RESUMEN

The destructive disease gray leaf spot, caused by Stemphylium solani, is prevalent in tomato plants in China. A variety of fungicides have been extensively used for controlling the disease, with a particular focus on succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs). However, there was a lack of information regarding the resistance of S. solani to boscalid (SDHI) and pyraclostrobin (QoI) in China. In this study, the sensitivity of S. solani to boscalid and pyraclostrobin was monitored. The EC50 values for boscalid ranged from 0.02 to 3.0 µg∙mL-1, with an average value of 0.62 µg∙mL-1, while the EC50 values for pyraclostrobin ranged from 0.21 to 14.71 µg∙mL-1, with an average value of 6.03 µg∙mL-1. Based on these findings, the frequencies of observed resistance were as follows: 36.7% for boscalid and 50% for pyraclostrobin; while the resistance frequency to both boscalid and pyraclostrobin in S. solani was 19.4%. The mutation associated with boscalid resistance in S. solani within tomato fields was identified as SdhB-H277Y, while the mutation related to pyraclostrobin resistance was found in cytochrome b, specifically Cytb-G143A. The resistant mutants displayed diminished fitness in terms of mycelial growth, yet their pathogenicity exhibited no significant disparities. To delay the development of resistance, it is advisable to employ a rotation strategy using alternative fungicides with different modes of action or mix with fungicides with multi-site-contact activity for disease management.

Asunto(s)

Ascomicetos , Compuestos de Bifenilo , Farmacorresistencia Fúngica , Fungicidas Industriales , Niacinamida , Enfermedades de las Plantas , Solanum lycopersicum , Estrobilurinas , Estrobilurinas/farmacología , Solanum lycopersicum/microbiología , Fungicidas Industriales/farmacología , Enfermedades de las Plantas/microbiología , Niacinamida/farmacología , Niacinamida/análogos & derivados , Farmacorresistencia Fúngica/genética , China , Compuestos de Bifenilo/farmacología , Ascomicetos/efectos de los fármacos , Ascomicetos/patogenicidad

RESUMEN

BACKGROUND: Plant diseases seriously decrease the yield and quality of agricultural crops. Fungicide treatments remain the main means of field fungi control. However, the residual activity of fungicides is rapidly reduced due to various factors in the natural environment, therefore the development of agents with novel modes of action is desirable. It is highly required to design and develop new fungicides to address the resistance issue. Designing low impact chemicals to safely and sustainably address needs of agriculture. RESULTS: In this work, we used the highly active fluxapyroxad and flutolanil as parent structures, to design and synthesize a series of pyrazole-4-carboxamide derivatives. Some of the pyrazole-4-carboxamide derivatives exhibit fungicidal activities that are comparable to or higher than those of the commercialized fungicides fluxapyroxad and bixafen. In particular, compounds TM-1, TM-2, TM-3, TM-4, TM-5, TM-7 and TM-8 showed excellent fungicidal activities against corn rust that were 2-4 times higher than those of fluxapyroxad and bixafen. Field trial results demonstrated that at the same dosage levels, compound TM-2 exhibited comparable field control efficacy against wheat rust as compared to triadimefon and pyrazophenamide. Molecular docking simulations reveal that compound TM-2 interacts with TRP 173 of succinate dehydrogenase (SDH) through hydrogen bonding, which could explain the probable mechanism of action between compound TM-2 and the target protein. CONCLUSION: These results indicate that compound TM-2 may be a promising fungicide candidate and provide valuable reference for further investigation. © 2024 Society of Chemical Industry.

RESUMEN

BACKGROUND: Gray mold caused by Botrytis cinerea is one of the most serious diseases affecting strawberry. Succinate dehydrogenase inhibitor (SDHI) fungicides have been used for more than a decade to control the disease. Monitoring resistance and improving in-depth understanding of resistance mechanisms are essential for the control of B. cinerea. RESULTS: In this study, resistance monitoring of a SDHI fungicide boscalid was conducted on B. cinerea isolated from strawberries in Korea during 2020 and 2021, with resistance rates of 76.92% and 72.25%, respectively. In resistant strains, mutations P225F/H and H272R were found in SdhB, with P225F representing the dominant mutation type. Simultaneous mutations G85A, I93V, M158V, and V168I in SdhC were detected in 54.84% of sensitive strains. Sensitivity profiles of different Sdh genotypes of B. cinerea strains to six SDHIs were determined in vitro and in vivo. In addition, the mutation(s) were genetically validated through in situ SdhB (SdhC) expression. Docking assays between SDHIs and AlphaFold model-based Sdh complexes revealed generally consistent patterns with their in vitro phenotypes. CONCLUSION: Resistance of B. cinerea to SDHI fungicide on strawberry was systematically investigated in this study. Deciphering of SDHI resistance through the genetic transformation system and AlphaFold model-based molecular docking will provide valuable insights into other target site-based fungicide resistance in fungal pathogens. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RESUMEN

