• Mediterranean grasslands;
  • herbage quality;
  • ozone damage;
  • global change;
  • ozone fluxes;
  • dehesa


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

A study was conducted on the effect of tropospheric ozone (O3) on soft brome (Bromus hordeaceus) and the modulation of its response by nitrogen (N). Two assays were conducted using open-top chambers (OTCs). Three O3 treatments were considered: filtered air, with concentrations below background levels (charcoal-filtered air), non-filtered air (NFA) that simulates ambient O3 concentrations, and unfiltered air to which 40 nL L−1 O3 above-ambient concentrations was added (NFA+), simulating elevated values recorded in natural areas of annual pastures in the Iberian Peninsula. Three N rates were used, simulating the increase in soil N through atmospheric deposition and excreta from livestock grazing. Ozone caused an augmentation in foliar senescence, whereas green biomass was not altered; consequently, an increased senescent/green biomass ratio was produced. A stronger O3 effect was detected in the second assay compared with the first. This was related to the estimated absorbed O3 fluxes, which were double the value calculated in the former. Increasing N input enhanced biomass production, but its effectiveness was greater in the first assay, under less-favourable weather conditions and lower plant growth. In the first assay, the O3 response was modulated by N availability, which mitigated the effects of O3 to medium concentration values. In the first assay, O3 reduced the aerial/subterranean biomass ratio, caused by a positive-trend effect on roots. Foliar concentration of lignin was increased by O3, and in vitro digestibility of aerial biomass and the plant cell wall fraction tended to decrease with increasing O3.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Chemical composition of the atmosphere, particularly the abundance of tropospheric ozone (O3) and its precursors, has experienced significant changes since the beginning of the industrial revolution in the mid-eighteenth century (Hauglustaine and Brasseur, 2001). Ozone is formed primarily from photochemical reactions between its precursors, mainly nitrogen oxides (NOx), hydrocarbons and volatile organic compounds (VOCs) generated by anthropogenic and natural activities. Its formation also requires favourable climatic conditions such as atmospheric stability with elevated solar radiation and temperature (Crutzen et al., 1999). Tropospheric O3 is considered a greenhouse gas (Mickley et al., 2001) that may contribute to global warming and climate change, but the singular aspect that has led to greatly increased scientific interest during the past 30 years is its high toxicity. It is now considered the most phytotoxic air pollutant due to the large number of plant species sensitive to its high oxidative capacity (Ashmore, 2005; Hayes et al., 2007). Its effects range from loss of production and quality in crop harvest (Mills et al., 2007; Booker et al., 2009) to reduction in forest and herbaceous vegetation growth (Davison and Barnes, 1998; Skärby et al., 1998), and alterations in the structure and diversity of natural ecosystems (Davison and Barnes, 1998; Pfleeger et al., 2010).

The Iberian Peninsula has experienced a progressive increase in emissions of O3 precursors due to extensive industrial development and transport since the 1950s. This trend continues today (MARM, 2009; Fernandez-Fernandez et al., 2011), despite several EU laws having been enacted in recent decades that aim to restrict emissions of O3 precursors. In addition, climatic and topographical conditions of the Peninsula favour the generation of this pollutant (Castell et al., 2008). All these circumstances mean that current values set by European legislation (2008/50/EC) for the protection of vegetation (MARM, 2009) are often exceeded, and they are sufficiently elevated to interfere with and disrupt production and operation of agroecosystems and natural ecosystems (Gimeno et al., 1999; Bermejo et al., 2003; Calvo et al., 2007; Calatayud et al., 2011).

In concert with increasing concentrations of O3, another environmental problem that also has had an effect at a global scale is the increased atmospheric deposition of nitrogen (N) in ecosystems (Phoenix et al., 2006). Increased use of fossil fuels in transport and industrial processes has been the main cause of emissions into the atmosphere of oxidized N compounds (NOx), while the adoption of increasingly intensive agricultural and livestock–farming systems has been responsible for the release of reduced nitrogen compounds (NH3). Both types of compounds are causing an alteration of the N cycle and an increase in forms of N that are biologically available to plants (Stevens et al., 2011). Nitrogen enrichment of anthropogenic origin is considered one of the main forces of change in Mediterranean ecosystems (Sala et al., 2000). Few experimental measurements of N deposition have been performed in the Iberian Peninsula, but Rodà et al. (2002) estimated it as between 15 and 22 kg N ha−1 year−1. This range exceeds the empirical critical load currently defined for the protection of many plant communities (Bobbink et al., 2010). Nitrogen deposition can cause, among other effects, changes in the susceptibility of species to biotic or abiotic factors (Jones et al., 2008), or nutrient imbalances (Elvira et al., 2006). When N deposition exceeds the assimilative capacity of ecosystems, it may cause an alteration of competitive relationships between species, thereby inducing alterations in the structure and diversity of the community (Van der Wal et al., 2003; Bobbink et al., 2010).

