Study On The Wettabable Powder Of Bacillus Amyloliquefaciens,a New Type Of Marine Microorganism Pesticide For Conctroling Cabbage Clubroot

The clubroot is one of the major global diseases of cruciferous crops,but it is very limited in the selection of fungicide control,and new,highly efficient and low toxic agent are urgently needed.Marine microorganisms,because of its special habitat,which often produce different metabolites from terrestrial microbial and have good control potential to difficult disease,have now become a new hotspot of microbial pesticide development.Microbial pesticide is usually guaranteed by high living bacteria content,so it is required to develop high density low foam culture technology.Marine microorganisms are prone to produce surface active substances,resulting in a large number of foam in fermentation culture,resulting in low liquid loading,escape and pollution.Therefore,the establishment of fermentation technology with high density and low foam is an important way to reduce the production cost of marine microbial fungicides.In this paper,a new marine habitat Bacillus amyloliquefaciens Txc2-1,which was screened by this research group,had a significant antagonistic effect on the clubroot of cruciferous cabbage,on the basis of previous work,screened the deformer for the cultivation process,optimized and enlarged the culture technology.At the same time,the spray drying process and preparation formulation of Bacillus amyloliquefaciens were optimized.The preliminary effect of wettable powder on cabbage clubroot prepared with fermentation broth of 5 L tank was evaluated in potted plant and field experiment.In addition,the mutant strain of Bacillus amyloliquefaciens with fluorescence labeled was established to observe the colonization of the root of Chinese cabbage.The defoamer D and E were obtained by the screening of ability with the instant defoaming and the continuous anti-foam,and the defoamer D with good biocompatibility with Txc2-1 was screened out.The optimal carbon source G and nitrogen source Q were determined by single factor experiment.The best culture medium formula was obtained by response surface optimization,in the 5 L tank at the end of fed batch culture,the living bacterium content reach to 2.54×1010 CFU/mL,increased 3.57 times more than primitive process at the end of fermentation,total spore time is 36h.In the 100 L fermentation tank,the amount of living bacteria was 1.81×1010 CFU/mL at the end of fermentation,which was 1.80 times higher than the original process,and the total spore time was 48h.Further amplified in the 600 L fermentation tank,the amount of living bacteria at the end of culture was 2.78×1010 CFU/mL,and the living bacteria content was 2.25 times higher than the original process at the end of culture,and the total spore time was 48h.The conditions of spray drying for Bacillus amyloliquefaciens wettable powders were optimized on a small spray drying tower with the dehydration capacity of 3 kg/h water,in which the best inlet temperature determined is 190?,and the best outlet temperature is 80?.The condition was verified by a large spray drying tower with the dehydration capacity of 800kg/h water.The preliminary results show that the physicochemical properties(water,live bacteria,suspension rate,death rate,etc)of the wettable powder prepared by large spray drying tower are similar with the small spray drying tower,and the wettability of wettable powders is better by large spray drying tower.The formulation optimization of wettability powder determined the best carrier:ZT-1,the best wetting agent ZJ-5,the best suspending agent ZJ-3.The optimum ratio after the mix formula design was 83:9:8,the results of amplification test on large spray drying tower are consistent with the predicted values.On the basis of the above process,the Txc2-1 wettable powder(Txc2-1WP)with the living bacteria content of 3 billion CFU/g useing 5 L tank culture medium and obtained by small drying tower carried on plant pot(40 days)and field experiment(60 days),the result showed that the control effect of Txc2-1WP on the clubroot of cruciferous cabbage was 70.53%and 66.84%,which was a bit better than the control effect of 10 billion CFU/g bacillus subtilis WP(the disease refers control effect was 68.76%and 65.38%,respectively),slightly lower than the control effect of 50%fluorine(the disease refers control effect was(76.49%and 72.45%,respectively),the corresponding blank disease index were 37.90 and 41.00 respectively.The Txc2-1-gfp mutant labeled with fluorescence was obtained by electroshock transformation and screening.The bacteria with fluorescent tags can be observed inside the root tissue and vascular bundle through inversion fluorescence microscope in the fourth day of root colonization.The amount of colonized living bacteria was 7.2×108 CFU/g root tissue,and after 30 days microbial colonization living bacteria remained in an order of magnitude of 105 CFU/g the root tissue.The results showed that Txc2-1 had good colonization ability in the body of cabbage.