【作者】 潘好芹；

【导师】 张天宇；

【作者基本信息】 山东农业大学 ， 植物病理学， 2009， 博士

【摘要】 土壤真菌是指以土壤作为活动场所完成全部或部分生活史的真菌,在土壤生态系统中发挥巨大作用,具有丰富的多样性。长江、黄河源头地区地处青藏高原腹地,自然环境极其严酷,主要表现在两个方面:一是高海拔,低气温,干旱;二是低植被,生态效能低。其特殊的地理条件蕴育了极其脆弱而低转化状态的高寒干旱的生态系统,特殊的生态环境蕴育了该地区特殊的土壤真菌物种多样性。本研究是我国土壤暗色丝孢真菌多样性研究的一部分,着重考察长江、黄河源头地区土壤中的暗色丝孢真菌及其生态分布特点,了解该地区土壤真菌种类,进一步丰富我国生物多样性方面的知识,为相关研究和真菌资源开发利用奠定基础。长期以来无性态真菌(Anamorphic Fungi)的分类主要以形态特征为根据,分类的依据主要包括:载孢体的类型(如载孢体菌丝状、孢梗束状、瘤座状、分生孢子器状等)、分生孢子的形态(包括单胞孢子、双胞孢子、多隔孢、砖格孢、线形孢、卷旋孢、星状孢)及分生孢子的形成方式与产孢梗(细胞)的延伸方式(如体生式、芽生式等)。本论文以自己分离到的34属暗色丝孢真菌为实材,在传统形态分类的基础上,进行ITS-rDNA序列分析,以丰富对无性态真菌分类信息的认识,探索此类真菌属、种级形态分类标准的分子依据。同时,对本实验室积累菌株较多的矛束霉属、毛束霉属和粘束孢属等三属真菌的代表性种,进行若干基因片段的分子生物学测定,为属的界定及属下种的划分,在形态分类的基础上寻求新的分子生物学分类标准。笔者等先后于2006-2007两年,从长江、黄河源头地区采集混合土壤样品200份,涉及的生境类型包括:森林、草原、农田、戈壁、沙漠、荒地、湿地、高山草甸等八类。采用稀释平板法、土壤平板法等分离培养,共获得700多个菌株。从中鉴定出暗色丝孢真菌35属,92种。其中,新种17个,新组合1个,中国新纪录种8个。所研究的属、种均作详细形态描述和分布地点、生境引证。对有关属、种级分类地位的变迁、范畴、分类标准等分类问题,主要是相似属、种之间的区别进行了简要讨论。一、暗色丝孢真菌物种多样性新种:球孢矛束霉Doratomyces globosae H.Q. Pan & T.Y. Zhang、到达漆斑霉土壤变种Myrothecium advena var. terrum H.Q. Pan & T.Y. Zhang、青海卷旋孢Cirrenalia qinghaiensis H.Q. Pan & T.Y. Zhang、大孢单格孢Monodictys macrosporum H.Q. Pan & T.Y. Zhang、黄河单格孢Monodictys huangheensis H.Q. Pan & T.Y. Zhang、多变粉枝孢Oidiodendron tshawytschae H.Q. Pan & T.Y. Zhang、矮小侧多格孢Pleurophragmium nanus H.Q. Pan & T.Y. Zhang、多变小鼻枝霉Rhinocladiella variabile H.Q. Pan & T.Y. Zhang、中华齿梗孢Scolecobasidium chinensis H.Q. Pan & T.Y. Zhang、光滑齿梗孢Scolecobasidium laevis H.Q. Pan & T.Y. Zhang、长孢齿梗孢Scolecobasidium longisporum H.Q. Pan & T.Y. Zhang、土生帚霉Scopulariopsis humicola H.Q. Pan & T.Y. Zhang、青霉状帚霉Scopulariopsis penicilli H.Q. Pan & T.Y. Zhang、刺突帚霉Scopulariopsis verruculosum H.Q. Pan & T.Y. Zhang、长孢葡萄穗霉Stachybotrys longisporum H.Q. Pan & T.Y. Zhang、小孢短梗蠕孢Trichocladium microsporium H.Q. Pan & T.Y. Zhang、戈壁维罗纳霉Veronaea gobica H.Q. Pan & T.Y. Zhang、圆孢维罗纳霉Veronaea latispora H.Q. Pan & T.Y. Zhang。未定名种(七个):枝顶孢属Acremonium sp.、头梗霉属Cephaliophora sp.、枝孢属Cladosporium sp.、帚霉属Scopulariopsis sp.1,Scopulariopsis sp.2、轮枝孢属Verticillium sp.、沃德霉属Wardomyces sp.。中国新纪录种(八个):花菱草顶孢Acroconidiella eschscholtziae (Harkn.) M.B. Ellis、棉絮状葡萄孢Botrytis byssoidea Walker、膨梗粘鞭霉Gliomastix inflate (C.H. Dickinson) W. Gams,、树状齿梗孢Scolecobasidium dendroides Pirozynski & Hodges、冬眠帚霉Scopulariopsis hibernica A. Mangan、喜粪维罗纳霉Veronaea coprophila (Subram. & Lodha) M.B. Ellis、异形沃德霉Wardomyces anomalus Brooks & Hansf.、土生沃德霉Wardomyces humicola Hennebert & G.L. Barron。其余为国内已知种。所有研究过的分离物标本(干制培养物)与活菌种均保存在山东农业大学植物病理学标本室(HSAUP)。在分类研究的基础上,以物种丰富度、种群优势度、Shannon-Wiener多样性指数、群落均匀度、生态位宽度及生态位重叠指数等为衡量指标,对黄河源头地区土壤中的暗色丝孢真菌物种多样性及其与生态类型间的相互关系进行了初步分析。研究发现,暗色丝孢真菌在不同生态类型中的数量和分布特征差异明显。不同生态类型中的优势物种(属级)不同,物种多样性与植被密切相关。植被丰富的森林、草原生境中,暗色丝孢真菌物种多样性明显高于植被稀疏的戈壁、沙漠。相对于我国土壤暗色丝孢真菌多样性考察研究中对低海拔地区的研究结果,长江、黄河源头地区的土壤暗色丝孢真菌物种数量显著较少,蠕形分生孢子真菌等大孢子无性态真菌种类较少,而小孢子物种相对较多。二、分离方法的改进暗色丝孢真菌人工培养常用的培养基有PDA,PCA,CMA,TWA+W等;培养条件一般为25℃,12h光照/12h黑暗,培养1-3周(培养时间根据不同的属、种产孢及孢子成熟所需时间的长短而定)。笔者根据土壤样品采集地点的高海拔、低气温特点,适当降低了培养温度,把培养温度调整到18℃,并根据每个样品土壤类型,在培养基中适当加入了原产地土壤浸出液,从而大大提高了分离效果。此外,机械损伤、适当的低温刺激等处理,有利于促进暗色丝孢真菌的产孢。三、基于5.8S rDNA-ITS序列的属级分类检索表分析通过5.8S rDNA-ITS序列分析,用Neighboring-joining方法对论文中涉及的32属构建系统发育树。