Demethylation Inhibitor Fungicides Research Articles

Sensitivity of soybean (Glycine max L.) pathogens Diaporthe aspalathi, D. caulivora, and D. longicolla to Difenoconazole and Fluopyram fungicides

Diseases of soybean (Glycine max L.) caused by species of Diaporthe have resulted in estimated yield losses totaling $248.2 million in the U.S. over the past 10 years. To effectively manage species of Diaporthe, it is important to use an integrated approach. In this study, we evaluated the in vitro sensitivity of isolates of the soybean pathogens Diaporthe aspalathi, D. caulivora, and D. longicolla from 18 U.S. states to difenoconazole (a demethylation inhibitor fungicide) and fluopyram (a succinate dehydrogenase inhibitor fungicide). The fungicides were incorporated into 2% water agar (WA) in petri dishes at various concentrations. A mycelial plug of each isolate (n=59 for difenoconazole and n=55 for fluopyram) obtained from a 7-day-old culture was placed at the center of the WA and incubated in the dark. After five days for D. caulivora and D. longicolla, and eight days for D. aspalathi, the colony diameter was measured, and the corresponding percent inhibition and effective concentration at which 50% mycelial growth was inhibited (EC50) were determined. Significant differences in EC50 values (P<0.0001) were observed among the isolates of D. aspalathi (0.227 µg/ml), D. caulivora (0.130 µg/ml), and D. longicolla (1.860 µg/ml) for difenoconazole. Similarly, for fluopyram, the EC50 values varied significantly (P<0.001) among the D. aspalathi (2.233 µg/ml), D. caulivora (1.610 µg/ml), and D. longicolla (0.347 µg/ml) isolates. This study established sensitivity profiles for difenoconazole and fluopyram fungicides for D. aspalathi, D. caulivora, and D. longicolla, and provides valuable information that may help in the development of Diaporthe disease management program.

Identification of Geotrichum citri-aurantii and G. candidum in citrus packinghouses in California, their sensitivity to cyproconazole, and incomplete cross-resistance to propiconazole.

High levels of sour rot on propiconazole-treated lemon fruit that were stored for extended times in some California packinghouses in 2020 and 2021 initiated surveys on fungicide sensitivity of the causal pathogen. In isolations from diseased fruit in 2020 to 2023, 157 isolates of Geotrichum spp. were obtained. Using species-specific primers, 143 were determined to be G. citri-aurantii and 15 were G. candidum. Isolates of G. citri-aurantii were either sensitive (EC50 0.06 to 0.34 μg/ml), moderately resistant (EC50 1.20 to 2.34 μg/ml), or highly resistant (EC50 >17.68 μg/ml) to propiconazole. There was incomplete cross resistance to cyproconazole, another demethylation inhibitor fungicide pending postharvest registration on citrus in the United States: isolates sensitive to propiconazole were sensitive, isolates moderately resistant to propiconazole were sensitive, and isolates highly resistant were moderately resistant to cyproconazole (EC50 0.11 to 0.63 μg/ml, 0.19 to 0.73 μg/ml, and 2.66 to 6.79 μg/ml, respectively). All except one isolate of G. candidum were highly resistant to both fungicides (EC50 >9.55). Isolates of both species were all considered sensitive to natamycin (EC50 1.18 to 5.01 μg/ml). In lemon fruit inoculations with G. candidum, the incidence of typical sour rot increased from 4.7% to 68.2% when inoculum was amended 2 µg/ml or 100 µg/ml cycloheximide, respectively, a compound known to suppress host defenses. In co-inoculations with 1:1 mixtures of the two Geotrichum spp., G. candidum was only recovered from the centers of decay lesions, whereas G. citri-aurantii was also obtained from the advancing margins. We conclude that G. candidum is a secondary pathogen of lemons, and its presence was favored by extended late-season storage of senescent fruit with reduced defense mechanisms.

CbCyp51-Mediated Demethylation Inhibitor Resistance Is Modulated by Codon Bias.

