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Seed coating with fungicides is a common practice in controlling seed-borne diseases, but conventional methods often result in high toxicity to plants and soil. In this study, a nanoparticle formulation was successfully developed using the metal-organic framework UiO-66 as a carrier of the fungicide ipconazole (IPC), with a tannic acid (TA)-Zn II coating serving as a protective layer. The IPC@UiO-66-TA-Zn II nanoparticles provided a controlled release, triggered and regulated by environmental factors such as pH and temperature. This formulation efficiently controlled the proliferation of Fusarium fujikuroi spores, with high penetration into both rice roots and fungal mycelia. The product exhibited high antifungal activity, achieving control efficacy rates of 84.09% to 93.10%, low biotoxicity, and promoted rice growth. Compared to the IPC flowable suspension formula, IPC@UiO-66-TA-Zn II improved the physicochemical properties and enzymatic activities in soil. Importantly, it showed potential for mitigating damage to beneficial soil bacteria. This study provides a promising approach for managing plant diseases using nanoscale fungicides in seed treatment., Competing Interests: Declarations Ethics approval and consent to participate We confirmed that all methods involving the plant and its material adhered with the relevant institutional, national, and international guidelines and legislation. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Mechanical forces have been shown to influence cellular decisions to grow, die, or differentiate, through largely mysterious mechanisms. Separately, changes in resting membrane potential have been observed in development, differentiation, regeneration, and cancer. We now demonstrate that membrane potential is the central mediator of cellular response to mechanical pressure. We show that mechanical forces acting on the cell change cellular biomass density, which in turn alters membrane potential. Membrane potential then regulates cell number density in epithelia by controlling cell growth, proliferation, and cell elimination. Mechanistically, we show that changes in membrane potential control signaling through the Hippo and MAPK pathways, and potentially other signaling pathways that originate at the cell membrane. While many molecular interactions are known to affect Hippo signaling, the upstream signal that activates the canonical Hippo pathway at the membrane has previously been elusive. Our results establish membrane potential as a central regulator of growth and tissue homeostasis.

Azo-incorporating was reported to be an effective strategy for increasing SDH inhibitory activity but for poor in vivo control effects. Herein, the azo-incorporated compounds were structurally optimized to retain a preferential conformation by replacing the azo bond with their bioisosteres. Interestingly, the 1,2,4-oxadiazole compound D2 displayed a broad fungicidal spectrum as well as fluxapyroxad. More excitedly, compound D2 showed excellent antifungal activities against rice sheath blight disease both in vitro (EC 50 = 0.001 μg/mL) and in vivo (EC 50 = 1.08 μg/mL, EC 95 = 4.67 μg/mL). In addition, an extra π-π interaction was found between the 1,2,4-oxadiazole ring of compound D2 and the phenyl ring of residue D_Y586, which might interpret the enhanced potency of compound D2 against Rhizoctonia solani . Further structural optimizations of the 1,2,4-oxadiazole compounds gave several analogues that made a breakthrough in controlling rice blast disease. These 1,2,4-oxadiazole compounds, derived from azobenzene derivatives, could be antifungal leads especially against R. solani and Magnaporthe grisea , exemplifying an interesting mode of pesticide discovery and providing theoretical guidance for innovation of the SDHI fungicide.

Fluopimomide, developed by Shandong Sino-Agri United Biotechnology Co., Ltd., is a pyridinylmethyl-benzamide fungicide with good activity against plant diseases caused by phytopathogenic oomycetes. However, there is uncertainty surrounding the resistance risk of fluopimomide and its resistance mechanism in Phytophthora capsici. In this study, the baseline sensitivity of P. capsici to fluopimomide was established, and 106 P. capsici isolates shown sensitive to fluopimomide, with a mean EC 50 value of 5.1892 ± 2.2613 μg/mL. Fungicide adaptation produced three fluopimomide-resistant P. capsici mutants, two of which exhibited considerably lower compound fitness index (CFI) than the parent strain, and one showed significantly improved CFI. While cross-resistance was observed between fluopimomide and fluopicolide, no cross-resistance was detected between fluopimomide and other fungicides. Overall, P. capsici presents a moderate resistance risk to fluopimomide. Two point mutations, G767E and K847R, were identified in the V-ATPase subunit a of P. capsici (PcVHA-a) in resistant mutants. These mutations were subsequently validated through site-directed mutagenesis and molecular docking assays, confirming their roles in conferring fluopimomide resistance in P. capsici., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Northern corn leaf blight (NCLB) infected by Setosphaeria turcica is a devastating disease of corn worldwide. Flusilazole is a broad-spectrum triazole fungicide. However, its resistance risk and field efficiency in controlling NCLB are still unknown. The present research evaluated the antifungal activity of flusilazole against 101 S. turcica isolates, and their EC 50 values ranged from 0.0013 to 0.0466 μg/mL, with a mean of 0.0157 μg/mL. Seven S. turcica mutants resistant to flusilazole were obtained from two wild-type isolates by fungicide adaptation. After 10 consecutive transfers on PDA medium without fungicide, their resistance decreased. Cross-resistance was not existed between flusilazole and fluazinam, pyraclostrobin, amobam, epoxiconazole, or fluxapyroxad. Compared to the wild-type isolates, seven flusilazole-resistant mutants showed reduced biological fitness. No point mutation was detected, however, over-expression of StCYP51 and StatrD genes were detected in the resistant mutants. In addition, in the field experiment, flusilazole exhibited over 85 % efficacy against NCLB, significantly higher than amobam. In summary, these results suggested that the resistance risk of S. turcica to flusilazole was low, and the over-expression of StCYP51 and StatrD might be related to the flusilazole resistance against S. turcica. Flusilazole showed great potential as an alternative fungicide for controlling NCLB., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phytophthora capsici, a pathogenic oomycete, poses a serious threat to global vegetable production. This study investigated the role of protein arginine methylation, a notable post-translational modification, in the epigenetic regulation of P. capsici. We identified and characterized five protein arginine methyltransferases (PRMTs) in P. capsici, with a focus on four putative type I PRMTs exhibiting similar functional domain. Deletion of PcPRMT3, a homolog of PRMT3, significantly affected mycelial growth, asexual spore development, pathogenicity, and stress responses in P. capsici. Transcriptome analyses indicated that absence of PcPRMT3 disrupted multiple biological pathways. The PcPRMT3 deletion mutant displayed heightened susceptibility to oxidative stress, correlated with the downregulation of genes involved in peroxidase and peroxisome activities. Additionally, PcPRMT3 acted as a negative regulator, modulating the transcription levels of specific elicitins, which in turn affects the defense response of host plant against P. capsici. Furthermore, PcPRMT3 was found to affect global arginine methylation levels in P. capsici, implying potential alterations in the functions of its substrate proteins., 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.)

Asparagine (Asn, N)-linked glycosylation is an abundant post-translational modification in which Asn, typically in N glyco -X-S/T; X ≠ P motifs, are modified with N-glycans. It has essential regulatory roles in multicellular organisms. In this study, we systematically investigate the function of three N-glycosylation motifs (N glyco -A, N glyco -D and N glyco -S) previously identified in Phytophthora sojae, through site-directed mutagenesis and functional assays. In P. sojae expressing glycosylation-dead variants pre-PsDMAP1 N70A (N glyco -A motif) or PsADF N64A (N glyco -D motif), zoospore release or cyst germination is impaired. In particular, the pre-PsDMAP1 N70A mutant reduces DNA methylation levels, and the PsADF N64A mutant disrupts the actin forms, which could explain the decrease in pathogenicity after N-glycosylation is destroyed. Similarly, P. sojae expressing PsNRX N132A (N glyco -S motif) shows increased sensitivity to H 2 O 2 and heat. Through autophagy or 26S proteasome pathway inhibition assays, we found that unglycosylated pre-PsDMAP1 N70A and PsADF N64A are degraded via the 26S proteasome pathway, while the autophagy pathway is responsible for PsNRX N132A clearance. These findings demonstrate that glycosylation of these motifs regulates the stability and function of glycoproteins necessary for P. sojae growth, reproduction and pathogenicity, which expands the scope of known N-glycosylation regulatory functions in oomycetes., 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.)

