Sensitivity of Botrytis cinerea from Vineyards to Boscalid, Isofetamid, and Pydiflumetofen in Shandong Province, China.

Succinate dehydrogenase inhibitor (SDHI) fungicides are the most commonly and effectively used class of fungicides for controlling gray mold. Among them, only boscalid has been registered in China for controlling grape gray mold, whereas isofetamid and pydiflumetofen are two new SDHI fungicides that have demonstrated high efficacy against various fungal diseases. However, the sensitivity of Botrytis cinerea isolates from vineyards in China to these three fungicides is currently unknown. In this study, the sensitivity of 55 B. cinerea isolates from vineyards to boscalid, isofetamid, and pydiflumetofen was determined, with the effective concentrations for inhibiting 50% of spore germination (EC50) values ranging from 1.10 to 393, 0.0300 to 42.0, and 0.0990 to 25.5 µg ml-1, respectively. The resistance frequencies for boscalid, isofetamid, and pydiflumetofen were 60.0, 7.2, and 12.8%, respectively. Three mutations (H272R, H272Y, and P225F) were detected in the SdhB subunit, with H272R being the most prevalent (75.7%), followed by H272Y (16.2%) and P225F (8.1%). All three mutations are associated with resistance to boscalid, and of them, H272R mutants exhibited high resistance. Only P225F and H272Y mutants exhibited resistance to isofetamid and pydiflumetofen, respectively. A weakly positive cross-resistance relationship was observed between boscalid and pydiflumetofen (r = 0.38, P < 0.05). Additionally, the H272R mutants showed no significant fitness costs, whereas the remaining mutants exhibited reduced mycelial growth (P225F) and sporulation (H272Y and P225F). These results suggest that isofetamid and pydiflumetofen are effective fungicides against B. cinerea in vineyards, but appropriate rotation strategies must be implemented to reduce the selection of existing SDHI-resistant isolates.

Do Growers Using Solo Fungicides Affect the Durability of Disease Control of Growers Using Mixtures and Alternations? The Case of Spot-Form Net Blotch in Western Australia.

Growers often use alternations or mixtures of fungicides to slow down the development of resistance to fungicides. However, within a landscape, some growers will implement such resistance management methods, whereas others do not, and may even apply solo components of the resistance management program. We investigated whether growers using solo components of resistant management programs affect the durability of disease control in fields of those who implement fungicide resistance management. We developed a spatially implicit semidiscrete epidemiological model for the development of fungicide resistance. The model simulates the development of epidemics of spot-form net blotch disease, caused by the pathogen Pyrenophora teres f. maculata. The landscape comprises three types of fields, grouped according to their treatment program, with spore dispersal between fields early in the cropping season. In one field type, a fungicide resistance management method is implemented, whereas in the two others, it is not, with one of these field types using a component of the fungicide resistance management program. The output of the model suggests that the use of component fungicides does affect the durability of disease control for growers using resistance management programs. The magnitude of the effect depends on the characteristics of the pathosystem, the degree of inoculum mixing between fields, and the resistance management program being used. Additionally, although increasing the amount of the solo component in the landscape generally decreases the lifespan within which the resistance management program provides effective control, situations exist where the lifespan may be minimized at intermediate levels of the solo component fungicide. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Effect of Fluopyram on Pratylenchus penetrans on Corn in the Field and In Vitro.

The effects of a fluopyram seed treatment on lesion nematodes (Pratylenchus spp.) and other plant-parasitic nematodes (PPNs) were evaluated on corn in multiple field locations in 2020 and 2021. The highest rate of fluopyram seed treatment (0.15 mg seed-1) reduced early season population density of lesion nematodes compared with the base treatment control in 2020 only. However, fluopyram did not affect late season lesion nematode population density and corn yields. Fluopyram seed treatment also had minimal or nonsignificant effects on other PPN species. Based on these results, the effects of fluopyram were tested in vitro on Pratylenchus penetrans. Results demonstrated that fluopyram severely affected motility in P. penetrans. The sensitivity of P. penetrans second-stage juveniles (J2s) to fluopyram was significantly higher than at J4 and adult, suggesting that sensitivity to fluopyram is dependent on developmental stage. In addition, the effects of fluopyram were reversible at an EC50 but were irreversible at the maximum concentration (25 µg/ml). Overall, our results indicate that fluopyram has potential for controlling P. penetrans, but its efficacy is variable depending on nematode developmental stage and chemical concentration. Further research is needed to determine if these impacts can translate to field scenarios.

