Screening And Identification Of Antagonistic Bacteria In Tobacco Anthracnose,optimization Of Fermentation Conditions And Study On Resistance Mechanism

In this study,the antagonistic bacteria of tobacco anthracnose bacteria were isolated from tobacco roots,stems and leaves,and the fermentation conditions and disease resistance mechanism of the resistant strains were optimized.The results were as follows:(1)A total of 137 endophytic bacteria were isolated from the healthy tobacco harvested from different tobacco planting areas in Yanbian state,and a strain C-4 with a remarkable control effect on tobacco anthrax bacteria was screened by plate confrontation method and fermentation broth re-screening,with a bacteriostasis rate of 72.14%.Through the analysis of culture shape,morphology observation,physiology and biochemistry and 16S rDNA sequence,the strain C-4 was finally identified as Bacillus velezensis.(2)Using Box-Behnken design,the three-factor three-level test optimization of fermentation conditions was carried out by the response surface method,and the regression equation was obtained by using Design-Expert 7.0 software:Y=-20.72177+1.30641 A+1.01 921B+0.054652C+0.01 1300AB+2.68750E-0.05AC-8.05000E-0.04BC-0.027221A2-0.087358B2-2.93581E-0.04C 2.According to the actual situation of production,the optimum conditions of C-4 strain fermentation were revised to the initial temperature of 25.5℃,pH of 7.1,and the liquid loading of 84.5 mL,fermentation time 48 h,shaker speed 180 r/min,inoculation 4%.After three repeated tests,the average actual OD600 value of the obtained fermentation broth was 1.859,which was 1.62 times before optimization,and the bacteriostasis rate was 8.4%higher after the bacteriostasis test than before.(3)The results of pot experiment showed that strain C-4 had induced disease resistance to tobacco,and the disease prevention effect was 74.02%.to further clarify the strain-induced disease resistance and its function,the activity of three major defense enzymes in tobacco leaves,POD,PPO,PAL,was determined.The results showed that the enzyme activity of C-4 solution was significantly higher than that of control,indicating that C-4 could induce systemic disease resistance.(4)The results of the test showed that the expression levels of the disease resistant defence genes NtLOX、NtPAL、PDF1.2、NtF6 genes in the leaves of strain C-4 were significantly up-regulated.Thus,the induced tobacco disease resistance was one of the mechanisms of strain C-4 to control tobacco anthracis.