Growth responses to ozone in plant species from wetlands
J. Franzaring *, A.E.G. Tonneijck, A.W.N. Kooijman, Th.A. Dueck
Plant Research International,P.O. Box 16,6700 AA Wageningen, The Netherlands
Received 20 October 1999; received in revised form 9 February 2000; accepted 12 February 2000
Abstract
Ten wet grassland species were fumigated with four concentrations of ozone (charcoal-filtered air, non-filtered air and non-filtered air plus 25 or 50 nl l−1ozone) in open-top chambers during one growing season to investigate the long-term effect of this air pollutant on various growth variables. OnlyEupatorium cannabinumshowed ozone-related foliar injury, while five species reacted with significantly ozone-enhanced senescence. Premature senescence was paralleled by a significant ozone-induced reduction of green leaf area in Achillea ptarmica, E. cannabinum and
Plantago lanceolata. At the intermediate harvest performed after 28 days shoot weights were significantly decreased by ozone inA.ptarmicaand increased inMolinia caerulea. At the final harvest performed at the end of the growing season two other species,Cirsium dissectumandE.cannabinumhad a significantly reduced shoot weight due to ozone. Root biomass was determined only at the intermediate harvest. The root:shoot ratio (RSR) was significantly reduced inC.dissectum, while it increased inM.caerulea. Seven of the species developed flowers during the experiment. While no significant ozone effects on flowering date and flower numbers were detected, flower weights were significantly reduced inE.cannabinumandP.lanceolata. © 2000 Elsevier Science B.V. All rights reserved.
Keywords:Cirsio-Molinietum; Foliar injury; Natural vegetation; Ozone sensitivity; Premature senescence
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1. Introduction
Concentrations of tropospheric ozone in rural areas are higher on average compared to urban areas, posing a phytotoxic risk to crops and natu-ral vegetations. In Europe, critical levels for ozone are currently being proposed within the frame-work of the UN/ECE to protect crops, forests and natural vegetation against adverse effects of rising concentrations of ozone (Fuhrer and
Achermann, 1999). This has resulted in broad research on the response of a significant number of plant species from the European flora to ozone. Some short-term experiments were performed in fumigation cabinets in the 1980s in which more than 200 European herbaceous species were ex-posed to elevated ozone concentrations (Cornelius et al., 1985; Ashmore et al., 1987). In such screen-ing experiments, visible injury has been frequently used as a reliable response parameter. However, chronic responses due to longer lasting episodes of elevated ozone may have greater ecological significance to a vegetation than responses to acute exposures to ozone.
* Corresponding author.
E-mail address: [email protected] (J. Fran-zaring).
J.Franzaring et al./En6ironmental and Experimental Botany 44 (2000) 39 – 48 40
A number of species from various European vegetation types have also been included in long-term experiments to date. Bergmann et al. (1996, 1998) and Pleijel and Danielsson (1997) studied the ozone sensitivity of a number of annual and biennial ruderals. Ashmore et al. (1996) concen-trated on a range of dry calcareous grassland species while Grub et al. (1997), Bungener et al. (1999) fumigated perennial species from managed pastures. No published information so far exists on the ozone sensitivity of species from wetlands (including fen meadows and mires) although rem-nants of such ecosystems are under special protec-tion throughout Europe. Changes in hydrology and eutrophication have already had negative ef-fects in the past decades (Joyce and Wade, 1998), but are there any effects known of the long-term impact of ozone on wetlands? The uptake of ozone and other air pollutants is inherently cou-pled to the gas exchange of a plant (Reich, 1987) and in studies on crops it has been shown that readily transpiring plants grown under moist soil conditions are more susceptible to ozone than plants grown under a slight drought stress. It can be hypothesised that vegetations from perma-nently wet environments are at a greater risk to adverse ozone concentrations than plants and vegetations growing in a dry habitat. Further-more, the importance of relative growth rates and leaf morphology in relation to the ozone sensitiv-ity of plant species requires testing.
