Selection and Amplification of Fungicide Resistance in Aspergillus fumigatus in Relation to DMI Fungicide Use in Agronomic Settings: Hotspots versus Coldspots

Aspergillus fumigatus is a ubiquitous saprophytic fungus. Inhalation of A. fumigatus spores can lead to Invasive Aspergillosis (IA) in people with weakened immune systems. The use of triazole antifungals with the demethylation inhibitor (DMI) mode of action to treat IA is being hampered by the spread of DMI-resistant “ARAf” (azole-resistant Aspergillus fumigatus) genotypes. DMIs are also used in the environment, for example, as fungicides to protect yield and quality in agronomic settings, which may lead to exposure of A. fumigatus to DMI residues. An agronomic setting can be a “hotspot” for ARAf if it provides a suitable substrate and favourable conditions for the growth of A. fumigatus in the presence of DMI fungicides at concentrations capable of selecting ARAf genotypes at the expense of the susceptible wild-type, followed by the release of predominantly resistant spores. Agronomic settings that do not provide these conditions are considered “coldspots". Identifying and mitigating hotspots will be key to securing the agronomic use of DMIs without compromising their use in medicine. We provide a review of studies of the prevalence of ARAf in various agronomic settings and discuss the mitigation options for confirmed hotspots, particularly those relating to the management of crop waste.

Molecular Characterization of Laboratory Mutants of Fusarium oxysporum f. sp. niveum Resistant to Prothioconazole, a Demethylation Inhibitor (DMI) Fungicide

Fusarium oxysporum f. sp. niveum (FON) is the causal agent of Fusarium wilt in watermelon, an international growth-limiting pathogen of watermelon cultivation. A single demethylation inhibitor (DMI) fungicide, prothioconazole, is registered to control this pathogen, so the risk of resistance arising in the field is high. To determine and predict the mechanism by which FON could develop resistance to prothioconazole, FON isolates were mutagenized using UV irradiation and subsequent fungicide exposure to create artificially resistant mutants. Isolates were then put into three groups based on the EC50 values: sensitive, intermediately resistant, and highly resistant. The mean EC50 values were 4.98 µg/mL for the sensitive, 31.77 µg/mL for the intermediately resistant, and 108.33 µg/mL for the highly resistant isolates. Isolates were then sequenced and analyzed for differences in both the coding and promoter regions. Two mutations were found that conferred amino acid changes in the target gene, CYP51A, in both intermediately and highly resistant mutants. An expression analysis for the gene CYP51A also showed a significant increase in the expression of the highly resistant mutants compared to the sensitive controls. In this study, we were able to identify two potential mechanisms of resistance to the DMI fungicide prothioconazole in FON isolates: gene overexpression and multiple point mutations. This research should expedite growers’ and researchers’ ability to detect and manage fungicide-resistant phytopathogens.

Investigating the sensitivity of Venturia inaequalis isolates to difenoconazole and pyraclostrobin in apple orchards in China

The resistance level of 90 single lesion conidial isolates of Venturia inaequalis collected from multiple commercial orchards in Shaanxi and Gansu Provinces and Xinjiang Autonomous Region of China to the demethylation inhibitor (DMI) fungicide difenoconazole and quinone outside inhibitor (QoI) fungicide pyraclostrobin was examined. The EC50 values of the 90 isolates to difenoconazole and pyraclostrobin ranged from 0.143 to 6.735 μg/mL and 0.084 to 2.026 μg/mL, respectively. Among the isolates, 19 had resistance, 66 had reduced sensitivity, and five had sensitivity to difenoconazole; eight had resistance, 81 had reduced sensitivity, and one had sensitivity to pyraclostrobin. Although a weak correlation between difenoconazole and pyraclostrobin was detected, four isolates were identified as resistant to difenoconazole and pyraclostrobin. However, isolates with practical resistance were not found widely in our study and were only sporadic in a few places, indicating that at present, difenoconazole and pyraclostrobin are still safe for disease management in the apple-growing areas of Shaanxi, Gansu and Xinjiang. However, the risk of fungicide resistance should be managed with caution, and yearly monitoring of resistance development is necessary.

Multiyear Evaluation of Fungicide Efficacy and Application Timing for Control of Southern Rust in Hybrid Corn in Arkansas

