Study On Mutagenesis, Optimization Of Fermentation Technics And Applying Formulation Of Biocontrol Bacterial Strain A

Treating the original Bacillus subtilis strain A(CGMCC No.1982) by compound mutation of LiCl and ultraviolet radiation,a mutagenesis bacterial strain with well genetic stability and antifungal activity was obtained.Subsequently,the growth of Strain J4-1 in different fluid cultural medium was studied.And then the fermentation parameters,including residual sugar,ammonia nitrogen,dissolved oxygen(DO) and pH,were screened in 20L fermentation cylinder.The applying formulation was developed to control the pathogens happened in vegetables and fruits.The results were as follows:1.The original Bacillus subtilis strain A(CGMCC No.1982) was treated by compound mutation of LiCl and ultraviolet radiation.A mutagenesis bacterial strain with well genetic stability and antifungal activity was screened out,named J4-1,using Bipolaris maydis as indicating fungus.The antifungal activity of J4-1 strain was 64.5%higher than that of the original strain.2.The optimizing medium of J4-1 strain was established by orthogonal test design.That was composed by 1%glucose,3%sucrose,3%yeast extract and peptone,2%(NH₄)₂SO₄. According to the growth curve,the pH change,the variation of residual sugar and ammonia nitrogen,the 42h fermentation time was detemined and feed batch fermentation which was used in before study was abandoned.In the condition of optimizing fermentation parameters, the concentration of J4-1 thalli could reach to(3.132±0.376)×10¹¹ cfu/mL.3.The applying formulation of J4-1 strain was developed.A liquid fungicide dosage form,which composed by 0.2‰Kathon,0.1%Methylparaben,1%Tween-20,0.5%sorbitol, 98.4%fermentation liquor,pH 8 was developed to control the pathogens happened in vegetables.The efficacy of this fungicide in field was tested by diluting ten times with water then spraying them to ordinary vegetables.The investigating results were as follows:(1) In greenhouse,the efficacy of this fungicide to green pepper blight was higher than that of chemical fungicide.The effect on powdery mildew was close to that of chemical pesticide.(2) In greenhouse,the efficacy of this fungicide to cucumber brown patch was no notable difference with that of chemical fungicide.(3) The efficacy of this fungicide to tomato botrytis reached to 65%that of chemical fungicide in greenhouse.(4) The efficacy of this fungicide on preventing grape brown patch and Downy Mildew existted no difference with that of chemical fungicide in field.4.A kind of antisepsis refreshing paper or bag was exploited by spreading fermentation liquor on the paper with a(4.62±0.293)×10⁸/cm² density of thalli or the bag with a (3.39±0.323)×10⁹cfu/cm² density of thalli.The pear refreshing experiment was conducted by packing pear with antisepsis refreshing paper and the results indicated that the weight-loss ratio was 26.84%and the decayed fruit ratio was 13.33%during 45d treatment at 25℃.These results were obviously lower than the untreated pears.The antisepsis refreshing bags were applied to grape production.The grape fruits packed by bags almost exhibited the same ability as the fruits sprayed by chemical fungicide in preventing the grape fruit anthrax.