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.     

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.     

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.     

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.     

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.     

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.     

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.     

Effects of acid mist on needles from mature Sitka spruce grafts. Part II. Influence of developmental stage, age and needle morphology on visible damage

Mature grafts of five Sitka spruce (Picea sitchensis (Bong.) Carr.) clones were exposed to simulated acid mist comprising an equimolar mixture of H(2)SO(4) and NH(4)NO(3) (1.6 and 0.01 mol m(-3)) at pH 2.5 and 5.0. Mist was applied to potted plants growing in open-top chambers on consecutive days, four times a week, at a precipitation equivalent of 1 mm per day. The total exposure to polluted mist was equivalent to three times that measured at an upland forest in SE Scotland. The aim of the experiment was to characterize the response of juvenile foliage produced by physiologically mature grafts (on seedling root stock) and compare it with the behaviour of juvenile foliage on seedlings. Development of visible foliar damage was followed through the growing season. Measurements of needle length, diameter, weight, surface area, surface was weight and wettability were made on current year needles to determine whether particular foliar characteristics increased susceptibility to injury. Significant amounts (> 10%) of visible needle damage was observed on only one of the five clones. Damage was most severe on the clone with the most horizontal branch and needle habit, but over the five clones there was no relationship between angle of branch display and damage. Likewise no combination of needle characteristics (length, width, area, amount of wax) was indicative of potential susceptibility. A comparison with previous acid misting experiments using seedlings suggests that juvenile foliage on physiologically mature trees is equally susceptible to visible injury as juvenile seedling foliage. Data of budburst differed among clones, and in this experiment exerted the over-riding influence on development of injury symptoms. Foliage exposed to a combination of strong acidity and high sulphate concentrations over the few weeks immediately following budburst suffered most visible injury. The absence of significant amounts of visible damage in UK forests probably reflects the general low susceptibility to visible injury of Sitka spruce exposed to acid mist.     

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.     

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.     

Impact of ozone, acid mist and soil characteristics on growth and development of fine roots and ectomycorrhiza of young clonal Norway spruce

As part of the joint 14-month exposure experiment on Norway spruce (Picea abies (L.) Karst.) sensitivity to pollution (two levels of ozone plus acid mist) of growth and development of the fine-root system and of mycorrhizae, have been investigated in two forest soils from areas showing forest decline. This study shows that differences in fine-root biomass and the occurrence of species of ectomycorrhizae were mainly due to prevailing conditions within the acid or calcareous substrate. The pollution treatment resulted in higher numbers of short root tips in only one soil, whereas the percentage of ectomycorrhizal roots with a well-developed mantle was low (10-23%) in both soils, irrespective of exposure of trees to ozone and acid mist. There was no consistent response, with the two clones examined, in terms of mycorrhizal frequency, beaded short roots and renewed growth. Regarding the effects on root growth, data cannot be used unrestrictedly for extrapolation to a more complex field situation.     

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 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.     

Effects of acid mist on mature grafts of Sitka spruce. Part I. Frost hardiness and foliar nutrient concentrations

Mature grafts of five clones of Sitka spruce (Picea sitchensis Bong. Sarg.) were exposed to simulated acid mist composed of an equimolar mixture of sulphuric acid and ammonium nitrate at pH 2.5 and pH 5.0 in open-top chambers from May to November 1991. Treatments were applied on consecutive days, four times a week. The pH 2.5 treatment provided an overall dose three times higher than that received by forests in upland areas of Britain. Frost hardiness was assessed in November by freezing detached current year shoots at a range of temperatures and assessing the rate of electrolyte leakage Foliar nutrient concentrations were determined on the same shoots. Acid mist at pH 2.5 significantly reduced frost hardiness in four of the five clones; the temperature causing 50% shoot death (LT50) was increased by 0 to 7 degrees C. The clones varied in their level of hardiness, one clone being exceptionally frost sensitive. The frost hardiness of the frost sensitive clone was found to be less perturbed by acid mist than the hardiness of the more frost resistant clones. Mature grafts showed a smaller reduction in hardiness at an equivalent dose than that found previously with Sitka spruce seedlings. Compared with seedlings, grafts had lower absolute concentrations of foliar sulphur. Exposure to acid mist at pH 2.5 increased %S in current year foliage by <0.05% compared with absolute increases of more than 0.10% in current year foliage of seedlings. We conclude that the effect of acid mist on frost hardiness is likely to be less on mature trees than on seedlings and that the increased frost risk to mature trees of Sitka spruce from occult deposition alone is small.     

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.     

Surface structures and epicuticular wax composition of spruce needles after long-term treatment with ozone and acid mist

Epicuticular wax structures and epicuticular wax composition were studied in needles of Picea abies (L.) Karst. plants after a long-term fumigation experiment. SEM observations showed aggregated as well as undamaged waxes without any relation to treatments. The chemical wax analysis revealed clone specific differences, but only one compound, the nonacosan-10-ol (C(29)-ol), was found enriched in ozone/acid mist treatments.     

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.     

Ozone Injury to Pinus Strobus

In a study of the ozone-induced needle blight of eastern white pine in central New York, acute injuries naturally induced on field trees during a year of relatively low ozone concentrations (1 966) were compared with injuries induced during a year of higher concentrations (1967). Injuries were more frequent and severe and were associated with higher mean ozone concentrations in 1967 than in 1966. Characteristic symptoms were induced on foliage of pine branches exposed to controlled doses of ozone as low as 7 ± 1 pphm for four hours or 3 ± 1 pphm for 48 hours. Such doses were equalled or exceeded two and four times, respectively, in the field during the 1 967 season. Ozone sensitivity of pine needle tissue was increased by fumigation in atmospheres containing water mist. High concentrations of ozone (40-60 pphm) caused general injury of foliage of both ozone-susceptible and resistant trees; the symptoms were unlike those found in the field or caused by fumigation with low concentrations of ozone.     

Limited compensation of ozone stress by potassium in Norway spruce

The effects of potassium fertilization and ozone stress were investigated in a clone of Picea abies (L.) Karst, by studies of the uptake of CO(2) by the crowns, the element content, on leaching of the youngest needles, and the longevity of the needles. All plants were exposed to 0.075 microl litre(-1) SO(2) from January to April 1986. The average ozone concentrations applied during the subsequent growing season (May-December) were 0, 0.027, 0.050 and 0.100 microl litre(-1). Half of the trees received liquid fertilizer applications from April to July 1986. CO(2) uptake by the crowns was significantly reduced in non-fertilized plants at ozone doses of 100-200 microl litre(-1) h, whereas similar reductions were recorded in fertilized plants only above an ozone dose of 300 microl litre(-1) h. Independent of the fertilization, however, the concentrations of calcium, magnesium and nitrogen in the needles increased in parallel with the ozone dose, whilst potassium, phosphorus and sulphur showed little response to ozone. In both nutrient regimes, the diffusive loss of elements from chloroform-washed needles was similar and tended to be reduced at the highest ozone concentration, when relating the leachate to the corresponding element content in the needles. Needles formed in the highest ozone treatment were significantly shed during the succeeding year, regardless of the nutrient supply. It appears that increased potassium supply has little compensating effect on ozone stress in spruce.