Infection of Various <i>Medicago sativa</i> Varieties by <i>Ascochyta medicaginicola</i> Triggers the Synthesis of Defensive Secondary Metabolites and Their Antifungal Mechanisms

Infection with <i>Ascochyta medicaginicola</i> triggers the production of defensive secondary metabolites in plants, with varying levels observed across two alfalfa varieties. Among the six metabolites identified, 3-indoleacrylic acid (YD) and 3-ethynylaniline (EL) exhibited antifungal activity, achieving inhibition rates of 82.21 and 66.36% at 200 μg/mL, respectively. YD and EL exerted protective and therapeutic effects, reducing alfalfa leaf lesion areas by more than 96.00% compared to the blank control. The antifungal mechanisms of YD and EL against <i>A. medicaginicola</i> included the destruction of cellular structure (mycelial deformity, increased membrane permeability, impaired cell integrity with leakage of cellular contents), the induction of oxidative stress (elevated levels of reactive oxygen species, hydrogen peroxide, and malondialdehyde), and the reduction of mitochondrial membrane potential in hyphae cells. These results suggest that EL and YD hold promise as chemical defensive metabolites for controlling alfalfa spring black stem disease.