Response to ambient ozone of two white clover (Trifolium repens L.cv. "Regal") clones, one resistant and one sensitive, grown in a Mediterranean environment

Two clones of white clover (Trifolium repens L.) differing in ozone tolerance were grown in southern Italy during 1997 and 1998 to study the effects of ambient ozone exposure on yield, leaf morphology and water use. Ambient ozone levels were high in both years with values exceeding the threshold for leaf injury reported in the literature. In both years ozone injury was observed on the sensitive clone (NC-S) but not on the resistant one (NC-R), and leaf and stolon dry matter production was significantly lower in NC-S than in NC-R. However, it cannot be excluded that other factors, such as high temperature, interacted with the effect of ozone on biomass production. The clones differed in morphological characteristics. Lower total leaf area in NC-S plants was due to a smaller number of leaves per plant, but the average area per leaf was higher in NC-S. Specific leaf weight and net assimilation rate were higher in the more productive clone (NC-R). Cumulative plant water use was higher in NC-R in each growth period because of the larger leaf area; by contrast, water use per unit leaf area was higher in NC-S, indicating higher leaf conductance to water vapour. The results suggest that ozone significantly reduces the yield of sensitive white clover plants under well-watered conditions, and that the difference in ozone tolerance between clover clones is related to differences in leaf morphology and water use.     

Ozone stress and antioxidant substances in Trifolium repens and Centaurea jacea leaves

Ozone-sensitive (NC-S clone) and resistant plants (NC-R clone) of Trifolium repens and Centaurea jacea were exposed to moderate ozone concentrations in ambient air. The aim of this study was the investigation of the relation between ozone-sensitivity and leaf concentrations of antioxidants (ascorbic acid, total phenolics and total antioxidant capacity). NC-R clone showed the highest concentrations of antioxidants with 50-70% more ascorbic acid than NC-S. NC-R had about 5 times more ascorbic acid in the young leaves and 9 times more in the old leaves than Centaurea. In a fumigation experiment with acute ozone stress (100 nl L(-1)) the antioxidant levels changed profoundly. The ozone-injured leaves of NC-S had 6-8 times more total phenolics than uninjured leaves. Generally older leaves had lower antioxidant concentrations and were more prone to ozone injury than younger leaves. Ascorbic acid concentrations were closer related to the appearance of visible ozone injury than the other antioxidative parameters.     

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.     

Development of a multi-factor model for predicting the effects of ambient ozone on the biomass of white clover

Results are presented from the UN/ECE ICP Vegetation (International Cooperative Programme on effects of air pollution on natural vegetation and crops) experiments in which ozone(O(3))-resistant (NC-R) and -sensitive (NC-S) clones of white clover (Trifolium repens cv. Regal) were exposed to ambient O(3) episodes at 14 sites in eight European countries in 1996, 1997 and 1998. The plants were grown according to a standard protocol, and the forage was harvested every 28 days for 4-5 months per year by excision 7 cm above the soil surface. Biomass ratio (NC-S/NC-R) was related to the climatic and pollutant conditions at each site using multiple linear regression (MLR) and artificial neural networks (ANNs). Twenty-one input parameters [e.g. AOT40, 7-h mean O(3) concentration, daylight vapour pressure deficit (VPD), daily maximum temperature] were considered individually and in combination with the aim of developing a model with high r(2) and simple structure that could be used to predict biomass change in white clover. MLR models were generally more complex, and performed less well for unseen data than non-linear ANN models. The ANN model with the best performance had five inputs with an r(2) value of 0.84 for the training data, and 0.71 for previously unseen data. Two inputs to the model described the O(3) conditions (AOT40 and 24-h mean for O(3)), two described temperature (daylight mean and 24-h mean temperature), and the fifth input appeared to be differentiating between semi-urban and rural sites (NO concentration at 17:00). Neither VPD nor harvest interval was an important component of the model. The model predicted that a 5% reduction in biomass ratio was associated with AOT40s in the range 0.9-1.7 ppm x h (microl l(-1) h) accumulated over 28 days, with plants being most sensitive in conditions of low NO(x), medium-range temperature, and high 24-h mean O(3) concentration.     

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.     