To study the ozone sensitivity of wetland spe-cies, an experiment was performed using common taxa from the Dutch flora. Ten perennial herbs and grasses from extensively managed wet grass-lands were used in a fumigation experiment with open-top chambers to investigate the ozone sensi-tivity in terms of growth responses. The results of the experiment will be presented and discussed in this paper.
2. Material and methods
2.1. Culti6ation and fumigation
Seeds of ten plant species (Table 1) were collected in the institute garden at Wageningen
in 1996. Plants in this garden originated from remnants of fen-meadows (Cirsio-Molinietum plant community, Schamine´e et al., 1996) in the Eastern Netherlands. Seeds were germinated in washed sand in a greenhouse in spring 1997. After 3 weeks one seedling per pot was transplanted into 3 l pots (for the intermediate harvest) and 5 l pots (for the final harvest). These pots were filled with a sand:potting mixture (1:2). The commercially available potting mixture (Lentse 3) had a pH of 5.5 and consisted of 70% peat and 30% river clay, to which 1.5 kg m−3 slow release NPK (12:14:24)
was added at the start of the experiment. No additional nutrients were supplied during the experiments.
The open-top chambers (OTCs) and the fumigation system were previously described by Dueck (1990). Four ozone concentrations were used in duplicate: charcoal-filtered air (CF), non-filtered air (NF), non-filtered air plus 25 nl l−1 O
3 (NF+) and non-filtered air plus 50 nl
l−1 O
3 (NF+ +). Chambers were arranged in
two blocks and the treatments were randomly placed within each block. Ozone was generated from pure oxygen via electric discharge (Sorbios generator) and added to the NF air from 10:00 to 19:00 CET using massflow controllers. Ozone-levels were measured sequentially in the OTCs with an ozone analyser (Monitor Labs model 8810). Ozone exposure levels are presented as seasonal daytime mean values and as accumulated exposures over a threshold of 40 nl l−1 (AOT40). The AOT40 is expressed as nl
l−1 h and is calculated as the sum of differences
between the hourly ozone concentrations and the cut-off threshold of 40 nl l−1 when the
global radiation exceeds 50 Wm−2 (Ka¨renla¨mpi
and Ska¨rby, 1996). The experiment commenced on 20 May 1997 with ten pots (five 5 l and five 3 l pots) per species randomly arranged in each of the eight chambers. The OTCs were 6 m2
2.2. Visual assessment and har6ests of plants
During the course of the experiment visual assessments of the plants were made once a week. Numbers of flowers and senescent leaves were counted and the plants were observed for visible injuries. After 28 days (intermediate harvest, 16 June) five plants per species and treatment were harvested to determine leaf numbers, leaf area and dry weights of leaves, stems, roots and flow-ers. Dry weights were determined by drying the plant material at 80°C for 48 h. Relative growth rates (RGR) and specific leaf areas (SLA) were determined according to Hendry and Grime (1993).
A final harvest was performed with the other five plants per species at a time depending on the species’ phenology (Table 1). For the seven spe-cies developing flowers, harvests were performed when seed ripening had begun. Succisa pratensis,
Carex nigra and Danthonia decumbens did not
produce flowers and were harvested between 10
September and 8 October. Areas of green leaves were measured in all species except C. nigra and
D.decumbens. Dry weights and numbers of green,
senescent and dead leaves were determined in all species, except the numbers of leaves in Achillea ptarmica,C.nigraandD.decumbens. The propor-tions of dead and senescent leaves in relation to total leaves (number and biomass) were consid-ered as senescence parameters. Dry weights of stems and flowers of all plants were determined.
2.3. Data processing and statistics
Data were processed separately for the two harvests. To test for significant ozone treatment effects, one-way analyses of variance (ANOVA) were performed on the untransformed data for each species. Data on percentage senescence and root:shoot ratios (RSR) was arc-sin transformed prior to the analysis according to Sokal and Rohlf (1981). Analyses followed a randomised block-de-sign with the four ozone treatments placed at random within each of the two blocks.
