Research On Lipopeptide Antibioticsfor Control Of Deoxynivalenol Pollution

Fusarium graminearum is the main causal pathogen of fusarium head blight in wheat. The fungus produces a number of mycotoxins including deoxynivalenol (DON) during the process of growth and invasion of grains. DON is designated as the most dangerous food contaminants by FAO and the World Health Organization. Several surveys about the mycotoxin contamination in wheat, corn and related products showed that the DON contamination rate was high, and the contents varied. Currently, chemical fungicide was the most used measure to control F. graminearum. The residual fungicide in natural environment threats the safety of ecological environment, agricultural products, human and animals. Some studies have highlighted the potential use of antagonistic microorganisms to prevent the hazards of fungi and toxins, which can reduce the amount of fungicide and the pollution of agricultural non-point pollution. Microbial pesticide is a kind of high-efficiency and environmental safety pesticide. However, only a few reports have described DON inhibition in harvested grains by antagonistic microorganisms. Therefore, the aim of this study was to identify some bacterial strains, isolated from shells, seeds and pods of peanut, with potential application in the prevention of fungal growth and mycotoxin formation in grains.In the dual culture plate test, forty-four bacterial strains showed more than40%inhibition of F. graminearum mycelia growth. The antifungal activity of bacterial cell-free culture supernatants was studied by tip-culture assay, and thirty-eight stains showed significant inhibition activity. The content of DON in F. graminearum infected wheat kernels after eighteen days of incubation was measured by HPLC. Result showed that fourteen strains with the DON inhibition rate more than50%, WPS4-1and WPP9with the highest inhibition rate of about90%. According to the morphological, physiological and biochemical analysis, and the16S rDNA and gyrA gene sequences analysis, the fourteen antagonistic bacterial strains with high antagonistic activities were identified as Bacillus amyloliquefaciens.The properties of culture supernatant were studied for the selection of suitable separation method. The bacterial culture supernatant was treated with different temperature (35°C,60°C and121°C), pH (1.0,3.0,5.0,9.0,11.0and13.0) and protease (trypsin, pepsin and proteinase K), and results showed that the antifungal substances were thermostable, stable in acidic conditions and not sensitive to trypsin, pepsin. The genomic DNA of antagonistic bacteria was amplified with nineteen primer pairs from antibiotic genes of Bacillus species. The fourteen positive amplified sequences were searched in the nucleotide blast program, and showed almost similar sequences with the known antibiotics genes. Therefore, there were genes involved in the synthesis of ericin, sublancin, iturins, surfactin, fengycin and bacilysin in the genome.Several methods such as ammonium sulfate precipitation, organic solvent precipitation, organic solvent extraction and HCl precipitation were used to separate the antifungal substances. The precipite with ammonium sulfate at the concentration of60%showed the inhibiton of78.07±3.21%against the mycelia growth of F. graminearum, and those were4.92±1.81%and9.61±1.04%for ethanol and aceton precipitate. The extracts of ethyl, n-butyl alcohol, benzene and chloroform showed the inhibiton of87.01±2.41%,78.11±5.97%,73.16±3.29%and46.17±4.42%respectively. The crude extract by HCl precipitation and methanol extraction showed the100%inhibition activity. Therefore, HCl precipitation was the most effective method for the separation of antifungal substances. Next macroporous resins were selected for the decoloration of bacterial culture supernatants. Results of adsorption assay showed that two macroporous resins D296R and D301R had higher adsorption capacity to pigments and almost no adsorption to antifungal substances. The decolored bacterial culture supernatant was acidified to pH3.0with7.0mol/L HCl, and the precipitation was extracted with methanol to obtain crude lipopeptide antibiotics. The decolored bacterial culture supernatant and crude extracts were analyzed by MALDI-TOF mass spectra, and the mass peaks were assigned as iturins and surfactins.The antifungal activity of crude lipopeptide extracts was discussed. The calculated50%inhibitory concentration (IC50) of crude lipopeptide antibiotics against F. graminearum mycelia was8.51μg/mL in tip culture assay. Lipopeptide antibiotics could delay or inhibit the germination of spores, reduce the production of DON. The lipopeptide antibiotics delayed the germination of spores, and made the hyphae malformation observed by light microscope and scanning electron microscope, and the inhibition activity enhanced as the concentration of lipopeptides increased. The conductivity of culture supernatant treated with lipopeptides increased, which meant that the cell membranes of hyphae were destroyed. These results showed that there might be targets of lipopeptides in the cell membranes of F. graminearum, making the pore formation and intracellular substances leakage resulting in the break of hyphae. The expression of Tri5gene in the mycelia treated with120μg/mL of lipopeptides, analyzed by fluorescence quantitative PCR amplification, was significantly lower than that of the control, which meant that lipopeptides achieved the activity through the inhibition of gene expression involved in DON synthesis.The production of lipopeptide antibiotics was influenced by the composition of culture medium and the condition of fermentation. The single factor and uniform design experiments obtained the best media components, that were13.7g/L of millet flour,2.5g/L of soybean cake powder and3.0g/L of sodium nitrate. The obtained culture supernatants showed the inhibition rate of90.38±1.33%, significantly higher than that of the original media. The best temperature was32°C and fermentation time was4days using10L fermenter determined by single factor experiment.The current study identified some bacterial strains with high antifungal activity, applied a method for identification and extraction of lipopeptide antibiotics, and explored the mechanism involved in the antifungal activity of lipopeptide antibiotics, thus providing a theoretical basis for the application of lipopeptide antibiotics as biocontrol agent in stored corns.