Abstract

Key Words

Biogeochemistry, pedogenesis, organic acids, chromosol, alfisol

Introduction

The rhizosphere is a narrow zone of the soil or regolith that is under the direct influence of plant roots (Eggleton 2001; Ryan et al. 2001). The root alters the physical, chemical and biological properties of the rhizosphere through concomitant processes of growth, nutrient uptake and organic anion exudation, and has been described as a zone of maximum biological activity within soils (Bertin et al. 2003; Curl and Truelove 1986; Hinsinger et al. 2003; Jones 1998; Jones et al. 2003). Many studies on the role of roots on regolith geochemistry focus on understanding and improving nutrient uptake (and crop yield) in agricultural and forestry settings (e.g. Egle et al. 2003; Hinsinger et al. 2003; Shaw 1960a, 1960b; van Hees et al. 1996; Vance et al. 2003; Zoyza et al. 1997). So while plant roots are recognised pedogenic agents there is limited understanding of the implications for mineral weathering and pedogenesis in the rhizosphere of Australian forest soils (see Ryan et al. 2001; Van Breeman et al. 2000; Waisel et al.1991; Zoysa et al. 1997). This pilot project is part of broader research study (see Leonard and Field 2003; Little and Field 2003) focused on understanding root-mediated weathering in the rhizosphere of a forest soil commonly observed on the Southern Tablelands of New South Wales. In particular the research aims to reach an understanding of how tree roots influence soil formation and geochemical change through nutrient uptake, the production of low molecular weight carboxylic acids, and in the provision of microbial habitat in a temperate Australian forest soil rhizosphere. We combine field and laboratory methods with knowledge of plant physiology, microbiology, and chemistry and mineralogy in soils and regolith in an effort to understand rhizosphere biogeochemistry in relation to mineral weathering and soil formation processes. First, samples were analysed in a compartment study of elemental accumulation in bulk soils, rhizosphere and < 3mm roots beneath two adjacent, mature trees, Eucalyptus mannifera and Acacia falciformis using standard geochemical techniques. Secondly, controlled dissolution experiments were undertaken using low concentrations of low molecular weight organic acids to investigate the potential influence of the production and exudation of soluble organic compounds on metal mobilisation in the rhizosphere of these soils.

General site characteristics

The field site is located approximately 9km north east of Bungendore, New South Wales, in a warm-temperate mixed-species dry sclerophyll forest. Specifically, the site is situated on highly weathered Devonian metasediments, on the East-facing lower-mid slope of some undulating to rolling low hills. Mature Eucalyptus mannifera and A. falciformis occur in the landscape as co-dominant trees. The soils can be classified as shallow Haplic Brown Chromosols (Isbell 1996), Dy 2.21 (Northcote 1971), or Yellow Podzolic soils (Stace et al. 1968), generally with a shallow (E. mannifera) to moderately thick (A. falciformis) sandy loam A₁ horizon, overlying a sandy clay (A. falciformis) to light clay (E. mannifera) B₂ horizon. According to the US Soil Taxonomy (Soil Survey Staff 1994), the soils might also be equated with Alfisols. The soil properties of the site are summarised in Table 1.

Table 1. Summary table, showing Bulk Density, Soil Moisture Content, Soil Organic Matter Content Cation Exchange Capacity (B – Bulk soil; R – Rhizosphere), and field pH, texture and thickness of the A₁ and B₂ soil horizons beneath two co-occurring trees.

	Eucalyptus mannifera		Acacia falciformis
	A1 horizon	B2 horizon	A1 horizon		B2 horizon
Thickness (cm)	5	10	25		10
field Texture	Sandy loam	Light clay	Sandy loam		Sandy clay
Bulk Density (g/cm3)	1.23	1.38	1.12		1.26
Soil Moisture Content (% Wt)	8.37	6.88	8.11		5.48
Field pH	4	6	3.5		4.5
Soil Organic Matter (% DWt)	5.79	3.39	7.32		3.97
Organic Carbon (% DWt)	3.42	2.00	4.32		2.30
CEC (mM+/kg)	22.6	14.3	B 44.7	R 55.0	B27.2	R 33.8

Methods

Because of the difficulties involved in separating root and microbial contributions to soil processes it was necessary to broaden our definition of the rhizosphere to include micro-organisms that are intimately associated with fine to medium sized (< 3 mm) roots, in the myco-rhizosphere (Barea et al. 2002; Van Breeman et al. 2000). Geochemical analysis of different soil fractions was used to identify differences observed in coarse soil chemical patterns beneath two adjacent mature trees, and to explain differences based on indicators of nutrient uptake in the rhizosphere of E. mannifera and A. falciformis.

