The effect of winter stress on Ilex aquifolium L.previously fumigated with ozone

European Holly (Ilex aquifolium) received either charcoal-filtered air (CFA) or CFA with 70 nl l(-1) ozone added for 7 h day(-1) over a 28 day period. Plants were then transferred into cooling incubators for hardening (4 degrees C day/2 degrees C night; day length 12 h) for 7 days and then to the frosting stage (2 degrees C day and -5, -10 or -15 degrees C night) for 4 days. The plants were then placed in ambient conditions. Treatment produced significant differences in chlorophyll fluorescence data. Stomatal conductance was significantly higher for the ozone treatments though both showed a general decline over all temperature regimes. Ozone also significantly increased electrolyte leakage and reduced winter survival. These results show that ambient concentrations of ozone can reduce the tolerance of I. aquifolium to freezing stress, which may have serious implications for its establishment and survival.