Deficit irrigation and rootstock: their effects on water relations, vegetative development, yield, fruit quality and mineral nutrition of Clemenules mandarin

Summary Differences between rootstocks, ‘Cleopatra’mandarin and ‘Carrizo’ citrange, in soil–plant water relationsand the influence of these factors on vigor, crop yield, fruitquality and mineral nutrition were evaluated in field-grownClemenules mandarin trees irrigated at 100% of potential sea-sonal evaporation (ETc) (control treatment), or irrigated at100% ETc, except during Phases I and III of fruit growth andpost-harvest when no irrigation was applied (deficit irrigation(DI) treatment), for 3 years. Differences between rootstocks inplant–soil water relations were the primary cause of differ-ences among trees in vegetative development and fruit yield.After 3 years of DI treatment, trees on ‘Cleopatra’ showedmore efficient soil water extraction than trees on ‘Carrizo’, andmaintained a higher plant water status, a higher gas exchangerate during periods of water stress and achieved faster recoveryin gas exchange following irrigation after water stress. The DItreatment reduced vegetative development more in trees on‘Carrizo’ than in trees on ‘Cleopatra’. Cumulative fruit yielddecreased more in DI trees on ‘Carrizo’(40%) than on ‘Cleopa-tra’ (27%). The yield component most affected by DI in ‘Cleo-patra’was the number of fruit, whereas in ‘Carrizo’it dependedon the severity of water stress reached in each phase (severewater stress in Phase I affected mainly the number of fruit,whereas it affected fruit size the most in Phase III). In the thirdyear of DI treatment, water-use efficiency decreased sharply intrees on ‘Carrizo’ (70%) compared to trees on ‘Cleopatra’(30%). Thus, trees on ‘Cleopatra’ were able to tolerate moder-ate water stress, whereas trees on ‘Carrizo’were more sensitiveto changes in soil water content.

Keywords: citrus rootstocks, fruit yield, gas exchange, vegeta-tive growth, water status, water-use efficiency.

Introduction

Citrus trees are widely cultivated in southeastern Spain, wherethe climate is semi-arid with dry hot summers and a high evap-

orative demand. In this region, scarcity of water resources isthe major factor limiting expansion of irrigated agriculture, es-pecially for high-water-consuming orchards such as citrus. Acommon approach to optimizing water resources in these areasis to employ a deficit irrigation (DI) strategy, such as sustainedDI, which involves the same total reduction in water supplythroughout the year based on potential seasonal evapotrans-piration (ETc), or regulated DI, which involves moderate or se-vere reductions in water supply during part or parts of the sea-sonal cycle of plant development, coinciding with periods oflow water-stress sensitivity (Lampinen et al. 1995). Deficit ir-rigation strategies are employed in semi-arid regions to savewater and to improve water-use efficiency with several Medi-terranean crops including olives (Olea europaea L.) (Alegre etal. 1999) and almonds (Prunus dulcis Mill. D.A. Webb.) (Ro-mero et al. 2004, Girona et al. 2005). Previous studies of defi-cit irrigation on Clementine citrus trees have reported reduc-tions in plant water status and gas exchange activity, decreasesin vegetative growth and yield, and changes in fruit quality de-pending on the severity of the water stress and on the pheno-logical stage of vegetative and reproductive growth when DIwas applied (Ginestar and Castel 1996, González-Altozanoand Castel 1999, 2000).

Citrus rootstocks have well-known effects on tree size, cropload and fruit size and quality (Castle et al. 1993), making thechoice of rootstock for a citrus orchard an important consider-ation. Inherent differences among rootstocks that affect plant–water relations are associated with differential fruit develop-ment and sugar accumulation of citrus fruit (Albrigo 1977,Barry et al. 2004, Koshita and Takahara 2004) and are consid-ered a primary cause of differences in vigor, crop load and fruitquality among citrus rootstocks (Albrigo 1977, Sinclair andAllen 1982, Castle 1995, Syvertsen et al. 2000, Barry et al.2004). Genetically determined rootstock characteristics thataffect plant water relations include root distribution (Castleand Krezdorn 1975), water- and nutrient-uptake efficiencies(Castle and Krezdorn 1977), vessel element anatomy (Vascon-

Tree Physiology 26, 1537–1548© 2006 Heron Publishing—Victoria, Canada

Deficit irrigation and rootstock: their effects on water relations,vegetative development, yield, fruit quality and mineral nutrition ofClemenules mandarin

P. ROMERO,1 J. M. NAVARRO,1 J. PÉREZ-PÉREZ,1 F. GARCÍA-SÁNCHEZ,2

A. GÓMEZ-GÓMEZ,1 I. PORRAS,1 V. MARTINEZ2 and P. BOTÍA1,3

1 Department of Citriculture, IMIDA, 30150 La Alberca, Murcia, Spain2 Department of Plant Physiology and Nutrition, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain3 Corresponding author ([email protected])

Received November 3, 2005; accepted February 15, 2006; published online September 1, 2006

cellos and Castle 1994) and associated differences in root hy-draulic conductivity (Sinclair and Allen 1982, Syvertsen andGraham 1985). The rootstock’s ability to supply water to theplant and to increase nutrient uptake may be the primary influ-ence on fruit development in citrus trees (Albrigo 1992), deter-mining the vigor of the scion cultivar and its tolerance to waterstress (Syvertsen and Lloyd 1994, Medina and Machado1998). Thus, the characteristics of the rootstock are also an im-portant consideration when determining the response of citrustrees to DI strategies.

We studied two citrus rootstocks that are widely used in theMediterranean area, an invigorating rootstock (‘Carrizo’ cit-range) and a less invigorating rootstock (‘Cleopatra’ manda-rin). Our objective was to study inherent rootstock differencesaffecting plant water relations to determine the influence ofthese factors on vigor, crop yield, fruit quality and mineral nu-trition of field-grown Clemenules citrus trees after applying aDI strategy for three years. Specifically, we examined root wa-ter uptake, plant water status and gas exchange regulation, andtheir relationships with vegetative and fruit growth, fruit qual-ity and mineral nutrition. We also evaluated the suitability ofthese rootstocks for Clemenules mandarin trees in a semi-aridenvironment, where scarcity of water resources is the mainlimiting factor and DI strategies are employed.

