The Dual O-Methyltransferase and FAD-Dependent Monooxygenase ESCB3 Is Essential for Elsinochrome Biosynthesis and Virulence in Elsinoë arachidis

The filamentous fungus Elsinoë arachidis is a major foliar pathogen responsible for peanut scab, which is a significant disease affecting commercial peanut cultivation. Elsinochrome (ESC), produced by numerous phytopathogenic Elsinoë species, is a non-host-selective polyketide phytotoxin with strong photosensitive activity and plays a crucial role in pathogenesis on host plants. In E. arachidis, a dual-domain enzyme encoded by the gene ESCB3, containing both O-methyltransferase and a FAD-dependent monooxygenase domain, has been identified. To elucidate the role of ESCB3, the biological function, expression pattern of the ESC biosynthesis gene cluster, and associated metabolomics analyses were investigated in the present study. An ESCB3 deletion mutant (Δ ESCB3) was created by targeted gene disruption. Notably, ESC production was completely blocked in the Δ ESCB3 mutant, and the expression of ESC biosynthetic genes, except for the polyketide synthase gene ESCB1, was significantly downregulated. Additionally, the Δ ESCB3 exhibited heightened sensitivity to multiple stress tolerance compared with the wild type, especially oxidative stress/H 2 O 2 , highlighting the crucial role of ESCB3 in growth, development, and ESC biosynthesis in E. arachidis. Pathogenicity assays revealed a significant reduction in the pathogenicity of the Δ ESCB3 mutant, suggesting a possible correlation with the suppressed biosynthesis of ESC. Metabolomic analyses further confirmed that ESCB3 is indispensable for the ESC biosynthetic process and acts as a key regulatory factor. Collectively, the results of this study provide significant insights into the molecular mechanisms governing ESCB3-mediated virulence and ESC production in E. arachidis, offering potential targets for disease control strategies in peanut scab. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .