Azithromycin, an Antimicrobial Agent, Targets Glycoside Hydrolase Family 35 and Exhibits Potent Curative Activity Against Tea Plant Diseases Caused by <i>Lasiodiplodia theobromae</i>

Tea disease caused by <i>Lasiodiplodia theobromae</i> is an emerging fungal disease that significantly reduces the yield and quality of tea in tea-producing regions owing to the lack of effective control methods. In this study, we evaluated the antifungal activity of azithromycin, a macrolide antibiotic, against <i>L. theobromae</i>. In vitro assays demonstrated strong inhibitory activity, with a half-maximal effective concentration (EC₅₀) of 140.61 μg/ml, and in vivo application resulted in a 54.30% lesion inhibition rate at 800 μg/ml against tea leaf spot. Morphological and ultrastructural analyses using optical and transmission electron microscopy revealed that azithromycin induced pronounced hyphal abnormalities, including cytoplasmic disorganization and membrane disruption. Integrated transcriptomic, proteomic, and metabolomic analyses indicated that azithromycin treatment led to systemic disruptions in <i>L. theobromae</i>, including dysregulation of genes involved in carbohydrate metabolism, protein degradation, glycoprotein and cell wall biosynthesis, ergosterol biosynthesis, mitochondrial function, and stress response pathways. Molecular docking and bioinformatic analyses identified glycoside hydrolase family 35 (GH35), a key protein in carbohydrate metabolism, as the principal target of azithromycin, exhibiting the most favorable binding energy of -7.9 kcal/mol. These findings demonstrate that azithromycin disrupts multiple metabolic and cellular processes in <i>L. theobromae</i>, primarily by targeting GH35, providing a robust multitarget approach for fungal inhibition and sustainable disease control strategies for tea cultivation.