| Rice blast caused by Magnaporthe oryzae spread all over the world.As one of three serious rice disease in china,this disease causes annual rice losses approximately 10%-30%.At present,rice blast control depends on the triazole pesticides.However,people pay more attention to residual toxicity and environmental pollution.Development environment friendly pesticide is becoming more important.Some biocontrol agents have been formulated into aqueous solutions and wettable powder.The successful delivery of biocontrol agents,shelf-life stability and effectiveness in commercial conditions depend largely on the dry flowable formulations.Bacillus amyloliquefaciens strain JT84 showed effective control to rice disease.The single factor test,Plackett-Burman experiments and response surface methodology were adopted to optimize the liquid fermentation medium and conditions of B.amyloliquefaciens JT84 for the development of dry flowable formulations.According to the evaluation by the response surface model established in this study,the optimum mass concentrations of the three components,glucose,soya peptone and K2HPO4 in fermentation medium were 3.59 g/l,21.89 g/l and 0.49 g/1 respectively.The fermentation condition determination tests indicated that temperature,liquid volume of shaking flask and inoculum volume were the major factors affecting JT84 yield.The optimized fermentation conditions were 30.7 0C,inoculation quantity 3%,loading volume 68 ml in 250 ml,shaking flask for 65.5 h,180 r·min-1 and initial pH 6.7.Under the conditions spore production was up to 1.67×109 cfu/ml.spores content increased by 159%and the width of antifungal activity is 9.8 mm,increased by 18.2%compared with the basal medium.Dry flowable formulations of B.amyloliquefaciens JT84 with fungicidal activity against the rice blast fungus Magnaporthe oryzae were synthesized by spray drying.The Plackett-Burman experiments and response surface methodology were applied to optimize the spray drying technology of B.amyloliquefaciens strain JT84.According to the evaluation by the response surface model established in this study,the optimum conditions were Inlet air temperature 118.9?,feed flow rate 25.7 r · min-1(680 ml/h),atomization caliber 0.7 mm,atomization pressure 0.2 MPa,hot air flow 28.56 M3/h.The storage stability test found that the vial spore density was 4.0 × 109cfu/ml after 180 days.When treated rice seeds with 500 times diluted dry flowable formulations supernatant,there were not only no toxic effects but also varying degrees of growth promotion.It was showed that control effects of B.amyloliquefaciens JT84 dry flowable formulations against rice blast were 79.3%in field trials.This effect was similar to the use of 75%tricyclazole wettable powder.To preliminarily study the mechanism of dry flowable formulations of B.amyloliquefaciens JT84 against the rice blast,the colonization dynamics was tested on leaves.JT84gfprif colonized rice leaves,reaching populations more than 1×104 cfu/cm2 after 10 days.Dual culturing experiment showed that inhibition zones of four Bacillus amyloliquefaciens JT84 dry flowable formulations against Magnaporthe oiyzae were 10.33 mm respectively.Inhibition zones of lipopeptides from dry flowable formulations against M.oryzae were 6.33 mm.Mycelium of M.oryzae exhibited obviously swelled by microscopic observations.Thus it can be seen that lipopeptides still exist in the dry flowable formulations.sfp,fenB,ituA or bamA,mycB were cloned in the genome of JT84 by four primers on the basis of lipopeptide synthesized genes.Three kinds of lipopeptides including surfactins,bacillomycinF,iturinA or mycosubtilin and fengycins were identified by HPLC-MS techniques.The antiactivity of the iturins-deficient mutant strain was obviously weakened,which suggested that iturins might play an important role in controlling the rice blast. |
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