Cercospora leaf spot, caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugarbeet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated with synonymous and nonsynonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/L144F_C) correlated with resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study offers an understanding of the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decision-making.

Pyrrolnitrin in Pseudomonas chlororaphis strain AFS009 metabolites reduces constitutive and DMI-induced MfCYP51 gene expression in Monilinia fructicola.

Brown rot, caused by Monilinia fructicola, is one of the most economically important diseases of peach. Demethylation inhibitor (DMI) fungicides play an important part in managing brown rot in the southeastern U.S., but over the last 20 years, reduced efficacy to DMIs has been reported in field isolates overexpressing the DMI target enzyme encoding MfCYP51 gene. Metabolites of the biocontrol agent (BCA) Pseudomonas chlororaphis strain AFS009 suppressed the MfCYP51 gene in sensitive and resistant M. fructicola isolates previousely, but it is not known what molecule was responsible. The goals of this study were to determine the presence and role of pyrrolnitrin (PRN), a common metabolite of P. chlororaphis and chemical analogue to fludioxonil with antifungal activity, in the suppression of the MfCYP51 gene and to investigate if MfCYP51 expression can also be suppressed by Bacillus subtilis. High-performance liquid chromatography (HPLC) detected pyrrolnitrin at 1.75 µg/mg in P. chlororaphis metabolites formulated as Howler EVO (Howler). PRN at 0.1 µg/ml, fludioxonil at 0.1 µg/ml, and Howler applied at a dose that contained 0.1 µg/ml PRN significantly reduced the MfCYP51 gene expression at similar levels in DMI-resistant isolates. Furthermore, MfCYP51 expression in DMI-sensitive and three DMI-resistant isolates treated with Howler (88.1 µg/ml), Theia (209.5 µg/ml), propiconazole (0.3 µg/ml), or the mixture of either Howler or Theia + propiconazole revealed that Howler significantly reduced the MfCYP51 target gene expression in two of three sensitive and all three resistant M. fructicola isolates. On the other hand, Theia showed no suppressive effect and even increased the MfCYP51 gene expression level in two of three resistant isolates. In detached fruit assays on apple with a DMI resistant isolate, only the mixture of Howler + 50 µg/ml propiconazole resulted in synergism. The results indicate that suppression of MfCYP51 target gene is BCA-dependent and can be induced by pyrrolnitrin.

Identification of Cercospora spp. on corn in North America and baseline flutriafol fungicide sensitivity.

Gray leaf spot (GLS) is an important corn disease reportedly caused by Cercospora zeae-maydis and C. zeina. Recently, flutriafol, a demethylation inhibitor (azole) fungicide received EPA registration as Xyway® LFR®, a product that is applied at planting for management of fungal diseases in corn, including suppression of GLS. In this study, 448 Cercospora spp. isolates were collected in 2020 and 2021 from symptomatic corn leaf samples submitted from the United States and Ontario, Canada. The Cercospora spp. were identified using multi-locus genotyping of the internal transcribe spacer (ITS), elongation factor 1-α (EF1), calmodulin (CAL), histone H3 (HIS), and actin (ACT) gene. Based on the multi-locus phylogenetic analyses, six species were identified; C. cf. flagellaris (n = 77), C. kikuchii (n = 4), C. zeae-maydis (n = 361), Cercospora sp. M (n = 2), Cercospora sp. Q (n = 1), and Cercospora sp. T (n = 3). In subsequent pathogenicity tests using selected isolates from each of these species, only C. zeae-maydis resulted in symptoms on corn with no disease symptoms observed after inoculation with C. cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T. While disease symptoms were observed on soybean following inoculation with C. cf. flagellaris, C. kikuchii, and Cercospora sp. Q, but not the other three species. Fungicide sensitivity of Cercospora spp. to flutriafol was assessed using a subset of 340 isolates. The minimum inhibitory concentration (MIC) to inhibit the growth of Cercospora spp. completely was determined based on growth of each species on flutriafol-amended clarified V8 agar at nine concentrations. The EC50 was also calculated from the same trial by measuring relative growth as compared to the non-amended control. Cercospora zeae-maydis was sensitive to flutriafol with mean MIC values of 2.5 µg/mL and EC50 values ranging from 0.016 to 1.020 µg/mL with a mean of 0.346 µg/mL. Cercospora cf. flagellaris, C. kikuchii, Cercospora sp. M, Cercospora sp. Q, and Cercospora sp. T had mean EC50 values of 1.25 µg/mL, 7.14 µg/mL, 2.48 µg/mL, 1.81 µg/mL, and 2.24 µg/mL respectively. These findings will assist in monitoring the sensitivity to the flutriafol fungicide in Cercospora spp. populations.

