Annals of Botany 103: 1239– 1247, 2009
doi:10.1093/aob/mcp074, available online at www.aob.oxfordjournals.org
Aluminium tolerance and high phosphorus efficiency helps Stylosanthes better
adapt to low-P acid soils
Yu-Mei Du1, Jiang Tian2, Hong Liao2, Chang-Jun Bai1, Xiao-Long Yan2 and Guo-Dao Liu1,*
1
Institute of Tropical Crop Genetic Resources, Chinese Academy of Tropical Agriculture Science, Danzhou 571737, China
and 2Root Biology Center, South China Agricultural University, Guangzhou 510642, China
Received: 30 December 2008 Returned for revision: 14 January 2009 Accepted: 27 February 2009 Published electronically: 26 March 2009
† Backgrond and Aims Stylosanthes spp. (stylo) is one of the most important pasture legumes used in a wide
range of agricultural systems on acid soils, where aluminium (Al) toxicity and phosphorus (P) deficiency are
two major limiting factors for plant growth. However, physiological mechanisms of stylo adaptation to acid
soils are not understood.
† Methods Twelve stylo genotypes were surveyed under field conditions, followed by sand and nutrient solution
culture experiments to investigate possible physiological mechanisms of stylo adaptation to low-P acid soils.
† Key Results Stylo genotypes varied substantially in growth and P uptake in low P conditions in the field. Three
genotypes contrasting in P efficiency were selected for experiments in nutrient solution and sand culture to
examine their Al tolerance and ability to utilize different P sources, including Ca-P, K-P, Al-P, Fe-P and
phytate-P. Among the three tested genotypes, the P-efficient genotype ‘TPRC2001-1’ had higher Al tolerance
than the P-inefficient genotype ‘Fine-stem’ as indicated by relative tap root length and haematoxylin staining.
The three genotypes differed in their ability to utilize different P sources. The P-efficient genotype,
‘TPRC2001-1’, had superior ability to utilize phytate-P.
† Conclusions The findings suggest that possible physiological mechanisms of stylo adaptation to low-P acid soils
might involve superior ability of plant roots to tolerate Al toxicity and to utilize organic P and Al-P.
Key words: Stylosanthes, phosphorus, P efficiency, organic P, Al toxicity, acid soil.
IN T RO DU C T IO N
Phosphorus (P) is an essential macronutrient, required for
many metabolic processes in plants. Low P availability is
one of the major factors limiting crop production on acid
soils (Barber, 1995). P fertilization is a conventional way to
amend soil P deficiency. Since supplied P is easily bound
either by organic or inorganic compounds into forms that are
unavailable to plants, high input of P fertilizer is not only
costly, but also inefficient and might result in environmental
pollution (Vance et al., 2003). Therefore, improvement of P
efficiency in crops would be more economical and efficient
than sole reliance on chemical P fertilization (Yan and
Zhang, 1997; Vance et al., 2003).
Plant P efficiency was broadly defined as having relatively
greater biomass at less optimal P level (Lynch, 1998; Liao
et al., 2008), including P acquisition efficiency (the ability to
acquire P from growth medium) and P utilization efficiency
(the ability to convert P into biomass and yield), which could
be separately reflected by P content and biomass produced by
unit P in plants (Graham, 1984; Clark and Duncan, 1991;
Batten, 1992). Since P is rarely mobile in soils, P acquisition
efficiency is mainly determined by the soil volume explored
to the roots as indicated by root morphology (i.e. root length
and root surface area) and root architecture (the spatial distribution of roots along soil profile; Yan and Zhang, 1997).
Accumulating results reveal that changes of root traits lead to
* For correspondence. E-mail: [email protected] or
[email protected]
increase of P acquisition efficiency, including modifying root
morphology and architecture, activating high affinity phosphate
(Pi) transporter(s), producing P-solubilizing root exudates, such
as organic acids and phosphatases to help release Pi from
bound-P pools in soils (especially Fe-P, Al-P and organic phosphate ester; Raghothama, 1999; Vance et al., 2003). All these
studies imply that root traits are vital for plants to efficiently
acquire P from the soils under P-limited conditions.
