| Wheat scab is a fungal disease caused by Fusarium graminearum,and is one of the main factors for wheat yield reduction.At present,the prevention and control of wheat scab mainly depends on chemical pesticides.The application of chemical pesticides will cause hidden dangers to human and animal health and environmental protection.Therefore,the use of environmentally friendly and harmless biological agents is an important way to solve this hidden danger.Based on this,the main research purpose of this paper is to develop the selected strains with potential commercial value advantages as the next generation of biocontrol bacteria.The following is the research content of this article.(1)Screening of active strains and antibacterial activity of fermentation brothPreliminary screening of the activity of the strains against wheat scab by plate confrontation showed that the average bacteriostatic rates of Bacillus amyloliquefaciens DEB-2 and D1 were 51.32% and 49.48%,respectively,which were significantly higher than the other 6 strains.Strains.In order to explore the minimum inhibitory concentration of fermentation broth,the growth rate method was used to test the concentration gradient of the fermentation broth of the dominant strains DEB-2 and D1.It was found that when the concentration of the fermentation broth was 64%,the fermentation broth of DEB-2 and D1 on wheat The inhibition rate of Gibberella pathogens is 100%.(2)Study on the stability of fermentation broth with biocontrol and antibacterial propertiesTo further clarify whether the dominant strain has the potential to develop as a biocontrol strain,the biocontrol effect of the strain was evaluated in combination with various environmental conditions.Therefore,the growth rate method was used to test the stability of the fermentation broth,including the stability test of temperature,acidbase,passage,and ultraviolet.In terms of temperature stability of the fermentation broth,at 0~100℃,DEB-2 The bacteriostatic rate of the fermentation broth is still maintained at 62.35%.In terms of the acid-base stability of the fermentation broth,the stability of the fermentation broth of both is good,and the differences between the groups of DEB-2 and D1 under different p H conditions are not significant.Under neutral conditions,the DEB-2 strain is fermented The maximum antibacterial rate of the liquid was 85.05%.It shows that it is more conducive to exert the best antibacterial effect of DEB-2 fermentation broth under neutral conditions.When pH=3~5,the fermentation liquid of D1 strain fermentation broth has the best bacteriostatic effect,which shows that it is more conducive to exert the best antibacterial effect of fermentation broth of D1 strain under moderately acidic conditions.In terms of the UV stability of the fermentation broth,the UV stability of the fermentation broth of the strains DEB-2 and D1 is relatively good,and the differences between the groups are not significant.Within 0-6 h,the maximum inhibition of the fermentation broth of the DEB-2 strain The bacterial rate and the minimum bacteriostatic rate were 82.80% and 81.72%,respectively,and the difference between the two was only 1.08%.The maximum and minimum inhibitory rates of the fermentation broth of D1 strain were 77.77% and 76.35% respectively,and the difference between the two was only 1.42%.In terms of the passage stability of the strain,after 30 consecutive transfers,it was found that the differences in the bacteriostatic rates of the DEB-2 and D1 fermentation broths between the groups were not significant.The maximum average bacteriostatic rate and the minimum bacteriostatic rate of DEB-2 were 83.22% and 81.77% respectively,and the difference between them was only 1.45%.The maximum average bacteriostatic rate and minimum bacteriostatic rate of D1 were 77.50% and 77.14% respectively,and the difference between them was only 0.36%.It shows that the genetic passage of DEB-2 and D1 strains are relatively stable.However,because the inhibition rate of DEB-2 strains against pathogens is better than that of D1 strains,and under extreme high temperature conditions(121℃),the stability of the fermentation broth of the DEB-2 strain is significantly higher than that of the D1 strain.Therefore,in antagonizing the pathogen of wheat scab,the antibacterial performance of the DEB-2 strain is particularly prominent.(3)Biocontrol experiment of isolated wheat earsIn order to investigate whether the dominant strain of DEB-2 has the potential for commercial development,the DEB-2 strain and the commercially available subtilis spore biocide were compared for the bacteriostatic effect of wheat wheat scab in vitro.The results showed that:(1)The activity of DEB-2 and commercially available Bacillus subtilis was tested by flat-pane confrontation method,and their active sizes were 51.32% and 30.02%,respectively(2)The commercially available biocontrol agent and laboratory of Bacillus subtilis were used.Comparison of the biocontrol effects of the isolated wheat scab in antagonistic strain DEB-2 fermentation broth.Using the method of first applying and then dyeing(preventing),the control effect of the fermentation broth of DEB-2 strain and the commercially available biofungal agent are 66.68% and 24.23% respectively;the method of first applying and then applying(curing)is effective They are 46.00% and 22.86% respectively.(3)Comparison of the biological control effect of the isolated wheat scab disease in the fermentation broth activated by the commercially available biocontrol agent Bacillus subtilis and the laboratory antagonist strain DEB-2 fermentation broth.Using the method of applying first and then dyeing(preventing),the control effects of the fermentation broth of the strain DEB-2 and the fermentation broth of commercially available Bacillus subtilis were 68.96% and 38.14% respectively;using the method of first dyeing and then applying(treatment),DEB The therapeutic effects of the fermentation broth of the strain-2 and the commercially available fermentation broth of Bacillus subtilis were 74.07% and 51.85%,respectively.This shows that DEB-2 has a better control effect than the currently commercially available biological agents,and it is an ideal strain developed as a nextgeneration biocontrol agent.(4)Separation of substances of antibacterial fermentation liquidIn order to clarify the active substances in the fermentation broth of the dominant strain DEB-2,the DEB-2 strain was expanded and fermented,and the fermentation broth was extracted with ethyl acetate and n-butanol to obtain the crude ethyl acetate layer extract.16.73 g;crude extract of n-butanol layer 110.13 g;crude extract of water layer 979.20 g.Through the activity tracking of the plate,the activity of the crude extract in the ethyl acetate layer was found to be 10.15%;the activity of the crude extract in the n-butanol layer was 23.42%;the crude extract in the water layer was inactive.In the follow-up experiment,two compounds and active fractions were separated from the ethyl acetate active layer by silica gel forward column chromatography.The active part of the ethyl acetate active layer and the n-butanol active layer were analyzed by GC-MS,and combined with the corresponding literature,it was concluded that the active substance of the fermentation broth of the strain DEB-2 was cyclo-(L-phenylalanine-L-Proline),which laid a material foundation for the subsequent chemical synthesis and modification of active substances. |
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