- Email: [email protected]
Field assessment of a snap bean ozone bioindicator system under elevated ozone and carbon dioxide in a free air system
Environmental Pollution 166 (2012) 167e171
Contents lists available at SciVerse ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Field assessment of a snap bean ozone bioindicator system under elevated ozone and carbon dioxide in a free air system Kent O. Burkey a, *, Fitzgerald L. Booker a, Elizabeth A. Ainsworth b, Randall L. Nelson c a
USDA-ARS, Plant Science Research Unit and Department of Crop Science, North Carolina State University, 3127 Ligon Street, Raleigh, NC 27607, USA USDA-ARS, Global Change and Photosynthesis Research Unit and Department of Plant Biology, University of Illinois, Urbana-Champaign, 1201 West Gregory, Urbana, IL 61801, USA c USDA-ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit and Department of Crop Sciences, University of Illinois, Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2011 Received in revised form 5 March 2012 Accepted 14 March 2012
Ozone-sensitive (S156) and -tolerant (R123 and R331) genotypes of snap bean (Phaseolus vulgaris L.) were tested as a plant bioindicator system for detecting O3 effects at current and projected future levels of tropospheric O3 and atmospheric CO2 under field conditions. Plants were treated with ambient air, 1.4 ambient O3 and 550 ppm CO2 separately and in combination using Free Air Concentration Enrichment technology. Under ambient O3 concentrations pod yields were not significantly different among genotypes. Elevated O3 reduced pod yield for S156 (63%) but did not significantly affect yields for R123 and R331. Elevated CO2 at 550 ppm alone did not have a significant impact on yield for any genotype. Amelioration of the O3 effect occurred in the O3 þ CO2 treatment. Ratios of sensitive to tolerant genotype pod yields were identified as a useful measurement for assessing O3 impacts with potential applications in diverse settings including agricultural fields. Published by Elsevier Ltd.
Keywords: Bioindicator Carbon dioxide Free air concentration enrichment (FACE) Ozone Snap bean
1. Introduction Ozone (O3) is a toxic component of air pollution, and plants are particularly sensitive to this pollutant. Present day atmospheric O3 concentrations are sufficiently high to damage sensitive species of native vegetation and cultivated plants in many regions worldwide (Booker et al., 2009). Crop losses in the United States alone are estimated to be billions of dollars annually with impacts of similar magnitude in other agricultural regions around the world (Van Dingenen et al., 2009). This problem is expected to continue, especially if projected increases in ambient O3 are realized (Dentener et al., 2005). For any given location and growing season, assessing the impact of O3 on vegetation is difficult because plant response is the integration of a complex set of factors that include ambient O3 concentration, environmental conditions that affect leaf gas exchange and thus O3 uptake, and the inherent genetic variability in O3 sensitivity within and between species. Except for situations where O3-induced foliar injury can be documented, O3 impacts are
* Corresponding author. E-mail addresses: [email protected] (K.O. Burkey), Fitz.Booker@ ars.usda.gov (F.L. Booker), [email protected] (E.A. Ainsworth), [email protected] (R.L. Nelson). 0269-7491/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.envpol.2012.03.020
not easily quantified in the absence of a “clean air control”. For many situations, such a control is neither feasible nor practical. However, bioindicator plants provide one approach to circumvent some of these challenges. Genetic variation in plant response to O3 stress can be utilized in the development of plant O3 bioindicator systems. The concept is based on identification of O3-sensitive and -tolerant clones or genotypes that exhibit similar growth and yield under low O3 conditions, but exhibit differential foliar injury or yield losses when grown in environments of increasing O3 concentrations. In this approach, the plants integrate the multiple factors contributing to the O3 response for a given time and location, and the result is reflected in a quantifiable parameter. The first O3 bioindicator system was developed in 1957 using sensitive and resistant varieties of tobacco (Heggestad, 1991). The sensitive cultivar BelW3 exhibits foliar injury in response to ambient O3 concentrations exceeding 40 ppb. It can be desirable, however, to have a measure of O3 effects more quantitative than foliar injury to assess the impact of ambient O3. One such system is based on the differential forage biomass production of O3-sensitive (NC-S) and O3-resistant (NC-R) white clover clones (Heagle et al., 1996). Although the yield potential of the two clones varied between environments, the NCS/NC-R yield ratio was found to be a good indicator of the O3 impact during the growth period being evaluated (Heagle et al., 1995). The clover system has been used in a number of studies as an O3
168
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
Table 1 Summary of seasonal O3 and CO2 exposures. Treatment
CO2 (12 h mean) (ppm)
O3 (8 h mean)a (ppb)
O3 (1 h max)b (ppb)
O3 (AOT40)c (ppm h)
O3 (SUM60)d (ppm h)
Ambient Air O3 CO2 O3 þ CO2
378 378 550 551
43 59 43 59
29e78 32e114 29e78 33e112
5.3 16.3 5.3 16.2
5.3 27.0 5.3 26.7
a b c d
Average hourly O3 concentrations measured between 0900 and 1700 h from May 25, 2006 through August 28, 2006. Range of daily 1-h maximum values measured between 0900 and 1700 h from May 25, 2006 through August 28, 2006. AOT40 is the hourly mean O3 concentration accumulated over a threshold O3 concentration of 40 ppb during daylight hours (Mauzerall and Wang, 2001). SUM60 is the sum of hourly O3 concentrations equal to or greater than 60 ppb during daylight hours (Mauzerall and Wang, 2001).
bioindicator species (Nali et al., 2009). NC-S and NC-R are clones, which must be maintained through vegetative propagation. This is a disadvantage in terms of shipping and planting compared to seed-propagated plants. Initial studies have been completed in the development of inbred snap bean genotypes as an alternative O3 bioindicator system. This project began with the development of a snap bean population derived from a cross between O3-sensitive and tolerant parents (Reinert and Eason, 2000), and from this population individual snap bean lines were developed that exhibit a range of biomass production when exposed to elevated O3 (Burkey and Eason, 2002). A two-year open-top chamber study showed that tolerant R123 and sensitive S156 snap bean lines grown in pots under optimal conditions could be used to detect effects of ambient O3 (Burkey et al., 2005). The pod yields of S156 and R123 were similar under charcoal-filtered air, resulting in yield ratios approximating 1.0 under a seasonal mean O3 concentration of 30 ppb. Significant declines in the yield ratio were observed when plants were subjected to a seasonal O3 concentration of approximately 50 ppb in either ambient air plots or chambers with nonfiltered air. A multi-year study in Long Island, NY showed that yields of S156 were significantly lower than in the tolerant R331 line when ambient O3 concentrations exceeded approximately 45 ppb under field conditions (Booker et al., 2009). In addition to O3, CO2 concentration in the atmosphere is steadily increasing (IPCC, 2007). Elevated CO2 has been shown to influence the response of many plants to O3 (Feng and Kobayashi, 2009; Fiscus et al., 2005; Morgan et al., 2003). The nature of the interaction, however, depends in part on the concentration of the gases, the sensitivities of the plants and other environmental conditions (Booker and Fiscus, 2005). For example, a previous open-top chamber study with snap beans grown in pots found that twice-ambient CO2 partially protected S156 from elevated O3 (Heagle et al., 2002). Objectives of this project were to test the capacity of this snap bean bioindicator system to detect O3 effects in field plots using standard agricultural practices and Free Air Concentration Enrichment (FACE) technology to create an elevated O3 treatment and to examine the effects of elevated CO2 on the response of these genotypes to O3 stress.
