Yabing Duan

Rice bakanae disease, caused by Fusarium fujikuroi, is a destructive seed-borne disease. Metconazole has excellent control potential, but its resistance risk and mechanism remain unreported. This study confirmed metconazole has strong antifungal activity, with EC50 values ranging from 0.0073 to 0.0468 μg/mL. After fungicide taming, we obtained seven metconazole-resistant mutants with low-level, genetically stable resistance. Biological fitness showed mycelial growth and virulence did not decrease significantly, but sporulation increased significantly. Cross-resistance found the mutants had cross-resistance to tebuconazole, prochloraz, triticonazole and difenoconazole, but not to prothioconazole, prothioconazole-desthio, epoxiconazole, ipconazole, propiconazole, triadimefon, fludioxonil, or pyraclostrobin. Sequence analysis detected the G464S substitution in FfCYP51B in all mutants, which site-directed mutagenesis confirmed reduces metconazole sensitivity. Molecular docking, dynamics simulation and MM/GBSA analysis showed the binding between metconazole and FfCYP51B-G464S was weakened, and the binding free energy was less favorable. In conclusion, the resistance risk of F. fujikuroi to metconazole is assessed as low.

Fludioxonil is a widely used phenylpyrrole fungicide. Previously, fludioxonil resistance was primarily associated with mutations in the histidine kinase Os1. However, highly resistant isolates without such mutations suggest the existence of additional mechanisms. This study identified a novel resistance pathway centered on the TGY motif of the terminal MAPK FgHog1 in Fusarium graminearum. Site-directed substitutions of phosphorylatable threonine and tyrosine residues in the TGY motif generated mutants FgHog1-T171G, FgHog1-Y173A, and double mutant FgHog1-T171G&Y173A. All these mutations abolished detectable FgHog1 phosphorylation and conferred extremely high fludioxonil resistance. Loss of FgHog1 phosphorylation also blocked fludioxonil-induced nuclear translocation of FgHog1. However, nonphosphorylatable mutants exhibited severe defects in vegetative growth, sporulation, virulence, glycerol accumulation, acetic acid utilization, and sexual development, demonstrating strong pleiotropy. These results indicate that FgHog1 phosphorylation governs fludioxonil sensitivity and multiple biological functions, while the associated fitness costs may limit resistance development in field populations.

Fusarium head blight (FHB), caused by Fusarium asiaticum , is a significant wheat disease in China, reducing yields and contaminating grain with deoxynivalenol (DON). Due to the absence of highly resistant wheat varieties, chemical control remains the primary method for managing FHB. This study identified two homologous SDHC genes in F. asiaticum , namely FaSDHC1 and FaSDHC2 . Deletion of FaSDHC2 impaired fungal sporulation, virulence and DON production, whereas the loss of FaSDHC1 increased DON production. Both genes were found to regulate sensitivity to SDHI fungicides—ΔFaSDHC1 showed decreased sensitivity, while ΔFaSDHC2 exhibited increased sensitivity. To explore the regulatory role of the SdhC subunit in SDHI fungicides sensitivity in F. asiaticum , this study obtained 11 boscalid‐resistant mutants from ΔFaSDHC2 through chemical taming. Genetic analysis identified six mutation genotypes, including two previously reported in Fusarium species (FaSdhB‐H248Y and FaSdhD‐E166K) and four novel mutations in the Fusarium genus (FaSdhC1‐H144Y, FaSdhC1‐H144N, FaSdhD‐H122Y and FaSdhD‐D133N). The site‐directed mutation verification and sensitivity assays revealed that mutants harbouring FaSdhC1‐H144Y/N, FaSdhD‐H122Y, FaSdhD‐D133N or FaSdhD‐E166K exhibited resistance to multiple SDHI fungicides (boscalid, fluopyram, pydiflumetofen, isopyrazam and benzovindiflupyr). In contrast, FaSdhB‐H248Y mutants remained sensitive to fluopyram while showing resistance to other SDHI fungicides. This study elucidates the important regulatory role of SdhC genetic differentiation to SDHI fungicide sensitivity in F. asiaticum . Furthermore, it confirms that specific variations within distinct subunits of the SDH complex endow F. asiaticum with resistance to SDHI fungicides. The findings advance understanding of SDHI fungicides resistance mechanisms and provide a basis for developing FaSdhC‐specific inhibitors and resistance management strategies.

