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.     

Leaf phenolic compounds in red clover (Trifolium pratense L.) induced by exposure to moderately elevated ozone

Red clover (Trifolium pratense L.), an important feed crop in many parts of the world, is exposed to elevated ozone over large areas. Plants can limit ozone-induced damages by various defence mechanisms. In this work, changes in the concentrations of antioxidant phenolic compounds induced by slightly elevated levels of ozone were determined in red clover leaves by high-performance liquid chromatography and mass spectrometry. 31 different phenolics were identified and the most abundant isoflavones and flavonoids were biochanin A glycoside malonate (G-M), formononetin-G-M and quercetin-G-M. Elevated ozone (mean 32.4 ppb) increased the total phenolic content of leaves and also had minor effects on the concentrations of individual compounds. Elevated ozone increased the net photosynthesis rate of red clover leaves before visible injuries by 21-23%. This study thus suggests that the concentrations of phenolics in red clover leaves change in response to slightly elevated ozone levels.     

Effects of ozone on managed pasture: II. Yield, species composition, canopy structure, and forage quality

Managed pasture composed of grasses, clover and weeds was exposed in open-top chambers to different levels of ozone (O(3)) during two consecutive seasons to study changes in yield, species composition, canopy structure, and forage quality. The pasture was established in 1990 and exposed in 1991 and 1992. Ozone treatments included charcoal-filtered air (CF), non-filtered air (NF), and two treatments with O(3) added to NF air during periods with global radiation >/= 400 W m(-2) (NF(+), NF(++)). The ratio between the 2-year cumulative, radiation-weighted O(3) concentration in ambient air (= 365 microl litre(-1) h) and in the different treatments was 0.50 (CF), 0.85 (NF), 1.11 (NF(+)), and 1.64 (NF(++)). Plots were harvested four times in 1991, and five times in 1992. The total forage yield for both seasons was modified little by O(3). The yield reduction in NF(++) was only 10% as compared to the CF treatment. Also, only marginal changes were observed in forage quality (Ca, crude protein, crude fibre), and in leaf area index and fractional light penetration. Ozone strongly reduced the yield of clover (Trifolium repens L. and Trifolium pratense L.). The O(3)-effect on clover growth was small after the first harvest and increased with each growth period. In NF, the 2-year cumulative clover yield was reduced by 24% relative to CF. In NF(++), clover growth almost ceased near the end of the second season. The reduction in clover yield with increasing O(3) was associated with a slight increase in the yield of grasses (mainly Dacytlis glomerata L). The increase in the proportion of invading species (weeds or herbs) (Taraxacum officinale L.) during the experiment was not significantly affected by O(3). A second order polynomial function was fitted to the data to establish an exposure-response model for the cumulative clover yield and the cumulative, radiation-weighted O(3) dose, and linear models were developed for total forage mass, grass yield and yield of weeds. Reducing O(3) from elevated levels (NF(+) and NF(++)) during the first season to near-ambient levels (NF) during the second season resulted in a significant recovery of clover yield after two re-growth periods. It is concluded that continuous exposure to ambient levels of O(3) negatively affects the yield of clover in frequently cut, managed pasture, but because of the relatively small proportion of clover, the shift in species composition only marginally affects total forage yield and forage quality. It is emphasised, however, that limitations of the experimental system must be taken into account before extrapolations to real field situations can be made.     

Arbuscular mycorrhiza enhanced arsenic resistance of both white clover (Trifolium repens Linn.) and ryegrass (Lolium perenne L.) plants in an arsenic-contaminated soil

In a compartmented cultivation system, white clover (Trifolium repens Linn.) and ryegrass (Lolium perenne L.), with their roots freely intermingled, or separated by 37 microm nylon mesh or plastic board, were grown together in an arsenic (As) contaminated soil. The influence of AM inoculation on plant growth, As uptake, phosphorus (P) nutrition, and plant competitions were investigated. Results showed that both plant species highly depended on mycorrhizas for surviving the As contamination. Mycorrhizal inoculation substantially improved plant P nutrition, and in contrast markedly decreased root to shoot As translocation and shoot As concentrations. It also showed that mycorrhizas affected the competition between the two co-existing plant species, preferentially benefiting the clover plants in term of nutrient acquisition and biomass production. Based on the present study, the role of AM fungi in plant adaptation to As contamination, and their potential use for ecological restoration of As contaminated soils are discussed.     

A modified glass bead compartment cultivation system for studies on nutrient and trace metal uptake by arbuscular mycorrhiza

A modified glass bead compartment cultivation system is described in which glass beads continue to be used in the hyphal compartment but are replaced by coarse river sand in the compartments for host plant roots and mycorrhizal hyphae. Arbuscular mycorrhizal (AM) associations were established using two host plant species, maize (Zea mays L.) and red clover (Trifolium pratense L.) and two AM fungi, Glomus mosseae and G. versiforme. When the standard and modified cultivation systems were compared, the new method yielded much more fungal tissue in the hyphal compartment. Using G. versiforme as the fungal symbiont, up to 30 mg of fungal dry matter (DM) was recovered from the hyphal compartment of mycorrhizal maize and about 6 mg from red clover. Multi-element analysis was conducted on samples of host plant roots and shoots and on harvested fungal biomass. Concentrations of P, Cu and Zn were much higher in the fungal biomass than in the roots or shoots of the host plants but fungal concentrations of K, Ca, Mg, Fe and Mn were similar to or lower than those in the plants. There were also significant differences in nutrient concentrations between the two AM fungi and these may be related to differences in their proportions of extraradical mycelium to spores. The high affinity of the fungal mycelium for Zn was very striking and is discussed in relation to the potential use of arbuscular mycorrhiza in the phytoremediation of Zn-polluted soils.     

Enhanced remediation of dioxins-spiked soil by a plant-microbe system using a dibenzofuran-degrading Comamonas sp. and Trifolium repens L

In this study, rhizoremediation technology was applied to dioxins-spiked soil. A dibenzofuran-degrading bacterium Comamonas sp. strain KD7, reported in the previous paper, was used in combination with white clover (Trifolium repens L.). First, the effect of strain KD7 on clover seed germination and root elongation was examined in the presence of dioxins compounds. As a result, the white clover seeds inoculated with strain KD7 exhibited a higher germination efficiency and increased root elongation compared with uninoculated white clover. Next, the recovery efficiency of two extraction methods were considered for analyzing the dioxin concentration in soil samples, then, the potential of the plant-microbe combination was evaluated for the remediation of dioxins-spiked soil. After 12 week of growth, significant reductions in the soil were confirmed for most compounds. Our results demonstrated that clover can function as a carrier in order to increase the dioxin-degrading activity of strain KD7. The association of clover and strain KD7 is considered to be a potential tool in the remediation of dioxin-contaminated soil.     

Physiological and foliar symptom response in the crowns of Prunus serotina, Fraxinus americana and Acer rubrum canopy trees to ambient ozone under forest conditions

The crowns of five canopy dominant black cherry (Prunus serotina Ehrh.), five white ash (Fraxinus americana L.), and six red maple (Acer rubrum L.) trees on naturally differing environmental conditions were accessed with scaffold towers within a mixed hardwood forest stand in central Pennsylvania. Ambient ozone concentrations, meteorological parameters, leaf gas exchange and leaf water potential were measured at the sites during the growing seasons of 1998 and 1999. Visible ozone-induced foliar injury was assessed on leaves within the upper and lower crown branches of each tree. Ambient ozone exposures were sufficient to induce typical symptoms on cherry (0-5% total affected leaf area, LAA), whereas foliar injury was not observed on ash or maple. There was a positive correlation between increasing cumulative ozone uptake (U) and increasing percent of LAA for cherry grown under drier site conditions. The lower crown leaves of cherry showed more severe foliar injury than the upper crown leaves. No significant differences in predawn leaf water potential (psi(L)) were detected for all three species indicating no differing soil moisture conditions across the sites. Significant variation in stomatal conductance for water vapor (g(wv)) was found among species, soil moisture, time of day and sample date. When comparing cumulative ozone uptake and decreased photosynthetic activity (P(n)), red maple was the only species to show higher gas exchange under mesic vs. drier soil conditions (P < 0.05). The inconsistent differences in gas exchange response within the same crowns of ash and the uncoupling relationship between g(wv) and P(n) demonstrate the strong influence of heterogeneous environmental conditions within forest canopies.     

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.     

