Effects of low-level long-term ozone fumigation and acid mist on photosynthesis and stomata of clonal Norway spruce (Picea abies (L.) Karst.)

Four clones of 3-year-old Norway spruce (Picea abies (L.) Karst.), grown on two soils, were from July 1986 to September 1987 exposed to ozone fumigation (50 microg m(-3) as a control, 100 microg m(-3) plus peaks between 130 and 360 microg m(-3) as treatment) and acid mist of pH 3.0 (versus mist pH 5.6 in the control). Climatic conditions, identical for both control and treatment, followed a diurnal and seasonal pattern characteristic of medium high altitudes of the Bavarian Forest, an area affected by the new-type forest decline. Gas-exchange measurements were carried out on the plants from December 1986 until the end of the 14-month's exposure using a series of climate-controlled minicuvettes. ANOVA of the four clones investigated towards the end of the experiment gave hints of a treatment-related depression of the photosynthetic capacity of the previous year's needles (age-class 1986). Within this age-class only one of the clones (11) showed a significant treatment effect, indicating an age-class dependence and a genetic influence of the treatment-related depression of the photosynthetic capacity. The current year's flush was not impaired through the ozone and acid mist exposure. Analysis also revealed clear effects of soil, clone and needle age on photosynthetic parameters.     

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.     

Persistent stimulation of CO2 assimilation and stomatal conductance by summer ozone fumigation in Norway spruce

CO(2) assimilation rate, stomatal conductance and chlorophyll content of current and previous years' needles of Norway spruce were measured in May 1988, 205 days after the cessation of ozone fumigation during the summer of 1987. Rates of assimilation were consistently higher for both needle year age classes for ozone fumigated trees in comparison to control trees, although only statistically significant for part of the day for current year's needles. A 26% and 48% stimulation, overall, in mean daily rates of assimilation for current and previous years' needles of ozone fumigated trees was observed. This was due to an enhanced apparent quantum yield and light saturated rate of assimilation of ozone fumigated trees. The temperature response regression of assimilation versus temperature was also greater, such that at any given temperature, assimilation was higher for ozone treated trees than control trees. Stomatal conductance was greater for ozone fumigated trees than the controls, but this was only marginally statistically significant. Moreover, there was a consistent increase in chlorophyll content in both year classes in ozone-treated trees. These results are discussed in relation to a possible long term effect of ozone fumigation upon the processes of conifer winter hardening and spring de-hardening.     

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).     

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.     

A cumulative ozone uptake-response relationship for the growth of Norway spruce saplings

Norway spruce saplings [Picea abies (L.) Karst.] were exposed during four growing seasons to different ozone treatments in open-top chambers: charcoal filtered air (CF), non-filtered air (NF) and non-filtered air with extra ozone (NF+, 1.4xambient concentrations). The CF and NF+ ozone treatments were combined with phosphorous deficiency and drought stress treatments. The total biomass of the trees was harvested at different intervals during the experimental period. The ozone uptake to current-year needles of the Norway spruce saplings was estimated using a multiplicative stomatal conductance simulation model. There was a highly significant correlation between the reduction of total biomass and the estimated cumulative ozone uptake, which did not vary when different thresholds were applied for the rate of ozone uptake. The reduction of the total biomass was estimated to 1% per 10 mmol m(-2) cumulated ozone uptake, on a projected needle area basis.     

Biochemical response of Norway spruce (Picea abies (L.) Karst.) towards 14-month exposure to ozone and acid mist: effects on amino acid, glutathione and polyamine titers

Two clones of Norway spruce were exposed to elevated ozone levels (100 microg m(-3) with episodes of 130-360 microg m(-3)) in combination with acidic mist (pH 3.0) for two vegetation periods. The plants did not exhibit any visible injury, but levels of several amino acids and polyamines were altered in comparison with control plants (50 microg m(-3) ozone, mist of pH 5.6), the changes being pronounced in clone 14. Total free amino acids as well as methionine titers were increased in clone 14. Asparagine was significantly increased in clone 11 and less so in clone 14. Arginine, which comprised more than 50% of the free amino acids in spruce needles, was not changed by the exposure regime applied. Reduced glutathione was significantly increased in all clones/soil/needle age combinations (average increase 50%). Free soluble putrescine was enhanced by 50-200% in clone 14, but remained unchanged in clone 11. Conjugated putrescine was significantly, and conjugated spermidime was slightly, increased in both clones, whereas other polyamines did not responde to the treatment.     