Development and implementation of a novel CAPS assay reveals high prevalence of a boscalid resistance marker and its co-occurrence with an azole resistance marker in Erysiphe necator.

Succinate dehydrogenase inhibitors (SDHIs), are frequently used against powdery mildew (PM) fungi, such as Erysiphe necator, the causal agent of grapevine PM. Fungicide resistance, however, hinders effective control. DNA-based monitoring facilitates the recognition of resistance. We aimed (i) to adapt an effective method to detect a widespread genetic marker of resistance to boscalid, a commonly used SDHI, and (ii) to study the co-occurrence of the marker with a marker of resistance to demethylase inhibitor (DMI) fungicides. Sequencing of the sdhB gene identified a non-synonymous substitution, denoted as sdhB-A794G, leading to an amino acid change (H242R) in the sdhB protein. In vitro fungicide resistance tests showed that E. necator isolates carrying sdhB-A794G were resistant to boscalid. We adopted a cleaved amplified polymorphic sequence-based method and screened more than 500 field samples collected from five Hungarian wine regions in two consecutive years. The sdhB-A794G marker was detected in all wine regions and in both years, altogether in 61.7% of samples, including 20.5% in which both sdhB-A794G and the wild-type were present. The frequency of sdhB-A794G was higher in SDHI-treated vineyards than in vineyards without any SDHI application. A significant difference in the presence of the marker was detected among wine regions; its prevalence ranged from none to 100%. We identified significant co-occurrence of sdhB-A794G with the CYP51-A495T (Y136F) mutation of the CYP51 gene, a known marker of resistance to DMIs. The monitoring of fungicide resistance is fundamental for the successful control of E. necator. Our rapid, cost-effective diagnostic method will support decision-making and fungicide resistance monitoring and management.

Resistance Profiles of Botrytis cinerea to Fluxapyroxad from Strawberry Fields in Shanghai, China.

Fluxapyroxad, a type of succinate dehydrogenase inhibitor fungicide, has been used to control the growth of gray mold on strawberry for more than 5 years in China. Selection for resistance to the causal agent Botrytis cinerea became a threat to the efficacy of fluxapyroxad. In total, 160 B. cinerea isolates collected from Shanghai during 2020 and 2021 were tested for their resistance to fluxapyroxad using mycelial growth inhibition. The results indicated that the curve of baseline sensitivity was unimodal, with an approximately normal distribution and a mean effective concentration of the fungicide that inhibited growth by 50% (EC50) of 0.18 ± 0.01 µg/ml. In total, 27.78 and 48.57% of isolates were resistant to fluxapyroxad in 2020 and 2021, respectively, where the lowest EC50 value of mycelial growth inhibition was 0.03 µg/ml and the highest value was 51.3 µg/ml. Resistance mechanism analysis showed that the succinate dehydrogenase subunit B (SdhB) N230I mutation could lead to resistance and P225F mutation could lead to higher resistance. These data suggest that the resistance frequency of B. cinerea isolates to fluxapyroxad increased in 2021 compared with 2020, which requires appropriate fungicide rotation strategies to be implemented in order to control gray mold on strawberry in the future.

A Rapid Molecular Detection System for Sdh Mutations Conferring Differential Succinate Dehydrogenase Inhibitor Resistance in Corynespora cassiicola.