Elevated atmospheric ozone increases concentration of insecticidal Bacillus thuringiensis (Bt) Cry1Ac protein in Bt Brassica napus and reduces feeding of a Bt target herbivore on the non-transgenic parent

Sustained cultivation of Bacillus thuringiensis (Bt) transgenic crops requires stable transgene expression under variable abiotic conditions. We studied the interactions of Bt toxin production and chronic ozone exposure in Bt cry1Ac-transgenic oilseed rape and found that the insect resistance trait is robust under ozone elevations. Bt Cry1Ac concentrations were higher in the leaves of Bt oilseed rape grown under elevated ozone compared to control treatment, measured either per leaf fresh weight or per total soluble protein of leaves. The mean relative growth rate of a Bt target herbivore, Plutella xylostella L. larvae was negative on Bt plants in all ozone treatments. On the non-transgenic plants, larval feeding damage was reduced under elevated ozone. Our results indicate the need for monitoring fluctuations in Bt toxin concentrations to reveal the potential of ozone exposure for altering dosing of Bt proteins to target and non-target herbivores in field environments experiencing increasing ozone pollution.     

Fine root dynamics of mature European beech (Fagus sylvatica L.) as influenced by elevated ozone concentrations

Fine root dynamics (diameter < 1 mm) in mature Fagus sylvatica, with the canopies exposed to ambient or twice-ambient ozone concentrations, were investigated throughout 2004. The focus was on the seasonal timing and extent of fine root dynamics (growth, mortality) in relation to the soil environment (water content, temperature). Under ambient ozone concentrations, a significant relationship was found between fine root turnover and soil environmental changes indicating accelerated fine root turnover under favourable soil conditions. In contrast, under elevated ozone, this relationship vanished as the result of an altered temporal pattern of fine root growth. Fine root survival and turnover rate did not differ significantly between the different ozone regimes, although a delay in current-year fine root shedding was found under the elevated ozone concentrations. The data indicate that increasing tropospheric ozone levels can alter the timing of fine root turnover in mature F. sylvatica but do not affect the turnover rate.     

Use of sap flow measurements to validate stomatal functions for mature beech (Fagus sylvatica) in view of ozone uptake calculations

For a quantitative estimate of the ozone effect on vegetation reliable models for ozone uptake through the stomata are needed. Because of the analogy of ozone uptake and transpiration it is possible to utilize measurements of water loss such as sap flow for quantification of ozone uptake. This technique was applied in three beech (Fagus sylvatica) stands in Switzerland. A canopy conductance was calculated from sap flow velocity and normalized to values between 0 and 1. It represents mainly stomatal conductance as the boundary layer resistance in forests is usually small. Based on this relative conductance, stomatal functions to describe the dependence on light, temperature, vapour pressure deficit and soil moisture were derived using multivariate nonlinear regression. These functions were validated by comparison with conductance values directly estimated from sap flow. The results corroborate the current flux parameterization for beech used in the DO₃SE model.     

Ozone fumigation (twice ambient) reduces leaf infestation following natural and artificial inoculation by the endophytic fungus Apiognomonia errabunda of adult European beech trees

In 2006, a controlled infection study was performed in the ‘Kranzberger Forst’ to address the following questions: (1) Will massive artificial inoculation with Apiognomonia errabunda override the previously observed inhibitory effect of chronic ozone? (2) Can biochemical or molecular markers be detected to account for the action of ozone? To this end six adult beech trees were chosen, three ozone fumigated (2× ozone) and three control trees (ambient = 1× ozone). Spore-sprayed branches of sun and shade crown positions of each of the trees, and uninoculated control branches, were enclosed in 100-L plastic bags for one night to facilitate infection initiation. Samples were taken within a five-week period after inoculation. A. errabunda infestation levels quantified by real-time PCR increased in leaves that were not fumigated with additional ozone. Cell wall components and ACC (ethylene precursor 1-amino cyclopropane-1-carboxylic acid) increased upon ozone fumigation and may in part lead to the repression of fungal infection.     

Suitability of Nicotiana tabacum 'Bel W3' for biomonitoring ozone in São Paulo, Brazil

Nicotiana tabacum 'Bel W3' is a widely used sensitive bioindicator for ambient ozone, but it is rarely used in tropical countries. Our goal was to determine the suitability of this plant for biomonitoring ozone in the city of S?o Paulo by evaluating the relationships between leaf necroses and ozone under field conditions and measurements of chlorophyll a fluorescence and antioxidants in plants exposed to different concentrations of ozone in closed chambers. While a weak linear relationship between leaf injury and ozone concentrations (R(2)=0.10) was determined in the field, a strong linear relationship was observed in the chamber experiments. Maximum leaf injury was observed in plants submitted to 40 ppb, which coincided with a significant decrease in fluorescence and total ascorbic acid. The relationship between leaf damage observed in the field and ozone was improved when the concentrations were limited to 40 ppb (R(2)=0.28).     

