¹Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, USA
²Department of Plant Science, The Pennsylvania State University, USA
³Department of Environmental Microbiology, The Pennsylvania State University, USA

*Corresponding author: Davis DD, Department of Environmental Microbiology, The Pennsylvania State University,University Park, PA, USA, Email: ddd2@psu.edu

Copyright: © Mercado ME, et al. 2021. 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: 11/03/2021; Published: 15/03/2021

Tropospheric ozone (O₃) is considered to be the most important phytotoxic air pollutant across many parts of the USA and continues to be of major concern in natural ecosystems worldwide. Milkweed species (Asclepias spp) are among the most O₃-sensitive plants that occur in natural ecosystems, as well as a weed in agricultural systems. Seven milkweed species were exposed to O₃ during 2020 within controlled-environment chambers in a greenhouse. Arizona, bloodflower (syn. ornamental), and common milkweed developed the greatest O₃-induced leaf injury (“stipple”) and were considered to be very sensitive to O₃. The response of purple milkweed was variable. Slimleaf milkweed exhibited low levels of leaf stipple and was considered tolerant. Horsetail and pineneedle Milkweed were considered resistant to O₃. Results were entered into a master table, which now lists the relative O₃-sensitivity of 27 of 76 (36%) milkweed species common in the USA.

Tropospheric ozone (O₃) is considered to be the most important phytotoxic air pollutant in many parts of the United States (USA) and continues to be of major concern in natural ecosystems worldwide [1,2]. O₃ is a secondary air pollutant formed when oxides of nitrogen (NOx) and volatile organic compounds (VOCs) react in the presence of sunlight. Tropospheric concentrations vary seasonally, generally being greater in summer and minimal in the winter across the USA [1,3]. O₃ levels also vary daily, typically following diurnal patterns with concentrations low in early morning, increasing in late afternoon, and minimal at night [1,3-5].

On 1 October 2015, the US Environmental Protection Agency (EPA) strengthened the USA National Ambient Air Quality Standard (NAAQS) for O₃, reducing the level of the standard from 75 to 70 ppb [6]. This more stringent NAAQS is based on the 4th highest daily maximum of an 8-hr average O₃ concentration across 3 consecutive years. The reduction makes the new O₃ NAAQS more stringent to help protect public health and welfare in the USA, including the health of O₃-sensitive plants such as milkweed.

Milkweeds (Asclepias spp) are classified within the family Asclepiadaceae. Agrawal [7] estimated that milkweed species in the Americas numbered ~130. Woodson [8,9] listed ~105 indigenous milkweed species in North America alone. The US Department of Agriculture (USDA) now lists 76 milkweed species found growing in the USA [10]. Scientific and common names of those 76 species are used in this paper (Table 1).

Milkweed species reported to be sensitive to O₃ include common [11], bloodflower (syn. tropical) [12], poke (syn. tall) [13,14], and swamp milkweed [15]. However, most milkweed species in the USA have not been evaluated for O₃ -sensitivity. Prior to this paper, only 22 of the 76 (29%) milkweed species listed in (Table 1) had been evaluated for O₃-sensitivity [16].

The objective of this study was to evaluate the O₃-sensitivity of 7 milkweed species and to determine or confirm their relative sensitivity or resistance by updating a master list previously compiled by the authors [1].

O₃ exposures were conducted within 16 continuous stirred tank reactor (CSTR) chambers [18] in a greenhouse on the University Park campus of The Pennsylvania State University (40°48’20”N, 77°51’08”W). O₃ concentrations within each chamber were monitored using a photometric OO₃ analyzer (Model 49,Thermo Environmental Corp., Franklin, MA). O₃ treatments were administered, monitored, and controlled via a computerized system during exposures, which also monitored and displayed temperature and relative humidity in each chamber [19].

