A Genomic DNA-Based Real-Time PCR Assay for Quantification of Ustilago maydis Biomass in Plant Samples.

Ustilago maydis is a basidiomycete fungus that causes common smut in maize, serving as a model system for studying smut fungi and biotrophic pathogens. Reliable quantification of fungal biomass in planta facilitates tracking fungal proliferation and disease development, evaluating fungal mutant virulence, and assessing host resistance. Traditional methods such as symptom scoring or microscopy after staining are semiquantitative and prone to subjectivity. In this chapter, a DNA-based quantitative real-time PCR (qPCR) assay for sensitive and reproducible measurement of U. maydis biomass in infected maize tissues is described, based on the relative amplification ratio of a fungal gene to a maize reference gene. This assay provides a robust and quantitative tool for investigating fungal virulence and plant resistance.