2008 NORTHEASTERN NATURALIST 15(1):57–66 Ozone Sensitivity of 28 Plant Selections Exposed to Ozone Under Controlled Conditions Lee J. Kline1, Donald D. Davis1,*, John M. Skelly1, James E. Savage1, and Jon Ferdinand1 Abstract - Ambient, ground-level ozone is the most important air pollutant affecting vegetation in the US. However, ozone-sensitive bioindicators need to be identified for use in field surveys to detect ozone-induced symptoms. To identify such bioindicators, 28 plant selections were exposed to ozone within greenhouse chambers during 2003 and 2004. Plants most sensitive to ozone included Asclepias incarnata (swamp milkweed), Asclepias syriaca (common milkweed), Cephalanthus occidentalis (buttonbush), Platanus occidentalis (American sycamore), Salix x cotteti (Bankers dwarf willow), Salix lucida (shining willow), Salix nigra (black willow), Salix sericea (silky willow), and Symphoricarpos albus (snowberry). Plants moderately sensitive included Aster novae-angliae (New England aster), Monarda didyma (bee-balm), Rhus aromatica (aromatic sumac), Salix discolor (pussy willow), Salix exigua (sandbar willow), Salix purpurea (basket willow), Sambucus ebulus (European dwarf elderberry), and Symphoricarpos spp. (mixture of “snowberries”). Plants more tolerant to ozone included Aster macrophyllus (bigleaf aster), Aster novi-belgii (New York aster), Cercis canadensis (redbud), Populus maximowizii x trichocarpa (hybrid poplar), Rudbeckia laciniata (cutleaf, or tall green-headed coneflower), Salix amygdaloides (peach-leaved willow), Salix eriocephala (diamond willow), Sambucus canadensis (American elder), Sambucus nigra (European elder), Symphoricarpos orbiculatus (coralberry), and Viburnum trilobum (highbush-cranberry). The incidence and severity of ozone-induced symptoms varied with species, ozone concentration, and length of exposure. Pigmented adaxial leaf surface stipple, bifacial necrosis, and premature defoliation were common foliar symptoms induced by ozone; leaf reddening, bronzing, and chlorosis occurred less frequently. Species that produced classic ozone-induced stipple in this study may serve as useful bioindicators in field surveys to confirm that ozone injury occurs in many parts of the country. Such findings may have a positive impact on establishing more stringent air-quality standards to protect vegetation and the environment. Introduction Ambient, ground-level ozone is the most important air pollutant affecting vegetation in the eastern US (Skelly 2000). Many universities and governmental organizations perform field surveys to determine if ozone is adversely impacting vegetation in remote areas, but lack suitable ozone-sensitive bioindicators for use in many parts of the country. 1Department of Plant Pathology and Penn State Institutes of the Environment and Energy, The Pennsylvania State University, University Park, PA 16802. *Corresponding author - ddd2@psu.edu. 58 Northeastern Naturalist Vol. 15, No. 1 The objective of such surveys is to document the incidence or severity of ozone-induced symptoms in relatively pristine areas, with the hope that results will be used in establishing more stringent ambient air-quality standards for ozone (US Congress 1977, US EPA 1996). Such surveys often utilize ozone-sensitive, broadleaved bioindicator plants (Coulston et al. 2003, US DOI 2003) that respond to ambient ozone by exhibiting foliar “stipple” (Richards et al. 1958), a characteristic symptom induced by excessive ambient ozone (Skelly 2000). During field surveys, crews often report ambiguous symptoms observed on additional broadleaved species. To confirm species sensitivity and to describe ozone-induced symptoms, these additional species must be exposed to ozone under controlled conditions, such as in continuously stirred tank reactor (CSTR) chambers (Heck et al. 1975). The objectives of this study were to evaluate the ozone sensitivity of 28 plant selections and to describe ozone-induced symptoms produced under controlled conditions. Methods Plant culture and exposure to ozone Most plant selections chosen for this study (Table 1) grow naturally throughout the midwest and northeast USA. Selections were suspected to be sensitive