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Environmental Pollution 193 (2014) 13e21

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Environmental Pollution

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Contrasting effects of water salinity and ozone concentration on twocultivars of durum wheat (Triticum durum Desf.) in Mediterraneanconditions

Giacomo Gerosa a, Riccardo Marzuoli a, *, Angelo Finco a, Robert Monga b, Isa Fusaro c,Franco Faoro b

a Universit�a Cattolica di Brescia, Dipartimento di Matematica e Fisica, Via dei Musei 41, Brescia, Italyb Universit�a degli Studi di Milano, Dipartimento di Scienze Agrarie e Ambientali, Via Celoria 2, Milano, Italyc Universit�a degli Studi di Teramo, Dipartimento di Scienze Alimentari, Viale Crispi 212, Teramo, Italy

a r t i c l e i n f o

Article history:Received 8 March 2014Received in revised form28 May 2014Accepted 30 May 2014Available online

Keywords:Triticum durumSaline irrigationOzoneStomatal conductanceGrain yield

* Corresponding author.E-mail address: riccardo.marzuoli@unicatt.it (R. M

http://dx.doi.org/10.1016/j.envpol.2014.05.0270269-7491/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

This paper reports the results of an Open-Top Chambers experiment on the responses of two durumwheatcultivars (Neodur and Virgilio) exposed to two different levels of ozone (charcoal-filtered air and ozone-enriched air) and irrigation water salinity (tap water as control and a 75 mM NaCl solution once a week).

The stomatal conductance of the flag leaves was measured on four dates during May. Flag leaf sampleswere collected to detect ozone visible leaf injuries. At the end of the growing season, the yield/biomassand grain quality parameters were assessed. Saline irrigation caused significant reductions in gs, yieldand grain quality in Neodur, while Virgilio was more tolerant. The yield response to ozone was almostnegligible, with Virgilio, despite the higher susceptibility to visible leaf injuries, being more productivethan Neodur.

The responses to the combined stress were not consistent, with the main tendencies undoubtedlydriven by the saline irrigation factor.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The shortage of water resources foreseen by actual climatechange scenarios for many areas worldwide, requires seriousattention to search for new water supplies for agriculture. There-fore, it is important to evaluate the use of water with increasingelectrical conductivity such as saline or brackish waters.

Moreover, increasing temperatures resulting from climatechange scenarios are expected to cause a rise of the sea level,threatening coastal areas and forcing agriculture to cope with theextension of salt-affected territories under the direct effect of seawater inundations or due to the increase of the sea level-connectedwater table of saline or brackish groundwater (Scharpenseel et al.,1990; V�arallyay, 1994).

Soil salinization is a quite common phenomenon in many agri-cultural contexts of Mediterranean regions where the poor quality

arzuoli).

of the irrigation water (often saline) is generally associated withhigh temperature and reduced rainfall (Maggio et al., 2004).

Moreover, in Mediterranean areas, climate change is expected toincrease the background levels of tropospheric ozone (O3), a strongphoto-oxidant pollutant that negatively affects the vegetation andcrop productivity of ecosystems (Piikki et al., 2008; Feng et al.,2008; Pleijel et al., 2007). Because soil salinity and troposphericO3 are among the most limiting factors in agriculture (Qadir et al.,2000; Fuhrer and Booker, 2003; FAO, 2007), it is important tostudy the potential combined effects of these two stressors inMediterranean environments, where the crop response to soilsalinization and O3 exposure typically overlap.

From a physiological point of view, salt accumulation the in rootzone of plants induces osmotic stress and disrupts cell ion ho-meostasis by inducing both the uptake inhibition of essential nu-trients, such as Kþ, Caþþ and NO3

� and the accumulation of toxiclevels of Naþ and Cl� ions. Osmotic stress is effective at thebeginning of salt exposure, while ion toxicity significantly affectsplant growth after prolonged exposure periods (Munns, 2002).From an agronomical point of view, the reduced water uptakecapability of roots as induced by osmotic stress will contribute to

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e2114

plant growth inhibition and to a reduction in the crop productivityduring long-term exposure to salt (Netondo et al., 2004).

In higher plants, water stress and metabolic imbalances, whichare caused by soil water salinity, often cause a general process ofoxidative injury (Nor'aini et al., 1997; Zhu, 2001).

Oxidative injury is also a common consequence of the O3 uptakeby plants (Kangasjarvi et al., 2005), which reduces plants photo-synthetic capacity (Morgan et al., 2003; Dermody et al., 2006;Zheng et al., 2000; Skotnica et al., 2005) and whole-plant growth(Akhtar et al., 2010), decreasing of the stomatal conductance(Wittig et al., 2007), alterations in the antioxidant systems (Leeet al., 2003; Herbinger et al., 2005), and accelerated leaf senes-cence (Massman et al., 2000; Wieser et al., 2000), although theextent of these effects varies among species and cultivars (US EPA,1986; Gerosa et al., 2009a; Gonzalez-Fernandez et al., 2010). Ozoneis thought to affect plants by generating reactive oxygen species(ROS), such as hydrogen peroxide (H2O2), superoxide radicals (O2.

