Effects of simultaneous ozone exposure and nitrogen loads on carbohydrate concentrations, biomass, growth, and nutrient concentrations of young beech trees (Fagus sylvatica)

Beech seedlings were grown under different nitrogen fertilisation regimes (0, 20, 40, and 80 kg Nha(-1)yr(-1)) for three years and were fumigated with either charcoal-filtered (F) or ambient air (O3). Nitrogen fertilisation increased leaf necroses, aphid infestations, and nutrient ratios in the leaves (N:P and N:K), as a result of decreased phosphorus and potassium concentrations. For plant growth, biomass accumulation, and starch concentrations, a positive nitrogen effect was found, but only for fertilisations of up to 40 kg Nha(-1) yr(-1). The highest nitrogen load, however, reduced leaf area, leaf water content, growth, biomass accumulation, and starch concentrations, whereas soluble carbohydrate concentrations were enhanced. The ozone fumigation resulted in reduced leaf area, leaf water content, shoot growth, root biomass accumulation, and decreased starch, phosphorus, and potassium concentrations, increasing the N:P and N:K ratios. A combined effect of the two pollutants was detected for the leaf area and the shoot elongation, where ozone fumigation amplified the nitrogen effects.     

Seasonal differences and within-canopy variations of antioxidants in mature spruce (Picea abies) trees under elevated ozone in a free-air exposure system

The effect of free-air ozone fumigation and crown position on antioxidants were determined in old-growth spruce (Picea abies) trees in the seasonal course of two consecutive years (2003 and 2004). Levels of total ascorbate and its redox state in the apoplastic washing fluid (AWF) were increased under double ambient ozone concentrations (2xO3), whilst ascorbate concentrations in needle extracts were unchanged. Concentrations of apoplastic and symplastic ascorbate were significantly higher in 2003 compared to 2004 indicating a combined effect of the drought conditions in 2003 with enhanced ozone exposure. Elevated ozone had only weak effects on total glutathione levels in needle extracts, phloem exudates and xylem saps. Total and oxidised glutathione concentrations were higher in 2004 compared to 2003 and seemed to be more affected by enhanced ozone influx in the more humid year 2004 compared to the combined effect of elevated ozone and drought in 2003 as observed for ascorbate.     

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.     

Alteration of physiological parameters in needles of cloned spruce trees (Picea abies (L.) Karst.) by ozone and acid mist

Three-year-old clonal spruce trees, kept in growth chambers, were treated with ozone and acid mist during a period of 14 months. One half of the trees were grown on an acidic sandy soil, the other half on a calcareous soil rich in carbonate. At the end of the fumigation period, carbohydrates (glucose, fructose, sucrose, raffinose, starch, glucose-1-phosphate and fructose-6-phosphate) and parameters of the energy status (ATP-, AdN-(ATP + ADP + AMP)- levels, ATP/ADP-ratios and adenylate-energy-charge-(AEC)-values) were determined in the current-year's needles. The results indicate that the metabolic status of a plant tissue is not only influenced by the nature of the air pollutants. Soil factors play an important role in metabolic changes within the plant and are thus of relevance in the manifestation of damage symptoms.     

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.     

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.     

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)).     

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.     

Shoot growth of mature Fagus sylvatica and Picea abies in relation to ozone

Epidemiological analysis of sequential growth data may be a tool in assessing ozone sensitivity of mature trees. Annual shoot growth of mature Fagus sylvatica in 83 Swiss permanent forest observation plots and of Picea abies in 61 plots was evaluated for 11 and 8 consecutive years, respectively, using branches harvested every 4 years. The data were assessed as annual deviation from average growth and related to fructification, ozone, meteorological parameters, and modelled soil water content using a mixed linear model. In beech, a significant association between ozone and shoot growth was observed which corresponded to a 7.4% growth reduction between 0 and 10 ppm h AOT40 (accumulated ozone over threshold 40). This is in the same order of magnitude as the response observed in experiments with seedlings. No interaction was found between ozone and drought parameters. In Norway spruce, shoot growth was neither associated with ozone nor with drought.     

