Ozone air pollution and foliar injury development on native plants of Switzerland

The objectives of this study were to examine the foliar sensitivity to ozone exposure of 12 tree, shrub, and herbaceous species native to southern Switzerland and determine the seasonal cumulative ozone exposures required to induce visible foliar injury. The study was conducted from the beginning of May through the end of August during 2000 and 2001 using an open-top chamber research facility located within the Lattecaldo Cantonal Forest Nursery in Canton Ticino, southern Switzerland (600 m asl). Plants were examined daily and dates of initial foliar injury were recorded in order to determine the cumulative AOT40 ppb h ozone exposure required to cause visible foliar injury. Plant responses to ozone varied significantly among species; 11 species exhibited visible symptoms typical of exposures to ambient ozone. The symptomatic species (from most to least sensitive) were Populus nigra, Viburnum lantana, Salix alba, Crataegus monogyna, Viburnum opulus, Tilia platyphyllos, Cornus alba, Prunus avium, Fraxinus excelsior, Ribes alpinum, and Tilia cordata; Clematis spp. did not show foliar symptoms. Of the 11 symptomatic species, five showed initial injury below the critical level AOT40 10 ppmh O3 in the 2001 season.     

Responses of hybrid poplar clones and red maple seedlings to ambient O(3) under differing light within a mixed hardwood forest

The responses of ramets of hybrid poplar (Populus spp.) (HP) clones NE388 and NE359, and seedlings of red maple (Acer rubrum, L.) to ambient ozone (O(3)) were studied during May-September of 2000 and 2001 under natural forest conditions and differing natural sunlight exposures (sun, partial shade and full shade). Ambient O(3) concentrations at the study site reached hourly peaks of 109 and 98 ppb in 2000 and 2001, respectively. Monthly 12-h average O(3) concentrations ranged from 32.3 to 52.9 ppb. Weekly 12-h average photosynthetically active radiation (PAR) within the sun, partial shade and full shade plots ranged from 200 to 750, 50 to 180, and 25 to 75 micromol m(-2) s(-1), respectively. Ambient O(3) exposure induced visible foliar symptoms on HP NE388 and NE359 in both growing seasons, with more severe injury observed on NE388 than on NE359. Slight foliar symptoms were observed on red maple seedlings during the 2001 growing season. Percentage of total leaf area affected (%LAA) was positively correlated with cumulative O(3) exposures. More severe foliar injury was observed on plants grown within the full shade and partial shade plots than those observed on plants grown within the sun plot. Lower light availability within the partial shade and full shade plots significantly decreased net photosynthetic rate (Pn) and stomatal conductance (g(wv)). The reductions in Pn were greater than reductions in g(wv), which resulted in greater O(3) uptake per unit Pn in plants grown within the partial shade and full shade plots. Greater O(3) uptake per unit Pn was consistently associated with more severe visible foliar injury in all species and/or clones regardless of differences in shade tolerance. These studies suggest that plant physiological responses to O(3) exposure are likely complicated due to multiple factors under natural forest conditions.     

Growth and nutritive quality of Poa pratensis as influenced by ozone and competition

Interspecific plant competition has been hypothesized to alter effects of early-season ozone (O3) stress. A phytometer-based approach was utilized to investigate O3 effects on growth and nutritive quality of Poa pratensis grown in monoculture and in mixed cultures with four competitor-plant species (Anthoxanthum odoratum, Achillea millefolium, Rumex acetosa and Veronica chamaedrys). Mesocosms were exposed during April/May 2000-2002 to charcoal-filtered air+25 ppb O3 (control) or non-filtered air+50 ppb O3 (elevated O3). Biomass production was not affected by O3, but foliar injury symptoms were observed in May 2002. Early-season O3 exposure decreased relative food value of P. pratensis by an average of 8%, which is sufficient to have nutritional implications for its utilization by herbivores. However, forage quality response to O3 was not changed by interspecific competition. Lack of injury and nutritive quality response in P. pratensis harvested in September may reflect recovery from early-season O3 exposure.     

