Ozone Dosage Response of Ponderosa Pine Seedlings

Photochemical oxidant injury to ponderosa pine (Pinus ponderosa Laws) is a severe problem in the southern California mountains. Three-year-old ponderosa pines fumigated in controlled environment chambers with ozone at 0.15, 0.30, or 0.45 ppm had apparent photosynthesis rates reduced by 10, 70, and 85%, respectively, after 30 days exposure. A fumigation with 0.30 ppm ozone for 33 days depressed the cold perchloric acid extracted polysaccharides of both current and one-year-old needles by 40%. The 80% ethanol soluble sugar concentration of current year, ozone-injured needles increased 16% and that of the one-year-old needles decreased slightly. Both carbohydrate fractions of control trees in carbon-filtered air increased moderately. Higher, endogenous concentrations of ascorbic acid in needles did not protect the tissue from ozone injury. Apparent photosynthesis rate was a sensitive index for ozone dosage response. Needle carbohydrate depletion probably induces premature abscission.     

Investigations on the photosynthetic system of spruce affected by forest decline and ozone fumigation in closed chambers

Investigations using chlorophyll fluorescence induction kinetics provide the parameters Rfd, L, and Ap to characterize different specifications of the photosynthetic system (PS). The application of ozone, with concentrations between 100 microg m(-3) and 2000 microg m(-3) to spruce in closed chambers yields a reduction of Rfd between 6% and 23% for the current year's needles, which indicates a reduced potential photosynthetic capacity. Further measurements on the current year's needles of spruce of the damage classes S0/S1, S2 and S3 show also a reduction of Rfd of between 7% and 14% in 2 successive years. In addition, the parameter L increases for damaged trees by between 11% and 49%, indicating a change of the chlorophyll content and of the internal energy distribution between PSI and PSII. As no effect of L can be observed with ozone fumigation, it is concluded that the change of some specifications of the PS can be simulated well by ozone fumigation (e.g. Rfd) while other specifications cannot (e.g. L).     

Analyses of enzyme activities and other metabolic criteria after five years of fumigation

Enzymatic activity (peroxidase, glutamate dehydrogenase, glutamine synthetase), foliage buffering capacity, soluble protein and nitrogen content were measured in current and previous year needles from young spruce (Picea abies) and fir (Abies alba). The trees were exposed to low levels of SO(2) and/or O(3) and simulated acidic precipitation (pH 4.0) in open-top chambers from 1983 through 1988. Needle samples were taken during March 1988 at the end of the five-year fumigation period. Exposure to SO(2) substantially increased sulphur content in both needle age classes of spruce and fir, and concomitantly reduced the foliage buffering capacity index (BCI), whereas the combined fumigation with SO(2) and O(3) had no effect on BCI. Peroxidase activity was markedly higher in year-old needles compared to current-year needles. However, trees from the SO(2) and SO(2) + O(3) treatments exhibited statistically significant stimulated peroxidase activities. Similarly, changes in the activities of the nitrogen-metabolizing enzymes indicated an altered cellular function of the trees after the long-term pollution stress. Levels of activity of both glutamate dehydrogenase and glutamine synthetase were increased by exposure to SO(2), especially in spruce. Although glutamate dehydrogenase in spruce was affected by all treatments, such changes in activity were found in fir only with the SO(2) treatment. The highest activity of glutamine synthetase, however, occurred in the older needles of trees exposed to SO(2) + O(3). Total nitrogen concentration was either unaffected by the pollutant treatments or decreased in spruce compared to the controls. No statistically significant changes due to the fumigation were found in soluble protein concentrations. Results indicated that chronic exposure to air pollutants lead to alterations in metabolic processes in conifer needles, detectable either by changes in typical stress indicating values or by increases in ammonium assimilation capacity.     

