organic acid behaviour in a calcareous soil implications for rhizosphere nutrient cycling
TRANSCRIPT
Organic acid behaviour in a calcareous soil implications
for rhizosphere nutrient cycling
Lena Stroma,*, Andrew G. Owenb, Douglas L. Godboldb, David L. Jonesb
aDepartment of Physical Geography and Ecosystems Analysis, GeoBiosphere Science Centre, Lund University, S-223 62 Lund, SwedenbSchool of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL572UW, UK
Received 12 November 2004; received in revised form 28 February 2005; accepted 23 March 2005
Abstract
Calcareous soils are frequently characterized by the low bioavailability of plant nutrients. Consequently, many vascular plant species are
unable to successfully colonize calcareous sites and the floristic composition of calcareous and acid silicate soils has been shown to differ
markedly. The root exudation of oxalate and citrate has been suggested to play a pivotal role in same nutrient acquisition mechanisms
operating in calcareous soils. The aim of this study was therefore to investigate the nutrient extraction efficiency of three individual organic
acids commonly identified in root exudates, i.e. citric, malic and oxalic acid. Our results clearly demonstrate the context dependent nature of
nutrient release by organic acids. The degree of P extraction was highly dependent on which organic acid was added, their concentration and
pH, and their contact time with the soil. P is generally more efficiently extracted by organic acids at a high pH and follows the series
oxalateOcitrateOmalate. The opposite relationship between pH and extraction efficiency was apparent for most other cations examined (e.g.
Zn, Fe), which are more efficiently extracted by organic acids at low pH. A serious constraint to the ecological importance of organic acid
exudation in response to P deficiency is, however, their very low P mobilization efficiency. For every mol of soil P mobilized, 1000 mol of
organic acid has to be added. It can, however, be speculated that in a calcareous soil with extremely low P concentrations it is still beneficial
to the plants to exude organic acids in spite of the seemingly high costs in terms of carbon.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Calcareous soil; Citrate; Malate; Organic acids; Oxalate; Phosphorus; Rendzina; Root
1. Introduction
Calcareous soils are frequently characterized by the low
bioavailability of plant nutrients and by a high base status
and pH between 7.5 and 8.5 depending on the quality and
quantity of carbonate minerals present (Chen and Barak,
1982; Marschner, 1995). Consequently, many vascular plant
species are unable to successfully colonize calcareous sites
and the floristic composition of calcareous and acid silicate
soils has been shown to differ markedly (Mabey, 1996;
Conti et al., 1999; Lee, 1999).
In calcareous soils, P is largely unavailable to plants due to
the formation of metal complexes (e.g. Ca–P and Mg–P),
rendering P only sparingly soluble. Furthermore, many
micronutrients (e.g. Fe, Mn, Cu and Zn) that are freely
0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2005.03.009
* Corresponding author. Tel.: C46 46 2223746; fax: C46 46 2224011.
E-mail address: [email protected] (L. Strom).
available in acid soils are only sparingly available in
calcareous soils, due to their poor solubility at high pH
(Brady and Weil, 1999). Experiments have shown calcifuge
plants (those which cannot establish well on calcareous soils)
to be primarily excluded from growth in calcareous soils due to
poor P use efficiency, and in a small proportion of species their
Fe use efficiency (Tyler, 1992, 1994; Kerley et al., 2001).
In order for plants to establish and grow successfully on
calcareous soils requires adaptations to overcome the nutrient
deficient conditions prevailing on these soils. A number of
mechanisms by which plants can adapt to nutrient deficient
soils have been suggested; (1) luxury uptake of nutrients
during periods of abundance, storage in roots, and release to
shoots in times of deficiency (Gupta and Rorison, 1975;
Veresoglou and Fitter, 1984); (2) developmental regulation
of plant C partitioning to maintain a high root-to-shoot ratio
(Fitter, 1997); (3) colonization of roots by effective
mycorrhiza to promote greater soil volume exploitation
and enhanced nutrient uptake (Goh et al., 1997); (4) exudation
of compounds from the root which promote mineral
Soil Biology & Biochemistry 37 (2005) 2046–2054
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Table 1
Characteristics of the calcareous soil
EC1:1, dS mK1 1.0
pH (1:1 H2O) 7.58
Carbonates as CaCO3, g kgK1 204
Moisture, g kgK1 392
Organic C, g kgK1 82
Total N, g kgK1 8.2
C:N ratio 10
Exchangeable cations, mmolc kgK1
Na 2.8
K 4.9
Ca 68
Mg 0.54
Extractable P, mmol kgK1 0.009
All values are the mean of two determinations.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–2054 2047
dissolution, organic matter mineralization, and root uptake of
barely soluble nutrient pools from the rhizosphere (Lipton
et al., 1987).