N6-methyladenosine (m6A), a vital post-transcriptional regulator, is among the most prevalent RNA modifications in eukaryotes. Nevertheless, the biological functions of m6A in oomycetes remain poorly understood. Here, we showed that the PsMTA1 and PsMTA2 genes are orthologs of human METTL4, while the PsMET16 gene is an ortholog of human METTL16. These genes are implicated in m6A modification and play a critical role in the production of sporangia and oospores, the release of zoospores, and the virulence of Phytophthora sojae. In P. sojae, m6A modifications are predominantly enriched in the coding sequence and the 3' untranslated region. Notably, the PsMTA1 knockout mutant exhibited reduced virulence, attributed to impaired tolerance to host defense-generated ROS stress. Mechanistically, PsMTA1-mediated m6A modification positively regulates the mRNA lifespan of DNA damage response (DDR) genes in reaction to plant ROS stress during infection. Consequently, the mRNA abundance of the DDR gene PsRCC1 was reduced in the single m6A site mutant ΔRCC1/RCC1A2961C, resulting in compromised DNA damage repair and reduced ROS adaptation-associated virulence in P. sojae. Overall, these results indicate that m6A-mediated RNA metabolism is associated with the development and pathogenicity of P. sojae, underscoring the roles of epigenetic markers in the adaptive flexibility of Phytophthora during infection., Competing Interests: 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 Zhang 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.)

Sulfur-containing compounds have diverse biological functions and are crucial in crop protection chemistry. In this study, a series of novel 1-methyl-1 H -pyrazol-5-amine derivatives incorporating disulfide moieties were synthesized and evaluated for their antimicrobial properties. In vitro bioassays demonstrated that compound 7f displayed potent antifungal activity against Valsa mali , with an EC 50 value of 0.64 mg/L, outperforming allicin (EC 50 = 26.0 mg/L) but lower than tebuconazole (EC 50 = 0.33 mg/L). In vivo experiments confirmed that compound 7f could effectively inhibit V. mali infection on apples at a concentration of 100 mg/L, similar to the positive control tebuconazole. Mechanistic studies revealed that compound 7f could induce hyphal shrinkage and collapse, trigger intracellular reactive oxygen species accumulation, modulate antioxidant enzyme activities, initiate lipid peroxidation, and ultimately cause irreversible oxidative damage to the cells of V. mali . Additionally, compound 7b exhibited notable antibacterial activity, particularly against Pseudomonas syringae pv. actinidiae , with a MIC 90 value of 1.56 mg/L, surpassing the positive controls allicin, bismerthiazol, and streptomycin sulfate. These findings suggest that 1-methyl-1 H -pyrazol-5-amine derivatives containing disulfide moieties hold promise as potent candidates for the development of novel antimicrobial agents.

Colletotrichum gloeosporioides is the causal pathogen for the devastating walnuts anthracnose. A novel quinone inside inhibitor (QiI) fungicide florylpicoxamid has strong inhibitory efficacy against C. gloeosporioides. This study looked into the resistance risk and mechanism of C. gloeosporioides to florylpicoxamid. The basal level sensitivity of C. gloeosporioides isolates (n = 102) to florylpicoxamid was established with an average 50% mycelial growth inhibition concentration (EC 50 ) value of 0.069 ± 0.035 μg/mL. Six stable florylpicoxamid-resistant mutants with resistance factors of >1000 were produced. The fitness of every mutant was much lower than that of their parental isolates. In general, the resistance risk of C. gloeosporioides to florylpicoxamid would be moderate. Molecular docking results revealed that the amino acid substitutions A37V, and S207L in CgCytb lead to a reduction in the binding affinity between florylpicoxamid and CgCytb, indicating that these two mutations (S207L and A37V in CgCytb) indeed confer florylpicoxamid resistance in C. gloeosporioides. These findings offer a fresh viewpoint on the mechanism underlying QiI fungicide resistance and could support the prudent application of florylpicoxamid in the future to combat walnut anthracnose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Fluoxapiprolin, a novel piperidinyl thiazole isoxazoline fungicide, was developed by Bayer Crop Science in 2012. Despite its well-documented inhibitory activity against plant pathogenic oomycetes such as Phytophthora capsici and Phytophthora infestans, limited information regarding its antifungal spectrum and protective and curative activity is available. Fluoxapiprolin exhibited strong inhibitory activity against Phytophthora spp. and several Pythium spp., with EC 50 values ranging from 2.12 × 10 -4 to 2.92 μg/mL. It was much more effective against P. capsici in inhibiting mycelial growth, sporangium production, and cystospore germination than at reducing zoospore release. Moreover, fluoxapiprolin displayed both protective and curative activity against P. capsici infection in pepper plants under greenhouse conditions, with systemic translocation capability confirmed by High Performance Liquid Chromatography (HPLC) analysis. The results demonstrated the strong inhibitory activity of fluoxapiprolin against economically important plant oomycete pathogens, including Phytophthora spp. and several Pythium spp., and its certain translocation activity in pepper plants., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Picarbutrazox is a new tetrazolyloxime fungicide discovered in 2014 by Nippon Soda. It is mostly used to protect against Phytophthora spp. and Pythium spp. However, little is known of its inhibition spectrum, protective and curative activity, and systemic translocation in plants., Results: While picarbutrazox did not show obvious antifungal activity, it exhibited significant activity against oomycetes, including Phytophthora spp., Pythium spp. and Phytopythium spp.. The effective concentration for 50% growth inhibition (EC 50 ) values of picarbutrazox against 16 oomycetes ranged from 3.1 × 10 -4 and 7.27 × 10 -3  μg mL -1 . Furthermore, picarbutrazox could markedly inhibited the mycelial development, sporangia production, zoospore release, and cyst germination of Phytophthora capsici, with EC 50 values of 1.34 × 10 -3 , 1.11 × 10 -3 , 4.85 × 10 -3 , and 5.88 × 10 -2  μg mL -1 , respectively. Additionally, under greenhouse conditions, the protective and curative activities of picarbutrazox at 200 mg L -1 (100%, 41.03%) against the P. capsici infection in peppers were higher than those of the reference fungicide dimethomorph at 200 mg L -1 (77.52%, 36.15%). High-performance liquid chromatography analysis confirmed that picarbutrazox showed excellent systemic translocation in pepper plants., Conclusion: The results showed that picarbutrazox markedly inhibited the important plant oomycete pathogens including Phytophthora spp., Pythium spp. and Phytopythium spp.. It also displayed excellent protective, curative and systemic translocation activity. Picarbutrazox thus has significant potential for preventing and controlling diseases caused by oomycetes. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Natural products are a valuable resource for the discovery of novel crop protection agents. A series of γ-butyrolactone derivatives, derived from the simplification of podophyllotoxin's structure, were synthesized and assessed for their efficacy against tobacco mosaic virus (TMV). Several derivatives exhibited notable antiviral properties, with compound 3g demonstrating the most potent in vivo anti-TMV activity. At 500 μg/mL, compound 3g achieved an inactivation effect of 87.8%, a protective effect of 71.7%, and a curative effect of 67.7%, surpassing the effectiveness of the commercial plant virucides ningnanmycin and ribavirin . Notably, the syn -diastereomer ( syn - 3g ) exhibited superior antiviral activity compared to the anti -diastereomer ( anti - 3g ). Mechanistic studies revealed that syn - 3g could bind to the TMV coat protein and interfere with the self-assembly process of TMV particles. These findings indicate that compound 3g , with its simple chemical structure, could be a potential candidate for the development of novel antiviral agents for crop protection.

In bacteria, algae, fungi, and plant cells, the wall must expand in concert with cytoplasmic biomass production, otherwise cells would experience toxic molecular crowding 1,2 or lyse. But how cells achieve expansion of this complex biomaterial in coordination with biosynthesis of macromolecules in the cytoplasm remains unexplained 3 , although recent works have revealed that these processes are indeed coupled 4,5 . Here, we report a striking increase of turgor pressure with growth rate in E. coli , suggesting that the speed of cell wall expansion is controlled via turgor. Remarkably, despite this increase in turgor pressure, cellular biomass density remains constant across a wide range of growth rates. By contrast, perturbations of turgor pressure that deviate from this scaling directly alter biomass density. A mathematical model based on cell wall fluidization by cell wall endopeptidases not only explains these apparently confounding observations but makes surprising quantitative predictions that we validated experimentally. The picture that emerges is that turgor pressure is directly controlled via counterions of ribosomal RNA. Elegantly, the coupling between rRNA and turgor pressure simultaneously coordinates cell wall expansion across a wide range of growth rates and exerts homeostatic feedback control on biomass density. This mechanism may regulate cell wall biosynthesis from microbes to plants and has important implications for the mechanism of action of antibiotics 6 .

