我国不同产区桃褐腐病病原鉴定与分析
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摘要
【背景】桃褐腐病是桃树的一种重要病害,给桃果生产带来了严重的经济损失。桃褐腐病病原种类较多。【目的】明确我国不同桃产区褐腐病菌的种类。【方法】采集不同产区桃褐腐病果,采用单孢分离法分离病菌,通过形态特征、分子生物学方法和接种试验对分离株进行种类鉴定和分析。【结果】从来自浙江丽水、四川成都、云南昆明、辽宁大连、河北石家庄、北京海淀、山东泰安和山东青岛等8个不同桃产区的褐腐病果上经单孢分离获得15株分离株。各分离株菌落形态略有差异,但来自云南的分离株hfyn与其他分离株明显不同。通过rDNA ITS序列和系统进化树分析,将云南分离株hfyn鉴定为云南丛梗孢(Monilia yunnanensis),来源于其他产区的14株分离株均鉴定为果生链核盘菌(Monilinia fructicola)。采用多对特异性引物PCR扩增分析,确证了上述鉴定结果。接种试验表明,不同产区来源的桃褐腐病菌分离株在桃果上的致病力存在差异,其中来自浙江丽水、河北石家庄、山东青岛的分离株致病力较强,而来自山东泰安和云南昆明的分离株致病力相对较弱。【结论】我国桃产区褐腐病菌主要以M.fructicola为主,但不同产区来源的菌株间存在形态和致病性差异,此结果可为我国桃褐腐病的治理提供科学依据。
[Background] Brown rot on peach and nectarine trees caused by Monilinia spp. is an important fungal disease, and produces serious economic losses frequently in China. There may exist more than one species of peach brown rot pathogens. [Objective] To clarify the species and pathogenicity of brown rot pathogens from different peach-growing areas in China. [Methods] The pathogens were isolated from peach brown rot fruits of different peach production areas, using the method of single spore isolation. Identification and analysis of isolates were carried out by morphological characteristics, molecular biology methods, and inoculation assay. [Results] In this study 15 brown rot pathogen isolates were obtained from 8 different peach-growing areas. The morphological characteristics of the isolates were slightly different from each other, but the isolate hfyn from Yunnan Province was significantly different from the other isolates. Based on its morphology, rDNA ITS sequence and phylogenetic analysis, the isolate hfyn was identified as Monilia yunnanensis, which was a newly found brown rot pathogen, and the other 14 isolates were identified as Monilinia fructicola. PCR amplification using several pairs of specific primers confirmed the morphological and rDNA ITS identification results. Inoculation tests showed that the pathogenicities on peach fruits of 15 isolates from different peach producing areas were different. The isolates from Lishui, Zhejiang Province,Shijiazhuang, Hebei Province, and Qingdao, Shandong Province had more virulent, and the virulences of isolates from Kunming, Yunnan Province and Tai'an, Shandong Province were relatively weak. [Conclusion] The results indicated that the major pathogen of brown rot in peach-growing areas in China was Monilinia fructicola, and there were differences in morphology and pathogenicity among these isolates from different peach-growing areas. The result from the present study may provide a scientific basis for managing peach brown rot epidemics.
[Background] Brown rot on peach and nectarine trees caused by Monilinia spp. is an important fungal disease, and produces serious economic losses frequently in China. There may exist more than one species of peach brown rot pathogens. [Objective] To clarify the species and pathogenicity of brown rot pathogens from different peach-growing areas in China. [Methods] The pathogens were isolated from peach brown rot fruits of different peach production areas, using the method of single spore isolation. Identification and analysis of isolates were carried out by morphological characteristics, molecular biology methods, and inoculation assay. [Results] In this study 15 brown rot pathogen isolates were obtained from 8 different peach-growing areas. The morphological characteristics of the isolates were slightly different from each other, but the isolate hfyn from Yunnan Province was significantly different from the other isolates. Based on its morphology, rDNA ITS sequence and phylogenetic analysis, the isolate hfyn was identified as Monilia yunnanensis, which was a newly found brown rot pathogen, and the other 14 isolates were identified as Monilinia fructicola. PCR amplification using several pairs of specific primers confirmed the morphological and rDNA ITS identification results. Inoculation tests showed that the pathogenicities on peach fruits of 15 isolates from different peach producing areas were different. The isolates from Lishui, Zhejiang Province,Shijiazhuang, Hebei Province, and Qingdao, Shandong Province had more virulent, and the virulences of isolates from Kunming, Yunnan Province and Tai'an, Shandong Province were relatively weak. [Conclusion] The results indicated that the major pathogen of brown rot in peach-growing areas in China was Monilinia fructicola, and there were differences in morphology and pathogenicity among these isolates from different peach-growing areas. The result from the present study may provide a scientific basis for managing peach brown rot epidemics.
