| The genus Bacillus is ubiquitous in many environments and is one of the most important plant growth promoting rhizobacteria (PGPR), which can control plant disease and promot plant growth effectively. Bacillus can produce endospores that are highly resistant to hostile environments, such as high temperature, drought, low temperature, UV and so on, thus a successful formulation of Bacillus is potentially more readily achieved than a formulation with a microorganism that does not produce a resistant life stage comparable to the Bacillus spore, and play an important role in biological control plant disease. HpaGooc, from rice pathogenic bacterium Xanthomonas oryzae pv. Oryzicola, is a member of the harpin group pf proteins, eliciting hypersensitive cell death in non-host plants, inducing insect, disease and drought resistance in plants, and enhancing plant growth.In this study, in order to further improve the growth and antagonistic ability of the Bacillus, and obtain higher efficiency strains, the genetically engineered strain 69HF was produced by transporting HpaGxooc encoding gene into 69 using genetic engineering methods, and biological seed coating agents were prepared with fermented liquid of 69 and 69HF as active ingredient. Various additives of the seed coating agent were selected, and the results showed that, the compound film former which was prepared by mixing a 4% polyvinyl alcohol (PVA) solution and 4% chitosan (CS) solution at the ratio of 8:2 (v/v) had no harmful effect to survival of biocontrol bacteria, besides it combined the advantage of PVA and CS, and had good film forming ability, water resistant property, swelling capacity, air and water permeability, thus we selected the compound film former as the optimal film former; 0.5% potassium sorbate was selected as the optimal antiseptic for it had no harmful effect to survival of biocontrol bacteria, and had good antiseptic property; 5% propylene glycol was selected as the optimal antifreeze for it had no harmful effect to survival of biocontrol bacteria, and had good property of resistance to frost.Biological seed coating agent was prepared by mixing the fermented liquid of biocontrol bacteria 69HF and various additives with a certain ratio, and content of active ingredient, pH value, viscosity, decrease of viable bacteria after heat storage and change of bacteria concentration in one year storage at room temperature of the agent were detected. The results showed that, the initial concentration of bacteria in seed coating agent were 5.0×108～8.0×108 cfu/ml, pH value of the agent was weak acidity, the viscosity was 255 mPa-s, decrease of viable bacteria after heat storage was 40.21%. Bacteria concentration can maintain at a high level in one year storage at room temperature, and the concentration was 5.34×106 cfu/ml in the twelfth month.Biocontrol effectiveness of the biological seed coating agents prepared with 69HF or 69 were determined in indoor and greenhouse experiments. The results showed that, seed coating agents which were prepared by 69HF or 69 were highly effective in promoting soybean growth and controlling root rot disease. Compared these agents, agent prepared with 69HF was more effective than agent prepared with 69, and they were both more effective than the chemical agent fludioxonil. In greenhouse condition, the height, fresh weight and root lenghth of the soybeans which were coated with agent prepared with 69HF enhanced by 67.94%,73.79%,63.84% compared with the control treatment, and the control efficiency to soybean root rot is 85.89%. The results demonstrated that the genetically engineered strain 69HF which was produced by transporting HpaGxooc encoding gene into 69 using genetic engineering methods was more effective than 69 in promot plant growth and control disease, and have a great potential in agricultural use.Besides,20 Bacillus strains, with biocontrol potential, were selected from many Bacillus strains which were isolated from rhizosphere soil of plants in Tibet. Through the experiment of dual-culture test in plate, we selected 11 strains which showed strong inhibitory to Fusarium oxysporum, and prepared biological seed coating agent by using their fermentation liquor as active ingredient, and adding a certain proportion of different additives which were selected by previous test. In order to screen the agents which can promote soybean growth, the indoor test was conducted. The results demonstrated that seed coating agent which were prepared with LSSC3 and SYST2 strains can enhance the germination and growth of soybean, and agents prepared with other strains had no effect to growth of soybean. To investigate whether these two agents had good biocontrol effectiveness in greenhouse, tests were conducted and the results showed that, in condition of inoculating with pathogenetic fungi in soil, the plant height, fresh weight and root length of soybeans which treated with these two biological agents were evidently higher than chemical agent and uncoated treatment. Besides, these tow biological seed coating agents can control soybean root rot disease effectively, with good control efficiency of 72.38% and 73.69%. |
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