Aluminium tolerance and high phosphorus efficiency helps Stylosanthes better
adapt to low-P acid soils
Yu-Mei Du
1
, Jiang Tian
2
, Hong Liao
2
, Chang-Jun Bai
1
, Xiao-Long Yan
2
and Guo-Dao Liu
1,
*
1
Institute of Tropical Crop Genetic Resources, Chinese Academy of Tropical Agriculture Science, Danzhou 571737, China
and
2
Root 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.
INTRODUCTION
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 distri-
bution of roots along soil profile; Yan and Zhang, 1997).
Accumulating results reveal that changes of root traits lead to
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 phos-
phate 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 aesti-
vum), 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 tox-
icity 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
* For correspondence. E-mail: liuguodao@scuta.edu.cn or
liuguodao2008@163.com
# The Author 2009. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org
Annals of Botany 103: 1239–1247, 2009
doi:10.1093/aob/mcp074, available online at www.aob.oxfordjournals.org

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 strat-
egies (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 effi-
ciency 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.
MATERIALS AND METHODS
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 19830
0
N, 109830
0
E at 149 m a.s.l.. The soil for the exper-
iment 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 ha
21
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 8Cto
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 gen-
otypes, including ‘Fine-stem’, ‘TPRC2001-1’ and ‘Verano’, dif-
fering in P efficiency were used in this study. Seeds were
pretreated as described above. Pretreated seeds were germinated
on filter paper moistened with 0
.
5mM CaSO
4
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 fol-
lowing macro- and micro-nutrients (in mM) as described by Liao
et al. (2006): 2
.
5KNO
3
,0
.
5KH
2
PO
4
,2
.
5 Ca(NO
3
)
2
.
4H
2
O,
4
.
57  10
23
MnCl
2
.
4H
2
O, 0
.
25 K
2
SO
4
,1
.
0 MgSO
4
.
7H
2
O,
0
.
38  10
23
ZnSO
4
.
7H
2
O, 1
.
57 10
24
CuSO
4
.
5H
2
O, 0
.
09
10
24
(NH
4
)
6
Mo
7
O
24
.
4H
2
O, 23
.
13 10
23
H
3
BO
3
,0
.
082
Fe-EDTA(Na). Al treatments were given the same solution con-
taining Al
3þ
as AlCl
3.
Three Al
3þ
levels were employed, ranging
from 0, 50 and 100 mM Al
3þ
. Twenty-four hours after Al treat-
ment, 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 sol-
ution with 0
.
5mM P or without P addition. Ninety days after trans-
planting, roots were harvested and measured for APase activity
TABLE 1. Soil chemical properties in the experimental field site
pH
Organic matter
(g kg
21
)
Total nitrogen
(g kg
21
)
Total phosphorus
(g kg
21
)
Total potassium
(g kg
21
)
Alkali hydrolytic
nitrogen
(mg kg
21
)
Available
phosphorus
(mg kg
21
)
Exchangeable
calcium
(cmol kg
21
)
Exchangeable
Al
(cmol kg
21
)
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, K
2
Cr
2
O
7
.
H
2
SO
4
digestion; total N
content, Kjedahl method; total P content, H
2
SO
4
.
HClO
4
digestion; total K content, NaOH fusion; available N content, alkaline diffusion; available P content,
Bray II method; available K content, 1 mol L
21
neutral NH
4
OAc extraction.
Du et al. — Stylosanthes adaptation to low-P acid soils1240