Table 1
Accumulated exposures over a threshold of 40 nl l−1 (AOT40, in
ml l−1 h)a and seasonal daytime mean ozone concentrations
(10:00–19:00 CET, in nl l−1) for exposures of 4 weeks (intermediate harvest) and a growing season (final harvest) of ten wet
grassland species
Exposure untilb Treatmentc
CF NF NF+ NF++
Intermediate harvest
0 7.5
16 June
All species 0.8 3.3
Final harvest
2.7
A.ptarmicaL. 19 August 0 11.0 25.1
2.9
C.nigra(L.) Reichard 30 September 0 12.5 29.3
26.3 11.5
2.9
C.dissecturm(L.) Hill 09 September 0
D.decumbens(L.) DC. 08 October 0 2.9 12.5 29.6
E.connabinumL. 26 August 0 2.9 11.4 26.1
7.2
1.3 17.7
0 29 July
L.flos-cuculiL.
2.3 9.7 22.9
L.salicariaL. 13 August 0
15 September 0
M.caerulea (L.) Moench 2.9 11.8 27.2
P.lanceolataL. 30 July 0 1.3 7.3 18.1
26.4
10 September 11.5
S.pratensisMoench 0 2.9
Seasonal 9 h daily means (nl l−1)
Intermediate harvest 16 June 4.5 35.5 58 77.5
8 October
Whole season 1997 3 33.5 53.5 77
aIdentical to AOT40 in ppm h−1.
bThe experiment commenced on 20 May 1997.
cCF, charcoal-filtered air; NF+, non-filtered air plus 25 nl l−1 O
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Fig. 1. Contribution (%) of senescent leaves to total leaf biomass in five plant species exposed to four levels of ozone for a growing season. Species are from left to rightA.ptarmica (black bars),C.nigra(striped bars),E.cannabinum(grey bars), M.caerulea (dotted bars) andP.lanceolata(white bars). For significance levels see Table 2.
nl l−1 O
3 in the second week of August. During
the whole season, AOT40 in the NF treatment remained below the 3 month critical level of 3 ml
l−1 h, which was proposed by the UN-ECE
(Ka¨renla¨mpi and Ska¨rby, 1996). The summer of 1997 thus represents a summer with low ozone exposure.
In the NF+ + treatments ozone concentra-tions occasionally reached 120 nl l−1in early June
and 150 nl l−1 in August. There were no
signifi-cant differences between the treatment replicates. The AOT40 and exposure duration at the final harvest differed between species (Table 1) and the AOT40 varied between 17.7 ml l−1 h for Lychnis
flos-cuculi and 29.6 ml l−1 h for D. decumbens in
the NF+ + treatments.
3.2. Visible injury and senescence
Foliar injury was first observed in Eupatorium
cannabinum in the NF+ + chambers 4 weeks
after the onset of the fumigation. The ozone-re-lated spots appeared on the upper surface of the first order leaves. Leaves that were produced later in the season did not show foliar injury. In the middle of the season, small whitish spots were observed in the centre of leaves of L. flos-cuculi. These rather un-specific symptoms occurred only in some plants from the NF+ + treatment and were accompanied by a structural change of the mesophyll, which appeared to be water-soaked.
Until the intermediate harvest no signs of ozone-enhanced senescence were observed at the weekly visual assessments. At the final harvest senescence appeared to be significantly affected by ozone in five species (Table 2, Fig. 1). Ozone-en-hanced senescence was paralleled by a significant reduction of green leaf area in A. ptarmica, E.
cannabinum and Plantago lanceolata (Fig. 2).
3.3. Growth responses
ANOVAs were calculated separately for the two harvests for each of the ten species (Table 2). Results of significant ozone treatment effects are presented for the response parameters shoot biomass, leaf area and number, and root and flower biomass.
Fig. 2. Green leaf area in three plant species exposed to four levels of ozone for a whole season. Species are from left to rightA.ptarmica(black bars),E.cannabinum(grey bars) and P.lanceolata(white bars). For significance levels see Table 2.
