A reproducible and efficient inoculation system for rice false smut based on conidia-germinated hyphae

Abstract Rice false smut, caused by the ascomycete fungus Ustilaginoidea virens , is one of the most severe diseases of rice. Conventional artificial inoculation methods for rice false smut rely mainly on conidia–hyphae mixtures (CH) as inocula, which frequently result in poor reproducibility and variable disease development across experiments. Here, we established a reproducible and efficient inoculation system for rice false smut using conidia-germinated hyphae (CGH) as inoculum, in which conidia are allowed to germinate before inoculation, yielding a more uniform and physiologically active inoculum. A rice leaf-based pre-culture step markedly improved conidial production and germination, and CGH showed higher infection efficiency and more stable disease symptoms than conventional CH under both controlled and field conditions. Time-course observations further revealed that CGH-based inoculation supports the typical infection progression of U. virens in rice spikelets. Importantly, this system enabled reliable phenotyping of U. virens mutants and effectively identified resistant and susceptible rice cultivars. In addition, the high rate of diseased spikelets achieved with CGH enabled consistent sample collection for downstream molecular and multiomics analyses. Overall, this study provides a refined inoculation system and a robust platform for functional studies of the U. virens –rice pathosystem.