Isolation Of Endophytic Actinomycetes From Mangrove Plants And Identification Of Novel Specieses Of Family Micromonosporaceae

The family Micromonosporaceae is the second largest group of secondary metabolites producer of actinomycetes. Endophytic actinomycetes have special characteristics for its unique niches. Mangrove forest, a special habitat located in intertidal zone is different from land and ocean and with diverse and unique microorganism resources. To study the diversity and bio-activity of endophytic Micromonosporaceae members from mangrove plants, total of82root samples were collected from14different plants of mangrove in Beihai, Haikou, Wenchang and Zhangjiang and selectively isolated for Micromonosporaceae. Bio-activity of fermentation products and secondary metabolites biosynthesis gene of the isolates were tested.Root samples were sterilized, crushed and preincubated, then sprayed on7kinds of selective agars. After purification, Total of380actinomycetes strains were obtained. Among the4sampling mangrove forests, Sanya account for the majority strains in total (267). Among the14different plants, Sonneratia apetala and Avicennia marina account for the majority strains in total (158,112respectively). Among the7different selective agars, SIM (Strptomyces Isolation Medium) got the most isolates in all (283). The isolates were divided into Miromonosporaceae-like.(365) and Miromonosporaceae-unlike (15) by morphologic and cultural characteristics.Fermentation was carried with300selected strains, and the broth was tested for bio-activities. Three strains showed activity of anti-Staphyloccocus aureus (ATCC45923),4strains showed activity of anti-Canidia Albicans (ATCC10231) and4strains showed activity of anti-Fusarium oxysporum f.sp.cubense. Secondary metabolism genes were detected by PCR with specific primers for126strains, with104(82%),97(77%) and102(81%) strains showed positive results in PKS I (polyketide synthase I), PKS II (polyketide synthase II) and NRPS (non-ribosomal peptide synthetases) gene.Analysis of16S rRNA gene sequences of147strains revealed that all strains distributed in4suborders,4families and5genus, thus132strains of Micromonospora (no less than15specieses),5strains of Microbispora,4strains of Pseudonorcadia,4strains of Streptomyces and2strains of Actinomycetospora.Polyphasic taxonomy identification including morphologic and cultural characteristics, physiological and biochemical characteristics, cell chemical constituents, genome DNA G+C mol%, DNA relatedness,16S rRNA and gyrB gene sequences phylogeny analysis and other tests were carried in strains of two suspected novel endophytic species to determin their taxonomy status. Strain274745was isolated from the root of Sonneratia apetala in Sanya mangrove forest. Strain268506was isolated from the root of Avicennia marina in Wenchang mangrove forest. Based on the differences of characteristics from reference type strains in identification, strain274745and268506represent two novel specieses in genus Micromonospora, for which the name Micromonospora sonneratiae sp. nov. and Micromonospora avicenniae sp. nov. are proposed.Strains2806702^270106and260305were isolated from mangrove soils. The polyphasic taxonomy identification showed they are novel specieses in genus of Micromonospora or Rugosimonospora, for which the name Micromonospora ovataspora sp. nov. Micromonospora acanthiterrae sp. nov. and Rugosimonospora mangrovi sp. nov.are proposed, respectively. Reclassification was carried out on Polymorphospora rubra TT97-42T. The phylogenetic, cell chemical constituents analysis results and microscopic morphologic characterestic are consistent with genus Micromonospora. So genus Polymorphospora should be combined into genus Micromonospora and P. rubra should be reclassified as Micromonospora rubra comb. nov..The results above showed a high diversity of Micromonospora the potential abilities for second metabolite material producing and the presence of novel taxa.