2009 NORTHEASTERN NATURALIST 16(2):307–313 Variation in Ozone Sensitivity Within Indian Hemp and Common Milkweed Selections from the Midwest Lee J. Kline1, Donald D. Davis1,* , John M. Skelly1, and Dennis R. Decoteau1 Abstract - Sixteen selections of Apocynum cannabinum (Indian Hemp) and nine of Asclepias syriaca (Common Milkweed) from midwestern USA were exposed to 40 or 80 ppb ozone under controlled conditions within greenhouse continuously stirred tank reactor (CSTR) chambers to evaluate their relative ozone sensitivity. The incidence and severity of ozone-induced symptoms on both species were directly related to ozone concentration and duration of exposure. The most common foliar symptom was classic, dark, adaxial stipple, similar to symptoms ascribed to ambient ozone in the field. Indian Hemp was more sensitive to ozone than Common Milkweed. Both species exhibited considerable intraspecific variation in ozone sensitivity. Variability in the data was too great to assign definitive ozone-sensitivity ratings within geographic regions from which seed was selected. However, two locations were identified as possible collection sites for ozone-sensitive selections of both species: Wabaunsee County, KS and Plattsmouth, NE for Indian Hemp; and Cloud County, KS and Swan Creek Lake Wildlife Area, NE for Common Milkweed. Plants derived from seed from these locations may serve as ozone-sensitive bioindicators. Introduction Ground-level, tropospheric ozone is the most significant air pollutant affecting native vegetation in the USA (US EPA 1996). Ozone concentrations high enough to cause visible symptoms on plants occur annually throughout rural portions of the Northeast (Comrie 1994, Coulston et al. 2003), including wildlife refuges (Davis 2007a,b; Davis and Orendovici 2006) and remote forested areas (Manning et al. 1996, Orendovici et al. 2007, Simini et al. 1992). Since the discovery that leaf stipple of grape was caused by ozone (Richards et al. 1958), adaxial stipple has been the classic symptom used to evaluate ozone injury on broadleaved bioindicators in the field (Skelly 2000, Skelly et al. 1987). Apocynum cannabinum L. (Indian Hemp) and Asclepias syriaca L. (Common Milkweed) have been listed as ozone-sensitive plants (bioindicators) for use in field surveys (US DOI 2003). However, the intraspecific variation in ozone sensitivity has not been reported for mid-western selections of these species. Potential bioindicators should be collected from the geographic region of interest, location of seed source identified, and resultant seedlings exposed to ozone under controlled conditions, such as in continuously stirred tank reactor (CSTR) chambers (Heck et al. 1975) to confirm ozone sensitivity and to describe ozone-induced symptoms. 1Department of Plant Pathology and Penn State Institutes of Energy and the Environment, The Pennsylvania State University, University Park, PA 16802. *Corresponding author - ddd2@psu.edu. 308 Northeastern Naturalist Vol. 16, No. 2 This paper is the third in which we report exposure of potential bioindicators to ozone within the same greenhouse/CSTR chambers and under similar environmental conditions (Kline et al. 2008, Orendovici et al. 2003). The objectives of this study were to evaluate the relative ozone sensitivity of two plant species grown from seed collected from several locations in mid-western USA, to describe foliar symptoms induced by ozone under controlled conditions, and to evaluate intraspecific variability in ozone sensitivity. Methods Seed from 16 selections of Indian Hemp and nine selections of Common Milkweed were collected at various locations within the Midwest by USDA Forest Service personnel and shipped to Penn State (Table 1). Seeds of Indian Hemp were collected in Illinois, Kansas, Nebraska, and Wisconsin. Common Milkweed seed was sent from Kansas and Nebraska. Seeds were placed in germination trays in a greenhouse; resultant seedlings were transplanted into 1.5-L pots containing Metromix 500® potting Table 1. Response of 16 selections of Indian Hemp and 9 selections of Common Milkweed exposed to 80 ppb ozone during 14 June–28 July 2005. Collection locationA No. plants exposed Average injuryB Indian Hemp Wabaunsee County, KS 12 25.22 a Plattsmouth, NE 8 22.78 ab Carlyle Lake State Park, IL 12 14.31 bc Fond du Lac County, WI 12 13.86 bc Clinton Lake, IL 12 11.60 cd Lake Farm County Park, WI 12 11.44 cd Swan Creek Lake Wildlife Area, NE 12 10.29 cd Mathissen State Park, IL 12 9.86 cd Plattsmouth, NE 