Study On The Interaction Between Epimedium Wushanense Seedlings And Dark Septate Endophytes From Roots

Epimedium wushanense T.S. Ying is traditional Chinese medicinal plant, which mainly distributes in Hubei, Sichuan and Guizhou province. In national pharmacopoeia (2010), E. wushang was recorded. There are not many published paper research on the endophytic fungus of E. wushanense plants. This paper studied the dark septate endophytes (DSE) isolated from the roots of E. wushanense plants and the interactions between the fungus and the E. wushanense seedlings. Also, the mechanism of dormancy and the breaking method for the seeds of E. wushanense was researched. The main results were showed as the following:(1) Anatomic observations of the infected roots showed that there were typical morphological structures of DSE in the roots of E. wushanense. Melanized, septate hyphae invaded the epidermal cells or cortical cells. The hyphae grew parallel to the main axis of the root and from cell to cell within the cortical cells, without causing apparent negative effects such as tissue disorganization. The intracellular hyphae formed "microsclerotia" within root cortical cells. The infection ratio of DSE was 78.3%.(2) 97 endophytic fungal strains were obtained from roots of E. wushanense, and ascribed to 7 DSE strains based on their macro- and microscopic features. The 7 strains were sterile, and no isolates appeared to produce any asexual or sexual reproductive structure. Sporulation assays were unsuccessful in all circumstances. The ITS1-5.8S-ITS2 nuclear ribosomal gene cluster of the 7 representative isolates was sequenced, and the sequences were recovered in 7 sequence groups. All of them were congeneric to ascomycetes. DSE8 and DSE5 were closely related to Leptodontidium. DSE10 could be placed in the Podospora. The remaining showed high homology to a large number of currently unassigned anamorphic ascomycete sequences.(3) The dormancy mechanism of E. wushanense seed was studied in order to find out the breaking method. The anatomy observation showed that:the seed had a tiny embryo, and the embryo rate was 15.39% only. When the seed harvested, the embryo was differentiated but underdeveloped. It developed cotyledon but need time to grow for permitting germination. The results showed that there was no obstacle of water permeability on the test of E. Wushanense seeds, after-ripening of embryogenesis and the germination inhibitor of the seed were the main reason for the seed dormancy. The cold stratification would be an effective way for breaking of the dormancy, which could significantly promote the seed embryogenesis and increase germination rate comparing to other methods. Soaking proper concentrations of GA3 pretreatment combine cold stratification could increase germination rate and germination capacity. The best treatment obtained in this experiment was that soaking with 300 g·mL-1 GA3 for 24 hours and then exposure to cold stratification at 5℃ for 90 days, which could raise germination rate and germination potential to 78.67% and 51.33%.(4) After 90 d of inoculation, epidermal cells and cortical cells of E. wushanense seedlings were colonized by melanized, septate hyphae, but no "microsclerotia" formed within host epidermal cells.(5) The results indicated different effect of the inoculation with different DSE strains on the growth of E. wushanense plants. The DSE5, DSE10, DSE8 and DSE1 were not pathogenic to seedlings. In contrast to the uninoculated controls, E. wushanense seedlings inoculated DSE8 had significant enhancements in plant height, root height, leaf area, new roots and leaves number, total flash weight and roots weight. Inoculated DSE8 could enhance the activity of protective enzyme (SOD, CAT) and reduce the contents of MDA of E. wushanense leaves. At the same time, Inoculated these strains proved better influence on increasing the contents of dissolved sugar, soluble protein, Icariin and total flavonoid. The results showed there were profoundly promotive effect of DSE8 on E. wushanense growth and intrinsic quality. The E. wushanense seedlings inoculated DSE5 or DSE2 were stunted. The biomass of these stunted seedlings were significantly decreased compared to uninoculated plants. The results indicated a neutral effect of the inoculation with DSE 10 on the growth of E. wushanense, except increasing the activity of CAT and the the contents of total flavonoid, as well reducing the leaf area and the contents of MDA. The leaves of plants inoculated the DSE1, DSE3 or DSE7 turned to chlorosis. After inoculation, the seedlings were wilting to death.