Journal of Plant Nutrition, 32: 618–628, 2009

Copyright © Taylor & Francis Group, LLC

ISSN: 0190-4167 print / 1532-4087 online
DOI: 10.1080/01904160802715430

Influence of Aluminum on the Growth and Organic

Acid Exudation in Alfalfa Cultivars Grown
in Nutrient Solution
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Howard Langer,1 Mara Cea,2 Gustavo Curaqueo,1 and Fernando Borie1

1
Departamento de Ciencias Quı́micas, Universidad de La Frontera, Temuco, Chile
2
Instituto de Agroindustria, Universidad de La Frontera, Temuco, Chile

ABSTRACT

A study was conducted to evaluate the effects of aluminum (Al) in nutrient solutions on
the dry weight (DW) yield, Al and phosphorus (P) contents, and organic acid exudation
in alfalfa (Medicago sativa L.). Four alfalfa cultivars (‘Robust’, ‘Sceptre’, ‘Aquarius’,
and ‘California-55’) were grown in nutrient solution at pH 4.5 and 6.0, with (50 and
100 µM) and without Al. The results revealed that Al caused a significant reduction
in DW, especially in pH 4.5 treatment. Organic acid exudation was affected by pH and
Al treatments. Citrate and succinate exudation increased with the high Al treatment at
pH 4.5. However, no relationship between pH and carboxylate exudation was observed
at pH 6.0. Accumulation of P and Al in roots suggests the existence of an exclusion
mechanism for Al in alfalfa. Selection of cultivars with enhanced organic exudation
capacity in response to Al might be useful for alfalfa production in moderately acidic
soils.

Keywords: aluminum, alfalfa, citrate, succinate, dry weight yield, nutrient solution

INTRODUCTION

Aluminum (Al) toxicity is considered to be a major environmental stress that

limits crop and forage production in acid soils (Mora et al., 1999). Aluminum
inhibits root cell elongation and division at the cellular level. The primary
symptom of Al toxicity in plants is a reduction in root growth, which reduces
water and nutrient uptake. Aluminum seems to be the most important species

Received 6 September 2007; accepted 27 February 2008.

Address correspondence to Howard Langer, Departamento de Ciencias Quı́micas,
Universidad de La Frontera, Casilla 54-D, Temuco, Chile. E-mail: langerh@ufro.cl

618
 Differential Response to Aluminum in Alfalfa 619

of rhizotoxic Al, although some authors indicate that other monomeric species,
such as AlOH2+ and Al(OH) 2 + , are even more toxic to soybean than Al3+
(Alva et al., 1986).
Plants have evolved two major Al mechanisms: Al tolerance, known as
internal tolerance and Al exclusion from the root apex (Matsumoto, 2000;
Barceló and Poschenrieder, 2002; Kochian et al., 2004; Kochian et al., 2005).
In this category, the exudation of Al-inducible organic acids has been reported
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to confer Al tolerance in several crop and pasture species.

Both the type and amount of organic acids released in response to Al
vary widely among plant species and even cultivars within species (Zheng
et al., 1998). For example, Dong et al. (2004) observed that soybeans exposed
to Al stimulated the release of citrate, whereas a phosphorus (P) deficiency
increased the exudation of malate and oxalate instead. Several studies have
reported the exudation of citrate in response to Al in some legumes species
such as soybean (Yang et al., 2000; Silva et al., 2001) and bean (Miyasaka
et al., 1991). On the other hand, the exudation of succinate has been reported in
response to P deficiency in alfalfa plants (Lipton et al., 1987) or as a result of
the overexpression of malate dehidrogenase enzyme, resulting in an increase
in the exudation of citrate, oxalate, malate, succinate and acetate (Tesfaye
et al., 2001).
In Chile, much of the alfalfa is grown on acidic Andisols (pH < 5.5)
displaying high levels of extractable Al. The inherent acidity combined with
the use of ammonia fertilizers contributes to increase the levels of Al in the
soil solution and thus, the Al phytotoxicity. The use of alfalfa cultivars with
an increased ability to exudate organic acids may improve the alfalfa yields in
moderately acidic soils.
To our knowledge, this is the first attempt to elucidate the role of organic
acid exudation in response to Al rhizotoxicity in alfalfa. The major aim of this
study was to determine the differential response in organic acid exudation by
four alfalfa (Medicago sativa L.) cultivars exposed to different Al levels and
pH values in nutrient solution experiments.

