|其他摘要||Mycorrhizae is a mutual symbiont produced during the long course of coevolution of plants and fungi. Ectomycorrhizae (EM) is one of the most important mycorrhizal types. In the rigorous alpine environment, plants have to confront adversities such as low temperature, low atmospheric pressure, sterile soils, low decomposed rate and short growing period, and paucity of nutrients. Ectomycorrhizae may be much more indispensable for plants in the alpine areas than in other regions. Alpine-meadow is one of the main vegetation types in China’s southwestern mountains. However, nothing has been known about ectomycorrhizae of herbs in the alpine-meadows. EMF diversities of four dominant herbs, i.e. Kobresia filicina, K. capillifolia, Polygonum macrophyllum and Potentilla stenophylla in Hongshan region were studied based on the analysis ITS rDNA sequence. In addition, structure and annual dynamics of mycorrhizae of four species of Cypripedium, i.e. C. flavum, C. yunnanense, C. tibeticum and C. guttatum in Shangri-la were studied. Coculture of C. flavum, C. guttatum with four strains of fungi isolated from the four Cypripedium species, and in situ culture of axenic seedlings of the two Cypripedium species were carried out. Symbiotic relationship between Cypripedium species and isolated fungi was preliminarily investigated. The main results are summarized as follows:
1. Diversity of EMF associated with four herbs in Hongshan alpine-meadow of southwestern China
Species diversity of EMF on Kobresia filicina、K. capilifolia、Polygonum macrophyllum and Potentilla stenophylla in Hongshan alpine-meadow of China’s southwestern mountains was estimated based on rDNA sequence analysis of ectomycorrhizal root tips. Phylogenetic analysis of LSU rDNA showed that EMF distributed in a series of families (genera) (Fig. 2-15). 233 ITS rDNA sequences were obtained from roots of 90 plots of the four plant species. Among them 176 were those of EMF, representing 100 EMF OTUs, which distributed to 13 genera of Basidiomycota and Ascomycota. Most of them belong to Basidiomycota, they are 87 OTUs distributed to 10 genera, species richness is high in Inocybe (23 OTUs) and Tomentella/Thelephora (24), followed by Cortinarius (9), Sebacina (8), Russula (9), and low in Laccaria (4), Lactarius (2), Hebeloma (3), Amanita (2) and Boletus (1). 13 OTUs belong to 3 genera in Ascomycota, they are Cenococcum geophilum complex (8), Hymenoscyphus (3) and Lachnum (2). EMF diversity of Inocybe and Tomentella/Thelephora was high for all the four plants and three seasons, members of the two genera were dominant mycobionts of the four plants, and many of them are widespread species. They may be well adaped to the environment, able to form mycorrhizae with diverse plants in diverse regions. In addition, 57 ITS rDNA sequences of other Ascomycota species were detected from ectomycorrhizal root tips of the four plants, representing 36 OTUs. They may be mycobionts (arbuscular mycorrhizal fungi, dark spetate fungi or EMF) coexisting in the roots of the plants.
Ours and former researches on EMF of alpine plants showed that diverse plants in different geographic regions share the same families and (or) genera of dominant mycobionts, while EMF of different regions differ at species level. Most of the EMF species detected in our study are endemic to southwestern China. Researches show that EMF diversity of alpine herbs in southwestern China is significantly higher than that of alpine plants of the same genera in Europe. Geological and historical events may dramatically have shaped the diversity.
Dauciform roots with EMF were detected on the two Kobresia species for the first time. 11 EMF were detected on them, 8 of them were also detected on ectomycorrhizal root tips. Both dauciform roots with EMF and ectomycorrhizae are produced when plants and fungi are adapting to the alpine environment.
Significant differences of average mycobiont species diversity per plot were not discovered among the three seasons and the four plant species in our study. Seasonal shifts of mycobiont dominance were documented by increase of species richness of Inocybe and Cortinarius in September compared to May and July, increase of that of Tomentella in May compared to July and September, and decrease of that of C. geophilum complex and Russula in July compared to May and September. As environmental conditions change among seasons, and groups of fungi are adapted to diverse environment, those more adapted to a given environment are more likely to become dominant mycoionts at that time.
2. Mycorrhizal structure of four Cypripedium species, coculture and in situ culture of tissue culturing seedlings of C. flavum and C. guttatum
Roots of four subalpine Cypripedium species (C. flavum, C. yunnanense, C. tibeticum and C. guttatum) were microscopically observed throughout their annual lifecycle. The common structure and annual changing pattern of their mycorhizae are as follows: 1) Fungi infect and form mycorrhizae with the Cypripedium species all the time, and four types of hyphae exist in the cortical cells of their roots: a. coffee-colored or yellow big peloton consisting of twisted hyphae, b. dust-colored or yellow big peloton consisting of fragmentary hyphae, c. colorless scattered hyphae, d. orange or yellow small peloton with blurry hyphae; 2) The four types of hyphae are detected in different phases of the infection-decomposition circle, which goes round and round in the plants’ grown period.
Axenic seedlings of C. flavum and C. guttatum were cocultured in sterilized autochthonous soil with four fungal strains isolated from the roots of Cypripedium species in laboratory. Two of the fungi were apparently shown to be deleterious to the two plant species, while the other two (fungi of Tulasnellaceae) facilitated growth of C. flavum to a certain extent, and may be mycorrhizae of Cypripedium species.
Tissue culturing seedlings of wild C. flavum and C. guttatum were planted in the autochthonous natural environments. It was found that none of the seedlings was infected by mycorrhizal fungi after plantation of 1, 2 or even 3 months,, which may suggest that the roots of the seedlings are anatomically unsuitable for fungal infection, or metabolites (especially the carbohydrates) they produced are not enough to supply for fungi.|