Plant Soil (2009) 320:91–102 DOI 10.1007/s11104-008-9873-0
REGULAR ARTICLE
Genotypic differences in phosphorus acquisition and the rhizosphere properties of Brassica napus in response to low phosphorus stress Haiwei Zhang & Yu Huang & Xiangsheng Ye & Lei Shi & Fangsen Xu
Received: 6 August 2008 / Accepted: 17 December 2008 / Published online: 20 January 2009 # Springer Science + Business Media B.V. 2009
Abstract Genotypic differences in acquiring immobile P exist among species or cultivars within one species. We investigated the P-efficiency mechanisms of rapeseed (Brassica napus L.) in low P soil by measuring plant growth, P acquisition and rhizosphere properties. Two genotypes with different P efficiencies were grown in a root-compartment experiment under low P (P15: 15 mg P kg−1) and high P (P100: 100 mg P kg−1) treatments. The P-efficient genotype produced more biomass, and had a high seed yield and high P acquisition efficiency under low P treatment. Under both P treatments, both genotypes decreased inorganic P (Pi) and organic P (Po) fractions in the rhizosphere soil. However there was no decrease in NaHCO3-Po at P100. For the P15 treatment, the concentrations of NaHCO3-Po and NaOH-Po were negatively correlated with soil acid phosphatase activity. The P-efficient genotype 102 differed from the P-inefficient genotype 105 in the
Responsible Editor: N. Jim Barrow. H. Zhang : Y. Huang : X. Ye Microelement Research Centre, Huazhong Agricultural University, Wuhan 430070, China L. Shi : F. Xu (*) National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China e-mail:
[email protected]
following ways. In the rhizosphere the soil pH was lower, acid phosphatase activity was higher, and depletion of P was greater. Further the depletion zones were wider. These results suggested that improving P efficiency based on the character of P efficiency acquisition in P-efficient genotype would be a potential approach for maintaining rapeseed yield potential in soils with low P bioavailability. Keywords Low-P . Brassica napus L . P efficiency . Rhizosphere properties . P fractions
Introduction Under P deficient conditions, plants have developed a number of adaptive strategies to take up and utilize soil P. To promote P uptake from the rhizosphere, root growth is favored rather than shoot growth (Nilsson et al. 2007). Root-associated factors such as root morphology, architecture, root hair density, nutrient absorption rate, ability to modify the rhizosphere and mycorrhizal symbiosis can strongly influence Pi acquisition (Raghothama 1999; López-Bucio et al. 2002; Ma et al. 2001; Wissuwa and Ae 2001; Schweiger et al. 2007). Root exudations by plant roots as amino acids, organic acids (Graham et al. 1981; Schwab et al. 1983; Li et al. 1997; Hinsinger 2001; Shane and Lambers 2005), H+ (Yan et al. 2002; Wang et al. 2006; George et al. 2002) and acid phosphatase (Tadano et al. 1993; Asmar et al. 1995)
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have also been shown to play important roles in increasing the availability of P. Through plant roots, microorganism activity and rhizosphere processes affect the biological, physical and chemical properties of rhizosphere soil (George et al. 2002; Liu et al. 2004; Marschner et al. 2007). A considerable increase in acid phosphatase activity was observed in the soil–root interfaces of four species compared to the bulk soil (Tarafdar and Jungk 1987). The depletion of Phosphate strongly declined with increasing distance from the root surface (Lewis and Quirk 1967; Hendriks et al. 1981; Zoysa et al. 1999; Marschner et al. 2007). Slicing technology using frozen samples, which scarcely alters the soil chemical properties, has been used to study the variety of nutrients in soil slices taken certain distances from the root surface. In addition, application of Hedley fractionation (Hedley et al. 1982a) in research of rhizosphere processes has given a further understanding of P form transformation resulting from root exudation and plant uptake. It is also very important for us to understand the degree to which the various P compounds can be utilized by plants. The Yangtze River valley in China is one of three rapeseed planting regions of the world. The area involved and the yield produced account for one third of the world’s production. However, low P concentration in soil solution seriously limits the production and quality of B. napus. To increase agricultural ecobenefits and product management, screening and identification of P-efficient genotypes of B. napus is important, as is exploration of mechanisms to utilize sparingly soluble P in soil. Earlier studies have shown that P-efficient genotypes or cultivars of B. napus could mobilize more sparingly soluble P and had higher biomass production and seed yield compared to P-sensitive types in P stressed soil (Foehse and Jungk 1983; Moorby et al. 1988; Hoffland et al. 1992). However, genotypic differences in soil biological and chemical properties as a result of root activities remain poorly understood. The aims of this paper therefore are to increase our understanding of the mechanisms of P efficiency by studying the differences in rhizosphere processes of two genotypes of B. napus. In line with this, the following are examined: (1) soil pH changes in the rhizosphere; (2) the distribution of soil acid phosphatase in the rhizosphere; and (3) the width of depletion zones
Plant Soil (2009) 320:91–102
and concentrations of P fractions in the rhizosphere and bulk soil.
