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.     

Mineral content in the soil and tree foliage

Seedlings of fir (Abies alba Mill.) and spruce (Picea abies L. Karst.) were fumigated with SO(2), O(3) and SO(2) + O(3) in open-top chambers (OTCs) for almost 5 vegetation periods. As background stress, simulated rain of pH 4.0 was applied. Nutrient content of soil, soil solutions, and trees was investigated and balanced. In the upper partition of the soil high concentrations of exchangeable Ca(2+) were found in all chambers. The SO(2) and SO(2) + O(3) treatments led to increased Ca(2+), Mg(2+) and Mn(2+) concentrations in soil solution and the pool of exchangeable protons increased. This response was most evident in the SO(2) and SO(2) + O(3) chambers and less clear in the filtered pH 5.0 control chamber. In the SO(2) treatment increased Mn and S levels were found in the needles. Ca content in the needles showed a decreasing trend. O(3) alone had no consistent effect on needle nutrient content.     

Chlorophyll, carotenoids and the activity of the xanthophyll cycle

Clone spruce trees (Picea abies L. Karst.) were exposed in the Hohenheim open-top chambers to low levels of O(3) and SO(2), singly and in combination, and to simulated precipitation of two pH treatments (Seufert et al., this volume). At the end of five years of continuous exposure, needles from the 13-year-old trees were sampled and analysed for pigments content by means of HPLC (high pressure liquid chromatography). The pigment content was determined for three needle age classes. Chlorophyll a content, measured on a dry weight basis, was similar for all needle age classes in the control chambers receiving only the simulated rain treatments at pH 5.0 or 4.0, and the chamber receiving O(3) and the rain treatment at pH 4.0. Also, no differences were noted in one-year-old needles in the chambers with SO(2) and simulated precipitation at pH 4.0 and SO(2) + O(3) and simulated precipitation at pH 4.0. Reductions of approximately 10 and 35% were measured in two-year-old needles from the chambers with SO(2) and precipitation at pH 4.0, and SO(2) + O(3) and precipitation at pH 4.0. The three-year-old needles from these chambers had 40% lower chlorophyll a content compared to the control chambers. No treatment effects were seen on the molar ratios of chlorophyll b, the carotenes, lutein, neoxanthin, and the sum of carotenoids involved in the xanthophyll cycle, violaxanthin + antheraxanthin + zeaxanthin, to chlorophyll [Formula: see text]. The xanthophyll cycle, assayed in one-year-old needles under defined light conditions (520 microE m(-2) s(-1), while light) was active in all samples. Needles from the control chambers and the chambers with SO(2) and with O(3) behaved similarly and differed from the SO(2) + O(3) treated needles by a 50% higher zeaxanthin content reached under light.     

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).     

Mycorrhizal populations and fine root development on Norway spruce exposed to controlled doses of gaseous pollutants and simulated acidic rain treatments

Effects after long-term fumigation and simulated acidic rain (pH 4.0) treatment in open-top chambers (OTC) during 5 vegetation periods on ectomycorrhizal (EM) frequency, fine root structure and distribution of short roots were recorded for Norway spruce. Simultaneous application of ozone and SO(2) resulted in a significant decrease (P< or =0.01) in the proportion of EM root tips and an increase in the numbers of necrotic short roots. The observed differences were partly due to more than additive effects of ozone and SO(2). On the basis of EM and non-mycorrhizal (NM) short roots in the irrigated controls (OTC-V, pH 5.6, Ah-horizon: EM + NM/cm=0.05) the acid treatments resulted in drastic effects (P< or =0.05) on the branching density and the proportion of EM root tips and NM short roots. Relative numbers of short roots infected by indigenous EM fungi with trees in the fumigation treatments at pH 4.0 were similar: 1.5-1.9 EM tips/cm and differed noticeably from the control: 5.2 EM tips/cm. Again, increasing numbers of necrotic and NM short roots were noted. Parallel measurements by the ingrowth-core technique confirm the argument, that EM formation seems to be a reliable indicator of pollution-induced stress.     

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.     

