Responce to NaCl salinity and excess boron in greenhouse tomato grown in semi-closed substrate...

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RESPONSE TO SODIUM CHLORIDESALINITY AND EXCESS BORON INGREENHOUSE TOMATO GROWN IN SEMI-CLOSED SUBSTRATE CULTURE IN AMEDITERRANEAN CLIMATEG. Carmassi a , M. Romani a , C. Diara a , D. Massa a , R. Maggini a ,L. Incrocci a & A. Pardossi aa Dipartimento di Biologia delle Piante Agrarie, University of Pisa,Pisa, ItalyAccepted author version posted online: 30 Jan 2013.Publishedonline: 18 Apr 2013.

To cite this article: G. Carmassi , M. Romani , C. Diara , D. Massa , R. Maggini , L. Incrocci & A.Pardossi (2013): RESPONSE TO SODIUM CHLORIDE SALINITY AND EXCESS BORON IN GREENHOUSETOMATO GROWN IN SEMI-CLOSED SUBSTRATE CULTURE IN A MEDITERRANEAN CLIMATE, Journal of PlantNutrition, 36:7, 1025-1042

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Journal of Plant Nutrition, 36:1025–1042, 2013Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904167.2013.766209

RESPONSE TO SODIUM CHLORIDE SALINITY AND EXCESS BORON

IN GREENHOUSE TOMATO GROWN IN SEMI-CLOSED SUBSTRATE

CULTURE IN A MEDITERRANEAN CLIMATE

G. Carmassi, M. Romani, C. Diara, D. Massa, R. Maggini, L. Incrocci,

and A. Pardossi

Dipartimento di Biologia delle Piante Agrarie, University of Pisa, Pisa, Italy

� The effects of sodium chloride (NaCl) salinity and boron (B) toxicity were investigated in green-house tomato (Solanum lycopersicum L.) plants grown in closed soilless culture under the typicalclimatic conditions occurring in the Mediterranean regions. The experiment was conducted undersemi-commercial conditions. Two NaCl (2.0 and 10.0 mol m−3) and B (27.8 and 185.0 mmolm−3) concentrations were combined to produce four different types of raw water used to prepare thenutrient solutions. The fertigation treatment did not affect significantly the uptake of water andmineral elements apart from that of sodium (Na), chloride (Cl), and B. The use of B-enriched wa-ter increased the accumulation of this element in the leaves, which showed marginal chlorosis andnecrosis within 35–40 days from planting. No or minor effects of NaCl and B concentrations in theirrigation water were found on leaf area development, biomass accumulation, crop yield and fruitquality.

Keywords: boron (B) toxicity, chlorophyll fluorescence, hydroponics, leaf burn,protected horticulture, Solanum lycopersicum L.

INTRODUCTION

Boron (B) is an essential element for plants and the importance of itsapplication in intensive cropping systems, such as greenhouse crops and soil-less cultures, is well recognized (Sonneveld and Voogt, 2009). On the otherhand, excess B may occur in soil naturally or as a result of over-fertilizationand/or irrigation with water rich in B (Nable et al., 1997). In some regions Bcontamination of groundwater represents a serious constraint to both agri-culture (Ben-Gal and Shani, 2002) and the production of drinking water(Weinthal et al., 2005).

Received 17 December 2010; accepted 3 January 2012.Address correspondence to G. Carmassi, Dipartimento di Biologia delle Piante Agrarie, University

of Pisa, Pisa 56124, Italy. E-mail: gcarmassi@agr.unipi.it

1026 G. Carmassi et al.

Excess B is often found in association with high salinity in soil and/orirrigation water, especially in arid or semi-arid regions (Ben-Gal and Shani,2003) or when reclaimed sewage water is used (Pescod, 1992). The possibleinteraction between salinity and B toxicity was investigated in many crops(e.g., Alpaslan and Gunes, 2001; Ben-Gal and Shani, 2002; Edelstein et al.,2005; Yermiyahu et al., 2008; Smith et al., 2010): generally, their effects werenot additive and salinity protected the plants from B by reducing its uptakeand accumulation in the shoot.

In greenhouse horticulture, closed soilless cultures (hydroponics),where the drainage nutrient solution (NS) is collected and recirculated,may reduce water consumption and minimize nutrient leaching (Massa et al.,2010). The salinity and/or the concentration of specific substances in theirrigation water create the main difficulties for the management of closedsystems, because any substance dissolved in the supplied NS at concentrationhigher than the plant uptake concentration (i.e., the ion/water uptake ratio)will accumulate in the root zone (Carmassi et al., 2007). Under these condi-tions, NS is generally re-circulated until electrical conductivity (EC) and/orthe concentration of some potential toxic ion [for instance, sodium (Na),chloride (Cl), or micronutrients] reaches a maximum acceptable thresholdvalue, afterwards it is discharged, at least partially (Carmassi et al., 2007).The term “semi-closed” is used for such systems.