Materials and methods

Plant material, experimental conditions and irrigationtreatments

The study was carried out from 2001 to 2003 in a 1-ha citrusorchard at the IMIDA experimental station in Torrepacheco(37°45′ N and 0°59′ E), Murcia, Spain. The soil is an aridisol,with 27.9% clay, 33.5% loam and 38.6% sand. The soil has anorganic matter content of 0.71% (dry soil), EC1–5 (electricconductivity) of 0.30 dS m–1, 17.50% active CaCO3 and apH 7.6. The weather is Mediterranean semi-arid, with a highmean daily solar radiation (> 200 W m– 2) and a high numberof daily solar hours (> 9 h), a mean annual air temperature ofaround 17 °C, low annual rainfall at the experimental site(283 mm), and a total annual reference evapotranspiration(ETo) of 1238 mm (Table 1), as calculated by the Penman-Monteith method (Doorenbos and Pruitt 1977, Allen et al.1998).

We studied 8-year-old Clemenules mandarin citrus trees(Citrus reticulata Blanco) grafted on either ‘Cleopatra’ man-darin (Citrus reshni Hort. ex Tanaka) or ‘Carrizo’ citrange(Citrus sinensis L., Osbeck × P. trifoliata L.) rootstocks. Theexperiment started in 2001 in trees with fully developed cano-pies and spaced 3 × 4 m apart. Two irrigation treatments wereapplied during three consecutive years: control and deficit irri-gation (DI). Trees in the control treatment were irrigated at100% ETc (crop evapotranspiration, ETc = (ETo)(Kc) over theyear; Table 1). Trees in the DI treatment were not irrigated dur-ing Phase I of fruit growth (initial fruit growth period, fromearly May to mid June) or Phase III of fruit growth (final fruitgrowth period, from early October to the end of November) or

during the post-harvest period (December–February), butreceived full irrigation (100% ETc) for the rest of the year(Figure 1).

The crop coefficients (Kc) applied were 1.0 in January andFebruary, and 0.7 from March to December, according toAmorós (1993), for citrus trees in the Mediterranean area. Pestcontrol practices and pruning were those commonly used bygrowers. Drip irrigation was applied daily, and was controlledand adjusted weekly according to soil matric potential (mea-sured by tensiometers located 25 cm from the drip head atdepths of 30, 60 and 90 cm), daily climatic data at the experi-mental site and neutron probe measurements. A drip line wasutilized in each tree row, with three self-compensating drip-pers (4 l h–1) per tree, 0.75 m apart. Irrigation was controlledautomatically by a head-unit programmer and electro-hydrau-lic valves. The amounts of water applied per treatment (Ta-ble 1) were measured with flow meters. The mean annualamount of fertilizers applied through the irrigation system dur-ing the experimental period was 260 kg ha–1 N, 103 kg ha–1

P2O5, 173 kg ha–1 K2O and 13.2 g Fe chelate per tree.The atmospheric parameters (mean air temperature, net so-

lar radiation, relative humidity, rainfall and wind velocity)were measured daily by a weather station located 2 km fromthe study site. Atmospheric vapor pressure deficit (VPD) wascalculated according to Allen et al. (1998).

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TREE PHYSIOLOGY VOLUME 26, 2006

Table 1. Annual applied water in the control (ETc) and deficit irriga-tion (DI; ETDI) treatments during the experimental period (2001–2003). Annual rainfall and reference evapotranspiration (ETo) at theIMIDA experimental station, Torrepacheco, Spain.

Water applied (mm)

Treatment Year Mean

2001 2002 2003

ETc (mm) Control 543 572 648 588ETDI DI 399 369 412 393% Reduction 27 35 36 33Rainfall (mm) 247 303 300 283ETo (mm) 1206 1257 1250 1238

Figure 1. Periods of irrigation cut-off during Phases I and III of fruitgrowth in each year in the deficit irrigation treatments. Black arrowsindicate the post-harvest date each year. Abbreviation: ETc = cropevapotranspiration.

The experimental design comprised three completely ran-domized selected plots. Each treatment consisted of nine trees(three trees per treatment and plot). In each row, border treeswere excluded from the study to eliminate potential bordereffects.

Plant water relations

Volumetric soil water content (θv) was measured periodicallywith a neutron probe (Model 3332, Troxler, Research TrianglePark, NC), previously calibrated at the experimental site. Asingle aluminum access tube was installed to a depth of100 cm. Readings were taken close to three trees per treatment,25 cm from the drip head and oriented perpendicularly to thedrip-lines, at 20-cm depth intervals to a maximum depth of100 cm.

From March to December of 2002 and 2003, midday xylemwater potential (Ψx) of one mature, fully expanded leaf fromthe outer mid-canopy of each of six trees per treatment wasmeasured periodically. The leaves were enclosed in foil-cov-ered plastic envelopes at least 1 h before the midday measure-ment (McCutchan and Shackel 1992). This procedure gave anestimate of the water potential in the xylem of the shoot, at thepoint of attachment of the petiole (Garnier and Berger 1985).Midday xylem water potential was measured at noon (1200–1400 h) with a pressure chamber (Model 3000, Soil MoistureEquipment, Santa Barbara, CA) according to the Schölanderet al. (1965) technique, with the recommendations of Turner(1988). The water-stress integral (SΨ; MPa day) was calcu-lated from the midday xylem water potential data as (Myers1988):

S c nΨ Ψ= ∑( i, i +1i =0

i =i

– ) (1)

where Ψi,i+1 is the mean water potential for any interval i, i +1,c is the maximum water potential measured during each period(a different value was used each year), and n is the number ofdays in the interval.

