Study Of Biocontrol Bacterium Strain A On Antifungal Spectrum And Mechanism, Separation And Purification Of Antifungal Protein

The disadvantages of chemical fungicides in entironment and food safety urged researchers to exploit new biocontrol methods to deal with pathogens.Bacillus subtilis,as a kind of biocontrol bacterium,has many advantages in being easily cultured,strong ability to adapt to environment and non-toxic to humans and animals etc.Presently,commercial formulation of Bacillus subtilis existed in civil or abroad market.These biocontrol agents were made by different strains of Bacillus subtilis and had different control objects.Strain A(CGMCC No.1982) showing strong antagonistic activities against Bipolaris maydis was isolated from diseased leaves of corn and identified as Bacillus subtilis by former researchers in the laboratory.In this study,further research of strain A on antifungal spectrum and mechanism,separation and purification of antifungal protein was executed.The results were as follows:(1) The antifungal spectrum and stability of antagonistic properties of Strain A were determinated.The inhibitory spectrum of Strain A to 20 kinds of phytopathogenic fungi and 8 kinds of edbile fungi was determinated by co-culture experiment.Strain A exhibited broad antifungal avtivities.Strain A intensively inhibited the growth of 11 kinds of phytopathogenic fungi,Cercospoia kaki,Venturia inaequalis,Dendrodochium lycopersici,and so on.Strain A intensively inhibited the growth of 3 kinds of edible fungi including Cordyceps militaris, Pholiota adipose and Auricularia auricula.Bipolaris maydis as indicator,the stability of antagonist properties of Strain A on temperature,pH and successive transfer culture were detected.The antagonistic activity of strain A had little change when the culture broth containing strain A thalli was treated by -20℃,-4℃,20℃,37℃,50℃for 7 d,respectively.The pH change of culture media from 1 to 11 did not affect the antagonistic activity of strain A.Under the condition of successive transfer culture,the antagonistic activity of 10th generation was 95.8%of the original Strain A.(2) The crude antifungal proteins were prepared by adding ammonium sulfate to culture filtrate with a saturation of 30%～60%.The separation and purification of the crude proteins were operated by a DEAE 52 anion exchange chromatography column and NaCl gradient elution method.The fraction with antifungal activity was obtained and named FV.FV showed a single band in SDS-polyacrylamide gel electrophoresis.The analysis of FV by matrix-assisted laser desporption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) suggested that FV be a kind of RNA-binding protein Hfq[Bacillus pumilus SAFR-032].And the relative molecular mass of the protein was 8478.5 and the isoelectric point(pI) was 8.09.(3) The antifungal mechanism of FV was studied using Bipolaris maydis as material.FV could affect the conidium germination and appressorium formation of B.maydis.The EC₅₀ of inhibiting spore germination was 138.32μg/mL while the EC₅₀ of inhibiting appressorium formation was 22.23μg/mL.The misshapen and swollened appressoria were detected when Bipolaris maydis treated by antifungal protein FV with the concentration of 17.29μg/mL.The misshapen mycelia and vesicles existing in top or middle of mycelia treated by FV were observed under optical microscope.The deformed mycelia became more obvious as the prolonged treating time.Under scanning electron microscope,the surface of deformed mycelia became rough and the appearance of swollen vesicle exhibited sags and crests and had some debris.(4) The effect of FV on the intracellular structure of mycelia was studied.Under transmission electron microscope,the protoplasm of mycelia treated by FV became "thin", "vacuolization" and the organelles decomposed.The cell wall became irregularly thick and accompanyed inordinately decomposing.Some treated mycelia existed "daughter mycelia".(5) The leakage of cell membrane of mycelia treated by FV was detected by electrical conductivity.The results that there was no significant difference between the value of electrical conductivity treated by FV before and after indicated the effect site of FV did not locate at cell membrane.