Effects of ozone, acid mist and soil characteristics on clonal Norway spruce (Picea abies (L.) Karst.)--an introduction to the joint 14 month tree exposure experiment in closed chambers

This paper introduces a series of publications referring to a single 14-month laboratory study testing the hypothesis that the recent decline of Norway spruce (Picea abies (L.) Karst.) at higher elevations of the Bavarian Forest and comparable forests in medium-range mountains and in the calcareous Alps is caused by an interaction of elevated ozone concentrations, acid mist and site-specific soil (nutritional) characteristics. The effect of climatic extremes, a further important factor, was not included as an experimental variable but was considered by testing of the frost resistance of the experimental plants. Results of these individual studies are presented and discussed in the following 14 papers. Plants from six pre-selected clones of 3-year-old Norway spruce (Picea abies (L.) Karst.) were planted in April 1985 in an acidic soil from the Bavarian Forest, or a calcareous soil from the Bavarian Alps. After a transition period, plants were transferred, in July 1986, into four large environmental chambers and exposed for 14 months to an artificial climate and air pollutant regime based on long-term monitoring in the Inner Bavarian Forest. The climatic exposure protocol followed realistic seasonal and diurnal cycles (summer maximum temperature, 26 degrees C; total mean temperature, 9.8 degrees C; winter minimum, -14 degrees C; mean relative humidity, 70%; maximum irradiance, 500 W m(-2); daylength summer maximum, 17 h; winter minimum, 8 h). Plants were fumigated with ozone, generated from pure oxygen (control: annual mean of 50 microg m(-3); pollution treatment: annual mean of 100 microg m(-3) with 68 episodes of 130-360 microg m(-3) lasting 4-24 h), and background concentrations of SO(2) (22 microg m(-3)) and NO(2) (20 microg m(-3)); windspeed was set at a constant 0.6 m s(-1). Plants were additionally exposed to prolonged episodes of misting at pH 5.6 (control) and pH 3.0 (treatment). Simulation of the target climatic and fumigation conditions was highly reliable and reproducible (temperature +/-0.5 degrees C; rh+/-10%; ozone+/-10 microg m(-3);SO(2) and NO(2)+/-15 microg m(-3)).     

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.     

No general effect of ozone on foliar nutrient concentrations in mature scions of grafted Picea abies trees

The effect of ozone (< 10, 200, or 400 microg m(-3)), on foliar nutrient concentrations of Picea abies were determined by fumigating potted grafts from mature trees (> 55 and 125 years), representing six clones, in open-top chambers at two locations in Norway. The concentration of nutrients in needles of grafted plants were significantly affected by clone and location. Generally, the concentrations of nutrients were not affected by ozone, but a significant increase in the concentrations of potassium and iron in two of the clones were found. These two clones were the only ones injured (yellow needles) by the fumigation.     

Influence of soil substrate and ozone plus acid mist on the pigment content and composition of needles from young Norway spruce trees

