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.     

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.     

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.     

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 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.     

Gravitational infusion of ethylenediurea (EDU) into trunks protected adult European ash trees (Fraxinus excelsior L.) from foliar ozone injury

Adult ash trees (Fraxinus excelsior L.), known to be sensitive or insensitive to ozone, determined by presence or absence of foliar symptoms in previous years, were treated with ethylenediurea (EDU) at 450 ppm by gravitational trunk infusion on six occasions at 21-day intervals in summer 2005 at Turin, Italy. At the end of the season, foliar ozone injury on EDU-treated trees was not complete, but was greatly and significantly reduced when compared to results from trees infused with water. Significant symptom reduction occurred at any crown level in the treated trees suggesting that EDU protected whole crowns. Gravitational infusion of EDU resulted in protection from ozone injury for ozone-sensitive ash trees. The amount of EDU needed to provide protection is assumed to be in the range 13-26 mg m(-2) leaf.     

Role of ethylene diurea (EDU) in assessing impact of ozone on Vigna radiata L. plants in a suburban area of Allahabad (India)

A field study was conducted to evaluate the suitability of ethylene diurea (N-[2-(2-oxo-1-imidazolidinyl)ethyl]-N'-phenylurea; EDU) in assessing the impact of O3 on mung bean plants (Vigna radiata L. var. Malviya Jyoti) grown in suburban area of Allahabad city situated in a dry tropical region of India. EDU is a synthetic chemical having anti-ozonant property. Mean monthly O3 concentration varied between 64 and 69 microg m(-3) during the experimental period. In comparison to EDU-treated plants, non-EDU-treated plants showed significant reductions in plant growth and yield under ambient conditions. Significant favourable effects of EDU-application were observed with respect to photosynthetic pigments, soluble protein, ascorbic acid and phenol contents. EDU-treated plants maintained higher levels of pigments, protein and ascorbic acid in foliage as compared to non-EDU-treated ones. The study clearly demonstrated that EDU alleviates the unfavourable effects of O3 on mung bean plants, and therefore can be used as a tool to assess the growth and yield losses in areas having higher O3 concentrations.     

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.     

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.     

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.     

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.     

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.     

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.     

Soluble carbohydrates in bean leaves transformed into oxidant-tolerant tissues by EDU treatment

Improved gas-liquid chromatography techniques were used to evaluate the effects of EDUa on soluble leaf carbohydrates in $\text{Phaseolus vulgaris}$ L. ‘Bush Blue Lake 290’. This snap bean cultivar is normally rather sensitive to 0₃ but becomes highly tolerant when treated systemically with EDU. Less than 24 h is required to induce the resistance. Standardized trifoliate leaves from EDU-treated and control plants were sampled 48 h after treatment. Additional plants were sampled 48 h after treatment. Additional plants were exposed to 0₃ one day after EDU soil application to assess the plant tolerance induced. The optimal dose required to enhance oxidant tolerance was 50 mg/pot.Major sugars in both EDU-treated (0₃-tolerant) and untreated (0₃-sensitive) leaves were glyceraldehyde, erythritol, fructose, glucose and sucrose. $\text{Myo}$-inositol, ribose and arbitol were present in lesser or trace amounts. EDU-treatment resulted in significant increases (35–62%) in all soluble carbohydrates except glyceraldehyde and $\text{myo}$-inositol. Implications relating to plant tolerance, to oxidants and stress-induced senescence are discussed.     

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.     

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.     

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.     

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.     

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.     

Chlorophyll Content of Soybean Foliage in Relation to Seed Yield and Ambient Ozone Pollution

A field test was conducted to determine if ozone pollution adversely affected the chlorophyll content and seed yield of soybean. Eight soybean cultivars were grown to maturity in test plots in central New Jersey; one-half of the plots was treated with an antioxidant (ethylenediurea) to protect the plants from the effects of ambient ozone and one-half was left untreated. Periodic chlorophyll measurements revealed no significant difference between EDU-treated and untreated plots during the major part of plant growth. The absence of a yield effect predicated on the normal chlorophyll content was corroborated by actual total seed measurement. Our results did not support predictive models that forecast a significant yield reduction from a 7-h seasonal mean of 0.058 ppm 0₃, but agreed with results obtained previously in Maryland and Georgia.