Analysis of Flutianil and OC56635 Residues in Hemp Cannabis Matrices by LC-MS/MS.

In this paper, we present analytical methodologies for the determination of the thiazolidine fungicide flutianil (trade name GATTEN) and its primary metabolite OC56635 in hemp cannabis matrices. A total of nine crop matrices were tested: whole seed, fiber, flower buds, hemp hearts, hemp seed oil, hemp meal, hemp flour, ethanol extracted CBD resin (CBD-E), and supercritical CO2 extracted CBD resin (CBD-C). Processing of the CBD-E and CBD-C crop fractions was carried out in-house using methods detailed herein. Field sample analysis utilized sequential extractions, stacked solid phase extraction (SPE) column cleanups, and evaporation to prepare the samples for LC-MS/MS quantitation. Method validations for each fraction were carried out using untreated hemp matrices over a minimum of three levels, with lowest levels of method validation (LLMV) of 0.010 µg/g for all fractions except the CBD resins, for which LLMV was 0.020 µg/g. Flutianil-treated samples from nine field sites were collected from several crop production regions and analyzed to determine the distribution of incurred flutianil and OC56635 residues within the different hemp matrices. This data was generated in support of nationwide registration with the United States Environmental Protection Agency (USEPA).

Computational simulations uncover enantioselective metabolism of chiral triazole fungicides by human CYP450 enzymes: A case study of tebuconazole.

Tebuconazole (TEB), a prominent chiral triazole fungicide, has been extensively utilized for plant pathogen control globally. Despite experimental evidence of TEB metabolism in mammals, the enantioselectivity in the biotransformation of R- and S-TEB enantiomers by specific CYP450s remains elusive. In this work, integrated in silico simulations were employed to unveil the binding interactions and enantioselective metabolic fate of TEB enantiomers within human CYP1A2, 2B6, 2E1, and 3A4. Molecular dynamics (MD) simulations clearly delineated the binding specificity of R- and S-TEB to the four CYP450s, crucially determining their differences in metabolic activity and enantioselectivity. The primary driving force for robust ligand binding was identified as van der Waals interactions with CYP450s, particularly involving the hydrophobic residues. Mechanistic insights derived from quantum mechanics/molecular mechanics (QM/MM) calculations established C2-methyl hydroxylation as the predominant route of R-/S-TEB metabolism, while C6-hydroxylation and triazol epoxidation were deemed kinetically infeasible pathways. Specifically, the resulting hydroxy-R-TEB metabolite primarily originates from R-TEB biotransformation by 1A2, 2E1 and 3A4, whereas hydroxy-S-TEB is preferentially produced by 2B6. These findings significantly contribute to our comprehension of the binding specificity and enantioselective metabolic fate of chiral TEB by CYP450s, potentially informing further research on human health risk assessment associated with TEB exposure.

Deciphering the role of nitric oxide in mitigation of systemic fungicide induced growth inhibition and oxidative damage in wheat.

Consento (CON) poses a significant environmental hazard as a systemic fungicide, adversely affecting the health of non-target organisms. Nitric oxide (NO), a signaling molecule, is known to play a crucial role in plant physiology and abiotic stress tolerance. However, whether NO plays any role to enhance fungicide CON tolerance in wheat seedlings is yet unclear. Therefore, we conducted a hydroponic experiment i) to investigate the morpho-physio-biochemical changes of wheat seedlings to fungicide CON stress, and ii) to examine the effects of NO and fungicide CON treatments on oxidative damage, antioxidant system, secondary metabolism and detoxification of systemic fungicide in wheat seedlings. The results showed that CON fungicide at the highest (4X) concentration significantly decreased wheat seedlings fresh weight (46.89%), shoot length (40.26%), root length (56.11%) and total chlorophyll contents (67.44%) in a dose response relationship. Moreover, CON significantly increased hydrogen peroxide, malondialdehyde, catalase, ascorbate peroxidase, glutathione-S-transferase, and peroxidase activities while decreased reduced glutathione (GSH) content. This ultimately impaired the redox homeostasis of cells, leading to oxidative damage in cell membrane. Under fungicide treatment, the addition of NO reduced the fungicide phytotoxicity, with an increase of over 60% in seedling growth. The NO application mitigated CON phytotoxicity as reflected by significantly increased chlorophyll pigments (69.88%) and decreased oxidative damage in wheat leaves. Indeed, the NO alleviatory effect was able to increase the tolerance of seedlings to fungicide, which resulted increments in antioxidant and detoxification enzymes activity, with the enhanced GSH level (78.54%). Interestingly, NO alleviated CON phytotoxicity through the phenylpropanoid pathway by enhancing the activity of secondary metabolism enzymes such as phenylalanine ammonia-lyase (47.28%), polyphenol oxidase (9%), and associated metabolites such as phenolic acids (77.62%), flavonoids (34.33%) in wheat leaves. Our study has provided evidence that NO plays a key role in the metabolism and detoxification of systemic fungicide in wheat through enhanced activity of antioxidants, detoxifications and secondary metabolic enzymes.

