Allelic Homogenization of Detoxification Genes RsCYP3A24 and RsGSTF5 Is Associated with Metabolic Resistance to SYP-14288 in Rhizoctonia solani

Abstract Fungicide resistance in multinucleate fungi, such as Rhizoctonia solani, poses a growing challenge for effective disease management. This study explores the molecular mechanisms underlying resistance to the fungicide SYP-14288 in R. solani, focusing on the role of two detoxification genes (RsCYP3A24 and RsGSTF5) and fungal nuclear heterogeneity. Genotyping analysis revealed that the parental R. solani strain harbors mixed allelic variants. Under fungicide stress, continuous subculturing reduced nuclear heterogeneity in resistant mutants, which progressively showed apparent allelic fixation of selected sequence variants, a genotypic shift toward homokaryotization. Transcriptomic and genomic analyses revealed significant upregulation and optimized gene composition of these detoxification genes in the resistant mutants. Functional validation through enzymatic activity assays confirmed that RsCYP3A24 participates in phase I oxidation, whereas RsGSTF5 mediates phase II glutathione-conjugation detoxification. Heterologous expression in Escherichia coli demonstrated that the detoxification proteins of resistant strains with fixed resistance-associated alleles possessed greater detoxification efficiency for SYP-14288. These genes were coordinately upregulated and functionally enhanced under fungicide selection pressure. This selective pressure altered the nuclear composition of heterokaryotic populations and promoted adaptive evolution characterized by allelic homogenization. Overall, this study provides new insights into the relationship between allelic variation and metabolic resistance in multinucleate fungi and offers valuable guidance for the sustainable management of fungicide resistance.