Many studies, in particular with tomato (e.g. Massa et al., 2010, 2011;Montesano et al., 2010; Varlagas et al., 2010), were published on the possibleeffect of sodium chloride (NaCl) salinity on the management of closedsoilless cultures. Nada et al. (2010) published a paper on the effect of highB concentration in the NS on tomato grown in hydroponics. However, weare not aware of any paper on combined effects of salinity and excess B inplants grown in closed soilless systems.

Therefore, a work was undertaken in 2009 to investigate how the con-centration of NaCl and B in the irrigation water influenced crop water andmineral relations, growth and fruit yield in semi-closed soilless (perlite)culture of tomato (Solanum lycopersicum L.) conducted in the greenhouseclimatic conditions occurring in the Mediterranean regions between latewinter and summer. Both macro- and micronutrients were determined inthe re-circulating nutrient solution. The experiment was carried out undersemi-commercial conditions given that: i) moderate concentrations of NaCl(2.0 and 10.0 mol m−3 corresponding to 116.88 and 584.43 mg L−1 and/orB (27.8 and 185.0 mmol m−3, corresponding to 0.30 and 2.0 mg L−1) wereused because greenhouse crops cannot be grown with highly saline irrigationwater for being profitable (Stanghellini et al., 2007); ii) the re-circulatingNS was discharged whenever EC exceeded 4.5 dS m−1 and nitrogen (N)-nitrate (NO3) concentration was below 1.0 mol m−3. Such value was chosenbecause 20 mg L−1 (1.42 mol m−3) is the maximum allowed concentration ofN-NO3 in wastewater discharged into surface water according to the current

B Toxicity in Tomato 1027

Italian legislation derived from European Nitrate Directive (Massa et al.,2010). This system gave rise to fluctuations in the mineral concentration ofNS rather than to steady values as used in most studies on plant response toconcurrent exposure to salinity and excess B.

Another experiment had been carried out in 2008, when the plants hadbeen cultivated for three months against nearly six months in 2009. A briefreport of this study was published in the proceedings of an internationalconference (Pardossi et al., 2009). Similar results were obtained in bothyears and only the second experiment is reported in this paper.

MATERIALS AND METHODS

Plant Material and Growing Conditions

Tomato (cv. ‘Caramba’) plants were grown for 169 days between springand early summer in an unheated glasshouse (240 m2) at the University ofPisa. Five-week old seedlings were planted on 4 March 2009 in 30-L perlitebags placed in plastic gullies with gently slope and irrigated with drippers.Crop density was 3.0 plants m−2 (six plants in each bag). The plants weregrown vertically and top-cut two leaves above the 12th truss; lateral shootsand the leaves below the bottommost truss with ripening fruits (up to the6th truss) were recurrently removed.

Experimental Treatments and Nutrient Solution Management

Four sources of raw water were tested: FWLB, fresh water (2.0 mol m−3

NaCl) with low B concentration (27.8 mmol m−3); FWHB, fresh water withhigh B concentration (185.0 mmol m−3); SWLB, saline water (10.0 mol m−3

NaCl) with low B concentration; SWHB, saline water with high B concen-tration. For this purpose, appropriate concentrations of NaCl and/or boricacid (H3BO3) were added to groundwater that also contained calcium (Ca)1.5 mol m−3, chloride (Cl) 2.2 mol m−3, magnesium (Mg) 0.75 mol m−3, Na1.5 mol m−3, B 10.1 mmol m−3, iron (Fe) 8.9 mmol m−3, manganese (Mn)2.7 mmol m−3, zinc (Zn) 2.1 mmol m−3.

Each treatment was applied 14 days after planting (DAP) till the end ofthe experiment (155 days of observations, in total) to three separate systems(replicates). Each system consisted of a 10 m2 bench with 30 plants and amixing tank of 80 L (8.0 L m−2), which collected the water drained from thesubstrate. The total volume of re-circulating NS, including the one retainedby the substrate (4 L m−2 at container capacity), was approximately 12 L m−2.

Whenever the water level in the mixing tank dropped off by approxi-mately 10 L, the tank was automatically refilled with a NS containing thedesired NaCl and B concentrations along with the following concentrationsof macro- and micro-nutrients: Ca 4.0 mol m−3; potassium (K) 7.0 mol m−3;

Mg 0.75 mol m−3; N-NO3 11.0 mol m−3; phosphorus (P)-H2PO4 1.2 mol m−3;sulfur (S)- sulfate (SO4) 2.41 mmol m−3; Fe 17.8 mmol m−3; Zn 5.0 mmolm−3; copper (Cu) 2.7 mmol m−3; manganese (Mn) 10.0 mmol m−3. The ECof refill NS prepared with FW and SW was, respectively, 1.90 and 2.64 dSm−1.