Root exudation has been suggested to play a central role in
some nutrient acquisition mechanisms operating in calcar-
eous soils (Strom, 1997; Jones, 1998). Calcicole plants (i.e.
those that can establish on calcareous soils) generally have
enhanced rates of root organic acid exudation, particularly
oxalate and citrate, in comparison to calcifuge plants (Strom
et al., 1994; Tyler and Strom, 1995; Strom, 1997).
After entering the soil, cations can react with the organic
acids to form organo-metallic complexes. If these com-
plexes are soluble they increase the availability of the
cation, protect it from precipitation and also provide a direct
route for cation uptake (e.g. Fe; Jones, 1998). If, on the other
hand, the organic acid-metal complex is insoluble this could
decrease the availability of the micronutrient (Brady and
Weil, 1999). Once exuded, organic acids may undergo
complexation reactions with target metals (e.g. Fe, Mn, Zn
and Cu or Ca, which enhances Ca–P mineral dissolution) or
non-target metals (e.g. Al, which does not mobilize much P;
Cline et al., 1982; Jones and Darrah, 1994). To fully
evaluate the importance of the proposed nutrient mobiliz-
ation mechanism requires a more detailed understanding of
the fate of exuded organic acids in calcareous soils.
The aim of this study was therefore to investigate the
nutrient extraction efficiency of three individual organic
acids commonly identified in root exudates of calcicole
plants, i.e. citric, malic and oxalic acid (Strom et al., 1994;
Tyler and Strom, 1995; Strom, 1997).
Table 2
Calcareous soil pH after extraction with three organic acid solutions at an initial
Addition (mM) Oxalate pHZ7.5 Oxalate pH!3.5 Malate pHZ
1.00 7.81G0.02 6.16G0.01 7.83G0.02
10.00 9.02G0.00 6.33G0.08 7.83G0.01
20.00 9.46G0.07 6.60G0.02 7.90G0.01
50.00 9.98G0.01 6.80G0.04 8.10G0.00
100.00 10.23G0.02 7.16G0.21 8.19G0.03
The initial soil pH was 7.58. Values represent meanGSEM.
2. Materials and methods
2.1. Soil
The soil represents a calcareous Typic Rendoll (Rendzic
leptosol), derived from Ordovician limestone and is located
on the ‘alvar’ of Oland in Sweden (568 40 0N, 168 30 0E). The
site has a mean annual rainfall of 388 mm, mean annual
temperature of 7.1 8C, slope of 2.48, elevation of 30 m and is
dominated by calcicole vegetation (e.g. Artemisia
campestris, Melica ciliata, Sedum album etc.).
The soil was collected from the Ah horizon (0–10 cm)
using a spade, sieved to pass 6 mm and kept field moist at
10 8C until required. Properties of the soil are provided in
Table 1.
2.2. Extraction efficiency of organic acids
To determine the concentration-dependent extraction
efficiency of the organic acids on P, Fe and Ca and several
other cations from the calcareous soil, 12.5 ml of organic
acid solution was added to 5.0 g of field moist soil contained
in 25 ml polypropylene tubes. Three individual organic
acids, citric, malic and oxalic acid were added to soil at
concentrations ranging from 1 to 100 mM to determine the
soil extraction efficiency (% release as mol ion per mol
organic acid added) of P, Fe and Ca. In addition, a single
concentration of 10 mM was used to determine the
efficiency of the acids in extracting P and cations. The
samples were orbitally shaken for 30 min, centrifuged
(16 000g, 10 min) and the supernatant examined for pH, P
and cations (Fe, Mn, Mg, Ca, Na, K, Cu and Zn; Jobin
Yvon Ultrace ICP-OES, Jobin Yvon SA, Longjumeau,
France).