- )and hydrogen radicals (OH$) (Elstner and Osswald, 1994). Theaccumulation of these ROS is probably the actual cause of cellularand foliar damage (Langebartels et al., 2002; Faoro and Iriti, 2009).

Moreover, the effects of O3 on biochemical and physiologicalprocesses may influence the plant response to other biotic orabiotic stresses, such as water salinity (Maggio and Fagnano, 2010)and plant pathogens (Manning & V. Tiedemann, 1995).

Among the different Mediterranean crops, which are oftenexposed to simultaneous O3 and saline water stress, durum wheathas recently been the subject of renewed interest, because of itsvaluable production and adaptation to low-rainfall conditions andsemiarid environments (Villegas et al., 2000; Araus et al., 2002). InMediterranean areas, durumwheat production is of great economicand environmental relevance because it is one of the most wide-spread crops. For example in 2010more than 72% of the agriculturalwheat areas in Italy were assigned to durum wheat cultivation(ISTAT, 2011).

The effects of the irrigation water salinity (Katerji et al., 2009;James et al., 2002) and tropospheric O3 (De la Torre, 2010; Picchiet al., 2010) on the growth and productivity of durum wheat havebeen studied separately by many authors, but the combined effectsof O3 and salt on durumwheat are not yet available in the literature.

An intensification of the soil salinity is expected to deplete thedefense system of durumwheat, potentially leading to an increasedsusceptibility to other oxidative stresses such as the exposure totropospheric ozone.

Themain objective of our study, therefore, was to investigate theeffects of salinity and O3, alone and in combination, on two durumwheat cultivars, in relation to the development of leaf visible in-juries, crop yield and grain quality.

2. Materials and methods

2.1. Experimental design and plant material

The experiment was performed at the Open-Top Chambers (OTC, Heagle et al.,1973) experimental site of Curno, Bergamo (Lat. 45� 4101700N, Long. 9�3604000 E,elev. 242 m a.s.l.) in Northern Italy. This site is equipped for the continuous moni-toring of the main agrometeorological variables, both in open field and within OTCs(Gerosa et al., 2008).

The seeds of two cultivars of durum wheat (Triticum durum), ‘Virgilio’ (O3 sen-sitive) and ‘Neodur’ (O3 tolerant), were sown on 24 February 2011 in 300 pots with 9seeds per pot.

The pots (30 cm diameter, 19.5 L) were filled with commercial soil (Terflor srl, I).After germination (10 days after sowing) some plants were uprooted in order toleave only 3 plants per pot. On 24March 2011, 224 potsweremoved into 8 OTCs (3mdiameter) for the experimental treatments.

The experiment was performed as a splitesplit plotwith a complete randomizedblock design.

The 8 OTCs were arranged in 4 randomized blocks of 2 OTCs according to theirorientation in the field. Within a single block, each OTC was randomly assigned toone of the 2 ozone treatments: CF-OTC (Charcoal-Filtered air treatment) and EN-OTC

(Ozone-Enriched air treatment). Then, within each OTC, 14 pots of cv “Virgilio” and14 pots of cv “Neodur” were placed. The pots of each cultivar were divided in twogroups towhich a different irrigation regimes were applied: one groupwas irrigatedwith saline water (Sþ) and the other group with tap water (S-).

Salinewaterwasobtained bydissolvingNaCl in tapwater to 75mM. This solutionhad a conductivity of 8.3 dS/m, and a saline concentration of 4382 ppm (mg/L),withinthe typical brackish water range of salinity (500e30,000 ppm). Irrigation was per-formed twice aweek during themain part of the growing season (between April andMay), in order to keep the plants well watered. The plants were irrigated until thefield capacity was reached. Sþ and S- pots received the same amount of water. Toavoid an initial excessive osmotic stress in young plants, increased NaCl concentra-tions were applied once a week during the first period of irrigation (14e28 April).

Salinity in the potted soils was measured using 12 microlysimeters that wereinserted into randomly chosen pots inside the OTCs. The electric conductivity (EC)and temperature of the water solution were measured with a portable con-ductimeter (Cond 6þ, EUTECH Instruments, USA) the day after each irrigationtreatment or rain event.

Inside the CF-OTCs, the air filtration system maintained O3 concentrations atapproximately 50% below the ambient air concentrations, while inside the EN-OTCsan O3 generator (TS-10, Ozone Solutions Inc., USA) and a control system maintainedthe O3 concentrations at approximately 35% higher than the ambient air every daybetween 9:00 AM and 5:00 PM starting from 15th April until the end of theexperiment.

2.2. Ozone measurements and exposure assessment

The O3 concentrations within the OTCs were continuously monitored using anO3 analyzer (model 1308 RS, Dasibi, USA) that was connected to a solenoid valves(TEQCOM, USA) switching system, which sampled the air inside the different OTCs atregular intervals.