The presence of elevated levels of O3 and N enrichment are two major components of global environmental change, the combination of which can seriously compromise current functioning of ecosystems. There are extensive rural areas on the Iberian Peninsula that are subject to this combination of factors, for instance in the dehesas at the base of the Sierra de Guadarrama, north of Madrid (Plaza et al., 1997; Alonso et al., 2009). The dehesas, which occupy about 4 million hectares on the Iberian Peninsula, are an agrosylvopastoral system comprising a low-density arboreal stratum, under which a pasture dominated by annual species serves as food for livestock and wildlife. The pastureland of the dehesa is characterized by strong seasonal and interannual variation in biomass production and nutritional quality that is strongly correlated with precipitation variability characteristic of the Mediterranean climate (Vazquez De Aldana et al., 2008). The most representative families of these pastures for both their species richness and importance for cattle grazing are Poaceae and Fabaceae (Buendía, 2000). In the last decade, several studies of species of both families have been conducted to characterize their sensitivity to O3 and to ascertain whether increased N in the substrate modulates their response (Sanz et al., 2005, 2007, 2011). These experiments have shown greater sensitivity of legumes than grasses based on parameters of biomass production, reproductive capacity and nutritional quality, and in the complexity of the interaction between O3 and N, depending on the parameter considered and O3 levels (Bermejo et al., 2003; Gimeno et al., 2004a,b). Another noteworthy result is the sensitivity to O3 of parameters such as nutritional quality or phenology in species that show no visible foliar damage or effects on biomass production (Sanz et al., 2011). The results indicate the potential sensitivity of these Mediterranean grasslands to the O3 and N factors considered, and this may cause changes in the competitive ability of species by altering their structure and composition or interfering with natural nutrient cycles. The loss of nutritional quality can have important implications for feeding cattle, or for wild herbivores (Krupa et al., 2004; Gilliland et al., 2012), although this is not currently considered as a parameter for defining critical O3 values for protecting semi-natural ecosystems (CLRTAP, 2010).

This study is framed within an extensive line of work devoted to analysing the sensitivity of Mediterranean grasslands to global change. It aims to determine which species/communities are most sensitive, how generalizable are the responses found, and which parameters must be considered for characterization of this sensitivity and which should be used to define the value limits of pollutants to ensure protection of plant communities (levels and critical loads of O3 and N). This article specifically presents the study conducted using Bromus hordeaceous, a characteristic annual grass of dehesa pastures. It starts with the assumption that B. hordeaceous would behave similarly to other previously analysed annual grasses and, therefore, should be relatively resistant to O3 considering biomass production parameters, although greater sensitivity to O3 of the parameters related to nutritional quality and a possible modulation of response by N might be expected.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Experimental design

The experiment was conducted in an experimental facility of OTCs, a system designed specifically to analyse effects of air pollutants on vegetation. The experimental field was located in a rural area distant from sources of pollution in the north-east of the Iberian Peninsula on the Ebro delta (latitude 40°41′N; longitude 0°47′E; 2 m elevation; Tarragona). An automatic and sequential system allowed continuous monitoring of concentrations of O3, SO2, NO and NO2 inside each of the chambers. The system allowed 10 min for sampling the pollution climate within each OTC before changing to the next. Nine NCLAN-type OTCs were used in the assays, 3 m height × 3 m diameter. For a detailed description of the facility, see Gimeno et al. (1999). The Fangar weather station (40°47′N, 0°46′E), belonging to the Catalan Meteorological Service network, was used to obtain the meteorological parameters considered for the description of the experiment (temperature, relative humidity, solar radiation). Two successive assays were conducted using the same experimental design and protocol, although under different environmental conditions resulting from the different implementation dates of the assays (Table 1).

Table 1. Timetable of events for the Bromus assays carried out in 2002 (DS indicates days from the start of the experiment).
  1. OTCs, open-top chambers.

First assay
Sowing27 May 
Emergence29 May 
N fertilization29 June 
Transplantation1 July 
Start of ozone exposure in OTCs2 July0
N fertilization15 July13
N fertilization29 July27
End of OTC exposure7 August36
Harvest7 August36
Second Assay
Sowing19 September 
Transplantation30 September 
Start of ozone exposure in OTCs1 October0
N fertilization2 October1
N fertilization21 October20
N fertilization8 November39
End of OTC exposure12 November43
Harvest14 November45

Plant material

Seeds originated from a collection campaign conducted during May–June 2001 in the Moncalvillo Dehesa, located in San Agustín de Guadalix, in the central area of the Iberian Peninsula (40°40′N, 0°47′E). Seeds were stored free of impurities under suitable environmental storage conditions until time of use. The seeds were sown in a substrate mixture of 50% vermiculite and 50% peat. Approximately 30 days later, the seedlings were transplanted individually into 2·5-L volume pots with a mixture of 50% peat, 20% vermiculite and 30% perlite, to which CaCO3 was added to adjust pH. All nutrients necessary for balanced development of the plants were provided through a nutrient solution prepared with a water-soluble, low-nitrogen fertilizer (Peters Professional®, Scotts, OH, USA; NPK: 4:25:35 plus microelements). Throughout the experiment, a drip irrigation system supplied water to the substrate. A nursery of 81 pots per assay was prepared to achieve an adequate number of samples per O3 and N treatment.

Ozone and nitrogen treatments

Nitrogen was applied in three doses at approximately 14-day intervals, based on various solutions prepared using NH4NO3 (35% N; Table 1). The volume of the applied solution per pot was the same, independent of the N dose. Three treatments with total N input were considered: 7·5 (N-low), 22·5 (N-medium) and 45 (N-high) kg N ha−1. These N-integrated doses were selected to reflect the natural variability that occurs in the annual N inputs to the dehesa soils of the Iberian Peninsula, considering both the N entering via excreta (Olea and San Miguel-Ayanz, 2006) and expected atmospheric N deposition (Rodà et al., 2002).

Ozone treatments were the following: charcoal-filtered air (CFA), non-filtered air (NFA) that simulates ambient O3 concentrations (NFA) and NFA to which 40 nL L−1 O3 was added from 07:00 until 17:00 (GMT) 5 days a week (NFA+), which simulates the elevated O3 values that occur in the natural growing areas of this species. Three replicates per O3 treatment (three OTCs per treatment) and three plants per N treatment within each OTC were used; therefore, there were nine plants for each combination of O3 and N.

