| The postharvest fruit disease during citrus storage is caused by P.italicum and P.digitatum,which is a big problem facing the citrus industry.Before storage,the prevention measures are to use chemical fungicides to soak the fruit,which is currently the common method.However,the long-term use of chemical fungicides is prone to drug resistance problems,and residual chemical fungicides also have safety issues that affect human health.In recent years,biocontrol strains such as Bacillus have been widely used in the control of postharvest pathogens,and have shown excellent control effects.Therefore,the development of antimicrobial agents has broad application prospects,which can be applied to the prevention and treatment of citrus postharvest diseases.Strain UB-201712 in this laboratory.isolation from citrus rhizosphere soil,which has biocontrol potential.In this study,the taxonomic status of Strain UB-201712 was determined using morphological and molecular biology methods,then determine the antibacterial spectrum of the Strain UB-201712,the antibiotic substance of the Strain UB-201712 was analyzed by PCR technology LC-MS etc.Finally,the spore production of this strain is optimized under liquid and solid fermentation conditions.The main results follows:(1)In order to determine the classification status and biological characteristics of the strain UB-201712,morphological,physiological,biochemical and molecular methods were used.The strain is rod-shaped,endophytic spore,and can produce,protease and cellulase.The homology of 16S rRNA with B.methylotrophicus,B.siamensis,B.amyloliquefaciens and B.velezensis was 100%.The homology of rpo B with B.velezensis was 99%.The amylase refore,it was determined as Bacillus velezensis ub-201712。The strain was aerobic and could grow at pH of 5-8 and NaCl concentration of 0-25g/l.(2)In order to determine the antibacterial spectrum of strain UB-201712,the dualculture method and the Oxford cup method were used to measure the antibacterial effect.The results showed that it had anti-pathogenic effect on 12 pathogenic fungi such as Rhizoctonia soloni,Magnaporthe grisea,P.italicum,P.digitatum.It had no inhibitory effect on 10 pathogenic bacteria,such as Xanthomon ascampestris pV.Citri,Staphylococcus aureus,Micrococcus kristinae etc.(3)In order to predict the antibiotic genes and explore the anti-pathogenic substances,PCR and LC/MC were used to detect them.PCR results showed that the strains UB-201712 present 10 different antibiotics genes,including Surfactin,Bacyllomicin,Fengycin,Biotin,Iturin,Bacylisin,Difficidin,Macrolactin,Bacillusaene and Bacillibactin.The results of LCMC showed that the crude methanol extract contained C15-17 Iturins,C15-17 Fengycin and C12-16 Surfactin.(4)In order to improve the spore yield of strain UB-201712 under liquid condition,single factor and response surface method were used to optimize the medium and culture conditions,and the best medium was obtained:soybean meal 4.88g/L,yeast extract 5.04g/L,MgSO4 2.37 g/L,bran 5 g/l,molasses 5 g/L and MnSO410mg/L.The optimal culture conditions were:fermentation temperature 37℃ pH value 6.5,inoculation amount 6%,liquid loading amount 60ml/250ml and culture time 54h.(5)In order to improve the spore production of strain UB-201712 under solid condition,the medium and culture conditions were optimized by single factor and uniform experiment.The results showed that the best ratio of solid material was soybean meal:corn meal:bran:mushroom residue=5:15:40:40.and added molasses 3%,peptone 1.4%,urea 0.3%,MnSO4 0.04%.The optimal culture conditions were:the culture time was 72 hours,the loading quantity was 35g/250ml,the inoculation quantity was 5%,and the materialliquid ratio was 1:1.5. |
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