Phylogeny Of Endophytic Actinobacteria Isolated From The Medicinal Plants In Sichuan

The endophtytic actinobacteria were isoloated and analyzed from 13 medicinal plants growing in six very different geographic locations in Sichuan, China.The results showed that the 210 actinobacteria were isolated from the medicinal plants including two genera: Streptomyces and Micromonospora, most of them belong to Streptomyces.Several new methods on the isolation of endophytic actinomycetes from plant tissues were studied and compared in this thesis. The preliminary investigation was carried out the effective surface-sterilization methods. Effective of different madia for isolation pure culure of endophytic actinomycetes were studied.The results indicated that the diversity of the isolates on HV medium and modified Gause II media were relative high; actinobacteria on Trehalose-proline media was less, but typical. Most of the actinobactera isolated from roots, about 68.6%; stem, 25.2%; leaf, 6.2%.The genetic diversity of 41 typical endophytic actinobacteria were investigated by using PCR-RFLP of the ribosomal 16S rRNA gene and the intergenic spacer (IGS) region between the 16S rRNA and 23S rRNA genes, BOX fingerprinting and full sequence analysis of the 16S rRNA gene. This paper investigated the diversity and phylogeny of the endophytic actinobacteria isolated from 13 commonly used medicinal plants in different geographical locations using genotypic approaches for the first time.The results revealed 29 genotypes from 16S rRNA genes PCR-RFLP data, which indicated considerable genetic diversity among the isolates. Based on the dendrograms, all the strains were repectively divided into 2 groups (Streptomyces and Micromonospora) at the similarity of 71%, and seperated into 4 groups at the similarity of 83%. The results revealed that the similarities of all strains did not correlate with the geographical sampling site and host medicinal plants.The results of IGS PCR-RFLP, BOX-PCR revealed even greater diversities within those endopytic actinobacteria. 40 genotypes from 16S-23S rDNA IGS PCR-RFLP data, and all the stains could be separated into 11 groups at the similarity of 80%. While the results of BOX-PCR fingerprinting revealed more diversities than IGS PCR-RFLP. The dendrogram showed that strains could be devided into 20 groups at the similarity of 83%. There were distinct differences among IGS PCR-RFLP and BOX-PCR. In dendrogram constructed using IGS PCR-RFLP, some strains tested were mainly grouped according to cultivars origins. Some actinobacteria correlated well with the geographical sites according to the analysis of BOX-PCR. BOX-PCR was more eficient in detecting minor diferences among closely related strains tested.