Fig. 3

Photograph and confocal laser scanning micrographs of Epichloë scottii – Melica uniflora association. a Photograph of E. scottii stroma bearing reproductive tiller of M. uniflora. Letters indicate the leaf parts, where samples were taken for CLS microscopy. b–h confocal laser scanning micrographs. Samples were treated with aniline blue diammonium salt to stain fungal and plant cell wall β-glucans (overlay of stain and autofluorescence of cytoplasm depicted in yellow, orange pseudo colors) and wheat germ agglutinin-Alexa Fluor 488 (WGA-AF488) to stain fungal chitin (blue pseudo color) and with propidium iodide (f, g, only) to stain plant and fungal nuclei (yellow pseudo color). (b) epibiotic hyphae of E. scottii on the surface of the stem 2 cm below the base of stroma, maximum projection of 150 µm z-stack. (c) transverse section of unfertilized stroma tissue (st), maximum projection of 35 µm z-stack. d Higher magnification of (c) showing densely colonized vascular bundle (vb) and hyper-proliferative hyphal growth on the cuticle. e epibiotic hyphae of E. scottii on the leaf sheath. f epibiotic hyphae at point of exit of endobiotic hypha (e = expressorium), maximum projection of 9.4 µm z-stack. Only after several hyphal compartments are formed, chitin can be visualized by staining with WGA-AF488, depicted in blue pseudo color. g endobiotic hyphae of E. scottii in leaf sheath epidermis, maximum projection of 144 µm z-stack. h endobiotic hyphae of E. scottii in leaf blade, the channel for blue pseudocolor is overexposed to visualize chitin in septa of endobiotic hyphae, maximum projection of 12 µm z-stack. Septa in F, G, and H are marked with asterisks (*) and nuclei in (f) and (g) with hashes (#). Bars = 20 µm (e–h), 50 µm (b, d) 200 µm (c)