除若干对相似属,如:矛束霉属与毛束霉属、粘鞭霉属与枝顶孢属、侧多格孢属与维罗纳霉属、顶孢属与弯孢属序列相似性为98%-100%外,其余各属均能在较明确遗传距离处完全分开。说明5.8S rDNA-ITS序列分析对土壤暗色丝孢真菌属级分类很有价值。特别是5.8S rDNA-ITS序列能够较好的反映出无性态真菌形态分类中,芽生式产孢这一属级分类标准,而对体生式产孢的特性,则未能明确地反映。全壁芽生式和内壁芽生式在系统发育树中明确划分,产孢方式为全壁芽生式的沃德霉属、矛束霉属、毛束霉属等聚为一支。同时系统发育树也能够很好的区分内壁芽生瓶梗式和内壁芽生孔生式的真菌。但也存在少数例外,产孢方式为全壁芽生和内壁芽生的个别属在聚类系统树中序列相似性较高,需要进一步的实验来求得合理的解释。四、对矛束霉属Doratomyces、毛束霉属Trichurus、粘束孢属Graphium代表性种,进行5.8S rDNA-ITS序列分析和mt-SSU基因序列分析,用Neighboring-joining方法构建聚类系统树的结果表明,粘鞭霉属Graphium能与其他两供试属明确区分,而矛束霉属Doratomyces和毛束霉属Trichurus供试菌株之间的5.8S rDNA-ITS序列相似性及mt-SSU基因序列相似性均达到97%-100%,两个片段序列在矛束霉属Doratomyces和毛束霉属Trichurus中具有高度保守性,不仅不能在属级水平上分开,在形态上差异显著的种间也不能进行区分。这一结果支持将矛束霉属和毛束霉属合并的观点。粘鞭霉属各供试菌株之间,通过5.8S rDNA-ITS序列比对构建系统发育树,可以划分为3个进化支,明确划分出拜迪粘束孢Graphium dubautiae,其他种间的界线不明显。在据mt-SSU基因序列构建的聚类系统树上,Graphium属内8个供试菌株被划分为4个进化支,进化支之间序列相似性仅为63%-94%,而同分支内各菌株之间序列相似性均为100%。分析结果与5.8S rDNA-ITS序列分析结果稍有差异,这可能与5.8S rDNA-ITS序列同mt-SSU基因序列进化速率不一致相关。由于5.8S rDNA-ITS序列和mt-SSU基因序列在三属内的高度保守性,由其构建的系统发育树未能将供试的属、种进行有效区分,预示仅通过这两个基因片段对矛束霉属、毛束霉属和粘束孢属及以下分类单位进行区分是不充分的,需要借助多基因分析手段才能真正明确供试属、种正确的分类地位。更多还原

【Abstract】 Soil fungi are those which have some parts or whole life cycle either in or directly associated with the soil environment. They play an important role in the soil ecosystem. The species diversity of fungi are the most abundant in soil, of which dematiaceous Hyphomycetes account for more than one half. The Yangtse River and Yellow River source area lie in the hinterland of the Qinghai-Tibetan Plateau. The natural environment is extremely harsh and the mailly show in two aspects: one is high altitude, low temperature and drought, the other is low vegetation and eco-efficiency. Its special geographical conditions breeds an extremely an fragile state and low conversion cold-arid ecosystems. Special ecological environment in these area gives a special species diversity of soil fungi. This study is a part of a whole set of diversity survey of dematiaceous Hyphomycetes in soil of China. It was aimed at investigating the species diversity and distribution of soil dematiaceous hyphomycetes in Yangtse River and Yellow River source area to enrich the biological diversity of China and lay a useful foundation for the development and utilization of fungi resources.The classification of mitosporic fungi have been maily based on morphology and lack of molecular information supporting. The morphological classification of mitosporic fungi are according to the cinidiomata type (hyphal, synnematal, sporodachial, pycnidial, etc.), conidial groups (amerosporae, didymosporae, phragmosporae, dictyosporae, scolicosporae, helicosporae and staurosporae) and formation pattern of conidia. Based on the 32 genera in the paper, phylogenetic relationships were studied to provide molecular support to taxonomic criteria at genus level of mitosporic fungi. Molecular systematics on represented species of Doratomyces, Trichurus and Graphium were also carried out.A total 206 soil samples were collected from the Yangtse River and