Materials and methods Plant and soil materials Two genotypes, 102 (P-efficient) and 105 (P-inefficient), of rapeseed (B. napus) screened from 149 recombined inbred lines (F9) derived from a cross between P-efficient cultivar 97081 and P-inefficient cultivar 97009 under low P soil were employed in this study. Genotype 102 shows no distinct symptoms of P deficiency while 105 shows severe symptoms at seedling stage (purple leaves and less biomass production) under low P condition. Eluvial soil (20 cm depth) from Shizi Mountain on the campus of Huazhong Agricultural University (Hubei province, China, 30.47°S 114.35°E), was used in this study; selected based on its low content of soluble P and high fixed P. It had a pH in water of 6.5 (1:2.5 w/w soil to water ratio), an organic matter content of 6.0 g·kg−1, total nitrogen content of 0.85 g·kg−1, total phosphate content of 0.21 g·kg−1, and available phosphate content of 1.78 mg·kg−1. Airdried soil samples were ground and passed through a 2 mm sieve then steam-sterilized for 3 h to minimize the effects of microorganisms. Plant growth conditions A rhizo-box (17 cm×12 cm×17 cm in length× width× depth) with three compartments made of PVC material was used to study the rhizosphere processes. The rhizo-box was divided into three parts vertically with two pieces of 30-μm mesh nylon cloth impenetrable by roots. The middle compartment was 2 cm in width while the two side compartments were 5 cm. Four PVC tubes with a diameter of 1.5 cm and 17 cm in length were fixed onto the four corners of the rhizo-box to supply deionized water. The compartments were filled with a total of 3 kg of the above mentioned soil. The soil was watered gravimetrically to field capacity (12%) every day. The seeds were surface sterilized for 1 min using 10% (w/v) NaOCl then washed four to five times with deionized water. Five to six seeds were sown in the
Plant Soil (2009) 320:91–102
middle compartment of the rhizo-box and covered with a small amount of fine soil in order to avoid evaporation. After 1 week, they were thinned to two uniform seedlings per rhizo-box. The status of plants growth was recorded throughout the experiment and shed leaves were collected and dried at 65°C. Two P treatments were employed: 15 mg P kg−1 (P15) and 100 mg P kg−1 (P100), added as KH2PO4. Nitrogen (N) and potassium (K) fertilizers were mixed with the soil at a rate of 200 mg N and K kg−1 soil in the form of urea and KCl before planting. Other nutrients were added as nutrient solution containing 25 mg·kg−1 MgSO4·7H2O; 2.86 mg·kg−1 H3BO3; 1.81mg·kg−1 MnCl2·4H2O; 0.22 mg·kg−1 ZnSO 4 ·7H 2 O; 0.08 mg·kg −1 CuSO 4 ·5H 2 O and 0.02 mg·kg−1 (NH4)6MO7O2·4H2O. At bud and pod stages, the plants were dressed with an additional 100 mg N and K kg−1 soil in solution form. The experimental treatments were replicated five times and conducted in a randomized complete block design in a glasshouse maintained at 2°C minimum and 26°C maximum temperatures in Huazhong Agricultural University. Five replicated rhizo-boxes without plants were also included as controls to determine the changes in rhizosphere soil properties during the study period. Plant and soil sampling Plant shoots were harvested after seven months growth. After being threshed, the samples were separated into stem, leaf, pod pericarp and seed. The stem, leaf and pod pericarp were defined as biomass production, mixed to weight and used for chemistry analysis. Soil samples were taken from the middle compartment of the rhizo-boxes then root systems were gently removed from the soil by holding the plants at the stem base. The roots were carefully shaken to remove excess soil, and clumps of soil trapped between roots were collected by brushing with a paintbrush. The soil adhering to the roots was defined as rhizosphere soil, while that in the two side compartments as bulk soil. Despite the care taken, some root breakage was unavoidable, and therefore, chemical analysis was only conducted on the aboveground samples in the experiment. In order to study chemistry changes in the soil slices induced by P deficiency with distance from the rhizoplane, soil from the side compartments was
93
removed and sliced into thin sections starting from the mesh boundary (the mesh surface) using a modified hand-made microtome. Samples were obtained at distances of 1, 2, 3, 4, 5 and 6 mm from the mesh surface. Soil from the control (unplanted) rhizo-box was dealt with in the same manner. Soil samples obtained from the two side compartments were mixed in accordance with their distance from the mesh surface as one sample. Plant and soil analyses All plant samples were dried for 48 h at 65°C, weighed then ground to < 1 mm in a stainless steel ball mill for P analysis. Total P was determined according to the vanadomolybdate method of Westerman (1990) after digesting the samples in concentrated H2SO4–HClO4 using a digestion block system. Soil pH was measured in water (1:2.5 w/w soil to water ratio) using a pHsensitive microelectrode. Determination of acid phosphatase activity Soil acid phosphatase activity was determined following the method of Tabatabai and Bremner (1969) as modified by Hedley et al. (1982b). Samples of approximately 0.3 g soil (< 2 mm) were used with 4 mL extraction buffer composed of 40 mM NaOAc– HAc (pH 6.5), and 2 mL of 15 mM p-nitrophenol phosphate as substrate. After incubation at 37°C for 1 h, the reaction was stopped with 1 mL of 1 M NaOH and absorbance was measured spectrophotometrically at 400 nm. One unit of acid phosphatase activity was defined as the activity per gram soil that produced 1 µmol p-nitrophenol/h. Soil P fractionation by sequential extraction The soil samples taken from the rhizo-boxes were used for P fractionation after plants were harvested. The soil P fractions of 0.5 g air-dried soil (sieved through a 2 mm mesh) were determined sequentially according to the procedure of Tiessen and Moir (1993) based on the method of Hedley et al. (1982a), which uses progressively more aggressive extractants to remove P. The sequential analytical procedure, the associated P fraction, and its proposed functional role in soil are described in Fig. 1. Four
94
Plant Soil (2009) 320:91–102
0.5g Air-dried soil H2 O
Water-Pi
Residue
Statistical analysis
0.5M NaHCO3
Extractant
NaCO3-Pi
Digestion NaCO3-Pt
Residue 0.1M NaOH
Extractant Digestion
Residue 1M HCl
Residue
digesting the soil residue in H2SO4–H2O2 (Tiessen and Moir 1993).
