Study On Diversity And Bioactivity Of Actinomycetes Isolated From Plants In The Mangrove Forest Of Zhanjiang, Guangdong Province

Objective: To study the diversity and bioactivity of actinomycetes isolated from mangrove plants in Zhanjiang of Guangdong Province, China. Methods: Samples were grinded into little pieces after surface sterilization, and then spread on the surface of agar of isolation media. Colonies were subcultures on ISP2 medium till pury strain was obtained. Purified strains were analyzed by comparison of 16 S rRNA gene sequence. Supernatants of fermentation broths were separated by centrifugator and extracted by ethyl acetate, respectively. Totally 14 pathogenic strains including drug-sensitive and some resistant strains of Candida albicans, Cryptococcus laurentii, Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa were used for the antimicrobial activity screening. The antifungal and antibacterial activities of the crude extracts were tested by disk diffusion method. We used C.elegans as a tool for the identification, the nematicidal activities were tested by 96 well plate fluid model. Screening of PKS I, PKS II and NRPS genes of the bioactive strains were carried out by PCR and agarose electrophoresis. Results: Totally, 161 strains were obtained, they distributed in 19 different genera affliated with 12 families as below: Sterptomycetaceae contains Streptomyces; Micromonosporaceae contains Micromonospora, Plantactinospora; Pseudonocardiaceae contains Pseudonocardia, Actinomycetospora; Geodermatophilaceae contains Blastococcus; Mycobacteriaceae contains Mycobacterium; Nocardiaceae contains Gordonia, Rhodococcus, Williamsia; Micrococcaceae contains Micrococcus, Kocuria; Microbacteriaceae contains Microbacterium, Curtobacterium; Intrasporangiaceae contains Phycicoccus; Nakamurellaceae contains Nakamurella; Kineosporiaceae contains Pseudokineococcus, Kineococcus; Thermomonosporaceae contains Actinoallomurus; The dominant genus was Streptomyces. The 16 S rRNA sequence similarity between strain IP6SC6 and the nearest strain Phycicoccus jejuensis KSW2-15T(DQ345443) in EzBioCloud database was 96.81%, the N-J phylogenetic tree based on 16 S rRNA gene sequences showed that the strain IP6SC6 formed a distinct branch. Phylogenetically, strain IP6SC6 is a new species of Phycicoccus. Antimicrobial assay showed that 46 among 88 strains exhibited positive results in antifungal or antibacterial assay, and the total positive rate was 52.27%. Amongst them, 23 strains showed inhibitory effects against the Gram-negative bacteria. Nematicidal assay showed that 27 among 88 strains exhibited positive results in fermentation broths, and the total positive rate was 30.68%, 19 among 88 strains exhibited positive results in ethyl acetate extracts, and the total positive rate was 21.59%. PCR results showed 36 in 47 bioactive strains have at least one antibiotics biosynthetic gene including NRPS, PKS I and PKS II, the total positive rate was 78.26%. 8 strains have all the three kinds of antibiotics biosynthetic genes. Conclusions: Plants of mangrove in Zhanjiang, Guangdong Province are rich of bioactive endophytic actinomycetes, which could be a potential source for the discovery of new antibiotics and new taxa.