Fungicide-Loaded Liposomes for the Treatment of Fungal Diseases in Agriculture: An Assessment of Botrytis cinerea.

In this work, liposomes loaded with the fungicide, Fludioxonil (FLUD), for the containment of fungal diseases in agriculture were developed. Three types of vesicles with different compositions were compared: (I) plain vesicles, composed of soy phosphatidylcholine and cholesterol; (II) PEG-coated vesicles, with an additional polyethylene glycol coating; and (III) cationic vesicles, containing didodecyldimethylammonium bromide. Nanometric-sized vesicles were obtained both by the micelle-to-vesicle transition method and by the extrusion technique, and encapsulation efficiency, drug loading content, and Zeta potential were determined for all the samples. The extruded and PEGylated liposomes were the most stable over time and together with the cationic ones showed a significant prolonged FLUD release capacity. The liposomes' biological activity was evaluated on conidial germination, germ tube elongation and colony radial growth of the ascomycete Botrytis cinerea, a phytopathogenic fungus affecting worldwide many important agricultural crops in the field as well as in the postharvest phase. The extruded and PEGylated liposomes showed greater effectiveness in inhibiting germ tube elongation and colony radial growth of the fungal pathogen, even at 0.01 µg·mL-1, the lowest concentration assessed.

Lignin/Surfactin Coacervate as an Eco-Friendly Pesticide Carrier and Antifungal Agent against Phytopathogen.

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.

Fungicide resistance in Botrytis cinerea and identification of Botrytis species associated with blueberry in Michigan.

Botrytis blossom blight and fruit rot, caused by Botrytis cinerea, is a significant threat to blueberries, potentially resulting in substantial economic losses if not effectively managed. Despite the recommendation of various cultural and chemical practices to control this pathogen, there are widespread reports of fungicide resistance, leading to decreased efficacy. This study aimed to characterize the resistance profile of B. cinerea isolated from blighted blossoms and fruit in 2019, 2020 and 2022 (n = 131, 40, and 37 for the respective years). Eight fungicides (fludioxonil, thiabendazole, pyraclostrobin, boscalid, fluopyram, fenhexamid, iprodione, and cyprodinil) were tested using conidial germination at specific discriminatory doses. Additionally, 86 isolates were phylogenetically characterized using the internal transcribed spacer regions (ITS) and the protein coding genes: glyceraldehyde-3-phosphate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and RNA polymerase II second largest subunit (RPB2). This revealed higher fungicide resistance frequencies in 2020 and 2022 compared to 2019. Over all 3 years, over 80% of the isolates were sensitive to fludioxonil, fluopyram, and fenhexamid. Pyraclostrobin and boscalid showed the lowest sensitivity frequencies (<50%). While multi-fungicide resistance was observed in all the years, none of the isolates demonstrated simultaneous resistance to all tested fungicides. Botrytis cinerea was the most prevalent species among the isolates (74) with intraspecific diversity detected by the genes. Two isolates were found to be closely related to B. fabiopsis, B. galanthina, and B. caroliniana and 10 isolates appeared to be an undescribed species. This study reports the discovery of a potentially new species sympatric with B. cinerea on blueberries in Michigan.

