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Background: Wheat crown rot (WCR), primarily caused by Fusarium pseudograminearum has become more and more prevalent in winter wheat areas in China. However, limited fungicides have been registered for the control of WCR in China so far. Pyraclostrobin is a representative quinone outside inhibitor (QoI) with excellent activity against Fusarium spp. There is currently limited research on the resistance risk and resistance mechanism of F. pseudograminearum to pyraclostrobin., Results: Here, we determined the activity of pyraclostrobin against F. pseudograminearum. The EC 50 values ranged from 0.022 to 0.172 μg mL -1 with an average EC 50 value of 0.071 ± 0.030 μg mL -1 . Four highly pyraclostrobin-resistant mutants were obtained from two sensitive strains by ultraviolet (UV) mutagenesis in the laboratory. The mutants showed decreased mycelial growth rate and virulence as compared with the corresponding wild-type strains, indicating that pyraclostrobin resistance suffered a fitness penalty in F. pseudograminearum. It was found that the high resistance of four mutants was caused by the G143S mutation in Cytb. Molecular docking analysis also further confirms that the G143S mutation in Cytb decreased the binding affinity between pyraclostrobin and Cytb., Conclusion: The resistance risk of F. pseudograminearum to pyraclostrobin could be low to medium. Although a mutation at the G143S position of Cytb could potentially occur, this mutation decreases the fitness of the mutant, which may reduce its survival in the environment. Therefore, the negative consequences of a possible mutation are lower. This makes pyraclostrobin a good candidate for controlling crown rot in wheat. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

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 EC 50 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 EC 50 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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Formally described in 2009, Phytophthora sansomeana is a pathogen of increasing interest in native, agricultural, and horticulturally important plant species. The objective of this study was to elucidate the symptomatic and asymptomatic host range of P. sansomeana on six agricultural crop species commonly used in field crop rotations in Michigan. In addition, sensitivity to oomicides commonly used in seed treatments, including oxathiapiprolin, mefenoxam, ethaboxam, and pyraclostrobin, was performed to aid in disease management recommendations. Plant biomass, quantity of P. sansomeana DNA in roots, and reisolations were used to assess pathogenicity and virulence of 18 isolates of P. sansomeana on each plant species using an inoculated seedling growth chamber assay. Isolates displayed varying levels of virulence to the hosts tested. Reisolations were completed for each plant species tested, and varying quantities of P. sansomeana DNA were found within all plant species root samples. Corn, wheat, soybean, dry bean, and winter cereal rye plants were symptomatic hosts with significant reduction observed in the total plant biomass. No significant reduction in total plant biomass was observed in oats, and oat roots harbored the least amount of P. sansomeana DNA. No P. sansomeana isolates were insensitive to the oomicide compounds tested with mean absolute inhibition (EC 50 ) values of fungicide required for 50% growth inhibition values of 7.8 × 10 -2 μg/ml for mefenoxam, 1.13 × 10 -1 μg/ml for ethaboxam, 2.6 × 10 -2 μg/ml for oxathiapiprolin, and 3.04 × 10 -1 μg/ml for pyraclostrobin. These results suggest that common crop rotations in Michigan may not be a viable option to reduce soilborne inoculum accumulation and oomicide seed treatments could be considered for early-season management of P. sansomeana ., Competing Interests: The author(s) declare no conflict of interest.

Tree peony black spot (TPBS), mainly caused by Alternaria suffruticosae , is a common leaf disease on the ornamental peony, which poses a great threat to the flower buds in the current year and the flowering quality in the next year. However, there is only one fungicide registered for the control of this disease, difenoconazole. In order to avoid the severe problem of pathogen resistance caused by long-term use of difenoconazole, it is necessary to screen more chemical fungicides for the prevention and control of TPBS. In this study, the biological activities of flutolanil, phenamacril, pyraclostrobin, and boscalid on mycelial growth, conidial germination, germ tube elongation, and sporulation quantity of A . suffruticosae were determined, and the field control efficacy was tested to evaluate the preventive and therapeutic activities. Difenoconazole was used as a control simultaneously. The results showed that pyraclostrobin had the strongest inhibitory effects on the conidial germination, mycelium growth, germ tube elongation, and sporulation quantity, with the average EC 50 values of 0.0517, 0.5343, 0.0008, and 0.8068 μg/ml, respectively. The inhibitory activity of flutolanil on the four developmental stages of A . suffruticosae was weaker than that of the other three fungicides. Compared with flutolanil, boscalid, the other succinate dehydrogenase inhibitor, had more strong inhibitory effects on the mycelial growth and sporulation quantity, with the average EC 50 values of 3.8603 and 1.4760 μg/ml, respectively. Phenamacril had a moderate inhibitory level and had more inhibitory activity on conidial germination and germ tube elongation, with the average EC 50 values of 31.5349 and 5.2597 μg/ml, respectively. All of the four fungicides had no significant effects on the shape of spores and germ tubes. The control fungicide difenoconazole had the strongest inhibitory activity on mycelial growth, and the average EC 50 value was only 0.3297 μg/ml. However, its inhibitory activity on the other three growth stages was not high. In the field trials, pyraclostrobin had high control efficacy on TPBS even at low concentrations, reaching a minimum of 62.6293%, which was higher than that of difenoconazole. The other three fungicides had higher control efficacy at high concentrations but decreased significantly at low concentrations. Considering the dosage and control efficacy, pyraclostrobin was the first choice for the control of TPBS. Pyraclostrobin is the preferred alternative fungicide to difenoconazole for the prevention and control of TPBS in production., Competing Interests: The author(s) declare no conflict of interest.

Cytochrome c oxidase (Cox) is a crucial terminal oxidase in the electron transport chain. In this study, we generated 14 Cox gene deletion or overexpression mutants in Fusarium graminearum . Fungicide sensitivity tests revealed that 11 Cox gene deletion mutants displayed resistance to pyraclostrobin, while 10 overexpression mutants showed hypersensitivity. RNA-Seq and RT-qPCR analyses demonstrated the upregulation of FgAox (alternative oxidase in F. graminearum ), FgAod 2, and FgAod 5 (alternative oxidase deficiency in F. graminearum ) in ΔFgCox4-2 and ΔFgCox17-75 mutants. In 11 Cox gene deletion mutants, FgAox expression was significantly upregulated, whereas in 10 Cox gene overexpression mutants, it was significantly downregulated. FgAox overexpression mutants exhibit resistance to pyraclostrobin, while FgAox deletion mutants show hypersensitivity to pyraclostrobin. FgAod2 and FgAod5 were identified as transcription factors for FgAox . Our findings reveal that FgCox influences pyraclostrobin sensitivity by regulating FgAox through FgAod2 and FgAod5. Understanding pyraclostrobin resistance mechanisms in F. graminearum could help develop better fungicide rotation and application strategies to manage resistance and guide the creation of new fungicides targeting different pathways.

Excessive usage of biologically toxic fungicides and their matrix materials poses a serious threat to public health. Leveraging fungicide carriers with inherent pathogen inhibition properties is highly promising for enhancing fungicide efficacy and reducing required dosage. Herein, a series of coacervates have been crafted with lignin and surfactin, both of which are naturally derived and demonstrate substantial antifungal properties. This hierarchically assembled carrier not only effectively loads fungicides with a maximum encapsulation efficiency of 95% but also stably deposits on hydrophobic leaves for high-speed impacting droplets. Intriguingly, these coacervates exhibit broad spectrum fungicidal activity against eight ubiquitous phytopathogens and even act as a standalone biofungicide to replace fungicides. This performance can significantly reduce the fungicide usage and be further strengthened by an encapsulated fungicide. The inhibition rate reaches 87.0% when 0.30 mM pyraclostrobin (Pyr) is encapsulated within this coacervate, comparable to the effectiveness of 0.80 mM Pyr alone. Additionally, the preventive effects against tomato gray mold reached 53%, significantly surpassing those of commercial adjuvants. Thus, it demonstrates that utilizing biosurfactants and biomass with intrinsic antifungal activity to fabricate fully biobased coacervates can synergistically combine the functions of a fungicide carrier and antifungal agent against phytopathogens and guarantee environmental friendliness. This pioneering approach provides deeper insights into synergistically enhancing the effectiveness of agrochemicals from multiple aspects, including fungicide encapsulation, cooperative antifungal action, and droplet deposition.