3. Results
3.1. Ozone concentrations
In the beginning of June, mean hourly concen-trations of ambient ozone exceeded 70 nl l−1 for
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Table 2
Effects of ozone treatment on various growth parameters of wet grassland species determined at two harvests (a) and plant traits of the ten wet grassland species determined in ozone-free air (b)a
Species a b
Final harvest (for harvest dates see Table 1)a Traits in O
3free air Intermediate harvest (after 28 days)a
Root
Shoot Leaf Leaf Root: RGR SLA Senescenceb Shoot Leaf Stem Flower Flower RGR SLA
(g g−1day−1)
numberd (cm−2g−1)
weight
areac weightd
weight weight area number weight shoot
Leaf Leaf Percent weight number weight
A.ptarmica 0.037 0.046 0.002 0.005 0.023 0.003 0.048 n.d. n.s. B0.001 n.s. 0.002 n.s. n.s. n.s. 0.197 236
n.s. 0.001 n.d. 0.001 B0.001 n.s. n.d. n.p.
n.s. n.p.
C.nigra n.s. 0.044 n.s. n.s. n.p. 0.123 150
n.s. n.s. n.s. n.s. n.s. 0.046
C.disseclum n.s. n.s. n.s. 0.027 0.001 n.s. n.s. n.s. n.s. 0.091 159
n.s. n.s. n.d. n.s. n.s. n.s. n.d. n.p.
n.s. n.p.
D.decumbens n.s. n.s. n.s. n.s. n.p. 0.070 147
n.s. n.s. B0.001 B0.001 0.002 0.006 B0.001 0.020
E.cannabinum n.s. n.s. n.s. n.s. n.s. n.s. 0.047 0.176 364
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.
n.d. n.s.
L.flos-cuculi n.s. n.s. n.s. n.d. n.s. 0.104 192
n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.158
L.salicaria n.s. n.s. n.s. 0.040 n.s. 244
0.001 n.s. 0.030 0.032 0.013 n.s. n.s. n.s.
0.044 n.s.
0.004 n.s. 0.087 202
M.caerulea 0.005 0.003 0.027
n.s. n.s. 0.002 B0.001 B0.001 n.s. 0.008 n.s. n.s. 0.007 0.110
P.lanceolata n.s. n.s. n.s. n.s. n.s. 167
n.s. n.s. 0.003 0.007 n.s. n.s. n.s. n.p. n.p. n.p. 0.090
n.s. 185
S.pratensis n.s. n.s. n.s. n.s.
aValues represent theP-values from ANOVAs indicating significant treatment effects. N.d., not determined; n.s., not significant; n.p., not present. bRefers to senescent and dead leaves and their proportion of leaf total.
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Fig. 3. The effect of ozone on shoot weights of four species at two harvests. The intermediate harvest (H1) was performed on half of the plants after 28 days and the
final harvest (H2) when seeds had ripened. Boxes represent treatment means, error bars represent +S.E. and stars indicate significant ozone treatment effects,
3.4. Shoot biomass
Four species showed significant shoot biomass responses to ozone. Growth reductions were ob-served inA. ptarmica at the intermediate harvest and inC.dissectumandE.cannabinumat the final harvest (Fig. 3). A significant growth stimulation occurred in M. caerulea at the intermediate har-vest. Moreover, P. lanceolata showed a trend towards growth reductions at both harvests and
C. nigra a trend towards growth stimulations at
the final harvest, but in both species these were not statistically significant.
3.5. Leaf area and number
Total leaf area could only be determined at the intermediate harvest before any senescence was observed. Leaf area was significantly affected by ozone in three species. In A. ptarmica it was reduced and in C. nigra and M. caerulea it was significantly increased by ozone (P-levels in Table 2). Leaf number was determined at the intermedi-ate harvest and was reduced by ozone in A.
ptarmicaand increased inM.caerulea(P-levels in Table 2).
3.6. Roots and flowers
Belowground biomass was determined only at the intermediate harvest. Root weights were sig-nificantly affected by ozone in A. ptarmica, C.
dissectum, L.salicariaandM.caerulea. However, RSR was affected in only three species. In A.
ptarmicaa strong reduction occurred in the NF+
treatment, while a significant decrease in RSR due to elevated ozone was observed in C. dissectum. An increase of RSR in the elevated ozone treat-ments was obtained in M. caerulea (Figs. 3 and 4).