Consumers are exposed to succinate dehydrogenase inhibitor (SDHI) pesticides through their diet. A cumulative dietary risk assessment for the French population has been performed with French monitoring data (2017-2021) and consumption data from INCA3. The calculation followed a two-tiered approach, using deterministic then probabilistic methods. It was carried out, using European health based guidance values (HBGV) derived for each active substance to characterise their toxicity. In Tier I, the calculated hazard index of 0.12 was below the threshold of 1 and in Tier II, the total margin of exposure at percentile 99.9 remains above the trigger value of 100 (1798 [1631-2311]). In Tier II, the three main risk drivers identified at the upper tail of the distribution were strawberries-fluopyram (19.1%), peaches-fluopyram (14.1%) and table grapes-boscalid (10.5%). Finally, the impact of the major sources of uncertainties was qualitatively evaluated. All together, they were considered of low impact on the outcomes. This work demonstrates the absence of unacceptable chronic risk related to the cumulative exposure of SDHI for French consumers during the 2017-2021 period.

Asunto(s)

Plaguicidas , Succinato Deshidrogenasa , Humanos , Francia , Medición de Riesgo , Plaguicidas/toxicidad , Succinato Deshidrogenasa/antagonistas & inhibidores , Adulto , Persona de Mediana Edad , Adulto Joven , Exposición Dietética , Femenino , Masculino , Contaminación de Alimentos/análisis , Adolescente , Anciano , Niño , Inhibidores Enzimáticos/toxicidad , Preescolar

RESUMEN

Bursaphelenchus xylophilus is a dangerous quarantine pest that causes extensive damage to pine ecosystems worldwide. Cyclobutrifluram, a succinate dehydrogenase inhibitor (SDHI), is a novel nematicide introduced by Syngenta in 2013. However, the nematocidal effect of cyclobutrifluram against plant-parasitic nematodes remains underexplored. Therefore, here, we aim to address this knowledge gap by evaluating the toxicity, effects, and mode of action of cyclobutrifluram on B. xylophilus. The result shows that cyclobutrifluram is the most effective agent, with an LC50 value of 0.1078 mg·L-1. At an LC20 dose, it significantly reduced the population size to 10.40 × 103 ± 737.56-approximately 1/23 that of the control group. This notable impact may stem from the agent's ability to diminish egg-laying and hatching rates, as well as to impede the nematodes' development. In addition, it has also performed well in the prevention of pine wilt disease, significantly reducing the incidence in greenhouses and in the field. SDH consists of a transmembrane assembly composed of four protein subunits (SDHA to SDHD). Four sdh genes were characterized and proved by RNAi to regulate the spawning capacity, locomotion ability, and body size of B. xylophilus. The mortality of nematodes treated with sdhc-dsRNA significantly decreased upon cyclobutrifluram application. Molecular docking further confirmed that SDHC, a cytochrome-binding protein, is the target. In conclusion, cyclobutrifluram has a good potential for trunk injection against B. xylophilus. This study provides valuable information for the screening and application of effective agents in controlling and preventing PWD in forests.

Asunto(s)

Antinematodos , Succinato Deshidrogenasa , Tylenchida , Animales , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Antinematodos/farmacología , Tylenchida/efectos de los fármacos , Tylenchida/genética , Tylenchida/fisiología , Pinus/parasitología , Simulación del Acoplamiento Molecular , Enfermedades de las Plantas/parasitología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo

RESUMEN

BACKGROUND: Succinate dehydrogenase inhibitor (SDHI) fungicides play important roles in the control of plant fungal diseases. However, they are facing serious challenges from issues with resistance and cross-resistance, primarily attributed to their frequent application and structural similarities. There is an urgent need to design and develop SDHI fungicides with novel structures. RESULTS: Aiming to discover novel potent SDHI fungicides, 31 innovative pyrazole ß-ketonitrile derivatives with diphenyl ether moiety were rationally designed and synthesized, which were guided by a 3D-QSAR model from our previous study. The optimal target compound A23 exhibited not only outstanding in vitro inhibitory activities against Rhizoctonia solani with a half-maximal effective concentration (EC50) value of 0.0398 µg mL-1 comparable to that for fluxapyroxad (EC50 = 0.0375 µg mL-1), but also a moderate protective efficacy in vivo against rice sheath blight. Porcine succinate dehydrogenase (SDH) enzymatic inhibitory assay revealed that A23 is a potent inhibitor of SDH, with a half-maximal inhibitory concentration of 0.0425 µm. Docking study within R. solani SDH indicated that A23 effectively binds into the ubiquinone site mainly through hydrogen-bonds, and cation-π and π-π interactions. CONCLUSION: The identified ß-ketonitrile compound A23 containing diphenyl ether moiety is a potent SDH inhibitor, which might be a good lead for novel fungicide research and optimization. © 2024 Society of Chemical Industry.

RESUMEN

Plant pathogenic fungi pose a significant threat to agricultural production, necessitating the development of new and more effective fungicides. The ring replacement strategy has emerged as a highly successful approach in molecular design. In this study, we employed the ring replacement strategy to successfully design and synthesize 32 novel hydrazide derivatives containing diverse heterocycles, such as thiazole, isoxazole, pyrazole, thiadiazole, 1,3,4-oxadiazole, 1,2,4-oxadiazole, thiophene, pyridine, and pyrazine. Their antifungal activities were evaluated in vitro and in vivo. Bioassay results revealed that most of the title compounds displayed remarkable antifungal activities in vitro against four tested phytopathogenic fungi, including Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, and Rhizoctonia solani. Especially, compound 5aa displayed a broad spectrum of antifungal activity against F. graminearum, B. cinerea, S. sclerotiorum, and R. solani, with the corresponding EC50 values of 0.12, 4.48, 0.33, and 0.15 µg/mL, respectively. In the antifungal growth assay, compound 5aa displayed a protection efficacy of 75.5% against Fusarium head blight (FHB) at a concentration of 200 µg/mL. In another in vivo antifungal activity evaluation, compound 5aa exhibited a noteworthy protective efficacy of 92.0% against rape Sclerotinia rot (RSR) at a concentration of 100 µg/mL, which was comparable to the positive control tebuconazole (97.5%). The existing results suggest that compound 5aa has a broad-spectrum antifungal activity. Electron microscopy observations showed that compound 5aa might cause mycelial abnormalities and organelle damage in F. graminearum. Moreover, in the in vitro enzyme assay, we found that the target compounds 5aa, 5ab, and 5ca displayed significant inhibitory effects toward succinate dehydrogenase, with the corresponding IC50 values of 1.62, 1.74, and 1.96 µM, respectively, which were superior to that of boscalid (IC50 = 2.38 µM). Additionally, molecular docking and molecular dynamics simulation results revealed that compounds 5aa, 5ab, and 5ca have the capacity to bind in the active pocket of succinate dehydrogenase (SDH), establishing hydrogen-bonding interactions with neighboring amino acid residues.

Asunto(s)

Diseño de Fármacos , Fungicidas Industriales , Compuestos Heterocíclicos , Hidrazinas , Enfermedades de las Plantas , Succinato Deshidrogenasa , Ascomicetos/efectos de los fármacos , Botrytis/efectos de los fármacos , Botrytis/crecimiento & desarrollo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fungicidas Industriales/síntesis química , Fungicidas Industriales/farmacología , Fusarium/efectos de los fármacos , Fusarium/crecimiento & desarrollo , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/farmacología , Hidrazinas/síntesis química , Hidrazinas/farmacología , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Enfermedades de las Plantas/microbiología , Rhizoctonia/efectos de los fármacos , Relación Estructura-Actividad , Succinato Deshidrogenasa/antagonistas & inhibidores