Cucumber leaf spot, caused by Corynespora cassiicola, is a serious disease of cucumbers in greenhouses. Due to the frequent application of succinate dehydrogenase inhibitors (SDHIs), resistance caused by point mutations in the SDHB/C/D gene has been reported. Different mutations lead to different resistance levels, and mutations vary over time and regions. This means that it is necessary to know the type of mutation in the field to select the appropriate SDHIs. Here, the sensitivity of mutations to SDHIs was determined, and eight resistance patterns were obtained: pattern I (BosVHR, FluoMR, PenHR, CarR); pattern II (BosMR, FluoSS, PenS, CarS); pattern III (BosVHR, FluoSS, PenLR, CarS); pattern IV (BosLR, FluoLR, PenS, CarR); pattern V (BosMR, FluoLR, PenS, CarS); pattern VI (BosMR, FluoLR, PenLR, CarS); pattern VII (BosVHR, FluoHR, PenHR, CarS); and pattern VIII (BosLR, FluoLR, PenLR, CarS). We successfully established nine allele-specific PCR (AS-PCR) assays that can detect mutation types. The sensitivity and specificity of AS-PCR were also determined. The sensitivity results showed that most of the detection thresholds of the AS-PCR assays were 100 pg/µl, while the AS-PCR assay of the B-H278R and D-G109V mutations exhibited high sensitivity, with 10 pg/µl. To validate the use of the developed AS-PCR assay, DNA from leaves inoculated with known mutations was extracted, detected by AS-PCR, and sequenced. The results showed good similarity between the two methods. Additionally, to rapidly detect mutations in the CcSdhD gene, we developed a single-tube multiplex allele-specific PCR (MAS-PCR) assay. In conclusion, AS-PCR and MAS-PCR were established for mutation detection and targeted control of CLS.

Risk Evaluation of Benzovindiflupyr Resistance of Verticillium dahliae Population in Maine.

Verticillium dahliae causes Verticillium wilt, resulting in significant losses to potato production. Benzovindiflupyr, a succinate dehydrogenase inhibitor, effectively controls V. dahliae. However, frequent applications of the chemical may expedite the development of fungicide resistance in the pathogen population. To evaluate the risk of benzovindiflupyr resistance, 38 V. dahliae strains were obtained from diseased potatoes in Maine. The sensitivity of the field population was determined based on effective concentration for 50% inhibition (EC50), which ranged from 0.07 to 11.28 µg ml-1 with a median of 1.08. Segregated clusters of EC50 values indicated that Maine V. dahliae populations have developed benzovindiflupyr resistance. By exposing conidia of V. dahliae to a high concentration of benzovindiflupyr, 18 benzovindiflupyr-resistant mutants were obtained. To examine their fitness, the mutants were continuously subculture-transferred for up to 10 generations. Mycelial growth, conidial production, competitiveness, pathogenicity, and cross resistance of the 10th generation mutants were examined. Results showed that 50% of the resistant mutants retained an adaptive level in mycelial growth, and 60% maintained conidial production similar to their parents. Pathogenicity did not change for any of the mutants. No cross resistance was detected between benzovindiflupyr and either azoxystrobin, boscalid, fluopyram, or pyrimethanil. Thus, the resistance risk in V. dahliae to benzovindiflupyr should be considered in Maine potato production.

Impact of Exposure to Pyraclostrobin and to a Pyraclostrobin/Boscalid Mixture on the Mitochondrial Function of Human Hepatocytes.

Fungicides are widely used in agriculture for crop protection. Succinate dehydrogenase inhibitors (SDHIs) and strobilurins inhibit mitochondria electron transport chain (ETC) in fungi, by blocking complex II and complex III, respectively. Questions regarding their selectivity of action for fungi have been raised in the literature, and we previously showed that boscalid and bixafen (SDHIs) alter the mitochondrial function of human hepatocytes. Here, we analyzed the impact of the exposure of human hepatocytes to pyraclostrobin, a fungicide belonging to the class of strobilurins. Using electron paramagnetic resonance (EPR), we observed a decrease in oxygen consumption rate (OCR) and an increase in mitochondrial superoxide levels after 24 h exposure to 0.5 µM concentration. As a consequence, the content in ATP amount in the cells was reduced, the ratio reduced/oxidized glutathione was decreased, and a decrease in cell viability was observed using three different assays (PrestoBlue, crystal violet, and annexin V assays). In addition, as SDHIs and strobilurins are commonly associated in commercial preparations, we evaluated a potential "cocktail" toxic effect. We selected low concentrations of boscalid (0.5 µM) and pyraclostrobin (0.25 µM) that did not induce a mitochondrial dysfunction in liver cells when used separately. In sharp contrast, when both compounds were used in combination at the same concentration, we observed a decrease in OCR, an increase in mitochondrial superoxide production, a decrease in the ratio reduced/oxidized glutathione, and a decrease in cell viability in three different assays.