Mixing Elemental Sulfur or Chlorothalonil with QoI and DMI Fungicides for Management of Late Leaf Spot of Peanut.

Reduced sensitivity to demethylation inhibitor (DMI) and quinone outside inhibitor (QoI) fungicides in Nothopassalora personata, the cause of late leaf spot of peanut (Arachis hypogaea) complicates management of this disease in the southeastern U.S. Mixtures with protectant fungicides may help preserve the utility of members of both DMI and QoI fungicide groups for leaf spot management. Field experiments were conducted in Tifton, GA from 2019 to 2021 and in Plains, GA during 2019 and 2020. The primary objective was to determine the effects of mixtures of DMI fungicides, tebuconazole and mefentrifluconazole, and QoI fungicides, azoxystrobin and pyraclostrobin, with micronized elemental sulfur on late leaf spot in fields with populations of N. personata with suspected reduced sensitivity to DMI and QoI fungicides. In four of the experiments, the efficacies of elemental sulfur and chlorothalonil as mixing partners were also compared. In most cases, standardized area under the disease progress curve (sAUDPC) and final percent defoliation were less for all DMI and QoI fungicides mixed with sulfur or chlorothalonil than for the respective fungicides alone. In most cases, sAUDPC and final percent defoliation were similar for sulfur and chlorothalonil when mixed with the respective DMI or QoI fungicide. These results indicate that mixtures of DMI or QoI fungicides with either micronized sulfur or chlorothalonil can improve control of late leaf spot compared to the DMI or QoI fungicide alone. These results also indicate that elemental sulfur has potential as an alternative to chlorothalonil in tank mixes where that protectant fungicide is currently being used as a mixing partner to improve leaf spot control.

Bioproducts of Pseudomonas chlororaphis Suppress DMI Fungicide-Induced CsCYP51A and CsCYP51B Gene Expression in Colletotrichum siamense and Generate Synergistic Effects with Metconazole and Propiconazole.

Mixtures of fungicides with different modes of action are commonly used as disease and resistance management tools, but little is known of mixtures of natural and synthetic products. In this study, mixtures of metabolites from the rhizobacterium Pseudomonas chlororaphis strain ASF009 formulated as Howler EVO with below-label rates (50 µg/ml) of conventional sterol demethylation inhibitor (DMI) fungicides were investigated for control of anthracnose of cherry (Prunus avium) caused by Colletotrichum siamense. Howler mixed with metconazole or propiconazole synergistically reduced disease severity through lesion growth. Real-time PCR showed that difenoconazole, flutriafol, metconazole, and propiconazole induced the expression of DMI target genes CsCYP51A and CsCYP51B in C. siamense. The addition of Howler completely suppressed the DMI fungicide-induced expression of both CYP51 genes. We hypothesize that the downregulation of DMI fungicide-induced expression of the DMI target genes may, at least in part, explain the synergism observed in detached fruit assays.

