Effect And Mechanism Of Bacillus Velezensis B9 Biofilm Formation On Reducing Cd Uptake In Lettuce

Vegetables in farmland soils can accumulate cadmium（Cd）and lead（Pb）in soil,which will not only reduce vegetable production,but also threaten human health and ecosystem security through food chain and its web.Microbial cells or their metabolites can precipitate or adsorb heavy metals in soil,thus reducing their bioavailability and reducing the absorption of heavy metals by vegetables.Therefore,it is becoming a research hotspot of bioremediation and vegetable safety production technology.Bacillus velezensis B9 strain is a beneficial rhizosphere bacterium isolated from the rhizosphere soil of Chinese cabbage,which has strong biofilm producing and Cd immobilizing ability,also has promoting plant-growth.The purpose of this thesis was to further explore the mechanisms of B9 wild strain,biofilm formation enhanced（Δywc C）and weakened（Δslr R）mutants and their complementary strains（B9S-h and B9Y-h）controlling the absorption of heavy metals.Our results will provide a theoretical basis and technical approach for the remediation of heavy metal pollution in farmland and the safe production of crops.A pot experiment was conducted to study the effects and mechanisms of inoculation by the tested strains B9,B9Δslr R,B9Δywc C,B9S-h and B9Y-h on the growth of lettuce and the absorption of Cd and Pb.The results showed that strains B9 and B9Δywc C significantly increased the shoot biomass of lettuce by 43.1%and 58.7%,respectively,compared with the control.Strains B9 and B9Δywc C reduced the contents of Cd and Pb in lettuce shoot（13.8～45.3%and 12.4～33.9%）,respectively.Also,Cd and Pb contents in lettuce root were decreased by 12.0～31.7%and 11.5～30.5%.Subsequently,the results showed that all the tested strains could colonize in the root and rhizosphere soil of lettuce.Inoculating strains B9 and B9Δywc C increased the p H and enzyme activity of rhizosphere soil,and significantly reduced the bioavailability of Cd and Pb in soil by 30.8～37.8%and24.3～31.6%,respectively.As a result,it was also found that the tested strains B9 and B9Δywc C could significantly improve the bacterial activity and biofilm formation in rhizosphere soils.The root surface biofilm was quickly constructed on the root surface of lettuce by dipping the root,and the morphological structure of the biofilm was characterized.The effects of the root surface biofilm on the growth of lettuce and the content of Cd in lettuce were evaluated,and the results were as follows:Firstly,the roots of lettuce were immersed in B9-gfp,B9-gfp,B9Δslr R-gfp,B9Δywc C-gfp,B9S-p-gfp,B9Y-p-gfp,B9S-h-gfp,B9Y-h-gfp suspensions and cultured for 4 days to form a stable biofilm structure on the root surface of lettuce.The results analyzed by Laser confocal microscope and scanning electron microscope showed that the biofilm formed by strains B9 and B9Δywc C on the root surface of lettuce was more compacter than B9Δslr R biofilm,the colonization ability of B9Δslr R was weaker,and the biofilm formed on the root surface was also scattered.The colonization of the bacterial cells mostly occurred in the mature zone,elongation zone and root hair of lettuce root.Meanwhile,the activity of the constructed biofilm on the root surface could be maintained for 30 days,and the colonization of the strain could also be detected in the roots and leaves of lettuce.Secondly,the results showed that the constructed root surface biofilm could significantly reduce the content of Cd in the root and shoot of lettuce,and the reduction rates were 21.1～53.2%and 19.3～38.7%,respectively,which significantly promoted the growth of lettuce.Finally,the root surface biofilm formed by B9 and B9Δywc C can effectively adsorb Cd in the solution,and the contents of Cd in the biofilm were 0.078 mg bottle^-1and 0.090 mg bottle^-1,respectively,and the content of Cd in the solution for 15 days was significantly reduced by 47.8～62.9%compared with the control group.In addition,we compared the differences of protein expression in lettuce root after strains B9,B9Δslr R and B9Δywc C colonized in the root system to form biofilm and the proteomic differences of the root surface biofilms formed by wild strains and mutant strains by label-free quantitative proteomics analysis technique,in order to reveal the key metabolic pathways that affects the colonization of the root of strain B9 and how the biofilm cells regulate the expression of their genes and proteins to improve their tolerance to heavy metals.The results were as follows:Firstly,proteomics data of lettuce root showed that among the differentially expressed proteins in the presence of strain B9,418 proteins were up-regulated and 238 proteins were down-regulated compared with the control;Among the differentially expressed proteins in root treated with B9Δslr R,393 proteins were up-regulated and 149 proteins were down-regulated;there are 460 protein were up-regulatedl and 201 proteins were down-regulated in B9Δywc C treatment.Simultaneously,GO and KEGG analysis showed that proteins in the inoculated treatments had functions related to organic acid metabolism,coenzyme metabolism,stress response,carbon metabolism,amino acid biosynthesis,secondary metabolite synthesis,glycolysis.Secondly,the root surface biofilms formed by B9、B9Δslr R and B9Δywc C were collected for proteomic analysis.The results showed that after inoculation with B9 and B9Δywc C,the proteins related to basic metabolism,energy metabolism,regulation and stress were all up-regulated,which enhanced the adaptation of lettuce to the external environment.In conclusion,B9、B9Δslr R、B9Δywc C could induce the expression of stress response related proteins in root of lettuce,and B9、B9Δywc C could improve the expression of basic metabolism-related proteins in lettuce,which could enhance the stress resistance of lettuce and promote its growth.