水稻纹枯病的综合治理研究

山东农业大学本科毕业生毕业论文学院：植物保护学院班级：09 级植检 1 班学号：20095017水稻纹枯病的综合治理研究Study on the integrated management of rice sheath blight作者：温东东 指导老师：刘爱新Author: Dongdong Wen The instructor: AiXin liuI目录中文摘要 .1关键词 .1Abstract .1Keywords .11. 前言 .31.1 水稻纹枯病的研究进展 .31.1.1 水稻纹枯病的病原及融合群 .31.1.2 水稻纹枯病的危害、防治及其存在问题 .31.1.2.1 抗病品种的选育 .31.1.2.2 生物防治 .41.1.2.3 化学防治 .41.1.3 水稻纹枯病菌（R.solani AG1-IA）的群体遗传学研究 .41.1.3.1 水稻纹枯病菌的遗传特性 .41.1.3.2 影响水稻纹枯病菌群体遗传结构变化的因素 .5（1） 随机遗传漂变 .5（2）基因流动 .5（3） 突变 .5（4） 重组 .5（5） 选择 .52 研究内容 .62.1 材料与方法 .62.1.1 材料 .62.1.1.1 菌株材料 .62.1.1.2 植物材料 .62.1.1.3 其它材料 .62.2 方法 .62.2.1 纹枯病菌分离培养 .62.2.2 纹枯病病菌致病力测定 .62.2.3 菌丝融合群测定 .72.2.4 供试菌株基因组 DNA 的提取 .72.2.5 AG1-IA 融合群 SSR 标记体系的建立及遗传多样性研究 .82.2.5.1 微卫星标记所需引物 .92.2.5.2 SSR-PCR 扩增反应体系 .102.2.5.3 SSR-PCR 反应程序 .102.2.5.4 聚丙烯酰胺凝胶电泳 .11（1） 所需试剂及配制 .11（2） 电泳 .11（3） 银染步骤 .122.2.6 数据统计 .123 结果与分析 .133.1 不同地区水稻纹枯病菌融合群鉴定 .133.1.1 水稻纹枯病菌菌丝融合鉴定 .133.1.2 5.8S rDNA-ITS 序列分析 .15II4 讨论 .174.1 关于 R. solani AG1-IA 水稻分离物的遗传分化 .174.2 菌株致病力与 SSR 多态性的关系 .17参考文献 .19致谢. . 21IIIDirectoryChinese abstract .1Chinese keywords .1Abstract .1Keywords .11.Preface.31.1 Research progress of rice sheath blight.31.1.1 The pathogen of rice sheath blight and fusion group.31.1.2 Harm, rice sheath blight and its control problems .31.1.2.1 Breeding resistant varieties .31.1.2.2 Biocontrol.41.1.2.3 Chemical control .41.1.3 Rhizoctonia solani (R.solani AG1-IA) Population Genetics .41.1.3.1 Genetic characteristics of Rhizoctonia solani .41.1.3.2 Rhizoctonia solani influence changes in the factors of population genetic structure.5（1）Random genetic drift .5（2）Gene flow .5（3）Mutation .5（4）Restructuring .5（5）Choose .52 Research .62.1 Materials and Methods.62.1.1 Material .62.1.1.1 Strain material .62.1.1.2 Plant material .62.1.1.3 Other materials.62.2 Method .62.2.1 Rhizoctonia solani isolated and cultured .62.2.2 Sheath blight pathogen virulence determination.62.2.3 Determination of anastomosis group .72.2.4 Strains tested genomic DNA extraction.72.2.5 AG1-IA fusion groups Establishment of SSR markers and genetic diversity of .82.2.5.1 Microsatellite marker primers required.92.2.5.2 SSR-PCR amplification reaction .102.2.5.3 SSR-PCR reaction procedure.102.2.5.4 Polyacrylamide gel electrophoresis .11（1） And preparation of the necessary reagents .11（2） Electrophoresis.11（3） Silver staining procedure .122.2.6 Statistics .123 Results and analysis .133.1Different regions of Rhizoctonia solani anastomosis group identification.133.1.1 Identification of Rhizoctonia solani anastomosis .133.1.2 5.8S rDNA-ITS sequence analysis.154 Discuss .174.1 About R. solani AG1-IA rice isolate genetic differentiation .174.2 Strain virulence and SSR polymorphism .17IVReferences.19Acknowledgements. .211中文摘要立枯丝核菌（Rhizoctonia solani）寄主范围广泛，可侵染危害 250 多种植物。R. solani 是一个复合种，该种至少包括 14 个融合群，每个融合群又可分为不同的融合亚群。R. solani AG1-IA 属于 AG1 融合群的 IA 亚群，该真菌可 危害 玉米、水稻和大豆等多种植物，在水稻形成典型的纹枯病症状，是水稻纹枯病的优势致病类群。