Diversity Of Mycorrhizal Fungi Isolated From Five Species Of The Genus Paphiopedilum

The genus Paphiopedilum is very well known as lady’s slipper orchid in horticultural science. Paphiopedilum orchids, as one of the most attractive flowers in the Orchidaceae, have numerous fans in the world. At present, wild populations of Paphiopedilum orchids are under great threat as a result of habitat destruction and over-collection for their beautiful and unique flowers. All species of this genus have been classified as endangered species in Appendix I of the CITES. Orchids are myco-heterotrophic plants. In nature, dust-seeds of orchid can germinate only when they are infected with appropriate symbiotic fungi which supply carbon and inorganic nutrients. Furthermore, orchid seedling development, adult growth and reproduction also need mycorrhizal fungi. Based on the different habitat, distribution, ecotype and classification status, P. purpuratum, P. dianthum, P. hirsutissimum, P. micranthum and P. malipoense were selected as samples. This study was tried to investigate the mycorrhizal structure, the mycorrhizal fungi diversity and the relationship between orchid distribution and symbiotic fungi. Meanwhile this study was also tried to establish more effective isolation method and symbiotic cultivation technology. The main results were as follows:1. The roots of P. purpuratum, P. micranthum and P. malipoense grew in the litter on the face of soil. The root external morphology of these three species was significantly different from the real terrestrial orchid. The roots of semi-epiphytic P. hirsutissimum and epiphytic P. dianthum grew in the litter on the surface of rocks and also different from the real epiphytic orchid. The basic structure of monocotyledonous plant roots was observed in these five species by optical microscopy and scanning electron microscopy. The exodermis structure, the number of cortical and vascular bundles of P. purpuratum, P. micranthum and P. malipoense was different from two lithophytic species. The exodermis structure of semi-epiphytic P. hirsutissimum was similar to three terrestrial species. The cortical and vascular bundles of this species, however, were similar to epiphytic P. dianthum. There were many pelotons that presented different forms in the cortical cells of these five species. The pelotons in the adjacent cortical cells linked through communication hyphae. A large number of starch grains were observed in cortical cells when the pelotons were digested.2. The technique of pelotons isolation from orchid mycorrhiza was improved. The four steps were as:(1) preparation of isolating medium disks,(2) treatment and selection of roots,(3) preparation of the suspension of pelotons,(4) isolation and purification of mycorrhizal fungi. The other three kinds of separation method and two kinds of the separation medium were compared in this study. More Rhizoctonia-like fungi were obtained using our technique of pelotons isolation and separation medium.3. Sixteen strains of Rhizoctonia-like fungi were isolated from the root pelotons of these five species and identified by morphological and molecular characteristics. All these strains belonged to Epulorhiza (anamorph of Tulasnella). Based on the phylogenetic analysis of the ITS-5.8S rDNA, sixteen isolates of Epulorhiza were separated into eight types. The fungi isolated from the roots of P. hirsutissimum and P. malipoense belonged to Type1and Type2. The fungi isolated from the roots of P. dianthum belonged to Type2and Type7. The strains associated with P. purpuratum were divided into Type3, Type4and Type8. The fungi isolated from the roots of P. micranthum belonged to Type5and Type6. These results showed the species in common habitats, like P. hirsutissimum, P. dianthum and P. malipoense, shared the same fungal taxa and P. purpuratum from distinctive habitat was associated with special fungal species of Epulorhiza. The molecular phylogenetic analyses of Cypripedioideae orchids and their mycorrhizal fungi indicated that specific relationship between them was complex. Cypripedium and Paphiopedilum species had its own unique mycorrhizal fungi. They also shared mycorrhizal fungi in the same clade.4. In situ and in vitro symbiotic seed germination of P. purpuratum, P. dianthum, P. hirsutissimum and P. malipoense were carried out. The results showed that seed germination of Paphiopedilum were difficult in nature. The germination rate of P. purpuratum in its habitat was1.06×10-4after buried12months. The germination rate of P. hirsutissimum in its habitat was3.8×10-5. The seeds placed in the site which grown Paphiopedilum adult plants were easier to germinate. The widespread P. hirsutissimum was generalist in its association with fungal symbionts compared to other three restricted species in vitro symbiotic seed germination test. This may be one of the reasons for its wide distribution.5. Several strains can promote seed germination of P. hirsutissimum in vitro symbiotic seed germination test, one of which has patent protection. It is the first report of obtaining Paphiopedilum symbiotic seedlings which can be used for transplanting by two kind of symbiotic seed germination techniques.