Genomic and Functional Characterization of an Alternaria brassicicola Isolate Causing Black Spot Disease on Broccoli Leaves

Alternaria brassicicola is a necrotrophic fungal pathogen causing black spot disease on cruciferous crops worldwide. In this study, we comprehensively characterized the pathogenic isolate Ab0920a from diseased broccoli in Shanghai using morphological, phylogenetic, host range, fungicide sensitivity, genomic, and functional analyses. Pathogenicity tests on 27 cruciferous varieties revealed a broad host range with varying resistance levels. Fungicide sensitivity assays showed that fluxapyroxad (EC50 = 0.0695 µg/mL), prochloraz (0.0711 µg/mL), and difenoconazole (0.0863 µg/mL) were highly effective, whereas fluazinam was least effective. Genome-wide annotation identified 941 secreted proteins (8.95% of the proteome) and 237 candidate effectors, including 31 small cysteine-rich secreted proteins and 68 homologs of known virulence factors. Conserved effector-associated motifs (e.g., RXLR, [Y/F/W]xxC) were detected, and carbohydrate-active enzyme CAZyme annotation revealed diverse families potentially involved in plant cell wall degradation. Functional validation using the pSUC2 yeast system confirmed that N-terminal signal peptides of tested effectors are competent for secretion. A PVX-based transient expression assay in Nicotiana benthamiana identified two effectors that suppress Bax-induced programmed cell death, suggesting their potential roles in modulating host immunity. Overall, this study provides comprehensive insights into the pathogen Ab0920a, offering resources for disease management and functional studies on necrotrophic fungal pathogenesis.