Fluoxastrobin Targets NAD[P]H:quinone oxidoreductase to Disrupt Membrane Redox Balance and Suppress <i>Lasiodiplodia theobromae</i> , an Emerging Tea Pathogen

Tea diseases caused by Lasiodiplodia theobromae are prevalent in many tea-producing countries, substantially affecting both yield and quality of tea. In this study, fluoxastrobin, a strobilurin fungicide, exhibited a half-maximal effective concentration (EC 50 ) of 143.44 μg/ml for inhibiting mycelial growth in vitro. At a dose of 800.0 μg/ml, it demonstrated 42.00% antifungal activity against leaf spot on detached tea twigs. Micromorphological observations revealed that fluoxastrobin induced hyphal abnormalities, including surface roughness, deformation, and swelling. Integrated transcriptomic, proteomic, metabolomic, and bioinformatic analyses indicated that fluoxastrobin significantly modulate the expression and abundance of genes, proteins, and metabolites associated with energy metabolism pathways. Fluoxastrobin notably reduced mitochondrial membrane potential, thereby inhibiting electron transport and impairing energy production. Molecular docking and molecular dynamics simulations suggested that fluoxastrobin binds to NAD(P)H quinone oxidoreductase (NQO), with a binding free energy of −10.1 kcal/mol. Microscale thermophoresis assay revealed a dissociation constant (Kd) of 0.121 μM for fluoxastrobin binding to NQO, which is significantly lower than that of azoxystrobin (0.319 μM). Surface plasmon resonance assay further confirmed the stronger binding affinity of fluoxastrobin to NQO, with a Kd value of 7.098 ×10 −5 M. To enhance disease control efficacy, the combinations of fluoxastrobin with pyrrolnitrin or osthole at a ratio of 1:2 was screened, yielding EC 50 values being 0.0036 and 15.25 μg/ml and synergistic ratio values being 1.29 and 4.00, respectively. Collectively, these findings suggest that fluoxastrobin targets quinone oxidoreductase to disrupt membrane redox balance and suppress L. theobromae, supporting its potential application in the management of tea diseases.