Magnaporthe oryzae is a rice blast pathogen that seriously threatens rice yield. Benzovindiflupyr is a succinate dehydrogenase inhibitor (SDHI) fungicide that effectively controls many crop diseases. Benzovindiflupyr has a strong inhibitory effect on M. oryzae; however, control of rice blast by benzovindiflupyr and risk of resistance to benzovindiflupyr are not well studied in this pathogen. In this study, six benzovindiflupyr-resistant strains were obtained by domestication induced in the laboratory. The MoSdhB H245D mutation was the cause of M. oryzae resistance to benzovindiflupyr, which was verified through succinate dehydrogenase (SDH) activity assays, molecular docking, and site-specific mutations. Survival fitness analysis showed no significant difference between the benzovindiflupyr-resistant and parent strains. Positive cross-resistance to benzovindiflupyr and other SDHIs and negative cross-resistance to azoxystrobin were observed. Therefore, the risk of benzovindiflupyr resistance in M. oryzae might be medium to high. It should be combined with other classes of fungicides (tebuconazole and azoxystrobin) to slow the development of resistance., (Copyright © 2024. Published by Elsevier Inc.)

Lignin, renowned for its renewable, biocompatible, and environmentally benign characteristics, holds immense potential as a sustainable feedstock for agrochemical formulations. In this study, raw dealkaline lignin (DAL) underwent a purification process involving two sequential solvent extractions. Subsequently, an enzyme-responsive nanodelivery system (Pyr@DAL-NPs), was fabricated through the solvent self-assembly method, with pyraclostrobin (Pyr) loaded into lignin nanoparticles. The Pyr@DAL-NPs shown an average particle size of 250.4 nm, demonstrating a remarkable loading capacity of up to 54.70 % and an encapsulation efficiency of 86.15 %. Notably, in the presence of cellulase and pectinase at a concentration of 2 mg/mL, the release of Pyr from the Pyr@DAL-NPs reached 92.66 % within 120 h. Furthermore, the photostability of Pyr@DAL-NPs was significantly improved, revealing a 2.92-fold enhancement compared to the commercially available fungicide suspension (Pyr SC). Bioassay results exhibited that the Pyr@DAL-NPs revealed superior fungicidal activity against Botrytis cinerea over Pyr SC, with an EC 50 value of 0.951 mg/L. Additionally, biosafety assessments indicated that the Pyr@DAL-NPs effectively declined the acute toxicity of Pyr towards zebrafish and posed no negative effects on the healthy growth of strawberry plants. In conclusion, this study presents a viable and promising strategy for developing environmentally friendly controlled-release systems for pesticides, offering the unique properties of lignin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The present study was aimed at assessing the impact of azoxystrobin-a fungicide commonly used in plant protection against pathogens (Amistar 250 SC)-on the soil microbiota and enzymes, as well as plant growth and development. The laboratory experiment was conducted in three analytical terms (30, 60, and 90 days) on sandy clay (pH-7.0). Azoxystrobin was applied to soil in doses of 0.00 (C), 0.110 (F) and 32.92 (P) mg kg -1 d.m. of soil. Its 0.110 mg kg -1 dose stimulated the proliferation of organotrophic bacteria and actinobacteria but inhibited that of fungi. It also contributed to an increase in the colony development index (CD) and a decrease in the ecophysiological diversity index (EP) of all analyzed groups of microorganisms. Azoxystrobin applied at 32.92 mg kg -1 reduced the number and EP of microorganisms and increased their CD. PP952051.1 Bacillus mycoides strain (P), PP952052.1 Prestia megaterium strain (P) bacteria, as well as PP952052.1 Kreatinophyton terreum isolate (P) fungi were identified in the soil contaminated with azoxystrobin, all of which may exhibit resistance to its effects. The azoxystrobin dose of 0.110 mg kg -1 stimulated the activity of all enzymes, whereas its 32.92 mg kg -1 dose inhibited activities of dehydrogenases, alkaline phosphatase, acid phosphatase, and urease and stimulated the activity of catalase. The analyzed fungicide added to the soil at both 0.110 and 32.92 mg kg -1 doses inhibited seed germination and elongation of shoots of Lepidium sativum L., Sinapsis alba L., and Sorgum saccharatum L.

Investigation of cultures of the basidiomycete Favolaschia minutissima TBRC-BCC 19434 led to the isolation of two undescribed β-methoxyacrylate metabolites, 9-methoxystrobilurins R (1) and S (2), and a degraded aldehyde derivative, favodehyde E (3). 9-Methoxystrobilurin derivatives 1 and 2 exhibited significant antimalarial activity against Plasmodium falciparum K1 (multidrug-resistant strain) with IC 50 values of 0.12 and 0.21 μM, respectively., (© 2024. The Author(s), under exclusive licence to the Japan Antibiotics Research Association.)

Stemphylium leaf spot of spinach, caused by Stemphylium beticola and S. vesicarium , is a disease of economic importance in fresh market, processing, and seed production. There have been increasing reports of difficulty managing the disease in the southern United States using fungicides in Fungicide Resistance Action Committee (FRAC) group 11. Isolates of S. beticola and S. vesicarium obtained from spinach leaves and seed from 2001 to 2020 were screened for resistance to azoxystrobin and pyraclostrobin in vitro, in vivo, and using PCR assays to detect mutations in cytochrome b associated with resistance in other fungi (F129L, G137R, and G143A). EC 50 values for mycelial growth and conidial germination of S. vesicarium isolates in vitro were significantly less (mean of 0.35 μg/ml) than that of S. vesicarium (mean of 14.17 μg/ml) with both fungicides. All isolates were slightly more sensitive to pyraclostrobin than azoxystrobin in both assays. In vivo assays of plants inoculated with the isolates of S. vesicarium demonstrated poor efficacy of fungicides with each of the two active ingredients. Only the G143A mutation was detected in all spinach isolates of S. vesicarium , including an isolate of S. vesicarium collected in 2003 and 82.9% of isolates from spinach seed lots harvested from crops grown in or after 2017 in Europe, New Zealand, and the United States. The FRAC 11 mutations were not detected in any isolates of S. beticola . The in vitro, in vivo, and DNA mutation assays suggest FRAC group 11 fungicide resistance is widespread in spinach isolates of S. vesicarium but not S. beticola., Competing Interests: The author(s) declare no conflict of interest.

