Molecular Systematic Studies Of Bipolaris And Its Similar Genera By Multiple Gene Aggregate Analysis

Bipolaris and its similar genera cause important diseases of theGramineae. In order to explicat e the phylogenetic relationship of Bipolarisand itssimilar genera and the taxonomic status of some unidentified species, on the basis oftraditional morphological classification criteria, this study analyzed and assessed thevariability and conservative property of the rDNA ITS, Gp,d ACT, CAL, EF-1α,Brn1gene in this group of fungal species. This study provided the molecular basisfor establishing standard of species identification, and esta blished molecularidentificatio n system in this group of fungal species by si ngle gene an d multiplegenes aggregate analysis.Some strains were collected fr om our laboratory and at the same time morethan1000specime ns were collected from several provinces of China. Then300strains of Bipolaris and its similar genera were isol ated by moisturizing cultivationand tissue isolation for all these spec imens. Adde d another70strains from domesticand foreign research institutions, the amount of Bipolaris and its similar generacollected in this study was nearly400strains. This study rechecked all these strainsvia morphological characteristics after the standard culture condition, and finally,22species of Bipolaris (including two unidentified species),5species of Drechsleraand4species of Exserohilum were identified.67strains with morphological stability were selected as the representativespecies from the genera and species which had been collected above. With theaddition of28strains of Curvularia that had all gene sequences, these95strainswere used as tested strains. The5.8S rDNA-ITS, Gpd, EF-1α, CAL, ACT, Brn1geneof all these tested strains were amplified and sequenced. After comprehensivelyanalyzed amplification success rate, variability and conservative of six candidategenes of all tested strains, we found that the amplification rates of5.8S rDNA-ITS,Gpd, and ACT genes were100%, as well as the conservative and variability of thesethree genes were relatively stable. On the other hand, the genetic sequence information of5.8S rDNA-ITS of these fungi is rich in GenBank, so5.8S rDNA-ITScan be used as the prior gene, next to Gpdand ACTgenes in th e molecularidentifying system of these fungi. Though CALand EF-1αgenes can explicitlydistinguish most Bipolaris species and its similar genera, however, theiramplification success rates are less than the5.8S rDNA-ITS, Gpdand ACTgenes, sothey can be used as complement genes in the molecular identifying systemof thesefungi. Amplification success rate of Brn1gene is less than50%, so Brn1genes werenot appropriate for the molecular identifying system of Bipolaris and its similargenera and species.Through analyzed and assessed the variability and conservative property of fivecandidate genes of Bipolaris and its similar genera and species, the results showedthat:(1) Determined taxonomic relations of Bipolaris and its similar genera and species.Drechslera has the farthest genetic relationship with other genera inHelminthosporioid fungi and a second farthest relationship with Exserohilum.Bipolaris was divided into two parts: one was called big spore Bipolaris and theother was small spore Bipolaris. The genetic relationship between big sporeBipolaris and small spore Bipolaris was more distant than that between bigspore Bipolaris and Curvularia, however, homology between small sporeBipolaris and Curvularia had reached to the interspecific homology level.Therefore, we suggest that put small spore Bipolaris into Curvularia.(2) By analyzing five candidate genes of Bipolaris, we found that there were alwayssome individual species could not be distinguished on each level of thesecandidate genes. Even if combined with5.8S rDNA-ITS, Gpd, and ACT genesanalysis, we still found that homology between Bipolaris maydis and Bipolarissacchari, Bipolaris spicifera and Bipolaris australiensis were very high. So weare think they are same species, suggest that B.sacchari as the synonym of B.maydisand B.australiensis as the synonym of B.spicifera.Based on the morphological and molecular identification, two unidentified strains ofBipolaris were identified as two new species. They were named as Bipolarisyunnanensis Meng Zhang&S.N. Xie, sp. nov.(ZM09362-2) and Bipolaris stipicolaMeng Zhang&S.N. Xie，sp. nov.(zm070031).5new combination species ofCurvularia are suggest. They are Curvularia spicifera（Subram）Meng Zhang&S. N.Xie comb. nov., Curvularia hawaiiensis（Uchida&Aragaki）Meng Zhang&S. N.Xie comb. nov., Curvularia prieskaensis（W. Q. Chen&W. J. Swart）Meng Zhang&S. N. Xie comb. nov., Curvularia nodulosa（Shoemaker）Meng Zhang&S. N.Xie comb. nov. and Curvularia cactivora（Alcorn）Meng Zhang&S. N. Xie comb.nov.. Anew leaf spot caused by Bipolaris peregianensison b ermu dagrass lawn wasdiscovered during this study. This study provided the foundation for the study ofdiseases bermudagrass and control methods of these diseases.This study established the mol ecular identifying system of Bipolaris and itssimilar gen era, at the same ti me, provided a method for accurate and rapi didentification of Bipolaris and its similar genera and spec ies, and provided molecularevidences for the systematic classification of such kind of fungi. In the meanti me, t hisstudy also laid the foundation for the study of this fungi group’s life barcode.