Fungicide Drives De Novo Evolution of Multidrug Resistance in the Plant Growth Promoting Rhizobacterium, Pseudomonas fluorescens.

Soil microbial communities underpin key ecosystem processes, including plant health, nutrient cycling and primary productivity. In agricultural soils, beneficial soil bacteria are often exposed to multiple stressors simultaneously, including fungicides, antibiotics, and warming temperatures. Despite their ecological importance, little is known about how beneficial soil bacteria respond to such combined stressors over evolutionary timescales. Here, we investigated how the model plant growth promoting rhizobacterium (PGPR) Pseudomonas fluorescens evolves resistance to the fungicide formulation Fubol Gold (metalaxyl-M + mancozeb), under ambient or warming soil conditions. Using a 16-week evolution experiment 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 resistance to chloramphenicol, sulphatriad and nalidixic acid antibiotics, likely through mutations in a mexS ortholog that may be associated with 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, so that population evolutionary rescue was less effective under dual stress. Our findings show that fungicide alone can drive multidrug resistance in beneficial soil bacteria, suggesting that non-antibiotic drivers of resistance in the environment should be incorporated into One Health frameworks for tackling AMR.