Directory UMM :Data Elmu:jurnal:T:Tree Physiology:vol17.1997:
Tree Physiology 17, 141--150
© 1997 Heron Publishing----Victoria, Canada
Citrus response to salinity: growth and nutrient uptake
DIONISIO RUIZ, VICENTE MARTÍNEZ and ANTONIO CERDÁ
Department of Plant Nutrition and Physiology, Centro de Edafologia y Biologia Aplicada del Segura, CSIC, Apdo 4195, 30080 Murcia, Spain
Received April 18, 1995
Summary To determine the effects of salinity on relative
growth rate (RGR), net assimilation rate on a leaf weight basis
(NARw), leaf weight ratio (LWR), and nutrient uptake and
utilization of citrus, we grew four citrus rootstocks (sour orange, Cleopatra mandarin, Carrizo citrange and Citrus macrophylla) in nutrient solutions containing 0, 10, 20, 40 or 80 mM
NaCl for 20, 40 or 60 days. For each element analyzed, specific
absorption rate (SAR) and specific utilization rate on a leaf
basis (SURL) were calculated for the period between Days 40
and 60. Relative growth rate decreased with time for all treatments and rootstocks. Salt treatment significantly reduced both
RGR and NARw, whereas LWR showed no definite trend. In
all rootstocks, NARw, but not LWR, was significantly correlated with RGR, indicating that NARw was an important factor
underlying the salinity-induced differences in RGR among the
citrus rootstocks. At Day 60, salinity had a significant effect on
leaf concentrations of Cl, Na, K, Ca, Mg, P, Fe, Mn and Zn and
on the SAR and SURL of most elements. In general, RGR was
correlated with SAR and SURL. Therefore, in addition to
osmotic effects and the inhibitory effects of high concentrations of Cl− and Na+, an imbalance of essential nutrients may
also contribute to the reduction in plant growth under saline
conditions.
Keywords: growth analysis, NaCl, net assimilation rate, rootstock, specific absorption rate, specific utilization rate.
Introduction
Secondary salinization from irrigation sources is a growing
problem in commercial agriculture. Citrus is grown preferentially in semiarid areas where irrigation is required to produce
maximum yield. In these areas, many soils and waters contain
amounts of salts that can inhibit the growth and yields of citrus
crops. Although Citrus species are classified as salt-sensitive
(Maas 1990, 1993), there is great variation in the ability of
citrus trees to tolerate salinity depending on rootstock (Cerdá
et al. 1977, Walker and Douglas 1983, Zekri and Parsons 1992)
and scion (Lloyd et al. 1989, 1990, Nieves et al. 1991).
Most attempts to correlate growth of different citrus rootstocks or rootstock scion combinations with the physiological
effects of salinity have been made on young seedlings at a
single harvest date. These comparative studies can be misleading because they do not consider the initial biomass of the
plant, which can influence the rate of growth and the size at
harvest (Hunt 1982, Cramer et al. 1990). To take account of the
initial biomass of the plant and thus provide a more realistic
comparison of the growth rates of different citrus cultivars
under similar saline conditions, we have expressed growth as
a relative growth rate (RGR) (Poorter 1989). Few studies have
employed plant growth analysis to determine the effects of
salinity on the morphological, physiological and biochemical
factors determining RGR (Curtis and Laüchli 1986, Shennan
et al. 1987, Wickens and Cheeseman 1988, Schachtman et al.
1989, Cramer et al. 1990, Romero and Marañón 1994).
Plants acquire essential nutrients from their root system
environment. In a saline habitat, the presence of NaCl alters the
nutritional balance of plants, resulting in high ratios of
Na+/Ca2+, Na+/K+, Na+/Mg2+, Cl−/NO −3 , and Cl−/H 2PO −4 (Grattan and Grieve 1992), which may cause reductions in growth.
Major saline ions can affect nutrient uptake through competitive interactions or by affecting the ion selectivity of membranes. Examples of these effects include Na+-induced Ca2+ or
K+ deficiencies, or both, and Ca2+-induced Mg2+ deficiencies
(Grattan and Grieve 1992).
The factors responsible for the effects of salinity on citrus
are complex. The role of different rootstocks, the causes of salt
injury and the interactions of soil salinity with other environmental stresses have been reviewed by Maas (1993). Although
there are several studies showing the effects of salinity on
macro and micronutrient concentrations (Nieves et al. 1990,
Bañuls et al. 1990, Zekri 1993), little is known about how
salinity interferes with nutrient uptake and translocation, or
how these changes are related to plant growth.
We have examined salt-tolerance mechanisms operating at
the whole-plant and cellular levels in four citrus rootstocks
with different abilities to exclude Cl− or Na+, or both, to
elucidate the mechanisms underlying such differences and to
identify those characteristics that can be applied to a breeding
program designed to enhance salt tolerance in citrus. In particular, we examined the effects of salinity on ion uptake and
its relation to growth. We also analyzed the effects of increasing salinity on the absorption rates and specific utilization rates
of mineral elements in four citrus rootstocks.
Materials and methods
Four citrus rootstocks were studied: sour orange (Citrus
aurantium (L.) (SO)), Cleopatra mandarin (C. reticulata
142
RUIZ, MARTÍNEZ AND CERDÁ
blanco (CM)), Carrizo citrange (C. sinensis (L.), Osbeck ×
P. trifoliata (L.) Ref. (CC)) and C. macrophylla wester (M).
Seeds of all rootstocks were germinated in trays of sterilized
vermiculite wetted with 0.5 mM CaSO4 in the dark at 29 °C.
When the radicles were 3--4 cm in length, the seedlings were
transferred to 15-liter containers filled with a continuously
aerated nutrient solution (6 mM KNO3, 4 mM Ca(NO3)2, 2 mM
KH2PO4, 2 mM MgSO4, 20 µM Fe3+ masquolate, 25 µM
H3BO3, 2 µM MnSO4.H2O, 2 µM ZnSO4, 0.5 µM CuSO4,
0.4 µM (NH4)6Mo7O24.H2O). The solutions were renewed
weekly and the pH was adjusted daily to 6.0--6.5. The plants
were grown in a controlled environment chamber at a
day/night temperature of 25/20 °C, a day/night relative humidity of 65/85% and a 16-h photoperiod. Photon flux density was
400 µmol m −2 s −2. Light was provided by a combination of
fluorescent tubes (Philips TLD 36 W/83, Sylvania F36
W/GRO) and metal halide lamps (Osram HQI. T 400 W).
Plants were grown in culture solution for 4 months before
the salinity treatments were initiated. Groups of 18 uniform
seedlings were selected for each rootstock per saline treatment.
The salt treatment consisted of adding NaCl daily to the nutrient solution in 10 mM increments to give final NaCl concentrations of 10, 20, 40 and 80 mM. Plants cultivated in the
nutrient solution without the addition of NaCl were used as
controls.
Plants were harvested after 20, 40 and 60 days of exposure
to the salinity treatments. Fresh and dry weights of roots,
shoots and leaves, the number of leaves and the root length of
six plants of each rootstock per treatment were measured just
before the addition of NaCl and after 20, 40 and 60 days. Plant
material was dried at 65 °C to a constant weight. Root length
was determined by the line intersect method (Tennant 1975).
Relative growth rate (RGR), net assimilation rate on a leaf
weight basis (NARw) and leaf weight ratio (LWR) were calculated from the dry weight values at the three harvests. Relative
growth rate was defined as the increase in plant weight per unit
of plant weight (W) per unit of time (t):
RGR = 1/W × dW/dt.
The NARw was defined according to Garnier (1991) as the
increase in plant weight per unit of leaf weight (LW) per unit
of time:
NAR w = 1/LW × dW/dt,
and LWR was calculated as the ratio between total leaf dry
weight and total plant dry weight. These parameters are related
by the following expression:
RGR = NAR w × LWR.
Leaf mineral nutrient analysis
Dried plant tissue was digested in a concentrated nitric/perchloric acid (2/1, v/v) mixture, and Na, K, Ca, Mg, Fe, Mn, Cu
and Zn contents were measured by atomic absorption spectrophotometry. Phosphorus was measured by the molybdenum-
blue method described by Dickman and Bray (1940). Chloride
was extracted from 0.1 g of ground material with 50 ml of
deionized water and measured by electrometric titration (Guilliam 1971).