Gas exchange measurements

Gas exchange was measured between 0900 and 1100 h weeklyfrom April to December in 2002 and 2003 on selected cleardays. Measurements were made on healthy, fully expandedmature leaves (one leaf on each of nine trees per treatment),exposed to the sun, from young branches in exterior mid-can-opy positions. Net CO2 assimilation rate (A), stomatal conduc-tance to water vapor (gs) and transpiration rate (E) weremeasured with a portable photosynthesis system (LI-6400,Li-Cor, Lincoln, NE) equipped with a broadleaf chamber(6.0 cm2). Leaf chamber temperature was maintained between28 and 32 °C, leaf to air VPD at 2.5 ± 0.5 kPa and the relativehumidity of the chamber at 30–40% during measurements.Molar air flow rate inside the leaf chamber was 300 µmolmol–1. All measurements were taken at a reference CO2 con-centration similar to ambient (370 µmol mol–1) and a saturat-ing photosynthetic photon flux of 1500 µmol m– 2 s–1 (Sinclair

and Allen 1982, Syvertsen 1984), by using a red/blue lightsource (6400-02B LED) attached to the leaf chamber. Gas ex-change parameters were calculated automatically by the inter-nal program of the LI-6400, based on the equations of vonCaemmerer and Farqhuar (1981).

Vegetative and fruit development measurements

Canopy volume (V) was calculated as (Turrel 1961):

V HD= 0 5238 2. (2)

where H is height and D is diameter of the tree canopy.Twenty-four fruit were selected per treatment (four fruits

per tree at four orientations and two trees per block); they weremarked at the initial fruit development stage in 2003 and fruitdiameter was measured weekly.

Relative growth rate (RGR) was calculated as (Tattini1995):

RGR =lnM M

t t2 1

2 1

–

–(3)

where M is the growth parameter measured and t is the timebetween measurements. Total RGR of the canopy volume wascalculated based on the total increment during the whole ex-perimental period (from 2001 to 2003).

Yield and fruit quality

Annually, in the last week of November during the experimen-tal period (2001–2003), individual tree yield was measured inthree trees per treatment and per replicate (nine trees per treat-ment). The number of fruit and the total fruit mass of each treewere measured. Water-use efficiency was calculated as the ra-tio of annual yield to water applied during the same period.

When the fruit reached commercial size, 15 fruit per treewere collected randomly from nine trees per treatment. Fruitmass and equatorial and longitudinal diameters were deter-mined. Fruit shape index was defined as the equatorial to lon-gitudinal length ratio. External color was determined at threepoints on the equatorial area with a tri-stimulus color differ-ence meter (Minolta CR 300, Konica-Minolta Sensing, Osaka,Japan). Each fruit was cut in the equatorial area and peel thick-ness (mm) was measured at three points. The fruit wassqueezed and filtered, and total soluble solids content (TSS)and titratable acidity (TA) measured. Maturity index (MI) wasexpressed as the soluble solids:acidity ratio. The TSS of thejuice was measured with a refractometer (at 25 °C) and TA wasdetermined by titration with 0.4 N NaOH and phenolphthaleinindicator (results are expressed as a percentage of citric acid inthe juice). All fruit fractions were separated, weighed and ex-pressed as juice, peel and pulp percentages.

Color index (C.I.) was calculated as:

C.I.=* 103a

Lb

×*

(4)

TREE PHYSIOLOGY ONLINE at http://heronpublishing.com

DEFICIT IRRIGATION AND ROOTSTOCK ON CLEMENULES MANDARIN 1539

where L indicates lightness and a* and b* are the chromaticitycoordinates.

The thickness index was calculated as (Cohen et al. 1968):

Thickness Index = (peel thickness (mm) 200)× ×

(fruit width (mm))–1(5)

Mineral analysis

Leaf samples were collected in November 2002 and 2003 formineral analysis. About 40 leaves from the spring flush werecollected from each of six trees per treatment. Leaves werewashed immediately, dried at 65 °C for one week and milled.Nitrogen concentration was determined in these samples bythe micro-Kjeldahl method. After HNO3:HClO4 (2:1) diges-tion, K, Mg, Ca, P, Fe, Mn, Zn and B were analyzed with an in-ductively coupled plasma optical emission spectrometer(Varian MPX Vista, Palo Alto, CA).

Statistical analysis

Data were subjected to analysis of variance (ANOVA) andmeans were separated by Duncan’s multiple range test. Rela-tionships between parameters were fitted to linear and nonlin-ear regressions with Sigma Plot 2000 (Systat, Richmond, CA).Schwartz’s Bayesion criterion index (SBC) was used to findthe best fit for the nonlinear regressions between parameters.

Results

Soil–plant water relations

Mean volumetric soil water content (θv) in the root zone

(0–60 cm) at the time of maximum water stress differed sig-nificantly between treatments for both rootstocks (Table 2). Atthe end of Phase I in both 2002 and 2003, DI-treated trees hadsignificantly reduced Ψx compared to control trees on bothrootstocks. In 2003, water stress during Phase I was more se-vere than in 2002, with mean Ψx values of –1.15 c, –2.59 a,–1.02 c and –1.99 b MPa, (different letters after mean valuesindicate significant differences at P < 0.05) for trees on ‘Car-rizo’ control, ‘Carrizo’ DI, ‘Cleopatra’ control and ‘Cleopatra’DI, respectively. Consequently, gas exchange was also moreaffected by water stress in 2003 than in 2002. In 2003, themaximum treatment differences in E, A and gs occurred at theend of Phase I with greater decreases in trees on ‘Carrizo’ thanin trees on ‘Cleopatra’ (Figure 2).

In Phase II, the recovery of θv beside trees on ‘Carrizo’ wascomplete after 6 days of re-irrigation (data not shown), with θv

values significantly higher in the DI treatment than in the con-trol treatment. In contrast, θv values beside DI-treated trees on‘Cleopatra’ were lower than values observed beside controltrees (Table 2), indicating that recovery of θv by trees on ‘Cle-opatra’ was not complete after 6 days of re-irrigation. On theother hand, recovery of Ψx was rapid (–1 MPa by about 7 days)in trees on both rootstocks in 2002 and 2003 (data not shown),with no significant differences in Ψx values between treat-ments or rootstocks.

