| Paenibacillus polymyxa is a spore producing Gram-positive bacteria, and classified as one of first level commercial species that for free to the security identification by the Ministry of Agriculture and the U.S. Environmental Protection Agency (EPA). Because it s safe to humans and animals relatively,environmentally friendly and non-pathogenic to plants. As a biocontrol bacteria, Paenibacillus polymyxa was been widely applied in the prevention of plants wilt and degradation of pesticides, but it’s lack of reports about the prevention of Fusarium disease and preparation of microbial inoculum.In this study, Paenibacillus polymyxa JSa-9, an antimicrobial peptides producing strain screened and preserved in our laboratory, was selected as the research object. First, by measuring JSa-9 inhibitory activity to fungi to determine its antimicrobial spectrum; Secondly, through optimizing fermentation medium and fermentation conditions to improve the spore production of JSa-9, and provide a theoretical method for preparing an effective biocontrol microbial agents; Finally, biocontrol test of cucumber in seedling stage provides a theoretical basis and technical support for biological control of Cucumber Fusarium Wilt by JSa-9. The results are as following:1. Identified the antimicrobial spectrum of JSa-9. Measured the antagonistic activity of JSa-9 to six harmful fungus by dual culture method, the results show, JSa-9 can produce significant antagonism band to the Fusarium oxysporum f.sp. cucumerinum, Fusarium moniliforme and Fusarium graminearum, the maximum inhibition zone was 8.56mm, 9.59mm,10.39 mm respectively, meanwhile the inhibition rates of broth to the three fungus was 43.20%,55.72%,43.76%. In addition, determined inhibiting activity of JSa-9 to the three fungus by growth rate method, the results show that, when the adding amout of JSa-9 fermentation concentrate was 16%, the inhibition rate was 99.15% to Fusarium oxysporum f.sp.cucumerinum, and it reached 100% both for Fusarium moniliforme and Fusarium graminearum.2. Optimized the sporulation medium of Paenibacillus polymyxa JSa-9. First, the single-factor test determined the optimum initial spore fermentation medium, carbon, nitrogen and inorganic salts was malt extract, corn steep liquor powder and CaCO3 respectively. On the basis of the single factor, through two sets of Taguchi for further optimization, suitable fermentation medium got:malt extract powder 15 g/L, corn steep liquor 15 g/L, CaCO35 g/L, MgCl2.6H2O0.5 g/L,MnSO4H2O 0.06g/L, Tween-801 mL/L. Under the conditions of inoculation amount 10%,initial pH 7.0,temperature 30℃, and the speed 180 rpm, the spore production and conversion of Paenibacillus polymyxa JSa-9 were achieve a higher level, reached 1.01×1010 cfu/mL and 90.99% respectively, which spores production improved 36.7 times than BPY.3. Optimized the sporulation conditions of Paenibacillus polymyxa JSa-9. Setting the optimized medium as the fermentation medium, by single factor tests to study impact of inoculation, initial pH and temperature on the spore production of JSa-9. The results showed that when the inoculum,initial pH and incubation temperature respectively was 5%, the 6.5 and 30℃, spore production and conversion rate of JSa-9 reaches the maximum, which was 1.46 x 1010 cfu/mL and 94.8%.4. Paenibacillus polymyxa JSa-9 biocontrol agents effects show that, JSa-9 spores was not only no significant effect (p> 0.05) on seed germination, but also have a role in promoting the growth of young roots. The biocontrol experiments of seedling Fusarium wilt has a significant effect and reached 69.02%, and has a significantl (p<0.05) role in promoting the growth of cucumber. |
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