Fungicide drives de novo evolution of multidrug resistance in the plant growth promoting rhizobacterium, Pseudomonas fluorescens

Plant growth-promoting rhizobacteria (PGPR) are key members of soil microbial communities, supporting nutrient cycling, plant health, and productivity. In agricultural soils, these beneficial bacteria are often exposed to multiple stressors simultaneously, including fungicides, antibiotics, and rising temperatures. Despite their ecological importance, little is known about how PGPR respond evolutionarily to such combined stressors. Here, we investigated how the model PGPR Pseudomonas fluorescens SBW25 evolves resistance to the fungicide formulation Fubol Gold (metalaxyl-M + mancozeb), under ambient or warming soil conditions. Using a 16-week experimental evolution in soil microcosms with a fully factorial design (fungicide {+/-} warming), we assessed the evolution of fungicide resistance via phenotypic assays and whole-genome sequencing. Fungicide exposure rapidly selected for increased fungicide resistance, detectable as early as week 4, and co-selected for multidrug resistance, likely through mutations in a mexS ortholog that cause efflux pump overexpression. Warming did not alter the evolution of fungicide resistance; however, populations subjected to both fungicide and warming stress went extinct more rapidly, indicating that population evolutionary rescue was less effective under dual stress. Our findings show that fungicides alone can drive multidrug resistance in beneficial soil bacteria, suggesting that strategies to tackle AMR in agriculture should also consider non-antibiotic drivers of resistance.