Ameliorative effects of gallic acid on tebuconazole–induced adverse effects in the cerebellum of adult albino rats: histopathological and immunohistochemical evidence

ABSTRACT Tebuconazole (TEB) is a common triazole sterol demethylation inhibitor fungicide utilized to manage a variety of diseases in crops like cereals, fruits, and vegetables. The aim of this work was to assess the effects of TEB on the structure of the cerebellum in adult albino rats and possible protective impact of co-administration of Gallic acid (GA). Four groups of forty adult male albino rats were randomly selected, and the rats in group I received corn oil through daily gavage for 4 weeks. Group II received GA dissolved in the normal saline at a dose of 100 mg/kg through daily gavage for 4 weeks, group III administered with TEB dissolved in corn oil at its acceptable daily intake dose (0.02 mg/kg body weight) through daily gavage for 4 weeks, group IV rats received both TEB and GA. For light microscopic, ultrastructural, and immunohistochemical investigations, cerebellar specimens were prepared. TEB exposure led to neuronal damage in the form of degenerated Purkinje cells with vacuolated cytoplasm, areas of lost Purkinje cells, the basket cells appeared vacuolated with degenerated neuropil, the granule cells clumped with congested areas between them, dilated cerebellar islands, weak positive bcl2 immunoreactions in the Purkinje cells, and numerous GFAP-positive astrocytes. GA mitigated TEB-mediated histological changes in the cerebellar cortex. We concluded that TEB caused Purkinje neurons in the rat cerebellar cortex to degenerate and undergo apoptosis. GA had a neuroprotective benefit against TEB toxicity in the rat cerebellar cortex.

Visual detection of fungicide resistance by combining RPA and CRISPR/Cas12a in peach Brown rot fungus Monilinia fructicola.

Peach brown rot caused by Monilinia fructicola severely affects the quality and yield of peach, resulting in large economic losses worldwide. Methyl benzimidazole carbamate (MBC) fungicides and sterol demethylation inhibitor (DMI) fungicides are among the most applied chemical classes used to control the disease but resistance in the target pathogen has made them risky choices. Timely monitoring of resistance to these fungicides in orchards could prevent control failure in practice. In the current study, we developed methods based on recombinase polymerase amplification (RPA) and CRISPR/Cas12a systems to detect MBC and DMI resistance based on the E198A mutation in the β-tubulin (MfTub2) gene and the presence of the Mona element in the upstream region of the MfCYP51, respectively. For MBC resistance, RPA primers were designed that artificially incorporated PAM sites to facilitate the CRISPR/Cas12a reaction. Subsequently, specific tcrRNAs were designed based on the E198A mutation site. For the detection of the Mona element, we designed RPA primers M-DMI-F2/M-DMI-R1 that in combination with crRNA1 detected 'Mona' and distinguished resistant from sensitive strains. Both methods exhibited high sensitivity and specificity, requiring only a simple isothermal device to obtain results within 1 h at 37 °C. The FQ-reporter enabled visualization with a handheld UV or white light flashlight. This method was successfully used with purified DNA from lab cultures and crude DNA from symptomatic fruit tissue, highlighting its potential for on-site detection of resistant strains in orchards. © 2024 Society of Chemical Industry.

Comparative Fitness of Monilinia fructicola Isolates with Multiple Fungicide-Resistant Phenotypes.

This study characterized 52 isolates of Monilinia fructicola from peach and nectarine orchards for their multiresistance patterns to thiophanate-methyl (TF), tebuconazole (TEB), and azoxystrobin (AZO) using in vitro sensitivity assays and molecular analysis. The radial growth of M. fructicola isolates was measured on media amended with a single discriminatory dose of 1 μg/ml for TF and AZO and 0.3 μg/ml for TEB. Cyt b, CYP51, and β-tubulin were tested for point mutations that confer resistance to quinone outside inhibitors (QoIs), demethylation inhibitors (DMIs), and methyl benzimidazole carbamates (MBCs), respectively. Eight phenotypes were identified, including isolates with single, double, and triple in vitro resistance to QoI, MBC, and DMI fungicides. All resistant phenotypes to TF and TEB presented the H6Y mutation in β-tubulin and the G641S mutation in CYP51. None of the point mutations typically linked to QoI resistance were present in the Monilinia isolates examined. Moreover, fitness of the M. fructicola phenotypes was examined in vitro and in detached fruit assays. Phenotypes with single resistance displayed equal fitness in vitro and in fruit assays compared with the wild type. In contrast, the dual- and triple-resistance phenotypes suffered fitness penalties based on osmotic sensitivity and aggressiveness on peach fruit. In this study, multiple resistance to MBC, DMI, and QoI fungicide groups was confirmed in M. fructicola. Results suggest that Monilinia populations with multiple resistance phenotypes are likely to be less competitive in the field than those with single resistance, thereby impeding their establishment over time and facilitating disease management.