In Phase II, A recovered more slowly than E and gs in treeson ‘Carrizo’ (Figures 2a, 2c and 2e), whereas in trees on ‘Cleo-patra’, A and gs recovered faster than E (Figures 2b, 2d and 2f).Gas exchange parameters recovered more slowly in trees on‘Carrizo’ than in trees on ‘Cleopatra’, which needed only 8, 15and 22 days for recovery of A, gs and E, respectively (Fig-ure 2). At 22 days after re-irrigation, DI trees on ‘Carrizo’ still

1540 ROMERO ET AL.


Table 2. Values of volumetric soil water content (θv) in the zone of highest root density (0–60 cm) for each treatment (control and deficit irrigation(DI)) and rootstock at four periods in 2002 and in 2003. For Phase I, the values presented correspond to the day of maximum water stress. For theother Phases (II, III and post-harvest), the values presented correspond to means for each period. Significance levels: ns = not significant; * = P <0.05; ** = P < 0.01; and *** = P < 0.001. Separation by Duncan’s multiple range test at the 95% confidence level. For each column, different lettersindicate significant differences at P < 0.05.

Rootstock Treatment Volumetric soil water content (%)

Phase I Phase II Phase III Post-harvest(June) (July–Sept) (Oct–Nov) (Dec–Feb)

Water-stress period Recovery period Water-stress period Water-stress period

2002 2003 2002 2003 2002 2003 2002 2003(June 12) (June 25) (pre-harvest) (post-harvest)

‘Carrizo’ Control 21.95 d 24.33 d 18.97 c 19.49 bc 20.92 17.09 ab 22.67 22.27 cDI 18.14 b 15.80 b 23.81 d 23.74 d 16.13 21.50 d 24.48 22.49 c

‘Cleopatra’ Control 19.48 c 20.39 c 16.93 b 18.62 ab 18.11 18.55 b 20.54 18.21 bDI 12.20 a 10.70 a 15.07 a 16.39 a 10.73 14.46 a 20.33 14.89 a

Analysis of varianceRootstock *** *** *** *** *** ** * ***Treatment *** *** * ns *** ns ns ***Rootstock × Treatment ** * *** *** ns *** ns ***

showed significant differences in gas exchange parameters(Figure 2). In 2002, gas exchange parameters in trees on‘Carrizo’ recovered more slowly than in trees on ‘Cleopatra’(data not shown).

During the pre-harvest period of Phase III of 2002 (wa-ter-stress period), θv values were significantly lower in DIplots than in control plots, and this decrease was greater forsoil beside trees on ‘Cleopatra’ than for soil beside trees on‘Carrizo’ (Table 2). The DI trees on ‘Cleopatra’ maintained ahigher plant water status than DI trees on ‘Carrizo’ (Ψx = –2and –3 MPa, respectively). Moreover, gs was significantlylower in DI trees on ‘Carrizo’ than in DI trees on ‘Cleopatra’(0.029 and 0.050 mol H2O m– 2 s–1, respectively). Both A and Ewere reduced by water stress, with no significant differencesbetween rootstocks (data not shown). During Phase III of2003, θv values were highest beside DI trees on ‘Carrizo’ (Ta-ble 2), which corresponded to a less negative value of Ψx andsignificantly higher E and gs values than in DI trees on ‘Cleo-patra’at the start of this phase (Figures 2a and 2e). However, as

water stress progressed, significantly lower A, E and gs valueswere observed in DI trees on ‘Carrizo’, with no significantchanges in A, E and gs in DI trees on ‘Cleopatra’ (Figure 2).

The θv values beside DI trees on ‘Carrizo’ recovered com-pletely in the post-harvest period of 2003, but they did notrecover beside the DI trees on ‘Cleopatra’ (Table 2). Neverthe-less, Ψx of DI trees on ‘Carrizo’ was significantly lower thancontrol trees on ‘Carrizo’ (–0.56 MPa and –0.70 MPa, respec-tively). On the other hand, during this period, all Ψx valueswere above –1 MPa (equivalent to well-irrigated values for cit-rus trees) mainly because of low evaporative demand and fre-quent rainfall. The relationship between θv and Ψx differedbetween rootstocks with distinct threshold values of soil water,below which Ψx decreased greatly in response to smallchanges in soil water content (Figure 3).

The rootstocks had different patterns of instantaneous andintrinsic gas-exchange efficiency (A/E and A/gs, respectively)(Figure 4). As water stress developed (gradual decrease in Ψx),gas-exchange efficiency increased significantly in trees on



Figure 2. Seasonal patterns of (a,b) transpiration rate (E); (c, d) CO2 as-similation rate (A); and (e, f ) stomatalconductance (gs) during the 2003growing season for each treatment(� = control; and � = deficit irrigation(DI)) and rootstock (‘Carrizo’ and‘Cleopatra’). Each value is the mean ofnine measurements. Vertical bars indi-cate the standard error of the mean.Long dashed lines indicate the start(May 5), the end of the stress period inPhase I (June 25) and the start of thewater stress in Phase III (October 1) in2003.

‘Carrizo’and remained relatively constant in trees on ‘Cleopa-tra’ (Figures 4a and 4b).

Vegetative and fruit development

During 2002–2003, DI reduced canopy volume relativegrowth rate of ‘Carrizo’ significantly more than that of ‘Cleo-patra’, with reductions of 33 and 18%, respectively (data notshown). There was a negative linear correlation between SΨ

for the 2002–2003 period and canopy volume RGR for thesame period (Figure 5). The DI treatment significantly re-duced fruit growth in Phase I and increased it in Phases II andIII for both rootstocks (Figure 6).

