Study The Diversity Of Acorus Calamus Endophytic Bacteria Under The Strategy Of Culture-dependent And -independent Methods

Plant body is regarded as the niche of endophytic bacteria.As such,the actual state of life in nature is a mixture of plant and symbiotic microorganism.The dominant Prokaryotic microorganisms colonized in plant tissues are Proteobacteria,Actinobacteria,Bacteroides,Firmicutes and the thick walled fungi.Due to the particularity of plant endogenous environment,diverse group of microorganisms with a unique and abundant metabolism are present.Acorus calamus is a perennial plant of the genus Anastraceae.It is an aromatic and an ancient medicinal plant,with antioxidant,anti-inflammatory,calcium inhibition and neuroprotective effects.It also plays an indispensable role in the plant's sewage treatment and eutrophication.This study focuses on the subject of Acorus calamus,by studying the distribution of endogenous bacteria in the root,the stem and the leaf with culture dependent and culture-independent methods for the endogenous flora distribution in the Acorus calamus endophytic bacteria.The study considered structure distribution characteristics by comparing two endophytic bacteria diversity differences under different diversity research strategy.Bacterial diversity provides the reference for the exploration and comparison of research methods.In this study,the"Nested"Crack Zone Optimization(NCZO)analysis model,was designed to extract two stages of the bacterial isolation process,namely,the different grinding inoculation methods and the separation medium formula.The results show that different treatment and separation media are different in the design of NCZO analysis model.Inoculation was best with a water solution,the design difference of the recipe of culture medium was that the formula of tissue base solution was better with A3 and A4,whereas the A5 effect was worse.The best nutrient solution was B5,B6,whereas B2 was the worst.In this paper,the"no"optimal theory of endogenous bacteria separation culture medium was put forward,which was designed with modularization to maximize the yield of endogenous microorganisms.This study was based on the isolation of endophytic bacteria from the root,the stem and the leaf of Acorus calamus plant by pure culture strategy.749 prokaryotic microorganisms were sampled,and 16S rRNA sequencing was performed to identify the taxonomic status to obtain 175 bacteria strains,which were distributed in 4 phyla,7classes,16 orders,36 families,71 genera and 175 species.Proteobacteria accounted for49.71%,Actinobacteria for 25.71%,Firmicutes for 21.71%,and Bacteroides accounted for less than 3%.Many potential taxa were found in 16 genera with the similarity rate<98.0%.Culture independent method,based on high-throughput analysis of 16S rRNA gene sequencing,was performed for the endophytic bacteria in the root,the stem,the leaf and rhizosphere soil of four ecological niche of Acorus calamus in five strains of full mature plant.The results showed that the Acorus calamus plant tissue at the leaf contains4-6 families,at the stem 12 to 18 families,at the root 22 to 56 families,and the rhizosphere consists of 113-121 families.The bacterial community diversity among the Acorus calamus plant tissues was low and the diversity in rhizosphere was much higher than that in plants.In the strategy analysis of Culture independent method,the results of species diversity and Culture dependent method converge,and the number of flora endophytic bacteria species decreases gradually with the distribution of Acorus calamus niche from the root to the leaf.In this paper,several novel taxa were identified in culture dependent method.One of the novel strain YIM X0211~T isolated from plant rhizosphere soil,was characterized by polyphasic taxonomy study,and based on phylogenetic,phentotypic and bicochemical characteristics,the strain YIM X0211~T is proposed to present as a novel species of the genus Sphingobacterium,and named Sphingobacterium soli sp.nov.