Effects And Mechanisms Of Bacillus Amyloliquefacien SQRT3 On Biological Control Of Bacterial Wilt In Tomato

Tomato(Lycopersicon esculentum Mill)is one of the main vegetables which broadly distributes around the world.The soil-born disease of tomato bacterial wilt is caused by Ralstonia solanacearum,which occurs in tropical,subtropical and some temperate regions,and causes a lot of economic loss every year and restrictes the development of the tomato production.The R.solanacearum from different countries and hosts have significant difference in serology,biovar and pathogenicty,so tomato bacterial wilt is difficult to be controlled.Traditional chemical pesticides control methods result in the environmental pollution and food safety problems,and also constantly enhance the resistance of pests and pathogens to chemical pesticides.Biological control of soil-born disease,due to its security and environmentally friendly way,is receiving more and more attention.In this study,one antagonistic strain SQRT3,isolated from tomato rhizosphere soil showed strong antagonist effect against R.solanacearum.The strain SQRT3 was used in pot experiments to assess their control efficiency of tomato wilt.To explore possible biocontrol mechanisms,the ability of colonization in tomato rhizosphere soil and inducing tomato systemic resistance of strain SQRT3 were studied.Besides,the phytase and growth hormone production of SQRT3 were determined.The main results obtained were listed as follows:1.In this study,the strain SQRT3 showed highly antagonistic effect against R.solanacearum ZJ3721 on NA medium plate,and its inhibiting zone ranged from 17 mm to 25 mm.Strain SQRT3 also could inhibite some fungal pathogens.The fractions of SQRT3 fermentation culture were separated by RP-HPLC.Then different fractions were examined by inhibiting R.solanacearum on NA plate and identified by the mass spectrometry.Found a new antagonistic substances,its molecular weight is 104.3.Based on the morphologic,physiological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene,SQRT3 was identified as B.amyloliquefaciens(GenBank accession number KM588088).B.amyloliquefaciens SQRT3 could produce protease,phytase and siderophore,and had abilities of biofilm formation and phosphate solubilization.Its maximum growth was recorded at 30-35?,pH 7.0 and 1.5%of NaCl concentration,And tryptone and sucrose were used as the most suitable nitrogen and carbon sources respectively.2.Application of organic fertilizers can significantly increase the numbers of antagonistic bacteria SQRT3 in the soil,and antagonistic bacteria SQRT3 number were more than 107 CFU·g-1 dry weight soil.The population of SQRT3 would decline repidly in soil if without application of manure organic fertilizers.Adding differernt proportions of raw pig feces in organic fertilizer had little effect on tomato biomass and the number of SQRT3 and spores in soil.The antagonistic bacteria SQRT3 could significantly reduce the disease incidence of tomato wilt.Different inoculation methods of SQRT3 could significantly affect biocontrol effects.With root-diping inoculation methed(Treatment 1),biocontrol efficiency reached 81.25%,which was better than that by drenching treatment with SQRT3(65.84%).The numbers of SQRT3 by root-diping inoculation methed were more than that by drenching method at different stages,and the largest number of SQRT3(7.69 log CFU g-1 DW)by root-diping inoculation methed reached at 17-day after inoculation.And the population of R.solanacearum in Treatment 1 was much less than that in Treatment 2.The antagonistic bacteria SQRT3 could significantly promote tomato growth.The tomato plant fresh weights,dry weights,stem diameter,root length,root volume,root area and choorophyll contents were significantly higher in SQRT3 treatment than in CK treatment in pot experiment.The ground dry weight was more 47%in SQRT3 treatment than in CK.The auxins in SQRT3 fermentation broth were identified by high performance liquid chromatography(HPLC).The results showed that indole-3-acetic acid,bibberellin A3 and indole-3-butytric acid were produced by SQRT3.3.Antagonistic bacterium B.amyloliquefaciens SQRT3 having phytase activity could degrade phytic acid to improve plant phosphorus in soil.The phytase activity in antagonistic bacterium SQRT3 was influenced by the fermentation temperature.The highest phytase activity was obtained at 37?and maximum ptytase activity was 16.33 U ml-1 at 48 h after fermentation.Phytase can degrade phytic acid in a wide pH range from pH 3.5 to pH 8.5,with optimal pH 5.5 in vitro.The phytase gene amplified from genomic DNA of SQRT3 was 1152 bp and encoded 383 amino acids,which showed 100%similarity with that in B.amyloliquefaciens subsp.Plantarum CAU B946.The phytase of SQRT3 have signal peptide,and the cleavage site was between position 26 and 27:26A-K27.Phytase had a molecular weight of 39.24 kDa predicted by software genious 3.8.Phytase gene was cloned into an expression vector pET28a(+)to obtain pET28a-phytase.Then,the recombinant plasmid was transformed into E.coli BL21(DE3)by electroporation.Phytase overexpression was induced by IPTG and purified by Ni-chelating affinity chromatography.It was approximately 40 kDa detected by SDS-PAGE.4.The shuttle vector pHAPII,which contain the green fluorescent protein gene(gfp), was transformed into SQRT3 by electroporation.Transformants SQRT3-GFP were obtained,which could emit bright green fluorescent under the UV microcopy.Strain SQRT3-GFP showed the same characteristics such as biofilm formation,growth speed and antagonistic activity as wild strain SQRT3.The plasmid pHAPII in SQRT3-GFP growing in LB liquid medium without antibiotics for 120-h was relatively stable,only 14.2%of plasmid pHAPII was lost.In the soil,the plasmid pHAPII stability was 66.3%at day 25 after inocubation.The distribution of SQRT3-GFP in tomato rhizosphere soil was observed in vermiculite by xonfocal laser scanning microscopy(CLSM).Results indicated that the colonization of SQRT3-GFP on tomato root(107 CFU·g-1 root)if SQRT3-GFP cells were suspensioned in 5%starch solution,then inoculating tomato root was much more than that (5.64 log CFU g-1 root)when SQRT3-GFP cells were suspensioned in PBS,The cells of SQRT3-GFP were mainly colonized on the elongation and differerntiation zones of root, respectively.While SQRT3-GFP cells were not observed along the root tip by CLSM.5.After the inoculation with R.solanacearum,PPO activity was always higher in the T3+RS treatment than that in the RS treatment,and the peak activity was 16.4%higher than that in the RS treatment at 72 h.POD activity showed similar trends as PPO activity in all treatments.The maximum POD activity in treatment of T3+RS was observed at 48 h,whereas it was at 72 h in RS treatment.Therefore,in the presence of R.solanacearum,SQRT3 strain can induce rapidly the synthesis of defence enzymes PPO and POD.In addition,the application of SQRT3 could reduce membrane lipid peroxidation in tomato leaves.The results in our study showed that SQRT3-mediated ISR(induced systematic resistance)in tomato is mainly dependent on the JA-pathway.In the T3+RS treatment,the expression of pin2 in JA-pathway quickly reached the maximum after inoculation of 24 h,which was more 31.52 folds than that in RS treatment at 24 h.But the expression of pin2 in RS treatment reached maximum at 48 h,and far less than the expression induced by SQRT3.