Non-triazole agricultural fungicides indirectly select for triazole resistance in the human pathogen Aspergillus fumigatus

The intensification of agriculture relies on chemical fungicides to manage crop disease ^1,2 , leading to the evolution of resistance in plant pathogens. ³ Fungicides have long half-lives, allowing them to remain active well beyond their intended targets and affect downstream ecosystems and agricultural practices. The saprophytic fungus Aspergillus fumigatus is an airborne ubiquitous fungus and an important human pathogen causing severe life-threatening invasive fungal disease. ⁴ Selection pressure from agricultural triazoles, demethylase inhibitors (DMIs), has led to cross-resistance to clinical triazoles, as they share the same target gene, cyp 51A. ^5,6 In the Netherlands ^7,8 , most triazole resistance arises from two cyp 51A haplotypes, the TR ₃₄ and TR ₄₆ . ^9,10 Genomic surveys of A. fumigatus have shown that these triazole-resistance alleles often co-occur with resistance alleles to non-DMI classes, these include some of the dominant fungicide classes used in Europe such as quinone outside inhibitors (QoIs), and succinate dehydrogenase inhibitors (SDHIs). ¹¹ Hypothesizing that agricultural environments with non-DMI fungicides can indirectly select for triazole resistance, we used grass mesocosms to compete A. fumigatus isolates. We found that already at low concentrations, commonly found in agricultural residues, DMI, SDHI, and QoI fungicides can each independently increase the proportions of triazole resistant alleles. We show that the resistance alleles for each class are not intrinsically cross-resistant, indicating that their co-occurrences in allelic combinations produce this multi-resistance selection. Consistent with our mesocosms results, environmental samples contained high phenotypic (>20%) triazole resistance in heaps with only non-DMI fungicides. This work provides the first experimental and field evidence of selection for triazole resistance by non-triazole fungicides via genomic hitch-hiking. We thus predict that when novel fungicides are used in the same selective environment, novel resistance alleles will most likely be selected in isolates that have already accumulated resistance alleles to other fungicide classes. Because of linked resistance alleles, tackling selection and spread of environmental triazole resistance will require consideration of all fungicide classes.