The Screening Of Potentials Of Biocontrol Bacteria Of Citrus Canker And The Study On Antibacterial Substance Of F9 Strain

Citrus canker is a world-wide-distributed disease caused by the Xanthomonas axonopodis pv.citri(Xac),which can infect plant branches,leaves,fruit,resulting in yields reduction and the decline of fruit quality.Its development also limits exports because of the quarantine status of the pathogen in some countries.C urrently,the most used strategies to control this disease is chemotherapy-dominant control system which usually brought a series of problems,such as chemical residua in product,and induction of drug resistance by pathogenic bacteria.Biocontrol measure will be an alternative way for disease prevention and treatment,as it is green,safe and non-pollution.In this study,poteintial biocontrol bacterial was isolated and identified by screening bacterial strains against Xac from rhizosphere soil.1.We collected the rhizosphere soil from the citrus orchard located in Jiangyong County of Hunan Province,and they were used to screenbacterial antagonistic to Xac.Totally 22 strains were identified with 2 Pseudomonas strains,1 strain belongs to the genus,19 strains belong to the genus Bacillus.2.F9 strain was further analysisas it has good antibacterial activity.The adapting 16 SrDNA PC R,ITS,physiological and biochemical,and specific primer PC R were used to identify and then to determine that biocontrol strain F9 is Bacillus amyloliquefaciens.3.According to our in-depth study,combined with previous reports of Bacillus amyloliquefaciens,the biocontrol substances of Bacillus amyloliquefaciens are mainly proteins and lipopeptides.In our experiments,the crude proteins precipitated from F9 fermentation supernatant were resista nt to high temperature and proteinase K treatment,which is not in line with the characteristics of protein substances,indicating that antimicrobial substances are most likely lipopeptide.This hypothesis was confirmed by the antibacterial substance extracted from crude proteins by acetone.It is therefore speculated that the antibacterial activity may be a lipopeptide.4.Crude lipid peptides Anion exchange chromatography columns "HiTrap DEAE FF"(GE Healthcare Life Sciences)HiTrap DEAE anion exchange columns have two peaks,one is through peak,the other one is elution peak.Active substances mainly concentrate in the elution peak.Collecting and testing the active substances of the elution peak at different time,we found the active substances were concentrated in the central of the peak.We collected the active components and separated them by Sephadex G-25 gel filtration.The separation results showed that there are three peaks,the active ingredient is located in the last peak.After purification and separation,the active components were identified by LC-MS.By comparing with the mass spectrum of the peptide,the antimicrobial substance may be Bacillomycin L.