NaOH-Pi NaOH-Pt HCl-Pi
H2SO4 + H2O2
The soil P fractions, acid phosphatase activity, pH and P uptake data were subjected to standard ANOVA. Means are presented with standard errors to indicate variation. When a significant (p<0.05) treatment effect was observed, the mean values were compared using the LSD test (p<0.05), and when values were not significantly different, but a clear pattern was apparent, this was noted as a ‘trend’.
Residue -P
Digestion
Fig. 1 The phosphorus fractionation scheme of Tiessen and Moir (1993) based on Hedley et al. (1982b)
inorganic P (Pi) and two organic P (Po) fractions were removed using distilled water, 0.5 M NaHCO3, 0.1 M NaOH and 1 M HCl consecutively. Water-P has been associated with plant-available P with respect to direct plant uptake. NaHCO3 extracted labile inorganic and organic P (Okalebo et al. 1993) while NaOH extracted moderately labile organic P and partially dissolved inorganic iron and aluminum phosphates. HCl-extractable P, on the other hand, is considered to be Ca-bound P. Total P in the NaHCO3 and NaOH extracts were determined by digesting aliquots of these filtrates in an autoclave at 103.5 kPa and 121°C (60 min for NaHCO3 and 90 min for the NaOH extract) using the acidified ammonium persulphate method (Greenberg et al. 1992). Orthophosphate P filtrates and digests of each soil extract were determined colorimetrically according to Murphy and Riley (1962). The difference between total P (Pt) and inorganic P in the extracts represented the organic P content. P not recovered in these successive extractions was the residual fraction, determined by
Results Plant growth and yield On the P15 treatment, both genotypes showed distinct P deficiency symptoms. They were small and stunted, and with darkish green old leaves; the latter was particularly evident in P-inefficient genotype 105. In contrast, both genotypes grew well under P100 treatment. At maturity, aboveground samples were divided into biomass production and seed yield in order to compare the difference in P utilization efficiency between two rapeseed genotypes. Both genotypes had significantly greater biomass production (total of stem, leaf and pod pericarp weight) and seed yield at P100 than P15 (p<0.05). Further, at P15 the seed yield of the P-efficient genotype 102, and the P efficiency coefficient was significantly higher than that of the P-inefficient genotype 105 (p <0.05; Table 1). We were unable to quantify the root weight, root hair density and length because of difficultly obtaining intact root system samples from the rhizo-box, but
Table 1 Biomass production, seed yield and the P efficiency coefficients (ratio of seed yield at P15 to P100) of two rapeseed genotypes under two P treatments Treatment
Genotype
Biomass production (g·plant−1)
Seed yield (g·plant−1)
P efficiency coefficient
P15
102 105 102 105
10.5±0.40b 8.9±0.44c 24.2±0.57a 25.0±1.25a
0.5±0.03c 0.4±0.01d 2.9±0.12a 2.6±0.05b
0.176 0.141 – –
P100
The data indicate means and standard errors, n=5, and different lower case letters indicate a significant difference at p<0.05
Plant Soil (2009) 320:91–102
95
abundant root hairs were visible through the nylon mesh at P100, but only a few at P15. Moreover, the root size and root hair density of genotype 102 were greater than those of genotype 105 under both P treatments.
105 decreased by 0.75. Fore the P15 treatments the decreases were 0.19 and 0.12. The decrease in pH was greater with genotype 102 than genotype 105 at the same distance from the rhizosplane (Fig. 2). Activity of acid phosphatase
P acquisition and utilization Both genotypes accumulated more total P under the P100 treatment than the P15 treatment (p<0.05) (Table 2). Further, under both P treatments, the P accumulation in seeds of P-efficient genotype 102 was significant higher and had a higher P translocation coefficient than those of the P-inefficient 105. The amount of P partitioned to seeds in P-efficient genotype 102 was higher than that in P-inefficient genotype 105 during low P stress, but it showed a lower P utilization efficiency than genotype 105 (Table 2). The P concentration of the biomass was significantly higher under P100 than P15, and the P concentration of both the biomass and seeds was significantly higher in genotype 102 than 105.
Acid phosphatase activity in the rhizosphere and bulk soils with rapeseed was significantly higher than that without rapeseed regardless of P treatments (p<0.05). The highest value was observed in rhizosphere soil, and decreased gradually with increasing distance from the rhizoplane (Fig. 3). A small change in soil acid phosphatase activity was observed with distance from the nylon mesh in controls without plants. The acid phosphatase activity under P100 treatment was higher than that under P15 at the same distance from the rhizoplane. The trend in soil enzyme activity was similar between two genotypes, but acid phosphatase activity at the root surface of P-efficient genotype 102 was significantly higher than that of P-inefficient genotype 105 (p<0.05; Fig. 3).