Biomass parameters

Aerial biomass was obtained by cutting the plant flush with the substrate after 37 and 45 days for the first and second assays, respectively, inside the chambers and exposed to the different O3 treatments. Senescent biomass, including both senesced leaves and senesced part of the leaves, was separated from green biomass to calculate the ratio between the two. Roots were collected by careful continuous washing in the substrate water. Aerial and subterranean biomass was dried at 60°C to constant weight.

Nutritional quality parameters

Nutritional quality parameters were analysed in the first assay based on samples collected for analysis of green biomass. Green biomass of three pots per chamber and N treatment were pooled for quality analysis. Crude protein (CP) concentration (% CP = % N × 6·25) was determined by the Kjeldahl method (Association of Official Analytical Chemists, 1995). Concentrations of neutral detergent fibre (NDF), acid detergent fibre (ADF) and lignin were determined sequentially following the procedures of Van Soest et al. (1991). Relative feed value (RFV) was calculated from ADF and NDF concentrations using prediction equations developed for cool-season-adapted C3 grasses and legumes (Linn and Martin, 1989). In vitro dry-matter digestibility (IVDMD) was determined according to the Van Soest et al. (1991) modification of the two-stage Tilley and Terry (1963) procedure, in which neutral detergent extraction of 48-hour fermentation residues was substituted for acid-pepsin digestion. In vitro NDF digestibility (IVNDFD) was calculated as [(NDFi − NDFr) ÷ NDFi] × 100%, where NDFi represents the initial mass of NDF in the sample and NDFr represents the final mass of NDF residue following extraction with neutral detergent solution. Ruminal fluid used for in vitro batch culture fermentation assays was obtained from a fistulated Holstein cow fed a mixed forage–grain concentrate diet.

Statistical analysis

The effect of fertilization with N and O3 concentrations on the parameters considered was analysed by a two-way analysis of variance for each parameter. The Tukey's test was used to analyse differences among means when anova indicated significant differences (< 0·05). Prior to the anova, homogeneity of variances was checked by means of the Levene's test and normality of the variables and their residues were analysed based on the corresponding graphs. The presence of influential points (leverage) was also analysed. Logarithmic transformation was performed for variables that did not meet some of these requirements.

Calculation of phytotoxic ozone dose

Ozone-absorbed fluxes inside the plant or phytotoxic O3 dose (POD) were calculated following the criteria developed under the Convention on Long-range Transboundary Air Pollution (CLRTAP, 2010). Stomatal conductance was modelled using a multiplicative approach (Jarvis, 1976) parameterized for B. hordeaceus by Alonso et al. (2007). In the stomatal conductance model, fphen and fSWP were set to 1, and gmax was transformed to mmol O3 m−2 s−1 using the coefficient of diffusivity factors of H2O and O3 in air, 0·663 (Massman, 1998). POD was accumulated from the start of the O3 fumigation treatments until the end of the experiment. No flux thresholds were considered for the calculation of the total POD.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Meteorological conditions and ozone levels

The mean values of meteorological parameters for the two assays are summarized in Table 2. In the first assay, the values of atmospheric vapour pressure deficit (VPD) were greater due to temperature, which maintained a mean value about 6°C above those recorded in the second assay for most of the day, whereas relative humidity (RH) conditions were similar in both (a mean value between 70 and 90% throughout the day). Another difference between assays was the total solar radiation (SR, Wm−2), as during the first assay, more hours of more intense sunlight were accumulated. These differences may be key factors in attempting to explain some of the differences observed between assays in response to O3 and N factors, because both VPD and SR are variables that strongly affect stomatal opening and therefore absorption of O3 and CO2 into the plant.

Table 2. Mean, maximum and minimum meteorological values and AOT40 index for the Bromus assays.
  1. RH, air relative humidity; SR, total solar radiation; VPD, atmospheric water vapour pressure deficit; CFA, charcoal-filtered air; NFA, non-filtered air; NFA+, non-filtered air + 40 nL L−1 O3.

First assay
Temperature (°C)24·532·017·7
RH (%)8310030
SR (Wm−2)2579480
VPD (Kpa)0·553·040
AOT40 (nL L−1 h) CFA39  
AOT40 (nL L−1 h) NFA1198  
AOT40 (nL L−1 h) NFA+10 891  
Second assay
Temperature (ºC)18·425·611·9
RH (%)8410051
SR (Wm−2)1427500
VPD (Kpa)0·361·520
AOT40 (nL L−1 h) CFA0  
AOT40 (nL L−1 h) NFA221  
AOT40 (nL L−1 h) NFA+6032  

Although the exposure period to O3 of the second assay was 7 days longer than in the first one, AOT40 accumulative exposure index values of O3 were significantly lower in this period (Table 2). Differences are due to different ambient O3 levels between assays. In the first assay, the AOT40 value registered in the NFA treatment, which reproduces ambient levels of the pollutant, was six times higher than in the second assay, whereas the treatment with higher levels of O3 (NFA+) was double. Values for the filtered control treatment (CFA) were consistently below 10 nL L−1 in both exposure periods, indicating proper operation of the filtration system. Ozone levels reproduced in the different treatments are within the seasonal and interannual variability recorded for this pollutant in the areas of distribution of annual grasslands in the central area of the Iberian Peninsula. Nitrogen dioxide and SO2 were always below very low levels and close to the detection limit of the monitors (data not shown). Total N deposition in the area, according to the EMEP model (EMEP MSC-W chemical transport model, 2012; was around 6·75 kg N ha−1 year−1 for the year of the experiment.

