Identification Of Antifungal Substances From Bacterial Strain B-FS01 And Their Inhibition To The Growth Of Fusrium Moniliforme And Production Of Fumonisin B1

Fusarium moniliforme Sheldon (Fusarium verticiIIioides) is a facultative and non-host necrotrophic pathogen, which broadly contaminates maize, sorghum, wheat, cotton, tomato, peanut, bananas, soybean, pimiento and feed. Fumonisins are the water-soluble metabolites produced by E moniliforme. They are acutely toxic to livestocks and cause diseses like leukoencephalomalacia in equines, pulmonary edema and hydrothorax in swine, and liver cancer in rats. In humans, the occurrence of fumonisin is correlated with higher incidence of esophageal cancer.In this study, an antagonistic bacterial strain B-FS01 was isolated from the rape stem infected by SIerotinia Sclerotiorum. Following analysis of its cultural, morphological, biochemical and physiological characters and partial sequencing of 16S rDNA, strain B-FS01 was identified as Bacillus subtilis. B-FS01 showed strong antagonism against F. moniIiforme and released antifungal substances to culture. The characterization of crude extractions containing antifungal substances indicated that the antifungal activity was thermostable, tolerant to basic pH, untolerant to acid pH, unsensible to proteinase K and pepsin.By approaches of ammonium sulphate precipitation, DE52 ion-exchange chromatography, Sephadex G100 gel filtration and HPLC, antifungal substances were able to be isolated and purified from the culture of B-FS01. The HPLC/ESI mass spectrum of antifungal substances revealed a cluster containing several molecules that was observed at [M+H] m/z =1,434.0; 1,435.9; 1,450.6; 1,464.0; 1,479.0; 1,492.9; 1,506.0 and 1,519.9. On the base of further CID spectrums of respective molecules, these compounds were identified as fengycin homologues containing Fengycins A, Fengycins B and a new fengycin type. In addition, by HC1 precipitation and silica gel column chromatography in the middle pressure preparative chromatography system, the purification of Fengycins from B-FS01 culture was greatly improved. The new approches are well suited for the semi-scale preparation of fengyicns.HPLC/MS is competent for analysis, but it is too expensive to be accepted by most labs. Also, SDS-PAGE is not a good approach for analysis of lipopeptide with low molecular weight. Fortunately, it was found that the Fengycins at the concentration upper critical micelle concentration (CMC) could be dyed as well as nucleic acid in the ultraviolet radiation. With this finding, we established a method for the rapid semi -quantitative detection of Fengycins by agar-gel electrophoresis.The bioassay of Fengycins against the growth of E moniliforme was performed. The results showed that IC₅₀ of AAC inhibiting the mycelial radial growth was 20μg/mL; MIC of AAC by agar diffusion inhibiting the spore growth was 0.78/μg per cup and IC₅₀ of AAC inhibiting the mycelial growth-weight was 48μg/mL. Additionaly, Fengycins could delay the germination of E moniliforme spores, and treatment with Fengycins significantly modified infection of maize seeds by ATCC 38932.Microscopic observation on morphology of F. moniliforme mycelia revealed that tips of partial mycelia were lysed, and intensive PI staining of Fengycins-treated F. moniliforme mycelia was observed under fluorescence microscope, indicating the toxin-mediated damage of cytoplasmic membrane. We also performed agar gel electrophoresis and found that Fengycins could bind the phosphatidylcholine. Fengycins are amphiphilic compounds and can aggregate at a concentration. So it can be hypothesized that Fengycins interacted with the memberane by binding the phosphatidylcholine, then formed Fengycins/phospholipid aggregates, and eventually caused the formation of ion-conducting pores as well as the damage of F. moniliforme mycelia membrane. Our hypothesis could be supported by the anatagonism of phosphatidylcholine aganist the antifungal activity of Fengycins in agar media. Recently studies demonstrate that phopholipase is closely relevant to the virulence of fungi, and is the virulence factor for many fungi. In the screening of antifungal agents using phopholipase as a target, inhibitors of this enzyme also could arrest the growth of many pathogens. It was noted that Fengycins could depress activity of PLA₂ secreted by F. moniliforme, which might contribute to the antifungal activity against F.moniliforme.In addition to the inhibitory activity against the growth of F. moniliforme, Fengycins was also shown to inhibit production of fumonisin B1 (FB1) . It was found that the mycotoxin-producd ability of F. moniliforme treated with 50/μg/mL Fengycins was significantly reduced, and the quantity of FB1 produced by unit mass mycelia decreased to 28% of the control. Genes involved in the fumonisins biosynthesis were identified and showed to be clustered in the genome of F. moniIiforme. Expression patern by reverse transcription-PCR (RT-PCR) showed that treatment of Fengycins did not affect transcriptional level of FUM1, but the level of FUM8 was down-regulated...