The re-circulating NS was checked almost daily for EC and pH; the latterwas frequently adjusted to 5.5–6.0 with sulphuric acid. Water uptake (W)was compensated with the refill NS and, when the EC of re-circulating NSreached 4.5 dS m−1, the mixing tank was refilled only with acidified rawwater until N-NO3 concentration dropped below 1.0 mol m−3, afterwards itwas emptied.

Determinations

In three circumstances (80, 120 and 160 DAP), chlorophyll fluorescencetransient was determined on leaves adapted to the dark for 30 min at am-bient temperature using a portable instrument (Plant Efficiency Analyser,PEA, Hansatech, Poole, UK). The measurements were taken on the upperleaf surface (4-mm diameter) exposed to a red light intensity of 600 W m−2

emitted by three diodes. Several quantities were measured with PEA, such asF0, Fm and Fv (Fm–F0), and different parameters were calculated includingFv/Fm and Performance Index (PI; Strasser et al., 2000). After measure-ments, the leaves were extracted with methanol for the determination ofchlorophyll concentration (Lichtenthaler, 1987).

Leaf area and dry mass of stem, leaves and fruits were determined in eachtreatment at 123 and 169 DAP; lateral shoots and basal leaves periodicallydetached from the plants were added to the total crop biomass. B concentra-tion (expressed as mg kg−1 DW) was measured in stems and leaves collectedfrom the lower, middle and upper third of the plants. Plant samples wererapidly washed with tap water, rinsed in deionised water, dried at 80◦C anddigested with a mixture of nitric and perchloric acid at 230◦C for 1 h; B wasdetermined by the Azomethine-H method.

Flowers and fruits were counted every 2–4 days on each truss. Ripe fruitswere harvested twice per week and several quality attributes were deter-mined in marketable berries: dry matter content, pH, EC, total soluble solids(◦Brix), titratable acidity (expressed as citric acid). B content was determinedalso in the dry residue of each fruit sample.

The occurrence of leaf burn (chlorotic and/or necrotic patches at themargins and tips) was evaluated at 40, 80, 123 and 169 DAP, as follows. Ateach sampling date, two compound leaves were harvested from the lower,middle and upper third (six leaves in total) of three individual plants ineach culture. The pictures of all leaflets in each leaf were taken with adigital camera and processed using Image Tool for Windows (version 3.0;Microsoft, Pullman, WA) to determine the percent ratio of injured area on

total area. In each sampled plant, the severity of leaf burn was estimated asthe area-weighted mean of percent necrosis in the six individual leaves.

Daily W was determined by recording with a volume meter the amount ofNS (or water) used to refill automatically the mixing tank. The evaporationfrom the substrate, which was wrapped in plastic bags, and water loss due toaccidental seepage were negligible.

Samples of re-circulating NS were collected frequently and in occasionof each flushing event for laboratory analysis of mineral elements. Boron wasalso determined in the substrate and in the root tissues at the end of culti-vation. After careful removal of main root filaments, an aliquot of substratefrom sampled bags was extracted for 2 h with aqua regia [nitric acid (HNO3)and hydrochloric acid (HCl) 1:3, v/v] at 130◦C or with distilled water at 60◦C.Boron was analyzed in the filtered solution as described previously.

Statistics

A completely randomized block design with a 2 × 2 factorial treatmentarrangement with three replicates was adopted. Two-way analysis of variance(ANOVA), least significant difference (LSD) test for mean separation, andregression analysis were conducted using Statgraphics Plus 5.1 (Manugistic,Rockville, MD).

RESULTS

Growing Conditions

As expected, air temperature (TAIR) tended to increase during the grow-ing season and in the last few weeks maximum daily values were gener-ally higher than 30◦C (seasonal average was 32.3◦C) and surpassed 40◦C(Figure 1); TAIR never dropped below 17◦C. Daily solar radiation ranged be-tween 1.39 and 14.99 MJ m−2 with an average of 9.33 MJ m−2 and a seasonallycumulated value of 1465.4 MJ m−2.

The re-circulating NS was discharged in three and seven occasions in thecultures conducted with FW and SW, respectively (Figure 2). The nutrientdepletion applied before discharge lasted seven to eight days during the firstthree months of cultivation and up to 10–15 days later on. This resultedin large oscillations in the EC and mineral concentrations of re-circulatingNS, as shown in Figure 2 where, for the sake of brevity, only EC and Bconcentrations are reported.

The different fertigation management imposed by the use of FW or SWresulted in moderate (Mg, Cu, Fe and Zn) or large (Ca, K, N-NO3 and Mn)differences among the treatments in the average ion concentrations of there-circulating NS, which generally were higher in FW treatments comparedto the others (Table 1). By contrast, Na and Cl concentrations were much

10 30 50 70 90 110 130 150 1700

30Mean T Min. TMax. T

Radiation

Days after planting

FIGURE 1 Air temperature and global radiation in the greenhouse during the experiment conductedin 2009 with tomato plants grown in semi-closed soilless culture.

