剂进一步进行林间药效试验。玉屏油茶叶枯病病原菌种类的鉴定及其防治药剂的筛选可为当地油茶抗性良种培育及病害防治提供理论依据。
In order to identify the pathogen species of Camellia oleifera leaf blight in Guizhou Yuping, evaluate the different reagents on the virulence of pathogenic bacteria, and to screen the effective fungicides. This study isolated and purified pathogenic bacteria in the leaves with typical leaf blight of Camellia oleifera in Yuping, and then obtained four pathogenic strains of Pestalotiopsis spp. by re-inoculation and morphological identification.Four pathogenic strains of Pestalotiopsis spp. were isolated from typical leaf blight leaves, which were identified as Pestalotiopsis microspore by ITS-TUB2 gene sequence. The inhibitory activity in vitro of 5 fungicides to P.microspore was determined by the inhibition of the 5 fungicides against mycelial growth and spore germination of the pathogen. Indoor toxicity determination indicated that the prochloraz and tebuconazole were better than remaining 3 fungicides. In mycelial growth test, the EC50 of prochloraz was 0.013 μg/mL and tebuconazole was0.055 μg/m L, and in spore germination test, the EC50 of prochloraz was 0.093 μg/m L and tebuconazole was0.082 μg/mL, respectively. Molecular identification of pathogens causing leaf blight of camellia oleifera in yuping and screening of fungicides might provide a theoretical basis for the cultivation and promotion of local cultivars and the prevention and control of the disease.
In order to identify the pathogen species of Camellia oleifera leaf blight in Guizhou Yuping, evaluate the different reagents on the virulence of pathogenic bacteria, and to screen the effective fungicides. This study isolated and purified pathogenic bacteria in the leaves with typical leaf blight of Camellia oleifera in Yuping, and then obtained four pathogenic strains of Pestalotiopsis spp. by re-inoculation and morphological identification.Four pathogenic strains of Pestalotiopsis spp. were isolated from typical leaf blight leaves, which were identified as Pestalotiopsis microspore by ITS-TUB2 gene sequence. The inhibitory activity in vitro of 5 fungicides to P.microspore was determined by the inhibition of the 5 fungicides against mycelial growth and spore germination of the pathogen. Indoor toxicity determination indicated that the prochloraz and tebuconazole were better than remaining 3 fungicides. In mycelial growth test, the EC50 of prochloraz was 0.013 μg/mL and tebuconazole was0.055 μg/m L, and in spore germination test, the EC50 of prochloraz was 0.093 μg/m L and tebuconazole was0.082 μg/mL, respectively. Molecular identification of pathogens causing leaf blight of camellia oleifera in yuping and screening of fungicides might provide a theoretical basis for the cultivation and promotion of local cultivars and the prevention and control of the disease.
引文