Biomass production

First assay

Mean values and results of statistical analyses of the first experiment for the various parameters of biomass analysed, by O3 and N treatments, are presented in Table 3. Exposure to O3 did not affect production of green biomass, but significantly increased the production of senescent biomass (< 0·005), which increased by 7 and 34% for NFA and NFA+ treatments, respectively, compared with CFA (mean values across N treatments), but differences with NFA were not significant. These results caused an imbalance in the senescent biomass/green biomass ratio (< 0·005), which increased 15 and 50% in plants grown in NFA and NFA+ respectively. Total aerial biomass (sum of green and senescent biomass) was not, however, significantly affected by the pollutant. Although the effect of O3 on subterranean biomass was not significant, a trend was observed (= 0·07) towards an increase in the NFA+ treatment, which caused a significant reduction (< 0·05) of 14% (mean across N treatments) in the aerial/subterranean biomass ratio in plants exposed to the highest levels of pollutant (< 0·05). The O3 increased the fresh green/dry weight ratio in NFA and NFA+ compared with the CFA control by 8% (mean value across N treatments), indicating greater water content in green aerial biomass.

Table 3. Growth-related parameters (means per plant ± s.e.) corresponding to the exposure of Bromus hordeaceus to the different O3 and N treatments on the first assay.
First assayGreen biomass (g dw)Senescent biomass (g dw)Senescent/green biomass ratioTotal aerial biomass (g dw)Subterranean biomass (g dw)Total biomass (g dw)Aerial/subterranean ratioFresh aerial biomass (g fw)Fresh/dry biomass ratio
  1. CFA, charcoal-filtered air; NFA, non-filtered air; NFA+, non-filtered air + 40 nL L−1 O3; Low-, Medium- and High-N represent N supply at 7.5, 22.5 and 45 kg ha−1 respectively. Different letters indicate significant differences among means in each nitrogen treatment when interactive effects were significant.

  2. The P values corresponding to the effect of each factor (O3 and N) are presented in the first part of the table and the values (mean ± s.e.) are presented below.

O3* Nitrogennsns<0·05nsnsnsnsnsns
N-low CFA0·95 ± 0·060·17 ± 0·020·19 ± 0·03a1·12 ± 0·061·26 ± 0·102·38 ± 0·150·92 ± 0·064·52 ± 0·224·47 ± 0·08
N-low NFA1·06 ± 0·080·17 ± 0·010·18 ± 0·03a1·24 ± 0·071·41 ± 0·052·64 ± 0·110·87 ± 0·036·37 ± 0·595·23 ± 0·19
N-low NFA+0·85 ± 0·090·25 ± 0·020·31 ± 0·03b1·09 ± 0·101·54 ± 0·102·64 ± 0·190·70 ± 0·054·92 ± 0·394·99 ± 0·11
N-medium CFA1·34 ± 0·100·17 ± 0·010·14 ± 0·01a1·52 ± 0·111·66 ± 0·093·18 ± 0·190·91 ± 0·046·93 ± 0·534·62 ± 0·10
N-medium NFA1·29 ± 0·110·17 ± 0·020·14 ± 0·01a1·46 ± 0·121·71 ± 0·113·17 ± 0·220·85 ± 0·046·98 ± 0·714·94 ± 0·17
N-medium NFA+1·39 ± 0·170·25 ± 0·020·21 ± 0·03ab1·64 ± 0·181·94 ± 0·153·58 ± 0·320·83 ± 0·058·18 ± 0·804·93 ± 0·12
N-high CFA2·12 ± 0·250·21 ± 0·020·11 ± 0·01a2·33 ± 0·262·19 ± 0·144·52 ± 0·371·05 ± 0·0811·63 ± 0·914·65 ± 0·05
N-high NFA1·55 ± 0·120·25 ± 0·040·17 ± 0·03a1·80 ± 0·112·00 ± 0·153·80 ± 0·240·93 ± 0·078·43 ± 0·614·72 ± 0·12
N-high NFA+2·19 ± 0·320·26 ± 0·020·13 ± 0·01a2·45 ± 0·332·43 ± 0·254·88 ± 0·571·00 ± 0·0513·01 ± 2·074·87 ± 0·13

Nitrogen stimulated growth progressively in all analysed biomass parameters commensurate with dose increase, except for senescent biomass that only responded to the highest dose of N. Total biomass (sum of aerial and root biomass) was increased by 30% for N-medium and 72% for N-high, as compared to that of N-low (mean across O3 treatments). The fertilizer elicited greater growth response by the aerial part than the subterranean part. Mean values for total aerial biomass were 34 and 90% greater for N-medium and N-high, respectively, than N-low (mean across O3 treatments); however, root biomass increased by 26 and 57%, respectively, in N-medium and N-high treatments compared with N-low. These imbalanced effects were reflected in the aerial biomass/root biomass ratio, which increased by 19% in N-high (< 0·05).

The senescent/green ratio decreased by 29 and 38% for N-medium and N-high, respectively, compared with the lowest dose of N (< 0·001; mean values across O3 treatments), thereby producing the opposite effect to that of O3, which caused an increase in this ratio (33% in NFA+). The only parameter in which a significant interaction (< 0·05) was observed among the O3 and N factors was the senescent/green biomass ratio, in that N attenuated the increase in the ratio induced by high O3 exposure levels (NFA+; Figure 1).


Figure 1. Mean values of senescent/green biomass ratio for the different ozone and nitrogen treatments (mean ± s.e.) for the first assay. CFA, charcoal-filtered air; NFA, non-filtered air; NFA+, non-filtered air + 40 nL L−1 O3; N-low, total N supply of 7·5 kg ha−1; N-medium, total N supply of 22·5 kg ha−1; N-high, total N supply of 45 kg ha−1. Different letters indicated significant differences among means for the NxO3 interaction.

