Phosphate solubilization by organic anion excretion from rice growing

New Phytol. (1999), 142, 185–200

Phosphate solubilization by organic anion excretion from rice growing in aerobic soil : rates of excretion and decomposition, effects on rhizosphere pH and effects on phosphate solubility and uptake G. J. D. K I R K*, E. E. S A N T O S    M. B. S A N T O S International Rice Research Institute, MCPO Box 3127, 1271 Makati City, The Philippines Received 2 July 1998 ; accepted 4 January 1999  Rice (Oryza sativa) plants were grown with their roots sandwiched between thin layers of phosphorus-deficient soil from which they were separated by fine mesh, and root-induced changes in the soil affecting phosphate solubility were measured. The concentrations of low molecular weight organic anions in the thin layers, particularly citrate, increased in the presence of the plants. Apparent rates of citrate excretion from the roots, calculated from the quantities in the soil and rates of decomposition calculated with a first order rate constant measured independently, varied from 337–155 nmol g−" root f. wt h−" over the course of plant growth, equivalent to 2–3% of plant d. wt. Rates of excretion were similar for NH + and NO −-fed plants. The soil pH decreased from % $ its initial value by up to 0.6 units for the NH +-fed plants and increased by up to 0.4 units for the NO −-fed ones. % $ The contribution of organic anion excretion to the pH changes was small compared with that of the inorganic cation-anion balance in the plants. The extent to which the observed excretion of citrate and root-induced pH changes could account for the observed phosphate solubilization and uptake was assessed using a mathematical model. Previous work had shown that phosphate solubilization by rice in this soil could not be explained by enhanced phosphatase activity in the rhizosphere, and the roots were not infected with mycorrhizas. The model allows for the diffusion of the solubilizing agent (citrate or H+) away from the roots, its decomposition by soil microbes (citrate only) ; its reaction with the soil in solubilizing phosphate and diffusion of the solubilized phosphate to the roots. The model contains no arbitrary assumptions and uses only independently measured parameter values. The agreement between the measured time course of phosphorus uptake and that predicted for solubilization by citrate was good. Root-induced acidification by NH +-fed plants resulted in additional % solubilization, the acidification enhancing the solubilizing effect of citrate. However, the final phosphorus uptake by NH +-fed plants was no greater than that of NO −-fed plants, presumably because the acidification inhibited % $ plant growth. The mechanism of solubilization by citrate involved formation of soluble metal-citrate chelates rather than displacement of phosphate from adsorption sites. Key words : citric acid, diffusion, rice (Oryza sativa), organic acid, phosphate, pH change, solubilization, rhizosphere.

 Certain plant species are particularly good at extracting phosphate from phosphorus (P)-deficient soils, and one of the main mechanisms behind this is thought to be the excretion of P-solubilizing organic anions from the roots. However only in a few extreme cases (Dinkelaker et al., 1989 ; Hoffland et al., 1989) have the amounts of organic anions *Author for correspondence (fax j63 2 845 0606 ; e-mail g.kirk!cgiar.org).

excreted and their P-solubilizing effects been shown to quantitatively explain the amounts of P solubilized and the resultant increase in P uptake. In this paper we measure organic anion excretion and P solubilization by rice plants growing in aerobic soil, and we use a mathematical model developed by Kirk (1999) to assess the extent to which the excretion can explain the observed P solubilization and uptake. We are interested in rice in aerobic soil both for upland conditions where the soil is unflooded, and for rainfed lowland conditions where the soil is

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186

G. J. D. Kirk et al.

flooded for most of the season but may be dry at planting or become dry mid-season. Rice is known to be adapted to acid P-deficient upland soils (Garrity et al., 1990). In earlier work (Hedley et al., 1994) with rice grown in a highly weathered P-deficient aerobic soil we found that the bulk of the P absorbed by the plants was solubilized from alkali-soluble inorganic forms. The solubilization could not be explained by root-induced pH changes, nor by increased mineralization of organic P because there was no net depletion of organic P. We subsequently found a substantial release of citrate from rice roots growing in nutrient solution (Kirk et al., 1999), as have others (Lin & You, 1989 ; Liu et al., 1990), and we postulated that this was responsible for the solubilization. As discussed by Kirk (1999), the excretion of organic anions from roots may solubilize P by changing the soil pH, by displacing P from adsorption sites, by chelating metal ions that would otherwise immobilize P, or by forming soluble metal-chelate complexes with P. Changes in pH may be important where the anion is excreted in association with protons and in large quantities. But in general pH changes in the rhizosphere are dominated by the inorganic cation–anion balance in the plant and the associated excretion of H+ or OH− from the roots. Likewise, displacement of adsorbed P will only be important where the amounts of organic anion adsorbed are comparable to the amounts of P desorbed. Unless very large amounts of organic anion are excreted, this would require strong adsorbtion of the anion on the soil ; it would therefore diffuse only very slowly through the soil with the effect confined to a narrow cylinder around the root. As we show later, chelation is likely to be the more important mechanism, at least in highly weathered soils containing large amounts of metal oxides. Kirk’s model allows for the diffusion of the organic anion away from a root, its reaction and decomposition in the soil, and diffusion of the solubilized P back to the root as well as away from it. He tested the model against measured concentration-distance profiles of P in the rhizosphere of rice plants growing in aerobic soil. But since organic anion excretion was not measured in the experiments in which the profiles were measured, he had to estimate the rates of citrate excretion. The agreement between observed and predicted P concentration profiles was very good. But since the rates of excretion were estimated this was only partial corroboration of the model. Measuring organic anion excretion into soil is complicated by : decomposition of the organic anion by soil microbes ; adsorption onto soil particles, making it necessary to extract the organic anion with an appropriate reagent ; and interference in the organic anion assay by other substances extracted. Also, as a result of adsorption and decomposition,

organic anions will diffuse only a short distance away from root surfaces, making it necessary to sample the soil very close to the roots. To obtain sufficient rhizosphere soil for detailed study, we grew plants with their roots sandwiched between two 3-mm-thick layers of soil connected to a nutrient solution reservoir via fibreglass filter paper wicks. All the soil could therefore be considered rhizosphere. The roots were separated from the soil by 24-µm pore-diameter nylon mesh sheeting which they could not penetrate but through which water and solutes could pass freely. In this paper we give the details of these measurements and we describe the development and application of the mathematical model. The specialist nomenclature in this field is described in Box 1.    Soil preparation The experimental system, which is a modification of that of Grinsted et al. (1982), is shown in Fig. 1a. The experimental soil is a highly weathered humic clay from Cavinti, Laguna, Philippines (described further by Hedley et al., 1994). It was air dried and ground to pass a 500 µm sieve, discarding large fragments of organic matter. Two P levels were established by mixing the dry soil with monocalcium phosphate at 100 and 1000 µg P g−" (hereafter referred to as P100 and P1000 soil), moistening to 60% by weight, incubating for 4 d at 40mC, and then drying at 60mC. The soil was resieved to 500 µm and mixed thoroughly by shaking end-over-end overnight. Portions (40 g) of air-dry soil were packed uniformly into the thin-layers, giving a bulk density of 1.16 kg dm−$ (oven-dry basis), and then moistened by connecting to water reservoirs held approx. 25 cm below the thin layers, via fibreglass filter paper wicks. The soil surface and the wicks were covered with black plastic sheeting to reduce water loss and algal growth. The thin layers and wicks were suspended in Perspex tanks containing water. The tanks were contained in polystyrene boxes to minimize temperature changes. The height of the thinlayers above the water was adjusted as necessary to keep the soil at field capacity. The thin layers were equilibrated in this way in a controlled-environment glasshouse for 4–5 wk ; then 2 d before planting, the water in the reservoirs was replaced with a P-free nutrient solution based on that of Yoshida et al. (1976). The full-strength nutrient solution was 2.9, 1.0, 1.0, 1.7 and 1.0 mM in N, K, Ca, Mg and Si, respectively, and 9.1, 0.52, 19, 0.15, 0.16 and 36 mM in Mn, Mo, B, Zn, Cu and Fe (as FeEDTA), respectively. The solution was either wholly NO −- or NH +-based. In the NO − solution $ % $ N, K and Ca were supplied as KNO , Ca(NO ) and $ $#


