【作者】 林晓民；

【导师】 李振岐；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2004， 博士

【摘要】 本研究包括大型真菌的生态多样性调查和菌丝体的分子鉴定两部分，现分述摘要如下： （一）大型真菌的生态多样性调查 调查了 422 种大型真菌的生境、生态，按生态习性将这 422 种大型真菌分为 12 个生态类型，即土壤腐生菌、木腐菌、落叶及腐草生菌、粪生菌、植物寄生菌、昆虫寄生菌、真菌寄生菌、地衣型真菌、外生菌根菌、昆虫共生菌、天麻共生菌和真菌共生菌。结合 94 幅典型照片对中国大型真菌的生态多样做了初步阐述，并对大型真菌生态多样性的研究方法进行了讨论。 （二）大型真菌菌丝体的分子鉴定 大型真菌的菌种一般都是菌丝体，而传统的真菌种类鉴定主要是依据子实体的特征，菌丝体不能直接用于种类鉴定。对于能用人工培养方法生产子实体的种类，可通过出菇试验对菌种进行种类鉴定，但这种鉴定方法花费的时间较长，往往需要一个月以上甚至一年的时间。对于尚不能用人工培养方法生产子实体的种类，则不能用这种常规方法进行菌种鉴定。分子鉴定技术可以对菌丝体进行种类鉴定，但过去文献上报道的大型真菌分子鉴定多是采用RAPD和ITS-RFLP等方法，这些方法除了精确性与重复性较差外，还需要以同种真菌的子实体作对照，不同条件下的研究结果缺乏可比性。 本研究是基于 rDNA ITS 区段的 DNA 序列对菌丝体进行物种鉴定，其方法是先扩增出待鉴定真菌 rDNA ITS 区段的 DNA，并测出其序列，然后与互联网上 DNA 序列数据库中的信息资源进行比较，从而得出物种鉴定的结论。 运用该方法首先对出菇试验证实了的 3 种栽培食用菌（桃红侧耳、白阿魏侧耳和杏鲍菇）的菌种进行了物种鉴定，分子鉴定结果与出菇试验的鉴定结果相一致，证明这种分子鉴定方法是切实可行的，鉴定结果是可靠的。然后进一步用该方法对分离自 6 种外生菌根菌子实体的菌株进行了分子鉴定，结果表明：分离自松口蘑（Tricholomamatsutake）子实体的 1 个菌株被证实为真正的松口蘑菌种，分离自其它 5 种外生菌根菌子实体的菌丝体均被证明不是原分离子实体所属物种的菌种。其中分离自褐环乳牛肝菌[Suillus luteus (L.:Fr.)Gray]子实体的 1 个菌株被证实是裂褶菌（S. commune），该分子鉴定结果进一步得到了出菇试验的支持；分离自干巴菌（Telephore ganbajun）子实体的 1 个菌株被证实为一种镰刀菌（Fusarium sp.），该分子鉴定结果进一步得到了形态鉴定的支持；分别分离自厚环乳牛肝菌[Suillus grevillei (Klotzsch.)Sing.]和玫红铆钉菇[G. roseus （Fr.）Karst.] 子实体的 2 个菌株经分子鉴定它们 ITS 区段的 DNA 序列完全相同，该 DNA 序列与互联网上 DNA 序列数据库中一种内生真菌（Endophytic fungi ）的相应序列具有高度的同源性 [96.61% （599/620）]，该内生<WP=7>ii 大型真菌的生态多样性及分子鉴定真菌分离自我国台湾的珍稀植物南方红豆杉（Taxus mairei），属于一种未鉴定出种类的担子菌；分离自污白疣柄牛肝菌[Leccinum holopus (Rostk. ) Watl.]子实体的 1 个菌株的 ITS 区段 DNA 序列与互联网上序列数据库中的所有 DNA 序列同源性均较低，最高同源性约为 78%，这些同源序列均属于子囊菌。 此外，以茯苓基因组 DNA 为模版，用通用引物 ITS1/ITS4 能扩增出了一条约 1700bp的特异性 DNA 片段，经 PCR 产物直接测序，测出了 2 条具有茯苓物种特异性的 DNA 序列，这两方面的 DNA 分子特征可作为茯苓物种鉴定的分子依据。更多还原

