A Practical Guide to Two-Stage Sporulation of Pyricularia oryzae: Method Comparison Using Strains from Diverse Grass Hosts

Background Pyricularia oryzae is a major fungal pathogen responsible for significant yield losses in rice. In recent years, diverse pathotypes have emerged as threats to other economically important grasses, including ryegrass, oats, wheat and foxtail millet. Research on host–pathogen interactions involving this species requires reliable spore production for inoculation. However, as a hemibiotrophic pathogen, P. oryzae often sporulates poorly on artificial media and typically requires specialized two-stage protocols for consistent spore production. Although several such methods have been developed, all were optimized for rice-derived strains and have not been systematically evaluated across strains from other hosts. Moreover, published protocols often lack detailed procedural information and troubleshooting guidance, posing challenges for researchers working in newly affected regions. Results We evaluated five two-stage sporulation protocols, including a novel method developed in this study. We then assessed their efficacy using 23 Pyricularia spp. strains collected from 13 Poaceae hosts, focusing on spore yield, technical feasibility and cost. The newly developed filter paper method consistently supported spore production and was compatible with cryopreservation at -40 °C, overcoming limitations associated with the short shelf life of fresh spores. Comparative analysis revealed strain-specific differences in sporulation levels across methods, with no apparent correlation with phylogenetic lineage. Step-by-step protocols with accompanying illustrations are also provided, along with practical recommendations for selecting suitable methods based on laboratory conditions, available resources, and research objectives. Conclusions This study provides a comparative evaluation of sporulation methods for P. oryzae strains from a range of grass hosts. The results serve as a practical reference for researchers conducting virulence profiling, resistance breeding, and disease management studies. These findings are particularly relevant for laboratories in regions that are currently affected by, or at risk of, outbreaks caused by this important plant pathogen.