Plant Culture: Plant culture followed protocols of Myers et al. [20]. Arizona, bloodflower, common, horsetail, pineneedle, purple, and slimleaf milkweed were selected based on availability of seeds.Common milkweed was included as a standard, since this species is known to be highly sensitive to O₃ [11,12]. During August 2020, milkweed seeds were placed in 2-L pots containing moist potting mix with ~1g slow-release fertilizer (15N-4P-10K) and watered as needed. Resultant seedlings were grown in a greenhouse containing air that passed through activated-charcoal filters, reducing the ambient greenhouse O₃ to ~5 ppb.

Plant exposure to O₃: O₃ exposures began in mid-September 2020. TwoO₃ treatments were used (elevated vs control). Our target value for the elevated O₃ concentration was 75 ppb since various milkweed species had exhibited classic O₃-induced leaf stipple following exposure to this concentration during previous studies conducted within these chambers [17,20]. However, the mean O₃ concentration within the exposure chambers was better controlled at ~77 ppb, which was thus used in this study. The average OO₃ concentrations in the control chambers were ~5 ppb, which was the lowest concentration that could be attained using the charcoal-filter system that scrubbed the air within both the greenhouse and control chambers. Fourteen chambers were maintained at ~77 ppb O₃ and 2 control chambers were kept at~5 ppb O₃. We had planned to put 2 plants/species in each chamber. However, due to reduced seed germination and decreased seedling survival, the number of plants/ species varied from 0-2/chamber.

Milkweed plants were exposed for 7 weeks, 6 days/week, 8h/ day (0900-1700 hours), in a square-wave design. During exposures, mean air temperature was ~25oC and relative humidity was ~44%. Once a week, plants were watered, redistributed randomly within each chamber, and O₃-induced foliar stipple evaluated. Following each weekly 6-day exposure, plants remained within the same CSTR exposure chambers with the chamber doors open until the next exposure began.

Leaf injury (stipple) evaluation: O3-induced foliar stipple on the upper leaf surface/plant was visually estimated [17,20] within each chamber using a modified Horsfall-Barratt scale [21]. Symptom rating classes in this scale ranged from 0 to 5, where 0 = no injury, 1 = 1-6% injury, 2 = 7-25%, 3 = 26-50%, 4 = 51-75%, and 5 = 76- 100% injury. To provide a single value for statistical analyses, the midpoint of each class was calculated as the mean of the minimum and maximum percentage.

Analyses of O₃ response data were conducted using a repeated measures analysis in a randomized complete block design as described in SAS PROC GLIMMIX [22]. Chamber was the block; random effects included chamber and plants nested in chamber. Since the (week x species) interaction was significant (P = 0.0001), the Least Squares means (LS means) for species within week were statistically compared using the SLICE DIFF Option at P = 0.05. LS means can be defined as a linear combination (sum) of the estimated effects (means) from the linear model. When the data contain no missing values, the LS means and arithmetic means are identical. When missing values occur, the two will differ and the LS means are preferred, because they reflect the model that was used to fit the data. When the data are extremely unbalanced, as in this study, the estimated LS means may differ greatly from the arithmetic means, because there are too few observations to make appropriate adjustments. Since the standard errors from the repeated measures are correct, the multiple comparisons for LS means in (Table 1) are correct, and the arithmetic means are presented in (Figure 1).

Results based on Least Squares means: Control plants did not exhibit O₃-induced foliar injury (data not shown). Foliar injury was not observed on any species following 1 week of exposure to ~77 ppb O₃. At 2 weeks, light stipple was noted on Arizona milkweed and extremely light stipple on horsetail milkweed (Table 2). Following 3 weeks of exposure, more severe O3 injury was observed on Arizona, bloodflower, common, purple, and slimleaf milkweed. At 3-4 weeks, stipple was noted on all species except horsetail and pineneedle milkweed. Following 5-7 weeks exposure, stipple occurred on all species except horsetail milkweed. Slimleaf milkweeds in both the control and exposure chambers began to die from unknown causes at 5 weeks and all were dead by 7 weeks.

