Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU): roles of biochemical changes and decreased stomatal conductance in enhancement of growth

Treatments with ethylenediurea (EDU) protect plants from ozone foliar injury, but the processes underlying this protection are poorly understood. Adult ash trees (Fraxinus excelsior), with or without foliar ozone symptoms in previous years, were treated with EDU at 450ppm by gravitational trunk infusion in May-September 2005 (32.5ppmh AOT40). At 30-day intervals, shoot growth, gas exchange, chlorophyll a fluorescence, and water potential were determined. In September, several biochemical parameters were measured. The protective influence of EDU was supported by enhancement in the number of leaflets. EDU did not contribute its nitrogen to leaf tissue as a fertiliser, as determined from lack of difference in foliar N between treatments. Both biochemical (increase in ascorbate-peroxidase and ascorbic acid, and decrease in apoplastic hydrogen peroxide) and biophysical (decrease in stomatal conductance) processes regulated EDU action. As total ascorbic acid increased only in the asymptomatic trees, its role in alleviating O(3) effects on leaf growth and visible injury is controversial.     

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.     

Use of the antiozonant ethylenediurea (EDU) in Italy: verification of the effects of ambient ozone on crop plants and trees and investigation of EDU's mode of action

Twenty-four experiments where EDU was used to protect plants from ozone (O₃) in Italy are reviewed. Doses of 150 and 450 ppm EDU at 2-3 week intervals were successfully applied to alleviate O₃-caused visible injury and growth reductions in crop and forest species respectively. EDU was mainly applied as soil drench to crops and by stem injection or infusion into trees. Visible injury was delayed and reduced but not completely. In investigations on mode of action, EDU was quickly (<2 h) uptaken and translocated to the leaf apoplast where it persisted long (>8 days), as it cannot move via phloem. EDU did not enter cells, suggesting it does not directly affect cell metabolism. EDU delayed senescence, did not affect photosynthesis and foliar nitrogen content, and stimulated antioxidant responses to O₃ exposure. Preliminary results suggest developing an effective soil application method for forest trees is warranted.     

Stem injection of Populus nigra with EDU to study ozone effects under field conditions

EDU or ethylenediurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea) has been used in experiments to assess ozone effects on vegetation under field conditions because it provides protection against oxidative damage. Tests have mainly been conducted on crop plants, but for woody species only few reports have provided evidence that it can be used in long-term experiments. In this study we tested the technique of stem injection of EDU to study the effects of ozone exposure on Populus nigra cv. Wolterson over one growing season. Cuttings of Populus nigra were grown in pots in the field and between mid-July and early September plants were repeatedly injected with EDU solution (5 mg/plant) or with water at 14-day intervals. Significant differences were found between EDU- and water-injected plants: water-treated plants had more foliar injury, more chlorotic leaves, and shedding of leaves started earlier, suggesting EDU was effective in preventing visible ozone injury and acceleration of senescence. Photosynthetic rates, measured for one leaf age, showed no differences but were mostly higher for the EDU-treated plants. At the end of the growing season diameter increment was 16% higher and there was a non-significant trend for above-ground biomass to be increased by 9% for the EDU-treated plants. This experiment has provided evidence that for this clone serious ozone damage occurs at relatively low concentrations and that EDU can provide protection against visible injury, as well as against longer term growth reductions.     

Effects of ethylenediurea (EDU) on ozone-induced acceleration of foliar senescence in potato (Solanum tuberosum L.)

Experiments were conducted to examine the effects of the anti-ozonant ethylenediurea (EDU) and chronic ozone (O3) exposure on leaf physiology and senescence in an O3-sensitive potato cultivar (Solanum tuberosum L. cv. Norland). A dose-response experiment showed that an EDU concentration of 15 mg l(-1) soil (given as a soil drench) provided complete protection from accelerated foliar senescence induced by exposure to 0.1 microl l(-1) O3 for 5 h day(-1) for 11 days. EDU doses of 45 and 75 mg active ingredient l(-1) soil also gave protection but were associated with symptoms of toxicity and delayed senescence. In further experiments, plants were given 0 or 15 mg EDU l(-1) soil and exposed to clean air or 0.1 microl l(-1) O3 for 5 h day(-1) for 14 days. Chronic O3 exposure in the absence of EDU resulted in accelerated foliar senescence, characterized by early declines in net photosynthesis and Rubisco quantity in O3-treated plants relative to controls. EDU in the presence of O3 gave complete protection against symptoms of accelerated senescence. Senescence was not delayed in plants that received EDU in the absence of O3, and no symptoms of EDU toxicity were evident. The results suggest that EDU-induced tolerance to O3 was not based on 'anti-senescent' properties of this anti-ozonant.     

