Inhibitor Fungicides Research Articles

Design, Synthesis, and Biological Evaluation of Novel Aryl Sulfonamide Derivatives as Potential Succinate Dehydrogenase Inhibitors Targeting Phytopathogenic Fungi.

In our pursuit of novel succinate dehydrogenase inhibitor (SDHI) fungicides for agriculture, we replaced the traditional amide structure with a sulfonamide framework and introduced various heterocyclic and aromatic rings at the sulfonamide's termini. This strategy yielded 30 synthesized compounds, which were screened for antifungal activity against eight phytopathogenic fungi. The biological assay results demonstrated that several target compounds exhibited significant in vitro antifungal activity. Notably, compound 2f showed remarkable antifungal activity against Valsa mali with an EC50 value of 0.56 mg/L, outperforming Boscalid (EC50 = 1.79 mg/L). In vivo experiments revealed that compound 2f provided significant protection to apple fruits, comparable to Boscalid. SEM analysis of compounds 2f and 3e showed that compound 2f disrupted the structure and morphology of fungal hypha analysis, which suggested that the terminal polyhalogen-substituted pyridine moieties might be pivotal regions influencing their antifungal efficacy. Molecular docking analysis revealed that compound 2f and Boscalid exhibited a comparable binding mode to SDH. Furthermore, detailed SDH inhibition assays confirmed the potential of 2f (IC50 = 2.51 μM) as prospective SDH inhibitors. RNA transcriptomic analysis indicated that the application of compound 2f could influence gene expression in fungi, thereby exerting a defensive effect against plant pathogenic fungi. Consequently, compound 2f shows promise for developing a novel and efficient agrochemical fungicide.

Combined effects of different-sized microplastics and fluindapyr on earthworm: Bioaccumulation, oxidative stress, histopathological responses and gut microbiota.

Soil is an important sink for microplastics (MPs) and pesticides. MPs can act as carriers for pesticides, thus induce direct and indirect effects on soil organisms. Fluindapyr (FIP), a novel succinate dehydrogenase inhibitors fungicides (SDHIs), may pose a serious threat to earthworms. However, few studies have evaluated the effects of joint exposure to MPs and FIP. Here, earthworms (Eisenia fetida) were jointly exposed to PMMA (polymethylmethacrylate) and PS (polystyrene) MPs of different sizes (0.1, 1 and 10μm) along with FIP for 28-day to investigate the toxic effects of single and joint exposure of FIP and MPs on earthworms. The results showed that joint exposure to 0.1 and 1μmMP promoted the accumulation of FIP in earthworms at the beginning of the experiment compared to the sole group, but the elimination of FIP from earthworms accelerated after 14d. In addition, the joint exposure caused more serious damages to the epidermis and intestine of earthworms and increased the severity of oxidative stress. The effects of joint exposure to FIP and MPs depended on the size of the MPs, and the strongest effects were observed in the treatment with the smallest size. The 16S rRNA sequencing results showed that the joint exposure to MPs and FIP didn't cause gut microbiota dysbiosis. However, the sole 0.1μmPS significantly altered the community diversity and richness of earthworm gut bacteria, and the relative abundance of Proteobacteria, Actinobacteria and Firmicutes was significantly changed. The obtained results inferred that MPs could influence environmental and toxicological behaviors of FIP and may provide data support for the risk assessments of MPs and FIP on soil ecosystems.

Ecotoxicological impact of succinate dehydrogenase inhibitor (SDHI) fungicides on non-targeted organisms: a review.