RESUMEN

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these agrochemicals, the interactions of 15 SDHIs with expression and activity of human cytochrome P-450 3A4 (CYP3A4), a major hepatic drug metabolizing enzyme, were investigated in vitro. 12/15 SDHIs, i.e., bixafen, boscalid, fluopyram, flutolanil, fluxapyroxad, furametpyr, isofetamid, isopyrazam, penflufen, penthiopyrad, pydiflumetofen and sedaxane, were found to enhance CYP3A4 mRNA expression in human hepatic HepaRG cells and primary human hepatocytes exposed for 48 h to 10 µM SDHIs, whereas 3/15 SDHIs, i.e., benzovindiflupyr, carboxin and thifluzamide, were without effect. The inducing effects were concentrations-dependent for boscalid (EC50=22.5 µM), fluopyram (EC50=4.8 µM) and flutolanil (EC50=53.6 µM). They were fully prevented by SPA70, an antagonist of the Pregnane X Receptor, thus underlining the implication of this xenobiotic-sensing receptor. Increase in CYP3A4 mRNA in response to SDHIs paralleled enhanced CYP3A4 protein expression for most of SDHIs. With respect to CYP3A4 activity, it was directly inhibited by some SDHIs, including bixafen, fluopyram, fluxapyroxad, isofetamid, isopyrazam, penthiopyrad and sedaxane, which therefore appears as dual regulators of CYP3A4, being both inducer of its expression and inhibitor of its activity. The inducing effect nevertheless predominates for these SDHIs, except for isopyrazam and sedaxane, whereas boscalid and flutolanil were pure inducers of CYP3A4 expression and activity. Most of SDHIs appear therefore as in vitro inducers of CYP3A4 expression in cultured hepatic cells, when, however, used at concentrations rather higher than those expected in humans in response to environmental or dietary exposure to these agrochemicals.

Asunto(s)

Citocromo P-450 CYP3A , Hepatocitos , Succinato Deshidrogenasa , Humanos , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP3A/genética , Hepatocitos/efectos de los fármacos , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Fungicidas Industriales/toxicidad , ARN Mensajero/metabolismo , ARN Mensajero/genética , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/toxicidad , Línea Celular

RESUMEN

Succinate dehydrogenase inhibitors (SDHIs) are widely-used fungicides, to which humans are exposed and for which putative health risks are of concern. In order to identify human molecular targets for these environmental chemicals, the interactions of 15 SDHIs with activities of main human drug transporters implicated in pharmacokinetics were investigated in vitro. 5/15 SDHIs, i.e., benzovindiflupyr, bixafen, fluxapyroxad, pydiflumetofen and sedaxane, were found to strongly reduce activity of the renal organic anion transporter (OAT) 3, in a concentration-dependent manner (with IC50 values in the 1.0-3.9 µM range), without however being substrates for OAT3. Moreover, these 5/15 SDHIs decreased the membrane transport of estrone-3 sulfate, an endogenous substrate for OAT3, and sedaxane was predicted to inhibit in vivo OAT3 activity in response to exposure to the acceptable daily intake (ADI) dose. In addition, pydiflumetofen strongly inhibited the renal organic cation transporter (OCT) 2 (IC50 = 2.0 µM) and benzovindiflupyr the efflux pump breast cancer resistance protein (BCRP) (IC50 = 3.9 µM). Other human transporters, including organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 as well as multidrug and toxin extrusion protein (MATE) 1 and MATE2-K were moderately or weakly inhibited by SDHIs, whereas P-glycoprotein, multidrug resistance-associated protein (MRP), OCT1 and OAT1 activities were not or only marginally impacted. Then, some human drug transporters, especially OAT3, constitute molecular targets for SDHIs. This could have toxic consequences, notably with respect to levels of endogenous compounds and metabolites substrates for the considered transporters or to potential SDHI-drug interactions. This could therefore contribute to putative health risk of these fungicides.

Asunto(s)

Succinato Deshidrogenasa , Humanos , Succinato Deshidrogenasa/antagonistas & inhibidores , Succinato Deshidrogenasa/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transporte Biológico/efectos de los fármacos , Fungicidas Industriales/toxicidad , Fungicidas Industriales/farmacología , Inhibidores Enzimáticos/farmacología , Estrona/análogos & derivados , Estrona/metabolismo , Células HEK293 , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/antagonistas & inhibidores , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico/antagonistas & inhibidores

RESUMEN

To promote the development of novel agricultural succinate dehydrogenase inhibitor (SDHI) fungicides, we introduced cinnamamide and nicotinamide structural fragments into the structure of pyrazol-5-yl-amide by carbon chain extension and scaffold hopping, respectively, and synthesized a series of derivatives. The results of the biological activity assays indicated that most of the target compounds exhibited varying degrees of inhibitory activity against the tested fungi. Notably, compounds G22, G28, G34, G38, and G39 exhibited excellent in vitro antifungal activities against Valsa mali with EC50 values of 0.48, 0.86, 0.57, 0.73, and 0.87 mg/L, respectively, and this result was significantly more potent than boscalid (EC50 = 2.80 mg/L) and closer to the specialty control drug tebuconazole (EC50 = 0.30 mg/L). Compounds G22 and G34 also exhibited excellent in vivo protective and curative effects against V. mali at 40 mg/L. The SEM and TEM observations indicated that compounds G22 and G34 may affect normal V. mali mycelial morphology as well as the cellular ultrastructure. Molecular docking analysis results indicated that G22 and boscalid possessed a similar binding mode to that of SDH, and detailed SDH inhibition assays validated the feasibility of the designed compounds as potential SDH inhibitors. Compounds G22 and G3 were selected for theoretical calculations, and the terminal carboxylic acid group of this series of compounds may be a key region influencing the antifungal activity. Furthermore, toxicity tests on Apis mellifera l. revealed that compounds G22 and G34 exhibited low toxicity to A. mellifera l. populations. The above results demonstrated that these series of pyrazole-5-yl-amide derivatives are promising for development as potential low-risk drug-resistance agricultural SDHI fungicides.

