The Diversity And Activities Of Actinomycetes In Xinghu Wetland

Being located in the transitional zone between terrestrial and aquatic ecosystems,Wetland is endowed with a unique ecological function as the soil is perennially soaked in water.Studies have shown that there are large quantities of special microorganisms living in wetlands.As an important taxa of microorganisms,the actinomycetes spread widely in a variety of wetland environments.The Xinghu Wetland,located in Zhaoqing City,Guangdong Province,can be considered as the typical representative of subtropical karst lakes in which seven peaks scatter and integrate with the water.There are a wealth of natural and biological resources in Xinghu Wetland.Therefore,it is of theoritical and practical importance to conduct systematic researches on actinomycetes in Xinghu Wetland,and it will be conducive to development and sustainable use of actinomycetes resources.Culture-independent techniques are used to analyze the diversity of actinomycetes in the sediment environment of Xinghu Wetland.The actinomycetes community of samples from ten locations were studyed based on Polymerase Chain Reaction--Density Gradient Gel Electrophoresis(PCR-DGGE)techniques.The DGGE profiles and results of the cloning sequences were analyzed combined with the physical and chemical properties,and The species diversity and spatial distribution characteristics were also analyzed.The Species richness(S),Shannon-wiener index(H?),evenness index(J?)of individual samples were different through PCR-DGGE fingerprints.When the similarity of actinobacteria communities of different locations were compared,it was found that the similarity coefficients of them were much related.The similarity was higher between closer samples.After being recycled,cloned and sequenced,the DGGE dominant bands showed that the dominant groups were mainly members of Streptomyces,Micromonospora,Nocardioides,Marmoricola,Cellulomonas,Isoptericola,Arthrobacter,Actinoplanes,Polymorphospora.The results of the canonical correspondence analysis showed that sediment WSOC and A-P were the key factors changing actinomycetes community composition in Xinghu Wetland.Species diversity was fairly high in Xinghu Wetland with abundant actinomycetes resources,which needs to be further explored.The actinomycetes of 26 samples in the 10 locations were isolated and cultured with culture-dependent techniques.New methods for isolation of actinomycetes were explored with isolating media compositions.Total 340 strains of actinomycete were isolated,among which 135 representative strains were selected.The population diversity of actinomycetes were analyzed with phylogenetic analysis method of 16 S rRNA gene sequences,and strains belonged to 7 orders,10 families,and 13 genera of actinomycetes.This showed that Xinghu Wetland was rich in actinomycetes resources with 13 genera,e.g.Amycolatopsis,Actinomadura,Dactylosporangium,Knoellia,Microbacterium,Micromonospora,Micrococcus,Nocardia,Nocardioides,Nonomuraea,Rhodococcus,Saccharopolyspora,and Streptomyces.Initial experiments on anti-microbial activity were carried with selected 83 actinomycetes strains.Three pathogenic bacteria,e.g.Staphylococcus aureus,Escherichia coli,and Salmonella,were used as the indicator bacteria.The result of this study showed that the inhibition percentage for Staphylococcus aureus and Escherichia coli were 24.09% and 4.8% respectively.The potentials of secondary metabolites of strains were evaluated.The selected representative of strains were used to detect genes for type I polyketide synthetase(PKS-I),type II polyketide synthetase(PKS-II),non-ribosomal peptide synthetase(NRPS),3-amino-5-hydroxybenzoic acid synthases(AHBA),3-Hydroxy-3-Methylglutaryl Coenzyme A(HMG CoA)by PCR with primers of K1F/M6 R,KS_?F / KS_?R,A3F/A7 R,P1/P2,HMGF/HMGR.The result showed that 16.7% of strains were positive for gene PKS-I,62.5% for gene PKS-II,16.7% for gene NRPS gene,12.5% for gene AHBA,29.2% for gene HMGA in 24 active strains.The inhibition and HPLC method showed that strains XD007,XD114 and XD128 could significantly inhibit activities of three pathogenic indicator bacteria,and produce plenty of secondary metabolites.The current studies revealed that abundant actinomycetes species exist in Xinghu Wetland sediments,and many strains show the high anti-microbial activities.The combination of culture-independent and culture-dependent techniques can greatly enhance the analyzing reliability of microbial diversity.It's of highly guiding significance to separate secondary metabolites through active genetic screening and HPLC techniques.