The diphenyl ether molecular pharmacophore has played a significant role in the development of fungicidal compounds. In this study, a variety of pyrazol-5-yl-phenoxybenzamide derivatives were synthesized and evaluated for their potential to act as succinate dehydrogenase inhibitors (SDHIs). The bioassay results indicate certain compounds to display a remarkable and broad-spectrum in their antifungal activities. Notably, compound 12x exhibited significant in vitro activities against Valsa mali , Gaeumannomyces graminis , and Botrytis cinerea , with EC 50 values of 0.52, 1.46, and 3.42 mg/L, respectively. These values were lower or comparable to those of Fluxapyroxad (EC 50 = 12.5, 1.93, and 8.33 mg/L, respectively). Additionally, compound 12x showed promising antifungal activities against Sclerotinia sclerotiorum (EC 50 = 0.82 mg/L) and Rhizoctonia solani (EC 50 = 1.86 mg/L), albeit lower than Fluxapyroxad (EC 50 = 0.23 and 0.62 mg/L). Further in vivo experiments demonstrated compound 12x to possess effective protective antifungal activities against V. mali and S. sclerotiorum at a concentration of 100 mg/L, with inhibition rates of 66.7 and 89.3%, respectively. In comparison, Fluxapyroxad showed inhibition rates of 29.2 and 96.4% against V. mali and S. sclerotiorum , respectively. Molecular docking analysis revealed that compound 12x interacts with SDH through hydrogen bonding, π-cation, and π-π interactions, providing insights into the probable mechanism of action. Furthermore, compound 12x exhibited greater binding energy and SDH enzyme inhibitory activity than Fluxapyroxad (Δ G cal = -46.8 kcal/mol, IC 50 = 1.22 mg/L, compared to Δ G cal = -41.1 kcal/mol, IC 50 = 8.32 mg/L). Collectively, our results suggest that compound 12x could serve as a promising fungicidal lead compound for the development of more potent SDHIs for crop protection.

The active splicing strategy has witnessed improvement in bioactivity and antifungal spectra in pesticide discovery. Herein, a series of simple-structured molecules ( Y1 - Y53 ) containing chloro-substituted benzyl esters were designed using the above strategy. The structure-activity relationship (SAR) analysis demonstrated that the fatty acid fragment-structured esters were more effective than those containing an aromatic acid moiety or naphthenic acid part. Compounds Y36 and Y41 , which featured a thiazole-4-acid moiety and trifluoromethyl aliphatic acid part, respectively, exhibited excellent in vivo curative activity (89.4%, 100 mg/L Y36 ) and in vitro fungicidal activity (EC 50 = 0.708 mg/L, Y41 ) against Botrytis cinerea . Determination of antifungal spectra and analysis of scanning electron microscopy (SEM), membrane permeability, cell peroxidation, ergosterol content, oxalic acid pathways, and enzymatic assays were performed separately here. Compound Y41 is cost effective due to its simple structure and shows promise as a disease control candidate. In addition, Y41 might act on a novel target through a new pathway that disrupts the cell membrane integrity by inducing cell peroxidation.

Small RNAs (sRNAs) are important non-coding RNA regulators that play key roles in the development and pathogenesis of plant pathogens, as well as in other biological processes. However, whether these abundant and varying sRNAs are involved in Phytophthora development or infection remains enigmatic. In this study, sRNA sequencing of 4 asexual stages of Phytophthora capsici ( P. capsici ), namely, as mycelia (HY), sporangia (SP), zoospores (ZO), cysts (CY), and pepper infected with P. capsici (IN), were performed, followed by sRNA analysis, microRNA (miRNA) identification, and miRNA target prediction. sRNAs were mainly distributed at 25-26 nt in HY, SP, and ZO but distributed at 18-34 nt in CY and IN. 92, 42, 176, 39, and 148 known miRNAs and 15, 19, 54, 13, and 1 novel miRNA were identified in HY, SP, ZO, CY, and IN, respectively. It was found that the expression profiles of known miRNAs vary greatly at different stages and could be divided into 4 categories. Novel miRNAs mostly belong to part I. Gene ontology (GO) analysis of known miRNA-targeting genes showed that they are involved in the catalytic activity pathway, binding function, and other biological processes. Kyoto Encyclopedia of Gene and Genome (KEGG) analysis of novel miRNA-targeting genes showed that they are involved in the lysine degradation pathway. The expression of candidate miRNAs was validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and miRNAs were downregulated in PcDCL1 or PcAGO1 mutants. To further explore the function of the detected miRNAs, the precursor of a novel miRNA, miR91, was knockout by CRISPR-Cas9, the mutants displayed decreased mycelial growth, sporangia production, and zoospore production. It was found that 503142 (Inositol polyphosphate 5-phosphatase and related proteins) can be predicted as a target of miR91, and the interaction between miR91 and 503142 was verified using the tobacco transient expression system. Overall, our results indicate that the diverse and differentially expressed sRNAs are involved in the development and pathogenesis of P. capsici., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhong, Zhang, Zheng, Zhang, Liu, Wang and Liu.)

The phytopathogenic oomycete Phytophthora litchii is the culprit behind the devastating disease known as "litchi downy blight", which causes large losses in litchi production. Although fluopimomide exhibits strong inhibitory efficacy against P. litchii, the exact mechanism of resistance is still unknown. The sensitivity of 137 P. litchii isolates to fluopimomide was assessed, and it was discovered that the median effective concentration (EC 50 ) of the fungicide had a unimodal frequency distribution with a mean value of 0.763 ± 0.922 μg/mL. Comparing the resistant mutants to the equivalent parental isolates, the resistance mutants' survival fitness was much lower. While there was no cross-resistance between fluopimomide and other oomycete inhibitors, there is a notable positive cross-resistance between fluopimomide and fluopicolide. According to the thorough investigation, P. litchii had a moderate chance of developing fluopimomide resistance. The point mutations N771S and K847N in the VHA-a of P. litchii (PlVHA-a) were present in the fluopimomide-resistant mutants, and the two point mutations in PlVHA-a conferring fluopimomide resistance were verified by site-directed mutagenesis in the sensitive P. capsici isolate BYA5 and molecular docking., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Black shank, a devastating disease in tobacco production worldwide, is caused by the oomycete plant pathogen Phytophthora nicotianae. Fluopicolide is a pyridinylmethyl-benzamides fungicide with a unique mechanism of action and has been widely used for controlling a variety of oomycetes such as Plasmopara viticola, Phytophthora infestans, Pseudoperonospora cubensis, P. nicotianae and Bremia lactucae. However, the fluopicolide-resistance risk and molecular basis in P. nicotianae have not been reported. In this study, the sensitivity profile of 141 P. nicotianae strains to fluopicolide was determined, with a mean median effective concentration (EC 50 ) value of 0.12 ± 0.06μg/mL. Five stable fluopicolide-resistant mutants of P. nicotianae were obtained by fungicide adaptation, and the compound fitness index of these resistant mutants were lower than that of their parental isolates. Additionally, cross-resistance tests indicated that the sensitivity of fluopicolide did not correlate with other oomycete fungicides, apart from fluopimomide. DNA sequencing revealed two point mutations, G765E and N769Y, in the PpVHA-a protein in the fluopicolide-resistant mutants. Transformation and expression of PpVHA-a genes carrying G765E and N769Y in the sensitive wild-type isolate confirmed that it was responsible for fluopicolide resistance. These results suggest that P. nicotianae has a low to medium resistance risk to fluopicolide in laboratory and that point mutations, G765E and N769Y, in PpVHA-a are associated with the observed fluopicolide resistance., (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_02g01260 and Bcin_12g06380 , along with the CarEs Bcin_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.

