Early events in Populus hybrid and Fagus sylvatica leaves exposed to ozone.

This paper aims to investigate early responses to ozone in leaves of Fagus sylvatica (beech) and Populus maximowiczii x Populus berolinensis (poplar). The experimental setup consisted of four open-air (OA) plots, four charcoal-filtered (CF) open-top chambers (OTCs), and four nonfiltered (NF) OTCs. Qualitative and quantitative analyses were carried out on nonsymptomatic (CF) and symptomatic (NF and OA) leaves of both species. Qualitative analyses were performed applying microscopic techniques: Evans blue staining for detection of cell viability, CeCl3 staining of transmission electron microscope (TEM) samples to detect the accumulation of H2O2, and multispectral fluorescence microimaging and microspectrofluorometry to investigate the accumulation of fluorescent phenolic compounds in the walls of the damaged cells. Quantitative analyses consisted of the analysis of the chlorophyll a fluorescence transients (fast kinetics). The early responses to ozone were demonstrated by the Evans blue and CeCl3 staining techniques that provided evidence of plant responses in both species 1 month before foliar symptoms became visible. The fluorescence transients analysis, too, demonstrated the breakdown of the oxygen evolving system and the inactivation of the end receptors of electrons at a very early stage, both in poplar and in beech. The accumulation of phenolic compounds in the cell walls, on the other hand, was a species-specific response detected in poplar, but not in beech. Evans blue and CeCl3 staining, as well as the multispectral fluorescence microimaging and microspectrofluorometry, can be used to support the field diagnosis of ozone injury, whereas the fast kinetics of chlorophyll fluorescence provides evidence of early physiological responses.

Visible leaf injury in young trees of Fagus sylvatica L. and Quercus robur L. in relation to ozone uptake and ozone exposure. An Open-Top Chambers experiment in South Alpine environmental conditions.

An Open-Top Chambers experiment on Fagus sylvatica and Quercus robur seedlings was conducted in order to compare the performance of an exposure-based (AOT40) and a flux-based approaches in predicting the appearance of ozone visible injuries on leaves. Three different ozone treatments (charcoal-filtered; non-filtered; and open plots) and two soil moisture treatments (watered and non-watered plots) were performed. A Jarvisian stomatal conductance model was drawn up and parameterised for both species and typical South Alpine environmental conditions, thus allowing the calculation of ozone stomatal fluxes for every treatment. A critical ozone flux level for the onset of leaf visible injury in beech was clearly identified between 32.6 and 33.6 mmolO3 m(-2). In contrast, it was not possible to identify an exposure critical level using the AOT40 index. Water stress delayed the onset of the leaf visible injuries, but the flux-based approach was able to take it into account accurately.

Measuring, modelling and testing ozone exposure, flux and effects on vegetation in southern European conditions--what does not work? A review from Italy.

Ozone (O3) exposure at Italian background sites exceeds UN/ECE concentration-based critical levels (CLe(c)), if expressed in terms of AOT40. Yet the occurrence of adverse effects of O3 on forests and crops is controversial. Possible reasons include (i) ability of response indicators to provide an unbiased estimate of O3 effects, (ii) setting of current CLe(c) in terms of cut-off value and accumulation level, (iii) response functions adopted to infer a critical level, (iv) environmental limitation to O3 uptake and (v) inherent characteristics of Mediterranean vegetation. In particular, the two latter points suggest that critical levels based on accumulated stomatal flux (CLe(f)) can be a better predictor of O3 risk than CLe(c). While this concept is largely acknowledged, a number of factors may limit its applicability for routine monitoring. This paper reviews levels, uptake and vegetation response to O3 in Italy over recent years to discuss value, uncertainty and feasibility of different approaches to risk assessment.

Air pollution, forest condition and forest decline in Southern Europe: an overview.

