Study On Diversity And Molecular Ecological Distribution Of Tolypocladium Species In Forest Soil Of Changbai Mountain

Changbai Mountain Nature Reserve, which is an intact ecosystem in the Eurasian, is a treasured place for species diversity. From June to September in 2013 and 2014, 324 soil samples were collected in zigzag route by means of multipoint sampling in different types of forest and soil within elevation of 800-2650 m in the North Slope of Changbai Mountain. Totally 133 strains of Tolypocladium, which were isolated from the soil samples, were obtained by method of soil-diluting plate and soil particle plate.The strains of the Tolypocladium we obtained were conducted by the method of statistic analysis, the characteristics of distribution of the Tolypocladium isolated from the Changbai Mountain was that:The number of the Tolypocladium we obtained increased gradually from the altitude of 1500-1800 m, reduced gradually from the altitude of 1800-200 m, and also increased gradually from the altitude, but no strain was isolated above the altitude of 2600 m. The number of the stains of the Tolypocladium was declined gradually from June to August, and raised again in September, but it was less than that of July.The identification and the genetic relationship of 133 strains of Tolypocladium were conducted by methods of molecular biology, the 18 S rDNA-ITS sequence analysis was compared with the elongation factor 1-α sequence analysis, the phylogenetic tree constructed by the 18 S rDNA-ITS sequences was not in accord with the results of the Blast, he phylogenetic tree constructed by the elongation factor 1-α sequences was more precise than the tree constructed by the 18 S rDNA-ITS sequences, so the result of the elongation factor 1-α sequence analysis was more reliable than that of 18 S rDNA-ITS sequence analysis.The genetic diversity of the Tolypocladium, which was isolated from the Changbai Mountain, was also estimated by the method of ISSR molecular markers. 10 among 40 ISSR universal primers were chosen for their reproducibility and high polymorphism. The results indicated that: high genetic polymorph was showed, and the polymorph bands was 79%,and the productions of the amplication had a good repeatability; The results also suggested that a low degree of genetic differentiation occurred in the Tolypocladium among the population, namely, the altitude and months had little influences on the genetic variation of the Tolypocladium isolated from the Changbai Mountain.The biological characteristics of three species of the Tolypocladium(Tolypocladium niveum, Tolypocladium cylindrosporum and Tolypocladium geodes), such as medium, pH, light, sources of nitrogen and carbon, were studied. It turned out that on the same condition, the optimum light conditions was light from darkness alternating, the most suitable medium was PDA, for the spore germination of the Tolypocladium, the optimal carbon source and nitrogen were glucose and ammonium sulfate, respectively. The differences among the three species were that: the growth vigour of the T.niveum was the best and its sporulation quantity was highest; the optimum medium of T.niveum was PDA; the optimum temperature was 25℃; T.niveum was not sensitive to pH, when pH was 7; its spore production quantity was the highest. The optimum source of carbon was glucose; the optimum source of nitrogen was ammonium citrate. The optimum medium of T.cylindrosporum was PDA; the optimum temperature was 25℃.