Two additional O3 analyzers (model 42M, Environnement Italia s.p.a.) were usedby the fumigation system to control in real time the O3 levels in the two differenttreatments.

The hourly mean O3 concentrations were recorded for each OTC, and at the endof the experiment, the AOT40 exposure index (Accumulated Ozone over a Thresholdof 40 ppb) was calculated. This index is the sum of the differences between thehourly O3 concentrations above 40 ppb and the value of 40 ppb, for each daylighthours (from 8:00 to 20:00) (UNECE, 2010).

The EU directive 2008/50/CE requires an integration period for the AOT40 indexcalculation between 1 May and 31 July for all the European crops. However, in ourcase an integration time between the beginning of the experiment (24 March) andthe harvest date (28 June) was chosen.

2.3. Stomatal conductance

During the growing period, the stomatal conductance (gs) was measured onsunny days on the upper side of the flag leaf of randomly selected plants in all of theOTCs. A total of 64 measurements were taken during each measuring cycle, i.e., 8 gsmeasurements per OTC. Threemeasuring cycles were performed on eachmeasuringday which occurred on 4, 11, 18 and 25 May, the first cycle being in the morning(8:00e10:00), the second at noon (12:00e14:00) and the third in the late afternoon(17:00e19:00).

The gswasmeasured using a Delta-T diffusion porometer (AP4, Delta-T, UK), thatwas calibrated before each measurement cycle.

2.4. Visible leaf symptoms

The plants in all of the OTCs were monitored weekly from the beginning of theO3 treatments to detect the onset of chlorosis and necrosis symptoms that aretypical of O3 injury on durumwheat (Picchi et al., 2010). Eight samples of flags leaveswere collected from two randomly chosen CF-OTCs and two EN-OTCs, three timesduring the growing season (4 and 31 May, and 20 June). In total, 24 leaves weredigitalized with a scanner and visually examined for the presence of macroscopicsymptoms.

The leaves that were collected on 31 May were also analyzed using Global Lab(Data Translation, Marlboro, MA, USA) to more accurately determine the percent ofchlorotic/necrotic area per cm2, excluding the leaf tips, which sometimes were burntby the saline water treatments.

2.5. Crop yield and grain quality

The plants were harvested 124 days after sowing (28 June). Each pot was har-vested individually, and 84 plants for each treatment were collected and measuredto investigate the crop yield and plant biomass. The aboveground biomass wasdivided into straw and ears. The straw and ears were dried in an oven at 80 �C for48e72 h and weighted pot-by-pot. The ears were shelled and the seeds from eachpot were weighed to assess the grain yield.

The harvest index for each pot was calculated as the ratio between the grainweight and the total aboveground dry biomass.

The hectoliter weight and gluten content of the grainwere determined using theNIT-method (Infratec 1241 Grain Analyzer). A scanning monochromator Infratec

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e21 15

1241 grain analyzer (Foss NIR Systems INC., Hoganas, Sweden) was used to measuretransmittance spectra from 850 to 1048 nm at every 2-nm intervals. The NIT spectrawere obtained for wheat samples using a rectangular sample transport module(15 cm length, 4.5 cmwidth, 3 cm depth). The path length of the top loading cuvettewas 18 mm.

The Kjeldahl method was used to determine crude protein content (AOAC,2000).

2.6. Statistical analysis

The statistical significance of the differences that were observed in the differenttreatments was assessed with an analysis of variance (ANOVA) by considering theOTC as the statistical unit within a splitesplit plot experimental design oncompletely randomized blocks with three factors. Ozone was set as the main (fixed)factor with 2 factor levels: CF-OTC and EN-OTC. Saline irrigation was set as a nestedfactor within each ozone treatment, with 2 factor levels: ‘saline’ (Sþ) and ‘tap-water’ (S-) irrigated plants. The cultivar (genotype) was set as a further nested factorwithin each ozone and saline treatment, with 2 factor levels: “Virgilio” and“Neodur”.

The normal distribution of the data of each parameter within each treatmentgroupwas verified by the ShapiroeWilkW Test and by normal probability plots. Theassumption of the homogeneity of the variances was verified for each parameter bythe Levene's test.

A contrast analysis with a-priori planned comparisons was used to test thesignificance of the response of each cultivar to ozone, to saline irrigation, and to thecombined effect (ozone � saline irrigation).

All of the tests were performed separately for each parameter with the GLM(General Linear Models) module of the STATISTICA 8.0 software (Tulsa, USA),

3. Results

3.1. Weather conditions, AOT40 and water salinity levels

The 2011 growing season presented unexpected weather withrespect to the thirty-year means of the location. The season wascharacterized by a hot and particularly dry April, with only 10 mmof total precipitation. Most of the rain events occurred during thefirst half of June, significantly increasing the relative humidityduring this month.