Rhizosphere soil pH
Soil phosphorus fractions
Soil pH decreased significantly in the rhizosphere of both genotypes for both P treatments compared to the unplanted controls (Fig. 2). The closer to the mesh, the more soil pH declined. There was no change in the soil pH greater than 6 mm from the root surface. The changes of soil pH in the rhizosphere and bulk were greater under P100 than that under P15. Moreover, the decrease in rhizosphere pH was greater for the efficient cultivar. For the P100 treatment, the rhizosphere pH of the P-efficient genotype 102 decreased by 0.95, that of the P-inefficient genotype
The largest fraction of the added P was extracted by NaOH (47%; Table 3). Large amounts were also extracted by NaHCO3–Pi (25%), residual-P (18%) and water-Pi (6%). However, organic P and HCl-Pi were little affected by P treatment. The amount of Pi fractions did not change significantly with distance from rhizoplane in control without plants under both P treatments (Fig. 4). Both genotypes decreased Pi fractions in the rhizosphere soil compared with the control. However, there was no significant decrease in HCl-Pi for 105 under P100
Table 2 P concentration, acquisition, P translocation coefficients (ratio of seed P acquisition to total plant P acquisition) and P utilization efficiency (ratio of seed yield to seed P acquisition) of two rapeseed genotypes under two P treatments P translocation P utilization Total P efficiency acquisition coefficient P concentration P acquisition P concentration P acquisition (g·mg−1) (mg·plant−1) −1 −1 −1 −1 (mg P·g dwt) (mg·plant ) (mg P·g dwt) (mg·plant )
Treatment Genotype Biomass
P15 P100
102 105 102 105
0.92b 0.68d 0.97a 0.87c
Seed
7.64b 6.30b 16.52a 14.98a
4.57c 3.26d 8.07a 5.98b
2.32c 1.21d 23.40a 15.72b
9.96c 7.52d 39.92a 30.70b
0.23 0.16 0.59 0.51
0.22 0.31 0.12 0.17
The data indicate means and standard errors, n=5, and different lower case letters indicate a significant difference at p<0.05
96 6.7
6.8
P15
6.6
6.6
P100
6.4 6.5
pH
Fig. 2 Effects of different rapeseed genotypes on the pH of the rhizosphere under two P treatments. Error bars show the standard error (n=5)
Plant Soil (2009) 320:91–102
6.2
6.4
6.0 5.8
6.3
105 102 Control
6.2
5.6 5.4 5.2
6.1 0
1
2
3
4
5
0
6
1
Distance from the rhizoplane
200
4
5
6
(mm)
Discussion Rhizosphere pH As a result of plant activity, the soil layer immediately surrounding the roots was very different from the bulk soil (Marschner et al. 1986). The pH change in the rhizosphere was dependent on the growth environment 240
P15
105 102 Control
220
P100
200 180
160
-1
-1
3
from the mesh boundary with genotype 102, and the depletion zones appeared similar to those of inorganic P. However, Under P100, there was a decreasing trend in NaHCO3-Po with distance away from the rhizoplane where the amount was highest. On the other hand, NaOH-Po changed less conspicuously with the distance. Residual-P was predominant P fraction under both P treatments, and there were no differences between two genotypes in residual-P depletion in the zones of side compartments. The residual-P in the rhizosphere, however, decreased slightly compared to the nonrhizosphere soil under P100. The residual-P content under P100 was higher than that under P15, possibly due to conversion of the P fertilizers by fixation (Fig. 6).
180 ( µg . g . h )
Fig. 3 Effects of different rapeseed genotypes on acid phosphatase activity in the rhizosphere. Error bars show the standard error (n=5)
Acid phosphatase activity
(Fig. 4). The lowest observed value of inorganic P fractions was observed in the rhizosphere soil adhering to the root surface for both P treatments. The content of inorganic P fractions gradually increased up to an amount equivalent to the control soil with increasing distance from the mesh boundary. Inorganic P fractions were depleted up to the first 3 or 4 mm under P15 but up to the first 5 mm under P100 (Fig. 4). The depletion in NaOH-Pi was the greatest regardless of genotypes and P treatments, followed by NaHCO3-Pi, HCl-Pi and water-Pi (Fig. 4), and more P was depleted from Pi fractions under P100 than P15. There was a general trend that at the same distance from the rhizoplane the P-efficient genotype 102 extracted more Pi fractions than the P-inefficient genotype 105, but HCl-Pi under P15 was not obvious (Fig. 4). The width of depletion zones of P-efficient genotype 102 was a little larger than that of Pinefficient genotype 105 in almost all Pi fractions under both P treatments. Regarding the depletion of organic P fractions, large differences were observed irrespective of P treatment and genotypes (Fig. 5). Under P15, the lowest content of NaHCO3-Po and NaOH-Po were observed in the rhizosphere and within a few mm
2
160 140
140 120
120
100 100
80 0
1
2
3
4
5
6
0
1
Distance from the rhizoplane
2 (mm)
3
4
5
6
Plant Soil (2009) 320:91–102 Table 3 Comparison of P fractions in the control soil
a
Transformation of P fertilizer (%)=(amount of P extracted from the soil under P100−amount extracted under P15)×100/(amount of P applied under P100− amount applied under P15). Data indicates the means and standard errors, n=5
97 P extracted from soil (µg·g−1 soil)
P fraction
Water-Pi NaHCO3-Pi NaHCO3-Po NaOH-Pi NaOH-Po HCl-Pi Residual-P Total P
P15
P100
2.9±0.3 7.8±0.7 8.1±0.6 54.9±1.7 22.0±1.1 15.4±0.9 110.0±2.6 221.0±7.9
7.5±0.5 29.4±0.7 7.1±1.2 95.2±1.9 24.3±3.3 16.8±1.1 126.3±4.3 306.8±13.1
Water-Pi (µg .
-1
g )
5
12
P15
10
4
P100
8
3
6 2 105 102 Control
1
4 2
0 10
0 40
9
35
8
30
7 25 20
4 70
15 110
65
100
60
90
HCl-Pi (µg .
-1
g )
-1
5
g )
6
NaOH-Pi (µg .
NaHCO3-Pi (µg .