Yellow River source area, including grassland, desert, gobi, alpine meadow, forest, wetland, farmland,wasteland eight ecosystem types from 2006 to 2007. From these samples, 700 fungi isolates of dematiaceous Hyphomycetes were isolated by using the soil plate and dilution plates methods. From them, 92 species in 35 genera were identified. Among them, 9 species in 3 genera are Stilbellales, 8 species in 2 genera belong to Tuberculariales and 74 species in 30 genera are Hyphomycetales, of which 17 are new species, 8 species are new records to China and 1 new combination. All genera and species are fully described and illustrated morphologically. Distribution sites and habitat are also cited. Dried and living cultures of all the fungi studied have been deposited in the Herbarium of Shandong Agricultural University: Plant Pathology (HSAUP).1 Species diversity of dematiaceous HyphomycetesNew species: Doratomyces globosae H.Q. Pan & T.Y. Zhang, Myrothecium advena var. terrum H.Q. Pan & T.Y. Zhang, Cirrenalia qinghaiensis H.Q. Pan & T.Y. Zhang, Monodictys macrosporum H.Q. Pan & T.Y. Zhang, Monodictys huangheensis H.Q. Pan & T.Y. Zhang, Oidiodendron tshawytschae H.Q. Pan & T.Y. Zhang, Pleurophragmium nanus H.Q. Pan & T.Y. Zhang, Rhinocladiella variabile H.Q. Pan & T.Y. Zhang, Scolecobasidium chinensis H.Q. Pan & T.Y. Zhang, Scolecobasidium laevis H.Q. Pan & T.Y. Zhang, Scolecobasidium longisporum H.Q. Pan & T.Y. Zhang, Scopulariopsis humicola H.Q. Pan & T.Y. Zhang, Scopulariopsis penicillin H.Q. Pan & T.Y. Zhang, Scopulariopsis verruculosum H.Q. Pan & T.Y. Zhang, Stachybotrys longisporum H.Q. Pan & T.Y. Zhang, Trichocladium microsporium H.Q. Pan & T.Y. Zhang, Veronaea gobica H.Q. Pan & T.Y. Zhang, Veronaea latispora H.Q. Pan & T.Y. Zhang.New records to China: Acroconidiella eschscholtziae(Harkn.) M.B. Ellis, Botrytis byssoidea Walker, Gliomastix inflate (C.H. Dickinson) W. Gams, Scolecobasidium dendroides Pirozynski & Hodges, Scopulariopsis hibernica A. Mangan, Veronaea coprophila (Subram. & Lodha) M.B. Ellis, Wardomyces anomalus Brooks & Hansf., Wardomyces humicola Hennebert & G.L. Barron.There are also 7 undeterminate species: Acremonium sp., Cephaliophora sp., Cladosporium sp., Scopulariopsis sp.1,Scopulariopsis sp.2, Verticillium sp., Wardomyces sp..The other 57 species have been known to China.Richness, Berger-Parker dominance index, Shannon-Wiener diversity index, evenness, niche breadth and niche overlap were used to analyze the community structure and eco-niche of soil dematiaceous hyphomycetes in different ecosystem-types.The results show that the amount and species distribution of soil dematiaceous hyphomycetes had an obvious variability in different ecosystem-types, and that the dominant genus species varied in the eight ecosystem-types studied. Species diversity was closely related to vegetation. The species diversity of soil dematiaceous hyphomycetes in forest and grassland was much higher than those in gobi and desert. The species of soil dematiaceous hyphomycetes in Yangtse