The role of arginine decarboxylase in modulating the sensitivity of barley to ozone

Polyamines (PA) are known to be involved in the areas of plant physiology and biochemistry which are related to the response of a plant to air pollution. This study examines the role of arginine decarboxylase (ADC), an important rate-limiting enzyme in polyamine synthesis, in barley plants exposed to ozone (O(3)). The activity of ADC increased significantly in O(3)-treated leaves when visible injury was hardly apparent. The increase in ADC activity may be a mechanism to increase the PA levels in O(3)-treated leaves and so minimize the damaging effects of O(3). Supporting this, foliar applications of DL-alpha-difluoromethylarginine (DFMA), a specific inhibitor of ADC, prevented the rise in ADC activity and visible injury was considerable on exposure to O(3). This damage was not due to the foliar sprays, as little visible injury was seen in leaves in the O(3)-free controls. The results are discussed in terms of the roles of PA in conferring O(3) resistance in plants.     

Effects of vapours of chlorpropham and ethofumesate on wild plant species.

Effects of vapours of two herbicides on plantlets of fourteen wild higher plant species and two bryophytes were screened in fumigation experiments using foliar injury, chlorophyll fluorescence and growth as response parameters. After vaporisation of the herbicides for 48 h, concentrations in the chambers reached 77 micrograms m-3 in the chlorpropham treatments and 184 ng m-3 in the ethofumesate treatments. Despite the higher concentrations of the volatile chlorpropham (vapour pressure, VP: 1.3 mP), plants showed no foliar injury, but vapours of this herbicide caused leaf crinkling in the agriophyte Agrostemma githago. The less volatile ethofumesate (VP: 0.56 mP) caused foliar injury in all higher species, with lowest no observed effect concentrations (NOECs) of 75 ng m-3. Chlorpropham affected growth only in Agrostemma, while ethofumesate reduced growth in one third of the higher plant species. Chlorophyll fluorescence proved to be a less suitable response parameter compared to foliar injury and growth. No adverse effects were observed in mosses, probably due to the slow growth and hence small doses of herbicides taken up. The extent of foliar injury due to ethofumesate showed a weak positive relationship to relative growth rates and specific leaf area in the tested higher plant species.     

Ozone exposure thresholds and foliar injury on forest plants in Switzerland

Canton Ticino in southern Switzerland is exposed to some of the highest concentrations of tropospheric ozone in Europe. During recent field surveys in Canton Ticino, foliar symptoms identical to those caused by ozone have been documented on native tree and shrub species. In Europe, the critical ozone level for forest trees has been defined at an AOT40 of 10 ppm.h O3 (10 ppm.h accumulated exposure of ozone over a threshold of 40 ppb) during daylight hours over a six-month growing season. The objective of this study was to determine the amount of ambient ozone required to induce visible foliar symptoms on various forest plant species in southern Switzerland. Species were grown within eight open-top chambers and four open plots at the Vivaio Lattecaldo Cantonal Forest Nursery in Ticino, Switzerland. Species differed significantly in terms of the ppb.h exposures needed to cause visible symptoms. The most to least symptomatic species grown within open-plots in this study rank as Prunus serotina, Salix viminalis, Vibrnum lantana, Rhamnus cathartica, Betula pendula, Rumex obtusifolius, Sambucus racemosa, Morus nigra, Prunus avium, Fraxinus excelsior, Rhamnus frangula, Alnus viridis, Fagus sylvatica and Acer pseudoplatanus. Similar rankings were obtained in the non-filtered chamber plots. The ranking of species sensitivity closely follows AOT values for the occurrence of initial symptoms and symptom progression across the remainder of the exposure season. Species that first showed evidence of foliar injury also demonstrated the most sensitivity throughout the growing season, with symptoms rapidly advancing over ca. 25-30% of the total plant leaf surfaces by the end of the observation period. Conversely, those species that developed symptoms later in the season had far less total injury to plant foliage by the end of the observation period (1.5 to < 5% total leaf area injured). The current European ambient ozone standard may be insufficient to protect native plant species from visible foliar injury, and many more native species may be sensitive to ozone-induced foliar injury than are currently known.     

Effects of rooting medium and fertilizer rate on response of white clover to tropospheric ozone

