Diversity Of Ericoid Mycorrhiza And The Related Fungi Isolated From Hair Roots Of Rhododendron Fortunei L.

Most members of family Ericaceae have a distinctive form of the symbiosis referred to as ericoid mycorrhiza (ERM), which appear to be able to mobilize nutrients from organic matter and alleviate certain environmental stresses, such as readily available metals, poor or very free drainge and high or tow temperatures et al., and so facilitate the establishment and survival of Ericaceae. And some ERM fungal strains were found beneficial for the growth of Ericaceous plants in vitro or nurseries. In order to understand and utilize the mycorrhizal potentials, and satisfy the requirement of ecology constructing and horticulture producing, ericoid mycorrhiza has attracted abroad attention in overseas. In China, various ericaceous species are distributed widely in various types of forest vegetation. However, no fungal isolates have been reported from the roots of those plants. Rhododendron fortunei L., a broadleaf rhododendron, is originated and distributed widely in China. The research focus on the diversity of ericoid mycorrhizal fungi isolated from the hair roots of R. fortunei in natural habitats in China. The main experimental results are as follows:The microstructure and colonial rate of mycorrhizal root of R. fortunei were observed with electron microscope scanning and fresh mount of hair root stained with trypan blue and resin sectioning. The results showed that mean percent root cell colonization of hair roots from natural sites were over 65%; the hair roots consisted of one layer of epidermis cells and 1-2 layers cortical cells surrounding the stele, they did not form root hair and were covered with loose or compact fungal mantle; the presence of the mycorrhizal fungus in epidermal cells did not appear to disrupt the primary tissue structure of the root, except that mature epidermal contain ericoid mycorrhizal hyphal coils; the fungal complex were mostly observed in epidermal cells and the cell wall of 90% epidermal cells with fungus were not obviously thickened; as judged by the size and structure of the fungal complex, at least two types of fungi appeared to form mycorrhizal coils in epidermal cells. One type was a thick mycelium fungus which formed tight coils, the other was a thin mycelium fungus formed loose coils.We successfully modified the method of isolation of ericoid mycorrhizal fungi according to Pearson & Read (1973) from the ericaceous hair roots. Firstly Martin agar medium was modified and introduced to ERM fungi isolation. So the contamination rate of bacterium and actinomycetes were greatly decreased. In total, 280 slow-growing fungal isolates were obtained from hair roots of R. fortunei from 4 natural habitats. The isolates were initially grouped as 17 types based on the results of ITS-RFLP analysis and the colonial characters. ITS sequences were obtained for representative isolates from each RFLP type and compared phylogenetically with sequences for known ericoid mycorrhizal endophytes and selected ascomycetes. Most of fungal isolates were also ascomycetes, and the sequence similarity is 89%-99%. 7 types of 14 RFLP types are related to known ericoid mycorrhizal endophytes, such as Oidiodendron maius,Epacris microphylla root associated fungus,Epacrid root endophyte sp.. Cryptosporiopsis ericae et al.; 2 types are Phialocephala-like strains; 2 types are unclassified ascomycetes; 1 type is similar to Cladosporium cladosporioides; 1 type is unknown fungal strains. Among the fungal strains, over 70% types were Simultaneously isolated from the hair roots of 2 natural sites. The fungal isolate types are also different among 4 natural sites. Phialocephala fortinii-like strains are the dominant fungal isolates from Huading and Mufu mountain forest parks, while Oidiodendron maius strains are dominant in the isolates from the other two Rhododendron fortunei habitats.In order to verify ericoid mycorrhiza status, fungal strains of each type were inoculated on the seedlings of R. fortunei in vitro. During the inoculation experiment the most seedlings remained healthy with well-developed root system. Based on the results of fresh mount of hair roots stained with trypan blue, 9 types, 33 fungal strains, were initially identified as ERM fungi with typical ericoid mycorrhizal coils in epidermal cells of hair roots; 8 types were not detected fungal coils in the hair roots of inoculated seedlings. Among the 9 types putative ERM strains, 5 types were identified as Oidiodendron maius and different Epacris root associated fungus which are ERM fungi from other ERM plants; 3 types were confirmed as Cladosporium cladosporioides, Cryptosporiopsis ericae and Phialocephala ftuminis; 1 fungal type was unknown strains.Most ERM fungal strains showed obviously beneficial to growth of the seedlings, normally the biomass of colonized seedlings increased by 20-250%, the highest being 400%. Some Epacris root associated fungus, C. ericae and unknown fungal isolates were firstly shown significantly positive effects on the colonized seedlings. At the same time, 5 ERM fungal strains were tested for the ability to provide their host with access to inorganic and organic N. Regardless of which mycorrhizal fungus colonized the roots and which nitrogen source was provided; mycorrhizal plants grew better than nonmycorrhizal plants. There were, however, differences among the fungi in use of the N source. Colonized seedlings on nitrate and organic nitrogen grew much better than on ammoniacal nitrogen and control medium, especially on BSA medium.In conclusion, we suggested that the presence of ericoid mycorrhizal associations play an important role in success of R. fortunei at natural habitats and the growth of the seedlings at artificial conditions. In vitro or nursery inoculating seedlings with ERM strains maybe a good applied technique to resolve some problems in Rhododendron propagation and cultivation.