The use of the fluorescent dye, 3,3′‐dihexyloxacarbocyanine iodide, for selective staining of ascomycete fungi associated with liverwort rhizoids and ericoid mycorrhizal roots

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Abstract
The lipophilic fluorochrome 3,3'dihexyloxacarbocyanine iodine [DiOC6 (3)], previously used to visualize mitochondria and ER in animal and plant tells, when applied at concentrations of 0.01-5 μg ml-1 selectively stains ascomycetous hyphae in ericaeous roots and in the rhizoids of liverworts in the families Lepidoziaceae (both tropical and temperate species), Calypogeiaceae, Adelanthaceae, Cephaloziaceae and Cephaloziellaceae. Basidio-mycetes forming endophytic associations with liverworts and ectomycorrhizas in seed plants, are stained with DiOC6 (3) only at concentrations at and above 50 μg ml-1 . VA mycorrhizal fungi in liverworts, pteridophytes and angiosperms fail to stain. Hyphae of the ericoid mycorrhizal fungus, Hymenoscyphus ericae, grown in axenic culture, are stained with much lower concentrations of DiOC6 (3) than are those of a range of ectomycorrhizal fungi, an orchid fungus and Oidiodendron griseum which has been reported occasionally to form ericoid mycorrhizal associations. In contrast to other fluorescent probes that recognize fungal wall components, DiOC6 (3) is a vital stain of fungal cytoplasm. Greater membrane permeability, compared to that in other fungi, is the likely basis for the selective staining of Hymenoscyphus ericae and the root and rhizoid-inhabiting ascomycetes with this dye. DiOC6 (3) offers a rapid means for identifying intracellular ascomycetous mycorrhizas and for determining the distribution of living hyphae within these associations.