Biodiversity And Physiologically Activities Of Actinobacteria From Rhizosphere Soil Of Mangrove Plant Acanthus Ilicifolius By Culture-dependent And-independent Approach And Identification Of A Novel Actinoallomurus

Rhizosphere is the dynamic interface between plant and soil. In this study, nine rhizosphere soil samples of plant Acanthus ilicifolius were collected from Wenchang and Beihai mangrove. Six media were used for selective isolation of actinomycetes. The distribution of actinomycetes in rhizosphere soils were analyzed with combination of16S rRNA genes clone library using actinomycetes-specific primer. Considering the high salt concentration and C/N ratio of mangrove soil, salt-tolerance and potential nitrogen-fixing capacity of these isolates were tested. Antibacterial activities were also evaluated. In addition, a presumed new species was identified by polyphasic taxonomy.From the16S rRNA genes clone library of9soil samples obtained using actinomycetes-specific primer, total of59genera were obtained, in which20genera could be found both in Wenchang and Beihai sample, but19genera were only found in Wenchang sample and20genera only in Beihai sample.Six media including RH, SM2, OA5, OA7, OA9,(Oat meal agar at pH of5,7,9) and1/10ATCC172were used for isolation. Total of295isolates obtained and most of the strains were from OA7(n=115) and OA9(n=128) media, followed by RH, SM2and1/10ATCC172media which were29,20and2strains, respectively. There was no strain isolated from the OA5medium. Fifty six reduplicative isolates were removed based on morphology. The16S rRNA gene of the remaining219isolates were sequenced and the similarity were analyzed via EzTaxon, which belong to15genera, under7families of7suborders, including Glycomycineae, Micrococcineae, Micromonosporineae, Pseudonocardineae, Propionibacterineae, Streptomycineae, and Streptosporangineae. One third of the genera were isolated from both of Wenchang and Beihai samples, including Actinomadura, Actinomycetospora, Microbispora, Micromonospora and Streptomyces; five genera were only isolated from Wenchang including Actinoallomurus, Actinopolymorpha, Cellulosimicrobium, Polymorphospora and Sphaerisporangium; five genus also were only isolated from Beihai including Actinaurispora、Actinoplanes、Glycomyces、Jishengella and Plantactinospora.Comparing with the distribution revealed by culture-dependant and culture-independent investigation approach, more number and much diverse actinomycetes were revealed by clone library analysis and the species aboundance are more correlated to the soil physical-chemical variables. While some actinomycetes groups were not obtained in the actinomycetes clone library which had obtained by selective isolation such as, two genera of Actinopolymorpha and Microbispora isolated from Wenchang, and four genera of Glycomyces, Actinaurispora, Actinomycetospora and Jishengella isolated from Beihai samples.Sodium chloride tolerance test for229isolates reflected their marine origin. Growth at15,10,6, and3.3%(w/v) of NaCl, were observed for2,7,40, and151strains, respectively. Five strains could not grow without salt but well at3.3%salinity and all tested strains could not grow at20%salinity. Nineteen isolates were deemed as potential nitrogen-fixing strains based on the Nfb media as well as Ashby media tests.Screen of antimicrobial activities on the232actinomycetes isolates and re-screening of91actinomycetes isolates showed that,26strains (11.21%) were active against Staphylococcus aureus ATCC51650;10strains (4.31%) were active against Methicillin-resistant Staphylococcus aureus;12strains (5.17%) were active against Pseudomonas eruginosa S001;18strains (7.76%) were active against Candida albicans ATCC10231; all of the strains didn’t show any activity against Klebsiella Aerogenes S008.Strain2614A723was isolated from rhizosphere soil of mangrove plant Acanthus ilicifolius collected in Wenchang. Phylogenetic analysis based on16S rRNA gene sequences showed that strain2614A723had the highest similarities with members of the genus Actinoallomurus, namely, Actinoallomurus spadix NBRC10499T (98.35%), Actinoallomurus purpureus TTN02-30(98.07%) and Actinoallomurus luridus TT02-15T (97.86%) and formed a distinct subclade with them. It is evident from the combination of genotypic and phenotypic data that strain2614A723represents a novel species of genus Actinoallomurus, for which the name Actinoallomurus acanthiterra sp. nov. is proposed.