Phosphate solubilization by organic anions

187

Soil buffer power for citrate, d[C]/d[CL] Soil buffer power for H+, d[H]/d[HL] Soil buffer power for ortho P, d[P]/d[PL] Soil buffer power for P*, (∂[P]/∂[PL* ])C Concentration of citrate in the whole soil Concentration of citrate in the soil solution Solute diffusion coefficient in free solution (subscripted C, H, P or P* for citrate, H3O+, ortho P and P*, respectively) Flux of citrate across root plane Flux of H+ across root plane Diffusion impedance factor

bc bH bP bp* [C] [CL] DL

FC FH f

[H] [HL ] k lh [P] [PL] [PL* ] α θ λH λC ρ

Concentration of soil acidity titratable to the original soil pH, mol dm–3 soil Concentration of H3O+ in the soil solution First order rate constant for citrate decomposition Length of root hairs Concentration of P in the whole soil Concentration of ortho P in the soil solution Concentration of all P species (ortho P and P complexed with C) in the soil Root absorbing power for P Volume fraction of soil water P–H interaction coefficient, (∂[PL]/∂[HL])P P–C interaction coefficient, (∂[PL* ]/∂[CL])P Soil bulk density

Box 1. Nomenclature (b)

13 cm

Nylon mesh Clamp

8 cm

Soil Fibreglass filter paper Backing plate

Root f. wt (g per system)

Wick connected to P-free nutrient solution

Plant P concentration (mg g–1) Plant d. wt (g per system)

(a)

16 P100-NH4 + P100-NO3 – P1000-NH4 + P1000-NO3 –

12 8 4 0 2.0 1.5 1.0 0.5 0.0 16 12 8 4 0

0

1 2 3 4 5 6 7 Time after transplanting (wk)

Fig. 1. Growth of Oryza sativa (rice) plants. (a) The experimental system. (b) Growth and P concentration over time. Four plants per system. Data are meanspSE of three replicates.

CaCl ; in the NH + solution they were supplied as # % (NH ) SO , K SO and CaCl . In both treatments, %# % # % # Mg was supplied as MgSO . % Plant growth A large number of seeds of Oryza sativa L. cv. Azucena were germinated by surface sterilizing with HgCl and imbibing with distilled water for 24 h, #

and then grown for 14 d in low P (0.065 mM) but otherwise full strength nutrient solution. Four seedlings with similar root lengths were transplanted into each system by spreading the roots across the face of one soil thin-layer and then placing the second thin layer on top. The plants were grown at day\night temperatures of 3mC\21mC and 70% rh with ambient light of c. 1000 µmol m−# s−" white light, 12 h photoperiod. The solution in the


188


reservoirs was changed twice weekly. For the first 2 wk half-strength solution was used ; subsequently full strength was used. Three replicates were harvested for each of the four treatments at weekly intervals from 1 to 6 wk after planting. Plant measurements At harvest, the thin-layer systems were dismantled and the plants separated from the soil and mesh. The combined root and shoot material was dried at 70mC for 48 h and then milled and weighed. Any roots escaping from the thin layers during plant growth were removed as they were produced and combined with the main root material at the following harvest. The combined dry root and shoot material was analysed as follows. N. Separate 500 mg samples (or as much as possible) were digested by micro-Kjeldahl distillation and analysed for N. Ca, Mg, K, S, P. Further 500 mg samples were digested in HNO and HClO (Jones & Case, 1990) $ % and analysed for Ca#+, Mg#+ and K+ by atomic absorption spectrometry, SO #− by inductively% coupled plasma emission spectrometry and P by colorimetry (Murphy & Riley, 1962). Cl. Further 500 mg samples were extracted in water and analysed for Cl− by ion exchange chromatography (Grunau & Swiader, 1986). Soil measurements At each of the six harvests, organic anions, soil solution P concentration, soil pH and extractable NH + and NO − were measured in sub-samples of % $ the combined soil from the two thin-layers per system (to be described). Before subsampling, the soil from the two thin-layers in each system was combined, moistened to 60% (w\w) and then thoroughly mixed. Organic anions. Triplicate 10 g subsamples of moist soil were shaken end-over-end overnight at 25mC in 40 cm$ of 5 mM H SO . The extracts were # % centrifuged and filtered, and the filtrates analysed for organic anions by HPLC. Organic anions in the solution in the mesh separating the roots and soil, and adsorbed on the mesh, were measured by shaking the mesh overnight in 20 cm$ of 5 mM H SO and analysing by HPLC. # % For the HPLC analysis, solution samples were filtered through 0.45 µm micropore filters. Organic anions were then separated on a Polyspher OAHY acid-specific column (Merck, Darmstadt, Germany) using an isocratic elution of 22.5 mM H SO at 0.8 # % cm$ min−" and column temperature of 30mC, and

detected at 210 nm using a Shimadzu SPD-6A UV spectrophotometric detector (Shimadzu, Kyoto, Japan). Peak areas and retention times were derived using a Shimadzu Chromatopac C-R6A data processor. Individual organic anions were identified by comparing retention times with those of standards prepared from free acids or alkaline salts obtained from Sigma Chemicals Ltd (St Louis, MO, USA). Soil solution P concentration, soil pH, and extractable NH + and NO −. Triplicate 3 g subsamples of moist $ % soil were shaken end-over-end for 1 h at 25mC in 30 cm$ of 0.01 M CaCl , and the pH of the suspension # taken with a combination electrode allowing 30 s for equilibration whilst stirring. The suspensions were then centrifuged and filtered through 0.4 µm nucleopore filters and the P concentrations determined by colorimetry (Murphy & Riley, 1962). Separate triplicate 2 g subsamples of moist soil were shaken end-over-end for 1 h at 25mC in 20 cm$ of 0.5 M H SO ; the suspension was then centrifuged and # % filtered, and NH + in the filtrates was measured % colorimetrically (Kempers & Zweers, 1986) and NO − by ion exchange chromatography (Grunau & $ Swiader, 1986). Subsidiary measurements Root hair length. Root hair length was measured by growing plants in thin layers of the experimental soil inclined at an angle so that the roots grew down a clear Perspex face, and then measuring the lengths of the hairs under a microscope with a graticule eyepiece. Mycorrhizal infection. Mycorrhizal infection of roots growing in the same soil under similar conditions to the thin layer experiment was measured by staining with trypan blue and examining under a microscope (Kokse & Gemma, 1989). Kinetics of citrate decomposition in the soil. P1000 soil was prepared by mixing soil and monocalcium phosphate as already described, incubating moist for 4 wk to allow complete reaction between the soil and MCP, and then air drying. Portions (100 g) of the air-dry soil were moistened to field capacity and incubated in a water-saturated aerated atmosphere for 1 wk at 25mC to restore microbial activity. The soil was then transferred to a 1 l flask and mixed with 200 cm$ of 10 mM CaCl solution containing 5 mM # H-citrate–Na-citrate at pH 4.3 (i.e. 10 mM citrate g−" soil). The flask was stoppered and shaken gently on a rotary shaker for 4 d at 25mC. Every 2 h starting immediately after mixing, 5 cm$ samples of suspension were withdrawn, centrifuged at 12 000 g for 5 min, filtered through 0.45-µm nucleopore filters and acidified to pH 2 with HCl. The filtrates were analysed for citrate by HPLC within 24 h.