Response of Bean and Tobacco to Ozone: Effect of Light Intensity,Temperature and Relative Humidity

Pinto bean (Phaseolus vulgaris, L.) and Bel W₃ tobacco (Nicotiana tabacum, L.) were grown and exposed to 40 pphm ozone for 1 hr under a range of light intensities, temperatures, and relative humidities. Foliar injury to the more sensitive plant leaves was determined on the third day after exposure. Each atmospheric factor was independently assessed. Two significant three-way interactions were found: exposure light intensity by growth light intensity by species, and growth temperature by exposure temperature by species. Three significant two-way interactions were found for humidity: growth humidity by exposure humidity, growth humidity by species, and exposure humidity by species. The sensitivity of each species to ozone changed with changes in each environmental condition.     

Antioxidant defense gene analysis in <Emphasis Type="Italic">Brassica oleracea</Emphasis> and <Emphasis Type="Italic">Trifolium repens</Emphasis> exposed to Cd and/or Pb

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.     

Toxicity of three halogenated flame retardants to nitrifying bacteria, red clover (Trifolium pratense), and a soil invertebrate (Enchytraeus crypticus)

Halogenated flame retardants have a high sorption affinity to particles, making soils and sediments important sinks. Here, three of the most commonly used flame retardants have been tested for sub-lethal toxicity towards soil nitrifying bacteria, a terrestrial plant (seed emergence and growth of the red clover, Trifolium pratense), and a soil invertebrate (survival and reproduction of Enchytraeus crypticus). Tetrabromobisphenol A (TBBPA) was quite toxic to enchytraeids, with significant effects on reproduction detected already at the 10 mgkg(-1) exposure level (EC(10)=2.7 mgkg(-1)). In contrast, decabromodiphenyl ether (DeBDE) was not toxic at all, and short-chain chloroparaffins (CP(10-13)) only affected soil nitrifying bacteria at the highest test concentration (EC(10)=570 mgkg(-1)). Exposure concentrations were verified by chemical analysis for TBBPA and DeBDE, but not for CP(10-13), as a reliable method was not available. Based on the generated data, a PNEC for soil organisms can be estimated at 0.3 mgkg(-1) for TBBPA and 57 mgkg(-1) for short-chain chloroparaffins. No PNEC could be estimated for DeBDE. Measurements of TBBPA in soil are not available, but measured concentrations in Swedish sludge are all lower than the estimated threshold value for biological effects in soil.     

Effects of elevated ozone on leaf delta13C and leaf conductance of plant species grown in semi-natural grassland with or without irrigation

Stable carbon isotope ratios (delta(13)C) and leaf conductance (g(s)) were measured (2002, 2003) in Holcus lanatus L., Plantago lanceolata L. Ranunculus friesianus (Jord.), and Trifolium pratense L. at two levels of ozone (O(3)) with or without irrigation. In non-irrigated control plots, R. friesianus showed the least negative delta(13)C, and the smallest response to the treatments. Irrigation caused more negative delta(13)C, especially in H. lanatus. Irrespective of irrigation, O(3) increased delta(13)C in relationship to a decrease in g(s) in P. lanceolata and T. pratense. The strongest effect of O(3) on delta(13)C occurred in the absence of irrigation, suggesting that under field conditions lack of moisture in the top soil does not always lead to protection from O(3) uptake. It is concluded that in species such as T. pratense plants can maintain stomatal O(3) uptake during dry periods when roots can reach deeper soil layers where water is not limiting.     

Effects of ozone on inter- and intra-species competition and photosynthesis in mesocosms of Lolium perenne and Trifolium repens

Trifolium repens and Lolium perenne were exposed as both monocultures and two-species mixtures to an episodic rural ozone regime in large, well-watered containers within solardomes for 12 weeks. There were reductions in biomass for T. repens, but not L. perenne, and the proportion of T. repens decreased in ozone-exposed mixtures compared to the control. In addition, leaf biomass of T. repens was maintained at the expense of biomass partitioning to the stolons. The decreased growth corresponded with decreased photosynthetic capacity for T. repens, however, by the end of the exposure there was also decreased photosynthetic capacity of L. perenne, a species previously considered insensitive to ozone. The observed decreases in photosynthetic efficiency and capacity in elevated ozone indicate that the ability of such ubiquitous vegetation to act as a sink for atmospheric carbon may be reduced in future climates.     

