Rhizoxin and 2,4-diacetylphloroglucinol Contribute to Biocontrol of Pseudomonas protegens Pf-5 Against Pea Ascochyta Blight Pathogen Didymella pinodes

Biological control using beneficial bacteria is a promising strategy for managing pea Ascochyta blight (AB), yet the underlying mechanisms remain poorly understood. In this study, we identified ten bacterial strains from four genera, including Bacillus, Paenibacillus, Peribacillus, and Pseudomonas, that significantly reduced the severity of AB caused by Didymella pinodes under greenhouse conditions. Most strains inhibited D. pinodes in vitro, suggesting antibiosis as a primary mode of action. To further elucidate the biocontrol mechanisms, we used Pseudomonas protegens Pf-5, which produces eight known antimicrobial compounds, as a model. While wild-type Pf-5 strongly inhibited D. pinodes in cultures and controlled AB in planta, a derivative ({Delta}8-fold mutant) lacking all eight compounds showed significantly compromised biocontrol efficacy. Individual complementation of biosynthetic genes for rhizoxin, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, or hydrogen cyanide partially restored inhibitory activity, confirming their roles in inhibition of D. pinodes. Notably, restoring rhizoxin and DAPG biosynthesis recovered the disease control capability of the {Delta}8-fold mutant in greenhouse trials. These results demonstrate that rhizoxin and DAPG are key metabolites driving the biocontrol activity of P. protegens against D. pinodes. SIGNIFICANCEAn advanced understanding of how beneficial bacteria control plant diseases can help us better use these microorganisms in agriculture. In this study, beneficial bacteria isolated from pea roots and soils effectively mitigated damages of pea Ascochyta blight caused by the fungal pathogen Didymella pinodes. Most of the identified beneficial bacteria inhibited the fungal pathogen in cultures, indicating antimicrobial compounds were likely produced by the bacteria to control the disease. Using the soil beneficial bacterium Pseudomonas protegens Pf-5 as a model, we demonstrated that four bacteria-derived antimicrobial compounds, rhizoxin and 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, and hydrogen cyanide play important roles in inhibiting D. pinodes growth. This study also showed that rhizoxin and DAPG produced by Pf-5 contribute to the suppression of AB development. These findings provided new insights into the molecular basis of beneficial bacteria-mediated disease suppression of pea Ascochyta blight.