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

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

Genotypic and phenotypic characterization of resistance to fenhexamid, carboxin, and, prochloraz, in Botrytis cinerea isolates collected from cut roses in Colombia.

Gray mold, caused by Botrytis sp., is a significant disease in Colombian rose crops and its control depends primarily on the intensive use of chemically synthesized fungicides. Despite the importance of this pathogen, there is limited information in Colombian floriculture about molecular taxonomy of species, fungicide resistance of populations and their genetic mechanism of resistance. In this study, we analyze 12 isolates of this fungus collected from rose-producing crops in the Department of Cundinamarca and conducted phylogenetic analysis using HSP60, G3PDH, and RPB2 gene sequences. Additionally, we realize phenotypic and genotypic characterization of resistance to the fungicides fenhexamid, carboxin, and prochloraz, evaluating the in vitro EC50 and presence of mutations of target genes of each isolate. All isolates were characterized as Botrytis cinerea in the phylogenetic analysis and presents different levels of resistance to each fungicide. These levels are related to mutations in target genes, with predominancy of L195F and L400F in the ERG27 gene to fenhexamid resistance, H272R/Y in the SDHB gene for carboxin resistance, and Y136F in the CYP51 gene for prochloraz resistance. Finally, these mutations were not related to morphological changes. Collectively, this knowledge, presented for the first time to the Colombian floriculture, contribute to a better understanding of the genetic diversity and population of B. cinerea from rose-producing crops in the department of Cundinamarca, and serve as a valuable tool for making informed decisions regarding disease management, future research, and improving crop management and sustainability in the Colombian floriculture industry.

Discovery of Benzothiazol-2-ylthiophenylpyrazole-4-carboxamides as Novel Succinate Dehydrogenase Inhibitors.

Succinate dehydrogenase (SDH) has been considered an ideal target for discovering fungicides. To develop novel SDH inhibitors, in this work, 31 novel benzothiazol-2-ylthiophenylpyrazole-4-carboxamides were designed and synthesized using active fragment exchange and a link approach as promising SDH inhibitors. The findings from the tests on antifungal activity indicated that most of the synthesized compounds displayed remarkable inhibition against the fungi tested. Compound Ig N-(2-(((5-chlorobenzo[d]thiazol-2-yl)thio)methyl)phenyl)-3-(difluoromethyl)-1-methyl-1H-yrazole-4-carboxamide, with EC50 values against four kinds of fungi tested below 10 µg/mL and against Cercospora arachidicola even below 2 µg/mL, showed superior antifungal activity than that of commercial fungicide thifluzamide, and specifically compounds Ig and Im were found to show preventative potency of 90.6% and 81.3% against Rhizoctonia solani Kühn, respectively, similar to the positive fungicide thifluzamide. The molecular simulation studies suggested that hydrophobic interactions were the main driving forces between ligands and SDH. Encouragingly, we found that compound Ig can effectively promote the wheat seedlings and the growth of Arabidopsis thaliana. Our further studies indicated that compound Ig could stimulate nitrate reductase activity in planta and increase the biomass of plants.

The neonicotinoid acetamiprid reduces larval and adult survival in honeybees (Apis mellifera) and interacts with a fungicide mixture.

Plant protection products (PPPs1), which are frequently used in agriculture, can be major stressors for honeybees. They have been found abundantly in the beehive, particularly in pollen. Few studies have analysed effects on honeybee larvae, and little is known about effects of insecticide-fungicide-mixtures, although this is a highly realistic exposure scenario. We asked whether the combination of a frequently used insecticide and fungicides would affect developing bees. Honeybee larvae (Apis mellifera carnica) were reared in vitro on larval diets containing different PPPs at two concentrations, derived from residues found in pollen. We used the neonicotinoid acetamiprid, the combined fungicides boscalid/dimoxystrobin and the mixture of all three substances. Mortality was assessed at larval, pupal, and adult stages, and the size and weight of newly emerged bees was measured. The insecticide treatment in higher concentrations significantly reduced larval and adult survival. Interestingly, survival was not affected by the high concentrated insecticide-fungicides-mixture However, negative synergistic effects on adult survival were caused by the low concentrated insecticide-fungicides-mixture, which had no effect when applied alone. The lower concentrated combined fungicides led to significantly lighter adult bees, although the survival was unaffected. Our results suggest that environmental relevant concentrations can be harmful to honeybees. To fully understand the interaction of different PPPs, more combinations and concentrations should be studied in social and solitary bees with possibly different sensitivities.