引文
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[3]Institute of Pomology/Citrus Research Institute,Chinese Academy of Agricultural Sciences.Chinese Fruit Pest[M].2nd ed.Beijing:China Agriculture Press,1994(in Chinese)中国农业科学院果树研究所/柑橘研究所.中国果树病虫志[M].2版.北京:中国农业出版社,1994
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[7]Batra LR.World species of Monilinia(fungi):Their ecology,biosystematics and control[J].Mycologia Memoirs,1991,16:1-246
[8]Chen LF,Xu JY.Agricultural Plant Pathology[M].4th ed.Beijing:China Agriculture Press,2015(in Chinese)陈利锋,徐敬友.农业植物病理学[M].4版.北京:中国农业出版社,2015
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[10]Hu MJ,Cox KD,Schnabel G,et al.Monilinia species causing brown rot of peach in China[J].PLoS One,2011,6(9):e24990
[11]van Leeuwen GCM,Baayen RP,Holb IJ,et al.Distinction of the Asiatic brown rot fungus Monilia polystroma sp.nov.from M.fructigena[J].Mycological Research,2002,106(4):444-451
[12]Luo CX.Advances and prospects on researches of brown rot disease on fruits[J].Acta Phytopathologica Sinica,2017,47(2):145-153(in Chinese)罗朝喜.果树褐腐病的研究现状及其展望[J].植物病理学报,2017,47(2):145-153
[13]Ji ZL,Zhang JJ,Xu JY,et al.Characteristics and competition of procymidone-resistant Botrytis cinerea isolates[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2007,28(2):65-68(in Chinese)纪兆林,张建军,徐敬友,等.抗腐霉利的灰葡萄孢菌株特性及其竞争能力[J].扬州大学学报:农业与生命科学版,2007,28(2):65-68
[14]Wei JC.Fungal Identification Manual[M].Shanghai:Shanghai Scientific&Technical Publishers,1979(in Chinese)魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979
[15]Lane CR.A synoptic key for differentiation of Monilinia fructicola,M.fructigena and M.laxa,based on examination of cultural characters[J].EPPO Bulletin,2002,32(3):489-493
[16]White TJ,Bruns T,Lee S,et al.Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes[A]//Innis MA.PCR Protocols:A Guide to Methods and Applications[M].New York:Academic Press,1990:315-322
[17]Ioos R,Frey P.Genomic variation within Monilinia laxa,M.fructigena and M.fructicola,and application to species identification by PCR[J].European Journal of Plant Pathology,2000,106(4):373-378
[18]Ma Z,Luo Y,Michailides TJ.Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California[J].Journal of Phytopathology,2003,151(6):312-322
[19]C?téMJ,Tardif MC,Meldrum AJ.Identification of Monilinia fructigena,M.fructicola,M.laxa,and Monilia polystroma on inoculated and naturally infected fruit using multiplex PCR[J].Plant Disease,2004,88(11):1219-1225
[20]Yin LF.Characterization of populations of Monilinia spp.on stone and pome fruits in China and genetic evolution of the Cyt b genes[D].Wuhan:Doctoral Dissertation of Huazhong Agricultural University,2015(in Chinese)尹良芬.中国核果/仁果类果树褐腐病菌种群结构及Cyt b基因的遗传进化研究[D].武汉:华中农业大学博士学位论文,2015
[21]Zhu XQ,Niu CW,Chen XY,et al.Monilinia species associated with brown rot of cultivated apple and pear fruit in China[J].Plant Disease,2016,100(11):2240-2250
