The Phylogenetic Taxonomy Of Botryosphaeria And Other Related Genus In China

Some Botryosphaeria species and their anamorphs were the pathogen of many treescanker diseases which included poplar canker, pines shoot blight, apple and pear trees ring-veindisease, stem canker and Eucalyptus canker. Botryosphaeria was ascomycetes with bitunicateasci in the storma or pseudothecium ascomata. The taxonomy of Botryosphaeria was largelydependent upon the Morphological and cultural characteristics of anamorphs for its verysimilar telomorphs characters. However, the taxonomy of Botryosphaeria is controversy andpromblematic for the changing taxa names and taxonomy status, varied anamorphs charactersand inconsistency between anamorph and telomorph. So the phylogenetic taxonomy study ofthose important pathogenic and diversity familiar fungi was necessary.In this study, using 158 isolates of Botryospaheria and related fungi collected from 59host plants in 34 areas of China as materials, the characteristics of those isolates in culture,growth rate and morphology were observed and identified. The phylogenetic relationship ofBotryospaheria were reviewed by rDNA-ITS,β-tubulin and EF1αsequence of allBotryosphaeria and other related fungi which come from GenBank database. Molecularmarkers of RAPD and ISSR were used to clarify the relationship and the genetic differentiationof those Botryospaheria. Phylogenetic taxonomy of our Botryospaheria isolates was studiedbased on 53 rDNA-ITS-LSU sequences and 35β-tubulin squences which we amplified andsequencing. Main results are as follows:1. Prunus serrulata Lindl., Prunus incisa C.P.Thunberg ex A.Murray, Amygdalus persicaL.var.compressa(Loud.)Yu et Lu, Cinnamomum burmannii (C G et Th Nees) B1., Citrusreticulate Blanco, Syzygium jambos (L.)Alston, Dillenia turbinata Finer et Gagnep, Terminaliachebula Retz., Bauhinia blakeana Dunn, Chrysalidocarpus lutescens H. Wendl., llliciumverum Hook. f., Albizia falcataria (L.) Fosberg, Cyclobalanopsis glaucoides Schotky, Alunscremastogyne Burkill, Betula spp., all of that 14 genus, 15 species plant was reported as newhosts of Botryosphaeria and other related fungi in China.2. Botryospaheria and related fungi isolates were classified to 7 major groups based ontheir colony morphology and growth rate under different temperatures on PDA medium. The fruit body morphological characters (conidiomata structure, morphology and size ofconidiogenous cell and conidia, conidia produce type, and develpmental of conidia) of 22isolates are described in detail. According to cultural and morphological characters, the isolatesin this study were identified as the following morphological species: B.dothidea, B.rhodina,B.parva, B.obtusa, B.stevensii, B.eucalyptorum, Fusicoccum mangiferum, Diplodia pinea.3. Phylogenetic analysis based on rDNA-ITS,β-tubulin and EF1αsequences of allBotryospaheria downloaded from GenBank was performed and the results showed that"Botryosphaeria"should be classified into 5 closely related monophylogenetic groups whichhomologous with genus Dothiorella, Fusicoccum, Neofusicoccum, Macrophomina andDiplodia/Lasiodiplodia complex anamorph genus. Moreover sequences data revealed a distinctdiversity between Diplodia and Lasiodiopldia. So, we concluded that the "Botryosphaeria"was a complex of 6 anamorphs genus.4. Clustering analysis of the Botryospaheria species and related fungi showed thatB.dothidea, B.rhodina, B.parva, B.obtusa, B.stevensii and D.pinea were different from eachother in RAPD and ISSR molecular markers. Isolates of B.dothidea clustered into groupⅠandⅢ. GroupⅡincluded B.rhodina, B.parva, B.obtusa, B.stevensii and D.pinea. FourteenB.dothidea isolates of groupⅢ, such as CXY120, CXY134 etc. had distinct PCR amplificationpatterns from the isolates in GroupⅠ. Clustering analysis showed that diversity in B.dothidea ishigher than the diversity among B.dothidea and other species.5. The isolates of Botryospaheria and related fungi in this study were identifed as 3 genus:Diplodia/Lasiodiplodia, Botryosphaeria and Neofusicoccum by the phylogetic analysis ofrDNA-LSU,rDNA-ITS andβ-tubulin gene. LSU analysis indicated that 6 isolates of B.rhodina(CXY616, CXY617, CXY611, CXY580, CXY585, CXY828) clustered as a monophyleticgroup with 99% bootstrap value. However, the phylogetic analysis of rDNA-ITS andβ-tubulinshowed that the B.rhodina identifed by morphology were classified into 2 distinct but closelyrelated phylogetic groups, CXY585, CXY617 etc. and CXY675 located in this two grouprespectively. Because lack of clear morphological distinction, we concluded that moleculardata analysis supported those groups denoted 2 new phylogenetic species. The analysis ofrDNA-LSU,rDNA-ITS abdβ-tubulin indicated that the B.dothidea isolates of RAPD/ISSRgroupⅢwere the same as other isolates in B.dothidea. For the different cultural, growth and host characters, distinct RAPD/ISS amplification patterns, the B.dothidea isolates ofRAPD/ISSR groupⅢwas defined as Rosaceae host plants forma specialis: B.dothidea deNot.f.sp. rosacola.