Diversity Of Endophytic Fungi In Three Cypripedium Species In Northern China And Their Effects On Protocorm Development

The genus of Cypripedium, belonging to the subfamily of Cypripedioideae in Orchidaceae, had been recognized as a global hot spot for its high economical, ornamental and medicinal values, most of the Cypripedium orchids have been endangered for over-collection. In the natural condition, the orchid plants need the participation of mycorrhizal fungi during all of their historical life cycle. The orchid fungi need to be infected by mycorrhizal fungi for sucessful germination, some of the adult orchid plant also need the mycorrhizal fungi to supply nutrition for growing. The study about the orchid and mycorrhizal fungi have practical fundation and broad development prospect.The northern China is the major production place of several Cypripedium species. So far, little attention has been paid to the compositions, species richness and seasonal fluctuates of endophytic fungi within the root of Cypripedium orchids. In the present study, four Cypripedium genera in three habitats were selected for this reaearch. the compositions and seasonal changes of endophytic fungi within the root of Cypripedium orchids were investigated using traditional tissue segments culture method and molecular biology techniques.In order to evaluate the effect of endophytic fungi isolates on orchid protocorm development and discuss the specificity between the orchid and the fungi, the protocorm of Cypripedium macranthos Sw. was inoculated with fungi strains isolated from adult orchids. The main results are as follows:33endophytic fungi strains were isolated and cultured from the root of three Cypripedium species,6strains belong to Basidiomycota,27strains belong to Ascomycota. About6Classes,12Orderes,23Families and30Genus were involved. Sordariales, Hypocreales, Helotiales had a high frequent in Ascomycetes. Only the Cantharellales was isolated within Basidiomycota.The different Cypripedium species in the same habitats were tend to isolated the similar endophytic fungi, but difference still exist in the total fungi species. The diversity of mycorrhizal fungi within the same Cypripedium species in the different habitats tend to form mutualistic symbiosis with several specific orders fungi, but the fungi species belongs to different orders for the effect of habitats.The isolation rate of endophytic fungi in roots of Cypripedium macranthos Sw. with the same age have great changes in different development stages, the isolation rate were as high as34.52%and55.95%at sprouting and growing stages respectively, whereas only about4%at blooming, fructification and decay stage. The identification result using molecular biology techniques showed that Tulasnellaceae was responsible for the seasonal fluctuates of endophytic fungi. Moreover, the variation of the isolation rate of Tulasnellaceae was synchronized with the growing seasons change of Cypripedium orchids.Symbiotic cultivation of in association with7fungi strains isolates showed that only3Tulasnellaceae of them had positive effects on promoting the protocorm development. Cadophora melinii, Fimetariella sp.,Xylaria sp., and Cylindrocarpon sp. were lethal or hinder for the protocorm development. Our results showed seemly evidence that specificity of fungi on Cypripedium orchids in symbiotic cultivation in virto. The Tulasnellaceae had significant effect on promoting the bud differentiation and decreasing the browning rate of Cypripedium macranthos Sw. protocorms.