question of whether this species is particularly sensitive to ozone remains to be established see
Barnes et al., 1999. However, ambient concentra- tions of the pollutant are known to be particularly
high in the Mediterranean region Gimeno et al., 1994 because of the high summer temperatures
and radiations, and the natural distribution of this species is coincident with the highest ground level
ozone
concentrations experienced
in Europe
Butkovic et al., 1990; Milla´n et al., 1996. Ac- cording to the UN-ECE standards Ka¨renlampi
and Ska¨rbi, 1996; Fuhrer et al., 1997; Fuhrer and Achermann, 1999, risks for forests are currently
evaluated in Europe with the index AOT40 accu- mulated hourly ozone exposure above 40 ppb
threshold. AOT40 values exceeding 10 000 ppb.h are considered dangerous for forest vegetation
Level I Critical Levels. The threshold of 40 ppb was chosen to exclude the non-anthropogenic
ozone, but this threshold has been seriously ques- tioned. Sutinen et al. 1990, for example, found
that considerably lower ambient ozone concentra- tions about 30 ppb caused ultrastructural alter-
ations on Picea abies needles.
Typical visible symptoms of ozone injury on the needles of P. halepensis have been observed in
California Ka¨renlampi, 1987 and in several parts of Southern Europe Gimeno et al., 1992; Velis-
sariou et al., 1992; Davison et al., 1995; Gimeno et al., 1995; Velissariou et al., 1996. These symp-
toms comprise chlorotic mottling that becomes visible towards the end of summer or early winter
Velissariou et al., 1996. Previous year C + 1 needles are primarily affected Ka¨renlampi, 1987;
Davison et al., 1995, though current year C needles can be affected if trees are exposed to
particularly high levels of the pollutant see Barnes et al. 1999. In the most severe cases, the
symptoms degenerate into necrosis and are associ- ated with premature needle loss. The chlorotic
mottle observed is considered a specific symptom of ozone injury, since the same symptoms have
been reproduced during several controlled or semi-controlled exposures of P. halepensis to
ozone Gimeno et al., 1992; Wellburn and Well- burn, 1994; Elvira et al., 1995; Anttonen et al.,
1998. Several ultrastructural studies have been car-
ried-out on symptomatic needles of P. halepensis in order to characterize the histological changes
induced by ozone Ka¨renlampi, 1986, 1987; Anttonen et al., 1994; Wellburn and Wellburn,
1994. Most of these studies have been performed on plant material exposed to ozone under con-
trolled or semi-controlled conditions Anttonen et al., 1994; Wellburn and Wellburn, 1994, although
one study was conducted on trees exposed in the field Ka¨renlampi, 1986, 1987. Findings have
been variable, presumably because of differences in the way in which trees have been exposed to
the pollutant cf. Anttonen et al., 1998 and the fact that several studies have focused on the ef-
fects on juvenile needles on young seedlings rather than effects on mature needles of forest trees
Kelly et al., 1995; Kolb et al., 1998.
In this study, we examined i the relationship between ambient concentrations of ozone at an
urban Mediterranean location and the appearance of visible symptoms on the needles of P. halepen-
sis, ii the impacts of ambient levels of ozone on needle ultrastructure and iii compared the ob-
served ultrastructural modifications with those de- scribed in the literature as typical of ozone
damage in this species.
2. Materials and methods
2
.
1
. Plant material and sites study Trees about 150 cm tall and 4 years old of P.
halepensis Mill. Aleppo pine field grown in the regional nursery of Roccastrada lat. 43°38 N
long. 11°10 E, in a remote and assumed as non-polluted forest area, were transplanted in
March 1996 and placed in individual pots in steam-sterilized soil: peat: Perlite 1:1:1. At the
beginning of the experiment May trees had cur- rent year C and previous year C + 1 needles.
C + 2 needles were being shed and were not con- sidered. Foliage did not show any biotic or abiotic
symptoms.
Between May and September of the same year, eight of these potted trees were placed at a site
lat. 43°47 N; long. 11°14 E within the urban
area of Florence central Italy where the ground level concentrations of ozone and sulphur dioxide
were constantly measured by ARPAT Regional Agency for Environmental Protection with state-
of-the-art instruments for further details see Grechi and Bruni, 1998. Air samplers were placed
at about the same height as the crowns. The index AOT40 was calculated.
Another eight plants ‘control trees’, were placed in a indoor chamber about 2 × 2 m wide,
ventilated with charcoal-Purafil
®
-filtered air, next the University of Pisa lat. 43°41 N long. 10°21 E.