Botrytis cinerea is one of the most destructive pathogens worldwide. It can damage over 200 crops, resulting in significant yield and quality losses. Cyclobutrifluram, a new generation of succinate dehydrogenase inhibitors, exhibits excellent inhibitory activity against B. cinerea. However, the baseline sensitivity and resistance of B. cinerea to cyclobutrifluram remains poorly understood. This study was designed to monitor the sensitivity frequency distribution, assess the resistance risk, and clarify the resistance mechanism of B. cinerea to cyclobutrifluram. The baseline sensitivity of B. cinerea isolates to cyclobutrifluram was 0.89 μg/mL. Cyclobutrifluram-resistant B. cinerea populations are present in the field. Six resistant B. cinerea isolates investigated in this study possessed enhanced compound fitness index compared to the sensitive isolates according to mycelial growth, mycelial dry weight, conidiation, conidial germination rate, and pathogenicity. Cyclobutrifluram exhibited no cross-resistance with tebuconazole, fludioxonil, cyprodinil, or iprodione. Sequence alignment revealed that BcSDHB from cyclobutrifluram-resistant B. cinerea isolates had three single substitutions (P225F, N230I, or H272R). Molecular docking verified that these mutations in BcSDHB conferred cyclobutrifluram resistance in B. cinerea. In conclusion, the resistance risk of B. cinerea to cyclobutrifluram is high, and the point mutations in BcSDHB (P225F, N230I, or H272R) confer cyclobutrifluram resistance in B. cinerea. This study provided important insights into cyclobutrifluram resistance in B. cinerea and offered valuable information for monitoring and managing cyclobutrifluram resistance in the future., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Proper transcription orchestrated by RNA polymerase II (RNPII) is crucial for cellular development, which is rely on the phosphorylation state of RNPII's carboxyl-terminal domain (CTD). Sporangia, developed from mycelia, are essential for the destructive oomycetes Phytophthora, remarkable transcriptional changes are observed during the morphological transition. However, how these changes are rapidly triggered and their relationship with the versatile RNPII-CTD phosphorylation remain enigmatic. Herein, we found that Phytophthora capsici undergone an elevation of Ser5-phosphorylation in its uncanonical heptapeptide repeats of RNPII-CTD during sporangia development, which subsequently changed the chromosomal occupation of RNPII and primarily activated transcription of certain genes. A cyclin-dependent kinase, PcCDK7, was highly induced and phosphorylated RNPII-CTD during this morphological transition. Mechanistically, a novel DCL1-dependent microRNA, pcamiR1, was found to be a feedback modulator for the precise phosphorylation of RNPII-CTD by complexing with PcAGO1 and regulating the accumulation of PcCDK7. Moreover, this study revealed that the pcamiR1-CDK7-RNPII regulatory module is evolutionarily conserved and the impairment of the balance between pcamiR1 and PcCDK7 could efficiently reduce growth and virulence of P. capsici. Collectively, this study uncovers a novel and evolutionary conserved mechanism of transcription regulation which could facilitate correct development and identifies pcamiR1 as a promising target for disease control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang 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.)

Background: SYP-34773 is a low-toxicity pyrimidine amine compound, which was synthesized by modifying the lead compound diflumetorim. Previous literature has shown that it can strongly inhibit the mycelial growth of several important plant pathogens, including Phytophthora litchii. However, the resistance risk of SYP-34773 has not been reported for P. litchii., Results: The mean effective concentration (EC 50 ) value of SYP-34773 against the mycelial growth of 111 P. litchii isolates was 0.108 ± 0.008 μg mL -1 , which can be used as the baseline sensitivity for SYP-34773 resistance detection in the future. Six mutants were obtained from two parental strain through fungicide induction, whose resistance factors fell between 194- and 687-fold, with stability. Results regarding mycelial growth, sporangial production, sporangial germination, zoospore release, cystspore germination, and pathogenicity showed that the mutants' compound fitness index values were significantly lower than those of their parental isolate. Furthermore, there was no cross-resistance between SYP-34773 and diflumetorim in P. litchii. Significant inhibition of the mitochondrial complex I enzyme activity in two wild-type P. litchii isolates, but not in mutants, was observed upon treatment with SYP-34773., Conclusion: The resistance risk of SYP-34773 in P. litchii is moderate, and resistance management strategies should be adopted in field use. SYP-34773 is a mitochondrial complex I inhibitor, and SYP-34773-resistant P. litchii isolates did not show cross-resistance against diflumetorim. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: Wheat Fusarium crown rot (FCR) is a serious problem primarily caused by Fusarium pseudograminearum, a pathogenic agent known to produce mycotoxins, including deoxynivalenol (DON). Cyclobutrifluram, a novel succinate dehydrogenase inhibitor devised by Syngenta, has immense potential to control both nematodes and Fusarium diseases. However, its efficacy in combating Fusarium species, its ability to prevent and reverse the detrimental effects of FCR, and its impact on the production of DON by F. pseudograminearum are yet to be fully ascertained., Results: Cyclobutrifluram exhibited substantial inhibitory activity against Fusarium species, with half-maximal effective concentration values ranging from 0.0021-0.0647 μg mL -1 . It demonstrated significant inhibitory activity toward three developmental stages of F. pseudograminearum, F. graminearum and F. asiaticum. Furthermore, cyclobutrifluram showed both protective and curative activities against FCR and was rapidly absorbed by roots and transported to wheat stems and leaves. Cyclobutrifluram could also decrease DON production by F. pseudograminearum., Conclusion: This investigation has revealed the potential of cyclobutrifluram as a formidable candidate fungicide, particularly in its ability to effectively combat FCR and other Fusarium-related ailments. This novel compound has exceptional pathogen-fighting capabilities, coupled with remarkable systemic translocation properties and a notable ability to reduce the production of DON. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: Brown rot disease, caused by Monilinia fructicola, poses a significant challenge to peach production in China. The efficacy of mefentrifluconazole, a new triazole fungicide, in controlling brown rot in peaches has been remarkable. However, the resistance risk and mechanism associated with this fungicide remain unclear. This study was designed to assess the resistance risk of M. fructicola to mefentrifluconazole and reveal the potential resistance mechanism., Results: The mean median effective concentration (EC 50 ) of 101 M. fructicola isolates to mefentrifluconazole was 0.003 μg mL -1 , and the sensitivity exhibited a unimodal distribution. Seven mefentrifluconazole-resistant mutants were generated from three parental isolates in the laboratory through fungicide adaption. The biological characteristics of the resistant mutants revealed that three of them exhibited enhanced survival fitness compared to the parental isolates, whereas the remaining four mutants displayed reduced survival fitness. Mefentrifluconazole showed strong positive cross-resistance with fenbuconazole, whereas no cross-resistance was observed with pyrimethanil, procymidone or pydiflumetofen. No overexpression of MfCYP51 gene was detected in the resistant mutants. Multiple sequence alignment revealed that three resistant mutants (MXSB2-2, Mf12-1 and Mf12-2) had a point mutation (G461S) in MfCYP51 protein. Molecular docking techniques confirmed the contribution of this point mutation to mefentrifluconazole resistance., Conclusion: The risk of M. fructicola developing resistance to mefentrifluconazole is relatively low-to-medium and point mutation G461S in MfCYP51 could confer mefentrifluconazole resistance in M. fructicola. This study provided essential data for monitoring the emergence of resistance and developing resistance management strategies for mefentrifluconazole. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Background: Sclerotinia stem rot caused by Sclerotinia sclerotiorum seriously endangers oilseed rape production worldwide, and the occurrence of fungicide-resistant mutants of S. sclerotiorum leads to control decline. Thus, it is critical to explore new green substitutes with different action mechanisms and high antifungal activity. Herein, the activity and the action mechanism of natamycin against S. sclerotiorum were evaluated., Results: Natamycin showed potent inhibition on the mycelial growth of S. sclerotiorum, and half-maximal effective concentration (EC 50 ) values against 103 S. sclerotiorum strains ranged from 0.53 to 4.04 μg/mL (mean 1.44 μg/mL). Natamycin also exhibited high efficacy against both carbendazim- and dimethachlone-resistant strains of S. sclerotiorum on detached oilseed rape leaves. No cross-resistance was detected between natamycin and carbendazim. Natamycin markedly disrupted hyphal form, sclerotia formation, integrity of the cell membrane, and reduced the content of oxalic acid and ergosterol, whereas it increased the reactive oxygen species (ROS) and malondialdehyde content. Interestingly, exogenous addition of ergosterol could reduce the inhibition of natamycin against S. sclerotiorum. Importantly, natamycin significantly inhibited expression of the Cyp51 gene, which is contrary to results for the triazole fungicide flusilazole, indicating a different action mechanism from triazole fungicides., Conclusion: Natamycin is a promising effective candidate for the resistance management of S. sclerotiorum. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

