Comparative Effects of Microbial Treatments Applied Before or After Pathogen Inoculation on Bacterial Wilt Development in Tomato

Bacterial wilt, caused by Ralstonia pseudosolanacearum, is a destructive soil-borne disease of tomato (Solanum lycopersicum) that results in severe agricultural and economic losses worldwide. Biological control offers a promising strategy to mitigate disease incidence and yield loss. Although fungal and bacterial biocontrol agents have been studied individually, their combined effects and the influence of inoculation timing remain poorly understood. This study evaluated the biocontrol potential of three isolates—Aspergillus japonicus, Aspergillus awamori, and Bacillus subtilis—applied either before (pre-treatment) or after (post-treatment) pathogen inoculation. In vitro, B. subtilis formed inhibition zones, while Aspergillus species overgrew and suppressed R. pseudosolanacearum colonies. In vivo experiments used a hybrid soil–hydroponic pot system to evaluate microbial effects while minimizing interference from soil microbiota. Pre-treatment with A. awamori and A. japonicus, individually or in combination, completely suppressed wilt symptoms and enhanced plant growth, achieving 0% disease incidence and severity. Under post-treatment conditions, A. awamori and B. subtilis, individually or in combination, reduced disease severity to 25% and incidence to 12% or less. Both pre-treatment and post-treatment strategies were associated with elevated levels of defense-related enzymes (PAL, POD, PPO), suggesting enhanced defense responses. Pre-treatment was associated with lower leaf H 2 O 2 accumulation than the Rs-only control, whereas post-treatment showed similar or slightly higher levels. These findings indicate that preventive microbial treatment is more effective than post-infection treatment and that both individual isolates and microbial combinations can contribute to plant defense. This study highlights the importance of applying microbial treatments at optimal times to suppress bacterial wilt in tomato.