Screen Of Antagonistic Microorganismon Gummosis Of Peach And Its Fermentation For Lipopeptide Production

The gummosis disease is one of the most important diseases of stone fruits worldwide and is caused by Botryosphaeria dothidea. Severe disease outbreaks significantly reduced both yield and fruit quality in orchards eventually resulting in tree death. As an alternative approach of synthetic chemical fungicides, biocontrol has aroused general concern to control plant diseases caused by phytopathogens. Bacillus strains were isolated from raw honeys and had the capacity to inhibit B. dothidea. This strain was identified by physiological and biochemical characteristics, 16 S r RNA and gyr B gene sequences. Mechanisms of this strain against gummosis of peach was explored. To improve lipopeptide yield and reduce cost, the submerged fermentation process and solid fermentation process were optimized. The major contents are attached as follows:?1? Twenty-three strains exhibited antifungal activity to B. dothidea. Strain SYBC H47 was selected for further study because of its most efficient antagonism characteristics. According to its physiological and biochemical characteristics, 16 S r RNA gene and gyr B gene analysis, the SYBC H47 strain was identified as B. amyloliquefaciens, and was preserved at the China Center for Type Culture Collection?CCTCC NO: M2013283?.?2? The fermentation supernatant of SYBC H47 has biosurfactant activity, emulsification activity against hydrocarbon, including n-hexane, methylbenzene, n-heptane, diesel, kerosene, and lubricating oil, and oil displacement ability against lubricating oil. The antifungal activity and emulsification activity of supernatant were lost at acidic condition?pH 1-5?, while the activities were stable at neutral and alkaline condition?pH 6-12?. The activities were insensitive to high temperature, which were stable from 30 to 100 °C. The results of antifungal spectrum showed both of bacterial suspension and supernatant had antagonistic activity against A. niger, M. racemosus, F. oxysporum, P. citrinum, C. albicans and B. dothidea. In total, inhibitory rate of bacterial suspension was higher than that of supernatant.?3? The mechanism of B. amyloliquefaciens SYBC H47 against B. dothidea was studied. There were three lipopeptides produced in supernatant, in which bacillomycin L?200 ?g·m L-1? had 100% suppression of conidial germination, while the other two lipopeptides, including fengycin and surfactin, had below 60% suppression of conidial germination at same concentration. The suppression of mycelial growth was about 45% by treated with fengycin, which was 5% higher than bacillomycin L treatment. However, surfactin had limited effect on the suppression of mycelial growth?approximately 10%?. Perhaps because of superimposed effect or synergistic effect, lipopeptide mixtures exhibited the maximum suppression on conidial germination and mycelial growth.?4? Catechins type siderophore was produced by B. amyloliquefaciens SYBC H47, which had also ability to inhibit the growth of B. dothidea, and could not be synthesize in medium containing ferric ion. The production of siderophore was 106 ?mol·L-1 in MM9 medium. Virulence regression equation of was y=1.432x+0.578, EC50 and EC90 were 0.395 g·L-1 and 3.099 g·L-1, respectively.?5? The results of disease resistance in vitro showed lipopeptide cell suspension and lipopeptide liquor had 100% control efficiency to peach gummosis. Disease index of gummosis was decreased to 22.5% by treated with lipopeptide cell suspension, which indicated that control efficiency of lipopeptide cell suspension was better than that of carbendazol in the field trial. The cause of control efficiency of in the field trial inferior to that of in vitro was that antifungal ingredients were diluted by the rain. So far, few studies were reported about controlling gummosis by B. amyloliquefaciens and Bacillus subtilis strains, our research providing a new approach to biocontrol gummosis.?6? The genome of SYBC H47 strain was sequenced. The length of circular chromosome is 3884433 bp. The access number of NCBI was CP017747. The genes encoding bacillomycin L, fengycin, surfactin and siderophore were obtained. In addition, there were some antibacterial polyketides gene clusters in genome, including bacillaene, difficidin and macrolactin.?7? Raising temperature and oxygen transfer coefficient were conducive to the surfactin production, while these conditions had negative effect to produce fengycin and bacillomycin L. The optimized parameters of medium were as followed?g·L-1?: glucose 42.65, NH₄NO₃ 6.19, MgSO₄·7H₂O 0.74, KH₂PO₄ 2.72, FeSO₄·7H₂O 0.274, MnSO₄·H₂O 0.0017, pH 7.0. The optimized parameters of culture conditions were as followed: relative filling volume 75 m L·250m L-1, 27 °C, rotate speed 250 r·min-1. After optimization, the production of lipopeptides reached 2.46 g·L-1, which was increased 70.83% than control. Productions of surfactin, fengycin and bacillomycin L were increased 38.98%, 106.77% and 133.33%, respectively.?8? The foam fractionation method was used to study lipopeptide content in foamate through 5 L fermenter. The lipopeptide products were increased to 2.91 g·L-1 at ventilation volume 0.6 vvm and 150 g·min-1, the fengycin and bacillomycin L was 25.6% and 44.2%, respectively. The lipopeptide yields were improved to 3.40 g·L-1 and the fengycin and bacillomycin L was 27.4% and 42.3%, respectively. However, after antifoaming agent addition, the lipopeptide contents enhanced to 1.63 g·L-1 and increased by 13.19% than Landy medium. In summary, agitation rate, relative filling volume and ventilation volume regulated the limited oxygen supply to selectively enhance the content of fengycin and bacillomycin L. Addition of Mg²⁺ and Fe²⁺ alleviated Mg²⁺ and Fe²⁺ chelation by lipopeptides and led to metal ion shortage in fermentation broth. Moreover, addition of Mg²⁺ and Fe²⁺ ensure cellular abundant metal ion for metabolization.?9? By single-factor experiments, the results showed that the four parameters including soybean oil, water content, inoculated quantity, and temperature had a significant effect on the yield of lipopeptides among multiple fermentation parameters. By the Box-Behnken design analysis, the best medium components were as followed: soybean flour 5 g, rice straw 5 g, soybean oil 0.336 g, glucose 0.3 g, Ca CO3 0.4 mg, water content 58.53%, inoculum size 15.29%, pH 7.0, 30°C and 96 h. The production of lipopeptides was 10.87 g·kgds-1, and increased by 80.86% than before optimization, and was 3.37 times than submerged fermentation.