Phosphate solubilization by organic anions Soil citrate buffer power. Portions (2.5 g) of air-dry P1000 soil were mixed with 5 cm$ of 10 mM CaCl # containing varying concentrations of H-citrate–Nacitrate in the range 0–5 mM at pH 4.3. The mixtures were shaken end-over-end at 25mC for 30 min, centrifuged at 12 000 g for 5 min, filtered through 0.45-µm nucleopore filters, acidified to pH 2 and stored in a refrigerator. The filtrates were analysed for citrate by HPLC within 24 h. Soil pH buffer power. Using a fine syringe, 2.0 cm$ of 0–0.5 M NaOH or HCl was sprayed onto 11.3 g portions of air-dry P100 and P1000 soil to give 21 samples for each soil covering the range j0.1 to k0.1 mmol H+ g−" dry soil. The soils were incubated for 12 d at 25mC in the dark, maintaining the moisture content by addition of water as necessary. Subsamples of 3 g were then shaken in 30 cm$ 0.01 M CaCl for pH determination as above. The # amounts of H+ added (mmol H+ g−" soil) were plotted against the measured pHs. Effect of pH on soil P desorption. Duplicate 2 g samples of P1000 soil were shaken end-over-end overnight at 25mC with volumes of 0.01 M CaCl at # the native soil pH (4.3) and at the native pHp0.5 units, with 0.18 mM HgCl to suppress microbial # activity. Seven volumes of CaCl were used (50, 100, # 250, 500, 1000, 2000 and 4000 cm$). The solutions were adjusted to the target pHs by addition of HCl and NaOH before adding them to the soil. After 30 min of shaking, the suspension pHs were rechecked and adjusted as necessary by further addition of HCl or NaOH. After shaking overnight, the suspensions were centrifuged at 12 000 g for 5 min and the supernatants filtered through 0.45 µm nucleopore filters. Phosphate concentrations in the filtrates were determined colorimetrically. The amount of P released from the soil for a given addition of H+ was assessed by shaking 1-g samples overnight in 20 cm$ of deionized water containing a strip each of HCO −-form anion exchange resin $ membrane and H+-form cation exchange resin membrane (Nos 55164 and 55165, BDH Ltd, Poole, Dorset, England). Each strip had 0.5 mmolc of exchange capacity. Phosphate was recovered from the anion resin by shaking in 20 cm$ of 0.5 M HCl for 1 h. Effect of citrate on soil P desorption. The effect of citrate on P desorption from the P1000 soil was measured as described by Kirk (1999). Soil samples were shaken in 0.01 M CaCl at pH 4.3 at a wide # range of soil :solution ratios (2 g soil :50, 100, 250, 500, 1000 and 2000 cm$ solution) and with different additions of citrate (initial concentrations 0, 0.1, 0.25, 0.5, 1.0 and 2.0 mM) in a factorial design. The P desorbed was calculated from the amount passing into solution.

189 Soil diffusion impedance factor. The diffusion impedance factor was measured by following the selfdiffusion of the Cl− ion in the soil using the pulse-labelling technique of Pinner & Nye (1982) with $'Cl.       :                                           ,       p Plant growth and P concentration Fig. 1b shows the changes in plant dry weight and root fresh weight with time and the corresponding changes in plant P concentration. In the P100 treatments little more P was absorbed than was in the plants at transplanting. In the P1000, P uptake continued throughout the experiment for the NO −$ fed plants and until after the fourth week for the NH +-fed plants. The critical minimum plant P % concentration for rice during vegetative growth is c. 1 mg g−". Therefore the plants were P-deficient from about the third week after transplanting in the P1000 and from the first in the P100. Organic anion accumulation in the soil The amounts of organic anions in the thin layers increased in the presence of plants. The main anion identified was citrate (Retention Time (RT) l 4.6 min under the HPLC conditions used ; identity confirmed with an enzymatic test kit (No. 139 0761996, Boehringer Mannheim 1996 Biochemicals Catalogue)). Smaller quantities of oxalate, malate, lactate and fumarate (RT l 4.2, 5.6, 7.3 and 8.9 min, respectively) were also detected, as well as quantities of two unidentified carboxylates (RT l 6.4 and 8.9 min) and unidentified ninhydrin-reactive amino acids (RT l 3.0 min). The P1000 treatments produced larger quantities of citrate than the P100, but the differences between the NH + and NO − treat% $ ments and between harvests were small. There were negligible quantities of citrate in the unplanted soil. The quantities recovered from the planted soil were 24.2p14.3 and 5.4p4.2 µmol per system in the P100-NH + and -NO − treatments and 51.3p20.6 % $ and 36.5p10.3 µmol per system in the P1000-NH + % and -NO − treatments. The corresponding figures $ for the mesh were 4.3p0.8, 3.5p0.4, 5.3p0.5 and 6.3p0.6 µmol per system. In preliminary experiments 20% of citrate added to the soil at relevant concentrations was recovered by the extraction procedure used. But with stronger extractants additional substances were extracted that interfered with the HPLC analysis. The procedure used was the best compromise between recovery and detectability. The recovery obeyed the relation : log[C]* l 0.733 log[Cextract]* j 0.752 (r# l 0.99) where [C]* l total citrate concentration (µmol g−") and [Cextract]* l citrate con-


190

G. J. D. Kirk et al. (a) P100-NH4 +

Citrate in thin layers (µmol per system)

500

(c) P1000-NH4 +

400 300

250

200 122 100 0

(d) P1000-NO3 –

(b) P100-NO3 –

500 400 300

215 200 100

39

0 1

2

3

7 0 5 6 1 2 Time after transplanting (wk)

4

3

4

5

6

7

10 (e) 1 y = 0.472–0.0644x r 2 = 0.98

0.1 0.01 0.001 0.0001 0 Citrate concentration on soil solid (µmol g–1)

Solution citrate concentration (mM)

0

10

20

30 40 Time (h)

50

60

10 (f ) 8 6 y = 7.257x 0.459 r 2 = 0.99

4 2 0 0.0

0.2 0.4 0.6 0.8 1.0 1.2 Citrate concentration in solution (mM)

1.4

Fig. 2. Soil citrate. (a–d) Amounts of citrate in the thin layers calculated from the amounts recovered from the soil (corrected for partial recovery) and mesh. Four plants and 80 g soil per system. Data are meanspSE of three replicates. Dotted lines indicate means over time. (e) Kinetics of citrate decomposition in non-rhizosphere soil measured in shaken soil suspensions. Data are meanspSE of three replicates. (f) Citrate adsorption data measured in shaken soil suspensions and fitted Freundlich equation. Data are meanspSE of two replicates.