In addition to P deficiency, aluminium (Al) toxicity is another
major factor limiting plant growth on acid soils. The phytotoxic
Al species are released to soil solution, resulting in inhibition of
root elongation by injuring the root apex (Foy, 1984; Delhaize
et al., 1993). Organic acid exudation is generally believed to
play critical roles in ameliorating Al toxicity through forming
non-toxic Al chelates, which has been well documented in
several species, such as malate release in wheat (Triticum aestivum), citrate exudation in bean (Phaseolus vulgaris), maize
(Zea mays), Cassia tora and soybean (Glycine max; Miyasaka
et al., 1991; Delhaize et al., 1993; Pellet et al., 1995; Ma
et al., 1997; Yang et al., 2001). Since P deficiency and Al toxicity commonly coexist on acid soils, it is assumed that plants
with good performance on acid soils might be both P efficient
and Al tolerant. Consistent with this assumption, recent
studies showed that P-efficient genotypes had great Al tolerance
in soybean and buckwheat (Fygopyrum esculentum) possibly
through precipitating or chelating toxic Al around roots
(Zheng et al., 2005; Liao et al., 2006).
Stylo (Stylosanthes spp.) is one of the most economically
important forage legumes and is widely distributed in the
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: [email protected]
1240
Du et al. — Stylosanthes adaptation to low-P acid soils
tropical areas, in which most soils are acid soils (Liu et al.,
1997; Miller et al., 1997; Chakraborty, 2004). Introduction
of stylo to improve animal production in tropical areas has
been successfully proved in northern Australia, South
America, Asia and Africa (Liu et al., 1997; Miller et al.,
1997; Ramesh et al., 1997; Chakraborty, 2004). For
example, in Queensland in Australia, stylo covers over one
million hectares, forming the basis of local beef production
(Chakraborty, 2004). Stylo is also widely used in a range of
agricultural systems as a cover crop to suppress weed
growth, and a pioneer crop to grow on infertile acid soils
(Ramesh et al., 1997; Chakraborty, 2004). Despite the superior
ability of stylo to adapt to acid soils, few studies have been
conducted to elucidate possible mechanisms underlying this
superior ability, especially for P efficiency and Al tolerance.
Recent results showed that one of the most widely sown
forages, signalgrass (Brachiaria decumbens), has novel strategies (i.e. low Al permeability of the plasma membrane) to
detoxify external Al stress, indicating how important it is to
use plants well adapted to Al stressful soils when investigating
the mechanism of Al tolerance (Wenzl et al., 2001). Earlier
studies found that substantial genotypic variations for P efficiency presented in stylo, and there was a positive relationship
between root acidification and P uptake (Yang and Yan, 1998).
However, no direct evidence has been reported whether
P-efficient stylo genotypes could utilize non-soluble P (i.e.
Al-P, organic P), and simultaneously have Al tolerance
because P deficiency and Al toxicity coexist in acid soils. In
this study, P efficiency of 12 stylo genotypes was surveyed
under field conditions with or without P application, followed
by sand and nutrient solution experiments with three selected
genotypes differing in P efficiency to elucidate the possible
mechanisms of stylo plants adapting to low-P acid soils
under both P deficiency and Al toxicity conditions.