8 9.10 cd Caryle Lake State Park, IL 11 8.30 cd Plattsmouth, NE 8 7.26 cd Shabbona Lake State Park, IL 12 7.13 cd Rend Lake, IL 12 5.93 cd Rend Lake, IL 12 5.42 cd Plattsmouth, NE 12 1.75 d Moraine View State Park, IL 12 1.24 d Common Milkweed Cloud County, KS 12 11.14 a Swan Creek Lake Wildlife Area, NE 4 10.27 a Pottawatomie County, KS 12 7.98 ab Plattsmouth, NE 12 2.66 bc Elm Creek, NE 8 2.21 bc Elm Creek, NE 12 1.82 bc Elm Creek, NE 12 0.73 c Kearney, NE 12 0.56 c Scotts Bluff, NE 6 0.00 c ASeed from locations with same name were collected at slightly different sites. BMeans followed by the same letter are not significantly different (P = 0.05) according to Duncan’s new multiple range test. 2009 L.J. Kline, J.M. Skelly, D.R. Decoteau, and D.D. Davis 309 soil (Scotts-Sierra Horticultural Products Co., Marysville, OH) supplemented with 5 g Osmocote® (15N:15P:15K) controlled-release fertilizer (Scotts-Sierra Horticultural Products Co., Marysville, OH). Seedlings were maintained on benches in a greenhouse receiving charcoal-filtered air (less than 8 ppb ozone daily hourly average) until placement into CSTR chambers for ozone treatments. Ozone exposures were conducted within 12 CSTR chambers, beginning on 14 June and ending on 28 July 2005. Six replications (chambers) were used for each of two concentrations of ozone. The number of individual plants/species/chamber varied slightly, depending upon plant condition, but usually involved two individual plants/genotype/chamber. Plants were exposed to 40 or 80 ppb ozone in a square-wave exposure for 7 hr/day, 5 days/ week (Monday–Friday). The level of 40 ppb was intended to approximate background ozone, and 80 ppb was near the secondary US National Ambient Air Quality Standard for ozone (US EPA 1996). Exposures began at 0900 hr and ended at 1600 hr daily. During non-exposure hours, all plants remained in the CSTR chambers with the chamber doors open and were exposed to the charcoal-filtered air and greenhouse environmental conditions. During overcast weather, each CSTR chamber received artificial supplemental lighting from an external overhead 1000-watt Lumalux lamp (GTE Products Corp., Sylvania Lighting Center, Danvers, MA) having a spectral distribution of 350–700 nm with peaks at 550 and 650 nm. Non-exposed plants were maintained on greenhouse benches in charcoal-filtered air. Ozone concentrations, light (photosynthetically active radiation, PAR), relative humidity, and temperature were monitored within each chamber for 1.5 min at 12-min intervals during each exposure. 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), calibrated at the beginning of the experiment. Ozone and environmental data were input to a data logger connected to a PC computer. Routine quality-control measures were maintained on monitoring equipment throughout the study. Each plant was rated as to amount of foliage injured (AMT) and the severity of the injured foliage (SEV). The assessments estimated the percentage injury to the plants and were assigned nominal values that reflect five broad classes of injury as follows: 0 = no injury, 1 = 1–6% injury, 2 = 7–25% injury, 3 = 26–50% injury, 4 = 51–75% injury, and 5 = 76–100% injury. These data were used to calculate an overall injury 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 injury 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. Experimental design was a split plot with 310 Northeastern Naturalist Vol. 16, No. 2 ozone treatments as the main plot and species as the subplot. A general linear model (GLM) was performed on the percentage data (%INJs), and significant (P = 0.05) differences between the two species and two ozone levels were examined using Duncan’s multiple range test (Minitab 2003). Statistical evaluations between and within species were conducted only on data from the 80 ppb ozone treatment, since few visible symptoms were induced by 40 ppb ozone. Results Ozone and environmental monitoring Mean ozone concentrations achieved for the target concentrations of 40 and 80 ppb during exposures were 37.5 and 73.0 ppb, respectively. The average temperature monitored within all exposure chambers was 31 °C, mean relative humidity was 75%, and average light (PAR) was 297.2 μmol m-2 s-1. The light level includes supplementation with artificial lights during periods of cloudy, overcast weather. Description of foliar symptoms Plants maintained on greenhouse benches in charcoal-filtered air (<8 ppb ozone daily hourly average) did not exhibit