centration in the extract (µmol g−"). Fig. 2a–d shows the total amounts of citrate in the thin layers at different times calculated from [Cextract]* values using this relation plus the citrate recovered from the mesh. The variability between replicates in the amounts of citrate recovered from the soil was large. In the

P1000 treatments the SE in the amounts recovered averaged over time was approx. 30% of the mean ; the corresponding figure for the mesh was 10%. This variability was probably largely because of errors in the extraction from the soil and in the assay by HPLC. The latter errors are large because of interference by other substances extracted. In par-


Phosphate solubilization by organic anions ticular, strongly ionized solutes, such as NO −, $ interfere with the more rapidly eluted ions such as citrate. However any systematic error resulting from this will have also affected the recovery curve and is therefore allowed for. A further cause of variability may have been fluctuations in O availability in the thin layers. # From the soil total porosity (l 1kρ\particle density l 1k1.16\2.56 l 0.55) and volumetric water content (l 0.46), the air-filled porosity was 0.09. Therefore on average conditions should have been aerobic, and this is confirmed by the high concentrations of NO − measured throughout the exper$ iment (Fig. 5). But there were possibly pockets of restricted O availability and consequently differing # rates of organic anion decomposition and synthesis. Some of the citrate extracted from the soil will have been extracted from root hair tissue left in the soil at harvest. Based on the root hair density (approx. 50 000 hairs dm−# root plane, each 500 mm long and 5 mm in diameter) and the root citrate concentration (approx. 250 nmol g−" root f. wt (Kirk et al., 1999)), the quantity in root hairs would have been 10 nmol per thin layer, which is negligible compared with the quantity in the soil. Kinetics of citrate decomposition in the soil Fig. 2e shows the kinetics of citrate decomposition measured in the shaken soil suspension experiment. The log of the concentration in solution, loge[CL], varied approximately linearly with time indicating first order kinetics : d[CL]\dt l kk[CL]

Eqn 1

The value of k obtained from the slope of the regression line in Fig. 2e is 4.16i10−& s−" (r# l 0.99), a half-life of 4.6 h. Distribution of citrate between the soil solid and solution Fig. 2f shows the distribution of citrate between the soil solid and solution and a Freundlich-type equation fitted to the data : [Cs] l a [CL]b

Eqn 2

([CS] l concentration in the soil solid (mol kg−" soil) and a and b are coefficients with values 0.173 and 0.459, respectively (r# l 0.99)). The concentration in the whole soil in the thin layers, [C], is therefore : [C] l θ[CL]jρa[CL]b

Eqn 3

Apparent rates of citrate excretion from the roots Organic anion excretion is generally found to be localized in the region of root tips (Hoffland et al., 1992 ; Delhaize et al., 1993 ; Pellet et al., 1995). Therefore the initial distribution of roots and the

191 distribution of subsequent new root growth determine the distribution of excretion across the root plane. New root growth was reasonably uniform both vertically and horizontally across the root plane, and we therefore consider that citrate excretion was uniform vertically and horizontally. However there will have been a gradient of citrate concentration away from the root plane across the soil, and since the rate of decomposition is concentration dependent it is necessary to allow for this gradient in calculating the net rate of decomposition. Considering the diffusion of citrate away from the roots, if decomposition of citrate adsorbed on the soil solid is slow (Boudot, 1992 ; Jones & Darrah, 1994) and decomposition of that in solution follows first order kinetics (Eqn 1), then from established theory for solute diffusion in soils we have : c[C]\ct l DLCθfc#[CL]\cx#kθk[CL]

Eqn 4

The term in [C] in Eqn 4 can be expressed in terms of [CL] by applying the chain rule : c[C]\ct l c[C]\c[CL];c[CL]\ct

Eqn 4a

and the derivative c[C]\cCL], the soil citrate buffer power, bC, can be found from Eqn 3. The following boundary conditions apply. The roots were not infected with mycorrhizas but root hairs penetrated the nylon mesh extending the boundary of the root plane (x l 0) into the soil by the root hair length, lh. We assume that citrate release occurred uniformly over lh ; Nye (1981) shows that for the similar problem of HCO − excretion from hairy roots, a $ more complicated treatment allowing for a gradient of excretion along the root hair length is not warranted. Therefore, for a constant flux, FC across the root plane : DLCθfd[CL]\dx l FC x l lh t0

Eqn 5

No citrate will have been transferred across the outer boundary (x l L), therefore : DLCθfd[CL]\dx l 0

xlL

t0

Eqn 6

We solved Eqn 4 subject to Eqns 5 and 6 using numerical methods as described by Kirk (1999). Thereby we obtained concentration-distance profiles of citrate for given values of FC, bC, k, θ and f. The total quantity of citrate in the soil in moles per system, Msoil, could then be calculated, and the values of FC corresponding to measured Msoil values inferred from the results. The necessary parameter values were obtained as follows. In the P1000 treatments, the citrate concentration reached an apparent steady state after 1 or 2 wk. In the P100 treatments the concentration was more variable and did not reach a steady state. From the average of the steady-state amounts in the P1000NH + and -NO − treatments, Msoil l 233 µmol per % $ system.



192

4.8

(a) P100

4.6

0.60 4.4 4.2 0.82

pH

4.0 3.8 4.8

(b) P1000

4.6

0.80

4.4 4.2 4.0

0.99

3.8 0

2 4 6 8 10 Plant d. wt (g per system)

12

Change in soil acidity (mmol H+ g–1 soil)

0.100 (c ) 0.075 0.050 0.025 0.000 –0.025 –0.050 –0.075 –0.100 –1.0 –0.5 0.0 0.5 1.0 Change in pH

1.5

2.0

Fig. 3. Changes in pH during plant growth. (a,b) Soil pH in the thin layers during plant growth. NH +, closed % circles ; NO −, open circles. Data are meanspSE of three $ replicates. (c) The pH buffer curve of the P100 (closed circles) and P1000 (open circles) soils.

The buffer power bC is concentration-dependent (Eqn 3). However over the range of concentrations across the thin layer it will not vary much and we may use an average value : bC l

&

[CL]L [CL]!

d[C]\d[CL];d[CL]\

&

[CL]L [CL]!

d[CL] l

θjρa([CL] bk[CL]Lb)\([CL] k[CL]L) ! !