Alternaria brassicicola is a part of the Alternaria complex that causes leaf blight and head rot (ABHR) in brassica crops. Infested broccoli seeds can play an important role in introducing A. brassicicola in transplant houses and production fields. However, characterization of natural seed infestation and seed-to-seedling transmission of A. brassicicola in broccoli is yet to be demonstrated. In this research, we characterized Alternaria spp. isolates from commercial broccoli seedlots for their species identity, pathogenicity, and aggressiveness on broccoli and their sensitivity to a quinone-outside inhibitor (QoI) fungicide (azoxystrobin). Two hundred commercial seedlots from two broccoli cultivars, Cultivar 1 (EC; n = 100 seedlots) and Cultivar 2 (ED; n = 100 seedlots) were, evaluated for the presence of A. brassicicola under in vitro conditions using a seedling grow-out assay. Alternaria spp. was detected in 31 and 28% of the commercial seedlots of Cultivar 1 and Cultivar 2, respectively. The seed-to-seedling transmission (%) varied considerably within each positive-infested seedlot, which ranged from 1.3 to 17.3%. Subsequent molecular identification of single-spore cultures ( n = 138) was made by sequencing four housekeeping genes: actin, the major allergen (Alta1), plasma membrane ATPase, and glyceraldehyde-3-phosphate dehydrogenase (GPD), and the sequences were concatenated and compared for the phylogenetic distance with diverse Alternaria species. Ninety-six percent ( n = 133) of the isolates formed a cluster with a known A . brassicicola based on a multigene phylogeny, which were later confirmed as A. brassicicola using a species-specific PCR assay. One hundred percent of the A. brassicicola seed isolates ( n = 133) were either highly or moderately aggressive on broccoli (cultivar Emerald Crown) based on a detached leaf assay. Sensitivity of representative A. brassicicola isolates ( n = 58) to azoxystrobin was evaluated using a spore germination assay, and the EC 50 values (effective fungicide concentration [ppm] at which germination of conidia of isolates were reduced by 50% compared to control) for each isolate was determined. A. brassicicola isolates from naturally infested commercial broccoli seeds were sensitive to azoxystrobin with considerably low EC 50 values in the range of <0.0001 to 0.33 ppm; however, there were a few isolates (14%) that showed 100-fold reduced sensitivity from the most sensitive isolate (EC 50 = 0.0001 ppm). Our results confirm that commercial broccoli seedlots can be naturally contaminated with pathogenic and aggressive A. brassicicola . We also provide evidence for the potential presence of A. brassicicola isolates with reduced azoxystrobin-sensitivity in naturally infested commercial broccoli seedlots, which has never been reported before. Together, these findings may have implications in considerations for seed-health testing, seed treatments, and greenhouse scouting to limit introduction of infested seedlots in commercial broccoli fields., Competing Interests: The author(s) declare no conflict of interest.

Phoma black stem (PBS), caused by Phoma macdonaldii Boerema (teleomorph Leptosphaeria lindquistii Frezzi), is the most common stem disease of sunflower ( Helianthus annuus L.) in the northern Great Plains region of the United States. However, the impact of PBS on sunflower yield in the United States is unclear, and a near complete absence of information on the impact of fungicides on disease management exists. The objectives of this study were to determine the impact of PBS on sunflower yield, the efficacy of available fungicides, the optimal fungicide application timing, and the economic viability of fungicides as a management tool. Fungicide timing efficacy was evaluated by applying single and/or sequential applications of pyraclostrobin fungicide at three sunflower growth stages in 10 field trials between 2017 and 2019. Efficacy of 10 fungicides from the Fungicide Resistance Action Committee (FRAC) groups 3, 7, and 11 were evaluated in four field trials between 2018 and 2019. The impact of treatments on PBS were evaluated by determination of incidence, severity, maximum lesion height, disease severity index (DSI), and harvested yield. Nine of the 10 fungicides evaluated and all fungicide timings that included an early bud application resulted in disease reductions when compared with the nontreated controls. The DSI was negatively correlated to sunflower yield in high-yield environments ( P = 0.0004; R 2 = 0.3425) but not in low- or moderate-yield environments. Although FRAC 7 fungicides were generally most efficacious, the sufficient efficacy and lower cost of FRAC 11 fungicides make them more economically viable in high-yielding environments at current market conditions., Competing Interests: The author(s) declare no conflict of interest.

Triazoles are compounds with various biological activities, including fungicidal action. They became popular through cholinesterase studies after the successful synthesis of the dual binding femtomolar triazole inhibitor of acetylcholinesterase (AChE, EC 3.1.1.7) by Sharpless et al. via in situ click chemistry. Here, we evaluate the anticholinesterase effect of the first isopropanol triazole fungicide mefentrifluconazole (Ravystar ® ), developed to overcome fungus resistance in plant disease management. Mefentrifluconazole is commercially available individually or in a binary fungicidal mixture, i.e., with pyraclostrobin (Ravycare ® ). Pyraclostrobin is a carbamate that contains a pyrazole ring. Carbamates are known inhibitors of cholinesterases and the carbamate rivastigmine is already in use for the treatment of Alzheimer's disease. We tested the type and potency of anticholinesterase activity of mefentrifluconazole and pyraclostrobin. Mefentrifluconazole reversibly inhibited human AChE and BChE with a seven-fold higher potency toward AChE ( K i = 101 ± 19 μM). Pyraclostrobin (50 μM) inhibited AChE and BChE progressively with rate constants of (t 1/2 = 2.1 min; k i = 6.6 × 10 3 M -1 min -1 ) and (t 1/2 = 1.5 min; k i = 9.2 × 10 3 M -1 min -1 ), respectively. A molecular docking study indicated key interactions between the tested fungicides and residues of the lipophilic active site of AChE and BChE. Additionally, the physicochemical properties of the tested fungicides were compared to values for CNS-active drugs to estimate the blood-brain barrier permeability. Our results can be applied in the design of new molecules with a lesser impact on humans and the environment.

Quinone outside inhibitor (QoI) has been applied to manage taro leaf blight caused by Phytophthora colocasiae in southeastern of China for many years. The risk of P. colocasiae to QoI and the potential resistant mechanism remain unknown. In this study, the 74 P. colocasiae strains were sampled from southeastern of China. Sequence analysis of the QoI target Cytb showed one nucleotide variant in the fragment of this gene in this population, producing two haplotypes. The nucleotide variant leads to codon change at 142 (GGT to GCT) producing A142 (alanine) and G142 (glycine) in Hap&#95;1 and Hap&#95;2 strains, respectively. The sensitivity differentiation to azoxystrobin of two haplotypes were observed in vitro. The Hap&#95;1 and Hap&#95;2 strains were confirmed resistant and sensitive by control efficacy of label rate fungicide application, which was 3.0% and 88.8% treated with 500 μg/mL azoxystrobin, respectively. In addition, 10.0 μg/mL azoxystrobin plus 50 μg/mL salicylhydroxamic acid (SHAM) supplemented in PDA medium was identified as a discriminatory dose for differentiation of these two phenotype strains. The azoxystrobin resistant frequency reached 86.5%, indicating prevalence of QoI resistance in the field. Further fitness related features showed that no significant difference in temperature sensitivity, mycelial growth rate, sporangia production, zoospore release and aggressiveness between azoxystrobin-resistant and sensitive strains indicating no potential fitness cost for azoxystrobin resistance. Taken together, azoxystrobin resistance need to be taken into consideration to manage taro leaf blight in southeastern of China., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plant pathogens have frequently shown multidrug resistance (MDR) in the field, often linked to efflux and sometimes metabolism of fungicides. To investigate the potential role of metabolic resistance in B. cinerea strains showing MDR, the azoxystrobin-sensitive strain B05.10 and -resistant strain Bc242 were treated with azoxystrobin. The degradation half-life of azoxystrobin in Bc242 (9.63 days) was shorter than that in B05.10 (28.88 days). Azoxystrobin acid, identified as a metabolite, exhibited significantly lower inhibition rates on colony and conidia (9.34 and 11.98%, respectively) than azoxystrobin. Bc242 exhibited higher expression levels of 34 cytochrome P450s (P450s) and 11 carboxylesterase genes (CarEs) compared to B05.10 according to RNA-seq analysis. The expression of P450 genes Bcin&#95;02g01260 and Bcin&#95;12g06380 , along with the CarEs Bcin&#95;12g06360 in Saccharomyces cerevisiae , resulted in reduced sensitivity to various fungicides, including azoxystrobin, kresoxim-methyl, pyraclostrobin, trifloxystrobin, iprodione, and carbendazim. Thus, the mechanism of B. cinerea MDR is linked to metabolism mediated by the CarE and P450 genes.

