Screening Of Biocontrol Bacteria And Study On Antibacterial Mechanism Of Peach Soft Rot Pathogen

The peach is ripe in the season of high temperature with high moisture content and crisp and tender tissue.Therefore,it is easy to cause damage due to collision in the process of picking and circulation.The pathogens then invade into the inside of the peach fruit and cause the disease of the peach.Among them,the soft rot caused by Rhizopus stolonifer（R.stolonifer）has brought great losses to the peach industry.In this paper,a biocontrol bacterium,which had antagonistic effects on R.stolonifer was isolated from the rhizosphere soil of the orchard.Firstly,the biocontrol bacterium was identified to clarify its taxonomic status.Then the effective antimicrobial substances in the antagonistic extracts of the biocontrol bacteria were isolated and identified,and the yield of secondary antimicrobial substances was increased by optimizing fermentation conditions.At the same time,in order to provide a theoretical foundation for the control of soft rot of peach,the biocontrol effect and antibacterial mechanism of the secondary antimicrobial substance were investigated.The main research contents and conclusions of this paper are as follows:1.104 strains of bacteria were isolated from the rhizosphere soil of the peach orchard in Jurong City.A strain with the best biocontrol effects on R.stolonifer was screened by confrontation culture method and named as UO.It was confirmed that UO was Paenibacillus polymyxa（P.polymyxa）through morphological observation and molecular biological identification.The bacteriostatic spectrum,growth curve,sporulation dynamics and stability of fermentation broth of UO strain were also analyzed.It is showed that P.polymyxa UO strain can effectively inhibit the growth of R.stolonifer and has broad antibacterial activity.When cultured in Nutrient Broth（NB）medium at 28℃,it was found that the stable period of P.polymyxa UO strain was 60～84 h.By this time,the spore number of P.polymyxa UO strain was the highest.In addition,the fermentation broth of P.polymyxa UO strain was stable in heat,UV-light,acid,alkali and protease.It is shown that P.polymyxa UO strain has the potential of industrial application.2.P.polymyxa UO strain fermented liquid was extracted with normal butanol to obtain the crude extracts of antibacterial substances.Through high performance liquid chromatography（HPLC）,the crude extract was isolated and purified,and the substances were collected for plate antagonism test.Finally,four kinds of relatively pure secondary antibacterial substances were obtained.The results of mass spectrometry showed that the four substances were Fusaricidins A,B,C and D.Among them,the antagonistic effect of Fusaricidin A was the best.To increase the production of Fusaricidin A,the fermentation conditions were optimized by single factor and orthogonal experiments.The results showed that the yield of Fusaricidin A could reach to 351.19 mg/L when 7.97 g/L yeast powder was used as nitrogen source,7.99 g/L glucose was used as carbon source,and 0.71 g/L Mn Cl₂was used as inorganic salt,which was nearly ten times higher than that before optimization.3.The biocontrol effects of Fusaricidin A in vitro and in vivo were investigated.The vitro experiments showed that Fusaricidin A could inhibit the growth of the silk of R.stolonifer,and the higher the concentration was,the more obvious the inhibition effect was.R.stolonifer could not grow when the concentration of Fusaricidin A was 4μg/m L.The surface of hyphae treated with Fusaricidin A was ridgy and curled irregularly,and the morphology of hypha was seriously deformed.In addition,Fusaricidin A could inhibit the germination of spores.When the concentration of Fusaricidin A was 2μg/m L,the spores hardly germinated.By fitting the virulence regression curve,the EC₅₀value was 1.48μg/m L.The vivo experiments showed that the MIC value of Fusaricidins to R.stolonifer was 80μg/m L,at which the area of the lesions were significantly lower than that of the control group,and the incidence index of it was only 22.2%,which was 2.25 times lower than that of the CK group.Through q RT-PCR,it was found that Fusaricidin A could significantly down-regulate the expression of genes related to hydrolases and metabolic enzymes of R.stolonifer,thus reducing its virulence.Meanwhile,Fusaricidins could also up-regulate the expression levels of defense genes such as Npr1,Wrky50,Pr1 and Chi in peach fruit,thus improving the resistance of peach.