Sampling Procedures

Soil and fine root samples were collected from pits dug 0.5m from the base of each of the adjacent E. mannifera and A. falciformis trees using 50mm-diameter bulk density cores. Two samples were collected from the A₁ and B₂ soil horizons respectively, placed into separate polyethylene sample bags and labelled; a total of four samples were collected from each soil pit. The field pH was estimated for each horizon using an INOCULO soil pH test kit. The field texture and thickness (cm) of the A₁ and B₂ horizons were described as well as the nature of the boundary between the two. We prepared the samples for two sets of analysis: (1); to determine baseline physical and chemical properties of the < 3mm roots, rhizosphere and bulk soils, (2); controlled dissolution experiments to identify potential interactions between low molecular weight organic acids (LMWOA) as root exudates and soil / regolith minerals.

Chemical content of Bulk soils, rhizosphere and < 3 mm roots

Samples used for analysis of the chemistry of bulk soil, rhizosphere and fine roots were prepared according to the following procedures:

1. The Bulk density core-samples were immediately weighed, dried at 105°C for 48 hours, then weighed again to determine the soil moisture content (% weight loss), and the bulk density (g/cm³) of the soils at the time of sampling.

2. The samples were separated into < 3 mm roots, rhizosphere and bulk soil compartments; first by shaking to remove the bulk soil (soil not adhered to the roots), then by further shaking which separated the roots (plus rhizoplane; i.e. the soil right at the root surface) from the rhizosphere soil. Careful washing of some of the < 3 mm roots was undertaken to remove the rhizoplane, for analysis of the element concentrations in the fine roots. The rhizosphere was considered to be the soil adhered to the roots after careful but vigorous shaking (Wang and Zabowski 1998), and the remainder was considered as the bulk soil.

3. The bulk soil, rhizosphere and root sample separates from each horizon were pooled and homogenised in order to ensure enough rhizosphere soil samples were available for analysis. The fine roots were so sparse beneath the E. mannifera tree that even after pooling, only enough rhizosphere sample was available for one analysis.

4. Bulk soil, rhizosphere and root samples were prepared for analysis of total chemical content using a nitric-perchloric acid (HNO₃, HClO₄) digest. Solutions were analysed using an Inductively Couple Plasma – Atomic Emissions Spectrometer (ICP-AES).

5. Rhizosphere and bulk soil samples were prepared for analysis of ammonium acetate (NH₄COOH, AAC) extractable and water (H₂O)-soluble elements. Three aliquots of either 25mL 1M AAC or deionised H₂O were added to extract elements from 1g soil and solutions were pooled. The samples were acidified to ~ 1% nitric acid (HNO₃) for analysis using ICP-AES.

6. We calculated Cation Exchange Capacity (CEC) for the bulk and rhizosphere soils using the sum of the AAC extractable Al³⁺, Ba²⁺, Ca²⁺, Fe^{2+ / 3+}, K⁺, Mg²⁺ and Na⁺ concentrations (Table 3).

Further analyses of trace element concentrations using ICP-MS were still to be interpreted at the time of writing this paper.