Specific absorption rate, SAR (mg g −1 day −1), an index of
the element uptake efficiency of roots, was calculated using the
formula SAR = 1/RDW ∂M/∂T, where RDW is the root dry
weight (g), M is the element amount (mg) in the whole plant
and T is the time of harvest in days. The specific utilization rate
on a leaf basis, SURL (g mg −1 day −1), an index of the efficiency
of the element in producing biomass, was calculated as the rate
of plant biomass production per unit of element in the leaves
(Hunt 1982, Romero et al. 1994). The relationships between
relative growth rate and SAR and SURL for the three harvests
were evaluated by regression equations. Because of the large
amount of data produced, leaf and root mineral nutrient concentrations, and SAR and SURL values are only presented for
the period between Days 40 and 60.
Statistical analyses
All measured parameters were statistically analyzed with the
STATGRAPHICS package (Manugistics, Inc., Rockville,
MD) for calculation of the standard error and regression lines.
Six replicates per salinity treatment per rootstock per harvesting date were used for analysis of the growth measurements.
The ANOVAs were calculated for each harvest based on five
salinity treatments and six replicates per treatment per rootstock with the SIGMASTAT package (Jandel Corporation, San
Rafael, CA).
Results
Growth analysis
Salinity resulted in decreased whole-plant biomass in all of the
rootstocks tested (Table 1). Differences in growth response
between salinized and unsalinized plants were evident after
40 days of treatment with 80 mM NaCl (data not shown).
Although differences became apparent at lower salt concentrations with increasing time of treatment, only data for the period
between Days 40 and 60 are presented. The organ that was
reduced most in biomass by the salt treatments varied with
rootstock. By the end of the experiment on Day 60, mean leaf,
stem and root dry weights of the SO rootstock that was least
affected by the 80 mM NaCl treatment were about 64, 49 and
33%, respectively, of the control values. For the CC rootstock,
in which seedlings of the most affected plants were totally
defoliated by the 80 mM NaCl treatment, stem and root dry
weights were 29 and 26% of the control values, respectively.
Reduced plant growth was associated with reductions in root
length (Figure 1A), stem growth, and new leaf production
(Figure 1B).
Relative growth rates of all plants decreased significantly in
response to salt treatment, although the effect varied with
rootstock (Figure 2A). Differences in RGR between unsalinized plants and plants treated with 80 mM NaCl were
evident after 60 days. Among rootstocks, RGR for control
plants ranged from 0.0479 to 0.0194 day −1, whereas RGR of
CITRUS AND SALINITY
143
Table 1. Effects of external NaCl on leaf (L), stem (S) and root (R) dry weights (g) of four citrus rootstocks (Citrus macrophylla, Carrizo citrange,
Cleopatra mandarin and Sour orange) following a 60-day exposure to salinity. Values are means of six replicates.
NaCl
Citrus macrophylla
(mM)
L
0
10
20
40
80
5.67
4.56
3.88
1.81
1.48
S
a
a
b
c
c
2.87
2.49
1.92
0.89
0.71
Carizo citrange
R
a
ab
b
c
c
3.32
2.36
2.27
1.22
0.82
L
a
b
b
c
c
1.69
1.14
0.96
0.48
--
Cleopatra mandarin
S
1
a
b
b
c
1.79
0.99
0.75
0.59
0.52
R
a
bc
c
c
2.18
1.48
1.10
0.97
0.69
L
a
b
c
cd
d
2.06
1.49
1.66
1.06
--
S
a
b
b
c
0.91
0.66
0.60
0.53
0.40
Sour orange
R
a
ab
ab
b
b
4.50
2.46
2.11
2.10
1.45
L
a
b
b
b
c
4.11
4.13
3.27
3.86
2.63
S
a
ab
ab
b
c
1.50
1.38
1.06
1.03
0.74
R
a
a
ab
ab
b
4.06
2.63
2.53
2.30
1.25
a
b
b
b
c
ANOVA, F-Values2
NaCl
Rootstock
NaCl × Rootstock
1
2
Leaves
20.0***
69.3***
2.9**
Stems
26.6***
36.0***
4.3***
Roots
77.6***
34.5***
1.7*
Means within a column followed by the same letter are not significantly different at P = 0.05, according to the Duncan’s test.
Significant effects are indicated by asterisks: * = P = 0.05, ** = P = 0.01 and *** = P = 0.001, NS indicates not significant at P = 0.05.
salinized plants varied between 0.0055 and − 0.028 day −1 (Figure 2A). In all rootstocks, the decline in RGR increased with
increasing salinity and with the period of exposure. For instance, after 40 days of exposure to 20 mM NaCl, RGR values
were 0.0205, 0.0142, 0.0359 and 0.0212 day −1 for the CM, CC,
M and SO rootstocks, respectively, whereas the corresponding
values at Day 60 were 0.0078, 0.0133, 0.0331, 0.0189 day −1.
Similar trends were observed in the other salt treatments.
Rootstocks showed differences in biomass accumulation over
time as a result of differences in initial size or RGR, or both.
During the study, NARw declined in all rootstocks, particularly in the salt-treated plants (data not shown); the decline
showed a similar trend to that of RGR, but was more intense
(Figure 2B, Table 2). In contrast, LWR showed no definite
trend with time (Figure 2C). Linear regessions of the relationships between RGR and NARw or LWR based on data from the
three harvests showed that NARw was significantly correlated
with RGR in all rootstocks. The determination coefficients, R2,
ranged from 0.87 for M rootstock to 0.71 for CM rootstock
(Figure 3). In contrast, LWR was not correlated with RGR,
except in the CM rootstock, (R2 = 0.30, data not shown).
Mineral nutrient concentrations
Figure 1. Effects of external NaCl on root length (A), number of leaves
(B) and weight per leaf (C) of four citrus rootstocks following a 60-day
exposure to NaCl. Error bars are ± SE (n = 6).
Tissue concentrations of Cl− and Na+ increased significantly in
response to the salt treatments (Table 3). The concentrations of
Cl− and Na+ increased in plants treated with ≤ 40 mM NaCl
until Day 20, and then remained constant for the remainder of
the study, whereas the concentrations of Cl− and Na+ in plants
treated with 80 mM NaCl increased slightly between Days 20
and 60 (data not shown). Saline-induced changes in the concentrations of the other elements analyzed varied with plant
organ and element (Table 3). Salinity lowered K+ concentrations in roots of all rootstocks and in leaves of the CM, SO and
CC rootstocks, whereas K+ concentrations increased in leaves
and roots of M rootstocks. Concentrations of Ca2+ and Mg2+
were reduced by salinity in all rootstocks, except M. In all
rootstocks, salinity increased P concentrations in leaves and Fe
144
RUIZ, MARTÍNEZ AND CERDÁ
Figure 3. Linear regressions between RGR and NARw for the three
harvests for four citrus rootstocks, SO (A), M (B), CM (C) and CC
(D). All determination coefficients are significant at P < 0.001.
Figure 2. Effects of external NaCl on RGR (A), NARw (B) and LWR
(C) of four citrus rootstocks following a 60-day exposure to NaCl.
Error bars are ± SE (n = 6).
Table 2. Analysis of variance of the RGR, NARw and LWR values
presented in Figure 2.
NaCl
Rootstock
NaCl × Rootstock
1
RGR
NARw
LWR
74.8***1
34.2***
3.4**
37.6***
4.1*
3.7**
12.2***
158.5***
6.1***
Significant effects are indicated by asterisks: * = P = 0.05, ** = P =
0.01 and *** = P = 0.001.
and Zn concentrations in roots, but no changes in Fe and Zn
concentrations were observed in leaves. The Mn response to
the salt treatments was variable. The absolute whole-plant
content of all elements analyzed increased with time (data not
shown).
Specific absorption rate
On Day 20, salinity had significantly increased the absorption
rates of Cl− and Na+ to about 4 and 3.5 mg g −1 root day −1,
respectively. After this time, the absorption rates with respect
to external salinity remained constant, although the absolute
values decreased with time (Figure 4).
On Day 20, SARs of K+, Ca2+ and Mg2+ were slightly
reduced by salt treatments in excess of 20 mM NaCl (data not
shown), but by Day 60, the SARs of these elements were
severely reduced by salinity in all rootstocks (Figure 4). Differences in SARs among rootstocks were evident after 60 days
of salt treatment. Values of SAR were higher for M and SO
rootstocks than for CC and CM rootstocks. Potassium SAR of
salinized SO and CC plants (40 mM NaCl treatment) ranged
from 3.81 to − 5.31 mg g −1 root day −1, respectively, and the
corresponding values for Ca2+ were 0.44 and − 0.16 mg g −1 root
day −1. The SAR for Mg2+ ranged between 0.030 and − 0.0043
mg g −1 root day −1 for M and CC rootstocks, respectively.