to ozone based on field symptoms, were within genera known to be ozone-sensitive (Innes et al. 2001, Skelly et al. 2000, VanderHeyden et al. 2000), or were fast-growing, intolerant plants. Fast-growing species are often sensitive to air pollutants (Harkov and Brennan 1979, Umbach and Davis 1984). Milkweed plants were grown from seed. Elderberry, poplar, and willow selections were grown from vegetative cuttings; remaining selections were obtained as bare-root seedlings. Plants were potted in Metromix 500® potting soil (Scotts-Sierra Horticultural Products Co., Marysville, OH) supplemented with a one-time application of 5 g Osmocote® controlled-release fertilizer (Scotts-Sierra Horticultural Products Co.) at time of potting. Potted plants were maintained on benches in a greenhouse supplied with charcoal-filtered air (less than 8 ppb ozone daily hourly average) until placement into CSTR chambers for exposure to ozone. Plants were routinely watered prior to, during, and after exposures. Four ozone concentrations (30, 60, 90, and 120 ppb) were generated in a square-wave fashion for 7 hrs/day, 5 days/week within the chambers. Experiments began at 0900 hrs and ended at 1600 hrs daily. The ozone level of 30 ppb was intended to approximate background ozone concentrations, 60 ppb reflected a typical daily 7–8 hr ambient ozone exposure during mid- to late-summer in central Pennsylvania, and 90 ppb was employed to induce symptoms on less sensitive species (Orendovici et al. 2003). Lastly, the 120 ppb concentration was selected to induce foliar symptoms on the least sensitive species. During non-exposure hours, plants remained in the chambers with the chamber doors open and were exposed to the charcoal-filtered 2008 L.J. Kline, D.D. Davis, J.M. Skelly, J.E. Savage, and J. Ferdinand 59 air and environmental conditions within the greenhouse. Control plants were maintained on greenhouse benches, outside of the CSTR chambers, and received charcoal-filtered air. In 2003, 12 plant species (Table 1) were used in two consecutive studies. Ozone exposures during the first study started on 14 July and ended Table 1. Ozone sensitivity of plants exposed in 2003 and 2004, based on a combined rating of percent symptomatic leaf tissue and severity of symptoms for each species. Average injury is the row mean calculated across the 30, 60, 90, and 120 ppb response for each species. # of %INJ by ozone level (ppb) Average Year/species plants 30 60 90 120 injuryA 2003 Platanus occidentalis L. (American sycamore) 8 0.00 0.06 17.00 44.44 15.38 a Asclepias syriaca L. (common milkweed) 8 0.00 0.00 16.78 33.44 12.56 ab Asclepias incarnata L. (swamp milkweed) 4 0.00 0.00 14.44 33.44 11.97 ab Symphoricarpos albus (L.) S.F. Blake 16 0.06 0.17 7.10 14.95 7.43 ab (snowberry) Rhus aromatica Aiton (aromatic sumac) 24 0.00 0.06 4.55 18.53 5.79 ab Symphoricarpos spp. (mixed snowberries) 44 0.01 0.77 1.48 13.67 3.98 ab Sambucus nigra L. (European elder) 24 0.02 0.04 0.10 6.43 1.65 b Sambucus canadensis L. (American elder) 28 0.00 0.02 1.33 4.48 1.46 b Cercis canadensis L. (redbud) 8 0.00 0.00 1.28 1.56 0.71 b Symphoricarpos orbiculatus Moench 20 0.00 0.00 0.21 1.23 0.24 b (coralberry) Viburnum trilobum Marshall 8 0.00 0.00 0.06 0.34 0.10 b (highbush-cranberry) Aster macrophyllus L. (bigleaf aster) 8 0.00 0.00 0.06 0.34 0.10 b 2004 Salix nigra Marshall (black willow) 12 B B 17.07 33.94 25.50C a Salix sericea Marshall (silky willow) 22 0.02 12.80 16.85 49.14 19.26 ab Salix lucida Muhl. (shining willow) 23 0.00 17.61 17.22 34.32 18.04 bc Salix x cotteti (Bankers dwarf willow) 24 0.00 2.83 25.42 40.73 17.24 bcd Cephalanthus occidentalis L. (buttonbush) 16 0.06 4.71 13.25 49.12 16.78 bcd Salix exigua Nutt. (sandbar willow) 24 3.38 8.93 11.11 31.81 13.81 bcde Salix purpurea L. (basket willow) 24 0.00 0.00 12.74 33.44 11.54 bcdef Aster novae-angliae L. 24 0.02 0.95 16.28 28.22 11.38 cdef (New England aster) Sambucus ebulus L. 19 0.02 6.50 18.15 15.04 9.89 def (European dwarf elderberry) Salix discolor Muhl. (pussy willow) 23 0.00 3.70 7.62 21.21 8.48 efg Monarda didyma L. (bee-balm) 18 0.04 1.77 9.30 16.04 6.78 efg Aster novi-belgii L. (New York aster) 24 0.08 2.67 2.07 21.51 6.58 efg Salix eriocephala