Aerial blight, caused by the fungus Rhizoctonia solani anastomosis group (AG) 1-IA, is an economically important soybean disease in the mid-Southern United States. Management has relied on fungicide applications during the season, but there is an increasing prevalence of resistance to commonly used strobilurin fungicides and an urgent need to identify soybean varieties resistant to aerial blight. Because the patchy distribution of the pathogen complicates field variety screening, the present study aimed to develop a greenhouse screening protocol to identify soybean varieties resistant to aerial blight. For this, 88 pathogen isolates were collected from commercial fields and research farms across five Louisiana parishes, and 77% were confirmed to be R. solani AG1-IA. Three polymorphic codominant microsatellite markers were used to explore the genetic diversity of 43 R. solani AG1-IA isolates, which showed high genetic diversity, with 35 haplotypes in total and only two haplotypes common to two other locations. Six genetically diverse isolates were chosen and characterized for their virulence and fungicide sensitivity. The isolate AC2 was identified as the most virulent and was resistant to both active ingredients, azoxystrobin and pyraclostrobin, tested. The six isolates were used in greenhouse variety screening trials using a millet inoculation protocol. Of the 31 varieties screened, only Armor 48-D25 was classified as moderately resistant, and plant height to the first node influenced final disease severity. The study provides short-term solutions for growers to choose less susceptible varieties for planting and lays the foundation to characterize host resistance against this important soybean pathogen., Competing Interests: The author(s) declare no conflict of interest.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Gray mold caused by Botrytis cinerea severely threatens the yield of ginseng ( Panax ginseng ). Various categories of fungicides have been utilized to control gray mold on this crop. In this study, the resistance of 102 isolates of B. cinerea from 11 commercial ginseng-growing regions in China to fungicides was examined. A total of 32.4% were resistant to boscalid, with EC 50 values that ranged from 12.26 to 235.87 μg/ml, and 94.1% were resistant to pyraclostrobin, with EC 50 values that ranged from 5.88 to 487.72 μg/ml. Except for sdh A and sdh D, the amino acid substitutions of P225F, P225L, N230I, H272Y, and H272R in the sdh B subunit from 24 (4 sensitive [S] and 20 resistant [R]), 5 (1 S and 4 R), 1 (S), 1 (R), and 8 (4 S and 4 R) strains, respectively, and the concurrent amino acid substitutions of G85A + I93V + M158V + V168I in the sdh C subunit from 5 (4 S and 1 R) strains were identified. A G143A substitution in cytochrome b was identified in 96 isolates that were resistant to pyraclostrobin and three that were sensitive to it. The Bcbi-143/144 intron was identified in the other three isolates sensitive to pyraclostrobin, but it was absent in the isolates that harbored the G143A mutation. The results showed that the populations of B. cinerea on ginseng have developed strong resistance to pyraclostrobin. Therefore, it is not recommended to continue using this fungicide to control gray mold on P. ginseng . Boscalid is still effective against most isolates. However, to prevent fungicide resistance, it is recommended to use a mixture of boscalid with other categories of fungicides., Competing Interests: The author(s) declare no conflict of interest.

Corynespora leaf spot, caused by Corynespora cassiicola, is a foliar disease in cucumber. While the application of quinone outside inhibitors (QoIs) is an effective measure for disease control, it carries the risk of resistance development. In our monitoring of trifloxystrobin resistance from 2008 to 2020, C. cassiicola isolates were categorized into three populations: sensitive isolates (S, 0.01 < EC 50  < 0.83 μg/mL), moderately resistant isolates (MR, 1.18 < EC 50  < 55.67 μg/mL), and highly resistant isolates (HR, EC 50  > 56.98 μg/mL). The resistance frequency reached up to 90% during this period, with an increasing trend observed in the annual average EC 50 values of all the isolates. Analysis of the CcCytb gene revealed that both MR and HR populations carried the G143A mutation. Additionally, we identified mitochondrial heterogeneity, with three isolates carrying both G143 and A143 in MR and HR populations. Interestingly, isolates with the G143A mutation (G143A-MR and G143A-HR) displayed differential sensitivity to QoIs. Further experiments involving gene knockout and complementation demonstrated that the major facilitator superfamily (MFS) transporter (CcMfs1) may contribute to the disparity in sensitivity to QoIs between the G143A-MR and G143A-HR populations. However, the difference in sensitivity caused by the CcMfs1 transporter is significantly lower than the differences observed between the two populations. This suggests additional mechanisms contributing to the variation in resistance levels among C. cassiicola isolates. Our study highlights the alarming level of trifloxystrobin resistance in C. cassiicola in China, emphasizing the need for strict prohibition of QoIs use. Furthermore, our findings shed light on the occurrence of both target and non-target resistance mechanisms associated with QoIs in C. cassiicola., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Corynespora cassiicola is the causal agent of cucumber Corynespora leaf spot, which affects many economically important plant species. Chemical control of this disease is hampered by the common development of fungicide resistance. In this study, 100 isolates from Liaoning Province were collected, and their sensitivity to 12 fungicides was determined. All the isolates (100%) were resistant to trifloxystrobin and carbendazim, and 98% were resistant to fluopyram, boscalid, pydiflumetofen, isopyrazam, and fluxapyroxad. However, none were resistant to propiconazole, prochloraz, tebuconazole, difenoconazole, and fludioxonil. The Cytb gene of trifloxystrobin-resistant isolates encoded the G143A mutation, whereas the β-tubulin gene of carbendazim-resistant isolates encoded the E198A and E198A and M163I mutations. Mutations in SdhB -I280V, SdhC -S73P, SdhC -H134R, SdhD -D95E, and SdhD -G109V were associated with resistance to the succinate dehydrogenase inhibitors (SDHIs). Trifloxystrobin, carbendazim, and fluopyram were barely effective on the resistant isolates, whereas fludioxonil and prochloraz were effective on the isolates that were resistant to the quinone outside inhibitors (QoIs), SDHIs, and benzimidazoles. Ultimately, this study demonstrates that fungicide resistance seriously threatens the effective control of Corynespora leaf spot., Competing Interests: The author(s) declare no conflict of interest.

Pear powdery mildew (PPM), caused by Phyllactinia pyri , is one of the most serious diseases affecting production in the Hebei pear-growing region of China. Iminoctadine trialbesilate and trifloxystrobin are known to have broad-spectrum activity against a wide range of plant pathogens, including P. pyri . A total of 105 P. pyri strains were isolated from 11 cities in Hebei Province from 2017 to 2019. Iminoctadine trialbesilate and trifloxystrobin significantly inhibited P. pyri growth. Microscopic observation showed that P. pyri mycelia had different degrees of desiccation and that the conidial cell contents had been released. The sensitivities of 60 P. pyri strains to iminoctadine trialbesilate and trifloxystrobin were determined in vitro, and the average EC 50 values were 0.5773 ± 0.0014 and 1.2038 ± 0.0010 μg/ml, respectively. The average EC 50 values for 85 and 75% of the strains with continuous single peak frequency distributions were 0.4534 ± 0.0012 and 0.8124 ± 0.0039 μg/ml, respectively. These data could be used as the baseline sensitivities of P. pyri to these two fungicides. The maximum difference multiples of the sensitivities of P. pyri strains from the different cities to iminoctadine trialbesilate and trifloxystrobin were 13.5- and 17.2-fold, respectively. Cluster analysis showed that there was no significant correlation between P. pyri sensitivity and geographical origin. The field efficacies in controlling PPM were higher than 85%. These findings can improve how we monitor iminoctadine trialbesilate and trifloxystrobin resistance and improve application efficiency., Competing Interests: The author(s) declare no conflict of interest.

Pyraclostrobin is a highly effective and broad-spectrum strobilurin fungicide. With the widespread use of pyraclostrobin to prevent and control crop diseases, its environmental pressure and potential safety risks to humans have attracted much attention. Herein, the toxicological risks of pyraclostrobin toward HepG2 cells and the mechanisms of intoxication in vitro were investigated. The liver toxicity of pyraclostrobin in zebrafish larvae was also evaluated. It was found that pyraclostrobin induced DNA damage and reactive oxygen species generation in HepG2 cells, indicating the potential genotoxicity of pyraclostrobin. The results of fluorescent staining experiments and the expression of cytochrome c, Bcl-2 and Bax demonstrated that pyraclostrobin induced mitochondrial dysfunction, resulting in cell apoptosis. Monodansylcadaverine staining and autophagy marker-related proteins LC3, p62, Beclin-1 protein expression showed that pyraclostrobin promoted cell autophagy. Furthermore, immunoblotting analysis suggested that pyraclostrobin induced autophagy accompanied with activation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)/mTOR signaling pathway. Visualization of zebrafish liver and oil red staining indicated that pyraclostrobin could induce liver degeneration and liver steatosis in zebrafish. Collectively, these results help to better understand the hepatotoxicity of pyraclostrobin and provide a scientific basis for its safe applications and risk control., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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.

Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and is managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola , has been reported to be resistant to quinone outside inhibitors (QoIs) and to have reduced sensitive to demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from fields in Foxhome, Minnesota, in 2017. Fitness of these resistant isolates was compared with that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI), and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused as high disease severity as the sensitive ones at 21 DAI. Although QoI- and/or DMI-resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices., Competing Interests: The author(s) declare no conflict of interest.

Strobilurins represent the most widely used class of fungicides nowadays andare considered relatively non-toxic to mammals and birds but highly toxic to aquatic biota. Dimoxystrobin is one of the novel strobilurins, recently included in the 3rd Watch List of the European Commission as available data indicate that it could pose a significant risk to aquatic species. As yet, the number of studies explicitly assessing the impact of this fungicide on terrestrial and aquatic species is extremely low, and the toxic effects of dimoxystrobin on fish have not been reported. Here we investigate for the first time the alterations induced by two environmentally relevant and very low concentrations of dimoxystrobin (6.56 and 13.13 μg/L) in the fish gills. morphological, morphometric, ultrastructural, and functional alterations have been evaluated using zebrafish as a model species. We demonstrated that even short-term exposure (96 h) to dimoxystrobin alters fish gills reducing the surface available for gas exchange and inducing severe alterations encompassing three reaction patterns: circulatory disturbance and both regressive and progressive changes. Furthermore, we revealed that this fungicide impairs the expression of key enzymes involved in osmotic and acid-base regulation (Na + /K + -ATPase and AQP3) and the defensive response against oxidative stress (SOD and CAT). The information presented here highlights the importance of combining data from different analytical methods for evaluating the toxic potential of currently used and new agrochemical compounds. Our results will also contribute to the discussion on the suitability of mandatory ecotoxicological tests on vertebrates before the introduction on the market of new compounds., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Phytophthora crown rot (PhCR) and leather rot (LR) caused by Phytophthora spp. are major threats to strawberry production worldwide. In the United States, these diseases are mainly caused by Phytophthora cactorum ; however, P. nicotianae has also been recently reported causing PhCR. Growers have relied on three different chemical products (i.e., mefenoxam and phosphites for PhCR and LR, and azoxystrobin for LR). Because resistance to mefenoxam and azoxystrobin has been reported, this study aimed to assess the in vitro sensitivity of Phytophthora spp. isolates from strawberry to phosphites and investigate its efficacy on in vivo assays. In vitro sensitivity of P. cactorum ( n = 128) and P. nicotianae ( n = 24) isolates collected from 1997 to 2018 was assessed for phosphite at 10, 50, 100, 150, and 300 µg/ml. Regardless of the Phytophthora sp. and isolation organ, most of the isolates (75% for P. cactorum and 54.2% for P. nicotianae ) had effective concentration that inhibits pathogen growth by 50% (EC 50 ) values ranging from 50 to 100 µg/ml. In vivo tests with strawberry fruit and plants revealed that commercial formulations of phosphite applied at the highest field rate controlled P. cactorum isolates but failed to control PhCR and LR caused by some isolates of P. nicotianae . In this study, EC 50 results from in vitro assay did not truly translate the efficacy of phosphites on controlling LR and PhCR caused by P. cactorum and P. nicotianae . Our findings support the hypothesis that the product acts in a dual way: direct on the pathogen and stimulating the plant immune system. Moreover, this has important implications for disease management, highlighting the importance of a correct diagnosis before phosphite recommendations, because its efficacy varies within Phytophthora spp., Competing Interests: The author(s) declare no conflict of interest.

Glomerella leaf spot (GLS) caused by Glomerella cingulata is a newly emerging disease that results in severe defoliation and fruit spots in apples. In China, the compound of pyraclostrobin and tebuconazole was registered to control GLS in 2018 and has achieved excellent control efficiency. In this study, we showed that the high-level resistant isolates of G. cingulata to pyraclostrobin, caused by the point mutation at codon 143 (GGT→GCT, G143A) in the cytochrome b gene, has appeared in apple orchards in Shandong Province in 2020, and the resistance frequency was 4.8%. Based on the genotype of the resistant isolates, we developed a loop-mediated isothermal amplification (LAMP) assay for detection of the pyraclostrobin resistance. The LAMP assay was demonstrated to have good specificity, sensitivity, and repeatability, and it exhibited high accuracy in detecting pyraclostrobin resistance in the field. This study reported the resistance status of GLS to pyraclostrobin in Shandong Province and developed a molecular tool for the detection of pyraclostrobin resistance, which is of practical significance for the scientific control of GLS.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance., Competing Interests: The author(s) declare no conflict of interest.

The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1 ) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.

Downy mildew is caused by Plasmopara viticola, an obligate oomycete plant pathogen, a devasting disease of grapevine. To protect plants from the disease, complex III inhibitors are among the fungicides widely used. They specifically target the mitochondrial cytochrome b (cytb) of the pathogen to block cellular respiration mechanisms. In the French vineyard, P. viticola has developed resistance against a first group of these fungicides, the Quinone outside Inhibitors (QoI), with a single amino acid substitution G143A in its cytb mitochondrial sequence. The use of QoI was limited and another type of fungicide, the Quinone inside Inhibitors, targeting the same gene and highly effective against oomycetes, was used instead. Recently however, less sensitive P. viticola populations were detected after treatments with some inhibitors, in particular ametoctradin and cyazofamid. By isolating single-sporangia P. viticola strains resistant to these fungicides, we characterized new variants in the cytb sequences associated with cyazofamid resistance: a point mutation (L201S) and more strikingly, two insertions (E203-DE-V204, E203-VE-V204). In parallel with the classical tools, pyrosequencing and qPCR, we then benchmarked short and long-reads NGS technologies (Ion Torrent, Illumina, Oxford Nanopore Technologies) to sequence the complete cytb with a view to detecting and assessing the proportion of resistant variants of P. viticola at the scale of a field population. Eighteen populations collected from French vineyard fields in 2020 were analysed: 12 showed a variable proportion of G143A, 11 of E203-DE-V204 and 7 populations of the S34L variant that confers resistance to ametoctradin. Interestingly, the long reads were able to identify variants, including SNPs, with confidence and to detect a small proportion of P. viticola with multiple variants along the same cytb sequence. Overall, NGS appears to be a promising method for assessing fungicide resistance of pathogens linked to cytb modifications at the field population level. This approach could rapidly become a robust decision support tool for resistance management in the future., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Cherrad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Since its reemergence in 2004, Pseudoperonospora cubensis , the causal agent of cucurbit downy mildew (CDM), has experienced significant changes in fungicide sensitivity. Presently, frequent fungicide applications are required to control the disease in cucumber due to the loss of host resistance. Carboxylic acid amides (CAA) and quinone outside inhibitors (QoI) are two fungicide groups used to control foliar diseases in cucurbits, including CDM. Resistance to these fungicides is associated with single nucleotide polymorphism (SNP) mutations. In this study, we used population analyses to determine the occurrence of fungicide resistance mutations to CAA and QoI fungicides in host-adapted clade 1 and clade 2 P. cubensis isolates. Our results revealed that CAA-resistant genotypes occurred more prominently in clade 2 isolates, with more sensitive genotypes observed in clade 1 isolates, while QoI resistance was widespread across isolates from both clades. We also determined that wild cucurbits can serve as reservoirs for P. cubensis isolates containing fungicide resistance alleles. Finally, we report that the G1105W substitution associated with CAA resistance was more prominent within clade 2 P. cubensis isolates while the G1105V resistance substitution and sensitivity genotypes were more prominent in clade 1 isolates. Our findings of clade-specific occurrence of fungicide resistance mutations highlight the importance of understanding the population dynamics of P. cubensis clades by crop and region to design effective fungicide programs and establish accurate baseline sensitivity to active ingredients in P. cubensis populations.