Visual assessments of the plants did not reveal any effects of ozone on the timing, duration and extent of flowering. At the final harvest seven species had produced flowers. Flower numbers were not affected by ozone, but significant ozone treatment effects on flower weight were observed inE.cannabinum andP.lanceolata. In the former species mean flower biomass was highest in the NF+ treatments, while in the latter plants from the NF treatments produced highest flower biomass.
4. Discussion
During the course of a whole growing season, premature senescence was the most common re-sponse to ozone in the present experiments, but there were significant growth responses after a shorter exposure to ozone. Although, none of the species showed signs of premature senescence in the first 4 weeks of the experiment, A. ptarmica
had already a significantly reduced shoot weight due to ozone at the intermediate harvest, whileM.
caerulea was significantly stimulated in growth
after the first 4 weeks. At this point, significant ozone effects on root biomass were observed in four species and RSR was affected in three spe-cies, indicating that the carbon allocation pattern of wet grassland plants might be altered by ozone. Such effects have before been noted in a number of tree and crop species, while for herbaceous plants from the native European flora, Reiling and Davison (1992a,b) were the first to show changes in RSR. A large proportion of
J.Franzaring et al./En6ironmental and Experimental Botany 44 (2000) 39 – 48 46
thates of grassland species is allocated to the belowground plant parts, which serves these spe-cies to cope with poor nutrient and water availability. Allocation patterns will also greatly influence the competition between species in wet grassland communities (Aerts et al., 1991). One could thus hypothesise that a decrease in root or shoot biomass (or the ratio between these) due to ozone, might cause a changed competitive ability of a species in a plant community. While a reduc-tion of root and shoot weights by ozone was observed inC.dissectum,M. caeruleashowed the opposite reaction, possibly increasing its competi-tive ability in plant communities under rising lev-els of ozone.
While the foliar injury observed in flowering plants of L. flos-cuculi could not be clearly at-tributed to ozone, the strong injury in E. can
-nabinum caused a substantial reduction of the
assimilating area. The loss of green leaf area may have resulted in the significantly reduced shoot biomass towards the end of the growing period. While the thin young leaves of Eupatorium ap-peared to be very sensitive to ozone in the early summer, later in the season plants produced ro-bust leaves, which did not show ozone-related injury. These findings demonstrate the need to account for seasonal and life stage related differ-ences in ozone sensitivity. Opposed to the findings of the present study, Carlsson et al. (1996) found young pea leaves to be less responsive than older leaves whereas Reiling and Davison (1992b) found that Plantago major reacted similarly to short ozone exposures at different developmental stages.
With regard to growth responses, the present experiments indicated differential effects in the same species depending on the exposure duration. The significant responses in shoot biomass of A.
ptarmicaandM.caeruleaobserved at the
interme-diate harvest were not significant at the final harvest, although the same trends still persisted.
InE. cannabinumand C.dissectum shoot weights
appeared to be significantly reduced only after a whole season of ozone exposure, indicating that in these species ozone effects were cumulative.
While vegetative plant parts were significantly affected in four wet grassland species, ozone
ef-fects on reproduction organs were less pro-nounced. Timing and duration of flowering and flower numbers remained unaffected in our exper-iments, indicating that ozone does not influence the phenology of wet grassland species. In con-trast to our results, direct effects of ozone on the flowering inBrassicaspecies were shown by Stew-art et al. (1996). However, flower weights were significantly affected by ozone in E. cannabinum
andP.lanceolatain the present study. The poten-tial impact of ozone on the biomass of reproduc-tive organs was previously shown in a number of short-lived European ruderals (Bergmann et al., 1998). While annuals depend on seed output, the survival of perennials like the wet grassland spe-cies in the present study depends on vegetative reproduction in the first place. However, the eco-logical significance of ozone effects on generative and vegetative reproduction has not been much accounted for because most fumigation studies were finished before flowering and seed ripening of the plants.
