Instrumental recording and biomonitoring of ambient ozone in the Greek countryside

Among eight commercial Greek varieties of tobacco (Nicotiana tabacum L.) tested for their ozone-sensitivity levels, the Zichnomirodata (KK6/5) variety was found to be the most sensitive, although less sensitive than the well-known super-sensitive Bel-W3. Besides qualitative differences in the appearance of macroscopic symptoms these two varieties can be used simultaneously as a reliable pair of ozone bioindicators. The occurrence of ozone in the Greek countryside was surveyed by biomonitoring in 14 rural regions over the country and by a simultaneous biomonitoring and instrumental recording of ozone concentrations at a single remote side (Pournaria, Arcadia). Phytotoxic symptoms were observed mainly on the leaves of Bel-W3 and occasionally on those of Zichnomirodata varieties, suggesting that ozone levels were high enough to affect at least sensitive species. The instrumental monitoring (during a total period of 912 h) revealed maximum hourly O3 concentration 62 ppb, while the thresholds of 30, 40 and 50 ppb were exceeded for 40%, 20% and 6% of the recording period, respectively. The accumulated exposure over 40 ppb (AOT40) for the daylight hours over the 38 monitored days was 680 ppb h.     

Effects of ozone on chlorophyll and quantum yield of tobacco (Nicotiana tabacum L.) varieties

Plants of Bel-W3 and of seven commercial tobacco varieties (Nicotiana tabacum L.) were exposed to two relatively low ozone concentrations (90 or 135 ppb) for 20 consecutive days, for 8 h per day. Ozone caused necrotic and chlorotic spots, acceleration of leaf senescence, depression of photosynthetic mechanism, chlorophyll diminution and greater destruction of chl a than of chl b. The higher sensitivity of chl a was also confirmed by exposure of segments of leaves in test tubes to high ozone concentration (>1000 ppb) as well as by bubbling of ozone in extracts of chlorophyll in vitro. The quantum yield (QY) of photosynthesis was positively correlated with the chlorophyll content and negatively correlated with the visible injury and the chl b/a ratio.     

Effects of ozone on chlorophyll and quantum yield of tobacco (Nicotiana tabacum L.) varieties

Plants of Bel-W3 and of seven commercial tobacco varieties (Nicotiana tabacum L.) were exposed to two relatively low ozone concentrations (90 or 135 ppb) for 20 consecutive days, for 8 h per day. Ozone caused necrotic and chlorotic spots, acceleration of leaf senescence, depression of photosynthetic mechanism, chlorophyll diminution and greater destruction of chl a than of chl b. The higher sensitivity of chl a was also confirmed by exposure of segments of leaves in test tubes to high ozone concentration (>1000 ppb) as well as by bubbling of ozone in extracts of chlorophyll in vitro. The quantum yield (QY) of photosynthesis was positively correlated with the chlorophyll content and negatively correlated with the visible injury and the chl b/a ratio.     

Ozone as a Tobacco Toxicant

Ozone is very toxic to tobacco, causing serious injury in greenhouses and fields. Certain varieties, differing in sensitivity, have proved useful as indicators of the presence, and to some extent the level, of air-polluting ozone. Environmental factors alter plant response to ozone and must be taken into account. What is now recognized as typical ozone injury to tobacco was first observed in Connecticut in 1951, Maryland in 1952, eastern North Carolina in 1953, and Ontario, Canada, in 1955. Cracking of rubber strips exposed daily at six locations in 1959 in the Connecticut Valley showed ozone concentrations were highest the day before a fleck outbreak, resulting in more than a million dollar loss. Measurement with a Mast ozone recorder from July to mid-October for two seasons at Beltsville, Md., six miles northeast of the District of Columbia, indicated 2.5 pphm as the average daily maximum value, 5 pphm as the level when plant injury is expected, and 10 pphm as very high and infrequent. The neutral buffered Kl method indicated higher ozone values, but about 15 pphm was maximum with this method. Shading portions of leaves for three hours in midday prevented ozone injury. Increased injury in the different tobacco areas was related to increased culture of more susceptible varieties. Although various antiozonants applied to the leaf, or even applied to cloth shade, reduce damage, the development of resistant tobaccos has proved the better means of control. Premature senescence and related chlorosis as well as fleck were prevented by carbon-filtered air, indicating that ozone may be a more important toxicant than previously recognized. Possibly oxidants other than ozone contributed to plant injury, but, if present, they were removed by carbon-filters. Growth of an ozone-sensitive tobacco Bel-W3, as indicated by dry weight, was only half as much in ambient as in carbonfiltered air in one experiment with chambers continued for 5 weeks. Evidence of synergistic action of sulfur dioxide and ozone is reported. Subthreshold concentrations of these toxicants produced injury following exposure of tobacco to mixed gases, whereas no injury resulted from the same concentration of the individual gases.     

