¹Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, USA
²Department of Plant Science, The Pennsylvania State University, USA

^*Corresponding author: Davis DD, Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, PA, USA; E-mail: ddd2@psu.edu

Copyright: © Davis DD, et al. 2020. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Article Information: Submission: 11/11/2020; Accepted: 14/12/2020; Published: 17/12/2020

During 1991 to 2016, we evaluated the annual incidence (percentage) of hybrid poplar trees exhibiting ozone (O₃)-induced leaf injury (stipple) within 15 planted field-study plots in southwestern Pennsylvania, USA. Incidence of stippled poplars decreased from a mean of 3.84% in 1991 to 0.00% in 2016. Mean annual 8-hr ambient O₃ concentrations, downloaded from a U.S. EPA monitoring site located ca 60 km west (upwind) from the closest study plot, decreased from 96 ppb in 1991 to 67 ppb in 2016. Regression analysis revealed that during the 26-year period, the annual incidence of poplar trees exhibiting ozoneinduced leaf stipple was significantly and linearly related to the mean annual O₃ concentration.

Air pollution; Ozone; O₃; Hybrid poplar trees; Leaf stipple

Tropospheric ozone (O₃) is the most important phytotoxic (plant damaging) air pollutant in the USA [1,2]. Ozone is a secondary air pollutant formed by photochemical reactions between Oxides of Nitrogen (NO_x) and Volatile Organic Compounds (VOCs), two precursors to O₃ formation [1,3,4]. Rural Pennsylvania (PA, USA) contains phytotoxic levels of ambient O₃ [2,5,6], especially in areas that experience long-range transport of NO_x and VOCs within slowmoving, stagnant, high-pressure systems [7]. Predominantly eastbound weather systems traverse the industrial Ohio River Valley on the western border of PA and the Pittsburgh industrial areas, collecting precursors to O₃ formation before entering our study area in southwestern PA. Elevated levels of ambient O₃ in the study area usually begin in May, increase through the summer, and often peak in August. Diurnal patterns often reflect greater ozone concentrations in mid-afternoon when O₃-forming reactions are driven by incoming solar radiation, and lowest O₃ levels generally occur after sunset, when O₃ is continuously converted back to NNO₂ and O₂ in the presence of NO [1,4,8].

The importance of O₃ as a significant phytotoxic air pollutant in the USA was first reported in the USA by Richards et al. in 1958 [9]. They had previously observed O₃-induced leaf injury on wild and cultivated grapes (Vitis spp.) as early as 1954 in the Los Angeles, CA air-basin. They described O₃-induced leaf symptoms as “stipple,” which were comprised of groups of dark brown-to-black, leaf palisade-mesophyll cells visible through the hyaline adaxial epidermis and overlying cuticle. Stipple on many species of broadleaved plants was later shown to be indicative of phytotoxic concentrations of ambient O₃, which could be used to identify O₃-sensitive bioindicator plants [2,10,11]. Early researchers observed that the incidence and severity of O₃-induced stipple varied among individuals within an open-pollinated species depended on their parentage [12]. However, variability could be minimized by using asexual, clonally propagated (e.g., from cuttings) bio-indicator plants [Davis, personal observations].

On 1 October 2015, the U.S. Environmental Protection Agency (EPA) enacted a more stringent National Ambient Air Quality Standard (NAAQS) for ground-level O₃ [13]. The new standard was based on the 4^th highest daily maximum O₃ concentration, averaged across 3 consecutive years for an averaging time of 8 hrs [14]. If that value exceeded 70 ppb, the relevant area was considered to be in nonattainment of the standard. Southwestern PA, including our study area, was in attainment of the new NAAQS for 2016 to 2018, the 3 most recent years for which EPA O₃ data were available at time of this writing (https://www3.epa.gov/airquality/greenbook/ map8hr_2015.html, accessed 24 September 2019).

