A Reproducible In Vitro Maintenance System for Physiologically Active and Pathogenic Phyllachora maydis Propagules

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 flour-based medium (ZeaMix agar) supplemented with zinc sulfate (ZnSO₄) and maize leaf extract under controlled conditions. Treatments consisted of three ZnSO₄ concentrations (0.014, 0.028, and 0.042 ppm) combined with maize leaf extract at 5, 10, or 15%. Propagule viability was assessed based on ascospore turgor, morphometry, and persistence over 7, 14, 21, and 28 days of incubation at 21°C. Fungal identity was confirmed by ITS1-ITS4 PCR amplification and Sanger sequencing. Pathogenicity was evaluated by inoculating maize plants at the V7 growth stage and quantifying stromatal severity and cirrus production. An intermediate ZnSO₄ concentration (0.028 ppm) combined with 10% maize leaf extract maximized ascospore turgor and size and resulted in the highest disease severity in greenhouse assays. This study describes a reproducible system for maintaining physiologically active, pathogenic P. maydis propagules in vitro, providing a useful platform for controlled studies of tar spot biology and host-pathogen interactions.