Validation of the stomatal flux approach for the assessment of ozone visible injury in young forest trees. Results from the TOP (transboundary ozone pollution) experiment at Curno, Italy

This paper summarises some of the main results of a two-year experiment carried out in an Open-Top Chambers facility in Northern Italy. Seedlings of Populus nigra, Fagus sylvatica, Quercus robur and Fraxinus excelsior have been subjected to different ozone treatments (charcoal-filtered and non-filtered air) and soil moisture regimes (irrigated and non-irrigated plots). Stomatal conductance models were applied and parameterised under South Alpine environmental conditions and stomatal ozone fluxes have been calculated.The flux-based approach provided a better performance than AOT40 in predicting the onset of foliar visible injuries. Critical flux levels, related to visible leaf injury, are proposed for P. nigra and F. sylvatica (ranging between 30 and 33 mmol O₃ m⁻²). Soil water stress delayed visible injury appearance and development by limiting ozone uptake. Data from charcoal-filtered treatments suggest the existence of an hourly flux threshold, below which may occur a complete ozone detoxification.     

Deciduous shrubs for ozone bioindication: Hibiscus syriacus as an example

Ozone-like visible injury was detected on Hibiscus syriacus plants used as ornamental hedges. Weekly spray of the antiozonant ethylenediurea (EDU, 300 ppm) confirmed that the injury was induced by ambient ozone. EDU induced a 75% reduction in visible injury. Injury was more severe on the western than on the eastern exposure of the hedge. This factor of variability should be considered in ozone biomonitoring programmes. Seeds were collected and seedlings were artificially exposed to ozone in filtered vs. not-filtered (+30 ppb) Open-Top Chambers. The level of exposure inducing visible injury in the OTC seedlings was lower than that in the ambient-grown hedge. The occurrence of visible injury in the OTC confirmed that the ozone sensitivity was heritable and suggested that symptomatic plants of this deciduous shrub population can be successfully used as ozone bioindicators. EDU is recommended as a simple tool for diagnosing ambient ozone visible injury on field vegetation.     

Spatial mapping of VOC and NOx-limitation of ozone formation in central California

Ambient aerometric data were used to predict whether ozone formation at specific times and locations in central California was limited by the availability of volatile organic compounds (VOC) or oxides of nitrogen (NO_x). The predictions were compared with differences between mean weekday and weekend peak ozone values. The comparison with weekend and weekday ozone levels provided a means for empirically investigating the effects of VOC and NO_x reductions on ozone formation, because the relative proportions and levels of ozone precursor species were significantly different on weekends than on weekdays. Weekend NO_x levels averaged 27 percent lower than weekday levels at the time of the peak ozone hour. Daytime weekend levels of VOC species were also consistently lower than weekday values throughout the region, though the differences between weekends and weekdays were not always statistically significant (p<0.05). Site-to-site differences between weekend and weekday mean peak hourly ozone were related to whether ozone formation was VOC- or NO_x-limited.     

Tibouchina pulchra (Cham.) Cogn., a native Atlantic Forest species, as a bio-indicator of ozone: visible injury

Tibouchina pulchra saplings were exposed to carbon filtered air (CF), ambient non-filtered air (NF) and ambient non-filtered air+40 ppb ozone (NF+O3) 8 h per day during two months. The AOT40 values at the end of the experiment were 48, 910 and 12,895 ppb h(-1), respectively, for the three treatments. After 25 days of exposure (AOT40=3871 ppb h(-1)), interveinal red stippling appeared in plants in the NF+O3 chamber. In the NF chamber, symptoms were observed only after 60 days of exposure (AOT40=910 ppb h(-1)). After 60 days, injured leaves per plant corresponded to 19% in NF+O3 and 1% in the NF treatment; and the average leaf area injured was 7% within the NF+O3 and 0.2% within the NF treatment. The extent of leaf area injured (leaf injury index) was mostly explained by the accumulated exposure of ozone (r2=0.89; p<0.05).     

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.     