The necrotrophic pathogen Botrytis cinerea infects a broad range of plant hosts and causes substantial economic losses to many crops. Although resistance to procymidone has been observed in the field, it remains uncertain why procymidone is usually involved in multidrug resistance (MDR) together with other fungicides. Nine mutants derived from the B. cinerea strain B05.10 through procymidone domestication exhibited high resistance factors (RFs) against both procymidone and fludioxonil. However, the fitness of the mutants was reduced compared to their parental strain, showing non-sporulation and moderate virulence. Furthermore, the RFs of these mutants to other fungicides, such as azoxystrobin, fluazinam, difenoconazole, and pyrimethanil, ranged from 10 to 151, indicating the occurrence of MDR. Transcriptive expression analysis using the quantitative polymerase chain reaction (qPCR) revealed that the mutants overexpressed ABC transporter genes, ranging from 2 to 93.7-fold. These mutants carried single-point mutations W647X, R96X, and Q751X within BcBos1 by DNA sequencing. These alterations in BcBos1 conferred resistance to procymidone and other fungicides in the mutants. Molecular docking analysis suggested distinct interactions between procymidone and Bos1 in the B. cinerea standard strain B05.10 or the resistant mutants, suggesting a higher affinity of the former towards binding with the fungicide. This study provides a comprehensive understanding of the biological characteristics of the resistant mutants and conducts an initial investigation into its fungicide resistance traits, providing a reference for understanding the causes of multidrug resistance of B. cinerea in the field.

To accelerate the development of novel fungicides, a variety of N -(pyrazol-5-yl)benzamide derivatives with a diphenylamine moiety were designed and synthesized using a pharmacophore recombination strategy based on the structure of pyrazol-5-yl-aminophenyl-benzamides. The bioassay results demonstrated that most of the target compounds had excellent in vitro antifungal activities against Sclerotinia sclerotiorum , Valsa mali , and Botrytis cinerea . In particular, compound 5IIIh exhibited remarkable activity against S. sclerotiorum (EC 50 = 0.37 mg/L), which was similar to that of fluxapyroxad (EC 50 = 0.27 mg/L). In addition, compound 5IIIc (EC 50 = 1.32 mg/L) was observed to be more effective against V. mali than fluxapyroxad (EC 50 = 12.8 mg/L) and comparable to trifloxystrobin (EC 50 = 1.62 mg/L). Furthermore, compound 5IIIh demonstrated remarkable in vivo protective antifungal properties against S. sclerotiorum , with an inhibition rate of 96.8% at 100 mg/L, which was close to that of fluxapyroxad (99.6%). Compounds 5IIIc (66.7%) and 5IIIh (62.9%) exhibited good in vivo antifungal effects against V. mali at 100 mg/L, which were superior to that of fluxapyroxad (11.1%) but lower than that of trifloxystrobin (88.9%). The succinate dehydrogenase (SDH) enzymatic inhibition assay was conducted to confirm the mechanism of action. Molecular docking analysis further revealed that compound 5IIIh has significant hydrogen-bonding, π-π, and p-π conjugation interactions with ARG 43, SER 39, TRP 173, and TYR 58 in the binding site of SDH, and the binding mode was similar to that of the commercial fungicide fluxapyroxad. All of the results suggest that compound 5IIIh could be a potential SDH inhibitor, offering a valuable reference for future studies.

2-Alkylpyridines are a privileged scaffold throughout the realm of organic synthesis and play a key role in natural products, pharmaceuticals, and agrochemicals. Herein, we report the first B-alkyl Suzuki cross-coupling of 2-pyridyl ammonium salts to access functionalized 2-alkylpyridines. The use of well-defined, operationally simple Pd-NHCs permits for an exceptionally broad scope of the challenging B-alkyl C-N cross-coupling with organoboranes containing β-hydrogen, representing a novel method for the discovery of highly sought-after molecules for plant protection.

Multi-fungicide resistance (MFR) is a serious environmental problem, which results in the excessive use of fungicides. Fitness penalty, as a common phenomenon in MFR, can partially counteract the issue of resistance due to the weakened vigor of MFR pathogens. Their underlying mechanism and relationship remain unexplained. By Oxford Nanopore Technologies sequencing and dot blot, we found that N6-methyloxyadenine (6mA) modification, the dominate epigenetic marker in Phytophthora capsici , was significantly altered after MFR emerged. Among the differently methylated genes, PcGSTZ1 could efficiently detoxify SYP-14288, a novel uncoupler, through complexing the fungicide with glutathione and induce MFR. Interestingly, PcGSTZ1 overexpression was induced by elevated 6mA levels and chromatin accessibility to its genomic loci. Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in P. capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici . In conclusion, the findings provide new insights into the biological role of 6mA as well as the mechanisms underlying the trade-off between MFR and fitness. These could also benefit disease control through the blockade of the epigenetic axis to resensitize resistant isolates.IMPORTANCEN6-methyloxyadenine (6mA) modification on DNA is correlated with tolerance under different stress in prokaryotes. However, the role of 6mA in eukaryotes remains poorly understood. Our current study reveals that DNA adenine methyltransferase 1 (DAMT1)-mediated 6mA modification at the upstream region of GST zeta 1 (GSTZ1) is elevated in the resistant strain. This elevation promotes the detoxification uncoupler and induces multifungicide resistance (MFR). Moreover, the overexpression led to reactive oxygen species burst and ferroptosis in SYP-14288-resistant mutants, which enhanced the resistance and induced fitness penalty in Phytophthora capsici through triggering low energy shock adaptive response. Furthermore, this study revealed that the 6mA-PcGSTZ1-ferroptosis axis could mediate intergenerational resistance memory transmission and enabled adaptive advantage to P. capsici . Overall, our findings uncover an innovative mechanism underlying 6mA modification in regulating PcGSTZ1 transcription and the ferroptosis pathway in P. capsici ., Competing Interests: The authors declare no conflict of interest.

Host-derived reactive oxygen species (ROS) are an important defense means to protect against pathogens. Although mitochondria are the main intracellular targets of ROS, how pathogens regulate mitochondrial physiology in response to oxidative stress remains elusive. Prohibitin 2 (PHB2) is an inner mitochondrial membrane (IMM) protein, recognized as a mitophagy receptor in animals and fungi. Here, we find that an ANK and FYVE domain-containing protein PsAF5, is an adapter of PsPHB2, interacting with PsATG8 under ROS stress. Unlike animal PHB2 that can recruit ATG8 directly to mitochondria, PsPHB2 in Phytophthora sojae cannot recruit PsATG8 to stressed mitochondria without PsAF5. PsAF5 deletion impairs mitophagy under ROS stress and increases the pathogen's sensitivity to H 2 O 2 , resulting in the attenuation of P. sojae virulence. This discovery of a PsPHB2-PsATG8 adapter (PsAF5) in plant-pathogenic oomycetes reveals that mitophagy induction by IMM proteins is conserved in eukaryotes, but with differences in the details of ATG8 recruitment., (© 2024. The Author(s).)

Soybean root rot is a worldwide soil-borne disease threatening soybean production, causing large losses in soybean yield and quality. Fusarium species are the most detrimental pathogens of soybean root rot worldwide, causing large production losses. Fusarium root rot has been frequently reported in Heilongjiang Province of China, but the predominant Fusarium species and the sensitivity of these pathogens to different fungicides remain unclear. In this study, diseased soybean roots were collected from 14 regions of Heilongjiang province in 2021 and 2022. A total of 144 isolates of Fusarium spp. were isolated and identified as seven distinct species: F. scirpi, F. oxysporum, F. graminearum, F. clavum, F. acuminatum, F. avenaceum, and F. sporotrichioide. F. scirpi and F. oxysporum had high separation frequency and strong pathogenicity. The sensitivity of Fusarium spp. to five different fungicides was determined. Mefentrifluconazole and fludioxonil showed good inhibitory effects, and the sensitivity to pydiflumetofen and phenamacril varied between Fusarium species. In particular, the activity of DMI fungicide prothioconazole was lower than that of mefentrifluconazole. Molecular docking showed that mefentrifluconazole mainly bound to CYP51C, but prothioconazole mainly bound to CYP51B. Furthermore, the sensitivity to prothioconazole only significantly decreased in ΔFgCYP51B mutant, and the sensitivity to mefentrifluconazole changed in ΔFgCYP51C and ΔFgCYP51A mutants. The results demonstrated that the predominant Fusarium species causing soybean root rot in Heilongjiang province were F. scirpi and F. oxysporum and DMI fungicides had differences in binding cavity due to the diversity of CYP51 proteins in Fusarium., (Copyright © 2023. Published by Elsevier Inc.)

