Artificial Inoculation of Epichloë festucae into Lolium perenne, and Visualisation of Endophytic and Epiphyllous Fungal Growth

Abstract

Natural hosts for the fungal endophyte <i>Epichloë festucae</i> include <i>Festuca rubra</i> (fine fescue) and <i>Festuca trachyphylla</i> (hard fescue). Some strains also form stable associations with <i>Lolium perenne</i> (perennial ryegrass). <i>L. perenne</i> is a suitable host to study fungal endophyte-grass interactions, such as endophytic fungal growth within the plant and epiphyllous growth on the plant surface. Here we provide a detailed protocol based on work by, for artificial inoculation of <i>E. festucae</i> into <i>L. perenne</i>, and newly developed staining and visualization techniques for observing endophytic and epiphyllous hyphae and the expressorium, an appressorium-like structure used by the fungus to exit the plant. The staining method uses a combination of glucan binding aniline blue diammonium salt (AB) and chitin binding wheat germ agglutinin-conjugated Alexa Fluor^®488 -(WGA-AF488). This protocol will be a useful tool to study <i>Epichloë</i>-grass interactions, particularly the comparison of different <i>Epichloë</i>-grass associations, various endophyte-host developmental stages, as well as the analysis of mutant <i>Epichloë</i> strains.