M AT E R IA L S A ND M E T HO DS
Field experiment
In the field experiment, 12 stylo genotypes from six species
were used as plant materials. Among them, ‘Reyan NO.2’,
‘Reyan NO.5’, ‘GC 1581’, ‘Mineirao’, ‘CIAT 1517’,
‘TPRC2001-1’ and ‘TPRC2001-2’ belong to Stylosanthes
guianensis. ‘Capica’, ‘Verano’, ‘Seca’, ‘Seabrana’ and
‘Fine-stem’ belong to Stylosanthes capitata, Stylosanthes
hamata, Stylosanthes scabra, Stylosanthes seabrana and
Stylosanthes hippocampoides, respectively. The field study
was conducted at the Tropical Pasture Center of the Chinese
Academy of Tropical Agriculture Science (CATAS). The site
is 198300 N, 1098300 E at 149 m a.s.l.. The soil for the experiment was a typical acid red soil deficient in available P
(Table 1). Seeds of stylo were soaked in hot water (80 8C)
for 2 min, and then rapidly cooled to room temperature to
facilitate germination. The pretreated seeds were germinated
on wet filter paper overnight in the dark at 28 8C and then
transferred to plastic pots filled with soil for seedling
growth. After 6 weeks, all plants were transferred to the
field. The 12 stylo genotypes tested were grown at high P
(120 kg P ha21 added as triple superphosphate) and low P
(without P fertilizer added) levels. Each treatment had three
replicates in a randomized complete block design. After
60 d, plants were harvested and dried in an oven at 75 8C to
determine plant dry weight. Phosphorus concentration in
plants was colormetrically measured using the method
described as before (Murphy and Riley, 1963). For measuring
total P content in seeds, 1000 seeds of each genotype were
dried in an oven at 75 8C with three replicates. Dry weight
and total P content of seeds were separately measured as the
method described above.
Al tolerance and acid phosphatase (APase) activity
measurement in nutrient solution culture
Based on the results from the field experiment, three stylo genotypes, including ‘Fine-stem’, ‘TPRC2001-1’ and ‘Verano’, differing in P efficiency were used in this study. Seeds were
pretreated as described above. Pretreated seeds were germinated
on filter paper moistened with 0.5 mM CaSO4 in a Petri dish
overnight in the dark at 28 8C. Three germinated seeds of each
genotypes with an emerging radical (0.5–1 cm in length) were
treated with Al. For P treatments, seedlings were precultured in
nutrient solution for 7 d. The nutrient solution contained the following macro- and micro-nutrients (in mM) as described by Liao
et al. (2006): 2.5 KNO3, 0.5 KH2PO4, 2.5 Ca(NO3)2.4H2O,
4.57 1023 MnCl2.4H2O, 0.25 K2SO4, 1.0 MgSO4.7H2O,
0.38 1023 ZnSO4.7H2O, 1.57 1024 CuSO4.5H2O, 0.09 1024 (NH4)6Mo7O24.4H2O, 23.13 1023 H3BO3, 0.082
Fe-EDTA(Na). Al treatments were given the same solution containing Al3þ as AlCl3. Three Al3þ levels were employed, ranging
from 0, 50 and 100 mM Al3þ. Twenty-four hours after Al treatment, the tap root length was measured using Image J software
(inspired by National Institutes of Health Image for the
Macintosh computer) and Al staining with haematoxylin as the
indicators of Al tolerance (Liao et al., 2006). Seedlings for
APase measurements were transplanted to the new nutrient solution with 0.5 mM P or without P addition. Ninety days after transplanting, roots were harvested and measured for APase activity
TA B L E 1. Soil chemical properties in the experimental field site
pH
Organic matter
(g kg21)
Total nitrogen
(g kg21)
Total phosphorus
(g kg21)
Total potassium
(g kg21)
Alkali hydrolytic
nitrogen
(mg kg21)
Available
phosphorus
(mg kg21)
Exchangeable
calcium
(cmol kg21)
Exchangeable
Al
(cmol kg21)
4.52
8.70
0.43
0.29
0.73
89.40
1.25
1.12
3.11
The chemical analysis was performed by the standard methods as follows: pH value, 2.5:1 (water/soil); organic matter, K2Cr2O7.H2SO4 digestion; total N
content, Kjedahl method; total P content, H2SO4.HClO4 digestion; total K content, NaOH fusion; available N content, alkaline diffusion; available P content,
Bray II method; available K content, 1 mol L21 neutral NH4OAc extraction.
Du et al. — Stylosanthes adaptation to low-P acid soils
1241
P kg21 sand was applied to the plants. Plants were watered
daily with P-deprived nutrient solution. Plants were harvested
60 d after planting. Dry weight and P concentration were determined as described as above. Total root length and total root
surface area were analysed using WinRhizo software (Regent
Instruments Inc., Quebec, Canada).
following Yan et al. (2001). The solution was well aerated and the
pH was adjusted to 4.2 with 1.0 mM HCl or NaOH. Each treatment was conducted with four replicates.