Pseudomonas chlororaphis Metabolites Reduce MfCYP51 Expression and Yield Synergistic Efficacy in Mixture with Reduced Rates of Propiconazole Against DMI-Resistant Monilinia fructicola Isolates.

Brown rot caused by Monilinia fructicola is one of the most important diseases affecting peach production in the southeastern United States. Management often involves the use of demethylation inhibitor (DMI) fungicides, but efficacy can be compromised because of overexpression of the MfCYP51 gene encoding the 14α-demethylase of the ergosterol biosynthesis pathway. This study aimed to investigate the influence of the biorational fungicide Howler EVO containing Pseudomonas chlororaphis ASF009 metabolites on the expression of MfCYP51 in M. fructicola and associated synergy with a DMI fungicide for control of DMI-resistant strains. Mycelia from two DMI-sensitive and three DMI-resistant M. fructicola isolates were exposed or not to propiconazole (0.3 μg/ml), Howler (88.1 μg/ml), or the combination propiconazole + Howler for 6 h prior to RNA extraction. Real-time PCR indicated that Howler reduced the constitutive expression of MfCYP51 in DMI-sensitive and two of three DMI-resistant isolates. Propiconazole-induced expression of the DMI target gene was significantly reduced by Howler and by the mixture of Howler plus propiconazole in all isolates. Detached fruit studies on apple revealed that the combination of Howler plus a reduced label rate of Mentor (50 μg/ml propiconazole) was synergistic against brown rot caused by a DMI-resistant isolate in high and low inoculum spore concentration experiments (synergy values of 40.1 and 4.9, respectively). We hypothesize that the synergistic effects against M. fructicola resistant to DMI fungicides based on MfCYP51 gene overexpression can be attributed to reduced 14α demethylase production due to transcription inhibition, which may necessitate fewer DMI fungicide molecules to arrest fungal growth. The use of Howler/DMI mixtures for brown rot control warrants further investigation because such mixtures could potentially allow for reduced DMI fungicide use rates in the field without compromising yield or increased resistance selection.

Influence of propiconazole and metconazole formulations on Bacillus subtilis vegetative cell growth and disease control of fruit crops.

Biological control agent (BCA) Bacillus subtilis formulated as Theia® is registered for control of fungal and bacterial diseases of fruit crops. Combinations of Theia® and strategic concentrations of two demethylation inhibitor (DMI) fungicides were investigated to explore potential synergisms. Bacteria were cultured in nutrient broth and combined with technical grades and two formulations of propiconazole (EC and WP) and metconazole (EC and WDG) at 0, 10, 50, 100, and 150 µg/ml active ingredient. After co-cultivation, optical density (OD600) and colony forming units (CFU/ml) were evaluated. In contrast to EC formulations, the WP or WDG formulations at 10 or 50 µg/ml of both DMIs did not affect vegetative cell growth. The mixture of Theia® and each formulated DMI at 50 µg/ml active ingredient resulted in a significant reduction of Monilinia fructicola lesion development on apple, Colletotrichum siamense lesion development on cherry, and Botrytis cinerea lesion development on cherry. The combination of Theia® with EC formulations showed weaker disease reduction due to antagonism. Only Theia® + non-EC formulated propiconazole and metconazole significantly reduced brown rot disease incidence of apple compared to the respective solo treatments and anthracnose disease incidence of cherry compared to the untreated control. Our results indicated that at least some DMI fungicides possess bactericidal effects depending on the formulation and concentration. The combination of Theia® with a lower than label rate concentration (50 µg/ml) of the DMI fungicides propiconazole and metconazole showed potential for synergistic effects especially when non-EC formulations were used.

