Screening Of Biocontrol Strains Against Vegetable Soft Rot And Study Of Their Biocontrol Effect

Vegetable soft rot is the most common bacterial disease with wide host range and long damage period.It can occur in all stages of vegetable production,storage and transportation,which restricts the development of vegetable industry.Under the background of vigorously developing green agriculture and fully implementing the double reduction of chemical fertilizers and pesticides,the use of beneficial microorganisms is an effective means to achieve green prevention and control of vegetable soft rot.The availability of efficient biocontrol strains is the basis of soft rot prevention and control and has application potential.Based on the frequent occurrence of vegetable soft rot in Heilongjiang Province,this study systematically studied the isolation,identification and pathogenicity analysis of pathogenic bacteria,screening and identification of broad-spectrum biocontrol strains,optimization of liquid fermentation conditions and study of biocontrol characteristics of biocontrol bacteria,and the in vitro and greenhouse control efficiency of biocontrol bacteria on vegetables such as cabbage,so as to provide new biocontrol strain resources and biological control technology for the prevention and control of vegetable soft rot.To help the development of green and organic vegetable industry.The main research results are as follows:1.Isolation and identification of vegetable soft rot bacteria.Tissue separation method and Koch’s postulates were used to isolate 11 strains of soft rot pathogens from the disease-like tissues of cabbage,onion,baby cabbage and other vegetables.The strains were identified by morphological,physiological and biochemical characteristics and 16S r DNA sequence analysis.In vitro artificial inoculation was used for pathogenicity analysis,and three strains with strong host widespread pathogenicity were obtained.They were Burkholderia gladioli YC1,Pectobacterium carotovorum BC2 and Pseudomonas sp.WWC2,respectively.2.Screening and identification of biocontrol bacteria for vegetable soft rot.1 012bacteria were isolated from 20 rhizosphere soil samples of vegetables such as cabbage,cucumber,eggplant and bean by using the diluted coating method.Three pathogenic bacteria YC1,BC2,and WWC2 were used as targets.Six strains with better antagonistic effects were screened using plate antagonistic method,including DJ1,MC1,SD2,TD1,XHS1,and SD1.Based on morphological,physiological and biochemical characteristics and molecular biology,all six strains were Bacillus,DJ1,MC1,SD2,TD1 SD1 was identified as Bacillus velezensis,and XHS1 was identified as Bacillus amyloliquefaciens.The antibacterial activity of strain DJ1 against YC1,BC2 and WWC2 was higher,and the antibacterial zone diameter was 10.60 mm,6.92mm and 3.92 mm,respectively.Therefore,DJ1 strain was selected as the object of subsequent biocontrol study.3.Study on biocontrol characteristics of strain DJ1.The biocontrol characteristics of strain DJ1 were studied in terms of inhibition spectrum,antibacterial substances and prediction of antibacterial substances based on genomic information on the degradation of quority-sensing signal factors and growth promotion of Chinese cabbage.Strain DJ1 has a wide antibacterial spectrum,which can inhibit the growth of Escherichia coli,Staphylococcus aureus and 8 plant pathogenic fungi in addition to 3strains of soft rot.Three kinds of antibacterial substances were obtained by hydrochloric acid precipitation,ethyl acetate extraction and ammonium sulfate precipitation,which showed strong antibacterial activity against BC2 strain.The antibacterial zone diameters were 22.30 mm,18.33 mm and 12.33 mm,respectively.Quantitative analysis was conducted on the crude protein precipitated by ammonium sulfate,and 96.60 mg of crude protein was obtained by precipitating 1 L of fermentation broth with 20%ammonium sulfate.The genome of strain DJ1 was sequenced and the complete genome map was obtained.The genome size was 3 929 792 bp.The G+C content was 46.5%,encoding 3942 genes.GO annotation had 3 023 genes related to cell composition,molecular functions and biological processes.KEGG analysis revealed 33 metabolic pathways,and anti SMASH software was used to analyze secondary metabolites,and it was found that DJ1 could produce lipopeptides,butanamicin,macrolides,bacitracin and bacitolysin antibacterial substances.Using Agrobacterium tumefaciens NT1 as the indicator strain,X-gal staining technique was used to determine that strain DJ1 can degrade the quorum sensing signal molecules N-hexanoyl-L-homoserine lactone and N-(Ketocaproyl)-L-homoserine lactone of BC2.The results indicated that strain DJ1 had the ability to degrade quorum sensing signal factors of Chinese cabbage soft rot pathogen.Chinese cabbage seeds of different concentrations of bacteria liquid processing after greenhouse cultivation,found 1×10~7 and 1×10~8 CFU/m L concentration of bacteria can promote the growth of Chinese cabbage seeds.Compared with the control,the root and stem lengths were significantly increased.The above studies showe that strain DJ1has a disease prevention and growth promoting effect,making it a good biocontrol strain.4.Study on optimization of liquid fermentation conditions and control effect of strain DJ1.Single factor and orthogonal test methods were used to optimize the medium formulation of strain DJ1.The optimal formula was maltose 2.5%,yeast powder 0.5%,sodium chloride 1.0%,manganese sulfate 0.02%and distilled water 1 000 m L.The combination of single factor methodology was used to optimize the optimal culture conditions for strain DJ1.Single factor was used to optimize the culture conditions of strain DJ1.The optimal culture conditions were culture time 48 h,p H 8,culture temperature 30℃,rotation speed 160 r/min,inoculation amount 0.5%.Using 1×10~8CFU/m L bacterial solution for in vitro and greenhouse control,the in vitro control effects on soft rot disease of Chinese cabbage,scallions,and baby cabbage were 84.30%,60.21%,and 69.96%respectively.The control effect on soft rot of Chinese cabbage in greenhouse potted plants was 70.36%,indicating that strain DJ1 has certain development and application value in the biological control of vegetable soft rot.