Yield and water-use efficiency

Trees on ‘Carrizo’had higher yield in 2002 than in 2001 due toan increase in fruit number, but not mass (Table 3). Controltrees on ‘Cleopatra’ and ‘Carrizo’ had similar fruit yield in thethird year. The DI treatment significantly decreased fruit pro-duction of trees on ‘Carrizo’ in 2002 (34% reduction in fruitmass) and 2003 (85% reduction in fruit number), whereas DIreduced yield of trees on ‘Cleopatra’ in 2003, only through adecrease in fruit number (54% reduction). In 2003, DI reducedyield of trees on both rootstocks, but the decrease was greaterfor trees on ‘Carrizo’ (81%) than for trees on ‘Cleopatra’(56%) (Table 3). There was a linear correlation between thenumber of fruit in 2003 and SΨ of Phase I in 2003 (Figure 7a),and between mean fruit mass in 2002 and SΨ of Phase III (Fig-ure 7b). The DI treatment significantly increased water-use ef-ficiency (WUE) in 2001 (49% for ‘Carrizo’ trees and 37% for‘Cleopatra’ trees), and the corresponding increases in 2001were 6 and 21%. In 2003, DI reduced WUE more for trees on‘Carrizo’ (70%) than for trees on ‘Cleopatra’ (30%). Over3 years of the study, mean WUE did not differ significantly be-tween treatments or rootstocks (data not shown).

Fruit quality

In 2002, fruit from DI trees had higher juice percentage, totalsoluble solids and titratable acidity, but a lower pulp percent-age, MI and external C.I. than fruit from control trees (Ta-ble 4). There were no treatment differences in fruit qualitybetween rootstocks in 2002. In 2003, the DI treatments in-creased fruit shape index (data not shown) and decreased ex-ternal C.I. in fruit from trees on ‘Carrizo’ (Table 4). Fruit fromDI trees on ‘Cleopatra’ had significantly decreased juice per-centage and thickness index, and increased peel percentage. In2003, fruit from DI trees on ‘Carrizo’ had significantly de-creased TSS, whereas TSS was increased in fruit from DI treeson ‘Cleopatra’.

Mineral nutrition

Trees on ‘Cleopatra’ had significantly higher leaf Ca, Mg andB concentrations than trees on ‘Carrizo’ (Table 5). In general,DI did not affect leaf mineral nutrition, although DI signifi-cantly decreased leaf N and Mg concentrations in 2002, andsignificantly increased leaf Mn concentration in 2003.

1542 ROMERO ET AL.


Figure 3. Relationship between soil water content (θv) and middayxylem water potential (Ψx) for trees on ‘Carrizo’ (y = –1363.38 (1 +x)–2.30, r = 0.71***) and trees on ‘Cleopatra’ (y = –54.36x–1.41, r =0.84***) rootstocks. Each θv value is a single measurement taken25 cm from the drip head in the root zone (0–60 cm), and midday xy-lem water potential was measured in the same trees for the years 2002and 2003. Abbreviation: DI = deficit irrigation.

Figure 4. (a) Relationship between instantaneous gas-exchange effi-ciency (A/E) and midday xylem water potential (Ψx) for trees on‘Carrizo’ (y = –4.68 – 7.37I, r = 0.81***) and ‘Cleopatra’ (y = 3.83 +0.23x, r = 0.56**) rootstocks. (b) Relationship between intrinsic gas-exchange efficiency (A/gs) and Ψx for trees on ‘Carrizo’ (y = –203.09– 295.08x, r = 0.8***) and ‘Cleopatra’ (y = 119.96 – 0.14x, r = 0.50*)rootstocks. Each value is a single measurement per tree for Ψx, andthe mean of two measurements for A/E and A/gs for the same trees.Measurements were made from April to June 2003.

Discussion

Root water uptake, plant water status and gas exchange

In 2002 and 2003, the ‘Cleopatra’ DI treatment decreased θv

significantly more than the ‘Carrizo’ DI treatment (Table 2),thus DI trees on ‘Cleopatra’ maintained a better tree water sta-tus during the water-stress periods (Ψx > –2 MPa) than DItrees on ‘Carrizo’ (Ψx < –2 MPa), indicating differences be-tween rootstocks in patterns of soil water extraction and rootuptake efficiency. The relationship between θv and Ψx (Fig-ure 3) indicated different threshold values of θv for eachrootstock, below which Ψx decreased rapidly as a result ofsmall changes in θv, inducing water stress (e.g., to reach Ψx <–1.5 MPa trees on ‘Cleopatra’needed θv < 12%, whereas treeson ‘Carrizo’ needed θv < 17%). These differences in plant wa-

ter status indicates that trees on ‘Cleopatra’ were more effi-cient in soil water extraction and exploited more of the avail-able soil water resources, maintaining a higher water status



Figure 5. Relationship between canopy volume relative growth rateand cumulative water-stress integral (SΨ) for the period 2002– 2003(y = 1.22 – 0.0021x, r = 0.74, P < 0.0001). Each value is a single mea-surement per tree for each treatment (control and deficit irrigation(DI)).

Figure 6. Mean values of fruit diameter relative growth rate (RGR) foreach treatment (control and deficit irrigation (DI)) in ‘Carrizo’ and‘Cleopatra’ rootstocks in 2003.Vertical bars represent the standard er-ror of the mean (n = 24).

Table 3. Influence of rootstock and amount of applied water on Clemenules mandarin fruit yield parameters during the experimental period(2001–2003). Significance levels: ns = not significant; * = P < 0.05; ** = P < 0.01; and *** = P < 0.001. Separation by Duncan’s multiple range testat the 95% confidence level. For each column, different letters indicate significant differences at P < 0.05. Abbreviation: DI = deficit irrigation.

Yield (kg tree–1) Fruit number Fruit mass (g)

2001 2002 2003 2001 2002 2003 2001 2002 2003

Treatment Control 63.9 75.1 77.8 591 674 708 108.8 111.6 111.3DI 64.6 53.3 29.4 593 621 210 110.6 87.9 136.2

Rootstock ‘Carrizo’ 69.1 68.1 45.6 649 731 415 107.7 92.5 128.3‘Cleopatra’ 59.4 60.2 57.1 536 564 502 111.7 107.0 119.3

Rootstock Treatment‘Carrizo’ Control 67.6 85.0 b 76.5 c 620 766 725 109.6 111.4 b 107.1 a

DI 70.6 51.2 a 14.7 a 677 695 105 105.7 73.5 a 149.4 b‘Cleopatra’ Control 60.2 65.1 a 79.1 c 562 582 690 107.9 111.7 b 115.5 a

DI 58.5 55.3 a 35.1 b 509 546 314 115.4 102.3 b 123.0 a

Analysis of varianceTreatment ns *** *** ns ns *** ns *** ***Rootstock * ns ns * * ns ns ** nsRootstock × Treatment ns * * ns ns ns ns *** *

than trees on ‘Carrizo’. This implies that the root system of‘Cleopatra’ is more effective at water uptake mainly becauseof a higher root density or a deeper and more densely branchedroot system compared to ‘Carrizo’(Davies and Albrigo 1994).

