Study On Biological Function And Fermentation Process Of Antagonist Bacterium Bacillus Mojavensis ZA1

In the study, the strain ZA1(Bacillus spp.) was screened from150strains of grassendophyte on the East Qilian Mountains Alpine grassland by Petri dishes confrontationmethod, which has the significant inhibition effect against potato gangrene pathogen (Phomafoveata). The biological function and antibacterial mechanism of the strain ZA1were studied,and the fermentation process of ZA1was optimized. The results were shown as follow:1Screening and determination of biology function of the strain ZA1The inhibiting rate of strain ZA1against P. foveata in Petri dishes confrontation was71.83%. Inhibition rate of strain ZA1against Alternaria solani, Stysanus stemonitis, Fusariumoxysporum and Colletotrichum coccodes were64.30%,41.05%,61.42%and74.92%,respectively. The concentration of IAA secreted by ZA1in the King medium with and withouttryptophan were12.17mg/L and9.75mg/L respectively. The strain ZA1possessed thecapacity of nitrogen fixation, however without the ability of phosphate solubilization.2Identification of the strain ZA1ZA1is rod-shaped with a size of0.5-0.7μm×1.5-3.15μm, Gram-positive, and producespore in the center or second terminal. Based on the phenotypic characteristics ofmorphological, physiological and biochemical characteristics of ZA1, and combined with16SrDNA and gyrB gene sequence analysis, the strain ZA1was identified as Bacillus mojavensis.3The antibacterial mechanism of strain ZA1ZA1shows a good antibacterial effect against the pathogen of potato gangrene (Phomafoveata). In the study, P. foveata was used as detected fungal pathogen, and then using themethod of petri dishes confrontation for culture condition optimization and stabilitydetermination of the antibiotic secreted by ZA1. The results showed that the optimum culturemedium of ZA1was made up of200gram potato,10gram peptone,20gram sucrose, and 1000milliliter tapwater. The optimum fermentation temperature of ZA1was17.8oC. Theoptimum pH value of ZA1’s culture medium was6.9. The optimum150ml triangle bottlevolume of ZA1was20ml. The optimum culture mode of ZA1was shaking cultivation indark for96hours. After the optimization, the EC50=0.1228μl/ml against P. foveata was37times higher than the EC50=4.5888μl/ml against P. foveata of preoptimization. Crudeextracting of bacteriostatic from ZA1showed the characteristic of high temperature resistanceand its relative activity could reach76.62%when it was treated with90oC for2h. Therelative activity of bacteriostatic crude extracting of ZA1was stable which could not bedestroyed under UV irradiation for30min. The bacteriostatic crude extracting of ZA1hadgood acid and alkali resistance when it was treated by pH=3and pH=11, the relative activitywas92.87%and85.11%respectively. ZA1was not sensitive to protease and heavy metal ionssuch as Ag+, Cu2+, Zn2+and Fe3+. ZA1’s relative activity could keep in86.93%under Ag+treatment condition.4Optimization of fermentation technology on Bacillus mojavensis ZA1In order to increase the biomass of strain ZA1, fermentation medium was optimized withmethods of the single factor experiments and box-behnken design by using shaking-flaskfermentation in the laboratory. The results showed that the optimum culture medium of ZA1was contained of14.25gram ammonium chloride,19gram corn flour,237gram potato,1000milliliter distilled water, and pH was7.7. The optimum cultivation conditions were containedof28℃,180revolutions per minute (r/min) and36hours fermentation time. In this condition,viable count of strain ZA1was4.12×1010colony-forming unit per millilier (CFU/ml). Fermentation technology of strain ZA1was optimized with methods of central compositedesign in10L fermentation tank. The optimum dissolved oxygen was60%and rotate speedwas180r/min. In the optimum culture condition, when fermentation time was36hours in the10L fermentation tank, viable count of strain ZA1was1.59×1011CFU/ml.5Effects of controlling disease and growth promotion of ZA1on potatoThe control efficiency of ZA1against potato gangrene was85.9%by spraying10timesdiluting fermentation broth on potato tubes in storage-period. The control efficiency of ZA1 against potato late blight was26.56%by seed dressing fermentation broth with diluting for20times on potato tubes under field condition. In field condition, the production growth ratios ofcommodity potato was increased by36.29%per hectare, as well as the production growthratios of potato was increaseed by33.88%per hectare. Pot experiments with the seed dressingpotatoes showed that the content of roots stems and chlorophyll were higher than the controlgroup. After treatment by ZA120times’ fermentation broth on potato tubes, the length of theroot, wet and dry weight of potato were increased by8cm,0.75g and5.07g, respectively. Inthe same time, the plant height, stem diameter, stem wet and dry weight and the chlorophyllcontent of potato were increased by2.74cm,0.27cm,0.52g,5.73g and0.54mg·g-1,respectively. The Root-Shoot ratios of wet and dry weight were increased by0.214and0.094,respectively. When spraying diluting fermentation broth of ZA1on potato leaves, the resultsindicated that the activity of CAT, PPO, PAL, SOD and POD of potatoes were increased.