NAD-Dependent Epimerase/Dehydratase Serves as a Novel Potential Target for Fluoxastrobin in <i>Pestalotiopsis trachicarpicola</i>, the Causal Agent of Tea Foliar Disease

Tea foliar disease, caused by the fungal pathogen <i>Pestalotiopsis trachicarpicola</i>, poses a significant threat to tea production and quality in China. The disease, which includes tea leaf spot and gray blight, severely impacts both tea yield and quality, partly due to the lack of effective epidemiological data and control strategies. Fluoxastrobin, a strobilurin fungicide, has shown promise in controlling various fungal diseases. This study evaluated the inhibitory activity of fluoxastrobin against multiple phytopathogenic fungi, revealing a half-maximal effective concentration of 13.64 μg/ml for <i>P. trachicarpicola</i> in vitro and a maximum in vivo curative activity of 81.63% against tea leaf spot caused by <i>P. trachicarpicola</i>. Observations from optical, scanning electron, and transmission electron microscopy revealed that fluoxastrobin induces severe hyphal deformation, including hyphal swelling, malformation, and rough hyphal surfaces. Integrated transcriptomic, metabolomic, and bioinformatic analyses indicated that fluoxastrobin affects the electron transfer process, therefore disturbing the energy metabolism of hyphae. Biochemical assays indicated that fluoxastrobin can inhibit ATP production in hyphae and increase the membrane potential of hyphae, with these effects being dose-dependent. Molecular docking and molecular dynamics simulations demonstrated that fluoxastrobin binds stably to NAD-dependent epimerase/dehydratase, a key enzyme in energy metabolism, with binding energies of -12.9 kcal/mol, suggesting that these may be potential target proteins in the fungus. Overall, this study demonstrates that fluoxastrobin disrupts electron transfer, reduces ATP production, and inhibits hyphal growth, likely by binding NAD-dependent epimerase/dehydratase. It shows potential for controlling tea foliar diseases in the future.