Figure 6. Endophytic and epiphyllous hyphae of E. festucae associated with perennial ryegrass.

Samples were taken two weeks after inoculation and stained with WGA-AF488 and AB (A, B, D, F) and WGA-AF488, AB and PI (C, E). A. Endophytic hyphae in the leaf sheath. The cell walls of endophytic hyphae are stained with AB and the chitin in septa is stained with WGA-AF488, shown in blue, and marked by asterisks. Autofluorescence of the plant chloroplasts is shown in orange. B. Endophytic hyphae in the leaf blade; note that the hyphae are not straight between the spongy mesophyll. Staining and pseudocolors are as in A. C. Endophytic hyphae in the blade, stained additionally with PI for visualization of nuclei. Fungal nuclei are in yellow and marked by arrows. Plant nuclei are shown in yellow and marked by hash symbols. D. Cell walls of the endophytic hyphae are not stained by WGA-AF488 and so are hyphae after emergence from the plant stained by AB, shown in red pseudocolor and marked by an arrow. Cell walls and septa of epiphyllous hyphae on the leaf sheath contain chitin stained with WGA-AF488, shown in blue, septa are marked by asterisks. E. Epiphyllous hyphae on the leaf blade, fungal nuclei are stained additionally with PI (arrows). Note that the hyphal cell walls are chitin-free shortly after emerging on the outer surface and therefore only septa bind WGA-AF488 and appear blue (asterisks), while the fungal cell wall is stained by AB and shown in red. F. Expressorium near blade meristem rupturing the cuticle, which shows green autofluorescence. Scale bars = 20 µm.