ozone-induced symptoms. Both Indian Hemp and Common Milkweed developed classic adaxial stipple, as well as premature defoliation, in response to ozone. Stipple was usually lightcolored during early weeks of exposure, but became darker with cumulative exposure. Premature defoliation occurred in the later stages of exposure. Sensitivity to ozone Both species exhibited statistically similar (P = 0.05) trace amounts of symptoms following exposure to 40 ppb ozone. Common Milkweed exhibited only 0.37% INJs at the lower concentration of ozone, whereas Indian Hemp was uninjured. In contrast, exposure to 80 ppb ozone elicited readily visible foliar symptoms on both species. Indian Hemp developed signifi- cantly more severe symptoms than Common Milkweed at the higher ozone level, with a mean rating of 10.17% INJs, whereas Common Milkweed had 3.97% INJs following exposure to 80 ppb ozone. Intraspecific response to ozone Since the 40 ppb ozone resulted in few foliar symptoms, response data from the 80 ppb exposures were used to rate sensitivity of the 16 Indian Hemp and nine Common Milkweed selections (Table 1). The two most sensitive selections of Indian Hemp were from Wabaunsee County, KS (25.22% INJs) and Plattsmouth, NE (22.78% INJs). These two selections were generally more sensitive than the remaining selections. The Carlyle Lake State Park, IL selection (14.31% INJs) ranked next in sensitivity, and the Fond du Lac County, WI, selection (13.86% INJs) ranked fourth. Mean % INJs on the remaining selections were statistically similar, including the least sensitive selections from Plattsmouth, NE (1.75 INJs) and Moraine View State Park, IL (1.24% INJs). 2009 L.J. Kline, J.M. Skelly, D.R. Decoteau, and D.D. Davis 311 Differences in sensitivity were also evident among the Common Milkweed selections from nine different sites (Table 1). Selections from Cloud County, KS (11.14 INJs), Swan Creek Lake Wildlife Area, NE (10.27 INJs), and Pottawatomie, KS (7.98% INJs) were most sensitive. Ozone injury values for plants from these three locations were greater than those from the remaining six selections, which ranged from 0.00 to 2.66% INJs, and were relatively insensitive to ozone. Discussion Several selections of Indian Hemp and Common Milkweed have potential as useful ozone bioindicators in the midwestern USA. All sensitive selections of both species exhibited classic adaxial leaf surface stipple (Richards et al. 1958) as the predominant symptom following ozone exposure. Ozone-induced stipple, as observed in these CSTR studies, was generally similar to foliar symptoms observed in the field and attributed to ambient ozone. Broadleaved species that produced classic stipple may serve as useful bioindicators when conducting field surveys to evaluate ozone injury. However, ozone may induce symptoms other than stipple, including occasional foliar reddening, chlorosis, premature defoliation, bronzing, and flecking. Such symptoms are not reliable tools when assessing ozone injury, since they could be caused by factors other than ozone (Orendovici et al. 2003). Such non-specific symptoms in response to ozone should not be utilized in field surveys. There was considerable intraspecific variability in ozone sensitivity among individual plants within the same species, likely due to the interaction of intraspecific genetic variations in ozone sensitivity, microsite differences in environmental factors, and levels of ambient ozone (Bennett et al. 2006; Berrang et al. 1989, 1991; Steiner and Davis 1978). There was a high degree of variability in ozone sensitivity expressed by the 16 selections of Indian Hemp and the nine selections of Common Milkweed. These significant intraspecific differences in response to ozone may represent genetic differences in ozone sensitivity among geographically scattered populations of the same species. Such differences may have arisen randomly, or were due to selection pressure from spatially different levels of ozone, which, over time, selected more ozone-tolerant plants in areas of greatest ambient ozone (Berrang et al. 1989, 1991). However, the dataset was too small to conduct robust spatial statistical analyses. Future studies using greater numbers of plants from geographic regions having different ambient ozone regimes are needed to verify these speculations. Additional research is also needed regarding development, confirmation, and symptom description for ozone-sensitive bioindicators. 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