To determine the pH effect on the organic acid extraction
efficiency the experiments described above was performed
both at an initial organic acid pH of 7.5 (adjusted by KOH)
and without pH adjustment, resulting in an initial pH of the
extractant solutions of between 2.5 and 3.5 (hereafter
denoted as pH!3.5).
To determine the time-dependence of organic acid
mediated P, Fe and Ca extraction, experiments were
performed as described above except that a single concen-
tration of organic acids was employed (10 mM) at high (7.5)
pH and shaking times varied between 2 min and 24 h.
pH of either 7.5 or !3.5 and at concentrations varying from 1 to 100 mM
7.5 Malate pH!3.5 Citrate pHZ7.5 Citrate pH!3.5
5.86G0.11 7.83G0.03 5.81G0.08
6.12G0.03 8.83G0.02 5.96G0.02
6.55G0.00 8.63G0.03 6.35G0.05
6.91G0.21 8.92G0.06 6.70G0.05
6.99G0.29 9.02G0.01 7.14G0.06
0
10
20
30
40
50
60P
ext
ract
ed (
µmol
kg–1
soi
l)pH 7.5 pH <3.5
Citrate
Malate
Oxalate
0 20 40 60 80 100
Organic acid addition (mM)0 20 40 60 80 100 120
Organic acid addition (mM)
Fig. 1. The efficiency of three organic acids (citrate, malate and oxalate) at extracting P from a calcareous soil. Organic acids were added to the soil at pH 7.5
(left panel) and at pH!3.5 (right panel) and at concentrations varying from 1 to 100 mM. Symbols represent meanGSEM.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–20542048
2.3. Statistical analysis
All treatments were performed in triplicate with same
treatments independently replicated on different days.
Statistical analysis (Ancova and Anova followed by the
Bonferroni method for significance level adjustments due to
multiple comparisons) was performed with the computer
program SPSS 10.0 (SPSS Inc., Chicago, ILL). Asterisks
represent statistical differences at the p%0.05 (*), p%0.0l
(**) and p%0.00l (***) level.
3. Results
0
2
4
6
8
10
12
14
0 10 20 30 40 50 60 1440Time (min)
**Citrate Malate Oxalate
P e
xtra
cted
(µm
ol k
g–1 s
oil)
Fig. 2. Time-dependent removal of P from a calcareous soil by three organic
acids (citrate, malate and oxalate). The initial concentration of the organic
acids added to the soil was 10 mM (pH 7.5). Symbols represent meanGSEM.
3.1. P extraction efficiency of organic acids
Soil extractions were performed with organic
acids normalized to pH 7.5 in order to simulate release
of KC-citrate from roots and at pH!3.5 (the intrinsic pH
of the organic acid when made up in distilled water) to
simulate the release of HC-citrate from roots. In all cases,
extraction of the soil with organic acid solutions at pH
7.50 resulted in an increase of extractant pH and in an
increase of the initial soil pH (7.58) by 0.23–2.65 units
(Table 2). In contrast, extraction of the soil with organic
acid solutions at pH!3.5 resulted in a decrease of initial
soil pH of between 0.17 and 1.77 units (Table 2). For both
the pH 7.5 and !3.5 extraction there was a strong trend
with gradually increasing pH with increasing extractant
concentration (Table 2).
The results clearly indicate that soil P extraction
efficiency is highly dependent upon the type and
concentration of organic acid as well on the pH of the
extractant solution (Fig. 1). At an extractant solution pH
of 7.5 the extraction of soil P generally followed the
series
oxalateOcitrateOmalate
while at pH!3.5, soil P extraction by the organic acids
followed the series
citrate Z malateOoxalate
At both solution pH’s, P removal from the soil was found to
be non-linear with organic acid concentration with the greatest
differences between organic acids observed at high extractant
concentrations (O10 mM). When comparing the acids at
individual concentrations (Anova) oxalate was more effective
than both malate and citrate at the two highest concentrations
of the extractant, e.g. 50 mM (*) and 100 mM (***), while at
100 mM citrate was also found to be more effective than
malate (**). The extraction efficiency for P was significantly
more effective both for oxalate (**, Ancova) and citrate
(*, Ancova) at pH 7.5 than at pH!3.5. For malate, however,
there was no statistically significant difference in P extraction
efficiency between the two pH’s of the extractant solution.