The influence of an ozone + acid mist treatment on photosynthetic pigments has been examined with the needles of the (Picea abies) clones 11, 14, 16, and 133 by spectrophotometric analysis of the total pigment extract and of single components upon HPLC separation (Part A), and in terms of a detailed pigment analysis of the 1987 and 1986 needles of clone 14 by TLC (Part B). Clone 14 had been already analysed prior to the onset of the experiment. At the end of the 14-month experiment, which incorporated frost events during a simulated winter period, neither symptoms corresponding to those of spruce Type I or IV decline, nor those of ozone damage could be observed. However, the 1986 needles of the trees on soil 1, which exhibit an adequate nutrient content, showed zonal chlorosis independently of the ozone + acid mist treatment. Analysis of variance of chlorophyll contents and needle ages showed a clear reduction to nearly 50% in the 1986 needles of clone 11, soil 1, and clone 16, soil 2. In contrast, clones 14 and 16 (soil 1) formed significantly more chlorophylls during the shorter exposure time in the 1987 flush. The detailed analysis of the individual pigment components of clone 14 needles provided no evidence for a destructive influence of the treatment on the chlorophylls, xanthophylls and beta-carotene in the two needle generations which had developed during the experiment, in spite of the distinct K deficiency of the 1986 needles of the trees on soil 2 and the common chlorosis of the needles of the trees on soil 1. The observed increase in violaxanthin content upon O(3)-treatment observed in clone 14 can be considered as an expression of the protective function of the xanthophylls against photooxidative processes. In conclusion, the observed differences in the chlorophyll and carotenoid contents are better correlated with the individual clone and soil character than with the ozone + acid mist treatment. Comparing the results of the pigment analyses of the needles the differences in the pigment concentrations reflect the N and K contents (Pfirrmann et al., 1990), which differ significantly between the clones. Thus it is not possible to pool the pigment data of all clones without considering the different nutrient levels.     

Norway spruce needles as bioindicator of air pollution in the area of influence of the Sostanj Thermal Power Plant, Slovenia

This paper reports the results of total sulphur content, photosynthetic pigments, ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) analysed in current-year needles of Norway spruce (Picea abies (L.) Karst.) in the area influenced by sulphur emissions from the Sostanj Thermal Power Plant (STPP), Slovenia, in the period 1991-2004. Ten differently polluted sampling sites in the emission area of STPP were selected. After desulphurization of emission gases from STPP total sulphur content in needles decreased and vitality parameters of needles increased. Moreover, a strong correlation between the average annual emissions of SO(2) from STPP and average annual sulphur content (increase) or average annual chlorophyll content (decrease) in current-year needles was found. The results showed that spruce needles may be an useful bioindicator for detecting changes in the emission rates of SO(2).     

Harmful effects of atmospheric nitrous acid on the physiological status of Scots pine trees

An open top chamber experiment was carried out in the summer of 2003 to examine the effect of nitrous acid (HONO) gas on the physiological status of Scots pine saplings (Pinus sylvestris). Four-year-old pine trees were exposed to two different levels of HONO gas (at ca. 2.5 ppb and 5.0 ppb) and a control (filtered air) from early evening to early morning (18:00-6:00), in duplicate open top chambers. Significant decreases in the ratios of chlorophylls a to b, an increase in the carbon to nitrogen (C/N) ratio, and a reduction of maximum yield of PS II (F(v)/F(m)) in pine needles were also observed after the 2 months' fumigation. Cation contents of pine needles were also decreased by the fumigation with HONO gas. The results could be explained by the harmful effects of OH radicals, generated from photolysis of HONO gas, and/or aqueous phase HONO (NO(2)(-)/HONO), on the photosynthetic capacity of pine needles.     

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.     

Biochemical response of Norway spruce (Picea abies (L.) karst.) towards 14-month exposure to ozone and acid mist: part II--Effects on protein biosynthesis

Four-year-old clonal Picea abies (L.) Karst. plants were treated with ozone (100 microm(-3) plus peaks of 130 to 360 microm(-3)) and acid mist (pH 3.0) during two vegetation periods. Pulse labelling experiments on shoots were performed with [(35)S]methionine in the second year of exposure. Extraction of soluble needle proteins in citric acid buffer of pH 2.8 revealed protein patterns on SDS polyacrylamide gels that differed from those of control needles fumigated with ambient levels of ozone (50 microg m(-3)) and mist of pH 5.6. New proteins of MW 16000 and 32000 were synthesized only in ozone-exposed needles and could not be detected in the controls.     

Monoterpene patterns of different tissues and plant parts of Norway spruce (Picea abies L. Karst.)

