Isolation, Identification and Bioactivity of Endophytic Actinomycetes from Mangrove Plants in Beilun River

Actinomycetes, as important sources of naturally occurred antibiotics, are strains for discovery of natural anti-infective agents. In order to isolate and purify the endophytic actinomycetes from mangrove plants in Beilun Estuary National Nature Reserve, Guangxi Zhuang Autonomous Region, explore their potential antimicrobial activities against pathogenic bacteria and fungi, and preliminarily clarify taxonomic status of bioactive strains, all 15 samples from different parts of 5 mangrove plants were cultured separately in 14 different liquid media for 4 weeks,and the enriched cultural media were then spreaded on the corresponding agar media with standard serial dilution plating method to isolate and purify the colonies. A total of 200 isolates were purified from samples including 62 strains from Avicennia marina, 43 strains from Kandelia candel,38 strains from Rhizophora stylosa, 29 strains from Clerodendrum inerme and 28 strains from Excoecaria agallocha, respectively. After removing the duplicate isolates by comparison of their cultural and morphological characterizations, 106 purified endophytic strains were tested for their antimicrobial activities against 6 strains of bacteria and 20 strains of Candida albicans by point planted method and disk diffusion method. A total of 52 isolates showed positive results and the positive rate was 49.1%. Among them,inhibitory rates against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteusbacillus vulgaris, Hemolytic streptococcus and Klebsiella pneumoniae were 16%, 15.1%, 4.7%, 7.5%, 11.3% and 3.8%,respectively; Inhibitory rates aganist drug- sensitive strains of Candida albicans and drug- resistant strains of Candida albicans were 16.0% and 29.2%, respectively. The bioactive strains were identified preliminarily as Streptomyces(21 strains), Nocardiopsis(26 strains), Pseudonocardia(2 strains), Agrococcus(2 strains) and Isoptericola(1 strain) by phylogenetic analysis based on 16 S r RNA gene sequences. Among them, the similarities of both strains UMBR1016 and UMBR1034 with Nocardiopsis alba DSM 43377 T were 98.27%and 98.37%, respectively; the similarity between strain UMBR 1008 and Nocardiopsis flavescens SA6 T, and between strain UMBR 1028 and Streptomyces pratensis ch24 T, was 98.36% and 98.49%, respectively. The results that similarities of 16 S r RNA gene sequences were less than 98.6% indicated 3 strains including UMBR1016, UMBR1034 and UMBR 1008 were potential new species of Nocardiopsis genus and UMBR1028 was potential new species of Streptomyces genus. The 38 and 35 isolates were found to show antimicrobial activity against at least one tested bacteria and one strain of Candida albicans, and the inhibitory rates were 35.8% and 33.0%, respectively. Abundant endophytic actinomycetes, including new species and potential antimicrobial activities found in the study, indicated that endophytic actinomycetes from mangrove plants in Beilun of Guangxi province could be a potential source for the discovery of new species and natural products and deserved further development in next step. The study will improve the conservation of mangrove plants in our country and further utilization of endophytic actinomycetes as important sources of drug discovery.