2.2. Snap bean planting plot management Seeds of R123, R331, and S156 snap bean (Phaseolus vulgaris L.) genotypes were planted on May 25, 2006 in each of two independent 1.9 1.9 m subplots within each ring. Three 1.5 m rows centered 0.5 m apart were established in the middle of each subplot to minimize shading from the surrounding soybeans. Each genotype was randomly assigned to one row within the subplot. At planting, 36 seeds were evenly distributed along the 1.5 m row, covered with soil, and immediately handwatered to enhance soil moisture for uniform emergence. All remaining water during the season was provided by natural rainfall events. Plants were thinned to 18e20 per row at three weeks after sowing, and the remaining plants subjected to season long exposures to the prescribed O3 and CO2 treatments. 2.3. Harvest protocol Plants were harvested during the three-day period of August 29e31 beginning at 96 days after planting at a stage when >80% of pods were characterized as fully mature brown pods. For each 1.5 m single row plot, plants were counted and pods were removed, counted, and dried to constant weight at 60 C. After drying, pods were weighed and the seeds were removed, counted, and weighed. 2.4. Statistics The number of plants recovered per row at harvest varied among individual plots (15e20). Positive relationships were observed between both pod number and pod weight per plot and plant number per plot. Therefore yields were calculated on a per plant basis. The results from the two subplots within a ring were averaged to create a ring mean that was used for statistical analysis. The experiment was analyzed as a 2 2 3 split-plot factorial with CO2 and O3 as whole plot factors and genotype as the subplot factor. There were four replicates of each treatment combination. A natural log transformation was applied to pod and seed weight data to stabilize the variance. The Mixed procedure in SAS was used to run the analysis (Littell et al., 1996) (SAS for Windows, Ver. 9.2). Pairwise mean comparisons were made using Tukey’s HSD test (Tukey, 1994), which were summarized by letter groupings using pdmix800 SAS macro (Saxton, 1998).
3. Results 3.1. Genotype effects on yield Yield based on pod dry weight per plant was 41% lower in S156 compared with R123 and R331 on average among all treatments (Table 2). Seed number and weight per seed were 23% lower in S156 than in R123 and R331 (Table 3). 3.2. Treatment effects on yield
2. Methods 2.1. Facility description and experimental treatments The study was conducted at the SoyFACE facility located on 32 ha near Champaign, IL, USA (40 020 N, 88 140 W, 228 m above sea level) using standard management practices for this site (http://www.igb.illinois.edu/soyface). The experiment was a randomized complete block design (n ¼ 4) with each block containing four treatments: 20 m diameter octagonal rings at ambient O3 and CO2 concentrations (the control for this study), elevated O3, elevated CO2, and the combination of elevated O3 and elevated CO2. The fumigation system was based on the design of Miglietta et al. (2001) for elevated CO2 and Morgan et al. (2004) for elevated O3. Targeted levels of 550 ppm CO2 and 1.4 ambient O3 in the elevated treatments were achieved (Table 1).
Pod dry weight per plant was not statistically significantly different among genotypes in the ambient air plots (Table 2). The elevated O3 treatments (Table 1) resulted in a significant main effect of O3 on yield and a significant genotype O3 interaction, reflecting the differential responses of O3-sensitive (S156) and -tolerant (R123 and R331) genotypes in the study (Table 2). Pod yields of R123 and R331 in the elevated O3 treatment were not significantly different from the ambient air control. In contrast, pod yield declined by 63% for S156. Similar results were found for yield based on seed dry weight per plant (Table 2). The S156 yield loss was associated with a 57% reduction in seed number per plant and
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
169
Table 2 Treatment effects on yield. Values are least squares means standard error. For pod weight or seed weight yields, different lower case letters within a column indicate significantly different treatment effects within a genotype whereas different upper case letters within a row indicate significantly different genotype responses within a treatment (P 0.05). Results from analysis of variance (ANOVA) are presented below the measured values. Treatment
Pod weight (g DW plant1)
Ambient Air O3 CO2 O3 þ CO2
6.9 5.8 7.0 9.3
ANOVA Source
Pod weight P
Seed weight P
Genotype O3 CO2 Genotype O3 Genotype CO2 O3 CO2 Genotype O3 CO2
<0.001 0.037 0.003 0.014 0.460 0.006 0.286
<0.001 0.042 0.003 0.008 0.387 0.007 0.255
R123
Seed weight (g DW plant1)
R331 1.0a,A 0.8a,A 1.0a,A 1.3a,A
7.5 4.7 8.4 8.5
S156 1.1a,A 0.7a,A 1.2a,A 1.2a,A
5.2 1.9 5.1 5.0
R123 0.7a,A 0.3b,B 0.7a,A 0.7a,A
4.7 4.1 4.8 6.5
R331 0.7a,A 0.6a,A 0.7a,A 0.9a,A
5.3 3.4 5.9 6.1
S156 0.8a,A 0.5a,A 0.8a,A 0.9a,A
3.7 1.3 3.7 3.5
0.5a,A 0.2b,B 0.5a,A 0.5a,A
the atmosphere. Pod yield ratios for S156/R123 and S156/R331 were 0.78 and 0.70 for ambient air plots, and declined to 0.35 and 0.41, respectively, under elevated O3 treatment (Table 4). Similar results were found for ratios based on seed yield (Table 4).
a 19% reduction in weight per seed in plants treated with elevated O3 compared with ambient air (Table 3). A significant main effect of elevated CO2 on yield (Table 2) and seed number (Table 3) was found as yields were 35% higher in plants exposed to 550 ppm CO2 compared to an ambient level of 378 ppm. However, this finding was partly a consequence of pooling yield data from the ambient air control and elevated O3 plots, where yields were reduced in the latter, and then making a comparison with the pooled yield data from the CO2 and CO2 þ O3 treatments where elevated O3 had no significant effect (see O3 CO2 interaction results below). When ambient air control plots were compared with the elevated CO2 plots only, the CO2 effect was not statistically significant (Table 2). All genotypes responded similarly to elevated CO2 as reflected by the absence of a significant genotype CO2 interaction. A significant O3 CO2 interaction was observed for both pod and seed yield (Table 2), seed number and seed size (Table 3) that related to amelioration of elevated O3 effects. For S156, yield under the combination treatment of elevated O3 þ CO2 was not significantly different from elevated CO2 (Table 2), demonstrating that 550 ppm CO2 completely protected this genotype from yield loss resulting from the elevated O3 treatment.
