【作者】 蔡悦；

【导师】 黄勃； 李增智；

【作者基本信息】 安徽农业大学 ， 森林保护学， 2012， 博士

【摘要】 球孢白僵菌是最广泛和最重要的虫生真菌，在持续控制害虫和维护生态平衡方面具不可替代的作用，故倍受国内外生物防治界的关注。由于球孢白僵菌以无性繁殖为主导的生殖方式和我们关于其有性型的发现，引发了其无性型流行的原因和有性型在自然界发生的分子机理的问题。本研究通过PCR-DGGE技术结合Bloc和TEF位点序列分析，对国内各有性型、无性型发生地的球孢白僵菌系统发育种进行鉴定，通过交配型基因（Mat）的扩增和SSR分子标记，对这些种群的交配型类型和基因流做深入分析，拟揭示球孢白僵菌有性型发生和无性型流行与自然种群交配型之间的关系，进一步分析不同的球孢白僵菌系统发育种存在有性型的可能性，最终为阐明该菌有性型发生及无性型流行的分子机理提供理论依据。主要结果如下：1、基于Bloc和TEF位点的序列，分别对国内6个地区共582株球孢白僵菌进行了系统发育分析。结果表明，随着地理位置和环境条件的不同，球孢白僵菌系统发育种在各地区的分布有很大差异。各地区系统发育种的数量由多到少依次顺序是鼎湖山（4）＞漫水河和牯牛降（3）＞宽阔水和琅琊山（2）＞净月潭（1）。综合来看，这些菌株可划分为5个不同的系统发育种，分别是Europe/N. Africa1（271株），AFNEO₁（27株），Asia3（60株），Asia4（212株）和N. America2（12株）。其中Asia4在6个地区均有分布，菌株数量占总数的36.4%，仅次于Europe/N. Africa1，表明该系统种在国内的分布区域很广。46.6%的菌株属于有性发生系统Europe/N.Africa1，在牯牛降、漫水河、琅琊山、宽阔水地区发现，这4个地区该系统种的菌株数量分别为120、61、60和30株。5%的菌株属于AFNEO₁，只在牯牛降和鼎湖山地区发现，分别为4和23株。其寄主范围相对较窄，多数侵染鞘翅目昆虫，暗示着菌株和寄主间可能存在潜在的关联。10%的菌株属于Asia3，只在漫水河和鼎湖山地区发现，分别为23和37株。该系统种全部由中国不同地区的菌株构成，为中国特有系统种。仅有2%的菌株属于于N.America2，仅在鼎湖山地区发现，而且均分离自土壤。2、对6个地区球孢白僵菌菌株交配型数量和比例进行了研究。582株球孢白僵菌菌株中存在两种交配型，MAT1或MAT2，不存在含有两种交配型基因的菌株，这些菌株的交配模式是异宗配合。从系统发育种的水平上看，大部分地区球孢白僵菌系统种内的交配型都不缺失（除净月潭）。有性型发生系统种Europe/N.Africa1的交配型比例都较均匀，分别为51:69（牯牛降）、37:24（漫水河）、39:21（琅琊山）、15:15（宽阔水），在净月潭和鼎湖山没有发现此系统种。不仅Europe/N. Africa1的交配型比例较均衡（142:129），但大部分无性系统种内的交配型比例也并不悬殊。AFNEO₁、Asia3和Asia4内的比值分别为11:16，33:27和125:87。鼎湖山地区的N. America2交配型比例不均衡（10:2）。就某一地区而言，有些系统种的交配型比例是不均衡的，如：Asia4在净月潭和鼎湖山的比例分别为39:1和2:7。因此在球孢白僵菌有性型产生的过程中交配型比例均匀只是必要条件，而不是充分条件。3、对6个地区球孢白僵菌群体的遗传多样性进行了初步调查。结果显示各地区球孢白僵菌种群Nei’s基因多样性指数（h）和Shannon信息指数（Is）由大到小依次为琅琊山（h=0.290，Is=0.449）＞净月潭（h=0.260，Is=0.402）＞漫水河（h=0.226，Is=0.361）＞鼎湖山（h=0.213，Is=0.349）＞宽阔水（h=0.175，Is=0.294）＞牯牛降（h=0.165，Is=0.271）。从有性系统种和其他系统种的遗传多样性比较来看，各地区Europe/N. Africa1系统种的遗传多样性水平往往不是最高的。可能的原因是在采集样品的过程中，取样过于集中于球孢虫草发生的核心区域，生态环境差异小，因此造成了种群的遗传多样性较低。4、研究了5个地区球孢白僵菌系统发育种间的基因流和遗传分化。从数据来看，存在有性型系统种的4个地区各系统发育种间的基因交流明显。其中宽阔水地区基因流最大（Nm=6.8987），而琅琊山最小（Nm=1.2126）。琅琊山地区（Gst=0.1709）各系统发育种间表现为高度分化，牯牛降（Gst=0.1447）和漫水河地区（Gst=0.0889）表现为中度分化，宽阔水地区（Gst=0.0350）各群体间则不存在分化。而鼎湖山地区各系统发育种的基因分化系数值（Gst=0.3157）超过了Wright重度分化阈值0.25，其中N. America产生的遗传分化很明显，可能是一个不同的生物种。5、综合分析发现，牯牛降和漫水河地区确实存在球孢虫草发生的必要条件，而宽阔水和琅琊山地区的球孢虫草资源有待进一步调查。依据Europe/N.Africa1系统种的交配型比例和遗传多样性指标，各地有性型产生的可能性由高到低次序为琅琊山＞牯牛降＞宽阔水＞漫水河。净月潭的系统种Asia4内MAT1/2=39:1，互补交配型的缺失会限制有性重组发生。鼎湖山发现的4个系统种均为无性系统种，因此无有性生殖。以上研究明确了存在有性系统发育种和交配型比例均匀是发生有性型的必要条件。6、基于核基因间区Bloc位点对中国不同地区的189株白僵菌进行了物种多样性分析，发现国内存在6个白僵菌种。分别为B. bassiana, B. brongniartii, B.australis, B. asiatica, B. pseudobassiana和B. caledonica。其中B. australis为国内新纪录种。这是首次对中国白僵菌属做出系统的分类研究。证明通过Bloc位点的引入，可以对白僵菌属内菌株进行准确鉴定。更多还原

【Abstract】 Beauveria bassiana is one of the most important and common species inentomopathogenic fungi. It plays important roles in regulating densities of pestpopulation and maintaining ecological balances in nature, and received considerableattention as a microbial control agent from many biocontrol researchers.Predominantly clonal reproduction for B. bassiana and the discovery of sexual speciesCordyceps bassiana lead us to explore the molecular mechanism of teleomorphproduction in nature as well as the cause of anamorphic prevailing. Phylogeneticspecies of B. bassiana from all over the country and places where