Measurement of Photochemical Air Pollution with a Sensitive Monitoring Plant

With newly developed monitoring techniques, investigators used the sensitive tobacco variety, Bel-W3, as a monitor for photochemical air pollution at different locations within 75 miles of Cincinnati, Ohio. Observations during the summers of 1966 through 1968 revealed almost daily injury to monitoring plants, fairly uniform total seasonal injury at all locations, and a marked variation in both oxidant and injury from one season to another within the city. The relationship between oxidant level and injury was not found to be consistent.     

Background ozone monitoring and phytodetection in the greater rural area of Corinth--Greece

Natural background ozone levels were monitored in three places within the greater rural area of Corinth, namely Bogdani Hill, Astronomical Observatory of Krionerion, and Kiato, and compared with ambient ozone monitored in the metropolitan area of Athens. Measurements were made sequentially, for a few weeks at each place, during the summer of 2000. In addition, ozone phytodetection, using tobacco (Nicotiana tabacum L.) plants of the Bel-W3 and Zichnomirodata varieties, was conducted in 12 places (the above included). Moreover, stomatal conductance was measured in the Bel-W3 plants, as well as in leaves of cultivated grape-vines (Vitis vinifera L.) and in needles of Aleppo pine (Pinus halepensis Mill.) trees and compared with the diurnal pattern of ozone concentrations.The 24 and 12 (08:00-20:00) hourly averages of ozone concentrations were high in Athens (37; 51 ppb), at Bogdani Hill (53; 56 ppb) and at the Astronomical Observatory (56; 55 ppb), but relatively low in Kiato (30; 34 ppb). Furthermore, the average daily AOT40 (accumulated exposure over 40 ppb for the daylight hours) (ppbh) was 193 in Athens, 212 at Bogdani Hill, 192 at the Astronomical Observatory and 47 in Kiato. Ozone concentrations exhibited the usual diurnal pattern in Athens (altitude 50 m), where they were maximum during midday and early afternoon hours, as well as at Bogdani Hill (300 m) and in Kiato (5 m) where, however, they were maximal 1-3 h later. At the Astronomical Observatory (altitude 920 m) ozone remained constant during both daylight and night hours. The differences in diurnal patterns are consistent with those in places of different elevation, reported elsewhere.The Bel-W3 plants were injured at all 12 places; Zichnomirodata plants exhibited lower injury and only in some of the places; probable ozone symptoms were also observed on vine plants and pine trees. The greatest injury was observed at the high altitude places of Astronomical Observatory and Mougostos. Stomatal conductance, in all three species, peaked during morning and early midday hours when ozone levels were higher in the high altitude, and lower in the low altitude, places.     

Tobacco clones derived from tissue culture with supersensitivity to ozone

At least two supersensitive tobacco somaclones were obtained from tissue culture (TC) , when this approach was used to asexually propagate Bel-W3 tobacco indicator plants. These somaclones can detect as low as 30 ppb ozone for a 4-h exposure duration both within CSTR exposure chambers and in ambient air. Comparison of the injury index and their coefficient of variance showed that the TC plantlets usually have more uniform performance in response to ozone in addition to their higher sensitivity. A quick regeneration procedure was established to preserve the supersensitive germplasm immediately when it was found. The TC plantlets will flower and produce seed similar to seed-grown tobacco. The TC approach proved to be a better propagation system for valuable indicator plant species. The mechanism that causes the variation and the possible difference in their genome from seed-grown tobacco is still unknown. Further studies are needed in the future to determine if factors in the TC system may be responsible for the sensitivity difference.     

Photochemical air pollutant levels and ozone phytotoxicity in the region of Mesogia–Attica, Greece

The levels of photochemical air pollutants: O₃, NO and NO₂, were monitored in Athens and in the neighbouring region of the Mesogia plain (Spata, Artemis and Markopoulo) from 1 May–31 August 2000. Phytodetection of ozone was also conducted using bioindicator plants of Bel-W3 and Zichnomirodata (KK6/5) tobacco varieties. The average maximum daily O3 concentration was 60–75 ppb, while the 24 hour average ranged from 40–65 ppb. The AOT40 (ppb hours) index was very high in Athens (16 679 over 121 days), Spata (16 325 over 110 days), Artemis (8093 over 22 days) and Markopoulo (18 646 over 113 days), suggesting potentially phytotoxic ozone levels. The ozone bioindicator plants of Bel-W3 and KK6/5 tobacco varieties were highly injured in all regions confirming the phytotoxicity of those ozone levels. The levels of NO and NO₂ recorded at the three stations, in the Mesogia plain, were considerably lower than those occurring in Athens. The data presented here provide important background information concerning pollution levels in the Mesogia plain shortly before the operation of the new international airport 'Eleftherios Venizelos' in this region (March 2001).     