Michx (diamond willow) 24 0.04 1.27 5.16 13.17 4.87 fg Populus maximowizii x trichocarpa 20 0.60 4.18 5.07 6.90 4.18 fg (hybrid poplar) Rudbeckia laciniata L. (cutleaf coneflower) 20 0.00 0.60 2.02 12.00 3.80 fg Salix amygdaloides Andersson 20 0.00 0.09 0.34 2.16 0.71 g (peach-leaved willow) AMeans in the final column (average injury) followed by the same letter are not sigificantly different. (p = 0.05) within that year, according to Duncan’s multiple range test. BBlank space indicates no data. CMean based only on 90 and 120 ppb data. 60 Northeastern Naturalist Vol. 15, No. 1 on 21 August. There were three replications (three chambers) for each ozone level (four concentrations), using 12 CSTR chambers. Exposures in the second study started on 9 September and ended on 30 September. Due to mechanical difficulties, only four CSTR chambers were utilized in the second 2003 exposure, involving one chamber for each level of ozone. In both studies, the number of individual plants varied slightly depending upon plant condition, but usually involved two plants/species/chamber. In 2004, 16 plant selections, including two hybrids (Table 1), were exposed during two consecutive studies, using methods generally as described above. In the first 2004 study, ozone exposures were conducted in eight chambers from 13 July to 10 August. In the second study, plants were exposed in 12 chambers from 27 August to 24 September. Ozone concentration, light, relative humidity, and temperature were monitored within each chamber for 1.5 min at 12-min intervals during all exposures. Ozone was sampled through Teflon tubing using a solenoid- driven sampling system connected to a TECO Model 49 photometric ozone analyzer (Thermo Environmental Corp., Franklin, MA). Ozone calibration quality-control measures for the TECO analyzer followed the standards documented by the Pennsylvania Department of Environmental Protection, Bureau of Air Quality, Harrisburg, PA. Ozone and environmental data were input to a data logger connected to a computer. Data collection and analyses Each plant was rated for amount of foliage injured (AMT: % leaves symptomatic) and the severity of the injured foliage (SEV: % area affected on symptomatic leaves) as described by Orendovici et al. (2003). The assessments estimated the percentage of symptomatic tissue and were assigned nominal values that reflect five broad classes: 0 = no symptoms, 1 = 1–6%, 2 = 7–25%, 3 = 26–50%, 4 = 51–75%, and 5 = 75–100% tissue symptomatic. These data were used to calculate an overall value for each plant, as well as a mean value for each species. The nominal values recorded for each plant were converted to percentage values representing the midpoint of each class as follows: 0 = 0%, 1 = 3.5%, 2 = 16%, 3 = 38%, 4 = 63%, and 5 = 88%. Percentage values were calculated per plant and per species: mean injury value (%INJp) per plant = AMT*SEV, and mean injury value (%INJs) per species = (AMT*SEV)/N, where N is the number of plants evaluated per species. The statistical design was a split plot, with ozone treatments as the main plot and species as the subplot. To increase the number of observations for statistical analyses, data from the two studies in each year were combined. A general linear model (GLM) was utilized to analyze the percentage data (%INJs) and means were separated using Duncan’s multiple range test (Minitab 2003). Control plants did not exhibit any symptoms similar to those induced by ozone on the plants exposed within the CSTR chambers. 2008 L.J. Kline, D.D. Davis, J.M. Skelly, J.E. Savage, and J. Ferdinand 61 Results Ozone and environmental monitoring 2003. In the first study, the mean ozone concentrations achieved in the chambers for target concentrations of 30, 60, 90, and 120 ppb were 27.9, 56.6, 84.3, and 113.8 ppb, respectively. Mean ozone concentrations for the four target levels in the second study were 26.3, 55.8, 83.3, and 113.8 ppb. Average temperatures monitored within the chambers in the two studies were 27.5 and 21.5 °C, and mean relative humidities were 84 and 83%, respectively. 