Comparative Transcriptome Analyses Reveal Conserved and Distinct Mechanisms of the SDHI Fungicide Benzovindiflupyr Inhibiting Colletotrichum.

Colletotrichum leaf disease (CLD) is an annual production concern for commercial growers worldwide. The succinate dehydrogenase inhibitor (SDHI) fungicide benzovindiflupyr shows higher bioactivity against CLD than other SDHIs. However, the mechanism underlying such difference remains unclear. In this study, benzovindiflupyr exhibits good inhibitory activity against Colletotrichum siamense and C. nymphaeae in vitro and in vivo. To reveal its mechanism for inhibiting Colletotrichum, we compared transcriptomes of C. siamense and C. nymphaeae under treatment with benzovindiflupyr and boscalid. Benzovindiflupyr exhibited higher inhibitory activity against SDH enzyme than boscalid, resulting in a greater reduction in the ATP content of Colletotrichum isolates. Most of the metabolic pathways induced in these fungicide-treated isolates were similar, indicating that benzovindiflupyr exhibited a conserved mechanism of SDHIs inhibiting Colletotrichum. At the same level of suppressive SDH activity, benzovindiflupyr activated more than three times greater gene numbers of Colletotrichum than boscalid, suggesting that benzovindiflupyr could activate distinct mechanisms against Colletotrichum. Membrane-related gene ontology terms, mainly including intrinsic components of membrane, were highly abundant for the benzovindiflupyr-treated isolates rather than boscalid-treated isolates. Only benzovindiflupyr increased the relative conductivities of hyphae, indicating that it could damage the cell membrane and increase mycelial electrolyte leakage. Thus, we proposed that the high bioactivity of benzovindiflupyr against Colletotrichum occurred by inhibiting SDH activity and damaging the cell membrane at the same time. The research improves our understanding the mode of action of SDHI fungicides against Colletotrichum.

Rapidly Increasing Boscalid Resistance in Corynespora cassiicola in China.

Corynespora leaf spot caused by Corynespora cassiicola is an important foliar disease in cucumber. Succinate dehydrogenase inhibitors are the main fungicides used to control this disease. With the application of succinate dehydrogenase inhibitors (SDHIs) in the field, boscalid-resistant isolates have been continuously detected in the field. Resistance monitoring programs were performed to investigate the frequency and genotypes of resistant isolates. In our resistance monitoring, the frequency of resistant isolates rapidly increased from 9.68 to 85.88% in 2005 to 2020. Nine genotypes conferring SDHI resistance were found in resistant isolates, with different levels of resistance to SDHIs: B-H278R, B-H278L, B-H278Y, B-I280V, C-N75S, C-S73P, D-D95E, D-H105R, and D-G109V. The first sdh mutation was detected in Hebei Province in China, conferring an amino acid substitution at codon 278 in the sdhB subunit from histidine to tyrosine (B-H278Y), and it was the dominant resistance genotype in 2014 to 2015. Subsequently, other genotypes were gradually detected in the field, and the dominant mutations varied across years and across regions. The newest genotype (B-H278L) conferring SDHI resistance was found in 2020. To the best of our knowledge, this is the first report of C. cassiicola in cucumber. To date, multiple resistance to SDHIs, quinone outside inhibitors, benzimidazole fungicides, and dicarboximide fungicides have been detected, accounting for 75.64% of SDHI-resistant isolates. Therefore, the above four fungicides must be strictly restricted, and further monitoring work in other provinces with more isolates should be performed in the future.

Aggressiveness of Small-Spored Alternaria spp. and Their Sensitivity to Succinate Dehydrogenase Inhibitor Fungicides.

