response of five citrus rootstocks to iron deficiency

2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.plant-soil.com

J. Plant Nutr. Soil Sci. 2011, 174, 837–846 DOI: 10.1002/jpln.201000341 837

Response of five citrus rootstocks to iron deficiencyMaribela Pestana1*, Pedro José Correia1, Manuela David1, Anunciación Abadía2, Javier Abadía2,and Amarilis de Varennes3

1 ICAAM, University of Algarve, FCT-DCBB, Ed. 8, Campus Gambelas, 8005–137 Faro, Portugal2 Department of Plant Nutrition, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas (CSIC), 50080 Zaragoza,

Spain3 Biosystems Engineering Center, Technical University of Lisbon (TULisbon), Tapada da Ajuda, 1349-017 Lisbon, Portugal

AbstractCitrus established in calcareous soils can be affected by iron (Fe)-deficiency chlorosis whichlimits yield and the farmers’ income. The degree of deficiency depends on the rootstock, but theresistance to Fe chlorosis still requires further investigation. To study physiological parametersof citrus rootstocks that could be used to evaluate resistance to Fe deficiency, plants of Troyercitrange (Citrus sinensis L. Osb. × Poncitrus trifoliata L. Raf.), Carrizo citrange, Volkamer lemon(Citrus volkameriana Ten. & Pasq.), alemow (Citrus macrophylla Wester), and sour orange(Citrus aurantium L.) were grown in nutrient solutions with 0, 5, 10, 15, or 20 lM Fe. For eachrootstock, plant height, root and shoot dry weights, and concentration of Fe in the shoots androots were measured at the end of the experiment. Chlorophyll (CHL) concentration was esti-mated throughout the experimental period using a portable CHL meter (SPAD-502) calibratedfor each rootstock. At the end of the experiment, CHL fluorescence parameters were measuredin each rootstock with a portable fluorimeter. Maximal and variable fluorescence values indi-cated that the photochemistry of Troyer was more affected by a low concentration of Fe in thenutrient solution than that of other rootstocks. To compare rootstocks, the absolute CHL concen-tration was converted into relative yield by employing a scaling divisor based on the maximumvalue of total CHL in plants without Fe-deficiency symptoms. Exponential models were devel-oped to determine the minimum Fe concentration in nutrient solution required to maintain leafCHL at 50% of the maximum CHL concentration (IC50). Models were also developed to assessthe period of time the rootstocks were able to grow under Fe-stress conditions before theyreached IC50. Volkamer lemon and sour orange needed the lowest Fe concentration(between 4 and 5 lM Fe) to maintain IC50, and Troyer citrange had the highest Fe requirement(14 lM Fe). Citrus macrophylla and Carrizo citrange required 7 and 9 lM of Fe, respectively.Similarly, Volkamer lemon and sour orange rootstocks withstood more days under total Fedepletion or with a low concentration of Fe (5 lM Fe in nutrient solution) until they reached IC50,compared to the other rootstocks. The approach used led to a classification of the rootstocksinto three categories, regarding their internal tolerance to Fe chlorosis: resistance (sour orangeand Volkamer lemon), intermediate resistance (C. macrophylla and Carrizo citrange), andreduced resistance (Troyer citrange).

Key words: chlorophyll / chlorophyll-fluorescence parameters / citrus-rootstocks resistance /exponential models / IC50

Accepted June 16, 2011

1 Introduction

Countries in Southern Europe, such as Portugal, Spain, Italy,and Greece, have large areas of calcareous soils with estab-lished orchards, where iron (Fe) chlorosis is a major factorthat limits fruit yield and farmers’ income. Calcium carbonateis present in high amounts in those soils, and the resultinghigh level of bicarbonate ions is the main cause of Fe defi-ciency in fruit trees (Álvarez-Fernández et al., 2006).

Iron deficiency is an important nutritional disorder in fruit treesthat results not only from a low level of Fe in soils but alsofrom impaired acquisition and use of the metal by plants. The

most evident effect of Fe deficiency is the decrease in photo-synthetic pigments, resulting in a relative enrichment of caro-tenoids over chlorophyll (CHL), and leading to the yellowcolor characteristic of chlorotic leaves (Abadía and Abadía,1993). The efficiency of photosystem II is only slightlyaffected by Fe deficiency in leaves of orange trees, sugarbeet, and pear (Morales et al., 1998, 2000; Pestana et al.,2001, 2005). Morales et al. (2006) concluded that, with theexception of severely chlorotic leaves, the photosyntheticapparatus in leaves of Fe-deficient fruit trees does not sufferany photoinhibitory damage, even at high densities of photo-

* Correspondence: Dr. M. Pestana; e-mail: [email protected]

synthetic photon flux and with mild water stress, which rep-resent the typical environmental conditions for crops growingin the Mediterranean area.

Iron chlorosis affects several metabolic processes and leadsto decreases in yield and quality of the fruits (Álvarez-Fernán-dez et al., 2006). In fruit trees, the resistance to Fe deficiencyis determined by the rootstock which influences factors suchas tree vigor, mineral nutrition, water balance, and finally fruityield and quality. Management of orchards using resistantrootstocks would represent a better alternative than commonremediation strategies. Unfortunately, there are no resistantrootstocks to Fe chlorosis in citrus with other favorable agro-nomical characteristics, such as resistance to pests and dis-eases such as the citrus Tristeza virus (CTV). For example,sour orange is a commonly used commercial rootstockbecause it leads to high-quality fruit and is resistant to severaldiseases and adverse conditions, including calcareous soils(Sudahono et al., 1994). However, the use of this citrus root-stock is limited to areas that are still free from CTV. Four newhybrids of Forner-Alcaide were recently identified and areunder field evaluation regarding their resistance to Fe chloro-sis (Gonzalez-Mas et al., 2009; Llosá et al., 2009).

Genetically improved chlorosis-resistant rootstocks may be along-term approach, still difficult to achieve and probablyinvolving genetic molecular manipulation. In consequence,screening methods based on physiological responses ofplants to Fe deficiency, which can be applied to young trees,have been extensively used. For example, Sudahono et al.(1994) concluded that SPAD-502 (a portable CHL metermeasuring leaf spectral characteristics) could be used forleaf-chlorosis assessment. However, in a preliminary experi-ment, Pestana et al. (2005) found that SPAD readings per sewere not sufficient to assess resistance to Fe deficiency incitrus, and that fluorescence parameters could help to grouprootstocks according to their resistance to bicarbonate-induced chlorosis.

The root Fe (III)-chelate reductase activity was used toscreen peach rootstocks (Gogorcena et al., 2000), grapehybrids (Dell’Orto et al., 2000), and citrus rootstocks (Llosáet al., 2009). Recently, Castle et al. (2009) screened a broadrange of plants across the orange subfamily Aurantioideae,growing both in nutrient solution and in soil, for their resis-tance to Fe stress. These authors also concluded that meas-uring the root Fe (III)-chelate reductase activity could be auseful screening tool.

The results obtained so far vary according to the physiologi-cal parameters studied, therefore raising the need to find amore refined and standardized protocol that could be appliedearly in breeding programs (Castle, 2010). Regression proce-dures have been used to evaluate salt resistance in severalcrops (Correia et al., 2010; Steppuhn et al., 2005) and couldbe tested to describe the response of crops to nutritionalstress, such as Fe depletion. Using this approach, the degreeof crop resistance to Fe deficiency would depend on theinherent ability of plants to withstand a low Fe availability inthe root zone and still produce a significant CHL yield. Thisapproach has not been used with citrus rootstocks, as far as

we know. To compare the tolerance of crops, CHL yieldsshould be standardized and expressed on a relative basis(Steppuhn et al., 2005).

In the present work, we used this new approach to evaluatethe physiological responses to Fe availability of five citrusrootstocks (Troyer citrange, Carrizo citrange, Citrus macro-phylla, Citrus volkameriana, and sour orange) that wereselected according to their level of resistance to Fe chlorosiscited in the literature. We adapted the model used in salinitystudies (Maas and Hoffman, 1977) to Fe chlorosis, replacingbiomass production by CHL concentration in leaves. Basedon this model, this work provides an integrative analysis ofthe concentration of Fe and the time of exposure to Fe stressrequired to reach the mid-yield CHL in leaves (IC50). Weevaluated the Fe-use efficiency of each rootstock as a short-term indicator of Fe resistance and grouped rootstocksaccording to their resistance to Fe-stress conditions.

2 Material and methods

2.1 Plant material and growth conditions

The five citrus rootstocks studied were: Troyer citrange(Citrus sinensis L. Osb. × Poncitrus trifoliata L. Raf.), Carrizocitrange, Volkamer lemon (Citrus volkameriana Ten. &Pasq.), alemow (Citrus macrophylla Wester), and sourorange (Citrus aurantium L.). Seeds of all rootstocks were ob-tained from Willits & Newcomb (Arvin, California, USA), andwere sterilized by immersion in a 15% sodium hypochloritesolution for 15 min and rinsed three times with running water.Seeds were germinated in the dark at 22°C in plastic trayswith sterilized moist vermiculite. After germination, the seed-lings were grown in moist vermiculite during 4 weeks in a con-trolled environment with day/night temperatures of 21°C/22°C, a relative humidity of 80%, and a 12 h photoperiod.The minimum photon flux density at plant level was 113 lmolquanta PAR (photosynthetic active radiation) m–2 s–1,provided by a combination of fluorescent and incandescentlamps. At the end of this period, groups of uniform seedlings(approximately [14 ± 2] cm long and with five to nine ex-panded leaves) were selected for each rootstock and placedin polystyrene boxes containing 20 L of a nutrient solutionwith the following composition: (in mM) 5.0 Ca(NO3)2,3.0 (NH4)2SO4, 1.4 KNO3, 1.0 MgSO4, 0.9 NaCl, 0.6 K2SO4,0.6 (NH4)2HPO4, 0.2 MgCl2, and (in lM) 41.8 H3BO3,6.9 MnSO4, 3.9 CuSO4, 3.8 ZnSO4, and 1.0 (NH4)6Mo7O24(Carpena, 1983).

Due to the size of the seedlings at the beginning of theexperiment, half-strength solutions with 5 lM Fe, added asFe (III) Na-EDHHA, were used during the first 2 months. Afterthis period, the solutions were then replaced with full-strengthsolutions and Fe was added to the solution as Fe (III) Na-EDHHA at five different concentrations (lM Fe): 0 (Fe0),5 (Fe5), 10 (Fe10), 15 (Fe15), and 20 (Fe20). These treat-ments were imposed during 5 weeks. The electrical conduc-tivity and the pH of the solutions were monitored once aweek. Full-strength nutrient solution was replaced when theelectrical conductivity dropped to 1.7 dS m–1. The pH of the


838 Pestana, Correia, David, Abadía, Abadía, de Varennes J. Plant Nutr. Soil Sci. 2011, 174, 837–846

new solutions was adjusted to 6.0 ± 0.1. During the experi-mental period, plants were grown in a glasshouse under nat-ural photoperiod conditions and temperature ≤ 25°C. Theexperiment was arranged as in a complete randomizeddesign, with 25 combinations of Fe concentrations and root-stocks and 6 replications (plants).

2.2 Leaf chlorophyll

Leaf CHL concentration (lmol m–2) was estimated using a por-table SPAD-502 meter (Minolta Co., Osaka, Japan). SPADreadings were taken in the two youngest fully expandedleaves of each plant at least four times during the experiment.Previously, a calibration curve was established for eachrootstock between the SPAD values and the colorimetricmeasurements of the pigments in leaf extracts (100%acetone and Na ascorbate) as in Abadía and Abadía (1993).The CHL was estimated according to the equations in Lich-tenthaler (1987). For each rootstock (Tab. 1), quadraticregression models (y = b0 + b1x + b2x2) were adjusted to thecalibration curves between SPAD (x) values and total CHLconcentration (y, lmol m–2). A strong relationship betweenSPAD readings and CHL contents (r2 > 0.94; p < 0.1%) wasobtained for all rootstocks.

2.3 Fluorescence parameters

At the end of experiment, CHL fluorescence parameters (F0,basal fluorescence; Fm, maximum fluorescence; Fv = Fm – F0,variable fluorescence) were measured with a portable fluori-meter (Plant Efficiency Analyser – PEA, Hansatech InstrumentsLtd., Norfolk, UK) in the second fully developed leaf of eachplant in a total of six plants from each rootstock. After 20 min ofdark adaptation, leaves were illuminated with a saturating pulseof 2100 lmol quanta m–2 s–1 for 5 s to induce fluorescence. TheFv : Fm parameter ratio was calculated, which reflects the maxi-mal quantum efficiency of photosynthesis.

2.4 Growth parameters

The height of each plant was measured at the beginning andat the end of the experiment and the relative growth rate(RGR, mm d–1) was calculated. Relative growth rate wasdefined as the increase in plant height per unit of time (t):

RGR � ln H2 � ln H1

t2 � t2� (1)

where H1and H2 are the plant height at initial and final daterespectively, and t2 – t1 is the number of days each rootstockgrew under the experimental conditions.

At the end of the experiment, each plant was separated intoshoots (stems and leaves) and roots. The dry weight of eachpart was determined after drying at 70°C during at least 48 huntil constant weight.

2.5 Iron shoot concentration

Plant material was rinsed with tap water, with distilled water con-taining 10 lM HCl, and finally three times with distilled water,dried and weighed as described above. Each sample consistedof two plants from each treatment, and three replicates per treat-ment were analyzed. Shoots (leaves and stems) were ground,ashed at 450°C, and dissolved in an acidic solution (1 mM HCl).The concentration of Fe was determined using atomic-absorp-tion spectrophotometry (M series, Pye Unicam, Cambridge,UK) following standard methods (A.O.A.C., 1990).

2.6 Response models and mid-yield chlorophyll

To model citrus-rootstock response to Fe availability, yield-re-sponse functions were established with the procedures of Maasand Hoffman (1977) developed to evaluate the resistance ofplants to salinity stress. Since Fe deficiency results in adecrease in the concentration of photosynthetic pigments inleaves (Abadía and Abadía, 1993), we used total leaf CHL asthe yield parameter in all tested models. Leaf CHL functionswere assessed for each rootstock in all Fe levels in nutrient solu-tion (0, 5, 10, 15, and 20 lM Fe). To compare rootstocks, theabsolute CHL was converted into relative yield (CHLr) byemploying a scaling divisor (CHLm) based on the maximumvalue of total CHL in leaves without Fe-deficiency symptoms(green leaves; Fe20 treatment). The CHLr value for eachrootstock was determined separately according to:

CHLr �CHL

CHLm� (2)

After this step, an exponential model was selected on thebasis of the best fit according to the square of the determina-tion coefficient (r2) for the equation:

CHLr � aeb Fei � (3)

where Fei is the Fe concentration in the nutrient solution, a isa constant that reflects the shape of the curve, and b definesthe slope of the model. For each coefficient (a and b), thestandard errors were also calculated. For each rootstock, theCHL concentration that was 50% of the maximum CHL(CHLr = 50%) was estimated by using Eq. 3, correspondingto the concentration of Fe in the nutrient solution needed toobtain a mid-yield of CHL (IC50) which may be related todifferences in Fe-use efficiency.

Rootstock resistance may also be related to the length of timethey are able to grow under stress conditions. In addition, we


Table 1: Quadratic regression coefficients (b0, b1, and b2) and thecoefficient of determination (r2) of calibration curves (y = b0+ b1x +b2x2) between SPAD (x) values and total CHL (y, lmol m–2) for eachrootstock. A global equation, considering all rootstocks together, wasalso included. Significant levels of the models are indicated by twoasterisks **, p < 1%.

Rootstock n b0 b1 b2 r2

Sour orange 29 0.33 3.8 0.064 0.96**

Carrizo citrange 39 –0.79 4.2 0.075 0.97**

C. macrophylla 30 55.09 –0.4 0.131 0.94**

Troyer citrange 37 –14.21 2.9 0.105 0.97**

Volkamer lemon 29 36.92 1.7 0.101 0.97**

Global equation 164 17.46 2.3 0.097 0.95**

J. Plant Nutr. Soil Sci. 2011, 174, 837–846 Citrus-rootstocks response to iron deficiency 839

tested another exponential model (best-fitted models) to calcu-late how many days it took for the CHL concentration equivalentto 50% of the maximum CHL under two level of stress, Fe deple-tion (Fe0) or the lowest level of Fe in the nutrient solution (Fe5):

CHLr � aeDaysi (4)

For each rootstock, Daysi is the number of days from thebeginning of the experiment until leaves had 50% of the maxi-mum CHL concentration in Fe20 plants.

2.7 Statistical analysis

Means were compared by analysis of variance and by using theDuncan multiple-range test at p < 5%. For each rootstock,

regression analysis was carried out between Fe level in nutrientsolution and relative shoot growth. Statistical analyses weredone with the STATISTICA software (Statsoft, 1995).

3 Results

3.1 Leaf chlorophyll and iron-chlorosis symptoms

Each rootstock presented different patterns of leaf CHL varia-tion during the experimental period (Fig. 1). In sour orange, aslight variation of CHL was observed and symptoms of ironchlorosis became apparent in Fe0 plants after 15 d of expo-sure to stress. Plants grown with 5, 10, and 15 lM Fe hadvisible symptoms only at the last date.


0

100

200

300

400

500

600

0 5 10 15 20 25 30 35 40

Sour orange

a

b

b,b,b

a

ab, ab, ab

b

0

100

200

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600

0 5 10 15 20 25 30 35 40

Carrizo citrange

b

c,c

a, a

a

ab, ab

b

c

0

100

200

300

400

500

600

0 5 10 15 20 25 30 35 40

C. macrophylla

a

b

c

dcd

a, a ,a

ab

b

0

100

200

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400

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600

0 5 10 15 20 25 30 35 40

Troyer citrange

b, b, b

a, a

a, a

b, b

a

Tota

l ch

loro

phy

ll / µ

mo

l m–2

0

100

200

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0 5 10 15 20 25 30 35 40

0

5

10

15

20

Volkamer lemon

a, a, a

μM Fe:

b

b

b

ab, ab

a, a

Days

Figure 1: Changes in total CHL concentration (lmol m–2) with time inrecently expanded fully developed leaves of five citrus rootstocksgrown with different Fe levels. Statistical analysis is presented for twodates: when symptoms of Fe chlorosis appeared and at the last date.For each date, means with the same letter were not significantlydifferent at p < 5%, using Duncan’s test.


Plants of the Fe20 treatment remained green during theexperimental period, and their CHL concentration was usedto calculate the percentage of CHL decrease in the othertreatments. In Carrizo, CHL was lower in the treatments Fe0,Fe5, and Fe10 with the largest decrease by the end of theexperiment (60% for Fe0 and Fe5; 45% for Fe10 plants),compared to Fe20 plants. After 20 d of exposure, plants of C.macrophylla grown with no Fe had a large decrease in CHL(70%). A similar decrease in CHL (60%) was observed inplants grown with Fe5, but only 30 d after the onset of thetreatments. In Troyer, CHL also decreased in plants of Fe0,Fe5, and Fe10 treatments, and apparently these plants weremore affected than Carrizo since symptoms of Fe chlorosisappeared 10 d after the onset of the treatments and wereaccentuated until the end of the experiment (75% decrease inCHL). Plants of the Fe15 and Fe20 treatments remainedgreen. In Volkamer lemon rootstock, two patterns could beidentified: green plants, which grew with higher Fe concentra-tions (10, 15, and 20 lM), and chlorotic plants (Fe0 and Fe5)with 62% less CHL only at the two last sampling dates.

In the absence of Fe, the number of days necessary toinduce symptoms of Fe chlorosis relative to Fe20 plants, dif-fered among rootstocks (Fig. 1): 10 d for Carrizo (50% of CHLdecrease) and Troyer (33% of CHL decrease), 15 d for sourorange (43% of CHL decrease) and Volkamer lemon (37% ofCHL decrease), and about 20 d for C. macrophylla (62% ofCHL decrease). However, after these periods of time, thedegree of chlorosis was severely enhanced in C. macrophyllaand Troyer rootstocks, moderately accentuated in Volkamerand Carrizo, and remained similar in sour orange until theend of the experiment.

3.2 Chlorophyll fluorescence parameters

The CHL fluorescence parameters obtained for each level ofFe at the end of the experiment are shown in Tab. 2. In sourorange, the Fv : Fm ratio was not affected by different levelsof Fe, but F0 was slightly decreased in the Fe20 treatment. InC. macrophylla and Carrizo rootstocks, only the most chloro-tic plants had lower values of this ratio resulting either fromlower Fm and/or greater F0. Similar values were obtained forVolkamer rootstock in the absence of Fe or with the lowestlevel of Fe. Troyer was the most affected rootstock, since theFv : Fm ratio decreased in the Fe0, Fe5, and Fe10 treatments,mainly due to lower Fm values. The Fe level in the nutrientsolutions did not affect the Fe concentration in shoots, exceptfor Troyer that presented the highest shoot Fe concentrationin the absence of Fe in solution (Tab. 2).

3.3 Growth parameters and iron in shoots

The level of Fe in the nutrient solution did not affect significantlythe shoot biomass of sour orange, Carrizo citrange, and Troyercitrange (Tab. 2). Plants of C. macrophylla grown in Fe15 hadthe highest shoot biomass. For Volkamer lemon, plants of theFe15 and Fe20 treatments had higher shoot biomass comparedto the other Fe concentrations. In roots, the differences wereless clear, but a significant increase in root biomass was foundin plants of C. macrophylla grown with 15 and 20 lM Fe.

The relative growth rate (RGR) was influenced by the level ofFe in nutrient solution (Fig. 2), and linear regression modelswere adjusted for each rootstock. The model for Volkamerrootstock had the steepest slope and the model for sourorange the lowest value. This meant that for the same incre-ment of Fe in the nutrient solution, the growth of Volkamerwas higher (about sixfold) than that of sour orange. The mod-els for C. macrophylla, Troyer, and Carrizo rootstocks had


Table 2: Shoot and root biomass, iron concentration in shoots, andchanges in photosystem II efficiency (Fv : Fm ratio; Fm, maximumfluorescence; F0, basal fluorescence) of citrus rootstocks grown withdifferent Fe concentrations in nutrient solution: 0, 5, 10, 15, and20 lM. For each rootstock and each parameter, means of threereplicates with the same letter were not significantly different atp < 5%, using Duncan’s test.

Treat-ments

Biomassper plant

Iron inshoots

Fluorescence parameters

(lM Fe) / g / g / mg kg–1 Fv : Fm Fm F0

shoots roots

Sour orange

0 1.25 a 0.33 b 57 a 0.68 a 2705 a 794 a

5 1.34 a 0.49 a 57 a 0.73 a 2903 a 791 a

10 1.51 a 0.47 ab 38 a 0.70 a 2787 a 816 a

15 1.33 a 0.60 a 36 a 0.71 a 2904 a 809 a

20 1.28 a 0.54 a 32 a 0.78 a 2919 a 628 b

Carrizo citrange

0 0.51 b 0.39 a 78 a 0.62 c 2430 b 763 a

5 0.77 ab 0.45 a 69 a 0.70 b 2749 a 691 ab

10 0.67 ab 0.39 a 51 a 0.77 a 2754 a 739 a

15 0.74 ab 0.44 a 57 a 0.81 a 2841 a 745 a

20 0.86 a 0.42 a 45 a 0.81 a 3007 a 561 b

C. macrophylla

0 0.63 c 0.24 b 51 a 0.56 b 2362 b 1013 a

5 0.60 c 0.17 b 50 a 0.75 a 2633 a 685 b

10 0.70 bc 0.26 b 46 a 0.76 a 2829 a 671 b

15 1.22 a 0.41 a 43 a 0.74 a 2788 a 666 b

20 0.95 b 0.34 a 71 a 0.80 a 2805 a 566 b

Troyer citrange

0 0.92 a 0.56 a 78 a 0.65 b 1672 c 716 a

5 1.00 a 0.56 a 38 bc 0.68 b 2141 bc 624 b

10 1.13 a 0.58 a 55 b 0.68 b 2317 b 586 b

15 1.44 a 0.63 a 52 b 0.83 a 3035 a 541 b

20 1.33 a 0.59 a 35 c 0.83 a 3085 a 552 b

Volkamer lemon

0 0.76 c 0.31 b 78 a 0.60 b 2000 b 615 a

5 0.92 bc 0.39 ab 69 a 0.63 b 2113 b 678 a

10 1.00 bc 0.39 ab 51 a 0.83 a 3035 a 526 b

15 1.27 ab 0.46 ab 57 a 0.82 a 2948 a 543 b

20 1.43 a 0.50 a 61 a 0.81 a 2986 a 555 b


similar slopes, and intermediate values between those ofVolkamer and sour orange rootstocks.

3.4 Response models and tolerance

At the end of experiment, the relative CHL variation (CHLr)was related to Fe treatments. The best models (those withthe lowest root mean square error and the highest coefficientof determination value; r2) obtained for each rootstock wereexponential equations. The CHLr increased exponentially asFe in nutrient solution increased (Fig. 3). The model forTroyer exhibited the most extended response lag but thesteepest slope. The highest accumulation of CHL in leaveswas observed in Troyer plants grown with more than 10 lMFe in nutrient solution.

Based on these models, the Fe level (lM) in nutrient solutionnecessary to obtain 50% (IC50) of the maximum CHL mea-sured in Fe20 for each rootstock was estimated (Fig. 4), andthe rootstocks ranked as follows: Volkamer lemon and sourorange needed the lowest Fe concentration in the nutrientsolution (between 4 and 5 lM Fe), and Troyer needed thehighest (14 lM Fe) to maintain 50% of maximum leaf CHL.Citrus macrophylla and Carrizo presented an intermediatebehavior, requiring 7 and 9 lM of Fe, respectively.

To study a possible effect of time of exposure to Fe depletion,new exponential models were established for the relation be-tween the variation of CHLr grown under stress conditions(Fe0 and Fe5) and the number of days of exposure (Fig. 5).In the last days of the experiment, all plants of both treat-ments were chlorotic. The best fitting equations for each root-stock and treatment are presented in Fig. 5. As expected, theCHLr of rootstocks grown under total absence of Fe wasmore affected than that obtained with 5 lM Fe in the nutrientsolution.

In Fig. 6, the number of days required by each rootstock toreach mid-yield of CHL (IC50) is presented (rootstocks are

ranked as for Fe derived IC50 in Fig. 4). Volkamer lemon andsour orange rootstocks withstood more days under Fe deple-tion (23 and 29 d, respectively), compared to other root-stocks. Moreover, in the presence of low Fe concentration(Fe5) the relative response was similar although delayed(35 d for Volkamer lemon and 42 d for sour orange). In theFe0 treatment, sour orange rootstocks withstood 16 d morethan Troyer to reach IC50, but in the Fe5 treatment this differ-


y = 0.0059x + 0.077r² = 0.92 **; T

y = 0.006x + 0.047r² = 0.79 *; C

y = 0.0067x + 0.038r² = 0.98 **; M

y = 0.0021x + 0.015r² = 0.86 *; S

y = 0.012x + 0.011r² = 0.95 **; V

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20

RG

R /

mm

d–1

Fe in nutrient solution / μM

Figure 2: Effect of Fe concentration in thenutrient solution on the relative growth rate(RGR; mm d–1) of each rootstock. The best fittedmodel is also presented for each rootstock.*, sour orange (S); �, Carrizo citrange (C); �,C. macrophylla (M); �, Troyer citrange (T); and�, Volkamer lemon (V). Significant levels of themodels are indicated by one asterisk *, p < 5%,or two asterisks **, p < 1%.

y = 43.85e0.035x

r² = 0.84*; S

y = 34.50e0.055x

r² = 0.91*; C

y = 25.57e0.074x

r² = 0.77*; M

y = 5.24e0.152x

r² = 0.83*; T

y = 42.30e0.052x

r² = 0.82*; V

0

20

40

60

80

100

120

140

160

0 5 10 15 20

Chl

r / %

Fe in nutrient solution / μM

Figure 3: Relationship between the Fe concentration in the nutrientsolution and the relative chlorophyll concentration (CHL) obtained bydividing absolute CHL by the maximum value of total CHL in leaveswithout Fe-deficiency symptoms (CHLr;%) 30 d after the beginning ofthe experiment. *, sour orange (S); �, Carrizo citrange (C); �, C.macrophylla (M); �, Troyer citrange (T); and �, Volkamer lemon (V).Significant levels of the models are indicated by one asterisk:* (p < 5%).


ence was larger (27 d). As a result, the Fe5 treatmentseemed to provide a better discrimination than the Fe0 treat-ment to screen the resistance of these rootstocks to Fe defi-ciency.

4 Discussion

Several authors have classified the Fe resistance of citrusrootstocks based on growth and other physiological para-meters (Castle et al., 2004, 2009; Gonzalez-Mas et al., 2009;Llosá et al., 2009; Pestana et al., 2005; Sudahono et al.,1994). In this experiment, we propose a new approach toestimate the ability of citrus rootstocks to cope with Fe defi-ciency.

When Fe is in short supply, Fe-efficient genotypes developshoot and/or root controlled responses, which include physio-logical, biochemical, and morphological changes (Schmidt,2006). At the shoot level, Fe is particularly important in CHLsynthesis and thylakoid stabilization (Abadía and Abadía,1993). The studied rootstocks had different CHL concentra-tions at the beginning of the experiment, even without pre-senting visual symptoms of Fe deficiency. These differencesof CHL concentration were probably related to different leafthickness and may also explain the different calibration equa-tions obtained for each rootstock, as previously reported forother citrus rootstocks (Pestana et al., 2005).

The conversion of photosynthetic energy is affected by Fechlorosis and may be assessed by fluorescence parameters(Morales et al., 2006). The CHL fluorescence parameters ob-

tained at the end of the experiment were considerablyaffected by Fe deficiency only in chlorotic leaves (Fe0 andFe5 treatments) for most of the rootstocks, with the exceptionof sour orange which did not reveal any significant impact inphotochemistry. Troyer was the most affected rootstock whenFe concentration was low. In general, the observed


Mid-Yield Chlorophyll (IC50)F

e L

evel

/ µ

M

Figure 4: Iron concentration in the nutrient solution required for mid-yield chlorophyll (IC50), considered as 50% of the maximumchlorophyll for each rootstock 30 d after the beginning of theexperiment. V, Volkamer lemon; S, sour orange; M, C. macrophylla;C, Carrizo citrange; and T, Troyer citrange. Columns with the sameletter were not significantly different at p < 5%, using Duncan’s test.

A – Fe0

y = 152.37e–0.084x

r² = 0.97**; Ty = 86.59e–0.031x

r² = 0.89**; C

y = 137.11e–0.055x

r² = 0.91**; My = 87.63e–0.018x

r² = 0.78*; Sy = 118.14e–0.032x

r² = 0.93*; V

0

20

40

60

80

100

120

140

160

0 10 20 30 40

Chl

r / %

Days

B – Fe5

y = 162.32e–0.078x

r² = 0.94**; Ty = 106.26e–0.034x

r² = 0.94**; C

y = 116.28e –0.034x

r² = 0.89**; My = 90.66e–0.013x

r² = 0.83*; Sy = 100.89e–0.019x

r² = 0.89*; V

0

20

40

60

80

100

120

140

160

0 10 20 30 40

Chl

r / %

DaysFigure 5: Variation of the relative chlorophyll concentration (CHL)obtained by dividing the absolute CHL by the maximum value of totalCHL in leaves without Fe-deficiency symptoms (CHLr;%) for two Feconcentrations in the nutrient solution (lM): Fe0 (A) and Fe5 (B). *,sour orange (S); �, Carrizo citrange (C); �, C. macrophylla (M); �,Troyer citrange (T); and �, Volkamer lemon (V). Significant levels ofthe models are indicated by one asterisk *, p < 5%, or two asterisks**, p < 1%.


decreases in Fv : Fm were more associated with slight in-creases in F0 than with decreases in Fm values. Increases inF0 values may indicate structural modifications of PSII, parti-cularly at the pigment level due to CHL breakdown or anten-nae reconfiguration, indicating lower absorption at CHLantenna. This kind of changes in Fv : Fm and in F0 areaccepted as good indicators of photoinhibition (Maxwell andJohnson, 2000). Lower Fm values in some Fe-deficient or Fe-deprived rootstocks (more evident in Troyer) could derivefrom increases in the dark reduction of the plastoquinonepool, as shown for sugar beet and other plant speciesaffected by Fe deficiency (Belkhodja et al., 1998). The ob-served effects will decrease citrus photosynthetic function, asboth down-regulation and photoinhibition were perceived, incontrast to chlorotic pear leaves where only down-regulationprocesses were observed but with no sustained photoinhibi-tion (Morales et al., 2000). In this study, we could not ascer-tain the kind or severity of the photoinhibition, sincedecreases in Fv : Fm values may be due to inaccurately largeF0 which is only prevented by far-red pre-illumination (Mor-ales et al., 2006).

