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.     

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 as a potential tool in evaluating ozone phytotoxicity: a review study on physiological,biochemical and morphological responses of plants

Present-day climate change scenario has intensified the problem of continuously increasing ground-level ozone (O₃), which is responsible for causing deleterious effects on growth and development of plants. Studies involving use of ethylenediurea (EDU), a chemical with antiozonant properties, have given some promising results in evaluating O₃ injury in plants. The use of EDU is especially advantageous in developing countries which face a more severe problem of ground-level O₃, and technical O₃-induced yield loss assessment techniques like open-top chambers cannot be used. Recent studies have detected a hormetic response of EDU on plants; i.e. treatment with higher EDU concentrations may or may not show any adverse effect on plants depending upon the experimental conditions. Although the mode of action of EDU is still debated, it is confirmed that EDU remains confined in the apoplastic regions. Certain studies indicate that EDU significantly affects the electron transport chain and has positive impact on the antioxidant defence machinery of the plants. However, the mechanism of protecting the yield of plants without significantly affecting photosynthesis is still questionable. This review discusses in details the probable mode of action of EDU on the basis of available data along with the impact of EDU on physiological, biochemical, growth and yield response of plants under O₃ stress. Data regarding the effect of EDU on plant ‘omics’ is highly insufficient and can form an important aspect of future EDU research.     

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.     

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.     

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.     

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.     

A comparison of North American and Asian exposure–response data for ozone effects on crop yields

Modelling-based studies to assess the extent and magnitude of ozone (O₃) risk to agriculture in Asia suggest that yield losses of 5–20% for important crops may be common in areas experiencing elevated O₃ concentrations. These assessments have relied on European and North American dose–response relationships and hence assumed an equivalent Asian crop response to O₃ for local cultivars, pollutant conditions and climate. To test this assumption we collated comparable dose–response data derived from fumigation, filtration and EDU experiments conducted in Asia on wheat, rice and leguminous crop species. These data are pooled and compared with equivalent North American dose–response relationships. The Asian data show that at ambient O₃ concentrations found at the study sites (which vary between ～35–75 ppb 4–8 h growing season mean), yield losses for wheat, rice and legumes range between 5–48, 3–47 and 10–65%, respectively. The results indicate that Asian grown wheat and rice cultivars are more sensitive to O₃ than the North American dose–response relationships would suggest. For legumes the scatter in the data makes it difficult to reach any equivalent conclusion in relative sensitivities. As such, existing modelling-based risk assessments may have substantially underestimated the scale of the problem in Asia through use of North American derived dose–response relationships.     

Tropospheric ozone decreases biomass production in radish plants (Raphanus sativus) grown in rural south-west Sweden

Potted plants of radish (Raphanus sativus L., cv. Cherry Belle) were grown in the ambient air for 5 weeks, with or without the application of a soil drench of the anti-ozonant ethylenediurea (EDU). The 24-h mean ozone concentration during the experimental period was 31 nl l(-1). Towards the end of the experiment two ozone episodes, with maximum concentrations around 70 and 115 nl l(-1), occurred. No visible injury that could be attributed to ozone was observed on any of the plants. Shoot and hypocotyl biomass were significantly lower in the non-EDU-treated plants than in the EDU-treated plants. The non-EDU-treated plants had a 32% lower hypocotyl biomass and a 22% lower shoot biomass. The shoot:hypocotyl ratio of the non-EDU-treated plants was higher than that of the EDU-treated plants, although the difference was not statistically significant. EDU treatment increased the leaf area and decreased the chlorophyll content of the leaves. These differences were, however, not statistically significant. It is suggested that the ambient rural ozone climate in southern Sweden has the potential to decrease biomass production in Cherry Belle radishes in the absence of visible injury.     

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.     

Evaluation of physiological, growth and yield responses of a tropical oil crop (Brassica campestris L. var. Kranti) under ambient ozone pollution at varying NPK levels

A field study was conducted to evaluate the impact of ambient ozone on mustard (Brassica campestris L. var. Kranti) plants grown under recommended and 1.5 times recommended NPK doses at a rural site of India using filtered (FCs) and non-filtered open top chambers (NFCs). Ambient mean O₃ concentration varied from 41.65 to 54.2 ppb during the experiment. Plants growing in FCs showed higher photosynthetic rate at both NPK levels, but higher stomatal conductance only at recommended NPK. There were improvements in growth parameters and biomass of plants in FCs as compared to NFCs at both NPK levels with higher increments at 1.5 times recommended. Seed yield and harvest index decreased significantly only at recommended NPK in NFCs. Seed quality in terms of nutrients, protein and oil contents reduced in NFCs at recommended NPK. The application of 1.5 times recommended NPK provided protection against yield loss due to ambient O₃.     

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

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

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.     