In these chambers the average daytime tempera- ture tracked external conditions mean daytime
temperature was 25 9 1°C and mean night-time temperature was 20 9 1°C; mean daily photosyn-
thetic photon flux density was 530 mmol m
− 2
s
− 1
and mean relative humidity was 85 9 3, see Sol- datini et al., 1998. All trees were watered to
field-capacity Fig. 1 shows monthly mean data for temperature
and precipitation at the field site over the experi- mental period. The displayed pattern is usual for
the considered period. Fig. 2 provides a summary of daily ground-level sulphur dioxide and ozone
concentrations over the same period. Sulphur diox- ide concentrations were generally low over the
experimental period. In contrast, monitoring data revealed that 7 h 10:00 – 17:00 h daily mean ozone
concentrations generally exceeded the 40 ppb threshold with peak hourly mean concentrations
approaching 100 ppb during six episodes in June and July. These peaks are quite high but not
unusual in Mediterranean conditions see Chalou- lakou et al., 1999. Hourly mean ozone data for
each month over the experimental period is shown in Fig. 3. These data revealed that between May
and September, mean hourly ozone concentrations exceeded the 40 ppb threshold for about one third
of the total exposure time 8 consecutive hours: 10:00 – 18:00 h-with the highest levels of ozone
experienced during July when there were 11 consec- utive h exceeding the 40 ppb threshold. The AOT40
was 20 000 ppb h
− 1
between May and September.
Fig. 1. Daily pattern of temperatures A and precipitations B at the urban field site during the experimental period.
Fig. 2. Ground-level sulphur dioxide A and ozone B concentrations at the urban field site during the experimental period. Sulphur dioxide concentrations expressed as the 24 h daily mean M24 and hourly maximum concentration MAX. Ozone
concentrations expressed as 24 h daily mean M24, 7 h 10.00 – 17.00 h daily mean concentration M7 and maximum hourly concentration MAX.
2
.
2
. Foliar symptoms and microscopical analyses At the end of the exposure period end of
September, 30 needles pairs of the previous year needles i.e. C + 1 were collected from each tree,
from the upper and outer part of the crown 120 – 150 cm from the soil, and the extent of
visible symptoms i.e. chorotic mottling evalu- ated. Additional needles ten pairs were sampled
from each tree for histological and ultrastructural analyses by means of light and transmission elec-
tron microscopy.
In order to assess the extent of visible injury, the percentage of the surface of each needle af-
fected by chlorotic mottling was visually esti- mated on each needle, using a 5 proportional
classes scale i.e. a scoring scale based on 5 increments of needle area affected. A chlorotic
mottle index CMI was calculated based on the mean area of each needle displaying visible injury.
Light microscopy and transmission electron mi- croscopy TEM were performed on the needles
from ‘control trees’ as well as needles showing visible injury i.e. chlorotic mottle. In this latter
case, we compared the chlorotic areas with the green areas of the same damaged needles. Obser-
vations were made on sections from ten needles per tree. Segments measuring about 2 cm in
length were removed from the central portion of each needle; these were then examined in primary
epifluorescence with a Leitz Dialux 22 microscope employing a 450 – 490 nm wide-band filter. Fur-
ther needle segments of the same length were fixed in a 4 solution of formalin, and then kept in a
1 saccharose solution for 24 h. Using a Reichert Jung freeze-microtome, 30 mm thick transverse
sections were prepared from each needle. These cross-sections were observed in primary fluores-
cence at 450 – 490 nm to determine the chlorophyll response Adams and Lintilhac, 1993; and at
340 – 380 nm to determine effects on lipids Adams and Lintilhac, 1993. Then, sections
were stained with either: Neutral RedAstra Blue-to highlight cell wall modifications Jensen,
1962 or Fluoral Yellow 088-to highlight lipid modifications Brundrett et al., 1991, and sec-
tions viewed with the aid of a Zeiss Axioplan microscope. Additional segments measuring 2 – 3
mm were removed from the central portion of each needle, fixed in formalin and then dehy-
drated by passing through an increasing ethanol series prior to pre-embedding sections in L.R.
White resin and absolute alcohol 1:1. Finally, sections were transferred to pure resin for 24 h
and oven-dried at 50°C. Transverse sections 0.5
m m were cut with a Reichert OM U3 micro-
tome, using a glass knife, and subsequently stained with Toluidine Blue 0 to examine overall
cell structure O’Brien and Mc Cully, 1981, with Schiff’s reagent PAS to examine starch
content. Samples destined for observation by TEM
were prefixed in phosphate buffer pH 7.2 con- taining 2.5 glutaraldehyde and 4 paraformal-
dehyde. After 20 h at 5°C, samples were rinsed twice 2 × 10 min in the same buffer, then post-
fixed 2 h in 2 osmium tetroxide prepared in the same buffer. Subsequently, samples were de-
hydrated in an increasing ethanol series 10 min at each stage of the fixation series. Finally, af-
ter two 5 min rinses in propylene oxide 100, the samples were embedded in resin, according
to Spurr’s procedure Spurr, 1969. A Reichert Ultracut S microtome was used to cut ultra-thin
sections 0.09 mm with a diamond knife. These sections were stained with uranyl acetate and
lead citrate, and then observed with a Philips EM-300 microscope.
Fig. 3. Hourly mean ground-level ozone concentrations over the course of the day at the urban field site. Data represent monthly averaged hourly concentrations over the experimental period May – September.
3. Results