Collection and analysis of executed organic acids
After seed germination, seedlings were grown in the normal
nutrient solution as described above for 2 weeks. Prior to collecting root exudation of plants subjected to Pi starvation,
plants were rinsed with 0.5 mM CaCl2 ( pH 5.8), and transferred to 45 mL of 0.5 mM CaCl2 ( pH 5.8) with (HP) or
without (LP) addition of 200 mM KH2PO4. To collect root exudation from plants subjected to Al treatments, seedlings were
rinsed with 0.5 mM CaCl2 ( pH 4.2), and separately transferred
into 45 mL of 0.5 mM CaCl2 ( pH 4.2) containing 0 (2Al) or
100 mM (þAl) AlCl3. After 24 h treatment, the collecting solution was separately stored at 220 8C, and concentrated using
a freezing dry vacuum system (Labconco, Kansas City, MO,
USA). Analysis of organic acids was conducted by reversedphase HPLC in the ion suppression mode following the
method described by Li et al. (2008). To identify organic
acids, the retention time and absorption spectra in samples
were compared with those of known standards.
Data analysis
The data of shoot dry weight, P content and all the root parameters were analysed using the SAS program (Statistical
Analysis Systems Institute, version 8.1).
R E S ULT S
Plant growth and P uptake in the field
To examine genotypic variations of stylo adaptation to low-P
acid soils, 12 genotypes were selected for the field experiments.
Genotypic variations for P content in the grain of the tested genotypes were observed, but P content in one grain of all the genotypes was ,1.5 mg (Fig. 1). All the stylo genotypes tested were
grown in the field with or without P application. Phosphorus
application significantly affected shoot dry weight, P content
and utilization efficiency of the 12 tested stylo genotypes
(Fig. 2). However, genotypes differed in response to P application. Shoot dry weight was significantly increased by P application in nine genotypes. Among them, ‘TPRC2001-1’ and
‘Reyan NO.5’ had the highest shoot dry weight with P application. But significant effects of P application on shoot dry
weight were not observed in the three genotypes, ‘Seca’,
‘Fine-stem’ and ‘Mineirao’ (Fig. 2A).
Similar to the results of shoot dry weight, P content was significantly increased by P application in the nine genotypes.
Among them, ‘TPRC2001-1’ had the highest P content.
However, P content was not affected in the three genotypes,
Sand culture experiment
Seeds of the selected three stylo genotypes were pretreated as
described above and germinated in a sand bed. Two seedlings
were transferred to a pot after 15 d and grown in a greenhouse
with a temperature of 25– 30 8C. Seedlings were supplied with
five forms of P: KH2PO4, CaHPO4, AlPO4, FePO4 and phytic
acid (C6H6O24P6Na12). Plants without P application were used
as a control. For convenience of description, the above P treatments were separately designated as K-P, Ca-P, Al-P, Fe-P,
phytate-P ( phy-P) and None-P. The equal amount of 200 mg
P content of seed ( g grain–1)
1·5
1·0
0·5
‘TPRC2001-2’
‘TPRC2001-1’
‘CIAT1517’
‘Mineirao’
‘GC1581’
‘Fine-stem’
‘Seabrana’
‘Seca’
‘Verano’
‘Capica’
‘Reyan NO.5’
‘Reyan NO.2’
0
F I G . 1. Total P content of a seed in tested stylo genotypes. Each column is the mean of three replicates with s.e. F-value of ANOVA: 141.93 for genotype
(P , 0.01).
