【作者】 瞿波；

【导师】 廖玉才；

【作者基本信息】 华中农业大学 ， 作物遗传育种， 2003， 博士

【摘要】 利用从中国小麦赤霉病主要发病区广泛收集的禾谷镰刀菌(F.graminearum)菌株，通过AFLP、SCAR、SSCP等先进的分子标记技术，分析了中国病原菌群体的DNA多态性和致病力，并与欧洲、美国和尼泊尔等不同大陆的禾谷镰刀菌菌系进行比较，首次从分子水平上揭示了中国禾谷镰刀菌的遗传多样性和分布状况及其与尼泊尔、欧美菌系的差别。 根据种类特异PCR的分析结果，不同大陆的禾谷镰刀菌存在6种类型(类型1-类型6)。中国的禾谷镰刀菌存在两种类型，即类型1和类型5。类型5属于温暖型菌系，是中国禾谷镰刀菌的主要群体，约占调查数的79％，分布在中国的大部分地区，尤其是温暖麦区；而类型1属于冷凉型菌系，是一个相对较小的群体，约占调查数的21％，其分布有明显的地区局限性，主要分布在较为冷凉的地区。西欧、北美等气候较冷凉地区的绝大部分菌株也是类型1，只在西北欧的少数地区发现了类型6菌株。这说明禾谷镰刀菌存在明显的生态地理类型。 SSCP分析首次更明确地区分出了这6种不同的类型。根据SSCP的多态性形态，可以进一步将类型1分为类型1A，类型1B和类型1C三个亚类。类型1A是主要的类型，大多数类型1菌株都是这一类型。其它几种类型没能进一步区分出亚类。用SSCP技术对中国禾谷镰刀菌群体所作的分析表明，中国的禾谷镰刀菌只有类型1(A)和类型5两种类型，在这两种类型中没有区分出亚类。 SCAR分析显示，特异性PCR产物类型是由于DNA序列的79-168和440-526区的核苷酸发生变化造成的，而其余380个核苷酸几乎完全一致，仅几个核苷酸位点发生了变化。对每一种类型而言，用不同来源的菌株扩增出的特异性DNA片段的核苷酸排列几乎完全相同。 AFLP分析结果表明，来自不同大陆的禾谷镰刀菌菌系存在丰富的变异。不同大陆的禾谷镰刀菌群体中没有发现AFLP带谱完全相同的两个个体，即使在同一来源地的菌株中也很难找到完全相同的个体，显示了禾谷镰刀菌群体中存在高度的遗传多样性。 AFLP分析首次明确显示出了禾谷镰刀菌两大类群与地理来源的关系。根据对来自不同大陆的禾谷镰刀菌的AFLP数据用UPGMA法所作的聚类分析，至少可将所有供试菌株明显地区分为两大类群，即以AFLP类群A为代表的欧美菌系和中国的类型1菌系，和以AFLP类群B为代表的亚洲菌系。前者包括除尼泊尔菌株N4(类型1C)外的所有类型1和类型6菌系；后者包括中国的类型5菌系，尼泊尔的类型2一类型5菌系，以及日本（类型5）菌系。禾谷镰刀菌的某些AFLP带型明确显示了类群内的菌系与地理位置间的关系，有的AFLP带型则在一定程度上显示了与致病力间的关系。 用UPGMA法对中国菌株的所作分析表明，中国的禾谷镰刀菌群体同样存在ALFP类群A和AFLP类群B两大类群。但中国的AFLP类群A只有类型l一种类型，AFLP类群B也只有类型5一种类型。中国禾谷镰刀菌AFLP类群可以进一步划分为几个亚类。从系统聚类分组来看，同一来源的菌系存在着较大的变异，不同来源菌系间菌株的变异似乎存在着平行关系。 选用来自不同大陆的禾谷镰刀菌菌株，从侵染力和扩展力两方面考查了病原菌对小麦幼苗的致病力。相关分析表明，病原茵扩展力与侵染力呈极显著正相关，相关系数r＝0．95，RZ吻．9025，说明本试验测定的侵染力和扩展力结果均能反映病原菌的致病力水平。 方差分析显示，各参试菌株对小麦幼苗的致病力存在极显著的差异0功．01人侵染力级别从最高5．2到最低1．4间存在一系列的差异，扩展力从50石mm上5．smm间也存在一系列的差异，说明不同菌株间的致病力存在广泛的变异。根据统计分析比较，结合习惯划分方法将致病力不同的菌株分成了强、中、弱三组，结果每组中都有同一来源或同一类型的菌株。多数病原菌的致病力表现为中或强。 综合来看，各地菌株或各种类型菌株的致病力均存在强弱分化，这与AFLP所揭示的各种类型的菌株或各地菌株间均存在广泛的遗传变异是一致的。但不同类型或生态地理来源的菌株与当地生态气候条件下的赤霉病病害流行有关。温暖型菌系是导致中国长江中下游等温暖麦区由禾谷镰刀菌引起的赤霉病害流行的主要病原菌，而在中国的北方或其他冷凉地区由禾谷镰刀菌导致的病害则主要是冷凉型菌系病原菌造成的。更多还原

