Progress 02/15/24 to 02/14/25

Outputs
Target Audience:The target audience for this year of the project included the project team members from Ohio. Ohio team members included Melanie Lewis Ivey and post-doctoral scientists Raees Paul, Timothy Frey and Sudharsan Sadhasivam. Additional targetaudience included the scientific community through a presentation at a national and international meeting and a poster presentation at the USDA-NIFA project directors meeting. Crop producers and OSU Extension Educators were also target audience during this reporting period. Changes/Problems:A no-cost extension was requested and approved to allow for completion of the studies and publication of the results. Due to the high costsand complications with importing the Aspergenius kits, we decided to use amplicon sequencing to confirm the presence of target resistance genes. Amplicon sequencing will allow us to identify fungicide resistance to other classes at no additional cost. What opportunities for training and professional development has the project provided?Dr. Paul presented a poster at the 2024NIAMRRE Annual Conference, attended a training hosted by the OSU MCIC on analyzing NextGen sequencing data from fungal genomes, andpresented research results toThe OhioState University Division of Infectious Diseases - College of Medicine. Dr. Paul mentored the post-docotoral fellows who joined the project in autumn 2024. How have the results been disseminated to communities of interest?Research results were presented to the scientific community at the2024NIAMRRE Annual Conference andto theThe OhioState University Division of Infectious Diseases - College of Medicine. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period the following activities will be conducted: 1. Complete amplicon sequencing and data analysis (Objective 1). 2. Collect additional samples from urban environments, process samples for resistance, and analyze data (Objective 1). 3. Write and submit manuscriptfor Objective 1. 4. Complete sample processing, resistance screening, and data analysis for strawberry field samples (Objective 2). 5. Write manuscript for Objective 2. 6. Submit an article to Scientia summarizing ARAf in the environment (Objective 3). 7. Complete ARAf dashboard build and make it public (Objective 3). 8. Complete and submitARAf review article to Open Forum Infectious Diseases (Objective 3) 9. Write white paper on azole stewardship in the environment (agricluture, urban, and naturalized).