RESUMEN

In an effort to promote the development of new fungicides, a series of 48 novel N-(1-methyl-4-thiocyanato-1H-pyrazol-5-yl)-benzamide derivatives A1-A36 and B1-B12 were designed and synthesized by incorporating a thiocyanato group into the pyrazole ring, and their fungicidal activities were evaluated against Sclerotinia sclerotiorum, Valsa mali, Botrytis cinerea, Rhizoctonia solani, and Phytophthora capsici. In the in vitro antifungal/antioomycete assay, many of the target compounds exhibited good broad-spectrum fungicidal activities. Among them, compound A36 displayed the best antifungal activity against V. mali with an EC50 value of 0.37 mg/L, which was significantly higher than that of the positive controls fluxapyroxad (13.3 mg/L) and dimethomorph (10.3 mg/L). Meanwhile, compound B6 exhibited the best antioomycete activity against P. capsici with an EC50 value of 0.41 mg/L, which was higher than that of azoxystrobin (29.2 mg/L) but lower than that of dimethomorph (0.13 mg/L). Notably, compound A27 displayed broad-spectrum inhibitory activities against V. mali, B. cinerea, R. solani, S. sclerotiorum, and P. capsici with respective EC50 values of 0.71, 1.44, 1.78, 0.87, and 1.61 mg/L. The in vivo experiments revealed that compounds A27 and B6 presented excellent protective and curative efficacies against P. capsici, similar to that of the positive control dimethomorph. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses showed that compound B6 could change the mycelial morphology and severely damage the ultrastructure of P. capsici. The results of the in vitro SDH enzymatic inhibition experiments indicated that compounds A27 and B6 could effectively inhibit the activity of P. capsici SDH (PcSDH). Furthermore, molecular docking analysis demonstrated significant hydrogen bonds and Pi-S bonding between the target compounds and the key amino acid residues of PcSDH, which could explain the probable mechanism of action. Collectively, these studies provide a valuable approach to expanding the fungicidal spectrum of pyrazol-5-yl-benzamide derivatives.

Asunto(s)

Fungicidas Industriales , Fungicidas Industriales/farmacología , Fungicidas Industriales/química , Antifúngicos/farmacología , Antifúngicos/química , Relación Estructura-Actividad , Simulación del Acoplamiento Molecular , Benzamidas/farmacología

RESUMEN

A series of novel pyrazole-4-carboxamides bearing an ether group were designed and synthesized on the basis of the structure of commercial succinate dehydrogenase inhibitor (SDHI) fungicide flubeneteram via scaffold hopping and evaluated for their antifungal activities against five fungi. The bioassay results showed that most of the target compounds exhibited excellent in vitro antifungal activity against Rhizoctonia solani and some compounds exerted remarkable antifungal activities against Sclerotinia sclerotiorum, Botrytis cinerea, Fusarium graminearum, and Alternaria alternate. Particularly, compounds 7d and 12b displayed outstanding antifungal activity against R. solani, with an EC50 value of 0.046 µg/mL, far superior to that of boscalid (EC50 = 0.741 µg/mL) and fluxapyroxad (EC50 = 0.103 µg/mL). Meanwhile, compound 12b also presented a broader fungicidal spectrum than other compounds. Moreover, in vivo anti-R. solani results showed that compounds 7d and 12b could significantly inhibit the growth of R. solani in rice leaves with excellent protective and curative efficacies. In addition, the results of the succinate dehydrogenase (SDH) enzymatic inhibition assay showed that compound 7d generated significant SDH inhibition, with an IC50 value of 3.293 µM, which was about 2 times better than that of boscalid (IC50 = 7.507 µM) and fluxapyroxad (IC50 = 5.991 µM). Furthermore, scanning electron microscopy (SEM) analysis indicated that compounds 7d and 12b significantly destroyed the typical structure and morphology of R. solani hyphae. The molecular docking study revealed that compounds 7d and 12b could embed into the binding pocket of SDH and form hydrogen bond interactions with TRP173 and TRY58 at the activity site of SDH, which was in line with fluxapyroxad, indicating that they had a similar mechanism of action. These results demonstrated that compounds 7d and 12b could be promising candidates of SDHI fungicides, which deserved further investigation.