Although SAR of P decreased with time, saline inhibition
was only evident on Day 60 (Figure 4). The maximum SAR of
P (1.71 mg g −1 root day −1) was observed in SO plants in the
40 mM NaCl treatment and the minimum SAR of P (− 0.44 mg
g −1 root day −1) was observed in CC plants in the 40 mM NaCl
CITRUS AND SALINITY
145
Table 3. Effects of external NaCl concentration on mineral composition (mmol kg−1) of leaves and roots of four citrus rootstocks. Data are for
plants harvested after a 60-day salinization period. Values are means of six replicate plants.
NaCl (mM)
Cl
K
Ca
P
Fe
Mn
Zn
12
73
183
511
667
1345
1500
1471
1654
1735
323
300
304
269
350
29
23
20
25
26
893
819
857
1139
1466
0.92
1.28
1.43
0.47
1.49
1.03
0.97
1.15
1.36
1.70
0.29
0.34
0.36
0.44
0.50
Carrizo citrange leaves
0
52
10
231
20
405
40
737
80
59
288
659
718
2892
2062
2866
2162
555
410
340
270
54
46
45
35
1303
1395
1415
1406
2.36
2.03
2.01
2.00
0.97
0.78
0.80
0.63
0.64
0.57
0.68
0.62
Cleopatra mandarin leaves
0
31
10
70
20
123
40
518
80
23
185
337
783
2042
1611
1871
1347
492
459
314
344
20
20
27
18
1261
1281
1163
1380
1.28
1.86
1.79
1.59
0.97
1.06
0.86
0.80
0.45
0.56
0.48
0.53
27
87
188
319
810
12
141
231
569
1159
2992
3269
2997
2758
2643
548
504
390
303
295
72
67
59
45
39
965
1013
1080
1301
1275
1.83
1.91
1.88
1.97
2.15
1.21
1.14
1.27
0.91
1.09
0.38
0.39
0.50
0.62
0.41
Citrus macrophylla roots
0
42
10
156
20
211
40
257
80
369
20
57
95
156
609
1784
1882
1513
742
934
186
199
178
222
231
92
87
95
113
109
831
699
768
649
690
3.73
3.97
3.07
5.70
7.10
2.24
2.36
2.03
3.82
6.48
3.28
3.45
3.01
5.68
8.15
Carrizo citrange roots
0
10
20
40
80
40
223
288
425
549
19
100
155
225
397
2496
2382
1980
1763
1206
150
150
112
113
112
48
41
44
46
49
1375
898
814
692
876
3.46
6.10
4.70
6.40
6.60
3.27
2.93
3.42
3.81
1.71
3.44
3.50
4.10
3.91
3.42
Cleopatra mandarin roots
0
14
10
241
20
332
40
311
80
299
21
129
189
133
300
2042
1611
1871
1347
831
221
242
272
235
221
86
106
131
106
99
1207
1186
1383
1210
1172
3.15
3.52
4.16
4.25
4.85
1.65
1.95
4.41
1.97
1.65
2.70
2.95
5.32
2.83
2.61
Sour orange roots
0
10
20
40
80
23
180
224
275
707
3265
3210
2566
1974
1816
631
748
665
550
656
77
78
73
64
83
1205
1260
1166
892
1008
3.98
5.04
5.07
5.01
7.04
2.76
3.00
3.85
1.76
1.09
3.77
3.90
4.55
4.08
7.21
Citrus macrophylla leaves
0
32
10
47
20
103
40
209
80
445
Sour orange leaves
0
10
20
40
80
29
338
390
444
609
Na
treatment. The SAR of the micronutrients showed a tendency
to decrease with increasing salinity, although the extent of the
response varied with both rootstock and micronutrient (Figure 4).
Mg
Specific utilization rate
Specific utilization rate on a leaf basis (SURL) showed similar
trends for all of the elements studied. There was a significant
decrease in SURL with both increasing salinity and time for all
146
RUIZ, MARTÍNEZ AND CERDÁ
Figure 4. Effect of external NaCl
concentration on specific absorption rates (SAR) in four citrus rootstocks at the 60-day harvest.
Values are means of six replicates.
elements examined, except for the SURL of P which was only
affected by salinity at the 60-day harvest (Figure 5). The SURL
for Mg2+ was higher than for K+, Ca2+, and P, and, among the
micronutrients, the highest SURL was for Zn. For all elements,
the SO and M rootstocks exhibited higher SURL values than
the CM and CC rootstocks.
Regression equations of RGR with SAR and with SURL of
all elements studied were calculated to evaluate the relative
importance of these parameters for each nutrient with respect
to their effects on RGR. The correlation coefficients are presented in Table 4.
Discussion
Growth analysis
Relative growth rates of the citrus rootstocks were less than
0.05 day −1; however, small changes in RGR may result in large
variations in growth (Hardwick 1984). Furthermore, the RGR
values are similar to those reported for tree seedlings of other
species (Grime and Hunt 1975). Differences in RGR observed
among the citrus rootstocks in response to salinity may be
associated with the growth characteristics of the rootstocks
(CC and CM were slow-growing rootstocks, whereas SO and
M rootstocks grew vigorously (Table 1)) and with the inherent
partitioning of biomass between shoot and roots. The SO and
M rootstocks favored a high biomass investment in leaves and
stems (Table 1), thus ensuring increased light interception, and
consequently, increased growth. In contrast, the CC and CM
rootstocks invested relatively more biomass in roots.
Inhibition of growth of CM and CC rootstocks in the 80 mM
NaCl treatment appeared to result from large increases in foliar
concentrations of Na+ or Cl−, or both. Because the CM and CC
rootstocks were less vigorous than the SO and M rootstocks
(Table 1), growth inhibition could be a consequence of a
greater concentration effect of these ions in the CM and CC
rootstocks than in the SO and M rootstocks. In many perennial
woody crops, the growth response to salt treatment varies with
rootstock (Maas 1993).
In many physiological studies on salinity, plant growth inhibition has been related to a reduction in photosynthesis
(Munns 1993). In all of our rootstocks, NARw was significantly
correlated with RGR, but not with LWR, suggesting that
growth of citrus rootstocks was affected more by a decline in
photosynthetic capacity than by a reduction in extension
growth. These observations are consistent with those reported
in Hordeum vulgare L. by Cramer et al. (1990) and in Melilotus
segetalis (Brot.) Ser. by Romero and Marañón (1994). In
contrast, Curtis and Läuchli (1986) and Shennan et al. (1987)
concluded that inhibition of growth in Hibiscus cannabinus L.
CITRUS AND SALINITY
147
Figure 5. Effect of external NaCl concentration on specific utilization rate
on a leaf basis (SURL) in four citrus
rootstocks at the 60-day harvest. Values are means of six replicates.
Table 4. Correlation coefficients of the regression equations for the relative growth rates (RGR) with the specific absorption rates (SAR) and the
specific utilization rates on a leaf basis (SURL) of mineral elements in citrus rootstocks in response to increasing salinity in the external solution.
Significant effects indicated by asterisks: * = P < 0.5, ** = P < 0.01 and *** = P < 0.001, and NS indicates not significant at P = 0.5.
Element
Cl
Na
K
Ca
Mg
P
N
Fe
Mn
Zn
Citrus macrophylla
Carrizo citrange
SAR
SURL
SAR
0.16NS
0.27NS
0.70**
0.80***
0.55*
0.61*
0.80***
0.47NS
0.36NS
0.40NS
0.88***
0.71**
0.80***
0.83***
0.71**
0.86***
0.86***
0.80***
0.87***
0.85***
0.69**
0.76***
0.95***
0.95***
0.96***
0.96***
0.95***
0.94***
0.78***
0.87***
Cleopatra mandarin
Sour orange
SURL
SAR
SURL
SAR
SURL
0.78***
0.77***
0.94***
0.91***
0.91***
0.94***
0.94***
0.92***
0.91***
0.93***
0.20NS
0.28NS
0.97***
0.89***
0.83***
0.84***
0.88***
0.82***
0.40NS
0.57*
0.58*
0.62*
0.75***
0.72**
0.81***
0.80***
0.78***
0.75***
0.81***
0.84***
0.23NS
0.23NS
0.90***
0.95***
0.93***
0.89***
0.95***
0.88***
0.92***
0.90***
0.74***
0.60*
0.97***
0.93***
0.95***
0.98***
0.98***
0.97***
0.95***
0.93***
and Aster tripolium L. under salt stress was caused by reductions in extension growth and leaf area development, rather
than by a decline in photosynthetic capacity.