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.     

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

This paper summarizes and evaluates the main findings of 14 preceding papers related to the joint 14-month tree-exposure experiment carried out by the 'Munich Working Party on Air Pollution' at the GSF, Munich, FRG, from July 1986 to September 1987. The experiment tested the hypothesis that an interaction of ozone/acid mist/soil/extreme climatic conditions is the cause of decline of Norway spruce (Picea abies (L.) Karst.) at higher altitudes of the Inner Bavarian Forest. The main findings of the individual studies are presented and their implications for the hypothesis are discussed. Clear effects of soil and genetic factors (differences between clones), for example on growth and frost resistance were found. Treatment with O(3)/acid mist was shown to have effects on plant biochemistry, physiology, histology/ cytology, and growth. The wide scattering of these effects, and the lack of a consistent pattern of response across all clones does not permits a firm conclusion on the validity of the experimental hypothesis. These effects were not confounded by the nutrient stresses imposed during the initial exposure period and were not found to be cumulative during repeated treatments, as was proposed by the hypothesis. It is concluded that the experimental evidence does not indicate that ozone/acid mist are major factors to explain the Norway spruce decline on acidic sites at higher altitudes of the Inner Bavarian Forest and probably similar forest areas.     

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.     

Biochemical response of Norway spruce (Picea abies (L.) Karst.) towards 14-month exposure to ozone and acid mist: part I--Effects on polyphenol and monoterpene metabolism

Three-year-old clonal Picea abies (L.) Karst. plants, grown either on a sandy (No. 1) or on a calcareous (No. 2) soil, were treated with ozone (100 microg m(-3) and peaks of up to 360 microg m(-3)) and acid mist (pH 3.0) over two vegetation periods. Needles of the current (1987) and previous (1986) year were analysed at the end of the experiment for biosynthetic enzymes and in vivo activity of the phenylpropanoid pathway, for products of polyphenol metabolism (clones 11 and 14), and for mono- and sesquiterpenes (clones 14 and 16). 1. Polyphenol metabolism. The activity of cinnamyl alcohol dehydrogenase, an enzyme of the lignin pathway, was increased by the treatment by up to 83% in the needles of both age classes. Chalcone synthase was measured in mature tree material for the first time. This enzyme, as well as phenylalanine ammonia-lyase activities and the content of catechin, astringin, isorhapontin, picein and p-hydroxyacetophenone, exhibited no significant treatment-dependent differences. However, soil and age-class dependent differences occurred. Pulse-labelling experiments with l[U-(14)C]phenylalanine and [2-(14)C]acetate were carried out at four different stages of shoot development and showed label incorporation into (+)-catechin and proanthocyanidins. There was no effect of the ozone and acid mist treatment. 2. Monoterpenes. The content of needle terpenes was generally diminished by the ozone and acid mist treatment. Other factors tested, such as clone, needle age and soil, exerted a highly significant influence on the content of most of the needle monoterpenes.     

Responses of spruce seedlings (Picea abies) to exhaust gas under laboratory conditions--I. Plant-insect interactions

The effects of motor vehicle exhaust gas on Norway spruce seedlings (Picea abies (L.) Karst) and plant-insect interaction of spruce shoot aphid (Cinara pilicornis Hartig) was studied. The exhaust gas concentrations in the fumigation chambers were monitored and controlled by measuring the concentration of nitrogen oxides (NO(x)) with a computer aided feedback system. The concentrations of major exhaust gas components (black carbon [BC], fine particles, VOCs and carbonyl compounds) in the chamber air were also measured. Responses of Norway spruce seedlings to a 2 and 3-week exhaust gas exposure and subsequent performance of spruce shoot aphid were studied using realistic exposure regimes; 50, 100 and 200 ppb NO(x). The feedback control system based on NO(x) concentrations proved an adequate and practical means for controlling the concentration of exhaust gases and studying plant responses in controlled environment chambers. The exhaust exposure resulted in increased concentrations of proline, glutamine, threonine, aspartic acid, glycine and phenylalanine and decreased concentration of arginine, serine, alanine and glycine in young needles. No changes in soluble N concentrations were observed. The results are interpreted as a stress response rather than use of NO(x) as a nitrogen source. No changes in total phenolics and only transient changes in some individual terpene concentrations were detected. The exhaust gas exposure stressed the exposed seedlings, but had no significant effect on N metabolism or the production of defence chemicals. Aphid performance was not significantly affected. Soluble N, secondary metabolism and aphid performance were not sensitive to exhaust gas exposure during shoot elongation in Norway spruce.     