The P extraction efficiency of organic acids as a function
of time is shown in Fig. 2. Oxalate extracted significantly
more P than malate and citrate (*, Ancova), whereas there
Table 3
The efficiency (% release as mol ion per mol organic acid added) of three organic acids (oxalate, malate and citrate) at extracting nutrients from a calcareous
soil
Element Oxalate pHZ7.5 Oxalate pH!3.5 Malate pHZ7.5 Malate pH!3.5 Citrate pHZ7.5 Citrate pH!3.5
Zn !0.001 0.006G0.0001 !0.001 0.006G0.0001 !0.001 0.007G0.001
Cu 0.004G0.001 !0.001 0.002G0.0001 !0.001 0.002G0.0004 !0.001
Mn 0.004G0.0002 !0.001 0.004G0.0001 !0.001 0.017G0.002 0.038G0.003
Fe 0.027G0.001 0.011G0.0001 0.020G0.0001 0.016G0.0002 0.105G0.003 0.168G0.018
Mg 0.02G0.004 1.41G0.158 0.38G0.025 2.68G0.216 0.78G0.022 2.99G1.001
Ca 0.32G0.056 9.33G0.307 28.52G3.246 74.84G2.099 40.26G2.276 84.28G0.565
Na 3.09G0.121 0.54G0.007 6.39G0.203 1.28G0.325 10.15G0.234 1.35G0.218
Sum cations 3.5 11.3 35.3 78.8 51.3 88.8
Phosphate 0.008G0.0003 0.001G0.0001 0.004G0.0001 0.003G0.0005 0.019G0.0011 0.002G0.0001
Organic acids (10 mM) were added to the soil at a pH of either 7.5 or !3.5. Values represent meanGSEM.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–2054 2049
was no statistically significant difference between malate
and citrate. This significance was, however, mainly a result
of the difference between oxalate and the other extractants at
two extractant times, i.e. 2 and 1440 min. In comparison to
malate and citrate, the rate of oxalate mediated P extraction
was greatest immediately following organic acid addition to
the soil, e.g. 2 min (***, Anova), at which point citrate was
also found to be more effective than malate (**, Anova), and
following 24 h of extraction (***, Anova). For the other
extractant times there was no significant difference between
the organic acids at an extractant concentration of 10 mM.
After 24 h, a net re-sorption or immobilization of P was
apparent for citrate and malate, as the amount of P in
solution had significantly decreased relative to that observed
after 60 min (Fig. 2). The amount of re-sorption tended to be
larger for malate (pZ0.054) than for citrate.
The extraction efficiency of the organic acids expressed
as mol P extracted per mol added organic acid (Table 3)
shows that the mol efficiency was very low with only less
than 1 mol of P extracted per 1000 mol added organic acid.
At an extractant concentration of 10 mM, citrate (pH 7.5)
and oxalate (pH 7.5) were more efficient in extracting P than
the other acids or pH’s.
0
20
40
60
80
100
120
140
160
0 20 40 60 80 100
Fe
extr
acte
d (µ
mol
kg–1
soi
l)
Organic acid addition (mM)
pH 7.5 Citrate
Malate
Oxalate
Fig. 3. The efficiency of three organic acids (citrate, malate and oxalate) at extracti
(left panel) and at pH!3.5 (right panel) and at concentrations varying from l to
3.2. Cation extraction efficiency of organic acids
The results show clear differences in cation extraction
efficiency between each of the individual organic acids. This
also appeared to be highly dependent upon the pH of the
extractant solution. The sum of extracted cations was, in all
cases, higher when the extractant solution had a pH!3.5
compared to that of pH 7.5 with citrate being 1.7 fold higher,
malate 2.2 fold higher and oxalate 3.2 fold higher (Table 3).
The mol of cations released per mol of organic acid added also
showed a clear difference in effectiveness between the acids
with citrate being the most effective acid in mobilizing cations
followed by malate and oxalate. This relationship was
apparent when the extractant solution pH was either !3.5
or pH 7.5. Furthermore, it is obvious that per mol addition of
organic acid a major part of the added acid results in extraction
of the cation that is dominating in the soil, i.e. Ca, except for
the oxalate pH 7.5 extractant (Table 3).