Transcriptome Analysis Reveals Potential Regulators of DMI Fungicide Resistance in the Citrus Postharvest Pathogen Penicillium digitatum.

Green mold, caused by Penicillium digitatum, is the major cause of citrus postharvest decay. Currently, the application of sterol demethylation inhibitor (DMI) fungicide is one of the main control measures to prevent green mold. However, the fungicide-resistance problem in the pathogen P. digitatum is growing. The regulatory mechanism of DMI fungicide resistance in P. digitatum is poorly understood. Here, we first performed transcriptomic analysis of the P. digitatum strain Pdw03 treated with imazalil (IMZ) for 2 and 12 h. A total of 1338 genes were up-regulated and 1635 were down-regulated under IMZ treatment for 2 h compared to control while 1700 were up-regulated and 1661 down-regulated under IMZ treatment for 12 h. The expression of about half of the genes in the ergosterol biosynthesis pathway was affected during IMZ stress. Further analysis identified that 84 of 320 transcription factors (TFs) were differentially expressed at both conditions, making them potential regulators in DMI resistance. To confirm their roles, three differentially expressed TFs were selected to generate disruption mutants using the CRISPR/Cas9 technology. The results showed that two of them had no response to IMZ stress while ∆PdflbC was more sensitive compared with the wild type. However, disruption of PdflbC did not affect the ergosterol content. The defect in IMZ sensitivity of ∆PdflbC was restored by genetic complementation of the mutant with a functional copy of PdflbC. Taken together, our results offer a rich source of information to identify novel regulators in DMI resistance.

Multiple resistance of Colletotrichum truncatum from soybean to QoI and MBC fungicides in Brazil

AbstractColletotrichum truncatum, the predominant fungal species associated with soybean anthracnose, is responsible for significant losses in this crop. Chemical control via fungicide application is the most effective strategy for the control of soybean foliar diseases. However, the increasing incidence of anthracnose in some regions of Brazil indicates that current chemical control is not effective against anthracnose. In this study, we evaluated the fungicide sensitivity of C. truncatum genetic lineages to the fungicides azoxystrobin, thiophanate‐methyl, difenoconazole, and fludioxonil using isolates representing two important regions of soybean production in Brazil. We characterized the molecular resistance to the quinone‐outside inhibitors (QoI), methyl benzimidazole carbamates (MBC), and demethylation inhibitor (DMI) fungicide groups based on amino acid sequences of the cytochrome b (cytb), β‐tubulin gene (β‐tub), and P450 sterol 14a‐demethylases (CYP51) genes. Multiple resistance of C. truncatum isolates to QoI and MBC was observed associated with mutation points in the β‐tub (E198A and F200Y) and cytb (G143A). Alternatively, low EC50 values were found for fludioxonil and difenoconazole indicating high efficacy. Analysis of C. truncatum genomes revealed two potential DMI targets, CYP51A and CYP51B, and higher genetic variability in the CYP51A gene. A positive correlation was found between genetic differentiation of C. truncatum populations and fungicide sensitivity (Student's t‐test <0.001). To our knowledge, this is the first report of multiple resistance to QoI and MBC fungicides in C. truncatum in Brazil.

Global Spread, Genetic Differentiation, and Selection of Barley Spot Form Net Blotch Isolates.

