Published in J. Environ. Qual. 33:1387-1392 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA
TECHNICAL REPORTS
Plant and Environment Interactions
In Situ Dynamics of Phosphorus in the Rhizosphere Solution of Five Species
Z. Y. Wanga,*,
J. M. Kellya and
J. L. Kovarb
a Department of Natural Resource Ecology and Management, 253 Bessey Hall, Iowa State University, Ames, IA 50011-1021
b USDA-ARS National Soil Tilth Laboratory, Ames, IA 50011-4420
* Corresponding author (wangz{at}lincolnu.edu).
Received for publication July 13, 2003.
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ABSTRACT
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Root activity can modify the chemistry of the rhizosphere and alter phosphorus (P) availability and uptake. However, until recently, relatively little was known about the dynamics of soil solution P at the root surface because of our inability to measure in situ changes in solution P at the plant root. A mini-rhizotron experiment with corn (Zea mays L. cv. Stine 2250), soybean [Glycine max (L.) Merr. cv. Pioneer 3563), cottonwood (Populus deltoids L.), smooth brome (Bromus inermis Leyss.), and switchgrass (Panicum virgatum L.) was conducted to measure the spatial and temporal dynamics of P in the rhizosphere solution of a fine silty, P-rich calcareous soil (solid-phase total P concentration = 62 mg kg–1, pH = 7.68) from western Iowa. Micro-suction cups were used to collect samples of soil solution from defined segments of the rhizosphere, and capillary electrophoresis (CE) was used to determine the P concentration of the soil solution. At the end of 10 d, a decreasing P concentration gradient in soil solution toward the root was observed in corn, cottonwood, and smooth brome. No clear rhizosphere effect was observed for soybean and switchgrass. Statistical analysis indicated significantly lower solution P concentrations in the rhizospheres of corn (p = 0.05), cottonwood (p = 0.01), and smooth brome (p = 0.01) compared with bulk soil solution. Results indicate that P depletion from rhizosphere soil solution depends on plant species. Under the conditions of this study, corn, cottonwood, and smooth brome were more effective in depleting solution P than soybean and switchgrass.
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INTRODUCTION
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PHOSPHORUS MOVEMENT into surface waters from a variety of diffuse sources can have negative effects on surface water quality (Hanrahan et al., 2001), leading in some cases to surface water eutrophication (Carpenter et al., 1998). Much more is known about the magnitude of loss and the options for control of the loss of particulate P (Withers and Jarvis, 1998) than for soil solution P. For example, riparian buffers can play an important role in the control of particulate loss, intercepting P associated with both organic and inorganic particles, as well as some of the dissolved P associated with runoff (Quinton et al., 2001). A diverse set of factors control losses of dissolved P (Torrent and Delgado, 2001), with plant uptake making a significant contribution to the reduction of dissolved P loss. However, species commonly planted in buffer areas vary greatly in both amount and timing of P removal. We know that roots can modify the chemistry of the rhizosphere and thus alter P availability and uptake (Darrah, 1993; Marschner, 1995; Hinsinger, 2001). However, our understanding of the in situ dynamics of soil solution P and the plant root needs to be improved to refine the models needed to develop more effective nutrient control strategies.
Plant uptake of P is strongly influenced by rhizosphere solution conditions (Morel et al., 2000). Previous studies indicate that root uptake significantly decreases solution-phase concentration of P in the rhizosphere, and there are obvious differences in P requirement among species (Asher and Loneragan, 1967; Jungk and Claassen, 1989; Zoysa et al., 1998). However, due to the absence of a dependable method for sampling in situ rhizosphere solution, little information is available on the spatial heterogeneity and temporal dynamics of P in the rhizosphere solution. In addition, little is known about differences in rhizosphere solution P dynamics among various plant species. Recent advances in soil solution sampling procedures (Göttlein et al., 1996; Wang et al., 2001) and in the analysis of micro-volume solution samples (Göttlein and Blasek, 1996) provide the means to overcome these problems.
The first objective of this study was to sample at high spatial resolution soil solution P in the rhizosphere of corn, soybean, cottonwood, smooth brome, and switchgrass seedlings. These plants represent a typical continuum of species occurring in a cropland–riparian buffer transition. The second objective of this study was to determine whether the technique for collecting soil solution using micro-suction cups in combination with capillary electrophoresis analysis is a suitable tool for P measurement in soil solution. Our results will begin to provide a clearer spatial and temporal picture of the concentration of solution-phase P present in the rhizosphere and the ability of these species to deplete rhizosphere P. This knowledge will contribute to model predictions of P uptake and movement.