-1
g )
6 25 0 47 3 2 18 101
1988). However, a major source of H+ fluxes in the rhizosphere was also related to the differential uptake of cations and anions by plant roots (Hinsinger et al. 2003; Jaillard et al. 2002; Marschner 1995; Tang et al. 1999). In the experiment, urea was the only nitrogen source and it was mainly taken up by plants as NH4+,
of the plants (Hinsinger et al. 2003). Our result showed that soil pH in the rhizosphere was significantly lower than the control (without plant; Fig. 2). This may be because rapeseed roots release H+ as a result of low P stress as reported by previous studies (Akhtar et al. 2008; Ruiz and Arvieu 1990; Moorby et al. 1985, Fig. 4 Concentration of Pi fractions in the rhizosphere with different rapeseed genotypes under P15 and P100 treatments. Error bars show the standard error (n=5)
Transformation of P fertilizer applied to the soil (%)a
55
80
50
70
45
60
40
50
35 20 18 16 14 12 10 8 6
25 20 15 10 5 0 0
1
2
3
4
5
6
0
1
2
Distance from the rhizoplane (mm)
3
4
5
6
NaHCO3-Po (µg . NaOH-Po (µg .
g-1)
Fig. 5 Concentration of different Po fractions in the rhizosphere with different rapeseed genotypes under P15 and P100 treatments. Error bars show the standard error (n=5)
Plant Soil (2009) 320:91–102 g-1)
98 11 10 9 8 7 6 5 4 40
20 18 16 14 12 10 8 105 6 102 4 Control 2 34 32 30 28 26 24 22 20 18 16 14 5 6
P15
30 20 10 0 0
1
2
3
4
P100
0
1
2
3
4
5
6
Distance from the rhizoplane (mm)
therefore the rapeseeds receiving NH4+ maybe release equivalent amounts of H+ in the rhizosphere to counterbalance the corresponding excess of positive charges entering the cell, thereby decreased the rhizosphere pH. Moreover, when supplying a plant with a K2SO4 solution, more cation such as K+ were taken up than the anions as SO42−, so H+ would be released into the rhizosphere soil to compensate for the excess of positive charges entering the cell (George et al. 2002). Organic acid anions are unlikely to cause acidification directly, but co-transport of H+ with organic acids release has often been referred to as a possible source of rhizosphere acidification (Loss et al. 1993). We were unable to obtain information about the organic acids excreted by the two genotypes, but earlier studies suggested that rapeseed releases malic acid, citric acid and other kinds of organic acids (Hoffland et al. 1992; Zhang et al. 1997; Akhtar et al. 2008). It is therefore possible that in this study the decrease in soil pH in the rhizosphere may be partly
Depletion of soil phosphorus fractions in the rhizosphere Some studies have reported that available P (water-Pi and NaHCO3-Pi) had a significant positive correlation with various growth parameters and P uptake by plants (Sharma and Subehia 2003; Verma et al. 2005). Water-Pi and NaHCO3-Pi existing in the soil solution may be initially absorbed by plants under P-deficient conditions, which resulted in a larger degree of depletion near the root surface compared with the other Pi fractions (Fig. 4). However, NaOH-Pi and HCl-Pi seemed to act as a buffering pool for NaHCO3-Pi, which was available to plants (Guo et al. 2000). Similar conclusions were also reported by many studies that the availability of NaOH-Pi and HCl-Pi were closely related to root exudation (Raghothama 1999; Dakora and Phillips 2002; Li et 160
140
P100
P15
g-1)
Residual-P (µg .
Fig. 6 Concentration of residual-P in the rhizosphere with different rapeseed under P15 and P100 treatments. Error bars show the standard error (n=5)
attributed to the co-transport of H+ with organic acid anion release.
120
140
100
120 105 102 Control
80
100 80
60 0
1
2
3
4
5
6
0
1
2
Distance from the rhizoplane (mm)
3
4
5
6
Plant Soil (2009) 320:91–102
99
r105 = -0.689* r102 = -0.869*
12
14
16
18
20
-1
180 170 160 150 140 130 120 110
-1
-1
-1
(µg g h )
Fig. 7 Relationship between acid phosphatase activity and NaOH-Po or NaHCO3-Po concentrations in the depletion zones of two rapeseed genotypes under P15 treatment (n=5; r105 and r102 are the correlation coefficients of genotypes 105 and 102, respectively. *Significant at p=0.05)
Acid phosphatase activity
It has been suggested that P acquisition efficiency is related to the mobilization and uptake of P from sparingly soluble inorganic or organic forms (Marschner 1995). Our results showed that rhizosphere chemistry was strongly and differentially modified by plant roots of the two rapeseed genotypes. The P-efficient genotype 102 depleted more NaOH-Pi than P-inefficient genotype 105 under both P treatments (Fig. 4). This may be explained by root-excreted organic acid
(µg g h )
Genotypic difference in the P acquisition efficiency
Acid phosphatase activity
