Cytological analysis of the effect of reactive oxygen species on sclerotia formation in Sclerotinia minor.

The Sclerotium is one of the most persistent organs in filamentous fungi. Control of sclerotial formation is promising in the prevention of sclerotial disease. In this study, cytological analyses of sclerotial development were conducted in Sclerotinia minor. Number and size of sclerotia were correlated with nutrient concentration of the media. Interruption of aeration by sealing with parafilm completely suppressed sclerotial formation. We also found that reactive oxygen species (ROS) generated two phases, i.e., hydrogen peroxide at sclerotial initial (SI) stage and O2 - at outer layer of sclerotial development/mature stages, during sclerotial formation. Ultrastructural analyses revealed that ROS was prominently produced at the outer layer of sclerotia in sclerotial mature (SM) phase. Although most of the inhibitors for ROS generation enzymes were ineffective for sclerotial formation, ascorbic acid, one of the scavengers of hydrogen peroxide, inhibited melanin biosynthesis during sclerotial maturation stage. The mycelia sealed with parafilm, when exogenously sprayed with hydrogen peroxide, could not produce sclerotium. These results indicated that ROS generation during sclerotial formation is mainly involved in the production of melanin layer.