| Plant growth-promoting rhizobacteria(plant growth-promoting rhizobacteria,PGPR)can promote plant growth and control soil-born disease through influencing nutrient acquisition efficiency of plant and suppressing phytopathogens.Bacillus,one of the most representative biocontrol agents in PGPR,is widely existing in soil,air,water,animals and plants.It has excellent characteristics such as secreting a large number of antimicrobial metabolites which can effectively inhibit the growth of Fusarium oxysporum,Aspergillus Niger,Botrytis cinerea and other pathogenic microbes,and can also induce system resistance of host plants.Thus,Bacillus is one of the biocontrol strains approved by the Ministry of Agriculture for agricultural production.At present,the development and utilization of Bacillus is mostly focused on agricultural production,such as the production of biological fertilizer,biological insecticide and seed coating agent,etc.,and the acquisition of sufficient Bacillus density is an important prerequisite to achieve this goal.The liquid fermentation technology is often used in production combined with the improvement of medium components and the modification of culture environment.Therefore,the high density liquid fermentation technology is necessary for increasing the bacterial density and accumulating secondary metabolites.In this study,Bacillus velezensis SQR9 was used as model strain,and the effective cell density in the fermentation broth was used as phenotype.We carried out a series of experiments to explore the optimal medium composition and fermentation process.And some other PGPRs were selected to test the fermentation effeciency of the novel medium and fermentation condition.We expect that this study could provide a reference for high-density fermentation of PGPRs.Bacillus velezensis SQR9 was used as the model strain,and the density of effective live bacteria in fermentation broth was taken as the phynotype in this experiment.The fermentation medium components and fermentation processes of Bacillus velezensis were optimized by single factor experiment combined with orthogonal test.The results showed that the optimal fermentation medium of SQR9 was as follows:soluble starch 1.5%,snowflake powder 2.00%,fish meal 2.50%,amino acid stock solution 0.50%and inorganic salt components such as sodium chloride 0.10%,potassium dihydrogen phosphate 0.05%,potassium dihydrogen phosphate 0.15%,magnesium sulfate seven hydrate 0.02%.The fermentation conditions were 190 r/min,37℃,2%inoculation amount,40%filling amount and p H 7.0.After growing for 12h,the SQR9 density in fermentation liquid from the shaking flask could reach 1.3×10~9CFU/m L,which was increased by 14 times and 6.6 times respectively compared with the conventional and optimized fermentation conditions of LB medium.Moreover,we used the medium and fermentation condition to grow other PGPRs like Bacillus sp.W19,Pseudomonas sp.PSE13 and Acinetobacter sp.Y40.And the results showed that the bacterial densities reached 1.00×10~9CFU/m L,1.01×10~8CFU/m L and4.58×10~9CFU/m L,respectively.Compared with the conventional and optimized fermentation conditions of LB medium,the bacterial densities were increased by 2.1 times9.2 times,2.6 times and 1.4 times,0.17 times and 1.7 times,respectively.Secondly,we compared the plant growth-promoting ability and pathogen suppression between optimized fermented medium and LB fermented medium.The results were showed as follows:(1)In terms of capacity for IAA secretion,the IAA production of optimized medium was 7.68 mg/L,and IAA production in LB medium was 4.29 mg/L,the IAA production infermented liquid of optimizing medium was 79.00%higher than LB medium.(2)In the growth-promoting pot experiment,compared with the treatment added with LB fermentation broth,the growth-promoting effection of the optimized fermentation broth in plant height,stem diameter,aboveground fresh weight,underground fresh weight,aboveground dry weight and underground dry weight were increased by 3.87%,1.83%,8.68%,6.77%,6.40%and 5.71%,respectively.(3)The optimizated fermentation medium and LB fermentation medium were both used for suppressing Fusarium oxysporum f.sp.cubense(FOC),Fusarium oxysporum f.sp.lycopersici(FOL),Fusarium solani(FOS),pathogenic Ralstonia solanacearum for tomato(RS for tomato)and Ralstonia solanacearum for peanut(RS for peanut).The results showed that under the condition of same volume,the suppression effects of optimized fermentation medium were higher than them of LB fermentation medium,and the suppressing ability to FOL was the most significant.(4)The pot experiment for suppressing tomato RS and FOL showed that the tomato plants biomass in treatment with optimized fermentation broth were higher than the treatment with LB fermentation broth and the control.Furthermore,the abundance of SQR9in rhizosphere with optimized fermentation broth inoculation was higher than that in LB fermentation broth,while the density of pathogens were showed the opposite.The optimized medium designed in this study can significantly increase the bacterial density,short the fermentation time,reduce the production cost,and have a general applicability to the cultivation of other microorganisms.Compared with the traditional medium,the optimized medium can increase the yield of IAA in fermentation broth and enhance the antagonistic ability to pathogens,which provides a good example for the fermentation of PGPR products. |
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