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MATERIALS AND METHODS
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Soils
All experiments were conducted with soil taken from the B horizon (0.5–1 m) of a Kennebec silt loam (fine-silty, mixed, superactive, mesic Cumulic Hapludolls) at the USDA-ARS Deep Loess Research Station located near Treynor, Iowa. The soil had a pH in water (1:2.5 w/w soil to water ratio) of 7.68 and a solid-phase total P concentration (McGrath and Cunliffe, 1985) of 62 mg kg–1. B horizon soil was used in this experiment to avoid (i) effects from the large volume of dormant seed typically found in the A horizon and (ii) artifacts that the use of soil sterilization to prevent seed germination might have on solution P. Soil was air-dried, sieved (<2 mm), and thoroughly mixed before being placed in the mini-rhizotrons.
Mini-Rhizotron Description
The mini-rhizotrons used in this study were 33 cm long, 11.5 cm wide, and 2.2 cm deep, with a volume of 330 cm3 (Fig. 1). One side of the mini-rhizotron was equipped with a 5- x 5-mm grid of holes for installation of micro-suction cups (Göttlein et al., 1996). The opposite side of the mini-rhizotron was equipped with a clear Plexiglas plate to allow the developing roots to be seen. The mini-rhizotron was positioned at an angle of 30° to force the roots to grow toward the Plexiglas plate. The transparent plate was covered with a black plastic sheet, which was removed only for the observation of root development. The micro-suction cups were inserted through the grid on the upper side of the mini-rhizotron and pushed toward the transparent Plexiglas plate. Micro-suction cup position as well as the distance to individual roots was thus clearly visible (Fig. 1).
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Fig. 1. Schematic drawing and photograph of the mini-rhizotron with an array of micro-suction cups installed. Each probe is connected to a discrete sample vial contained in the vacuum box. A computer-regulated vacuum pump is attached to the vacuum box and a tension of –100 kPa is applied to each micro-suction cup through the vacuum box.
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The micro-suction cups were ceramic P80 material with a porosity of 48% by volume, and a maximum pore size of 1 µm (KPM, Berlin, Germany). The tip of the micro-suction cup was sealed with melted glass using a Bunsen burner. The cup was glued to a 5-cm length of 1.59-cm polyetheretherketone (PEEK) tubing (inside diameter = 0.75 mm). Each micro-suction cup was individually tested for leaks. To install the micro-suction cups into the soil, a steel scoop having the same dimensions as the micro-suction cups was inserted into the soil to the desired depth and then removed. This produced a small channel that prevented damage to the suction cup during installation. Each micro-suction cup was inserted carefully and fixed to a stainless steel tube with shrink wrap tubing (Göttlein et al., 1996).
Irrigation System
To avoid concentration gradients caused by irrigation, the rhizotron was irrigated with a solution having an ionic composition similar to the equilibrium soil solution. The solution was obtained from a batch soil-water equilibration (soil to water ratio = 1:3 w/w). The irrigation solution was collected by vacuum extraction and passed through a 0.45-µm filter. After degassing, it was introduced into the rhizotron by four porous polymer tubes (inside diameter = 7 mm, length = 10 cm) (Eijkelkamp, Atlanta, GA), which were adjusted to a water potential of –80 kPa by adjusting the height between the rhizotron and the bottle of irrigation solution (Fig. 1). To establish an equilibrium within the soil matrix of the mini-rhizotron, this solution was percolated through each mini-rhizotron before its use as irrigation solution.
Plant Culture
All plants were grown in a controlled-environment chamber. Relative humidity in the chamber was maintained at 80%, and the day and night (14 and 10 h) temperatures were 25 and 20°C, respectively. Light intensity was set at 120 µE m–2 s–1. Corn, soybean, and cottonwood plants, each having individual root lengths of 2 cm, were transplanted into the mini-rhizotrons. Smooth brome and switchgrass plants were started from seed germinated directly in the mini-rhizotron soil. Each mini-rhizotron contained two chambers, and each chamber contained one to three plants. Two mini-rhizotrons were used for each species (N = 4 chambers).
Solution Collection
We installed dense grids of micro-suction cups (15 cups for each root system) in front of the developing root system in each chamber of the mini-rhizotron. The micro-suction cups were connected to a vacuum collection box, which allowed the solution from each micro-suction cup to be collected in an individual sample vial (Göttlein et al., 1996). The micro-suction cups sampled continuously under a vacuum of –100 kPa and samples were collected at 24-h intervals for 10 d (16 d for switchgrass). A computer-regulated vacuum pump attached to the vacuum box maintained a constant –100 kPa tension. The volume collected from each micro-suction cup was typically in the range of 100 to 200 µL.