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Second assay

In the second assay, biomass production of Bhordeaceous was nearly double that collected in the first assay (Table 4). In this case, the negative effect of O3 significantly affected fewer parameters related to biomass production compared with the first assay. Neither subterranean biomass nor the aerial/subterranean biomass–parameters ratio responded to increasing levels of the pollutant. Nevertheless, damage severity (percentage of effect compared with control) of affected parameters was somewhat greater than that observed in the first growth cycle. As in the first assay, O3 caused an increase in senescent biomass, which achieved 54% (mean across N treatments) in NFA+ compared with both NFA and CFA, without affecting production of green aerial biomass. Consequently, there was a 58% increase (< 0·001) in senescent/green biomass ratio in the treatment with the greatest concentration of pollutant (NFA+) compared with the other two. No effect from O3 was found in this assay in relation to the hydric status of biomass.

Table 4. Growth-related parameters (means per plant ± s.e.) corresponding to the exposure of Bromus hordeaceus to the different O3 and N treatments on the second assay. (Abbreviations: refer to footnote of Table 3).
Second assayGreen biomass (g dw)Senescent biomass (g dw)Senescent/green biomass ratioTotal aerial biomass (g dw)Subterranean biomass (g dw)Total biomass (g dw)Aerial/subterranean ratioFresh aerial biomass (g fw)Fresh/dry biomass ratio
  1. CFA, charcoal-filtered air; NFA, non-filtered air.

O3 * Nitrogennsnsnsnsnsnsnsnsns
N-low CFA2·89 ± 0·120·23 ± 0·030·08 ± 0·013·12 ± 0·122·82 ± 0·165·94 ± 0·191·14 ± 0·0814·97 ± 0·735·17 ± 0·10
N-low NFA2·76 ± 0·120·20 ± 0·010·07 ± 0·002·97 ± 0·132·76 ± 0·175·73 ± 0·291·09 ± 0·0414·06 ± 0·575·10 ± 0·07
N-low NFA+3·00 ± 0·160·36 ± 0·070·13 ± 0·033·36 ± 0·142·99 ± 0·116·36 ± 0·171·14 ± 0·0715·88 ± 1·185·24 ± 0·12
N-medium CFA3·12 ± 0·160·23 ± 0·020·07 ± 0·013·36 ± 0·152·94 ± 0·186·30 ± 0·211·18 ± 0·0917·30 ± 0·735·51 ± 0·14
N-medium NFA3·09 ± 0·120·22 ± 0·020·07 ± 0·003·30 ± 0·123·28 ± 0·166·59 ± 0·231·02 ± 0·0516·51 ± 0·585·37 ± 0·12
N-medium NFA+2·96 ± 0·120·35 ± 0·030·12 ± 0·013·31 ± 0·113·00 ± 0·126·31 ± 0·151·12 ± 0·0516·01 ± 0·715·30 ± 0·05
N-high CFA3·88 ± 0·060·29 ± 0·030·07 ± 0·004·17 ± 0·063·57 ± 0·177·74 ± 0·211·19 ± 0·0521·18 ± 0·505·45 ± 0·08
N-high NFA3·79 ± 0·130·31 ± 0·010·08 ± 0·004·10 ± 0·123·73 ± 0·187·83 ± 0·211·12 ± 0·0620·03 ± 0·705·29 ± 0·11
N-high NFA+3·65 ± 0·140·43 ± 0·020·12 ± 0·014·07 ± 0·133·31 ± 0·137·39 ± 0·231·24 ± 0·0519·62 ± 0·955·37 ± 0·09

Nitrogen fertilization also caused a clear response in B. hordeaceous and increased the values of all biomass parameters analysed, although differences found between doses of N were less than those observed in the first assay; in fact, no significant differences were observed between N-low and N-medium. The N-high treatment similarly increased production of green and senescent biomass by 30% (mean across O3 treatments) compared with the N-medium/N-low mean. Hence, no alteration in the senescent/green biomass ratio was observed.

Although N more strongly stimulated the aerial part of the plant (31% greater in total biomass of N-high compared with N-low) with respect to the subterranean portion (24% increase), variability among samples does not permit detection of a significant effect on aerial/subterranean biomass ratio as clearly detected in the first cycle. Plants exposed to the medium and high doses of N increased the hydration of their green biomass relative to the N-low plants, as the fresh green/dry weight ratio displayed a significant average increase of 4% for these treatments compared with N-low (< 0·05). No significant interaction between O3 and fertilizer was found in this second assay.

Nutritional quality

The mean values within O3 and N treatments for nutritional quality parameters analysed in the first assay are presented in Table 5. Neither O3 nor N affected foliar concentrations of CP, ADF or NDF. Concentrations of lignin in plants grown in NFA were 50% lower (mean across N treatments) than NFA+, but there was no difference between the control (CFA) and NFA+ treatments. The predicted RFV was not altered by either O3 or N, but IVDMD and IVNDFD both tended to decrease with increasing O3.

Table 5. Ozone (O3) and nitrogen-supply (N) effects on nutritive quality-related parameters of Bromus hordeaceus at the end of the exposure period (30 days) for the first assay.
First assayCP (%)NDF (%)ADF (%)Lignin (%)RFVIVDMD (%)IVNDFD (%)
  1. The upper part of the table shows the results of the two-way anova test that was carried out to evaluate the effects of O3 and N, singly and in combination, on the assessed parameters. The lower part indicates the mean values and their standard errors corresponding to the different treatments.

  2. CP, crude protein; NDF, neutral detergent fibre; ADF, acid detergent fibre; RFV, relative feed value (standardized by reference to a medium-quality forage containing 53% NDF, 41% ADF and RFV of 100); IVDMD, in vitro dry-matter digestibility; IVNDFD, in vitro NDF digestibility; CFA, charcoal-filtered air; NFA, non-filtered air; NFA+ is non-filtered air + 40 nL L−1 O3; N-low is total N supply of 5 kg ha−1; N-Medium is total N supply of 15 kg ha−1; N-high is total N supply of 30 kg ha−1.