30FWLB

10FWLB

0 20 40 60 80 100 120 140 1600

30 FWHB

Days from planting

0 20 40 60 80 100 120 140 160 1800

Days from planting

FIGURE 2 Boron concentration and electrical conductivity (EC) in the re-circulating nutrient solutionin semi-closed soilless cultures of greenhouse tomato conducted using different sources of irrigationwater. See Table 1 for abbreviations. Mean (±SE) values of three replicates. The asterisks near theabscissa in the EC graphs indicate when the nutrient solution was discharged.

TABLE 1 Electrical conductivity (EC) and the concentration of different mineral elements in therecirculating nutrient solution in semi-closed soilless cultures of tomato conducted using differentsources of irrigation water

Nutrient FWLB FWHB SWLB SWHB

EC (dS m−1) 3.60 ± 0.32 3.50 ± 0.35 4.99 ± 0.29 4.52 ± 0.25Ca (mol m−3) 8.58 ± 0.65 8.71 ± 0.65 4.45 ± 0.21 4.00 ± 0.18Cl (mol m−3) 4.78 ± 0.45 5.14 ± 0.25 32.36 ± 2.31 25.75 ± 2.31K (mol m−3) 9.61 ± 0.57 8.79 ± 0.52 7.07 ± 0.28 6.08 ± 0.28Mg (mol m−3) 1.89 ± 0.19 1.73 ± 0.16 2.16 ± 0.12 1.80 ± 0.09Na (mol m−3) 4.48 ± 0.25 4.44 ± 0.25 29.26 ± 1.31 27.45 ± 1.27N-NO3 (mol m−3) 11.78 ± 0.56 11.16 ± 0.50 9.39 ± 0.60 7.33 ± 0.49P-PO4 (mol m−3) 0.97 ± 0.06 0.87 ± 0.07 0.66 ± 0.04 0.64 ± 0.04B (mmol m−3) 94.48 ± 7.03 636.34 ± 46.09 72.36 ± 3.18 488.38 ± 22.79Cu (mmol m−3) 13.46 ± 0.98 13.21 ± 1.05 10.21 ± 0.61 10.05 ± 0.65Fe (mmol m−3) 24.80 ± 1.46 28.73 ± 1.90 22.70 ± 1.04 21.97 ± 1.08Mn (mmol m−3) 7.57 ± 0.41 7.15 ± 0.41 4.66 ± 0.25 4.15 ± 0.27Zn (mmol m−3) 8.71 ± 2.66 7.57 ± 2.75 7.95 ± 0.35 7.02 ± 0.25

FWLB, fresh water (2.0 mol m−3 NaCl) with low B concentration (27.8 mmol m−3); FWHB, freshwater with high B concentration (185.0 mmol m−3); SWLB, saline water (10.0 mol m−3 NaCl) with lowB concentration; SWHB, saline water with high B concentration. Mean values (±SE) of 20 (fresh water)or 28 (saline water) determinations performed in triplicate during the growing season.

lower in FWLB and FWHB than in SWLB and SWHB (Table 1). Boron con-centration was higher in the re-circulating NSs prepared with FW comparedto SW (Table 1).

In SWLB and SWHB treatments, EC averaged 4.99 ± 0.29 dS m−1 against4.52 ± 0.25 dS m−1 in other treatments (Table 1). The progressive increaseof EC was mainly attributable to the accumulation of Na and Cl (data notshown); a significant correlation was found between EC and the concentra-tion of both ions (R2 = 0.925, for Na; R2 = 0.885, for Cl). Boron concentra-tion in the re-circulating NS tended to increase in all treatments (Figure 2);on average, it was much lower in the SWHB culture than in the FWHB cul-ture, while there were not important differences between FWLB and SWLBtreatments (Table 1).

Water and Mineral Relations

The composition of irrigation water did not affect significantly dailyW (data not shown), which oscillated between 0.59 and 5.14 L m−2 day−1

Seasonal W averaged 509.1 ± 15.5 L m−2.The apparent uptake of both macro- and micro-nutrients was estimated

by the mass balance method, which is by subtracting the quantities containedin the drainage water (including the residual NS at the end of cultivation)from those delivered to the crop with the refill NS. Boron and NaCl con-centration in the irrigation water did not affect the cumulative uptake of allnutritive ions, apart from B. The uptake of Na and Cl was not influenced

TABLE 2 Boron content in different vegetative tissues of greenhouse tomato plants grown in semi-closed soilless cultures using different sources of irrigation water. See Table 1 for abbreviations.Samples were taken from lower, middle and upper third of the plants harvested at the end of growingseason (169 days after planting)

ANOVA1

Position FWLB FWHB SWLB SWHB NaCl B NaCl × B

Stem Lower 56.2 ab 62.5 a 48.4 b 59.4 a ns ns nsMiddle 51.7 b 65.2 a 47.3 b 67.2 a ∗ ∗∗ ∗Upper 51.2 b 56.1 ab 53.2 ab 58.1 a ns ns ns

