【作者】 陶刚；

【导师】 刘作易；

【作者基本信息】 华中农业大学 ， 微生物学， 2009， 博士

【摘要】 兰科植物是被子植物最大科之一,是重要的观赏花卉和药用植物,这类植物要与特定真菌建立共生关系,才能完成正常的生长发育,这些独特的特征使兰科植物成为植物与真菌协同进化、真菌多样性和专一性等方面研究的重要材料。本研究选择贵州境内普遍分布的兰科植物黄花白及(Bletilla ochracea Schltr.)为研究材料,采用传统培养分离方法与分子生物学方法相结合,通过对分布在不同地区的白及(B.ochracea)材料的叶和根组织内生真菌的分离频率、种类组成、多样性指数和主成分分析等研究,揭示白及叶和根组织内生真菌类群的多样性组成及生态分布,探讨地理环境因素对内生真菌类群多样性和生态分布的影响。通过对贵州5个地区采集的32份白及(B.ochracea)植株材料的1800个根和叶组织段(块)分离内生真菌,共分离菌株1028株,根据形态学特征和形态型菌株的rDNA的5.8S基因和ITS序列的相似性比较和分子系统发育分析确定为90个分类单元,其中33个鉴定到种,42个鉴定到属,15个鉴定到科及以上水平。从叶组织共分离775株,鉴定为46个分类单元,分别属于14个属36种和10个只鉴定到目及以上水平的分类单元;从根组织共分离253株,鉴定为47个分类单元,分别属于19个属42种和5个鉴定到科及以上水平的分类单元。其中,2个兰科菌根真菌属(Epulorhiza和Sebacina)14个种类在分子水平上与其他地区的差异明显,即Epulorhiza sp1-sp5、Epulorhiza sp7、Sebacina sp1-sp8。内生真菌类群中能够产生孢子及相关鉴定特征的有697株,包含38个分类单元,属于12个属36个种,2个鉴定到科的水平,占分离菌株总数的67.8%,产孢菌株中有221株分离自根组织,有476株分离自叶组织。其中,有19种子囊菌,19种担子菌,担子菌全部分离自根组织。不能产孢及鉴定特征的形态型菌株有331株,占分离菌株总数的32.2%,划分为51个形态型。通过ITS区序列的相似性比较和分子系统发育分析等方法鉴定为39个分类单元,属于21个属,17个鉴定到种,21个鉴定到属,1个鉴定到目,其中2个属于担子菌,其他37个属于子囊菌,还有12个形态型菌株无法鉴定。通过ITS-PCR、随机克隆、DGGE和系统发育分析等方法检测和鉴定了贵州清镇白及(B.ochracea)材料的根和叶组织中内生真菌类群组成和多样性。从叶组织共得到203个内生真菌的ITS区序列的克隆,通过分子鉴定为18个不同的OTUs,包括15种子囊菌(91%),3种担子菌(9%);其中Mycosphaerella、Alternaria、Colletotrichum、Leptosphaerulina和Dioszegia属以及1个未鉴定的子囊菌Ascomycetesp.2为优势类群;来自根组织内生真菌的ITS区序列的克隆为211个,属于10个不同OTUs,包括9种子囊菌(54%)和1种担子菌(46%),其中Sebacina、Fusarium、Gibberella和Nectria属种类为优势类群。结果表明,叶组织中内生真菌的多样性指数(H′,2.354)要高于根组织的(H′,1.560),而且叶和根组织中内生真菌是两个完全不同的类群;与传统培养分离方法比较,分子方法检测到的内生真菌类群组成差别明显,有些优势类群和非优势类群不能在两种方法的结果中同时体现。对贵州5个地区的白及(B.ochracea)根和叶组织内生真菌类群的生态分析表明:根组织内生真菌的优势类群主要属于Epulorhiza、Ceratorhiza和Sebacina 3个担子菌属(56.40%相对分离频率,RF)以及子囊菌(10.74%RF)的Phomopsis和Fusarium属,而叶组织内生真菌的优势类群主要属于Colletotrichum(68.26%RF)、Guignardia(14.10%RF)和Cercospora(7.36%RF)3个属;这些优势类群在不同地理位置的生态分布是有差别和不均匀的;不同地理位置和环境因素对内生真菌类群的丰度和分布有明显的影响;不同地理位置的内生真菌类群多样性(H′)趋势是根组织的要比叶组织的高;不同地理位置的根内生真菌类群间相似性系数(Cs)很低,而不同地区的叶内生真菌类群间相似性系数(Cs)要明显高于根的,这与叶内生真菌类群在不同地区的生态分布差别和不均匀要小于根的结果相一致;根和叶组织内的真菌类群是两个完全不同的群落,来自根和叶组织的真菌类群的相似性系数(Cs)接近于零;根组织的内生真菌多样性与经度、海拔和纬度都有一定的相关性,但在叶组织中则没有体现相关性。本研究首次对不同地理环境的白及(B.ochracea)根和叶组织的内生真菌类群分布及多样性进行较为系统的研究,研究结果有助于全面了解兰科植物内生真菌的类群组成和生态分布规律,为兰科植物内生真菌这一重要资源的认识和利用提供理论依据和资源基础,具有一定的理论和实践意义。更多还原