O3 * Nitrogennsnsnsnsnsnsns
N-low CFA11·86 ± 1·2451·23 ± 1·2126·03 ± 0·690·84 ± 0·05124·91 ± 8·5185·88 ± 0·7672·42 ± 1·38
N-low NFA11·06 ± 1·2051·66 ± 0·6726·46 ± 0·340·48 ± 0·07123·12 ± 2·5585·62 ± 0·5872·13 ± 1·14
N-low NFA+9·25 ± 0·8651·49 ± 0·7926·71 ± 0·630·59 ± 0·04123·14 ± 5·7784·80 ± 0·0069·87 ± 0·00
N-medium CFA10·14 ± 0·7550·85 ± 0·5426·32 ± 0·520·82 ± 0·03125·21 ± 4·2087·02 ± 0·2574·48 ± 0·31
N-medium NFA11·16 ± 0·8051·49 ± 0·7425·77 ± 0·640·42 ± 0·03124·49 ± 5·7486·07 ± 0·4072·97 ± 0·61
N-medium NFA+10·03 ± 0·2952·62 ± 0·4326·93 ± 0·200·93 ± 0·08120·10 ± 1·7884·26 ± 0·5670·35 ± 1·20
N-high CFA9·89 ± 0·6150·82 ± 0·5726·54 ± 0·560·62 ± 0·04125·00 ± 4·5887·71 ± 0·4675·76 ± 1·15
N-high NFA9·54 ± 0·3350·51 ± 0·8425·56 ± 0·540·48 ± 0·08127·20 ± 6·1886·40 ± 0·5473·08 ± 0·89
N-high NFA+11·15 ± 0·4850·51 ± 1·0025·90 ± 0·661·19 ± 0·04126·75 ± 7·2586·05 ± 1·5170·97 ± 2·50

Phytotoxic ozone dose

POD values for both assays are presented in Table 6. During the first assay, POD values reached 7·8 and 13·0 mmol O3 m−2 s−1 for NFA and NFA+ respectively. Higher values were found during the second assay. In this case, POD was 14·2 and 23·2 mmol O3 m−2 s−1 for NFA and NFA+ respectively. The O3 absorbed fluxes during the second assay almost doubled the dose absorbed during the first assay, with an average flux increment of 80% for NFA and NFA+ in the second assay compared with the first one.

Table 6. Phytotoxic ozone dose (POD) accumulated from the start of the O3 exposure till the end of the harvest for the different O3 treatments. (CFA, charcoal-filtered air; NFA, non-filtered air; NFA+, non-filtered air + 40 nL L−1 O3.) No thresholds were considered for the calculation of the total POD (POD0).
Bromus hordeaceus 1st assay2nd assay
POD0 (mmol O3 m−2 s−1)1·77·813·02·514·223·2


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

The response of Bhordeaceous to O3 followed a similar pattern in the two assays. In neither case did the pollutant alter the green aerial biomass or total aerial biomass of the species. This response concurs with results from other studies with Poaceae, whether annual species belonging to the same community and ecological characteristics, such as Briza maxima (Sanz et al., 2011), or perennial species, such as Poa pratensis, Anthoxantum odoratum, Lolium perenne or Dactylis glomerata (Bender et al., 2006; Gonzalez-Fernandez et al., 2008; Dawnay and Mills, 2009; Wyness et al., 2011). These results reinforce the generalization of lower sensitivity to O3 in grasses than in legumes and forbs (Wilbourn et al., 1995; Gimeno et al., 2004a,b; Hayes et al., 2010). However, increased O3 did cause elevated premature leaf senescence, the mean increase in which across the two assays was 44% in the highest O3 treatment. The increase in O3-induced senescent biomass is a frequently observed response in many herbaceous species pertaining to different plant families (Franzaring et al., 2000; Bermejo et al., 2003; Sanz et al., 2007). It has been considered a particularly important response parameter for defining sensitivity of grasses to O3, which often do not develop other more specific foliar symptoms (Bermejo et al., 2003; Hayes et al., 2010). In the present study, O3-enhanced leaf senescence caused an imbalance between the senescent biomass/green biomass ratio, increasing the mean value over both assays by 54% in plants grown in NFA+. Interestingly, a significant interaction between O3 and N was found in this parameter; hence, in the first assay, the fertilizer compensated for the increased senescent/green ratio induced by exposure to elevated O3 (NFA+). A similar response has been found in other herbaceous annuals belonging to the legume and grass annuals (Sanz et al., 2005, 2011), perennial grasses (Jones et al., 2010) and even trees (Bielenberg et al., 2001), linking the acceleration of foliar senescence induced by O3 to the low availability of soil N. The importance of this relatively generalized effect at the ecosystem level of organization is yet to be defined, but its impact on soil metabolic processes and nutrient cycling of the system can be significant.

Modulation of the response to O3 by N, favouring plant development in polluted environments, has also been found in other highly important parameters for annual species such as floral biomass and seed production (Sanz et al., 2007). The mechanisms by which N counterbalances the O3 effects are still under study. The synthesis of antioxidant compounds at the cellular scale to prevent the oxidative processes induced by O3 inside the plant tissues, or the processes related to the repair and replacement of structures affected by the pollutant, demand a metabolic cost which might be supported by the soil N availability (Andersen, 2003). In other cases, however, the interaction between O3 and N was negative for herbaceous species, increasing the O3-induced effects on quality parameters (Sanz et al., 2005) or in the root system (Wyness et al., 2011). Further studies are needed to investigate the combined effects of O3 and N, due to the complexity of their effects.