【Abstract】 1.Investigation on the Ecological Diversity of Macrofungi The Ecological characteristics of 422 species of macrofungi have been investigated.These macrofungi were divided into 12 ecological types, they are saprophytic fungi on wood,saprophytic fungi on straws and fallen leaves, saprophytic fungi on dung, saprophytic fungion soil, fungi as plant parasites, fungi as insect parasites, fungi as fungus parasites,ectomycorrhizal fungi, fungi forming lichen, symbiotic fungi associated with insects,symbiotic fungi associated with Gastrodia elata Bl and fungi associated with other fungi insymbiotic relation. The ecological diversity of macrofungi in China and its study methodswere discussed. Also, 94 photographs showing the ecological diversity of macrofungi inChina were provided. 2.Molecular Identification of Mycelia in Macrofungi Traditional method of species identification in fungi is mainly based on characteristics ofsporocarps. Spawn of macrofungi is commonly mycelium. Therefore, the speciesidentification of spawn cannot be directly conducted. To identify spawn of domesticmacrofungi, they can be cultivated to obtain fruiting body, but the way takes longer time,generally, it spends more than a month, it need even a year for some species. Moreover, theway is not useful for untamed macrofungi. The molecular method can identify species offungi using mycelium. In past study, RAPD and ITS-RFLP were major techniques applyingto molecular identification of macrofungus mycelium. However, RAPD is less reliable,ITS-RFLP lacks enough accuracy to distinguish some species. In addition, both mycelia andtheir original sporocarps were necessary for molecular identification in most of past study. In this study, species identification of macrofungus mycelium was conducted based onDNA sequences in the ITS region of rDNA. The method writes as follows: ITS regions ofrDNA of fungi identified are amplified and sequenced, the sequences determined werecompared with those registered in DNA sequence database on Internet, then identificationresults were obtained according to comparing results and other biological information inDNA sequence database on Internet. Firstly, 3 isolates from sporocarps belonging respectively to Pleurotus eryngii, P.nebrodensis, and P. salmoneostramineus, which were previously proved to be spawn of this 3domestic edible fungi by cultivating identification, were identified using above molecularmethod, the results are consistent with that obtained by cultivating identification. It indicatesthat the method of molecular Identification is feasible and its identification result is reliable.<WP=9>iv 大型真菌的生态多样性及分子鉴定Then 6 isolates from sporocarps belonging respectively to 6 species of ectomycorrhizal fungiwere identified, the results show that every one of these isolates, except the isolate from thesporocarp of Tricholoma matsutake, were not the spawn of their original sporophores. The isolate from sporocarp of Suillus luteus was proved to belong to Schizophyllumcommune by both molecular and cultivating identifications. The isolate from sporocarp ofTelephore ganbajun was proved to belong to Fusarium sp. by both molecular andmorphological identifications. Both isolates from Suillus grevillei and Gomphidius roseushave same sequence in the ITS region of rDNA. The sequence and that of an endophyticfungus registered in GenBank have higher homology. That is, about 97% (599 of 920) of theirnucleotide sequence is consistent. The endophytic fungus was isolated from Taxus mairei inTaiwan, which belongs to basidiomycetes. The ITS sequence of the isolate from sporocarp ofLeccinum holopus has less similarity with those in DNA database on Internet (less than 78%). In addition, a DNA fragment was amplified using the universal primers ITS1/ITS4 andthe gDNA of Poria cocos, which is about 1700bp and obviously longer than the ITS region ofother fungi. The sequences of two parts in the frag更多还原