Ambient ozone (O3) and adverse crop response: a unified view of cause and effect

This paper presents a cohesive view of the dynamics of ambient O(3) exposure and adverse crop response relationships, coupling the properties of photochemical O(3) production, flux of O(3) from the atmosphere into crop canopies and the crop response per se. The results from two independent approaches ((a) statistical and (b) micrometeorological) were analyzed for understanding cause-effect relationships of the foliar injury responses of tobacco cv Bel-W3 to the exposure dynamics of ambient O(3) concentrations. Similarly, other results from two independent approaches were analyzed in: (1) establishing a micrometeorological relationship between hourly ambient O(3) concentrations and their vertical flux from the air into a natural grassland canopy; and (2) establishing a statistical relationship between hourly ambient O(3) concentrations in long-term, chronic exposures and crop yield reductions. Independent of the approach used, atmospheric conditions appeared to be most conducive and the crop response appeared to be best explained statistically by the cumulative frequency of hourly ambient O(3) concentrations between 50 ppb and 90 ppb (100 and 180 microg m(-3)). In general, this concentration range represents intermediate or moderately enhanced hourly O(3) values in a polluted environment. Further, the diurnal occurrence of this concentration range (often approximately between 0900 and 1600 h in a polluted, agricultural environment) coincided with the optimal CO(2) flux from the atmosphere into the crop canopy, thus high uptake. The frequency of occurrence of hourly O(3) concentrations > 90 ppb (180 microg m(-3)) appeared to be of little importance and such concentrations in general appeared to occur during atmospheric conditions which did not facilitate optimal vertical flux into the crop canopy, thus low uptake. Alternatively, when > 90 ppb (180 microg m(-3)) O(3) concentrations occurred during the 0900-1600 h window, their frequency of occurrence was low in comparison to the 50-90 ppb (100-180 microg m(-3)) range. Based on the overall results, we conclude that if the cumulative frequency of hourly ambient O(3) concentrations between 50-62 ppb (100-124 microg m(-3)) occurred during 53% of the growing season and the corresponding cumulative frequency of hourly O(3) concentrations between 50-74 ppb (100-148 microg m(-3)) occurred during 71% of the growing season, then yield reductions in sensitive crops could be expected, if other factors supporting growth, such as adequate soil moisture are not limiting.     

Relationship between leaf antioxidants and ozone injury in Nicotiana tabacum ‘Bel-W3’ under environmental conditions in São Paulo,SE – Brazil

Previous studies have reported that the extent of leaf injury in Nicotiana tabacum “Bel-W3” exposed to environmental conditions in the city of São Paulo is influenced by weather conditions. This influence may occur by means of antioxidant responses. Thus, this study aimed to evaluate whether daily antioxidant responses to environmental variations interfere on the progression of leaf injury on plants of this cultivar during their exposure in a state park of São Paulo and to determine a linear combination of variables, among antioxidants and environmental factors, which mostly explain this visible response. Plants were exposed at the mentioned site for 14 days in four different experiments. During each experiment, three plants were daily sampled to determine the accumulated percentage of leaf area affected by necrosis and antioxidant responses (concentrations of total ascorbic acid (AA) and activity of superoxide dismutase (SOD) and peroxidases (POD)). Ozone concentrations and weather conditions were also daily measured. Pearson correlations and multivariate analyses assessed the relationship between biological and environmental variables. Leaf injury appeared between the 3rd and 6th days of exposure and increased over the exposure periods. The daily concentrations of AA tended to decrease with time of exposure in all experiments, but the activity of SOD and POD oscillated during plant exposure. Positive correlations were observed between AA or SOD and O₃ concentrations, as well as negative correlations between AA and air temperature. The increasing percentage of leaf necrosis across the whole period was explained by decreasing levels of AA 2 days before injury estimation and by higher O₃ concentrations 5 days before (R² = 0.36; p < 0.001). The use of N. tabacum Bel-W3 as a bioindicator can be restricted by leaf antioxidant responses to both atmospheric contamination and weather conditions.     

