Diversity Of Endophytic Actinomycetes From Sophora Alopecuroides L.and Excavating Antibiotic Synthetase Genes

Endophytic actinomycetes was in the same category of microbial which were living in plants all or a part of their lives, they were widespread in the all kinds of terrestrial and aquatic plants which had currently been found. Their diversity was influenced by many factors, such as different areas or site, climate conditions, and vegetation. Researchs had showed that endophytic actinomycetes from medical plants had better developmental potential and commercial value, which has close ecological relationship with the host. The secondary metabolites produced by the plant endophytic actinomycetes had gotten more and more significant application in medicine, plant disease biology controls, and become the important direction in finding new active natural products.In this paper, we chose Sophora alopecuroides L. as the object, which is the important medical psammophyte of Baijitan National Nature Reserve in Ningxia. Endophytic actinomycetes from S. alopecuroides L. were isolated and identified, its diversity and ecological distribution were analyzed and studied. Soon afterwards that, antibacterial active strains of them were screened by biological method and strains which had PKS and NRPS functional genes were screened from them,and the types of antibiotics were identified which were produced from endophytic actinomycetes.And the phylogenetic of functional strains were analysis. The main conclusions were as follows:1.111 endophytic actinomycetes were separated and cultivated from 30 samples of healthy roots、 stems、 leaves and seeds of Sophora alopecuroides L. by tissue homogenate in six sample plots, where were sampled from different vegetations and soil types in Baijitan National Nature Reserve of Ningxia. Quantities of endophytic actinomycetes from roots and seeds of Sophora alopecuroides L. were more than ones of stems and leaves for the same plant. In different vegetations types, the quantities of endophytic actionmycetes from Sophora alopecuroides L. were most significantly in light sierozem and least in sandy soil. Endophytic actionmycetes were classified according to their morphological characteristics of culture、bacterial colonies and spores and 16S rDNA sequences analysis. The results had showed they belong to Streptomyces、Nocardiopsis、Prauserella、Actinophytocola、 Microbacterium、Actinosynnema、Thermoleophilum、Glycomyces and Saccharothrix respectively.Streptomyces and Nocardiopsis were dominant genera and the others were rare genera. Streptomyces was distributed in every sample spots.2. Taking 15 kinds of plant pathogenic fungi as target bacteria, antibacterial activity of 19 endophytic actionmycetes strains which were isolated from S. alopecuroides L. were tested. Cultivated about 7 days in culture dishes and observed the results. The results showed that 17 strains had strong inhibiting effects agaisted Fulvia fulva.16 strains had strong inhibiting effects agaisted Rhizoctonia solan and Fusarium graminearm.The inhibition zones of YWZKDS4 against Alternaria japonica reached 17.58 mm and YCZKDS4 against Pyricularia oryzae reached 15.62 mm. Fermentation broth of strain YWZKDS4 was prepared to spray the diaeased grape fruit which was suffered from anthracnose, then tested in a field and found that the average control effect of strain YWZKDS4 was 61.27%.3.19 endophytic actinomycetes strains which were highly inhibitory activity of S. alopecuroides L. were specific amplification by PKS Ⅰ, PKS Ⅱ and NRPS genes primers. The results showed that 11 strains were positive strains. The rate of PKS Ⅰ,PKS Ⅱ and NRPS positive strains were 47.4%、10.5% and 21.1% respectively. According to their morphological characteristics and phylogeny analysis,10 strains belong to Streptomyces and only 1 belong to Nocardiopsis of positive strains.4. Experimental materials were analyzed by TLC and HPLC analysis, and they were extracts of zymotic fluid from 9 positive strains which had PKS, PKS Ⅱ or NRPS genes, and standard substances of macrolide antibiotics, aromatic polyketide antibiotics and non ribosomal peptide antibiotics which number were 3,2 and 2 respectively. The results showed that 9 strains can produce medemycin, YWZKDS3 can produce 4 antibiotics, they were medemycin, acetylspiramycin, clarithromycin and tetracycline respectively.NDZKDS69 also can produce 4 antibiotics, they were medemycin, acetylspiramycin, oxytetracycline and teicoplanin respectively.