Isolation, Diversity And Antibiotic Activity Of Actinomycetes From Apis Cerana Cerana Fabricius

Actinomycete is a kind of microbial resources with the great use value. Because it can produce a large variety of biologically active compounds such as antibiotic, enzyme inhibitor, organic acid, vitamin and the like, it has a wide range of applications in medicinal field, agricultural field, food production field, etc. Hence, there is no doubt that exploring new acitnomycete resources and new bioactive compounds is a significant research work. Insects have more species than any other creature in the ecosystem, and all of them possess the symbiotic relationship with microorganisms. Nevertheless, there is very few relevant research on endosymbiotic actinomycetes of insects, so they have great exploration potential. In this study, Apis cerana cerana Fabricius was the research sample. The objective of the research is to isolate and incubate cultivable endosymbiotic actinomycetes from Apis cerana cerana Fabricius, to study the diversity and antimicrobial activity of them, and explore more new microbial resources.In the experiment,0.2% CIO2 for 60 s was utilized as the surface disinfection method, and seven media were chosen as isolation media. As a result,161 endosymbiotic actinomycetes were isolated from Apis cerana cerana Fabricius samples. Different isolation media showed the different isolation effect.48 strains were isolated from HV media which showed the best isolation effect. M7 media showed the highest isolation rate of rare actinomycetes. Based on the morphology of the colonies and cells of the new isolates,84 representative strains were selected from 161 isolated strains for genetic diversity research and antibiotic activity research.Four kinds of restriction enzymes, which were Hha I, HaeⅢ, Msp I and Hint I were chosen for 16S rRNA-RFLP analysis. As a result,18 kinds of 16S rRNA genotypes were produced. According to UPGMA dendrogram which was constructed from the 16S rRNA-RFLP fingerprints, all of the representative strains were together at the similarity level of 57%, and at the similarity level of 93%,84 representative strains could be grouped into 10 different genetic type groups. The group 2 was the biggest that included 50 strains. Combining with the colony characteristics of the 84 representative strains and the result of the 16S rRNA-RFLP analysis,16 strains were selected from these 10 genetic type groups for 16S rRNA gene sequencing. The result showed that actinomycetes isolated from Apis cerana cerana Fabricius samples belonged to 3 orders,4 families and 4 genera, they were Streptomyces, Micromonospora, Gordonia and Nocardiopsis, and SCAUEB4, SCAUED23 and SCAUEB32 were identified as potential new novel species.2 Pathogenic bacteria and 2 pathogenic fungus were used in antimicrobial test for exploring the antimicrobial activity of representative strains. The results of the antibiotic test showed that 31.0%,48.8%,27.4% and 16.7% of the representative strains presented antimicrobial activity to Escherichia coli, Bacillus subtilis, Curvularia lunata and Fusarium oxysporum,71.4% of the representative strains presented antimicrobial activity to at least one pathogen, and in which 14.3% of them presented antimicrobial activity to three or more than three pathogens.The results of this study indicate that the choice of the isolation method has a great impact on isolation result. And there are great diverse actinomycetes inside Apis cerana cerana Fabricius and they have great antimicrobial activity, which show a great potential for researching new bioactive compounds.