Light and scanning electron microscopic observations of calcium oxalate crystals produced during growth of Sclerotium rolfsii in culture and in infected tissue

Abstract

Crystals produced during growth of Sclerotium rolfsii on cellophane overlaying water agar were identified as calcium oxalate based on their solubility characteristics in certain acids, positive staining reaction with silver nitrate – dithiooxamide, and characteristic energy-dispersive X-ray emission spectrum. Addition of calcium salts to water agar enhanced crystal formation. Similar crystals of calcium oxalate were observed with the light and scanning electron microscopes in sugar beet and carrot leaf tissues infected by Sclerotium rolfsii. They frequently formed along the infecting hyphae or were associated with hyphal aggregates and were observed in abundance within the tissue. Addition of oxalic acid solutions to leaf discs resulted in necrosis of the tissue, and crystals similar in all respects to those produced in infected tissues were formed. These observations indicate that the oxalic acid produced by Sclerotium rolfsii in culture or in diseased tissue may sequester available calcium to form calcium oxalate, and provide evidence for the role of oxalic acid in pathogenesis.