The saline-induced decrease in NARw could be associated
with a decrease in photosynthetic rate, an increase in respiration rate, or an increase in the relative amount of non-photosynthetic tissue participating in respiration (Poorter 1989). In
citrus, the primary effect of salinity on photosynthesis is stomatal closure, which leads to decreased mesophyll capacity for
CO2 assimilation (Lloyd et al. 1989, 1990). The extent of the
saline-induced reduction in photosynthesis varies with both
scion and rootstock (Walker et al. 1982, Lloyd et al. 1989,
1990, Bañuls and Primo-Millo 1992). Irrespective of the primary cause of reduced CO2 assimilation, high foliar concen-
148
RUIZ, MARTÍNEZ AND CERDÁ
trations of both Na+ and Cl− ions are capable of inducing a
reduction in CO2 assimilation (Lloyd et al. 1989, 1990, Bañuls
and Primo-Millo 1992, Garcia-Legaz et al. 1993).
The decrease in NARw with salinity in the citrus rootstock
may also be associated with an increase in respiration. Salinity
may increase whole-plant respiration (Richardson and McCree
1985), thereby inducing a higher carbohydrate requirement
(Schwarz and Gale 1981). Additional carbohydrates presumably provide the additional energy required for increased rebuilding of organelles and compounds that are disrupted by
salinity. A saline-induced increase in respiration would presumably occur at the expense net CO2 fixation, resulting in
reduced overall growth. However, Curtis et al. (1988) found an
increase in respiration in mature kenaf leaves with increasing
salinity, but no evidence that it resulted in a reduced amount of
available carbohydrate in the growing tissue. The lower NARw
values in the CC and CM rootstocks at the two highest salt
concentrations may result from their relatively high root
weights per unit leaf area, which would induce an increase in
respiration (Poorter 1989).
The effects of salinity on LWR suggest that allocation of
biomass to leaves was maintained or increased under saline
conditions. Although specific leaf area was not determined, we
observed morphological differences among leaves of the rootstocks, indicating that this LAR component may have been
affected by salt treatment. For example, in the control treatment, SO seedlings had few but large leaves (177 mg per leaf ),
M seedlings had the highest number of leaves with 75.7 mg per
leaf, whereas the individual leaf weight of CM and CC rootstocks were 64.1 and 48.2 mg, respectively (Figure 1C). The
fast-growing species (SO and M) formed large thin leaves with
a large amount of water per unit of leaf weight (Poorter 1989).
Leaf water content per unit dry weight increased with salinity
in all rootstocks.
Thus, for citrus seedlings growing in saline conditions,
NARw is the most important factor explaining differences in
RGR, whereas LWR is of secondary importance. However,
changes in specific leaf area may also be involved. We conclude that: (a) citrus species are among the most salt-sensitive
horticultural crops, because a 60-day exposure to 10 mM NaCl
had a significant impact on the relative growth rate of the
rootstocks tested, and (b) there are marked differences in the
response of RGR to salinity among rootstocks, with the SO and
M rootstocks being less sensitive to salt than the CC and CM
rootstocks.
Nutrient uptake and utilization
Much attention has been devoted to understanding adverse
effects of Na+ and Cl− on physiological and biochemical processes and how these ions contribute to plant growth inhibition
(Munns and Termaat 1986, Maas 1993, Munns 1993). Both
uptake and accumulation of Na+ and Cl− in leaves increased
with increasing concentrations of these ions in the external
solution, and the increases were paralleled by decreases in
RGR and NARw. These elements had a higher utilization rate
in control plants than in salt-stressed plants. Similar findings
in Melilotus segitalis have shown that low concentrations of
saline ions that have a minimal nutritional requirement can
stimulate growth, whereas high salt concentrations in the external solution have toxic effects (Romero et al. 1994). The
rapid decline in SURL of Na+ and Cl− with increasing time and
salinity, especially for Cl−, may underlie the inhibitory effects
of salinity on relative growth rate in citrus.
An imbalance of essential nutrients may also be a factor
involved in the salt-induced decrease in photosynthesis and
consequently in plant growth reduction. We found that the salt
treatments altered mineral nutrient distribution and decreased
both absorption rates (Figure 3) and specific utilization rates
(Figure 4) of all of the nutrients studied. The decrease in
element uptake may be partly a result of a reduction in their
activities caused by high concentrations Cl− and Na+ in the
nutrient solution (Cramer et al. 1986). Uptake may also have
been reduced as a result of competition with the salt ions in the
external solution (Grattan and Grieve 1992).
A decrease in K+ uptake and accumulation in salt-treated
plants is likely an important growth limiting factor because this
element plays an essential role in many plant processes
(Marschner 1986). The NaCl treatments decreased K+ uptake
to a higher degree in CM and CC rootstocks than in M and SO
rootstocks, which may explain the RGR differences observed
among these rootstocks. The regulation of K+ uptake and
transport in these citrus rootstocks appears to involve different
mechanisms. In M and SO rootstocks, the decreased K+ concentration in roots may be attributed to an exchange between
Na+ and K+ in the basal stem and proximal root and a further
release of K+ into the xylem (Walker and Douglas 1983,
Walker 1986). In CM and CC rootstocks, root and leaf K+
concentrations decreased with increasing salinity, indicating
that the reduction in K+ SAR results from a competitive process (Janzen and Chang 1987, Subbarao et al. 1990) or is
induced by changes in membrane integrity caused by the
displacement of Ca2+ by Na+ (Cramer et al. 1985).
Salinity reduced Ca2+ absorption rates, resulting in a high
Na+/Ca2+ ratio that may have restricted root growth (Kent and
Läuchli 1985, Hansen and Munns 1988). In all rootstocks, the
root was the organ most affected by salinity. The decrease in
Ca 2+ SAR paralleled root growth and may explain the constant
concentration of Ca2+ in this organ. Except in M rootstock, the
foliar concentration of Ca2+ was reduced by salinity, indicating
that Ca2+ translocation was inhibited. Similar findings have
been reported for other plant species (Maas and Grieve 1987,
Lazof and Läuchli 1991).
Magnesium is the central atom of the chlorophyll molecule
and has a fundamental influence on the size, structure and
function of chloroplasts (Marschner 1986). Thus, Mg2+ deficiency may lead to decreased photosynthesis and NAR, and
could contribute to reduced growth rates because RGR was
significantly correlated with NARw. In all rootstocks, except in
M plants grown at 80 mM NaCl, there was a reduction in Mg2+
leaf concentration at the final harvest compared with the control. Magnesium deficiency in citrus occurs at a leaf concentration of 0.083 mmol gdw−1 (Del Amor et al. 1985). Plants
grown in 80 mM NaCl showed a leaf Mg2+ concentration of
0.039 mmol gdw−1 at the final harvest as a result of reduced
CITRUS AND SALINITY
absorption rates and reduced translocation. The absorption
rates of Mg2+ were highly correlated with RGR for all treatments (Table 4). The utilization rates of this element were
much higher than for K+ and Ca2+.
The uptake of phosphate was not impaired by a 60-day
exposure to NaCl, so P imposed no limitation to plant growth
under the experimental conditions of our study.
The influence of salinity on micronutrient concentrations in
plants is highly variable (Grattan and Grieve 1992). In citrus,
the specific absorption and utilization rates of all micronutrients examined were reduced by salinity, indicating that the
saline-induced reduction in NARw could be associated with the
disturbed absorption of these elements, which are all directly
or indirectly involved in photosynthesis (Marschner 1986).
We conclude that citrus nutrition is altered by salinity. Relative growth rate was correlated with saline-induced declines in
NARw and in the SAR and SURL of all mineral elements. Thus,
in addition to the toxic effects of high concentrations of Na+
and Cl− in plant tissue, the saline-induced changes in mineral
nutrient uptake and utilization likely contributed to the reduction in plant growth. In support of this argument, a greater
growth reduction was observed in rootstocks CM and CC,
which have low SAR and SURL values.
Acknowledgments
The authors thank J. Abrisqueta and A. Aragon for technical assistance. This work was supported by the Comision Interministerial de
Ciencia y Tecnologia. Project No. AGR91-1096-C03-02 (Spain).