Leaf rachis Lower 52.5 bc 78.6 a 48.0 c 68.4 ab ns ns ∗and petioles Middle 36.9 b 66.9 a 40.8 b 73.8 a ∗∗ ∗∗ ∗∗

Upper 57.0 b 96.1 a 48.2 b 83.6 a ns ns ns

Leaf lamina Lower 140.0 c 326.7 a 135.0 c 280.0 b ∗∗ ∗∗ ∗∗Middle 97.5 c 362.6 a 98.8 c 256.0 b ∗∗ ∗∗ ∗∗Upper 106.2 c 335.2 a 95.3 c 253.5 b ∗∗ ∗∗ ∗∗

Mean (n = 3) values followed by the same letters were not significantly different (LSD test; P < 0.05).1NS, not significant; ∗, significant at 5% level; ∗∗, significant at 1% level.

by B concentration and was much higher in the plants grown with SW (datanot shown).

Leaf lamina contained invariably more B than petioles and stems(Table 2). The use of HB water increased noticeably the B concentra-tions in leaf lamina regardless of node position, but it had no or minoreffects on the B content of other plant organs (Table 2). Compared to theFWHB plants, B content of leaf lamina was significantly lower in the SWHBplants regardless of leaf position (Table 2). Similar results were found at 123DAP, when somewhat lower values were determined in all organs (data notshown).

Root B content was determined only at the end of cultivation and was54.3 ± 0.7, 70.6 ± 0.7, 49.3 ± 1.1 and 50.3 ± 3.8 mg kg−1 DW in FWLB,FWHB, SWLB and SWHB treatment, respectively. Root dry mass was similarin all treatments and averaged 0.05 kg m−2; therefore, the amount of Baccumulated in these organs was little (few mg m−2).

The total shoot B content was estimated by adding the dry mass ofdifferent organs (including the leaves and lateral shoots removed duringthe cultivation) times their respective B concentration. The use of HB wa-ter augmented significantly shoot B accumulation compared to LB water(Figure 3). Shoot B content was higher in the FWHB plants compared to theSWHB plants, but the difference was not significant due to relatively smallincidence of leaf lamina on total aerial biomass (including fruits). Using thedata recorded in all treatments at 123 and 169 DAP, a highly significant mul-tiple regression was computed for the relationship of shoot B accumulation([B]shoot) against its mean concentration in the re-circulating NS ([B]NS;

FWLB FWHB SWLB SWHB0.0

ANOVA Acc. Upt. conc.

NaCl ns nsB ** **NaCl x B ns ns

Uptake concentrationUptake

Source of irrigation water

Uptake concentration (m

FIGURE 3 Total B uptake and uptake concentration in greenhouse tomato plants grown in semi-closedsoilless cultures using different sources of irrigation water. See Table 1 for abbreviations. The results ofANOVA are shown inside the graph. Mean (n = 3) values followed by the same letters (upper case forcentral lamina; lower-case for leaf margins) were not significantly different (LSD test, P < 0.05).

mmol m−3) and the cumulative W (L m−2) in the period between plantationand sampling:

[B]shoot = 0.0001789 [B]NS + 0.0001696 W(R2 = 0.992; n = 8; P < 0.001)

NaCl salinity did not influence B uptake concentration, as calculated as theratio between the total accumulation of B in plant tissues (including roots)and W, which was significantly higher in the plants grown with HB waterthan in those grown with FW (Figure 3).

The comparison between the values of genuine (based on tissues anal-ysis) and apparent (derived from mass balance) uptake suggested that con-siderable amounts of B deposited in the substrate, when HB water was used.This was confirmed by the results of B determination in the substrate sam-pled at the end of cultivation and extracted with hot aqua regia. In fact, largeB amounts were found in the substrate sampled from FWHB (0.33 ± 0.09 gm−2) and SWHB (0.28 ± 0.04 g m−2) cultures, while B content was muchlower in the substrate sampled from FWLB and SWLB cultures (it averaged0.03 ± 0.01 g m−2).When water was used for substrate extraction, B contentwas 3–4 fold lower compared to the values determined using hot aqua regia(data not shown). As the amount of B measured in the substrate accountedby approximately 58% (FWHB) or 51% (SWHB) of the difference betweengenuine and apparent B uptake, most likely B precipitation also occurred inthe gullies and in the mixing tank.

B Toxicity

FWHB and SWHB plants displayed evident signs of B toxicity within35–40 DAP. Leaf burn appeared first on the older leaves and its severityincreased with time, to a larger extent in the FWHB plants (Figure 4). At theend of the experiment, all leaves of FWHB and SWHB plants were affectedby marginal burn while no symptoms were observed in other treatments.

