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A lack of winter hardiness limits the utility of perennial ryegrass (Lolium perenne L.) as a turf and forage grass in northern latitudes. The fungal endophyte Epichloë festucae var. lolii is commonly associated with perennial ryegrass and is believed to enhance stress tolerance in some environments. The effect of E. festucae var. lolii on freezing tolerance was assessed using seven diverse perennial ryegrass entries. Freezing tolerance was used as a proxy for winter hardiness, and most entries had been previously field tested to confirm this supposition. Three experiments were designed to isolate the effect of the endophyte from confounding effects from the grass host and the endophyte removal process. Experiment 1 compared hosts with (E+) or without (E−) endophytes that were either genetically identical or non-isogenic, but from the same entry. Isogenic E+ and E− plants did not differ in freezing tolerance; however, some non-isogenic populations differed in freezing tolerance. Experiment 2 used additional populations of non-isogenic hosts to confirm the association between freezing tolerant hosts and endophyte infection. Isogenic hosts were polycrossed and the half-sib E+ and E− progeny were used in Exp. 3, eliminating any effect of the endophyte removal process. Freezing tolerance did not vary between related families differing in endophyte infection, confirming that highly related hosts were not affected by endophyte. These results strongly suggest that native E. festucae var. lolii has no direct effect on freezing tolerance but may be found in higher frequencies in freezing-tolerant hosts. G.C. Heineck and E. Watkins Dep. of Horticultural Science, Univ. of Minnesota, 1970 Folwell Ave., St. Paul, MN; N.J. Ehlke, Dep. of Agronomy and Plant Genetics, Univ. of Minnesota, 1991 Upper Buford Circle, St. Paul, MN. Received 14 Dec. 2017. Accepted 1 Apr. 2018. *Corresponding author ([email protected]). Assigned to Associate Editor Geunhwa Jung. Abbreviations: E+, endophyte infected; E−, endophyte free; LT50, lethal temperature 50. Published in Crop Sci. 58:1788–1800 (2018). doi: 10.2135/cropsci2017.12.0731 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Published June 7, 2018