The controlled dissolution experiment

The purpose of this experiment was to examine the influence of low concentrations (~1 mM) of LMWOAs on soil material dissolution in a controlled environment. The di-and tri-carboxylic acids in root exudates are of particular interest because of the roles they play in nutrient uptake, though the implications of these for mineral weathering in the rhizosphere are still poorly understood. Stone (1997) however, provides a comprehensive discussion of ligand exchange and metallo-organic complex formation. Studies that have examined the relationships between mineral weathering and organic acids have generally focused on pure minerals (e.g. Barker et al. 1998; Welch and Ulmann 2000; Welch et al. 2002) with a greater emphasis on micro-organisms rather than the roots (see for example, Banfield et al. 1999; Gadd 2004), and with little focus on more complex soil and regolith materials (see for example review by Barker et al. 1997). Citric, malic and oxalic acids were chosen because of their established associations with the mobilisation of such nutrient elements as phosphorus (P), iron (Fe), calcium (Ca), and others such as aluminium (Al), and the ability of their anions (citrate^3-, malate^2-, oxalate^2-) to form stable, 6- and 5- ring chelates (see Ryan et al. 2001; Stone 1997) with these elements (e.g. Bolan et al. 1997; Gottlein et al., 1999; Markewitz and Richter 1998; Neumann and Martinoia 2002; Stone 1997; Watt and Evans 1999). We examined the dissolution of mineral elements from soils in dilute concentrations (~1 mmol L^-1) of low molecular weight di- and tri-carboxylic acids commonly exuded by plant roots and micro-organisms. Citric (C₆H₈O₇), malic (C₄H₆O₅) and oxalic (C₂H₂O₄) acids were chosen because of their strong metal-chelating abilities (Gottlein et al. 1999; Markewitz and Richter 1998; Stone 1997; van Hees et al. 1996) and their known association with nutrient uptake by both fine roots and micro-organisms in the myco-rhizosphere (e.g. Curl and Truelove 1986; Hinsinger et al. 2003 ; Vance et al. 2003). The controlled dissolution experiment was established for the soil materials using the following procedure:

1. Sub-samples of the bulk soils from each soil horizon were autoclaved (110⁰C for ½ hr) and then redried.

2. The experiment was designed so that 4, 1g sub-samples were exposed to 4 treatments, and each treatment was undertaken in triplicate.

3. Initial solutions were ~ 1 mM citric acid, oxalic acid and malic acid and an inorganic sodium chloride (NaCl) control solution; initial pH was adjusted to 4.5 in an effort to mimic the naturally occurring pH in the field soils.

4. About 200 mL of the 1 mM solution was added to the soil samples, bottles were sealed, and left to react.

5. Aliquots of the supernatant, approximately 15mL, were collected by syringe, and passed through a 0.20µm milli-pore filter into centrifuge tubes after days 1, 8 and 15.

6. The pH of 5mL sub-samples was measured using an ORION 290A+ pH / Eh meter and recorded before acidifying the remainder of the sample solutions to ~ 0.1% HNO₃.

7. The sample solutions were stored in a cool dry place prior to analysis using ICP-AES

At this stage all interpretations are purely observational and no statistical significance is implied from the data.

Results and Discussion

The results of major and trace elemental analyses of the different soil fractions collected from the regolith (bulk soil, rhizosphere and fine root; Table 2) conforms to other studies that show distribution of mineral elements is influenced by plant roots (see, Zoysa et al. 1997). The highly leached and weathered nature of the landscape have, over time, led to the development of soils deficient in most biologically important mineral elements (Table 2).

Element dynamics in bulk soils, rhizosphere and fine roots

The patterns of accumulation or depletion were most evident when the total and AAC extractable element concentrations were examined together. Table 2 shows a decline in Al and Fe concentrations from the rhizosphere to the < 3 mm roots beneath the A. falciformis and E. mannifera, and may be an indication that this element is being excluded from uptake. The Al concentration in the root and rhizoplane is a factor of 1/10 the rhizosphere concentration, and the < 3 mm roots contain only 1/50 of the Al compared to the rhizosphere and bulk soils. The change in Fe concentrations is at least as clear.

The biologically important elements, Ca, K and Mg also exhibit interesting rhizosphere dynamics, in what are highly leached and weathered soils. Calcium and K are particularly deficient in these soils (compare Tables 2 and 3), though in different ways. Nearly 80% and 25% of the total Ca in the A₁ soil horizons beneath the A. falciformis and E. mannifera respectively, can be accounted for by AAC extractable forms, indicating that it is available for uptake, even though the results suggest little Ca is contained in the parent materials. The results indicate that Ca is being recycled by both species through litter fall and resultant decomposition. More distinct is the order of magnitude decline in AAC extractable Ca in the B2 horizon rhizosphere of the E. mannifera, in addition to the two- to five-fold increase in Ca concentrations from the rhizosphere to the roots for both species, which might indicate that the fine roots of both species are actively taking up this vital element.

