Defining the pathways of symbiotic Epichloë colonization in grass embryos with confocal microscopy

ABSTRACT

Asexual cool-season grass endophytes of the genus Epichloë (Ascomycota: Clavicipitaceae) are strictly vertically disseminated. The hosts of these mutualistic fungi express no symptoms during the fungal lifecycle that takes place entirely within the plant, while their hosts receive beneficial outcomes. These fungi are distributed in two major locations within the mature seeds of their hosts; namely, within the embryo (including the scutellum, coleoptile, plumule, radicle, and coleorhiza tissues) and between the aleurone and pericarp layers, with the latter hyphae playing no role in transmission of the fungus to the next plant generation. Conflicting evidence remains in the literature on the timing of embryo colonization. In a detailed investigation, utilizing confocal microscopy to observe the distribution of Epichloë coenophiala strain AR601 in tall fescue (Lolium arundinaceum), we tracked endophyte hyphal colonization in the ovary (pre-fertilization) through to the fully mature seed stage. Confocal microscopy images revealed that at the early and mature developmental stages of the embryo sac, before host grass fertilization, there were large quantities of endophyte mycelium present, especially around the antipodal cells, indicating that this endophyte enters the embryo sac before the fertilization stage. After host fertilization, fungal hyphae could be seen in the true embryo and early nonstarchy endosperm. Understanding the mechanisms of transmission to the seed is important for commercial seed producers and end users.

KEY WORDS:

• confocal microscopy
• endophyte
• Epichloë coenophiala
• fluorescent dye
• Lolium arundinaceum

Acknowledgments

This study had financial support from Grasslanz Technology Ltd., PGG Wrightson Seeds Ltd., AgResearch Core Funding, and the China Scholarship Council (for a PhD scholarship for the first author). We thank Natasha Forester and Brian Tapper of AgResearch Ltd. for valuable discussion and technical support. We thank Christine Voisey and David Hume of AgResearch Ltd. for reviewing the manuscript. We also thank Pauline Hunt of AgResearch Ltd. for assisting with preparing the figures.

Supplemental material

Supplemental data for this article can be accessed on the publisher’s Web site at www.tandfonline.com/umyc.