Asunto(s)

Antifúngicos , Fungicidas Industriales , Antifúngicos/farmacología , Antifúngicos/química , Relación Estructura-Actividad , Éter , Succinato Deshidrogenasa , Simulación del Acoplamiento Molecular , Fungicidas Industriales/química , Rhizoctonia , Pirazoles/farmacología , Pirazoles/química

RESUMEN

Due to the single target but extensive application of commercialized succinate dehydrogenase inhibitors (SDHIs), resistance problems have gradually become apparent in recent years. To solve this problem, a series of novel N-thienyl-1,5-disubstituted-1H-4-pyrazole carboxamide derivatives were designed and synthesized in this work based on the active skeleton 5-trifluoromethyl-4-pyrazole carboxamide. The bioassay results indicated that some target compounds exhibited excellent in vitro antifungal activities against the eight phytopathogenic fungi tested. Among them, the EC50 values of T4, T6, and T9 against Nigrospora oryzae were 5.8, 1.9, and 5.5 mg/L, respectively. The in vivo protective and curative activities of 40 mg/L T6 against rice infected with N. oryzae were 81.5% and 43.0%, respectively. Further studies revealed that T6 not only significantly inhibited the growth of N. oryzae mycelia but also effectively hindered spore germination and germ tube elongation. Morphological studies using scanning electron microscopy (SEM), fluorescence microscopy (FM), and transmission electron microscopy (TEM) found that T6 could affect the mycelium membrane integrity by increasing cell membrane permeability and causing peroxidation of cellular lipids, and these results were further verified by measuring the malondialdehyde (MDA) content. The IC50 value of T6 against succinate dehydrogenase (SDH) was 7.2 mg/L, lower than that of the commercialized SDHI penthiopyrad (3.4 mg/L). Further, ATP content detection and the results after docking T6 and penthiopyrad suggested that T6 was a potential SDHI. These studies demonstrated that active compound T6 could both inhibit the activity of SDH and affect the integrity of the cell membrane at the same time via a dual action mode, which is different from the mode of action of penthiopyrad. Thus, this study provides a new idea for a strategy to delay resistance and diversify the structures of SDHIs.

Asunto(s)

Ascomicetos , Fungicidas Industriales , Succinato Deshidrogenasa , Antifúngicos/farmacología , Pirazoles/farmacología , Pirazoles/química , Relación Estructura-Actividad , Fungicidas Industriales/química , Simulación del Acoplamiento Molecular

RESUMEN

The identification of succinate dehydrogenase inhibitor (SDHI) fungicides bearing a novel scaffold is of great importance to control pathogenic fungi. Difluoromethyl-pyrazole ß-ketonitrile derivatives were rationally designed through an innovative amide-ß-ketonitrile bioisosteric replacement strategy and evaluated for their antifungal activities. In preliminary fungicidal screening, our new ß-ketonitrile compounds showed outstanding in vitro activity. Compounds A7 and A14 exhibited EC50 values of 0.116 and 0.165 µg/mL against Sclerotinia sclerotiorum, respectively, and A14 also displayed an EC50 of 0.0774 µg/mL against Rhizoctonia solani. Furthermore, A14 exhibited moderate in vivo protective activity against rice sheath blight on rice plants. Results from SDH enzymatic assays demonstrated that A14 possesses significant inhibitory effect toward porcine heart SDH, with an IC50 value of 0.183 µM, which was 20-fold more potent than that of fluxapyroxad (IC50 = 3.76 µM). A docking study indicated that H-bonds, cation-π interactions, and edge-to-face π-π interactions play key roles in the binding of A14 with R. solani SDH. The CoMSIA model guided the approach to further structural optimizations and indicated that hydrophobic and steric substituents on the benzene ring have decisive effects on the fungicidal activity against R. solani. The present work describes for the first time the successful bioisosteric replacement of the common SDHI amide moiety by a ß-ketonitrile group and highlights the potential of ß-ketonitriles as an innovative novel SDHI subclass.

