Many symbiotic Epichloë species are seed-transmitted in their grass hosts. For a detailed investigation of Epichloë festucae colonization throughout the life cycle of its host, the authors transformed strain Fl1 with a fungal-active gene for enhanced cyan-fluorescent protein (eCFP), introduced it into perennial ryegrass (Lolium perenne), and used confocal microscopy to track its growth in the shoot apex, floral primordium, floral organs, seeds, and seedlings. Hyphae intercellularly colonized leaf sheaths, blades, true stems, and leaf primordia, and among floral primordia the endophyte exhibited different levels of colonization. In preanthesis florets, E. festucae colonized the pistil and stamen, but not pollen grains, and ramified throughout the ovule nucellus, but not the integument or embryo sac. Generally, only a single hypha was observed extended from the ovary placenta into the ovule. Within 4 d after anthesis, fungal hyphae had ramified throughout the developing seed and embryo. As the embryo matured, fungal hyphae became abundant between the testa and aleurone layer, and around the shoot apex and radical of the embryonic axis. During germination, hyphae accumulated in the mesocotyl and invaded the newly formed shoot apex near the meristem. In this host-fungus symbiosis, transmission to seedlings averaged 41% in 2010 and 76% in 2011. Each year, the frequency of ovary infection was similar to the frequency of infecting embryos and seedlings, indicating that colonization of the ovary and embryo was required for seed transmission.
Keywords: Autofluorescent protein; Clavicipitaceae; Poaceae; confocal microscopy; plant symbiont; vertical transmission.