Although the response of B. hordeaceous to O3 showed a similar pattern in the two experiments, the most pronounced effects on the senescence-related parameters were found in the second experiment (i.e. senescent biomass in the first assay was 34% in the NFA+ treatment, but increased to 54% in the second). In the second assay, O3 levels were lower and plants grew nearly double in size compared with the first assay. Increased O3 effects in this assay could be explained by differences in environmental conditions under which the two experiments were conducted. In the first assay, weather conditions were more stressful for plant growth: higher temperature, solar radiation and VPD and lower relative humidity values would be more limiting to stomatal opening, thereby reducing gas exchange to optimize plant water use and reduce oxidative stress caused by photoinhibition. This behaviour has been described in herbaceous annuals that cohabit with B. hordeaceous in dehesa grasslands (Alonso et al., 2007; González-Fernández et al., 2010). This limitation of gas exchange would produce a reduction in the availability of carbon for the photosynthetic process and, consequently, reduced plant growth. Alternatively, the restriction of gas flow would have reduced O3 uptake into the interior of cells, thereby leading to diminished negative effect of O3. This hypothesis is supported by the estimated POD values for the two assays, showing 80% higher POD associated with more severe O3 effects in the second assay. Studies conducted in recent decades indicate that effects caused by O3 on vegetation are more related to the actual flux of the pollutant absorbed through stomata and not on atmospheric pollutant concentrations (Pleijel et al., 2007; CLRTAP, 2010). The results of this study support this conclusion.

Ozone exposure frequently modifies the distribution of assimilated carbon, inducing an imbalance in the aerial/subterranean biomass ratio of the plant. However, the O3 response of this ratio does not follow a common pattern among the different herbaceous species. Some species, such as Briza maxima, did not show O3 effects on root biomass (Sanz et al., 2011), whereas the perennial D. glomerata decreased root biomass, but without significant impact on aerial/subterranean biomass ratio (Wyness et al., 2011). Although in a previous screening study with annuals, no O3 effects were found in Bromus regarding this ratio (Gimeno et al., 2004a), in the first assay of the present study O3 induced a 14% reduction. This imbalance is associated with increased root biomass in plants subjected to high concentrations of O3 without affecting significantly the total aerial biomass. This response can give B. hordeaceous some competitive advantage taking into account the opposite response found for some legumes belonging to the same natural community: the annual clover Trifolium subterraneum increased the aerial/subterranean ratio up to 39% caused by the more pronounced effect of O3 on roots (Sanz et al., 2007; Vollsnes et al., 2010). Moreover, exposure to O3 enhanced the foliar hydric content of B. hordeaceous, another contrasted response compared with other more O3-sensitive species from the same habitat, which showed no response (Sanz et al., 2011), or responded in the opposite direction (Sanz et al., 2005). If future studies confirm the pattern found for B. hordeaceous, it would be of interest to consider the potential competitive advantage that this species could have in environments subjected to chronic O3 contamination vs. legumes with which it competes, especially considering that it is a plant community characterized by a high diversity of species in an environment of limited water and resources (Olea and San Miguel-Ayanz, 2006).

Another aspect of great interest and importance is the analysis of the O3 and N effects in relation to the nutritive quality of B. hordeaceous for herbivores. The effect of O3 on the diet of herbivores via intake of grass exposed to elevated O3 concentrations is a recently emerging line of research, results of which link the effects of O3 on vegetation with its impact on the food chain (Krupa et al., 2004; Gilliland et al., 2012). In temperate pastures, B. hordeaceous is of relatively low nutritive value and has limited acceptability to grazing herbivores (Peeters, 2004). In the context of Mediterranean grasslands, however, it can be a useful forage species and it matures later than many other annual grasses. Furthermore, as its seeds do not readily shatter, cattle will graze it well into summer and derive additional nutritional benefit even after seeds have matured. Exposure to O3 did not affect the chemical composition or nutritional quality of the leaves as indicated by the RFV index, which is calculated by reference to a digestible DM intake of a mature forage containing 53% NDF and 41% ADF equivalent to an RFV of 100 (Linn and Martin, 1989). Among all the chemical compositional parameters analysed that are related to nutritional quality (i.e. CP, ADF, NDF and lignin), only lignin was responsive to O3. The negative relationship that exists between lignin concentration and nutritive quality is not reflected explicitly in calculation of RFV, but it is implied from the negative relationship that exists between lignin concentration and digestibility of plant cell-wall constituents (i.e. NDF and ADF) of which lignin is a structural and analytical component. Use of mixed-batch cultures of ruminal microorganisms in the bioassay of nutritive quality enables detection of possible effects of lignin that are not readily evident from its quantification by detergent fractionation, but to which fibrolytic ruminal microorganisms and enzyme systems might be sensitive (Powell et al., 2003). In vitro digestibility of both the plant total DM (aerial biomass) and the cell-wall fraction (NDF) tended to decrease with increasing O3. In previous studies, increase in foliar lignin concentration has been clearly and directly related with exposure to the pollutant (Muntifering et al., 2000, 2006; Sanz et al., 2005, 2011; Bender et al., 2006), highlighting the importance of considering effects on quality as well as those of production when analysing O3 effects. Results of the current study with B. hordeaceous are in agreement with previous observations of an inhibitory effect of O3 on digestibility, both in vitro and in vivo, even in the absence of visible foliar injury (Powell et al., 2003) or increased concentrations of detergent fibre fractions that are indicative of accelerated foliar senescence (Muntifering et al., 2006; Gilliland et al., 2012). The pattern of lignin response to O3 in B. hordeaceous is similar to that of other grasses tested, the nutritional-quality parameters of which did not respond in a consistent manner to exposure levels to the pollutant (Gonzalez-Fernandez et al., 2008). It is also important to consider that O3 might be affecting the nutritional quality of pastures indirectly due to its ability to increase the competitiveness of the grasses. A consequent reduction in the proportion of legumes might result in the loss of digestible nutrients, the main factor that determines the quality of Mediterranean pastures (Olea and San Miguel-Ayanz, 2006). This effect could affect ingestive and processing behaviours in grazing cattle because of preferential selection of clover over grass (Rook et al., 2002). Also, considering the relationship between ruminant diet and digestibility on CH4 outputs (Misselbrook et al., 2013), there may be consequences to patterns of enteric gas emissions from cattle grazing dehesa pastures.