Asunto(s)

Fungicidas Industriales , Animales , Porcinos , Fungicidas Industriales/química , Relación Estructura-Actividad , Ácido Succínico , Succinato Deshidrogenasa , Succinatos , Amidas

RESUMEN

Pyrazole carboxamides are a class of traditional succinate dehydrogenase inhibitors (SDHIs) that have developed into a variety of commercialized fungicides. In the present work, a series of novel 1,5-disubstituted-1H-pyrazole-4-carboxamide derivatives were designed and synthesized based on the active backbone of 5-trifluoromethyl-1H-4-pyrazole carboxamide. Bioassay results indicated that some target compounds exhibited excellent in vitro antifungal activities against six phytopathogenic fungi. Notably, the EC50 values of Y47 against Gibberella zeae, Nigrospora oryzae, Thanatephorus cucumeris, and Verticillium dahliae were 5.2, 9.2, 12.8, and 17.6 mg/L, respectively. The in vivo protective and curative activities of Y47 at 100 mg/L against G. zeae on maize were 50.7 and 44.2%, respectively. Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis revealed that the large steric hindrance and electronegative groups on the 5-position of the pyrazole ring were important for the activity. The IC50 value of Y47 against succinate dehydrogenase (SDH) was 7.7 mg/L, superior to fluopyram (24.7 mg/L), which was consistent with the docking results. Morphological studies with fluorescence microscopy (FM) and scanning electron microscopy (SEM) found that Y47 could affect the membrane integrity of mycelium by inducing endogenous reactive oxygen species (ROS) production and causing peroxidation of cellular lipids, which was further verified by the malondialdehyde (MDA) content. Antifungal mechanism analysis demonstrated that the target compound Y47 not only had significant SDH inhibition activity but could also affect the membrane integrity of mycelium, exhibiting obvious dual action modes. This research provides a novel approach to the development of traditional SDHIs and their derivatives.

Asunto(s)

Fungicidas Industriales , Fungicidas Industriales/farmacología , Antifúngicos/farmacología , Succinato Deshidrogenasa , Relación Estructura-Actividad , Relación Estructura-Actividad Cuantitativa , Pirazoles/farmacología , Simulación del Acoplamiento Molecular

RESUMEN

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.

Asunto(s)

Cucumis sativus , Fungicidas Industriales , Ácido Succínico , Succinato Deshidrogenasa/genética , Fungicidas Industriales/farmacología , Mutación , Succinatos

RESUMEN

A series of pyrazol-5-yl-benzamide derivatives containing the oxazole group were designed and synthesized as potential SDH inhibitors. According to the results of the bioassays, most target compounds displayed moderate-to-excellent in vitro antifungal activities against Valsa mali, Sclerotinia scleotiorum, Alternaria alternata, and Botrytis cinerea. Among them, compounds C13, C14, and C16 exhibited more excellently inhibitory activities against S. sclerotiorum than boscalid (EC50 = 0.96 mg/L), with EC50 values of 0.69, 0.26, and 0.95 mg/L, respectively. In vivo experiments on rape leaves and cucumber leaves showed that compounds C13 and C14 exhibited considerable protective effects against S. sclerotiorum than boscalid. SEM analysis indicated that compounds C13 and C14 significantly destroyed the typical structure and morphology of S. scleotiorum hyphae. In the respiratory inhibition effect assays, compounds C13 (28.0%) and C14 (33.9%) exhibited a strong inhibitory effect on the respiration rate of S. sclerotiorum mycelia, which was close to boscalid (30.6%). The results of molecular docking indicated that compounds C13 and C14 could form strong interactions with the key residues TRP O:173, ARG P:43, TYR Q:58, and MET P:43 of the SDH. Furthermore, the antifungal mechanism of these derivatives was demonstrated by the SDH enzymatic inhibition assay. These results demonstrate that compounds C13 and C14 can be developed into novel SDH inhibitors for crop protection.

Asunto(s)

Rhizoctonia , Succinato Deshidrogenasa , Succinato Deshidrogenasa/metabolismo , Antifúngicos/farmacología , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Oxazoles/farmacología , Benzamidas/farmacología

