Isolation,Identification Of Active Components Produced By Strain Bna05 And Their Prevention Effect On Paddy Mildew

Mildew is the main cause of losses both in quality and quantity in grains during storage.Because of the high temperature and humidity growth environment,paddy easily gets mildew and accumulates mycotoxins.Due to influences of traditional chemicals on environment and the pathogens' increasing drug resistance,it's necessary for sustained agriculture development to explore new type biological anti-mildew agents.In this paper,Bacillus subtilis natto Bna05,which was isolated from natto in our previous studies and showed high antifungal activity,was used for production of antifungal metabolites.In order to assess the main components in the antifungal metabolites and to understand their functions,the fermentation process was optimized,active components was isolated and identified,the antifungal mechanisms were analyzed and paddy mildew prevention effect of the antifungal metabolites was studied.The result would give valuable references to the development of broad spectrum,safety and effective anti-mildew agent for paddy storage.Liquid fermentation process was optimized through single factor test and response surface design.Suitable conditions for Bna05 producing antifungal compounds were as follow:temperature 30 ?,culture duration 37 h,medium initial pH 6.6.Suitable medium formula was:soy peptone(or tryptone or peptone)1.0 g,glucose 2.0 g,beef extract 0.3 g,Na2HPO4 0.15 g,KH2PO4 0.1 g,yeast extract 0.3 g,glutamate 0.06 g,aspartic acid 0.06 g,H2O 100 mL.The fungal inhibition rete(FIR%)against Aspergillus niger of the supernatant after fermentation optimization was 87.02%,which was enhanced by 65.69%compared with the FIR%of the supernatant before optimization.Specific primers for biosynthetic genes of several antibiotic lipopeptide synthetases were used to amplify the genomic DNA of strain Bna05,PCR products were sequenced and analyzed for similarity through Blast analysis,the result showed that strain Bna05 was positive for srfAA and sfp,no ituC?ituD?fenD?fenACE?bymB and bymC genes were detected.The solid phase extraction of liquid supernatant(SPEL)of Bna05 was analyzed by reversed phase high performance liquid chromatography(RP-HPLC),3 group of active fractions F2?F3 and F4 were isolated.The MIC50 of F2?F3 and F4 against A.niger were detected to be 50,200 and 100 ?g/mL separately using a microtiter plate colorimetry method.No lipopeptide was detected in F2,2 V7-surfactin variants in F3 and 3 I/L7-surfactin variants in F4 were identified by MS analysis.The result of LC/MS/MS and mascot analysis showed that several proteins in F2 had high similarity to the reported antifungal proteins,including subtilisin?fibrinolytic enzyme and serine alkaline protease,these low molecule proteins might be the main antifungal components in F2.To assess the antifungal mechanisms,the tested fungus was analyzed by PI staining,leakage detection of nucleotide acid and protein,morphology observation by light microscope and scanning electron microscope(SEM).PI staining analysis showed that most of the A.niger spores were damaged under the treatment of F2,yet no cell disruption observed.However,the treatment of F3?F4 induced some of the A.niger spores to disruption,other unbroken spores were almost undamaged.Leakage of nucleotide acid and protein of A.niger was detected after the treatment of F2,F3 and F4 separately,the OD260 and OD280 of extracellular fluids increased rapidly in samples treated for the first 3 hours.Observation of light microscope showed that A.niger spore germination was delayed and hyphal growth inhibited under treatment of either of the three RP-HPLC isolates.The spores and mycelium trended to assemble together in the presence of F2,and some drop like materials were observed around the mycelium in the presence of either F3 or F4,these drop like things might be formed by the leakage of cell content of the fungus,and this drop phenomenon induced by F4 was more obvious than induced by F3.SEM revealed that the hypha treated by either of the three isolates became shriveling and distortion,severe collapse could be seen in the spore head.In contrast,the actively growing hyphae was plump and exuberant in untreated cultures.The preliminary antifungal synergy study revealed that the combinations of either V7-surfactin or I/L7-surfactin with F2 showed synergistic antifungal effect,there was no synergy effect between V7-surfactin and I/L7-surfactin.The antifungal spectrum and stability under different conditions of sample SPEL were analyzed,most of the tested fungi,including A.niger?A.flavus?Rhizopus sp.?Penicillium expansum and A.fumigatus,were sensitive to SPEL.SPEL was tolerable to high temperature and a wide range of pH value,yet temperature higher than 80?.strong acid and alkali?some metal ions such as Mg2+?Zn2+?Fe2+ and Ca2+ should be avoid.The in vitro antifungal effect of SPEL on paddy was studied,the result showed that SPEL with an additive content of 300 ?g SPEL/g paddy could reduce the number of molds in the control from severe hazard level to critical level,fatty acid value was reduced from the standard of seriously unsuitable for storage to standard of suitable for storage.And the content of aflatoxin Bi was reduced by 74.0%.It's also found that SPEL could inhibit paddy germination effectively.These results suggest that SPEL are valuable for exploration of new antifungal agent for paddy storage.