Impacts
What was accomplished under these goals? Objective 1 (Research)We sampled 62 sites across Ohio including agricultural settings (n=38; 61.46%), naturalized environments (n= 15; 23.93%) and urban settings (n= 9; 14.61%), for presence of azole-resistant A. fumigatus (ARAf). Agricultural environments includedvineyards (n = 10), vegetable farms (n = 10), flower nurseries (n = 8), apple farms (n = 7), corn and soybean fields (n = 3), and berry farms (n = 2). Naturalized environments included: Deciduous woodlands (n = 8), Prairies (n = 3), Lake beach (n = 2), Public Park (n = 1) and Wildlife reserve (n = 1). Urban environments includedgolf clubs (n = 4), sports fields (n = 3), city public park (n = 1), and residential lawns (n = 1). Replicate air (n=342), soil (n=311), and compost (n=32) samples were collected from the study environments. ARAfwas detected in air, soil and compost samples using culture-based and non-culture-based methods, as detailed in the previous progress report. A descriptive analysis was conducted on 397 air samples, including 55 air samples from 2023. Among these samples, 141 (35.52%) carried wild-type cyp51A (WT), 71 (17.88%) only a TR genotype, 37 (9.32%)both WT and TR genotypes, and 148 (37.28%) were negative for A. fumigatus DNA. The distribution of cyp51A genotypes among the environmental categories did not differ(Pearson's Chi-Square p-value = 0.69). Similarly, no significant differences in cyp51A genotype distribution were observed among environmental subcategories (Pearson's Chi-Square p-value = 0.26). Out of 43 agricultural environments sampled (including five sites from the earlier study period), 34 (79.1%) had a history of demethylase inhibitor (DMI) fungicide use in their spray program during the current or previous growing season. The distribution of air samples from these sites with DMI exposure included propiconazole (32.93%), myclobutanil (23.95%), mefentrifluconazole (13.17%), triflumizole (11.98%), tetraconazole (11.98%), difenoconazole (2.99%), and tebuconazole (2.99%). There were no significant differences in the distribution of cyp51A genotypes with the use of DMI (p= 0.79) or generic DMI used (p= 0.26). A. fumigatus culture positivity in air samples was noted at 18.12% while nested PCR showed presence of A. fumigatus DNA in 62.72% of samples. The sensitivity of culture to capture ARAf from air samples was low at 0.76% while nested PCR showed ARAf presence in 27.2% of samples. Binary logistic regression analysis showed TR presence in air samples was independent of A. fumigatus culture-positivity [OR (95%C.I.); 1.41(0.5-3.7), p=0.47], ARAf culture-positivity (p=0.99), environmental category [1.1(0.71-1.67), p=0.68], environmental subcategory [1.1(0.97-1.26), p=0.12] or DMI use [1.14(0.94-1.36), p=0.174]. 352 replicate soil and 42 in-house compost samples (collected from all except four sites) grew A. fumigatus in 41.4% (163/394) of samples of which 9.2% (15/163) samples from six sites were positive for ARAf using the replica plate method. A. fumigatus culture positivity was higher in compost samples compared to soil samples (83.3% vs 36.4%, P<0.01) and statistically non-significant among environmental categories (P=0.8). The ARAf positive samples included nine compost samples and six soil samples. The ARAf positive soil sample sites included a strawberry farm (3/5 samples ARAf positive), a flower farm (3/5 samples ARAf), a golf course (6/14 samples ARAf), an apple orchard (1/8 ARAf), a city public park (1/5 ARAf), and a wildlife area (1/5 ARAf). ARAf were significantly isolated more from compost samples than soil samples (21.4%) % vs 1.7%, P<0.01). The presence of ARAf was more common in agricultural (2.8%) and urban (9.5%) environments compared to naturalized settings (1.4%), though the difference was statistically non-significant (P=0.06). The exposure and no-exposure to DMI was recorded in 5.3% and 1.3% of ARAf samples (P=0.096). To improve diagnostic rigor and develop a novel modality for detecting A. fumigatus cyp51A TR mutations directly from DNA extracts, we are optimizing a targeted amplicon sequencing approach as mentioned previously. For this purpose, we prepared 18 mock DNA mixtures containing defined proportions of WT, TR34/L98H, and TR46/Y121F/T289A A. fumigatus culture DNAs. Additionally, we included DNA extracts from 4 air samples, 3 soil samples, and 4 compost samples. We amplified the 235 bp cyp51A promoter region using cyp51A-specific primers ligated to Illumina adapters at 5'end. Amplicon library preparation involved steps: bead cleaning, indexing, gel electrophoresis, additional bead cleaning, pooling, and quality control checks. Sequencing was conducted on the Illumina NextSeq1000 platform with PE P1 chemistry. The paired-end FASTQ files were quality-checked using FASTQC, and adapter sequences were trimmed with Cutadapt. A customized R script was developed using BioStrings package of the Bioconductor project to calculate the relative proportions of WT, TR34 bp, TR46 bp, and TR53 bp sequencing reads in each sample. We are facing a technical challenge in sequencing the amplicons from mock A. fumigatus DNA mixture samples as the quality of the reads were suboptimal due to low-diversity library in those samples. We are still working on optimizing the library for mock DNA samples. However, optimal read quality was noted in amplicons from environmental samples. Of the 4 air samples, one sample had presence of WT, TR34 and TR46 reads in the proportions: 80%, 7.5%, 10.5%, respectively, while 2% of the reads were unclassified. The remaining three samples had presence of only WT reads with unclassified read proportions as 1.5%, 1.4%, and 1.25%. Among the 7 soil samples/compost samples, four samples had WT reads only with minor fractions of unclassified reads. 3 samples had mixed presence of WT, TR34 and TR46 reads in the proportions of 76.5%, 11.8%, and 9.4%; 32.2%, 48.4, and 19.1%; 81.3%, 8.2%, and 9.7%, respectively. The remaining fractions in each sample were unclassified reads. Objective 2 (Research)A replicated field trial on strawberry was repeated to determine if propiconazole used for fungal disease control exerts selection pressure for ARAf. Soil samples were collected before and after the fungicide applications. Fruit were sampled twice. TReplica plating was used to screen for resistance. In general, there was a reduction in A. fumigatus CFU in treatment rows that received 3 exposures (T1) compared to those that received 0 (T3) and 1 (T2) applications. A. fumigatus burden in the soil was highest at the baseline time point, and lowest at TP4 across all treatment-defined rows, though no particular trend in A. fumigatus burden was noted in the intervening timepoints. Three putative ARAf were isolated from one soil sample from the T1 row in one among 6 replicate blocks and 1 ARAf isolate was isolated from T3 row in one among 6 replicate blocks. Plant debris from snapbean and strawberry from each treatmentwas heaped into cull piles and samples were collected at 0-, 3- and 6-month time points. Duplicate samples from each treatment were analyzed for presence of A. fumigatus and ARAf using the replica plate method. At 6 months, strawberry compost from T1 and T3 treatments yielded confluent growth of A. fumigatus but ARAf was not recovered. Samples from T2 treatment rows had low A. fumigatus counts. Snapbean compost from T1 and T2 treatment were positive for A. fumigatus growth, but no ARAf were detected in those samples. A. fumigatus was not recovered from the strawberry fruit samples. Objective 3 (Extension)The mobile application AspTrack was developed using Clappia (www.clappia.com/en-us) to collect meta data from field sample collection in real-time. Clappia was used to build a temporary dashboard. Digital source data was transferred to Tableau, which will host the public facing dashboard. Additional collaborators were recruited to contribute to the ARAf review article and a first draft was completed.

Publications

• Type: Other Status: Published Year Published: 2023 Citation: Paul, R., Miller, S.A., Paul, P. and Lewis Ivey, M.L. (2024) Identifying the environmental niches and mechanism of azole-resistant Aspergillus fumigatus. [Poster]. NIAMRRE Annual Conference, Columbus, OH.

Progress 02/15/23 to 02/14/24

Outputs
Target Audience:The target audience for the first year of the project included the project team members from Ohio and Oregon. Ohio team members included Drs. Pierce Paul, Sally Miller, and Melanie Lewis Ivey, post doctoral scientisit Dr. Raees Paul, and MS graduate student Doyeon Park. Oregon team members include Drs. Walt Mahaffee and Virginia Stockwell. Additional target audience included the scientific community through a presentation at an international meeting and the publication of a manuscript. Crop producers, IPM consultants and managers, andOSU Extension Educators were also target audience during this reporting period. Changes/Problems:There is no optimal method yet for detecting ARAf directly from sample DNA extracts, especially in samples containing both ARAf and non-ARAf spores. The mixed population of ARAf and non-ARAf spores in a sample may confound the detection of ARAf samples due to stochastic inhibition. AsperGenius multiplex qPCR kit though claimed to perform optimally on patient samples where often the patient is often infected with either an ARAf or non-ARAf A. fumigatus population, doesn't seem to work optimally in environmental samples that often have a mixed population of A. fumigatus spores. Therefore, we started to develop and optimize an amplicon sequencing-based method to determine the relative proportions of ARAf and wild type spores in DNA extracts. We expect this technique to be superior to the AsperGenius kit for detecting TR mutations in environmental DNA samples. Some of the funds budgeted for theAsperGenius multiplex qPCR kit will be diverted to amplicon sequencing. The graduate student on the project resigned in December 2023 and the contract of the post doc on the project ends in September 2024. Therefore, recruitment for a post doc to replace the graduate student and post was initiated. Co-PI Miller retired December 31, 2023. Dr. Miller will continue to advise on the project as Professor Emeritus. What opportunities for training and professional development has the project provided?The graduate student (D. Park) submitted an abstract to APS 2023 (Denvor, CO) and presented a poster on the resesarch pertaining to objective 2. She also presented her research at a department symposium for plant health students. Dr. R. Paul (post doc) attended a workshop on amplicon sequencing and bioinformatics hosted by the Molecular Cellular and Imaging Center (The Ohio State University). How have the results been disseminated to communities of interest?I was an invited speaker at the International Congress of Plant Pathology, Lyon, France. Research results were presented. Poster presentations were made at the annual APS meeting and internally at the department of plant pathology graduate student association. A one page project summary targeting crop producers, IPM consultants and managers, and Extension Educators was written to assist with recruiting locations to sample. What do you plan to do during the next reporting period to accomplish the goals?During the second year of the project the following activies are planned: 1. Continue tooptimizean amplicon sequencing-based method to determine the relative proportion of ARAfand wild-type (WT) spores in the sample DNA extracts. (Objective 1) 2. Apply treatments, collect and process samples from the 2024 strawberry field trial.(Objective 2) 3. Finish processing strawberry samples collected in 2023.(Objective 2) 4. Collect air samples from environments across Ohio.(Objective 1) 5. Write and submit a review articleon fungicide stewardship for mitigatingARAf resistance.(Objective 3) 6. Hire a post doc to replace current post doc.(Objective 1 and 2) 7. Present results at National Institute for Antimicrobial Resistance Research and Education(Objective 3) 8. Prepare and submit third year report and request no cost extension (Project management) 9. Attend required project meeting in California (Project management)

