Unravelling the Phytotoxin 5-Methylmellein as a Key Virulence Mechanism of Walnut Branch Blight Caused by Phomopsis capsici

Phomopsis capsici has recently been identified as the fungal pathogen responsible for walnut (Juglans regia) branch blight, a disease that significantly reduces yield and causes substantial economic losses. Despite the known role of secondary metabolites in fungal pathogenicity, the specific toxic components of P. capsici and their mechanisms of action remain poorly understood. This study aimed to isolate and characterise the toxic substances produced by this pathogen to elucidate its phytotoxic effects. Two non-protein toxins, PC-1 and PC-2, were isolated and purified from P. capsici using HPLC, mass spectrometry and NMR. Structural analysis identified PC-1 as 5-methylmellein (C₁₁H₁₂O₃), a known phytotoxin, and PC-2 as tyrosol (C₈H₁₀O₂), a compound with antibacterial and antioxidant properties. Biological assays revealed that PC-1 exhibited strong toxicity, causing severe necrosis and cell death in walnut branches, while PC-2 showed minimal toxic effects. Further investigation demonstrated that PC-1 disrupts cellular homeostasis by inducing excessive accumulation of reactive oxygen species (ROS), leading to membrane damage and cell death. This study also observed significant increases in the activities of defence enzymes, superoxide dismutase and peroxidase, in response to PC-1, highlighting the role of oxidative stress in disease progression. This research represents the first isolation and characterisation of toxic components from P. capsici, providing critical insights into its virulence mechanisms. The findings contribute to a broader understanding of Phomopsis species as both endophytes and pathogens, offering a foundation for developing targeted control strategies for walnut branch blight and other fungal diseases in agriculture, with implications for ensuring global food security.