Cai L.,Hyde K.D.,Taylor P.W.J.,Weir B.S.,Waller J.M.,Abang M.M.,Zhang J.Z.,Yang Y.L.,Phoulivong S.,Liu Z.Y.,Prihastuti H.,Shivas R.G.,McKenzie E.H.C.,and Johnston P.R.,2009,A polyphasic approach for studying Colletotrichum,Fungal Divers.,39:183-204
Cui C.Y.,Wang Y.X.,Jiang J.X.,Ou Y.H.,Qin S.L.,and Huang T.,2015,Identification of the pathogen causing brown spot disease of Acer rubrum'October Glory',Linye Kexue(Scientia Silvae Sinicae),51(10):142-147(崔朝宇,王园秀,蒋军喜,欧阳慧,秦双林,黄婷,2015,美国红枫褐斑病病原菌鉴定,林业科学,51(10):142-147)
Dai D.J.,Song H.M.,Zhang C.Q.,Shen Y.,and Shen Y.,2017,Application and screening of fungicides for controlling anthracnose in Dendrobium candidum caused by Colletotrichum gloeosporioides,Nongyao(Pesticides),56(7):524-527,534(戴德江,宋会鸣,张传清,沈瑶,沈颖,2017,铁皮石斛炭疽病防治药剂的室内筛选与应用技术,农药,56(7):524-527,534)
Ding R.,Wang Y.L.,Li B.X.,and Xie C.P.,2010,Identification and characterization of mango leaf blight disease pathogen Pestalotiopsis mangiferae,Zhongguo Nanfang Guoshu(South China Fruits),39(4):20-24(丁榕,王延丽,李博勋,谢昌平,2010,杧果拟盘多毛孢叶枯病菌鉴定及其生物学特性研究,中国南方果树,39(4):20-24)
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 D-NA ITS序列的谱系分析,基因组学与应用生物学,34(4):830-835)
Gao Y.Y.,He L.F.,Li B.X.,Lin J.,Mu W.,and Liu F.,2017,I-dentification of the pathogen causing pepper anthracnose in Shandong Province and screening of highly effective fungicides,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),50(8):1452-1464(高杨杨,禾丽菲,李北兴,林琎,慕卫,刘峰,2017,山东省辣椒炭疽病病原菌的鉴定及高效防治药剂的筛选,中国农业科学,50(8):1452-1464)
Jeewon R.,Liew C.Y.E.,and Hyde K.D.,2004,Phylogenetic evaluation of species nomenclature of Pestalotiopsis in relation to host association,Fungal Divers.,17:39-55
Jeon Y.H.,Kim S.G.,and Kim Y.H.,2007,First report on leaf blight of Lindera obtusiloba caused by Pestalotiopsis microspora in Korea,Plant Pathol.,56(2):349
Keith L.M.,Velasquez M.E.,and Zee F.T.,2006,Identification and characterization of Pestalotiopsis spp.causing scab disease of guava,Psidium guajava in Hawaii,Plant Dis.,90(1):16-23
Liao W.J.,Zou D.X.,Wu Y.J.,Deng Y.,and Huang N.X.,2015,Identification of pathogenic Camellia oleifera seedling of leaf blight in Guangxi,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),28(6):2537-2540(廖旺姣,邹东霞,吴耀军,邓艳,黄乃秀,2015,广西油茶苗期叶枯病病原菌鉴定,西南农业学报,28(6):2537-2540)
Lu D.S.,Huang X.H.,and Dai B.,2012,Survey and identification of Camellia oleifera diseases in Xinyang area,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),40(5):83-85,111(卢东升,黄新华,代兵,2012,信阳市油茶病害及其病原鉴定,东北林业大学学报,40(5):83-85,111)
Metz A.M.,Haddad A.,Worapong J.,Long D.M.,Ford E.J.,Hess W.M.,and Strobel G.A.,2000,Induction of the sexual stage of Pestalotiopsis microspore,a taxol-producing fungus,Microbiology,146(8):2079-2089
Wang G.J.,Xiong J.W.,and Chen L.J.,2016,Evaluation of control effects several fungicides on tea anthracnose in Xinyang,Nongyao(Pesticides),55(8):604-606(王国君,熊建伟,陈利军,2016,几种杀菌剂对信阳茶树炭疽病的防治效果评价,农药,55(8):604-606)