The monthly mean concentrations of O3 had its maximumduring May (47 ppb), while the maximum hourly mean value was

Table 1Ozone exposure (AOT40) and electric conductivity (EC) of the circulating solution in the pcumulated from the beginning of each phenological stage to the beginning of the followinphonological stage. CF-OTC ¼ Charcoal-Filtered Open-Top Chambers; EN-OTC ¼ O3-Enrictreatment.

recorded in June (123 ppb). Unusually, many peak episodes of theO3 concentration were also recorded during April, due to the hightemperatures and dry weather.

The seasonal durumwheat exposure to O3 in CF-OTCs remainedfar below 3000 ppb h, the long-term objective for vegetation pro-tection established by the European Directive 2008/50/CE, reachinga final mean value (calculated as the mean of 4 CF-OTCs) of699 ppb h. In contrast, the AOT40 critical level was greatly exceededin the EN-OTCs, reaching a final mean value of 23,509 ppb h(calculated as the mean of four replicates OTCs).

Table 1 reports the AOT40 values that cumulated during eachphenological stage in the two different O3 treatments, and therange of EC values of the water solution in the soil during the sameperiods.

It is worth noting that more than half of the total exposure valuein the EN-OTCs was accumulated during the grain filling period (i.e.from anthesis to harvest, 12,074 ppb h).

Also significant is the AOT40 that cumulated during the earlytillering period (2626 ppb h) and during the earing period(3865 ppb h).

The salinity of the circulating solution in the pots of the saline-irrigated plants was, on average, 13.2 dS/m corresponding to the“strongly-saline soil” class according to the classification of theUSDA Salinity Laboratory (Richards, 1954). This parameterincreased during the growing season, reaching amaximumvalue of23 dS/m during anthesis and then decreasing with the main rainevents.

In contrast, the conductivity level in the pots with the controlplants (S-) was less than 1 dS/m (0.68 dS/m on average).

3.2. Stomatal conductance

The mean gs that were recorded throughout the entiremeasuring period (4e25 May) at different daytime hours are re-ported in Table 2. The gs measured during the afternoon cycles was

ots, related to the different phenological stages of durumwheat. AOT40 values wasg stage. EC values are expressed as the range of conductivity experienced during eachhed Open-Top Chambers; Sþ ¼ Saline irrigation treatment; S-¼ Tap water irrigation

Table 2Mean values of the stomatal conductance to water (gs) during the measuring period (4e25 May) at different daytime. CFS� ¼ Charcoal-Filtered OTC without saline irrigation;CFSþ ¼ Charcoal-Filtered OTC with saline irrigation; ENS� ¼ O3-enriched OTC without saline irrigation; ENSþ ¼ O3-enriched OTC with saline irrigation; SD ¼ StandardDeviation.

Treatment Morning Midday Afternoon

Mean gsmmol m�2 s�1

SD Mean gsmmol m�2 s�1

SD Mean gsmmol m�2 s�1

SD

Neodur CFS� 386 ±40 399 ±134 287 ±122ENS� 456 ±153 389 ±81 204 ±47CFSþ 242 ±105 214 ±65 105 ±85ENSþ 246 ±152 192 ±42 114 ±33

Virgilio CFS� 273 ±139 382 ±95 156 ±73ENS� 418 ±193 412 ±126 223 ±106CFSþ 250 ±40 315 ±102 182 ±68ENSþ 237 ±77 289 ±60 174 ±53

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e2116

always lower than that of morning and midday in both of thecultivars.

The trend of gs during the measuring period was similar in bothof the cultivars in the control treatments (CFS�) at midday (be-tween 12:00 and 14:00, Fig. 1 uppermost graphs), when the lightconditions can be considered uniform throughout the differentmeasurement days. Nevertheless, the mean gs of Neodur wasslightly higher than that of Virgilio (þ3.2% on average).

The effects of the different treatments on the gs are shown in thelowermost graphs of Fig. 1, which are referred to the middaymeasurements.

Saline irrigation reduced the gs in both of the cultivars. However,Neodur presented a more significant decrease (�57% on average,p ¼ 0.001) than did Virgilio (�22%, p ¼ 0.09), and the latter had an

Fig. 1. Midday stomatal conductance (gs) to H2O of the two cultivars Virgilio (right) and Neodthe lower graphs show, for each treatment, the percentage variation of gs respect to controlwith saline irrigation; ENS� ¼ O3-enriched OTC without saline irrigation; ENSþ ¼ O3-enri

irregular gs seasonal trend, with an early stimulation (at low ECvalues) and a progressive decrease in values (at high EC) followedby a final recoverywhen the EC values dropped below 10 dS/m aftercertain rain events.

An ANOVA test (data not shown) confirmed the statistical sig-nificance of the negative effect of saline irrigation on gs even for themorning (p ¼ 0.001) and afternoon (p ¼ 0.004) measurements inNeodur.

In contrast, no significant effect related to the O3 treatment wasdetected in either cultivar.

