Studies On Detecting Methods And Microstructure Of Epichlo(e|") Endophytes Of Grasses

Using Achnatherum inebrians, Festuca sinensis, Hordeum brevisubulatum and Elymus dahuricus as experimental plants in this study, on the basis studies of light microscopy, transmission electron microscopy on microstucture of Epichloe endophytes, we used tissue print immunoblotting test and high performance liquid chromatography of alkaloids detection to detect Neotyphodium species, asexul Epichloe endophytic fungi, in different grasses qualitatively. The study offered the basis of quantitative determination of endophytic fungi. The main results are as follows:1` By observing the internodal region of stems, sheaths, blades and seeds of the four gramineous grass species under the light microscopy and transmission electron microscopy, we found that the endophyte of each species were common associated in the internodal region of stems, sheaths, blades and seeds. In the internodal region of stems, sheaths and blades, hyphae characterization of the four species were similar; the hyphae were densely distributed in the internodal region of stems and followed by sheaths, but were sparse in blades; they were long, thin and unbranched, and longitudinally orientated in the cell extension direction, straight or curved. In the seeds, endophyte was widespread in the aleurone layers of F. sinensis, H. brevisubulatum and E.dahuricus, but rarely observed in the aleurone layers of A. inebrians. We could obviously observe mycelia in the seed coats and embryo of all the four grasses.2. We uesd double-antibody sandwich tissue print immunoblotting test to detect the endophyte in A. inebrians and H. brevisubulatum respectively. The results indicated that the antiserum of Lolium perenne also could be used in A. inebrians and H. brevisubulatum. Moreover, the sensitivity of endophyte detection of tissue print immunoblotting test was higher than that of the traditional method, especially in the initial stage of seed germination.3. Detecting the variation of contents of ergine and ergonovine from A. inebrians by HPLC during different growth stages, we found that there were huge variations between individuals, and that the alkaloids content of both two species achieved the peak in the middle stage of the grass life, then gradually decreased along with the growth of the grass. This result could reflect indirectly the change of endophytic fungi development, which may grow as seedlings develop and stop expanding when the grass mature.