Wei J.G.,Xu T.,Huang W.H.,Guo L.D.,and Pan X.H.,2008,Molecular phylogenetic investigation on relationship of endophytic and pathogenic Pestalotiopsis species,Zhejiang Daxue Xuebao(Journal of Zhejiang University(Agriculture&Life Sciences)),34(4):367-373(韦继光,徐同,黄伟华,郭良栋,潘秀湖,2008,从分子系统发育探讨内生拟盘多毛孢与病原拟盘多毛孢的关系,浙江大学学报(农业与生命科学版),34(4):367-373)
Weir B.S.,Johnston P.R.,and Damm U.,2012,The Colletotrichum gloeosporioides species complex,Stud.Mycol.,73(1):115-180
Wu J.R.,Lin M.,Mu L.J.,Yang G.F.,Duan C.B.,and Zhang D.H.,2012,Phytopathogen identification,susceptibility and the biological characteristics of brown spot disease in Camellia reticulata f.simpex Sealy,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),28(13):81-84(伍建榕,林梅,穆丽娇,杨光富,段成波,张东华,2012,滇西红花油茶褐斑病病原鉴定、致病性及主要生物学特性研究,中国农学通报,28(13):81-84)
Zhang S.C.,Li Y.F.,Huang R.Z.,Lv Z.H.,and Huang Q.Z.,2017,Identification of the pathogen causing processing type pepper anthracnose and its indoor toxicity in Chongqing,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1933-1937(张世才,李怡斐,黄任中,吕中华,黄启中,2017,重庆加工型辣椒炭疽病病原鉴定及防治药剂室内毒力测定,分子植物育种,15(5):1933-1937)
Zhang S.Y.,Li L.,Chen X.L.,Qi Q.Q.,Lou H.Z.,and Zhang Z.Q.,2017,Identification of brown spot on fruit of Torreya grandis and screening of fungicides,Zhiwu Baohu Xuebao(Acta Phytophylacica Sinica),44(5):817-825(张书亚,李玲,陈秀龙,戚钱钱,楼焕泽,张传清,2017,香榧果实褐斑病病原菌鉴定及防治药剂筛选,植物保护学报,44(5):817-825)
Zhao H.H.,Yue Q.H.,and Liang C.,2014,The pathogen causing Pestalotiopsis twig dieback of blueberry,Junwu Xuebao(Mycosystema),33(3):577-583(赵洪海,岳清华,梁晨,2014,蓝莓拟盘多毛孢枝枯病的病原菌,菌物学报,33(3):577-583)
Cui C.Y.,Wang Y.X.,Jiang J.X.,Ou Y.H.,Qin S.L.,and Huang T.,2015,Identification of the pathogen causing brown spot disease of Acer rubrum'October Glory',Linye Kexue(Scientia Silvae Sinicae),51(10):142-147(崔朝宇,王园秀,蒋军喜,欧阳慧,秦双林,黄婷,2015,美国红枫褐斑病病原菌鉴定,林业科学,51(10):142-147)
Dai D.J.,Song H.M.,Zhang C.Q.,Shen Y.,and Shen Y.,2017,Application and screening of fungicides for controlling anthracnose in Dendrobium candidum caused by Colletotrichum gloeosporioides,Nongyao(Pesticides),56(7):524-527,534(戴德江,宋会鸣,张传清,沈瑶,沈颖,2017,铁皮石斛炭疽病防治药剂的室内筛选与应用技术,农药,56(7):524-527,534)
Ding R.,Wang Y.L.,Li B.X.,and Xie C.P.,2010,Identification and characterization of mango leaf blight disease pathogen Pestalotiopsis mangiferae,Zhongguo Nanfang Guoshu(South China Fruits),39(4):20-24(丁榕,王延丽,李博勋,谢昌平,2010,杧果拟盘多毛孢叶枯病菌鉴定及其生物学特性研究,中国南方果树,39(4):20-24)
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 D-NA ITS序列的谱系分析,基因组学与应用生物学,34(4):830-835)
Gao Y.Y.,He L.F.,Li B.X.,Lin J.,Mu W.,and Liu F.,2017,I-dentification of the pathogen causing pepper anthracnose in Shandong Province and screening of highly effective fungicides,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),50(8):1452-1464(高杨杨,禾丽菲,李北兴,林琎,慕卫,刘峰,2017,山东省辣椒炭疽病病原菌的鉴定及高效防治药剂的筛选,中国农业科学,50(8):1452-1464)
Jeewon R.,Liew C.Y.E.,and Hyde K.D.,2004,Phylogenetic evaluation of species nomenclature of Pestalotiopsis in relation to host association,Fungal Divers.,17:39-55