The combined effect of saline irrigation and O3 (ENS þ plants)generally caused amarked decrease of the gs in Neodur plants, evenif not statistically significant, driven by the saline treatment, whichwas the prevailing factor.

ur (left). The upper graphs reports the average gs in the control treatment (CFS�) while. CFS� ¼ Charcoal-Filtered OTC without saline irrigation; CFSþ ¼ Charcoal-filtered OTCched OTC with saline irrigation.

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e21 17

3.3. Leaf symptoms

In the leaves that were collected on 4 of May visible symptomswere present as mild chlorotic spots only on the leaves of the S-plants of Virgilio and, to a lesser extent, of Neodur that werecollected from EN-OTCs. Instead, symptoms were absent from theSþ plants growing in the same OTCs, as well as in all of the plantsfrom CF-OTCs, independent of the treatment. The symptom in-tensity in the S- plants growing in EN-OTCs increased gradually inthe following weeks, and at the end of May, these symptoms alsoappeared in Virgilio leaves as chlorotic lesions, often coalescingeach other, and sometimes turning into necrotic lesions. Thesesymptoms covered 31.38% ± 4.99 of the leaf, excluding the tip(Fig. 2). On the same date, Neodur leaves were spotted by several

Fig. 2. Leaf symptoms in Virgilio (V) and Neodur (N) plants growing in EN-OTCs and CF-OTCstreatment, or barely detectable in CF-OTCs. These symptoms, enlarged in the lower panel,sometimes necrotizing, arrow) than in Neodur, at least, in plants from EN-OTCs, while it wpresence of tip burnt, particularly severe in Virgilio. CF-OTCs ¼ Charchoal-filtered Open-treatment; S- ¼ Tap water irrigation treatment.

small chlorotic or necrotic lesions involving 4.49% ± 1.33 of the leafarea (Fig. 2). Interestingly, both Virgilio and Neodur plants growingin the same EN-OTC but that were treated with saline water irri-gation did not shown any of the above symptoms, except forintensely burnt tips, possibly due to salt treatment, as thesesymptoms were also present in the corresponding plants that weregrown in CF-OTCs. The lack of O3 symptoms in the S- plants couldbe explained by the decreased uptake of O3 because of the reduc-tion of the gs that was caused by saline irrigation (particularly inNeodur). In CF-OTCs the Sþ plants remained symptomless, exceptfor the above-mentioned burnt tips, while the S- plants showedmild chlorotic spots, possibly more frequent in Neodur(1.89% ± 0.40 of the leaf area) than in Virgilio (0.87% ± 0,18 of theleaf area) (Fig. 2). Finally, in the middle of June, the symptom

, at May 31. Typical symptoms of ozone injury are more evident in S- plants of EN-OTCsappear more severe in Virgilio (chlorotic spots coalescing into larger chlorotic lesions,as the opposite in CF-OTCs. Sþ plants appeared always symptomless, except for the

Top Chambers; EN-OTCs ¼ O3-enriched Open-Top Chambers; Sþ ¼ Saline irrigation

Table 3Mean values and standard deviation of crop yield and quality parameters measured in the 4 replicates of each ozone and saline treatment, for the two studied cultivars ofdurumwheat. CFS�¼ Charcoal-Filtered OTCwithout saline irrigation; CFSþ¼ Charcoal-Filtered OTCwith saline irrigation; ENS�¼ O3-enriched OTCwithout saline irrigation;ENSþ ¼ O3-enriched OTC with saline irrigation.

Parameter Unit Neodur Virgilio

CFS� CFSþ ENS� ENSþ CFS� CFSþ ENS� ENSþTotal grain weight g 570 ±69 493 ±66 566 ±36 509 ±42 650 ±61 643 ±46 629 ±6 649 ±27Total number of ears n� 340 ±46 290 ±5 329 ±21 279 ±7 338 ±20 268 ±24 336 ±19 271 ±18Average grain weight per ear g 1.99 ±0.17 1.93 ±0.15 1.98 ±0.19 2.08 ±0.12 2.27 ±0.12 2.50 ±0.04 2.19 ±0.13 2.51 ±0.09Total number of empty ears n� 51 ±16 32 ±17 38 ±16 33 ±17 48 ±20 10.0 ±9 44 ±15 12 ±9Total dry weight of stems g 609 ±80 498 ±47 713 ±81 437 ±127 513 ±54 424 ±68 498 ±39 484 ±32Total dry weight g 1410 ±164 1194 ±98 1502 ±122 1145 ±91 1389 ±49 1282 ±125 1343 ±30 1346 ±53Average plant height cm 65.0 ±2.8 67.7 ±4.1 64.8 ±1.6 68.6 ±2.1 58.7 ±1.2 65.7 ±3.1 63.7 ±3.3 67.5 ±2.2Harvest index % 28.5 ±3.3 29.0 ±3.3 27.3 ±2.9 30.6 ±4.3 31.7 ±3.0 33.4 ±1.2 31.8 ±2.0 32.5 ±1.6Hectolitre weight Kg/hl 82.3 ±0.6 80.4 ±1.1 82.2 ±0.2 80.5 ±0.9 79.8 ±0.6 79.5 ±0.54 79.6 ±0.2 78.9 ±0.4Protein content % 14.2 ±2.2 16.7 ±0.9 14.9 ±0.5 16.2 ±0.3 14.2 ±0.8 15.1 ±0.7 14.5 ±0.8 15.5 ±0.5Gluten content % 11.0 ±2.2 13.5 ±0.9 11.7 ±0.5 13.0 ±0.3 11.1 ±0.8 11.8 ±0.9 11.0 ±0.5 12.3 ±0.5

