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 10 bacterial strains from 4 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 cultures, indicating a production of antimicrobial metabolite(s) by these bacteria. To identify the antimicrobial compounds responsible for inhibition of D. pinodes and investigate 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 (Δ8-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 Δ8-fold mutant in greenhouse trials. These results demonstrate that rhizoxin and DAPG are key metabolites driving the biocontrol activity of P. protegens Pf-5 against D. pinodes .