Identification, Pathogenicity, and Fungicide Sensitivity of Colletotrichum Species Associated with Anthracnose on Italian Ryegrass in Southwestern China.

Italian ryegrass (Lolium multiflorum L.) is widely cultivated as an important forage worldwide because of its high nutritional value and good palatability. Anthracnose caused by Colletotrichum species was a common and new emerging disease of Italian ryegrass. In this study, 88 Colletotrichum isolates were collected from diseased leaves of Italian ryegrass planting regions in Sichuan, Chongqing and Guizhou provinces of southwestern China between 2019 and 2022. By pure culture technique, 15 representative single-spore isolates were obtained for further study. Multi-locus phylogenetic analysis coupled with morphological features showed that these isolates were finally identified as six new record species: C. cereale of the C. graminicola species complex, C. fioriniae and C. nymphaeae of the C. acutatum species complex, C. boninense and C. citricola of the C. boninense species complex, and C. nageiae. Pathogenicity tests indicated that all species could induce anthracnose symptoms; of these, C. cereale was more invasive than other species, followed by C. fioriniae, C. nageiae, C. citricola and C. boninense; C. nymphaeae was weakest pathogenic to Italian ryegrass plants (P ≤ 0.05). Fungicide sensitivity assays showed that iprodione, propineb and oxime·tebuconazole had strong inhibitory effect on the mycelial growth of six Colletotrichum species; in addition, azoxystrobin and fludioxonil also significantly inhibited the mycelial growth of C. nymphaeae and C. fioriniae, respectively. These results provide the basis for the diagnosis and detection in the field, pathogen identification and management of anthracnose on Italian ryegrass.

Peer review of the pesticide risk assessment for the active substance difenoconazole in light of confirmatory data submitted.

The conclusions of the EFSA following the peer review of the initial risk assessment carried out by the competent authority of the rapporteur Member State, Spain, for the pesticide active substance difenoconazole are reported. The context of the peer review was that requested by the European Commission following the submission and evaluation of confirmatory information with regard to the consumer risk assessment. The conclusions were reached on the basis of the evaluation of the representative uses of difenoconazole as a fungicide on pome fruit, carrot, wheat, barley, triticale, rye and oats. The reliable endpoints concluded as being appropriate for use in regulatory risk assessment, derived from the available studies and/or literature in the dossier peer reviewed, are presented. Concerns were not identified.

Morinda citrifolia Essential Oil: A Plant Resistance Biostimulant and a Sustainable Alternative for Controlling Phytopathogens and Insect Pests.

With the growing demand for sustainable and safe agricultural practices, plant compounds emerge as a solution for biological activities. Here, we evaluated the potential of using Morinda citrifolia essential oil to induce plant resistance and to control phytopathogens (Curvularia lunata) and insect pests (Daubulus maidis). We conducted a chromatographic analysis to unveil the essential oil components. We also quantified the activity levels of antioxidant enzymes and chitinase for resistance induction. The antifungal action was evaluated through disease progression and the inhibition of mycelial growth in addition to in silico studies that made it possible to predict the interaction site between the fungal protein and the compounds. We assessed the toxicity and repellent actions towards the D. maidis. Octanoic acid (58.43%) was identified as the essential oil major compound. Preventive treatment with essential oil and octanoic acid (25.0 µL mL-1) increased not only the plant defense activities (i.e., the activity of the enzymes superoxide dismutase, catalase, phenol peroxidase, ascorbate peroxidase, and chitinase) but also controlled Curvularia leaf spot. The stable interactions between octanoic acid and tyrosine-tRNA ligase from C. lunata suggested protein synthesis inactivation. The essential oil inhibited 51.6% of mycelial growth, and this effect was increased to 75.9% with the addition of adjuvants (i.e., angico gum). The essential oil reduced 76% of the population of D. maidis adults and repelled 50% of the number of D. maidis after 48 h under field conditions. The repellency effect in the field reduced the population of D. maidis adults, transmitters of the stunting complex, by 50%. The results highlight the potential of M. citrifolia as a resistance activator, fungicide, insecticide, and an effective biorational alternative.