Protection of palak (Beta vulgaris L. var Allgreen) plants from ozone injury by ethylenediurea (EDU): Roles of biochemical and physiological variations in alleviating the adverse impacts

Ameliorative effects of ethylenediurea (N-[2-(2-oxo-1-imidazolinidyl) ethyl]-N′ phenylurea, abbreviated as EDU) against ozone stress were studied on selected growth, biochemical, physiological and yield characteristics of palak (Beta vulgaris L. var Allgreen) plants grown in field at a suburban site of Varanasi, India. Mean eight hourly ozone concentration varied from 52 to 73 ppb which was found to produce adverse impacts on plant functioning and growth characteristics. The palak plants were treated with 300 ppm EDU at 10 days after germination at 10 days interval up to the plant maturity. Lipid peroxidation in EDU treated plants declined significantly as compared to non-EDU treated ones. Significant increment in F_v/F_m ratio in EDU treated plants as compared to non-EDU treated ones was recorded. EDU treated plants showed significant increment in ascorbic acid contents and reduction in peroxidase activity as compared to non-EDU treated ones. As a result of the protection provided by EDU against ozone induced stress on biochemical and physiological characteristics of palak, the morphological parameters also responded positively. Significant increments were recorded in shoot length, number of leaves plant⁻¹, leaf area and root and shoot biomass of EDU treated plants as compared to non-EDU treated ones. Contents of Na, K, Ca, Mg and Fe were higher in EDU treated plants as compared to non-EDU treated ones. The present investigation proves the usefulness of EDU in partially ameliorating ozone injury in ambient conditions.     

Ethylenediurea (EDU): a research tool for assessment and verification of the effects of ground level ozone on plants under natural conditions

Ethylenediurea (EDU) has been widely used to prevent ozone (O₃) injury and crop losses in crop plants and growth reductions in forest trees. Successful use requires establishing a dose/response curve for EDU and the proposed plant in the absence of O₃ and in the presence of O₃ before initiating multiple applications to prevent O₃ injury. EDU can be used to verify foliar O₃ symptoms in the field, and to screen plants for sensitivity to O₃ under ambient conditions. Despite considerable research, the mode of action of EDU remains elusive. Additional research on the mode of action of EDU in suppressing O₃ injury in plants may also be helpful in understanding the mode of action of O₃ in causing injury in plants.     

Influence of ozone and ethylenediurea (EDU) on growth and yield of bean (Phaseolus vulgaris L.) in open-top field chambers

Greenhouse and ambient air experiments have shown ethylene diurea (EDU) to be a strong and specific protective suppressant of ozone injury in plants. To examine how EDU affects plant responses to various ozone (O(3)) levels under controlled field conditions, Phaseolus vulgaris L. cv. Lit was treated with 150 ppm EDU every 14 days and exposed in open-top chambers to charcoal-filtered air (CF), nonfiltered air (NF) or two cf treatments with ozone added. The ozone treatments were proportional additions of one (CF1) and two (CF2) times ambient ozone levels. The mean ozone concentrations in the CF, NF, CF1 and CF2 treatments were 0.98, 14.1, 14.98 and 31.56 nl litre(-1). A two-way split plot ANOVA revealed that shoot dry weight was significantly reduced by ozone. EDU treatment was highly significant for leaf dry weight, root dry weight and shoot dry weight, but not for pod dry weight; leading to a higher biomass of EDU-treated plants. Ozone/EDU interactions were significant for root weight only, indicating that EDU reduced growth suppression by ozone. These results show that EDU action on plant biomass could be interpreted as a delay in senescence since EDU-treated plants showed a significant decreased biomass loss even in the CF treatment.     

Black cherry growth response to ambient ozone and EDU

Field-grown black cherry (Prunus serotina Ehrh.) seedlings were treated with the antioxidant ethylenediurea (EDU) to evaluate height, diameter, and above-ground dry-weight biomass growth response to ambient ozone over four years. Nine blocks with 44 trees/block were used in a randomized complete block design with three foliar spray treatments: (1) 1000 ppm EDU mixed with a surfactant and water; (2) surfactant mixed with water; and (3) water only. In each growing season treatments were applied seven times at approximately 10-day intervals. Repeated measures analysis of variance indicated significant (P< or =0.05) treatment and year effects for log-transformed height and diameter growth over the four-year period. After four years, EDU-treated trees were approximately 17% taller and stem diameters were 21% greater than non-EDU-treated trees. Total above-ground dry-weight biomass at the end of four years was 47% greater for EDU-treated trees compared to non-EDU-treated trees.     