4. Discussion Yields of the three snap bean genotypes were not significantly different under ambient air conditions in this study where the O3 concentration averaged 43 ppb (Tables 1 and 2). An O3 concentration in the range of 40 ppb is considered the threshold level for observing injury in many sensitive plants and was selected as the cutoff value for the AOT40 metric (AOT40 is the hourly mean O3 concentration accumulated over a threshold O3 concentration of 40 ppb during daylight hours) (Fuhrer et al., 1997). A critical level of a 3 month AOT40 of 3 ppm h was derived for crop yields, including bean, for plants adequately supplied with water and nutrients (Fuhrer et al., 1997; Mills et al., 2007). The AOT40 value of 5.3 ppm h in this study (Table 1) suggested that ambient O3 concentrations were at a marginal level for detecting statistically significant impacts on yield, especially for bean where large variability among cultivars has been observed (Mills et al., 2007; Feng and Kobayashi, 2009). The management practices employed at SoyFACE include reliance on rainfall for maintaining soil moisture so it is possible that reduced stomatal conductance may have limited O3 uptake and reduced the impact of ambient O3, although 2006 was not especially dry or hot (Eastburn et al., 2010). There is evidence that
3.3. Yield ratios Yield ratios were calculated to assess the potential of the snap bean genotypes to distinguish between contrasting O3 concentrations in
Table 3 Treatment effects on seed number and weight per seed. Values are least squares means standard error. Different lower case letters within a column indicate significantly different treatment effects within a genotype whereas different upper case letters within a row indicate significantly different genotype responses within a treatment (P 0.05). Results from analysis of variance (ANOVA) are presented below the measured values. Treatment
Seed number plant1 R123
Weight per seed (mg) R331
3a,A 3a,A 3a,A 3a,A
28 20 33 35
S156 3a,A 3a,A 3a,A 3a,A
23 10 26 26
R123 3a,A 3b,B 3a,A 3a,B
200 192 187 189
R331 6a,A 6a,A 6a,A 6a,A
Ambient Air O3 CO2 O3 þ CO2
24 22 28 35
ANOVA Source
Seed number P
Weight per seed P
Genotype O3 CO2 Genotype O3 Genotype CO2 O3 CO2 Genotype O3 CO2
0.001 0.290 0.001 0.060 0.895 0.026 0.808
<0.001 0.028 0.209 0.202 0.736 0.031 0.392
194 171 182 178
S156 6a,A 6b,B 6a,A 6a,A
162 132 141 141
6a,B 6b,C 6b,B 6b,B
170
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
Table 4 Yield ratios. Values are least squares means standard error. Treatment
Ambient Air O3
Pod weight ratios
Seed weight ratios
S156/R123
S156/R331
S156/R123
S156/R331
0.78 0.05 0.35 0.05
0.70 0.05 0.41 0.05
0.81 0.05 0.34 0.05
0.71 0.05 0.40 0.05
yield differences between S156 and R123 can be detected at O3 concentrations as low as 48 ppb with optimal nutrition and water (Burkey et al., 2005). The predicted sensitivity for the genotypes was observed when plants were subjected to an elevated O3 treatment of 59 ppb with an AOT40 of 16.3 ppm h (Table 1). For the two tolerant genotypes, R123 and R331, pod yields were not significantly different in the elevated O3 treatment relative to the ambient air control (Table 2). The S156 genotype predicted to be the most sensitive line showed a significant pod yield loss of 63% in the elevated O3 treatment (Table 2). There is evidence for R123 having greater O3 tolerance than R331 (Flowers et al., 2007) from a study where these three genotypes were compared in outdoor plant environment chambers under optimal growth conditions. This suggests that comparative yields among genotypes are influenced by plant culture practices. The application of the yield ratio concept to the snap bean bioindicator system appears to be useful for identifying effects at O3 concentrations characteristic of present day pollution levels. The yield effects were nearly identical in this study whether measured on a pod weight or seed weight basis. Given that less labor is involved in processing pod samples than in threshing pods to recover seeds, using pod mass to calculate yield ratios would be a more efficient approach. A synthesis of the available data suggests that the minimum O3 concentration at which an effect can be detected will depend upon the management practices employed. Highly managed experiments where plants are grown with adequate nutrients and water will probably exhibit greater sensitivity than studies where inputs, particularly water, are more variable or restricted (Fuhrer et al., 1997). Even then, environmental interactions may alter responses at ambient O3 concentrations typical of present day levels. For example, significant yield differences between S156 and R123 were observed at ambient O3 concentrations of approximately 48 ppb that resulted in S156/R123 ratios of 0.4e0.5 (Burkey et al., 2005), but a study of similar design found no apparent differences in yield between S156 and R123 grown in ambient air (Elagoz and Manning, 2005). Pod yields for S156, R123 and R331were not significantly different in the ambient air treatment of 43 ppb O3 at the SoyFACE field site during 2006 (see Table 2, ambient air treatment). The resulting S156/R123 and S156/R331 ratios of 0.70e0.78 (Table 4) indicated little O3 effect. The snap bean system did detect a significant differential impact on yield in the field when plants were subjected to an elevated O3 treatment of 59 ppb with pod yield differences for the sensitive and tolerant lines resulting in S156/R123 and S156/R331 ratios of 0.35 and 0.41, respectively (Table 4). The implication from these various locations is that the snap bean bioindicator system requires managed experiments that include irrigation if the purpose is to detect effects of ambient O3 concentrations less than 50 ppb. This study provides evidence that the snap bean system can detect O3 effects in a non-irrigated field when seasonal mean O3 concentrations averaged 59 ppb. If similar results are found in follow-up field tests, the snap bean bioindicator system could be developed as an approach for detecting potential O3 impacts in agricultural settings where O3 effects on the crop of interest cannot be readily evaluated through the use of an appropriate clean-air control or chemical protectant. Recognizing that bean is a relatively O3-sensitive species (Mills et al., 2007; Feng and Kobayashi, 2009), yield losses
detected by the snap bean bioindicator system may be greater than losses for less sensitive crops grown in the same location. Establishing criteria for relating snap bean yield ratios to crop loss is one aspect to be addressed in future research. Based on the data from this study and other published results (Burkey et al., 2005; Booker et al., 2009), a decline in the snap bean S/R yield ratio to values of 0.5 or below is indicative of environments where O3 has probably impacted sensitive vegetation. Elevated CO2 at 550 ppm ameliorated the O3 effect on S156 yield (Table 2). Based on this observation, the snap bean system may indicate a diminishing impact of ambient O3 as atmospheric CO2 levels rise. However, there appears to be a limit on the ability of CO2 to protect S156 from O3 stress. A higher CO2 concentration of 697 ppm provided little protection against pod yield loss when S156 was exposed to 72 ppb O3 (Heagle et al., 2002). With further testing, the complex interaction between CO2 and O3 and the associated impact on the snap bean system could be better defined. The snap bean bioindicator system should remain a useful tool for detecting potential O3 effects on crops and other vegetation as climate conditions change. In our study, the effect on yield of elevated CO2 in ambient air was not statistically significant (Table 2). While CO2 generally stimulates vegetative biomass production in C3 crops, effects on yield have been more variable and often smaller than biomass enhancements (Ainsworth and Long, 2005; Fiscus et al., 2005; Reid and Fiscus, 2008; Ziska et al., 1998). For example, yields of rice and wheat at 550 ppm CO2 were not significantly different from ambient air in some studies (Ainsworth and Long, 2005; Heagle et al., 2000). In the previous CO2 O3 study with snap bean (Heagle et al., 2002), 697 ppm CO2 increased pod yield of S156 by 24% in clean air. The increase in pod yield of the O3-resistant Tenderette genotype, however, was only 5% in the same experiment. There is clear evidence for intraspecific variation in growth and yield responses to elevated CO2 in a number of crop species (Ainsworth and Long, 2005; Ziska et al., 1998, 2004). Yield responses to CO2 are influenced by other environmental factors, such as nutrient and water availability, O3 concentrations and plant competition (Ainsworth and Long, 2005; Fiscus et al., 2005; Reid and Fiscus, 2008). This suggests that resource limitations can be important influences on yield responses to CO2 (Fiscus et al., 2005). Yield responses of snap bean to elevated CO2 in our experiment suggested factors such as intraspecific variation, concentrationeresponse relationships, resource limitations and other abiotic influences affected the expected yield stimulation with CO2, but they did not prevent the occurrence of O3 amelioration. Reduced O3 uptake due to decreased stomatal conductance at elevated CO2 would be a reasonable explanation for the protective effects observed (Booker and Fiscus, 2005; Fiscus et al., 1997). 