【作者】 王玉娥；

【导师】 赵之伟； 李涛；

【作者基本信息】 云南大学 ， 生物物理学， 2012， 硕士

【摘要】 西南桦(Betula alnoides)是中国西南地区生长较快的阔叶树种,在造林上具有很大的发展潜力。云南省西双版纳普文试验林场的西南桦人工林面积26.67hm2,海拔高度为860-910m。原生地主要是山地雨林,长期以来因受人为破坏,形成次生林,其林相极度不整齐。从1987年开始建造西南桦人工试验林,各试验林地的立地条件基本一致,目前有5个不同生长阶段的西南桦人工林群落,分别为23年生、18年生、8年生、6年生和3年生。本研究以5个不同生长阶段的西南桦人工林作为研究对象,首先,对5个样地中深色有隔内生真菌(Dark Septate Endophyte, DSE)和丛枝菌根真菌(Arbuscular Mycorrhizal Fungi, AMF)在西南桦根系上的定殖情况进行了调查；其次,对西南桦根内分离的纯培养的DSE菌株进行鉴定；最后,对3个不同生长阶段的西南桦根系内生真菌的物种多样性进行了分子生物学研究。研究取得的主要结果和结论如下：1、用碱解离、酸性品红染色法对西双版纳人工林西南桦根内的DSE和AMF定殖特征进行了研究。研究结果表明,在所调查的5个不同生长阶段的西南桦根内都有DSE和AMF的结构。通过F-检验和T-检验发现AMF的菌丝与菌丝圈有显著的正相关性,DSE的菌丝与微菌核也存在显著的正相关性,但是DSE或AMF的定殖率与西南桦不同生长阶段的相关关系不大。2、从西南桦人工林各个样地中所采集的植物根样中分离、纯化,获得DSE纯培养菌株为208株。样地23(23yr)分离得到真菌36株、样地18(18yr)21株、样地8(8yr)52株、样地6(6yr)51株、样地3(3yr)48株。其中产孢的菌株为79株,而不产孢的菌株为129株。通过形态学和分子生物学鉴定后,结果显示,Cladosporium属真菌为西双版纳普文试验林场中西南桦根内的优势类群。3、在以上两部分的研究基础上,以样地18、样地8和样地3的西南桦根样为研究对象,通过nested PCR技术构建植物根内内生真菌ITS rDNA的基因文库,进一步研究西南桦根内内生真菌的多样性。通过克隆测序获得完整的、可靠的、有效的序列998条,去除嵌合序列和非真菌序列后,最终获得909条序列。用Dotur软件进行分析后,得到156个操作分类单元(operational taxonomic unit, OTU),表明西双版纳的普文试验林场中,人工种植的西南桦林根内的内生真菌具有极高的分子多样性。从样地18中获得序列301条,OTU74个,表现出较高的分子多样性,担子菌门的优势目是红菇目(Russulales),子囊菌门的优势目是柔膜菌目(Helotiales);从样地8中获得序列304条,OTU28个,主要是子囊菌门和担子菌门的真菌,在目以下分类单元水平上无明显的优势类群；从样地3获得序列304条,OTU69个,根内真菌的多样性较高,担子菌门的优势目是革菌目(Thelephorales),子囊菌门的优势目是柔膜菌目(Helotiales),其中还有1个OTU的内生真菌属于球囊菌门(Glomeromycota)。上述结果表明,西双版纳普文林场不同林龄的西南桦根内定殖着多种内生真菌。其中,AMF和DSE在不同林龄的西南桦根内普遍定殖；从不同林龄的西南桦根内分离到的DSE表现出较高的物种多样性；三个不同生长阶段西南桦根内真菌分子多样性研究表明,子囊菌门和担子菌门的真菌是根内真菌的主要类群,其中子囊菌门中柔膜菌目(Helotiales)的真菌(大多数DSE属于柔膜菌目真菌)在18年和3年林龄的根内是优势类群,与分离培养的DSE菌株多样性得到相互映证。更多还原

【Abstract】 Betula alnoides is considered as one of the most fast-growing broad-leave trees with great forestation potential in development of mountainous areas, southwest China. Puwen Betula alnoides Forestry Experimental Farm is locaed in Puwen, Xishuangbanna, with a plantations area of26.67hm2, at the altitude of860-910m. Due to human destruction for a long time, the formation of secondary forest is rather irregular. Betula alnoides plantations were constructed in1987with the same site conditions basically. There are five different growing ages of Betula alnoides plantations for23-year-old,18-year-old,8-year-old,6-year-old and3-year-old. In this study, five plantations of Betula alnoides were targeted. The colonization status of DSE (Dark Septate Endophyte) and AMF (Arbuscular Mycorrhizal Fungi) in the roots was surveyed, some DSE strains were isolated and identified; and the molecular diversity of endophytic fungi colonizing in the roots of Betula alnoides with three different growing ages was investigated. The major results and conclusions are as follows:1. The colonization status of DSE and AMF in the roots of Betula alnoides plantations were examined by alkaline lysising and acid fuchsin staining. The results showed that typical DSE and AMF structures were formed in all root samples of Betula alnoides. T-test and F-test showed that there is a significant positive correlation between hyphal and hyphal coils of AMF, hyphal and microsclerotia of DSE, however, the correlation between the colonization of DSE or AMF and the different growing stages of Betula alnoides is not obviously.2. In total,208DSE strains were isolated and purified from the root samples of Betula alnoides.Among which,36DSE strains were obtained from sample site23(refers to the plantation is23-year-old),21strains from sample site18,52strains from sample site8,51strains from sample site6, and48strains from sample site3, respectively. In the208DSE strains, there are79strains of sporulation, and129strains are sterile. Identification of these DSE strains based on morphological and molecular characters showed that fungi in the Cladosporium were in the dominant.3. Three plantations of Betula alnoides with18-year-old,8-year-old and3-year-old were selected as samples sites. Molecular diversity of root associated endophytic fungi was evaluated by internal transcribed spacer (ITS1and ITS2) and5.8S ribosomal RNA genes. In total,998sequences of endophytic fungi were amplified by nested polymerase chain reaction (PCR), cloned and sequenced, and909ITS1-5.8S-ITS2sequences were obtained excluding chimeric sequences and non-endophytic fungi. At last, we got156OTU (operational taxonomic unit) of endophytic fungi from Betula alnoides by Dotur analysis. In the18-year-old plantation, the301sequences were belong to74OTU, Russulales was the dominant order of Basidiomycota and Helotiales was the dominant order of Ascomycota; In the8-year-old plantation, the304sequences were belong to28OTU, and in the3-year-old plantations, the304sequences were belong to69OTU, Thelephorales was the dominant order of Basidiomycota and Helotiales was the dominant order of Ascomycota, beside, an OTU belonging to Glomeromycota was also founded in this plantation.The above results suggested that endophytic fungi were colonization in the roots of Betula alnoides plantations. DSE and AMF structures were formed in all root samples of Betula alnoides. DSE strains were isolated from the root samples of Betula alnoides had high species diversity. Molecular diversity of root associated endophytic fungi was showed that the Ascomycota and Basidiomycota fungi are the main group in the root. Helotiales (most DSE strains belong to Helotiales) was the dominant order of Ascomycota in18yr and3yr, which was mutual reflection of DSE diversity.更多还原