As the global population continues to grow, the use of pesticides to increase food production is projected to escalate. Pesticides are critical in plant protection, offering a powerful defense against fungal diseases such as apple scab, leaf spot, sclerotinia rot, damping off, sheath blight, and root rot, which threaten crops like cereals, corn, cotton, soybean, sugarcane, tuberous vegetables, and ornamentals. Succinate Dehydrogenase Inhibitor (SDHI) fungicides represent a novel class essential for controlling fungal pathogens and bolstering food security. However, the impact of SDHIs on non-target organisms, including freshwater and terrestrial invertebrates, crustaceans, and oligochaetes, remains insufficiently understood. Empirical studies indicate that SDHIs can induce mortality, mitochondrial dysfunction, oxidative stress, and developmental delays in non-target organims. Additionally, the environmental persistence of these compounds raises concerns about their potential for ecological disruption. The effects of SDHIs on pollinating species and the possible transgenerational transmission of harmful effects warrant further investigation. Comprehensive transcriptomic analyses are necessary to elucidate the molecular disturbances and adverse outcome pathways triggered by SDHIs. Furthermore, there are emerging concerns about the endocrine-disrupting potential of SDHIs in aquatic organisms. For the first time, this review aims to synthesize existing knowledge on the ecotoxicological impacts of SDHIs on non-target organisms and identify critical research directions to address the ecological challenges posed by their use.

Sensitivity to the Demethylation Inhibitor Difenoconazole Among Baseline Populations of Various Penicillium spp. Causing Blue Mold of Apples and Pears.

Difenoconazole (DIF), a demethylation inhibitor fungicide, was registered in 2016 for the control of postharvest diseases of pome fruits. In this study, 162 isolates from P. expansum (n = 31) and 13 other "non-expansum" Penicillium spp., i.e., P. solitum (n = 52), P. roqueforti (n = 32), P. commune (n = 15), P. paneum (n = 9), P. psychrosexuale (n = 8), P. crustosum (n = 5), P. carneum (n = 3), P. palitans (n = 2), along with one isolate each of P. citrinum, P. griseofulvum, P. raistrickii, P. ribium, and P. viridicatum, were collected from multiple packinghouses in the U.S. Pacific Northwest. In vitro sensitivity assays showed similar sensitivities of spores and mycelia across species with the mean EC50 values ranging from 0.01 for P. psychrosexuale (n = 8) to 1.33 μg mL-1 for P. palitans (n = 2), whereas the mean EC50s were 0.03, 0.12, 0.19, and 0.51 μg mL-1 for P. expansum (n = 31), P. paneum (n = 9), P. solitum (n = 52), and P. crustosum (n = 5), respectively. The recommended rate of DIF controlled P. expansum and P. roqueforti isolates but not all isolates of four other Penicillium spp. on Fuji apples after five months at 1.5 °C. The mixture of DIF + fludioxonil (FDL) (AcademyTM) controlled all the dual-sensitive isolates (DIFSFDLS) and DIF single-resistant (DIFR) isolates among the six species tested but not the FDLR and dual DIFRFDLR isolates. Notable polymorphism was detected in the CYP51 gene of the "non-expansum" species with four mutations located at four residues. Although the isolates analyzed in this study had not previously been exposed to DIF, the findings indicate variable sensitivity levels among the Penicillium spp.

Apple Bitter Rot: Biology, Ecology, Omics, Virulence Factors, and Management of Causal Colletotrichum Species.

Colletotrichum species-Domain Eukaryota, Kingdom Fungi, Phylum Ascomycota, Class Sordariomycetes, Order Glomerellales, Family Glomerellaceae, Genus Colletotrichum. Hemibiotrophic pathogen with a wide host range that establishes a biotrophic interaction where it penetrates host plants using appressoria followed by a switch to necrotrophy causing rot symptoms. Cercosporin, colletotrichins, colletotric acid, ferricrocin. The host range varies by species but largely occurs on dicotyledonous plants and is less prevalent on monocots as well as gymnosperms, ferns, mosses and animals (e.g., insects). Symptoms often manifest as flat to sunken necrotic areas on fruit. Lesions on leaves and fruit can have concentric rings with abundant pathogen sporulation. Colletotrichum spp. are primarily managed by single-site quinone outside inhibitor (Qol), methyl benzimidazole carbamate (MBC), demethylation inhibitor (DMI) fungicides, and multisite dithiocarbamate and phthalimide fungicides. Susceptibility may vary with species, strain specificity, or geographic region. Other management options include clean stock production, cultural practices, resistance breeding, and biological control through the introduction of protective or competing microorganisms.

Genomic Insights into Fusarium verticillioides Diversity: The Genome of Two Clinical Isolates and Their Demethylase Inhibitor Fungicides Susceptibility.