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© 1997 Heron Publishing----Victoria, Canada
Citrus response to salinity: growth and nutrient uptake
DIONISIO RUIZ, VICENTE MARTÍNEZ and ANTONIO CERDÁ
Department of Plant Nutrition and Physiology, Centro de Edafologia y Biologia Aplicada del Segura, CSIC, Apdo 4195, 30080 Murcia, Spain
Received April 18, 1995
Summary To determine the effects of salinity on relative
growth rate (RGR), net assimilation rate on a leaf weight basis
(NARw), leaf weight ratio (LWR), and nutrient uptake and
utilization of citrus, we grew four citrus rootstocks (sour orange, Cleopatra mandarin, Carrizo citrange and Citrus macrophylla) in nutrient solutions containing 0, 10, 20, 40 or 80 mM
NaCl for 20, 40 or 60 days. For each element analyzed, specific
absorption rate (SAR) and specific utilization rate on a leaf
basis (SURL) were calculated for the period between Days 40
and 60. Relative growth rate decreased with time for all treatments and rootstocks. Salt treatment significantly reduced both
RGR and NARw, whereas LWR showed no definite trend. In
all rootstocks, NARw, but not LWR, was significantly correlated with RGR, indicating that NARw was an important factor
underlying the salinity-induced differences in RGR among the
citrus rootstocks. At Day 60, salinity had a significant effect on
leaf concentrations of Cl, Na, K, Ca, Mg, P, Fe, Mn and Zn and
on the SAR and SURL of most elements. In general, RGR was
correlated with SAR and SURL. Therefore, in addition to
osmotic effects and the inhibitory effects of high concentrations of Cl− and Na+, an imbalance of essential nutrients may
also contribute to the reduction in plant growth under saline
conditions.
Keywords: growth analysis, NaCl, net assimilation rate, rootstock, specific absorption rate, specific utilization rate.
Introduction
Secondary salinization from irrigation sources is a growing
problem in commercial agriculture. Citrus is grown preferentially in semiarid areas where irrigation is required to produce
maximum yield. In these areas, many soils and waters contain
amounts of salts that can inhibit the growth and yields of citrus
crops. Although Citrus species are classified as salt-sensitive
(Maas 1990, 1993), there is great variation in the ability of
citrus trees to tolerate salinity depending on rootstock (Cerdá
et al. 1977, Walker and Douglas 1983, Zekri and Parsons 1992)
and scion (Lloyd et al. 1989, 1990, Nieves et al. 1991).
Most attempts to correlate growth of different citrus rootstocks or rootstock scion combinations with the physiological
effects of salinity have been made on young seedlings at a
single harvest date. These comparative studies can be misleading because they do not consider the initial biomass of the
plant, which can influence the rate of growth and the size at
harvest (Hunt 1982, Cramer et al. 1990). To take account of the
initial biomass of the plant and thus provide a more realistic
comparison of the growth rates of different citrus cultivars
under similar saline conditions, we have expressed growth as
a relative growth rate (RGR) (Poorter 1989). Few studies have
employed plant growth analysis to determine the effects of
salinity on the morphological, physiological and biochemical
factors determining RGR (Curtis and Laüchli 1986, Shennan
et al. 1987, Wickens and Cheeseman 1988, Schachtman et al.
1989, Cramer et al. 1990, Romero and Marañón 1994).
Plants acquire essential nutrients from their root system
environment. In a saline habitat, the presence of NaCl alters the
nutritional balance of plants, resulting in high ratios of
Na+/Ca2+, Na+/K+, Na+/Mg2+, Cl−/NO −3 , and Cl−/H 2PO −4 (Grattan and Grieve 1992), which may cause reductions in growth.
Major saline ions can affect nutrient uptake through competitive interactions or by affecting the ion selectivity of membranes. Examples of these effects include Na+-induced Ca2+ or
K+ deficiencies, or both, and Ca2+-induced Mg2+ deficiencies
(Grattan and Grieve 1992).
The factors responsible for the effects of salinity on citrus
are complex. The role of different rootstocks, the causes of salt
injury and the interactions of soil salinity with other environmental stresses have been reviewed by Maas (1993). Although
there are several studies showing the effects of salinity on
macro and micronutrient concentrations (Nieves et al. 1990,
Bañuls et al. 1990, Zekri 1993), little is known about how
salinity interferes with nutrient uptake and translocation, or
how these changes are related to plant growth.
We have examined salt-tolerance mechanisms operating at
the whole-plant and cellular levels in four citrus rootstocks
with different abilities to exclude Cl− or Na+, or both, to
elucidate the mechanisms underlying such differences and to
identify those characteristics that can be applied to a breeding
program designed to enhance salt tolerance in citrus. In particular, we examined the effects of salinity on ion uptake and
its relation to growth. We also analyzed the effects of increasing salinity on the absorption rates and specific utilization rates
of mineral elements in four citrus rootstocks.
Materials and methods
Four citrus rootstocks were studied: sour orange (Citrus
aurantium (L.) (SO)), Cleopatra mandarin (C. reticulata
142
RUIZ, MARTÍNEZ AND CERDÁ
blanco (CM)), Carrizo citrange (C. sinensis (L.), Osbeck ×
P. trifoliata (L.) Ref. (CC)) and C. macrophylla wester (M).
Seeds of all rootstocks were germinated in trays of sterilized
vermiculite wetted with 0.5 mM CaSO4 in the dark at 29 °C.
When the radicles were 3--4 cm in length, the seedlings were
transferred to 15-liter containers filled with a continuously
aerated nutrient solution (6 mM KNO3, 4 mM Ca(NO3)2, 2 mM
KH2PO4, 2 mM MgSO4, 20 µM Fe3+ masquolate, 25 µM
H3BO3, 2 µM MnSO4.H2O, 2 µM ZnSO4, 0.5 µM CuSO4,
0.4 µM (NH4)6Mo7O24.H2O). The solutions were renewed
weekly and the pH was adjusted daily to 6.0--6.5. The plants
were grown in a controlled environment chamber at a
day/night temperature of 25/20 °C, a day/night relative humidity of 65/85% and a 16-h photoperiod. Photon flux density was
400 µmol m −2 s −2. Light was provided by a combination of
fluorescent tubes (Philips TLD 36 W/83, Sylvania F36
W/GRO) and metal halide lamps (Osram HQI. T 400 W).
Plants were grown in culture solution for 4 months before
the salinity treatments were initiated. Groups of 18 uniform
seedlings were selected for each rootstock per saline treatment.
The salt treatment consisted of adding NaCl daily to the nutrient solution in 10 mM increments to give final NaCl concentrations of 10, 20, 40 and 80 mM. Plants cultivated in the
nutrient solution without the addition of NaCl were used as
controls.
Plants were harvested after 20, 40 and 60 days of exposure
to the salinity treatments. Fresh and dry weights of roots,
shoots and leaves, the number of leaves and the root length of
six plants of each rootstock per treatment were measured just
before the addition of NaCl and after 20, 40 and 60 days. Plant
material was dried at 65 °C to a constant weight. Root length
was determined by the line intersect method (Tennant 1975).
Relative growth rate (RGR), net assimilation rate on a leaf
weight basis (NARw) and leaf weight ratio (LWR) were calculated from the dry weight values at the three harvests. Relative
growth rate was defined as the increase in plant weight per unit
of plant weight (W) per unit of time (t):
RGR = 1/W × dW/dt.
The NARw was defined according to Garnier (1991) as the
increase in plant weight per unit of leaf weight (LW) per unit
of time:
NAR w = 1/LW × dW/dt,
and LWR was calculated as the ratio between total leaf dry
weight and total plant dry weight. These parameters are related
by the following expression:
RGR = NAR w × LWR.
Leaf mineral nutrient analysis
Dried plant tissue was digested in a concentrated nitric/perchloric acid (2/1, v/v) mixture, and Na, K, Ca, Mg, Fe, Mn, Cu
and Zn contents were measured by atomic absorption spectrophotometry. Phosphorus was measured by the molybdenum-
blue method described by Dickman and Bray (1940). Chloride
was extracted from 0.1 g of ground material with 50 ml of
deionized water and measured by electrometric titration (Guilliam 1971).