Spot form net blotch, caused by Pyrenophora teres f. maculata, is a significant necrotrophic disease of barley that spread worldwide in the twentieth century. Genetic relationships were analyzed to determine the diversity, survival, and dispersal of a diverse collection of 346 isolates from Australia, Southern Africa, North America, Asia Minor, and Europe. The results, based on genome-wide DArTseq data, indicated that isolates from Turkey were the most differentiated with regional sub-structuring, together with individuals closely related to geographically distant genotypes. Elsewhere, population subdivision related to country of origin was evident, although low levels of admixturing was found that may represent rare genotypes or migration from unsampled populations. Canadian isolates were the next most diverged, and Australian and South African the most closely related. With the exception of Turkish isolates, multiple independent Cyp51A mutation events (which confer insensitivity to demethylation inhibitor fungicides) between countries and within regions was evident, with strong selection for a transposable element insertion at the 3' end of the promoter and counterselection elsewhere. Individuals from Western Australia shared genomic regions and Cyp51A haplotypes with South African isolates, suggesting a recent common origin. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Barley powdery mildew control in Western Australia and beyond

AbstractAustralia is one of the largest barley exporters in the world, with Western Australia accounting for some 40% of national production. The crop is predominantly grown in the south and south‐west of the state in winter and spring, where temperate conditions and higher rainfall levels are more suited to barley than northern and eastern regions. Between 2007 and 2013, prolonged outbreaks of barley powdery mildew (BPM) occurred. This was brought about by a combination of the extensive use of susceptible cultivars and an over‐reliance on a small number of single mode‐of‐action demethylation inhibitor fungicides, which select for mutations in the C14α‐demethylase (Cyp51A) gene. This review highlights the steps taken to reduce losses to BPM, breeding efforts to introduce resistance into cultivars and the success of pre‐breeding research to find new and durable resistance genes. We also draw comparisons with powdery mildew in Australian wheat, where similar factors are leading to substantial outbreaks.

Reevaluation of Sensitivity of Monilinia fructicola Isolates to the DMI Fungicide Propiconazole in the Southeastern United States and Investigation of the Genetic Element Mona.

Sterol demethylation inhibitor (DMI) fungicides continue to be essential components for the control of brown rot of peach caused by Monilinia fructicola in the United States and worldwide. In the southeastern United States, resistance to DMIs had been associated with overexpression of the cytochrome P450 14α-demethylase gene MfCYP51 as well as the genetic element Mona, a 65 bp in length nucleotide sequence located upstream of MfCYP51 in resistant isolates. About 20 years after the first survey, we reevaluated sensitivity of M. fructicola from South Carolina and Georgia to propiconazole and also evaluated isolates from Alabama for the first time. A total of 238 M. fructicola isolates were collected from various commercial and two experimental orchards, and sensitivity to propiconazole was determined based on a discriminatory dose of 0.3 μg/ml. Results indicated 16.2, 89.2, and 72.4% of isolates from Alabama, Georgia, and South Carolina, respectively, were resistant to propiconazole. The detection of resistance in Alabama is the first report for the state. All resistant isolates contained Mona, but it was absent from most sensitive isolates. It was unclear if the resistance frequency had increased in South Carolina and Georgia. However, the resistance levels (as assessed by the isolate frequency in discriminatory dose-based relative growth categories) did not change notably, and no evidence of other resistance genotypes was found. Analysis of the upstream MfCYP51 gene region in the resistant isolate CF010 revealed an insertion sequence described for the first time in this report. Our study suggests that current fungicide spray programs have been effective against increasing resistance levels in populations of M. fructicola and suppressing development of new resistant genotypes of the pathogen.

Workflows for detecting fungicide resistance in net form and spot form net blotch pathogens.