Solution Analysis
Solution samples were analyzed for dissolved P by capillary electrophoresis using a P/CE System MDQ from Beckman (Fullerton, CA). A buffer solution consisting of 8 mM tris (hydroxymethyl) aminomethane (TRIS) (99.9%), 2 mM 1,2,4-benzenetricarboxylic (trimellitic acid, TMA) (99%), and 0.3 mM tetradecyltrimethylammonium bromide (TTAB) (99%) at a pH of 7.6 (Westergaard et al., 1998) was used for all P analyses. Separations were performed in a fused-silica capillary with a 75-µm i.d. (Beckman) and an applied separation voltage of –30 kV at a temperature of 20°C and a detection wavelength of 254 nm. Pretreatment of the capillary between each run included rinsing with a 0.1 M sodium hydroxide solution for 1 min, water for 0.5 min, followed by preconditioning with the electrolyte for 5 min. An IQ (San Diego, CA) Model 200 scientific pH meter was used to measure pH.
Data Analysis
To simplify data presentation and enable a comparison of the different micro-suction cup matrices, the sampling events were classified according to day number, with the first day that a root entered the micro-suction cup array assigned as Day 0. Distance from a suction cup to the root was classified as being in one of three classes: <1 mm, 1 to 8 mm, and >8 mm, depending on the location of the sampling point. Preliminary evaluation indicated that grouping sampling points into these three distance categories would provide enough data points in each category for further statistical analysis. Analysis of variance (SPSS Version 10.0; SPSS, 2002) was used to test the effects of species, time, and distance on solution P concentration. Mean values of P concentration were obtained by averaging all values with the same day number and the same class of distance to the root. The number of samples in the same class of distance to the root varied each day, depending on root growth.
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RESULTS
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For each of the five plant species studied, P concentration varied with distance from the root (Fig. 2). For corn, cottonwood, and smooth brome, from Day 7 on, the P concentration decreased (p = 0.05 or 0.01) at the sampling points near the roots (<1 mm) as compared with those at greater distances (1–8 mm, >8 mm). This decrease was less prominent in the soybean and switchgrass rhizospheres.
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Fig. 2. Time course of P concentration in the soil solution for three different root distance classes for corn (A), soybean (B), cottonwood (C), smooth brome (D), and switchgrass (E) plants. Individual points in each figure are the means of all sampling points falling within each distance class on that day. The number of sampling points within each distance class can change with sample day. Note the difference in length of study period for switchgrass.
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A typical solution P concentration (mg L–1) at each sampling point in a mini-rhizotron chamber after 10 d of cottonwood root development is shown in Fig. 3. The root reached Suction Cups 4, 8, and 12 at Days 4, 5, and 6, respectively. Phosphorus concentrations for these three sampling points were 0.47, 0.68, and 0.73 mg L–1 at Day 10, illustrating the reduction in P concentration in the rhizosphere as a function of time. Comparison of these values with those observed at sampling points at a greater distance from the roots illustrates the effect of distance from the root on solution concentration.
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Fig. 3. Photograph of cottonwood roots growing in a mini-rhizotron. The circular white points arrayed across the photograph are the micro-suction cups. Numbers in parentheses are used to identify each sampling point. Values shown are solution P concentrations (mg L–1) observed after 10 d of root growth. Micro-suction cups near the top of the photograph have been influenced by root uptake for a longer period of time. Lower values near roots emphasize the effect the plant has had in decreasing solution concentration near the root compared with those in bulk solution. The sample volume at Point 2 was not sufficient for analysis of P concentration.
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Soil solution P concentrations varied with both distance from the root and plant species for the last two sampling dates (Days 9 and 10 for corn, soybean, cottonwood, and smooth brome; Days 13 and 16 for switchgrass) (Table 1). There was a 22% reduction in solution P concentration at a distance of <1 mm from the root of the corn, cottonwood, and smooth brome as compared with the solution concentration at a distance of >8 mm based on the Day 10 observations. These data demonstrate a change in P concentration over a 10-d period. No clear rhizosphere P depletion was observed for soybean and switchgrass after 10 and 16 d of growth, respectively (Table 1). Comparing the measured rhizosphere solution concentration observed at a distance of <1 mm for the five plant species, the relative P concentrations are: cottonwood = smooth brome < corn < soybean < switchgrass (Table 2).