Pyraclostrobin (Pyra) is a fungicide in the strobilurin class and has proven to be very toxic to organisms primarily aquatic species. Resveratrol (Res) is a phytoalexin that exhibits multiple bioactivities as anti-oxidative, anti-inflammatory, cardiovascular protective, and anti-aging and is found in plant species such as mulberry, peanut, and grape. This study aimed to determine the protective effect of Res against Pyra-induced lipid peroxidation, oxidative stress, and DNA damage in rats. For this purpose, a total of 48 male rats divided into 6 groups - 8 in each group - were exposed to 30 mg/kg Pyra by oral gavage once a day for 30 days and to three different concentrations of Res (5, 10, and 20 mg/kg) together with Pyra. Pyra administration increased liver enzyme parameters and malondialdehyde (MDA) levels whereas decreased glutathione (GSH) levels and activities of superoxide dismutase (SOD) and catalase (CAT). Also, Pyra treatment increased pro-apoptotic (Bax), apoptotic (Caspase-3, Caspase-8, and Caspase-9), pro-inflammatory (NFκB), cancer (CYP2E1), and cell regulatory (p53) gene expressions and decreased anti-apoptotic (Bcl-2) gene expression in the liver. Furthermore, DNA damage in blood and histopathological changes in the liver and kidney were observed with Pyra administration. In contrast, Res administrations in a dose-dependent manner improved Pyra-induced lipid peroxidation, oxidative and DNA damages, expression levels of these genes in the liver, and histopathological changes in the liver and kidney. Consequently, the treatment of Res, known for its anti-oxidant and protective properties, exhibited a protective effect on Pyra-induced lipid peroxidation, oxidant/anti-oxidant status, gene expressions, and DNA damage in rats., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Quinone outside inhibitor (QoI) fungicides have not been effective in controlling the wheat blast disease [Pyricularia oryzae Triticum lineage (PoTl)] in Brazil. The first report of resistance of PoTl to QoIs in this country occurred in 2015. This study aimed to test hypotheses about the changes in fitness parameters and competitive advantage of the QoI-resistant (R) PoTl isolate group compared to the sensitive (S) isolate group. Mycelial growth on PDA medium and in vivo conidial production, incubation period and disease severity were analyzed as fitness parameters. The competitive ability was measured on wheat leaves and heads inoculated with mixtures of R:S isolates at the following proportions: 0S:100R, 20S:80R, 50S:50R, 80S:20R, 100S:0R, and 0S:0R., Results: The QoI-R isolate group had significantly higher fitness than the sensitive isolate group, considering both in vitro and in vivo parameters. The highest in vivo conidial production on wheat leaves and the highest leaf and head disease severity were detected when resistant strains were predominant in the isolate's mixtures (20S:80R or 0S:100R proportions), in the absence of fungicide pressure. Conidia harvested from wheat blast lesions on leaves inoculated with 20S:80R and 0S:100R mixtures were resistant to QoIs in vitro assays based on discriminatory doses of the fungicide., Conclusion: Therefore, QoI resistance facilitated a higher fitness and a competitive advantage in PoTl, which contrasts with the evolutionary theory that associates a fitness cost to fungicide resistance. We discuss the evolutionary and ecological implications of the higher fitness as found in the fungicide-resistant adapted populations of the wheat blast pathogen. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: For the first time, the biological activity of slow-release fungicide formulations for suppressing potato pathogens deposited in a degradable poly-3-hydroxybutyrate/sawdust base has been obtained and investigated., Results: The slow-release fungicide formulations (azoxystrobin, azoxystrobin + mefenoxam, and difenoconazole) were studied in vitro and in vivo in comparison with commercial analogues. In in vitro cultures of phytopathogens, the deposited fungicides showed an inhibitory effect comparable to commercial analogues, limiting the growth of colonies of Phytophthora infestans, Alternaria longipes, Rhizoctonia solani and Fusarium solan (2.0-2.3 times relative to the negative control). In laboratory experiments, the use of deposited fungicides was accompanied by earlier germination and more active growth of potatoes against the background of a decrease in the area of plant damage and an increase in yield. In the field experiment, the deposited fungicides suppressed the development of Phytophthora and Alternariosis in the rhizosphere during the entire growing season and reduced the area of plant damage by pathogens by 10-15%, which is two times less than in the groups of plants treated with commercial preparations. The higher biological activity of the embedded fungicides ensured the maximum number of tubers undamaged by pathogens and the total yield of 22-23 t ha -1 , which exceeded the yields in the groups with commercial fungicides (18.4-20.8 t ha -1 )., Conclusion: The slow-release fungicide formulations deposited in a degradable P(3HB)/sawdust base are effective in protecting potatoes from pathogens and increasing yields and have an advantage over commercial counterparts. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: Rice blast, caused by Magnaporthe oryzae, is a destructive disease threatening the production of staple foods worldwide. Quinone outside inhibitors (QoIs) are a group of chemicals exhibiting excellent activity against a majority of plant pathogens, with the disadvantage that pathogens can easily develop resistance to QoIs., Results: Here, we investigated the activity of picoxystrobin against M. oryzae, which showed a great inhibitory effect on 100 strains of M. oryzae with half-maximal effective concentrations (EC 50 ) ranging from 0.0251 to 0.1337 μg ml -1 . The EC 50 values showed a continuous unimodal distribution that was identical to the normal distribution, suggesting the potency of our study to represent baseline sensitivity. In addition, nine resistant mutants were obtained by exposing M. oryzae to a high dosage of picoxystrobin in the laboratory; all of them showed cross-resistance to the other five QoI fungicides. Although some mutants showed a decreased resistance factor after ten successive cultures on fungicide-free medium, the resistance to picoxystrobin was still inheritable. Amino acid substitution of G143S was detected in eight of nine picoxystrobin-resistant mutants, and G143A was detected in only one of nine mutants. A fitness penalty was found in the mutants carrying G143S rather than G143A., Conclusion: Our findings suggested that M. oryzae had a mid to high risk of resistance to picoxystrobin. Considering this, we should be vigilant to the resistance risk and apply picoxystrobin sensibly in the field. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Background: While several agricultural fungicides are known to directly affect invertebrate pests, including aphids, the mechanisms involved are often unknown. One hypothesis is that fungicides with antibacterial activity suppress bacterial endosymbionts present in aphids which are important for aphid survival. Endosymbiont-related effects are expected to be transgenerational, given that these bacteria are maternally inherited. Here, we test for these associations using three fungicides (chlorothalonil, pyraclostrobin and trifloxystrobin) against the bird cherry-oat aphid, Rhopalosiphum padi, using a microinjected strain that carried both the primary endosymbiont Buchnera and the secondary endosymbiont Rickettsiella., Results: We show that the fungicide chlorothalonil did not cause an immediate effect on aphid survival, whereas both strobilurin fungicides (pyraclostrobin and trifloxystrobin) decreased survival after 48 h exposure. However, chlorothalonil substantially reduced the lifespan and fecundity of the F1 generation. Trifloxystrobin also reduced the lifespan and fecundity of F1 offspring, however, pyraclostrobin did not affect these traits. None of the fungicides consistently altered the density of Buchnera or Rickettsiella in whole aphids., Conclusions: Our results suggest fungicides have sublethal impacts on R. padi that are not fully realized until the generation after exposure, and these sublethal impacts are not associated with the density of endosymbionts harbored by R. padi. However, we cannot rule out other effects of fungicides on endosymbionts that might influence fitness, like changes in their tissue distribution. We discuss these results within the context of fungicidal effects on aphid suppression across generations and point to potential field applications. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

The uptake, translocation, and subcellular distribution of oxathiapiprolin and famoxadone in tomato plants were investigated using hydroponic experiments. Oxathiapiprolin and famoxadone mainly accumulated in the tomato roots with limited translocation capacity from the roots to the upper part. The root absorption and inhibitor results noted the dominance of the apoplastic and symplastic pathways in the oxathiapiprolin and famoxadone uptake by the tomato roots, respectively. Furthermore, the uptake process for the two fungicides followed passive and aquaporin-dependent transport. Insoluble cell components (cell organelles and walls) were the dominant storage compartments for oxathiapiprolin and famoxadone. In the protoplast, oxathiapiprolin in the soluble fraction had a higher proportion than that of famoxadone. Finally, the uptake and distribution of the two fungicides by the tomato plants was accurately predicted using a partition-limited model. Thus, this study provides an in-depth understanding of the transfer of oxathiapiprolin and famoxadone from the environment to tomato plants.