Brown leaf spot of potato is caused by a number of small-spored Alternaria spp. Alternaria alternata sensu stricto, A. arborescens, and A. tenuissima have been reported with increasing frequency in commercial potato fields. Potato cultivars with resistance to small-spored Alternaria spp. have yet to be developed; therefore, the application of foliar fungicides is a primary management strategy. Greenhouse inoculation assays demonstrated that isolates of these three small-spored Alternaria spp. were pathogenic. Significant differences in aggressiveness were observed across isolates; however, there was no trend in aggressiveness based on species. Significant fungicide by isolate interactions in in vitro fungicide sensitivity and significant differences between baseline and nonbaseline isolates were observed in all three small-spored Alternaria spp. The ranges of in vitro sensitivity of A. alternata baseline isolates to boscalid (EC50 <0.010 to 0.89 µg/ml), fluopyram (<0.010 to 1.14 µg/ml) and solatenol (<0.010 to 1.14 µg/ml) were relatively wide when compared with adepidyn (<0.010 to 0.023 µg/ml). The baseline sensitivities of A. arborescens and A. tenuissima isolates to all four fungicides were <0.065 µg/ml. Between 10 and 21% of nonbaseline A. alternata isolates fell outside the baseline range established for the four succinate dehydrogenase inhibitor (SDHI) fungicides evaluated. In A. arborescens, 10 to 80% of nonbaseline isolates had higher sensitivities than the baseline. A. tenuissima isolates fell outside the baseline for boscalid (55%), fluopyram (14%), and solatenol (14%), but none fell outside the baseline range for adepidyn. Evaluations of in vivo fungicide efficacy demonstrated that most isolates were equally controlled by the four SDHI fungicides. However, reduced boscalid efficacy was observed for four isolates (two each of A. arborescens and A. tenuissima) and reduced fluopyram control was observed in one A. alternata isolate. Results of these studies demonstrate that isolates of all three species could be contributing to the brown leaf spot pathogen complex and that monitoring both species diversity and fungicide sensitivity could be advantageous for the management of brown leaf spot in potatoes with SDHI fungicides.

Comparison of Current Peanut Fungicides Against Athelia rolfsii Through a Laboratory Bioassay of Detached Plant Tissues.

Southern stem rot of peanut, caused by Athelia rolfsii, is an important fungal disease that impacts peanut production worldwide. Foliar-applied fungicides are used to manage the disease, and several fungicides have been recently registered for southern stem rot control in peanuts. This study compared fungicidal, residual, and potential systemic activity of current fungicides against A. rolfsii using a laboratory bioassay. Peanut plants grown in the field were treated with eight fungicides approximately 90 days after planting, and plants were collected for the laboratory bioassay weekly for 5 weeks following application. Peanut plants were separated into the newest fully mature leaf present at sample collection, the second newest fully mature leaf present at the time of fungicide application, the upper stem, and the crown tissues. Each plant tissue was inoculated with A. rolfsii then incubated at 30°C for 2 days. Lesion length was measured, and percent inhibition of fungal growth by each fungicide relative to the control was calculated. All fungicides provided the greatest inhibition of A. rolfsii on leaf tissues that were present at the time of fungicide application, followed by the newly grown leaf and upper stem. Little inhibition occurred on the crown. Fungal inhibition decreased at similar rates over time for all fungicides tested. Succinate dehydrogenase inhibitors provided less basipetal protection of upper stems than quinone outside inhibitor or demethylation inhibitor fungicides. Properties of the fungicides characterized in this study, including several newly registered products, are useful for developing fungicide application recommendations to maximize their efficacy in controlling both foliar and soilborne peanut diseases.

Effect of In-Furrow Application of Fluopyram on Leaf Spot Diseases of Peanut.