R. solani AG1-IA 群体结构组成和变化对该类病害的发生和流行起着非常重要的作用。本研究中，我们利用 SSR 分子标记，对我国山东和广东 4 个主要产稻区的 61 个 R. solani AG1-IA 分离物的群体遗传结构进行了分析, 旨在明确 AG1-IA 群体在不同地区间的遗传多样性组成及演化动态，希望为进一步制定水稻纹枯病的综合治理措施提供理论依据。1. 纹枯病菌采集、分离及融合群确定从山东临沂、东营和济阳采集标本，经分离培养，通过菌丝融合测定、ITS 序列测定等鉴定所属的融合群，将 51 株鉴定为 AG1-IA 的水稻分离物用于本研究，还有 10 株来自广东的菌株。2 R. solani AG1-IA 水稻分离物遗传多样性分析利用 10 对 SSR 引物对山东 3 个水稻产区的 51 株 AG1-IA 水稻分离物和 10 株广州分离物进行SSR 遗传多样性分析，共检测到 32 条多态性条带，每对引物能扩增出 25 条多态性条带。采用UPMGA 法进行 SSR 聚类分析结果表明，61 个分离物在 0.50 到 1.00 遗传相似系数范围内，被分为不同聚类组。来自不同地区的水稻纹枯病菌，多以地理来源聚为一类，表明 SSR 分组与菌株的地理来源有一定的相关性。Neis 遗传多样性指数在广东省达到最大，为 0.4228，Shannons 指数在广东省达到最高，为0.7309，表明来自广东省的水稻纹枯病菌具有复杂的遗传结构。其中，来自广东和济阳的菌株遗传距离最远，临沂和济阳的菌株遗传距离最近。对群体的遗传分化进行分析，群体内和群体间都存在一定的遗传分化，群体间的变异约占总变异的 17.4%，群体内的变异占总变异的 82.6%，因此，遗传变异主要发生在群体内。关键词：水稻纹枯病；SSR；遗传多样性AbstractRhizoctonia solani (Rhizoctonia solani) wide host range and can infect endanger more than 250 kinds of plants. R. solani is a species complex, which includes at least 14 species of fusion groups, each group can be divided into different fusion fusion subsets. R. solani AG1-IA IA belong AG1 fusion group subsets, the fungus can be hazardous to corn, rice and soybeans and other plants, in the form of a typical rice sheath blight symptoms are the advantages of rice sheath blight pathogenicity groups. R. solani AG1-IA changes in population structure and composition of the class of the incidence and prevalence of disease plays a very important role. In this study, we used SSR markers on Chinas Shandong and Guangdong four major producing rice for 61 R. solani AG1-IA isolates were analyzed genetic structure, aimed at clarifying AG1-IA groups in different regions between the composition and evolution of the dynamics of genetic diversity, hope for the further development of rice sheath blight comprehensive measures provide a theoretical basis.1 R. solani collection, separation and fusion group identified2From Linyi, Shandong Dongying and Jiyang collect specimens, the isolation and culture, through hyphal fusion determination, ITS sequencing and other identification of an integration group, the 51 identified as AG1-IA rice isolates used in this study, as well as 10 strains from Guangdong.2 R. solani AG1-IA rice genetic diversity of isolatesThe use of 10 pairs of SSR primers Shandong three rice-producing areas 51 AG1-IA rice isolates and 10 Guangzhou isolates SSR analysis of genetic diversity were detected 32 polymorphic bands per primer pair can expand increase of 2 to 5 polymorphic bands. SSR clustering method using UPMGA analysis showed that 61 isolates in the 0.50 to 1.00 range of genetic similarity coefficient, is divided into different cluster groups. From different regions of Rhizoctonia solani, and more to the geographical origin clustered together, indicating that the SSR grouping and geographical origin of strains have some relevance.Neis genetic diversity index in Guangdong province reaches the m