Analysis On Diazotrophic Communities In The Rizosphere Of Beach Plant Jerusalem Artichock And The Mechanism Of Action Of Its Endophytic Bacteria

Instead of living solely in nature,plants coordinate with microbeings and formate unique plant associative environment on the surface and inside of plant.Bacteria is one of the most important residents in plant associative environment with the highest population.In rhizosphere the main interface for energy and substance exchange of plant,bacteria could form various communities with the infection of different biotic and abiotic factors.Bacteria communities in the rhizosphere indicate the health status of the environment and play important roles in practical applications.Plant associative bacteria could promote the growth of plants utilizing strategies including biological nitrogen fixation,IAA secretion and activation of insoluble inorganic nutrient.Plant growth promoting bacteria has been widely used in agriculture and forestry.Enodophytic bacteria could live within healthy plant tissues without causing disease.Interior of plant,endophytes are protected from stresses of external enviroment and could promote growth of host plant via different mechanism.Endophytic bacteria could interact with host plant as well as other microorganisms including endophytes and pathogens.Quorum sensing is one of the most commonly and widely studied intra-and inter-species communication of bacteria.It was reported to regulate various colonial activities,such as motility,symbiosis and biological toxicity.To study the community of diazotrophic bacteria in rhizosphere,we optimized a molecular fingerprint technique using specific gene of nitrogen fixing bacteria.From the root of halophytic plant Jerusalem artichoke,we isolated an endophytic bacteria strain with plant growth promoting abilities.Its plant growth promoting effect and colonization dynamics in planta were investigated.To elaborate quorum sensing regulating endophytic bacteria,we employed E.toletana which is endophytic bacteria of olive knot the model of interspecies community study.1.In rhizosphere,nitrogen fixation bacteria were dominated with difficult-cultural,non-cultural and un-cultural bacteria,which made traditional culture dependent method unsuitable for the study of community of diazotrophic bacteria community.Nowadays molecular fingerprint techniques including LH-PCR and T-RFLP have been widely applied with the advantages of being easily operational and highly reproducible.Targeting nitrogenase coding gene nifH which has the same phylogenetic status as 16s rDNA,we detected the community of nitrogen fixing bacterial in the rhizosphere soil of Jerusalem artichoke.Compared with different molecular fingerprint techniques,we found that T-RFLP with MnlI restriction enzyme showed the most abundant OTU.With the influence of root activities,nitrogen fixing bacteria in rhizosphere soil of Jerusalem artichoke exhibited higher abundance and diversity than blank soil.High nitrogen fertilizer input could decrease the diversity of nitrogen fixation bacteria and influence the structure of nitrogen fixation bacteria community in a negative way.2.Within the endophytic diazotrophic bacteria strains we isolated from the root of Jerusalem artichoke,Ochrobactrum sp.Mnl showed the highest nitrogenase activity which is 207.34 nmol-mL-1·h-1 detected with acetylene reduction method.Ochrobactrum sp.Mnl exhibits high IAA and siderophore secretion with the ability of activation insoluble phosphorus.Endophytic diazotrophic bacteria Ochrobactrum sp.Mnl displayed growth promoting effect on Jerusalem artichoke under greenhouse condition and also in field trails.Plant growth promoting effect of Ochrobactrum sp.Mnl has many aspects.With biological nitrogen fixation ability,it contributed about 20%of total nitrogen content.IAA produced by Ochrobactrum sp.Mnl could significantly optimize root structure of host plant.With the help of siderophore,Jerusalem artichoke could improve its characteristics of absorbs and utilize nutrient like P,K,Fe and Mg.Utilizing green fluorescent protein and re-isolate technique,we explored the colonization dynamics of Ochrobactrum sp.Mnl in different plant tissues.Under fluorescence microscope,we observed that Ochrobactrum sp.Mnl mainly colonized interior of root and stem tissues.The highest colonized number of Ochrobactrum sp.Mnl was detected in root tissue which is about 105 CFU per gram.3.Olive knot cause by P.savastanoi has been recognized as a model of intra-and inter-species interaction study with collaboration of host plant,pathogen bacteria and endophytic bacteria.AHLs quorum sensing played momentous roles in this model including biological toxicity of P.savastanoi and interspecies interaction between P.savastanoi and endophytic bacteria E.toletana.Via RNAseq analysis,we discovered that AHLs synthesized by etoI gene of E.toletana mediates a series physiological activity such as bacteria motility,plasmid conjugation and antibiotic resistance.Apart from secondary metabolism,AHLs quorum sensing is capable to regulate primary metabolism of E.toletana including catabolism of different carbon sources like maltose and D-glucarate.E.toletana possesses a complete inositol operon and could use inositol as single carbon source,which is an important structural and signal substrate in plant.We discovered for the first time that 9 genes in inositol operon were down regulated by AHL quorum sensing system.