Specific absorption rate, SAR (mg g −1 day −1), an index of
the element uptake efficiency of roots, was calculated using the
formula SAR = 1/RDW ∂M/∂T, where RDW is the root dry
weight (g), M is the element amount (mg) in the whole plant
and T is the time of harvest in days. The specific utilization rate
on a leaf basis, SURL (g mg −1 day −1), an index of the efficiency
of the element in producing biomass, was calculated as the rate
of plant biomass production per unit of element in the leaves
(Hunt 1982, Romero et al. 1994). The relationships between
relative growth rate and SAR and SURL for the three harvests
were evaluated by regression equations. Because of the large
amount of data produced, leaf and root mineral nutrient concentrations, and SAR and SURL values are only presented for
the period between Days 40 and 60.
Statistical analyses
All measured parameters were statistically analyzed with the
STATGRAPHICS package (Manugistics, Inc., Rockville,
MD) for calculation of the standard error and regression lines.
Six replicates per salinity treatment per rootstock per harvesting date were used for analysis of the growth measurements.
The ANOVAs were calculated for each harvest based on five
salinity treatments and six replicates per treatment per rootstock with the SIGMASTAT package (Jandel Corporation, San
Rafael, CA).
Results
Growth analysis
Salinity resulted in decreased whole-plant biomass in all of the
rootstocks tested (Table 1). Differences in growth response
between salinized and unsalinized plants were evident after
40 days of treatment with 80 mM NaCl (data not shown).
Although differences became apparent at lower salt concentrations with increasing time of treatment, only data for the period
between Days 40 and 60 are presented. The organ that was
reduced most in biomass by the salt treatments varied with
rootstock. By the end of the experiment on Day 60, mean leaf,
stem and root dry weights of the SO rootstock that was least
affected by the 80 mM NaCl treatment were about 64, 49 and
33%, respectively, of the control values. For the CC rootstock,
in which seedlings of the most affected plants were totally
defoliated by the 80 mM NaCl treatment, stem and root dry
weights were 29 and 26% of the control values, respectively.
Reduced plant growth was associated with reductions in root
length (Figure 1A), stem growth, and new leaf production
(Figure 1B).
Relative growth rates of all plants decreased significantly in
response to salt treatment, although the effect varied with
rootstock (Figure 2A). Differences in RGR between unsalinized plants and plants treated with 80 mM NaCl were
evident after 60 days. Among rootstocks, RGR for control
plants ranged from 0.0479 to 0.0194 day −1, whereas RGR of
CITRUS AND SALINITY
143
Table 1. Effects of external NaCl on leaf (L), stem (S) and root (R) dry weights (g) of four citrus rootstocks (Citrus macrophylla, Carrizo citrange,
Cleopatra mandarin and Sour orange) following a 60-day exposure to salinity. Values are means of six replicates.
NaCl
Citrus macrophylla
(mM)
L
0
10
20
40
80
5.67
4.56
3.88
1.81
1.48
S
a
a
b
c
c
2.87
2.49
1.92
0.89
0.71
Carizo citrange
R
a
ab
b
c
c
3.32
2.36
2.27
1.22
0.82
L
a
b
b
c
c
1.69
1.14
0.96
0.48
--
Cleopatra mandarin
S
1
a
b
b
c
1.79
0.99
0.75
0.59
0.52
R
a
bc
c
c
2.18
1.48
1.10
0.97
0.69
L
a
b
c
cd
d
2.06
1.49
1.66
1.06
--
S
a
b
b
c
0.91
0.66
0.60
0.53
0.40
Sour orange
R
a
ab
ab
b
b
4.50
2.46
2.11
2.10
1.45
L
a
b
b
b
c
4.11
4.13
3.27
3.86
2.63
S
a
ab
ab
b
c
1.50
1.38
1.06
1.03
0.74
R
a
a
ab
ab
b
4.06
2.63
2.53
2.30
1.25
a
b
b
b
c
ANOVA, F-Values2
NaCl
Rootstock
NaCl × Rootstock
1
2
Leaves
20.0***
69.3***
2.9**
Stems
26.6***
36.0***
4.3***
Roots
77.6***
34.5***
1.7*
Means within a column followed by the same letter are not significantly different at P = 0.05, according to the Duncan’s test.
Significant effects are indicated by asterisks: * = P = 0.05, ** = P = 0.01 and *** = P = 0.001, NS indicates not significant at P = 0.05.
salinized plants varied between 0.0055 and − 0.028 day −1 (Figure 2A). In all rootstocks, the decline in RGR increased with
increasing salinity and with the period of exposure. For instance, after 40 days of exposure to 20 mM NaCl, RGR values
were 0.0205, 0.0142, 0.0359 and 0.0212 day −1 for the CM, CC,
M and SO rootstocks, respectively, whereas the corresponding
values at Day 60 were 0.0078, 0.0133, 0.0331, 0.0189 day −1.
Similar trends were observed in the other salt treatments.
Rootstocks showed differences in biomass accumulation over
time as a result of differences in initial size or RGR, or both.
During the study, NARw declined in all rootstocks, particularly in the salt-treated plants (data not shown); the decline
showed a similar trend to that of RGR, but was more intense
(Figure 2B, Table 2). In contrast, LWR showed no definite
trend with time (Figure 2C). Linear regessions of the relationships between RGR and NARw or LWR based on data from the
three harvests showed that NARw was significantly correlated
with RGR in all rootstocks. The determination coefficients, R2,
ranged from 0.87 for M rootstock to 0.71 for CM rootstock
(Figure 3). In contrast, LWR was not correlated with RGR,
except in the CM rootstock, (R2 = 0.30, data not shown).
Mineral nutrient concentrations
Figure 1. Effects of external NaCl on root length (A), number of leaves
(B) and weight per leaf (C) of four citrus rootstocks following a 60-day
exposure to NaCl. Error bars are ± SE (n = 6).
Tissue concentrations of Cl− and Na+ increased significantly in
response to the salt treatments (Table 3). The concentrations of
Cl− and Na+ increased in plants treated with ≤ 40 mM NaCl
until Day 20, and then remained constant for the remainder of
the study, whereas the concentrations of Cl− and Na+ in plants
treated with 80 mM NaCl increased slightly between Days 20
and 60 (data not shown). Saline-induced changes in the concentrations of the other elements analyzed varied with plant
organ and element (Table 3). Salinity lowered K+ concentrations in roots of all rootstocks and in leaves of the CM, SO and
CC rootstocks, whereas K+ concentrations increased in leaves
and roots of M rootstocks. Concentrations of Ca2+ and Mg2+
were reduced by salinity in all rootstocks, except M. In all
rootstocks, salinity increased P concentrations in leaves and Fe
144
RUIZ, MARTÍNEZ AND CERDÁ
Figure 3. Linear regressions between RGR and NARw for the three
harvests for four citrus rootstocks, SO (A), M (B), CM (C) and CC
(D). All determination coefficients are significant at P < 0.001.
Figure 2. Effects of external NaCl on RGR (A), NARw (B) and LWR
(C) of four citrus rootstocks following a 60-day exposure to NaCl.
Error bars are ± SE (n = 6).
Table 2. Analysis of variance of the RGR, NARw and LWR values
presented in Figure 2.
NaCl
Rootstock
NaCl × Rootstock
1
RGR
NARw
LWR
74.8***1
34.2***
3.4**
37.6***
4.1*
3.7**
12.2***
158.5***
6.1***
Significant effects are indicated by asterisks: * = P = 0.05, ** = P =
0.01 and *** = P = 0.001.
and Zn concentrations in roots, but no changes in Fe and Zn
concentrations were observed in leaves. The Mn response to
the salt treatments was variable. The absolute whole-plant
content of all elements analyzed increased with time (data not
shown).
Specific absorption rate
On Day 20, salinity had significantly increased the absorption
rates of Cl− and Na+ to about 4 and 3.5 mg g −1 root day −1,
respectively. After this time, the absorption rates with respect
to external salinity remained constant, although the absolute
values decreased with time (Figure 4).
On Day 20, SARs of K+, Ca2+ and Mg2+ were slightly
reduced by salt treatments in excess of 20 mM NaCl (data not
shown), but by Day 60, the SARs of these elements were
severely reduced by salinity in all rootstocks (Figure 4). Differences in SARs among rootstocks were evident after 60 days
of salt treatment. Values of SAR were higher for M and SO
rootstocks than for CC and CM rootstocks. Potassium SAR of
salinized SO and CC plants (40 mM NaCl treatment) ranged
from 3.81 to − 5.31 mg g −1 root day −1, respectively, and the
corresponding values for Ca2+ were 0.44 and − 0.16 mg g −1 root
day −1. The SAR for Mg2+ ranged between 0.030 and − 0.0043
mg g −1 root day −1 for M and CC rootstocks, respectively.
