Screening,Identification And Optimization Of Culture Conditions Of Biocontrol Strains Of Bacillus Velezensis And Brevibacillus Laterosporus

Bacillus is a kind of spore-forming Gram-positive bacteria,which is widely used in the prevention and treatment of plant diseases because of its strong resistance to stress,broad spectrum of antibacterial activity and fast growth and metabolism.It can produce a variety of bacteriostatic substances.Unlike traditional antibiotics,it mainly acts through the cell membrane,which causes the leakage of substances in the membrane,and even causes the cells to dissolve directly to cause cell death.In this study,a new strain with antagonistic effect on Alternaria solani and a bacteriostatic width of 5.45 mm was screened from the saline-alkali soil of Tianjin Binhai New Area by using the plate screening method with Aspergillus flavus and Alternaria solani as indicator bacteria.Subsequently,a fermentation broth of more than 10,000 kinds of samples was established.Using a high-throughput 96-well plate method,Bacillus cereus and Pseudomonas syringae pv.lachrymans were used as indicator strains to screen a strain which inhibited Bacillus cereus,with a bacteriostatic rate of 91.00% and a plate punching diffusion method with a diameter of 11.76 mm.Then,the two strains were identified as Bacillus velezensis and Brevibacillus laterosporus and named as WN015 and WN11079,respectively by colony morphology observation,microstructure observation,physiological and biochemical tests and 16 S r DNA identification.The single factor test was used to optimize the culture condition of strain WN015 with its antibacterial efficacy as the response value and the inhibition zone width of strain WN015 increased from 5.45 mm to 6.7825 mm.On the basis of it,the response surface method was used to further optimize the culture conditions.Firstly,the Plackket Burman test was carried out on the strain WN015.The results showed that the initial p H value,fermentation temperature and inoculum of the medium had significant effects on the antibacterial activity of the strain WN015.Secondly,according to the PB test and the steepest climbing test results,the design center combination(CCD)test shows that when the initial p H of the medium is 7.55,the fermentation temperature is 32.47?,and the inoculum is 4.2%,the strain WN015 has the strongest antibacterial activity,and the inhibition zone width is 12.68 mm,which is 2.33 times that of the unoptimized one.The crude extract extraction test showed that the antibacterial substance had the best precipitation when the ammonium sulfate saturation was 80%.The sensitization test on the temperature,p H and protease of the fermentation supernatant showed that the antibacterial activity of the fermentation supernatant was about 42.35% after autoclaving at 121 ? which was not sensitive to weak acid and weak alkali environment,sensitive to strong acid and alkali.The fermentation supernatant after treatment with proteinase K decreased the bacteriostatic activity by about 31.3%.The antibacterial efficacy of WN11079 was optimized by the same method.The results of PB test showed that the initial p H value of the medium,the shaking speed and the fermentation temperature had significant effects on the antibacterial activity of WN11079.The results of the center combination(CCD)test showed that when the initial p H value was 7.75,the shaking speed was 212.35 rpm,and the fermentation temperature was 34.41 °C,its antibacterial effect is the strongest,and the diameter of the inhibition zone is 24.48 mm,which is 2.08 times that before optimization.The crude extract extraction test results showed that the antibacterial substance had the best precipitation effect when the ammonium sulfate saturation was 40%.The test results of temperature,p H and protease sensitivity of the fermentation supernatant showed that the fermentation supernatant was not sensitive to temperature and acidic environment,sensitive to alkaline environment,and not sensitive to protease.In this paper,the antibacterial activity of Bacillus velezensis WN015 and Brevibacillus laterosporus strain WN11079 was preliminarily explored,which provided a theoretical basis for the follow-up study of antibacterial substances in these two antagonistic strains and its application in plant disease prevention and control.