Tree size may also influence the rate of water extraction intrees on different rootstocks (Castle and Krezdorn 1977).Trees on ‘Carrizo’ experienced more severe water stress and agreater reduction in tree size in response to the DI treatmentthan did trees on ‘Cleopatra’. As a consequence, water uptakeby trees on ‘Carrizo’, following the relief of water stress, wasless than that of trees on ‘Cleopatra’. This could explain thesignificantly higher θv values observed beside DI trees on‘Carrizo’compared with control trees after the water-stress pe-riods.

Despite differences in the severity of water stress experi-enced by trees on the two rootstocks, plant water status recov-ered quickly when stressed trees were re-watered, and a week

1544 ROMERO ET AL.


Figure 7. (a) Relationship between the water-stress integral (SΨ) dur-ing Phase I of fruit growth (early May–mid-June) and the number offruit per tree at harvest for Clemenules mandarin in 2003 (y =922.15 – 15.15x, r = 0.85, P < 0.0001). (b) Relationship between thewater-stress integral during Phase III of fruit growth (October–mid-November) and the single fruit mass at harvest for Clemenules man-darin in 2002 (y = 127.4 – 0.51x, r = 0.77, P < 0.0001). Each value is asingle measurement per tree for each treatment (control and deficit ir-rigation (DI)) (n = 6).

Tabl

e4.

Cle

men

ules

man

dari

nfr

uitc

hara

cter

istic

sin

rela

tion

toth

ero

otst

ock,

‘Cle

opat

ra’o

r‘C

arri

zo’,

and

the

amou

ntof

appl

ied

wat

er(c

ontr

olan

dde

fici

tirr

igat

ion

(DI)

)in

2002

and

2003

.Jui

ce,p

ulp

and

peel

cont

ents

are

expr

esse

din

perc

entw

eigh

twith

rela

tive

toth

ew

hole

frui

tmas

s.T

SS(°

Bri

x),T

Aan

dM

Iare

the

cont

ento

ftot

also

lubl

eso

lids,

the

titra

tabl

eac

idity

,and

the

TSS

*10

/TA

ratio

inth

efr

uitj

uice

(mat

urity

inde

x),r

espe

ctiv

ely.

Indi

ces

anal

yzed

wer

eth

ickn

ess

(Th.

I.)a

ndex

tern

alco

lor(

Ex.

I.C

.).S

igni

fica

nce

leve

ls:n

s=

nots

igni

fica

nt;*

=P

<0.

05;**

=P

<0.

01;a

nd**

*=

P<

0.00

1.Se

para

tion

byD

unca

n’s

mul

tiple

rang

ete

stat

the

95%

conf

iden

cele

vel.

For

each

colu

mn,

diff

eren

tlet

ters

indi

cate

sign

ific

antd

iffe

renc

esat

P<

0.05

.

2002

2003

Juic

e(%

)Pu

lp(%

)Pe

el(%

)T

SSTA

MI

Th.

I.E

x.I.

C.

Juic

e(%

)Pu

lp(%

)Pe

el(%

)T

SSTA

MI

Th.

I.E

x.I.

C.

Tre

atm

ent

Con

trol

47.0

7.7

45.4

12.8

7.8

16.5

8.4

6.2

42.3

4.3

49.3

12.5

8.9

14.2

9.1

12.3

DI

48.7

6.1

45.3

13.9

9.7

14.5

8.8

4.0

42.1

4.5

49.9

12.2

8.9

13.9

8.7

8.7

Roo

tsto

ck‘C

arri

zo’

48.7

6.4

45.0

13.5

9.0

15.2

8.3

5.2

43.2

3.6

49.7

12.1

8.5

14.2

8.7

10.6

‘Cle

opat

ra’

47.0

7.5

45.6

13.2

8.5

15.8

8.9

5.1

41.2

5.3

49.5

12.6

9.2

13.9

9.1

10.5

Roo

tsto

ckTr

eatm

ent

‘Car

rizo

’C

ontr

ol47

.67.

045

.412

.98.

115

.98.

06.

242

.3b

3.3

50.5

b12

.6bc

8.7

14.5

8.6

a13

.1c

DI

49.7

5.7

44.6

14.0

9.8

14.4

8.6

4.1

44.1

b3.

848

.9ab

11.5

a8.

313

.98.

7a

8.0

a‘C

leop

atra

’C

ontr

ol46

.38.

445

.312

.67.

417

.08.

86.

242

.3b

5.3

48.1

a12

.3b

9.0

13.9

9.6

b11

.5b

DI

47.6

6.5

45.9

13.8

9.5

14.6

9.0

3.9

40.0

a5.

250

.8b

12.9

c9.

413

.88.

6a

9.4

ab

Ana

lysi

sof

vari

ance

Tre

atm

ent

***

ns**

***

***

*ns

**ns

nsns

nsns

ns*

***

Roo

tsto

ck*

*ns

nsns

nsns

ns**

*ns

***

nsns

nsns

Roo

tsto

ck×

Tre

atm

ent

nsns

nsns

nsns

nsns

**ns

****

*ns

ns*

*

after irrigation, there were no differences in Ψx between treat-ments or rootstocks. Fereres et al. (1979) also observed a rapidrecovery of Ψx in orange trees after irrigation. The quick re-covery of Ψx from a minimum of below –3 MPa in DI trees on‘Carrizo’could be partly a result of the high hydraulic conduc-tivity of the ‘Carrizo’ root system (Syvertsen and Graham1985).