5. Conclusions An initial field study suggested that genetic differences in O3 sensitivity between the sensitive (S156) and tolerant (R123, R331) genotypes of snap bean can be utilized as an O3 bioindicator system to detect the presence of O3 effects at a given location following season-long exposures to ambient O3. Ratios of yields from sensitive and tolerant lines were found to be a good measurement for assessing O3 impacts. The system has the potential for adaptation in diverse settings, but ambient O3 effects detectable by the system will likely depend on the management practices employed. Highly managed plots with irrigation and adequate nutrients may be required to detect O3 impacts in environments where seasonal O3 concentrations are less than approximately 50 ppb. In agricultural fields without irrigation, higher O3 concentrations may be needed for the bioindicator system to detect O3 effects on sensitive vegetation. Further testing of this snap bean system under field conditions is required to
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
assess the general applicability as an O3 bioindicator, and future atmospheric CO2 concentrations will influence the system performance. Acknowledgments The authors thank Renee Tucker for excellent technical assistance during this project and the dedicated staff at SoyFACE for maintaining the exposure and data acquisition systems at the field site. References Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist 165, 351e372. Booker, F.L., Fiscus, E.L., 2005. The role of ozone flux and antioxidants in the suppression of ozone injury by elevated CO2 in soybean. Journal of Experimental Botany 56, 2139e2151. Booker, F., Muntifering, R., McGrath, M., Burkey, K., Decoteau, D., Fiscus, E., Manning, W., Krupa, S., Chappelka, A., Grantz, D., 2009. The ozone component of global change: potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. Journal of Integrative Plant Biology 57, 337e351. Burkey, K.O., Eason, G., 2002. Ozone tolerance in snap bean is associated with elevated ascorbic acid in the leaf apoplast. Physiologia Plantarum 114, 387e394. Burkey, K.O., Miller, J.E., Fiscus, E.L., 2005. Assessment of ambient ozone effects on vegetation using snap bean as a bio-indicator species. Journal of Environmental Quality 34, 1081e1086. Dentener, F., Stevenson, D., Cofala, J., Mechler, R., Amann, M., Bergamaschi, P., Raes, F., Derwent, R., 2005. The impact of air pollutant and methane emission controls on tropospheric ozone and radiative forcing: CTM calculations for the period 1990e2030. Atmospheric Chemistry and Physics 5, 1731e1755. Eastburn, D.M., Degennaro, M.M., Delucia, E.H., Dermody, O., McElrone, A.J., 2010. Elevated atmospheric carbon dioxide and ozone alter soybean diseases at SoyFACE. Global Change Biology 16, 320e330. Elagoz, V., Manning, W.J., 2005. Responses of sensitive and tolerant bush beans (Phaseolus vulgaris L.) to ozone in open-top chambers are influenced by phenotypic differences, morphological characteristics, and the chamber environment. Environmental Pollution 136, 371e383. Feng, A., Kobayashi, K., 2009. Assessing the impacts of current and future concentrations of surface ozone on crop yield with meta-analysis. Atmospheric Environment 43, 1510e1519. Fiscus, E.L., Reid, C.D., Miller, J.E., Heagle, A.S., 1997. Elevated CO2 reduces O3 flux and O3-induced yield losses in soybeans: possible implications for elevated CO2 studies. Journal of Experimental Botany 48, 307e313. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant, Cell and Environment 28, 997e1011. Flowers, M., Fiscus, E.L., Burkey, K.O., Booker, F.L., Dubois, J.-J.B., 2007. Photosynthesis, chlorophyll fluorescence, and yield of snap bean (Phaseolus vulgaris L.) genotypes differing in sensitivity to ozone. Environmental and Experimental Botany 61, 190e198. Fuhrer, J., Skarby, L., Ashmore, M., 1997. Critical levels for ozone effects on vegetation in Europe. Environmental Pollution 97, 91e106. Heagle, A.S., Miller, J.E., Chevone, B.I., Dreschel, T.W., Manning, W.J., McCool, P.M., Morrison, C.L., Neely, G.E., Rebbeck, J., 1995. Response of a white clover indicator
171
system to tropospheric ozone at eight locations in the United States. Water Air Soil Pollution 85, 1373e1378. Heagle, A.S., Reinert, R.A., Miller, J.E., 1996. Response of white clover to ozone in different environments. Journal of Environmental Quality 25, 273e278. Heagle, A.S., Miller, J.E., Pursley, W.A., 2000. Growth and yield responses of winter wheat to mixtures of ozone and carbon dioxide. Crop Science 40, 1656e1664. Heagle, A.S., Miller, J.E., Burkey, K.O., Eason, G., Pursley, W.A., 2002. Growth and yield responses of Phaseolus vulgaris to mixtures of carbon dioxide and ozone. Journal of Environmental Quality 31, 2008e2014. Heggestad, H.E., 1991. Origin of Bel-W3, Bel-C, and Bel-B tobacco varieties and their use as indicators of ozone. Environmental Pollution 74, 264e291. IPCC, 2007. Climate change 2007: the physical science basis. In: Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, UK, p. 996. Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., 1996. SAS System for Mixed Models. SAS Institute, Inc., Cary, NC. Mauzerall, D.L., Wang, X., 2001. Protecting agricultural crops from the effects of tropospheric ozone exposure: reconciling science and standard setting in the United States, Europe and Asia. Annual Review of Energy and the Environment 26, 237e268. Miglietta, F., Peressotti, A., Vaccari, F.P., Zaldei, A., deAngelis, P., ScarasciaMugnozza, G., 2001. Free-air CO2 enrichment (FACE) of a poplar plantation: the POPFACE fumigation system. New Phytologist 150, 465e476. Mills, G., Buse, A., Gimeno, B., Bermejo, V., Holland, M., Emberson, L., Pleijel, H., 2007. A synthesis of AOT40-based response functions and critical levels of ozone for agricultural and horticultural crops. Atmospheric Environment 41, 2630e2643. Morgan, P.B., Ainsworth, E.A., Long, S.P., 2003. How does elevated ozone impact soybean? A meta-analysis of photosynthesis, growth and yield. Plant, Cell and Environment 26, 1317e1328. Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long, S.P., 2004. An in vivo analysis of the effect of season-long open-air elevation of ozone to anticipated 2050 levels on photosynthesis in soybean. Plant Physiology 135, 2348e2357. Nali, C., Francini, A., Lorenzini, G., 2009. White clover clones as a cost-effective indicator of phytotoxic ozone: 10 years of experience from central Italy. Environmental Pollution 157, 1421e1426. Reid, C.D., Fiscus, E.L., 2008. Ozone and density affect the response of biomass and seed yield to elevated CO2 in rice. Global Change Biology 14, 60e76. Reinert, R.A., Eason, G., 2000. Genetic control of O3 sensitivity in a cross between two cultivars of snap bean. Journal of the American Society of Horticultural Science 125, 222e227. Saxton, A.M., 1998. A macro for converting mean separation output to letter groupings in proc mixed. In: Proc. 23rd SAS Users Group Intl. SAS Institute, Cary, NC, pp. 1243e1246. Tukey, J.W., 1994. The Collected Works of John W. Tukey. In: Multiple Comparisons: 1948e1983, vol. VIII. Chapman and Hall, New York. Van Dingenen, R., Dentener, F.J., Raes, F., Krol, M.C., Emberson, L., Cofala, J., 2009. The global impact of ozone on agricultural crop yields under current and future air quality legislation. Atmospheric Environment 43, 604e618. Ziska, L.H., Bunce, J.A., Caulfield, F.A., 1998. Intraspecific variation in seed yield of soybean (Glycine max) in response to increased atmospheric carbon dioxide. Australian Journal of Plant Physiology 25, 801e807. Ziska, L.H., Morris, C.F., Goins, E.W., 2004. Quantitative and qualitative evaluation of selected wheat varieties released since 1903 to increasing atmospheric carbon dioxide: can yield sensitivity to carbon dioxide be a factor in wheat performance? Global Change Biology 10, 1810e1819.
