| French researcher Fery from French Academy of Agricultural Sciences had constructed engineered Hrp- mutant of Ralstonia solanacearum by insertion of an ?-Km interposon within the hrp gene cluster of each pathogenic strain. Hrp- mutants as potential biocontrol agents have advantages of safety and stability, and showed long period protection from tomato bacterial wilt, and the protection rate higher than 75%.Furthermore,there was evidence that soil environment and soil microorganisms had little effect on the ability of Hrp- mutants to control bacterial wilt, because Hrp- mutants could invade host plants and grow in the plants, which have also conquered the limitation of unstable control effect under field condition. In conclusion, since Hrp- mutants as potential biocontrol agents of tomato bacterial wilt have high commercial value, it is important to study the fermentation condition optimization and formulation of Hrp- mutants.Medium optimization of Hrp- mutant of Ralstonia solanacearum was conducted applying full factorial design, orthogonal analysis and single factorial design under the condition of flask fermentation. The best formula of fermentation medium was gained, which was glucose(g/L)8g, fine corn flour 5g, bean cake powder 35g, (NH4)2SO4 2g ,yeast extract 1g , casamino acids 1g.The fermented bacterial biomass could reach from 1.34×1010 cfu/ml to 3.45×1010 cfu/ml. Plackett-Burman design and response surface analysis were used to optimize the fermentation conditions of Hrp- mutant. the results showed that the best inoculation time was 10h, the optimum temperature was 20℃, most appropriate primary pH was 8.75, the inoculation content was 6.25%,the flask column was 100ml/500ml. The production increased from 1.34×1010cfu/ml to 4.42×1010cfu/ml under the optimized fermentation condition,increasing by 329.9%.Based on the results of shaking flasks experiment, Hrp- mutant was scaled up in a 30L fermentor. The changes of fermentation indices of Hrp- mutant in the 30L fermentor, including pH, reduced sugar and ammonia nitrogen, biomass and dissolved oxygen were investicated. It was established that the fermentation period was 54h.The results of three consecutive fermentation showed that the production of Hrp- mutant reached 1.28×1011cfu/ml, increasing by 289.6%.Study on solid formulation of Hrp- mutant was explored. We have worked out the preparation method of Hrp- mutant solid formulation through screening various carriers, protection agents and miscellaneous microbe inhibitors including bactericide and fungicide. The detailed method was as follows: Bacterial cultures after fermentation were collected by centrifugation at 5000r/min for 10min in an centrifuge tube, washed twice in sterile water and suspended in mineral oil,followed by dispersing evenly by surging for 15-20min.The solid formulation was prepared by mixing the suspension with sterilized peat with the proportion of 1:1. Nisin and Natamycin were added into the mixture to inhibit other miscellaneous microbes. The solid agent was supposed to preserve with the temperature of 25℃, pH of 5.3 and water content of 35%, but without vacuumization. The survival rate after 6 months could sustain at 44.4%,and the biomass could reach 1.12×1010cfu/g.The field control effect of Hrp- agent of solid formulation have been investigated. The best application method was soaking the root before translocation, by what means the field experiment for the control effect was conducted. The result showed that when the tomatoes were treated by Hrp- solid formulation agent dilution before translocation, the disease incidence was 40.6% and the control efficacy sustained at 58.6%。... |
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