Compared with trees on ‘Carrizo’, trees on ‘Cleopatra’ sup-plied more water to leaves, and this was reflected in higher gasexchange rates under water-stressed conditions and faster re-covery of gas exchange after re-watering. Trees on ‘Carrizo’recovered more slowly (one month in Phase II) than trees on‘Cleopatra’ (one week). The difference in recovery time mayreflect the severity of water stress reached in Phase I (Ψx =–3 MPa in ‘Carrizo’ trees and –2 MPa in ‘Cleopatra’ trees) be-cause Ψx was significantly related to A, E and gs (data notshown), as has been observed in other field-grown tree species(Romero et al. 2004). Fereres et al. (1979) found that, after irri-

gation, orange trees with Ψx < –5 MPa did not recover gs fortwo months.

The rootstocks showed different patterns of leaf-level intrin-sic and instantaneous gas-exchange efficiency (A/gs and A/E,respectively) (Figure 4). As water stress developed (gradualdecrease in Ψx), gas-exchange efficiency increased in trees on‘Carrizo’, but remained relatively constant in trees on ‘Cleopa-tra’ (Figures 4a and 4b). Trees on ‘Carrizo’ (with lower leafwater status) had a stronger stomatal regulation of gas ex-change (lower gs and E) than trees on ‘Cleopatra’, therebyavoiding high water losses by transpiration and increasingA/gs. Stomatal limitation also limited A more in DI trees on‘Carrizo’ than on ‘Cleopatra’, when compared to their respec-tive control values. When DI was applied, E and A decreasedto a similar extent in trees on ‘Cleopatra’ (56%) but not in treeson ‘Carrizo’ (84% and 73%, respectively). Thus, trees on ‘Cle-opatra’ appear to have the ability to tolerate moderate waterstress, whereas trees on ‘Carrizo’citrange are more sensitive to



Table 5. Leaf nutrient concentrations of Clemenules mandarin in relation to the rootstock, ‘Cleopatra’ or ‘Carrizo’, and the amount of water ap-plied (control and deficit irrigation (DI)) in 2002 and 2003. Data are expressed as mmol kg DM

–1. Significance levels are shown for each year sepa-rately: ns = not significant; * = P < 0.05; ** = P < 0.01; and *** = P < 0.001. Separation by Duncan’s multiple range test at the 95% confidence level.

N P K Ca Mg Fe Mn Zn B

2002

Rootstock ‘Carrizo’ 1318 30.8 160 952 116 1.95 0.40 0.34 13.3‘Cleopatra’ 1377 30.3 211 1154 131 1.83 0.38 0.29 18.8

Treatment Control 1382 31.7 200 1038 129 2.03 0.41 0.33 16.4DI 1309 29.5 172 1068 117 1.75 0.37 0.30 15.7

Rootstock TreatmentCarrizo Control 1367 32.6 188 964 119 2.17 0.42 0.33 13.3

DI 1261 29.0 133 939 113 1.73 0.37 0.35 13.3Cleopatra Control 1396 30.7 211 1113 140 1.89 0.40 0.32 19.6

DI 1357 30.0 210 1196 121 1.77 0.36 0.25 18.1

Analysis of varianceRootstock * ns *** *** *** ns ns * ***Treatment * ns ns ns *** ns ns ns nsRootstock × treatment ns ns ns ns ns ns ns ns ns

2003

Rootstock ‘Carrizo’ 1405 40.3 241 983 117 1.25 0.55 0.37 16.7‘Cleopatra’ 1369 32.2 231 1202 146 1.67 1.09 0.49 21.8

Treatment Control 1376 35.3 229 1067 130 1.46 0.58 0.46 18.8DI 1398 37.2 243 1118 133 1.47 1.06 0.41 19.7

Rootstock TreatmentCarrizo Control 1396 38.5 227 959 116 1.31 0.42 0.38 16.0

DI 1414 42.2 256 1007 119 1.20 0.68 0.36 17.3Cleopatra Control 1355 32.1 232 1174 145 1.62 0.74 0.54 21.6

DI 1382 32.2 230 1230 147 1.73 1.45 0.45 22.0

Analysis of varianceTreatment ns ns ns ns ns ns * ns nsRootstock * *** ns *** *** * * * **Rootstock × treatment ns ns ns ns ns ns ns ns ns

changes in soil water content.

Vegetative and fruit growth

Compared with trees on ‘Cleopatra’, a reduction in totalphotosynthetic capacity, as a result of the higher water stress,in trees on ‘Carrizo’could be responsible for the greater reduc-tion in vegetative growth. The negative linear correlation be-tween SΨ and canopy volume RGR (Figure 5) suggests that thehigher SΨ effect in trees on ‘Carrizo’ induced a lower can-opy-volume RGR than in trees on ‘Cleopatra’. Vegetativegrowth in citrus is highly sensitive to drought and can be re-duced by drought more than fruit growth (Hilgeman and Sharp1970, Levy et al. 1978).

The reduction in fruit growth in Phase I in response to DI(Figure 6) may be associated with translocation of water fromthe fruit to the transpiring leaves, leading to turgor loss in thefruit (Huang et al. 2000). The compensatory growth of fruit inPhase II may reflect a more negative water potential in thefruit, creating a greater water uptake force (Huang et al. 2000).Citrus fruit on trees that have been water stressed can growfaster after re-watering than fruit on regularly watered trees(Goell et al. 1981, Huang et al. 1986, Cohen and Goell 1988).