Aircraft measurement of ozone turbulent flux in the atmospheric boundary layer

In May 1995, the “Chimie-Creil 95” experiment was undertaken in the north of France. The field data are first used to validate the methodology for airborne measurement of ozone flux. A certain number of methodological problems due to the location of the fast ozone sensor inside the airplane are, furthermore discussed. The paper describes the instrumentation of the ARAT (Avion de Recherche Atmosphérique et de Télédétection), an atmospheric research and remote-sensing aircraft used to perform the airborne measurements, the area flown over, the meteorological conditions and boundary layer stability conditions. These aircraft measurements are then used to determine ozone deposition velocity and values are proposed for aerodynamic, bulk transfer coefficients (ozone and momentum). The paper also establishes the relationship between the normalised standard deviation and stability parameters (z/L) for ozone, temperature, humidity and vertical velocity. The laws obtained are then presented.     

Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU)

Cutleaf coneflower (Rudbeckia laciniata L.) seedlings were placed into open-top chambers in May, 2004 and fumigated for 12 wks. Nine chambers were fumigated with either carbon-filtered air (CF), non-filtered air (NF) or twice-ambient (2×) ozone (O₃). Ethylenediurea (EDU) was applied as a foliar spray weekly at 0 (control), 200, 400 or 600 ppm. Foliar injury occurred at ambient (30%) and elevated O₃ (100%). Elevated O₃ resulted in significant decreases in biomass and nutritive quality. Ethylenediurea reduced percent of leaves injured, but decreased root and total biomass. Foliar concentrations of cell-wall constituents were not affected by EDU alone; however, EDU × O₃ interactions were observed for total cell-wall constituents and lignocellulose fraction. Our results demonstrated that O₃ altered the physiology and productivity of cutleaf coneflower, and although reducing visible injury EDU may be phytotoxic at higher concentrations.     

Nitrogen mediates above-ground effects of ozone but not below-ground effects in a rhizomatous sedge

Ozone and atmospheric nitrogen are co-occurring pollutants with adverse effects on natural grassland vegetation. Plants of the rhizomatous sedge Carex arenaria were exposed to four ozone regimes representing increasing background concentrations (background-peak): 10-30, 35-55, 60-80 and 85-105 ppb ozone at two nitrogen levels: 12 and 100 kg N ha⁻¹ yr⁻¹. Ozone increased the number and proportion of senesced leaves, but not overall leaf number. There was a clear nitrogen × ozone interaction with high nitrogen reducing proportional senescence in each treatment and increasing the ozone dose (AOT40) at which enhanced senescence occurred. Ozone reduced total biomass due to significant effects on root biomass. There were no interactive effects on shoot:root ratio. Rhizome tissue N content was increased by both nitrogen and ozone. Results suggest that nitrogen mediates above-ground impacts of ozone but not impacts on below-ground resource translocation. This may lead to complex interactive effects between the two pollutants on natural vegetation.     

Ozone uptake by citrus trees exposed to a range of ozone concentrations

The Citrus genus includes a large number of species and varieties widely cultivated in the Central Valley of California and in many other countries having similar Mediterranean climates. In the summer, orchards in California experience high levels of tropospheric ozone, formed by reactions of volatile organic compounds (VOC) with oxides of nitrogen (NO_x). Citrus trees may improve air quality in the orchard environment by taking up ozone through stomatal and non-stomatal mechanisms, but they may ultimately be detrimental to regional air quality by emitting biogenic VOC (BVOC) that oxidize to form ozone and secondary organic aerosol downwind of the site of emission. BVOC also play a key role in removing ozone through gas-phase chemical reactions in the intercellular spaces of the leaves and in ambient air outside the plants. Ozone is known to oxidize leaf tissues after entering stomata, resulting in decreased carbon assimilation and crop yield. To characterize ozone deposition and BVOC emissions for lemon (Citrus limon), mandarin (Citrus reticulata), and orange (Citrus sinensis), we designed branch enclosures that allowed direct measurement of fluxes under different physiological conditions in a controlled greenhouse environment. Average ozone uptake was up to 11 nmol s^?1 m^?2 of leaf. At low concentrations of ozone (40 ppb), measured ozone deposition was higher than expected ozone deposition modeled on the basis of stomatal aperture and ozone concentration. Our results were in better agreement with modeled values when we included non-stomatal ozone loss by reaction with gas-phase BVOC emitted from the citrus plants. At high ozone concentrations (160 ppb), the measured ozone deposition was lower than modeled, and we speculate that this indicates ozone accumulation in the leaf mesophyll.