Over the last decades much of the work on the impact of air pollution on forests in Europe has concentrated on central and northern countries. The southern part of Europe has received far less attention, although air pollutants-especially the photochemical ones-can reach concentrations likely to have adverse effects on forest vegetation. Although international forest condition surveys present serious problems where data consistency is concerned, they reveal considerable year-by-year species-specific fluctuations rather than a large-scale forest decline. Cases of obvious decline related to environmental factors are well circumscribed: (1) the deterioration of some coastal forests due to the action of polluted seaspray; (2) the deterioration of reforestation projects, especially conifers, mainly due to the poor ecological compatibility between species and site; and (3) the decline of deciduous oaks in southern Italy and of evergreen oaks in the Iberian peninsula apparently due to the interaction of climate stresses and pests and diseases. However, besides obvious deterioration, changes in environmental factors can provoke situations of more subtle stress. The most sensitive stands are Mediterranean conifer forests and mesophile forests of the Mediterranean-montane plane growing at the edges of the natural ecological distribution. Evergreen sclerophyllous forests appear less sensitive to variations in climatic parameters, since they can adapt quite well to both drought and the action of UV-B rays. Several experiments were carried out to test the sensitivity of Mediterranean forest species to air pollutants. Most of those experiments used seedlings of different species treated with pollutant concentrations too high to be realistic, so it is difficult to derive adequate information on the response of adult trees in field conditions. Ozone has been proved to cause foliar injury in a variety of native forest species in different Southern European countries, while the effects of other pollutants (e.g. nitrogen, sulphur, acidic deposition) are less obvious and likely to be very localized. In the case of ozone, visible symptoms were almost completely missed by large-scale surveys and-at the same time-non-visible symptoms are suspected to be even more widespread than the visible ones. Owing to this and to the complex relationships existing between species sensitivity, ozone exposure and doses, length of the vegetative periods, influence of climatic and edaphic condition on the tree's response, the impacted areas are yet to be identified. Therefore, the large-scale impact of air pollutants on the forests of Southern Europe remains largely unknown, until more specific investigations are carried out.

Assessment of ozone visible symptoms in the field: perspectives of quality control.

The second UN/ECE ICP-Forests Intercalibration Course on the Assessment of Ozone Injury on European Tree Species was carried out in August 2001 at Lattecaldo (Canton Ticino, CH) and Moggio (Lombardy, I). Forty-eight experts from several European countries participated in the exercises and assessed visible symptoms of ozone injury both in open-top chambers (OTC) (Lattecaldo) and under open field (Moggio) conditions. Evaluation of the results indicated a large variability among the teams and call for adequate training of the observers prior to symptom assessment for quality assurance purposes. Highest variability was found for the species developing unclear symptoms which could be confused with senescence processes; such species should not be used in the field. The authors provide suggestions to improve the reliability of the ozone injury assessment on forest plant species.

Response of Quercus pubescens leaves exposed to geothermal pollutant input in southern Tuscany (Italy).

The paper reports a case of evident and widespread leaf damage on trees in southern Tuscany (Central Italy) attributed to the input of pollutants produced in a geothermal area. The main potentially phytotoxic substances are boron and hydrogen sulphide. Trees affected are conifers as well as both evergreen and deciduous broadleaves. In the present study the possible impact of geothermal pollutants on Quercus pubescens leaves has been considered. Leaf samples coming from three sampling locations (S1 inside the geothermal area; S2 on the margins; S3 outside) and three consecutive dates (June, July and August) were analyzed for the following parameters: sulphur and boron concentration; leaf area; leaf mass per area; chlorophyll fluorescence (Fv/Fm); chlorophyll a, chlorophyll b and carotenoid concentrations. Anatomical and ultrastructural observations were also performed. In all sampling location sulphur and boron concentrations are greater than the background values recorded in southern Tuscany in a previous survey. The sulphur concentration in leaves was higher in S1 than S2 and S3, but did not increase throughout the survey period. Boron reached the greatest concentrations in S2 and showed a continuous increase over the study period. Leaves subjected to a higher load of pollutants were smaller in size (in terms of leaf area), but were more sclerophyllous. Damaged chloroplasts and reduced Fv/Fm values were observed at S1 and S2, but chlorophyll concentration values were higher at S1. Such an apparent anomaly can possibly be explained by the onset of compensation and recovery mechanisms. Foliar injuries appeared to be related to boron concentration.