Bixafen, Prothioconazole, and Trifloxystrobin Alone or in Combination Have a Greater Effect on Health Related Gene Expression in Honey Bees from Nutritionally Deprived than from Protein Supplemented Colonies.

The aim of this study was to evaluate whether alterations in food availability compromise the metabolic homeostasis of honey bees exposed to three fungicides alone or together. Ten honey bee colonies were used, with half receiving carbohydrate-protein supplementation for 15 weeks while another five colonies had their protein supply reduced with pollen traps. Subsequently, forager bees were collected and exposed by contact to 1 or 7 µg of bixafen, prothioconazole, or trifloxystrobin, either individually or in combination. After 48 h, bee abdomens without the intestine were used for the analysis of expression of antioxidant genes (SOD-1, CAT, and GPX-1), detoxification genes (GST-1 and CYP306A1), the storage protein gene vitellogenin, and immune system antimicrobial peptide genes (defensin-1, abaecin, hymenoptaecin, and apidaecin), through real-time PCR. All fungicide treatments induced changes in gene expression, with bixafen showing the most prominent upregulation. Exposure to 1 µg of each of the three pesticides resulted in upregulation of genes associated with detoxification and nutrition processes, and downregulation of immune system genes. When the three pesticides were combined at a dose of 7 µg each, there was a pronounced downregulation of all genes. Food availability in the colonies affected the impact of fungicides on the expression of the studied genes in forager bees.

Development of an RNA Nanostructure for Effective Botrytis cinerea Control through Spray-Induced Gene Silencing without an Extra Nanocarrier.

Spray-induced gene silencing represents an eco-friendly approach for crop protection through the use of double-stranded RNA (dsRNA) to activate the RNA interference (RNAi) pathway, thereby silencing crucial genes in pathogens. The major challenges associated with dsRNA are its limited stability and poor cellular uptake, necessitating repeated applications for effective crop protection. In this study, RNA nanoparticles (NPs) were proposed as effectors in plants and pathogens by inducing the RNAi pathway and silencing gene expression. RNA structural motifs, such as hairpin-loop, kissing-loop, and tetra-U motifs, were used to link multiple siRNAs into a long, single-stranded RNA (lssRNA). The lssRNA, synthesized in Escherichia coli, self-assembled into stable RNA nanostructures via local base pairing. Comparative analyses between dsRNA and RNA NPs revealed that the latter displayed superior efficacy in inhibiting spore germination and mycelial growth of Botrytis cinerea. Moreover, RNA NPs had a more robust protective effect on plants against B. cinerea than did dsRNA. In addition, RNA squares are processed into expected siRNA in plants, thereby inhibiting the expression of the target gene. These findings suggest the potential of RNA NPs for use in plant disease control by providing a more efficient and specific alternative to dsRNA without requiring nanocarriers.

Is the Biopesticide from Tea Tree Oil an Effective and Low-Risk Alternative to Chemical Pesticides? A Critical Review.

The use of chemical pesticides in agriculture contributes to soil, water and air pollution, biodiversity loss, and injury to non-target species. The European Commission has already established a Harmonized Risk Indicator to quantify the progress in reducing the risks linked to pesticides. Therefore, there is an increasing need to promote biopesticides, or so-called low-risk pesticides (LRP). Tea tree oil (TTO) is known for its antiseptic, antimicrobial, antiviral, antifungal, and anti-inflammatory properties. TTO has been extensively studied in pest management as well as in the pharmaceutical and cosmetic industry; there are already products based on its active substances on the market. This review focuses on the overall evaluation of TTO in terms of effectiveness and safety as a biopesticide for the first time. The collected data can be an added value for further evaluation of TTO in terms of the authorization extension as a fungicide in 2026.

Effect of Combining Wuyiencin and Pyrimethanil on Controlling Grape Gray Mold and Delaying Resistance Development in Botrytis cinerea.

