| Geodorum G. Jacks.is a typical perennial terrestrial orchid genus of Orchidaceae. Yachang Orchid Nature Reserve in Guangxi is one of distribution areas of Geodorum plants. With the destruction of human activity and habitat fragmentation, population of Geodorum in this area is becoming quite low and the species Geodorum eulophioides is hanging by a thread and waiting for protection. In this research we take3species of Geodorum which live in Guangxi Yachang Orchid Nature Reserve as research objects, try to explore the symbiotic mycorrhizal fungi of Geodorum and their specificity by isolation and identification of mycorrhizal fungi, and try to obtain seedling of3species of Geodorumby in situ germination. This research aims at the recovery of the population of Geodorum plants in Yachang and protects the resource of Geodorum plants.Results went as follows:(1)431isolates were isolated from root samples of adult Geodorum plants by single peloton isolation method, in which one isolate comes from a single peloton in plant roots, and plenty of them own the same morphology features. After screened,20isolates were identified by both morphology and molecular methods. Result showed that those isolates belongs to Epulorhizasnd Fusarium, in which Epulorhiza(teleomorph Tulasnella) is the most common taxon of mycorrhizal fungi.(2) An analysis on sequence of ribosomal DNAintemal transcribed spacer (ITS) regions was done in those15Epulorhiza isolates, and phylogenetic trees were constructed with mycorrhizal fungi of wide distributed terrestrial orchids. Result showed that the relationship between Epulorhizaisolates and reference sequences were quite close, and isolates from the same root sample have very close relationships with each other; the specificity inside the genus Geodorumis very strong, and it is unrelated to geographic location of plants; the specificity of G. densiflorum, G. eulophioides is stronger than that of G. recurvum, which may be one of reasons of distribution restriction.(3) We improved a method of surface disinfection of orchid seeds usingseed bags made of nylon mesh. The disinfection effect of2%NaClO solution with processing time6min, which the germination rate of Geodorum eulophioides reached33.88%, and it had no significant effect on the germination rate.The total germination rates of seeds of3species (G. recurvum, G.densiflomm, G. eulophioides) reached38.22%,48.01%and23.95%respectively, and protocorms successfully established seedlings after transferred into light condition and differential medium for17months. Different hormone combination of medium has impact on seed germination, which1.0mg/L GA3enhanced the germinate of G. eulophioides, which germination rate raised from12.29%to30.67%. (4) In symbiotic germination test, few seeds of3species had the ability to absorb moisture and none of them germinated with fungi isolated from adult Geodorum plants. Those isolates have no influence on seed germination.The probable reason is that the fungi used in this test were isolated from adult plant which cannot promote the growth of germination stage.Ex situ test were not successful since the original living condition of orchids were harsh, no seedling were observed during23months of test.(5) In mycorrhization test of G. densiflorumand G. eulophioides, the growth rate of protocorms between experimental and control groups were not significant. No pelotons were found in cross section of rhizome, which means that symbiotic relationship was not built between fungi and protocorms.On the other side, the mycorrhizal fungi isolated from adult plants of Geodorum cannot promote germination of seeds and differentiation of protocorms, which indicates the specificity of Geodorom when they choose their symbiotic partner. |
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