Although SAR of P decreased with time, saline inhibition
was only evident on Day 60 (Figure 4). The maximum SAR of
P (1.71 mg g −1 root day −1) was observed in SO plants in the
40 mM NaCl treatment and the minimum SAR of P (− 0.44 mg
g −1 root day −1) was observed in CC plants in the 40 mM NaCl
CITRUS AND SALINITY
145
Table 3. Effects of external NaCl concentration on mineral composition (mmol kg−1) of leaves and roots of four citrus rootstocks. Data are for
plants harvested after a 60-day salinization period. Values are means of six replicate plants.
NaCl (mM)
Cl
K
Ca
P
Fe
Mn
Zn
12
73
183
511
667
1345
1500
1471
1654
1735
323
300
304
269
350
29
23
20
25
26
893
819
857
1139
1466
0.92
1.28
1.43
0.47
1.49
1.03
0.97
1.15
1.36
1.70
0.29
0.34
0.36
0.44
0.50
Carrizo citrange leaves
0
52
10
231
20
405
40
737
80
59
288
659
718
2892
2062
2866
2162
555
410
340
270
54
46
45
35
1303
1395
1415
1406
2.36
2.03
2.01
2.00
0.97
0.78
0.80
0.63
0.64
0.57
0.68
0.62
Cleopatra mandarin leaves
0
31
10
70
20
123
40
518
80
23
185
337
783
2042
1611
1871
1347
492
459
314
344
20
20
27
18
1261
1281
1163
1380
1.28
1.86
1.79
1.59
0.97
1.06
0.86
0.80
0.45
0.56
0.48
0.53
27
87
188
319
810
12
141
231
569
1159
2992
3269
2997
2758
2643
548
504
390
303
295
72
67
59
45
39
965
1013
1080
1301
1275
1.83
1.91
1.88
1.97
2.15
1.21
1.14
1.27
0.91
1.09
0.38
0.39
0.50
0.62
0.41
Citrus macrophylla roots
0
42
10
156
20
211
40
257
80
369
20
57
95
156
609
1784
1882
1513
742
934
186
199
178
222
231
92
87
95
113
109
831
699
768
649
690
3.73
3.97
3.07
5.70
7.10
2.24
2.36
2.03
3.82
6.48
3.28
3.45
3.01
5.68
8.15
Carrizo citrange roots
0
10
20
40
80
40
223
288
425
549
19
100
155
225
397
2496
2382
1980
1763
1206
150
150
112
113
112
48
41
44
46
49
1375
898
814
692
876
3.46
6.10
4.70
6.40
6.60
3.27
2.93
3.42
3.81
1.71
3.44
3.50
4.10
3.91
3.42
Cleopatra mandarin roots
0
14
10
241
20
332
40
311
80
299
21
129
189
133
300
2042
1611
1871
1347
831
221
242
272
235
221
86
106
131
106
99
1207
1186
1383
1210
1172
3.15
3.52
4.16
4.25
4.85
1.65
1.95
4.41
1.97
1.65
2.70
2.95
5.32
2.83
2.61
Sour orange roots
0
10
20
40
80
23
180
224
275
707
3265
3210
2566
1974
1816
631
748
665
550
656
77
78
73
64
83
1205
1260
1166
892
1008
3.98
5.04
5.07
5.01
7.04
2.76
3.00
3.85
1.76
1.09
3.77
3.90
4.55
4.08
7.21
Citrus macrophylla leaves
0
32
10
47
20
103
40
209
80
445
Sour orange leaves
0
10
20
40
80
29
338
390
444
609
Na
treatment. The SAR of the micronutrients showed a tendency
to decrease with increasing salinity, although the extent of the
response varied with both rootstock and micronutrient (Figure 4).
Mg
Specific utilization rate
Specific utilization rate on a leaf basis (SURL) showed similar
trends for all of the elements studied. There was a significant
decrease in SURL with both increasing salinity and time for all
146
RUIZ, MARTÍNEZ AND CERDÁ
Figure 4. Effect of external NaCl
concentration on specific absorption rates (SAR) in four citrus rootstocks at the 60-day harvest.
Values are means of six replicates.
elements examined, except for the SURL of P which was only
affected by salinity at the 60-day harvest (Figure 5). The SURL
for Mg2+ was higher than for K+, Ca2+, and P, and, among the
micronutrients, the highest SURL was for Zn. For all elements,
the SO and M rootstocks exhibited higher SURL values than
the CM and CC rootstocks.
Regression equations of RGR with SAR and with SURL of
all elements studied were calculated to evaluate the relative
importance of these parameters for each nutrient with respect
to their effects on RGR. The correlation coefficients are presented in Table 4.
Discussion
Growth analysis
Relative growth rates of the citrus rootstocks were less than
0.05 day −1; however, small changes in RGR may result in large
variations in growth (Hardwick 1984). Furthermore, the RGR
values are similar to those reported for tree seedlings of other
species (Grime and Hunt 1975). Differences in RGR observed
among the citrus rootstocks in response to salinity may be
associated with the growth characteristics of the rootstocks
(CC and CM were slow-growing rootstocks, whereas SO and
M rootstocks grew vigorously (Table 1)) and with the inherent
partitioning of biomass between shoot and roots. The SO and
M rootstocks favored a high biomass investment in leaves and
stems (Table 1), thus ensuring increased light interception, and
consequently, increased growth. In contrast, the CC and CM
rootstocks invested relatively more biomass in roots.
Inhibition of growth of CM and CC rootstocks in the 80 mM
NaCl treatment appeared to result from large increases in foliar
concentrations of Na+ or Cl−, or both. Because the CM and CC
rootstocks were less vigorous than the SO and M rootstocks
(Table 1), growth inhibition could be a consequence of a
greater concentration effect of these ions in the CM and CC
rootstocks than in the SO and M rootstocks. In many perennial
woody crops, the growth response to salt treatment varies with
rootstock (Maas 1993).
In many physiological studies on salinity, plant growth inhibition has been related to a reduction in photosynthesis
(Munns 1993). In all of our rootstocks, NARw was significantly
correlated with RGR, but not with LWR, suggesting that
growth of citrus rootstocks was affected more by a decline in
photosynthetic capacity than by a reduction in extension
growth. These observations are consistent with those reported
in Hordeum vulgare L. by Cramer et al. (1990) and in Melilotus
segetalis (Brot.) Ser. by Romero and Marañón (1994). In
contrast, Curtis and Läuchli (1986) and Shennan et al. (1987)
concluded that inhibition of growth in Hibiscus cannabinus L.
CITRUS AND SALINITY
147
Figure 5. Effect of external NaCl concentration on specific utilization rate
on a leaf basis (SURL) in four citrus
rootstocks at the 60-day harvest. Values are means of six replicates.
Table 4. Correlation coefficients of the regression equations for the relative growth rates (RGR) with the specific absorption rates (SAR) and the
specific utilization rates on a leaf basis (SURL) of mineral elements in citrus rootstocks in response to increasing salinity in the external solution.
Significant effects indicated by asterisks: * = P < 0.5, ** = P < 0.01 and *** = P < 0.001, and NS indicates not significant at P = 0.5.
Element
Cl
Na
K
Ca
Mg
P
N
Fe
Mn
Zn
Citrus macrophylla
Carrizo citrange
SAR
SURL
SAR
0.16NS
0.27NS
0.70**
0.80***
0.55*
0.61*
0.80***
0.47NS
0.36NS
0.40NS
0.88***
0.71**
0.80***
0.83***
0.71**
0.86***
0.86***
0.80***
0.87***
0.85***
0.69**
0.76***
0.95***
0.95***
0.96***
0.96***
0.95***
0.94***
0.78***
0.87***
Cleopatra mandarin
Sour orange
SURL
SAR
SURL
SAR
SURL
0.78***
0.77***
0.94***
0.91***
0.91***
0.94***
0.94***
0.92***
0.91***
0.93***
0.20NS
0.28NS
0.97***
0.89***
0.83***
0.84***
0.88***
0.82***
0.40NS
0.57*
0.58*
0.62*
0.75***
0.72**
0.81***
0.80***
0.78***
0.75***
0.81***
0.84***
0.23NS
0.23NS
0.90***
0.95***
0.93***
0.89***
0.95***
0.88***
0.92***
0.90***
0.74***
0.60*
0.97***
0.93***
0.95***
0.98***
0.98***
0.97***
0.95***
0.93***
and Aster tripolium L. under salt stress was caused by reductions in extension growth and leaf area development, rather
than by a decline in photosynthetic capacity.