Fungicides are an effective way to control gray mold of grapes, but the pathogen Botrytis cinerea can develop resistance, overcoming the effectiveness of a fungicide that is repeatedly applied. More importantly, the emergence of multidrug resistance (MDR) in the field, where multiple fungicides with different modes of action simultaneously lose their efficacies, is a significant concern. MDR is associated with ATP-binding cassette (ABC) transporters of the pathogen, and certain plant secondary metabolites (PSMs) stimulate the upregulation of ABC transporters, we hypothesized that the pathogen's preadaptation to PSMs might contribute to MDR development. To test this in B. cinerea, ten PSMs, namely, resveratrol, reserpine, chalcone, flavanone, eugenol, farnesol, anethene, camptothecin, salicylic acid, and psoralen, were selected based on their association with ABC transporters involved in fungicide resistance. B. cinerea strain B05.10 was continuously transferred for 15 generations on potato dextrose agar amended with a PSM (PDAP), and sensitivities to PSMs and fungicides were examined on the 5th, 10th, and 15th generations. RNA was extracted from B. cinerea from the selected generations. After 15 generations of culture transfers, an up-regulation was observed in the expression of ABC transporter-encoding genes BcatrB, BcatrD, and BcatrK using quantitative polymerase chain reaction (qPCR). This upregulation was found to contribute to MDR of B. cinerea against two or more fungicides, among azoxystrobin, boscalid, fludioxonil, difenoconazole, prochloraz, and pyrimethanil. This finding was confirmed through genetic transformation. The decreased sensitivity of B. cinerea to fungicides was confirmed as a subsequent MDR phenotype after exposure to camptothecin, flavanone, and resveratrol. Besides, transcriptome analysis also revealed the upregulation of transcription factors related to ABC expression following resveratrol exposure. This suggests that PSMs contributed to inducing preadaptation of B. cinerea, leading to subsequent MDR.IMPORTANCEThe emergence of MDR in plant pathogens is a threat to plant disease management and leads to the use of excessive fungicides. Botrytis cinerea is of particular concern because its MDR has widely emerged in the field. Understanding its genesis is the first step for controlling MDR. In this study, the contribution of PSMs to MDR has been examined. Effective management of this pathogen in agroecosystems relies on a better understanding of how it copes with phytochemicals or fungicides., Competing Interests: The authors declare no conflict of interest.

Structural diversity derivatization from natural products is an important and effective method of discovering novel green pesticides. Cinnamic acids are abundant in plants, and their unparalleled structures endow them with various excellent biological activities. A series of novel cinnamic oxime esters were designed and synthesized to develop high antifungal agrochemicals. The antifungal activity, structure-activity relationship, and action mechanism were systematically studied. Compounds 7i , 7u , 7v , and 7x exhibited satisfactory activity against Gaeumannomyces graminis var. tritici , with inhibition rates of ≥90% at 50 μg/mL. Compounds 7z and 7n demonstrated excellent activities against Valsa mali and Botrytis cinerea , with median effective concentration (EC 50 ) values of 0.71 and 1.41 μg/mL, respectively. Compound 7z exhibited 100% protective and curative activities against apple Valsa canker at 200 μg/mL. The control effects of 7n against gray mold on tomato fruits and leaves were all >96%, exhibiting superior or similar effects to those of the commercial fungicide boscalid. Furthermore, the quantitative structure-activity relationship was established to guide the further design of higher-activity compounds. The preliminary results on the action mechanism revealed that 7n treatment could disrupt the function of the nucleus and mitochondria, leading to reactive oxygen species accumulation and cell membrane damage. Its primary biochemical mechanism may be inhibiting fungal ergosterol biosynthesis. The novel structure, simple synthesis, and excellent activity of cinnamic oxime esters render them promising potential fungicides.

Lychee downy blight (LDB), a common disease caused by the oomycete Phytophthora litchii, poses a significant threat to both pre- and post-harvest stages, leading to substantial economic losses. Famoxadone, a quinone outside inhibitor fungicide, was registered for controlling LDB in China in 2002. However, limited information is available regarding the risk, mechanism, and impact on lychee fruit quality associated with famoxadone resistance. In this study, we determined the sensitivity of 133 P. litchii isolates to famoxadone, yielding a mean EC 50 value of 0.46 ± 0.21 μg/mL. Through fungicide adaption, we derived resistant mutants with M124I and Y131C substitutions in PlCyt b (Cytochrome b in P. litchii) from wild-type isolates. In vitro assessments revealed that the fitness of the resistant mutants was significantly lower compared to the parental isolates. These laboratory findings demonstrate a moderate resistance risk of P. litchii to famoxadone. Molecular docking analyses indicated that the M124I and Y131C alterations disrupted hydrogen bonds and weakened the binding energy between famoxadone and PlCyt b. This indicates that the M124I and Y131C changes do indeed confer famoxadone resistance in P. litchii. Infection caused by famoxadone-resistant mutants exhibited a decreased or comparable impact on the characteristic traits of lychee fruit compared to the sensitive isolate. For future detection of famoxadone-resistant strains, AS-PCR primers were designed based on the M124I substitution., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fusarium head blight in wheat is caused by Fusarium graminearum, resulting in significant yield losses and grain contamination with deoxynivalenol (DON), which poses a potential threat to animal health. Cyclobutrifluram, a newly developed succinate dehydrogenase inhibitor, has shown excellent inhibition of Fusarium spp. However, the resistance risk of F. graminearum to cyclobutrifluram and the molecular mechanism of resistance have not been determined. In this study, we established the average EC 50 of a range of F. graminearum isolates to cyclobutrifluram to be 0.0110 μg/mL. Six cyclobutrifluram-resistant mutants were obtained using fungicide adaptation. All mutants exhibited impaired fitness relative to their parental isolates. This was evident from measurements of mycelial growth, conidiation, conidial germination, virulence, and DON production. Interestingly, cyclobutrifluram did not seem to affect the DON production of either the sensitive isolates or the resistant mutants. Furthermore, a positive cross-resistance was observed between cyclobutrifluram and pydiflumetofen. These findings suggest that F. graminearum carries a moderate to high risk of developing resistance to cyclobutrifluram. Additionally, point mutations H248Y in FgSdhB and A73V in FgSdhC 1 of F. graminearum were observed in the cyclobutrifluram-resistant mutants. Finally, an overexpression transformation assay and molecular docking indicated that FgSdhB H248Y or FgSdhC 1 A73V could confer resistance of F. graminearum to cyclobutrifluram., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Ipconazole is a broad-spectrum triazole fungicide that is highly effective against Fusarium pseudograminearum. However, its risk of developing resistance and mechanism are not well understood in F. pseudograminearum. Here, the sensitivities of 101 F. pseudograminearum isolates to ipconazole were investigated, and the average EC 50 value was 0.1072 μg/mL. Seven mutants resistant to ipconazole were obtained by fungicide adaption, with all but one showing reduced fitness relative to the parental isolates. Cross-resistance was found between ipconazole and mefentrifluconazole and tebuconazole, but none between ipconazole and pydiflumetofen, carbendazim, fludioxonil, or phenamacril. In summary, these findings suggest that there is a low risk of F. pseudograminearum developing resistance to ipconazole. Additionally, a point mutation, G464S, was seen in FpCYP51B and overexpression of FpCYP51A, FpCYP51B and FpCYP51C was observed in ipconazole-resistant mutants. Assays, including transformation and molecular docking, indicated that G464S conferred ipconazole resistance in F. pseudograminearum., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Mefentrifluconazole, a triazole fungicide, exhibits remarkable efficacy in combating Fusarium spp. The mean EC 50 value of mefentrifluconazole against 124 isolates of Fusarium pseudograminearum was determined to be 1.06 μg/mL in this study. Fungicide taming produced five mefentrifluconazole-resistant mutants with resistance factors ranging from 19.21 to 111.34. Compared to the original parental isolates, the fitness of three resistant mutants was much lower, while the remaining two mutants displayed enhanced survival fitness. There was evidence of positive cross-resistance between tebuconazole and mefentrifluconazole. Mefentrifluconazole resistance in F. pseudograminearum can be conferred by FpCYP51B L144F , which was identified in four mutants according to molecular docking and site-directed transformation experiments. Overexpression of FpCYP51s was also detected in the resistant mutants. In conclusion, mefentrifluconazole has a low-to-medium resistance risk in F. pseudograminearum , and the L144F mutation in FpCYP51B and the increased expression level of FpCYP51s may be responsible for mefentrifluconazole resistance in F. pseudograminearum .