【Abstract】 Fusarium graminearum isolates collected from China, Nepal, Europe and USA were selected for this study. It was the first time to analysis the isolates at molecular level with AFLP, SCAR, SSCP and species-specific PCR technology to reveal genetic diversity of the isolates and compare the difference of the isolates from several continents.Species-specific PCR revealed that there were six types of PCR fragments based on their size among the isolates analysed (type 1 to 6) from different continent. There were only two types (types 1 and 5) present in the Chinese isolates. The type 5 was a main F. graminearum population in China, about 79% of the isolates were typeS, especially in the warm area. It was divided into ’warm-type’ isolates. But the type 1 only was a small F. graminearum population, about 21%, and existed in the cool area in China. It was divided into ’cool-type’ isolates. Most isolates from Northwest Europe and North America where it is cool were type 1, whereas a small proportion was type type 6. The results indicate that there were various types of F. graminearum isolates related to geographical environment and ecosystems in F. graminearum.SSCP analyses not only confirm six types identified by species-specific, but also further divided the type 1 into three subtypes (1A, IB and 1C). All the isolates from China and Europe, and most isolates from USA belonged to type 1A, while some American isolates were type IB and Nepalese isolates were type 1C. There were no subtypes within each of other five types detected with SSCP. SCAR analyses of the twenty isolates indicated that the six types contain almost identical sequences of about 380bp and the differences among the six types were mainly due to variation within the regions from nucleotides 79 to 168 and 440 to 526. There were only several single nu-cleotide mutations in the sequences outside of these two regions.AFLP revealed much more molecular diversity among the isolates and there were high polymorphisms even among the isolates from the same geographic locations. The results suggest the presence of a high degree of genetic diversity in the F. graminearum population, implying that sexual recombination ofF. graminearum exist in the fields.AFLP revealed for the first time the correlation between the two F graminearum classes and the geographic origin. Dendrogram analyses of AFLP patterns with UPGMAinprogram showed two main classes in F. gramineantm populations. Class I represented the isolates of Europe, USA and the Chinese type 1, which consisted of types 1 and 6 ; Class II represented the Asian isolates, it was consisted of Chinese type 5, Nepalese types 2 to type 5, and the Japanese (type 5) isolates. Some AFLP markers indicated a clear correlation between the isolates and the origin in the classes, while other AFLP bands suggested possible association to pathogenesis of the isolates.Chinese isolates were also divided into two classes by dendrogram analyses. But there was only typel in Chinese class I, and only type 5 in Chinese class II. Each class further consists of many subclasses.Pathogenicity test was carried out with 76 isolates from different continents by scoring penetration ability and scaling spreading rate based on the necrotic symptoms of the coleoptiles and leaves. The results showed that the isolates varied significantly in their pathogenicity towards wheat. Correlation analysis showed a significantly direct correlation co-efficiency (r = 0.95, R2 = 0.9025), indicating that both penetration ability and spreading rate can be used for evaluation of the pathogenicity of F. graminearum isolates.The high degree of variation of pathogenicity among the isolates is in accordance with the diverse genetic diversity observed in AFLP analysis. Different types or origination of the isolates may be associated with ecological conditions and local climates. The warm-type isolates are the predominant fungal pathogens causing epidemics in Yangtze river valleys where the climates is warm. In cooling regions for wheat growing such as更多还原