There were no or minor differences among the treatments in the Bcontent of central portions of leaf lamina, which invariably remained greenand contained much less B compared to the 10-mm outer parts (Figure 5).Boron concentration at leaf margins was much higher in the plants grownwith HB water and in the FWHB plants than those irrigated with SWHB.Using the data determined in the FWHB and SWHB plants at 123 and 169DAP, a significant linear regression was calculated between percent injuredleaf area (ILA) and leaf B content (([Bleaf]; mg kg−1 DW) X):

ILA = 0.134 [Bleaf] − 23.09(R2 = 0.994; P< 0.01; n = 4)

Chlorophyll fluorescence was determined with handy PEA in the asymp-tomatic areas of leaves in the morning and at midday, when TAIR ranged,respectively, between 25 and 32◦C and between 30 and 42◦C. Invariably,Fv/Fm ratio and PI were affected neither by irrigation treatment nor byleaf position (data not shown). However, both parameters were significantly

0 30 60 90 120 150 1800

30FWHB

Days from planting

FIGURE 4 Evolution of leaf burn due to B toxicity in greenhouse tomato plants grown in semi-closedsoilless cultures of greenhouse tomato using different sources of irrigation water. See Table 1 for abbre-viations. Mean (±SE) values of three replicates. The asterisk indicates a significant difference (LSD test,P < 0.05) between FWHB and SWHB.

FWLB FWHB SWLB SWHB0

Central parts

Margins

ANOVA Central Margins

NaCl ns nsB *** ***NaCl x B ns ns

Source of irrigation water

FIGURE 5 B content in the central and 10-mm outer parts of mature leaves sampled from greenhousetomato plants grown in semi-closed soilless cultures using different sources of irrigation water. See Table 1for abbreviations. The results of ANOVA are shown inside the graph. Mean (n = 3) values followed by thesame letters (uppercase for central lamina; lowercase for leaf margins) were not significantly different(LSD test, P < 0.05).

lower at midday (Fv/Fm = 0.76 ± 0.01; PI = 0.68 ± 0.12) than in the morning(Fv/Fm = 0.81 ± 0.01, PI = 2.85 ± 0.58).

Irrigation water composition, leaf position and sampling time did notinfluence significantly leaf chlorophyll content (data not shown), whichaveraged 111.7 ± 17.3 µg cm−2.

Crop Growth and Yield

Dry biomass accumulation (including roots and fruits) did not differamong the four fertigation treatments (data not shown) and averaged 2.06± 0.05 kg m−2. The estimated water accumulated in plant tissues averaged17.6 ± 1.0 kg m−2 and accounted for about 3% of total W. The evolution ofleaf area index (LAI) was almost identical in all cultures till 123 DAP (datanot shown). Nevertheless, at the end of cultivation LAI was significantly lowerin HB cultures (1.70 ± 0.20, on average) compared to those irrigated withLB water (2.14 ± 0.18). At this time, in all cultures LAI was lower than at 123DAP because the detachment of basal leaves had continued after top-cutting.

The quality of irrigation water did not have any important influence onthe number of flowers and fruits brought by each truss and on the timing ofblooming and fruit ripening (data not shown). In all treatments, floweringtook place regularly between 30 and 125 DAP; the harvest started 78 DAP(mid-May) and continued for roughly three months. In contrast, the number

of both flowers and fruits, the weight of individual fruits and, hence, the yieldof each truss depended on its position on the stem with a clear tendency todecrease from the bottom to the top (Figure 6). Conversely, there was noclear effect of truss position on fruit set (i.e., the fruit/flower percent ratio),which ranged from 48% (11th truss) to 65% (6th truss; Figure 6).

The use of HB water did not influence significantly the total fruit yieldand average fruit weight, which however were reduced slightly but signifi-cantly by SW (Table 3). In all treatments, very few berries were not marketablebecause of small size, cracking, blossom-end rot or discoloration.

100Flowers Fruits Fruit-set (%)

Fruit set (%

1 2 3 4 5 6 7 8 9 10 11 120

Mean fruit weigth Truss yield

Truss number

) Truss yield (kg)

FIGURE 6 Number of flowers and fruits, fruit set (i.e. the percent ratio between the number of flowersand fruits), mean fruit weight and whole yield on each truss of greenhouse tomato plants grown insemi-closed substrate culture using different sources of irrigation water. No significant differences wereobserved between irrigation treatments and mean values (±SE) values of 12 replicates are shown.

