Study On The Preparation And Preservation Technique Of Liquid And Freeze-Dry Formulations Of Biocontrol Agents

Biological control becomes very important in agriculture and environmental protection, and many different species of biocontrol bacteria have tremendous potential. However, the various environmenal factors have a strong influence on the biocontrol agents. It is not convenient for the practical application of the biocontrol agents. In this study, the factors for the preparation of freeze-dried formulation of several biocontrol agents from the genus Bacillus sp., Pseudomonas sp. and Pantoea sp. Which isolated by our lab biocontrol agents were discussed, we also studied the impact of cells concentration in application and storage of biocontrol agents.The effect of different protective agents and rehydration media on the viabilities of biocontrol agent Pantoea ananatis strain YT11was investigated when it was subjected to freeze-drying, and the subsequent survival rates during storage under different temperatures were examined. The results indicated that5%sucrose gave the greatest protection to YT11during freeze-drying, the viability of cells was more than90%when YT11was rehydrated at25℃, and the highest survival of the biocontrol agent was reached100%when5%non-fat skim milk as rehydration media. Freeze-dried cells stored at4℃provided a higher survival viability than the sample at22℃, and after24weeks storage, the YT11powder formulation stored at4℃and22℃both maintained biocontrol efficacies,71.57%and50.98%respectively. It was proved that freeze-drying can be used for preparation of effective biocontrol agent YT11powder formulation.To study the feasibility for preparation of biocontrol from different genus by freeze-drying method, the effect of different protective agents and rehydration media on the viabilities of three freeze-dried biocontrol agents Bacillus sp. strain AR156, Pseudomonas sp. strain2R37and Pantoea sp. strain2Re40were investigated, and the subsequent survival rates during storage were also examined. The results indicated that the most suitable protective agent of different biocontrol agents were not same. The cellobiose (5%) and D-galactose (5%) gave maximum viability of strains2R37and2Re40(98.01%and54.31%respectively) when they were rehydrated at at25℃while the perfect one (100%) of strain AR156was obtained with sucrose (5%) during freeze-drying, and the highest survival of the three biocontrol agents was reached when they were rehydrated with10%NFSM. In the following storage at room temperature (25℃), the tracing survival rates showed that AR156could still reach50%after12months, while2R37and2Re40were thoroughly dead after3months or less.At the present the most extensive formulation of biocontrol agents used is still the liquid formulation, and the concentration of viable cells is the one of most basic and primary factors of the shelf life and biocontrol efficacies of biocontrol agent. So, the effect of different concentrations on biocontrol efficacies and storage stabilities of three biocontrol agents, Bacillus sp. strain AR156, Pantoea sp. strain YT11and Pseudomonas sp. strain ABc9, which were isolated to control tomato bacterial wilt caused by Ralstonia solanacearum was studied. The most suitable concentrations of viable biocontrol agents for using and storage were discussed. The results indicated that the biocontrol efficacies did not increase along with the enhancement of the concentration of biocontrol agents. When the concentrations were higher than109CFU/ml, the biocontrol efficacies were showed to weak. The treatment of fresh bacterial suspension, with the bacterial concentration of109CFU/ml of the strain ABc9,108CFU/ml of AR156and YT11, showed the best biocontrol efficacy63.06%,49.55%and52.25%respectively. Moreover, the treatment with higher initial bacterial concentration brought the higher mortality in storage, and the most suitable concentration of the biocontrol agents for preservation was found to be10CFU/ml for YT11and109CFU/ml for AR156and Abc9.In conclusion, stored at4℃,the freeze-dried formulation of Pantoea ananatis strain YT11with5%sucrose as protective agent and5%skim milk as rehydration media, can be effective applied to practical and maintain the biocontrol efficacy of biocontrol agent. The study also compared affects of freeze drying to three different genuses of biocontrol agents. It was indicated that different genera of biocontrol agents showed different sensitivity to freeze-drying, and there were also some differences of the greatest protective agents and rehydration agents for freeze-dried biocontrol agents. It also showed biocontrol efficacies and storage of biocontrol agents were not positively correlated with the concentrations of viable cells; too high concentrations of biocontrol agents would get a weaker biocontrol efficacy.