Performance of some growth variables

European beech (Fagus sylvatica L.), Norway spruce (Picea abies L. Karst.) and Silver fir (Abies alba Mill.) were exposed to low concentrations of ozone (O(3)) and sulfur dioxide (SO(2)), alone and combined, and simulated acid rain (pH 4.0) in sheltered open-top chambers in Hohenheim (Southwest Germany) for almost five years. The concentrations of O(3) and SO(2) used were related to annual ambient average found in southern West Germany. Two control chambers were ventilated with charcoal filtered air and rainfall was simulated at pH 4.0 and 5.0. Because of large dense plant growth in the chambers it was only possible to measure uncompleted growth of shoots in the upper canopy. Therefore, growth analysis was restricted to this area. The treatment with acidic precipitation decreased the annual shoot growth of beech and reduced leaf surface area of those trees. Exposure to SO(2), O(3) alone and in combination resulted in further reduction of shoot length and leaf surface area. Fumigation with SO(2) and O(3) + SO(2) caused insignificant decreases of shoot length, total dry weight and needle surface area of spruce. The lateral leader shoot growth of spruce exposed to O(3) was significantly reduced only in the last year of the experiment. Growth rates of the spruce exposed to charcoal filtered air and non-acidic precipitation were reduced more than those of beech and fir. Growth variables determined for fir reflected different rates of incremental change. Exposure to O(3) resulted in the largest dry matter production of all fir groups but those exposed to charcoal filtered air and non-acidic precipitation responded with the best lateral leader shoot growth, lowest specific leaf area (SLA) and leaf area ratio (LAR) respectively indicating best metabolic efficiency. At the conclusion of this study a classification of sensitivity was developed for the tree species.     

The individual and combined effects of ozone and simulated acid rain on growth, gas exchange rate and water-use efficiency of Pinus armandi Franch

The seedlings of Pinus armandi Franch. were exposed to ozone (O(3)) at 300 ppb for 8 h a day, 6 days a week, and simulated acid rain of pH 3.0 or 2.3, 6 times a week, alone or in combination, for 14 weeks from 15 June to 20 September 1993. The control seedlings were exposed to charcoal-filtered air and simulated rain of pH 6.8 during the same period. Significant interactive effects of O(3) and simulated acid rain on whole plant net photosynthetic rate were observed, but not on other determined parameters. The exposure of the seedlings to O(3) caused the reductions in the dry weight growth, root dry weight relative to the whole plant dry weight, net photosynthetic rate, transpiration rate in light, water-use efficiency and root respiration activity, and increases in shoot/root ratio, and leaf dry weight relative to the whole plant dry weight without an appearance of acute visible foliar injury, but did not affect the dark respiration rate and transpiration rate in the darkness. The decreased net photosynthetic rate was considered to be the major cause for the growth reduction of the seedlings exposed to O(3). On the other hand, the exposure of the seedlings to simulated acid rain reduced the net photosynthetic rate per unit chlorophyll a + b content, but did not induce the significant change in other determined parameters.     

Structural changes in spruce and fir needles

Needles from spruce and fir trees were analyzed for histological changes induced by long-term exposure in open-top chambers to SO(2) and/or O(3) combined with acid rain. Light and electron microscopical evaluation revealed initial structural changes in the vascular bundle of fir needles, with an increased number of crushed sieve cells in the phloem. In addition the walls of young, adaxial sieve cells lacked the typical thickening usually observed in naturally aged needles. These findings may indicate restricted assimilate translocation. The presence of SO(2) in any treatment led to thylakoidal swellings and membrane reductions in the chloroplasts of mesophyll cells near the vascular bundle. This damage pattern resembled alterations caused by nutrient deficiency rather than by the direct action of gaseous pollutants. In general, fir appears to be more sensitive to environmental stress than spruce; this substantiates the findings of previous studies.     

Effects of SO2, NO2, and O3 on population development and morphological and physiological parameters of native herb layer species in a beech forest

The native ground vegetation of a beech forest was fumigated with moderate doses of SO(2), NO(2), and O(3) in open-top chambers. The effects of fumigants on growth and above-ground development were dependent on species. The treatments caused early senescence in several of the species present. The epicuticular waxes were attacked by fumigation, which was shown by higher wettability of the leaf surfaces and by leaching of ions. Interspecific differences were observed in the responses of transpiration and photosynthesis to fumigation. Similar patterns of effects on transpiration and photosynthesis, however, were found in the same species. Carbohydrate metabolism was altered by fumigation, leading to starch accumulation in the leaves. Besides effects on higher plants, fumigations also resulted in alterations of the soil microflora. The bacteria/fungi ratio was depressed at the fumigation plots.     

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.     

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.     

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.     

Nitrate reductase activity of needles of Norway spruce fumigated with different mixtures of ozone, sulfur dioxide, and nitrogen dioxide

Four-year-old spruce clones (Picea abies (L.) Karst.) cultivated in sand and provided with a complete nutrient solution, or a solution deficient in magnesium and calcium, were exposed to the pollutant mixtures SO(2)/NO(2), O(3)/NO(2), and O(3)/SO(2)/NO(2), at realistic concentrations for 32 weeks. Fumigation caused a slight increase of total N contents in current year needles, whereas in one-year-old needles N concentrations did not change. The response of nitrate reductase activity to pollutant stress depended on needle age and nutrient supply, respectively. In one-year-old needles fumigation resulted in a significant inhibition of enzyme activity, particularly in Mg and Ca deficient trees. The combination of all three components proved to be most effective in causing a decrease by 60% compared to the control. In contrast, nitrate reductase activity was stimulated in current year needles, especially by O(3)/NO(2) and O(3)/SO(2)/NO(2). Changes in the activity of nitrate and nitrite reductases are considered as a factor contributing to the high phytotoxic potential of pollutant combinations with NO(2).     

