Shikonin Targets Dihydroorotase to Inhibit the Growth and Virulence of Phytopathogenic Fungi

Fungal diseases seriously threaten global crop production, highlighting the need for fungicides with novel modes of action. Here, we show that shikonin exerts antifungal activity by targeting dihydroorotase (DHOase). Shikonin significantly inhibited mycelial growth, spore germination, and appressorium formation in representative phytopathogenic fungi in vitro . Biophysical analyses showed that shikonin directly binds to the DHOase of Magnaporthe oryzae (MoPyr4), with dissociation constants of 3.084, 1.22, and 1.26 μM determined by MST, SPR, and ITC, respectively. Molecular docking and mutagenesis identified R90, N178, and H320 as key binding residues. Consistently, deletion of MoPYR4 in M. oryzae reduced sensitivity to shikonin. In planta, shikonin restricted invasive hyphal growth in rice cells and showed both protective and curative activities against rice blast disease. Shikonin also bound to and inhibited the DHOases of Fusarium graminearum and Fusarium oxysporum, indicating broad-spectrum antifungal potential. These findings identify fungal DHOase as a promising target for fungicide development.