Impacts
What was accomplished under these goals? Determine the prevalence and mechanisms of azole resistant Aspergillus fumigatus in agricultural, urban, commercial, and naturalized systems. (Research) A mobileapplication for internal use was developed to collect meta data associated with sample colelction.The data collected through the mobile app can be transferred directly to a spreadsheet, whihc will then be used for development of the dashboard. We surveyed 12 diverse sites and collected 108 samples, including 63 air samples, 37 soil samples, and 8 compost samples from environments such as food crop sites, an arboretum, a flower farm, a nursery, a golf club, and a commercial composting plant. Azole-resistant A. fumigatus (ARAf) was identified in air and soil samples using non-culture and culture-based approaches. In the non-culture-based method, DNA extracts from air and soil samples were directly probed for the presence of ARAf associated cyp51A TR signature mutations using a nested PCR assay and the AsperGenius® real-time PCR multiplex assay in a subset of samples. The culture-based method involved growing suspensions of samples on a propiconazole-amended Sabouraud dextrose agar (SDA) replica plate method. This propiconazole-SDA replica method was optimized to screen samples for ARAf presence, using sterile soil samples spiked with serial dilutions of spore suspensions of control A. fumigatus isolates with TR34/L98H (AR0733), TR46/Y121F/T289A (AF-385) mutations, and WT cyp51A (AR0740). This assay had a limit of detection (LOD) of 1 resistant conidium/mL of soil suspension. Putative ARAf colonies were confirmed according to the Clinical Laboratory Standards Institute reference broth microdilution method (M38) and approved clinical cut-off values for azole resistance. The resistant isolates were analyzed for cyp51A tandem repeat (TR) signatures (TR34 and TR46) through PCR and sequencing of the upstream 235 promoter sequence. We identified ARAf in 12% (13/108) of the samples, corresponding to 50% (6/12) of the surveyed environments. Evaluation of ARAf directly from the sample DNA extracts of 55 air samples revealed the presence of A. fumigatus in 61.8% and TR ARAf signature mutations in 7.3% of the air samples. The AsperGenius Resistance kit detected cyp51A resistance markers only in samples with confluent growth on replica resistance plate screening. Soil or compost samples from 7 out of 12 sites were tested for ARAf presence using a breakpoint concentration of propiconazole (5 mg/L), determined based on broth microdilution propiconazole sensitivity testing, which showed an upper propiconazole minimum inhibitory concentration (MIC) of 4 mg/L for non-ARAf isolates and MIC > 4 for ARAf isolates. Soil samples from 5 sites were found to harbor ARAf. Of the 8 compost samples collected from the golf club (n = 7) and an apple orchard (n = 1), 7 tested positive for ARAf, with both TR34 and TR46 mutations present, though TR46 was predominantly found. Three sites had records of DMI fungicide use in their fungal pest management protocols: a golf club (Mefentrifluconazole, Tebuconazole, Propiconazole), an apple orchard (Mefentrifluconazole, Fenbuconazole), and a flower farm (Tetraconazole). In the spring of 2024, we surveyed 5 public parks in Ohio, collecting 26 air samples and 25 soil samples. Putative ARAf was detected in one public park's soil sample using the replica plate method, though sample DNA extracts have not yet been probed for TR mutations. We inititated a study tooptimiziean amplicon sequencing-based method to determine the relative proportion of ARAfand wild-type (WT) spores in the sample DNA extracts. This method is expected to be a superior diagnostic modality for detecting TR mutations in sample DNAs. Determine if agricultural azoles exert fungicide resistance selection pressure on Aspergillus fumigatus populations in vegetable and fruit production systems. (Research) Replicated field trials were conducted to determine if propiconazole used for disease control exerts selection pressure for ARAf in snap bean and strawberry fields. Propiconazole was applied 0, 1 or 3 times at labelled rates. Soil and foliar samples were collected before and after the fungicide applications with the sampling timing corresponding to the plots that received three applications of propiconazole. Bean pods and strawberry fruit were sampled twice. Putative A. fumigatus was isolated from the bean samples and screened for resistance to the clinical azoles (itraconazole, voriconazole, posaconazole) using the EUCAST protocol and to propiconazole using a MIC bioassay (0-5 mg/L). None of the 61 isolates recovered were resistant to the clinical azoles. Three of the isolates, from pods, were resistant to propiconazole (MIC >5 mg/L) and the remaining isolates were sensitive to propiconazole at≥2 mg/L.Of the three resistant isolates, two were from the plots treated with three applications of propiconazole, and one was from a nontreated plot. Samples collected from the strawberry field trial were stored and resistance screening is in process. Plant debris from the strawberry and bean field was cut and compost piles were made. Samples were collected at 0,3, and 6 months and stored for processing. Provide a Knowledge Exchange to allow in-person and virtual information sharing by researchers, industry members, and government on antifungal resistance and fungicide stewardship. (Extension) A spreadsheet was designed to input data fromobjective 1 so that the data can be coded to develop a dashboard. Third party sources were investigated to assist with the dashboard development. A collaboration with the National Academy of Science and Univ. Georgia was established to write an review article on fungicide stewardship for mitigatingARAf resistance, an outline was written.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Jimenez Madrid, A.M., Paul, R.A., Rotondo, F., Deblais, L., Rajashekara, G., Miller, S.A., and Lewis Ivey, M.L. 2024. Triazole resistance in Aspergillus fumigatus isolated from a tomato production environment exposed to propiconazole. AEM. 90:e00017-24. https://doi.org/10.1128/aem.00017-24
• Type: Conference Papers and Presentations Status: Other Year Published: 2023 Citation: Park, D., Miller, S.A., Paul, P., Paul, R., and Lewis Ivey, M.L. (2023) The effect of propiconazole applications for disease control in snap beans and strawberry on clinical azole resistance development in Aspergillus fumigatus. [Abstract]. Annual Plant Pathology Spring Symposium, Wooster, OH
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Park, D., Miller, S.A., Paul, P., Paul, R., and Lewis Ivey, M.L. (2023). The effect of propiconazole applications for disease control in snap beans on clinical azole resistance development in Aspergillus fumigatus. [Abstract/Poster]. Phytopathology. 11:S3.1-S3.197
• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Lewis Ivey, M.L.; Paul, R.; Paul, P.; Miller S.A. (2023). The Hunt for Killer Aspergillus fumigatus in the environment - Surveillance of tomato and corn fields in Ohio. International Congress of Plant Pathology, Lyon, France