2004. In the first study, the respective mean ozone concentrations for targets of 30, 60, 90, and 120 ppb were 27.1, 57.9, 87.3, and 117.4 ppb, respectively. In the second study, the respective monitored ozone levels were 29.3, 59.0, 87.8, and 119.7 ppb. Temperatures within the exposure chambers in the two studies were 26.2 and 28.3 °C, and mean relative humidities were 84 and 77%, respectively. Foliar symptoms 2003. Adaxial leaf-surface stippling, the classic foliar response of broadleaved plants to ozone (Richards et al. 1958, Skelly 2000), was observed on sensitive plant selections. Stipple progressed from light to dark in color with increasing length of exposure or increasing ozone concentration. Classic dark stipple was most evident on the more mature leaves that were present later in the exposures. Premature defoliation was also noted on sensitive plants. Ozone induced other occasional symptoms including tan stipple, chlorotic or necrotic spotting, general chlorosis, and reddening, but these symptoms were not rated. Plant species that exhibited classic, dark stipple in response to ozone included American elder, American sycamore, aromatic sumac, common milkweed, highbush-cranberry, redbud, and snowberry. On swamp milkweed, the stipple was initially tan, but became darker as exposures progressed. Common symptoms on coralberry included general chlorosis, followed by the classic dark stipple that intensified with increasing exposure. The predominant leaf symptom on bigleaf aster was a grayish-white stipple that was fine and “grainy” in appearance. European elder consistently exhibited a white bifacial fleck. Foliage of species with high levels of red leaf pigments has a tendency to develop a reddish color upon exposure to ozone (Orendovici et al. 2003). Aromatic sumac most consistently exhibited foliar reddening, in addition to stipple, at higher exposures. 2004. Adaxial leaf-surface stippling was observed on several species. As in 2003, stipple often was initially light-colored, and became darker with increasing ozone. Other ozone-induced symptoms were occasionally observed, including tan stipple, chlorotic spotting, chlorosis, reddening, bronzing, necrosis, and premature defoliation. 62 Northeastern Naturalist Vol. 15, No. 1 Plant species that most commonly developed the classic dark stipple were Bankers dwarf willow, bee-balm, and occasionally cutleaf coneflower. In addition, coneflower exhibited a dark-gray to dark-brown, irregular discoloration on upper leaf surfaces, and buttonbush exhibited a tan stipple that became darker with length of exposure time. A whitish-tan stipple occurred on the upper leaf surfaces of basket willow, European dwarf elderberry, peach-leaved willow, and silky willow. Foliar reddening occurred on Bankers dwarf willow and buttonbush at greater ozone exposures. Ozone-induced symptoms on most species of willows appeared as orange, tan, or rust-colored angular spots, usually on the edges or tips of leaves. Other symptoms on willow included tip burn, bifacial edge necrosis, chlorosis, and premature defoliation. Black willow, pussy willow, and silky willow exhibited the most severe symptoms of the willows. Foliage of New England aster and New York aster slowly faded in luster and color during exposure, until leaves eventually became light tan and then entirely necrotic. Large areas of black bifacial necrosis and premature defoliation occurred on hybrid poplar. Chlorosis was evident to some degree on the foliage of most sensitive species. Response to different levels of ozone 2003. Following exposure to 30 ppb, the lowest ozone concentration utilized, a few individual European elder, snowberry, and the mixture of snowberries exhibited only traces of ozone-induced symptoms (Table 1). At 60 ppb, these species plus American elder, American sycamore, and aromatic sumac developed symptoms. All species exhibited some symptoms at 90 ppb ozone. American sycamore, common milkweed, and swamp milkweed were clearly symptomatic at 90 ppb, but bigleaf aster, redbud, European elder, and highbush-cranberry were only slightly symptomatic at this concentration. At 120 ppb ozone, some individuals within all selections exhibited symptoms. American sycamore, common milkweed, and swamp milkweed exhibited considerable foliar symptoms, but approximately half of the selections were somewhat insensitive to 120 ppb, showing only light symptoms. Bigleaf aster and highbush-cranberry were least sensitive. Exposure to highter concentrations of ozone generally resulted in increased symptoms on most species. 