The Control Efficacy And Mechanism Of Bacillus Subtilis M-23 On Citrus Melanose

In recent years,Citrus Melanose is becoming more and more serious,which has caused a huge economic loss to China’s citrus industry.In order to solve this problem facing the citrus industry,it is urgent to study the biological control methods of Citrus Melanose.Healthy Citrus leaves were collected from Citrus orchard where Citrus Melanose occurred,and a biological control strain M-23 with good inhibitory effect on Citrus Melanose was screened by means of dilution separation and plate confrontation.The traditional morphology,physiological and biochemical reaction and molecular biology were used to identify the bacteria.The optimal ratio of each component of the liquid fermentation medium and fermentation conditions were optimized by single factor control method and multi-factor orthogonal analysis.Subsequently,the antibacterial mechanism,field control effect and the influence of strain M-23 on the phyllosphere microbial diversity of citrus were investigated.The main results were as follows:（1）A total of 72 strains of bacteria were isolated from the endophytic bacteria in healthy citrus leaves.The bacteria M-23 with the highest inhibition rate,was screened by the method of plate confrontation with Diaporthe citri as the target strain,and the inhibition rate of the strain M-23 on Diaporthe citri was 72.67%and 61.80%,respectively.（2）Through traditional morphological observation and detection of physiological and biochemical reactions,the results showed that strains M-23 basically matched the basic characteristics of Bacillus.The strain M-23 was identified as Bacillus subtilis by analysis of16S r DNA sequence.（3）Through single factor control method and multi-factor orthogonal analysis,the optimal fermentation medium composition ratio of strain M-23 was determined as follows:glucose 15 g/L,Yeast extract fermentation15 g/L,Na Cl 3.5 g/L,K₂HPO₄ 1.5g/L;Optimum fermentation conditions:28℃,180 r/min,p H=7.5,fermentation time was 72h.（4）It was found that strain M-23 contained four genes producing antibacterial lipopeptide,including Surfactin,Fengycin,Bacillomycin and Iturin.Lipopeptide crude extract inhibited the germination of mycelia and spores.（5）In the field experiment of M-23 fermentation broth,the control effect of diluted30-fold fermentation broth was 79.09%,and that of diluted 50-fold fermentation broth was68.97%.The results of high-throughput sequencing analysis of the phyllosphere microbial community diversity showed that the citrus phyllosphere bacteria were mainly composed of Proteobacteria,Firmicutes,Bacteroidetes and Actinobacteria,and the phyllosphere fungi were mainly composed of Ascomycota and Basidiomycota.The community richness of phyllosphere microbial decreased after the spraying of the fermentation broth,but the phyllosphere microbial community structure was optimized,and Bacillus subtilis M-23could colonize in phyllosphere.The relative abundance of Bacillus and Pseudomonas in the phyllosphere bacterial community increased,while that of Cladosporium in the phyllosphere fungal community increased significantly.