Tar spot of maize, caused by the obligately biotrophic fungus Phyllachora maydis, has become an increasingly important constraint to maize production throughout the Americas. The inability to culture P. maydis on synthetic media has limited experimental research on its biology and pathogenicity. In this study, we developed a reproducible in vitro maintenance system that supports the survival and pathogenicity of P. maydis propagules without axenic cultivation. Stromata were maintained on a maize