Photosynthetic and stomatal conductance responses of Norway spruce and beech to ozone, acid mist and frost--a conceptual model

Two-year-old beech and Norway spruce seedlings were exposed to a combination of ozone and acid mist treatments in open-top chambers in Scotland during the months of July through to September 1988. Replicate pairs of chambers received charcoal-filtered air (control), ozone-enriched air (140 nl ozone litre(-1)) or 140 nl ozone litre(-1) plus a synthetic acid mist (pH 2.5) composed of ammonium nitrate and sulphuric acid. Field measurements of assimilation and stomatal conductance were made during August. In addition, measurements of assimilation and conductance were made during September in the laboratory. Light response curves of assimilation and conductance were determined using a GENSTAT nonrectangular hyperbolic model. During February 1988/9 the Norway spruce were subject to a four day warming period at 12 degrees C and the light response of assimilation determined. The same plants were then subject to a 3-h night-time frost of -10 degrees C. The following day the time-course of the recovery of assimilation was determined. It was found that ozone fumigation did not influence the light response of assimilation of beech trees in the field, although stomatal conductance was reduced in the ozone-fumigated trees. The rate of light-saturated assimilation of Norway spruce was increased by ozone fumigation when measured in the field. Measurements of assimilation of Norway spruce made during the winter showed that prior to rewarming there was no difference in the rate of light-saturated assimilation for control and ozone-fumigated trees. However, the ozone plus acid mist-treated trees exhibited a significantly higher rate. The 4-day period of warming to 12 degrees C increased the rate of light-saturated assimilation in all treatments but only the ozone plus acid mist-treated trees showed a significant increase. Following a 3-h frost to -10 degrees C the control trees exhibited a reduction in the rate of light-saturated assimilation (Amax) to 80% of the pre-frost value. In comparison, following the frost, the ozone-fumigated trees showed an Amax of 74% of the pre-frost value. The ozone plus acid mist-treated trees showed an Amax of 64% of the pre-frost trees. The time taken for Amax to attain 50% of the pre-frost value increased from 30 min (control) to 85 min for ozone-fumigated trees to 190 min (ozone plus acid mist). These results are discussed in relation to the impact of mild, short-term frosts, which are known to occur with greater frequency than extreme, more catastrophic frost events. A simple conceptual framework is proposed to explain the variable results obtained in the literature with respect to the impact of ozone upon tree physiology.     

Effects of simultaneous ozone exposure and nitrogen loads on carbohydrate concentrations, biomass, and growth of young spruce trees (Picea abies)

Spruce saplings were grown under different nitrogen fertilization regimes in eight chamberless fumigation systems, which were fumigated with either charcoal-filtered (F) or ambient air (O3). After the third growing season trees were harvested for biomass and non-structural carbohydrate analysis. Nitrogen had an overall positive effect on the investigated plant parameters, resulting in increased shoot elongation, biomass production, fine root soluble carbohydrate concentrations, and also slightly increased starch concentrations of stems and roots. Only needle starch concentrations and fine root sugar alcohol concentrations were decreased. Ozone fumigation resulted in needle discolorations and affected most parameters negatively, including decreased shoot elongation and decreased starch concentrations in roots, stems, and needles. In fine roots, however, soluble carbohydrate concentrations remained unaffected or increased by ozone fumigation. The only significant interaction was an antagonistic effect on root starch concentrations, where higher nitrogen levels alleviated the negative impact of ozone.     

Ozone-induced nitrate formation in needles and leaves of Picea abies, Fagus sylvatica and Quercus robur

