Root Colonization Of Nitrogen-fixing Pseudomons Stutzeri A1501 And Its Effect On Plant Growth Promotion

Root exduates plays an important role in the interaction between plant host and the nitrogen-fixing microorganisms.As induced by root exduates,the nitrogen-fixing microorganisms can be chemotactic,swimming toward to colonize and achieve aggregates by forming biofilm,thereby gaining a competitive advantage in the rhizosphere.However,the molecular mechanisms involved in these processes remain largely unknown.The nitrogen-fixing bacterium Pseudomonas stutzeri A1501 was originally isolated from rice rhizosphere,with capacity of chemotaxis,biofilm formation and plant growth promotion.Thus,it is an ideal model strain for the root colonization research.In our previous studies,a series of genes related to biofilm formation based on the analysis at genomic level have been identified,including the genes involved in regulating the secondary metabolism Gac/Rsm system(RNA-binding gene rsmA,non-coding RNA rsmYZ and gacA gene in two-component system for the secondary metabolic regulation),sigma factor(?~544 factor rpo N and?~700 ECF factor algU genes),cyclic diguanylate degrading gene bifA,cyclodiguanylate synthetase gene sad C,bacterial cellulose synthase gene bcsA,and extracellular polysaccharide synthase gene pslA.The regulatory mechanism of biofilm formation in the nitrogen-fixing microorganisms has been systematically studied in pure culture experiment,providing a good foundation for further research on the mechanism of biofilm formation-related genes during root colonization.By using competitive inoculation coupled with resistance screening and plate counting method,the main object of this study is to figure out the function of biofilm-related genes in competitive colonization of rice root surface and promotion of maize plant growth.The main results are as follows:1.Under simulated rhizosphere oligotrophic conditions,compared with wild-type,the biofilm formation of the RNA-binding gene rsmA mutant strain increased by 17%,indicating rsmA gene may played a certain role in inhibiting the formation of biofilm in the rhizosphere environment.While the biofilm formation of the pslA,gacA,rpoN,sadC,algU,rsmY and rsmZ genes mutant strains lead to a decline of 35-70%against with wild-type,indicating their positive role in the biofilm formation.In addition,little effect of cyclic diguanylate degrading gene bifA mutant strain on the biofilm formation was observed.2.In the hydroponic culture with controlled temperature and light intensity,the wild-type A1501was mixed in pairs with each mutant strain in equal proportion as inocula to the rhizosphere of rice seedlings.By competing with wild-type,the coloning density of rsmA and rsmZ gene mutant strain significantly increasd by 55%and 65%.However,the coloning density of algU,pslA,sadC and bcsA gene mutant strains significantly decreased by 30-73%.Besides,the coloning density of rpoN,gac A and rsmY genes mutant strains showed no significant difference compared with wild-type.The result of competitive colonization of mutant strains was consistent with those of biofilm formation under simulated rhizosphere oligotrophic conditions,suggesting that these genes were involved in the regulation of biofilm formation which was vital for the effective colonization on root surface.3.By using the sterile vermiculite as soil matrix,maize seedlings were inoculated with the wild-type,rsmA,algU,sadC,pslA genes mutant strains applied as single-strain or mixed inocula.In both single-strain and mixed inocula treatments,inoculation of maize with rsmA gene mutant strain caused a significant increase in plant height and root weight(4.4%and 12.8%on average).While inoculation of maize with pslA or algU gene mutant strain caused a significant decrease in plant height and root weight(26.1%and 6.4-21.8%on average).Furthmore,maize inoculated with a mixture of rsmA and sad C gene mutant strains increased by 5.8%in the plant weight and by 4.9%in the root weight,comparing with the maize inoculated with a mixture of algU and pslA gene mutant strains.The results above indicated that the plant biomass was highly associated with the genes related to biofilm formation in the nitrogen-fixing bacteria.In summary,this study demonstrated the biofilm-forming related genes in Pseudomonas stutzeri A1501 play an important role in the root colonization and plant growth promotion.These results will provide an important theoretical basis for revealing the regulation mechanism of root colonization and biofilm formation,and exploring the molecular mechanism of the interaction between crops and nitrogen-fixing microorganisms.