中国气-水两生型丝孢真菌资源与系统分类研究

Aero-aquatic fungi are a group of anamorphic hyphomycetes, which are generally growing on the plant litters at the bottom of water, producing propagules on water surface. Distinctive habitat and sporulation mode of these fungi show an important value on researching fungal phylogeny and envolution.In addition, the ability of decomposing plant litters of these fungi at the condition of low temperature, anaerobic and high sulfur dioxide concentration, indicate a great significance on resource screening. Presently, it is difficult not only to collect and culture these fungi but also to induce their sporulation on culture media, and also most of their teleomorphs are unknown. This project, firstly, intend the taxonomic investigations on these fungi in China, to clarify their kinds of species, distribution, habitat and diversity etc., to seek after the optimal isolation and cultivation method, to analyse propagules growth and forming mechanism, to induce the production of teleomorph, to clarify morphological characteristics . Further, by international exchange, loaning the specimens and isolates, and multi-gene sequence analyses, the taxonomic position of the questionable genera and species can be clarified, the connection between teleomorph and anamorph can be built. These will adapt to the new fungal nomenclature for name revision. At last, combined with morphology, ecology, molecular data and teleomorph information, phylogeny and evolution relationship of these fungi can be further evaluated, a natural phylogenetic tree can be constructed. At the same time, collected strains can be used for further resource exploitation and application.

气-水两生型真菌是一类通常定居在水中的植物残体上，在水下生长，水面产生中空的孢子球体的无性型丝孢菌。其独特的生存环境和产孢特点，在真菌的系统发育与水陆两生进化上具有重要的研究价值；另外，其在低温、缺氧、高H2S的水底能够降解植物残体，在资源筛选方面意义重大；但目前还存在采集、分离培养困难，人工培养难以产孢，有性型缺乏等问题。本项目拟首先通过资源调查和形态分类的方法，摸清我国该类真菌的种类、分布、基质生境、多样性等，探索适宜的分离培养方法，解析孢子体发育形成机制，诱导有性型产生，明确形态分类特征；进一步通过国际交换、借阅标本和菌株，采用多基因序列分析的手段，确定疑难属、种的分类地位，建立有性型与无性型的联系，为适应新的真菌命名法规进行名称修订做准备；最后，结合形态、生态、分子、有性型等特征，阐明系统演化关系，构建趋于自然的分类系统；同时，积累菌株为进一步资源开发利用奠定基础。

气-水两生丝孢真菌独特的生境特点及孢子体形态在真菌资源及系统演化上有重要研究价值。项目对我国该类真菌及其相关类群进行系统采集分类研究，完成预期目标，取得如下结果：（1）通过系统的资源调查与标本采集，明确了该类真菌的生境特点、分布和多样性。缓水溪流、林间池塘、水温11-17℃，PH 5.5-6.0，半浸水枯枝、腐木，部分沉水落叶等生境适合其分布；获得标本400余份，鉴定描述60余种，发现新种5个，中国新记录属13个，新纪录种32个，分离菌株近百号。（2）探索获得该类真菌适宜的分离培养方法及产孢条件，生长范围15-30ºC，21ºC PDA培养基对菌丝体生长有利，不利产孢；模拟自然生境CMA加木质浸渍液培养基，低温16-20ºC，及光暗厌氧刺激，利于形成孢子繁殖体（propagules）。（3）解析了孢子繁殖球体的种类和发育方式。笼格状，由中心细胞通过重复的二杈或三杈分枝以丝状向四周延伸集结成团网状的球形孢子体；团球状，分生孢子丝由中心细胞分枝延伸并向内卷曲聚集成球状的孢子体；念珠簇球状，以多分枝的念珠球状小细胞向四周延伸并密集连结，形成球状或椭球状中空的孢子体；三维卷旋，分生孢子丝在立体空间内螺旋2-3周以上或盘旋上升成桶状中空孢子体。（4）对不易分离培养，菌体孢子量少的种类，探索获得了微量菌体孢子（mg级）快速灵敏提取DNA的方法，达到PCR扩增检测量级，实现形态疑难属、种的免培养分子鉴定，该方法计划申请专利。（5）ITS和gpd基因系统发育分析显示，气-水两生真菌及相关类群主要分为三组，Zalerion-Spirosphaera组，Helicomyces-complex组和Zalerion-complex组，每组内部种类分布分散，非单系起源。gpd不适合作为该类真菌的第二标记条形码基因。（6）参加“菌物命名法与多样性编目研讨会”，“真菌分子生态学新技术培训班”等高水平学术会议，与国内外顶级专家Hawksworth、Kirk等进行广泛深入的学习讨论交流。对国际菌物命名法规一些重大条款的变革，有了进一步的了解和认识，为所从事类群真菌的系统分类研究提供重要参考。培养研究生5名，毕业2名，发表SCI论文4篇。