Two white clover (Trifolium repens L.) clones with varying sensitivity to O(3) are being developed as a system to indicate effects of ambient concentrations of tropospheric O(3) on plants. One clone (NC-S) is highly sensitive to O(3) and the other (NC-R) is highly resistant. The system relies on periodic measurement of foliar injury, foliar chlorophyll, and forage production of NC-S and NC-R grown in 15-liter pots throughout a summer season. Relative amounts of foliar injury and ratios (NC-S/NC-R) for chlorophyll and forage weight can be used to estimate biologically effective ambient O(3) concentrations. The effect of variation in rooting media formulation and fertilizer rate on response of NC-S and NC-R to ambient O(3) was determined in the present study. In the rooting medium experiment, clover was grown in three mixtures of sandy loam topsoil:course washed sand:Metro Mix 220 (ratios (by volume) of 2:1:1, 2:1:5, and 6:1:1). In the fertilizer experiment, clover was grown in the 2:1:1 medium at four fertilizer rates (soluble 5-11-26 (N-P-K) at 0.0, 0.5, 1.0, or 2.0 g per pot). Ozone caused more foliar injury, more chlorosis, and a greater decrease in forage production of NC-S than of NC-R in all studies. Rooting media treatments affected both clones similarly and occasional clone x media interactions were judged to be random. Forage production by NC-S, relative to that of NC-R, was generally greater in the 0.0 fertilizer treatment, but the forage ratios were similar at all other fertilizer treatments. The relative response of NC-S and NC-R to O(3) is fairly stable under cultural conditions that support normal plant growth.     

Effects of air quality on foliar injury, growth, yield, and quality of muskmelon

An evaluation of the effects of ambient ozone (O3) on muskmelon was conducted with the use of open-top chambers (OTCs). 'Superstar' muskmelons grown in charcoal-filtered (CF) chambers compared to those grown in nonfiltered (NF) chambers showed significant differences in the severity of visible foliar O3 injury. Furthermore, plants grown in NF conditions had significantly less (21.3%) marketable fruit weight and fewer (20.9%) marketable fruit number than those from CF chambers. No differences were found in early biomass production, leaf area, or number of nodes after 3 weeks of exposure to treatment conditions. Ambient O3 did not affect soluble solids content of mature fresh fruit nor foliage fresh weight at final harvest. Results indicate that ambient concentrations of O3 in southwestern Indiana caused significant foliar injury and yield loss to muskmelons.     

Physiological and foliar injury responses of Prunus serotina,Fraxinus americana,and Acer rubrum seedlings to varying soil moisture and ozone

Sixteen black cherry (Prunus serotina, Ehrh.), 10 white ash (Fraxinus americana, L.) and 10 red maple (Acer rubrum, L.) 1-year old seedlings were planted per plot in 1997 on a former nursery bed within 12 open-top chambers and six open plots. Seedlings were exposed to three different ozone scenarios (ambient air: 100% O3; non-filtered air: 98% ambient O3; charcoal-filtered air: 50% ambient O3) within each of two different water regimes (nine plots irrigated, nine plots non-irrigated) during three growing seasons.During the 1998 and 1999 growing season, leaf gas exchange, plant water relations, and foliar injury were measured. Climatic data,ambient- and chamber-ozone-concentrations were monitored. We found that seedlings grown under irrigated conditions had similar (in 1998) but significantly higher gas exchange rates (in 1999) than seedlings grown within non-irrigated plots among similar ozone exposures. Cherry and ash had similar ozone uptake but cherry developed more ozone-induced injury (< 34% affected leaf area, LAA) than ash (<5% LAA), while maple rarely showed foliar injury, indicating the species differed in ozone sensitivity. Significantly more severe injury on seedlings grown under irrigated conditions than seedlings grown under non-irrigated conditions demonstrated that soil moisture altered seedling responses to ambient ozone exposures.     

Dynamics of biomass partitioning in field-grown radish varieties, treated with ethylenediurea

During the growing season of 1990, five staggered crops of radish (Raphanus sativus L.) were grown in the field, using the cultivars 'Cherry Belle', 'Red Prince', and 'Red Devil B'. Half of the plants received a soil drench (100 ml plant(-1); 100 mg litre(-1) of ethylenediurea (EDU) once, early in plant development. Destructive harvests were carried out at 2-day intervals during vegetative development. Non-linear growth kinetics, derived from Richards' function, were fitted to the dry weight data of the total plant, main organs (shoot and hypocotyl) and to the dry weight ratio between below-ground and above-ground organs. Estimating the parameters of these non-linear functions and testing their differences between EDU-treated and untreated plants unveiled biologically meaningful information on the impact of different levels of ambient ozone (O(3)) during the growth periods. The modified function which was applied to the data of biomass partitioning between the major plant parts was more powerful in detecting transient alterations in assimilate allocation compared to the growth dynamics of individual plant organs. At low levels of O(3), biomass partitioning towards the below-ground sink organs was slightly delayed and finally restricted in EDU-treated plants. When ambient O(3) reached moderate levels, which did not cause visible foliar injury, assimilate partitioning between organs was only insignificantly altered during early growth when EDU-treatments were compared. As growth progressed, however, less assimilates were allocated towards the hypocotyl and roots in the plants not protected by EDU. This pattern was similar in all cultivars tested, but was smallest in 'Cherry Belle', which is known to be sensitive to O(3) with respect to foliar injury. During the 15- to 19-day periods of rapid growth, the O(3)-exposure >80 nl litre(-1) ranged from 0.015 to 0.209 microl litre(-1) O(3) h, which corresponds to 7 h d(-1) mean values between 40 and 50 nl litre(-1) O(3), confirming that ambient ozone did not exceed a moderate level in this study.     

