Studies On Fermentation Conditions Optimization, Field Experiment And BIO-Control Mechanism Of BIO-Pesticide AR156

Plant growth-promoting rhizobacteria (PGPR) widely exist in the plant rhizosphere soil. They compete with the pathogens for nutrition, colonization situs, or by producing some antibiotic substances to stop the infection, and promoting the growth of plants. The fermentation optimizing and the field experiment are important stages for the commercialization of the potential PGPRs, and necessarily for the progressing of the bio-pesticide. So more and more researchers around the world focus on how to make good use of the potential biological control strains and establish a beneficial research model, which includ the commercialization, field experiment and the mechanism research. This study was focused on development of an effective research strategy of bio-pesticide AR156, and it was expected that the study can give some suggestions on establishing a research patterns for biological strains.1. Optimization of Medium Components and Cultural Conditions of Bacillus cere us AR156The single factor experiments and orthogonal experiments were adopted to optimize the liquid fermentation medium and conditions of the biocontrol agnet of Bacillus cereus AR156. The results indicated that the constituents of medium, the filling volume of shaking flask, the rotation speed and the temperature were the major factors which affected the yield of AR156. The composition of maltose0.25%, maizena0.5%, soybean flour0.5%, tryptone0.5%, CaCl2H2O0.05%, MnSO4H2O0.05%and K2HPO40.1%was selected to be the medium formula, and the fermentation conditions were filling volume200mL in1L shaking flask, inoculum5%,28℃, shaking at200rpm/min for48h, and initial pH at7.0respectively. Under the optimized fermentation conditions, the production of spores was up to1.03×109CFU/mL, and the rate of spores was more than97%.2. Effect of bio-pesticide AR156and AR156combination against root-knot nematodes in cucumberIn preceding in vitro and greenhouse experiment, AR156and AR156combination showed good biocontrol efficacy against root-knot nematodes. From2008to2009, we carried out a field experiment at town Dingji in Huai’An, Jiangsu province.In the field experiment the data showed that AR156strain and AR156combination had great inhibition to the gall formation on cucumber roots, meanwile the growth of the plant and the yield had increased a lot. The average biocontrol efficacy and the yield increasement of the treatment AR156were53.70%and31.49%respectively when investigated at the end of the cropping season. The AR156combination treatment had a higher biocontrol efficacy up to83.33%and yield increased31.49%. Both of them were much better than the3%Carbofuran Drug granules.3. Study on rhizosphere population patterns of bio-pesticide AR156Bacillus spp. strain AR156and SM21, Serratia sp. strain XY21are three potential biocontrol bacteria screened and stored in our lab. Kanamycin, streptomycin and rifampin were used to label AR156, SM21and XY21respectively, the resistance of the mutants to the propotional antibiotic are all200μ/mL. We did some verification tests and picked the antibiotic mutant AR-km2, SM-Strl, XY-rifl. In the following experiment, the ability of these antibiotic mutants colonize in the tomato rhizosphere were tested as single, both and three of them. The result showed that the three potential biocontrol strains could colonize well in the tomato rhizosphere, no matter they were single, both or all of them. The colonization of the two Bacillus spp. strain AR156and SM21in tomato rhizosphere first decreased, then increased and stabilized at about10CFU/g soil in the rhizosphere after inoculated for60d. Serratia sp. strain XY21decreased gradually and stabilized at about104-105CFU/g soil after inoculated for60d in tomato rhizosphere.4. ConclusionIn this study, we optimized the fermentation medium components and cultural conditions of Bacillus cereus AR156. The biocontrol efficacy and yield increasment of the AR156and AR156combination were significant in controlling the cucumber root-knot nematodes during the field experiments. Based on these results, some researches on population patterns of bio-pesticide AR156we did in tomato rhizosphere, to reveal the biocontrol mechanisml.