TABLE 3 Fruit yield and quality in greenhouse tomato plants grown in semi-closed soilless culturesusing two B and NaCl concentrations in the irrigation water. See Table 1 for abbreviations

ANOVA1

FWLB FWHB SWLB SWHB NaCl B NaCl × B

Yield (kg m−2) 21.9 a 20.9 a 19.7 b 19.6 b ∗ ns nsFruit number (m−2) 117.3 a 117.5 a 116.2 a 115.6 a ns ns nsMean fruit fresh

weight (g)186.7 a 177.6 ab 169.9 b 169.6 b ∗ ns ns

pH 4.02 a 4.04 a 4.01 a 4.02 a ns ns nsDry residue (% FW) 5.54 b 5.79 b 6.11 a 5.61 b ∗ ns ∗Total soluble solids

(◦Brix)4.00 b 4.00 b 4.50 a 4.08 b ∗ ∗ ∗

Titratable acidity (%) 0.57 a 0.57 a 0.62 a 0.63 a ns ns nsB content (mg kg−1

DW)40.3 b 46.3 b 38.0 b 49.5 a ns ∗∗ ns

Mean (n = 3) values followed by the same letters were not significantly different (LSD test; P < 0.05).1NS, not significant; ∗, significant at 5% level; ∗∗, significant at 1% level.

The influence of fertigation regime on some quality attributes was inves-tigated in marketable fruits picked from the 4th or 5th truss. No differencesin any measured quantity were found between the two positions (data notshown) and, thus, ANOVA was conducted with pooled data. Water composi-tion did not influence fruit pH and titratable acidity, whereas the use of SWincreased both dry residue and the content of total soluble solids, but onlyin SWLB plants (Table 3).

Fruit B concentration was not affected by NaCl salinity and was slightly,but significantly, higher in the FWHB and SWHB plants (47.9 ± 2.0 mg kg−1,on average) compared to the others (39.2 ± 1.5 mg kg−1). One would haveto eat daily more than 3.6 kg of tomatoes grown with FWHB water or more ofthose grown with other sources of irrigation water, to exceed the maximumdaily B intake (10 mg) established for adult people by European Food SafetyAuthority (EFSA, 2004).

DISCUSSION

In our work, the main result of using HB water was the appearance ofleaf burn, which was less severe in the plants irrigated with SW and neveraffected the plants grown with LB water (Figure 5). Several authors (Ferreyraet al., 1997; Ben-Gal and Shani, 2002; Edelstein et al., 2005; Yermiyahu et al,2008; Smith et al., 2010) reported that NaCl salinity alleviated the effect ofexcess B by reducing W and the accumulation of B in shoot tissues. In ourwork, neither NaCl salinity nor B level influenced W and the protectionof SW against B toxicity was attributable to the different fertigation regimeimposed by the use of this water source. Due to the progressive accumulation

of NaCl (Table 1), the EC of NS tended to increase more rapidly in the SWcultures and, therefore, the NS was discharged more frequently than inthe FW cultures; this resulted in a lower B concentration in the root zone(Figure 2; Table 1), thus restricting its uptake and translocation to the leaves(Table 2). Plant B content was similar in FWHB and SWHB treatments(Figure 3), because B concentration differed only in leaf lamina (Table 2),which accounted for 28% (on average) of total crop biomass. Tomato rootscontained much less B than leaf tissues in agreement with Kaur et al. (2006).

In the FWHB and SWHB cultures, B accumulation in the re-circulatingNS was limited by its deposition in the substrate, probably due to the for-mation of insoluble Ca metaborate complex as suggested by the high Caconcentration in the NS (Tariq and Mott, 2007).

Boron is absorbed by plant roots as boric acid (H3BO3) and, under con-ditions of adequate or excessive availability, this element has been thoughtto be absorbed by the roots primarily through a passive process involving Bdiffusion across the lipid bilayer, to move into the plants via the transpiration-driven water flow and to accumulate in the shoot, especially in the leaves(Miwa and Fujiwara, 2010). However, there is also evidence that energy-dependent mechanisms may facilitate B uptake under deficiency conditionsor limit its accumulation in the shoot in the presence of high B concentrationin the growing medium (Miwa and Fujiwara, 2010). Based on the compari-son between actual B uptake and the one predicted from plant transpirationand the B concentration in the growing medium, Smith et al. (2010) con-cluded that, under excess B conditions, broccoli plants showed a mechanismthat restricted B uptake and accumulation in the shoot. In our experiment,B uptake concentration was significantly higher in tomato plants grown withHB water (on average, 39.1 mmol m−3; Figure 3) than in those grown withLB water (21.8 mmol m−3), but it was invariably much lower than B concen-tration in the re-circulating NS (Table 1). In fact, the ratio between B uptakeconcentration and its concentration in the NS (Table 1) was 0.237, 0.068,0.292 and 0.072 in FWLB, FWHB, SWLB and SWHB treatment, respectively.These findings suggest the presence of a mechanism limiting B uptake intomato plants, which appeared more efficient when the plants were irrigatedwith HB water.

Absorbed B accumulated principally in leaf lamina, while a much lowercontent was found in other organs(Table 2 and Figure 3) in agreementwith other findings (e.g., Ben-Gal and Shani, 2002, 2003; Yermiyahu et al.,2008; Nada et al., 2010). The extent to which tomato fruits are isolatedfrom the xylem stream (Davies et al., 2000) was the reason for the limited Baccumulation in these organs, which in all treatments remained well belowthe level representing a potential risk for human health, taking a realisticdaily consumption of tomato.

