【作者】 程燕林；

【导师】 张星耀；

【作者基本信息】 中国林业科学研究院 ， 森林保护学， 2012， 博士

【摘要】 葡萄座腔菌科（Botryosphaeriaceae）（子囊菌门Ascomycota，座囊菌纲Dothideomycetes）真菌分布广泛，种类繁多，寄主类型多样，在我国引起多种重要的经济树木枝条或主干发生溃疡病，包括杨树和多种果树等。长期以来病原真菌的命名多根据病害和寄主来命名，造成了同菌异名、同名异菌的问题，引起了该科真菌的命名和分类混乱的问题。该属模式种Botryosphaeria dothidea是该科最常见的种之一，是引起树木溃疡病最重要的病原，在我国多个气候区均有发生，阐明该种不同地理群体间的遗传多样性和遗传分化，对病害的检测和防治具有重要意义。为了澄清我国树木溃疡病病原真菌的种类和调查葡萄座腔菌科真菌在我国分布的多样性，针对引起杨树和蔷薇科果树病害的葡萄座腔菌科病原进行研究，采用形态学和系统发育学的方法，从我国南北方的5个典型气候区分离到58株寄主为杨树和37株寄主为蔷薇科果树的试验菌株，结合形态学和系统发育分析方法，鉴定葡萄座腔菌科5个属8（或9）种真菌。Botryosphaeria dothidea种群具有中度偏高的遗传多样性，Nei’s基因多样性指数（H）为0.6777。利用形态学结合ITS、β-tubulin和EF1-α序列比较分析表明，发生于杨树上的种类包括Botryosphaeria dothidea、Neofusicoccum parvum、Diplodia seriata、D. mutila、Dothiorellaviticola和一个未定名新种Fusicoccum sp.1，其中，共有53株为B. dothidea，其它种各1株；发生于蔷薇科果树上的有B. dothidea、Lasiodiplodia pseudotheobromae、Neof.parvum/Neof. ribis复合种和另一个未定名新种Fusicoccum sp.2，其中，B. dothidea15株，L. pseudotheobromae和Neof. parvum/Neof. ribis各有3株， Fusicoccum sp.2有6株。上述种类中，D. seriata在我国是第一次报道发生在杨树上；Do. viticola是第一次报道于滇青冈。根据β-tubulin序列系统发育关系研究，对B. rhodina与其无性型L. theobromae的对应关系提出质疑，认为B. rhodina的无性型应为L. pseudotheobromae。研究表明，除了L. pseudotheobromae只发生于南亚热带外，其余绝大多数试验菌株在我国的分布与气候区没有直接的联系。培养特征的比较和LSU序列、ITS/EF1-α序列联合建立的系统发育关系进一步支持了两个未定名种Fusicoccum sp.1和Fusicoccum sp.2的系统地位，属于Botryosphaeriacomplex。Fusicoccum sp.1与Fusicoccum fabicercianum表现出最近的亲缘关系，Fusicoccumsp.1和Fusicoccum sp.2与Botryosphaeria complex其它种存在着显著的培养特征上的区别：它们的菌落均呈现环状，且培养至第10天气生菌丝仍未变灰。Botryosphaeria dothidea为两类寄主上的优势种类，其分布区域横跨南亚热带到中温带，显示出对不同地理和寄主的适应性。为了分析B. dothidea种群的遗传分化和遗传多样性，从文献记载的标记中筛选出来10个简单序列重要（SSR）标记用于分析来自5个地区种群77个试验菌株的遗传多样性和种群遗传结构，分别来自5个气候区：中温带亚湿润型气候大区、暖温带亚湿润型气候大区、北亚热带湿润型气候大区、中亚热带湿润型气候大区和南亚热带湿润型气候大区。结果表明各种群的多样性指数均为中度偏高（H=0.6777）；群体间的遗传分化程度较低（平均Fst=0.0758），且遗传差异大多来自群体内部，而群体间的遗传差异较小。除中温带亚湿润型气候大区种群外，其余4个种群都有自己特定的等位基因，其特有等位基因数依次为21、2、2、3个。77个试验菌株构成的不同地理群体间的分化程度低，基因流动较大，可能与菌株来源于人为活动频繁的地区有关，并且与寄主的反复迁移有直接关系，尤其是杨树。杨树和蔷薇科寄主的菌株没有表现也寄主分化特征。但同时也发现了不同种群特定的等位基因，包括北亚热带湿润型气候大区种群的407bp和425bp，中亚热带湿润型气候大区种群的424bp和429bp，南亚热带湿润型气候大区种群的234bp、408bp和416bp及暖温带亚湿润型气候大区种群的特定等位基因313bp、333bp等，这对于一些潜伏于寄主组织内的特定B. dothidea地理种群的监测具有重要作用。更多还原