Fig. 3 shows the relationship between Fe mobilizing
efficiency and the amount of added organic acid as well as the
effect of pH. At an extractant solution pH of !3.5, citrate was
the most effective mobilizer of Fe (***, Ancova) followed by
malate and oxalate (malate and oxalate are significantly
0 20 40 60 80 100 120Organic acid addition (mM)
pH <3.5
ng Fe from a calcareous soil. Organic acids were added to the soil at pH 7.5
100 mM. Symbols represent meanGSEM.
0
50
100
150
200
250
300
350
0 10 20 30 40 50 60 1440Time (min)
Fe
extr
acte
d (µ
mol
kg–1
soi
l)
Citrate
Malate
Oxalate
Fig. 4. Time-dependent removal of Fe from a calcareous soil by three
organic acids (citrate, malate and oxalate). The initial concentration of the
organic acids added to the soil was 10 mM (pH 7.5). Symbols represent
meanGSEM.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–20542050
different from each other at the ** level, Ancova). At an
extractant pH of 7.5, the pattern of organic acid-mediated Fe
mobilization was not simple. Citrate was clearly more
effective at the lower concentrations (***, Ancova) but at
extractant solution concentrations above 20 mM the Fe
mobilizing capacity of citrate decreased (Fig. 3).
Over an extended extraction period, citrate consistently
mobilized significantly more Fe (***, Ancova) than malate
and oxalate (2–24 h). Comparing the amounts of Fe
mobilized by citrate at 2 min to that mobilized after 24 h
showed an increase of approximately 10-fold (Fig. 4).
Fig. 5 shows the relationship between Ca mobilizing
efficiency and the amount of added organic acid as well as
the effect of extractant pH on Ca extraction efficiency. At all
concentrations and for all three acids the pH!3.5 extractant
extracted more Ca than the pH 7.5 extractant (citrate
pZ0.004, malate and oxalate p!0.001, Ancova). As
occurred for Fe, the extraction efficiency of Ca by citrate
0
5000
10000
15000
20000
25000
30000
CitrateMalateOxalat
pH 7.5Citrate
Malate
Oxalate
Ca
extr
acte
d (µ
mol
kg–1
soi
l)
0 20 40 60 80 100
Organic acid addition (mM)
Fig. 5. The efficiency of three organic acids (citrate, malate and oxalate) at extracti
(left panel) and at pH!3.5 (right panel) and at concentrations varying from l to
tended to decrease at the higher extractant concentrations,
whereas the efficiency of malate increased with increasing
addition. There was no statistically significant difference
between citrate and malate in their ability to mobilize Ca at
either pH 7.5 or !3.5, whereas, oxalate extracted much
lower amounts of Ca than either of the other two organic
acids in both cases (***, Ancova). Over an extended
extraction period (2 min to 24 h) the extraction efficiency
followed the series
citrateOmalateOoxalate
(***, Ancova), with oxalate again extracting low
amounts of Ca in comparison to citrate and malate (Fig. 6).
4. Discussion
Typically, Fe and P are the two main nutrients that limit
plant growth on calcareous soils (Marschner, 1995).
However, whilst the total P and Fe content of calcareous
and silicate soils often are in the same order of magnitude,
the easily exchangeable and soil solution concentrations
are often very low in calcareous soils (Tyler and Olsson,
1993; Strom, 1997). Furthermore, liming of an acid soil
can result in a decreased plant uptake of many elements,
e.g. P (Tyler and Olsson, 2001). Due to its very low
availability P deficiency is one of the main factors limiting
plant growth and survival in calcareous soils (Tyler, 1992,
1994). It has been documented that calcicole plants have
increased amounts of organic acids, mainly of oxalate and
citrate, in their root vicinity (Strom et al., 1994; Tyler and
Strom, 1995; Strom, 1997). Organic acids have been
hypothesised by many authors to be involved in the
mobilization of nutrients within the rhizosphere (Gardner
et al., 1983; Hoffland et al., 1989; Hoffland, 1992;
Marschner, 1995; Farrar and Jones, 2000). In correspon-
dence with these suggestions we have hypothesized
e
pH <3.5
0 20 40 60 80 100 120
Organic acid addition (mM)
ng Ca from a calcareous soil. Organic acids were added to the soil at pH 7.5
100 mM. Symbols represent meanGSEM.