An assessment of the impact of ambient ozone on field-grown crops in New Jersey using the EDU method: part 1-white potato (Solanum tuberosum)

Six potato cultivars were grown to maturity in field plots in New Brunswick, New Jersey, according to standard commercial practices over a 5-year period. One-half of the plots were given a periodic soil drench of an antioxidant (EDU) which has the capacity to protect foliage against ozone toxicity. Based upon visible foliar injury and total tuber yield, the cultivars Norland and Norchip proved significantly more sensitive to ambient ozone pollution than Green Mountain, Irish Cobbler, Belrus or Superior. When foliar injury was less than 20%, no impact on tuber yield was detected. However, when 75% of the foliage exhibited O(3) toxicity symptoms, tuber yield was reduced 25% and 31%, respectively, in 'Norland' and 'Norchip'. A review of results from studies in the US and Canada utilizing different assessment methodologies provides evidence that ambient ozone causes significant tuber yield reduction in sensitive white potato genotypes when foliar injury exceeds 20 to 40%.     

Foliar response of black cherry (Prunus serotina) clones to ambient ozone exposure in central Pennsylvania

During late summer of 1996 and 1997 we examined ozone-induced foliar injury in a plantation of 111 black cherry trees (ramets) comprising 15 clones originating from wild ortets growing in the Allegheny National Forest, Pennsylvania, and the Monongahela National Forest, West Virginia. The experimental plantation was a clonal seed orchard in Centre County, Pennsylvania, started in 1971 using ortet buds grafted onto seedling rootstocks of mixed origin. Clones differed significantly in severity of foliar injury symptoms (F=31.83, p<0.001). One clone (R-12) had significantly more foliar injury with >50% leaf area affected than other clones during both years. In contrast, clone R-14, which is from the same area in northcentral Pennsylvania as R-12, exhibited significantly less injury (LAA<6%). Although ambient O(3) concentrations were similar in both years, foliar injury was significantly greater (15.7%) in 1996 than in 1997 (9.9%). This is probably explained by lower stomatal conductance in 1997 caused by drier and hotter weather patterns in June and July of that year. Despite very different weather patterns and overall levels of injury in 1996 and 1997, mean clonal injury was significantly correlated between both years of assessment (r=0.92, p<0.001). Within tree crowns, foliage in lower and inner crown positions was significantly more injured than foliage in upper and exterior crown positions. There was no evidence of geographically based population differences in sensitivity to foliar O(3) injury. On the contrary, results demonstrate that wild genotypes of proximal geographic origin may differ greatly in sensitivity.     

Influence of light fleck and low light on foliar injury and physiological responses of two hybrid poplar clones to ozone

Five-month old hybrid poplar clones NE388 and NE359 were exposed to square-wave 30, 55, and 80 ppb O(3) (8 h/day, 7 day/week) under constant high light (HL) and light fleck (LF) during 28 May-29 June 1999, and exposed to 30 and 55 ppb O(3) under HL, LF, and constant low light (LL) during 22 May-28 June 2000 within Continuously Stirred Tank Reactors (CSTR) in a greenhouse. Ramets of these two hybrid clones received similar total photosynthetically active radiation (PAR) within the LF and LL treatments. Visible foliar symptoms, leaf gas exchange, and growth were measured. More severe O(3) induced foliar symptoms were observed on ramets within the LF and LL treatments than within the HL treatment for both clones. The LF treatment resulted in significantly greater foliar injury than the LL treatment for NE388. The LF and LL treatments generally resulted in lower photosynthetic rates (Pn) for both clones, but did not affect stomatal conductance (g(wv)); therefore, the ratios of g(wv)/Pn and the O(3) uptake/Pn were greatest in plants grown under the LF treatment, followed by those grown under LL treatment; plants grown under HL had the lowest ratios of g(wv)/Pn and O(3) uptake/Pn. Greater ratios of g(wv)/Pn and O(3) uptake/Pn were consistently associated with more severe visible foliar symptoms. The negative impacts of the LF treatment on growth were greater than those of the LL treatment. Results indicate that not only the integral, but also the pattern of photo flux density, may affect carbon gain in plants. Increased foliar injury may be expected under light fleck conditions due to the limited repair capacity as a result of continuity of O(3) uptake while photosynthesis decreases under LL conditions.     