In peanut (Arachis hypogaea) production, in-furrow applications of the premix combination of the succinate-dehydrogenase-inhibitor (SDHI) fungicide and nematicide fluopyram and the insecticide imidacloprid are used primarily for management of nematode pests and for preventing feeding damage on foliage caused by tobacco thrips (Frankliniella fusca). Fluopyram is also active against many fungal pathogens. However, the effect of in-furrow applications of fluopyram on early leaf spot (Passalora arachidicola) or late leaf spot (Nothopassalora personata) has not been characterized. The purpose of this study was to determine the effects of in-furrow applications of fluopyram + imidacloprid or fluopyram alone on leaf spot epidemics. Field experiments were conducted in Tifton, GA in 2015, 2016, and 2018 to 2020. In all experiments, in-furrow applications of fluopyram + imidacloprid provided extended suppression of early leaf spot and late leaf spot epidemics compared with the nontreated control. In 2020, there was no difference between the effects of fluopyram + imidacloprid and fluopyram alone on leaf spot epidemics. Results indicated that fluopyram could complement early-season leaf spot management programs. Use of in-furrow applications of fluopyram should be considered as an SDHI fungicide application for resistance management purposes.

Impact of SDH Mutations in Alternaria solani on Recently Developed SDHI Fungicides Adepidyn and Solatenol.

Early blight, caused by Alternaria solani, is observed annually in all midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, A. solani has developed reduced sensitivity or resistance to many single-site fungicides such as quinone outside inhibitor (QoI, FRAC group 11), succinate dehydrogenase inhibitor (SDHI, FRAC group 7), demethylation inhibitor (DMI, FRAC group 3), and anilinopyrimidine (AP, FRAC group 9) fungicides. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides used commercially on a variety of crops, including potato. Five SDH mutations have been characterized previously in A. solani that affect the efficacy of boscalid while only one of these mutations has been demonstrated to negatively affect fluopyram efficacy. Conidial germination assays were used to determine if a shift in sensitivity has occurred in these SDHI fungicides. A. solani isolates collected prior to the commercial application of SDHI fungicides (baseline) were compared with recently collected isolates (nonbaseline). Greenhouse evaluations were conducted also to evaluate the efficacy of boscalid, fluopyram, solatenol, and adepidyn on A. solani isolates possessing individual SDH mutations. Additionally, field trials were conducted to determine the effects of application of these SDHI fungicides on the frequency of SDH mutations. Fluopyram, solatenol, and adepidyn had high intrinsic activity against A. solani when compared with boscalid, based on in vitro assays. The application of adepidyn and solatenol resulted in greater early blight control than the application of boscalid and fluopyram in greenhouse experiments. Molecular characterization of A. solani isolates collected from the field trials determined that the frequency of the H134R-mutation can increase in response to more recently developed SDHI fungicides. In contrast, the H278R/Y- and H133R-mutations decreased to the point of being nearly absent in these field experiments.

Sensitivity of Rhizoctonia solani Anastomosis Group 2-2 Isolates from Soybean and Sugar Beet to Selected SDHI and QoI Fungicides.

Rhizoctonia solani causes root and stem diseases on soybean and sugar beet, and fungicides are commonly used to manage these diseases. Quinone outside inhibitor (QoI) fungicides (pyraclostrobin and azoxystrobin) have been used for in-furrow and postemergence application since 2000. Succinate dehydrogenase inhibitor (SDHI) fungicides (sedaxane, penthiopyrad, and fluxapyroxad) became popular seed treatments after their registration in Minnesota and North Dakota between 2012 and 2016. Periodic monitoring of sensitivity to these fungicides in R. solani anastomosis group (AG) 2-2 is important to detect potential shifts in sensitivity over time. R. solani AG 2-2 isolates (n = 35) collected from soybean and sugar beet in Minnesota and North Dakota were evaluated in vitro for sensitivity. Isolates were considered as baseline or nonbaseline for the above-mentioned fungicides based on previous potential exposure. The effective concentration (EC50) required to suppress radial fungal growth by 50% was determined. The mean EC50 values for sedaxane, penthiopyrad, fluxapyroxad, and pyraclostrobin were 0.1, 0.15, 0.16, and 0.25 (µg ml-1), respectively. The mean EC50 value for azoxystrobin for 22 isolates was 0.76 to 1.56 µg ml-1; and EC50 could not be determined for 13 isolates because of <50% inhibition at the highest concentrations used. The EC50 values for the QoI fungicides did not differ significantly between baseline and nonbaseline isolates. EC50 values for SDHI fungicides were significantly higher for isolates collected from soybean than from sugar beet, and isolates collected from both crops had similar EC50 values for pyraclostrobin. All SDHI fungicides and pyraclostrobin effectively suppressed R. solani isolates from soybean and sugar beet at low concentrations in vitro.