0
5000
10000
15000
20000
25000
0 10 20 30 40 50 60
Ca
extr
acte
d (µ
mol
kg–1
soi
l)
Time (min)
CitrateMalateOxalate
Fig. 6. Time-dependent removal of Ca from a calcareous soil by three
organic acids (citrate, malate and oxalate). The initial concentration of the
organic acids added to the soil was 10 mM (pH 7.5). Symbols represent
meanGSEM.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–2054 2051
previously that calcicole plants exude increased amounts of
organic acids from their roots in order to solubilize P and
overcome P deficiency. Organic acids have been reported
to strongly enhance the mobilization of P from soils
(Gerke, 1992, 1993, 1994; Gerke et al., 1994) and oxalate
is often reported to be particularly efficient in this respect
(Lopez-Hernandez et al., 1986; Fox et al., 1990; Fox and
Comerford, 1992; Bolan et al., 1994; Strom, 1997). We
show that P is more efficiently solubilized by oxalate and
citrate than malate and that oxalate is much more efficient
than the other organic acids over time, when extracted by a
high pH solution (Figs. 1, 2 and Table 3). Thus, increased
or generally high exudation of primarily oxalate but also of
citrate from calcicole plants could be beneficial to the P
uptake of these plants when growing in P deficient
calcareous soil.
Gerke et al. (2000) showed that P mobilization was
negligible or small below an oxalate or citrate concentration
of 10 mmol gK1 soil. We find a marked increase in the P
extraction efficiency above an extractant concentration of
10 mM (corresponds to 25 mmol extractant added gK1 soil)
for citrate and oxalate at pH 7.5 and above 20 mM
(50 mmol gK1 soil) for the malate (pH 7.5) and all the
pH!3.5 extractants (Fig. 1). These findings indicate that
rhizospheric concentrations of organic acids in this order of
magnitude might be necessary to have a considerable effect
on P mobilization and plant uptake in calcareous soils. The
organic acid concentrations in the rhizosphere or in soil
solutions reported in the literature vary greatly and can
range from just a few mM to over 80 mM (Jones, 1998;
Veneklaas, 2003). For white lupin growing in P deficient
calcareous soil Dinkelaker et al. (1989) found citrate
concentrations as high as 48 mmol gK1 rhizospheric soil.
Thus, the concentration range used in our experiment are
within the range of where naturally occurring rhizospheric
concentrations could be of importance to P uptake in natural
ecosystem. Rhizospheric production of organic acids can for
P deficient plants drain 5–25% of the carbon assimilated
through photosynthesis (Jones, 1998). The photosynthetic
rate on the ‘alvar’ of Oland was measured in 2003 and
ranged between 1 and 7 mmol CO2 assimilated mK2 hK1
(Strom unpublished results). The above ground biomass was
simultaneously determined to between 3 and 12 g mK2 and
using a shoot to root ratio of 1.0 (G0.15 SE, mean of 80
alvar species, Hickler unpublished results) allows us to
estimate the potential root release of organic acids on the
‘alvar’. Using the above assumptions, the organic acid
production in the rhizosphere would amount to between 18
and 138 mmol C gK1 root hK1. Although the true rate of
exudation on the ‘alvar’ needs to be determined in situ to
validate these calculations they indicate that the root release
on these P deficient calcareous soils could be well within the
range necessary to have a substantial effect on P
mobilization (Gerke et al., 2000) and presumably also on
plant uptake. Furthermore, they indicate that the extractant
concentrations used in our experiment are within the range
of where natural concentrations could occur.
In addition, plants respond to deficiency of many other
nutrients with increased root exudation, for example to, K
(Kraffczyk et al., 1984), Zn (Zhang et al., 1989), Cu
(Nielsen, 1976) and Fe (Treeby et al., 1989; Zhang et al.,
1991). Since organic acids efficiently solubilize/mobilize
many metal cations [e.g. Ca, K, Mg (Jones and Darrah,
1994), Al and Fe (Gerke, 1992, 1993, 1994; Gerke et al.,
1994; Jones and Darrah, 1994) and Mn (Jauregui and
Reisenauer, 1982)], the purpose of this increased exudation
could be to increase the solubilization of deficient nutrients.
