Mycorrhizal Microstructure And Fungal Endophyte Diversity Of Cypripedium Japonicum Thunb.

The Orchidaceae is one of the largest and most diverse families of angiosperms. Due to their high ornamental and medical values, a great number of orchids have been over-collected from natural habitats, which, in addition to rapid loss of habitats, resulted in considerable reduction in species and individual number, thus brought them to the verge of extinction and desiring conservation. In nature, orchids are well known to form mycorrhizal associations with fungi and show strong dependence on the symbiosis for their survival throughout lifetime. Therefore a profound knowledge of orchid mycorrhiza would help to explore the mechanism of adaptation to the environment so as to conserve these orchids effectively. In this paper, fresh mycorrhizal roots of Cypripedium japonicum Thunb. were collected from natural habitats for structural observation and fungal isolation. Then the mycorrhizal microstructure, the ways mycorrhizal fungi infection and the characteristics of pelotons were investigated, at the same time, mycorrhizal fungi were isolated and identified. The objectives of this work were to explain the mechanism by which the mycorrhizae of C. japonicum formed as well as to examine the specificity and diversity of mycorrhizal fungi compatible with this orchid. Consequently further research in the mycorrhizal associations between the orchid and fungal isolates could be carried out, by which beneficial strains could be screened out. In addition, this work could enrich the species of mycorrhizal fungi resources in China and provide reference for habitatal evaluation during in situ and ex situ conservation of orchids.Mycorrhizal microstructure were observed through paraffin sections and aniline blue staining tabletting under the microscope. The result showed that the root of C. japonicum consisted of velamen, cortex and stele. The velamen was formed by a layer of cell, and the cortex covered most of the root cross-section. A large number of starch grains, which usually concentrated around the nucleus, existed in the cortical cells. Acicular crystals existed in several outer cortical cells. The primary xylem were hexarch, and the endodermis cells adjacent to the phloem showed horseshoe-shaped thickening. As the typical features of orchid mycorrhizae, mycorrhizal fungi formed pelotons in the cortical cells of C. japonicum. The pelotons were connected through the hyphae penetrated cell wall and formed a network within the root. Infection and pelotons formation were noticed in the cortical cells, especially in the outer cortical cells, however hyphae had never be seen in the endodermis, stele or root tip cells. Entry of the fungus into the cortex was always through damaging the velamen. The hyphae spread into the cortex intracellularly, or formed pelotons with thick hyphae in several live cells of the velamen. Vesicles could be noticed in some velamen cells, which were connected with each other by thin hyphae. We found that either vesicles or pelotons formed with extremely thick hyphae existed in the velamen cells.All the isolates from fresh mycorrhizal roots of C. japonicum were initially classified based on morphological observations and 13 final strains were obtained. We sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal DNA from 13 isolates and identified 10 strains as 8 species, i.e. Fusarium solani, Neonectria radicicola, Chaetomium globosum, Alternaria alternate, Phialosimplex caninus, Ceriporia lacerate, Trichoderma asperellum, Debaryomyces hansenii. Furthermore, another strain could be identified to genus Coprinellus, and two strains identified to families Tricholomataceae or Trichocomaceae. Phylogenetic analysis showed that these mycorrhizal isolates belonged to more than 10 genera,10 families,8 orders,5 classes, and 3 subphyla, containing 3 classes,4 orders,4 families,4-5 genera of ascomycota,3 genera,2 families,1 order,1 class of basidiomycota and 3 genera,2 families,1 order,1 class of deuteromycotina. The results indicated a wide range of fungi might form mycorrhizal associations with C. japonicum and a high diversity of mycorrhizal fungi, which consisted with the status as a widely dispersed species.