Size-mediated foliar response to ozone in black cherry trees

Local ozone concentration and visible foliar injury were measured over the 1994 growing season on open-grown black cherry (Prunus serotina Ehrh.) trees of varying size (age) within forest stands and adjacent openings at a site in north-central Pennsylvania. Relationships were determined between visible ozone injury and ozone exposure, as well as calculated between injury and ozone uptake expressed as the product of stomatal conductance and ozone concentration. In addition, simultaneous measurements of visible symptoms and leaf gas exchange were also conducted to determine the correlation between visible and physiological injury and ozone exposure. By September, the amount of leaf area affected by visible foliar ozone injury was greatest for seedlings (46%), followed by canopy trees (20%) and saplings (15%). A large amount of variability in foliar ozone symptom expression was observed among trees within a size class. Sum40 and Sum60 (ozone concentration > 40 and > 60 nl liter(-1)) cumulative exposure statistics were the most meaningful indices for interpretation of foliar injury response. Seedlings were apparently more sensitive to ozone injury than larger trees because their higher rates of stomatal conductance resulted in higher rates of ozone uptake. Seedlings also had higher rates of early leaf abscission than larger trees with an average of nearly 30% of the leaves on a shoot abscised by 1 September compared to approximately 5% for larger trees. However, per unit ozone uptake into the leaf, larger trees exhibited larger amounts of foliar injury. The amount of visible foliar injury was negatively correlated (r(2) = 0.82) with net photosynthetic rates, but was not related to stomatal conductance. Net photosynthesis and stomatal conductance thus became uncoupled at high levels of visible foliar injury.     

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%.     

Assessing plant response to ambient ozone: growth of ozone-sensitive loblolly pine seedlings treated with ethylenediurea or sodium erythorbate

One-year-old seedlings from an ozone-sensitive half-sib family of loblolly pine (Pinus taeda L.) were transplanted into replicated plots in blocks in a large forest clearing near Nacogdoches, Texas. Seedlings were either non-treated (controls) or treated bi-weekly with foliar sprays of ethylenediurea (EDU), at 150, 300 or 450 ppm or sodium erythorbate (NaE), at 515, 1030, or 1545 ppm, for three growing seasons. Results from the final third year harvest indicated that both EDU and NaE increased all growth parameters, with significant differences only for EDU at 450 ppm. Both EDU and NaE would be useful for long-term studies on assessing the effects of ambient ozone on established native plants.     

Dose-response studies with the antiozonant ethylenediurea (EDU), applied as a soil drench to two growth substrates, on greenhouse-grown varieties of Phaseolus vulgaris L

To study plant growth and yield effects of the antiozonant ethylenediurea (EDU), which is frequently used for ozone crop loss assessments, dose-response studies were carried out with potted bean plants under greenhouse conditions in winter and spring. Two cultivars of Phaseolus vulgaris L., differing in sensitivity to ozone (O(3)), were grown in unfiltered air on a sandy loam rich in organic matter and on a vermiculite-clay mixture. Four treatments of EDU at concentrations from 300 to 800 mg liter(-1) were given as a soil drench during plant development. Foliar symptoms of EDU phytoxicity were observed at all doses, and plant biomass, particularly pod dry weight, was considerably reduced to increasing doses of EDU. Primary and first trifoliate leaf weight in EDU-treated plants increased as did the number of buds, indicating an extension of vegetative growth and a delay of reproductive processes. 'BBL 290' beans, which are O(3)-sensitive, were injured by EDU more than the O(3)-tolerant 'BBL 274'. The phytotoxic effects of EDU were more pronounced in the synthetic growth substrate than in field soil. In a second experiment, EDU was applied in concentrations from 100 to 400 mg liter(-1) to 'BBL 290' plants, exposed to filtered air or simulated levels of O(3) pollution. In field soil, plant growth and biomass partitioning in filtered air was only slightly altered by EDU, although leaf injury due to EDU occurred. In the vermiculite-clay mix, the biomass of most plant organs, particularly that of roots, was linearly reduced with increasing EDU doses. O(3) did not cause any alteration in plant biomass in field soil-grown and EDU-treated plants. Ozone leaf injury, which affected 67% of primary leaf area in non-treated plants, was completely suppressed by EDU doses as low as 100 mg liter(-1). This indicates that low concentrations of EDU, which do not affect plant growth in field soil, provide sufficient protection from O(3) injury. The need for careful EDU dose-response studies prior to field assessments is emphasized.     

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.     