The general objective of this study was to evaluate temporal and spatial patterns of incidence (percentage) of planted hybrid poplar trees that exhibited O₃-induced stipple during 1991 to 2016 on study plots in southwestern PA. The specific objective was to determine if annual ambient ozone levels were statistically related to the annual incidence of ozone-induced poplar stipple during the 26-year period.

Material and Methods Annual mean 8-hr ambient O₃ concentrations (ppb) for 1991 to 2016 were downloaded from EPA O₃ monitoring site #42-003-00008 (40°27’55”N, 79°57’38”W), located ~40 km westward and upwind from the study area.

The study area is located in a rural portion of the Appalachian Plateau Physiographic Province in southwestern PA (Figure 1), as described by McClenahen et al. [15]. The area has a rolling dissected topography with ridges oriented in a southwest-northeast direction and elevations ranging from ~400 - 800 m. The climate is continental, average annual precipitation is ~100 cm, and prevailing winds are from the west. Although the study area is mainly rural, it contains scattered industries and is located downwind from the Pittsburgh and the Ohio River Valley industrial areas, which are major sources of precursors to O₃ formation. Johnstown, which has a 100-year history of air pollutant emissions from steel mills and coke works [16], lies slightly downwind from the southeastern edge of the study area and may be a source of O₃ and precursors during winds from the southeast.

In 1964, Hutnik et al. established a network of ecological plots in former agricultural fields within the study area to study effects of sulfur and SO₂ on coniferous plants [17]. However, during the late 1960s and early 1970s, air pollution effects research in PA shifted emphasis from primary pollutants such as SO₂, often localized near point sources, to the widespread secondary air pollutant O₃ [Davis, personal observations]. Therefore during 1972 to 1976, Hutnik and colleagues established 20 rooted cuttings of an O₃-sensitive hybrid poplar clone (Populus maximowizii x trichocarpa USDA Forest Service Northeastern Forest Experiment Station Clone NE 388) on each of 22 50x50 m plots as bioindicators of phytotoxic levels of ambient O₃ [18]. During 1981 to 1990, the years with the most complete datasets at the time, the incidence of O₃ injury on the poplar trees was significantly (P = 0.05) correlated with the number of days having hourly O₃ concentrations > 40 ppb [18]. By 1991 several original plots had been lost due to change of land ownership and various land-use changes. Therefore, we selected 15 of the remaining intact study plots for use in this 26-year study beginning in 1991 (Figure 1). Large poplars were removed to minimize plot shading and were replaced with new rooted cuttings of the same clone; data were not taken on the new cuttings until 1 year after establishment.

Beginning in 1991, each poplar tree on each plot was evaluated for presence or absence of visible O₃-induced leaf stipple (Figure 2), which was shown to be caused by O₃ in controlled studies [18]. Symptom evaluations were conducted in early to mid-August, usually the time of greatest O₃ injury on native broadleaved plants in the study area [5,7,19]. Stipple on each tree was classified as present or absent, and incidence (percentage) of poplar trees exhibiting stipple/ plot/year calculated until the study was terminated in 2016.

Polynomial regression was performed with SAS’s PROC REG (SAS for Linear Models, 4^th Ed.) to evaluate the relationship between O₃-induced leaf injury and mean annual ambient ozone concentration. However, only the linear term was significant and residual plots indicated the quadratic term did not improve the model. Therefore, linear models were fit in order to model changes in O₃ ambient concentrations and leaf injury over the 26-year study period.

Regarding general temporal patterns, ambient O₃ data downloaded from the EPA monitoring site revealed that the annual mean 8-hr O₃ concentration decreased from 96 ppb in 1991 to 67 ppb in 2016, a 30% decrease (Figure 3). Mean annual percentage of hybrid poplar trees exhibiting O₃-induced stipple decreased from 3.84% in 1991 to 0.00% in 2016, the last year of the study (Table 1). For the entire 26-year period, the mean annual ambient O₃ concentrations and mean annual percentage of O₃ injury on poplars were significantly and positively correlated in a linear manner (n = 26, R² = 0.39, P = 0.001). The complete lack of leaf stipple in 2016 was possibly due to ambient O₃ levels being below a general level needed to induce O₃ injury, or to environmental conditions that were not conducive to O₃ uptake and subsequent development of foliar injury.