An estimated 240 fungicides are presently in use, but the direct targets for the majority remain elusive, constraining fungicide development and efficient resistance monitoring. In this study, we found that Pcα-actinin knockout did not influence the sensitivity of Phytophthora capsici to fluopicolide, which is a notable oomycete inhibitor. Using a combination of Bulk Segregant Analysis Sequencing and Drug Affinity Responsive Target Stability (DARTS) assays, the vacuolar H + -ATPase subunit a (PcVHA-a) was pinpointed as the target protein of fluopicolide. We also confirmed four distinct point mutations in PcVHA-a responsible for fluopicolide resistance in P. capsici through site-directed mutagenesis. Molecular docking, ATPase activity assays, and a DARTS assay suggested a fluopicolide-PcVHA-a interaction. Sequence analysis and further molecular docking validated the specificity of fluopicolide for oomycetes or fish. These findings support the claim that PcVHA-a is the target of fluopicolide, proposing vacuolar H + -ATPase as a promising target for novel fungicide development.

In this study, we determined the sensitivity of 148 Phytophthora litchii isolates to cyazofamid, yielding a mean EC 50 value of 0.0091 ± 0.0028 μg/mL. Through fungicide adaptation, resistant mutants (RMs) carrying the F220L substitution in PlCyt b were derived from wild-type isolates. Notably, these RMs exhibited a lower fitness compared with the parental isolates. Molecular docking analysis further revealed that the F220L change contributed to a decrease in the binding energy between cyazofamid and PlCyt b. The total phenol and flavonoid contents in the litchi pericarp treated with cyazofamid on day 5 were significantly higher than in other treatments. Overall, the laboratory assessment indicated a moderate risk of cyazofamid resistance in P. litchii , but the emergence of the F220L change could lead to a high level of resistance. Thus, cyazofamid represents a promising agrochemical for controlling postharvest litchi downy blight and extending the shelf life of litchi fruits.

To achieve a stable size distribution over multiple generations, proliferating cells require a means of counteracting stochastic noise in the rate of growth, the time spent in various phases of the cell cycle, and the imprecision in the placement of the plane of cell division. In the most widely accepted model, cell size is thought to be regulated at the G1/S transition, such that cells smaller than a critical size pause at the end of G1 phase until they have accumulated mass to a predetermined size threshold, at which point the cells proceed through the rest of the cell cycle. However, a model, based solely on a specific size checkpoint at G1/S, cannot readily explain why cells with deficient G1/S control mechanisms are still able to maintain a very stable cell size distribution. Furthermore, such a model would not easily account for stochastic variation in cell size during the subsequent phases of the cell cycle, which cannot be anticipated at G1/S. To address such questions, we applied computationally enhanced quantitative phase microscopy (ceQPM) to populations of cultured human cell lines, which enables highly accurate measurement of cell dry mass of individual cells throughout the cell cycle. From these measurements, we have evaluated the factors that contribute to maintaining cell mass homeostasis at any point in the cell cycle. Our findings reveal that cell mass homeostasis is accurately maintained, despite disruptions to the normal G1/S machinery or perturbations in the rate of cell growth. Control of cell mass is generally not confined to regulation of the G1 length. Instead mass homeostasis is imposed throughout the cell cycle. In the cell lines examined, we find that the coefficient of variation (CV) in dry mass of cells in the population begins to decline well before the G1/S transition and continues to decline throughout S and G2 phases. Among the different cell types tested, the detailed response of cell growth rate to cell mass differs. However, in general, when it falls below that for exponential growth, the natural increase in the CV of cell mass is effectively constrained. We find that both mass-dependent cell cycle regulation and mass-dependent growth rate modulation contribute to reducing cell mass variation within the population. Through the interplay and coordination of these 2 processes, accurate cell mass homeostasis emerges. Such findings reveal previously unappreciated and very general principles of cell size control in proliferating cells. These same regulatory processes might also be operative in terminally differentiated cells. Further quantitative dynamical studies should lead to a better understanding of the underlying molecular mechanisms of cell size control., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu 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.)

The cucumber target spot, caused by Corynespora cassiicola, is a major cucumber disease in China. Mefentrifluconazole, a new triazole fungicide, exhibits remarkable efficacy in controlling cucumber target spot. However, the resistance risk and mechanism remain unclear. In this study, the inhibitory activity of mefentrifluconazole against 101 C. cassiicola isolates was determined, and the results indicated that the EC 50 values ranged between 0.15 and 12.85 μg/mL, with a mean of 4.76 μg/mL. Fourteen mefentrifluconazole-resistant mutants of C. cassiicola were generated from six parental isolates in the laboratory through fungicide adaptation or UV irradiation. The resistance was relatively stable after ten consecutive transfers on a fungicide-free medium. No cross-resistance was observed between mefentrifluconazole and pyraclostrobin, fluopyram, prochloraz, mancozeb, or difenoconazole. Investigations into the biological characteristics of the resistant mutants revealed that six resistant mutants exhibited an enhanced compound fitness index (CFI) compared to the parental isolates, while others displayed a reduced or comparable CFI. The overexpression of CcCYP51A and CcCYP51B was detected in the resistant mutants, regardless of the presence or absence of mefentrifluconazole. Additionally, a two-way mixture of mefentrifluconazole and prochloraz at a concentration of 7:3 demonstrated superior control efficacy against the cucumber target spot, achieving a protection rate of 80%. In conclusion, this study suggests that the risk of C. cassiicola developing resistance to mefentrifluconazole is medium, and the overexpression of CcCYP51A and CcCYP51B might be associated with mefentrifluconazole resistance in C. cassiicola. The mefentrifluconazole and prochloraz two-way mixture presented promising control efficacy against the cucumber target spot., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ametoctradin is mainly used to treat plant oomycetes diseases, but the mechanism and resistance risk of ametoctradin in Phytophthora sojae remain unknown. This study determined the ametoctradin sensitivity of 106 P. sojae isolates and found that the frequency distribution of the median effective concentration (EC 50 ) of ametoctradin was unimodal with a mean value of 0.1743 ± 0.0901 μg/mL. Furthermore, ametoctradin-resistant mutants had a substantially lower fitness index compared with that of wild-type isolates. Although ametoctradin did not show cross-resistance to other fungicides, negative cross-resistance to amisulbrom was found. In comparison to sensitive isolates, the control efficacy of ametoctradin to resistant mutants was lower, implying a low to moderate ametoctradin resistance risk in P. sojae. All ametoctradin-resistant mutants contained a S33L point mutation in PsCytb. A system with overexpression of PsCytb in the nucleus was established. When we ectopically overexpressed S33L-harboring PsCytb, P. sojae developed ametoctradin resistance. We hypothesized that the observed negative resistance between ametoctradin and amisulbrom could be attributed to conformational changes in the binding cavity of PsCytb at residues 33 and 220., (Copyright © 2023. Published by Elsevier Inc.)