The saline-induced decrease in NARw could be associated
with a decrease in photosynthetic rate, an increase in respiration rate, or an increase in the relative amount of non-photosynthetic tissue participating in respiration (Poorter 1989). In
citrus, the primary effect of salinity on photosynthesis is stomatal closure, which leads to decreased mesophyll capacity for
CO2 assimilation (Lloyd et al. 1989, 1990). The extent of the
saline-induced reduction in photosynthesis varies with both
scion and rootstock (Walker et al. 1982, Lloyd et al. 1989,
1990, Bañuls and Primo-Millo 1992). Irrespective of the primary cause of reduced CO2 assimilation, high foliar concen-
148
RUIZ, MARTÍNEZ AND CERDÁ
trations of both Na+ and Cl− ions are capable of inducing a
reduction in CO2 assimilation (Lloyd et al. 1989, 1990, Bañuls
and Primo-Millo 1992, Garcia-Legaz et al. 1993).
The decrease in NARw with salinity in the citrus rootstock
may also be associated with an increase in respiration. Salinity
may increase whole-plant respiration (Richardson and McCree
1985), thereby inducing a higher carbohydrate requirement
(Schwarz and Gale 1981). Additional carbohydrates presumably provide the additional energy required for increased rebuilding of organelles and compounds that are disrupted by
salinity. A saline-induced increase in respiration would presumably occur at the expense net CO2 fixation, resulting in
reduced overall growth. However, Curtis et al. (1988) found an
increase in respiration in mature kenaf leaves with increasing
salinity, but no evidence that it resulted in a reduced amount of
available carbohydrate in the growing tissue. The lower NARw
values in the CC and CM rootstocks at the two highest salt
concentrations may result from their relatively high root
weights per unit leaf area, which would induce an increase in
respiration (Poorter 1989).
The effects of salinity on LWR suggest that allocation of
biomass to leaves was maintained or increased under saline
conditions. Although specific leaf area was not determined, we
observed morphological differences among leaves of the rootstocks, indicating that this LAR component may have been
affected by salt treatment. For example, in the control treatment, SO seedlings had few but large leaves (177 mg per leaf ),
M seedlings had the highest number of leaves with 75.7 mg per
leaf, whereas the individual leaf weight of CM and CC rootstocks were 64.1 and 48.2 mg, respectively (Figure 1C). The
fast-growing species (SO and M) formed large thin leaves with
a large amount of water per unit of leaf weight (Poorter 1989).
Leaf water content per unit dry weight increased with salinity
in all rootstocks.
Thus, for citrus seedlings growing in saline conditions,
NARw is the most important factor explaining differences in
RGR, whereas LWR is of secondary importance. However,
changes in specific leaf area may also be involved. We conclude that: (a) citrus species are among the most salt-sensitive
horticultural crops, because a 60-day exposure to 10 mM NaCl
had a significant impact on the relative growth rate of the
rootstocks tested, and (b) there are marked differences in the
response of RGR to salinity among rootstocks, with the SO and
M rootstocks being less sensitive to salt than the CC and CM
rootstocks.
Nutrient uptake and utilization
Much attention has been devoted to understanding adverse
effects of Na+ and Cl− on physiological and biochemical processes and how these ions contribute to plant growth inhibition
(Munns and Termaat 1986, Maas 1993, Munns 1993). Both
uptake and accumulation of Na+ and Cl− in leaves increased
with increasing concentrations of these ions in the external
solution, and the increases were paralleled by decreases in
RGR and NARw. These elements had a higher utilization rate
in control plants than in salt-stressed plants. Similar findings
in Melilotus segitalis have shown that low concentrations of
saline ions that have a minimal nutritional requirement can
stimulate growth, whereas high salt concentrations in the external solution have toxic effects (Romero et al. 1994). The
rapid decline in SURL of Na+ and Cl− with increasing time and
salinity, especially for Cl−, may underlie the inhibitory effects
of salinity on relative growth rate in citrus.
An imbalance of essential nutrients may also be a factor
involved in the salt-induced decrease in photosynthesis and
consequently in plant growth reduction. We found that the salt
treatments altered mineral nutrient distribution and decreased
both absorption rates (Figure 3) and specific utilization rates
(Figure 4) of all of the nutrients studied. The decrease in
element uptake may be partly a result of a reduction in their
activities caused by high concentrations Cl− and Na+ in the
nutrient solution (Cramer et al. 1986). Uptake may also have
been reduced as a result of competition with the salt ions in the
external solution (Grattan and Grieve 1992).
A decrease in K+ uptake and accumulation in salt-treated
plants is likely an important growth limiting factor because this
element plays an essential role in many plant processes
(Marschner 1986). The NaCl treatments decreased K+ uptake
to a higher degree in CM and CC rootstocks than in M and SO
rootstocks, which may explain the RGR differences observed
among these rootstocks. The regulation of K+ uptake and
transport in these citrus rootstocks appears to involve different
mechanisms. In M and SO rootstocks, the decreased K+ concentration in roots may be attributed to an exchange between
Na+ and K+ in the basal stem and proximal root and a further
release of K+ into the xylem (Walker and Douglas 1983,
Walker 1986). In CM and CC rootstocks, root and leaf K+
concentrations decreased with increasing salinity, indicating
that the reduction in K+ SAR results from a competitive process (Janzen and Chang 1987, Subbarao et al. 1990) or is
induced by changes in membrane integrity caused by the
displacement of Ca2+ by Na+ (Cramer et al. 1985).
Salinity reduced Ca2+ absorption rates, resulting in a high
Na+/Ca2+ ratio that may have restricted root growth (Kent and
Läuchli 1985, Hansen and Munns 1988). In all rootstocks, the
root was the organ most affected by salinity. The decrease in
Ca 2+ SAR paralleled root growth and may explain the constant
concentration of Ca2+ in this organ. Except in M rootstock, the
foliar concentration of Ca2+ was reduced by salinity, indicating
that Ca2+ translocation was inhibited. Similar findings have
been reported for other plant species (Maas and Grieve 1987,
Lazof and Läuchli 1991).
Magnesium is the central atom of the chlorophyll molecule
and has a fundamental influence on the size, structure and
function of chloroplasts (Marschner 1986). Thus, Mg2+ deficiency may lead to decreased photosynthesis and NAR, and
could contribute to reduced growth rates because RGR was
significantly correlated with NARw. In all rootstocks, except in
M plants grown at 80 mM NaCl, there was a reduction in Mg2+
leaf concentration at the final harvest compared with the control. Magnesium deficiency in citrus occurs at a leaf concentration of 0.083 mmol gdw−1 (Del Amor et al. 1985). Plants
grown in 80 mM NaCl showed a leaf Mg2+ concentration of
0.039 mmol gdw−1 at the final harvest as a result of reduced
CITRUS AND SALINITY
absorption rates and reduced translocation. The absorption
rates of Mg2+ were highly correlated with RGR for all treatments (Table 4). The utilization rates of this element were
much higher than for K+ and Ca2+.
The uptake of phosphate was not impaired by a 60-day
exposure to NaCl, so P imposed no limitation to plant growth
under the experimental conditions of our study.
The influence of salinity on micronutrient concentrations in
plants is highly variable (Grattan and Grieve 1992). In citrus,
the specific absorption and utilization rates of all micronutrients examined were reduced by salinity, indicating that the
saline-induced reduction in NARw could be associated with the
disturbed absorption of these elements, which are all directly
or indirectly involved in photosynthesis (Marschner 1986).
We conclude that citrus nutrition is altered by salinity. Relative growth rate was correlated with saline-induced declines in
NARw and in the SAR and SURL of all mineral elements. Thus,
in addition to the toxic effects of high concentrations of Na+
and Cl− in plant tissue, the saline-induced changes in mineral
nutrient uptake and utilization likely contributed to the reduction in plant growth. In support of this argument, a greater
growth reduction was observed in rootstocks CM and CC,
which have low SAR and SURL values.
Acknowledgments
The authors thank J. Abrisqueta and A. Aragon for technical assistance. This work was supported by the Comision Interministerial de
Ciencia y Tecnologia. Project No. AGR91-1096-C03-02 (Spain).
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