[22]Zhou F.Population structure and pathogenicity of Monilinia in Shanxi Province[D].Taigu:Master’s Thesis of Shanxi Agricultural University,2015周芳.山西省褐腐病菌种群结构及致病性研究[D].太谷:山西农业大学硕士学位论文,2015
[23]Yan DQ.Genetic diversity of Monilia yunnanensis by using RAPD analysis[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2016(in Chinese)闫大琦.果树褐腐病菌Monilia yunnanensis遗传多样性的RAPD分析[D].武汉:华中农业大学硕士学位论文,2016
[24]Zhu XQ,Zheng HH,Fang YL,et al.A method to induce significant production of conidia from Monilinia fructigena,Monilia polystroma,and Monilia yunnanensis[J].Australasian Plant Pathology,2014,43(5):531-533
[25]Fan JY,Zhu XQ,Guo LY,et al.Comparison of three molecular identification methods for Monilinia species on stone and pome fruits[J].Acta Phytophylacica Sinica,2007,34(3):289-295(in Chinese)樊锦艳,朱小琼,国立耘,等.褐腐病菌三种分子鉴定方法的比较[J].植物保护学报,2007,34(3):289-295
[26]Wang JR,Guo LY,Xiao CL,et al.Detection and identification of six Monilinia spp.causing brown rot using TaqMan real-time PCR from pure cultures and infected apple fruit[J].Plant Disease,2018.DOI:10.1094/PDIS-10-17-1662-RE
[2]Xiang WN.Chinese Mycology and Plant Pathology Literature[M].Beijing:Science Press,1957(in Chinese)相望年.中国真菌学与植物病理学文献[M].北京:科学出版社,1957
[3]Institute of Pomology/Citrus Research Institute,Chinese Academy of Agricultural Sciences.Chinese Fruit Pest[M].2nd ed.Beijing:China Agriculture Press,1994(in Chinese)中国农业科学院果树研究所/柑橘研究所.中国果树病虫志[M].2版.北京:中国农业出版社,1994
[4]Zhu XQ,Chen XY,Luo Y,et al.First report of Monilinia fructicola on peach and nectarine in China[J].Plant Pathology,2005,54:575
[5]Hu MJ,Chen Y,Chen SN,et al.First report of brown rot of peach caused by Monilinia fructicola in southeastern China[J].Plant Disease,2011,95(2):225
[6]Li SF,Chen C.Incidence and management of the peach fruit brown rot[J].Plant Protection,2009,35(2):134-139(in Chinese)李世访,陈策.桃褐腐病的发生和防治[J].植物保护,2009,35(2):134-139
[7]Batra LR.World species of Monilinia(fungi):Their ecology,biosystematics and control[J].Mycologia Memoirs,1991,16:1-246
[8]Chen LF,Xu JY.Agricultural Plant Pathology[M].4th ed.Beijing:China Agriculture Press,2015(in Chinese)陈利锋,徐敬友.农业植物病理学[M].4版.北京:中国农业出版社,2015
[9]Harada Y,Nakao S,Sasaki M,et al.Monilia mumecola,a new brown rot fungus on Prunus mume in Japan[J].Journal of General Plant Pathology,2004,70(6):297-307
[10]Hu MJ,Cox KD,Schnabel G,et al.Monilinia species causing brown rot of peach in China[J].PLoS One,2011,6(9):e24990
[11]van Leeuwen GCM,Baayen RP,Holb IJ,et al.Distinction of the Asiatic brown rot fungus Monilia polystroma sp.nov.from M.fructigena[J].Mycological Research,2002,106(4):444-451
[12]Luo CX.Advances and prospects on researches of brown rot disease on fruits[J].Acta Phytopathologica Sinica,2017,47(2):145-153(in Chinese)罗朝喜.果树褐腐病的研究现状及其展望[J].植物病理学报,2017,47(2):145-153