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Table 1. Concentration of PO3–4 ion in soil solution at two distances from the root for the last two sampling days (Days 9 and 10 for corn, soybean, cottonwood, and smooth brome; Days 13 and 16 for switchgrass).
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Table 2. Statistical comparison of mean PO3–4 concentration in rhizosphere (<1 mm from root surface) solution as a function of plant species, based on samples collected on two sampling days (Days 9 and 10 for corn, soybean, cottonwood, and smooth brome; Days 13 and 16 for switchgrass).
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DISCUSSION
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In the present short-term study, the dynamics of P concentration in soil solution were compared at three different distances from the surface of a growing root for five plant species (Fig. 2). A typical gradient of decreasing P concentration in soil solution toward the root was observed in corn, cottonwood, and smooth brome. Since mass flow generally contributes only a small proportion of the P taken up (Barber, 1995), the depletion of P due to uptake by growing roots is mainly controlled by amount and rate of orthophosphate diffusion in soil solution. This depletion generates a concentration gradient toward the root. Autoradiography of labeled P provided the first direct evidence of P depletion occurring in the vicinity of roots (Lewis and Quirk, 1967). Other approaches have also provided ample evidence of rhizosphere depletion of water-soluble P (Morel and Hinsinger, 1999), Olsen P (Pecqueux et al., 1998), resin P (Zoysa et al., 1999), NaOH-P (Zoysa et al., 1999), and acid-soluble P (Bertrand et al., 1999; Zoysa et al., 1999).
Comparing the five plant species, the roots of corn, cottonwood, and smooth brome were more effective in removing P from solution than soybean and switchgrass roots under these conditions (Table 2). The differences in the ability of various species to deplete soil inorganic P have often been cited (Zoysa et al., 1998; Bertrand et al., 1999). Further research will be needed to investigate soil P depletion by the roots of other plant species or varieties and/or clones within a species.
Kirk (1999) and Geelhoed et al. (1999) observed that possible interactions between P ions and other solutes, such as protons or organic anions, may affect the solubility of soil P. Soil pH changes can induce dramatic changes in P concentration in soil solution (Morel et al., 1994). Excretion of organic anions or acids from roots is thought to be one of the main mechanisms by which plants mobilize less readily available soil phosphates from non-acidic soils (Marschner, 1995). Nevertheless, for many plant species, the flux of organic anion exudates is rather small (Jones, 1998), compared with the uptake of major nutrients. The pattern and amount of root exudates also varies considerably among various species (Neumann and Römheld, 1999; Kirk et al., 1999). One could speculate that in this experiment, excretion of organic acid by soybean roots might have increased the P concentration in the rhizosphere sufficiently to offset any uptake. This could account for the observed lack of change in solution P concentration. However, no change in solution pH was observed in this study (data not shown), possibly due to the calcareous and thus strongly buffered nature of the study soil. For switchgrass, the lack of solution P depletion might be due to the plant's slow growth rate and a low P requirement. We are not aware of any other reports on the dynamics of P in the rhizosphere solution of soybean and switchgrass that might aid us in interpreting the observed response. Both of these species can be mycorrhizal, which makes P uptake from greater distances away from the root possible. However, for a B horizon soil high in P, the likelihood of a mycorrhizal contribution is probably low.
Our results, although based on a relatively short period of observation, suggest that roots of commonly used riparian buffer species such as cottonwood and smooth brome may be more effective than switchgrass roots in intercepting solution-phase P before it has a chance to exit the buffer. To more fully understand changes in solution P depletion within the rhizosphere, further investigation of the relationships among soil solution, soil solid phase, soil exploration, root exudation, and plant nutrient status is needed under both controlled and field conditions. With these additional insights, we can better assess the ability of individual species to deplete solution P, and the role plant uptake plays in controlling loss of dissolved P from cropland, pasture, and riparian buffers. This enhanced understanding will allow us to develop better model representations of P uptake and solution-phase chemistry. Better models will be important as we seek to develop more effective nutrient control strategies that are tailored to land use, soil conditions, application rate, and the type of plant cover.
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ACKNOWLEDGMENTS
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Journal paper of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA, Project no. 3905. Supported in part by Mcintire-Stennis and State of Iowa funds as well as grants from the Leopold Center for Sustainable Agriculture (2002-30) and the USDA-ARS (SCA#58-3625-2-125). The authors wish to express their gratitude to Dr. Dan Armstrong of the ISU Chemistry Department for providing access to the capillary electrophoresis instrument.
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ABSTRACT
INTRODUCTION