Fungicide resistance in Pyrenophora teres f. maculata and P. teres f. teres has become an important disease management issue. Control of the associated barley foliar diseases, spot form and net form net blotch, respectively, relies on three major groups of fungicides, demethylation inhibitors (DMIs), succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, resistance has been reported for the DMI and SDHI fungicides in Australia. To enhance detection of different resistance levels, phenotyping and genotyping workflows were designed. The phenotyping workflow generated cultures directly from lesions and compared growth on discriminatory doses of tebuconazole (DMI) and fluxapyroxad (SDHI). Genotyping real-time polymerase chain reaction (PCR) assays were based on alleles associated with sensitivity or resistance to the DMI and SDHI fungicides. These workflows were applied to spot form and net form net blotch collections from 2019 consisting predominantly of P. teres f. teres from South Australia and P. teres f. maculata from Western Australia. For South Australia the Cyp51A L489-3 and SdhC-R134 alleles, associated with resistance to tebuconazole and fluxapyroxad, respectively, were the most prevalent. These alleles were frequently found in single isolates with dual resistance. This study also reports the first detection of a 134 base pair insertion located at position-66 (PtTi-6) in the Cyp51A promoter of P. teres f. maculata from South Australia. For Western Australia, the PtTi-1 insertion was the most common allele associated with resistance to tebuconazole. The workflow and PCR assays designed in this study have been demonstrated to efficiently screen P. teres collections for both phenotypic and genetic resistance to DMI and SDHI fungicides. The distribution of reduced sensitivity and resistance to DMI and SDHI fungicides varied between regions in south-western Australia, suggesting the emergence of resistance was impacted by both local pathogen populations and disease management programmes. The knowledge of fungicide resistance in regional P. teres collections will be important for informing appropriate management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sensitivity of the Causal Agent of Northern Leaf Blight of Corn, Exserohilum turcicum, to the Demethylase-Inhibiting Fungicide Flutriafol

Fungicides including active ingredients in the demethylation inhibitor group (Fungicide Resistance Action Committee Group 3) are used to manage corn diseases such as northern corn leaf blight, caused by Exserohilum turcicum. This study assessed the level of sensitivity to the demethylation-inhibiting fungicide flutriafol on a population of 81 E. turcicum isolates collected from across the United States in 2020. Sensitivity to flutriafol for E. turcicum was assessed using fungicide-amended media to determine the effective concentration needed to limit hyphal growth by 50%. The mean absolute EC50 values of the overall population calculated using the four-parameter log-logistic and Weibull2 four-parameter models were 2.1881 and 3.2643 µg/ml, respectively. The growth of most isolates ( n = 70, 86.4%) was completely inhibited by 1 µg/ml of flutriafol. However, the growth of seven isolates (8.6%) was only completely inhibited at 10 or 100 µg/ml, and complete inhibition was not observed at 100 µg/ml for four isolates (4.9%). These results suggest that there may be isolates of E. turcicum in the United States that are resistant to flutriafol. Additional fungicide sensitivity screening is critical to monitor the development of fungicide resistance in U.S. populations of E. turcicum.

Evaluation of foliar fungicides for frogeye leaf spot control and yield protection in soybean in Arkansas

The efficacy of fifteen foliar fungicides to control frogeye leaf spot (caused by Cercospora sojina K. Hara) and protect soybean grain yield was investigated over three years (2014–2016) in two field experiments. Experiments were divided into two fungicide groups: premix fungicides, which included combinations of quinone outside inhibitors (QoI), demethylation inhibitors (DMI), succinate dehydrogenase inhibitors (SDHI), or methyl benzimidazole carbamates (MBC), and solo fungicides, all DMI fungicides. Fungicides were applied once at the full pod (R4) in 2014 and 2015 and at the beginning seed (R5) growth stage in 2016. A very susceptible frogeye leaf spot cultivar, Armor 48R40, was used. Frogeye leaf spot severity at fungicide application timing average 1.0, 0.5, and 8.0% in 2014, 2015, and 2016, respectively. Final disease severity averaged 21.7, 14.5, and 15.1% in 2014, 2015, and 2016, respectively on the non-treated control. Azoxystrobin provided no disease control, suggesting the presence of QoI-resistant C. sojina. Three fungicides: azoxystrobin + difenoconazole, flutriafol, and thiophanate-methyl + tebuconazole applied at R4 provided effective disease control compared to the non-treated control. Fungicides applied at R5 provided no disease control. No fungicide provided significant grain yield protection in either experiment. Overall, the average yield protection trend by fungicides applied at R4 and R5 was 6.8 and 3.0%, respectively compared to the non-treated control. These data suggest that although foliar fungicides can provide effective frogeye leaf spot control, yield protection may be limited when applied at the R4 growth stage, particularly when disease severity exceeds 0.5% on a very susceptible soybean cultivar.