湖北省桑椹腐烂病病原菌鉴定
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摘要
为明确引起桑椹腐烂病的病原菌种类,2017年从湖北省采集病果,采用常规组织分离法进行分离,用分离纯化的菌株进行菌丝块针刺和孢子液注射接种健康果均可致病,形态学鉴定发现其形态学特征与灰霉菌(Botrytis cinerea)一致。在GenBank进行Blast比对,该菌株的rDNA-ITS序列与Botrytis cinerea和Botrytis fabae相似率最高为99%,用区分Botrytis cinerea和Botrytis fabae的特异性引物进行检测,同时构建分子系统进化树进行分子生物学鉴定,进一步表明该菌为Botrytis cinerea。致病性测定、形态学及分子生物学鉴定结果表明,引起湖北省桑椹腐烂病的病原菌为Botrytis cinerea。
In order to understand pathogens associate with mulberry rot disease,fruit with rot symptoms were collected from Hubei province in 2017,and the conventional tissue separation method was used to obtain the isolates. Healthy fruits inoculated with purified strain by mycelium plugs puncture and conidia injection both can cause diseased symptoms. Morphological identification showed that the morphological features of the strain were similar to Botrytis cinerea. The Blast search of rDNA ITS sequence of the strain showed 99% sequence identity with Botrytis cinerea and Botrytis fabae in GenBank. The specific primers were used for distinguish Botrytis cinerea and Botrytis fabae,and the construction of molecular phylogenetic tree further confirm the strain was Botrytis cinerea. The results of pathogenicity test,morphological and molecular identification indicated that the pathogen of mulberry rot in Hubei province was Botrytis cinerea.
In order to understand pathogens associate with mulberry rot disease,fruit with rot symptoms were collected from Hubei province in 2017,and the conventional tissue separation method was used to obtain the isolates. Healthy fruits inoculated with purified strain by mycelium plugs puncture and conidia injection both can cause diseased symptoms. Morphological identification showed that the morphological features of the strain were similar to Botrytis cinerea. The Blast search of rDNA ITS sequence of the strain showed 99% sequence identity with Botrytis cinerea and Botrytis fabae in GenBank. The specific primers were used for distinguish Botrytis cinerea and Botrytis fabae,and the construction of molecular phylogenetic tree further confirm the strain was Botrytis cinerea. The results of pathogenicity test,morphological and molecular identification indicated that the pathogen of mulberry rot in Hubei province was Botrytis cinerea.
引文
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[2]李祖发,叶建美.对湖北蚕桑产业发展的再认识[J].北方蚕业,2008,29(3):1-3.
[3] MURRAY M G,THOMPSON W F. Rapid isolation of high molecular weight plant DNA[J].Nucleic Acids Research,1980,8(19):4321-4326.
[4] GLASS N L,DONALDSON G C. Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes[J].Applied and Environmental Microbiology,1995,61(4):1323-1330.
[5] MIRZAEI S,GOLTAPEH E M,SHAMSBAKHSH M,et al. Identification of Botrytis spp. on plants grown in Iran[J].Journal of Phytopathology,2010,156(1):21-28.
[6] RIGOTTI S,GINDRO K,RICHTER H,et al. Characterization of molecular markers for specific and sensitive detection of Botrytis cinerea Pers.:Fr. in strawberry(Fragaria×ananassa Duch.)using PCR[J].Fems Microbiology Letters,2002,209(2):169-174.
[7] THOMPSON J D,GIBSON T J,PLEWNIAK F,et al. The CLUSTAL_X windows interface:Flexible strategies for multiple sequence alignment aided by quality analysis tools[J].Nucleic Acids Research,1997,25(25):4876-4882.
[8] ZHU Z,ZHENG L,PAN L,et al. Identification and characterization of Fusarium species associated with wilt of Eleocharis dulcis(Chinese water chestnut)in China[J].Plant Disease,2014,98(7):977-987.
[9]中国科学院中国孢子植物志编辑委员会.中国真菌志第二十六卷葡萄孢属柱隔孢属[M].北京:科学出版社,2006.
[10] MARTINEZ F,BLANCARD D,LECOMTE P,et al. Phenotypic differences between vacuma and transposa subpopulations of Botrytis cinerea[J].European Journal of Plant Pathology,2003,109(5):479-488.
[11]张静.湖北省灰霉病病菌区系和灰葡萄孢菌多样性研究[D].武汉:华中农业大学,2010.
[12]黄燕,段灿星,陆鸣,等.蚕豆赤斑病病原菌鉴定[J].植物保护,2012,38(6):115-118.
[13]顾振芳.桑树灰霉病菌Botrytis cinerea Pers.的生物学特性[J].浙江大学学报(农业与生命科学版),1986(1):104-106.
[14]王泽林,宋友全.桑灰霉病的防治措施[J].蚕学通讯,2014(3):30-31.
[15]韩蓓蓓.桑椹病原真菌的鉴定和采后微生物保鲜的研究[D].江苏镇江:江苏科技大学,2016.