Much attention has been paid to ozone as a major cause of novel forest decline in Europe. In combination with acidic mist, O(3) has been observed to increase ion leaching. Besides cations lake Mg(2+), Ca(2+), K(+), NH(4)(+), considerable amounts of nitrate were found to be leached by acidic mist from needles of Norway spruce. Controlled fumigation experiments, with 100, 300, and 600 microg O(3)m(-3) over 22 days continuously, have led to a nitrate accumulation of 94.1 +/- 14.8, 119.4 +/- 28.7 and 198.9 +/- 14.9 microg NO(3)(-1) g(-1) FW, respectively, in leaves of Quercus robur. Similar values were found in leaves of Fagus sylvatica and current and previous year needles of Picea abies. Nitrate levels of controls receiving charcoal filtered air were well below 40 microg NO(3)(-) g (-1) FW. Statistically significant elevated nitrate levels were observed after only 48 h of continuous fumigation with 600 microg O(3)m(-3), in all tree species tested, and after 144 h in the 100 microg O(3)m(-3) treatment. In another experiment, trees of Picea abies were kept in two charcoal (C) and two Purafil plus charcoal (P/C) ventilated chambers, and fumigated with O and 500 microg O(3)m(-3) in cabinets of each filter-type in order to eliminate NO(x) from chamber air. After 29 days of continuous ozone fumigation, NO(3)(-) accumulation in needles amounted to 102.0 +/- 37.7 and 137.4 +/- 40.5 microg g(-1) FW in P/C and C-filtered chambers, respectively. Nitrate contents of controls were below 30 microg NO(3)(-)g(-1) FW at the end of the experiment. No significant differences in NO(3)(-) accumulation between filter treatments were observed. Since NO(x) was reduced by more than 95% in the Purafil/charcoal versus the charcoal treatment, NO(3)(-) accumulation in needles can be attributed predominantly to the influence of ozone and not to direct NO(2) uptake of needles by the possible oxidation of NO to NO(2) in the presence of ozone.     

Fatty acids and ultrastructure of ozone-exposed Aleppo pine (Pinus halepensis Mill.) needles

Fifteen or 18-month-old Aleppo pine seedlings were fumigated with different concentrations and doses of ozone over a period of 2-16 days in controlled-environmental growth chambers. The total fatty acid content and ultrastructure of the current year needles were subsequently analysed. In acute, high concentration exposures, significant reductions in the levels of linolenic acid were detected. Increases in myristic or palmitic acid were common in needles exposed to lower concentrations of ozone. Ultrastructural studies revealed reductions in chloroplast size and a darkening of stroma at low ozone exposures while at high concentrations disruption of the chloroplast membranes was also identified.     

Persistent effects of ozone on needle water loss and wettability in Norway spruce

Four-year-old, seed-grown trees of Norway spruce (Picea abies (L.) Karst.) were exposed in open-top chambers to charcoal-filtered air (8 h daily mean 54 microg O(3) m(-3)) over three consecutive summers (1986-1988). In mid-May 1988, before the third season of fumigation and more than 7 months after exposure to ozone the previous summer had terminated, daily rates of transpiration from intact shoots and water loss from excised needles were measured together with the amount of wax on the needle surface. In mid-July, 92 days after the beginning of the third year of exposure, the wettability of needles was assessed by measuring the contact angle of water droplets on the surface of needles. Exposure to 156 microg O(3) m(-3) resulted in a 16% increase in daily transpiration in current year's needles and a 28% increase in 1-year old needles. These effects were associated with slower stomatal closure in response to increasing water deficit in the needles previously exposed to 156 microg m(-3) ozone. The long-lasting nature of such ozone-induced effects could predispose trees to drought and winter desiccation. No significant effects of ozone were found on the amount of wax covering the needle surface, but a marked increase in the wettability of needles exposed to ozone was observed. The far reaching physiological consequences of these effects in the field and the possibility that similar disturbances may contribute to the decline of high-altitude forests of Norway spruce in Europe are discussed.     

Effects of reactive hydrocarbons and hydrogen peroxide on antioxidant activity in cherry leaves

One-year-old cherry trees were fumigated with propene and gas-phase hydrogen peroxide, singly and in combination, in controlled-environment chambers for an 8-week period during the summer season. A UV light source was included with the combined propene and hydrogen peroxide regime to provide a source of hydroxyl radicals and ozone, and thus all the constituents of a photochemical smog. Measurements were made of soluble protein concentration and of glutathione reductase activity in leaf extracts from two or three leaf classes in plants from each treatment regime at the end of each fumigation period. Significant increases in soluble protein concentration with respect to the controls were found in plants fumigated with propene and hydrogen peroxide. The occurrence and extent of these differences depended on the leaf class and on the timing of the fumigation period over the summer with respect to bud break. The activity of glutathione reductase was found to be significantly increased in mature lower leaves of plants which had been fumigated with hydrogen peroxide. This effect was independent of the timing of fumigation with respect to bud break. Enzyme activity was also increased in propene and in propene plus hydrogen peroxide treatments, but only when plants were fumigated early in the growth season.     

