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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 ...
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

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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

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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)

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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. L I T E R AT U R E C I T E D Asmar F, Gahoonia T, Nielsen N. 1995. Barley genotypes differ in activity of soluble extracellular phosphatase and depletion of organic phosphorous in the rhizosphere soil. Plant and Soil 172: 117–122. Barber SA. 1995. Soil nutrient bioavailability: a mechanistic approach. New York, NY: John Wiley and Sons, 202– 230. Batten GD. 1992. A review of phosphorus efficiency in wheat. Plant and Soil 146: 163–168. Chakraborty S. 2004. High-yielding anthracnose-resistant stylosanthes for agricultural systems. CSIRO, Australia, 27– 28. Clark RB, Duncan RR. 1991. Improvement of plant mineral nutrition through breeding. Field Crops Research 27: 219– 240. Delhaize E, Ryan PR, Randall PJ. 1993. Aluminum tolerance in wheat (Triticum aestivum L.). II. Aluminum-stimulated excretion of malic acid from root apices. Plant Physiology 103: 695– 702. Foy CD. 1984. Physiological effects of hydrogen, aluminum, and manganese toxicities in acid soil. In: Adams Fed. Soil acidity and liming. Madison, WI: American Society of Agronomy, 57– 97. Graham RD. 1984. Breeding for nutritional characteristics in cereals. Advances in Plant Nutrition 1: 57– 102. Helal HM. 1990. Varietal difference in root phosphatase activity as related to the utilization of organic phosphates. Plant and Soil 123: 161– 163. Kochian LV, Hoekenga OA, Pineˇros MA. 2004. How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorus efficiency. Annual Review of Plant Biology 55: 459– 493.

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