Development of a bioassay-guided genome mining approach for antifungal natural product discovery from pseudomonads.

Zymoseptoria tritici causes Septoria Leaf Blotch disease of wheat and has evolved to overcome most chemical and genetic control methods. As such, new tools are required for future disease control. We identified Pseudomonas isolates that antagonise Z. tritici through the production of secreted secondary metabolites, using a novel in vitro antagonism assay. Quantitative assessment identified variation in sensitivity of Z. tritici isolates to antagonism by Pseudomonas isolates. Genome assemblies of 3 strongly antagonistic Pseudomonas isolates contain a predicted Biosynthetic Gene Cluster (BGC) with high similarity to a reference BGC encoding the biosynthesis of known antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG). Mutagenesis of the core biosynthetic gene phlD resulted in loss of 2,4-DAPG production in Pseudomonas isolate Roth82, and loss of Z. tritici inhibition in vitro. These results demonstrate that our in vitro antagonism assay can be used to identify, quantify, and mechanistically characterise bacterial antagonism of Z. tritici through the production of secondary metabolites. This is the first study to putatively identify that resistance to bacterial antagonism exists as a quantitative trait within natural Z. tritici populations. Our approach of in vitro phenotype-guided genome mining can also prioritise candidate BGCs for discovery of antifungal natural products effective against Z. tritici.