Field Assessment of Six Point-Mutations in SDH Subunit Genes Conferring Varying Resistance Levels to SDHIs in Clarireedia spp.

Dollar spot, caused by Clarireedia spp. (formerly Sclerotinia homoeocarpa F.T. Bennett), is the most economically important turfgrass disease causing considerable damage on golf courses. While cultural practices are available for reducing dollar spot infection, chemical fungicide use is often necessary for maintaining optimal turf quality. Since the release of boscalid in 2003, the succinate dehydrogenase inhibitor (SDHI) class has become an invaluable tool for managing dollar spot. However, resistance to this class has recently been reported in Clarireedia spp. and many other plant pathogenic fungi. After SDHI field failure on four golf courses and one university research plot, a total of six unique SDH mutations conferring differential in vitro sensitivities to SDHIs have been identified in Clarireedia spp. In 2018 and 2019, turf research plots were inoculated with sensitive, non-mutated isolates of Clarireedia spp., as well as resistant isolates harboring each unique identified mutation. Fungicide efficacy trials were conducted on inoculated plots to assess differential sensitivity to five SDHI active ingredients (boscalid, fluxapyroxad, isofetamid, fluopyram, and pydiflumetofen) across mutations under field conditions. Results indicate unique mutations are associated with distinct SDHI field efficacy profiles as shown in in-vitro sensitivity assays. Isolate populations with B subunit mutations (H267Y/R) were more sensitive to fluopyram, whereas isolate populations with C subunit mutations (C-G91R, C-G150R) showed resistance to all SDHIs tested. Mutation-associated differential sensitivity observed under field conditions indicates a need for a nation-wide survey and frequent monitoring of SDHI sensitivity of dollar spot populations on golf courses in the USA. Further, the information gained from this study will be useful in providing sustainable management recommendations for controlling site-specific resistant populations of Clarireedia spp.

Baseline and Temporal Changes in Sensitivity of Zymoseptoria tritici Isolates to Benzovindiflupyr in Oregon, U.S.A., and Cross-Sensitivity to Other SDHI Fungicides.

Zymoseptoria tritici is the causal agent of Septoria tritici blotch (STB), a disease of wheat (Triticum aestivum) that results in significant yield loss worldwide. Z. tritici's life cycle, reproductive system, effective population size, and gene flow put it at high likelihood of developing fungicide resistance. Succinate dehydrogenase inhibitor (SDHI) fungicides (FRAC code 7) were not widely used to control STB in the Willamette Valley until 2016. Field isolates of Z. tritici collected in the Willamette Valley at dates spanning the introduction of SDHI (2015 to 2017) were screened for sensitivity to four SDHI active ingredients: benzovindiflupyr, penthiopyrad, fluxapyroxad, and fluindapyr. Fungicide sensitivity changes were determined by the fungicide concentration at which fungal growth is decreased by 50% (EC50) values. The benzovindiflupyr EC50 values increased significantly, indicating a reduction in sensitivity, following the adoption of SDHI fungicides in Oregon (P < 0.0001). Additionally, significant reduction in cross-sensitivity among SDHI active ingredients was also observed with a moderate and significant relationship between penthiopyrad and benzovindiflupyr (P = 0.0002) and a weak relationship between penthiopyrad and fluxapyroxad (P = 0.0482). No change in cross-sensitivity was observed with fluindapyr, which has not yet been labeled in the region. The results document a decrease in SDHI sensitivity in Z. tritici isolates following the introduction of the active ingredients to the Willamette Valley. The reduction in cross-sensitivity observed between SDHI active ingredients highlights the notion that careful consideration is required to manage fungicide resistance and suggests that within-group rotation is insufficient for resistance management.

The Short-Term Exposure to SDHI Fungicides Boscalid and Bixafen Induces a Mitochondrial Dysfunction in Selective Human Cell Lines.

