Conditions for sporangiospore formation in Syzygites megalocarpus and their application to long-term preservation

Syzygites megalocarpus, a mycoparasitic mucoromycetous fungus, is the only currently recognized species of the genus Syzygites and known to be difficult to preserve over the long term. This is because the formation of sporangiospores, which are suitable for long-term preservation and have high germination rates, decreases during serial transfers or preservation. However, the factors that influence sporangiospore formation are poorly understood. In this study, we investigated the optimal conditions for sporangiospore formation using multiple strains to improve the long-term preservation of this species. Our results revealed that S. megalocarpus produced abundant sporangia when cultured on hyperosmotic media. Specifically, mycelia containing sporangia, grown on Potato Dextrose Agar (PDA) supplemented with 5.0–7.5% NaCl at 20–25 °C, were stably preserved for 1 y at -80 °C. In contrast, mycelia or zygosporangia lacking sporangia cultured on Malt Extract Agar or PDA without salt could not be reliably revived after cryopreservation. These findings indicate that the induction of sporangiospore formation enables stable preservation of S. megalocarpus at -80 °C and may contribute to the stable long-term preservation of other mucoromycetous fungi.