The temporal pattern of O₃ injury incidence peaked in 1998. However, this peak did not appear to be strongly related to a peak in ambient O₃ concentration in (Figure 3). Therefore, the 1998 peak may have been influenced by environmental factors such as ideal soil moisture levels that were conducive to maximum O₃ uptake that occurred concurrently with environmental conditions conducive for maximum foliar injury. Foliar O₃ injury precipitously decreased in 1999, but the decline did not appear related to a similar decline level of ambient O₃ (Figure 3).

However, injury-threshold values are only general estimates [20]. Onset of O₃-induced foliar injury can be influenced by multiple interacting factors, including genetic (inherent species O₃ sensitivity or tolerance), physiologic (detoxification capacity, defense mechanisms), physiographic (elevation), environmental (drought), seasonal, and others. These confounding factors and their interactions make it difficult to determine meaningful O₃ injury threshold values [20].

Relatedly, Smith et al. reported a similar temporal trend in O₃ injury in a large study involving the incidence of O₃-induced stipple on milkweed bioindicator plants (n = 65,448) in the northeastern USA from 1994 to 2010 [21]. Similar to our findings, they also reported a peak of O₃ injury in 1998 followed by low stipple levels in 1999 that they related to a widespread drought that occurred in 1999 within northeastern USA. This drought also encompassed our study area, as verified by the Palmer Drought Severity Index for southwestern PA (Figure 4) [22]. Figure 4 also illustrates that two severe drought periods occurred in southwestern PA during the period from 1999 to 2003. During this time interval, the drought likely caused soil moisture stress, resulting in closed stomata and reduced O₃ uptake, which in turn could reduce O₃ injury on sensitive bioindicators [21,23]. The low level of O₃-injury on poplars then persisted through 2016, except for small peaks of injury in 2001 and 2012 (Table 1 and Figure 3). These small peaks may have been partially related to subtle changes in O₃ concentrations in combination with subtle changes in soil moisture (Figure 3). Interestingly, the similar temporal patterns in O₃ injury between our study involving poplars and that of Smith et al. involving milkweeds, suggest similar environmental factors for the two species that influenced the percentage of O₃ injury [21]. Such common factors may influence O₃ injury in other various plant species [21,23].

Regarding spatial patterns, during 1991 - 2003 and 2010 - 2013, mean O₃-induced leaf symptoms on poplars appeared to be slightly greater on plots in the west-southwest (generally upwind) portion of the study area and becoming less severe with distance downwind (to the northeast) (Figure 1 and Table 1). This upwind-todownwind decreasing pattern is likely attributable, at least partially, to O₃ precursors (NO_x and VOCs) originating from industries in southwestern PA and the Ohio River Valley. However, during the later time period from 2014 to 2016, O₃ injury was minimal and therefore spatial patterns could not be discerned (Table 1 and Figure 3). In 2016. for the first time since the study began, O₃ injury was not observed on ozone-sensitive hybrid poplar.

The incidence of O₃-induced stipple on hybrid poplar trees on field-study plots in southwestern PA decreased from a mean of 3.4% in 1991 to 0.00% in 2016. Mean annual 8-hr ambient O₃ concentrations, downloaded from a nearby U.S. EPA monitoring site likewise decreased during the 26-year study. During 1991 to 2016, the mean annual incidence of poplars exhibiting stipple induced by ambient ozone was significantly (P = 0.001) correlated with the mean annual ozone concentration.

This work was supported by the USDA National Institute of Food and Agriculture (NIFA) and Federal Appropriations under Project PEN04564, Accession number 1002837; and by the owners of the Keystone and Conemaugh electric generating stations.