Du et al. — Stylosanthes adaptation to low-P acid soils
Shoot dry weight (g per 3 plants)
1242
150
A
Low P
High P
100
50
B
400
200
0
0·9
C
0·6
‘TPRC2001-2’
‘TPRC2001-1’
‘CIAT1517’
‘Mineirao’
‘GC1581’
‘Fine-stem’
‘Seabrana’
‘Seca’
‘Verano’
‘Capica’
0
‘Reyan NO.5’
0·3
‘Reyan NO.2’
P utilization efficiency (kg mg–1)
P content (mg per 3 plants)
0
600
F I G . 2. Plant growth and P uptake in the field at two P levels. Each column is the mean of three replicates with s.e. (A) Shoot dry weight, F-value of ANOVA:
9.99 for genotype (P , 0.01), 42.16 for P level (P , 0.01). (B) P content, F-value of ANOVA: 12.55 for genotype (P , 0.01), 116.72 for P level (P , 0.01). (C)
P utilization efficiency, F-value of ANOVA: 20.4 for genotype (P , 0.01), 207.88 for P level (P , 0.01).
‘Seca’, ‘Fine-stem’ and ‘Mineirao’ (Fig. 2B). P utilization efficiency of all the tested genotypes was significantly decreased
by P addition, but the differences among P utilization efficiency were not as much as the shoot biomass and P content
at two P levels (Fig. 2C).
Effects of Al levels on tap root elongation and Al accumulation
Al tolerance of the three selected stylo genotypes differing in
P efficiency was further examined by measuring relative tap root
growth and haematoxylin staining. ‘TPRC2001-1’ was a
P-efficient genotype, ‘Fine-stem’ was a P-inefficient genotype
and ‘Verano’ was intermediate. Tap root growth was significantly inhibited in stylo genotypes with increasing Al concentration in the nutrient solution. However, various responses to
Al toxicity were observed among the three genotypes
(Fig. 3A). Relative tap root growth in ‘Fine-stem’ was 56 %
and 59 % at 50 and 100 mM Al3þ levels, respectively.
However, relative tap root growth of ‘TPRC2001-1’ was not
severely inhibited by Al toxicity, which was 89 % and 82 % at
50 and 100 mM Al3þ levels, respectively. This finding was
further confirmed by haematoxylin staining, which showed
that the P-efficient genotype, ‘TPRC2001-1’ accumulated less
Al at 50 mM and 100 mM Al3þ levels than the P-inefficient genotype ‘Fine-stem’ (Fig. 3B).
Acid phosphatase activity of stylo
The response of root APase activity in stylo to P availability
varied with genotypes. Except for the P-efficient genotype,
Du et al. — Stylosanthes adaptation to low-P acid soils
120
1243
A
‘Fine-stem’
Relative taproot length (%)
‘TPRC2001-1’
‘Verano’
80
40
0
B
‘Fine-stem’ ‘Verano’ ‘TPRC2001-1’
‘Fine-stem’ ‘Verano’ ‘TPRC2001-1’
0
50
‘Fine-stem’ ‘Verano’ ‘TPRC2001-1’
100
Concentration of Al3+ (µM)
F I G . 3. Stylo root growth was affected by different Al concentrations: (A) relative tap root growth; (B) roots stained with haematoxylin to visualize root Al
content. Plants were grown in nutrient solution at three Al3þ levels: 0, 50 or 100 mM as AlCl3. Relative tap root growth was calculated as the percentage of
tap root growth for roots grown at different Al3þ levels at pH 4.2 relative to the tap root growth without Al addition at pH 5.8. Each column is the mean
and s.e. of four replicates. F-values of ANOVA: 12.24 for genotype (P , 0.0001), 28.68 for Al treatments (P , 0.0001).
‘TPRC2001-1’, no significant effect of P levels on the root
APase activity in stylo was found (Fig. 4). For
‘TPRC2001-1’, the total APase activity in roots was obviously
increased by Pi starvation. Under low P conditions, root APase
activity of ‘TPRC2001-1’ was about 60 % higher than that at
high P level (Fig. 4).
Root exudated orgainc acids under Pi starvation and
Al toxicity conditions
Malic, tartaric and lactic acids were secreted by the three
stylo genotypes under both Pi starvation and Al toxicity conditions (Figs 5 and 6). However, effects of Pi starvation and
Al stress had different effects on the secretion patterns of
organic acids. It showed that Pi starvation did not significantly
increase efflux of the organic acids from the three genotypes
(Fig. 5). Furthermore, significant differences of the secreted
lactic acid were not observed at two P levels among the
three genotypes. However, it was observed that
‘TPRC2001-1’ secreted more malic acid than ‘Fine-stem’ at
the low P level (Fig. 5). Al toxicity did not affect the tartaric
acid secretion among the three genotypes (Fig. 6). Malic acid
secretion was significantly increased in ‘Fine-stem’ subjected
to Al toxicity, but there was no genotypic variation among
the three genotypes (Fig. 6). Increased secretion of lactic
acid was observed under Al toxicity conditions in
‘Fine-stem’ and ‘Verano’, not in ‘TPRC2001-1’ (Fig. 6).
