Studies On Dark Septate Endophytes(DSE) In Special Ecosystems Of Yunnan

Dark septate endophytes (DSE) are a miscellaneous group of root-associated ascomycetes with dark septate hyphae which widely exsit in the plant roots. Their omnipresent distribution and potential ecological functions similar as mycorrhizae, especially their positive roles in stressful environment unfravorable for mycorrhizae, make them to be valuable for the ecological remediation and conservation, the vegetational recovery and reconstruction as well as the biological control etc. In order to study the resources of DSE in the tropical rainforest of Xishaungbanna, the dry-hot valley-type savanna of Jinsha River (Yuanmou, valleys of Pudu River-Xiao River in Dongchuan) and the metal-polluted areas (mining areas of Huize and Lanping) inYunnan, southwest China, we investigated the colonization status of DSE in plant roots in these areas. DSE isoaltes were obtained from the plant roots in the tropical rainforest and the metal-polluted areas. According to the molecular and the traditional morphological classification and identification, the sequences of ITS1-5.8S-ITS2rDNA of fungal isolates were analyzed and the morphologic characters of fungal isolates were observed. Their taxonomic affinity was affirmed. The resistance to heavy metals of some fungal isolates was determined by the resistance screening which was carried out on solid medium with metals of different concentrations. The main results were as follows:1. Totally575plant root samples, representing256plant species and80plant families were investigated in the tropical rainforest of Xishuangbanna, the dry-hot valley-type savanna of Jinsha River and the metal-polluted area in Yunnan. It was found that DSE were ubiquitous in the root samples. The colonization frequency of plant species in the three ecosystems were98.8%,95.0%,89.2%respectively, and the colonization frequency of plant samples were87.9%,70.5%,83.4%respectively. The colonization extent of DSE hyphae in plant roots of each ecosystem were9.7±0.9%,14.4±1.4%.6.7±0.7%respectively, while the colonization extent of DSE microsclerotia in plant roots of each ecosystem were0.4±0.1%,1.4±0.3%,0.8±0.2%respectively. The total colonization frequency in all the sampling sites was80.5%, and the average colonization extent of hyphae was10.9±1.6%, the average colonization extent of microsclerotia was1.0±0.3%. The morphology of DSE presented in the plant roots was typical and diverse.2. According to molecular and morphological evidence, DSE strains isolated from plant roots were composed of a variety of genus, including the well-known DSE groups, such as Exophiala and Phialophora, as well as ascomycetes which were not included in the range of DSE yet. It was shown by the molecular identification that fungal stains isoalted from plant roots in the tropical rainforest of Xishuangbanna mainly belonged to Colletotrichum, Ochroconis and Phoma, while the majority of fungal stains isolated from plant roots in the mining areas of Huize were falled in the genera of Exophaila and Phialophora, and fungal strains closely related with Phialophora and Lophiostoma were dominant in the mining areas of Lanping. The taxonomic affinity to Exophiala and Phialophora of fungal strains was supported by morphologic identification, but taxonomic affinity of other fungal strains was still to be determined. Fungal strains of Exophiala and Phialophora were dominant in the metal-polluted area.3. Fungal isolates with better resistance to heavy metals Pb, Zn and Cd were obtained according to the resistance screening and the value of the half maximal effective concentration (EC50) was calculated to show their metal resistance. It was indicated that, except for the isolates susceptible for Cd, all the experimental isolates were resistant to Pb, Zn and Cd. Exophiala isolates from Huize were generally resistant to the metals and were specially resistant to Pb and Cd. The average EC50value of Exophiala isolates for Pb, Zn and Cd was1037.7±177.1mg/L,914.1±165.8mg/L and245.6±66.9mg/L respectively. Phialophora isolates were noticed for their intensive resistance to Pb. The average EC50value for Pb of Phialophora isolates from Huize was1043.9±60.7mg/L. A group of isolates with microspores and phylogenetically related with Exophiala (H5) from Huize were resistant to excessive Pb, Zn and Cd and their resistant ability to Zn was far more notable than other isolates. Their average EC50value for Pb. Zn and Cd were1068.5±344.1mg/L,2285.7±1262.4mg/L and138.6±95.9mg/L respectively. Among the fungal strains isolated from root samples in mining areas of Lanping, resistant ability to Pb, Zn and Cd of Phialophora isolates was medius, their average EC50value for Pb, Zn and Cd were773.9±37.8mg/L,248.8±23.6mg/L and24.1±1.7mg/L respectively. Fugal group L2with microspores from Lanping showed strong resistance to metals Pb, Zn and Cd, especially to Pb, their average EC50value for Pb was1478.1±329.9mg/L. And fugal group L3with claviform spores and phylogenetically related with Ployscytalum from Lanping were noticeable for their resistance to Zn and Cd, the average EC50value for Zn and Cd was1138.7.1±91.6mg/L and256.4±90.4mg/L. Meanwhile the non-sporulating fungal isolates of group L4from Lanping showed apparent resistance to Cd, their average EC50value for Cd was233.2±174.4mg/L. Morphological variation of representative isolates of Exophiala and Phialophora in metalliferous medium implied their different mechanism of metal resistance.The results of the thesis indicated that:DSE were ubiquitous in the plant roots in the tropical rainforest of Xishuangbanna, the dry-hot valley-type savanna of Jinsha River and the metal-polluted area in Yunnan province; The molecular identification indicated that fungal stains isoalted from plant roots in the tropical rainforest of Xishuangbanna mainly belonged to Colletotrichum, Ochroconis and Phoma, while fungal stains isoalted from plant roots in the mining areas of Huize were consider to be mainly Exophaila and Phialophora, and fungal stains isoalted from plant roots in the mining areas of Lanping were mainly Lophiostoma and Phialophora; Metal resistance research of fungal isolates from metal-polluted area showed that most of the fungal isolates were resistant to Pb, Zn and Cd, some of which were highly resistant. Our research carried out the resource investigation of DSE and collection of DSE strains in the special ecosystems, which provided a foundation for the research of ecological functions of DSE in these ecosystems. Moreover, a group of metal resistant strains were selected from fungal isolates from metal-polluted areas, which supplied fungal strains for the seek of resistant genes.