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).     

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.     

Injury and physiological responses of Larrea tridentata (DC) Coville exposed in situ to sulphur dioxide

The response of shrubs of Larrea tridentata (DEC) Coville (creosotebush) exposed to sulphur dioxide (SO(2)) was evaluated using in situ plants of the Majove Desert. Larrea was exposed to acute levels of 0.3 to 2.0 microl litre(-1) SO(2) for periods up to 13 days using field chambers or an open-air fumigation system. Plants exposed in the spring exhibited considerable leaf injury (necrosis and defoliation) when exposed to 2.0 microl litre(-1) SO(2), and in the autumn had leaf injury when exposed to >0.4microl litre(-1) SO(2). Injured plants had higher transpiration rates, less negative water pressure potentials, and/or lower photosynthetic rates than control plants. It is likely that Larrea would not be injured by the typically low SO(2) concentrations and dry environmental conditions of the Mojave Desert. However, if injury were to occur, it would be accompanied by changes in plant-water relations and photosynthesis, followed by recovery after the SO(2) stress was removed.     

Fine root studies in situ and in the laboratory

The fine roots and myocorrhizae of beech, spruce and fir trees exposed to ozone, sulphur dioxide and simulated acid precipitation in open-top chambers (OTC) were examined both in situ by rhizoscopy and in the laboratory using root samples from soil cores. Prior to measurements the trees were treated for about one year. During the second year of treatment the fine root production of all three tree species was determined rhizoscopically. The OTC experiments were concluded after an additional three years at which time fine root and small root dry matter as well as the absolute and relative frequencies of mycorrhizae of spruce and fir were determined from soil cores. The vitality of spruce mycorrhizae was examined by fluorescein diacetate staining. In addition total contents of essential cations of spruce mycorrhizae were measured. Long-term exposure to SO(2), SO(2) + O(3), and simulated acid precipitation led to an increased mycorrhizal production by fir. On spruce, a decreased number of mycorrhizae was found in the chambers polluted with SO(2), but a high proportion of dead fine roots indicated an increased root production with an intensified turnover or a delayed decomposition of spruce mycorrhizae. The cation analyses showed an accumulation of Ca(2+) and Zn(2+) in the mycorrhizae of spruce exposed to ozone.     

Effects of ozone on foliar leaching in Norway spruce (Picea abies L. Karst): confounding factors due to NOx production during ozone generation

Previous experiments with conifers fumigated with O(3), produced by air-operated electric discharge ozonators, have provided evidence that O(3) increases the leaching of NO(3)(-), NH(4)(+), K(+), Ca(2+), Mg(2+) and some other cations from needles, when the trees are treated with acid mist. This evidence has provided the foundation of the ozone-acid mist hypothesis of spruce decline. We report experiments with Norway spruce saplings fumigated with purified and unpurified O(3). The results show that the accumulation of NO(3)(-) in the needles arises from the rapid deposition of N(2)O(5) and HNO(3) formed from N(2) in the ozonator. An increase in removal of NH(4)(+), Na(+), Ca(2+), Mg(2+), Zn(2+) and Mn(2+) from the needles during soaking in H(2)SO(4), pH3, was also observed, which was related to the increase in NO(3)(-) but was independent of O(3) concentration. It is concluded that results of previous experiments cited in support of the ozone-acid mist hypothesis arose from effects which were at least partly caused by N(2)O(5) produced as a contaminant, and were incorrectly attributed to ozone. Other effects, such as growth stimulations, visible symptons, enhanced frost sensitivity, and infestation by pests or pathogens, which have been attributed to O(3) generated by electric discharge in air, should be interpreted with caution. Future experiments with ozone must eliminate this problem by either using O(2)-driven ozonators, or by purifying the output from air-driven ozonators using cold and/or water traps.     

Deriving ozone dose-response of photosynthesis in adult forest trees from branch-level cuvette gas exchange assessment

Branch-level gas exchange provided the basis for assessing ozone flux in order to derive the dose-response relationship between cumulative O3 uptake (COU) and carbon gain in the upper sun crown of adult Fagus sylvatica. Fluxes of ozone, CO2 and water vapour were monitored simultaneously by climatized branch cuvettes. The cuvettes allowed branch exposure to an ambient or twice-ambient O3 regime, while tree crowns were exposed to the same O3 regimes (twice-ambient generated by a free-air canopy O3 exposure system). COU levels higher than 20mmolm(-2) led to a pronounced decline in carbon gain under elevated O3. The limiting COU range is consistent with findings on neighbouring branches exposed to twice-ambient O3 through free-air fumigation. The cuvette approach allows to estimate O3 flux at peripheral crown positions, where boundary layers are low, yielding a meso-scale within-crown resolution of photosynthetic foliage sensitivity under whole-tree free-air O3 fumigation.