【Abstract】 The species-rich family, Botryosphaeriaceae (Ascomycota, Dothideomycetes), has acosmopolitan distribution and causes canker and die-back on branches and stems of variouseconomically important trees including poplar and fruit trees in China. The pathogens arecommonly nominated according to diseases and host plants for long, which brings the sameorganism with different names or different organisms with the same name, and confusion ofnomination and taxonomy of Botryosphaeriaceae. These problems hindered the effectivecontrol and management of the disease. The type species, Botryosphaeria dothidea, is one ofthe most common speices in this family. This species has been found in many climate regions.It is significant to find out genetic diversity and differerciation of B. dothidea populations fordetecting and managing disease.This study investigate the pathogens of canker and die-back and the diversity ofBotryosphaeriaceae in China, especially the botryosphaeriaceous fungi on poplar androsaceous fruit trees. The morphological and phylogenetic analyses showed that the58isolateson poplar and37isolates on rosaceous fruit trees from five typical climate regions in Chinawere considered as8or9species of Botryosphaeriaceae afflicting to five genera.Botryosphaeria dothidea populations had moderate genetic diversity (Nei’s genetic diversityindex H=0.6777).B. dothidea, Neofusicoccum parvum, Diplodia seriata, D. mutila, Dothiorella viticola andan undescribed new species, Fusicoccum sp.1on poplar were identified based on morphologycombined comparisons of DNA sequences for the ITS, β-tubulin and EF1-α regions. Fifty-threeisolates were regarded as B. dothidea. B. dothidea, Lasiodiplodia pseudotheobromae, Neof.parvum/Neof. ribis complex and another undescribed new species, Fusicoccum sp.2on fruittrees were identified. There were15isolates were reported as B. dothidea,3isolates as L.pseudotheobromae, Neof. parvum/Neof. ribis complex each, and the other6isolates asFusicoccum sp.2. D. seriata was firstly reported on poplar in China, and Dothiorella viticola was observed on from Cyclobalanopsis glaucoides in this study as well as on poplar in aprevious study. There was reason to suspect the corresponding relation between B. rhodina andL. theobromae acorrding to the phylogenetic results based on β-tubulin sequence, and considerthat the anamorphic B. rhodina is L. pseudotheobromae. Distribution of most isolates had nodirect correlation with climate regions, except that L. pseudotheobromae was only found insouth subtropical in China.Comparison of cultural characteristics and phylogenetic relationships based on LSUregion and ITS/EF1-α combined sequences supported the phylogenetic position of Fusicoccumsp.1and Fusicoccum sp.2belonging to Botryosphaeria complex. Fusicoccum sp.1was theclosest to Fusicoccum fabicercianum. Fusicoccum sp.1and Fusicoccum sp.2appearedobviously different from the other species in Botryosphaeria complex based on culturalcharacteristics.Botryosphaeria dothidea, the dominant species on both poplar and fruit trees, was locatedfrom mid-temperate to south semitropical zone, which indicated the differentiation potencyadapting to different host and geography. To find out genetic differences and genetic diversityof B. dothidea population, ten polymorphic SSR (Simple Sequence Repeat) loci were selectedfor investigating genetic diversity and structure of77isolates from5climate regions, namelymid-temperate sub-humid climate region (12B), warm-temperate sub-humid climate region(13B), north-subtropical humid climate region (21A), mid-subtropical sub-humid climateregion (22A) and south-subtropical humid climate region (23A). In this study on the77isolates,5populations were found to have a moderate gene diversity and a low genetic variation(Fst=0.0758). Most of genetic variation was from inner population, while those betweenpopulations were low. Except12B, the other4populations had unique alleles, and the numberwas21,2,2and3. The reason for low genetic differentiation and high gene flow in5populations were probably that the locations of77isolates were the places with frequenthuman activities, and were directly assisted by movement with their host plant, especiallypoplar. Meanwhile, the unique alleles were found in different populations, including407bp and425bp of21A,424bp and429bp of22A,234bp,408bp and416bp of23A, and313bp,333bp, etc. of13B. These unique alleles were very significant for detecting B. dothidea when latent intissues.更多还原