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e2118

pattern remained almost unchanged, although the plants that weregrown in EN-OTCs showed an advanced stage of senescencecompared to the plants from CF-OTCs (not shown).

3.4. Crop yield and grain quality

The post-harvest effects on the crop yield and quality parame-ters of the two durumwheat cultivars in relation to different O3 andsaline irrigation treatments are shown in Table 3, while Table 4reports the results of the ANOVA test for the entire splitesplitplot experiment.

According to Table 4, the genotype did not significantly influ-ence the response of durum wheat to ozone (except for oneparameter, average grain weight per ear); in contrast this influencewas often significant and stronger on many responses to salineirrigation, involving the total grainweight, average grainweight perear, total dry weight and hectoliter weight.

Table 5 shows the planned comparisons on the ANOVA results ofTable 4 to highlight the significance of the response of each specificcultivar under the different treatments.

Saline irrigation significantly decreased the total number of earsin both of the cultivars (�14.9% in Neodur, p ¼ 0.0015; �20.0% inVirgilio, p ¼ 0.0001) and significantly decreased the number ofempty (i.e., unfertile) ears in Virgilio (�75.9%, p ¼ 0.0008). Theseeffects caused a significant reduction of the grain weight in Neodur(�11.8%, p ¼ 0.0047) and a negligible loss in Virgilio (ns), which incontrast showed an increase of the mean grain weight per ear(þ12.3%, p ¼ 0.0046).

The hectoliter weight was significantly reduced in Neodur(�2.2%, p ¼ 0.0008), while protein and gluten contents

Table 4ANOVATest of significance for different crop yield and quality parameters considering threirr. ¼ saline irrigation).

Ozone cv cv � ozone

Total grain weight 0.9653 0.0001*** 0.5829Total number of ears 0.5689 0.0896 0.1004Average grain weight per ear 0.3353 0.0000*** 0.0210*Total number of empty ears 0.2646 0.0295* 0.5014Total dry weight of stems 0.5936 0.0043** 0.9868Total dry weight 0.8200 0.3149 0.8100Average plant height 0.1874 0.0729 0.2501Harvest index 0.6475 0.0001*** 0.3967Hectolitre weight 0.4205 0.0000*** 0.2163Protein content 0.1598 0.1119 0.7668Gluten content 0.3111 0.0719 0.8872

*p � 0.05.**p � 0.01.***p � 0.001.

increased: þ13.0% (p ¼ 0.078) and þ16.7% (p ¼ 0.0084), respec-tively. The increase in the protein (þ7.0%) and gluten (þ8.8%)contents that were observed in Virgilio were not significant.

Saline irrigation was also responsible for general reductions ofthe total dry weight (�19.7%, p < 0.0001) and the stem biomass(�29.2%, p ¼ 0.0001) in Neodur and for a slightly significant in-crease in the harvest index (þ6.8%, p ¼ 0.0369).

However, the height increase in these plants (þ5%, p ¼ 0.0152)may be related to a thinner stem structure, with a consequentlower quality. The O3 treatment had no significant effects (Tables 3and 4), with the only exception of the plant height in Virgilio, whichsignificantly increased by þ5.4% (p ¼ 0.0114).

The combined effect of the two treatments was strongly drivenby saline stress. However, only the total dry weight of the stemswas significantly reduced by the combination of the two stressorsin Neodur, while no significances emerged for Virgilio.

4. Discussion

In general, Virgilio cultivar was more productive than Neodur(þ14% of the total grain weight, confronting only the plants underthe CFS� conditions), also presenting higher values of the harvestindex in all of the different applied treatments.

Even if both of the cultivars had the same values of abovegroundbiomass, Virgilio presented a lower stem dry weight, indicating astronger investment in the grain-filling processes than Neodur.

The saline irrigation effects on durumwheat were more markedthan O3 effects, especially in Neodur. Corresponding results havealso been reported byMastrorilli et al. (1995) and Araus et al. (1998)for other Triticum durum cultivars.

e factors (cultivar, ozone, saline irrigation) and their combinations. (cv¼ cultivar; sal.

Sal. irr. ozone � sal. irr. cv � sal. irr. cv � ozoneX sal. irr.