anions and the desorption of soil P induced by the acidification. Organic acid anions like malate or citrate released by plant roots were responsible for the mobilization of NaOH-Pi through ligand exchange, and the genotypic variations in the kinds and quantity of organic acids also resulted in the different Pi depletion in the rhizosphere (Hoffland et al. 1989; Ohwaki and Hirata 1992; Ae et al. 1990). On the other hand, P fractionation described by Tiessen and Moir (1993) is a sequential analytical procedure, so the NaOH-extractable Pi includes the various fractions not only on the mineral surface but also in the interior of the soil particles. Since the sorption and desorption of phosphate were pH dependent (Nanzyo and Watanabe 1982), to a certain extent the acidification in the rhizosphere may favor the desorption of phosphate from the soil particles and increase the availability of P fractions (Barrow 1983, 1984), which also resulted in a decrease of the NaOHextractable Pi. The differences in utilization of NaHCO3-Po and NaOH-Po between genotypes 102 and 105 reflected the capacity of each to mineralize organic P (Fig. 5). This could be explained by the differences in rhizosphere acid phosphatase activity in the depletion zones (Fig. 3). As shown by previous studies (Asmar et al. 1995; Chen 2003; Radersma and Grierson 2004), significantly negative correlation between acid phosphatase activity and the content of organic P fractions was observed (Fig. 7). Higher acid phosphatase activity in rhizosphere soil with genotype 102 resulted in the greater depletion of NaHCO3-Po and NaOH-Po in the rhizosphere soil than genotype 105. Acid phosphatase activity can therefore be used as an index to identify more P-efficient genotypes or species in soils where organic P is enriched. Our results showed that P-efficient genotype 102 had higher P concentration, P acquisition and P translocation coefficients than P-inefficient genotype
al. 2007). Figure 4 suggested that the acidification or root exudation maybe play an important role in the depletion of HCl-Pi and NaOH-Pi in the study. The importance of the mineralization of organic P compounds in regulating the supply of plant-available P has been reported by Scherer and Sharma (2002). Nuruzzaman et al. (2006) and Tarafdar and Jungk (1987) also reported that significant depletion of NaHCO3-Po and NaOH-Po occurred in the rhizosphere, and the depletion depended on acid phosphatase activity. As shown in Fig. 7, significantly negative correlation was observed between soil acid phosphatase activity and content of organic P fractions under P15 treatment, confirming the above conclusions. However, contradictory results were found in the present study. NaHCO3-Po fractions near the root surface of both genotypes accumulated at P100 (Fig. 5). This may be attributed to the strong activity of microorganisms which immobilize inorganic P into organic P in the rhizosphere. NaHCO3-Po accumulation in the rhizosphere under P100 could be explained by the fact that the immobilization of inorganic P into organic P by microorganisms was higher than organic P hydrolysis by phosphatase in the rhizosphere of plants. Similar results have been reported by Zoysa et al. (1998) and George et al. (2002).
180 170 160 150 140 130 120 110
22
6.0 6.5
-1
NaOH-Po (µg g )
r105 = -0.842* r 102 = -0.936*
7.0
7.5
8.0 -1
105
102
NaHCO3-Po (µg g )
8.5
100
105 under both P treatments (Table 2). Correspondingly, the depletion of soluble P fractions (water-Pi and NaHCO3-Pi) in the rhizosphere with genotype 102 were greater and the width of the depletion zones was larger than with genotype 105 (Fig. 4), which indicated that P-efficient rapeseed genotypes had the capacity to take up more available P from the soil and translocate it into shoot than P-inefficient genotypes. The conclusion was consistent with that of Osborne and Rengel (2002). In conclusion, our results indicated that significant differences in the capacity to take up soil P in Pdeficient soil existed between the two B. napus genotypes tested. High P efficiency was shown by changing their root physiological and biochemical mechanisms and taking up greater amounts of P from the soil. In contrast to P-inefficient genotypes, Pefficient genotypes under P-deficient conditions may be able to (1) acidify rhizosphere soil, which increased the availability of sparingly soluble Ca-P and to a certain extent induced the desorption of P from the soil particles; (2) induce higher phosphatase activity, resulting in hydrolyzation and mineralization of soil organic P into inorganic form and (3) show greater ability of uptake and translocation P, which resulting in uptake of more available P by plant from the rhizosphere soil. These results suggested the possibility for breeding P-efficient rapeseed cultivars by using the character of P efficiency acquisition in Pefficient genotype for maintaining rapeseed yield potential in soils with low P bioavailability. Acknowledgements This work was supported by grants from the National Basic Research and Development Program (2005CB120905), National 863 High Technology Program (2006AA10A112), and Specialized Research Fund for the Doctoral Program of Higher Education (20050504009), China.
References Ae N, Arihara J, Okada K, Yoshihara T, Johansen C (1990) Phosphorus uptake by pigeon pea and its role in cropping systems of the Indian subcontinent. Science 248:477–480 doi:10.1126/science.248.4954.477 Akhtar MS, Oki Y, Adachi T (2008) Genetic variability in phosphorus acquisition and utilization efficiency from sparingly soluble P-sources by Brassica cultivars under P-stress environment. J Agron Crop Sci 194:380–392 doi:10.1111/j.1439-037X.2008.00326.x Asmar F, Gahoonia TS, Nielsen NE (1995) Barley genotypes differ in activity of soluble extracellular phosphatase and
