In vitro culture and draft genome assembly of Monographella maydis predict a molecular driver of its sexual fitness in maize tar spot disease

Background Tar Spot Complex (TSC) is a devastating foliar disease of maize that threatens global food security, causing yield losses of up to 50%. While the disease is initiated by Phyllachora maydis , the “fisheye” necrosis that intensifies disease severity is attributed to Monographella maydis . Historically, the precise role of M. maydis has remained elusive due to a lack of reliable in vitro culture systems and genomic resources. This study reports the first successful isolation and reproducible in vitro culture of M. maydis , identifying V8 agar as an optimal medium for mycelial growth and sporulation. Results Using PacBio long-read sequencing technology, we generated a high-quality 39.8 Mb genome assembly for M. maydis , predicting 10,228 protein coding genes, 227 non-coding RNAs, 175 tRNAs and 45 rRNAs. Functional annotation identified a diverse repertoire of secreted proteins, likely involved in the tar spot disease pathosystem. Phylogenomic analysis confirmed that M. maydis is distinct from P. maydis , supporting a synergistic rather than evolutionary relationship between the two fungi. Furthermore, the identification of a conserved adenosine deaminase-acting-on-tRNA residue (H352) suggests potential for A-to-I RNA editing during sexual reproduction. Finally, we report the use of ITS1 and ITS4 primer pair for successful detection of M. maydis from infected leaf tissues. Conclusion This study establishes the first robust in vitro culture system and high-quality reference genome for Monographella maydis , providing foundational resources for investigating its biology and role in the tar spot disease complex. These genomic and molecular tools will facilitate future studies on fungal pathogenicity, host–pathogen interactions, and the development of improved disease management strategies.