Eqn 7

([CL] and [CL]L are the concentrations at x l 0 and ! x l L). With the other parameter values as below, the value calculated by iteration was 5.1. The other parameter values were ρ l 1.16 kg dm−$, θ l 0.46, f l 0.40, DLC l 6.9i10−) dm# s−" (Darrah, 1991), lh l 0.5 mm, L l 3 mm. The time courses of citrate accumulation in the thin layers calculated with these parameter values and different values of FC indicate that steady state

was reached within 2–3 wk of planting, and a steadystate accumulation of 233 µmol per system is obtained with FC l 3.3i10−"! mol dm−# s−". The corresponding concentrations in the soil solution are 0.83 and 0.49 mM at x l 0 and x l L. This flux compares with 3.7i10−"! mol dm−# root surface s−" for malate excretion from the apices of aluminium (Al)-stressed wheat roots measured by Delhaize et al. (1993), and approx. one fifth of this for citrate excretion from the apices of Al-stressed maize roots measured by Pellet et al. (1995). Considering that the surface area of the root plane was at least an order of magnitude smaller than the total root surface area in the plane, our fluxes are therefore reasonable. The excretion per unit root weight is 2AFC\W where A l cross-sectional area of root plane (note there are two root-planes per system) and W l root weight. With FC l 3.3i10−"! mol dm−# s−", this gives 337–155 nmol g−" root f. wt h−" or 2288–852 nmol g−" root d. wt h−" for the second to sixth harvests of the P1000 treatments, corresponding to 2–3% of plant d. wt. This compares with 100–200 nmol g−" root f. wt h−" found by Kirk et al. (1999) for P-deficient rice grown in nutrient solutions, with excretion measured by periodically submerging the roots in water for 30 min and analysing the water for citrate. The corresponding citrate concentrations in the roots were approx. 250 nmol g−" root f. wt. It also compares with 740 nmol g−" root d. wt h−" for citrate and malate excretion from P-deficient maize in sterile nutrient solutions measured by Jones & Darrah (1995), and 23% of plant d. wt for citrate excretion from white lupin measured by Dinkelaker et al. (1989). We have assumed that the citrate recovered from the rhizosphere was all released from roots. But it is possible that at least part of the citrate was generated by microbes subsisting on root-derived carbon. However, because this microbial citrate production would have been root-induced, the result in terms of root-induced P solubilization is the same. Jones & Darrah (1994) estimated a half-life for citrate in solution of 11.7p3.0 h. This is somewhat larger than the value obtained in our shaken soil suspensions (4.6 h). They added a cocktail of glucose, glycine and citrate to their soils to simulate ‘ root exudate ’, and the apparent half life of citrate may have been extended either because citrate was synthesized from the other compounds or because decomposition was impeded by them. If the rate of decomposition in the thin layers was slower than indicated by the measured k value, then FC will have been correspondingly overestimated. The dependence of FC on k can be appreciated from the following simple solution of Eqn 4 for a semi-infinite block of soil at steady state with [C] l bC[CL] : Msoil l AbcFC\θk (from Kirk, 1999, Eqn 16). Thus, at steady state, overestimation of k results in a



193

0.6 (c) P1000-NH4 +

(a) P100-NH4 +

0.5 0.4 Ion concentration (mmolc g–1)

K 0.94 0.3

K 0.95

Na 0.93

Na 0.98

0.2

S 0.28 Mg 0.94 Ca 0.86 CI 0.92 P 0.95

S 0.49 Mg 0.94 CI 0.92 Ca 0.78 P 0.87

0.1 0.0 (b) P100-NO3 –

(d) P1000-NO3 –

0.5 0.4 K 0.88 0.3

K 0.77 Na 0.95

Na 0.93

0.2

S 0.30

S 0.40 Mg 0.64 Ca 0.62 CI 0.98 P 0.95

0.1 0.0 1

2

3

4

5

6

7

Mg 0.86 Ca 0.80 CI 0.88 P 0.92 0

2

4

6

8

10

12

Plant d. wt (g per system)

Fig. 4. Ion concentrations in the plants versus plant dry weight. Lines are best fits to the data ; numbers next to curves are r# values for the fits.

proportionate overestimation of FC. However in the calculation of P solubilization by citrate excretion given below, overestimates of FC and k will cancel each other. The citrate concentration in the rhizosphere peaked 3–4 wk after transplanting in the P100 treatment and reached an apparent steady state 1 or 2 wk after transplanting in the P1000 treatment. The concentration at a particular time will depend on the rate of excretion from the roots, the rate of decomposition, and the rate of synthesis by rhizosphere microbes subsisting on other organic compounds. Each of these will be time-dependent and there are likely to be interactions with other factors. For example changes in P-availability in the rhizosphere may influence both excretion rates and the activity of microbes. Therefore, though we have applied first order kinetics to citrate decomposition in our system, we note that more complicated kinetics may well apply. Root-induced pH changes in the soil, and plant cation–anion balances The changes in pH in the thin layers during plant growth are shown in Fig. 3. The pH tended to increase during plant growth in the NO treatments $ and decrease in the NH treatments. This is expected % from the differences in the inorganic cation–anion balances in the plants and the consequent export of H+ or OH− from the roots. We calculate the extent to which the pH changes can be explained solely by the

inorganic cation–anion balances as follows. Because some NH + and NO − are produced in the soil, N $ % intake will not have been wholly as NH + or NO − in % $ either the NH + or NO − treatments, and the % $ proportions of N taken up as NH + and NO − are % $ therefore uncertain. We estimated these proportions from the acid–base changes in the rhizosphere and the intakes of other cations and anions, as follows. First, the observed pH changes in the thin layers were regressed against plant dry weight (lines in Fig. 3a, b). The changes in titratable acidity required to produce these pH changes, ∆HS (mmol per system), were then calculated from the regressions using the soil pH buffer power (Fig. 3c). Second, the contents of individual nutrients other than N in the plants (mmol g−" plant d. wt) were regressed against plant dry weight (Fig. 4) and the regressions used to calculate the intakes of non-N cations, ΣCatnon-N (mmolc per system) (Ca#+, Mg#+, K+, Na+) and non-N anions, ΣAnnon-N (mmolc per system) (SO #−, Cl−, H PO −). % # % Third, the regressions were used to calculate the apparent difference between NH + and NO − intakes % $ (CatNkAnN, mmol per system) from (CatNkAnN) l ∆HSk(ΣCatnon-NkΣAnnon-N). Finally the NH + intake was calculated from % Ca tN l ( ( C a tNj A nN) j ( C a tNk A nN) ) \ 2 l (N j (CatNkAnN))\2 where N l (CatNjAnN), and thence the NO − intake. $ The proportions of total N uptake as NH + and % NO − so calculated and the corresponding measured $ extractable NH + and NO − contents of the soil are % $


G. J. D. Kirk et al. 1.0 0.8 Fraction of total N uptake as NH4+ or NO3–

(c) P1000-NH4 +

(a) P100-NH4 +

0.6

NH4+

0.4

–

3.0 NH4+

2.4 1.8

NO3

1.2 NO3–

0.2

0.6 0.0

0.0

(d ) P1000-NO3 –

(b) P100-NO3 –

NO3–

0.8 –

NO3

3.0 2.4

0.6 1.8 0.4

1.2

NH4+

Extractable NH4+ or NO3– in soil (µmol g–1)

194

NH4+

0.2

0.6

0.0 1

2

3

4

5

6

8 0 2 4 7 Plant d. wt (g per system)

6

8

10

12

0.0 14

Fig. 5. Calculated fractions of total N uptake as NH + and NO − to account for observed rhizosphere pH % $ changes (solid and dashed lines respectively), and extractable NH + and NO − concentrations in the soil (closed % $ and open symbols respectively).

shown in Fig. 5. It will be seen that for the NH + % treatments, comparable intakes of NH + and NO − % $ are required to explain the observed pH changes. But for the NO − treatments, an intake of some 90% $ of the N as NO − is sufficient to explain the observed $ pH changes. These differences are reasonable in view of the differences in extractable NH + and % NO − in the soil. Note that although the extractable $− NO concentration is lower than the NH + con$ % centration, the concentrations in the soil solution and hence rates of delivery to roots may be similar because some of the NH + is adsorbed on the soil % solid. We conclude that the inorganic cation and anion intakes satisfactorily explained the observed soil pH changes. Export of organic anions from roots will tend to lower the pH of the rhizosphere to the extent that (a) the export is accompanied by an export of protons ; (b) the pKs of the conjugate acids are less than the rhizosphere pH, or the anions are adsorbed by the soil leaving protons in solution, or both ; and (c) they are not metabolized by microbes, whether to CO or # carbohydrates and proteins. Because the cytoplasmic pH (6–7) is maintained well above the pKs of the acids in question, they are present in the root cells mainly in the dissociated salt form, not the acid form. But, depending on the electrochemical potential gradient across the root cell membrane, export of the anion from the root may be coupled with H+ export. However there can only be a net export of H+ from the roots if the equivalents of organic anions exported less inorganic anions imported exceeds the equivalents of cations imported.