Jeon Y.H.,Kim S.G.,and Kim Y.H.,2007,First report on leaf blight of Lindera obtusiloba caused by Pestalotiopsis microspora in Korea,Plant Pathol.,56(2):349
Keith L.M.,Velasquez M.E.,and Zee F.T.,2006,Identification and characterization of Pestalotiopsis spp.causing scab disease of guava,Psidium guajava in Hawaii,Plant Dis.,90(1):16-23
Liao W.J.,Zou D.X.,Wu Y.J.,Deng Y.,and Huang N.X.,2015,Identification of pathogenic Camellia oleifera seedling of leaf blight in Guangxi,Xinan Nongye Xuebao(Southwest China Journal of Agricultural Sciences),28(6):2537-2540(廖旺姣,邹东霞,吴耀军,邓艳,黄乃秀,2015,广西油茶苗期叶枯病病原菌鉴定,西南农业学报,28(6):2537-2540)
Lu D.S.,Huang X.H.,and Dai B.,2012,Survey and identification of Camellia oleifera diseases in Xinyang area,Dongbei Linye Daxue Xuebao(Journal of Northeast Forestry University),40(5):83-85,111(卢东升,黄新华,代兵,2012,信阳市油茶病害及其病原鉴定,东北林业大学学报,40(5):83-85,111)
Metz A.M.,Haddad A.,Worapong J.,Long D.M.,Ford E.J.,Hess W.M.,and Strobel G.A.,2000,Induction of the sexual stage of Pestalotiopsis microspore,a taxol-producing fungus,Microbiology,146(8):2079-2089
Wang G.J.,Xiong J.W.,and Chen L.J.,2016,Evaluation of control effects several fungicides on tea anthracnose in Xinyang,Nongyao(Pesticides),55(8):604-606(王国君,熊建伟,陈利军,2016,几种杀菌剂对信阳茶树炭疽病的防治效果评价,农药,55(8):604-606)
Wei J.G.,Xu T.,Huang W.H.,Guo L.D.,and Pan X.H.,2008,Molecular phylogenetic investigation on relationship of endophytic and pathogenic Pestalotiopsis species,Zhejiang Daxue Xuebao(Journal of Zhejiang University(Agriculture&Life Sciences)),34(4):367-373(韦继光,徐同,黄伟华,郭良栋,潘秀湖,2008,从分子系统发育探讨内生拟盘多毛孢与病原拟盘多毛孢的关系,浙江大学学报(农业与生命科学版),34(4):367-373)
Weir B.S.,Johnston P.R.,and Damm U.,2012,The Colletotrichum gloeosporioides species complex,Stud.Mycol.,73(1):115-180
Wu J.R.,Lin M.,Mu L.J.,Yang G.F.,Duan C.B.,and Zhang D.H.,2012,Phytopathogen identification,susceptibility and the biological characteristics of brown spot disease in Camellia reticulata f.simpex Sealy,Zhongguo Nongxue Tongbao(Chinese Agricultural Science Bulletin),28(13):81-84(伍建榕,林梅,穆丽娇,杨光富,段成波,张东华,2012,滇西红花油茶褐斑病病原鉴定、致病性及主要生物学特性研究,中国农学通报,28(13):81-84)
Zhang S.C.,Li Y.F.,Huang R.Z.,Lv Z.H.,and Huang Q.Z.,2017,Identification of the pathogen causing processing type pepper anthracnose and its indoor toxicity in Chongqing,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(5):1933-1937(张世才,李怡斐,黄任中,吕中华,黄启中,2017,重庆加工型辣椒炭疽病病原鉴定及防治药剂室内毒力测定,分子植物育种,15(5):1933-1937)
Zhang S.Y.,Li L.,Chen X.L.,Qi Q.Q.,Lou H.Z.,and Zhang Z.Q.,2017,Identification of brown spot on fruit of Torreya grandis and screening of fungicides,Zhiwu Baohu Xuebao(Acta Phytophylacica Sinica),44(5):817-825(张书亚,李玲,陈秀龙,戚钱钱,楼焕泽,张传清,2017,香榧果实褐斑病病原菌鉴定及防治药剂筛选,植物保护学报,44(5):817-825)
Zhao H.H.,Yue Q.H.,and Liang C.,2014,The pathogen causing Pestalotiopsis twig dieback of blueberry,Junwu Xuebao(Mycosystema),33(3):577-583(赵洪海,岳清华,梁晨,2014,蓝莓拟盘多毛孢枝枯病的病原菌,菌物学报,33(3):577-583)