Screening Of Fusarium Graminae Antagonists And Study On Bacteriostatic Mechanism

Fusarium graminearum is the main pathogen causing Fusarium head blight(FHB)in wheat,which leads to wheat yield reduction and can also produce Mycotoxins,such as Zearalenon(ZEA)and deoxynivalanol(DON)endanger food and feed safety.In this study,F.graminearum biocontrol strains were screened and its antagonistic substances were further explored to provide a theoretical basis for F.graminearum biocontrol.Specific experimental results are as follows:1.Screening of Fusarium graminae antagonists and analysis of its antagonistic abilityA strain with 66.43% inhibition rate against F.graminearum was selected from wheat soil and named ASAG 010.The strain was identified as Bacillus subtilis by physiological,biochemical and 16 S r DNA sequencing.Scanning electron microscope showed that ASAG 010 fermentation liquid made F.graminearum mycelium membrane shrink.In addition,ASAG 010 fermentation liquid had better protective effect on wheat grains and malt contaminated by F.graminearum.In addition,ASAG 010 not only has a broad spectrum of antagonistic fungi,but also has a high degradation rate(86.95%)to the toxic secondary metabolite ZEA produced by F.graminearum.2.Stability of the supernatant of Bacillus subtilis ASAG 010 and response surface analysis of the strain culture conditionsIt was found that the active component of Bacillus subtilis ASAG 010 antagonizing F.graminearum was located in the supernatant.The stability of the antibacterial substance in the fermentation supernatant was studied,and the active substance was more sensitive to acid and ultraviolet(P<0.05),it have a certain tolerance to high temperature,pepsin,trypsin,protease K,and longer storage time will not affect its antibacterial effect(P>0.05).The study on the culture conditions of Bacillus subtilis ASAG 010 showed that the culture temperature was 32～37°C,p H was 5.0～7.0,fermentation time was 24～48 h,and liquid content was 20～50 m L.The antagonistic activity against F.graminearum was higher.The response surface of fermentation conditions was optimized with antibacterial rate as response value and fermentation time,liquid loading,temperature and p H as variables.The results showed that the fermentation time of ASAG 010 was 46 h,the temperature was 36.5°C,the p H was 6.5,the liquid volume was 54 m L,and the predicted bacteriostasis rate was 67.59%.It was proved that the actual antibacterial rate was 66.43%,reaching 98.28% of the predicted value,which proved that the model had high reliability.3.Extraction,isolation and purification of antagonistic fungi active substances and study on bacteriostatic mechanismIn order to further determine the antifungal substances in the supernatant of Bacillus subtilis ASAG 010,different organic solvents were used to extract the active ingredients and it was found that the active substances dissolved in methanol had antibacterial properties.The active ingredient was predicted to be a lipopeptide by TLC and synthesis related gene detection.The Surfactin and Fengycin of lipopeptide were identified by high performance liquid chromatography(HPLC)and matrix assisted laser desorption time of flight mass spectrometry(MALDI-TOF-MS).Subsequently,a higher active Fengycin was isolated and purified,and it was found that it can effectively destroy the cell membrane of F.graminearum and reduce the content of ergosterol in the cell membrane,so as to play an antagonistic role.In conclusion,a strain of Bacillus subtilis ASAG 010,which can effectively inhibit the growth of F.graminearum,was screened out in this study,and its ability to antagonize fungi was verified.Based on the localization,extraction,isolation and purification of the antagonist and the further study of its bacteriostatic mechanism,it was found that the antagonist could destroy the cell structure reducing ergosterol content on the cell membrane surface of F.graminearum.The results laid the foundation for the study of F.graminearum biocontrol agent.