Study On Endophytic Insecticidal Activity And Diversity Of Achnatherum Inebrians

Endophytes were isolated from roots, stems, leaves and seeds of health A inebrians which growth in different trophophase of Nanshan Mountain in Xinjiang.Taking different separating culture mediu and grinding separation method to isolate from roots, stems, leaves and seeds of health A. inebrians;they were identified by a dual approach of morphological and physiological observation and 16S rDNA gene (for bacteria) and ITS sequence (for fungi) based molecular identification. Then, those endophytes were inoculated into liquid media for fermentation and the crude extracts were used to insecticidal activities tests by slide disc immersion and nebulization methods. The community diversity of endophytic bacteria in A inebrians was investigated by using culture-independent and culture-dependent approache. Analysis of the endophyte community of A.inebrians.by Terminal Restriction Fragment Length Polymorphism and the similarity of microbial community in sample. Major results were as follows:91 endophyte were isolated from 12 strains A. inebrians .Most of them were Baeillus, of the total there were 2 fungi and 2 actinomycetes classified into 10 genera.In this study the insecticidal activities of 91 endophyte strains from A. inebrians were assayed.The result showed that the strains GA,PF-2,2N153,2N185,2P118 were high mortality to cotton aphis after primary and second screenings. Of them, the strain Streptomyces rochei (GA) and Claviceps purpurea (PF-2) were showed more than 85% of mortality to cotton aphis.Samples were strictly surface-sterilized followed by CTAB-SDS DNA Extraetion Protocol to obtain their genome DNA respectively.To construct 16S rDNA clone libraries,primers 799f and 1492r were selected to amplify 16S rDNA gene. Fifty seven OTUs,including 249 Positive clones, were assigned according to their band pattern similarity in A.inebrians clone library.The representatives of each OUT were sequenced.The results showed that Proteobacteria was the dominant group(143 OTUs,57% of the total clones),followed byα-roteobacteria(16% of the total clones),β-proteobacteria(28% of the total clones),γ-proteobacteria(8% of the total clones), δ-proteobacteria(2% of the total clones),ε-proteobacteria(3% of the total clones). Firmicutes was the second dominant group(31 OTUs,12% of the total clones).The clones were highly related to the known bacteria in the genus Bacillus(>98% sequence similarity). Bacillus is the most dominant species,it was identical with the result of culture-dependent. 7% of the total clones were belong to Actinobacteria and 11% were belong to Bacteroidetes.The results of T-RFLP analysis program indicate that fungies in seed of Achnatherum inebrians. were the most abundant.All the predominant bacterial population were Bacillus(above 29%) in different organism of Achnatherum inebrians.. The predominant fungal population were Mycosphaerella (6.5%),Teratosphaeria(4.5%),Fragum(1.1%),Sebacina(11.3%) in seed,leaf,stem and root.The investigated that bacterial population relationship between root and stem is similar,and it is similar between leaf and seed. Otherwise, the fungal population relationship is closer between root and seed,then is the stem,the last is the leaf.In culture-independent study also identified there were potentially exist new strain source. Culture-dependent method result showed that Bacillus is the most dominant species of endophytic bacterial in Achnatherum inebrians.