The efficacy and timing of eight foliar fungicides to manage southern rust of corn (caused by Puccinia polysora Underwood) was investigated over 4 years in three field experiments. Each experiment consisted of one-, two-, or three-fungicide application timings at tassel, milk, or dent growth stages with quinone outside inhibitor (QoI), demethylation inhibitor (DMI), or QoI + DMI fungicides. Each year trace amounts of southern rust were observed in the field at tassel, except in 2018, when rust was not observed until physiological maturity. Southern rust severity on ear leaf and two leaves above the ear leaf was approximately 50, 35, 75, and 0% at dent in 2015, 2016, 2017, and 2018, respectively. Applications that contained a QoI or QoI + DMI fungicide provided greater southern rust control than DMI fungicides, with little variation within fungicide classes. Applications of QoI or QoI + DMI fungicides applied at tassel provided greater disease control (52.5%) than those applied at milk (5.8%) or dent (1.4%), and greater yield protection (40.4%) than those applied at milk (23.7%) or dent (2.6%) when final rust development was severe (>40%). When rust development increased later in the season, after milk growth stage, a trend of better disease control was observed with fungicides applied at milk (57.8%) compared with tassel (35.2%), but grain yield protection was similar, with an average yield protection of 7.4%. There was no yield benefit with fungicides applied in the absence of disease or at the dent growth stage. Southern rust was most effectively managed with QoI or QoI + DMI fungicides applied at tassel when southern rust was present and environmental conditions favored rust development.

Cross-Resistance Among Demethylation Inhibitor Fungicides With Brazilian Monilinia fructicola Isolates as a Foundation to Discuss Brown Rot Control in Stone Fruit

Despite the resistance problems in Monilinia fructicola, demethylation inhibitor fungicides (DMIs) are still effective for the disease management of brown rot in commercial stone fruit orchards in Brazil. This study aims to investigate the sensitivity of M. fructicola isolates and efficiency of DMIs to reduce brown rot. A set of 93 isolates collected from Brazilian commercial orchards were tested for their sensitivities to tebuconazole, propiconazole, prothioconazole, and myclobutanil. The isolates were analyzed separately according to the presence or absence of the G461S mutation in MfCYP51 gene, determined by allele-specific test. The mean EC50 values for G461S mutants and wild-type isolates were respectively 8.443 and 1.13 µg/ml for myclobutanil, 0.236 and 0.026 µg/ml for propiconazole, 0.115 and 0.002 µg/ml for prothioconazole, and 1.482 and 0.096 µg/ml for tebuconazole. The density distribution curves of DMI sensitivity for both genotypes showed that myclobutanil and prothioconazole curves were mostly shifted toward resistance and sensitivity, respectively. Incomplete cross-resistance was detected among propiconazole and tebuconazole in both wild-type (r = 0.45) and G461S (r = 0.38) populations. No cross-sensitivity was observed among wild-type isolates to prothioconazole and the others DMIs tested. Fungicide treatments on detached fruit inoculated with M. fructicola genotypes showed significant DMI efficacy differences when fruit were inoculated with wild-type and G461S isolates. Protective applications with prothioconazole were more effective for control of both G461S and wild-type isolates compared with tebuconazole. Curative applications with tebuconazole were most effective in reducing the incidence and lesion size of G461S isolates. Sporulation occurred only for G461S isolates treated with tebuconazole under curative and preventative treatments. The differences found among the performance of triazoles against M. fructicola isolates will form the basis for recommendations of rational DMI usage to control brown rot in Brazil.

Baseline Sensitivity and Toxic Action of the Sterol Demethylation Inhibitor Flusilazole Against Botrytis cinerea

In the present study, a total of 95 Botrytis cinerea single-spore strains collected from different hosts in Shaanxi Province of China were characterized for their sensitivity to the sterol demethylation inhibitor fungicide flusilazole. The effective concentration for 50% inhibition of mycelial growth (EC50) of flusilazole ranged from 0.021 to 0.372 µg/ml, with an average value of 0.093 µg/ml. Cross-resistance between flusilazole and commonly used fungicides was not detected, and no flusilazole-resistant mutants were induced. Both on detached strawberry leaves and in greenhouse experiments, flusilazole was more effective than the commonly used fungicide carbendazim at reducing gray mold. After culture on PDA plates or detached strawberry leaves, no difference in sclerotia production or pathogenicity was detected between two strains, WG12 (most sensitive to flusilazole) and MX18 (least sensitive to flusilazole). After treatment with flusilazole, however, the two strains lost the ability to produce sclerotia, and oxalic acid and ergosterol contents in mycelium decreased. Interestingly, the inhibition rate of ergosterol content in MX18 was significantly lower than that in WG12. Expression of Cyp51, BcatrD, and Bcmfs1 genes all increased after treatment with flusilazole, especially the Cyp51 and BcatrD genes. However, the expression of Cyp51 gene or BcatrD gene in WG12 and MX18 were significantly different from each other after treatment with flusilazole. In addition, no point mutations in Cyp51 gene were found in MX18. These data suggest flusilazole is a promising fungicide for resistance management of gray mold and also provided novel insights into understanding the resistance mechanism of flusilazole against plant pathogens.

Survey and genetic analysis of demethylation inhibitor fungicide resistance in Monilinia fructicola from Michigan orchards

Resistance to sterol demethylation inhibitor fungicides (DMIs) in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern U.S., is associated with a sequence element termed ‘Mona’ that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009-2011 and in 2019, and determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays but the ‘Mona’ insert was not uniformly detected, and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI-resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the ‘Mona’ element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of ‘Mona’ or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the ‘Mona’ insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.