In B-injured leaves, the asymptomatic areas showed the same fluores-cence transient as in healthy leaves of FWLB and SWLB plants (data not

shown). Moreover, the lack of any significant differences among the treat-ments in W, which was accounted for about 97% by transpiration, suggestedthat neither NaCl salinity nor B concentration influenced stomatal con-ductance. Therefore, the photosynthesis of healthy leaf tissues was hardlyaffected by NaCl and B concentration in the irrigation water and it was con-cluded that B toxicity manifested basically by reducing leaf expansion. Bastıaset al. (2004) found that in maize leaf water relations, growth and chlorophyllcontent were influenced by salinity, but not by excess B. In contrast, Nadaet al. (2010) observed that leaf photosynthesis and stomatal conductancedecreased in tomato plants grown hydroponically with excessive B concen-tration (4 mg L−1 or higher) in the NS.

Notwithstanding leaf burn, excess B did not influence significantlybiomass production and fruit yield, while the latter was slightly decreasedby NaCl salinity in reason of a small reduction of fruit size (Table 3). Dryresidue and total soluble solids were increased by the use of SW; both quan-tities were lower in the SWHB plants than in the SWLB plants (Table 3). Thepositive effect of salinity on dry matter and sugar content of tomatoes is wellknown (Dorais et al., 2001). Recently, Nada et al. (2010) found that excess Bdecreased fruit yield and total soluble solids in hydroponically-grown tomatoand this result was ascribed to reduced availability of photoassimilates forfruit development.

Two reasons could explain why, in this study, the reduction of photosyn-thetically active leaf area in B-stressed plants did not have important effectson plant growth and fruit yield.

Firstly, the appearance of leaf burn was progressive and, at the end ofcultivation, the percent injured area was roughly 22% and 12% in FWHBand SWHB culture, respectively, (Figure 4). This reduction probably wasnot large enough to depress overall plant photosynthesis in reason of acompensation mechanism. Many crop plants were found to compensate forpartial defoliation, for instance due to damages from herbivores (Straussand Agrawal, 1999), through an increase in the photosynthetic rate of theremaining leaf area. For instance, removing 25% of leaf area did not in-fluence significantly fruit yield in tomato (Stacey, 1983) or whole-plant drymatter accumulation in cucumber (Ramirez et al., 1988).

Moreover, in our experiments tomato plants were exposed to stressfultemperature conditions, in particular in the second half of the season (Fig-ure 1), which may have equalized the plant response to B toxicity. TheFv/Fm ratio represents the PSII functional status and is considered a markerfor plant stress (Strasser et al., 2000); values below 0.80, as those observedaround noon in our work, are generally considered indicative of stressedplants (Maxwell and Johnson, 2000). Heat stress was likely to be responsi-ble also for the low fruit-set (about 60%) and the progressive reduction offlower numbers from the bottom to the upper trusses (Figure 6). Fruit-setin tomato is quite sensitive to high temperature (Picken, 1984), which is

considered one of the major limits to greenhouse tomato production insouthern regions (Peet and Welles, 2005). As found in crops exposed tocombined excess B and salinity or drought (e.g., Ben-Gal and Shani, 2002,2003; Yermiyahu et al., 2008; Bastıas et al., 2010; Smith et al., 2010), theeffects of the two stressors are not additive and, if one stress (heat stress, inthis case) is particularly severe, crop response to a second stress (B toxicity)will be a minor consequence. Under more favorable climate, without sourceand/or sink limitations of fruit production, and/or in case of longer cultiva-tions, such as those realized in other regions (e.g., Sicily in Italy; Andalusiain Spain; Westland in the Netherlands), the use of water rich in B mighthave a greater impact on crop performance.

CONCLUSIONS

In semi-closed culture of tomato conducted under the typical green-house conditions of Mediterranean regions, moderate NaCl salinity and Bconcentration in the irrigation water did not have important effects on cropuptake of water and minerals (apart from Na and B) and on growth andfruit yield, which appeared to be reduced by both source (reduced photo-synthesis) and sink (poor fruit set) limitations. The severity of B-inducedleaf burn was alleviated by the use of SW as a result of higher frequencyof NS discharge; this reduced the accumulation of B in the recycling waterand its accumulation in the shoot, which was dependent on both W andmean B concentration in the re-circulating NS. Apparently, B depositionin the growing medium contributed to reduce its accumulation in the re-circulating NS in the cultures irrigated with HB water. In all treatments, fruitB content remained well below the level representing a potential risk for theconsumers.

ACKNOWLEDGMENTS

This work was funded by the European Commission, Directorate Generalfor Research (7th Framework RTD Programme; Project EUPHOROS) and byMIUR-PRIN 2009 (Ministero dell’Istruzione, dell’Universita e della Ricerca,Italy, Project “Physiological response of vegetables crops to boron excess”).

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