In the present case the plant inorganic cation– anion balance evidently far exceeded the accumulation of acidifying organic acids (excretion less decomposition). The accumulation of citrate was approx. 0.23 mmol per system in the P1000 treatments, but the total cation–anion imbalance, calculated from the change in soil acidity, was 7 mmol per system. If organic anion excretion had contributed significantly to the cation–anion balance, then the estimated NH + intakes would be too high and the % NO − intakes too low. But the calculated contri$ butions of NH + and NO − intake to the cation–anion % $ balances appear reasonable in view of the extractable NH + and NO − contents of the soil. % $       :                            We now consider the extent to which the measured rates of citrate excretion and the measured pH changes are sufficient to account for the measured P solubilization and uptake. The work by Hedley et al. (1994) mentioned earlier, in which rice plants grown in a highly weathered soil obtained their P by solubilization from inorganic pools and not by increased mineralization of organic P, used the same soil as here and similar conditions. We therefore exclude enhanced mineralization of organic P as the main means of P acquisition. Rates of P uptake The cumulative P uptake by the plants over time is shown in Fig. 6a. In the NO −-fed plants, uptake $



P in plants (µmol per system)

300 (a)

250 200

NO3 NH4

150 100 50 0

P concentration in CaCl2 extract (µM)

(b) 0.8 NH4

0.6 0.4 0.2

NO3 0.0

0


Fig. 6. Total P uptake by the plants over time (a) and the corresponding P concentrations in CaCl extracts of the # soil (b). Data are meanspSE of three replicates.

continued throughout the experiment and in the NH -fed plants it continued until after the fourth % week. Uptake by the NH -fed plants was slightly but % consistently greater than by the NO -fed plants until $ the fourth week. The decline after the fourth week coincided with a decrease in the plant growth rate but not in P content (Fig. 1b). It also coincided with the soil pH falling below 4.0 (Fig. 3c). Possibly the low pH induced toxic concentrations of Al or Mn or both in the soil solution, and this impaired growth and P uptake. The corresponding P concentrations in the soil CaCl extracts are shown in Fig. 6b. These reflect P # concentrations in the soil solution averaged across the thin layer. Upper estimates of the concentrations in the soil solution are obtained by multiplying by the ratio of the soil : solution ratio of the extract (1 : 10) to soil : solution ratio of the thin-layer soil (1 : 0.4). This gives [PL] values in the range 5–20 µM. These compare with initial concentrations in solution in the thin layers, uninfluenced by roots, of 0.5 µM, estimated from the soil P desorption isotherms at the native soil pH in Fig. 7 extrapolated to ∆[P] l 0. The adjustment according to soil : solution ratios overestimates [PL] because it does not allow for P desorbed from the soil solid into the extract. But in general it shows that the concentrations in solution were larger than the initial values and they were maintained over the course of plant growth, in spite of the large removal of P by the plants. Calculated uptake without solubilization The maximum rate of uptake attainable without solubilization can be calculated as follows. Con-

195 sidering the diffusion of P through the soil to the roots, established theory gives : bPc[PL]\ct l DLPθfc#[PL]\cx#

Eqn 8

In solving Eqn 8 the following boundary conditions apply. The root-plane (x l 0) is extended into the soil by root hairs (x l lh), and P depletion across the root hair zone will be uniform. The roots were not infected with mycorrhizas. The inner boundary condition is therefore : DLPθfd[PL]\dx l kα[PL]

x l lh t0

Eqn 9

(α l ‘ root absorbing power ’ for P (Nye & Tinker, 1999)). At the outer boundary (x l L), there will be no transfer of P. Therefore : DLPθfd[PL]\dx l 0 x l L

t0

Eqn 10

We solved Eqn 8 subject to these boundary conditions by numerical methods described by Kirk (1999). The soil parameter values were [PL]initial l 0.5 µM ; bP l 2000, estimated from the initial slope of the P desorption isotherm (Fig. 7) and ρ (l 1.16 kg dm−$) ; θ l 0.46 ; f l 0.40. We assigned α a value greater than the minimum for mildly P-deficient plants (10−% dm s−" (Nye & Tinker, 1999)) ; the calculation was insensitive to it at and above this value. Also we assumed there was no uptake for the first week following transplanting because the plants suffered a ‘ transplanting shock ’ lasting about a week. With these values the calculated maximum uptake in 6 wk without solubilization is 26 µmol per system. The observed uptake was 190 µmol per system. The sensitivity of this calculation to the input parameters can be appreciated from the following approximate solution of Eqn 8 which assumes that the thin layer is semi-infinite (from Crank, 1975, Eqn 3.15) : MP l bP[PL]initial(lhj2NDPt\π)

Eqn 11

(MP l amount absorbed in moles per unit area and DP l DLPθf\bP). Thus MP varies in proportion to [PL]initial and approximately in proportion to the square root of bP. The estimate of bP used above is not entirely reliable, but given that MP varies only as NbP the error in MP will be small. Clearly therefore, to account for the roughly seven-fold larger P uptake measured, the plants must have solubilized P. Effect of pH on soil P desorption The absence of large differences in P uptake between the NH + and NO − treatments, in spite of the % $ associated opposing pH changes in the rhizosphere (Fig. 3c), indicates that P solubilization by pH changes was not the main mechanism causing enhanced P uptake. However, the persistently larger uptake in the NH + treatments, where the pH was %



Change in P concentration in whole soil (µmol g–1)

196

P concentration in solution (µM) 0.0 0.2 0.4 0.6 0.8 1.0 0.0 4.88 3.98 3.45 4.81 4.78 4.08 4.00 3.52 4.87 4.15 4.87 3.64 4.23 3.77 –0.2 4.80

DDLθfd[PL]\dx l kα[PL], DLHθfd[HL]\dx l FH

4.31 3.87

x l lh t0 Eqn 14

–0.4

DDLθfd[PL]\dx l 0, DLHθfd[HL]\dx l 0

4.71

xlL

3.99

t0 Eqn 15

We evaluate λH in Eqn 12 as follows. Following Nye (1984) we define the ‘ solubilizing effect ’ of H+ on P as the quantity of P that must be removed from the soil for a given uniform addition of H+ to leave the concentration of P in solution unchanged, or (k∆[P]\∆[H])[P ]. From Nye (1984) Eqn 15 :