Plant Soil (2009) 320:91–102 depletion of organic phosphorus in the rhizosphere soil. Plant Soil 172(1):117–122 doi:10.1007/BF00020865 Barrow NJ (1983) A mechanistic model for describing the sorption and desorption of phosphate by soil. Eur J Soil Sci 34(4):733–750 Barrow NJ (1984) Modelling the effects of pH on phosphate sorption by soils. Eur J Soil Sci 35(2):283–297 doi:10.1111/j.1365-2389.1984.tb00283.x Chen HJ (2003) Phosphatase activity and P fractions in soils of an 18-year-old Chinese fir (Cunninghamia lanceolata) plantation. For Ecol Manage 178:301–310 doi:10.1016/ S0378-1127(02)00478-4 Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient environments. Plant Soil 245:35–47 doi:10.1023/A:1020809400075 Foehse D, Jungk A (1983) Influence of phosphate and nitrate supply on root hair formation of rape, spinach and tomato plants. Plant Soil 74(3):359–368 doi:10.1007/BF02181353 George TS, Gregory PJ, Robinson JS, Buresh RJ (2002) Changes in phosphorus concentrations and pH in the rhizosphere of some agroforestry and crop species. Plant Soil 246:65–73 doi:10.1023/A:1021523515707 Graham JH, Leonard RT, Menge JA (1981) Membrane mediated decrease in root exudation responsible for phosphorus inhibition of vesicular–arbuscular mycorrhiza formation. Plant Physiol 68:548–552 doi:10.1104/pp.68.3.548 Greenberg AE, Clesceri LS, Eaton AD (1992) Standard methods for the examination of water and waste water. American Public Health Association, Washington, DC Guo F, Yost RS, Hue NV, Evensen CI, Silva JA (2000) Changes in phosphorus fractions in soils under intensive plant growth. Soil Sci Soc Am J 64:1681–1689 Hedley MJ, Stewart JWB, Chauhan BS (1982a) Changes in inorganic and organic soil phosphorus fractions induced by cultivation practices and by laboratory incubations. Soil Sci Soc Am J 46:970–976 Hedley MJ, White RE, Nye PH (1982b) Plant induced changes in the rhizosphere of rape (Brassica napus var. Emerald) seedlings. III. Changes in L value, soil phosphate fractions and phosphatase activity. New Phytol 91:45–56 doi:10.1111/j.1469-8137.1982.tb03291.x Hendriks L, Claassen N, Jungk A (1981) Phosphatverarmung des wurzelnahen Bodens und Phosphataufnahme von Mais und Raps. Z Pflanzenern Bodenkd 144:486–499 doi:10.1002/jpln.19811440507 Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195 doi:10.1023/A:101335 1617532 Hinsinger P, Plassard C, Tang CX, Jaillard B (2003) Origins of root-mediated pH changes in the rhizosphere and their responses to environmental constraints: a review. Plant Soil 248:43–59 doi:10.1023/A:1022371130939 Hoffland E, Boogaard RVD, Nelemans JA, Findenegg GR (1992) Biosynthesis and root exudation of citric and malic acids in phosphate-starved rape plants. New Phytol 122:675–680 Hoffland E, Findenegg GR, Nelemans JA (1989) Solubilization of rock phosphate by rape. II. Local root exudation of organic acids as a response to P-starvation. Plant Soil 113:161–165
Plant Soil (2009) 320:91–102 Jaillard B, Plassard C, Hinsinger P (2002) Measurements of H+ fluxes and concentrations in the rhizosphere. In: Rengel Z (ed) Handbook of Soil Acidity. Marcel Dekker, New York (in press) Lewis DG, Quirk JP (1967) Phosphate diffusion in soils and uptake by plants. II. Uptake by wheat plants. Plant Soil 26:119–128 doi:10.1007/BF01978679 Li M, Shinano T, Tadano T (1997) Distribution of exudates of lupin roots in the rhizosphere under phosphorus deficient conditions. Soil Sci Plant Nutr 43:237–245 Li L, Li SM, Sun JH, Zhou LL, Bao XG, Zhang HG, Zhang FS (2007) Diversity enhances agricultural productivity via rhizosphere phosphorus facilitation on phosphorus-deficient soils. Proc Natl Acad Sci USA 104:11192–11196 doi:10.1073/pnas.0704591104 Liu Y, Mi GH, Chen FJ, Zhang JH, Zhang FS (2004) Rhizosphere effect and root growth of two maize (Zea mays L.) genotypes with contrasting P efficiency at low P availability. Plant Sci 167:217–223 doi:10.1016/j. plantsci.2004.02.026 López-Bucio J, Hernández-Abreu E, Sánchez-Calderón L, Nieto-Jacobo MF, Simpson J, Herrera-Estrella L (2002) Phosphate availability alters architecture and causes changes in hormone sensitivity in the Arabidopsis root system. Plant Physiol 129:244–256 doi:10.1104/ pp.010934 Loss SP, Robson AD, Ritchie GSP (1993) H+/OH− excretion and nutrient uptake in upper and lower parts of lupin (Lupin angustifolius) root systems. Ann Bot (Lond) 72:315–320 doi:10.1006/anbo.1993.1113 Ma Z, Bielenberg DG, Brown KM, Lynch JP (2001) Regulation of root hair density by phosphorus availability in Arabidopsis thaliana. Plant Cell Environ 24:459–467 doi:10.1046/j.1365-3040.2001.00695.x Marschner H (1995) Mineral nutrition of higher plants, 2nd edn. Academic, London Marschner H, Römheld V, Horst WJ, Martin P (1986) Rootinduced changes in the rhizosphere: importance for the mineral nutrition of plants. Z Pflanzenern Bodenkd 149 (4):441–456 doi:10.1002/jpln.19861490408 Marschner P, Solaiman Z, Rengel Z (2007) Brassica genotypes differ in growth, phosphorus uptake and rhizosphere properties under P-limiting conditions. Soil Biol Biochem 39:87–98 doi:10.1016/j.soilbio.2006.06.014 Moorby H, Nye PH, White RE (1985) The influence of nitrate nutrition on H+ efflux by young rape plants. Plant Soil 84:403–413 doi:10.1007/BF02275477 Moorby H, White RE, Nye PH (1988) The influence of phosphate nutrition on H+ efflux from the roots of young rape plants. Plant Soil 105:247–256 doi:10.1007/ BF02376789 Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36 doi:10.1016/S0003-2670(00)88444-5 Nanzyo M, Watanabe Y (1982) Diffuse reflectance infrared spectra and ion-adsorption properties of the phosphate surface complex on goethite. Soil Sci Plant Nutr 28 (3):359–368 Nilsson L, Müller R, Nielsen TH (2007) Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant
101 Cell Environ 30:1499–1512 doi:10.1111/j.1365-3040. 2007.01734.x Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2006) Distribution of carboxylates and acid phosphatase and depletion of different phosphorus fractions in the rhizosphere of a cereal and three grain legumes. Plant Soil 281:109–120 doi:10.1007/s11104-005-3936-2 Ohwaki Y, Hirata H (1992) Differences in carboxylic acid exudation among P-starved leguminous crops in relation to carboxylic acid contents in plant tissues and phospholipid level in roots. Soil Sci Plant Nutr 38(2):235–243 Okalebo JR, Gathua KW, Woomer PL (1993) Laboratory methods of soil and plant analysis: a working manual. Tropical Soil Biology and Fertility Programme, Nairobi, Kenya Osborne L, Rengel Z (2002) Genotypic differences in wheat for uptake and utilisation of P from iron phosphate. Aust J Agric Res 53:837–844 doi:10.1071/AR01101 Radersma S, Grierson PF (2004) Phosphorus mobilization in agroforestry: organic anions, phosphatase activity and phosphorus fractions in the rhizosphere. Plant Soil 259:209–219 doi:10.1023/B:PLSO.0000020970.40167.40 Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol Plant Mol Biol 50:665–693 doi:10.1146/annurev. arplant.50.1.665 Ruiz L, Arvieu JC (1990) Measurement of pH gradients in the rhizosphere. Symbiosis 9:71–75 Scherer HW, Sharma SP (2002) Phosphorus fractions and phosphorus delivery potential of aluvisol derived from loess amended with organic material. Biol Fertil Soils 35:414–419 doi:10.1007/s00374-002-0488-y Schwab SM, Menge JA, Leonard RT (1983) Quantitative and qualitative effects of phosphorus on extracts and exudates of Sudan grass roots in relation to vesicular–arbuscular mycorrhiza formation. Plant Physiol 73:761–765 doi:10.1104/pp.73.3.761 Schweiger PF, Robson AD, Brarrow NJ, Abbott LK (2007) Arbuscular mycorrhizal fungi from three genera induce two-phase plant growth responses on a high P-fixing soil. Plant Soil 292:181–192 doi:10.1007/s11104-0079214-8 Shane MW, Lambers H (2005) Cluster roots: a curiosity in context. Plant Soil 274:101–125 doi:10.1007/s11104-0042725-7 Sharma SP, Subehia SK (2003) Effects of twenty-five years of fertilizer use on maize and wheat yields and quality of an acidic soil in the western Himalayas. Exp Agric 39:55–64 doi:10.1017/S0014479702001035 Tabatabai MA, Bremner JM (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase activity. Soil Biol Biochem 1:301–307 doi:10.1016/0038-0717(69)90012-1 Tadano T, Ozawa K, Sakai H, Osaki M, Matsui H (1993) Secretion of acid phosphatase by the roots of crop plants under phosphorus-deficient conditions and some properties of the enzyme secreted by lupin roots. Plant Soil 56:95–98 doi:10.1007/BF00024992 Tang C, Unkovich MJ, Bowden JW (1999) Factors affecting soil acidification under legumes III. Effects of nitrate supply. New Phytol 143:513–521 doi:10.1046/j.14698137.1999.00475.x Tarafdar JC, Jungk A (1987) Phosphatase activity in the rhizosphere and its relation to the depletion of soil organic
102 phosphorus. Biol Fertil Soils 3:199–204 doi:10.1007/ BF00640630 Tiessen H, Moir JO (1993) Characterization of available P by sequential extraction. In: Carter MR (ed) Soil sampling and methods of analysis. Canadian Society of Soil Science. CRC, Lewis, Boca Raton, FL, pp 75–86 Verma S, Subehia SK, Sharma SP (2005) Phosphorus fractions in an acid soil continuously fertilized with mineral and organic fertilizers. Biol Fertil Soils 41:295–300 doi:10.1007/s00374-004-0810-y Wang Z, Shen J, Zhang F (2006) Cluster-root formation, carboxylate exudation and proton release of Lupinus pilosus Murr. as affected by medium pH and P deficiency. Plant Soil 287:247–256 doi:10.1007/s11104-006-9071-x Westerman RL (1990) Soil testing and plant analysis, 3rd edn. Soil Science Society of America, Madison, WI Wissuwa M, Ae N (2001) Genotypic variation for tolerance to phosphorus deficiency in rice and the potential for its exploitation in rice improvement. Plant Breed 120:43–48 doi:10.1046/j.1439-0523.2001.00561.x
Plant Soil (2009) 320:91–102 Yan F, Zhu YY, Müller C, Zörb C, Schubert S (2002) Adaptation of H+-pumping and plasma membrane H+ATPase activity in proteoid roots of white lupin under phosphate deficiency. Plant Physiol 129:50–63 doi:10.1104/pp.010869 Zhang FS, Ma J, Cao YP (1997) Phosphorus deficiency enhances root exudation of low-molecular weight organic acids and utilization of sparingly soluble inorganic phosphates by radish (Raghanus satiuvs L.) and rape (Brassica napus L.) plants. Plant Soil 196:261–264 doi:10.1023/A:1004214410785 Zoysa AKN, Loganathan P, Hedley MJ (1998) Effects of form of nitrogen supply of mobilisation of phosphorus from a phosphate rock and acidification in the rhizosphere of tea. Aust J Soil Res 36:373–387 doi:10.1071/S97079 Zoysa AKN, Loganathan P, Hedley MJ (1999) Phosphorus utilisation efficiency and depletion of phosphate fractions in the rhizosphere of three tea (Camellia sinensis L.) clones. Nutr Cycl Agroecosyst 53:189–201 doi:10.1023/ A:1009706508627