Fusarium verticillioides is an important plant pathogen in maize and other cereals that is seldom detected as the cause of human fusariosis. Here, we provide the analysis of the available diversity of F. verticillioides sequenced worldwide and report the first two genome assemblies and annotations (including mitochondrial DNA) of Fusarium verticillioides from clinical settings. Fusarium verticillioides 05-0160 (IUM05-0160) and Fusarium verticillioides 09-1037 (IUM09-1037) strains were obtained from the bone marrow and blood of two immunocompromised patients, respectively. The phylogenomic analysis confirmed the species identity of our two strains. Comparative genomic analyses among the reannotated F. verticillioides genomes (n = 46) did not lead to the identification of unique genes specific to the clinical samples. Two subgroups in the F. verticillioides clade were also identified and confirmed by a mitochondrial diversity study. Clinical strains (n = 4) were positioned in the multigene phylogenetic tree without any correlation between the host and the tree branches, grouping with plant-derived strains. To investigate the existence of a potential fitness advantage of our two clinical strains, we compared demethylase inhibitor fungicides susceptibility against the reference Fusarium verticillioides 7600, showing, on average, lower susceptibility to agricultural and medical-used antifungals. A significant reduction in susceptibility was observed for itraconazole and tetraconazole, which might be explained by structural changes in CYP51A and CYP51C sequences. By providing the first two annotated genomes of F. verticillioides from clinical settings comprehensive of their mitogenomes, this study can serve as a base for exploring the fitness and adaptation capacities of Fusarium verticillioides infecting different kingdoms.

Sensitivity Analysis of Pyrenophora tritici-repentis to Quinone-Outside Inhibitor and 14α-Demethylase Inhibitor Fungicides in Latvia.

Tan spot caused by Pyrenophora tritici-repentis is a severe threat to wheat production in all major wheat-growing regions. Sustainable tan spot control can be achieved by an integrated approach, including responsible management of fungicide sprays. The data about the sensitivity of P. tritici-repentis to various fungicides in the Baltic Sea region are rare. In this study, we described the variation of P. tritici-repentis sensitivity to four fungicide active ingredients to detect the formation of resistance to the most commonly used quinone-outside inhibitor (QoI) and 14α-demethylase inhibitor (DMI) fungicides in the pathogen's population in Latvia. The effect of prothioconazole, mefentrifluconazole, pyraclostrobin, and azoxystrobin on 93 P. tritici-repentis strains from various hosts was tested in vitro by assessing mycelium linear growth inhibition at three different active ingredient concentrations (0 0.01, 0.1 and 0.5 mg L-1). Pathogen sensitivity significantly (p < 0.001) varied between the fungicide active ingredients and strains. The prothioconazole (concentration 0.5 mg L-1) had the most significant effect, with a median mycelial growth inhibition of 70.34%, followed by pyraclostrobin (47.02%), azoxystrobin (24.24%), and mefentrifluconazole (11.11%). Mutation G143A was detected in cytb gene sequences and confirmed the resistance formation in Latvia's P. tritici-repentis population, while F129L and G137R mutations were absent. This study provided insight into P. tritici-repentis population's sensitivity to active ingredients of DMI and OoI fungicide groups, helping to fill the knowledge gap about the pathogen fungicide sensitivity in this region.

Demethylation Inhibitor Fungicides Have a Significantly Detrimental Impact on Population Growth and Composition of Nectar Microbial Communities

Demethylation inhibitor (DMI) fungicides are a mainstay of modern agriculture due to their widespread use for crop protection against plant-pathogenic fungi. However, DMI residues can disperse and persist in the environment, potentially affecting non-target fungi. Previous research has demonstrated that DMIs and other fungicides inhibit yeast growth in floral nectar microbial communities and decrease fungal richness and diversity of exposed flowers with no apparent effect on bacteria. Nevertheless, the effect of DMIs on the population growth of different species of nectar inhabitants and the dynamics of these microbial communities remains understudied. To address these issues, in this study we created synthetic microbial communities including yeasts (Metschnikowia reukaufii and Metschnikowia pulcherrima) and bacteria (Rosenbergiella epipactidis and Comamonas sp.) and propagated them in culture media containing different DMIs (imazalil, propiconazole, and prothioconazole) at different doses or no fungicide. Our results showed that DMIs have a significant impact on some of the most common microbial inhabitants of floral nectar by favoring the growth of bacteria over yeasts. Furthermore, habitat generalists such as M. pulcherrima and Comamonas sp. were more impacted by the presence of fungicides than the nectar specialists M. reukaufii and R. epipactidis, especially upon dispersal across habitat patches. Future research should determine if the patterns observed in the present study hold true for other species of nectar microbes and explore the interaction between growth limitation due to fungicide presence, dispersal limitation, and other mechanisms involved in community assembly in floral nectar.