–0.6 4.38

–0.8 4.15

L

–1.0

Fig. 7. The effect of pH on P desorption from the P1000 soil. Initial suspension pHs were 3.8 (triangles), 4.3 (circles), 4.8 (squares). Numbers beside data are final suspension pHs. Data are meanspSE of two replicates.

lowered, and the larger apparent P concentrations in the soil solution in the NH + treatments, indicate that % some P was solubilized by the reaction of H+ with the soil. Fig. 7 shows the results of the shaken soil suspension experiments for the effect of pH changes on P desorption. The amounts P desorbed (-∆[P]) were inferred from the initial and final P concentrations in solution in the suspensions and the solution : soil ratios. The final suspension pHs are also shown ; these differ from the target pHs as a result of changes in the solution H+ buffer power with solution volume. The figure shows that addition of base slightly lowered the concentration of P in solution, whereas addition of acid increased it. Calculated uptake with solubilization by H+ We calculate the additional P absorbed as a result of solubilization by H+ using the theory of Nye (1983) for the diffusion of two interacting solutes in soil. We have previously used this theory to study P solubilization by rice growing in anaerobic soil, which also involves root-induced acidification (Kirk & Saleque, 1995 ; Saleque & Kirk, 1995). From Nye’s Eqns 10 and 11 we have for the diffusion and interaction of P and H+ : bPc\ct([PL]kλH[HL]) l DLPθfc#[PL]\cx# b c[H ])\cct l D θfc#[H ]\cx# H

Also, over the relevant pH range, H+ ions are carried through the soil predominantly by the movement of the acid–base pair H O+–H O−, the contribution $ # made by other acid–base pairs, such as H CO –HCO −, will be small. In solving Eqns 12 # $ $ and 13 the following boundary conditions apply, analogous to those for Eqn 8 :

L

LH

L

Eqn 12 Eqn 13

Note that [HL][PL] and so the diffusion of H+ will not be significantly affected by the diffusion of P.

(k∆[P]\∆[H])[P ] l λHbP\bH Eqn 16 L The quantity (k∆[P]\∆[H])[P ] can be estimated L from the data in Fig. 7. From the data for the suspensions that were initially acidified to pH 3.8, some 0.5 µmol P g−" was removed from the soil to leave the concentration of P in solution at its initial value ([PL] l 0.5 µM), and the final pH was approx. 4.0. From the soil pH buffer curve (Fig. 3c), the quantity of H+ that reacted with the soil in changing the pH from 4.3 (the initial value) to 4.0 was 28 µmol g−". Therefore (k∆[P]\∆[H])[P ] $0.018 mol P per L mol H+. As a check on this, approx. 9 µmol g−" of P was extracted from the soil by anion exchange resin in the presence of 500 µmol H+ g−" of H+-form cation exchange resin. Assuming all the H+ reacted with the soil, k∆[P]\∆[H] $0.018 mol P per mol H+. From the soil pH buffer curve over the range of pH in the P1000-NH + treatment, bH l k0.06i1.16\ % (10−%.$k10−$.)) l 642. Therefore, with bP l 2000, λH l 0.018i642\2000 l 0.0058. We calculated FH from the net acidity change in the thin layers in the P1000-NH + treatment (0.06 % mol kg−" from Fig. 3) by assuming a constant flux over the 5 wk following the transplanting shock. Thus, considering the thin layer thickness (0.03 dm) and bulk density (1.16 kg dm−$), FH l 0.06i1.16i 0.03\(5i7i24i60i60) l 6.9i10−"! mol dm−# s−". The other parameter values are as already given. The calculated increase in P uptake over time as a result of solubilization by H+ is shown in Fig. 8. The dotted lines give the calculated uptake without solubilization. It will be seen that while the increase in P uptake is significant, it is less than a third of the total solubilization after 6 wk and only a fifth of that after 4 wk. So although the changes in [P] in the thin layers (3.2 µmol g−") are small in comparison with the changes in [H] (60 µmol g−"), the solubilizing effect of H+ is not sufficient to explain the observed solubilization.



desorbed (-∆[P]) were inferred from the initial and final solution P concentrations in the shaken suspensions, multiplied by the solution : soil ratios. The final solution citrate concentrations are also indicated ; these are the result of both adsorption on the soil solid and decomposition by microbes during shaking. Fig. 9 is complicated because it reflects the combined effects of changing solution volume and citrate concentration. Kirk (1999) gives a full discussion of this. In short, over the range of conditions shown, sorption is influenced both by the original sorption isotherm for the soil and the isotherm for the soil modified by the reaction of citrate. For a given citrate concentration, as the solution :soil ratio increases, sorption is increasingly dominated by the citrate-influenced isotherm.

(a)

250 P in plants (µmol per system)

197

200 150 100 λH = 0

50 (b)

4.4

1.0 [PL]

0.8

4.2 pH

0.6 4.0

0.4 pH

3.8 0


[PL] (µM)

0

0.2 0.0

Fig. 8. (a) Comparison of the measured time courses of P uptake in the P1000–NH + treatment with that calculated % for solubilization by H+. Points are observed data and lines calculated ; dotted line gives calculated uptake without solubilization. (b) The corresponding calculated soil pHs and P concentrations in solution, and the measured soil pHs.

Change in P concentration in whole soil (µmol g–1)

0

P concentration in solution (µM) 10 20 30 40 50

0

0.48 0

0.14

0.64

1.33

0.15

–2

1.50

0.22

0.22

bP*c\ct([PL ]kλC[CL]) l DLP*θfc#[PL ]\cx#

Eqn 17

bCc[CL]\ct l DLCθfc#[CL]\cx#kθk[CL]

Eqn 18

(Note, as for H+, because [CL][PL ], the diffusion of C is not affected by the diffusion of P.) The following boundary conditions apply, analogous to those for Eqns 12 and 13 : DLP*θfd[PL ]\dx l kα[P ], DLCθfd[CL]\dx l FC, L DLP*θfd[PL ]\dx l 0, DLCθfd[CL]\dx l 0

0.83

0.08

The following two equations, analogous to Eqns 12 and 13, describe the diffusion and interaction of P and citrate in the soil (Kirk, 1999, Eqns 15, 16) :

x l lh t0 Eqn 19

0.19

–4

–6

60

Calculated uptake with solubilization by citrate

xlL

0.31

0.94

0.40

–8

0.99

0.43

1.76

1.96

We obtained values of the P-C interaction coefficient, λC, from the curves in Fig. 9 as follows. By analogy with Eqn 16, the solubilizing effect of C on P is given by : (k[P]\∆[C])[P] l λCbP*\bC L

1.00 –10

2.00

2.00 –12

Fig. 9. The effect of citrate on P desorption from the P1000 soil. Common symbols indicate common initial citrate concentrations (0, 0.1, 0.25, 0.5, 1.0 and 2.0 mM). Numbers beside data are citrate concentrations (mM) measured in the equilibrium solutions. Data are meansp SE of two replicates.

Effect of citrate on soil P desorption The effect of citrate on P desorption from the soil is shown in Fig. 9. As in Fig. 7, the quantities of P

t0 Eqn 20

Eqn 21

In Fig. 9, ∆[P] for given values of ∆[C] and [PL ] is found from the difference in ∆[P] between the appropriate lines at [PL ]. The corresponding value of ∆[C], the quantity of C reacting with the soil per unit soil volume, is found from ∆[CL]ibC. The mean calculated P and C concentrations in solution in the thin layers were c. 8 µM and 0.7 mM respectively (Fig. 10). With [PL ] $8 µM, comparing the value of ∆[P] at [CL] l 0.40 mM with that at 0.99 mM in Fig. 9, the quantity of P that must be removed from the soil to leave [PL ] unchanged as [CL] increases from 0.40 to 0.99 mM is c. k2.3i1.16 mmol dm−$ soil (note ρ l 1.16 kg dm−$). Therefore ∆[P]\∆[C] $k2.3i1.16\(5.1i(0.40k0.99)) l 0.89 mol P per mol C reacting (note bC l 5.1). From



198

P in plants (µmol per system)

parameter values are as in Fig. 10a, b. Uptake increases in proportion to both FC and λC. Thus if λC is doubled, a given uptake can be achieved with half the citrate excretion.

(a)

250 200 150

λC = 0

100

   

50 0 (b)

10 [P*L]

5

8

4

6

3

[C]

4

2 2

1 0

P uptake (µmol per system)

[PL*] (µM)

[C] (µmol g–1)

6

500

0


0

(c)

400

λC = 0.05

300

0.025

200

0.01

100 0 0.0

0.1

0.2 0.3 0.4 0.5 FC (nmol cm–2 s–1)

0.6

Fig. 10. P uptake and citrate. (a) Comparison of the measured time course of P uptake from the P1000 thin layers with that calculated for solubilization by citrate. Points are observed data (NH +, closed symbols ; NO −, % $ open symbols) and lines calculated ; dotted line gives calculated uptake without solubilization. (b) The corresponding calculated concentrations of citrate in the whole soil and P in the soil solution (lines), and the measured mean citrate concentrations in the soil (plus signs). (c) Sensitivity of the calculated P uptake 6 wk after planting to the flux of citrate across the roots (FC) and the soil P-citrate interaction coefficient (λC). Other parameter values as in (a, b).

the slope of the curve at [CL] l 1 mM, where P sorption will be dominated by the isotherm for the soil modified by the reaction of citrate (see previous section), bP* l (c[P]\c[PL ])C $200. Therefore λC l 0.025. To calculate the extent of solubilization by citrate in the thin layer we use the above values of bP*, bC and λC with FC l 3.3i10−"! mol dm−# s−" and k l 4.16i10−& s−", and the other parameter values given earlier. Fig. 10 compares the time courses of P uptake so calculated with the observed results. The dotted lines give the calculated uptake without solubilization. It will be seen that the model accurately predicts the time course of P uptake within the limits of experimental error. Fig. 10c shows the sensitivity of the calculated P uptake at 6 wk after planting to FC and λC. The other

The results show that the rates of citrate excretion into the thin layers and the P-solubilizing effect of citrate are sufficient to explain P uptake by the plants. No other mechanism could explain the observed solubilization and uptake. In the thin-layer system the diffusion of citrate away from the roots is constrained by the width of the thin layer, and citrate therefore tends to accumulate close to the roots. The zone of solubilization is correspondingly close to the roots and a large proportion of the P solubilized is taken up. But for an isolated root in a large volume of soil, citrate might diffuse far from the root and a smaller proportion of the P solubilized would then be taken up. Kirk (1999) shows how the importance of this effect varies with the values of the soil citrate and phosphate diffusion coefficients. He also shows that in cylindrical geometry, as appropriate for an individual root, the increase in P uptake for a given release of citrate is smaller than in planar geometry. However, in a system of many roots, neighbouring roots will benefit from P solubilized by each other to an extent depending on root density and the localization of excretion along the root length, and this will tend to obviate the effect of cylindrical geometry. We saw that in the absence of citrate a decrease in pH tends to increase the concentration of P in the soil solution (Fig. 7). In addition pH changes will alter the P-solubilizing effect of citrate. Various processes are involved in this. On the one hand a decrease in pH will tend to increase the soil surface positive charge as the surface takes up protons, making it less likely to adsorb citrate from the soil solution. On the other a decrease in pH will cause increased protonation of citrate in solution tending to decrease its adsorption on the solid. It will also tend to decrease the ability of citrate to chelate metal ions. The net effect in our soil appears to be increased P solubilization by citrate at low pH. Thus the concentration of P in the CaCl extracts was larger in # the NH + treatments in Fig. 6 as was the initial P % uptake. Jones & Darrah (1994) also found increased solubilization by citrate at low pH ; solubilization by H-citrate was 40% greater than could be accounted for by the effects of H+ and citrate alone. However such effects will vary greatly between soils. It would be possible to allow for pH–organic anion interactions in the model by modifying Eqn 17 and calculating the pH profile with Eqn 13. But evidently this would be an undue complication for the present case.


Phosphate solubilization by organic anions The concentration of citrate in the soil, averaged across the thin layers, was comparable to the change in P concentration ($3 µmol g−" at the final harvest). But since the soil citrate buffer power was much smaller than the P buffer power, the amounts of citrate adsorbed were much smaller than the amounts of P desorbed. Therefore displacement of P by citrate was unimportant and the main mechanism by which citrate solubilized P is likely to have been through chelation of metal ions, principally Fe, Mn and Al. This may involve either the removal of metal ions that would otherwise immobilize P, or the formation of soluble C-metal-P complexes, or both. The model does not distinguish between these two possibilities. However if soluble C-metal-P chelates are involved, the assumption implicit in Eqn 19, that complexed P arriving at a root surface is readily absorbed, may be invalid. It is possible that Cmetal-P complexes are absorbed directly. But in that case complexed Al would enter the root and this would negate the supposed Al-detoxifying role of organic anions. Alternatively the complex may be dissociated before the P is absorbed, possibly by a root-mediated process, such as Fe reduction for citrate-Fe-P complexes (Marschner, 1995). In any case the fact that the model predicts the time course of P uptake satisfactorily without taking account of such processes indicates that they do not limit the rate of uptake, at least for the present example.  $

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Citrate excretion from rice roots at moderate rates was sufficient to account for the observed P solubilization and uptake. The model presented accurately predicted the time-course of P uptake using only independently measured parameter values. It also accurately predicted concentration-distance profiles of P in the rhizosphere (Kirk, 1999). We conclude that the model provides a satisfactory description of the processes involved. The apparent rates of citrate excretion into the soil per unit root fresh weight were comparable to rates measured for roots growing in nutrient solution. The apparent half-life of citrate in the soil solution was 5 h. The half-life is a function of both rates of decomposition by rhizosphere microbes and rates of de novo synthesis from other substances excreted. The reaction of H+ with the soil also solubilized P, and the net solubilizing effect of citrate was greater at lower pH. However, at very low pH, plant growth was inhibited and the additional P solubilized was not taken up. Solubilization by displacement of P from adsorption sites on the soil was unimportant. The main mechanism of solubilization involved chela-

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tion of metal ions that would otherwise immobilize P or formation of soluble citrate-metal-P complexes or both. Much more needs to be known about the factors controlling the release of organic anions from plant roots, their longevity in the rhizosphere, and their P solubilizing effects in different soils.

               We thank Erma Lallana for assistance in the HPLC analyses, Joey Solivas for measuring the diffusion impedance factor and Rene Carandang for general laboratory assistance.

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