Utilization of different P sources in stylo
The three genotypes were also selected to determine their
ability to utilize different P sources, including K-P, Ca-P,
Al-P, Fe-P and phy-P. Results showed that there were significant
variations in shoot dry weight and P content with different
sources of P among the three stylo genotypes in the sand
culture experiment (Fig. 7). The three genotypes had the
highest shoot dry weight and P content when they were supplied
with K-P and Ca-P (Fig. 7A, B). However, genotypic variations
in shoot dry weight and P content were observed when plants
were supplied with Al-P and phy-P. ‘TPRC2001-1’ had a
higher shoot dry weight and P content than ‘Verano’ and
‘Fine-stem’ under phy-P application conditions (Fig. 7A, B).
Furthermore, significant decreases in shoot dry weight and P
content were observed among the three genotypes with Fe-P
or without P application (Fig. 7A, B).
Root length and total root surface area were also significantly affected by different P sources (Table 2). The three
stylo genotypes tested had greater root length and total root
1·8
APase activity in roots
(µmol mg protein–1 min–1)
Low P
a
High P
ab
1·2
b
b
ab
b
0·6
0·0
‘Fine-stem’
‘Verano’
‘TPRC2001-1’
Root secreted malic acid Root secreted tartaric acid
(µmol g–1 h–1)
(µmol g–1 h–1)
0·20
Root secreted lactic acid
(µmol g–1 h–1)
F I G . 4. Acid phosphatase (APase) activity in the roots of the three stylo genotypes at two P levels. Plants were treated with or without P addition for 90 d,
and then roots were harvested for APase activity measurement as described by
Yang et al. (2001). Each column is the mean of four replicates with s.e.
Different letters represent significant difference at the 0.05 level.
0·09
A
Low P
a
a
0·15
High P
0·10
b
b
b
b
0·05
0
5
4
a
B
Root secreted malic acid Root secreted tartaric acid
(µmol g–1 h–1)
(µmol g–1 h–1)
Du et al. — Stylosanthes adaptation to low-P acid soils
0·08
Root secreted lactic acid
(µmol g–1 h–1)
1244
0·08
A
–Al
0·06
a
+Al
0·04
ab
b
b
0·02
b
b
0
9
a
B
6
ab
ab
3
b
b
b
0
a
C
0·06
b
0·04
0·02
c
c
c
c
0
‘Fine-stem’
‘Verano’
‘TPRC2001-1’
F I G . 6. Secreted tartaric (A), malic (B) and lactic acids (C) in the three stylo
genotypes under Al toxicity conditions. Each column is the mean of four replicates with s.e. Different letters represent significant difference at the 0.05 level.
ab
ab
3
ab
2
1
D IS C US S IO N
ab
Genotypic variations for P efficiency in stylo
b
0
a
C
a
0·06
a
a
a
0·03
a
0
‘Fine-stem’
‘Verano’
‘TPRC2001-1’
F I G . 5. Secreted tartaric (A), malic (B) and lactic acids (C) from stylo plants
at two P levels. Each column is the mean of four replicates with s.e. Different
letters represent significant difference at the 0.05 level.
surface area under K-P and Ca-P application conditions. When
plants were supplied with Al-P, significant decreases of root
length and total root surface area were only detected in
‘Fine-stem’, not in ‘TPRC2001-1’ or ‘Verano’, compared
with plants under K-P or Ca-P applied conditions.
‘TPRC2001-1’ had the greatest root length and root surface
area when supplied with phy-P.
