【作者】 丁平；

【导师】 王克荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 硕士

【摘要】 栗疫病(Chestnut blight)是栗属(Castanea)植物上最常见、最重要的病害，也是世界著名的森林病害之一。本文对栗疫病菌的群体结构进行了研究。 本研究用标准交配型菌株对中国东部11个省的114个栗疫病菌进行了交配型测定，结果表明，只要条件适宜，各种植区栗疫病菌均可以形成有性阶段的子囊壳。本研究克隆了我国栗疫病菌菌株133和293的MAT-1交配型的部分基因，序列分析表明，133和293菌株与GeneBank的MAT-1的序列同源性为94％和97％。用PCR技术对中国东部栗疫病菌交配型进行了快速分子测定，结果发现MAT-1和MAT-2菌株各占50％，多数种植区栗疫病菌同时存在MAT-1、MAT-2两种交配型；用分子测定不同子囊壳栗疫病菌子囊孢子后代的交配型分离情况多数分离比为1：1。分子测定与生物学测定交配型结果吻合的有87个菌株，占75.7％；不吻合的有26株，占22.6％。结果提示有性杂交导致栗疫病菌的遗传变异仍然是栗疫病菌遗传多样性的重要原因。 采用RAPD方法对来源于栗疫病菌8个不同子囊壳的子囊孢子后代和无性生殖的对照群体各23个菌株进行了群体结构的比较。从RAPD(random amplified polymorphic DNA)随机引物中筛选出扩增多态性丰富的4条引物，共扩增出条带73条，多态性检测率为100％。研究结果表明，在8个子囊壳和无性生殖群体中的基因多样性，64.27％由群体内部引起，只有35.73％的多样性由群体之间的基因差异引起。各子囊壳群体间存在的基因流动很小(Nm=0.8994)。有性群体和无性群体之间的遗传距离为0.1389，基因流动值为3.4212，说明子囊壳群体和无性生殖群体之间可能存在一定的关系。分析表明栗疫病菌子囊孢子后代在自然界的传播对自然界病菌的多样性起重要的作用。 利用紫外诱变方法得到了栗疫病菌的突变体菌株。突变体菌株和初始菌株相比，在生长速度、产生分生孢子数量、色素的产生、代谢相关的酶及致病性上都有明显的差异。在含有0.5％单宁酸的马铃薯培养基(PDA)上生长时生长速度比在PDA上生长快；RAPD分析表明，A59与A59-2在DNA指纹图谱上没有差异，但寄主基因组的改变对表型的改变有一定的作用。含有病毒的突变体的培养特征类似其原养型突变体：与一般低毒力菌株相比，色素产生较多，产孢量高，具有作为生物防治栗疫病材料的潜力。更多还原

【Abstract】 Chestnut blight epidemic is associated with devastation ofCastanea, Chestnut blight also one of an important disease in the forests. The population diversity of Cryphonectria parasitica was studied in this thesis.One hundred and fourteen isolates of C. parasitica collected from 11 provinces in the east of China were tested for their mating type with four standard tested isolates. Results showed that both mating types, MAT-1 and MAT-2, exist in C. parasitica population of China. MAT-1 partial mating-type idiomorph of C. parasitica isolates 133 and 293 were cloned and sequenced. Analysis of partial sequences of MAT-1 idiomorph of C. parasitica isolates 133 and 293, found the sequence of partial idiomorphs are 94% and 97% identical to the partial sequence from GeneBank. A PCR assay was carried out to determine the mating-type of C. parasitica isolates obtained from eastern China, it was found that MAT-1 and MAT-2 was 50% in the 266 isolates from eastern China. In most of isolates from the same location the tested locations, two mating types existed together. Ascospore progeny strains from the fungal perithecia segregated for mating type at ratio of 1:1. The test results of mating type to 87 of 114 isolates by PCR and crossing in pair to standand isolates were consistant, but the other 26 of 114 isolates were different suggestting that the sexual stage occurred in natural condition, the sexual recombination might be a potential resource for the genetic diversity of C. parasitica.The population structure of C. parasitica was analyzed using random amplified polymorphic DNA (RAPD). 23 isolates of each of eight perithecia and the fungal asexual populations as the samples collected from natural garden, and assayed for genetic diversity by using 4 RAPD primers with abundant polymorphisms. 73 RAPD mark bands were obtained and 100% of them were polymorphic. Ascospore strains from peritheria showed much more variability among themself, in contrast with field asexual isolates showing lower genetic diversity. Genetic diversity and UPGMA dendrogram were analyzed by use of Popgen Version 1.31,and the results showed that the genetic variation between perithecia were rich: 64.27% was attributed to diversity within populations, as compared with 35.73% among populations. Gene flow value (Nm) was 0.8994 between different peritheria while the genetic distance among sexual and asexual populations was 0.1389, but the gene flow estimate was 3.4212更多还原