Abstract: Dactylobotrys graminicola (Dgr), a necrotrophic fungal pathogen, has recently been identified as the causative agent of a novel sheath-rot disease in hulless barley, causing substantial reductions in yield and quality. Research has mainly focused on early detection and fungicide screening, while the resistance mechanisms in hulless barley remain poorly understood. This knowledge gap substantially impedes the breeding of resistant varieties and the development of environmentally sustainable control technologies. In this study, RNA sequencing and metabolomic analyses were conducted on hulless barley varieties exhibiting contrasting resistance to Dgr infection. These analyses revealed that the phenylpropanoid biosynthesis pathway was markedly upregulated in the resistant cultivar Z257. Notably, accumulation and secretion of coniferyl aldehyde (CA) were elevated in Z257 compared to the susceptible cultivar Z251. In vitro antifungal assays demonstrated that CA strongly inhibited Dgr growth. Drug affinity responsive target stability and microscale thermophoresis assays identified pyruvate kinase (PK) as CA's target. Molecular docking and enzymatic activity assays indicated that CA disrupts the binding of the substrate phosphoenolpyruvate to the active site of PK, reducing enzymatic activity. In conclusion, hulless barley enhances its resistance against Dgr by secreting CA, which inhibits the pathogen's PK, establishing an effective defense mechanism.

Key words: antifungal, coniferyl aldehyde, Dactylobotrys graminicola, phenylpropanoid biosynthesis, pyruvate kinase