0.0481* 0.4072 0.0193* 0.89650.0000*** 0.9380 0.3284 0.88190.0232* 0.3026 0.0394* 0.77480.0012** 0.4262 0.0589 0.71610.0003*** 0.3710 0.0133* 0.0302*0.0001*** 0.7996 0.0017** 0.05190.0002*** 0.5176 0.2072 0.20720.0215* 0.3998 0.5429 0.10610.0015** 0.8661 0.0448* 0.64420.0049** 0.5452 0.3069 0.50830.0056** 0.7747 0.3001 0.3393

Table 5Planned comparison test of significance for different crop yield and quality parameters in Neodur and Virgilio cultivars. (cv ¼ cultivar; sal. irr. ¼ saline irrigation).

Neodur Virgilio

Cv � ozone cv � sal. irr. cv � ozone � sal. irr. cv � ozone cv � sal. irr. cv � ozone � sal. irr.

Total grain weight 0.7729 0.0047** 0.6170 0.6987 0.7296 0.4965Total number of ears 0.3810 0.0015** 0.9601 0.9521 0.0001*** 0.8728Average grain weight per ear 0.3693 0.8410 0.3519 0.6697 0.0046** 0.5892Total number of empty ears 0.4677 0.1555 0.4143 0.9253 0.0008*** 0.7551Total dry weight of stems 0.5437 0.0001*** 0.0339* 0.5317 0.1593 0.3014Total dry weight 0.6121 0.0000*** 0.1131 0.8329 0.2351 0.2043Average plant height 0.7587 0.0152* 0.6458 0.0114* 0.0005*** 0.1827Harvest index 0.8690 0.0369* 0.0833 0.6104 0.1933 0.5572Hectolitre weight 0.9762 0.0008*** 0.8348 0.3638 0.2149 0.6560Protein content 0.8375 0.0078** 0.3745 0.5681 0.1194 0.9672Gluten content 0.8395 0.0084** 0.3804 0.7160 0.1324 0.6283

*p � 0.05.**p � 0.01.***p � 0.001.

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e21 19

Virgilio, in contrast, seemed to better tolerate saline irrigation,despite the larger extension of burnt tip symptoms. In fact,although the total number of ears was significantly reduced, thetotal grain yield remained almost unchanged, as well as the hec-toliter weight. Because this result was related to a decrease in thenumber of empty ears and to a significant increase in the specificgrain yield per ear, it reveals that the salt stress induced in Virgilio agreater investment in the reproductive efficiency, a plant responsewhich has been generally reported for other abiotic stresses, suchas O3 (Gerosa et al., 2009b; Black et al., 2007; Wang et al., 2008) .

The reduction in the stem weight in both of the cultivars,revealed a negative effect of saline irrigation during the risingphenological phase. During this phase, the increasedwater demandby plants, which is exacerbated by the saline stress, led to adecreased emission of the stems. Then, the activation of osmoreg-ulation and of the antioxidant defense systems decreased theavailable energy, resulting in a lower accumulation of plantbiomass.

The protein content in the grain significantly increased in theplants that were subjected to saline irrigation (Table 3) as a directconsequence of the increased in gluten content, as gluten repre-sents approximately 80% of the proteins in the caryopsis. This resultagrees with other studies investigating with different type ofstresses, such as high temperature, drought and nitrogen deposi-tion inwinter wheat (Triticum aestivum) (Gooding et al., 2003; Zhaoet al., 2009; Saint Pierre et al., 2008) and durum wheat (Li et al.,2013).

In both of the cultivars the saline-irrigated plants had a reducedstomatal conductance.

Similar effects have been reported for Triticum durum by Katerjiet al. (2003) and by other authors for several other species (e.g.,Huang et al., 1994 on winter wheat; Katerji et al., 1997 on sugarbeet; Maggio et al., 2009 on alfalfa).

Regarding the O3 treatment, no statistically significant effects ofthis stressor on the grain yield and biomass production wereobserved in either cultivar, while this treatment seemed to havecaused a slight but non-significant increase in the gs values in theVirgilio cultivar.

Many authors have reported that O3 tends to reduce the gs incrops (Bou Jaoud�e et al., 2008; Fagnano and Merola, 2010). How-ever, increased gs at higher O3 concentrations were observed inmany forest trees by Braun et al. (2010), McLaughlin et al. (2007),Bussotti and Ferretti (1998) and Paoletti et al. (2008).

The meaning of this response has been attributed to anO3�induced damage of the stomatal guard cells with a consequentimpairment of the stomata functionality and/or closure mecha-nisms (Paoletti and Grulke, 2005; Wittig et al., 2007; Pearson and

Mansfield, 1993). Our results confirm these latter findings, as evi-denced also by a previousmicroscopic analysis demonstrating H2O2accumulation around the stomata not exceeding the cell deaththreshold in guard cells but possibly enough to impair the closuremechanisms (Faoro and Iriti, 2009).