The DNA methyltransferase 1-associated protein (DMAP1) was initially identified as an activator of DNA methyltransferase 1 (DNMT1), a conserved eukaryotic enzyme involved in diverse molecular processes, including histone acetylation and chromatin remodeling. However, the roles and regulatory mechanisms of DMAP1 in filamentous pathogens are still largely unknown. Here, employing bioinformatic analysis, we identified PsDMAP1 in P. sojae, which features a canonical histone tail-binding domain, as the ortholog of the human DMAP1. A phylogenetic analysis of DMAP1 protein sequences across diverse eukaryotic organisms revealed the remarkable conservation and distinctiveness of oomycete DMAP1 orthologs. Homozygous knockout of PsDMAP1 resulted in the mortality of P. sojae. Furthermore, silencing of PsDMAP1 caused a pronounced reduction in mycelial growth, production of sporangia and zoospore, cystospore germination, and virulence. PsDMAP1 also played a crucial role in the response of P. sojae to reactive oxygen species (ROS) and osmotic stresses. Moreover, PsDMAP1 interacted with DNA N6-methyladenine (6 mA) methyltransferase PsDAMT1, thereby enhancing its catalytic activity and effectively regulating 6 mA abundance in P. sojae. Our findings reveal the functional importance of PsDAMP1 in the development and infection of P. sojae, and this marks the initial exploration of the novel 6 mA regulator PsDMAP1 in plant pathogens., 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 B.V. All rights reserved.)

The suboptimal efficiency in pesticide utilization may elevate residues, posing safety risks to human food and non-target organisms. To address this challenge, delivery systems, such as pathogen infection stimuli-responsive carriers, can be employed to augment the efficiency of fungicide utilization. The bursting of reactive oxygen species (ROS) is a common defense response of host plants to pathogenic infections. In this study, ROS-responsive mesoporous silica nanoparticles (MSN) modified with phenyl sulfide (PHS) as azoxystrobin (AZOX) carrier (MSN-PHS-AZOX) were fabricated. Results demonstrated that MSN-PHS-AZOX exhibited fungicide release kinetics dependent on ROS. In vitro inhibition experiments confirmed the fungicidal effect of MSN-PHS-AZOX on Botrytis cinerea , relying on external ROS. In vivo leaf experiments showcased the superior persistence of MSN-PHS-AZOX in compared to AZOX SC. Furthermore, MSN-PHS-AZOX exhibits favorable biosafety and lower toxicity to aquatic zebrafish compared to AZOX SC, with no adverse impact on cucumber leaf growth. These findings suggest the potential application of this ROS-responsive nano fungicide in managing plant disease in agricultural fields.

Background: Plant diseases caused by phytopathogenic fungi and oomycetes pose a serious threat to ensuring crop yield and quality. Finding novel fungicidal candidates based on natural products is one of the critical methods for developing effective and environmentally friendly pesticides. In this study, a series of salicylaldehyde derivatives containing an α-methylene-γ-butyrolactone moiety were designed, synthesized, and their fungicidal activities were evaluated., Results: The bioassay studies indicated that compound C3 displayed an excellent in vitro activity against Rhizoctonia solani with a half-maximal effective concentration (EC 50 ) value of 0.65 μg/mL, higher than that of pyraclostrobin (EC 50  = 1.44 μg/mL) and comparable to that of carbendazim (EC 50  = 0.33 μg/mL). For Valsa mali and Phytophthora capsici, compound C3 also showed good fungicidal activities with EC 50 values of 0.91 and 1.33 μg/mL, respectively. In addition, compound C3 exhibited promising protective in vivo activity against R. solani (84.1%) at 100 μg/mL, which was better than that of pyraclostrobin (78.4%). The pot experiment displayed that compound C3 had 74.8% protective efficacy against R. solani at 200 μg/mL, which was comparable to that of validamycin (78.2%). The antifungal mode of action research indicated that compound C3 could change the mycelial morphology and ultrastructure, increase cell membrane permeability, affect respiratory metabolism by binding to complex III, and inhibit the germination and formation of sclerotia, thereby effectively controlling the disease., Conclusion: The present study provides support for the application of these salicylaldehyde derivatives as promising potential pesticides with remarkable and broad-spectrum fungicidal activities against phytopathogenic fungi and oomycetes in crop protection. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Tomato early blight is a significant disease that causes substantial losses to tomato yield and quality. Mefentrifluconazole, an isopropanol-azole subgroup of triazole fungicides, has been registered in China for controlling various plant diseases, including tomato early blight, grape anthracnose, and apple brown spot. However, limited information is available on the mefentrifluconazole resistance risk and mechanism in plant pathogens. The sensitivity to mefentrifluconazole of 122 isolates of Alternaria alternata, one of the causal agents of tomato early blight, collected from different provinces in China, was evaluated. The results showed a unimodal curve for the sensitivity frequency, with an average EC 50 of 0.306 μg/mL. Through fungicide adaption, six resistant mutants (N4, N5, T4, T5, NG1, and NG10) were obtained from three parental isolates, with a mutation frequency of 3.28 × 10 -4 and resistance factors ranging between 19 and 147. The survival fitness of the resistant mutants, except for NG1, was significantly lower than that of their parental isolates. Positive cross-resistance was observed between mefentrifluconazole and difenoconazole or fenbuconazole, whereas no cross-resistance was found with three non-DMI fungicides. Furthermore, three distinct point mutations were detected in the AaCYP51 protein of the resistant mutants: I300S in T4 and T5; A303T in N4, NG1, and NG10; and A303V in N5. Compared to the parental isolates, the AaCYP51 gene was overexpressed in all six resistant mutants when treated with mefentrifluconazole. In summary, the resistance risk of A. alternata to mefentrifluconazole was low, and point mutations and overexpression of the AaCYP51 gene were identified as contributing factors to mefentrifluconazole resistance in A. alternata., (Copyright © 2023 Elsevier Inc. All rights reserved.)

In an effort to promote the development of new fungicides, a series of 48 novel N -(1-methyl-4-thiocyanato-1 H -pyrazol-5-yl)-benzamide derivatives A1 - A36 and B1 - B12 were designed and synthesized by incorporating a thiocyanato group into the pyrazole ring, and their fungicidal activities were evaluated against Sclerotinia sclerotiorum , Valsa mali , Botrytis cinerea , Rhizoctonia solani , and Phytophthora capsici . In the in vitro antifungal/antioomycete assay, many of the target compounds exhibited good broad-spectrum fungicidal activities. Among them, compound A36 displayed the best antifungal activity against V. mali with an EC 50 value of 0.37 mg/L, which was significantly higher than that of the positive controls fluxapyroxad (13.3 mg/L) and dimethomorph (10.3 mg/L). Meanwhile, compound B6 exhibited the best antioomycete activity against P. capsici with an EC 50 value of 0.41 mg/L, which was higher than that of azoxystrobin (29.2 mg/L) but lower than that of dimethomorph (0.13 mg/L). Notably, compound A27 displayed broad-spectrum inhibitory activities against V. mali , B. cinerea , R. solani , S. sclerotiorum , and P. capsici with respective EC 50 values of 0.71, 1.44, 1.78, 0.87, and 1.61 mg/L. The in vivo experiments revealed that compounds A27 and B6 presented excellent protective and curative efficacies against P. capsici , similar to that of the positive control dimethomorph . Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses showed that compound B6 could change the mycelial morphology and severely damage the ultrastructure of P. capsici . The results of the in vitro SDH enzymatic inhibition experiments indicated that compounds A27 and B6 could effectively inhibit the activity of P. capsici SDH ( Pc SDH). Furthermore, molecular docking analysis demonstrated significant hydrogen bonds and Pi-S bonding between the target compounds and the key amino acid residues of Pc SDH, which could explain the probable mechanism of action. Collectively, these studies provide a valuable approach to expanding the fungicidal spectrum of pyrazol-5-yl-benzamide derivatives.

In this investigation, the antifungal activity, its influence on the quality of apples, and the molecular mechanism of natamycin against Colletotrichum fructicola were systematically explored. Our findings indicated that natamycin showed significant inhibition against C. fructicola . Moreover, it efficaciously maintained the apple quality by modulating the physicochemical index. Research on the antifungal mechanism showed that natamycin altered the mycelial microstructure, disrupted the plasma membrane integrality, and decreased the ergosterol content of C. fructicola . Interestingly, the exogenous addition of ergosterol weakened the antifungal activity of natamycin. Importantly, natamycin markedly inhibited the expression of Cyp51A and Cyp51B genes in C. fructicola , which was contrary to the results obtained after treatment with triazole fungicide flusilazole. All these results exhibited sufficient proof that natamycin had enormous potential to be conducive as a promising biopreservative against C. fructicola on apples, and these findings will advance our knowledge on the mechanism of natamycin against pathogenic fungi.