Biochemical changes in needles of Pinus radiata D. Don trees in relationship to long-term ozone exposure indices

Ambient concentrations of ozone in Europe are high enough to cause negative effects on vegetation. Therefore, many efforts have been made to determine exposure indices and critical levels for protection of vegetation. In this context, the choice of a suitable attribute to determine the pollutant effect is of paramount importance. Until now, much of the work has been done with attributes such as biomass or growth. In the present work correlation factors have been established between biochemical parameters (peroxidase activity, ascorbate and sulfhydryl contents) of Pinus radiata trees and exposure indices of ozone. Our results show that peroxidase cannot be used as an indicator of effects of long-term exposure to ozone but still remains as an excellent indicator of short-term ozone fluctuations in the field. Ascorbate may act as an intermediate indicator responding to both short fluctuations and long-term exposures to ozone. Finally, sulfhydryl may be used as a long-term indicator in relation to the AOT (average over threshold) exposure index. Our results also point to the fact that Pinus radiata may be affected by ozone at AOT values lower than 10 ppm.h as already observed with other tree species.     

Atmospheric phenanthrene pollution modulates carbon allocation in red clover (Trifolium pratense L.)

The influence of atmospheric phenanthrene (PHE) exposure (160 μg m⁻³) during one month on carbon allocation in clover was investigated by integrative (plant growth analysis) and instantaneous ¹³CO₂ pulse-labelling approaches. PHE exposure diminished plant growth parameters (relative growth rate and net assimilation rate) and disturbed photosynthesis (carbon assimilation rate and chlorophyll content), leading to a 25% decrease in clover biomass. The root-shoot ratio was significantly enhanced (from 0.32 to 0.44). Photosynthates were identically allocated to leaves while less allocated to stems and roots. PHE exposure had a significant overall effect on the ¹³C partitioning among clover organs as more carbon was retained in leaves at the expense of roots and stems. The findings indicate that PHE decreases root exudation or transfer to symbionts and in leaves, retains carbon in a non-structural form diverting photosynthates away from growth and respiration (emergence of an additional C loss process).     

The effects of soil and Trifolium repens (white clover) on the fate of estrogen

In this study, we investigated the behavior of estrogens in the rhizosphere of white clover (Trifolium repens, clover hereafter) with two different pot tests, using soil and agar as growth media. In a pot test using agar spiked with estrogen, the estrogen concentration in the agar with clover decreased to non-detectable levels within one month, while in the agar without clover, 60% of initially added estrogen remained after one month. The half-lives of estrone (E1) and 17β -estradiol (E2) in the agar with clover were 2.4–3.8 and 13.2 d, respectively. The dissipation of E1 followed first-order rate law, while that of E2 fitted a zero-order reaction, indicating that they had different mechanisms of dissipation from agar. In the soil pot test, the behavior of E1 and E2 was not influenced by clover. An initial rapid decrease in the amount of estrogen extracted by methanol/acetic acid was followed by persistence for 1–3 months, regardless of presence of clover. Moreover, in three weeks E1 and E2 were only partly degraded by microbes extracted from the soil used in the pot test. In this study, abiotic degradation of estrogens and sorption of estrogen to soil, rather than the effects of soil microbes and clover, contributed to the initial rapid dissipation of estrogens in the soil. However, the results of the agar pot test suggested that vegetation such as clover may significantly contribute to removal of estrogens when estrogens in aqueous phase are discharged with surface runoff and preferential flow after heavy rain in agricultural fields, or when present in soils with low estrogen sorptivity.     

Predicting molybdenum toxicity to higher plants: Estimation of toxicity threshold values

Four plant species (oilseed rape, Brassica napus L.; red clover, Trifolium pratense L.; ryegrass, Lolium perenne L.; and tomato, Lycopersicon esculentum L.) were tested on ten soils varying widely in soil properties to assess molybdenum (Mo) toxicity. A larger range (66-fold-609-fold) of added Mo concentrations resulting in 50% inhibition of yield (ED₅₀) was found among soils than among plant species (2-fold-38-fold), which illustrated that the soils differed widely in the expression of Mo toxicity. Toxicity thresholds based on soil solution Mo narrowed the variation among soils compared to thresholds based on added Mo concentrations. We conclude that plant bioavailability of Mo in soil depends on Mo solubility, but this alone did not decrease the variability in observed toxicity enough to be used in risk assessment and that other soil properties influencing Mo toxicity to plants need to be considered.     