Table 2. Total chemical composition (⁺g / kg, or mg / kg) of the fine root, rhizosphere soil and bulk soil compartments in the soil profiles beneath trees.

Tree species	Sample	Horizon	+Al	+Fe	+Ca	+K	+Na	+Mg	Ba	Cu	Mn	Sr	Zn
A. falciformis	Soil	A1	12.5	7.8	0.6	2.5	0.2	1.0	61	6	36	12	15
	Rhizosphere		17.2	11.5	0.8	5.0	0.3	1.4	100	11	56	14	43
	Fine root + RP		9.2	4.2	2.4	3.1	0.5	1.2	80	17	66	37	31
	*< 3mm root		0.2	0.1	3.3	4.0	0.7	0.5	14	20	49	29	53
	Soil	B2	20.8	13.8	0.5	5.5	0.2	1.6	99	2	54	10	16
	Rhizosphere		22.6	11.3	0.6	5.9	0.3	1.7	115	5	62	12	21
	Fine root + RP		8.5	3.2	1.4	2.0	0.5	1.4	99	8	25	33	27
E. mannifera	Soil	A1	19.4	9.8	0.6	4.9	0.2	1.4	104	13	60	10	21
	Rhizosphere		20.9	17.1	0.6	5.8	0.4	1.6	204	13	883	11	35
	Fine root + RP		5.3	2.3	2.3	3.4	0.5	1.4	75	8	149	22	19
	Soil	B2	27.0	13.2	0.3	6.7	0.2	1.8	125	3	60	8	30
	Rhizosphere		29.4	24.7	0.3	7.2	0.3	2.2	200	3	89	9	32
	Fine root + RP		10.6	2.8	1.3	2.2	0.6	1.3	250	14	30	22	41
	*< 3mm root		0.8	0.3	4.0	7.0	0.8	1.2	28	18	98	28	53

*denotes previously unpublished data provided by Leonard and Field (2004); RP – rhizoplane

The results are less clear for the E. mannifera, however there is still good evidence that the fine roots of this tree were involved in actively scavenging for elements, especially Ca, K and Mg, indicated by relatively high root concentrations of the elements, but the rhizosphere dynamics are yet to be elucidated. In contrast there is relatively little AAC extractable Al or Fe in either the bulk soils or rhizosphere (Table 3), despite the high total concentrations, reflecting their abundance as structural components in soil minerals and / or that mechanisms are employed by fine roots to minimise movement of these elements through the rhizosphere. Further discussion on elemental dynamics in the rhizosphere of these co-occurring trees is given in Little et al. (2004).

Dissolution of soil materials in 1mmol L^-1 Low Molecular Weight Organic Acid solutions

Within one week of commencing the experiment there were clearly visible signs that the content of metal ions, especially Fe and Al in solution and suspension had increased. This was evidenced by a marked colour change in solution from clear to yellow-brown and an initially rapid increase in pH in the oxalic acid and malic acid treatments (Figure 1a). A much greater effort was required to filter the oxalic and malic acid solution samples as the reaction progressed, suggesting the formation of stable suspended nano-particulate insoluble complexes with clay minerals and metal ions in these treatments (van Hees et al. 1996). In contrast, the citric acid treatments appeared to show greater dissolution efficiency, with only a slight increase in insoluble complexes suspended in solution. The sum of major and trace elements measured in solution over time in the three organic acid solutions was at least an order of magnitude greater than the inorganic control. This was most apparent for the major ions, Fe and Al. The solubilisation of Al and Fe showed the following order, citric acid > oxalic acid > malic acid ~ sodium chloride control (Figure 1b and 1c). At pH environments between 4.5 and 7 Al and Fe^III are usually considered to be relatively immobile, however citric acid was particularly effective at solubilising these two elements (see Gottlein et al. 1999), with solution concentrations at day 15 of the reaction being greater than for all other elements (see Figure 1). While the concentrations of Al and Fe liberated into solution by the oxalic and malic acid anions were relatively low, they were an order of magnitude greater than for essentially all other elements analysed. Interestingly, the solubilisation of Al and Fe was greatest in the B₂ soil horizons beneath the E. mannifera tree, where there were potentially more clay and sesquioxide materials. Aluminium and Fe might be adsorbed on or complexed with soil organic matter (especially humus) in the A₁ soil horizons, especially under the A. falciformis tree, which could lead to lower concentrations solubilized in this experiment (van Hees et al. 1996).

