Diversity Of Endophytic Fungi Colonizing The Roots Of Betula Alnoides Plantations In Xishuangbanna

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.