Shoot biomass, P content and shoot biomass produced by unit
P could represent the P efficiency, P acquisition and utilization
efficiency of the tested stylo genotypes in the field and greenhouse experiments according to Batten (1992). Both the
present field and greenhouse studies demonstrated substantial
genotypic variations for P efficiency among the tested stylo
genotypes, as indicated by shoot dry weight and P content
(Figs 2 and 7). Among them, ‘TPRC2001-1’ was a
P-efficient genotype, ‘Fine-stem’ was a P-inefficient genotype
and ‘Verano’ was intermediate. Although genotypic differences of total P content were observed in the seeds of the
tested stylo genotypes (Fig. 1), total P content in plants at
low P level was .1000 times of that in seeds (Fig. 2B), indicating that P stored in seeds could not explain the genotypic
differences in P efficiency among the stylo genotypes.
Genotypic differences of P efficiency might be mainly
due to the capacity of plants acquiring P from soils, which
was further supported by the present sand experiment
results, in which the P-efficient genotype ‘TPRC2001-1’,
had superior ability to extract P from phy-P. The results
were consistent with other results, in which P efficiency of
stylo was mainly determined by P acquisition efficiency
(Yang and Yan, 1998).
Du et al. — Stylosanthes adaptation to low-P acid soils
1245
1·8
A
‘Fine-stem’
Shoot d. wt (g per 2 plants)
‘TPRC2001-1’
‘Verano’
1·2
0·6
0
4·5
P content (mg per 2 plants)
B
3·0
1·5
0
K-P
Ca-P
Al-P
Phy-P
Fe-P
No P
F I G . 7. Plant growth and P uptake in sand culture supplied with different P sources. Each column is the mean and s.e. of four replicates. (A) Shoot dry weight,
F-value of ANOVA: 10.16 for genotype (P , 0.0001), 105.81 for P treatment (P , 0.0001). (B) P content, F-value of ANOVA: 8.28 for genotype (P , 0.0001),
133.71 for P treatment (P , 0.0001).
P-efficient genotype had high Al tolerance
Al toxicity and low P availability often coexist in acid soils
(Kochian et al., 2004). Therefore, it is assumed that
P-efficient genotypes might also have high Al tolerance. This
was further proved in the present study. It was found that tap
root growth of the P-efficient genotype, ‘TPRC2001-1’, was
less affected by Al toxicity than a P-inefficient genotype,
‘Fine-stem’ (Fig. 3A). It has been indicated that P could ameliorate Al toxicity possibly through precipitation of Al in the rhizosphere. Under P-limited conditions, P-efficient genotypes might
acquire more P from soils and transport more P to the tap root
tips resulting in an increase in Al tolerance (Zheng et al.,
2005), which is consistent with the present results from the
sand culture experiment. The P-efficient ‘TPRC2001-1’ was
Al tolerance, and had superior ability to extract P from Al-P
(Fig. 7). Secretion of organic acid from roots is considered to
be one of the major mechanisms of Al tolerance in plants
(Pellet et al., 1995; Ma et al., 2001; Kochian et al., 2004;
Ligaba et al., 2004). It has been reported that secretion of
citrate, tartrate and acetate was related to mobilization of Al-P
in common bean (Shen et al., 2002). In the study, secreted tartaric, malic and lactic acids have been detected in the three stylo
genotypes subjected to Al stress, but ‘TPRC2001-1’ did not
secrete more organic acids than any other stylo genotypes
(Fig. 6), indicating secreted organic acids could not explain
the superior ability of ‘TPRC2001-1’ to tolerate Al, which
might be involved in other Al tolerance mechanisms.
P-efficient genotype had superior ability to utilize organic P
In soil, 30– 80 % of the total P is in organic form (Tarafdar
and Claassen, 2005). Particularly, phytate, as well as its
derivatives, accounts for 20– 50 % of the total soil organic P,
which is poorly utilized by plants (Richardson et al., 2001).
Root growth and the ability of three stylo genotypes differing
in P efficiency to utilize P from phytate were examined.
Among them, ‘TPRC2001-1’ had the highest capacity to
extract P from phy-P, as indicated by the highest shoot dry
weight, P content, and superior root length and surface area,
respectively (Fig. 7 and Table 2). These findings suggest that
12.69***
38.01***
6.60***
202.08 + 13.35
194.00 + 23.83
214.63 + 32.70
198.93 + 5.91
43.35 + 1.87
55.33 + 13.31
264.48 + 33.68
263.48 + 41.91
168.55 + 10.47
41.03 + 6.50
63.98 + 11.55
70.35 + 13.87
150.00 + 8.42
135.35 + 11.76
128.73 + 17.92
71.98 + 17.83
55.75 + 8.51
63.50 + 12.22
‘TPRC2001-’
‘Fine-stem’
efficient utilization of organic P may be another factor
accounting for the genotypic variations for P efficiency in
stylo grown on low-P acid soils. The present results were consistent with other studies, where organic P could be utilized by
different plant species, including bean, wheat and barley
(Helal, 1990; Asmar et al., 1995). It is generally believed
that most organic P could not be directly used by plants
unless being hydrolysed by acid phosphatases (APases)
(Tarafdar and Claassen, 2005). A positive relationship has
been reported between root APase activity and P uptake
from organic P in bean (Helal, 1990) and barley (Asmar
et al., 1995). The present results also showed that the
P-efficient genotype, ‘TPRC2001-1’ had higher root APase
activity under low P conditions (Fig. 4), suggesting that the
P-efficient genotype had higher root APase activity resulting
in efficient utilization of organic P.
21.92***
26.04***
5.57***
Each value in the table is the mean of four replicates with s.e.
* 0.05 . P . 0.01; ** 0.01 . P . 0.001; *** P , 0.001.
†
G, Genotypes used in this research; P, phosphorus forms; G P, interaction effects between genotypes and P sources.
6.19**
31.25***
5.12***
1034.20 + 133.09
970.00 + 63.32
1010.55 + 138.48
574.58 + 149.49
445.75 + 56.47
568.83 + 138.24
1202.28 + 72.06
1554.10 + 173.20
1423.48 + 256.93
1278.83 + 51.12
313.15 + 19.87
386.20 + 79.56
1702.38 + 205.77
1714.73 + 258.67
1085.68 + 51.68
348.28 + 46.93
479.23 + 80.41
502.45 + 93.97
0.17 + 0.02
0.25 + 0.04
0.20 + 0.02
0.18 + 0.03
0.04 + 0.01
0.06 + 0.01
0.21 + 0.03
0.28 + 0.05
0.18 + 0.01
0.05 + 0.02
0.07 + 0.01
0.07 + 0.01
K-P
Ca-P
Al-P
Phy-P
Fe-P
None-P
F values†
G
P
GP
0.09 + 0.01
0.10 + 0.01
0.10 + 0.01
0.07 + 0.01
0.05 + 0.00
0.07 + 0.01
‘Verano’
‘TPRC2001-1’
‘Fine-stem’
‘Verano’
‘TPRC2001-1’
‘Fine-stem’
Form of P
Root length (cm per 2 plants)
Conclusions
Root dry weight (g per 2 plants)
TA B L E 2. Root parameter of stylo as affected by P sources in sand culture experiment
‘Verano’
Du et al. — Stylosanthes adaptation to low-P acid soils
Total root surface area (cm2 per 2 plants)
1246
The results demonstrate that there are genotypic variations
for adaptation to low-P acid soils in stylo. The P-efficient genotype, ‘TPRC2001-1’, not only had higher Al tolerance, but
also had superior ability to utilize organic P sources, indicating
that the mechanisms underlying stylo adaptation to low-P acid
soils might involve a superior ability to tolerate Al and utilize
organic P efficiently.
ACK N OW L E DG E M E N T S
We thank Jonathan P. Lynch for critical reading of the manuscript and helpful comments and Dr Li Haigang for helping
with organic acids analysis. This research was supported by
funds from National Basic Research Program (973 Program)
of China (2007CB108903 to G.L. and 2005CB120902 to
X.Y. and H.L.) and the McKnight Foundation Collaborative
Crop Research Program (05-780) and the National Natural
Science Foundation of China to H.L.
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