Contents lists available at SciVerse ScienceDirect
Environmental Pollution journal homepage: www.elsevier.com/locate/envpol
Field assessment of a snap bean ozone bioindicator system under elevated ozone and carbon dioxide in a free air system Kent O. Burkey a, *, Fitzgerald L. Booker a, Elizabeth A. Ainsworth b, Randall L. Nelson c a
USDA-ARS, Plant Science Research Unit and Department of Crop Science, North Carolina State University, 3127 Ligon Street, Raleigh, NC 27607, USA USDA-ARS, Global Change and Photosynthesis Research Unit and Department of Plant Biology, University of Illinois, Urbana-Champaign, 1201 West Gregory, Urbana, IL 61801, USA c USDA-ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit and Department of Crop Sciences, University of Illinois, Urbana-Champaign, 1101 West Peabody Drive, Urbana, IL 61801, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 November 2011 Received in revised form 5 March 2012 Accepted 14 March 2012
Ozone-sensitive (S156) and -tolerant (R123 and R331) genotypes of snap bean (Phaseolus vulgaris L.) were tested as a plant bioindicator system for detecting O3 effects at current and projected future levels of tropospheric O3 and atmospheric CO2 under field conditions. Plants were treated with ambient air, 1.4 ambient O3 and 550 ppm CO2 separately and in combination using Free Air Concentration Enrichment technology. Under ambient O3 concentrations pod yields were not significantly different among genotypes. Elevated O3 reduced pod yield for S156 (63%) but did not significantly affect yields for R123 and R331. Elevated CO2 at 550 ppm alone did not have a significant impact on yield for any genotype. Amelioration of the O3 effect occurred in the O3 þ CO2 treatment. Ratios of sensitive to tolerant genotype pod yields were identified as a useful measurement for assessing O3 impacts with potential applications in diverse settings including agricultural fields. Published by Elsevier Ltd.
Keywords: Bioindicator Carbon dioxide Free air concentration enrichment (FACE) Ozone Snap bean
1. Introduction Ozone (O3) is a toxic component of air pollution, and plants are particularly sensitive to this pollutant. Present day atmospheric O3 concentrations are sufficiently high to damage sensitive species of native vegetation and cultivated plants in many regions worldwide (Booker et al., 2009). Crop losses in the United States alone are estimated to be billions of dollars annually with impacts of similar magnitude in other agricultural regions around the world (Van Dingenen et al., 2009). This problem is expected to continue, especially if projected increases in ambient O3 are realized (Dentener et al., 2005). For any given location and growing season, assessing the impact of O3 on vegetation is difficult because plant response is the integration of a complex set of factors that include ambient O3 concentration, environmental conditions that affect leaf gas exchange and thus O3 uptake, and the inherent genetic variability in O3 sensitivity within and between species. Except for situations where O3-induced foliar injury can be documented, O3 impacts are
* Corresponding author. E-mail addresses: [email protected] (K.O. Burkey), Fitz.Booker@ ars.usda.gov (F.L. Booker), [email protected] (E.A. Ainsworth), [email protected] (R.L. Nelson). 0269-7491/$ e see front matter Published by Elsevier Ltd. doi:10.1016/j.envpol.2012.03.020
not easily quantified in the absence of a “clean air control”. For many situations, such a control is neither feasible nor practical. However, bioindicator plants provide one approach to circumvent some of these challenges. Genetic variation in plant response to O3 stress can be utilized in the development of plant O3 bioindicator systems. The concept is based on identification of O3-sensitive and -tolerant clones or genotypes that exhibit similar growth and yield under low O3 conditions, but exhibit differential foliar injury or yield losses when grown in environments of increasing O3 concentrations. In this approach, the plants integrate the multiple factors contributing to the O3 response for a given time and location, and the result is reflected in a quantifiable parameter. The first O3 bioindicator system was developed in 1957 using sensitive and resistant varieties of tobacco (Heggestad, 1991). The sensitive cultivar BelW3 exhibits foliar injury in response to ambient O3 concentrations exceeding 40 ppb. It can be desirable, however, to have a measure of O3 effects more quantitative than foliar injury to assess the impact of ambient O3. One such system is based on the differential forage biomass production of O3-sensitive (NC-S) and O3-resistant (NC-R) white clover clones (Heagle et al., 1996). Although the yield potential of the two clones varied between environments, the NCS/NC-R yield ratio was found to be a good indicator of the O3 impact during the growth period being evaluated (Heagle et al., 1995). The clover system has been used in a number of studies as an O3
168
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
Table 1 Summary of seasonal O3 and CO2 exposures. Treatment
CO2 (12 h mean) (ppm)
O3 (8 h mean)a (ppb)
O3 (1 h max)b (ppb)
O3 (AOT40)c (ppm h)
O3 (SUM60)d (ppm h)
Ambient Air O3 CO2 O3 þ CO2
378 378 550 551
43 59 43 59
29e78 32e114 29e78 33e112
5.3 16.3 5.3 16.2
5.3 27.0 5.3 26.7
a b c d
Average hourly O3 concentrations measured between 0900 and 1700 h from May 25, 2006 through August 28, 2006. Range of daily 1-h maximum values measured between 0900 and 1700 h from May 25, 2006 through August 28, 2006. AOT40 is the hourly mean O3 concentration accumulated over a threshold O3 concentration of 40 ppb during daylight hours (Mauzerall and Wang, 2001). SUM60 is the sum of hourly O3 concentrations equal to or greater than 60 ppb during daylight hours (Mauzerall and Wang, 2001).
bioindicator species (Nali et al., 2009). NC-S and NC-R are clones, which must be maintained through vegetative propagation. This is a disadvantage in terms of shipping and planting compared to seed-propagated plants. Initial studies have been completed in the development of inbred snap bean genotypes as an alternative O3 bioindicator system. This project began with the development of a snap bean population derived from a cross between O3-sensitive and tolerant parents (Reinert and Eason, 2000), and from this population individual snap bean lines were developed that exhibit a range of biomass production when exposed to elevated O3 (Burkey and Eason, 2002). A two-year open-top chamber study showed that tolerant R123 and sensitive S156 snap bean lines grown in pots under optimal conditions could be used to detect effects of ambient O3 (Burkey et al., 2005). The pod yields of S156 and R123 were similar under charcoal-filtered air, resulting in yield ratios approximating 1.0 under a seasonal mean O3 concentration of 30 ppb. Significant declines in the yield ratio were observed when plants were subjected to a seasonal O3 concentration of approximately 50 ppb in either ambient air plots or chambers with nonfiltered air. A multi-year study in Long Island, NY showed that yields of S156 were significantly lower than in the tolerant R331 line when ambient O3 concentrations exceeded approximately 45 ppb under field conditions (Booker et al., 2009). In addition to O3, CO2 concentration in the atmosphere is steadily increasing (IPCC, 2007). Elevated CO2 has been shown to influence the response of many plants to O3 (Feng and Kobayashi, 2009; Fiscus et al., 2005; Morgan et al., 2003). The nature of the interaction, however, depends in part on the concentration of the gases, the sensitivities of the plants and other environmental conditions (Booker and Fiscus, 2005). For example, a previous open-top chamber study with snap beans grown in pots found that twice-ambient CO2 partially protected S156 from elevated O3 (Heagle et al., 2002). Objectives of this project were to test the capacity of this snap bean bioindicator system to detect O3 effects in field plots using standard agricultural practices and Free Air Concentration Enrichment (FACE) technology to create an elevated O3 treatment and to examine the effects of elevated CO2 on the response of these genotypes to O3 stress.