In calcareous soils the most limiting nutrient second to P is
typically Fe (Tyler, 1992, 1994). Our results show that
citrate or malate generally are more efficient in extracting
cations from the calcareous soil used in our study than
oxalate, especially at pH!3.5 of the extractant (Figs. 3–6
and Table 3). Thus, the higher amounts of citrate found in
the root vicinity of calcicole plants compared to calcifuge
plants (Strom et al., 1994; Tyler and Strom, 1995; Strom,
1997) could be released in response to Fe deficiency and,
presumably, result in a promoted Fe nutrition of calcicole
plants.
However, our results clearly demonstrate the context
dependent nature of nutrient release by organic acids. The
outcome of the extraction is highly dependent on which acid
is added and at what concentration and pH. P is generally
more efficiently extracted by an extractant solution of pH
7.5, whereas, the opposite relationship exists between pH
and extraction efficiency for most other cations, which are
more efficiently extracted by pH!3.5 solution (Figs. 1–6
and Table 3). These results are in agreement with that of
Strom (1997) for two contrasting Swedish calcareous soils.
In the Strom (1997) study, using a single extractant
concentration of 15 mM for oxalate and 10 mM of citrate,
an identical relationship was observed between pH and
oxalate and citrate and their respective P and Fe extraction
efficiency as demonstrated in the present study.
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–20542052
The large differences in extraction efficiency of the
organic acids are probably related to pH, both in the soil and
in the extracts and to pH-related properties of Fe, Ca and P.
We show that the pH!3.5 extraction with citrate is by far
the most efficient extractor of Fe (Figs. 3 and 4 and Table 3).
In a soil with a pH around 8 the concentration of Fe3C in the
soil solution decreases 1000-fold for each unit increase in
pH (Barber, 1995). Thus, the pH lowering effect of the
pH!3.5 extractant solution (Table 2) is clearly important to
the solubility of Fe in a limestone soil such as the one used
here (pH 7.58). However, the importance of Fe complexa-
tion by citrate and the possession of a high stability constant
is also demonstrated since the other extractants of an equally
low pH (i.e. pH!3.5 extraction with malate and oxalate)
had a lower extraction efficiency (Fig. 3 and Table 3).
Subsequently, our results indicate that the Fe-citrate chelate
is soluble and increases the availability of this cation.
Presumably chelation prevents Fe from being re-precipi-
tated and might provide a direct route for Fe uptake at least
by dicotyledonous plants (Jones, 1998).
At a solution pH of 7.5, the most powerful extractors of P
in the calcareous soil were citrate (at low extractant
concentrations, Table 3, Fig. 1) and oxalate (at higher
concentrations and over a prolonged period of time, Figs. 1
and 2). Both these extractants resulted in a pH increase from
the initial soil pH of 7.58 (pH 7.81–10.23 for oxalate; pH
7.83–9.02 for citrate; Table 2). The pH increase during these
extractions is probably largely due to the reaction between
CaCO3 and the oxalate and citrate ions. The resultant
formation of Ca-oxalate and Ca-citrate will release CO2K3
leading to an increase in solution pH. Following extraction
with pH!3.5 oxalate and citrate, the initial soil pH was
lowered in relation to initial soil pH to between 6.16 to 7.16
for oxalate and 5.81 to 7.41 for citrate (Table 2). The pKa
values of oxalic acid are 1.23 and 4.19 and of citric acid are
3.14, 4.77 and 6.39 (Weast, 1989). This indicates that oxalic
acid after a very short extraction time became fully
dissociated and citric acid fully or two steps dissociated
during both the low and high pH extraction. The effect of the
oxalate and citrate ion in chelating Ca and, thereby,
releasing HPO2K4 should therefore be similar in both
extractions. However, at pH!7, CaCO3 starts to dissolve
(H2CO3; pKa1Z6.38, pKa2Z10.32) and the free Ca2C can
precipitate HPOK24 previously released by oxalate and
citrate. Therefore, it may be hypothesised that the effect
of the metal complexing ion is approximately equal at
extraction with both high and low pH extractants. During
the pH!3.5 extraction, however, we hypothesize that the P
released becomes re-precipitated as insoluble Ca–P and is
therefore not recovered in the extract.
Anions of organic acids need to be exuded with a positive
counter-ion in order for electroneutrality to be maintained.