2004. There was only a trace of foliar symptoms following exposure to 30 ppb ozone (Table 1). An exception was shining willow, which consistently exhibited rust-colored angular spots, usually on the edges or tips of leaves, following exposure to 30 ppb. All species except basket willow showed symptoms at 60 ppb, although symptoms on peach-leaved willow were extremely light. Due to the low number of plants available, black willow was only exposed to 90 and 120 ppb. Most species were clearly symptomatic following exposure to 90 and 120 ppb ozone, but symptoms on peach-leaved willow were very light. 2008 L.J. Kline, D.D. Davis, J.M. Skelly, J.E. Savage, and J. Ferdinand 63 Ranking of species sensitivity 2003. American sycamore was most sensitive to ozone, followed in descending order by common milkweed, swamp milkweed, and snowberry (Table 1). The level of symptoms on these four species was similar (p = 0.05). Aromatic sumac, followed by mixed snowberries, European elder, and American elder, ranked next and were generally similar in ozone-sensitivity. Redbud was somewhat insensitive to ozone. Coralberry, bigleaf aster, and highbush-cranberry were least sensitive. However, the mean response values of the last nine selections (Table 1) were not significantly different, likely due to the high variability in the data. 2004. Among the willows, black willow was very sensitive to ozone (Table 1). However, due to the small number of available plants, black willow was only exposed at the two greater levels of ozone, resulting in inflated response values. Nevertheless, if one compares the symptom values for this species following exposure to 90 and 120 ppb with that induced on other species, black willow still ranks among the more sensitive willows. Shining willow, silky willow, and Bankers dwarf willow were also quite sensitive to ozone, as were sandbar willow and basket willow. Pussy willow showed few ozone-induced symptoms, diamond willow exhibited fewer symptoms, and peach-leaved willow was insensitive. Among plants other than willows, buttonbush was most sensitive, followed by New England aster and European dwarf elderberry. Bee-balm, cutleaf coneflower, New York aster, and hybrid poplar were less sensitive. Discussion The main objectives of this study were to evaluate the relative ozone sensitivity of different plant selections and to describe ozone-induced foliar symptoms under controlled conditions in CSTR chambers. The most sensitive plants included American sycamore, aromatic sumac, basket willow, Bankers dwarf willow, bee-balm, black willow, buttonbush, common milkweed, European dwarf elderberry, New England aster, sandbar willow, shining willow, silky willow, snowberry, and swamp milkweed. These species are potential bioindicators for use in ozone surveys. However, ozone caused marginal and tip leafburn on the willows, symptoms that would be very difficult to attribute to ozone in the field. Therefore, we propose that the most useful bioindicators are American sycamore, aromatic sumac, beebalm, buttonbush, common milkweed, European dwarf elderberry, New England aster, snowberry, and swamp milkweed. Our study confirmed earlier reports that the following species were sensitive to ozone: swamp milkweed (Orendovici et al. 2003), common milkweed (Duchelle and Skelly 1981), American sycamore (Davis and Coppolino 1976, Neufeld and Renfro 1993), European dwarf elderberry (Orendovici et al. 2003), and snowberry (Davis and Coppolino 1976). We previously reported hybrid poplar to be sensitive to ozone when exposed within growth chambers in a laboratory (Davis et al. 1993), but the same clone was rather 64 Northeastern Naturalist Vol. 15, No. 1 insensitive in the study reported herein. This discrepancy was likely due to different exposure/environmental conditions between the two studies. Cutleaf coneflower is very sensitive to ambient ozone in the field (Chappelka et al. 2003), but our selection was quite