teleomorphic andanamorphic strains occurred will be identified by PCR-DGGE combined with Blocand TEF sequence data. Using molecular technique of SSR and amplification formating gene, the mating types and gene flow in B. bassiana populations are studied toreveal the relationship between mating types in natural population and teleomorphoccurring and anamorphic prevailing, and to speculate possibility of telemorphoccurring in B. bassiana phylogenetic species. The scientific basis for molecularmechanism for teleomorph production in nature and the cause of anamorphicprevailing will be provided finally. The main results are summarized as follows:1. Phylogenetic analysis of582isolates of B. bassiana from6different areas inChina was conducted based on Bloc and TEF locus. The results showed that therewere significant differences in the distribution of B. bassiana phylogenetic species indifferent locality. The numbers of phylogenetic species from high to low in the areawere: Dinghu Mountain （4）＞Manshuihe and Guniujiang （3）＞Kuankuoshui andLangya Mountain （2）＞Jingyuetan （1）. In general, these isolates could be dividedinto5different phylogenetic species: Europe/N. Africa1（271）, AFNEO₁（27）, Asia3（60）, Asia4（212） and N. America2（12）. Asia4including212isolates （36.4%） wasjust fewer than that in Europe/N. Africa1, which is a phylogentic species observed in6areas with national distribution in China.271isolates（46.6%） belong to Europe/N.Africa1, teleomorphic phylogenetic species, were found in4sites inculdingGuniujiang （120）, Manshuihe （61）, Langya Mountain （60） and Kuankuoshui （30）.27isolates（5%） isolated mostly from Coleoptera insect belong to AFNEO₁which wereobserved in Guniujiang （4） and Dinghu Mountain （23）, which suggests that thephylogenetic species have potentially relationships with the Coleoptera insect. Asia3consists of60isolates （10%） and was found in Manshuihe （23） and Dinghu Mountain （37）. All the isolates were collected from China, indicating the phylogeneitc species isendemic to China. All12strains （2%） isolated from soil only in Dinghu Mountainbelong to N. America2.2. The number and proportion of mating types of six Beauveria bassianapopulations were studied. The PCR-based mating-type assay demonstrated thatindividual isolates of B. bassiana possessed either MAT1or MAT2, and weresupposed to be heterothallic. From the perspective of phylogenetic species, bothmating types were detected in most of phylogenetic species. The ratio of mating typesin Europe/N. Africa1（teleomorphic phylogenetic species） was not skewed inGuniuijang, Manshuihe, Langya Mountain and Kuankuoshui with ratio of MAT1toMAT2of51:69,37:24,39:21and15:15, respectively. The ratios were relativelybalance not only in Europe/N. Africa1（142:129）, but also in most asexualphylogenetic species. Ratios of MAT1to MAT2in AFNEO₁、Asia3and Asia4were11:16,33:27and125:87, respectively. Mating types ratio of MAT1to MAT2ofN. America2（10:2） in Dinghu Mountain were skewed. In a certain area, mating typesratio in some phylogenetic species were also skewed. For example, Ratios of MAT1to MAT2of Asia4in Jinigyuetan and Dinghu Mountain