This cation has been suggested to be HC (Marschner, 1995),
however, it now appears that HC release and organic acid
release are two biochemically separate transport events with
enhanced HC excretion under P deficiency coming from an
upregulation of the HC-ATPase (Jones, 1998). Furthermore,
for many organic acid release situations the counter ion has
yet to be identified. It is therefore difficult to speculate on
whether organic acid exudation in calcareous soils will lead
to an increase or decrease in the rhizospheric pH. However,
a high exudation of organic acids does not necessarily lead
to a measurable lowering of pH in the rhizosphere soil
(Strom, 1997). It is clear from our study that different plant
strategies would be beneficial depending on the nature of the
nutrient limitation. In a P deficiency situation a counter ion
such as KC, resulting in increase in the rhizosphere pH,
would be beneficial, whereas, in a cation deficiency
situation, e.g. Fe, it would clearly be beneficial to decrease
rhizosphere pH and, thus, use HC as the counter ion.
A serious objection to the ecological importance of
organic acid exudation in response to P deficiency is the
very low mobilization efficiency. At an organic acid
extractant concentration of 10 mM at pH 7.5 the resulting
P concentration in solution was only 1.9 mM for citrate and
0.8 mM for oxalate (Table 3), equating to a 1000-fold
organic acid ‘loss’. Can organic acid exudation really be of
any benefit to the plant when it involves such a large carbon
loss? The calcifuge plants used in the studies by Strom et al.
(1994), Tyler and Strom (1995) and Strom (1997) were all
species that grow in open land and, thus, they are rarely
carbon limited. Furthermore, their growth rates are
generally low. It can be speculated, therefore, that these
plants invest a large amount of assimilated carbon into
organic acid exudation and, subsequently, place a lower
amount of carbon into biomass production. If a plant
contains approximately 0.2% P on a dry weight basis the
total vegetation P content on the ‘alvar’ would amount to
0.4–1.5 mmol P mK2, assuming an above ground biomass
of 3–12 g mK2 (Strom unpublished results) and a shoot to
root weight ratio of 1.0 (Hickler unpublished results). If
organic acids are solely responsible for mobilizing P and if
we assume an oxalate exudation of 5% of the C fixed
through photosynthesis, we crudely estimate that it would
take between 369 and 645 days for the plants to take up the
total P content of the biomass. The plants on the ‘alvar’ are
in general perennial and have a slow growth rate, traits that
in combination with an effective translocation and storage
of P are an absolute necessity if such a low rate of P
acquisition should ever result in a sustainable development
of the vegetation. However, plants cannot grow without a
reliable supply of P and it might be speculated that the
benefits of exudation exceed the costs. Stearns (1992) states
that ‘an adaptation is a change in a phenotype that occurs in
response to a specific environmental signal and has a clear
functional relationship to that signal that results in an
improvement in growth, survival or reproduction. Other-
wise it does not appear’. In a previous study with 33P, we
showed that the direct addition of oxalate to the soil can
double the plant uptake of P from the calcareous soil used in
this study, whereas citrate is much less efficient in this
respect (Strom et al., 2002). We show that oxalate extracts P
L. Strom et al. / Soil Biology & Biochemistry 37 (2005) 2046–2054 2053
more efficiently than malate and citrate over time (Fig. 2).
Furthermore, citrate and malate are biodegraded to a much
higher extent than oxalate. Twenty-four hours after addition
to the calcareous soil used in our experiment only 7% of the
added oxalate had been degraded, 30% of the added citrate
and 33% of the added malate (Strom et al., 2001). It can
therefore be speculated that in a calcareous soil where
available P concentrations are extremely low, it is beneficial
to the plants to exude large quantities of organic acids in
spite of the seemingly high costs in terms of carbon.
Furthermore, it can be assumed that oxalate would be
particularly efficient in this respect since it possesses a
greater P extraction efficiency and is biodegraded to a lesser
extent. Further work is therefore required to examine the
organic acid nutrient extraction efficiency across a wider
range of calcareous and non-calcareous sites and to
investigate in situ whether the concentrations of organic
acids in soil solutions are sufficient to mobilize significant
amounts of P.
Acknowledgements
The Leverhulme Trust, Overseas Development Program,
supported this work. We would also like to thank Germund
Tyler for his experimental support.
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