2.2. Snap bean planting plot management Seeds of R123, R331, and S156 snap bean (Phaseolus vulgaris L.) genotypes were planted on May 25, 2006 in each of two independent 1.9 1.9 m subplots within each ring. Three 1.5 m rows centered 0.5 m apart were established in the middle of each subplot to minimize shading from the surrounding soybeans. Each genotype was randomly assigned to one row within the subplot. At planting, 36 seeds were evenly distributed along the 1.5 m row, covered with soil, and immediately handwatered to enhance soil moisture for uniform emergence. All remaining water during the season was provided by natural rainfall events. Plants were thinned to 18e20 per row at three weeks after sowing, and the remaining plants subjected to season long exposures to the prescribed O3 and CO2 treatments. 2.3. Harvest protocol Plants were harvested during the three-day period of August 29e31 beginning at 96 days after planting at a stage when >80% of pods were characterized as fully mature brown pods. For each 1.5 m single row plot, plants were counted and pods were removed, counted, and dried to constant weight at 60 C. After drying, pods were weighed and the seeds were removed, counted, and weighed. 2.4. Statistics The number of plants recovered per row at harvest varied among individual plots (15e20). Positive relationships were observed between both pod number and pod weight per plot and plant number per plot. Therefore yields were calculated on a per plant basis. The results from the two subplots within a ring were averaged to create a ring mean that was used for statistical analysis. The experiment was analyzed as a 2 2 3 split-plot factorial with CO2 and O3 as whole plot factors and genotype as the subplot factor. There were four replicates of each treatment combination. A natural log transformation was applied to pod and seed weight data to stabilize the variance. The Mixed procedure in SAS was used to run the analysis (Littell et al., 1996) (SAS for Windows, Ver. 9.2). Pairwise mean comparisons were made using Tukey’s HSD test (Tukey, 1994), which were summarized by letter groupings using pdmix800 SAS macro (Saxton, 1998).
3. Results 3.1. Genotype effects on yield Yield based on pod dry weight per plant was 41% lower in S156 compared with R123 and R331 on average among all treatments (Table 2). Seed number and weight per seed were 23% lower in S156 than in R123 and R331 (Table 3). 3.2. Treatment effects on yield
2. Methods 2.1. Facility description and experimental treatments The study was conducted at the SoyFACE facility located on 32 ha near Champaign, IL, USA (40 020 N, 88 140 W, 228 m above sea level) using standard management practices for this site (http://www.igb.illinois.edu/soyface). The experiment was a randomized complete block design (n ¼ 4) with each block containing four treatments: 20 m diameter octagonal rings at ambient O3 and CO2 concentrations (the control for this study), elevated O3, elevated CO2, and the combination of elevated O3 and elevated CO2. The fumigation system was based on the design of Miglietta et al. (2001) for elevated CO2 and Morgan et al. (2004) for elevated O3. Targeted levels of 550 ppm CO2 and 1.4 ambient O3 in the elevated treatments were achieved (Table 1).
Pod dry weight per plant was not statistically significantly different among genotypes in the ambient air plots (Table 2). The elevated O3 treatments (Table 1) resulted in a significant main effect of O3 on yield and a significant genotype O3 interaction, reflecting the differential responses of O3-sensitive (S156) and -tolerant (R123 and R331) genotypes in the study (Table 2). Pod yields of R123 and R331 in the elevated O3 treatment were not significantly different from the ambient air control. In contrast, pod yield declined by 63% for S156. Similar results were found for yield based on seed dry weight per plant (Table 2). The S156 yield loss was associated with a 57% reduction in seed number per plant and
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
169
Table 2 Treatment effects on yield. Values are least squares means standard error. For pod weight or seed weight yields, different lower case letters within a column indicate significantly different treatment effects within a genotype whereas different upper case letters within a row indicate significantly different genotype responses within a treatment (P 0.05). Results from analysis of variance (ANOVA) are presented below the measured values. Treatment
Pod weight (g DW plant1)
Ambient Air O3 CO2 O3 þ CO2
6.9 5.8 7.0 9.3
ANOVA Source
Pod weight P
Seed weight P
Genotype O3 CO2 Genotype O3 Genotype CO2 O3 CO2 Genotype O3 CO2
<0.001 0.037 0.003 0.014 0.460 0.006 0.286
<0.001 0.042 0.003 0.008 0.387 0.007 0.255
R123
Seed weight (g DW plant1)
R331 1.0a,A 0.8a,A 1.0a,A 1.3a,A
7.5 4.7 8.4 8.5
S156 1.1a,A 0.7a,A 1.2a,A 1.2a,A
5.2 1.9 5.1 5.0
R123 0.7a,A 0.3b,B 0.7a,A 0.7a,A
4.7 4.1 4.8 6.5
R331 0.7a,A 0.6a,A 0.7a,A 0.9a,A
5.3 3.4 5.9 6.1
S156 0.8a,A 0.5a,A 0.8a,A 0.9a,A
3.7 1.3 3.7 3.5
0.5a,A 0.2b,B 0.5a,A 0.5a,A
the atmosphere. Pod yield ratios for S156/R123 and S156/R331 were 0.78 and 0.70 for ambient air plots, and declined to 0.35 and 0.41, respectively, under elevated O3 treatment (Table 4). Similar results were found for ratios based on seed yield (Table 4).
a 19% reduction in weight per seed in plants treated with elevated O3 compared with ambient air (Table 3). A significant main effect of elevated CO2 on yield (Table 2) and seed number (Table 3) was found as yields were 35% higher in plants exposed to 550 ppm CO2 compared to an ambient level of 378 ppm. However, this finding was partly a consequence of pooling yield data from the ambient air control and elevated O3 plots, where yields were reduced in the latter, and then making a comparison with the pooled yield data from the CO2 and CO2 þ O3 treatments where elevated O3 had no significant effect (see O3 CO2 interaction results below). When ambient air control plots were compared with the elevated CO2 plots only, the CO2 effect was not statistically significant (Table 2). All genotypes responded similarly to elevated CO2 as reflected by the absence of a significant genotype CO2 interaction. A significant O3 CO2 interaction was observed for both pod and seed yield (Table 2), seed number and seed size (Table 3) that related to amelioration of elevated O3 effects. For S156, yield under the combination treatment of elevated O3 þ CO2 was not significantly different from elevated CO2 (Table 2), demonstrating that 550 ppm CO2 completely protected this genotype from yield loss resulting from the elevated O3 treatment.
