Brefeldin A, an effector of Ascochyta medicaginicola, mediates virulence.

Alfalfa spring black stem, caused by Ascochyta medicaginicola, severely affects global alfalfa production. This study isolates and identifies brefeldin A (BFA) from pathogen fermentation products and demonstrates its strong phytotoxicity. BFA inhibits leaf photosynthesis and seed germination in a dose-dependent manner at concentrations ≥10-4 mol. Significant variation in BFA sensitivity is observed among cultivars, with 'Longdong' showing the highest susceptibility and 'Spyder' displaying relative resistance. LC-MS/MS analysis detects BFA in naturally infected plants, confirming its presence during disease development. BFA treatment increases leaf relative electrolyte leakage, reduces chlorophyll content, and induces chloroplast structural damage, including shrinkage and disintegration of grana lamellae. Furthermore, BFA enhances defense-related enzyme activities and alters the expression of 953 genes. Gene set enrichment analysis reveals that photosynthesis pathways are significantly down-regulated, whereas defense responses are up-regulated. These results identify BFA as a key virulence effector and provide a basis for disease-resistant breeding and ecological management of spring black stem disease.