Studies On Relationship Between Reproduction And Mating Types In Nature Populations Of Beauveria Bassiana

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.