All biomass-related parameters increased with addition of N in both assays, although the response patterns differed. In the first assay, biomass production was higher for N-medium treatments, followed by the N-high treatment. However, this pattern was not observed in the second experiment in which the N-low and N-medium treatment did not induce a significant response in B. hordeaceous, and only the N-high treatment stimulated its growth. Thus, the N-use efficiency was higher in the first assay because the same amount of N caused a greater increase in biomass than in the second one. Moreover, the senescent/green ratio decreased with fertilization during the first assay, whereas no effect was observed in the second. In the same way, an imbalance in the aerial/subterranean ratio caused by a greater stimulation of the aerial part was also found only in the first assay, following the common response in grasses by addition of N, just as lack of N fertilizer enhances development of the root system (Jones et al., 2010). These differences in the response to N between assays could be explained by the different meteorological conditions, due to the different times when they were conducted which would have directly affected plant growth. Conditions more favourable for the development of B. hordeaceous during the second assay encouraged greater plant growth. In turn, greater plant growth would have required higher amounts of N to achieve the same effect on biomass percentage as that observed in the first cycle. These differences in the N effectiveness between assays might also be related to the absence of O3 × N interaction in the second assay regarding the senescent/green biomass ratio. The results also indicate the importance of considering plant phenology linked with meteorological conditions when assessing the response of Mediterranean annual plant communities to N inputs in the system.

The long-term objective of the European Directive (2008/50/EC) and the 3-month cumulative critical level for annual communities defined by the LRTAP Convention (CLRTAP, 2010) to prevent O3 damage to sensitive species have been set at 3000 nL L−1 h as a 3-month cumulative threshold value. When the O3 effect on total biomass is considered as the response parameter, following the CLRTAP criteria, B. hordeaceous can be classified as a relatively O3-tolerant species and would be protected under the currently defined thresholds. Other O3 effects (aerial/subterranean rate, leaf hydric status), potentially giving B. hordeaceous some competitive advantage, would support its classification as a relatively tolerant species. This conclusion coincides with that of other annual Poaeceae species, as analysed in previous studies, such as Briza maxima (Bermejo et al., 2003; Sanz et al., 2011). However, taking into account senescence-related parameters, B. hordeaceous could be classified as a species that is moderately sensitive to O3. The exposure-response functions based on the relative response of the senescence parameters indicate that 10% of the effect was reached with O3 exposures around 1500 nL L−1 h (y = 0·0041x + 103·68, R² = 0·65 for senescent/green biomass ratio). Thus, the current critical level set for annual plant communities would not protect B. hordeaceous from O3-induced effects on the senescent/green biomass ratio.

The results of this study, taken with previously published findings, indicate that species of Poaceae are potentially competitive with legumes and generally more sensitive to O3 (Wilbourn et al., 1995; Bermejo et al., 2003; Gimeno et al., 2004b) in areas subjected to chronic O3 pollution, or in a future scenario where background O3 levels are increased as physicochemical atmospheric models predict. From an ecological perspective, the marked difference in sensitivity to O3 between grasses and legumes is an important risk factor for conservation of the diversity and structure of Mediterranean pasturelands. With respect to pasture nutritive quality, increasing the competitiveness of grasses vs. legumes would entail a loss of quality of Mediterranean pastures.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

Ozone induces an increase in foliar senescence in B. hordeaceus with the consequent imbalance in the senescent/green biomass ratio. This response was modulated by N availability in the first assay, which mitigated the effect of the pollutant for medium O3 levels. The intensity of this response to O3 is related more to the O3 absorbed fluxes inside the plant (POD) than to the atmospheric concentration of the pollutant. Bromus hordeaceus showed some positive responses to O3 related to root growth and hydric content of the leaves that might give this species a competitive advantage in a high-biodiversity community with limited water and resources, such as in annual Mediterranean pastures. No significant effect on total biomass or the RFV nutritional quality index was found, although bioassay with mixed cultures of ruminal microorganisms revealed a trend towards decreased digestibility of foliar DM and cell-wall fraction associated with increased concentration of lignin.

Considering that no O3-effects were found on green and total biomass, and some positive effects on aerial/subterranean biomass ratio or the leaf water content, B. hordeaceus can be classified as an O3-tolerant species; but its sensitivity increases if senescence-related parameters are taken into account. The application of N increased biomass production in general, but intensity of response varied between experimental cycles. The greatest response to fertilizer N occurred under the growing conditions that were least favourable for the plant.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References

This research was funded by MONTES (CONSOLIDER-INGENIO CSD2008-00040, Spain), AGRISOST (S2009AGR-1630, Spain), EDEN (CGL2009-13188-C03-02, Spain) NEREA (AGL2012-37815-C05-03) and ECLAIRE (FP7-ENV) projects eliminate. Thanks are given to MIGJORN S.A. farm and Modesto Mendoza for the support and care of the OTCs facility. Appreciative acknowledgement is extended to the anonymous referees who helped with the improvement of the manuscript.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusions
  8. Acknowledgments
  9. References
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