Study On The Isolation, Identification Of Endophytic Actinomycetes From Melia Toosendan

Microorganisms produced more than20000kinds of metabolic active substances (e.g. antibiotics, etc.), among which,60%-80%was produced by actinomycetes. In recent years, due to the abuse of antibiotics, drug resistant strains increased dramatically. Besides, some newly pathogenic microorganisms were discovered, such as SARS, H5N1, H7N9, which are a serious threat to human health. As a result, it become more and more difficult to screen new bioactive substances and research new drug. However, microbial secondary metabolites is still an important source of new drugs for pharmaceutical industry. The reserch results on soil microorganisms, rhizosphere microorganisms and microorganisms which are attached to the plant surface are aboundant. But endophyte research is in its infancy. Due to the diversity of topography and climate in Sichuan province, the resources of medicinal plant are rich. Melia toosendan, as a kind of authentic Chinese herbal medicinal plant in Sichuan, containing a variety of biological active substances, is important raw material to make new plant pesticide, which has broad application prospects. So far, the report of endophytic actinomycetes from Melia toosendan is rare, therefore, research on endophytic actinomycetes from Melia toosendan is of great significance.This study regard Melia toosendan collected from regions of Sichuan province as the research object. Seven kinds of selective separation medium were used to isolate endophytic actinomycetes from the roots, stems, leaves, bark and fruit of Melia toosendan. A total of403strains of endophytic actinomycetes were isolated and purified. The number of isolates were varied with different sampling sites, medium and paint tissues. Eighty-six strains were obtained from the samples in Guangyuan, which was the highest. The least was samples of Mianyang, only12strains. One hundred and forty-eight strains of actinomycetes were isolated from the bark, accounting for36.7%of the total strains. Only31strains were isolated from the fruit, accounting for7.6%of the total strains. Although the quantity which isolated from the roots was rarely, the isolation frequncy was the highest. The most ideal medium were number5and number3, which indicated that this two media were most suitable for the growth of endophytic actinomycete. The combination of50mg/1potassium dichromate and25mg/1nalidixic acid, showed the best effect to inhibit the growth of other bacteria and fungi, and just had little effect on the growth of actinomyces.Among the403isolates, one hundred and ninety-one strains with different morphological characteristics were selected for gene diversity analysis. The restriction enzymes Haeâ…¢ and Hha I were chosen for16S rDNA PCR-RFLP analysis. Haeâ…¢ and Hha I produced9and12kinds of restriction patterns, respectively. According to the enzyme belt type, sixty-nine strains showed different16S rDNA PCR-RFLP, which divided into10ARDRA genetic types at the85%similarity level. Thirty-seven representative strains from each cluster were chosen for16S rDNA sequencing. The results of sequencing showed that the tested strains belong to were Streptomyces, Kitasatospora, Arthrobacter and Kribbella. Among them, Streptomyces was dominant, accounting for91%of the total number of strains, and only9%of those were rare actinomycetes. In Streptomyces, different strains formed different branches in the phylogenetic tree, which indicated that there was a great diversity of the endophytic actinomycetes in Melia toosendan.