Although PROC GLIMMIX estimated the LS means for slimleaf milkweed on week 7, the value is questionable since there were no living slimleaf milkweed plants at this time (Table 2). After the 7th and final rating, O₃ injury based on LS mean mid-point ratings of the various milkweed species, from greatest to least, was: [Arizona (36.9%)>common (29.8%)=bloodflower (27.5%)=purple (25.2%)>pineneedle (0.1%)=horsetail (0.0%), and slimleaf (undetermined due to 100% mortality)] (Table 2).

Results based on mid-point arithmetic means: Results based on mid-point arithmetic means: Injury ratings for Arizona milkweed were greater than all other species as early as week 2, and increased nearly linearly through the study (FIgure 1). The injury ratings for common and blood flower milkweeds increased slowly for the first 5 weeks before increasing more rapidly in the final 2 weeks

Seven milkweed species were exposed to O₃ during 2020. As shown in (Table 1), Arizona, bloodflower, and common milkweeds developed the greatest O₃-induced leaf injury (“stipple”) and were classified as “Very Sensitive”. The response of purple milkweed was variable between (Table 2) (Figure 1) and Figure 1 and was classified as “Tolerant”, but will be exposed again in the next study using more plants. Slimleaf milkweed exhibited very low levels of leaf stipple and was considered “Tolerant”. Horsetail and pineneedle milkweed exhibited either a trace or no symptoms and were classified as “Resistant”. Slimleaf milkweeds in both the control and exposure chambers began to die from unknown cause(s) at 5 weeks (Figure 1). However, there was a trace of accumulated stipple on living slimleaf milkweeds after 4 weeks of exposure, so the species was classified as slightly “Sensitive” to O₃ (Table 1).

Milkweed species confirmed to be sensitive to O₃ based on previous reports include common[11] and bloodflower [12]. Common milkweed is a widely used bioindicator of phytotoxic levels of tropospheric O₃ across the USA [11,17]. Bloodflower milkweed is likewise very sensitive to O₃, but may not be acceptable to use as a bio indicator in some regions of the USA since it may contain a protozoan that is pathogenic to some butterflies, including the popular monarch butterfly [7,17].

An important finding in this study, is that we reported for the first time that Arizona milkweed is very sensitive to O₃. The species was the most sensitive of the 7 milkweed species from week 2 to week 7 (Figure 1). Arizona milkweed is rare in the USA, being found only in southern Arizona (and into northern Mexico), where it grows in arroyos, canyons, along streambeds, and on slopes (http://southwestdesertflora.com/WebsiteFolders/All_Species/ Asclepiadaceae/Asclepias%20angustifolia,%20Arizona%20).

The high O₃-sensitivity of Arizona milkweed to O₃ is significant, since it was injured by ~77 ppb O₃, a concentration that occurs in the USA troposphere. In fact, in 2020 the US EPA reported measuring ambient O₃ concentrations as high as 90 ppb in Denver, CO, likely a result of O_{311,7]. Arizona milkweed may be an excellent, new bio indicator to detect phytotoxic levels of O3 in the arid Southwest, if its high sensitivity to O3 is maintained in dry regions. In dry areas, droughts can induce stomatal closure, which in turn reduce gas uptake, including uptake of O3 and subsequent foliar injury [22], confounding O3 injury surveys.}

Findings from this study added 5 new milkweed species to (Table 1) and confirmed the ratings of 2 others. Milkweed species, such as Arizona milkweed, which are classified as “Very Sensitive” to O₃, should be considered for possible use as bio indicators to detect phytotoxic concentrations of ambient O₃. (Table 1) now lists the relative O₃-sensitivity of 27 of 76 (36%) milkweed species. We present this updated list in hopes that other air pollution researchers will expand and improve the ratings by evaluating the O₃ sensitivity of additional milkweed species during field surveys used in combination with controlled O₃ exposures [17].

We acknowledge funding from the Pennsylvania Department of Environmental Protection, Bureau of Air Quality, Harrisburg, PA, USA and The Pennsylvania State University, University Park, PA, USA. This work was also supported by the USDA National Institute of Food and Agriculture (NIFA) and Federal Appropriations under Project PEN04564, Accession number 1002837.