Clone and soil effects on the growth of young Norway spruce during 14 months exposure to ozone plus acid mist

Five clones of 3-year old Norway spruce (Picea abies [L.] Karst), planted in a soil from the Bavarian Forest (pH 4.4) or a soil from the Calcareous Bavarian Alps (pH 6.9), were exposed for two successive vegetation periods, in closed environmental chambers, to a pollution treatment consisting of acidic mist (pH 3.0) plus ozone levels of 100 microg m(-3) with episodes of 130-360 microg m(-3); control trees were exposed to mist of pH 5.6 and ozone levels of 50 microg m(-3). Climatic and pollution protocols followed the diurnal and seasonal pattern characteristic for the Inner Bavarian Forest in Southern Germany, an area affected by the new-type forest decline. Biometric parameters were strongly related to clone and soil. Pollution treatment had a limited effect on only a few growth parameters. The stem diameter growth increment of two clones was reduced by pollution treatment in both soils, a third clone was affected in the acidic soil only. Two other clones were not affected at all. Stem volume increment of three clones, calculated as D(2)H, was reduced by pollution treatment in the neutral soil, a fourth clone was affected in the acidic soil only. Bud break was either delayed (two clones) or accelerated (two other clones) by treatment. Depending on soil and clone, needle yellowing was observed in previous years' needles in both treatment and control trees exposed to increased light intensities. The 'spotted' yellowing was not identical to symptoms found in forest decline areas and was most likely a consequence of nutrient deficiencies during the vegetation period preceding the experiment. The results of this experiment are discussed with regard to field observations and forest productivity. The complex pattern of growth responses resulting from interactions between air pollution, soil and genetic factors is considered to reflect different susceptibilities of trees to air pollutants.     

Photolysis of polycyclic aromatic hydrocarbons adsorbed on spruce [Picea abies (L) Karst] needles under sunlight irradiation

Photolysis of polycyclic aromatic hydrocarbons (PAHs) sorbed on surfaces of spruce [Picea abies (L.) Karst.] needles under sunlight irradiation was investigated. PAHs were produced by combustion of polyvinyl chloride (PVC), wood, high-density polyethylene (HDPE), and styrene in a stove. The factors of sunlight irradiation on the surfaces of spruce needles were taken into consideration when investigating the kinetic parameters. The photolysis of the 18 PAHs under study follows first-order kinetics. The photolysis half-lives range from 15 h for dibenzo(a,h)anthracene to 75 h for phenanthrene. Photolysis of some PAHs on surfaces of spruce needles may play an important role on the fate of PAHs in the environment.     

Responses of spruce seedlings (Picea abies) to exhaust gas under laboratory conditions--II. Ultrastructural changes and stomatal behaviour

