Fungicides remain a critical tool used by farmers to limit crop losses due to fungal disease. One of the most widely used classes of fungicides are the demethylation inhibitors (DMIs) that target fungal C14-demethylase enzymes involved in the synthesis of the essential sterol ergosterol. For example, in Iowa, an estimated 12.7 million acres were planted to corn in 2022, with another 10.3 million acres planted to soybeans. The most recent surveys within Iowa show that DMI fungicides were applied to 22% of corn acreage during 2021 and to 16% of soybean acreage in 2020. Despite this broad usage, there is a growing regulatory threat that at its extreme could result in a ban on the use of DMI fungicides for crop protection. The basis of this threat is rooted in chemistry; namely, the DMI fungicides share an identical mode-of-action with the triazole class of human anti-fungal drugs. The latter represent the primary therapeutic approach for the treatment of invasive fungal infections of humans. These include infections caused by the cosmopolitan filamentous fungus Aspergillus fumigatus, which are responsible for an estimated 15,000 hospitalizations per year in the US and are a significant cause of mortality to transplant recipients and occupants of intensive care units. Moreover, many cases of “farmer’s lung” are likely attributable to infectious molds such as A. fumigatus. Recent studies focused primarily in Europe suggest that the application of DMI fungicides has inadvertently selected for triazole resistance amongst populations of A. fumigatus that reside in soil or plant debris. This link between fungicide usage and clinical triazole resistance has caused the CDC to place azole-resistant A. fumigatus on the watch list for its 2019 Antibiotic Resistance Threats Report. Of greater concern, the EPA has issued a Concept Note as a step towards implementing restrictions on the use of DMI fungicides so as to protect the efficacy of clinical triazoles. Any such restriction would have enormous implications for the ability Iowa’s soybean growers to manage crop disease while maintaining yields and profit margins

At this time, the extent to which triazole-resistance in A. fumigatus can be traced to fungicide use has not been thoroughly assessed in the United States. For example, despite the dominant agricultural landscape found throughout the US Midwest, no attempt has yet been made to systematically assess the potential threat posed by triazole-resistant A. fumigatus in this region. The lack of clinical reports from state and regional public health agencies further limits our ability to determine the extent of risk. To address this knowledge gap and ensure that potential regulatory considerations are data-driven, we aim to determine the abundance and distribution of triazole resistant isolates of A. fumigatus in Iowa agricultural settings. Samples of soil, crop residue, silage, and hay will be collected from a variety of locations that span the diversity of soil types and landscapes present in the state of Iowa. Locations will include research and demonstration farms that are owned or affiliated with the Iowa State University College of Agriculture, as well as private farm operations to which we have granted permission to access. The majority of these locations are devoted to rotating corn and soybean production or the sole production of corn. At each location, multiple sites where fungicides have been applied at least once over the past two growing seasons will be sampled along with untreated control sites. Standardized protocols will be used for the; (i) collection of samples and metadata at each site, (ii) plating and recovery of A. fumigatus isolates, (iii) testing of recovered isolates for azole resistance, and (iv) genomic analyses to determine the genetics basis of any observed resistance. Preliminary results from two ISU research farms have revealed the presence of A. fumigatus in corn residue and in silage, though it remains to be determined whether any of these isolates are azole resistant.

We anticipate two possible outcomes from our survey. If azole-resistant A. fumigatus is detected, we strongly suspect that it will be very localized with our results helping to guide potential approaches that limit farmer exposure to the pathogen (e.g., use of an N95 mask while handling large volumes of silage). Alternatively, failure to detect azole-resistant A. fumigatus would presumably provide sound evidence that blanket restrictions on the use of DMI fungicides are not necessary.

Updated May 30, 2024:
Our initial sampling efforts have focused on the following sites; the ISU NWRF, the ISU McNay farm, and from a private farm (location has been de-identified). Sampling was performed in Oct/Nov 2023 and March/April 2024. Material sampled includes soil, crop residue (both soybean and corn), and silage. All samples were processed as described in the proposal. From these samples, 173 putative isolates of Aspergillus fumigatus have been recovered. The majority of these came from silage and from soybean residue. Of these presumptively resistant isolates, 51 have been tentatively identified as azole resistant based on plate assays. The majority of these isolates (44/62) came from silage recovered from two different sites. Follow-up tests are underway to confirm azole resistance and to verify the identity of the isolates as A. fumigatus. In parallel, additional sampling is also underway.

Updated February 12, 2025:
Additional sampling efforts (June/July 2024) focused on silage, compost, and crop residue available on ISU farms and sites located near Ames as well as the NWRF and SERF. We were not able to access any wastewater sites during the project period, but plan to sample such sites in 2025. To date we now have >500 isolates tentatively classified as A. fumigatus based on growth characteristics and colony morphology. These isolates consistently are most predominant in silage samples. Preliminary screening using SAB plates supplemented with Tebuconzole identified ~80 isolates as possibly being azole resistant. However, subsequent tests using SAB plates supplemented with Itraconazole or Voriconazole revealed that only eight isolates exhibit resistance to all tested azoles. Additional quantitative testing using e-strips is currently underway in an attempt to confirm the qualitative plate tests. To save time and resources, DNA extractions for diagnostic confirmation of isolates as A. fumigatus were delayed until we could confirm azole resistance. These are now being done for the smaller subset that exhibit potential resistance. Overall, results from our sampling suggest that azole resistance is not highly prevalent amongst populations of A. fumigatus in Iowa. However, our sampling efforts have resulted in the identification of other fungi that are potential human pathogens and do display more prevalent resistance to azoles.

Our preliminary results based on one season of primarily sampling silage, compost, and crop residue suggest that although A. fumigatus is very prevalent as an environmental fungus, levels of azole resistance within populations of this fungus appear to be very low.

(1) Determining the extent to which azole-resistant Aspergillus fumigates might pose a threat to farmer health and well-being. (2) Providing sound scientific evidence that will help guide practical decisions regarding the use of DMI fungicides.