BACKGROUND:The ascomycete Magnaporthe oryzae is a destructive phytopathogenic fungus that causes rice blast disease and can greatly reduce rice yields. Due to the widespread use of synthesized chemical fungicides and the frequent occurrence of fungicide resistance and environmental pollution as a result, it has become increasingly urgent to develop environmentally friendly biocontrol alternatives, including plant crude extracts. RESULTS:In this study, the crude extracts of the tropical plant Millettia pachyloba were screened out and exhibited excellent preventive effects against M. oryzae at 200 μg mL-1. Bio-guided isolation of M. pachyloba was conducted and three isoflavonoids, rotenolone, durmillone, and durallone, whose chemical structures were determined using spectroscopic and spectrometric analyses, were obtained. The three isoflavonoids exhibited antifungal activities on conidial germination and appressorium formation in M. oryzae. Rotenolone had the strongest effect, with EC50 values of 57.81 μg mL-1 on conidial germination and 19.14 μg mL-1 on appressorium formation. Comparative metabolomics showed that differential metabolites were enriched in ABC transporter pathways and amino acid metabolic pathways when M. oryzae conidia were treated with rotenolone, suggesting that rotenolone interferes with amino acid transportation. Moreover, the M. pachyloba crude extract also effectively inhibited the infection of other fungal pathogens on tomato, apple, wheat, and maize. CONCLUSION:The results suggest that isoflavones extracted from M. pachyloba prevent rice blast by inhibiting the infection-related morphogenesis of M. oryzae and may interfere with amino acid transport, demonstrating that M. pachyloba crude extract exhibits potential as a bio-fungicide for controlling fungal diseases in plants. © 2025 Society of Chemical Industry.