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GROWTH AND MINERAL NUTRITION ARE AFFECTED BY
SUBSTRATE TYPE AND SALT STRESS IN SEEDLINGS OF TWO
CONTRASTING CITRUS ROOTSTOCKS
Vicente Gimenoa; James P. Syvertsenb; Francisco Rubioa; Vicente Martíneza; Francisco García-Sáncheza
a
Centro de Edafología y Biología Aplicada del Segura-CSIC, Campus Universitario de Espinardo,
Murcia, Spain b Citrus Research and Education Center, University of Florida, Lake Alfred, Florida, USA
Online publication date: 25 June 2010
To cite this Article Gimeno, Vicente , Syvertsen, James P. , Rubio, Francisco , Martínez, Vicente and García-Sánchez,
Francisco(2010) 'GROWTH AND MINERAL NUTRITION ARE AFFECTED BY SUBSTRATE TYPE AND SALT STRESS
IN SEEDLINGS OF TWO CONTRASTING CITRUS ROOTSTOCKS', Journal of Plant Nutrition, 33: 10, 1435 — 1447
To link to this Article: DOI: 10.1080/01904167.2010.489982
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Journal of Plant Nutrition, 33:1435–1447, 2010
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Copyright ISSN: 0190-4167 print / 1532-4087 online
DOI: 10.1080/01904167.2010.489982
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
GROWTH AND MINERAL NUTRITION ARE AFFECTED
BY SUBSTRATE TYPE AND SALT STRESS IN SEEDLINGS
OF TWO CONTRASTING CITRUS ROOTSTOCKS
Vicente Gimeno,1 James P. Syvertsen,2 Francisco Rubio,1 Vicente Martı́nez,1
and Francisco Garcı́a-Sánchez1
1
Centro de Edafologı́a y Biologı́a Aplicada del Segura-CSIC, Campus Universitario de
Espinardo, Murcia, Spain
2
Citrus Research and Education Center, University of Florida, Lake Alfred, Florida, USA
2
We evaluated plant growth and leaf and root mineral nutrient responses to salinity of twomonth-old citrus rootstock seedlings growing in four types of container growth media: aerated hydroponic solution, river washed sand, perlite, or a native clay-loam soil. Seedlings of Cleopatra mandarin (Citrus reticulata Blanco; Cleo, relatively salt tolerant) and Carrizo citrange [C. sinensis
(L.) Osb. Poncirus trifoliate L.; Carr, salt sensitive] were grown in a controlled-environment
chamber using Hoagland’s nutrient solution containing either 0 mM (Control) or 50 mM sodium
chloride (NaCl; salt). Without salt, seedlings in solution culture and sand grew the most and
seedlings in perlite and clay-loam grew the least. The salinity treatment decreased growth in both
Cleo and Carr seedlings in solution and sand but not in smaller seedlings in perlite and clay-loam
soil. Cleo seedlings had lower leaf chloride (Cl −) concentration and higher leaf sodium (Na +) concentration than Carr seedlings. In the salinized clay-loam soil, Cl − and Na + concentrations tended
to be highest in leaves but lowest in roots. Salt treatment generally reduced leaf calcium (Ca 2+) concentration in Cleo seedlings in all substrates and in Carr seedlings in solution and perlite. Based on
total plant dry weight, seedlings grown in solution culture and sand were more salt tolerant than
those grown in perlite and clay-loam soil. Since the reduced growth in clay-loam soil and perlite
negated the effects of the salt treatment, salt tolerance was not linked to leaf Cl − concentration.
Keywords:
citrus, rootstock, salt tolerance, mineral nutrition, leaf water relations
INTRODUCTION
Citrus has been classified as a salt-sensitive crop as saline irrigation water
reduces citrus tree growth and fruit yield (Prior et al., 2007; Grieve et al.,
Received 24 August 2008; accepted 25 July 2009.
Address correspondence to Dr. Francisco Garcı́a-Sánchez, Centro de Edafologı́a y Biologı́a Aplicada del Segura-CSIC, Campus Universitario de Espinardo, 30562, Espinardo, Murcia, Spain. E-mail:
[email protected]
1435
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
1436
V. Gimeno et al.
2007; Garcı́a-Sánchez et al., 2006). Citrus rootstock species control the uptake of chloride (Cl−) and/or sodium (Na+) and the relative tolerance of
citrus rootstocks has been based on the accumulation of Cl− in leaves (Levy
and Syvertsen, 2004). In Spain, sour orange (Citrus aurantium L.) historically has been an excellent rootstock for citrus trees under saline conditions.
However, sour orange is highly susceptible to the tristeza disease (Salibe,
1973) so this rootstock has been replaced by Cleopatra mandarin and Carrizo citrange. Cleopatra mandarin is a good Cl−-excluder whereas ‘Carrizo’
citrange is considered to be a Cl− accumulator but a good Na+ excluder
(Bañuls and Primo-Millo, 1995; Levy et al., 1999). The Cl− restriction mechanism in Cleopatra relative to Carrizo citrange could be linked to either the
low absorption of chloride per volume of water, a less efficient root system
for water uptake or to a high shoot-to-root ratio (Moya et al., 2003). However,
Na+ restriction mechanisms in Carrizo citrange are still unknown.
Citrus responses to salinity can depend on growth conditions, amount of
irrigation water, climate or soil type (Levy and Syvertsen, 2004; Murkute et al.,
2005). For example, under saline conditions, relative yield was increased in
Fino 49 lemon on C. macrophylla due to a reduction of the average rootzone
salinity by a 25% increase in the amount of water applied (Garcı́a-Sánchez
et al., 2003). Arid or semi-arid climates with large vapor pressure deficits
can increase leaf Cl− concentration by increasing foliar transpiration (Moya
et al., 1999). In addition, root morphology and ion uptake by roots in solution culture can be different from roots growing in soil (Storey, 1995, Levy
and Syvertsen, 2004). There is little information how soil type can influence
in the salt tolerance of citrus but soil ion exchange capacity, mechanical
impedance to root growth or the effect of soil matric potential could influence the availability of salt ions and thus, change tolerance to salinity
(Villagra and Cavagnaro, 2005). In this experiment, we grew the relatively
salt-tolerant Cleopatra mandarin and the more salt-sensitive Carrizo citrange
citrus rootstock seedlings in four contrasting substrate types: hydroponic
solution, river washed sand, perlite, and clay-loam soil. Plant growth responses and leaf and root mineral nutrient status were used as indices of salt
tolerance.
MATERIALS AND METHODS
Plant Material and Growing Conditions
Seeds of Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliate L.,
Carr] and Cleopatra mandarin (Citrus reticulata Blanco, Cleo) were germinated in containers containing vermiculite wetted with 0.5 mmol L−1 calcium
sulfate (CaSO4 ). When seedlings were two-month-old, they were supported
in 1-L containers of continuously aerated Hoagland’s complete nutrient
solution, or transplanted into 1-L containers with drainage filled with river
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
Citrus Seedlings under Saline Conditions
1437
washed sand, perlite or a native clay-loam soil. Hoagland’s solution contained
6 mM potassium nitrate (KNO3 ), 4 mM calcium nitrate [Ca(NO3 )2 ], 2 mM
monopotassium phosphate (KH2 PO4 ), 2 mM magnesium sulfate (MgSO4 ),
20 µM iron (Fe+3) masquolate, 25 µM boric acid (H3 BO3 ), 2 µM manganese
sulfate (MnSO4 .H2 O), 2µM zinc sulfate (ZnSO4 ), 0.5 µM copper sulfate
(CuSO4 ), 0.4 µM ammonium molybdate [(NH4 )6 Mo27 O24 .H2O ]. The solution was renewed weekly and the pH was adjusted to 6.0–6.5. The river
washed sand was 98% sand, 2% silt with no clay or organic matter content, a cation exchange capacity of 0.2 meq 100g−1, extractable phosphorus (P) and calcium (Ca2+) of 7 mg kg−1 and 700 mg kg−1, respectively,
and with no extractable potassium (K) (Mehlich 3 extraction). The Perlite
substrate was an inert media with particle size of 3–5 mm, pH of 7, and
containing extractable P, K, and Ca (mg kg−1) of 7.4, 89.9, and 395, respectively. Clay-loam soil was 24.1% sand, 48.70% silt, and 27.2% clay with
1.10% organic matter, cation exchange capacity of 14 meq 100g−1, active calcium carbonate of 11.10% and extractable P and K of 14 and 257 mg kg−1,
respectively.
Plants were grown in a controlled-environment chamber with 16/8 h
light/dark cycle and air temperature of 25◦ C/21◦ C day/night. The relative
humidity was 65% (day) and 85% (night), and the photon flux density at
plant height was 550 µmol m−2 s−1. Plants in perlite, sand, and clay-loam
soil were watered every other day with 50 mL of the Hoagland nutrient
solution sufficient to leach from the bottom of all pots. Two weeks after
transplanting, 50 mM NaCl (Salt) was added to the nutrient solution of half
of the seedlings in each treatment while the other half got 0 mM (Control).
To avoid an osmotic shock, salinity was increased in increments of 10 mM
NaCl per day until 50 mM NaCl was achieved. The experimental design was
a 2 × 2 × 4 factorial of two rootstocks (Cleo and Carr), two salt treatments (0
mM NaCl or 50 mM NaCl) and four different substrates (solution culture,
sand, perlite and clay-loam soil) with six replicate plants in each treatment.
Growth and Leaf Nutrient Concentration
Eight weeks after initiating the salinity treatments, plants were harvested
and separated into leaves, stem and root. Tissues were briefly rinsed with
deionized water, oven-dried at 60◦ C for at least 48 h, weighed and ground
to a fine powder. Subsamples of leaf and root tissues were extracted with
deionized water. Tissue chloride concentration was measured using a silver
ion titration chloridometer (Corning 926 Chloridometer; Sherwood, UK).
Tissue Na+, K +, magnesium (Mg2+), Ca+2 and P concentrations were determined by inductively coupled plasma spectrometry (Iris Intrepid II, Thermo
Electron Corporation, Franklin, MA, USA) after a previous hot acid digestion in nitric acid (HNO3 ): hydrogen peroxide (H2 O2 ) (5:3) in a microwave
1438
V. Gimeno et al.
reaching 200◦ C in 20 minutes and holding at this temperature for two hours
(Mars Xpress, CEM, Matthews, NC, USA).
Electrical Conductivity and Cl− Concentration in the Drainage
Solution
Electrical conductivity (EC) and Cl− concentration was measured at the
end of the experiment in the hydroponic or leached drainage solutions from
each pot. The leachate was collected after watering with 50 mL of Hoagland
nutrient solution. Electrical conductivity was measured with a Crison EC
meter and Cl− concentration was measured with the chloridometer as above.
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
Statistical Analysis
Data were subjected to analysis of variance using two salinity treatments ×
four substrates as main effect for each rootstock, and six replicate plants per
treatment. When salinity treatment × substrate interactions were significant
(P < 0.05), means were separated using Duncan’s multiple range test.
RESULTS
Growth
Overall, Carr seedlings were larger than Cleo seedlings. Plant growth
of the non-salinized seedlings was significantly affected by substrate type as
total plant dry weight was greatest in seedlings of both rootstocks from solution culture and the lowest in seedlings from clay-loam soil. Salt treatment
reduced total plant growth of plants from hydroponic and sand but not of
the already smaller plants in perlite and clay-loam soil (Figures 1a, 1c). In
Carr seedlings, total plant reductions were about a 29% in both hydroponic
and sand, and in Cleo, salinity-induced reductions were about a 24% and
35% for hydroponic and sand, respectively. Carr seedlings in solution culture and sand allocated more growth to shoots than to roots such that their
shoot to root dr wt ratios were higher than those from perlite and clay-loam
(Figure 1b). Cleo seedlings in sand had the higher shoot to root dw than
those from the other substrates (Figure 1d). The salt treatment reduced root
and shoot growth similarly as shoot to root dr wt ratio was not affected in
Carr or Cleo.
Chloride and Sodium Concentration in Leaves and Roots
Salinity increased the concentration of Cl− in both leaves and roots of
Cleo and Carr seedlings regardless of substrate (Figure 2). In both Carr
and Cleo, the highest leaf Cl− concentrations were in seedlings from the
clay-loam soil and at least numerically lowest in seedlings from perlite
1439
Citrus Seedlings under Saline Conditions
Carrizo
2.0
7
Substrate ***
Salt ***
Soil x Sallt *
a
(a)
Substrate **
Salt ns
Soil x Sallt ns
A
(b)
A
1.6
b
5
bc
B
cd
4
B
de
1.2
de
ef
3
f
0.8
Shoot to root
Total plant d.w (g)
6
Control
Salt
2
0.4
1
0.0
0
Hydroponic
Perlite
Sand
Soil
Hydroponic
Perlite
Sand
Soil
Cleopatra
2.8
Substrate **
Salt ns
Soil x Sallt ns
(d)
2.4
A
b
b
2.0
B
bc
c
B
1.6
B
cd
1.2
de
1
Shoot to root
2
(c)
Substrate ***
Salt **
Soil x Sallt **
a
Total plant d.w (g)
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
3
0.8
e
0.4
0.0
0
Hydroponic
Perlite
Sand
Substrate
Soil
Hydroponic
Perlite
Sand
Soil
Substrate
FIGURE 1 Effects of substrate (hydroponic, perlite, sand or clay-loam soil) and salt treatment (control =
0 mM NaCl, or salt = 50 mM NaCl) on mean (n = 6) total plant dry weight (a, c) and shoot to root dw ratio
(Shoot/Root; b, d) of Carr (a, b) and Cleo (c, d) rootstock seedlings. Different lower case letters within
each figure indicate significant differences at P < 0.05 among substrate × salt treatments. Differences
between substrate types are indicated by different upper case letters. Ns, ∗,∗∗,∗∗∗ indicate non-significant
or significant differences at P < 0.05, 0.01, or 0.001 respectively, for the two way interaction salt × soil
treatments for each rootstock.
(Figure 2a). Root Cl− concentration was lowest in seedlings from clay-loam
soil in both Carr and Cleo. Concentration of Cl− in Cleo roots was the highest in seedlings from solution culture, whereas perlite-grown seedlings had
significantly lower root Cl− than those from sand (Figure 2d).
Salinity increased the concentration of Na+ in leaves and roots of both
Cleo and Carr seedlings regardless of substrate except in the already low Na+
in Cleo roots (Figure 3). The highest concentration of Na+ in Carr leaves was
in seedlings from clay-loam soil and solution culture, and the lowest from
those in perlite (Figure 3a). In Cleo, the highest leaf Na+ concentration
also was in seedlings from clay-loam soil and the lowest in those from perlite
(Figure 3c). Concentration of Na+ in roots was highest in seedlings from
solution culture and lowest in roots from clay-loam soil in both Carr and
Cleo (Figure 3b, d).
1440
V. Gimeno et al.
Carrizo
3.0
6
Substrate ***
Salt ***
Soil x Sallt ***
(a)
a
(b)
2.5
2.0
4
b
a
a
a
3
c
c
2
1.0
1
d
1.5
b
d
Perlite
Sand
d
d
d
d
d
Cl- root (% dw)
5
Cl- leaf (% dw)
Substrate ***
Salt ***
Soil x Sallt ***
Control
Salt
0.5
d
0.0
0
Hydroponic
Soil
Hydroponic
Perlite
Sand
Soil
Cleopatra
3.0
Substrate ***
Salt ***
Soil x Sallt ***
a
(d)
2.5
a
4
2.0
b
3
b
b
2
1.5
c
1.0
Cl- root (% dw)
(c)
Substrate ***
Salt ***
Soil x Sallt **
5
Cl- leaf (% dw)
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
6
d
c
1
d
d
d
d
Perlite
Sand
Soil
e
e
e
0.5
e
0.0
0
Hydroponic
Hydroponic
Substrate
Perlite
Sand
Soil
Substrate
FIGURE 2 Effects of substrate (hydroponic, perlite, sand or clay-loam soil) and salt treatment (control
= 0 mM NaCl, or salt = 50 mM NaCl) on mean (n = 6) leaf Cl− concentration (a, c) and root Cl−
concentration (b, d) of Carr (a, b) and Cleo (c, d) rootstock seedlings. Different lower case letters within
each figure indicate significant differences at P < 0.05 among substrate × salt treatments. Differences
between substrate types are indicated by different upper case letters. Ns, ∗,∗∗,∗∗∗ indicate non-significant
or significant differences at P < 0.05, 0.01, or 0.001 respectively, for the two way interaction salt × soil
treatments for each rootstock.
Other Nutrients in Leaves and Roots
In the non-salinized control, Carr and Cleo leaves from sand tended to
have the highest leaf Ca+2 concentration, and seedlings from perlite and
sand tended to have the highest leaf K + concentrations (Table 1). Cleo
and Carr seedlings from perlite had the highest leaf Mg2+ concentration
while Carr seedlings from solution culture and sand, and Cleo seedlings in
solution had the lowest. Carr seedlings from solution culture and sand, and
Cleo seedlings from solution culture, had the highest leaf P concentration
whereas the lowest concentration was observed in clay-loam soil for Cleo and
Carr seedlings. The salt treatment in Carr decreased leaf Ca2+ concentration
in seedlings from solution culture and sand. Salinity increased the leaf K +
concentration in Carr seedlings from all four substrates, and increased the
leaf P concentration in seedlings from perlite and clay-loam soil. In Cleo
1441
Citrus Seedlings under Saline Conditions
Carrizo
1.50
2.5
Na+ leaf (% dw)
2.0
(a)
a
ab
Substrate ***
Salt ***
Soil x Sallt ***
1.25
1.00
a
b
1.5
(b)
0.75
b
1.0
b
c
c
0.5
0.25
d
d
d
d
Perlite
Sand
0.50
Na+ root (% dw)
Substrate ***
Salt ***
Soil x Sallt **
Control
Salt
d
d
d
d
Perlite
Sand
0.00
0.0
Hydroponic
Soil
Hydroponic
Soil
Cleopatra
1.50
a
2.0
(d)
Substrate ***
Salt ***
Soil x Sallt ***
1.25
1.00
b
b
0.75
1.5
a
c
1.0
0.50
b
Na+ root (% dw)
(c)
Substrate ***
Salt ***
Soil x Sallt ***
2.5
Na+ leaf (% dw)
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
3.0
b
0.5
d
d
d
d
Perlite
Sand
Soil
c
c
c
c
Perlite
Sand
0.25
c
0.00
0.0
Hydroponic
Substrate
Hydroponic
Soil
Substrate
FIGURE 3 Effects of substrate (hydroponic, perlite, sand or clay-loam soil) and salt treatment (control
= 0 mM NaCl, or salt = 50 mM NaCl) on mean (n = 6) leaf Na+ concentration (a, c) and root Na+
concentration (b, d) of Carr (a, b) and Cleo (c, d) rootstock seedlings. Different lower case letters within
each figure indicate significant differences at P < 0.05 among substrate × salt treatments. Differences
between substrate types are indicated by different upper case letters. Ns, ∗,∗∗,∗∗∗ indicate non-significant
or significant differences at P < 0.05, 0.01, or 0.001 respectively, for the two way interaction salt × soil
treatments for each rootstock.
seedlings, the salt treatment decreased the leaf Ca2+ concentration in all
four substrates, but leaf K +, Mg2+ and P concentration were not affected.
Carr roots from non-salinized clay-loam soil and solution culture treatment, and Cleo roots from non-salinized soil treatment had the highest Ca2+
concentration (Table 2). Again, root Ca2+ tended to be lowest in those plants
from perlite in both Cleo and Carr. The highest root K + concentration was
observed in Carr seedlings from sand and perlite, and in Cleo seedlings from
solution culture whereas the lowest K + concentration was observed in clayloam soil for both Cleo and Carr. The highest root Mg2+ concentration was
in Carr seedlings from perlite and in seedlings from perlite and clay-loam
soil for Cleo. The highest P concentration was in solution culture and the
lowest in clay-loam soil for both Cleo and Carr seedlings. In Carrizo, root
K + concentration was decreased significantly by salt treatment in seedlings
from all four substrates. In Cleo, root Ca2+ concentration was decreased by
1442
V. Gimeno et al.
TABLE 1 Effects of growth substrate (hydroponic solution culture, perlite, sand or clay-loam soil) and
salinity treatment (control = 0 mM NaCl or salt = 50 mM NaCl) on mean (n = 6) leaf Ca2+, K +, Mg2+
and P concentration of Carrizo citrange and Cleopatra mandarin rootstock seedlings
Rootstock
Carr
Substrate
Salinity
Ca2+
Hydroponic
Control
Salt
Control
Salt
Control
Salt
Control
Salt
1.93 bc
1.40 e
1.70 cd
1.64 d
2.38 a
1.69 cd
1.82 bcd
2.00 b
Perlite
Sand
Soil
Downloaded By: [Garcia-Sanchez, Francisco] At: 13:40 30 June 2010
Anova
Cleo
Anova
Substrate
Salt
Substrate × Salt
Hydroponic
Control
Salt
Perlite
Control
Salt
Sand
Control
Salt
Soil
Control
Salt
Substrate
Salt
Substrate × Salt
∗∗∗
K+
1.76 B
2.57
2.39 A
3.29
3.00 A
3.21
1.47 B
2.43
∗∗∗
∗∗∗
∗∗∗
1.59 B
1.27
1.55 B
1.28
1.99 A
1.54
1.66 AB
1.61
∗∗∗
Mg+2
(% dw)
0.18 C
0.12
0.65 A
0.64
0.24 C
0.17
0.31 B
0.29
∗∗∗
∗∗∗
P
0.28 a
0.27 ab
0.18 c
0.24 b
0.25 ab
0.24 b
0.12 d
0.16 c
∗∗∗
∗
ns
2.22 B
2.65
3.11 A
2.93
2.74 AB
2.89
2.40 B
2.50
ns
ns
0.13 D
0.12
0.61 A
0.51
0.20 C
0.23
0.30 B
0.31
0.24 A
0.27
0.20 B
0.20
0.18 B
0.18
0.15 C
0.14
ns
ns
ns
ns
ns
ns
∗∗
ns
∗
∗∗∗
∗
∗∗∗
Significant differences among substrate × salt treatments are indicated by different lower case letters,
whereas within each column, means followed by the same letters are not significant different at P < 0.05.
Significant differences between substrate type are indicated by different upper case letters. ns, ∗,∗∗,∗∗∗
indicate non-significant or significant differences at P < 0.05, 0.01, or 0.001 respectively.
salt treatment only in seedlings from sand and clay-loam soil, and root K +
concentration was decreased by salt treatment in all four substrates.
Electrical Conductivity and Cl− Concentration in Solution
The EC and Cl− concentration in the salinized leached solution was
highest for clay-loam soil whereas the EC and Cl− in the leachate from
perlite and sand was lowest (Figure 4). The EC and Cl− concentration in
nutrient solution from solution culture treatment was significantly lower
than those of the others substrates.
DISCUSSION
Effect of Substrate Type without Salinity
Both Cleo and Carr seedlings grew the most in solution culture and sand.
The fine texture of the compact native clay-loam soil with poor drainage may
1443
Citrus Seedlings under Saline Conditions
TABLE 2 Effects of growth substrate (hydroponic solution culture, perlite, sand or clay-loam soil) and
salinity treatment (control = 0 mM NaCl or salt = 50 mM NaCl) on mean (n = 6) root Ca2+, K +, Mg2+
and P concentration of Carrizo citrange and Cleopatra mandarin rootstock seedlings
Rootstock
Carr
Substrate
Salinity
Ca2+
Hydroponic
Control
Salt
Control
Salt
Control
Salt
Control
Salt
1.42 A
1.80
0.50 B
0.53
0.95 B
1.02
1.76 A
2.30
Perlite
Sand
Soil
Anova
Mg+2
P
(% dw)
1.86 B
0.13 C
1.48
0.21
1.88 AB
0.46 A
1.70
0.42
2.23 A
0.14 C
1.59
0.15
1.43 C
0.28 B
1.18
0.29
∗∗∗
Substrate
Salt
Substrate × Salt
Hydroponic
Control
Salt
Perlite
Control
Salt
Sand
Control
Salt
Soil
Control
Salt
Substrate
Salt
Substrate × Salt
Cleo
∗∗∗
∗
∗∗∗
∗∗∗
Ns
0.94 B
0.82 Bc
0.54 D
0.57 D
0.92 B
0.70 Cd
1.19 A
0.63 Cd
Ns
1.89 A
1.45
1.33 B
0.98
1.55 B
1.05
0.94 C
0.52
∗∗∗
∗∗∗
∗∗∗
∗∗∗
Ns
Ns
0.23 B
0.23
0.35 A
0.32
0.21 B
0.20
0.31 A
0.31
Ns
Ns
0.31 A
0.27
0.27 C
0.25
0.23 B
0.20
0.10 D
0.12
Ns
Ns
Ns
Ns
∗∗∗
∗∗∗
∗∗
0.59 A
0.51
0.24 B
0.24
0.23 B
0.27
0.14 C
0.10
Ns
∗∗∗
Significant differences among substrate × salt treatments are indicated by different lower case letters,
whereas within each column, means followed by the same letter are not significant different at P < 0.05.
Significant differences between substrate type are indicated by different upper case letters. ns, ∗,∗∗,∗∗∗
indicate non-significant or significant differences at P < 0.05, 0.01, or 0.001 respectively.
16
12
(a)
Substrate ***
Salt ***
Soil x Sallt ***
b
b
(b)
Substrate ***
Salt ***
Soil x Sallt ***
Control a
Salt
a
b
80
b
10
60
c
8
100
c
40
6
Cl- (mM)
14
EC (dS m-1)
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Anova
K+
d
e
4
f
ef
20
2
d
0
Hydroponic
Perlite
Sand
Substrate
Soil
Hydroponic
d
d
d
0
Perlite
Sand
Soil
Substrate
FIGURE 4 Effects of substrate (hydroponic, perlite, sand or clay-loam soil) and salt treatment (control
= 0 mM NaCl, open bars or salt = 50 mM NaCl, shaded bars) on mean (n = 6) electrical conductivity
(EC; a) and Cl− concentration (b) in the drainage solution from Carrizo citrange seedlings. Different
lower case letters within each figure indicate significant differences at P < 0.05 among substrate × salt
treatments. Differences between substrate types are indicated by different upper case letters. Ns, ∗,∗∗,∗∗∗
indicate non-significant or significant differences at P < 0.05, 0.01, or 0.001 respectively, for the two way
interaction salt × soil treatments for each rootstock.
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1444
V. Gimeno et al.
have negatively affected to the growth in seedlings. The low air-filled porosity could have increased root penetration resistance and reduced oxygen
diffusion to the roots (Mcafee et al., 1989). Reduction in soil oxygen (O2 )
concentration from water logging can decrease citrus seedling growth along
with stomatal conductance and net assimilation of carbon dioxide (CO2 )
(Garcı́a-Sánchez et al., 2007), while increasing oxidative damage (Arbona
et al., 2008). Drought stress in the perlite substrate also could have limited
growth because irrigation every other day may have been inadequate. Perlite
has been reported to support excellent growth of tomato seedlings when
watered more frequently and maintaining a good water status (Aroiee et al.,
2006).
These two citrus rootstocks have different abilities to uptake water and
nutrients (Castle et al., 1993) as leaves from Carr seedlings had higher Ca2+
and lower K + concentration than leaves from Cleo seedlings. Substrate type
also influenced leaf Mg2+ concentration as leaves from perlite, with the
highest extractable, had higher leaf Mg+2 concentration than those from
sand. However, others factors such as substrate texture which can influence
water availability, root production, root to shoot ratio, root morphology and
extractable nutrient concentration, could have influenced leaf Ca2+ and K +
concentration. For example, clay-loam soil had a greater extractable Ca+
concentration than sand but leaf Ca2+ concentration was similar for Cleo
seedlings in both substrates. Leaf Ca2+ concentration in Carr seedlings was
higher in sand than in clay-loam soil. We did not evaluate root morphology
but second order lateral roots, greater branching and root tip numbers in
citrus roots can enhance the capacity to absorb nutrients such as nitrate from
nutrient solution (Sorgonà et al., 2005).
Effect of Substrate Type on Salt Tolerance
Citrus trees are considered salt sensitive mainly due to the high growth
and yield reductions caused by the Cl− and/or Na+ toxicity in the leaves
(Garcı́a-Sánchez et al., 2000). Salinity in the nutrient solution reduced
growth in seedlings from solution culture and sand but not in those from
perlite or clay-loam soil. However, the highest leaf Cl− concentration was
observed in Cleo and Carr seedlings from clay-loam soil. Thus, in this experiment growth reduction by salt treatment could not be linked directly to leaf
Cl− and Na+ concentration because physical characteristic of clay-loam soil
or drought stress perlite that could have been more limiting to the growth
than the effects of salt treatment. Based on absolute values of total plant
dry weight from salinized seedlings, the most salt tolerant seedlings were
grown in solution culture and the least salt tolerant seedlings were grown in
clay-loam soil for both Cleo and Carr.
Differences in the leaf mineral nutrient concentrations in the different
substrate types could have also influenced salt tolerance. Salinity reduced
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Citrus Seedlings under Saline Conditions
1445
total plant growth in Carr seedlings from solution culture and sand similarly
(38% relative to control treatment) despite the fact that Carr seedlings from
sand had higher leaf Cl− concentration (3.13% dw) than those from solution culture (2.20% d.w). Thus, the higher Ca2+ and K + concentration that
occurred in leaves from sand could have alleviated the negative effect of the
high leaf Cl− concentration. High leaf Ca2+ concentration in citrus ameliorated salt induced leaf abscission so Ca2+ has an intrinsic role in improving
growth of salinized plants besides maintaining a balance in Na+/Ca2+ ratio
(Romero-Aranda et al., 1998). High leaf Ca2+ concentration in plum increased salt tolerance by maintaining membrane permeability (Bolat et al.,
2006). In addition, improving K + nutrition of plants under salt stress can
greatly lower ROS production by reducing activity of NAD(P)H oxidases and
maintaining photosynthetic electron transport (Cakmak, 2005).
In general leaf Cl− concentration was higher in seedlings from Carr
and leaf Na+ concentration was higher in seedlings from Cleo supporting
the well known idea that Carrizo citrange is considered a Cl− accumulator
and Cleopatra mandarin in a Na+ accumulator (Storey and Walker, 1999).
In addition, the highest leaf Cl− and Na+ concentration was observed in
seedlings from clay-loam soil. In contrast to leaves, the root Cl− and Na+
concentrations tended to be the lowest in seedlings from clay-loam soil
suggesting that growing Carr and Cleo seedlings in clay-loam soil decreased
the ability of roots to sequester salt ions and thus increased salt accumulation
in leaves (Garcı́a-Sánchez and Syvertsen, 2006). The low leaching fraction of
this substrate could have increased the salt ion accumulation as supported by
the highest electrical conductivity and chloride concentration in the leachate
from salinized clay-loam soil. In addition, the low shoot growth in clay-loam
soil could have also increased the leaf Cl− concentration in seedlings by a
concentrating effect. It is also possible that some aspect of root growth in
the different substrates could have had a direct effect on the Cl− and/or
Na+ uptake. Carr and Cleo seedlings grown in sand had higher leaf Cl−
concentration than those grown in perlite despite the higher shoot to root
ratio in seedlings grown in sand. This too, could have been a function of
drought stress in perlite since Cl− accumulation in leaves can be diminished
by reducing plant water use (Moya et al., 2003; Garcı́a-Sánchez et al., 2006).
Leaf Ca2+ concentration in Cleo seedlings from all four substrates and
in Carr seedlings from solution culture and clay-loam soil was decreased by
salinity. Thus, salt treatment also altered mineral nutrient concentration in
leaves, although these alterations depended on the seedlings and the type
of substrate. Uptake and translocation of Ca2+ from roots to leaves in citrus
can be inhibited by salinity (Cámara et al., 2003) and translocation of Na+ to
the leaves in C. auratium can lead to a displacement of apoplastic Ca2+ (Zid
and Grignon, 1985). Leaf K + concentration was increased by salt treatment
in Carrizo citrange seedlings but not in Cleo seedlings. We found similar
responses in salinized ‘Sunburst’ mandarin trees grafted on Carrizo citrange
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1446
V. Gimeno et al.
(Garcı́a-Sánchez et al., 2002). Since Carrizo citrange is a good Na+ excluder
from leaves, increasing K + within cells could be a regulatory mechanism to
maintain osmotic balance against the high levels of Cl− under saline stress.
In seedlings from perlite and clay-loam soil with the lowest leaf P concentration, salinity increased P concentration in leaves of Carr. The increased
P accumulation in the shoot is presumably controlled at the root level and
salinity can enhance P uptake by roots (Grattan and Grieve, 1992).
In conclusion, solution culture and sand media supported more growth
in both Cleo and Carr seedlings than the perlite or clay-loam soil substrates.
Salt treatment reduced plant growth in solution culture and sand but not in
perlite or clay-loam soil, suggesting that plant growth was more limited by the
substrate than the salt effect. Seedlings from clay-loam soil had the highest
leaf Cl− and Na+ concentration as a consequence of low shoot growth, high
Cl−, and Na+ concentration in the growing media and a low ability of the
roots to sequester Cl− and Na+. The lowest leaf Cl− and Na+ concentration
in seedlings from perlite was perhaps due to decreasing the Cl− uptake
by roots. Substrate-mediated growth and nutrient uptake responses can be
more important than Cl− uptake in determining salinity tolerance in citrus
rootstock seedlings. Comparative studies of relative salt tolerance should
consider potential contributions of growth substrate to salinity responses.
ACKNOWLEDGMENTS
V. Gimeno is a PhD student supported by the Fundación Seneca (Región
de Murcia). Funding for this research came from the Ministerio de Ciencia
e Innovación (Gobierno de España), Project Plan Nacional AGL2007-65437C04-02/AGR. The authors wish to thank to Dr. Walter E. Pereira for the help
in the statistical analysis of the data.
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