MATERIALS AND METHODS

Growth Conditions and Solutions

Seeds of four alfalfa cultivars (‘Robust’, ‘Sceptre’, ‘Aquarius ‘and ‘California-

55’) were selected for shape, color, and size. Seeds were surface sterilized with
ethanol (70% v/v) and sodium hypoclorite (4%), and germinated on wet filter
paper in the dark for 4 d. Eight seedlings were transplanted to polypropylene
pots containing 1 L of aireated Taylor and Foy nutrient solution (Taylor and
Foy, 1985) with the following composition in macro- and micronutrients (mM):
ammonium nitrate (NH 4 NO 3 ), 120; sodium chloride (NaCl), 150; magnesium
 620 H. Langer et al.

chloride (MgCl 2 6H 2 O), 162; monopotassium phosphate (KH 2 PO 4 ), 120;

ammonium chloride (NH 4 Cl), 66; sodium nitrate (NaNO 3 ), 252; sodium
sulfate (Na 2 SO 4 10H 2 O), 72; potassium nitrate (KNO 3 ), 330; calcium nitrate
[Ca(NO 3 ) 2 4H 2 O], 762; boric acid (H 3 BO 3 ), 3.96; zinc sulfate (ZnSO 4
7H 2 O), 0.36; manganese sulfate (MnSO 4 H 2 O), 1.44; ammonium molybdate
[(NH 4 ) 6 Mo 7 O 24 4H 2 O], 0.06; iron (Fe)-ethylenediaminetetraacetic acid
(EDTA), 10.74; copper sulfate (CuSO 4 5H 2 O), 0.12. Nutrient solutions were
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renewed every 5d. Three Al levels (0, 50, and 100 µM) added in the form of
aluminum chloride (AlCl 3 ) and two levels of pH (4.5 and 6.0) were tested.
The pH was kept constant by daily addition of 0.1 M hydrochloric acid (HCl)
or sodium hydroxide (NaOH) as required. The experiment was conducted in
a controlled-environment growth chamber for 31 d at 22◦ C with a 50 to 60%
relative humidity and a 16 h light photoperiod.

Collection and Analysis of Root Exudates

Collection of exudates was performed according to Rosas et al. (2007). After

fifteen days of growth, roots of eight intact plants that had been treated with 0
to 100 µM Al were submerged for 1 h in aerated deionized water (50 mL) and
the resulting solution containing exudates was stored at −20◦ C. A short elution
time was selected in order to reduce possible degradation of organic acids by
microorganisms (Jones et al., 1996).
In order to quantify the concentration of organic acids (citrate and succi-
nate), root exudates were lyophilized and the residue re-dissolved in 5 mL of
deionized water, filtered (0.45 µm) and used for HPLC analysis. Separation
was achieved on a 250 × 4 mm reverse phase column (Merck, LiChrospher
100 RP-18, 5-µm particle size). Sample solutions (20 µL) were injected into
the column, and a 200 mM ortho-phosphoric acid (pH 2.1) solution was used
for isocratic elution at a flow rate of 1 mL min−1 with UV detection at 210 nm.
Preliminary studies with standard organic acids indicated that recovery of the
organic anions was about 98%.
Aluminum speciation and the formation of Al-complexes in the nutrient so-
lutions was evaluated using the modified GEOCHEM v.2.0. computer program
(Parker et al., 1987). The total amount of organic acid exuded in 1 L of nutrient
solution was considered for chemical speciation (8 plants per treatment).