River and Yellow River source area were less compared with these area in low altitude. There were rich of soil dematiaceous hyphomycetes with smaller conidia and lack of helminthosporioid fungi.2. The experience and improvement on taxonomic theory and methodsAssociated with identification work undertaken, the researcher paid great attention to the basal taxonomic theory at generic and species ranks of dematiaceous hyphomycetes and the improvement the fungal isolation and culture methods.(ⅰ) Taxonomic criteria at genus levelThe patterns of conidia formation and conidiophores (conidiogenous cells) proliferation are the main criteria used to separat diferent genera in dematiaceous Hyphomycetes. The conidiogensis patterns of almost all fungal genera isolated from soil in this study are holoblastic or enteroblastic. But the proliferation patterns of conidiophores (conidiogenous cells) are various, for example sympodial, phialidic, annellidic etc.. The morphological characters of conidiogenous cells and conidiophores, patterns of conidia germination and the order of septum (distoseptum) ontogeny are very important in differentiate those genera whose patterns of conidia formation and conidiophores proliferation are the same. The characters, color and growth traits of colonies are useful assistant characteristics to differentiate some genera while absence or presence of setae is also helpful to differentiate a few genera.(ⅱ) Taxonomic criteria at species levelThe morphological characters of conidia, especially the shape, size, color, septum and ornamentation etc. are important characters in identificating species. Aiming to reducing the influence of environment, all species in one genus should be cultured at the same or being close to the same conditions. The conidia shape and size of some species in some genera are very easily influenced by environment factors, it should be considered that the culture conditions used by the origional author(s) should be followed as closely as possible. The characters of conidiophores and conidia are also very useful in identificating species in some genera. Characters of setae may be used to separat species in a few genera, such as Trichurus Clements & Shear and Myrothecium Tode. Molecular methods are also used in identificate the species with less differences between each other in one genus.(ⅲ) The improvements of isolateting and culture methodsThe mainly used culture media are PDA, PCA, CMA and TWA+W in isolating and culturing the soil dematiaceous Hyphomycetes. The suitable conditions for soil dematiaceous Hyphomycetes is cultured for about 1-2 weeks under 20-25℃with 12h /12h alternate light and dark. According to the different altitude and temperature in Yangtse River and Yellow River source area, the author reduced the culture temperature to 18℃and added the soil extract into the medium. Mechanical damnification and stimulation with lower temperature etc. were also helpful in stimulating the fungus sporulation.3 Analysis of classification at genus