【Abstract】 All the 331 non-sporulated strains (32.2% to the total isolates) were grouped into 51 fungal morphotypes according to similar cultural characters. These 51 morphotypes were identified to 39 taxa, of which 17 were identified to species, 21 to genus, 1 to order, and 12 were unidentified. Of the 39 taxa above, there were 37 Ascomycota and 2 Basidiomycota.Based on methods of ITS-PCR, random cloning, DGGE and phylogenetic analysis, fungal communities were detected and identified within leaves and roots of B. ochracea collected from Qingzhen city in Guizhou. A total of 203 ITS clones were obtained from leaves and were identified to 18 different OTUs, including 15 Ascomycota (91% to total clones) and 3 Basidiomycota (9%), among of these, Mycosphaerella, Alternaria, Colletotrichum, Leptosphaerulina, Dioszegia and Ascomycete sp.2 were dominant species. Correspondingly, the 211 clones were discovered from roots and were identified to the 10 OTUs, contained 9 taxa of Ascomycota (54%) and 1 taxon of Basidiomycota (46%), of these, Sebacina, Fusarium, Gibberella and Nectria were dominant species. The result showed that fungal diversity in leaf tissues (H’, 2.354) were higher than that in root tissues (H’, 1.560), and also revealed that fungal communities within leaves and roots were significantly different from each other. Comparing with traditional method, fungal communities detected by molecular method were quite different, and some dominant species or common species could not be detected simultaneously by both methods.The results of ecological analysis of fungal communities within roots and leaves from 5 geographic regions in Guizhou showed that species of Epulorhiza, Ceratorhiza and Sebacina genera in Basidiomycota were dominant with 56.40% to total relative frequency (RF), and species of Phomopsis and Fusarium genera in Ascomycota were also common with 10.74% to total RF in root tissues. And within leaves, the dominant species were in the Colletotrichum (68.26%) , Guignardia (14.10%) and Cercospora (7.36%) genera. However, ecological distribution of these dominant species were a bit different among sampling sites; Richness and distribution of endophytic fungi were significantly affected by different sampling sites; Shannon-Wieiner diversity index (H’) of endophytic All the 331 non-sporulated strains (32.2% to the total isolates) were grouped into 51 fungal morphotypes according to similar cultural characters. These 51 morphotypes were identified to 39 taxa, of which 17 were identified to species, 21 to genus, 1 to order, and 12 were unidentified. Of the 39 taxa above, there were 37 Ascomycota and 2 Basidiomycota.Based on methods of ITS-PCR, random cloning, DGGE and phylogenetic analysis, fungal communities were detected and identified within leaves and roots of B. ochracea collected from Qingzhen city in Guizhou. A total of 203 ITS clones were obtained from leaves and were identified to 18 different OTUs, including 15 Ascomycota (91% to total clones) and 3 Basidiomycota (9%), among of these, Mycosphaerella, Alternaria, Colletotrichum, Leptosphaerulina, Dioszegia and Ascomycete sp.2 were dominant species. Correspondingly, the 211 clones were discovered from roots and were identified to the 10 OTUs, contained 9 taxa of Ascomycota (54%) and 1 taxon of Basidiomycota (46%), of these, Sebacina, Fusarium, Gibberella and Nectria were dominant species. The result showed that fungal diversity in leaf tissues (H’, 2.354) were higher than that in root tissues (H’, 1.560), and also revealed that fungal communities within leaves and roots were significantly different from each other. Comparing with traditional method, fungal communities detected by molecular method were quite different, and some dominant species or common species could not be detected simultaneously by both methods.The results of ecological analysis of fungal communities within roots and leaves from 5 geographic regions in Guizhou showed that species of Epulorhiza, Ceratorhiza and Sebacina genera in Basidiomycota were dominant with 56.40% to total relative frequency (RF), and species of Phomopsis and Fusarium genera in Ascomycota were also common with 10.74% to total RF in root tissues. And within leaves, the dominant species were in the Colletotrichum (68.26%) , Guignardia (14.10%) and Cercospora (7.36%) genera. However, ecological distribution of these dominant species were a bit different among sampling sites; Richness and distribution of endophytic fungi were significantly affected by different sampling sites; Shannon-Wieiner diversity index (H’) of endophytic fungi from roots was higher than that from leaves; Sorensen index (Cs) of endophytic fungi from roots was very low, and Cs of endophytic fungi from leaves much higher than that from roots. Fungal communities within roots and leaves were dramatically different from each other, and their Cs was nearly to zero; Shannon-Wieiner diversity index of endophytic fungi from roots had a certain correlation with longitude, altitude and latitude, however, there was no correlation within leaves.This is the first detailed investigation of fungal communities, distribution and diversity within roots and leaves of B. ochracea from different sampling sites, and the results above will help us for understanding comprehensively fungal diversity within plants and their geographical distribution. It is significant for us to realise and utilize important resources of endophytic fungi from orchid plants in this study.更多还原