Study On The Effect Of Manganese Ion On Biofilm Formation And Banana Root Colonizaiton Of Bacillus Subtilis Strain R31

Bacillus subtilis strain R31 is an endophytic bacteria with broad-spectrum antifungal function,it had certain control effects on the banana fusarium wilt in pot and field experiments,but the control efficiency was not very stable,it was influenced by the concentration of inoculated bacteria and the initial incidence of the banana fusarium wilt.The manganese ion could promote the bio film formation of R31 and deactivate the effect of fusaric acid,which is the major virulence in coursing the banana fusarium wilt,on R31 bioflim formation.It could be considered that adding manganese as synergistic agent to increase the stability of the banana fusarium wilt control efficiency of R31 and decrease the usage of R31.Firstly,the effect of different concentration of manganese ion on R31 growth and biofilm formation were evaluateed in DSM₂ culture medium.Then the expression change of genes related to biofilm formation and multiple cell types of R31 with different concentrations of manganese ion treatments were measure with real-time quantitative PCR technique.Thirdly,the effect on banana?Musa spp.AAA cv Baxi?roots MnSOD activity and H2O2 burst of different concentration of manganese ion and R31 treatments were tested.And green fluorescent protein?GFP?tagged R31 strain was used to monitor the influence of different concentration manganese ion on R31 colonization in banana roots.Finally,the effect of 120 ?g/mL MnSO₄ and 9 ?g/mL fasrium acid and different concentriaon of R31 on the roots MnSOD activity and H2O2 burst were tested.The results could offer the scientific guides for R31 used in banana fusarium bio-control in different fields.The results were as follows:1.Manganese ion promoted R31 growth and biofilm formation in DSM₂ medium.R31 could not generate pellicle and spreading colony when cultured in DSM₂ medium,but adding different concentrations manganese sulfate in DSM₂ medium could promoted R31 pellicle generation in DSM₂ liquid medium and spreading colony in DSM₂ solid medium,120 ?g/mL manganese sulfate had the significantly promotion effect.When R31 were shaking cultured in DSM₂ liquid medium,they grew slowly and the lagphase lasted up to 15 h,but after manganese sulfate added,R31 growth was promoted and the lagphase was shorten to 2 h.There were no differences among the promotion effects of different concentrations of manganese sulfate.2.The effect of manganese ion on the expression change at different points of genes related to R31 biofilm formation,motility and extracellular material production.The key genes contained md-PTR?Manganese-dependent protein-tyro sine phosphatase encoding exopolysaccharides?EPS?production??md-IP?Manganese-dependent inorganic pyrophosphatase??epsG?Exopolysaccharides?EPS?production??aprE?Encoding alkaline serine proteases??tasA?Encoding amyloid protein??flgG?flagellum biosynthesis??degU?A master regulator of differentiation?and sfp?Encoding surfactin?.Results showed that the expression of md-PTR were inhibited by manganese ion in R31 early growth?4-8 h?,but promoted significantly at mid of growth?12 h?,80 ?g/mL manganese sulfate could promote md-PTR expression at every time point.The expression of epsG were promoted at all points,the difference existed in the different concentrations of manganese sulfate treatment,but epsG expression of all treatments arrived the peak value at 12 h.Manganese ion promote the expression of sfp appeared at 12 h,but inhibited expression of sfp in the early growth of R31.The expression of aprE also significantly affected by manganese ion,the promoting effects lasted from 8 h to 48 h.Manganese ion showed promoting degU expression only at 48 h,and there was no significant effect on the expression in early time.The expression of flgG were inhibited at all points.The expression changes of tasA and md-IP were not found.To conclude,manganese ion could promote the expression of aprE in the early growth,promote the expression of md-PTR?epsG?sfp at mid of growth,which is relate to biofilm formation,and promote the expression of degU in the late growth but inhibit the expression of flgG.3.The effect of manganese ion on the banana roots H2O2 and MnSOD activity caused by R31 colonization:R31 inoculated at 10⁶ cfu/mL and 10⁷ cfu/mL concentration could cause the banana roots MnSOD activity increasing and reaching the highest level at 4 h,the roots H2O2 levels also increased significantly.The MnSOD activity of roots inoculated by 10⁸ cfu/mL R31 were stable at different time points,and the activity was about 4 U/g.In general,manganese sulfate had weak effect on the stabilization of the roots MnSOD activity and H2O2 levels with 10⁶ cfu/mL R31 treatments,but low concentration of manganese sulfate was benefit for the roots MnSOD activity and the H2O2 levels keeping stable,the effect of 80 ?g/mL MnSO₄ treatment is best.Combination of 80 ?g/mL manganese sulfate and 10⁷ cfu/mL R31 or only treating with 10⁸ cfu/mL R31 were most beneficial to stabilize the roots MnSOD activity and the H2O2 levels.4.The effect of manganese ion on the R31 colonization on banana roots:the fluorescence intensity of 10⁸ cfu/mL R31-gfp treated roots was higher than 10⁷cfu/mL R31-gfp treatment,but the fluorescence were not observed on roots treated by 10⁶ cfu/mL R31-gfp.40 ?g/mL manganese sulfate increased the fluorescence intensity of 10⁷ cfu/mL R31-gfp treated roots.When manganese sulfate concentration was increased to 80 ?g/mL,the luminescence intensity of treated roots significantly increased.But with the in increasing of manganese sulfate up to 100 ?g/mL or more higher,the fluorescence intensity would weaken.Combination of 80 ?g/mL manganese sulfate and 10⁷ cfu/mL R31 was most beneficial for R31 colonizing on Baxi roots.5.The effect of manganese ion on passivating the toxin of fusaric acid and the banana roots H2O2 and MnSOD activity caused by R31 colonization:The roots MnSOD activity of fusaric acid treatment increased only slightly at 4 h,but level of H2O2 increased significantly at 8 h.The effect of fusaric acid on the changes of H2O2 and MnSOD activity treated by 10⁶ cfu/mL and 10⁷ cfu/mL R31 at the same time were not obvious,but it could improve H2O2 level and MnSOD activity increasing which treated with 10⁸ cfu/mLR31 at the same time.120 ?g/mL manganese sulfate was benefit for stabilization the roots MnSOD activity and the H2O2 level with 9 ?g/mL fusairum acid and 10⁷ cfu/mL R31,but could improve MnSOD activity and H2O2 level increasing with 9?g/mL fusairum acid and 10⁸ cfu/mL R31.Manganese ion could stimulate the gene expression related to R31 extracellular secretion and promote R31 growth and biofilm formation by some unknown way.80?g/mL manganese sulfate was beneficial to maintaining the MnSOD activity and the H2O2 level,it was similar to the 10⁸ cfu/mL R31 treatment.80 ?g/mL manganese sulfate significantly promoted 10⁷ cfu/mL R31-gfp formatting fluorescent biofilm on the surface of Baxi roots.9 ?g/mL fusairum acid could stimulate the H2O2 level increasing sharply after 8 h of irrigating,combination of 120 ?g/mL manganese sulfate and 10⁷ cfu/mL R31 was beneficial to remaining the stable of plant immune response when fusairum acid existed According to these results,it could be taken into account that taking manganese ion especially manganese sulfate as synergistic agent for helping R31 to control banana fusarium wilt.