Screening And Identification Of Methylotrophic Bacillus ZH-9 And Biological Control Of Anthracnose Disease

Cucumber anthracnose is a fungal disease caused by the Colletotrichum orbiculare.Cucumber anthracnose is common in China,causing serious harm,resulting in large-scale production cuts.Farmers have to spray chemical pesticides in multiple times,which is harmful to sustainable disease control,and leads to chemical pesticide residues.This puts a burden on the development of environmentally friendly agriculture.In order to solve the problems effectively,this paper has developed and researched high-efficiency biocontrol strains that antagonize cucumber anthracnose.The aim is to find out microbial control materials with good biocontrol effect on cucumber anthracnose,and provide basis for its biological control.In this study,20 samples were randomly sampled from Qinling forest soil,and 143strains of bacteria were isolated and purified.Then the strain of ZH-9 with good inhibitory activity against cucumber anthracnose was selected by plate confrontation method and growth rate method.The results showed that the sterile fermentation broth of ZH-9 had a bacteriostatic rate of 74.28%against cucumber anthracnose.In addition,ZH-9 had good broad-spectrum biological activity.Then combined with morphological identification,physiological and biochemical analysis to identify ZH-9.The results showed that ZH-9were basically the same as those of Bacillus strains.Combined with molecular biological identification?16S r DNA sequence analysis?,ZH-9 was identified to be Bacillus methylotrophicus.Combining the single factor variable analysis method and the multi-factor variable orthogonal test,the fermentation conditions of ZH-9 sterile filtrate were optimized.The results showed that the components?carbon source,nitrogen source,trace elements?and the ratio of the optimal medium were 1.5%Soluble starch,1%yeast extract,0.2%K₂HPO₄,0.15%Mg Cl₂.The best culture conditions:p H is 7,culture temperature is,shaker speed is 200r/min,and culture time is 36h.The optimized ZH-9 fermentation broth had an strain fermentation broth had an OD₆₀₀ value of 0.83 and a viable bacterial volume of 5×10⁸cfu/m L.It shows that the optimization effect is remarkable.Therefore,after optimizing the fermentation conditions of ZH-9,the living bacterial quantity of ZH-9 strain and the antagonistic activity against cucumber anthracnose were improved.The biocontrol activity of ZH-9 fermentation broth against cucumber anthracnose was tested in greenhouse pot experiment and field experiment.The potting results in the greenhouse showed that the protective and therapeutic effects of ZH-9 were 72.69%and65.94%,respectively.The protective effect was greater than the therapeutic effect.In terms of application time,ZH-9 is suitable for application before the onset of disease.The disease has a better preventive effect.The results of the field test under the field test showed that the field control effect of ZH-9 reached 60.34%,proving that it has antagonistic activity against cucumber anthracnose under field conditions and can control the spread of the disease.Therefore,ZH-9 can be used as a biocontrol agent for the prevention of cucumber anthracnose,and has very good application prospects.This article starts with collecting Qinling forest soil samples,through separation and purification,screening of antagonistic strains and identification.Then through optimization of fermentation medium and culture conditions,to determine the actual control effect of ZH-9 fermentation broth on cucumber anthracnose.Bacillus methylotrophicus ZH-9,which has good control effect on cucumber anthracnose,is obtained.This paper has practical significance for the organic and environmentally friendly prevention of the anthracnose disease.