were39:1and2:7,respectively. Therefore, the balance ratio of MAT1to MAT2in B. bassianapopulation wasn’t sufficient condition for teleomorph occurring, but only therequirement.3. The genetic diversity of six populations from six collections cites wereanalyzed. The result indicated that Nei’s genetic diversity index （h） and Shannon’sinformation index （Is） followed by Langya Mountain （h=0.290， Is=0.449）＞Jingyuetan （h=0.260，Is=0.402）＞Manshuihe （h=0.226，Is=0.361）＞DinghuMountain （h=0.213，Is=0.349）＞Kuankuoshui （h=0.175，Is=0.294）＞Guniujiang（h=0.165， Is=0.271）. Compared teleomorphic phylogenetic species to asexalphylogenetic species, Europe/N. Africa1was not the highest at the genetic diversitylevel. The main reason is samples of teleomorphic phylogenetic species collectedfrom the small core area of teleomorphic occurrence, which led to the low geneticdiversity for the population.4. Genetic differentiation （Gst） and gene flow （Nm） of five collection sites wereinvestigated. The analysis showed that gene flow was obvious among differentphylogenetic species in4area where existed sexual phylogenetic species, and it washighest in Kuankuoshui （Nm=6.8987） while lowest in Langya Mountain （Nm=1.2126）. Genetic differentiation （Gst） was0.1709in Langya Mountain indicating there washeight differentiation between phylogenetic species,0.1447in Guniujiang and0.0889in Manshuihe indicating moderate differentiation and0.0350in Kuankuoshuiindicating no differentiation among phylogenetic species. In Dinghu Mountain, thegenetic differentiation （0.3157） exceeded the threshold value of severe differentiation（0.25）, indicating N. America may be a different species in B. bassiana because ofreproduction isolation.5. Comprehensive analysis showed that conditions necessary for the occurring ofC. bassiana did exist in Guniujiang and Manshuihe area, and further investigation ofC. bassiana collection is necessary in Kuankuoshui and Langya Mountain. Accordingto ratios of mating types and genetic diversity of Europe/N. Africa1, possibility ofsexual reproduction were Langya Mountain＞Guniuijang＞Kuankuoshui＞Manshuihe. The ratio of mating types in Jingyuetan was39:1of MAT1to MAT2,suggesting that reproduction is constrained to asexual modes due to lack ofcomplementary mating types in this locality. Four phylogenetic species found inDinghu Mountain were all asexual phylogenetic species, indicating that sexualreproduction hardly occurred. This study indicated that the presence of teleomorphicphylogenetic species and the balance ratio of MAT1to MAT2are necessary forteleomorph occurrence.6. A phylogenetic diversity survey of189exemplar isolates of Beauveria wasconducted based on Bloc nuclear intergenic region. The phylogenetic analysisassigned the Chinese Beauveria isolates to six species lineages corresponding to B.bassiana, B. brongniartii, B. australis, B. asiatica, B. pseudobassiana and B.caledonica. B. australis was reported for the first time in China. This studyrepresented the first phylogenetic analysis of Beauveria species diversity in China anddemonstrates an effective screening strategy to identify Beauveria genetic resourcesbased on Bloc loci.更多还原