Protection of plants from ambient ozone by applications of ethylenediurea (EDU): A meta-analytic review

A meta-analysis was conducted to quantitatively assess the effects of ethylenediurea (EDU) on ozone (O₃) injury, growth, physiology and productivity of plants grown in ambient air conditions. Results indicated that EDU significantly reduced O₃-caused visible injury by 76%, and increased photosynthetic rate by 8%, above-ground biomass by 7% and crop yield by 15% in comparison with non-EDU treated plants, suggesting that ozone reduces growth and yield under current ambient conditions. EDU significantly ameliorated the biomass and yield of crops and grasses, but had no significant effect on tree growth with an exception of stem diameter. EDU applied as a soil drench at a concentration of 200-400 mg/L has the highest positive effect on crops grown in the field. Long-term research on full-grown tree species is needed. In conclusion, EDU is a powerful tool for assessing effects of ambient [O₃] on vegetation.     

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.     

Translocation and persistence of EDU (ethylenediurea) in plants: the relationship with its role in ozone damage

Bean plants (Phaseolus vulgaris L. cv Lit) were treated with N-[2-(2-oxo-1-imidazolinidyl)ethyl]-N'-phenylurea (EDU) (150 microg ml(-1)) in hydroponic conditions. The EDU concentration in different plant tissues was measured by HPLC. EDU accumulated in leaves and persisted for more than 10 days showing a slow degradation. Using five different EDU concentrations, a significant relationship between EDU concentration in nutrient solution, ozone tolerance and EDU concentration in leaves was shown. Leaves which contained more EDU were less sensitive to ozone damage. Investigations on protoplasts and cell cultures showed that EDU did not enter the cells. Possible implications of EDU accumulation in the leaf apoplast are discussed.     

Dose-response studies with ethylenediurea (EDU) and radish

There is some concern that the antiozonant ethylenediurea (EDU), used for crop loss assessment due to ambient ozone (O3) may per se affect plant growth and yield. In view of this, and to provide knowledge for later field experiments, dose-response studies with EDU and O3 were carried out in greenhouses in winter and spring 1989, using radish (Raphanus sativus L.) cv. 'Cherry Belle' and 'Red Prince', grown in two different substrates. EDU was applied as a single or repeated soil drench in concentrations ranging from 300 to 800 mg litre(-1) in the first, and from 100 to 400 mg litre(-1) in the second trial. In the second experiment, plants were exposed to a chronic level of O3, mimicking ambient patterns, or to filtered air after the EDU-treatment. When applied in concentrations above 300 mg litre(-1), EDU reduced growth, thereby affecting the development of the thickened hypocotyl far more than the shoot growth that was partially stimulated by lower doses of EDU. Phytotoxic symptoms on the leaves, attributable to EDU, were observed at concentrations above 200 mg litre(-1), but complete protection from visible O3-injury was provided by a single application of EDU at a concentration as low as 100 mg litre(-1). Significant interactions on growth characters measured between O3-exposure and EDU application were observed only in one of the substrates. While these results demonstrate the need for careful dose-response studies prior to field assessments, they also provide evidence of a dosage that is effective in protecting radish from O3 damage without interfering with plant growth itself.     

Use of ethylenediurea (EDU) to ameliorate ozone effects on purple coneflower (Echinacea purpurea)

Purple coneflower plants (Echinacea purpurea) were placed into open-top chambers (OTCs) for 6 and 12 weeks in 2003 and 2004, respectively, and exposed to charcoal-filtered air (CF) or twice-ambient (2x) ozone (O3) in 2003, and to CF, 2x or non-filtered (NF), ambient air in 2004. Plants were treated with ethylenediurea (EDU) weekly as a foliar spray. Foliar symptoms were observed in >95% of the plants in 2x-treated OTCs in both years. Above-ground biomass was not affected by 2x treatments in 2003, but root and total-plant biomass decreased in 2004. As a result of higher concentrations of select cell wall constituents (% ADF, NDF and lignin) nutritive quality was lower for plants exposed to 2x-O3 in 2003 and 2004 (26% and 17%, respectively). Significant EDU x O3 interactions for concentrations of cell wall constituents in 2003 indicated that EDU ameliorated O3 effects on nutritive quality. Interactions observed in 2004 were inconsistent.     

Assessing the impact of ambient ozone on growth and productivity of two cultivars of wheat in India using three rates of application of ethylenediurea (EDU)

Three rates of ethylenediurea were used to assess the impact of ambient ozone on growth and productivity of wheat (Triticum aestivum L) cultivars "Malviya 533" (M 533) and "Malviya 234" (M 234) at a suburban site near Varanasi, India, beginning in December. Wheat plants were treated with EDU at 0, 150, 300 and 450 ppm as soil drenches at 10-day intervals. EDU treatment affected plant growth, with effects varying with cultivar, age, and EDU concentration. Seed yield was improved for M 533 at 150 ppm EDU, while yield improved for M 234 at 300 and 450 ppm EDU. M 533 appears to be more resistant to ozone than M 234. Overall results confirmed that EDU is very useful in assessing the effect of ambient ozone in India.