Effect of Bloom Sprays on Alternaria Rot in California Mandarins and Fungicide Efficacy Against Colletotrichum.

This study aimed to provide new insights into the management of Alternaria rot and Colletotrichum dieback in mandarins. Field trials were conducted to evaluate the impact of bloom sprays on latent infections, disease progression, and the influence of bloom inoculations on disease development. Detached citrus leaves were used to assess the efficacy of commercial fungicides in reducing infections by Colletotrichum spp. C. karstii was more sensitive than C. gloeosporioides, as evidenced by the overall probabilities of infection. Preventative treatments containing quinone outside inhibitor fungicides, such as Pristine, Quadris Top, and Luna Sensation, consistently reduced infections. Curative treatments with Pristine and Luna Sensation also demonstrated efficacy. Bloom inoculations with Colletotrichum species did not induce visible symptoms on flowers and twigs, nor did they lead to dieback until harvest, suggesting that bloom infections may not play a significant role in the disease cycle. For Alternaria, the frequency of latent infections in flowers increased significantly during the bloom period. Shoots inoculated with Alternaria species during bloom reduced the number of fruits per shoot at harvest. Fungicide treatments during bloom exhibited a dual effect, reducing Alternaria infections in fruitlets and influencing disease progression, ultimately reducing preharvest fruit drop. This study provides valuable insights into the role of flower infections in the development of Alternaria rot and Colletotrichum dieback in California mandarins. Furthermore, it offers insights into the efficacy of commercial fungicides in these pathosystems.

The transcription factor FgCreA modulates ergosterol biosynthesis and sensitivity to DMI fungicides by regulating transcription of FgCyp51A and FgErg6A in Fusarium graminearum

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease that severely affects crop yield and quality worldwide. The catabolite responsive elements A (CreA) plays a critical role in numerous cellular processes in eukaryotes. In this study, we performed a functional characterization of CreA in F. graminearum. Deletion of FgCreA led to the transcriptional upregulation of FgCyp51A and FgErg6A, consequently resulting in elevated levels of ergosterol. This augmented ergosterol content was observed to improve cellular membrane integrity and diminished sensitivity to demethylation inhibitors (DMI) fungicides in F. graminearum. FgCreA is an essential element in carbon catabolite repression (CCR), consequently, the ΔFgcreA mutant was compromised in radial growth on various carbon sources, sexual/asexual development, and deoxynivalenol toxin biosynthesis, which was suppressed due to transcriptional downregulation of the biosynthesis-required enzymes. These results unveil a novel regulatory mechanism of FgCreA, influencing both the pathogenicity of F. graminearum and its sensitivity to DMI fungicides.

Analysis of the stereoselective fate and toxicity of penflufen in the water-sediment system for risk reduction