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

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

Beech (Fagus sylvatica) response to ozone exposure assessed with a chlorophyll a fluorescence performance index

This paper describes a relationship between ozone exposure, biomass, visual symptoms and a chlorophyll a fluorescence performance index for young beech trees (Fagus sylvatica). The plants were exposed to four levels of ozone in open-top fumigation chambers (50, 85, 100% of ambient, and 50% of ambient+30 nl l(-1) ozone) that fluctuated in parallel with ambient ozone during a single growing season. The trees were fumigated in the four treatments with ozone levels corresponding to an AOT40 (accumulated exposure above a threshold of 40 nl l(-1)) of 0.01, 3.35, 7.06 and 19.70 microl l(-1) h, respectively. Highly significant differences were found between the 50% of ambient+30 nl l(-1) ozone treatment and all other treatments, with a 70.5% reduction in primary photosynthetic performance, as measured with the PI index. The reduction of the PI values demonstrated a high correlation with visual symptom development (r(2)=0.98), and by the end of September with biomass loss (r(2)=0.99). A significant ozone exposure-response relationship was found between AOT40 and primary photochemistry (r(2)=0.97). Thus, analysis of PI provides an alternative method for regional monitoring of tree health within the context of the currently employed AOT40.     

Influence of soil substrate and ozone plus acid mist on the frost resistance of young Norway spruce

The needles of clonal Norway spruce grown in environmental chambers on two different soils (an acidic soil 1 and a calcareous soil 2) and exposed to two levels of ozone fumigation (a low level combined with neutral mist = control, and an elevated one combined with acid mist = treatment) were analyzed for their frost hardiness. No effect of ozone was observed on either the development of frost resistance during the hardening phase or on the decrease in frost resistance during the dehardening phase. The preliminary results of Brown et al. (1987) and Barnes and Davidson (1988), which indicated that ozone treatment predisposes plants to winter injury, could thus not be confirmed. Frost resistance was, however, distinctly influenced by the content of the mineral nutrients of the soils. The pronounced K(+) deficiency of the needles of the trees growing on the neutral soil (Alps) had less effect on the development of frost resistance than did the Ca(++) and Mg(++) deficiency of the needles of the trees grown on the acidic soil 1 (Bavarian Forest). The variability of frost resistance between the different clones on soil 1 was partly attributed to fluctuations in the mineral nutrient content of the needles, rather than to a genetic predisposition.     

Influence of ozone and acid mist on the contents of gibberellic acid (GA(3)) in spruce needles (Picea abies) (L.) Karst

The phytohormone GA(3) in needles from 4-year-old Norway spruce trees was analyzed after treatment with ozone and acid mist in environmental chambers under controlled conditions. GA(3) was extracted with methanol from the lyophilized material. Subsequent purification steps included the use of polyvinylpyrollidone (PVP), cartridge reversed-phase purification, ethylacetate extraction and HPLC. The GA(3) was determined in the methylated form by means of a highly specific and sensitive enzyme immunoassay. Higher GA(3) contents were detected in young needles (year 1987) as compared to older ones (year 1986). However, no statistically significant differences were found in the GA(3) levels between the controls and the needles of trees which were treated with increased levels of ozone and acidic mist.     

Secondary effects of air pollution: ozone decreases brown rust disease potential in wheat

Young wheat plants were fumigated with 170 microg m(-3) ozone for 3 days, or with 210 microg m(-3) ozone for 7 days, for 7 hours a day. At the end of the fumigation period the plants were inoculated with brown rust (Puccinia recondita f. sp. tritici) uredospores. The development of new uredospore pustules on fumigated and control plants was evaluated as a measure of rust disease potential. The number of pustules on the ozone fumigated plants was greatly reduced in comparison with the number of plants treated with charcoal-filtered air.     

