A cytosolic fluorescence-based platform for robust high-throughput fungicide screening in phytopathogenic fungi

Widespread use of conventional fungicides has decreased effectiveness due to the emergence of resistance in plant fungal pathogens, making the development of new fungicides essential for disease management. Traditional chemical screening relies on plate-based assays measuring hyphal growth or conidial germination rate, typically quantified by optical density (OD). However, OD-based measurements are often unreliable because irregular hyphal growth causes inconsistent readings, melanization interferes with accurate signal detection, and OD does not directly reflect fungal viability. Thus, a more robust and reliable high-throughput screening system is needed for filamentous fungi. We developed cytosolic fluorescence-expressing strains for seven phytopathogenic fungi with diverse biological characteristics and established a fluorescence-based high-throughput screening platform. Compared with conventional OD-based measurements, the fluorescence-based platform exhibited lower coefficient of variation, indicating improved measurement consistency and reproducibility. Furthermore, we extended this fluorescence-based platform by developing a dual-fluorescence marker system that enabled direct comparison of wild-type and mutant strains within the same well, demonstrating its potential for mode-of-action-oriented chemical screening. Our fluorescence-based platform overcomes the inherent limitations of conventional OD-based assays, providing a more accurate and scalable system for screening candidate fungicides and establishing a versatile framework for future mechanism-based screening strategies.