Differential protection of ethylenediurea (EDU) against ambient ozone for five cultivars of tropical wheat

The antiozonant EDU (ethylenediurea) was used to assess the impact of ambient O₃ under field conditions on five cultivars of tropical wheat (Triticum aestivum L.). EDU solution (0 ppm and 400 ppm) was applied as soil drench (100 ml plant^?1) 10 days after germination (DAG) at an interval of 12 days. EDU-treated plants showed significant increments in stomatal conductance, photosynthetic rate, variable fluorescence, total chlorophyll, ascorbic acid, proline and protein contents and protective enzymes (POX, SOD and APX) activities in HUW468, HUW510 and HUW234 cultivars, while, a reverse trend was observed for lipid peroxidation. EDU application restored grain yield significantly by maintaining higher levels of antioxidants, metabolites and enzymes in cultivars HUW468 and HUW510. Sonalika and PBW343 showed least response of measured parameters under EDU treatment suggesting their greater resistance to O₃. EDU, thus proved its usefulness in screening suitable wheat cultivars for areas experiencing elevated concentrations of O₃.     

An assessment of the impact of ambient ozone on field-grown crops in New Jersey using the EDU method: part 2-soybean (Glycine max (L.) Merr.)

The yields of eleven commercially grown soybean cultivars were compared in ethylenediurea (EDU)-treated and non-treated field plots in New Brunswick, New Jersey, over a 4 year period. No statistically significant difference between treatments was found for any cultivar; the inference being ambient ozone did not adversely affect soybean yield. Succeeding field experiments supported this interpretation of the data. 'Sanilac' white bean, a legume known to be more sensitive to O(3) than soybean, was found to produce a significantly greater yield in EDU-treated than non-treated plots, unlike a companion planting of 'Williams 82' soybean which did not exhibit the differential response. The results indicated that the specific EDU protocol used in the soybean experiments is capable of detecting an ozone effect in a legume. Moreover, in a concurrent greenhouse experiment the yield of EDU-treated Sanilac white bean was not significantly different from non-treated plants in the absence of ozone pollution. In a dose-response field experiment during a year of unusually high O(3) pollution, yield of 'Williams 82' increased slightly with each EDU increment up to 500 ppm and decreased at 1000 ppm. The difference between non-treated and EDU-treated plants, however, was not statistically significant. There was no evidence to suggest that the EDU concentration (500 ppm) used in previous soybean experiments reduced seed yield. Fortuitously, the tolerance of commercially-grown soybean to ambient ozone is at least partially conditioned by the practce of not irrigating the crop. The New Jersey results are in agreement with reports from Maryland, Georgia and Tennessee in which an adverse impact of ambient O(3) was not found in soybean, but contrary to a current predictive model.     

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.     

Elevated ozone phytotoxicity ameliorations in mung bean {Vigna radiata (L.) Wilczek} by foliar nebulization of silicic acid and ascorbic acid

The present work provides an insight into the development of biochemical adaptations in mung beans against ozone (O₃) toxicity. The study aims to explore the O₃ stress tolerance potential of mung bean genotypes under exogenous application of growth regulators. The seeds of twelve mung bean genotypes were grown in plastic pots under controlled conditions in the glasshouse. Six treatments, control (ambient ozone level 40–45 ppb), ambient O₃ with ascorbic acid, ambient ozone with silicic acid, elevated ozone (120 ppb), elevated O₃ with ascorbic acid (10 mM), and elevated ozone with silicic acid (0.1 mM) were applied. The O₃ fumigation was carried out using an O₃ generator. The results revealed that ascorbic acid and silicic acid application decreased the number of plants with foliar O₃ injury symptoms in different degrees, i.e., zero, first, second, third, and fourth degrees; whereas 0–4 degree symptoms represent, no symptoms, symptoms occupying?<?1/4, 1/4–1/2, 1/2–3/4, and?>?3/4 of the total foliage area, respectively. Application of ascorbic acid and silicic acid also prevented the plants from the negative effects of O₃ in terms of fresh as well as dry matter production, leaf chlorophyll, carotenoids, soluble proteins and ascorbic acid, proline, and malondialdehyde (MDA) contents. Overall, silicic acid application proved more effective in reducing the negative effects of O₃ on mung bean genotypes as compared to that of the ascorbic acid. Three mung bean genotypes (NM 20–21, NM-2006, and NM-2016) were identified to have a better adaptive mechanism for O₃ toxicity tolerance and may be good candidates for future variety development programs.

     

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 the impact of ambient ozone on growth and yield of a rice (Oryza sativa L.) and a wheat (Triticum aestivum L.) cultivar grown in the Yangtze Delta, China, using three rates of application of ethylenediurea (EDU)

Foliar applications of ethylenediurea (abbreviated as EDU) were made at 0, 150, 300 or 450 ppm to field-grown rice and wheat in the Yangtze Delta in China. Rice and wheat responded differently to ambient ozone and EDU applications. For wheat, some growth characteristics, such as yield, seed number per plant, seed set rate and harvest index, increased significantly at 300 ppm EDU treatment, while for rice no parameters measured were statistically different regarding EDU application. The reason may be that the wheat cultivar used may be more sensitive to ozone than the rice cultivar. EDU was effective in demonstrating ozone effects on the wheat cultivar, but not on the rice cultivar. Cultivar sensitivity might be an important consideration when assessing the effects of ambient ozone on plants.     

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

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