[13]Ji ZL,Zhang JJ,Xu JY,et al.Characteristics and competition of procymidone-resistant Botrytis cinerea isolates[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2007,28(2):65-68(in Chinese)纪兆林,张建军,徐敬友,等.抗腐霉利的灰葡萄孢菌株特性及其竞争能力[J].扬州大学学报:农业与生命科学版,2007,28(2):65-68
[14]Wei JC.Fungal Identification Manual[M].Shanghai:Shanghai Scientific&Technical Publishers,1979(in Chinese)魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979
[15]Lane CR.A synoptic key for differentiation of Monilinia fructicola,M.fructigena and M.laxa,based on examination of cultural characters[J].EPPO Bulletin,2002,32(3):489-493
[16]White TJ,Bruns T,Lee S,et al.Analysis of phylogenetic relationships by amplification and direct sequencing of ribosomal RNA genes[A]//Innis MA.PCR Protocols:A Guide to Methods and Applications[M].New York:Academic Press,1990:315-322
[17]Ioos R,Frey P.Genomic variation within Monilinia laxa,M.fructigena and M.fructicola,and application to species identification by PCR[J].European Journal of Plant Pathology,2000,106(4):373-378
[18]Ma Z,Luo Y,Michailides TJ.Nested PCR assays for detection of Monilinia fructicola in stone fruit orchards and Botryosphaeria dothidea from pistachios in California[J].Journal of Phytopathology,2003,151(6):312-322
[19]C?téMJ,Tardif MC,Meldrum AJ.Identification of Monilinia fructigena,M.fructicola,M.laxa,and Monilia polystroma on inoculated and naturally infected fruit using multiplex PCR[J].Plant Disease,2004,88(11):1219-1225
[20]Yin LF.Characterization of populations of Monilinia spp.on stone and pome fruits in China and genetic evolution of the Cyt b genes[D].Wuhan:Doctoral Dissertation of Huazhong Agricultural University,2015(in Chinese)尹良芬.中国核果/仁果类果树褐腐病菌种群结构及Cyt b基因的遗传进化研究[D].武汉:华中农业大学博士学位论文,2015
[21]Zhu XQ,Niu CW,Chen XY,et al.Monilinia species associated with brown rot of cultivated apple and pear fruit in China[J].Plant Disease,2016,100(11):2240-2250
[22]Zhou F.Population structure and pathogenicity of Monilinia in Shanxi Province[D].Taigu:Master’s Thesis of Shanxi Agricultural University,2015周芳.山西省褐腐病菌种群结构及致病性研究[D].太谷:山西农业大学硕士学位论文,2015
[23]Yan DQ.Genetic diversity of Monilia yunnanensis by using RAPD analysis[D].Wuhan:Master’s Thesis of Huazhong Agricultural University,2016(in Chinese)闫大琦.果树褐腐病菌Monilia yunnanensis遗传多样性的RAPD分析[D].武汉:华中农业大学硕士学位论文,2016
[24]Zhu XQ,Zheng HH,Fang YL,et al.A method to induce significant production of conidia from Monilinia fructigena,Monilia polystroma,and Monilia yunnanensis[J].Australasian Plant Pathology,2014,43(5):531-533
[25]Fan JY,Zhu XQ,Guo LY,et al.Comparison of three molecular identification methods for Monilinia species on stone and pome fruits[J].Acta Phytophylacica Sinica,2007,34(3):289-295(in Chinese)樊锦艳,朱小琼,国立耘,等.褐腐病菌三种分子鉴定方法的比较[J].植物保护学报,2007,34(3):289-295
[26]Wang JR,Guo LY,Xiao CL,et al.Detection and identification of six Monilinia spp.causing brown rot using TaqMan real-time PCR from pure cultures and infected apple fruit[J].Plant Disease,2018.DOI:10.1094/PDIS-10-17-1662-RE