Dry Weight and Chemical Analysis of Plants

After 45 d, the plants were harvested, separated into roots and shoots,
oven-dried (60◦ C for 48 h) and weighed. Plant samples were ashed at 500◦ C
for 8 h and then analysed for Al and phosphorus (P) content following
digestion with 2 M HCl. Aluminum was measured by using atomic absorption
 Differential Response to Aluminum in Alfalfa 621

spectrophotometry (AAS) and P was measured colorimetrically according to

the vanadophosphomolybdate method of Sadzawka et al. (2004).

Statistics

The experimental design was a randomized complete block with three repli-
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cations. The results were analyzed by ANOVA and were compared using the
Tukey Test (P ≤ 0.05).

RESULTS AND DISCUSSION

Effects of Al and pH on the Growth of Alfalfa Cultivars

Aluminum reduced the shoot and root DW of alfalfa growing in nutrient so-
lutions, especially at pH 4.5 (Figure 1). The plant roots exposed to higher Al
levels were brown and poorly developed, with the major effect of Al exposure
observed in shoot DW. This clearly indicates that Al affected nutrient uptake,
and reduced the plant growth. However, the magnitude of growth reduction
depended of the alfalfa cultivar. When the plants were exposed to 50 µM Al,

Figure 1. Effects of Al on shoot and root dry weights (g dry weight pot−1 ) in four
alfalfa cultivars in nutrient solutions at two pH levels. Mean of three replicates. Bars in
columns indicates standar error (SE).
 622 H. Langer et al.

Table 1
Relative growth reduction (%), related to the control without Al, in plants exposed to
Al in nutrient solutions at pH 4.5 and 6.0

pH 4.5 pH 6.0

Cultivars 50 µM Al 100 µM Al 50 µM Al 100 µM Al

California 55 21 52 67 50
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Robust 55 61 24 29
Sceptre 38 63 17 46
Aquarius 44 55 20 0

the shoot DW was reduced 21 and 55% for California 55 and ‘Robust’, respec-
tively, in relation to the control treatments without Al. At higher Al levels (100
µM) the reduction increased to 52 and 63% for ‘California 55’ and ‘Sceptre’,
respectively. Differential response in shoot DW in absence of Al likely reflects
different yield potentials among alfalfa cultivars (Table 1). Similar variations
in cultivar response to Al have been previously reported in some crop species
(Foy, 1988). The use of alfalfa cultivars that show a high degree of Al tolerance
under nutrient solutions may increase forage production under field conditions.
Although Al3+ is the predominant aqueous species at low pH as shown in
Table 2, other Al monomeric species such AlOH2+ , Al(OH) 2 + , and Al(OH) 4 −
and the polymer Al 13 polymer present at higher pH values have been implicated
in the reduction of plant yields (Kinraide, 1991). It is interesting to mention
that nutrient levels in solution remained adequate to ensure the plant growth,
so they were not strongly affected by the addition of Al to the nutrient solution
(data not shown), which concentrates the possible toxicity of Al-OH forms at
pH 6.0, more than a deficiency in some other nutrient.
Levels of Al and P in the shoots of the alfalfa cultivars growing at different
pH levels are shown in Table 3. Levels of P in shoots were not affected by Al
treatments at both pH levels. However, an increase in Al concentrations in the
shoot tissues for ‘California 55’ and ‘Robust’ was observed. On the other hand,
an increase in root concentration of Al and P was observed in the plants exposed
to Al. Higher levels of Al in roots were achieved by plants growing at pH 4.5
with Al (100 µM), varying from 6.35 to 7.84 g Al kg−1 DW in ‘Aquarius’ and
‘California 55’ cultivars, respectively. Levels of P in roots were from 0.28 to
0.38% in ‘Aquarius’ and ‘Sceptre’ cultivars, respectively.
Our results are in agreement with those reported by Zheng et al. (2005), who
studied the accumulation of Al with P in two cultivars of Fygopyrum esculentum
expressing different degrees of Al tolerance, showing high concentrations of
P and Al in the roots of the tolerant cultivar. These results suggest that the
precipitation of Al with P in root tissues is one mechanism of reducing Al
toxicity. This hypothesis is also supported by the fact that accumulation of P
 Differential Response to Aluminum in Alfalfa 623