Norway spruce saplings (Picea abies L. Karst.) were exposed for five years to controlled concentrations of ozone and/or sulphur dioxide in open-top chambers. The monoterpenes of needles, twigs (bark and wood), bark and buds harvested from these trees were qualitatively and quantitatively analysed by combined gas chromatography-mass spectrometry. Different tissues and plant parts, showed notable differences in their percentage distribution. However, comparison of the monoterpene patterns of corresponding plant material obtained from trees which had been exposed to different fumigation regimes showed no significant effects of these pollutants.     

Effects of ozone fumigation on epiphytic macrolichens: ultrastructure, CO2 gas exchange and chlorophyll fluorescence

The lichen species Anaptychia ciliaris, Collema nigrescens, Evernia prunastri, Hypogymnia bitteri, Lobaria pulmonaria, Pseudevernia furfuracea and Usnea rigida s.l. were fumigated with site-relevant concentrations (for Central Europe) of ozone over 80 days (180 microg m(-3) during daytime, 80 microg m(-3) during the night). Chlorophyll fluorescence measurements revealed a significant reduction of Fv/Fm after ozone fumigation in five of the species investigated, indicating severe stress on photosystem II due to ozone. The physiological impairment paralleled our fine structural investigations, revealing a significantly higher percentage of collapsed photobiont cells. This indicates that the effects of ambient ozone concentrations under experimental conditions included biophysical and physiological, as well as structural impairment in the lichens studied.     

Chronic effects of vapour phase di-n-butyl phthalate (DBP) on six plant species

A fumigation experiment was performed in which six plant species representing the European flora were exposed to a range of DBP concentrations. Controlled amounts of DBP-saturated air were injected into the ingoing air-streams of plant fumigation chambers, maintaining constant concentrations there for a period of up to 76 days. The target concentrations were a control, 0.8, 1.5, 3.5, and 10.0 microg m(-3). The variation in sensitivity between plant species to atmospheric DBP was quantified on the basis of whole plant biomass in order to derive no-observed-effect-concentrations (NOECs). Significant dose-response relationships, based on realised concentrations, were thus derived using non-linear regression, resulting in NOECs of 0.51 microg m(-3) for Trifolium repens, 0.96 microg m(-3) for Brassica campestris, 1.87 microg m(-3) for Phaseolus vulgaris and 2.21 microg m(-3) for Plantago major. A significant effect was also observed for Holcus lanatus at 12.4 microg m(-3) DBP, but due to the variation at lower levels of DBP exposure, no dose-response relationship could be derived. No significant effect on growth of current year needles in Picea abies was observed, even at the highest level of DBP, 13.7 microg m(-3). Based on statistical extrapolation according to Aldenberg and Slob [Ecotox. Environ. Safety, 25 (1993) 48], an overall predicted no-effect concentration (PNEC) for the plant-atmosphere compartment of 0.33 microg m(-3) DBP was calculated. The PNEC was calculated using the mean and standard deviation of the NOEC for four of the tested species and an extrapolation factor. In addition to changes in leaf colour, leaf crinkling and growth reduction, a number of not quantified observations are described, indicating that DBP affects the physiology as well as the morphology of these species.     

Ozone-induced changes in the chloroplast structure of conifer needles, and their use in ozone diagnostics

Ozone induces characteristic symptoms in the chloroplasts of the needles of several coniferous species. Chloroplasts are (1) reduced in size and (2) the stroma is electron dense. Moreover, (3) these chloroplast alterations are more pronounced in the outer mesophyll cell layers and in the upper side of the needle compared to the inner layers and lower side. The syndrome, including the three symptoms (1)-(3), is found in the green needles of Scots pine and Norway spruce not only in the experimental fumigations, but also in mature trees in the field, and has potential for diagnosis of ozone stress. For sound ozone diagnostics all three symptoms must be present in the samples studied. The symptoms in relation to needle anatomy and physiology is discussed, and recommendations for sampling and analysis are given.     

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.