| Mangrove forest, growing in the coastal intertidal zone, is a special ecosystem from land to sea. As one of the highest productive marine ecosystems, mangrove is a precious resource for discovering novel microorganisms. Bamenwan Mangrove Forest is located at Qinglan Mangrove Reserve (110°30'-110°02'E,19°15'-20°09'N) in Hainan Province, where there are the most mangrove species in China and multiple and rare microbial resources.In this study, high-throughput sequencing and analyses of microorganism isolated from the soils of five habits (intertidal zone, transition zone, bruguiera sexangula mangrove zone, Xylocarpus mekongensis mangrove zone, land) were carried out. The results showed that microorganism resources in mangrove ecosystem had abundant and high genetic diversity. In order to explore the actinomycetes resources further, actinomycetes stains were isolated, the diversities of the culture level were analysed, and polyphasic taxonology identifications of three potential novel species were proceeded with. The results were as follows:Diversities of actinobacteria, baceria, archaea and fungi in soil from different habits were analyzed by454high-through sequencing. The amount of sequences, diversity index and coverage were all high. The numbers of OTUs was high,25527OTUs for actinobacteria,24402OTUs for bacteria,10514OTUs for archaea, and3432OTUs for fungi. Index of Ace and Chao1had the same trend with that of the numbers of OTUs, actinobacteria>bacteria>archaea>fungi. Shannon and Simpson index were2.72-7.9and0.1342-0.001, respectively. Coverage was0.75-0.99, with0.9-0.98for archaea,0.95-0.99for fungi,0.86-0.91for actionbacteria and0.75-0.84for bacteria?Amounts of unclassified OTUs were obtained in this study, which were similar with the results of rarefaction curve, suggesting that amounts of unclassified OTUs were obtained in this study, which were similar with the results of rarefaction curve, suggesting that numbers of uncultured microbes were in the soils in mangrove ecosystem.For bacteria,23populations were detected at the level of family, mainly including Deltaproteobacteria, Alphaproteobacteria, Gammaproteobacteria and Acidobacteria, which had different proportions in different samples. Little differences were observed in samples between intertidal zone and transition zone and samples between two mangrove zone. Composition of populations in land sample presented much difference with those of the other four samples, which mainly due to the higher percents of Actinobacteria and Thermoleophilia in land samples. For archaea,22populations were detected at the level of family, mainly including Halobacteria, Thermoplasmata, Miscellaneous Crenarchaeotic Group (MCG), Marine Benthic Group B (MBG), Terrestrial Group, Group C3, Soil Crenarchaeotic Group(SCG) and Methanomicrobia, and amounts of unclassified populations were detected, which suggested that much potential uncultured archaea were worthy to explored in the future. For fungi,15populations were detected at the level of family, mainly including Agaricomycetes, Sordariomycetes, Eurotiomycetes, Dothideomycetes and Orbiliomycetes. The compositions of population in different samples were different, with the higher diversities of samples in intertidal zone and transition zone than those of samples of mangrove zones. For actinobacteria,12populations were detected at the level of order, mainly including Frankiale (30.9%), Corynebacteriales (15.1%), Micromonosporales (13.2%), Propionibacteriales (12.7%), Micrococcales (7.4%), Pseudonocardiales (6.6%), Streptomycetales (5.9%), Kineosporiales (4.3%), Streptosporangiales (2.3%), PeM15(1.2%), Catenulisporales (0.09%) and Elev-16S-976cluster (0.04%). The populations among the five samples were similar while the percents of populations were different. The percents Corynebacteriales in samples of intertidal zone and transition zone were higher than those other samples, and Kineosporiales in transition zone had much higher percentage (21.6%) than other samples (2.2%,2.9%,2.8%and1.5%, respectively). The populations in two mangrove zone samples were similar. Population composition in land sample presented much difference with those other four samples, mainly due to the percentage of Frankiales (49.7%).A total of210actinomycete isolates were searated from the soil of Bamenwan Margrove Forest by dilution-plate method. They were distributed in twelve genera based on16S rRNA gene, namely