Ustilago maydis Seedling Infection Assay and Scoring.

This protocol outlines a reproducible method for assessing the virulence of Ustilago maydis strains in Zea mays seedlings through targeted syringe inoculation of the leaf whorl. Using seedlings at the V3-V4 growth stage ensures optimal susceptibility and developmental uniformity. The method can be used with both solopathogenic strains and compatible mating-type combinations, enabling comparative analyses of infection efficiency and symptom development. Fungal cultures are prepared under controlled conditions to maintain virulence, and post-infection humidity is regulated to enhance colonization success. Symptom progression is monitored over 10-12 days and quantified using an ordinal disease scoring system. This assay provides a robust tool for evaluating effector mutants, analyzing host-pathogen interactions, and comparing strain virulence under standardized conditions.