By screening the compounding combination of Wuyiencin and chemical agents, this study aims to delay the emergence of chemical agent resistance, and provide a technical reference for scientific and rational fungicides technology. This study investigated the impacts of the antibiotic wuyiencin derived from Streptomyces albulus var. wuyiensis and its combination with pyrimethanil on the inhibition of Botrytis cinerea. Treatment with wuyiencin (≥80 µg mL-1) strongly inhibited the pathogenicity of B. cinerea and activated the plant defense response against B. cinerea. Application of 80-100 µg mL-1 wuyiencin effectively controlled grape gray mold (by 57.6-88.1% on leaves and 46.7-96.6% on fruits). Consequently, the application of 80-100 µg mL-1 wuyiencin effectively mitigated grape gray mold incidence, leading to a substantial reduction in disease symptoms to nearly imperceptible levels. When wuyiencin (at the median effective concentration [EC50]) was combined with pyrimethanil (EC50) at a ratio of 7:3, it exhibited the highest efficacy in inhibiting B. cinerea growth. This combination was significantly more potent (p < 0.05) than using wuyiencin or pyrimethanil alone in controlling gray mold on grape leaves and fruits. Furthermore, the combination effectively delayed resistance development in gray mold. The experimental results show that wuyiencin can delay resistance development by affecting the expression of methionine biosynthesis genes and reducing the activity of the cell wall-degrading enzyme activity.

Transcriptional dynamics of Fusarium pseudograminearum under high fungicide stress and the important role of FpZRA1 in fungal pathogenicity and DON toxin production.

Fusarium pseudograminearum, the causal agent of Fusarium crown rot, poses a significant threat to cereal crops. Building upon our previous investigation of the transcriptional response of this pathogen to four key fungicides (carbendazim, phenamacril, pyraclostrobin, and tebuconazole), this study delves into the impact of elevated fungicide concentrations using RNA-seq. Global transcriptomic analysis and gene clustering revealed significant enrichment of genes involved in the ABC transporter pathway. Among these transporters, FPSE_06011 (FpZRA1), a conserved gene in eukaryotes, exhibited consistent upregulation at both low and high fungicide concentrations. Targeted deletion of FpZRA1 resulted in reduced sporulation, spore germination, and tolerance to cell wall stress, osmotic stress, and oxidative stress. Furthermore, the FpZRA1 knockout mutants exhibited decreased pathogenicity on wheat coleoptiles and reduced production of the mycotoxin deoxynivalenol (DON), as evidenced by the markedly down-regulated expression of TRI5, TRI6, and TRI10 in the RT-qPCR analysis. In summary, our findings highlight the impact of fungicide concentration on transcriptional reprogramming in F. pseudograminearum and identify FpZRA1 as a critical regulator of fungal development, stress tolerance, and pathogenicity.

CbCyp51-mediated DMI resistance is modulated by codon bias.

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

Novel Insights into Stereoselective Reproductive Toxicity Induced by Mefentrifluconazole in Earthworms (Eisenia fetida): First Report of Estrogenic Effects.

Widespread use of the new chiral triazole fungicide mefentrifluconazole (MFZ) poses a threat to soil organisms. Although triazole fungicides have been reported to induce reproductive disorders in vertebrates, significant research gaps remain regarding their impact on the reproductive health of soil invertebrates. Here, reproduction-related toxicity end points were explored in earthworms (Eisenia fetida) after exposure for 28 d to soil containing 4 mg/kg racemic MFZ, R-(-)-MFZ, and S-(+)-MFZ. The S-(+)-MFZ treatment resulted in a more pronounced reduction in the number of cocoons and juveniles compared to R-(-)-MFZ treatment, and the expression of annetocin gene was significantly downregulated following exposure to both enantiomers. This reproductive toxicity has been attributed to the disruption of ovarian steroidogenesis at the transcriptional level. Further studies revealed that MFZ enantiomers were able to activate the estrogen receptor (ER). Indirect evidence for this estrogenic effect is provided by the introduction of 17ß-estradiol, which also induces reproductive disorders through ER activation.