Quinofumelin, a quinoline‐based fungicide, demonstrated potent antifungal activity against Fusarium graminearum , the pathogen responsible for Fusarium head blight (FHB) in wheat. Previously, we elucidated that quinofumelin targets dihydroorotate dehydrogenase (DHODH), an indispensable enzyme in the de novo pyrimidine biosynthesis pathway. To explore its resistance mechanism, five FgDHODHII site‐directed mutants with distinct genotypes were generated using the homologous recombination strategy. Sensitivity assays identified varying resistance levels: FgDHODHII‐A94V remained sensitive to quinofumelin, FgDHODHII‐D155T conferred low‐level resistance, FgDHODHII‐N281A conferred moderate‐level resistance, and FgDHODHII‐V179E/V179D conferred high‐level resistance. Quinofumelin inhibition was reversed by the addition of uridine monophosphate (UMP), uridine or uracil. Although the mutants did not exhibit growth defects, quinofumelin‐resistant mutants displayed reduced sporulation and virulence. Quinofumelin showed cross‐resistance to ipflufenoquin, but no cross‐resistance to other common fungicides (phenamacril, carbendazim, tebuconazole, pydiflumetofen, prothioconazole, fluopyram, benzovindiflupyr, pyraclostrobin and azoxystrobin). Molecular docking, molecular dynamics (MD) simulations and microscale thermophoresis (MST) revealed that D155T, V179E, V179D and N281A mutations altered the binding mode of quinofumelin, resulting in diminished binding affinity. This study provides key insights into its mode of action and resistance mechanism of quinofumelin, facilitating its application in sustainable FHB management.

Fusarium head blight (FHB), primarily caused by the Fusarium graminearum species complex (FGSC), reduces cereal yields and causes substantial economic losses globally. In China, Fusarium asiaticum is the predominant FGSC species, causing yield losses and deoxynivalenol contamination. Pydiflumetofen, a succinate dehydrogenase inhibitor, was widely used in China for FHB control. Among 5176 field isolates collected in 2023 from Hubei, Anhui, and Jiangsu provinces, five pydiflumetofen-resistant F. asiaticum isolates were identified. These isolates exhibited reduced sporulation and virulence. Site-directed mutagenesis confirmed that the FaSDHC2-A83V substitution conferred high resistance while impairing sporulation. No cross-resistance occurred with pyraclostrobin, phenamacril, or tebuconazole, but strong positive cross-resistance was observed with fluopyram and enpyracymid, and negative cross-resistance with benzovindiflupyr, a novel finding with potential implications for agricultural and clinical antifungal strategies. This study reports the first field-derived pydiflumetofen-resistant F. asiaticum mutants and identifies FaSDHC2-A83V as a key resistance marker, aiding monitoring and sustainable FHB management.

Modern fungicides have made significant contributions to crop disease management, but the development of resistant fungal strains has caused their failure in disease control. Therefore, developing fungicides with novel action mechanisms is the most effective measure to manage resistance. Quinofumelin, a novel quinoline fungicide, exhibits exceptional antifungal activity against phytopathogens. However, there is currently no available information on its mechanism of action. Here, we used transcriptome and metabolome analysis to observe a co-enrichment pattern of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) within pyrimidine biosynthesis pathway (PBP), identifying down-regulation of dihydroorotate dehydrogenase (DHODH). Exogenous uridine monophosphate (UMP), uridine, or uracil (metabolites in PBP) successfully restored quinofumelin-induced inhibition of mycelial growth in Fusarium graminearum and Fusarium asiaticum. Additionally, the deletion of FgDHODHII was determined to be lethal; however, mycelial growth of ΔFgDHODHII mutants could be restored by adding UMP, uridine, or uracil. These findings indicate that the deficiencies in FgDHODHII are functionally equivalent to complete inhibition of its activity by quinofumelin. Finally, molecular docking, surface plasmon resonance (SPR), and microscale thermophoresis (MST) results strongly support the precise interaction between quinofumelin and FgDHODHII. Collectively, these findings provide compelling evidence for the involvement of de novo uracil biosynthesis as a mechanism of action for quinofumelin while identifying FgDHODHII as its specific target.

Modern fungicides have made significant contributions to crop disease management, but the development of resistant fungal strains has caused their failure in disease control. Therefore, developing fungicides with novel action mechanisms is the most effective measure to manage resistance. Quinofumelin, a novel quinoline fungicide, exhibits exceptional antifungal activity against phytopathogens. However, there is currently no available information on its mechanism of action. Here, we used transcriptome and metabolome analysis to observe a co-enrichment pattern of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) within pyrimidine biosynthesis pathway (PBP), identifying down-regulation of dihydroorotate dehydrogenase (DHODH). Exogenous uridine monophosphate (UMP), uridine or uracil (metabolites in PBP) successfully restored quinofumelin-induced inhibition of mycelial growth in Fusarium graminearum and Fusarium asiaticum. Additionally, the deletion of FgDHODHII was determined to be lethal; however, mycelial growth of ΔFgDHODHII mutants could be restored by adding UMP, uridine or uracil. These findings indicate that the deficiencies in FgDHODHII are functionally equivalent to complete inhibition of its activity by quinofumelin. Finally, molecular docking, surface plasmon resonance (SPR) and microscale thermophoresis (MST) results strongly support the precise interaction between quinofumelin and FgDHODHII. Collectively, these findings provide compelling evidence for the involvement of de novo uracil biosynthesis as mechanism of action for quinofumelin while identifying FgDHODHII as its specific target.