Biodiversity Of Actinomyces Isolated From Froest Soil In The Gaowangjie National Natural Reserve

In this study, the culturable antinomyces in the froest soil samples collected from the Gaowangjie National Natural Reserve(28°39′02.3 "~28°42′36.8" N, 110°03′24.4 "~110°10′34.3" E), Hunan Privnce, PR China, were isolated by using culture-dependent method. After purification, the actinomyces diversity was investigated by using phylogenetic analysis based on 16 S r RNA gene sequence comparisons. Then, the antibacterial ability of of the fermentation broth extracts from the isolates was detected on eight sensitive microorganisms by using the agar diffusion method. Further more, the biological characteristics of some active strains were characterized.A total of 249 antinomyces strains were isolated from the forest soil samples on a variety of media(ISP2, humic acid agar, LSV-SVF, MA5, trehalose-asparagine agar, chitin agar, trehalose- proline agar, Gauss’ s synthesis). Based on the results of their morphological, size, aerial hyphae, substrate hypha, and soluble pigment characteristics, 169 isolates were selected for further study.Based on contrast analysis of morphological characters, 66 representive strains were selected to perform the phylogenetic analysis based on 16 S r RNA gene sequences. The results demonstrated that the strains belonged to 8 genera(Streptomyces, Nocardia, Kitasatospora, Rhodococcus, Nocardiopsis, Pseudonocardia, Actinocorallia, Polymorphospora) of 6 families(Micromonosporaceae, Nocardiaceae, Nocardiopsaceae, Pseudonocardiaceae, Streptomycetaceae, Thermomonosporaceae) in the phylum Actinobacteria. The most abundant and diverse isolates were within the genus Streptomyces(54 strains, 81.8%), followed by the genera Nocardia(4 strains, 6.1%), Nocardiopsis(2 strains) and Rhodococcus(2 strains). The other four genera, Kitasatospora, Pseudonocardia, Actinocorallia and Polymorphospora only had 1 isolate, respectively. The 66 represented strains could be classified as 38 genomic species, and there were obvious genetic divergences between most isolates and their closestly related type strains(sequence similarities, 98.71% to 99.93%). Out of 66 isolates, at least 3 strains(JSM 147604, JSM 147645, JSM 147773) should be representatives of 3 potential novel species of 2 recognized genera(Streptomyces, Actinocorallia): JSM 2147604 should be a member of the genus Streptomyces, being related most closely to the type strain of Streptomyces cavourensis(sequence similarity 98.75%), and these two strains formed an independent branch on the evolutionary tree; strain JSM 147773 was related most closely to the type strain of Streptomyces xiamenensis(sequence similarity 98.59%); strain JSM 147645 was related most closely to the type strain of Actinocorallia libanotica(98.90%), but it formed an independent branch on the evolutionary tree.The antibacterial activities of 169 isolates were screened by using an agar diffusion method on eight sensitive microorganisms, i.e. three G+ bacteria(Bacillus subtilis, Sarcina lutea, Staphylococcus aureus), three G- bacteria(Aerobacter aerogenes, Escherichia coli, Proteus vulgaris) and two fungi(Asperillus niger, Candida albican). The results investigated that the antimicrobial activities of 47 isolates were positive(27.8%), in which 6 strains(JSM 147612、JSM 147786、JSM 147823、JSM 147831、JSM 147842、JSM 147846) exhibited strong antimicrobial activity.Biological characteristics of above 6 antibacterial-activity-positive strains were studied by using conventional methods. The results showed that, most of the strains grew well in the range of temperature 20°C–40°C and p H 6–10. All the strains tested cold produce at least one enzyme among the five exoenzymes observed(amylase, protease, urease, gelatinase, esterase). The results of culture and morphological characteristics showed that, the 6 strains exhibiting strong antimicrobial activity(JSM 147612, JSM 147786, JSM 147823, JSM 147831, JSM 147842, JSM 147846) grew well on most tested media, producing exuberant vegetative hyphae and aerial hyphae, These 6 strains were studied by using a phylogenetic analysis method based on 16 S r RNA gene sequences, and the results suggested that they were all members of the genus Streptomyces, belonging to five recognized species of this genus.In conclusion, there is an abundant actinomycete diversity and some potential new taxa in the forest soil collected from Gaowangjie National Natural Reserve in Hunan Province, China, and that a number of isolates exhibited antibacterial activity.