Detecting ozone and demonstrating its phytotoxicity in forested areas of Poland: a pilot study

Ambient concentrations of ozone (O(3)) were measured and O(3) phytotoxicity to tobacco (Nicotiana tabacum L.) was demonstrated in several forest locations in Poland during a pilot study from July-October, 1991. At southern and central locations in Poland, the 24-hour average O(3) concentrations measured with a UV absorption photometer were in the range of 32-55 ppb, and the corresponding 1-hour maxima in the range of 39-83 ppb. At these locations longer period (four to fifteen days) average concentrations were determined using O(3) passive samplers (DGA, Inc.) and were reaching 60 ppb, while at Bialowieza in eastern Poland O(3) concentrations averaged less than 40 ppb. In Szarow, near the Niepolomice Forest in southern Poland, 1-hour O(3) maxima estimated from the data obtained using passive samplers were about 105 ppb in early September. At several locations in southern and central Poland, extensive O(3) injury was determined on O(3)-sensitive Bel W-3 tobacco plants; such injury did not occur in the Bialowieza Forest of eastern Poland. The results of this pilot study indicate that O(3) is present at phytotoxic levels in southern and central Poland.     

Interaction of tobacco etch or tobacco vein mottling virus and ozone on biomass changes in burley tobacco

Burley tobacco is susceptible to several different types of virus diseases that suppress plant growth and development. Two viruses, tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), are particularly damaging to burley. Burley tobacco cultivars resistant to these two viruses are currently being developed. Some of these cultivars also show differential sensitivity to ozone (O3). Recent field observations have suggested that burley tobacco infected with TEV and TVMV was more sensitive to O3 than non-virus-infected tobacco. Experiments were designed to identify interactions between O3 and each of the two virus diseases. Three cultivars, Burley 21, Burley 49, and Greeneville 131, which were differentially sensitive to O3 and both virus diseases, were grown in a charcoal-filtered greenhouse environment. Tobacco plants of each cultivar were inoculated with TEV or TVMV, and virus infected and virus-free plants were exposed to 0.0, 0.05, 0.2, and 0.4 ppm O3 (1 ppm of O3 is equivalent to 1960 microg m(-3)), 3h day(-1), 5 days week(-1) for 3 weeks in continuous-stirred tank reactor exposure chambers in the greenhouse. Exposures were begun after systemic virus symptoms were expressed in inoculated plants. The suppression of lead and stem dry weight by increasing O3 concentrations was less in TEV-infected burley cultivars than in noninfected burley cultivars. Tobacco vein mottling virus infection enhanced biomass suppression by O3 on Burley 21 and on Greeneville 131, but not on Burley 49. Thus, the interactions with O3 were dependent on specific virus-cultivar combinations.     

Responses of bean (Phaseolus vulgaris L. cv. Pros) to chronic ozone exposure at two levels of atmospheric ammonia

Plants of bean (Phaseolus vulgaris cv. Pros) were exposed to a range of O3 concentrations up to 70 nl litre(-1) for 9 h day(-1) in the presence (45 nl litre(-1)) and absence (21 nl litre(-1)) of enhanced NH3 in 12 open-top chambers. Treatment effects on visible injury, growth and yield were assessed after 49 (intermediate harvest) and 62 days of exposure (final harvest). The proportion of leaves with visible injury at final harvest increased with increasing concentrations of O3. Enhanced NH3 did not cause any symptoms and did not affect injury by O3. The estimated seasonal mean concentration corresponding with 5% injury was circa 23 nl litre(-1) O3. Biomass production and green pod yield decreased with increasing concentrations of O3 and were generally stimulated by enhanced NH3 at both harvests. Adverse effects of O3 on biomass and pod yield did not depend on the NH3 level. Relative yield response to increasing 9-h daily mean O3 concentrations was nonlinear and yield losses of 5 and 10% were calculated to occur at seasonal daytime mean concentrations of 27 and 33 nl litre(-1) O3, respectively. Linear regression showed that the Accumulated exposures Over a Threshold of 30 (AOT30) and 40 nl litre(-1) (AOT40) O3 performed equally well. The estimated accumulated O3 exposures corresponding with a yield loss of 5% were 1600 nl litre(-1) h for AOT30 and 400 nl litre(-1) h for AOT40. The results are discussed in relation to the long-term critical level that is used as a guideline to protect crops against adverse effects by O3.     