Table 3. Ammonium Acetate (AAC) extractable cation content (⁺g / kg; or mg / kg soil) of the bulk soils and rhizosphere under adjacent Eucalyptus mannifera and Acacia falciformis trees.

Spp.	Horizon	Compartment	+Al	+Fe	+Ca	+Na	+K	+Mg	Ba	Mn	Sr	Zn
A. falciormis	A	Soil	0.04	0.03	0.4	0.08	0.08	0.1	20	9	7	5
		Rhizosphere	0.04	0.03	0.6	0.1	0.09	0.2	23	12	9	3
	B	Soil	0.04	0.02	0.2	0.06	0.05	0.1	14	2	3	3
		Rhizosphere	0.04	0.02	0.2	0.08	0.08	0.2	14	3	3	3
E. mannifera	A	Soil	0.02	0.02	0.2	0.05	0.08	0.07	18	6	2	2
	B	Soil	0.04	0.02	0.03	0.04	0.09	0.04	26	1	1	2

Dissolution of soil materials in 1mmol L^-1 Low Molecular Weight Organic Acid solutions

Within one week of commencing the experiment there were clearly visible signs that the content of metal ions, especially Fe and Al in solution and suspension had increased. This was evidenced by a marked colour change in solution from clear to yellow-brown and an initially rapid increase in pH in the oxalic acid and malic acid treatments (Figure 1a). A much greater effort was required to filter the oxalic and malic acid solution samples as the reaction progressed, suggesting the formation of stable suspended nano-particulate insoluble complexes with clay minerals and metal ions in these treatments (van Hees et al. 1996). In contrast, the citric acid treatments appeared to show greater dissolution efficiency, with only a slight increase in insoluble complexes suspended in solution. The sum of major and trace elements measured in solution over time in the three organic acid solutions was at least an order of magnitude greater than the inorganic control. This was most apparent for the major ions, Fe and Al. The solubilisation of Al and Fe showed the following order, citric acid > oxalic acid > malic acid ~ sodium chloride control (Figure 1b and 1c). At pH environments between 4.5 and 7 Al and Fe^III are usually considered to be relatively immobile, however citric acid was particularly effective at solubilising these two elements (see Gottlein et al. 1999), with solution concentrations at day 15 of the reaction being greater than for all other elements (see Figure 1). While the concentrations of Al and Fe liberated into solution by the oxalic and malic acid anions were relatively low, they were an order of magnitude greater than for essentially all other elements analysed. Interestingly, the solubilisation of Al and Fe was greatest in the B₂ soil horizons beneath the E. mannifera tree, where there were potentially more clay and sesquioxide materials. Aluminium and Fe might be adsorbed on or complexed with soil organic matter (especially humus) in the A₁ soil horizons, especially under the A. falciformis tree, which could lead to lower concentrations solubilized in this experiment (van Hees et al. 1996).