Yield and water-use efficiency

Differences in yield between 2002 and 2003 occurred in re-sponse to differences in the severity of water stress reached inthe different fruit growth phases during the two years. In treeson ‘Carrizo’, the more severe water stress reached in Phase I in2003 (–2.6 MPa) than in 2002 (–1.5 MPa) and the cumulativeeffect of severe water stress in Phase III of 2002 could havenegatively affected flower bud formation and fruit develop-ments, and induced fruit abscission (Phase I), producing alower fruit number in 2003. The significant linear relationshipbetween the water-stress integral (SΨ) during Phase I of fruitgrowth and fruit load (Figure 7a) indicates that, the higher theaccumulated water stress during Phase I, the greater the reduc-tion in fruit load at harvest. In citrus, water stress applied dur-ing flowering, fruit set and Phase I of fruit growth reduces thenumber of fruit because of increased fruit drop (Kriedemannand Barrs 1981, Doorenbos and Kassam 1986, Ginestar andCastel 1996). The percentage fruit fall has also been correlatedwith the severity of water stress (Barbera and Carimi 1988,Gónzalez-Altozano 1998). However, a greater water stress inPhase III in 2002 in ‘Carrizo’(–3.00 MPa) than in 2003 (–0.97MPa) mainly reduced fruit mass (Table 3; Figure 7b). A simi-lar yield response to DI-induced drought has been observedpreviously in Clemenules mandarin trees on ‘Carrizo’rootstock (Ginestar and Castel 1996, González-Altozano andCastel 1999). Although, in DI trees on ‘Cleopatra’, the waterstress reached in Phase I in 2003 (–2.0 MPa) also reduced fruitnumber and, consequently, total yield; this yield reduction wassignificantly lower than for DI trees on ‘Carrizo’ because ofthe lower water stress reached by the latter. Similarly, thelower water stress in Phase III in 2002 (–2.1 MPa) in trees on‘Cleopatra’ did not reduce the fruit mean mass. The cumula-tive effect of water stress on fruit yield during the 3-year study

progressively reduced WUE in trees on both rootstocks, andthe reduction was greater in trees on ‘Carrizo’ than in trees on‘Cleopatra’, as indicated by the greater reduction in fruit yield(Table 3).

Fruit quality

Fruit quality differences occurred between years as a result ofthe different effects of water stress on fruit development be-tween years. Regardless of the rootstock, the severe waterstress in Phase III of 2002 affected some fruit ripening pro-cesses, increasing juice percentage, TSS and TA and delayingfruit ripening (shown by a decrease in the MI and external C.I.)(Table 4). However, when water stress in Phase III was slight(in 2003), increases in TSS and TA were not achieved. Thus,the timing and severity of water deficit have important effectson juice quality in citrus (cf. Barry et al. 2004).

An increase of TSS in response to water deficit has been ob-served previously in citrus trees (Levy et al. 1979, Bielorai etal. 1985, Chartzoulakis et al. 1999, Moon et al. 2004). A re-duction in irrigation during Phase III is reported to increaseTSS and TA in citrus (Sánchez-Blanco et al. 1989, Shalhevetand Levy 1990, Gónzalez-Altozano and Castel 2003), but withno effect on the MI. The increase in TSS in response to regu-lated DI could be due to a dehydration effect (González-Altozano 1998) or to active osmotic adjustment in fruit(Huang et al. 2000, Barry et al. 2004). In our study, the in-creases in TSS and TA in fruit resulting from the DI treatmentsin 2002 were not associated with a reduction in juice volume,because the DI treatment significantly increased juice percent-age (Table 5). The increases may be the result of increasedsynthesis of organic solutes, rather than a concentration effectin water-stressed trees, implicating a possible osmotic adjust-ment. In 2003, the slight decrease in TSS of fruit from DI treeson ‘Carrizo’ was probably a dilution effect (slight increase injuice volume), and fruit from DI trees on ‘Cleopatra’ had in-creased TSS perhaps because of a concentration effect (lowerjuice volume) (Table 4).

Mineral nutrition

As has been reported previously (Wutscher and Shull 1976,Georgiou 2002), rootstock influenced the nutritional status ofClemenules trees (Table 5), likely reflecting differences in thecapacities of the rootstocks for water and mineral uptake andin the physical characteristics of the root systems (Castle andKrezdorn 1975). The low N values corroborated findings forvarious mandarin-type scions (Smith 1975, Wutscher andShull 1976, Georgiou 2000). A severe water stress in Phase III(2002) reduced foliar N concentrations in DI trees, as reportedpreviously (Castel and Buj 1990, Peng and Rabe 1998); how-ever, foliar N concentrations completely recovered in 2003(Table 5). Mean fruit mass and number of set fruit increasewith increasing leaf N concentrations in the low range (Legazet al. 1995). We found a positive correlation (P < 0.01) be-tween N concentration and mean fruit mass in 2002, and nocorrelation with fruit yield or growth parameters in 2003.Magnesium is important for fruit growth because it determines

1546 ROMERO ET AL.


total yield and the fruit mass (Maksoud et al. 1994), so thelower mean fruit mass and fruit yield on DI trees in 2002 (Ta-ble 3) could have been caused by the low leaf Mg concentra-tions because these parameters were correlated positively (P <0.05) (data not shown).

Based on our data and the cited studies, we conclude thatthe rootstock differences affecting plant–soil water relationswere the primary cause of the differences found in vegetativedevelopment and fruit yield after application of a 3-year deficitirrigation treatment in Clemenules mandarin trees. Clemen-ules trees on ‘Cleopatra’ were more efficient in soil water ex-traction than trees on ‘Carrizo’ under low soil water condi-tions, and these trees maintained a better water status and morerapid gas exchange during water stress as well as a faster re-covery in gas exchange after periods of water stress. The lowerdegree of water stress reached in trees on ‘Cleopatra’ stimu-lated greater vegetative growth and a higher fruit yield com-pared with trees on ‘Carrizo’, which showed a greater cumula-tive water stress effect. Fruit quality was little affected byrootstock, although it was improved by severe water stress inPhase III. Leaf mineral nutrition was affected by bothrootstock and irrigation treatment; DI reduced leaf N and Mgconcentrations. We conclude that ‘Cleopatra’ mandarin root-stock has a higher tolerance to water stress than ‘Carrizo’citrange, and is a better rootstock for field-grown Clemenulesmandarin when long-term regulated DI in Phase I and PhaseIII of fruit growth is employed under semi-arid conditions.

Acknowledgments

The authors thank I.M. García-Oller, M. Sánchez-Baños, and A.J.Tristán for help in both field and lab work. We also thank Dr. DavidWalker for correction of the English. This work was supported by theComisión Interministerial de Ciencia y Tecnología, through projectsAGL2000-2015-C03-03 and AGL2003-08502-C04-04.

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Deficit irrigation and rootstock: their effects on water relations, vegetative development, yield, fruit quality and mineral nutrition of Clemenules mandarin