Chiral succinate dehydrogenase inhibitor (SDHI) fungicides are widely used in agricultural production, but there is insufficient research on their environmental risk in water–sediment ecosystems. Here, the stereoselective fate and toxic effects of the chiral SDHI fungicide, penflufen, in the water–sediment system were investigated. The results showed that S-penflufen is more persistent in water, sediment, and zebrafish. Additionally, the sorption coefficient (Koc) in sediment and uptake rate constant (Ku) in zebrafish of S-penflufen were higher than those of R-penflufen. The acute toxicity of S-penflufen to zebrafish, Daphnia magna and Chironomus kiiensis were 32-, 6.1-, and 8.9-fold higher than those of R-penflufen. The AlphaFold2 and molecular docking results showed that S-penflufen had stronger binding capability with SDH in the three water–sediment organisms than R-penflufen. Therefore, S-penflufen induced stronger sub-chronic toxic effects on zebrafish than R-penflufen, even at 0.05 mg/L. The results of multi-omics analysis showed that S-penflufen affected the tricarboxylic acid cycle in zebrafish and induced antioxidant, detoxification, and immune system responses, ultimately affecting zebrafish metabolic processes and cellular function. The overall results indicate that S-penflufen has a higher risk in water–sediment systems. Moreover, combining multi-omics and AlphaFold2 techniques facilitates the elucidation of the molecular mechanism of the stereoselective toxic effects of chiral pesticides.

Sensitivity of Meloidogyne incognita and Rotylenchulus reniformis to Cyclobutrifluram.

Cyclobutrifluram, a succinate dehydrogenase inhibitor fungicide, is being evaluated as a seed-applied nematicide in cotton and soybean to manage plant-parasitic nematodes. Currently, there is no information on the toxicity, ovicidal activity, nematode recovery, or effects on nematode infection for Meloidogyne incognita or Rotylenchulus reniformis after exposure to low concentrations of cyclobutrifluram. Nematode toxicity assays were performed in aqueous solutions of cyclobutrifluram, and root infection assays were conducted on tomato. Nematode paralysis was observed after 2 h of exposure to 0.5 μg/ml cyclobutrifluram for both nematode species. Based on an assay of nematode motility, the 2-h effective concentration of fungicide required for 50% growth inhibition (EC50) value for M. incognita and R. reniformis was 0.48 and 1.07 μg/ml, respectively. In a comparable assay with a similar nematicide, continuous exposure to 0.5 μg/ml cyclobutrifluram for 24 h resulted in at least 45% more immotile nematodes for both species compared with those treated with 0.5 μg/ml fluopyram. Continuous exposure to concentrations greater than 1.0 μg/ml suppressed hatching for both species compared with the water control. Nematode recovery from paralysis was greater than 80% for M. incognita and R. reniformis 24 h after nematodes were rinsed and removed from a 1-h treatment to their respective 2-h EC50 concentrations. Nematode infection of tomato roots was reduced following a 1-h treatment with aqueous solutions of cyclobutrifluram, ranging from 0.12 to 0.48 μg/ml for M. incognita and 0.27 to 1.07 μg/ml for R. reniformis. Overall, the toxicity of cyclobutrifluram to these nematode species was greater than that of fluopyram, and although the effects of cyclobutrifluram were reversible, low concentrations were effective at reducing the ability of both nematodes to infect tomato roots.

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&lt;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&lt;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.

Fungicide Sensitivity Survey of Pecan Scab in Texas Pecan Orchards

A fungicide sensitivity survey of pecan scab ( Venturia effusa) from eight different locations in seven pecan orchards in Texas was conducted during the summer of 2021. Using a rapid fungicide sensitivity assay based on single discriminatory concentrations, sensitivities to five fungicides (dodine, fentin hydroxide, thiophanate-methyl, propiconazole, and tebuconazole) were evaluated. Pecan orchards with frequent fungicide use tended to have greater levels of reduced sensitivities to demethylation inhibitor (propiconazole and tebuconazole; &gt;40% of conidial germination at 1 µg/ml of active ingredient) and fentin hydroxide fungicides (&gt;64% of conidial germination at 3 µg/ml of active ingredient) compared to those with fewer fungicide applications in the past. All populations tested in this survey exhibited a high level of resistance to the thiophanate-methyl fungicide but no resistance to dodine. Thiophanate-methyl did not inhibit conidial germination for six scab populations, while dodine consistently inhibited conidial germination for scab populations tested at the discriminatory concentration. The cytochrome b gene was sequenced for determining mutations associated with quinone outside inhibitor (QoI) fungicides. The mutation that results in a substitution of glycine 137 to serine (G137S) in the cytochrome b gene and is related to high resistance to QoI was not found in the representative isolates of scab populations tested in this survey. Our findings are the first report of fungicide resistance development in Texas pecan orchards. Information about fungicide sensitivities of scab populations in orchards will help pecan producers to prepare proper fungicide treatment plans.