Despite the relatively high concentration of Fe and Al in the dissolution experiments, Si concentration was approximately an order of magnitude less in all the treatments. This might be an indication that most of the Fe and Al being solubilised are unlikely to come from dissolution or leaching of primary silicate minerals, but rather from secondary aluminosilicates and metal oxides (compare Figure 1b, 1c and 1d). Although organic ligands can form stable complexes with Si, the major effect of organic acids is to solubilise Fe and Al, which weakens the mineral structure, catalysing Si release to solution (see for example, Barker et al. 1997 and references therein). The general trend in affectiveness of the organic acids to solubilise Si was similar to that for Al and Fe, citrate ≥ oxalate > malate ≥ sodium chloride, with the exception of the A₁ horizon, where oxalate was the most effective. Most other elements were found in concentrations near the detection limits of the ICP-AES. Nevertheless repeatable trends in dissolution were observed. The concentrations of most elements measured were greater in the organic acid solutions compared to the inorganic control. However, in contrast to the major framework elements, the organic acids had little effect on Ca release to solution, in fact, Ca concentrations were ~ 10-20% greater in the inorganic control solutions than in the organic acid experiments. The ionic strength of Ca²⁺ is quite high compared with the Na⁺ ions in solution, and dissolution of these elements is probably quite rapid even though their base concentrations are low (Table 4). The dissolution of Ca in oxalate and malate appears to be pH dependent and solution concentrations decrease as pH increases (compare Figure 1a with 1b). This may be in part due to the formation of insoluble Ca-oxalate mineral phases (eg Welch et al. 2002). However this cannot account for Ca concentrations in citrate where the dissolution reaction appears to be buffered somewhat against pH changes. Similarly, dissolution of Sr (Figure 1f) generally followed the order, oxalate > sodium chloride ≥ malate > citrate.

Figure 1. General trends of solution concentrations, (mg / kg solution) for 5 elements and pH, when ~1g field soil samples were exposed to ~200mL 1mmol L^-1 Citric acid, Malic acid, Oxalic acid and sodium chloride (starting pH 4.5) in isolation, in controlled laboratory dissolution experiments: (a) pH, (b) aluminium (Al), (c) iron (Fe), (d) silicon (Si), (e) calcium (Ca), and (f) strontium (Sr).

Conclusions and Future Directions

Results of this study show differences in the geochemistry of fine roots, and rhizosphere verses bulk soil, and suggest increased mineral weathering in the presence of root exudates, indicating that plant roots can have large effects on soil geochemistry and element mobility. Particularly good indicators of the rhizosphere element dynamics were seen on examination of the bulk soil, rhizosphere soil and < 3 mm root compartments (Table 2 and Table 3). Failure to remove the rhizoplane soil from the roots led to an apparent lessening of this effect, though one might argue that there are mechanisms leading to a decline in concentration for elements in the rhizoplane, especially for those elements either not required by the trees, or in excess supply (Table 2 and Table 3). Additionally, the small sample size from the E. mannifera rhizosphere was a result of the sparse distribution of fine roots throughout the soil profile. Larger sample sizes will be collected in future and future root samples will be washed to remove the rhizoplane to minimise contamination issues and to maximise opportunities to examine real changes in rhizosphere soils.

The results are promising, and warrant further investigations into the rhizosphere, and root weathering in these co-occurring tree species; E. mannifera and A. falciformis. The soils are highly leached and weathered, and as a result are deficient in many biologically important elements, especially calcium. While the adjacent eucalypt and acacia take up a suite of similar nutrients, the location of uptake can vary and in some cases elements appear to be excluded from uptake. For the A. falciformis it is apparent that rhizosphere activity is greatest in the surface, organic rich horizons where nutrient elements are in much greater abundance and in forms more readily plant accessible than those usually observed in the deeper mineral horizons. Evidence is also present for uptake of elements such as Ca, Mg and Sr in the B₂ horizons beneath both trees, where these elements are more likely to occur in inorganic forms. The dissolution experiments confirm the highly weathered nature of the soils, but Al and Fe are dissolved by citric acid in concentrations much greater than for any other element, including silicon.

These results suggest that most Fe and Al are being dissolved and organic acid exudates will potentially dissolve secondary aluminosilicate minerals and metal oxides in the rhizosphere. Low levels of dissolved Ca in all acid solutions provide another strong suggestion that the Mulloon Creek soils are highly leached and weathered. More results suggesting the importance of LMWOA root exudates in mineral weathering were seen in the ability of citric acid, a common root exudate, to dissolve and mobilise Al and Fe (Figure 1) in conjunction with the apparent depletion of these elements in the rhizoplane compared with the rhizosphere and bulk soils (Table 2 and 3).

Further experimentation will be undertaken using XRD-mineralogy techniques to identify the minerals present in the soils, and to determine LMWOA content in rhizosphere soils under the co-occurring trees. A combination of broader-scale comparative and spatial investigations in rhizosphere chemistry and mineralogy for these species will contribute to a growing knowledge of how trees interact with soils in typically harsh Australian conditions.

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