【作者】 张燕；

【导师】 赵之伟； 李涛；

【作者基本信息】 云南大学 ， 微生物学， 2012， 博士

【摘要】 深色有隔真菌(Dark septate endophytes, DSE)是一类在分类学上多样的、广泛存在于在植物根内并形成深色有隔菌丝的的子囊真菌。DSE极为广泛的生态分布和类似菌根的潜在生态学功能,特别是在不利于菌根发育的胁迫环境中的积极作用,使其在生态修复和保护、植被恢复和重建以及生物防治等多个领域具有潜在的应用价值。本研究以云南西双版纳热带雨林、金沙江干热河谷中河谷型萨瓦纳植被区(元谋、东川普渡河-小江河谷)和重金属污染的铅锌矿区(兰坪-会泽矿区)三类特殊生境中植物根内的DSE为研究对象,对DSE在植物根内的定殖状况进行了调查。在此基础上,进一步从西双版纳热带雨林和重金属污染的铅锌矿区植物根内分离和培养DSE菌株；采用分子生物学方法结合传统形态学分类方法,对分离菌株ITS1-5.8S-ITS2区DNA序列进行扩增测序,通过诱导产孢观察其产孢结构和孢子形态学特征,对分离菌株进行分类学鉴定；在纯培养条件下,以含有不同浓度重金属离子的固体培养基对重金属污染的铅锌矿区来源的菌株进行抗性筛选,初探其对重金属的抗性。研究得到以下结果：1.通过对云南西双版纳热带雨林,金沙江干热河谷中河谷型萨瓦纳植被区以及重金属污染的铅锌矿区不同生态环境中,DSE在80科256种植物,共575个植物根样的定殖情况的调查发现,DSE普遍存在于这几种生态系统的植物根内。三种类型样地内植物DSE感染率分别为98.8%、95.0%、89.2%。其中,菌丝平均感染程度分别为9.7±0.9%、14.4±1.4%、6.7±0.7%；微菌核感染程度分别为0.4±0.1%、1.4±0.3%、0.8±0.2%。三类样地植物物种总感染率为93.0%,样本总感染率为80.5%,菌丝平均感染程度为10.9±1.6%,微菌核平均感染程度为1.0±0.3%。DSE在植物根内所呈现的形态也呈现出典型性、多样性的特征。2.通过分子生物学结合形态学方法对分离培养自根内的DSE菌株进行的分类鉴定表明,所分离的菌株涵盖多个种属,包括已经被广泛公认为DSE类群的Exophila、Phialophora,以及尚未被列入DSE范畴的子囊菌。分子鉴定显示分离自西双版纳热带雨林植物根样中的菌株以Colletotrichum、Ochroconis、 Phoma为主,而分离自会泽矿区植物根样中的菌株以Exophila和Phialophora为主,兰坪矿区植物根样中的分离菌株则以Phialophora和Lophiostoma为主。形态学研究支持了Exophila和Phialophora的分类地位,其它菌株的分类地位有待确认。Exophila、Phialophora真菌成为重金属污染的铅锌矿区根内DSE的优势类群。3.通过对重金属污染的铅锌矿区来源的真菌菌株的抗性筛选,以半抑制浓度EC50值表征菌株的重金属抗性的强弱,得到对Pb、Zn、Cd三种重金属具有较强抗性的菌株。抗性筛选实验显示,除Cd敏感菌株外,矿区分离得到的菌株都对Pb、Zn、Cd三种重金属具有一定的抗性。分离自会泽矿区的Exophiala菌株对三种重金属具有普遍的抗性,以对Pb和Cd的抗性尤为突出,该属真菌菌株对Pb、Zn、Cd的EC5o的平均值分别为1037.7±177.1mg/L、914.1±165.8mg/L245.6+66.9mg/L；会泽来源的Phialophora真菌菌株也显示出较强的Pb抗性,该属真菌菌株对Pb的EC50的平均值为1043.9±60.7mg/L；分离自会泽矿区的一类产小孢子的,在分子鉴定中显示与Exophiala有密切关系的菌株(类型H5)则能够耐受高浓度的Pb、Zn、Cd,此类菌株对Zn的耐受力更是远远超出其它菌株。该类型真菌菌株对Pb、Zn、Cd的EC50的平均值分别为1068.5±344.1mg/L、2285.7±1262.4mg/L、138.6±95.9mg/L。而分离自兰坪矿区的菌株中,Phialophora真菌菌株对Pb、Zn、Cd三种重金属的抗性处于中间水平,菌株对Pb、Zn、Cd的EC50的平均值分别为773.9±37.8mg/L、248.8±23.6mg/L、24.1±1.7mg/L;兰坪的一类产小孢子(类型L2)真菌菌株显示出对三种重金属较强的抗性,尤其是对Pb的抗性,此类真菌菌株对Pb的EC50的平均值为1478.1±329.9mg/L；兰坪的另一类产棒状孢子的,在分子鉴定中显示与Ployscytalum有亲缘关系的(类型L3)真菌菌株显示出很强的Zn和Cd抗性,此类菌株对Zn和Cd的EC50的平均值分别为1138.7.1±91.6mg/L和256.4±90.4mg/L；而兰坪的非产孢(类型L4)真菌菌株则具有较强Cd抗性,对Cd的EC50的平均值为233.2±174.4mg/L。对Exophiala和Phialophora真菌代表菌株重金属胁迫下的形态变化的研究提示了两种真菌可能以不同的机制抵抗重金属的毒害。上述研究结果表明：DSE普遍定殖于西双版纳热带雨林,金沙江干热河谷河谷型萨瓦纳植被区以及重金属污染的铅锌矿区三种特殊生境中植物根内；对分离菌株的分子鉴定表明,热带雨林植物根上定殖的DSE以Colletotrichum、 Ochroconis和Phoma为主,而分离自会泽矿区植物根样中的菌株以Exophila和Phialophora为主,兰坪矿区植物根样中的分离菌株则以Phialophora和Lophiostoma为主：重金属矿区来源的DSE重金属抗性研究表明,大部分菌株对Pb、Zn、Cd都具有一定的抗性,其中不乏高抗菌株。论文对云南三种特殊环境中的DSE资源进行了普查和收集,分离保存了来自上述三种特殊生态环境中的DSE菌株,并对其分类学地位进行了初步的鉴定,为开展DSE在上述三类特殊生境中的生态学功能研究打下了基础；与此同时,本研究从重金属污染的铅锌矿区来源的DSE菌株中初步筛选得到了一批对Pb、Zn、Cd具有一定抗性的DSE菌株,为从重金属污染区的DSE中发掘抗性基因提供了菌种保障。更多还原

【Abstract】 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.更多还原