Shoot Fe was not affected by treatments. In fact, a higher Feconcentration was registered in shoots of Troyer under Fedepletion, which may be attributed to a possible dilutioneffect. In conclusion, shoot Fe does not seem to be a goodparameter to diagnose Fe deficiency and seems to be regu-lated by genetic differences rather than by Fe availability.

The rootstocks used in this study may be divided into twogroups: nontrifoliate species (Volkamer lemon, sour orange,and C. macrophylla) as the resistant group with the highestrates of reductase activity, and the hybrids of trifoliate (Car-rizo citrange and Troyer citrange) as the susceptible groupwith the lowest values of ferric chelate reductase activity(Castle et al., 2009). According to the results of the presentstudy, evaluation of the tolerance of these rootstocks couldbe refined based on the Fe-use efficiency and the number of

days that each rootstock was able to grow under low Fe con-centrations (Fe0 and Fe5) before the CHL concentrationdecreased to 50% of that of plants that remained green(Fe20). Given that a total absence of Fe is not found in natureand the Fe5 level was more effective in distinguishing root-stocks, the classification proposed is as follows (IC50Fe;IC50days(Fe5)):

(1) resistance: sour orange (5; 42) > Volkamer lemon (4; 35),

(2) intermediate resistance: C. macrophylla (7; 24) > Carrizocitrange (9; 22),

(3) reduced resistance: Troyer citrange (14; 15).

The tolerance of sour orange is well-documented, and basedon our results it is possible to assume that there is some rela-tionship between resistance and growth rate. In fact, thecitrus rootstocks tested seemed to have different strategiesto adapt shoot elongation to Fe depletion. This fact was moreobvious in sour orange and Volkamer lemon, as the RGR ofthe former was always lower, while Volkamer lemon alwayshad the highest increase per unit of Fe supplied to nutrientsolution. This means that for the same increment of Fe in thenutrient solution, the growth of Volkamer was higher (aboutsixfold) than that of sour orange. Based on this rate, it waspossible to consider sour orange and Volkamer lemon root-stocks as slow-growing and fast-growing species, respective-ly. Slow-growing species have low photosynthesis per leafweight and low ion-uptake rates (Lambers et al., 2008; Lam-bers and Poorter, 1992) and therefore incorporate less photo-synthates and nutrients into structural biomass. Plants grow-ing under shortage of nutrients are expected to conservethem (Lambers et al., 2008), and it is possible to presumethat sour orange followed a conservative strategy leading to abetter Fe-use efficiency. This is supported by the fact that thisrootstock had the lowest Fe concentration in the shootscompared with all other rootstocks. Moreover, sour orange


Mid-Yield Chlorophyll (IC50)

A - Fe0 B - Fe5

Figure 6: Number of days toreach mid-yield chlorophyll con-sidered as 50% of the maximumchlorophyll (IC50) for eachrootstock under two level ofstress, Fe depletion (Fe0; A)and the lowest level of Fe innutrient solution (Fe5; B). V,Volkamer lemon; S, sourorange; M, C. macrophylla; C,Carrizo citrange; and T, Troyercitrange. Columns with thesame letter were not signifi-cantly different at p < 5%, usingDuncan’s test.


displays a high plastic response of root architecture to lownitrate availability (Sorgonà et al., 2005).

The behavior of Volkamer lemon may be more difficult toexplain, since the high RGR was linked to better resistance.The fast-growing Volkamer lemon plants presented a highergrowth rate when Fe was present in the nutrient solution,which probably indicates efficient nutrient and carbon acquisi-tion by roots and leaves. This genetic potential leads to a fasttransport of Fe to sink organs, such as new leaves, suggest-ing an “Fe-spending” strategy rather than a conservative one.The models for C. macrophylla, Troyer citrange, and Carrizocitrange rootstocks had similar slopes and intermediate be-tween those observed for Volkamer lemon and sour orangerootstocks. The ecological meaning of these two strategiestowards Fe-use efficiency needs to be further investigated.However, based on the models established for CHLr, bothstrategies seem to be efficient in this artificial growing sys-tem.

The proposed methodology, using CHL models with time andIC50 calculations, may be applied to other species to screenrootstocks for Fe-chlorosis resistance. For example, inSouthern Portugal the citranges are arbitrarily used, althoughwe showed that Troyer citrange is more affected by Fe stressthan is Carrizo citrange. In future, similar studies must be car-ried out with the addition of calcium carbonate to nutrientsolutions to mimic the rhizosphere environment characteristicof calcareous soil, which is the most prevalent cause of ironchlorosis in field.

Acknowledgments

This study was funded by two Projects from the PortugueseFoundation for Science and Technology (PTDC/AGR-ALI/66065/2006 and PTDC/AGR-AAM/100115/2008) and by theSpanish Ministry of Science and Innovation ProjectAGL2009-09018 co-financed by FEDER.

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response of five citrus rootstocks to iron deficiency

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