Role Of Biofilm Formation By Bacillus Pumilus HR10 In Biocontrol Against Pine Seedling Damping-off Disease

Biological control mediated by PGPR control diseases through varieties of mechanisms.Biofilm is an significant form of evolution in the development of PGPR.It can occupy the niche to strengthen colonization and resist adverse environments.Elucidating biofilm formation,colonization and its association with other mechanisms is an important aspect of exploring biocontrol mechanisms.Bacillus pumilus HR10 has inhibitory activity against Rhizoctonia solani causing pine seedling damping-off disease,and it can produce a robust biofilm structure.In this study,B.pumilus HR10 was mutated and its mutants were screened by colony morphology.The biofilm-related characteristics of strains were measured and the colonization abilities were evaluated by several ways.The experiments of induction of pine seedling resistance and the biocontrol of pine seedling damping-off disease were performed by inoculation with Pinus massoniana seedlings.In addition,in order to better exert the potential of growth-promoting,resistance to disease and stress of B.pumilus HR10 in the wild,the effects of different culture conditions on its biofilm formation was researched.The main results are as follows:1.After mutation,the different phenotype of biofilm-deficient mutants of B.pumilus HR10were screened.From the results of colony morphology and biofilm formation on the liquid-air surface,it was found that the HR10 WT strain form a significant three-dimensional fold biofilm structure.The biofilm by MA15 obtained the lowest degree of fold,and its formation and development are relatively slow.Biofilm on the liquid-gas surface was more robust than MA15while its colony structure was relatively simple with that of MA15,indicating that biofilm had certain adaptability in a certain living environment.MA4828 formed the most complete biofilm among the mutants screened,which was almost close to the level of WT strain.2.The content of biofilm matrix,swarming motility and the biofilm-related gene expression of 4 strains were detected.It was found that the content of biofilm matrix by the mutant strains were all reduced.The matrix content and related gene expression were positive correlation with biofilm formation.Relatedly,the secretion of extracellular substances made biofilm differentiate variously.The more secreted extracellular substances,the more dense and complex biofilm was.The results of qualitative swarming motility and RT-qPCR gene quantitative measurement showed that the reduction of the swarming ability led to the slow formation of biofilm.The expression of flagella movement fliG gene was consistent with swarming motility to some extent.The expressions of sfp gene of MA15 and MA1330 at 24 h and 48 h were different from that of WT strain,indicating that the synthesis of surfactin from B.pumilus HR10 may also regulate the formation of biofilm.3.The colonization on P.massoniana seedling root of 4 strains was tested using crystal violet staining and plate counting and observed under scanning electron microscope.It was found that 4 strains could colonize on the roots,and colonization of 3 mutants were declined,especially MA15.It is speculated that the adhesion and swarming motility of MA15 were weak and the rate of aggregation to form biofilm was slow and the level is low.No matter in the rhizosphere or on the root surface of pine seedlings,colonization was significantly weaker than colonization of other 3 strains.MA1330 showed the fastest degradation rate in the colonization stage of the later 10-12 days,which is basically consistent with the trend of the fastest degradation of biofilm on the liquid-gas surface.4.Combining the inhibitory activity of 4 strains against R.solani and the detection of previous biofilm-related gene expression,it is further speculated that surfactin may be involved in regulating the biofilm formation and inhibitory activity of B.pumilus HR10 strains.After inoculation with the strains,the expression of the defense genes of the pine seedling was tested.It was found that 4 strains can significantly enhance the expression of PR2,the SA pathway related gene,which indicates that the strains may trigger the SA signaling pathway.The PR2expression of pine seedlings inoculation with MA15 was significantly lower than that inoculation with HR10 WT strain treatment,showing that the defect of biofilm formation of MA15 resulted in the weakening of its ability to induce plant resistance.However,the expressions of PAL,COI and GPX showed no significant change,demonstrating that the pine seedling defense pathway induced by strains was specific.The inoculation test showed that four strains could significantly reduce the incidence of damping-off disease,and the control effect of WT strain reached upto 76.88%,which verified its effective biocontrol activity.The biocontrol effects of mutants were reduced,of which MA15 showed the lowest effect.It is speculated that the defect of biofilm formation and inducing resistance both caused the lowest control effect.5.Different culture conditions had different effects on the biofilm formation of B.pumilus HR10.The biofilm formed by B.pumilus HR10 exhibited good performance at different temperatures.At 20-40°C,HR10 strain formed biofilm structure instead of planktonic state.Despite the significant difference,HR10 strain was capable of forming biofilm at pH 5.5-9.0.The addition of several sugar sources made biofilm more robust and complete,especially glucose.Low concentrations of Fe²⁺,Ca²⁺,K⁺and Na⁺promoted biofilm formation of HR10strain,while Cu²⁺,Mn²⁺and Zn²⁺significantly reduced biofilm foramtion,especially Zn²⁺which even affected the normal growth of HR10 strain.