This study examines the effects of exhaust gas exposure on the epistomatal wax structure and mesophyll ultrastructure in needles of Norway spruce (Picea abies (L.) Karst.) seedlings. Stomatal diffusive resistance was also measured. Two independent exhaust gas fumigations were performed: 100 and 200 ppb measured as NO(x), for 10 days and 50, 100 and 200 ppb NO(x) for 19 days. The obstructive effect of exhaust gas exposure on epistomatal wax tubules was apparent. The stomata became covered by flat and solid wax resulting from the structural degradation of the wax crystalloids. Increasing the exhaust gas concentration in the chamber atmosphere exacerbated the degradation of the wax structure. Exhaust gas exposure induced aggregation and electron translucence of plastoglobuli, swelling of thylakoids, increase of cytoplasmic lipids and slight increase of vesiculation of cytoplasm in mesophyll cells of current and previous year needles. These changes were exemplified in current year needles. Damage to the epicuticular waxes and mesophyll ultrastructure of spruce needles most likely reflects the NO(x) and volatile hydrocarbon fraction. The alterations in epicuticular waxes and mesophyll ultrastructure can be related to accelerated senescence of the youngest, photosynthetically active, needle generation. The exhaust gas also resulted in decreased diffusive stomatal resistance at night which indicates that the exhaust gas exposure disturbed the gas exchange of spruce seedlings. The results show that even relatively short-term exposure to realistic concentrations of exhaust gas in the atmosphere can induce rather severe injuries to the needle surface structure as well as ultrastructure at the cellular level.     

Effects of deicing salt on the vitality and health of two spruce species,Picea abies Karst., and Picea glehnii Masters planted along roadsides in northern Japan

In northern Japan, the growth of Picea abies Karst., and Picea glehnii Masters, which have been planted along the highways, is often suppressed due to several environmental stresses. To examine the adverse effects of deicing salt, the primary source of stress,we measured needle life span, photosynthetic capacity, and water potential and transpiration rate of the two spruce species at a site with damaged trees, near the roadside and a site with healthy trees, located far from the highway. Results from the analysis showed large amounts of sodium and chlorine in the soil and snow at the damaged site. These elements had accumulated in the needles of the spruce. Moreover, physiological traits of the spruce, at the damaged site were also affected. Therefore, we concluded that poor physiological traits might be attributed to an accumulation of deicing salt in the needles, resulting in the suppression of tree growth.     

Ozone exposure-response relationships for biomass and root/shoot ratio of beech (Fagus sylvatica), ash (Fraxinus excelsior), Norway spruce (Picea abies) and Scots pine (Pinus sylvestris)

Current-year seedlings of beech, ash, Norway spruce and Scots pine were exposed during one growing season to different, but moderate, ozone (O(3)) scenarios representative for Switzerland (50, 85, 100% ambient, 50% ambient+30 nl l(-1)) in open-top chambers (OTCs) and to ambient O(3) concentrations in the field. Biomass significantly decreased with increasing O(3) dose in all species except for spruce. Losses of 25.5% (ash), 17.4% (beech), 9.9% (Scots pine) were found per 10 microl l(-1) h accumulated O(3) exposure over a threshold concentration of 40 nl l(-1) during daylight hours (AOT40). Ratios of root/shoot biomass (RSR) also significantly decreased with increasing AOT40 levels in beech and ash, but not in Norway spruce and Scots pine. The data show that the deciduous species beech and ash were more susceptible to O(3) with respect to RSR and biomass than the coniferous species Norway spruce and Scots pine.     

The effects of excess nitrogen deposition on young Norway spruce trees. Part II the vegetation

The effects of wet-deposited nitrogen on soil acidification and the health of Norway spruce were investigated in a pot experiment using an open-air spray/drip system. Nitrogen was applied as ammonium ((NH(4))(2)SO(4)) or nitrate (HNO(3)/NaNO(3)) in simulated rain to either the soil or the foliage. Symptoms of forest decline as observed in the field were not reproduced, and there was no evidence of direct toxicity. Treatments did, however, have significant effects on tree nutrition. Both NH(+)(4) and NO(-)(3) treatment applied to the foliage lowered foliar K concentrations. NH(+)(4) to a greater extent. Soil-applied NH(+)(4) reduced foliar Mg concentrations and increased foliar Al and Fe. Soil-applied NO(-)(3) significantly reduced foliar P concentrations, and at high doses prevented the alleviation of P deficiency by fertiliser. These effects could be important in some field situations. Ammonium deposition is predicted to be more damaging than nitrate deposition, although the latter may be critical for forests where P status is marginal, such as in parts of the British uplands.