Rice bakanae disease (RBD) caused by Fusarium fujikuroi is a widespread and destructive disease of rice. It is urgent to introduce a new class of fungicide to manage the fungicidal resistance problem and effectively control the disease. Azoxystrobin (AZO) is an active fungicide with a broad antifungal spectrum, while its activity against F . fujikuroi is not well investigated. In this study, the baseline sensitivity of F . fujikuroi to AZO was established by testing the sensitivity of 100 isolates, collected from Anhui Province of China. The mechanism of resistance to AZO was also investigated. AZO exhibited a strong activity against the 100 F . fujikuroi isolates with EC 50 values of 0.822 ± 0.285 and 0.762 ± 0.283 μg/mL for mycelial growth and conidial germination, respectively, and both of the baseline sensitivity curves were validated as unimodal curves. To investigate the resistance mechanism, six mutants with resistance factor (RF) values >50 were generated from wild-type sensitive strains through UV mutagenesis, and sequence analysis showed that mutation G143A in cyt b conferred the resistance to AZO. Mycelial growth, conidia production, pathogenicity, and ATP production were decreased in all six resistant mutants as compared to the parental strains, indicating the fitness penalties in this phenotype of resistance mutation. In addition, the cross-resistance assay showed that there was no cross-resistance between AZO and carbendazim, prochloraz, phenamacril, or pydiflumetofen. AZO can be an efficient candidate to control RBD in China with moderate to low fungal resistance risk, but continuous resistance monitoring should be performed during the application of this fungicide.

Colletotrichum acutatum is a species complex that causes anthracnose fruit rot and root necrosis on strawberry. The major and minor species within the complex that affect strawberry production are C. nymphaeae and C. fioriniae , respectively. The disease can significantly reduce yield under conducive weather, and its management has greatly relied on quinone-outside inhibitor fungicides (QoI). However, due to the emergence of resistant isolates, such products are no longer effective. Therefore, alternative fungicides were investigated. C. nymphaeae and C. fioriniae isolates were collected from multiple strawberry fields in the United States from 1995 to 2017. The sensitivity of benzovindiflupyr, penthiopyrad, pydiflumetofen, fluazinam, fludioxonil, and cyprodinil was assessed by in vitro and in vivo assays. Both Colletotrichum species were sensitive to benzovindiflupyr, penthiopyrad, fluazinam, and fludioxonil based on mycelial growth assays. Interestingly, of these products, only penthiopyrad did not inhibit conidial germination at 100 µg/ml. For cyprodinil, C. nymphaeae was sensitive based on the mycelial growth, whereas C. fioriniae was not inhibited. Neither species was inhibited by pydiflumetofen in mycelial growth, conidial germination, nor detached fruit assays. The prepackaged mixtures fludioxonil + cyprodinil and fludioxonil + pydiflumetofen were effective in a field trial; however, their use should be carefully considered because of the lack of efficacy of one of the compounds in the mixture. This study sheds light on the potential registration of products alternative to QoIs, such as benzovindiflupyr and fluazinam, which could improve the management of strawberry anthracnose.

Acetamiprid (neonicotinoid insecticide) and azoxystrobin (fungicide) are widespread pesticides used for pest management, but they have the potential for toxicity to mammals. The goal of this study was to look for oxidative stress, metabolic alterations, and reproductive problems in male rats' serum after 2 months of exposure to sub-lethal dosages of acetamiprid and azoxystrobin. Seven classes of male rats were formed: control, 3 groups of acetamiprid (1/10, 1/20, 1/40 LD 50 ), and 3 groups of azoxystrobin (1/10, 1/20, 1/40 LD 50 ) and were orally daily treated (n = 8/group). Our findings revealed that acetamiprid and azoxystrobin disrupted oxidative and metabolic processes in the examined rats throughout 30 and 60 days of testing. The levels of nitric oxide increased significantly, while catalase, a superoxide dismutase enzyme, and glutathione reductase activity were reduced. Serum levels of sex hormones, calcium, and total protein have all dropped substantially in rats. In comparison to the control group, the testis and liver structure, as well as spermatozoa parameters, had distinct histological characteristics. In conclusion, acetamiprid and azoxystrobin exhibit dose- and time-dependent effects on oxidative parameters that cause testis damage., (© 2021. The Author(s).)

All- trans retinoic acid-based differentiation therapies have succeeded in the treatment of acute promyelocytic leukemia, which is a rare subtype of acute myeloid leukemia (AML). Their clinical efficacy is negligible, however, for other subtypes of AML. Here, we showed that strobilurin derivatives, a well-established class of inhibitors of mitochondrial electron transport chain (ETC) complex III, possessed differentiation-inducing activity in AML cells. Impairment of mitochondrial ETC activity was involved in the differentiation effects of strobilurin derivatives, where reactive oxygen species generation appeared unnecessary. Conversely, strobilurin derivative-mediated differentiation was triggered by pyrimidine deficiency, which resulted from the inhibition of the mitochondrial-coupled dihydroorotate dehydrogenase enzyme. Moreover, strobilurin derivative-mediated pyrimidine depletion led to the activation of the Akt/mTOR cascade, which was required for the differentiation. Our study provided evidence that strobilurin derivatives may represent a novel class of differentiation-inducing agents for the treatment of AML.