Progress 02/15/22 to 02/14/23

Outputs
Target Audience:The target audience for the first year of the project included the project team members from Ohio and Oregon. Ohio team members included Drs. Pierce Paul, Sally Miller, and Melanie Lewis Ivey, post doctoral scientist Dr. Raees Paul, and MS graduate student Doyeon Park. Oregon team members include Drs. Walt Mahaffee and Virginia Stockwell. Changes/Problems:Recruitment and hiring of a post doctoral scientist took nearly 9 months. This was due to a lack of acceptable candidates in the first round and hiring obstacles due to immigration procedures. As a result there was a delay in initiating activities proposed in Objective 1. Procurement of key reagents and supplies continues to be a problem. What opportunities for training and professional development has the project provided?Dr. Raees Paul travelled to Oregon State University and was trained on using the cyclone air sampler to collected spore samples from the air. All Ohio team members participated in equity and diversity training and Title IX training as required by Ohio State University. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?During the second year of the project the following activities are planned: Further characterize A. fumigatus isolates collected in Year 1 (Objective 1) Identify air sampling sites in Ohio and begin sampling (objective 1) Repeat the bean field study (objective 2) Apply treatments to the strawberry field and process samples (objective 2) Write and submit abstract for 2023 and 2024 ICPP Meeting (Objectives 1 and 2) Finalize advisory panel members and invite them to participate in the project (Project management) Prepare and submit second annual report(Project management) Assemble expert team and develop plan for systematic review (Obj. 3)