Visible and microscopic injury in leaves of five deciduous tree species related to current critical ozone levels

Because the current critical level of ozone (O(3)) for forest trees is based only on one species, the responses of five deciduous tree species were differentiated in a climate chamber experiment. The number of symptomatic leaves per tree was significantly increased, and stomatal conductance was decreased under 50% ambient+30 nl l(-1) O(3) as compared to 'normal' senescence at 50% ambient [O(3)]. Species with a high stomatal conductance did not show earlier or more leaf injury symptoms. The additional 30 nl l(-1) O(3) induced specific pectinaceous cell wall protrusions, phenolic cell wall incrustations, tonoplast vesicles, and inhomogeneous, condensed/precipitated phenolic material in the vacuoles. Due to added O(3), cell senescence was accelerated with increased electron-density of the cytoplasm, and initial chloroplast degeneration. The slow degeneration process started in mesophyll cells, and expanded into epidermal and finally guard cells. Because of the large variance in biomass between individuals and species, the current critical level is supported by the assessment of visible leaf symptoms rather than growth reduction.     

Comparison of calculated and measured foliar O3 flux in crop and forest species

We designed a new gas exchange system that concurrently measures foliar H2O, O3, and CO2 flux (HOC flux system) while delivering known O3 concentrations. Stomatal responses of three species were tested: snapbean, and seedlings of California black oak (deciduous broadleaf) and blue oak (evergreen broadleaf). Acute O3 exposure (120-250 ppb over an hour) was applied under moderate light and low vapor pressure deficits during near steady state conditions. The rate of stomatal closure was measured when the whole plant was placed in the dark. An adjacent leaf on each plant was also concurrently measured in an O3-free cuvette. Under some conditions, direct measurements and calculated foliar O3 flux were within the same order of magnitude; however, endogenously low gs or O3 exposure-induced depression of gs resulted in an overestimation of calculated O3 fluxes compared with measured O3 fluxes. Sluggish stomata in response to light extinction with concurrent O3 exposure, and incomplete stomatal closure likewise underestimated measured O3 flux.     

Effects of atmospheric ammonia (NH3) on terrestrial vegetation: a review

At the global scale, among all N (nitrogen) species in the atmosphere and their deposition on to terrestrial vegetation and other receptors, NH3 (ammonia) is considered to be the foremost. The major sources for atmospheric NH3 are agricultural activities and animal feedlot operations, followed by biomass burning (including forest fires) and to a lesser extent fossil fuel combustion. Close to its sources, acute exposures to NH3 can result in visible foliar injury on vegetation. NH3 is deposited rapidly within the first 4-5 km from its source. However, NH3 is also converted in the atmosphere to fine particle NH4+ (ammonium) aerosols that are a regional scale problem. Much of our current knowledge of the effects of NH3 on higher plants is predominantly derived from studies conducted in Europe. Adverse effects on vegetation occur when the rate of foliar uptake of NH3 is greater than the rate and capacity for in vivo detoxification by the plants. Most to least sensitive plant species to NH3 are native vegetation > forests > agricultural crops. There are also a number of studies on N deposition and lichens, mosses and green algae. Direct cause and effect relationships in most of those cases (exceptions being those locations very close to point sources) are confounded by other environmental factors, particularly changes in the ambient SO2 (sulfur dioxide) concentrations. In addition to direct foliar injury, adverse effects of NH3 on higher plants include alterations in: growth and productivity, tissue content of nutrients and toxic elements, drought and frost tolerance, responses to insect pests and disease causing microorganisms (pathogens), development of beneficial root symbiotic or mycorrhizal associations and inter species competition or biodiversity. In all these cases, the joint effects of NH3 with other air pollutants such as all-pervasive O3 or increasing CO2 concentrations are poorly understood. While NH3 uptake in higher plants occurs through the shoots, NH4+ uptake occurs through the shoots, roots and through both pathways. However, NH4+ is immobile in the soil and is converted to NO3- (nitrate). In agricultural systems, additions of NO3- to the soil (initially as NH3 or NH4+) and the consequent increases in the emissions of N2O (nitrous oxide, a greenhouse gas) and leaching of NO3- into the ground and surface waters are of major environmental concern. At the ecosystem level NH3 deposition cannot be viewed alone, but in the context of total N deposition. There are a number of forest ecosystems in North America that have been subjected to N saturation and the consequent negative effects. There are also heathlands and other plant communities in Europe that have been subjected to N-induced alterations. Regulatory mitigative approaches to these problems include the use of N saturation data or the concept of critical loads. Current information suggests that a critical load of 5-10 kg ha(-1) year(-1) of total N deposition (both dry and wet deposition combined of all atmospheric N species) would protect the most vulnerable terrestrial ecosystems (heaths, bogs, cryptogams) and values of 10-20 kg ha(-1) year(-1) would protect forests, depending on soil conditions. However, to derive the best analysis, the critical load concept should be coupled to the results and consequences of N saturation.     