Table 2
Distribution of Al species in nutrient solutions at different levels of Al and pH as
response to organic acid exudation from alfalfa roots as calculated by GEOCHEM

pH 4.5 pH 6.0
Al treatment (µM) Al treatment (µM)

Complexes 50 100 50 100

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California 55
+3
Al 17.5 8.8 — —
Al-PO 4 −3 30.4 15.2 0.03 0.02
Al-citrate 43.1 8.5 7.8 19.7
Al-OHa 7.0 66.6 92.23 80.32
Robust
Al+3 13.1 8.8 — —
Al-PO 4 −3 23.8 15.2 0.03 0.02
Al-citrate 58.3 4.3 16.4 1.63
Al-OHa 3.2 70.8 83.53 98.32
Sceptre
Al+3 17.5 8.8 — —
Al-PO 4 −3 30.3 15.2 0.03 0.02
Al-citrate 22.1 20.4 18.9 11.8
AlOHa 28.0 54.7 81.03 88.22
Aquarius
Al+3 17.5 8.8 — —
Al-PO 4 −3 30.3 15.2 0.03 0.02
Al-citrate 24.5 9.4 19.9 11.0
Al-OHa 25.5 65.7 80.13 89.02
a
Al complexed in solution and precipitated.

in roots increases in plants exposed to higher levels of Al. Precipitation of Al

with P (as phosphates) in roots has been described as an important mechanism
that confers Al tolerance in plants (Gaume et al., 2001). Those authors report
increased precipitation of Al and a high capacity to utilize P, due to a mechanism
for the active transport of Al-P complexes from the cell wall to the vacuole
(Vásquez et al., 1999).
The Al-dependent efflux of organic acids into the rhizosphere has been
widely described as an important mechanism in plant species to minimize the
toxic effects of Al in acid soils (Ma et al., 2001; Ryan et al., 2001; Barceló
and Poschenrieder, 2002; Kochian et al., 2004). Our results indicate that the
exudation of citrate increased significantly in response to Al, the magnitude
of which was dependent on the alfalfa cultivar (Figure 2). Citrate exudation
was higher in cultivars ‘California 55’ and ‘Robust’, especially under Al stress
 624 H. Langer et al.

Table 3
Effects of pH and aluminum on the phosphorus (%) and aluminum (g kg−1 ) contents
in shoots and roots in four alfalfa cultivars in nutrient solutions

pH 4.5 pH 6.0

Shoot Root Shoot Root

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Al P) Al P Al P Al P Al
(µM) (%) (g kg−1 ) (%) (g kg−1 ) (%) (g kg−1 ) (%) (g kg−1 )

California 55
0 0.14 abA 0.05 abA 0.23 0.23 0.13 abA 0.03 cdB 0.29 0.08
50 0.15 abA 0.09 aA 0.32 6.08 0.13 abA 0.06 abB 0.36 3.70
100 0.14 abA 0.10 aA 0.32 7.84 0.14 abA 0.07 aB 0.30 3.04
Robust
0 0.17 abA 0.03 cA 0.29 0.30 0.16 abA 0.02 dA 0.35 0.04
50 0.15 abA 0.03 cA 0.31 6.83 0.16 abA 0.06 abcA 0.40 3.66
100 0.18 aA 0.05 abA 0.30 7.03 0.15 abA 0.07 aA 0.35 3.10
Sceptre
0 0.13 abA 0.04 abA 0.27 0.27 0.11 abA 0.03 dA 0.27 0.02
50 0.17 abA 0.05 abA 0.32 7.08 0.17 aA 0.03 cdB 0.31 2.48
100 0.15 abA 0.09 aA 0.38 7.20 0.15 abA 0.03 cdB 0.29 2.21
Aquarius
0 0.12 bA 0.04 abA 0.23 0.28 0.10 bA 0.02 dA 0.32 0.05
50 0.14 abA 0.06 bA 0.26 5.46 0.13 abA 0.03 dB 0.34 2.96
100 0.14 abA 0.06 bA 0.28 6.35 0.13 abA 0.04 bcdA 0.27 2.32