Impacts
What was accomplished under these goals? Determine the prevalence and mechanisms of azole resistantAspergillus fumigatusin agricultural, urban, commercial, and naturalized systems. (Research) A postdoctoral scientist was recruited and hired. Dr. Raees Paul started in October 2022. Dr. R. Paul traveled to Oregon on 12/4/2022 and had an in-person meeting with Dr Walt Mahaffee and Dr. Virginia Stockwell from 12/5/22 to 12/6/22 to discuss the AFRI proposal, the challenges, and the feasibility of surveilling agricultural settings for azole-resistant A. fumigatus (ARAF), molecular detection methods, etc. using the vacuum-assisted, hand-held, cyclonic air-sampler spore sampling. To optimize sampling using the vacuum-assisted, hand-held, cyclonic air-sampler spore sampling for azole-resistant A. fumigatus (ARAf) was performed in a commercial open-windrow composting facility in OARDC. Spore samples were collected three times (11/23/22, 12/1/22, and 2/13/23). At the first sampling time the compost piles were not disturbed. For the second and third sampling times the piles were disturbed to create a dust plume. A total of 55 A. fumigatus isolates were recovered from the spore samples. A. fumigatus isolates from the first two sample sets (n=17 and n=11 respectively) were screened for ARAf on 4-well RPMI-1640 agar amended with clinical breakpoint concentrations of itraconazole (4µg/mL), voriconazole (2 µg/mL) and posaconazole (0.5 µg/mL). Isolates from the third sample set (n=27) were pre-screened for resistance by plating on SDA and SDA amended with 3 mg/L propiconazole. No ARAf isolates were recovered from the first sampling. Five isolates were recovered from the second sample set and the minimum inhibitory concentration of triazoles against these isolates were determined using broth microdilution method. Four isolates exhibited higher MIC for itraconazole (>16 µg/mL), voriconazole (1-2 µg/mL) and posaconazole (0.5 µg/mL), while one isolate reduced azole- sensitivity but below the breakpoint concentration (itraconazole, 1; voriconazole, 2; and posaconazole 0.25). Four of the 5 ARAf isolates harbored TR mutation as determined by amplification of the 100 bp cyp51A promoter element (Spiess et al, 2012). From the third sampling, two isolates had a resistance phenotype and further characterization of these isolates is ongoing. All the A. fumigatus isolates were preserved in silica gel at -80. Determine if agricultural azoles exert fungicide resistance selection pressure onAspergillus fumigatuspopulations in vegetable and fruit production systems. (Research) A summer undergraduate student was hired to assist with the field trials and a graduate student was enrolled to conduct the research proposed for objective 2. Isolation of Aspergillus fumigatus from environmental samples such as soil and plant tissue is challenging due to the more competitive growth of mucormycetes fungi. Sabouraud dextrose agar (SDA) and Flamingo medium (FM) were evaluated for selective isolation of A. fumigatus and inhibition of mucormycetes fungi at mesophilic and thermophilic temperatures. Sabourauddextrose agar (SDA), Flamingo medium (FM),and Flamingomediumwithoutdichloran(FM-D) wereevaluated for their specificity toA. fumigatusgrowth at three different temperatures (35°C ,37°C and 43°C).Flamingomediumwithoutdichloran with incubation at 43°Cwas the most efficient growth temperatures for the selection of A. fumigatus from organic samples. A snap bean (bush type; cv.Tender Green Improved Bush bean) field was established at The Ohio State University CFAES-Wooster (Wooster, OH) on Wooster silt loam soil in 2022. Each plot consisted of three treatments: T1 propiconazole (295 ml/HA) applied three times at 14-day intervals beginning at flowering; T2 propiconazole (295 ml/HA) applied once at flowering and; T3 non-treated control. Soil and foliar samples (n=5) were randomly collected from each plot prior to the first fungicide treatment and every 14 days thereafter. Snap beans were sampled three times once the beans were mature. Putative A. fumigatus was isolated from the samples and the isolates (n=61) were purified and screened for resistance to the clinical triazoles (itraconazole, voriconazole, and posaconazole) using the EUCAST protocol. In addition, the isolates were screened for resistance to propiconazole using a MIC bioassay. None of the isolates were resistant to the clinical triazoles. XX isolates were resistant to propiconazole at X mg/L. An annual production strawberry field was planted from bareroot Earliglow plants at The Ohio State University CFAES-Wooster (Wooster, OH) on Wooster silt loam soil in 2022. Each plot consisted of seven rows (3 treatment and 4 guard rows) and were replicated six times. Flowers were removed from the plants during bloom to promote vegetative growth only. Plots were hand weeded and straw was applied to the rows in the fall for winter protection. A composite straw sample for each replicated plot was collected prior to applying the straw and putative A. fumigatus was isolated from the samples and the isolates (n=5) were purified and screened for resistance to the clinical triazoles (itraconazole, voriconazole, and posaconazole) using the EUCAST protocol. In addition, the isolates were screened for resistance to propiconazole using a MIC bioassay. None of the isolates were resistant to the clinical triazoles. XX isolates were resistant to propiconazole at X mg/L. Fungicide treatments will be applied in 2023. Provide a Knowledge Exchange to allow in-person and virtual information sharing by researchers, industry members, and government on antifungal resistance and fungicide stewardship.(Extension) Nothing to report. Project Management Quarterly team meetings were scheduled and the first was held in October 2022 and the second in February 2023. A list of candidates for the Advisory Panel was compiled. Drs. Ivey and Paul have mentored the graduate student during this time. Dr. Ivey recruited and hired and supervised the postdoctoral scientist and undergraduate student.?

Publications