Role Of Biofilm Formation By Bacillus Amyloliquefaciens JK-JS8 In Biocontrol Against Sphaeropsis Sapinea

Biocontrol bacteria can exert their biological control functions by inhibiting the growth of plant pathogenic bacteria,inducing plant systemic resistance and competing with plant pathogenic bacteria for ecological niche.Biofilm is an important form of biocontrol bacteria occupying ecological niche,which can help them resist adverse environment and build a"wall"to prevent the invasion of plant pathogenic microorganisms.The role of biocontrol bacteria biofilm in the antagonism of pathogen resistance is an important direction to explore the biocontrol mechanism of bacteria.Previous studies showed that Bacillus amyloliquefaciens JK-JS8 had antagonistic effect on Sphaeropsis sapinea（the pathogen of pine shoot disease）,and the strain coμLd produce obvious biofilm structure after fermentation.However,it remains to be seen whether the formation of biofilm plays a role in the antagonistic process during the strain JK-JS8 against pathogens.Therefore,in this study,the antagonistic ability of JK-JS8 against Sphaeropsis sapinea in the absence of biofilm was investigated by inhibiting the formation of JK-JS8 biofilm under Zn stress,and the relationship between the decrease of antagonism and biofilm formation of JK-JS8 strain was explained in several aspects.At the same time,in order to better play the biocontrol function of JK-JS8 strain in the forest,we also investigated the effects of different media and environmental conditions on the formation of this strain biofilm.Our main findings are as follows:1.The ability of Bacillus amyloliquefaciens JK-JS8 to form biofilm was different in different media,among which YSP（Yeast Extract Sucrose Peptone）medium was the best medium for biofilm formation.Environmental conditions had a great influence on biofilm formation of JK-JS8strain,and the optimal condition was to stand at 30℃for 48 h.It was not suitable for biofilm formation in both too acidic or too alkaline environment,and the neutral condition（p H=7）was the most suitable for biofilm formation.At the same time,the study also found that except for Cu²⁺,the other four metal ions have different degrees of inhibition on the formation of JK-JS8 biofilm,among which Zn²⁺and Mn²⁺have the strongest inhibition effect on the biofilm,while Mg²⁺and K⁺have weaker inhibition effect.2.Firstly,the formation of JK-JS8 biofilm was inhibited by ZnCl₂.Then,the antagonistic ability of JK-JS8 against Sphaeropsis sapinea in the absence of biofilm was detected.Finally,the changes of lipopeptides before and after biofilm deletion were identified by High Performance Liquid Chromatography-Mass Spectrometry（HPLC-MS）.The results showed that the production of bacteriostatic lipopeptide Bacillomycin D decreased after Zn stress caused the disappearance of biofilm.We also found that Bacillomycin D was one of the main antifungal substances against pine shoot pathogen.3.We detected the transcription level of JK-JS8 biofilm-related genes after ZnCl₂ treatment by RT-q PCR.The results showed that there was no significant difference in the transcription level of eps A（extracellμLar polysaccharide related genes）and sip W（signal peptidase synthesis gene）.However,tas A（protein biofilm amyloid associated genes）,srf AA（synthesis of surfactant associated genes）,spo0A（key regμLatory genes for biofilm formation）and bam D（key synthesis genes of Bacillomycin D）were down-regμLated in different degrees.By constructing the spo0A deletion mutant,we found that the loss of spo0A gene affected the formation of JK-JS8 biofilm and Bacillomycin D,and reduced its ability to antagonize Sphaeropsis sapinea.However,adding Bacillomycin D did not restore the biofilm character.It is suggested that the inhibition of JK-JS8under Zn stress may be due to the down-regμLation of spo0A,which inhibits biofilm formation and affects the synthesis of antimicrobial peptide Bacillomycin D.4.The function of Tas A in JK-JS8 was analyzed by bioinformatics and prokaryotic expression system.The results showed that Tas A had contact inhibitory activity against the pathogen of pine shoot back disease.It is further indicated that biofilm formation plays an important role in antagonistic process of JK-JS8 against Sphaeropsis sapinea.