No general ozone-specific indicator among the hexane- and dichloromethane-soluble components of Picea abies needles exposed to ozone in open-top chambers

The effect of ozone (< 10, 200 or 400 microg m(-3) on hexane- and dichloromethane-soluble components of Picea abies needles was determined by fumigating potted grafts from mature trees. The trees (>55 and 125 years, 2.5 m high), representing six clones of Norway spruce, were fumigated in open-top chambers at two locations in Norway for one growth season. The needles were extracted with hexane and dichloromethane; 142 compounds from the hexane extract and 164 silylated compounds from the dichloromethane extract were analysed by gas chromatography although no identifications were made. The concentration of four of the compounds from the hexane extract changed with ozone dose in a way that made them promising as indicators, but the present analytical method could not verify this possibility. None of the other 302 compounds qualified as a general indicator of ozone stress in Norway spruce, as none changed its concentration with ozone dose consistently in all romets of all clones. Most of the variation in the experiment is mainly attributable to genetic variation and to climate.     

Changes in pigment concentration and composition in Norway spruce induced by long-term exposure to low levels of ozone

Rametes of Norway spruce were fumigated with 30 ppb (nl litre(-1)) ozone above ambient level for 4 years in open-top chambers. They were grown under different light conditions, because some of the chambers received approximately 10% less light than the others. Samples from three age classes were analyzed for nitrogen and pigments using HPLC. It could be demonstrated that the ozone treatment reduced the concentration of chlorophyll (a) and (b), alpha- and beta-carotene, but increased the concentration of antheraxanthin. A significant decrease was found for the violaxanthin/antheraxanthin ratio following the ozone treatment. The concentration of all the pigments and of nitrogen were significantly related to the age classes, and a similar relationship was found for the light levels, except for antheraxanthin and total carotenoids. The ratio of chlorophyll a/b was only significantly related to the age classes.     

Role of climate, crown position, tree age and altitude in calculated ozone flux into needles of Picea abies and Pinus cembra: a synthesis

Ozone (O(3)) flux into Norway spruce (Picea abies) and cembran pine (Pinus cembra) needles was estimated under ambient conditions at six rural sites between 580 and 1950 m a.s.l. We also assessed age-related differences in O(3) flux by examining changes in leaf conductance across the life span of Norway spruce. At the leaf level O(3) flux into the needles was effectively controlled by stomatal conductance and, hence by factors such as temperature, irradiance and humidity, which control stomatal conductance. Seasonal variations in O(3) flux were mainly attributed to the course of the prevailing temperature. During the growing season, however, data have emphasised leaf-air vapour pressure difference as the environmental factor most likely to control stomatal conductance and O(3) flux into the needles. In the sun crown stomatal conductance averaged over the growing season decreased with increasing tree age from 42.0+/-3.5 mmol O(3) m(-2) s(-1) in 17-year-old trees to 7.1+/-1.0 mmol O(3) m(-2) s(-1) in 216-year-old trees, indicating that O(3) concentration in the substomatal cavities is higher in young than in old trees. Independent from tree age stomatal conductance and O(3) flux were approximately 50% lower in shade needles as compared to sun-exposed needles. Stomatal conductance was also greater in the current flush (24+/-5.6 mmol O(3) m(-2) s(-1)) and in 1-year old needles (16+/-4 mmol O(3) m(-2) s(-1)) than in older needle age classes (12+/-1 mmol O(3) m(-2) s(-1), averaged across the four older needle age classes). In trees similar in age (60-65 years old) average O(3) flux into sun needles increased from 0.55+/-0.36 nmol m(-2) s(-1) at the valley floor to 0.9 nmol m(-2) s(-1) in 1950 m a.s.l. Cumulative O(3) uptake during the vegetation period increased from 11.4+/-1.7 mol m(-2) in the valley to 14 mol m(-2) at the alpine timberline. Although stomatal conductance provides the principal limiting factor for O(3) flux, additional field research is necessary in order to improve our understanding concerning the quantitative 'physiological threshold dose' which internally can be active and can have adverse effects of O(3) on forest trees.     