4.1. Possible causes and effects of different ozone sensiti6ity
Half of the species showed premature senes-cence due to ozone. The occurrence of ozone-en-hanced senescence has been reported several times, but the physiological mechanisms for it remain unclear. Senescence seems to be a com-plex, highly regulated process in the life of a leaf and results in the re-mobilisation of compounds to other parts of the plant (Buchanan-Wollaston, 1997). In the individual plant premature senes-cence due to ozone might have strong implica-tions, eventually shortening the vegetative phase and reducing the general vitality. In a plant com-munity a differential response in terms of en-hanced senescence might lead to disadvantages for some and advantages for other species.
com-petitive species have larger gas exchange rates, which might result in larger ozone responses. The concept of strategies is based on Grime et al. (1988), who classified plant species as competitors (C), stress tolerators (S) and ruderals (R). In our experiments, the slow-growing and most sclero-phyllous (lowest RGR and SLA, see Table 2, right-hand side)D.decumbenswas the most ozone tolerant species as it did not show any ozone-re-lated symptoms in terms of premature senescence or growth reductions. These observations are well in line with Grime et al. (1988) who classified
Danthonia as a stress tolerator, but contrast
re-sults of Ashmore et al. (1996), who found a growth stimulation due to ozone. Another species,
S.pratensiswas also classified as a stress tolerator by Grime et al. (1988) and our results support this with regard to the ozone effects as this species did not show any significant growth responses. Again, the low RGR and SLA of this species suggest that plant traits, as expressed for example by growth strategy sensu Grime, to a certain extent deter-mine the relative ozone sensitivity of plant species. This relationship could also be observed in the case of the two most responsive species, E. can
-nabinumandA.ptarmica. The thin leaves (highest SLA) of the fast growing (high RGR)Eupatorium
might be the morphological and physiological trigger of strong leaf injury and consequently for the ozone-induced reduction in shoot biomass. Likewise, the high RGR inA.ptarmica accompa-nied by a high SLA could have determined the adverse ozone effects present in this species. Both ozone sensitive species are classified as competi-tive ruderals (CR/CSR) by Grime et al. (1988) indicating that species with a strong ruderal com-ponent might be strongly affected by ozone. Gen-erally speaking, our experiments indicate that fast growing thin leaved taxa might take up larger doses of ozone than slow growing sclerophyllous species.
Two of the species in our experiments, M.
caeruleaandC.nigraare classified as competitive stress tolerators (Grime et al., 1988). The first had a significantly increased shoot biomass in re-sponse to ozone in the present experiment while the second did not significantly respond to ozone. It is interesting to note that althoughMoliniahad
a significantly increased shoot weight due to ozone at the intermediate harvest a higher rate of senescence was observed at the end of the season. The growth stimulation due to ozone in this species raises the question if Molinia will have a competitive advantage over other species under rising levels of ozone. Although, the species grows relatively slowly, its strong competitive ability in semi-natural vegetation may be brought about by its growth form (monoclonal spread via rhi-zomes), longevity and stress tolerance. While N-and P-eutrophication were often found to be the main cause for an increased competitiveness ofM.
caerulea throughout Europe (Egloff, 1987; Heil
and Bruggink, 1987; Aerts, 1989; Aerts and de Caluwe, 1989; Berendse, 1994), the results of our study indicate that increasing ozone concentra-tions might be an additional factor to consider. However, the possible influences of gaseous air pollutants on community structures and plant biodiversity need to be tested in field-trials in order to establish such a relationship.
Overall, the results of this fumigation experi-ment indicate a diverse reaction pattern of wet grassland species to ozone. While premature senescence is a common response to ozone, growth reductions seem to be less widespread and may in part be related to ecological plant strategy and growth rates. However, growth stimulations due to ozone were only observed in M. caerulea. Comparable responses were observed earlier in other grass species (Ashmore et al., 1996; Grub et al., 1997), but the physiological mechanisms for these and ecological consequences are not yet understood.
Acknowledgements
J.Franzaring et al./En6ironmental and Experimental Botany 44 (2000) 39 – 48 48
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