High-throughput Screening of Antagonistic Bacteria against Tobacco Bacterial Wilt and Biocontrol Effects of Compound Microbial Agents

It is crucial to construct high-efficiency compound microbial agents for the biological control of tobacco bacterial wilt. Using the tobacco variety Yanyan-97 with high resistance to tobacco bacterial wilt pathogen, Ralstonia. solanacearum, a high-throughput screening of tobacco rhizo-bacteria was performed. R. solanacearum chemotactic antagonistic bacteria to rhizo-bacteria were screened by plate confrontation assay and chemotaxis gene PCR detection. The antagonistic single strains were selected by heuristic algorithms and combined to form biocontrol microbial consortia. The efficacy of consortia on disease resistance with susceptible variety of Honghuadajinyuan was validated by pot experiments. Fluorescence quantitative PCR was utilized to detect R. solanacearum quantities in the rhizosphere soil. Biolog carbon chip technology were used to analyze the impact of the assembled microbial consortia on tobacco rhizosphere microbiome. In total, 109 antagonistic bacteria were screened. The inhibition zone diameter of 25 strains was over 20 mm. Three mutually compatible antagonistic strains possessing chemotaxis genes (CheA) and biofilm formation ability were selected and combined to synthesize the compound microbial consortia (Y364+Y832+Y878). Y364, Y832 and Y878 were identified as Pseudomonas nicosulfuronedens, Pseudomonas delhiensis, and Bacillus altitudinis, respectively by 16SrDNA gene sequencing. Ultimately, pot experiments showed that the disease control efficacy of compound microbial agent treatment was 79.75% and 36.22 percentage points higher than that of individual strain treatment. The disease incidence of compound microbial agent treatment was 33.33, 45.68 and 35.80 percentage points lower than that of single Y364, Y832 and Y878 treatments, respectively. It also decreased the population of R. solanacearum and increased rhizosphere microbial functional diversity in rhizosphere soil. In conclusion, the composite consortium possesses potential synergistic effects and rhizosphere colonization ability, providing more biocontrol resources for the biological control of tobacco bacterial wilt.