Virgilio is considered an O3 ‘sensitive’ cultivar (Picchi et al.,2010), while Neodur is a ‘tolerant’ one (Faoro, personal communi-cation), and this characteristic was assigned based on the onset ofspecific visible leaf injuries. From this point of view, Virgilio wasconfirmed as sensitive because the diffusion of ozone-like symp-toms was markedly more severe than in Neodur (Fig. 2), at least inthe presence of high O3 levels (EN-OTCs). Curiously, in CF-OTCs,Neodur was slightly more susceptible than was Virgilio. Thisdiscrepancy could be attributed to the higher gs of Neodur underlow O3 conditions and to the consequent greater O3 uptake.Considering that the gs was not significantly different betweenthese cultivars in EN-OTCs, it could be speculated that the Neodurantioxidant activity increases with the increasing oxidative stressto a greater extent than in Virgilio, thereby better counteracting thenegative effects of ROS, and leading to a cell-death restraint andless-severe visible symptoms (Faoro and Iriti, 2005).

However, Virgilio's greater sensitivity was reflected only byweak (and not significant) reductions of the total grain yield andplant biomass (these reductions are negligible in Neodur). In anycase, it is worth noting that even under O3 stress the overall yield ofVirgilio is still markedly higher (þ11%) than in Neodur, a distinctiveagronomic feature that makes trivial the onset of visible injuries onflag leaves.

This conclusion agrees with the US EPA (1986) observations andwith other studies (Barnes et al., 1990; Simini et al., 1987; Picchiet al., 2010) that reported that visible foliar injuries on plants donot always result in crop yield losses and that there is little or nocorrelation between visible O3 damage and reduced yield. This factsuggests a revision of the O3 ‘tolerance’ and ‘sensitivity’ definitions,as it is evident that the O3 sensitivity shown by plants for a certainparameter (e.g. foliar symptoms) is not always related to otherparameters (e.g., agronomic yield). It is, therefore, necessary todevelop an integrated approach that considers all of the aspects. Inthe case of durum wheat, where the economic aspects are morerelevant than the ecological aspects, it seems more sound to basethis definition on the agronomical yield/loss.

Regarding other yield parameters, it is important to note thatthe production of straw (total stem dry weight) increased in both ofthe cultivars under O3 conditions.

Similar interesting results were also found by Pleijel et al. (1991,1995) and by Kohut et al. (1987) on Triticum aestivum, whileopposite findings have also been reported by other authors, e.g.,

G. Gerosa et al. / Environmental Pollution 193 (2014) 13e2120

Fuhrer et al. (1989, 1990) and Ollerenshaw and Lyons (1999), on thesame species.

The relative O3 tolerance of durumwheat which arises from thisexperiment and from other studies (Barnes et al., 1990;Reichenauer et al., 1998) clearly contrasts with the results re-ported by the UN-ECE on Triticum aestivumwhere, at O3 exposuresequal to those in this study, yield losses averaging 25% wereobserved (Mapping manual UN-ECE, 2004; Fuhrer et al., 1997). AsTriticum aestivum is used as a sensitive reference crop at the Eu-ropean level to estimate the O3 effects on the continental area, thischoice could be inappropriate for Mediterranean countries wherethe production of durum wheat is largely prevalent. These coun-tries are likely to suffer excessive overestimation of the O3 risks anddamages on crops, which may not occur in the field.

The combination of these two stresses caused contrasting ef-fects in the two cultivars in terms of yield and grain quality, even ifnot univocally clear and defined. Ozone and saline irrigationgenerally have a small antagonistic action in Virgilio and a slightsynergic effect in Neodur, at least regarding the above parameters.If the synergistic effects can be easily understood as the addition oftwo different stressors, the antagonistic effect of salt and O3 seen inVirgilio could be explained by the fact that both stressors stimulatethe expression of the antioxidant enzymes, which also may help incoping with additional oxidative stress. For example, Sandermannet al. (1998) reported that O3 is an abiotic elicitor of defensemechanisms against other biotic stresses (pathogens), whileMaggio et al. (2007) found that salinity increased the antioxidantproduction in tomato plants.

In any case, it must be stressed that saline irrigation greatlyreduced the ozone visible symptoms in both of the cultivars butparticularly in Virgilio. This result can be easily explained by thereduction in gs and the consequent lower O3 uptake, limiting theoxidative stress around stomata, at least under a cell-deaththreshold. The latter explanation agrees with the work of Maggioet al. (2009) regarding experiments on the cross effects of O3 andsalinity on alfalfa and with Hassan (2004) in similar experimentthat were conducted on bread wheat.

Acknowledgments

The authors are grateful to Dr. Mladen Todorovic and theCIHEAM-MAI (International Centre for Advanced MediterraneanAgronomic Studies, Mediterranean Agronomic Institute) of Bari(Italy) for their support, to Dr. Simone Mereu for his useful sug-gestions and to the Agricultural Research Council (CRA-SCV) of S.Angelo Lodigiano (Italy) for their support in the harvestingmeasurements.

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