MELANIN PRODUCTION BY A FILAMENTOUS SOIL FUNGUS IN RESPONSE TO COPPERAND LOCALIZATION OF COPPER SULFIDE BY SULFIDE-SILVER STAINING

Gaeumannomyces graminis var. graminis, a filamentous soil ascomycete, exhibited enhanced cell wall melanin accumulation when exposed to as l ittle as 0.01 mM CuSO4 in minimal broth culture. Because its synthesis was inhibited by tricyclazole, the melanin produced in response to co pper was dihydroxynaphthalene melanin. An additional hyphal cell wall layer was visualized by electron microscopy when hyphae were grown in the presence of copper and fixed by cryotechniques. This electron-dens e layer was between the outer cell wall and the inner chitin layer and doubled the total wall thickness. In copper-grown cells that were als o treated with tricyclazole, this electron-dense layer was absent. Ato mic absorption spectroscopy demonstrated that up to 3.5 mg of Cu per g of fungal mycelium was adsorbed or taken up by hyphae grown in 0.06 m M CuSO4. A method for silver enhancement was developed to determine th e cellular location of CuS. CuS was present in cell walls and septa of copper-grown hyphae. Electron microscopy of silver-stained cells sugg ested that CuS was associated with the melanin layer of cell walls.