Microscopic Characterization of the Infectious Process, ROS Production, and Fungi Cellular Death of Alternaria alternata on Tangerine Resistant to QoIs.

Quinone outside inhibitor (QoI) fungicide resistance in Alternaria alternata populations was reported in Brazil for the first time in 2019, in São Paulo orchards, and the mutation G143A in cytochrome b (cytb) was found in resistant isolates. Our study investigated the infectious process, production of reactive oxygen species (ROS), and fungal cell death in resistant (QoI-R) and sensitive (QoI-S) A. alternata pathotype tangerine (Aapt) isolates. Morphological characterization of Aapt isolates was performed using confocal laser scanning microscopy (CLSM). Alternaria brown spot (ABS) symptoms were produced by Aapt isolates on tangelo cv. BRS Piemonte. Germination of QoI-R conidia and production of germ tubes on tangelo leaflets treated with 100 μg mL-1 of pyraclostrobin 18 h after inoculation (hai) was observed using scanning electron microscopy (SEM). At the same time, QoI-S conidial germination was inhibited on tangelo leaflets treated with pyraclostrobin. ROS production and cell death in Aapt isolates at high fungicide concentrations were observed using CLSM. QoI-S conidia exhibited high ROS production, indicating high oxidative stress. When dyed with propidium iodate (PI), QoI-S conidia emitted red fluorescence, showing cell death and confirming their sensitive phenotype. In contrast, QoI-R conidia neither produced ROS nor exhibited red fluorescence, indicating no cell death and confirming their resistant phenotype. Therefore, our findings evidence that microscopic techniques may help characterize events during fungi-plant interactions, ROS production, cell death, and Aapt phenotypes resistant and sensitive to QoIs using fluorometric protocols.

Derivation of ecological safety thresholds and risk assessment of new SDHI fungicides in farmland system

Succinate dehydrogenase inhibitor (SDHI) fungicides have become some of the top-selling fungicides in recent years. As the utilization of these fungicides intensifies, the corresponding potential risks to the environment proportionately increase. However, there is still limited knowledge about their toxic effects on ecosystems. In this study, acute toxicity data from laboratory assessments of the springtail Folsomia candida, alongside collected data from terrestrial and aquatic non-target species, were utilized to construct a species sensitivity distribution (SSD) model for both terrestrial and aquatic non-target organisms. Subsequently, we derived ecological baseline values for diverse scenarios within ecosystems. The results indicated that benzovindiflupyr exhibited the highest 7-day median lethal concentration (7d-LC50) to Folsomia candida at 2.0 μg cm−2, while the toxicity levels of other SDHI fungicides varied, ranging from 99 to 304 μg cm−2. In agricultural environments, the Hazard Concentration for 5 % of species (HC5) values for fluxapyroxad, boscalid, sedaxane, and isopyrazam were determined to be 8.0, 1240, 12.97, and 25.37 g ha−1, respectively. In aquatic environments, the HC5 values for benzovindiflupyr, fluxapyroxad, boscalid, sedaxane, isopyrazam, and carboxin were 0.0013, 0.022, 1.76, 0.372, 0.013, and 0.161 mg L−1, respectively. In an evaluation of typical agricultural scenarios within China, SDHI fungicides were found to exert substantial ecological risks to terrestrial non-target fauna and aquatic ecosystems around agricultural fields. Specifically, isopyrazam and fluxapyroxad were identified as posing heightened ecological risks to Typhlodromus pyri and Aphidius rhopalosiphi. Moreover, the application of benzovindiflupyr, carboxin, isopyrazam, and fluxapyroxad in paddy field environments is associated with unacceptable risks to groundwater. The findings of this study contribute significantly to the environmental risk evaluation of SDHI fungicides within farmland system, thereby informing the development of policy frameworks for their scientifically grounded application.

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.

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.

Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

Pepper anthracnose, caused by &lt;i&gt;Colletotrichum&lt;/i&gt; spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of &lt;i&gt;Colletotrichum&lt;/i&gt; spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.