Streptomyces, Micromonospora, Saccharopolyspora, Pseudonocardia, Nonomuraea, Microbiospora, Streptosporangium, Verrucosispora, Tsukamurella, Nocardiopsis, Isoptericola and Jishengella. Among the strains, Streptomyces won the largest population, accounting for65.7%(138strains); rare actinomycetes accounted for34.2%(72strains), of which Micromonospora won the largest population,18.6%(39strains), and the other ten genera,33isolates occupied15.7%.On the base of the principle of polyphasic taxonomy, systematic studies had been conducted on the identification of three potential novel actinomycetes (strain HA12301, HA12420and HA12415) isolated from soil samples at Bamenwam Mangrove, and they represented as novel species of the genus Streptomyces, Pseudonocardia and Nocardiopsis, respectively. The descriptions are as follows:Strain HA12301T:Gram-positive and acerobic actinomycete which forms spiral aerial mycelia, the spore is ovoid or cylindric with short spine surface. Growth occurs at28-43?(optimally37?), pH6-9(optimally pH8) and1-5%(optimally1%, w/v) NaCl. Able to degrade starch and cellulose. Negative for decomposition of urea, liquefaction of gelatin and oxidase test, and positive for reduction of nitrate and catalase. Melanim and H2S are not produced. The major fatty acids are i-C16:0, ai-C15:0, C14:0and iso-C16:1H. The predominant polar lipid profiles are PE, PG, DPG, PI, PC and PIM. The DNA G+C content is72.6%. The highest homology with Streptomyces sanyensis GIMN4.003T is99.0%. and followed by S.atacamensis C60T (98.8%), S.radiopugnans R97T (98.6%) and S. nanhaiensis SCSIO01248T (98.5%) based on16S rRNA gene sequence analysis. Phylogenetic analysis indicated that the novel strain formed a distinct subclade and clustered with them. The DNA-DNA hybridization value of strain HA12301T with the four type strains are57.2%,49.1%,45.5%and41.8%, respectively. The results of the phenotypic, genotypic, biochemical and physiological analysis clearly indicate that strain HA123011should be assigned to a novel species in the genus Streptomyces, for which the name Streptomyces bamenwan sp. nov. is proposed.Strain HA12415T:Gram-positive and acerobic actinomycete which forms fractured aerial mycelia, the spore is rod with smooth surface. Do not produce soluble pigment. Growth occurs at14-28?(optimally25?), pH6-11(optimally pH7) and1-5%(optimally3%, w/v) NaCl. Don't reduce nitrate, degrade starch and produce H2S. Negative for Milk coagulation and peptonization. The major polar lipid profiles are DPG, PME, PC PI and PIM. The DNA G+C content is74.9mol%. The highest homology with Pseudonocardia carboxydivorans Y8T is97.1%, and followed by P.ammonioxydans H9T (97.1%), P.tropica YIM61452T (97.0%), P.antarctica DV5alT (97.0%) and P.parietis04-St-002T (97.0%). The six strains were clustered into a larch barch in the phylogenetic tree. The DNA-DNA hybridization value of strain HA124151with the five type strains are48.6%,45.1%,41.5%,30.2%and28.4%, respectively. The results clearly indicate that strain HA12415T should be assigned to a novel species in the genus Pseudonocardia, for which the name Pseudonocardia mangrovi sp. nov. is proposed.Strain HA12420T:Gram-positive and acerobic actinomycete which forms long, straight or irregular, zig-zag shaped aerial mycelia, the spore is oval or cylindric with smooth surface. Do not produce soluble pigment. Growth occurs at4-43?(optimally37?), pH6-12(optimally pH7) and0-5%(optimally1%, w/v) NaCl. Positive for nitrate reduction, negative for milk coagulation or peptonization. Melanim and H2S are not produced. Degrade starch, but not cellulose and gelatin. The major cellular fatty acids are iso-C14:0, ant-C15:0, iso-C16:0, C16:0and C16:1?9c. The major polar lipid profiles are PE, PIM, PC and GL. The predominant isoprenoid quinone is MK-10(H2, H4). The DNA G+C content is73.7mol%. The highest homology is99.0%with Nocardiopsis trehalosi VKM Ac-942T based on16S rRNA gene sequence analysis, and positioned on the same branch in the hylogenetic tree. The DNA-DNA hybridization relatedness between them was34.8%. The results of the phenotypic and genotypic analyses clearly indicate that strain HA124201should be assigned to a novel species in the genus Nocardiopsis, for which the name Nocardiopsis wenchangensis sp. nov. is proposed. The soil of mangrove forest is rich in bacteria, archaea and fungi resources, especially actinomycetes, which have high genetic diversity and new actinomycete species. It is worth further exploring, developing and utilizing. |
PDF Full Text Request