level baced on rDNA-ITS sequencePhylogenetic relationships among 32 genera involved in this paper were studied based on 5.8S rDNA-ITS sequences by NJ (Neighboring-joining). The resultes showed the conidiogensis pattern‘blastic’could be easily reflected in the phylogenetic tree while the the conidiogensis pattern‘thallic’could not be reflected. The conidiogensis patterns‘holoblastic’and‘enteroblastic’were distincted in the phylogenetic tree. Also two major distinct clades were found within the genera whose conidiogensis patterns were enteroblastic-phialidic and enteroblastic-tredic. The similarity of 5.8S rDNA-ITS sequences between several genera with different conidiogensis patterns was much higher because of the different evolutionary rate between them.Taxonomic criteria at genus level, such as holoblastis, which used in morphology, could be well reflected in molecular systematics. Multi-gene sequences analysis could be used to reveal the conidiogensis patterns‘arthric’and other patterns of‘holoblastic’.4 Molecular systematics on represented species of Doratomyces, Trichurus and GraphiumIn this study, phylogenetic analysis of 5.8S rDNA-ITS and mt-SSU sequence data sets by NJ (Neighboring-joining)with a broad taxon sampling of Doratomyces, Trichurus and Graphium were conducted for inferring evolutionary relationships between the three genera. The results of analysis with 5.8S rDNA-ITS and analysis with SSU-rDNA were basically the same. The genus Graphium could be divided with Doratomyces and Trichurus on the phylogenetic tree. And the genus Doratomyces and Trichurus could not be divided from each other. The similarity of 5.8S rDNA-ITS sequences and SSU-rDNA sequences between all the species in Doratomyces and Trichurus were between 97% and 100%. These two genes were highly conservative in Doratomyces and Trichurus and the species with different morphology couldn’t be distinguished by analysis of 5.8S rDNA-ITS and SSU-rDNA sequences.Based on 5.8S rDNA-ITS sequences analysis, three distinct clades were found within the genus Graphium, of which Graphium dubautiae divided with other species. Four clades were found on the phylogenetic tree based on SSU-rDNA sequences. The similarity between the species were 100% and the similarity between the four clades were only between 63% and 94%. There was a little difference between the results of 5.8S rDNA-ITS and SSU-rDNA sequences analysis. The morphological results were not supported by the molecular data. More gene regions should be used with multi-gene molecular phylogeny to inferring evolutionary relationships between the three genera. Because 5.8S rDNA-ITS and mt-SSU sequence were highly conserved in the three genera, not enough variability was present to distinguish genera and species clearly, which implied that 5.8S rDNA-ITS and mt-SSU sequence were not enough when used them to study the lower taxa such as at genus and species leval. Multi-gene genealogy were suggested to ues in testing the correct taxonomic status of these species.更多还原