Impact of increased springtime O3 exposure on Scots pine (Pinus sylvestris) seedlings in central Finland

Three-year-old Scots pine (Pinus sylvestris L.) seedlings were exposed to ambient or elevated ozone (O(3)) concentrations in open-air exposure fields in central Finland in 1995-97. Three different treatments were applied in 1996 and 1997: ambient air, elevated O(3) (1.3-1.5xambient) during the growing season (June-September) and elevated O(3) in March-September, i.e. the growing season including the springtime O(3) exposure. The ambient mean O(3) concentrations were 40% higher in springtime (March-May) compared to the concentrations during the growing seasons. Maximum O(3) concentrations were measured in April or early May, whereas a clear increase in the stomatal activity of the seedlings was observed by the middle of May. This suggests a low intake of O(3) by conifers despite the higher O(3) concentrations in spring. Stomatal conductance, and contents of chlorophyll and ribulosebisphosphate carboxylase/oxygenase (Rubisco) in current-year needles were not significantly affected by any O(3) treatment. Only a slight decrease in current-year shoot growth, slight increase in the abscission of 2-year-old needles and increased electron density of chloroplast stroma by springtime O(3) exposure suggest a rather small contribution of elevated springtime O(3) concentrations to total O(3) damage under current climatic conditions in Finland. However, the increases in springtime O(3) concentrations may enhance the cumulative effects of O(3) during long-term O(3) exposures.     

Apoplastic ascorbate contributes to the differential ozone sensitivity in two varieties of winter wheat under fully open-air field conditions

We studied leaf apoplastic ascorbates in relation to ozone (O₃) sensitivity in two winter wheat (Triticum aestivum L.) varieties: Yangfumai 2 (Y2) and Yangmai 16 (Y16). The plants were exposed to elevated O₃ concentration 27% higher than the ambient O₃ concentration in a fully open-air field from tillering stage until final maturity. The less sensitive variety Y16 had higher concentration of reduced ascorbate in the apoplast and leaf tissue by 33.5% and 12.0%, respectively, than those in the more sensitive variety Y2, whereas no varietal difference was detected in the decline of reduced ascorbate concentration in response to elevated O₃. No effects of O₃ or variety were detected in either oxidized ascorbate or the redox state of ascorbate in the apoplast and leaf tissue. The lower ascorbate concentrations in both apoplast and leaf tissue should have contributed to the higher O₃ sensitivity in variety Y2.     

Integrated biomonitoring of air quality with plants and lichens: a case study on ambient ozone from central Italy

A biennial integrated survey, based on the use of vascular plants for the bioindication of the effects of tropospheric ozone together with the use of automatic analysers of ozone, as well as the mapping of lichen biodiversity was performed in the area of Castelfiorentino (Tuscany, central Italy). Photochemically produced ozone proved to be a fundamental presence during the warm season, with maximum hourly means reaching 114 ppb, exceeding the information threshold as fixed by EU: the use of supersensitive tobacco Bel-W3 confirmed the opportunity of carrying out detailed cost-effective monitoring surveys. The potential for didactical and educational implications of this methodology are appealing. Critical levels set up for the protection of vegetation have exceeded considerably. The comparison of biomass productivity in sensitive and resistant individuals (NC-S and NC-R white clover clones, in the framework of an European network) provided evidence that ambient ozone levels are associated with relevant reduction (up to 30%) in the performance of sensitive material; effects on flowering were also pronounced. The economic assessment of such an impact deserves attention. Mapping of epiphytic lichen biodiversity – which has been used to monitor air quality worldwide – was not related to ozone geographical distribution as depicted by tobacco response.     

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.     

Differential response of CO2 uptake parameters of soil- and sand-grown phaseolus vulgaris (L.) plants to absorbed ozone flux

Shoots of a soil- or sand-grown dwarf bean variety were exposed to O(3) concentrations in the range of 500 to 900 ppb for up to 5 h. The measured exchange rates of water vapor and CO(2) during exposures were used to calculate stomatal and mesophyll conductances averaged across all leaves. Changes in conductances were related to exposure duration and absorbed O(3) totals (AOT). Both conductances were more sensitive to AOT in sand-grown plants, which also had more visible injury under comparable AOT values. Measurements of the relationship between CO(2) exchange and internal CO(2) concentration of single leaflets of treated plants also showed greater sensitivity of CO(2)-saturated photosynthesis in sand-grown plants. Diffusional processes were not likely to have been the cause of dissimilar responses because the O(3) absorption rate was lower in sand-grown plants. A difference in the scaveninng capacities in cells is suggested to be the cause of the differences in sensitivity to acute O(3) exposure.