Fungicides are used to suppress the growth of fungi for crop protection. The most widely used fungicides are succinate dehydrogenase inhibitors (SDHIs) that act by blocking succinate dehydrogenase, the complex II of the mitochondrial electron transport chain. As recent reports suggested that SDHI-fungicides could not be selective for their fungi targets, we tested the mitochondrial function of human cells (Peripheral Blood Mononuclear Cells or PBMCs, HepG2 liver cells, and BJ-fibroblasts) after exposure for a short time to Boscalid and Bixafen, the two most used SDHIs. Electron Paramagnetic Resonance (EPR) spectroscopy was used to assess the oxygen consumption rate (OCR) and the level of mitochondrial superoxide radical. The OCR was significantly decreased in the three cell lines after exposure to both SDHIs. The level of mitochondrial superoxide increased in HepG2 after Boscalid and Bixafen exposure. In BJ-fibroblasts, mitochondrial superoxide was increased after Bixafen exposure, but not after Boscalid. No significant increase in mitochondrial superoxide was observed in PBMCs. Flow cytometry revealed an increase in the number of early apoptotic cells in HepG2 exposed to both SDHIs, but not in PBMCs and BJ-fibroblasts, results consistent with the high level of mitochondrial superoxide found in HepG2 cells after exposure. In conclusion, short-term exposure to Boscalid and Bixafen induces a mitochondrial dysfunction in human cells.

A revised model of fungicide translaminar activity.

Translaminar redistribution is valuable for fungicide activity but difficult to measure and predict. The translaminar activity of 38 fungicides active against cucumber powdery mildew was measured experimentally and used to develop a QSAR (Quantitative structure-activity relationship) model of translaminar movement from calculated parameters. Over 300 physiochemical parameters generated from energy-minimized 3D structures were considered and one-parameter, two-parameter, and five-parameter models were developed. The one-parameter lipophilicity model explained 39% of variability in translaminar activity in the full dataset but none of the variability in the small succinate dehydrogenase inhibitor (SDHI) set. Adding a polar surface area parameter to the lipophilicity parameter improved predictability to 52% and explained over 70% of the variability in the SDHI class. The expanded model with five physiochemical parameters explained more than 80% of the variability in overall translaminar redistribution. The three additional parameters were correlated with molecular size and reactivity. The models were validated with a Leave-One-Out method that showed excellent robustness (r2adj = 0.83, q2 = 0.79, p < .0001) for the five-parameter model. Because the models require only calculated parameters from a 3D chemical structure, they could enable the design or selection of compounds likely to be translaminar.

In vitro toxicity of fungicides with different modes of action to alfalfa anthracnose fungus, Colletotrichum destructivum.

Sensitivity of 24 isolates of Colletotrichum destructivum O'Gara, collected from alfalfa plants in Serbia, to eight selected fungicides, was investigated in this study. Molecular identification and pathogenicity test of isolates tested were also performed. Fungicide sensitivity was evaluated in vitro, using mycelial growth assay method. All isolates exhibited significant pathogenicity, causing necrosis at the alfalfa seedling root tips two days after inoculation. Using the primer pair GSF1-SR1 and by comparing the amplified fragments of the tested isolates with the marker (M), the presence of the amplicon of the expected size of about 900 bp was determined for all isolates. The isolates tested in this study showed different sensitivity towards fungicides in vitro. Mycelial growth was highly inhibited by QoI (quinone outside inhibitors) fungicide pyraclostrobin (mean EC50=0.39 µg mL-1) and by DMI (demethylation-inhibiting) fungicide tebuconazole (mean EC50=0.61 µg mL-1), followed by azoxystrobin (mean EC50=2.83 µg mL-1) and flutriafol (mean EC50=2.11 µg mL-1). Multi-site fungicide chlorothalonil and MBC (methyl benzimidazole carbamate) fungicide thiophanate-methyl evinced moderate inhibition with mean EC50=35.31 and 62.83 µg mL-1, respectively. Thirteen isolates were sensitive to SDHI (succinate dehydrogenase inhibitors) fungicide boscalid and fluxapyroxad, (mean EC50=0.49 and 0.19 µg mL-1, respectively), while the rest of isolates were highly resistant.