tolerant. Our coneflower seed was collected from the field in southeastern Pennsylvania, within an area that has very high levels of ambient ozone. Coneflower populations that exhibited ozone-induced symptoms, such as discolored leaves and reduced growth, were likely not collected, and thus, ozone-tolerant populations of cutleaf coneflower may have been inadvertently selected by seed collectors. Genetic variation in sensitivity among different populations of plants, as well as intraspecific variability in ozone sensitivity among individual plants within the same species, is well known (Karnosky et al. 2005). In our study, this high degree of variability often limited statistical analysis when testing for within-species differences in sensitivity. Thus, the rankings in this paper must be considered only as a general guide. Sensitive species other than willows often exhibited classic adaxial leaf surface stippling as the predominant symptom following ozone exposure. Stipple was first described as being a symptom of ozone response by Richards et al. (1958) on grapes and has since been defined as the classic symptom of ozone-induced symptoms on broadleaved species (Skelly 2000). Ozone-induced stipple, as observed in these CSTR studies, was often similar to foliar symptoms attributed to ambient ozone in the field. The broadleaved species that produced classic stipple in this study may serve as useful bioindicators. However, nearly half of the sensitive selections exhibited at least one non-specific symptom, such as foliar reddening, premature defoliation, bronzing, flecking, bleaching, leaf-tip burn, chlorosis, necrosis, and spotting. The bifacial fleck that we observed on European elder was similar to that reported by VanderHeyden et al. (2000) during field studies in Switzerland. Such symptoms are not reliable tools when assessing ozone-induced symptoms, since they could be caused by biotic and abiotic factors other than ozone (Orendovici et al. 2003). Species that routinely exhibit non-specific symptoms in response to ozone may not be useful bioindicators. However, induction of such symptoms indicates that ozone may be causing adverse symptoms on vegetation that have not previously been recognized as being induced by ozone. Many plant species in this study were selected for their potentially high sensitivity to ozone (US DOI 2003). Thus, even the “insensitive” species as defined by this study may be symptomatic in the field, since they often developed symptoms following exposure to 60 ppb ozone, below the proposed national ambient air quality standard. However, extrapolation of these CSTR results to the field must be done carefully, since CSTR/greenhouse conditions may influence ozone response and are not representative of natural environmental conditions. Additional studies are needed wherein potential bioindicator species are exposed to ozone in the field under more realistic conditions, such as in open-top chambers. Nevertheless, across much 2008 L.J. Kline, D.D. Davis, J.M. Skelly, J.E. Savage, and J. Ferdinand 65 of the US, phytotoxic levels of ozone occur during each growing season, and ozone-induced injury to native vegetation in these areas will likely continue in future years. Sensitive bioindicators are useful tools to confirm the occurrence of ozone injury in many parts of the country, including areas previously thought to be “pristine.” Such findings may have a positive impact on establishing more stringent air-quality standards to protect vegetation and the environment, ultimately resulting in cleaner air. Acknowledgments The authors acknowledge financial support from the USDA Forest Service and the University of Massachusetts, and technical assistance from T. Orendovici-Best. The authors also thank the USDA Forest Service and their cooperators for supplying plant material. Literature Cited Chappelka, A., H. Neufeld, A. Davison, G. Somers, and J. Renfro. 2003. Ozone injury on cutleaf coneflower (Rudbeckia laciniata) and crown-beard (Verbesina occidentalis) in Great Smoky Mountains National Park. Environmental Pollution 125:53–59. Coulston, J.W., G.C. Smith, and W.D. Smith. 2003. 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