Screening Of Antifungal Bacteria Against Aspergillus Flavus, Antifungal Mechanism, Fermentation Technological Conditions And Application Of Aspergillus Flavus

Aflatus contamination poses a great threat to the safety of crops.Studies have shown that it is a common problem that crops are infected by Aspergillus flavus before harvest.Therefore,it is an effective measure to prevent agricultural products from polluting Aspergillus flavus by controlling from the source,that is,eliminating Aspergillus flavus from soil.This study focuses on the biological control of Aspergillus flavus in soil,laying a foundation for the control of crop Aspergillus flavus pollution.In this paper,the high efficient strains antagonistic to Aspergillus flavus were obtained,the species and genus of the strain were identified,and the biological characteristics of the strain were studied.650 antifungal strains stored in the laboratory were screened to obtain the strains with strong activity antagonistic to Aspergillus flavus by growth-confrontation method.Through morphological observation,physiological and biochemical and biological experiment,16S rDNA sequence analysis and development tree construction,the species and genus of the strain were identified.The optimum growth conditions of the strains in shaking-bed culture were determined by single factor test.The results showed that a highly active strain B10-6-1 antagonistic to Aspergillus flavus was obtained,and the inhibitory zone was up to 0.95 cm;The strain was identified as Bacillus amyloliquefaciens.The optimum conditions for shaking-bed culture of strain B10-6-1:carbon source was maltose,nitrogen source was soybean cake powder,C/N ratio was 8:1,metal ion was Ca²⁺.The initial pH of culture medium was 6.0,the culture temperature was 30?,the concentration of NaCl was 1%,inoculation quantity was 5%,and the volume of medium was 50 mL/250mL triangle flask.The fermentation conditions for producing antagonistic substances by strain B10-6-1were optimized.The optimal carbon source,N source and inorganic salt of the strain were determined by single factor test.According to the response surface analysis test,the composition of fermentation medium and fermentation conditions,including initial pH value of medium,seed age of shake flask,volume of liquid in shake bottle,inoculum quantity,fermentation time,etc,were optimized.The optimal fermentation conditions were as follows:corn meal 3.0%,powder 2.0%,KCl 0.13%,initial pH 7.5,seed age 13 h,liquid volume 100 mL/250 mL triangle flask,inoculation quantity 8%,fermentation time 72 h.Under these conditions,the inhibiting circle diameter of the supernatant of the fermentation broth reached 2.97 cm,and the antifungal activity increased 36.77%compared with that before the optimization.Then,the species of antagonistic substances of antagonistic strain B10-6-1 were investigated,the structure of lipopeptide antibiotics was identified by HPLC-ESI-MS,and the properties and antifungal mechanism of the antagonistic strain B10-6-1 were also studied.The synthetic gene was cloned and analyzed.The crude protein extracts,lipopeptide extracts and sugar substance were extracted from the fermentation broth of antagonistic bacteria B10-6-1,and the antifungal test was carried out to determine the antagonistic substances.The effects of temperature,pH value,protease,metal ions,EDTA and other factors on the antagonistic activity of the substance were investigated.The genomic DNA of antagonistic strain B10-6-1 used as template,DNA amplification of ItuA,ItuB,ItuC,ItuD,FenA,FenB,FenD,Sfp,bmyA,bmyB,bmyC synthetic gene were carried out by PCR.The crude extracts of lipopeptide antibiotics were obtained from fermentation broth by centrifugation,acid precipitation and methanol extraction.The elution peak components were separated by high performance liquid chromatography?HPLC?,and the antifungal activity of the eluted peak components was tested in vitro.The structure of the components with antifungal activity was analyzed by HPLC-ESI-MS.Finally,the effects of antagonists on spores and hyphae of Aspergillus flavus were investigated.The results showed that the antifungal substances of strain B10-6-1 were crude protein extracts and lipopeptide crude extracts.The results showed that the minimum inhibitory concentration of lipopeptide was 0.5 g·L^-1,and its properties were stable.Temperature,pH value,protease,metal ions and EDTA had no obvious effect on its antifungal activity,and its antifungal spectrum was wide.It has obvious inhibitory effect on nine strains of fungi,such as Brassica campestris,Rhizoctonia aestivum and so on.The results of PCR amplification showed that the ItuA,ItuC,ItuD,FenA,FenD,Sfp,bmyA,bmyB,bmyC synthesis gene could be amplified from the strain B10-6-1 antagonistic to Aspergillus flavus,but the ItuB,FenB synthesis gene could not be amplified from the strain B10-6-1antagonistic to Aspergillus flavus.Seven components were collected by HPLC and tested in vitro.Components 5 and 7 had antifungal activity.By liquid chromatography,components 5 and 7 were lipopeptide antibiotics C14bacillocnycin D and C15bacillocnycin D,respectively.In addition,the crude lipopeptide extracts could inhibit the spore germination of Aspergillus flavus,promote the dissolution of hypha cell wall,aggravate the swelling and distortion of hypha,melt the protoplast,finally rupture and even melt the body wall of mycelium.In order to apply the obtained antagonistic strains to production practice,the technology of producing spores by liquid fermentation was studied.The spore yield measured by the plate counting method was taken as the index of determination,The fermentation medium and culture conditions were optimized by single factor and response surface method,and the results were as follows:the medium formula was 1.5%lactose,1.5%lactose,4.0%peptone,0.05%calcium chloride,0.07%sodium chloride.The culture conditions were 7.28 initial pH,6.26%inoculum,37?,49.17 h,and 4.0%peptone,0.05%calcium chloride and 0.07%sodium chloride.Under these conditions,the final sporulation rate was 97.52%and the final biomass was 3.2×10^-9 CFU·mL^-1.Finally,the application effect of antagonistic agent B10-6-1 in peanut soil polluted by Aspergillus flavus was investigated.The antagonistic bacterium B10-6 was applied to peanut potted soil system.First,1 mL Aspergillus flavus spore solution with concentration of 10⁸ CFU·mL^-1 was added to the soil.Next 20 ml antagonistic bacteria B10-6-1 with a concentration of 10⁵ CFU·mL^-1,10⁶ CFU·mL^-1,10⁷ CFU·mL^-1,10⁸ CFU·mL^-1 were added to experimental group 1⁴ to investigate the effects of fungicide use on the amount of Aspergillus flavus in soil and its effects on plant morbidity,as well as its effects on indigenous microorganisms,enzymes and plants growth.The results showed that the inhibition rate of Aspergillus flavus in peanut group was 89.6%?P<0.01?.In soil group and peanut group,the amount of fungi decreased by 27.78%?P<0.01?,33.33%?P<0.01?,the amount of bacteria increased by 93.30%?P<0.01?,71.43%?P<0.01?,respectively.Fungicide use had little effect on actinomycetes.Meanwhile,the activity of sucrase and urease increased by 106.25%?P<0.01?and 74.94%?P<0.01?in the soil group and 18.99%?P<0.01?and 175.33%?P<0.01?in the peanut group,respectively.It has little effect on catalase and cellulase.Moreover,the bacterial solution significantly reduced the probability of crop infection with Aspergillus flavus.The infection rate of peanut control group was 42.9%,and the infection rate of peanut treated with antagonistic bacteria solution was 0.The application of antagonistic bacteria solution made little change in root length and increased stem length and dry weight of peanut.