Twenty-one novel 3,4-dichloroisothiazolocoumarin-containing strobilurins were rationally designed and synthesized. Preliminary bioassay showed that compounds 7c, 7h and 7l exhibited over 80% inhibitory rate against Sclerotinia sclerotiorum at 50 μg/mL, 7c exhibited good activity against S. sclerotiorum with median effective concentration (EC 50 ) of 4.08 μg/mL, while the positive control coumoxystrobin showed EC 50 of 1.00 μg/mL. In addition, 7d showed better fungicidal activity with a lower EC 50 value of 7.65 μg/mL against Botrytis cinerea than that of positive control trifloxystrobin with its EC 50 value of 21.96 μg/mL. This study indicated that 3,4-dichloroisothiazolocoumarin-containing strobilurin was a promising fungicide lead deserved for further study., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Acquired resistance is a threat to antifungal efficacy in medicine and agriculture. The diversity of possible resistance mechanisms and highly adaptive traits of pathogens make it difficult to predict evolutionary outcomes of treatments. We used directed evolution as an approach to assess the resistance risk to the new fungicide fenpicoxamid in the wheat pathogenic fungus Zymoseptoria tritici. Fenpicoxamid inhibits complex III of the respiratory chain at the ubiquinone reduction site (Qi site) of the mitochondrially encoded cytochrome b, a different site than the widely used strobilurins which inhibit the same complex at the ubiquinol oxidation site (Q o site). We identified the G37V change within the cytochrome b Q i site as the most likely resistance mechanism to be selected in Z. tritici. This change triggered high fenpicoxamid resistance and halved the enzymatic activity of cytochrome b, despite no significant penalty for in vitro growth. We identified negative cross-resistance between isolates harbouring G37V or G143A, a Q o site change previously selected by strobilurins. Double mutants were less resistant to both QiIs and quinone outside inhibitors compared to single mutants. This work is a proof of concept that experimental evolution can be used to predict adaptation to fungicides and provides new perspectives for the management of QiIs., (© 2021 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background/aim: Azoxystrobin (AZOX), a methoxyacrylate derivative, has potent antimicrobial and antitumor activities. Here, we report the anticancer effects of AZOX on the p53-negative human myelogenous leukemia cell line HL-60RG and the p53 positive human T-cell leukemia cell line MOLT-4F., Materials and Methods: Using both leukemia cells, the anticancer effect of AZOX treatment was analyzed throughout the cell cycle., Results: AZOX damaged both cell lines dose-dependently, and the cell damage rates were almost the same in both lines. Cell cycle distribution analysis showed that the treated MOLT-4F cells arrested at the S phase, whereas HL-60RG cells increased during the subG1 phase, suggesting that cell death was occurring. AZOX-induced cell death in HL-60RG was inhibited with the addition of uridine, which is used as a substrate for the salvage pathway of pyrimidine nucleotides., Conclusion: AZOX has p53-independent anticancer effects in leukemia cells, but the mechanisms underlying the damage differ between cell lines., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The widespread use of conventional chemical antifungal agents has led to worldwide concern regarding the selection of resistant isolates. In this scenario, antimicrobial photodynamic treatment (APDT) has emerged as a promising alternative to overcome this issue. The technique is based on the use of a photosensitizer (PS) and light in the presence of molecular oxygen. Under these conditions, the PS generates reactive oxygen species which damage the biomolecules of the target organism leading to cell death. The great potential of APDT against plant-pathogenic fungi has already been reported both in vitro and in planta, indicating this control measure has the potential to be widely used in crop plants. However, there is a lack of studies on environmental risk with ecotoxicological assessment of PSs used in APDT. Therefore, this study aimed to evaluate the environmental toxicity of four phenothiazinium PSs: i) methylene blue (MB), ii) new methylene blue N (NMBN), iii) toluidine blue O (TBO), and iv) dimethylmethylene blue (DMMB) and also of the commercial antifungal NATIVO®, a mixture of trifloxystrobin and tebuconazole. The experiments were performed with Daphnia similis neonates and zebrafish embryos. Our results showed that the PSs tested had different levels of toxicity, with MB being the less toxic and DMMB being the most. Nonetheless, the environmental toxicity of these PSs were lower when compared to that of NATIVO®. Furthermore, estimates of bioconcentration and of biotransformation half-life indicated that the PSs are environmentally safer than NATIVO®. Taken together, our results show that the toxicity associated with phenothiazinium PSs would not constitute an impediment to their use in APDT. Therefore, APDT is a promising approach to control plant-pathogenic fungi with reduced risk for selecting resistant isolates and lower environmental impacts when compared to commonly used antifungal agents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Fungicides often cause genotoxic stress and neurodevelopmental disorders such as autism (ASD). Fungicide-azoxystrobin (AZOX) showed acute and chronic toxicity to various organisms, and remained a concern for ill effects in developing neurons. We evaluated the neurotoxicity of AZOX in developing mouse brains, and observed prenatal exposure to AZOX reduced neuronal viability, neurite outgrowth, and cortical migration process in developing brains. The 50% inhibitory concentration (IC50) of AZOX for acute (24 h) and chronic (7 days) exposures were 30 and 10 μM, respectively. Loss in viability was due to the accumulation of reactive oxygen species (ROS), and inhibited neurite outgrowth was due to the deactivation of mTORC1 kinase activity. Pretreatment with ROS scavenger- N-acetylcysteine (NAC) reserved the viability loss and forced activation of mTORC1 kinase revived the neurite outgrowth in AZOX treated neurons. Intra-amniotic injection of AZOX coupled with in utero electroporation of GFP-labelled plasmid in E15.5 mouse was performed and 20 mg/kg AZOX inhibited radial neuronal migration. Moreover, the accumulation of mitochondria was significantly reduced in AZOX treated primary neurons, indicative of mitochondrial deactivation and induction of apoptosis, which was quantified by Bcl2/Bax ratio and caspase 3 cleavage assay. This study elucidated the neurotoxicity of AZOX and explained the possible cure from it.

Pomegranate crown rot caused by Coniella granati is one of the most severe diseases of pomegranate. No fungicides have been registered for controlling this disease in China. Pyraclostrobin, belonging to strobilurin fungicides, has a broad spectrum of activity against many phytopathogens. In this study, based on the mycelial growth and conidial germination inhibition methods, we investigated the biological activity of pyraclostrobin against C. granati in the presence of 50 μg/ml of salicylhydroxamic acid using 80 isolates collected from different orchards in China from 2012 to 2018. The EC 50 (50% effective concentration) values ranged from 0.040 to 0.613 μg/ml for mycelial growth and 0.013 to 0.110 μg/ml for conidium germination. Treated with pyraclostrobin, the hyphae morphology changed and conidial production of C. granati decreased significantly. The result of transmission electron microscope showed that treatment of pyraclostrobin could make the cell wall thinner and lead to ruptured cell membrane and formation of intracellular organelle autophagosomes. The pyraclostrobin showed good protective and curative activities against C. granati on detached pomegranate fruits. In field trials, pyraclostrobin showed excellent control efficacy against this disease, in which the treatment of 25% pyraclostrobin EC 1,000× provided 92.25 and 92.58% control efficacy in 2019 and 2020, respectively, significantly higher than that of other treatments. Therefore, pyraclostrobin could be a candidate fungicide for the control of pomegranate crown rot.

Sclerotium rolfsii is a fungi pathogen of southern blight with broad host range. The quinone outside inhibitor fungicide pyraclostrobin was officially approved for controlling many diseases in 2015. In this study, baseline sensitivity of S. rolfsii to pyraclostrobin was established by measuring the 50% effective concentration (EC 50 ) values of 155 isolates of S. rolfsii . The EC 50 values ranged from 0.0291 to 1.0871 µg/ml, with a mean EC 50 of 0.4469 ± 0.2490 µg/ml (mean ± standard deviation). In a preventive fungicide in vitro experiment and a field experiment, pyraclostrobin preventive efficacy reached 90% and 80%, respectively, which were much higher than that of the control agent carbendazim. Curative efficacy of pyraclostrobin was significantly lower than its preventive efficacy. Pyraclostrobin at 0.1, 0.5, and 2 μg/ml significantly reduced the number of sclerotia produced on potato dextrose agar medium, but had no significant influence on their total weight. Pyraclostrobin had no significant influence on mycelial cell membrane permeability, but it significantly reduced oxalate secretion and protein synthesis of S. rolfsii . Our findings are of great significance for resistance monitoring of S. rolfsii and also provide new insight into the action mechanism of pyraclostrobin against S. rolfsii .

Long-term use of a single fungicide increases the resistance risk and causes adverse effects on natural ecosystems. Controlled release formulations of dual fungicides with different modes of action can afford a new dimension for addressing the current issues. Based on adjustable aperture and superhigh surface area, metal-organic frameworks (MOFs) are ideal candidates as pesticide release carriers. This study used Al 3+ as the metal node and 2-aminoterephthalic acid as the organic chain to prepare aluminum-based metal-organic framework material (NH 2 -Al-MIL-101) with "cauliflower-like" structure and high surface area of 2359.0 m 2 /g. Fungicides of azoxystrobin (AZOX) and diniconazole (Dini) were simultaneously encapsulated into NH 2 -Al-MIL-101 with the loading content of 6.71% and 29.72%, respectively. Dual fungicide delivery system of AZOX@Dini@NH 2 -Al-MIL-101 demonstrated sustained and pH responsive release profiles. When the maximum cumulative release rate of AZOX and Dini both reached about 90%, the release time was 46 and 136 h, respectively. Furthermore, EC 50 values as well as the percentage of inhibition revealed that AZOX@Dini@NH 2 -Al-MIL-101 had enhanced germicidal efficacy against rice sheath blight ( Rhizoctonia solani ), evidenced by the synergistic ratio of 1.83. The present study demonstrates a potential application prospect in sustainable plant protection through co-delivery fungicides with MOFs as a platform.