4. Discussion Yields of the three snap bean genotypes were not significantly different under ambient air conditions in this study where the O3 concentration averaged 43 ppb (Tables 1 and 2). An O3 concentration in the range of 40 ppb is considered the threshold level for observing injury in many sensitive plants and was selected as the cutoff value for the AOT40 metric (AOT40 is the hourly mean O3 concentration accumulated over a threshold O3 concentration of 40 ppb during daylight hours) (Fuhrer et al., 1997). A critical level of a 3 month AOT40 of 3 ppm h was derived for crop yields, including bean, for plants adequately supplied with water and nutrients (Fuhrer et al., 1997; Mills et al., 2007). The AOT40 value of 5.3 ppm h in this study (Table 1) suggested that ambient O3 concentrations were at a marginal level for detecting statistically significant impacts on yield, especially for bean where large variability among cultivars has been observed (Mills et al., 2007; Feng and Kobayashi, 2009). The management practices employed at SoyFACE include reliance on rainfall for maintaining soil moisture so it is possible that reduced stomatal conductance may have limited O3 uptake and reduced the impact of ambient O3, although 2006 was not especially dry or hot (Eastburn et al., 2010). There is evidence that
3.3. Yield ratios Yield ratios were calculated to assess the potential of the snap bean genotypes to distinguish between contrasting O3 concentrations in
Table 3 Treatment effects on seed number and weight per seed. Values are least squares means standard error. Different lower case letters within a column indicate significantly different treatment effects within a genotype whereas different upper case letters within a row indicate significantly different genotype responses within a treatment (P 0.05). Results from analysis of variance (ANOVA) are presented below the measured values. Treatment
Seed number plant1 R123
Weight per seed (mg) R331
3a,A 3a,A 3a,A 3a,A
28 20 33 35
S156 3a,A 3a,A 3a,A 3a,A
23 10 26 26
R123 3a,A 3b,B 3a,A 3a,B
200 192 187 189
R331 6a,A 6a,A 6a,A 6a,A
Ambient Air O3 CO2 O3 þ CO2
24 22 28 35
ANOVA Source
Seed number P
Weight per seed P
Genotype O3 CO2 Genotype O3 Genotype CO2 O3 CO2 Genotype O3 CO2
0.001 0.290 0.001 0.060 0.895 0.026 0.808
<0.001 0.028 0.209 0.202 0.736 0.031 0.392
194 171 182 178
S156 6a,A 6b,B 6a,A 6a,A
162 132 141 141
6a,B 6b,C 6b,B 6b,B
170
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
Table 4 Yield ratios. Values are least squares means standard error. Treatment
Ambient Air O3
Pod weight ratios
Seed weight ratios
S156/R123
S156/R331
S156/R123
S156/R331
0.78 0.05 0.35 0.05
0.70 0.05 0.41 0.05
0.81 0.05 0.34 0.05
0.71 0.05 0.40 0.05
yield differences between S156 and R123 can be detected at O3 concentrations as low as 48 ppb with optimal nutrition and water (Burkey et al., 2005). The predicted sensitivity for the genotypes was observed when plants were subjected to an elevated O3 treatment of 59 ppb with an AOT40 of 16.3 ppm h (Table 1). For the two tolerant genotypes, R123 and R331, pod yields were not significantly different in the elevated O3 treatment relative to the ambient air control (Table 2). The S156 genotype predicted to be the most sensitive line showed a significant pod yield loss of 63% in the elevated O3 treatment (Table 2). There is evidence for R123 having greater O3 tolerance than R331 (Flowers et al., 2007) from a study where these three genotypes were compared in outdoor plant environment chambers under optimal growth conditions. This suggests that comparative yields among genotypes are influenced by plant culture practices. The application of the yield ratio concept to the snap bean bioindicator system appears to be useful for identifying effects at O3 concentrations characteristic of present day pollution levels. The yield effects were nearly identical in this study whether measured on a pod weight or seed weight basis. Given that less labor is involved in processing pod samples than in threshing pods to recover seeds, using pod mass to calculate yield ratios would be a more efficient approach. A synthesis of the available data suggests that the minimum O3 concentration at which an effect can be detected will depend upon the management practices employed. Highly managed experiments where plants are grown with adequate nutrients and water will probably exhibit greater sensitivity than studies where inputs, particularly water, are more variable or restricted (Fuhrer et al., 1997). Even then, environmental interactions may alter responses at ambient O3 concentrations typical of present day levels. For example, significant yield differences between S156 and R123 were observed at ambient O3 concentrations of approximately 48 ppb that resulted in S156/R123 ratios of 0.4e0.5 (Burkey et al., 2005), but a study of similar design found no apparent differences in yield between S156 and R123 grown in ambient air (Elagoz and Manning, 2005). Pod yields for S156, R123 and R331were not significantly different in the ambient air treatment of 43 ppb O3 at the SoyFACE field site during 2006 (see Table 2, ambient air treatment). The resulting S156/R123 and S156/R331 ratios of 0.70e0.78 (Table 4) indicated little O3 effect. The snap bean system did detect a significant differential impact on yield in the field when plants were subjected to an elevated O3 treatment of 59 ppb with pod yield differences for the sensitive and tolerant lines resulting in S156/R123 and S156/R331 ratios of 0.35 and 0.41, respectively (Table 4). The implication from these various locations is that the snap bean bioindicator system requires managed experiments that include irrigation if the purpose is to detect effects of ambient O3 concentrations less than 50 ppb. This study provides evidence that the snap bean system can detect O3 effects in a non-irrigated field when seasonal mean O3 concentrations averaged 59 ppb. If similar results are found in follow-up field tests, the snap bean bioindicator system could be developed as an approach for detecting potential O3 impacts in agricultural settings where O3 effects on the crop of interest cannot be readily evaluated through the use of an appropriate clean-air control or chemical protectant. Recognizing that bean is a relatively O3-sensitive species (Mills et al., 2007; Feng and Kobayashi, 2009), yield losses
detected by the snap bean bioindicator system may be greater than losses for less sensitive crops grown in the same location. Establishing criteria for relating snap bean yield ratios to crop loss is one aspect to be addressed in future research. Based on the data from this study and other published results (Burkey et al., 2005; Booker et al., 2009), a decline in the snap bean S/R yield ratio to values of 0.5 or below is indicative of environments where O3 has probably impacted sensitive vegetation. Elevated CO2 at 550 ppm ameliorated the O3 effect on S156 yield (Table 2). Based on this observation, the snap bean system may indicate a diminishing impact of ambient O3 as atmospheric CO2 levels rise. However, there appears to be a limit on the ability of CO2 to protect S156 from O3 stress. A higher CO2 concentration of 697 ppm provided little protection against pod yield loss when S156 was exposed to 72 ppb O3 (Heagle et al., 2002). With further testing, the complex interaction between CO2 and O3 and the associated impact on the snap bean system could be better defined. The snap bean bioindicator system should remain a useful tool for detecting potential O3 effects on crops and other vegetation as climate conditions change. In our study, the effect on yield of elevated CO2 in ambient air was not statistically significant (Table 2). While CO2 generally stimulates vegetative biomass production in C3 crops, effects on yield have been more variable and often smaller than biomass enhancements (Ainsworth and Long, 2005; Fiscus et al., 2005; Reid and Fiscus, 2008; Ziska et al., 1998). For example, yields of rice and wheat at 550 ppm CO2 were not significantly different from ambient air in some studies (Ainsworth and Long, 2005; Heagle et al., 2000). In the previous CO2 O3 study with snap bean (Heagle et al., 2002), 697 ppm CO2 increased pod yield of S156 by 24% in clean air. The increase in pod yield of the O3-resistant Tenderette genotype, however, was only 5% in the same experiment. There is clear evidence for intraspecific variation in growth and yield responses to elevated CO2 in a number of crop species (Ainsworth and Long, 2005; Ziska et al., 1998, 2004). Yield responses to CO2 are influenced by other environmental factors, such as nutrient and water availability, O3 concentrations and plant competition (Ainsworth and Long, 2005; Fiscus et al., 2005; Reid and Fiscus, 2008). This suggests that resource limitations can be important influences on yield responses to CO2 (Fiscus et al., 2005). Yield responses of snap bean to elevated CO2 in our experiment suggested factors such as intraspecific variation, concentrationeresponse relationships, resource limitations and other abiotic influences affected the expected yield stimulation with CO2, but they did not prevent the occurrence of O3 amelioration. Reduced O3 uptake due to decreased stomatal conductance at elevated CO2 would be a reasonable explanation for the protective effects observed (Booker and Fiscus, 2005; Fiscus et al., 1997). 