3种杓兰属植物菌根真菌系统发育和多样性分析

兰科植物菌根真菌(Orchid mycorrhizal fungi, OrMF)在兰科植物种子萌发和后续生长发育过程中具有重要作用。该研究采用培养(菌丝团分离)和非培养(克隆文库)2种方法获得同一栖息地3种不同杓兰属植物根中菌根真菌ITS序列并划分可操作分类单元(Operational taxonomic units, OTUs)，分析其系统发育关系和多样性。结果表明:(1)所有根段中都有菌丝团定植,共分离出菌根真菌64株，其中63株为胶膜菌科(Tulasnellaceae)真菌，1株为角担菌科(Ceratobasidiaceae)真菌；可划分为7个OUT，每个OTU代表菌株的菌丝都能形成OrMF典型的近球形或椭球形链状排列的念珠状细胞；分离出来的菌根真菌均为无性型菌丝且不产生无性孢子。(2) 非培养法得到的3种杓兰属植物的根中OrMF分别隶属于胶膜菌科(Tulasnellaceae)，腊壳菌科(Sebacinaceae)、角担菌科(Ceratobasidiaceae)和革菌科(Thelephoraceae)，其中胶膜菌科OTU在种类和数量上占有绝对优势，培养和非培养2种方法得到的OrMF OTU类型和数量均为西藏杓兰(Cypripedium tibeticum)>无苞杓兰(C. flavum)>黄花杓兰(C. bardolphianum)，但培养法少于非培养法。(3)对胶膜菌进行系统发育分析显示，优势和非优势OTU均分布在系统发育树的3个不同分支上，这种与多种亲缘关系较远的OrMF共生的现象可能与杓兰属植物对环境的适应性有关，且不同杓兰的OrMF物种丰富度没有显著差异，但群落结构存在差异。

Phylogenetic and Diversity Analysis of Mycorrhizal Fungi in Three Cypripedium Species

Orchid mycorrhizal fungi (OrMF) play an important role in seed germination and growth of orchids. In this study, the operational taxonomic units (OTUs) of ITS sequences of mycorrhizal fungi in the roots of three Cypripedium species in the same habitat were obtained by culture dependent (peloton isolation) and culture independent (clone library) method, to study the phylogenetic relationship and diversity of OrMF. The results showed that: (1) the pelotons distributed in all of the root fragments; 64 OrMF were isolated, including 63 Tulasnellaceae strains and 1 Ceratobasidiacea strain; 7 OTUs were found, and the representative isolate of every OTU formed typical subglobose or ellipsoid monilioid mycelia in chain arrangement. All of the isolated fungi were asexual hyphae and did not produce asexual spores. (2) OrMF obtained from roots of three Cypripedium species by culture independent method were affiliated to Tulasnellaceae, Sebacinaceae, Ceratobasidiaceae and Thelephoraceae; The OTUs of Tulasnellaceae had an absolute predominance in type and quantity; It was Cypripedium tibeticum>C. flavum>C. bardolphianum in OTU type and quantity of OrMF acquired by both culture dependent and culture independent approach; And the types and quantity of mycorrhizal fungal OTUs obtained by the culture dependent method were less than the culture independent one. (3) The phylogenetic analysis of OTUs of Tulasnellaceae showed that the dominant and non dominant OTUs were distributed in three different clades of the phylogenetic tree; This phenomenon that orchids symbiose with many kinds of relatively distant relatives of OrMF may be related to the environmental adaptability of Cypripedium species; There was no significant difference in the OrMF species richness of different Cypripedium species, but there was a differentiation in the community structure.