Response of cellular antioxidants to ozone in wheat flag leaves at different stages of plant development

The effect of ozone (O3) on growth, yield and foliar antioxidants of spring wheat (Triticum aestivum L. cv. Turbo) was investigated in 1990 and 1991 in Braunschweig, Germany. Plants were grown full-season in pots in open-top chambers ventilated with charcoal-filtered (CF) air to which one or two levels of O3 were added. Mean 8 h day(-1) (10.00-18.00 h) O3 concentrations in the CF and CF + O3 treatments were 5.9, 61.2 and 92.5 nl litre(-1) in 1990, and 4.7 and 86.4 nl litre(-1) in 1991. Plants that received the high O3 level showed symptoms of premature senescence of the oldest leaves and yield reductions in both growing seasons. The contents of ascorbate and glutathione and the enzyme activities of ascorbate peroxidase and glutathione reductase were measured in symptomless flag leaves in weekly intervals before and after the beginning of anthesis. Leaf age had a significant effect on all antioxidants investigated. The O3 exposures of about 90 nl litre(-1) increased the activity of ascorbate peroxidase and the concentration of glutathione, but there were no pollutant effects on ascorbate content and glutathione reductase activity. Measurements of the antioxidant levels throughout one day showed no clear indications of diurnal changes in the antioxidative capacity in wheat flag leaves. The results are discussed in relation to the role of antioxidants in O3 detoxification.     

Assessment of the ozone tolerance of two soybean cultivars (Glycine max cv. Sambaíba and Tracajá) cultivated in Amazonian areas

Brazilian soybean cultivars (Glycine max Sambaíba and Tracajá) routinely grown in Amazonian areas were exposed to filtered air (FA) and filtered air enriched with ozone (40 and 80 ppb, 6 h/day for 5 days) to assess their level of tolerance to this pollutant by measuring changes in key biochemical, physiological, and morphological indicators of injury and in enzymatic and non-enzymatic antioxidants. Sambaíba plants were more sensitive to ozone than Tracajá plants, as revealed by comparing indicator injury responses and antioxidant stimulations. Sambaíba exhibited higher visible leaf injury, higher stomatal conductance, and a severe decrease in the carbon assimilation rate. Higher ozone level (80 ppb) caused an increase in cell death in both cultivars. Levels of malondialdehyde and hydrogen peroxide also increased in Tracajá exposed under 80 ppb. Sambaíba plants exhibited decreases in ascorbate and glutathione levels and in enzymatic activities associated with these antioxidants. The higher tolerance of the Tracajá soybean appeared to be indicated by reduced physiological injuries and lower stomatal conductance, which might decrease the influx of ozone and enhance oxidation-reduction reactions involving catalase, ascorbate peroxidase, ascorbate, and glutathione, most likely stimulated by higher hydrogen peroxide.     

Effects of ethylenediurea and ozone on the antioxidative systems in beans (Phaseolus vulgaris L.)

To study the biochemical mechanism of EDU protection against ozone injury, peroxidase, ascorbate-dependent peroxidase, and catalase activities, and the contents of ascorbic acid, dehydroascorbic acid, malondialdehyde and soluble protein were measured in Phaseolus vulgaris L. cv. Lit exposed to ozone and ethylenediurea (EDU) in open-top chambers. Plants not treated with EDU showed foliar bronzing due to ozone, while EDU-treated plants were not affected. EDU application modified the reaction of biochemical parameters to ozone. Soluble protein content was elevated by EDU. Peroxidase activity increased with ozone exposure in untreated plants only, while ascorbate-dependent peroxidase activity was lower in EDU treated plants. Catalase activity decreased in EDU-untreated plants. The ratio of ascorbic acid to dehydroascorbic acid was significantly increased in EDU treated plants. These results suggest that EDU might induce ascorbic acid synthesis and therefore provide the plant with a very potent antioxidant. Or the content of hydrogen peroxide was reduced due to other unknown processes and caused a delay in foliar senescence, regardless of whether these processes were ozone-induced or due to natural aging processes.