Within columns, means followed by the same lower case letter were not significantly
different (P ≤ 0.05) according to ANOVA and Tukey’s test. Mean values within rows
for each variable with the same upper case letter were not significantly different
(P ≤ 0.05) according to Student’s t-test. Values of Al and P in shoots are means of
three replicates, except for a single value in roots.

conditions. Previous studies performed in soybean have shown the high-Al

binding capacity of citrate (Ginting et al., 1998) and the existence of varietal
differences in the amount of citrate exuded from roots in response to Al exposure
(Silva et al., 2001).
Although plants are certainly able to concentrate succinate, however, the
chemical speciation results performed by GEOCHEM indicate that succinate is
not capable of forming strong complexes with Al that could potentially reduce
its toxicity (Figure 3). In addition, previous have confirmed that succinate is a
weak ligand with a low capacity to detoxify Al (Hue et al., 1986). Nevertheless,
the exudation of organic acids including succinate from alfalfa roots has been
 Differential Response to Aluminum in Alfalfa 625
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Figure 2. Effect of Al on the exudation of citrate (µmol h−1 g−1 root dry weight) in four
alfalfa cultivars under nutrient solutions at pH 4.5. Mean of three replicates. Columns
followed by different letters indicate significant differences (P ≤ 0.05) according to
Tukey test. Bars in columns indicates standar error (SE).

reported by Lipton et al. (1987) as a response to limited P availability in nutrient

solutions.

Figure 3. Effect of Al on the exudation of succinate (µmol h−1 g−1 root dry weight)
in four alfalfa cultivars under nutrient solutions at pH 4.5. Mean of three replicates.
Columns followed by different letters indicate significant differences (P ≤ 0.05) ac-
cording to Tukey test. Bars in columns indicates standar error (SE).
 626 H. Langer et al.

Both the quality and the quantity of the organic acid exuded by plant roots
are critical to reducing Al toxicity. Some estimations suggest that an equimolar
concentration of citrate is required to avoid the root elongation in maize, which
is considered a useful criteria for estimating Al toxicity (Zheng et al., 1998).
This means that the concentration of citrate required in the nutrient solution in
order to overcome Al toxicity might in the range of 50 or 100 µM.
Aqueous chemical speciation of Al is largely dependent on pH. Estimates
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of Al speciation using GEOCHEM predicted levels of Al3+ between 8.8 and

17.5 µM at pH 4.5. However, calculations indicate that Al3+ was not present in
nutrient solutions at pH 6.0 because it was mainly present in the form of Al-OH
complexes. This finding is consistent with the results from similar evaluations
performed by Nair and Prenzel (1978) who found that at neutral to high pH,
complex formation of Al with phosphate and organic acids might reduce Al
activity. In the current system, speciation estimates indicate that much of the
Al in solution was complexed with citrate, varying from 22 to 58%, especially
at pH 4.5.
High Al concentrations in nutrient solutions resulted in poor growth of
alfalfa plants. However, chemical speciation analysis showed that nutrient levels
in solutions were not affected by adding Al (data not shown). Despite the fact
that Al3+ was the most abundant species at pH 4.5, other Al species such as
AlOH2+ , Al(OH) 2 + , or Al(OH) 4 − and polymeric Al 13 likely control Al toxicity
at pH 6.0 (Kinraide, 1991).
In conclusion, the present study suggests that citrate exudation by alfalfa
cultivars exposed to Al is an important mechanism for reducing the rhizotoxicity
of Al. However, differences in DW yield that were not fully explained by citrate
exudation suggest that multiple mechanisms are important in controlling Al
tolerance and increasing P acquisition in alfalfa plants growing on acid soils.

ACKNOWLEDGMENTS

This work was supported by Fundación Andes (Chile) C-13755-28 and

DIUFRO Grant number 160602.

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