【作者】 赵嘉平；

【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， 2007， 博士

【摘要】 葡萄座腔菌属(Botryosphaeria)多种真菌及相关无性型是引起杨树溃疡病，松树枯梢病，苹果、梨树等果树轮纹、干腐病，桉树溃疡病等多种树木枝干溃疡的致病真菌。Botryosphaeria是在子座或假囊壳型囊实体中产生双囊壁子囊的真菌的总称。由于真菌的有性型形态特征极其相似，Botryosphaeria属真菌的分类主要依据无性型的形态学及培养学特征。但是由于在历史上相关真菌名称、分类地位多次变更，具有众多形态各异的无性型，以及无性型与有性型不对应等原因，Botryosphaeria属真菌分类一直存在很多矛盾与争议。因此，作为一种主要的树木病原真菌，Botryosphaeria的系统分类具有重大的生物学及病理学意义。本研究以在我国17个省(区、市)、34个县(市)，19科，31个属，59个种、品种或无性系的寄主植物上分离到的158株Botryospaheria及其相关真菌为材料，根据培养学特征、生长速率特征、形态学特征对试验菌株进行了分类鉴定；收集GenBank中全部Botryosphaeria及其相关真菌的rDNA-ITS、β-tubulin及EF1α基因序列，对于Botryospaheria及相关真菌的系统发育地位重新进行了分析；应用RAPD、ISSR分子标记研究了Botryospaheria及其相关真菌的相互关系及遗传分化；依据本研究获得的53条rDNA-ITS～LSU序列，35条β-tubulin基因，对我国部分地区的Botryospaheria及其相关真菌的系统发育进行了研究。主要研究结果如下：1．通过对于我国树木病害病原研究的分析，初步确定樱花(Prunus serrulata Lindl．)、大豆樱(Prunus incisa C．P．Thunberg ex A．Murray)、蟠桃(Amygdalus persica L．var．compressa(Loud．)Yüet Lu)、阴香(Cinnamomum burmannii(C G et Th Nees)Bl．)、柑桔(Citrus reticulate Blanco)、水蒲桃(Syzygium jambos(L．)Alston)、大花五桠果(Dillenia turbinata Finet et Gagnep)、诃子(Terminalia chebula Retz．)、红花羊蹄甲(Bauhinia blakeana Dunn)、散尾葵(Chrysalidocarpus lutescens H．Wendl．)、八角(Illicium verum Hook．f．)、南洋楹(Albizia falcataria(L．) Fosberg)、滇青冈(Cyclobalanopsis glaucoides Schotky)、水冬瓜(Aluns cremastogyne Burkill)、桦树(Betula spp．)等14属，15种植物是Botryosphaeria及相关真菌的新的寄主植物类型。2．根据PDA培养基上的菌落特征和不同温度下的生长速率特征，将Botryospaheria及相关真菌分为7个主要类群；对其中的22个菌株的子实体特征(结构、壁的类型、产分生孢子细胞和分生孢子的形态、大小、颜色、分隔、分生孢子的产生方式、分生孢子的发育变化特征等)进行了详细描述；依据菌落特征、生长速率以及子实体特征，将试验菌株确定为B．dothidea、B．rhodina、B．parva、B．obtusa、B．stevensii、B．eucalyptorum、Fusicoccum mangiferum、Diplodia pinea等8个形态学种。3．应用GenBank中rDNA-ITS、β-tubulin及EF1α基因序列，对Botryospaheria相关真菌的系统分类地位进行了分析，研究表明，GenBank中的Botryosphaeria真菌可以分为5个紧密相关的单系类群，这些类群和Fusicoccum、Dothiorella、Neofusicoccum、Macrophomina属以及Diplodia／Lasiodiplodia复合体相对应。序列分析显示Diplodia与Lasiodiplodia之间也有显著的分子差异，因此，传统意义上Botryospaheria真菌可以认为是由这6个属构成的属的复合体。4．RAPD／ISSR聚类分析明确解析出B．dothidea、B．rhodina、B．parva、B．obtusa、B．stevensii、D．pinea等真菌的差异；B．dothidea分化为两个类群Ⅰ、类群Ⅲ，主要以蔷薇科多种植物为寄主的类群Ⅲ菌株(CXY120、CXY134等14个)具有截然不同的PCR扩增模式，分析表明，类群Ⅰ、Ⅲ的种内遗传距离大于类群Ⅰ与类群Ⅱ的种间遗传距离。5．rDNA-LSU、rDNA-ITS及β-tubulin分析均将试验菌株归于Diplodia／Lasiodiplodia、Fusicoccum、Neofusicoccum属；LSU分析表明CXY616、CXY617、CXY611、CXY580、CXY585、CXY828共6个B．rhodina菌株构成一个具有99％自展支持度的单系类群；rDNA-ITS及β-tubulin分析表明形态学确定的B．rhodina真菌分化为紧密相关但截然不同的两个系统发育群，CXY585、CXY617等6个菌株和CXY576分别位于两个发育群中，由于没有观察到CXY585、CXY617等菌株与CXY576之间的明确地形态学差异，因此将其根据分子数据作为两个不同系统发育学种(phylogenetic species)；RAPD／ISSR类群Ⅲ的B．dothidea菌株具有和其他的B．dothidea真菌相同的序列特征，但是由于培养特征、生长特性、寄主类型以及RAPD／ISSR扩增模式与典型的B．dothidea真菌明显不同，确定RAPD／ISSR类群Ⅲ菌株是B．dothidea真菌的蔷薇科植物寄主专化型：B．dothidea de Not．f．sp．rosacola。更多还原

【Abstract】 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.更多还原