RESUMEN

Succinate dehydrogenase inhibitors (SDHIs) are one of the most frequently used fungicides in cucumber fields in China. Our previous studies indicated that the sensitivity profile of Corynespora cassiicola, the causal agent of Corynespora leaf spot, to different SDHIs varied greatly; however, the underlying mechanism remains unclear. The 50% effective concentration (EC50) values of boscalid, fluopyram, fluxapyroxad and isopyrazam in C. cassiicola collected from 2017 to 2020 shifted, with resistance frequencies of 79.83%, 78.43%, 83.19% and 49.86%, respectively. The sequence alignment of sdhB/C/D of resistant strains revealed that eight single amino acid mutations (B-H278Y/L, B-I280V, C-S73P, C-N75S, C-H134R, D-D95E and D-G109V), and three dual-mutations (B-I280V&C-S73P, B-I280V&C-N75S and C-S73P&C-N75S) conferred various SDHI resistance levels and cross-resistance profiles. The expression level of the sdhB/C/D gene and succinate dehydrogenase (SDH) activity in the mutants were significantly altered by the presence of SDHIs, compared with the wild type strain. Additionally, molecular docking results suggested that the missense mutation influenced the crystal structure of SDH and subsequently interfered with the interaction bonds and bond distances among the target protein and chemicals. In brief, amino acid mutations altered the fungicide response of target gene expression, SDH activity and the binding features of SDH-ligand complexes and subsequently conferred multiple resistance levels and complex cross-resistance patterns to SDHIs in C. cassiicola. The evaluation of C. cassiicola resistance to SDHIs provided a significant foundation for efficient chemical development and integrated CLS management strategies.

Asunto(s)

Fungicidas Industriales , Succinato Deshidrogenasa , Aminoácidos , Ascomicetos , Farmacorresistencia Fúngica/genética , Fungicidas Industriales/farmacología , Simulación del Acoplamiento Molecular , Mutación , Enfermedades de las Plantas/genética , Succinato Deshidrogenasa/genética

RESUMEN

Fluindapyr and penthiopyrad are two new succinate-dehydrogenase-inhibitor fungicides both employed as racemic mixtures of enantiomers to control various fungal pathogens. In the present work, a robust and highly-sensitive method for simultaneous determination of fluindapyr and penthiopyrad enantiomers in plant-origin foods (cereals, fruits and vegetables) was developed using UPLC-MS/MS combined with a chiral stationary phase. Rapid baseline chiral separation of four stereoisomers of fluindapyr and penthiopyrad was obtained within 4.2 min on chiral MX(2)-RH column under reversed-phase conditions (with the eluent of acetonitrile/0.1% formic acid in water =70/30 (V:V) and column temperature maintained at 30 °C). The plant-origin samples were extracted quickly with acetonitrile and purified with multi-walled carbon nanotubes. Excellent linearity for the target analytes was observed in the concentration ranging from 1 to 250 µg/L with regression coefficient no less than 0.9967. The mean recoveries of fluindapyr and penthiopyrad enantiomers from six matrices were 77.1-107.2%, with all relative standard deviations values lower than 9.1%. The limit of quantification of four stereoisomers of two target chiral fungicides was 5 µg/kg. The analysis of real samples reveal that the developed method is suitable for the simultaneous chiral determination of fluindapyr and penthiopyrad residues in cereals, fruits and vegetables samples at enantiomeric level and can support their further investigation on enantioselective environmental behaviors and residue surveillance.

Asunto(s)

Fungicidas Industriales , Nanotubos de Carbono , Acetonitrilos/análisis , Cromatografía Líquida de Alta Presión/métodos , Cromatografía Liquida/métodos , Fungicidas Industriales/análisis , Estereoisomerismo , Ácido Succínico , Espectrometría de Masas en Tándem/métodos , Verduras/química

RESUMEN

With the further application of succinate dehydrogenase inhibitors (SDHI), the resistance caused by double mutations in target gene is gradually becoming a serious problem, leading to a decrease of control efficacy. It is important to assess the sensitivity and fitness of double mutations to SDHI in Corynespora cassiicola and analysis the evolution of double mutations. We confirmed, by site-directed mutagenesis, that all double mutations (B-I280V+D-D95E/D-G109V/D-H105R, B-H278R+D-D95E/D-G109V, B-H278Y+D-D95E/D-G109V) conferred resistance to all SDHI and exhibited the increased resistance to at least one fungicide than single point mutation. Analyses of fitness showed that all double mutations had lower fitness than the wild type; most of double mutations suffered more fitness penalties than the corresponding single mutants. We also further found that double mutations (B-I280V+D-D95E/D-G109V/D-H105R) containing low SDHI-resistant single point mutation (B-I280V) exhibited higher resistance to SDHI and low fitness penalty than double mutations (B-H278Y+D-D95E/D-G109V) containing high SDHI-resistant single mutations (B-H278Y). Therefore, we may infer that a single mutation conferring low resistance is more likely to evolve into a double mutation conferring higher resistance under the selective pressure of SDHI. Taken together, our results provide some important reference for resistance management.