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 disease1,2, 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, cyp51A.5,6 In the Netherlands7,8, most triazole resistance arises from two cyp51A haplotypes, the TR34 and TR46.9,10 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/728463v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@259848org.highwire.dtl.DTLVardef@30bfdeorg.highwire.dtl.DTLVardef@84bb35org.highwire.dtl.DTLVardef@1eb39d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LITriazole-resistance alleles often co-occur with resistance alleles to non-DMI fungicide classes such as SDHI and QoI. C_LIO_LIDMI, SDHI, and QoI all independently increase the proportions of triazole resistant alleles at low concentrations (0.1 mg/kg) commonly found in agricultural residues. C_LIO_LIThe DMI resistance allele TR46 has a high fitness only under fungicide selective conditions, whereas TR34 maintains its fitness also under fungicide free conditions. C_LIO_LIBeneficial alleles for one fungicide class increase in frequency and hitch-hike with resistance alleles to other fungicide classes. C_LIO_LIResistance alleles to novel fungicide classes will more likely be selected in isolates that already accumulated other fungicide resistance alleles. C_LI