Studies On Relationships Between Swainsonine And Fungal Endophytes In Four Populations Of Oxytropis Glabra

Oxytropis glabra is a kind of plant that widely distributes in grassland and desert regions of Inner Mongolia. Some contain alkaloids called swainsonine which causes locoism by inhibiting the activity of mannosidase in cell. Livestock is poisoned and then suffered locoism when taking certain amount of O.glabra. With the poisoning plant spreading, it causes significant loss for animal husbandry on grassland. It has great theoretical and applying values to study the poisoning mechanism of swainsonine in O.glabra. In addition, O.glabra has important medicinal and ecological values. It is excellent forage when removing swainsonine. The swainsonine is a potential anticancer medicament as well.This research used the part of stem of O.glabra. as explant to cultivate fungal endophytes and to study the morphologic characters of the fungal colony. The 5.8SrDNA/ITS sequences of the fungal endophytes which cultured in vitro were amplified and then sequenced, and then the sequences were submitted to GenBank and compared with the published sequences of fungal endophytes from O.glabra, and other locoweed by blastn program. The populations of fungal endophytes were analyzed according to the results of molecular biological and microbiological studies. The fungal specific sequences of 5.8SrDNA/ITS in individual plants from which the fungal endophytes were not cultured were also sequenced. The swainsonine was extracted both from individual plant of O.glabra and the fungal endophytes. The swainsonine contents in samples were determined by gas chromatography. To study the relationship between swainsonine from O.glabra and the fungal endophytes in O.glabra, we optimize the medium for culturing fungal endophytes to analyse the influences of different medium on the composing of swainsonine. The results were as follows:1.22 fungal endophytes from O.glabra were isolated and cultured. 21 of them were same to the 10 fungal endophytes from O.glabra and the Embellisia sp. L12 in published papers in morphologic characteristics of the colonies. The sequences of the 5.8SrDNA/ITS of our fungal endophytes in O.glabra were totally the same as that of published fungal endophytes in O.glabra. Compared to the sequence of Embellisia sp.L12, there was only one variation site in 5.8SrDNA region. Therefore the fungal endophytes from O.glabra were considered to belong to Embellisia. Another fungous called OTQ1.15 was slightly different from fungal endophytes mentioned above in morphologic characters. Since its sequence of 5.8SrDNA/ITS were 99.0% of identity compare to the sequences mentioned above from O.glabra, and 98.8% of identity compare to the sequence of Embellisia sp.L12, the fungal endophyte of OTQ1.15 was also considered to belong to Embellisia.2. The fungal specific bands were amplified from all 60 plants of O.glabra and 51 of them contained swainsonine. The plant which swainsonine was detected contained fungal endophyte in it. The contents of swainsonine in fungal endophytes cultured in vitro were higher than that of swainsonine in their host plants.3. Four kinds of media including potato-sugar-agar, potato-dextrose-agar, yellow bean sprouts extract agar and Czapek's were used to culture fungal endophytes from O.glabra. The growth rate of fungal endophytes was fastest in potato- sucrose -agar medium, and then in potato-dextrose-agar medium. The lowest growth rate was in the yellow bean sprouts extract agar medium. The content of swainsonine in mycelium was highest in potato-dextrose-agar medium, and then in potato-sucrose-agar medium. The lowest content was in yellow bean sprouts extract agar medium.4. The fungal endophytes were inoculated in liquid medium. The fungal endophyte grew slowly at first and the mycelium.pellets formed in one or two weeks later. The mycelium pellets were white, and the diameters of them were generally about 0.5cm to lcm. The mycelium pellets stopped growing after 20 to 30 days, and the yellow fermentation liquid became dark, and then changed from gray, dark gray to dark black. The contents of swainsonine in mycelium pellets were lower than that of swainsonine both in the host plants and in mycelium cultured on solid media.