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 Z. tritici antagonism assay. In addition to high-throughput qualitative assessment of Pseudomonas antagonism of Z. tritici, a quantitative assessment identified variation in the sensitivity of Z. tritici isolates to antagonism by a subset of Pseudomonas isolates. Genome assemblies of 3 strongly antagonistic Pseudomonas isolates were found to contain a predicted Biosynthetic Gene Cluster (BGC) with high sequence similarity to a reference BGC encoding the biosynthesis of the known antifungal compound 2,4-diacetylphloroglucinol (2,4-DAPG). Mutagenesis of the core biosynthetic gene phlD resulted in a loss of 2,4-DAPG production in Pseudomonas isolate Roth82, and a loss of Z. tritici inhibition in the antagonism assay. These results demonstrate that the described 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 find significant differences in the response of genetically diverse isolates of Z. tritici isolates to bacterial antagonists, suggesting sensitivity to bacterial antagonism exists as a quantitative trait within natural Z. tritici populations. Our approach can be used to identify and characterise putatively novel BGCs that encode natural products with antifungal activity against Z. tritici.