CO2/H2O gas exchange parameters of one- and two-year-old needles of spruce and fir

Gas exchange was characterized in one- and two year-old spruce (Picea abies L. Karst.) and fir seedlings (Abies alba Mill.) which had been exposed to low levels of ozone, sulfur dioxide and simulated rain or a combination of all three variables in open top chambers from 1983 through 1988. The gas exchange measurements were carried out in March 1988 at the end of the five year experiment. The twigs examined did not exhibit any visible sign of injury, specifically no differences were apparent between trees under the treatments of simulated acidic rain at pH 5.0 and pH 4.0. The study of carbon dioxide response curves showed different effects of the pollutants on the tree species. One-Year-old spruce needles treated with O(3) and simulated acidic precipitation pH 4.0 showed noticeable reduction of net photosynthetic rate. Exposure to the combination O(3) and SO(2) at pH 4.0 resulted in a significant depression of photosynthesis in two-year-old needles Transpiration rate was not decreased to a similar extent. No changes either in photosynthesis or transpiration were found in spruce under fumigation with SO(2) alone. These results indicate that ozone is the principal cause of changes in photosynthetic performance of spruce. It alters mesophyll response rather than reducing stomatal conductance. The specific changes that occur in the mesophyll could be diagnosed as inactivation of a carbon fixing enzyme as well as damage of the electron transport system. Fir seem to be more tolerant to ozone. No changes in photosynthesis and transpiration following exposure to O(3) alone were found. However, SO(2) fumigation, alone or in combination with O(3), resulted in a marked decrease of photosynthetic performance. Particularly, carboxylation efficiency and also maximum carboxylation velocity were depressed indicating a reduction in carbon fixing enzyme activity. No differences between single and combined fumigation treatments regarding these variables were determined. However, parameters measured to determine changes in electron transport rate showed a higher depression in the presence of both pollutants. Transpiration also was reduced by SO(2).     

Effects of ozone exposure in open-top chambers on poplar (Populus nigra) and beech (Fagus sylvatica): a comparison

Rooted cuttings of poplar (Populus nigra) and seedlings of beech (Fagus sylvatica) were exposed to ozone in open-top chambers for one growing season. Three treatments were applied: charcoal-filtered (CF), non-filtered (NF) and non-filtered air plus 30 ppb (nl l(-1)) ozone (NF+). Extra ozone was only added on clear days, from 09:00 until 17:00-20:00. The AOT40s (accumulated exposure over a threshold of 40 ppb), calculated from April to September were 4055 ppb.h for the NF and 8880 ppb.h for the NF+ treatments. For poplar ozone exposure caused highly significant reductions in growth rate, light-saturated net CO(2) assimilation rate, stomatal conductance, F(v)/F(m) and chlorophyll content. The largest effects were observed in August at which time ozone concentrations were elevated. A reduction was noticed in new leaf production, while accelerated ageing and visible damage to leaves caused high leaf losses. For beech the responses were similar but less pronounced: ozone exposure resulted in non-significant growth reductions, slight changes in light-saturated photosynthesis and accelerated leaf abscission. The chlorophyll content of beech leaves was not affected by the ozone treatments. The results confirmed previous observations that fast-growing tree species, such as most poplar species and hybrids, are more sensitive and responsive to tropospheric ozone than slower-growing species, such as beech. The growth reductions observed and reported here for beech were within the range of those reported in relationship to the AOT40 (accumulated exposure over a threshold of 40 ppb) critical level for ozone.     

Free-air fumigation of mature trees. A novel system for controlled ozone enrichment in grown-up beech and spruce canopies

A novel system for continuous and controlled free-air fumigation of mature tree canopies with ozone is described. Ozone generated from oxygen is diluted with air in a pressurized tank and conducted into the canopies by a system of 100 PTFE tubes hanging down from a grid fixed above the crowns. With 45 calibrated outlets per tube providing a constant flow of 0.3 l/min each, a total volume of about 10*10*15 m3 comprising 5 beech and 5 spruce canopies is fumigated. The spatial ozone distribution in the fumigated volume as well as surrounding reference tree canopies is controlled by continuous measuring instruments installed at 4 levels and a dense array of passive samplers. The system will later be used for CO2 fumigation as well. Results of the first year of continuous operation, with 2 * ambient ozone levels having been achieved, are reported.