Impacts of urban levels of ozone on Pinus halepensis foliage.

Between May and September, 1996, seedlings of Pinus halepensis were placed at a site adjacent to an automated air pollution monitoring station within the urban area of Florence. Additional 'control' plants were placed in chambers ventilated with charcoal/Purafil(R)-filtered air. All trees were well watered throughout the whole experimental period. During the exposure period, ambient levels of sulphur dioxide were very low, whilst the accumulated hourly exposure to ozone above 40 ppb (i.e. AOT40) exceeded 20000 ppb h(-1) - peak hourly ozone concentrations rising to levels above 100 ppb. Trees exposed to ambient levels of air pollution exhibited typical symptoms of ozone damage (chlorotic mottle) on previous year needles toward the end of the summer. Similar symptoms were not observed on equivalent trees exposed to filtered-air, nor were visible symptoms accompanied by insect or pest infestation. Anatomical and ultrastructural observations made on symptomatic needles revealed degeneration in mesophyll cells bordering sub-stomatal cavities and alterations in chloroplast ultrastructure (fat accumulation, starch and tannin pattern modifications). These observations are consistent with the known effects of air pollutants (namely ozone) recorded in the literature. Findings are discussed in relation to the impacts of ozone on P. halepensis in the Mediterranean region.

Interactions and competition processes among tree species in young experimental mixed forests, assessed with chlorophyll fluorescence and leaf morphology.

Chlorophyll a fluorescence (ChlF) and leaf morphology were assessed in two sites in Europe (Kaltenborn, Germany, and Satakunta, Finland) within a forest diversity experiment. Trees at Satakunta, planted in 1999, form a stratified canopy, while in Kaltenborn the trees are 7 years old, with no apparent canopy connection among broadleaf species. The following ChlF parameters from measured OJIP transient curves were examined: F(V)/F(M) (a proxy for maximum quantum yield); ΨEo (a proxy for efficiency in transferring an electron from reduced QA to the electron transport chain); I-P phase (a proxy for efficiency of reducing final acceptors beyond PSI); and PItot (total performance index for potential energy conservation from photons absorbed by PSII to reduction of PSI end acceptors). At Satakunta F(V)/F(M) and ΨEo in Betula pendula were higher in monocultures and lower in mixed plots, perhaps due to increasing light availability in mixed plots, which can induce photoinhibition. The opposite trend was observed in Picea abies, which was shaded in mixed plots. At Kaltenborn F(V)/F(M) decreased in Fagus sylvatica and P. abies in mixed plots due to competition both above- and belowground. At Satakunta LMA increased in B. pendula leaves with increasing species richness. Leaf area of ten leaves was reduced in F. sylvatica in mixed plots at Kaltenborn. By up-scaling the overall fluorescence response to plot level (PItot_plot ), a significant positive correlation with tree diversity was found at Kaltenborn, but not at Satakunta. This could suggest that competition/facilitation processes in mixed stands play a significant role in the early stages of forest establishment, but then tend to be compensated in more mature stands.

Validation of the stomatal flux approach for the assessment of ozone visible injury in young forest trees. Results from the TOP (transboundary ozone pollution) experiment at Curno, Italy.

This paper summarises some of the main results of a two-year experiment carried out in an Open-Top Chambers facility in Northern Italy. Seedlings of Populus nigra, Fagus sylvatica, Quercus robur and Fraxinus excelsior have been subjected to different ozone treatments (charcoal-filtered and non-filtered air) and soil moisture regimes (irrigated and non-irrigated plots). Stomatal conductance models were applied and parameterised under South Alpine environmental conditions and stomatal ozone fluxes have been calculated. The flux-based approach provided a better performance than AOT40 in predicting the onset of foliar visible injuries. Critical flux levels, related to visible leaf injury, are proposed for P. nigra and F. sylvatica (ranging between 30 and 33 mmol O(3) m(-2)). Soil water stress delayed visible injury appearance and development by limiting ozone uptake. Data from charcoal-filtered treatments suggest the existence of an hourly flux threshold, below which may occur a complete ozone detoxification.