Fermentation Optimization And Preparation Of Biocontrol Agent Bacillus Velezensis HMB28023 Against Grape Gray Mold

Gray mold caused by Botrytis cinerea is the most serious disease in grape production and storage,which can damage many grape tissues.Fungicide-resistant pathogen resulting from long-term use of chemical pesticides and the high recurrence rate of this disease in the field seriously threatens grape industry.Bacillus velezensis HMB28023 is an bacterial isolate screened by our laboratory from soil collected in Jiangzi,Tibet,which shows better antagonism against many pathogenic fungi including Botrytis cinerea,Valsa sordida,Botryospuaeria berengeriana,Sphaeropsis sapinea,Dothiorella gregaria and Cercospora insulana.Especially in the control of grape gray mold,this bacterial isolate has been determined with excellent control efficacy and further application value.In order to further promote the industrialization of the Biocontrol bacterial isolate,the key fermentation conditions and preparation of the isolate were studied in this paper.The main results are as following:1.Optimization of fermentation condition.The composition of the medium suitable B.velezensis HMB28023 was optimized by L9(34)four-factor and three-level orthogonal test.The optimized medium was determined to includ corn powder 30 g,soybean powder 10 g,glucose 2 g,yeast powder 2 g.disodium hydrogen phosphate 2 g,potassium dihydrogen phosphate 0,6 g,calcium carbonate 0.2 g,potassium chloride 0.15 g,magnesium sulfate 0.15 g,ammonium nitrate 0.45 g.manganese sulfate 0.06 g,water 1000 mL.The suitable inoculation amount was 4%and the seed age was 24 h.under these conditions,the fermentation level of-this isolate reaches 4.1×109 CFU/mL in shake flask experiment,which was 95.23%higher than that before optimization,while the fermentation level reaches 8.6×109 CFU/mL in scale-up test in 50 L fermentation tank,and spore-forming time reduces 30 h than that under the shaking flask experiment.2.Development the processing techniques of 12 billion CFU/mL liquid technical material and 15 billion CFU/g dry powder of technical material for B.velezensis HMB28023.The original liquid crude drug of 8.6×109CFU/mL was obtained by fermentation and concentrated 1.5 times by centrifugation.The liquid crude drug of 1.2×1010CFU/mL was obtained.After 2 times centrifugal concentration,the carrier material was added and dried at 37℃ and crushed by air flow pulverizer to obtain 1.5×1010 CFU/mL dry powder.3.Formula of B.velezensis HMB28023 Dust powder.The wetting and dispersing agent of B.velezensis HMB28023 dust powder was screened by flow point method,and the carrier was screened with sedimentation rate.The formula of 10 billion CFU/g B.velezensis HMB28023 dust powder is determined to include calcium carbonate 10-20%,wetting dispersant LT-522W 3%,and dry powder of technical material supplement to 100%.4.Formula of B.velezensis HMB28023 aqueous suspension concentrate(SC).The types and dosage of wetting dispersant,thickener and preservative of B.veleznisis HMB28023 SC were screened,and the formula of 10 billion CFU/mL B.veleznisis HMB28023 SC was determined as wetting dispersant LT-522W 3%,thickener xanthan gum 0.3%,preservative potassium sorbate 2%,and liquid technical material supplement to 100%.5.Evaluation of two preparations to control grape gray mold.The in-vitro-leaf method was used to evaluate the control efficacy of the tested preparation of B.velezensis HMB28023 to control grape gray mold.The results showed that HMB28023 dust powder can completely control grape gray mold after 7 days post inoculation,while the control efficacy of HMB28023 SC reaches at 82.1%.It shows good application potential.