Egyptian plant species as new ozone indicators

The aim of this study was to test and select one or more highly sensitive, specific and environmentally successful Egyptian bioindicator plants for ozone (O3). For that purpose more than 30 Egyptian species and cultivars were subjected to extensive screening studies under controlled environmental and pollutant exposure conditions to mimic the Egyptian environmental conditions and O3 levels in urban and rural sites. Four plant species were found to be more sensitive to O3 than the universally used O3-bioindicator, tobacco Bel W3, under the Egyptian environmental conditions used. These plant species, jute (Corchorus olitorius c.v. local), clover (Trifolium alexandrinum L. c.v. Masry), garden rocket (Eruca sativa c.v. local) and alfalfa (Medicago sativa L. c.v. local), ranked in order of decreasing sensitivity, exhibited typical O3 injury symptoms faster and at lower 03 concentrations than Bel W3. Three variables were tested in search of a reliable tool for the diagnosis and prediction of O3 response prior to the appearance of visible foliar symptoms: pigment degradation, stomatal conductance (g(s)) and net photosynthetic CO2 assimilation (Pnet). Pigment degradation was found to be unreliable in predicting species sensitivity to O3. Evidence supporting stomatal conductance involvement in 03 tolerance was found only in tolerant species. A good correlation was found between g(s), restriction of O3 and CO2 influx into the mesophyll tissues, and Pnet. Changes in Pnet seemed to depend largely on fluctuations in g(s).     

Foliar injury and leaf diffusive resistance of rice and white bean in response to SO2 and O3, singly and in combination

Plants of rice (Oryza sativa) and white bean (Phaseolus vulgaris) were exposed to 524 microg m(-3) SO2, 392 microg m(-3) O3 and a mixture of both gases, i.e. 524 microg m(-3) SO2 and 392 microg m(-3) O3 to determine the visible foliar injury and leaf diffusive resistance. Response of leaf diffusive resistance was measured on upper and lower surfaces of leaves, i.e. the two unifoliate leaves of bean and the first, second and third primary leaves of rice. The difference in the response may be due to sensitive guard cells causing stomatal closure in the presence of O3, whilst a low concentration of SO2 caused the stomata to open. Thus, SO2 alone is known to decrease, and O3 tends to increase leaf diffusive resistance. However, exposure to both gases increases or decreases the resistance, depending on the species response.     

Ozone-induced foliar injury in saplings of Psidium guajava 'Paluma' in São Paulo, Brazil

Psidium guajava 'Paluma' was evaluated under field conditions as a tropical bioindicator species of ozone (O(3)). Three exposures of 90 days each were performed (June 21, 2004-March 19, 2005). In each one of them, saplings of 'Paluma' (n=30) were exposed to ambient air at a site in S?o Paulo (Brazil) with high ozone concentrations, and in a greenhouse with charcoal-filtered air. Ozone-like visible foliar injuries were observed during the winter, spring and summer exposures, when AOT40 reached 6166ppbh, 3504ppbh and 4828ppbh, respectively. No injuries were observed in the plants kept under filtered air. The injuries consisted in red stippling on adaxial leaf surfaces. They did not cover the veins and appeared first in older leaves, becoming more intense as the exposure period increased. Injury incidence, severity, and the cumulative exposure threshold at injury onset varied among the exposure periods. AOT40 explained partly the incidence, severity and leaf injury index LII (r(2)=0.52, 0.39, 0.38, respectively, p=0.002). The results confirm the potential of the species as an O(3)-sensitive bioindicator.