5. Conclusions An initial field study suggested that genetic differences in O3 sensitivity between the sensitive (S156) and tolerant (R123, R331) genotypes of snap bean can be utilized as an O3 bioindicator system to detect the presence of O3 effects at a given location following season-long exposures to ambient O3. Ratios of yields from sensitive and tolerant lines were found to be a good measurement for assessing O3 impacts. The system has the potential for adaptation in diverse settings, but ambient O3 effects detectable by the system will likely depend on the management practices employed. Highly managed plots with irrigation and adequate nutrients may be required to detect O3 impacts in environments where seasonal O3 concentrations are less than approximately 50 ppb. In agricultural fields without irrigation, higher O3 concentrations may be needed for the bioindicator system to detect O3 effects on sensitive vegetation. Further testing of this snap bean system under field conditions is required to
K.O. Burkey et al. / Environmental Pollution 166 (2012) 167e171
assess the general applicability as an O3 bioindicator, and future atmospheric CO2 concentrations will influence the system performance. Acknowledgments The authors thank Renee Tucker for excellent technical assistance during this project and the dedicated staff at SoyFACE for maintaining the exposure and data acquisition systems at the field site. References Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air CO2 enrichment (FACE)? A meta-analytic review of the responses of photosynthesis, canopy properties and plant production to rising CO2. New Phytologist 165, 351e372. Booker, F.L., Fiscus, E.L., 2005. The role of ozone flux and antioxidants in the suppression of ozone injury by elevated CO2 in soybean. Journal of Experimental Botany 56, 2139e2151. Booker, F., Muntifering, R., McGrath, M., Burkey, K., Decoteau, D., Fiscus, E., Manning, W., Krupa, S., Chappelka, A., Grantz, D., 2009. The ozone component of global change: potential effects on agricultural and horticultural plant yield, product quality and interactions with invasive species. Journal of Integrative Plant Biology 57, 337e351. Burkey, K.O., Eason, G., 2002. Ozone tolerance in snap bean is associated with elevated ascorbic acid in the leaf apoplast. Physiologia Plantarum 114, 387e394. Burkey, K.O., Miller, J.E., Fiscus, E.L., 2005. Assessment of ambient ozone effects on vegetation using snap bean as a bio-indicator species. Journal of Environmental Quality 34, 1081e1086. Dentener, F., Stevenson, D., Cofala, J., Mechler, R., Amann, M., Bergamaschi, P., Raes, F., Derwent, R., 2005. The impact of air pollutant and methane emission controls on tropospheric ozone and radiative forcing: CTM calculations for the period 1990e2030. Atmospheric Chemistry and Physics 5, 1731e1755. Eastburn, D.M., Degennaro, M.M., Delucia, E.H., Dermody, O., McElrone, A.J., 2010. Elevated atmospheric carbon dioxide and ozone alter soybean diseases at SoyFACE. Global Change Biology 16, 320e330. Elagoz, V., Manning, W.J., 2005. Responses of sensitive and tolerant bush beans (Phaseolus vulgaris L.) to ozone in open-top chambers are influenced by phenotypic differences, morphological characteristics, and the chamber environment. Environmental Pollution 136, 371e383. Feng, A., Kobayashi, K., 2009. Assessing the impacts of current and future concentrations of surface ozone on crop yield with meta-analysis. Atmospheric Environment 43, 1510e1519. Fiscus, E.L., Reid, C.D., Miller, J.E., Heagle, A.S., 1997. Elevated CO2 reduces O3 flux and O3-induced yield losses in soybeans: possible implications for elevated CO2 studies. Journal of Experimental Botany 48, 307e313. Fiscus, E.L., Booker, F.L., Burkey, K.O., 2005. Crop responses to ozone: uptake, modes of action, carbon assimilation and partitioning. Plant, Cell and Environment 28, 997e1011. Flowers, M., Fiscus, E.L., Burkey, K.O., Booker, F.L., Dubois, J.-J.B., 2007. Photosynthesis, chlorophyll fluorescence, and yield of snap bean (Phaseolus vulgaris L.) genotypes differing in sensitivity to ozone. Environmental and Experimental Botany 61, 190e198. Fuhrer, J., Skarby, L., Ashmore, M., 1997. Critical levels for ozone effects on vegetation in Europe. Environmental Pollution 97, 91e106. Heagle, A.S., Miller, J.E., Chevone, B.I., Dreschel, T.W., Manning, W.J., McCool, P.M., Morrison, C.L., Neely, G.E., Rebbeck, J., 1995. Response of a white clover indicator
171
system to tropospheric ozone at eight locations in the United States. Water Air Soil Pollution 85, 1373e1378. Heagle, A.S., Reinert, R.A., Miller, J.E., 1996. Response of white clover to ozone in different environments. Journal of Environmental Quality 25, 273e278. Heagle, A.S., Miller, J.E., Pursley, W.A., 2000. Growth and yield responses of winter wheat to mixtures of ozone and carbon dioxide. Crop Science 40, 1656e1664. Heagle, A.S., Miller, J.E., Burkey, K.O., Eason, G., Pursley, W.A., 2002. Growth and yield responses of Phaseolus vulgaris to mixtures of carbon dioxide and ozone. Journal of Environmental Quality 31, 2008e2014. Heggestad, H.E., 1991. Origin of Bel-W3, Bel-C, and Bel-B tobacco varieties and their use as indicators of ozone. Environmental Pollution 74, 264e291. IPCC, 2007. Climate change 2007: the physical science basis. In: Solomon, S., Qin, D., Manning, M., Chen, Z., Marquis, M., Averyt, K.B., Tignor, M., Miller, H.L. (Eds.), Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge University Press, Cambridge, UK, p. 996. Littell, R.C., Milliken, G.A., Stroup, W.W., Wolfinger, R.D., 1996. SAS System for Mixed Models. SAS Institute, Inc., Cary, NC. Mauzerall, D.L., Wang, X., 2001. Protecting agricultural crops from the effects of tropospheric ozone exposure: reconciling science and standard setting in the United States, Europe and Asia. Annual Review of Energy and the Environment 26, 237e268. Miglietta, F., Peressotti, A., Vaccari, F.P., Zaldei, A., deAngelis, P., ScarasciaMugnozza, G., 2001. Free-air CO2 enrichment (FACE) of a poplar plantation: the POPFACE fumigation system. New Phytologist 150, 465e476. Mills, G., Buse, A., Gimeno, B., Bermejo, V., Holland, M., Emberson, L., Pleijel, H., 2007. A synthesis of AOT40-based response functions and critical levels of ozone for agricultural and horticultural crops. Atmospheric Environment 41, 2630e2643. Morgan, P.B., Ainsworth, E.A., Long, S.P., 2003. How does elevated ozone impact soybean? A meta-analysis of photosynthesis, growth and yield. Plant, Cell and Environment 26, 1317e1328. Morgan, P.B., Bernacchi, C.J., Ort, D.R., Long, S.P., 2004. An in vivo analysis of the effect of season-long open-air elevation of ozone to anticipated 2050 levels on photosynthesis in soybean. Plant Physiology 135, 2348e2357. Nali, C., Francini, A., Lorenzini, G., 2009. White clover clones as a cost-effective indicator of phytotoxic ozone: 10 years of experience from central Italy. Environmental Pollution 157, 1421e1426. Reid, C.D., Fiscus, E.L., 2008. Ozone and density affect the response of biomass and seed yield to elevated CO2 in rice. Global Change Biology 14, 60e76. Reinert, R.A., Eason, G., 2000. Genetic control of O3 sensitivity in a cross between two cultivars of snap bean. Journal of the American Society of Horticultural Science 125, 222e227. Saxton, A.M., 1998. A macro for converting mean separation output to letter groupings in proc mixed. In: Proc. 23rd SAS Users Group Intl. SAS Institute, Cary, NC, pp. 1243e1246. Tukey, J.W., 1994. The Collected Works of John W. Tukey. In: Multiple Comparisons: 1948e1983, vol. VIII. Chapman and Hall, New York. Van Dingenen, R., Dentener, F.J., Raes, F., Krol, M.C., Emberson, L., Cofala, J., 2009. The global impact of ozone on agricultural crop yields under current and future air quality legislation. Atmospheric Environment 43, 604e618. Ziska, L.H., Bunce, J.A., Caulfield, F.A., 1998. Intraspecific variation in seed yield of soybean (Glycine max) in response to increased atmospheric carbon dioxide. Australian Journal of Plant Physiology 25, 801e807. Ziska, L.H., Morris, C.F., Goins, E.W., 2004. Quantitative and qualitative evaluation of selected wheat varieties released since 1903 to increasing atmospheric carbon dioxide: can yield sensitivity to carbon dioxide be a factor in wheat performance? Global Change Biology 10, 1810e1819.