Mycorrhizal Fungi Diversity Of The Dominant Orchids In The Huanglong Valley, Sichuan

Orchid mycorrhizas are mutualistic interaction between fungi and members of the Orchidaceae, one of the world's largest plant families. Considering the huge temperate zone and alpine mountains, China has rich terrestrial orchid resources. The Huanglong Valley in Sichuan province, at an elevation of 3100-3569 meter and 3.5 kilometers long, is the largest travertine region in the world. Rich terrestrial orchid diversity of 30 orchid species in 19 genera was found in this narrow valley. Cypripedium flavum, Corallorhiza trifida, Galearis diantha, Malaxis monophyllos, Neottia acuminate, Ponerorchis chusua, Phaius delavayi, Platanthera minutiflora and Tipularia szechuanica are the dominant wild temperate terrestrial orchids from two habitats in the Huanglong Valley. From conservation perspective, this study was tried to investigate the mycorrhizal fungi diversity and ecological characteristic of mycorrhizosphere. The main results were as follows:1. The mycorrhizal fungi were isolated in tissue blocks cultivation and identified by using microstructure examination and ITS gene sequence analysis methods, the phylogenetic diversity of fungi isolates was also investigated. Sixty-six fungi strains were isolated from the samples. The molecular phylogeny indicated that the fungi of Basidiomycota mainly belonged to Tulasnellaceae, and the fungi of Ascomycota which was predominant mainly belonged to Helotiales, Hypocreales and Xylariales. Mycorrhizal fungi specificity at the species level has not been found except for Galearis diantha, which was associated with genus Hypocrea only through out the whole growing season. Species number and Simpson diversity index (D) of mycorrhizal fungi from the orchids growing in a forest habitat were higher than those in a travertine habitat. The variation patterns of mycorrhizal fungi diversity from both habitats were synchronized with the season changing pattern of the Huanglong Vally.2. Thirty-three endophytic bacteria strains associated with nine species orchids were isolated and identified by Grams staining and sequencing 16S rDNA gene. The most commonly isolated genus was Pseudomonas and Bacillus, follwed by Antarctic bacterium, the first time that psychrotrophic bacteia have been found in wild terrestrial orchids. The total number of colony formine units varied with the growth stage of host orchids and habitat ecological conditions.3. The average temperature of soil in forest habitat was lower than in open habitat. Properties of soil differences existed for the distinct orchid species and habitats. Fifty-eight fungi strains were isolated from rhizophere soil. Colony number of saprophytic fungi raised as the growing seasons, and it was relative common for the rhizophere soil of night orchids to share similar species during the same growing period or habitat. While in non-orchids area, soil fungi belong to saprophytic type entirely. 4.22 endophytes fungi were use in symbiotic germination in vitro which was to investigate the specificity between orchids and mycorrhizal fungi. Seeds of seven orchid species germinated with most fungi strain except Cypripedium flavum and Tipularia szechuanica. In fact, seeds of Cypripedium flavum got high viability according to TTC staining examination. On the contrary, Corallorhiza trifida, Phaius delavayi and Platanthera minutiflora display relative low viability. The embryo all seeds germinated swelled, or few of them developed radical hairs. The results showed that seed germination rate related to orchid mycorrhizal specificity more than seed viability.5. In order to investigate the specificity of tropical orchid mycorrhizal association,22 endophytic fungi mentioned above were inoculated with the seeds, protocorms and seedlings of tropical orchid species Doritis pulcherrima in flask. After twenty weeks, the embryo swelled just with two fungi strains in symbiotic germination. The protocorms differentiated into leaves or roots obviously with nine strains. In the seedling-fungi symbiotic cultivation, eleven fungi strains could make the fresh mass of seedling higher than the control, especially as for the treatment with fungi Mm-1. The results suggest that some variation of the specificity exist as orchid grows. It might connect with survival strategy of paticular orchid species.