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A quantitative assessment of phosphorus forms in some Australian soils.

Introduction

Australian soils are generally characterised by low phosphorus (P) concentrations by world standards, primarily because they are generally highly weathered and many are derived from sedimentary rocks, including sandstone, which are generally low in P (Beadle 1962; Holford 1997). Plant-available P in soil is an important determinant of soil fertility (Norrish and Rosser 1983); consequently, application of P fertilisers is usually required to ensure adequate crop production in Australian soils (Cornish 2009a). The long-term application of P fertilisers in southern Australia has exceeded P removal such that substantial quantities of P have accumulated in some soils (McLaughlin et al. 1991). This not only represents an inefficient and unsustainable use of P fertilisers but also increases the potential for nutrient leaching to the off-farm environment and can cause eutrophication of waterways (Sharpley et al. 1994).

Ineffectiveness of P fertilisers is due to the fixation of P by sorption, complexation, and precipitation reactions with soil constituents. Generally, 10-30% of applied fertiliser P is recovered by crops in the year following application. The remainder of P necessary for plant growth is obtained from residual inorganic P and recalcitrant forms of organic P or 'unavailable' P forms (Bolland and Gilkes 1998; Bunemann et al. 2006). Recent research has examined ways to reduce the input of soluble P fertilisers and to better use existing soil P (e.g. Conyers and Moody 2009; Cornish 2009b; Evans and Condon 2009; Guppy and McLaughlin 2009; Richardson et al. 2009). Potential strategies to increase plant-available P include the selection or modification of plants with favourable root architecture (fine roots, lots of root hairs) and effective root exploration (Richardson et al. 2009), the mutualistic association of plant roots with mycorrhizal fungi (Smith et al. 2003), and the direct application of manufactured extracellular enzymes (Conyers and Moody 2009; Evans and Condon 2009). However, the success of any technique that aims to increase the availability of soil P and implement a more sustainable approach to P management in Australian soils will rely on an understanding of the P forms (including organic P forms) that exist within the soil.

Early work on the characterisation of soil organic P used extraction and chromatographic techniques. These techniques indicated myo-inositol hexakisphosphate (phytate), a P-rich compound derived predominantly from plant seeds (Reddy et al. 1989), to be a dominant form of soil organic P. Initial investigations of soil 'phytate fractions' in three Australian soils were undertaken by Cosgrove (1962, 1963) and Cosgrove and Tate (1963), where they were able to show the presence of myo- + DL, neo-, and scyllo-inositol hexaphosphates. Cosgrove (1963) provided approximate concentrations of some inositol phosphate components, with estimates of myo-+DL-inositol hexaphosphate equating to 10-13% of total organic phosphate and scyllo-inositol hexaphosphate ranging from 2 to 3%. Williams and Anderson (1968) undertook the first comprehensive characterisation of Australian soils and focused on identifying the inositol-phosphate concentrations of 47 surface soils from eastern Australia. The concentrations varied widely (0.4-38% of total organic P) but the authors did not identify other forms of organic P. Irving and Cosgrove (1982) detected chiro-, neo-, myo-, and scyllo-inositol pentakis-and hexakisphosphates in four soils from New South Wales, the two most abundant species being the myo- and scyllo-hexakisphosphates with concentrations ranging from 12 to 167 mg P/kg. Similarly, Steward and Tate (1971) reported that inositol polyphosphates comprised 2 31% of soil organic P in four South Australian (Urrbrae) and four eastern Australian soils.

More recently, solution 3,p nuclear magnetic resonance (NMR) spectroscopy has become the most widely used technique for the speciation of soil P (McDowell et al. 2005; Turner et al. 2005; Murphy et al. 2009). However, the application of solution 31p NMR spectroscopy to Australian soils and therefore a more detailed characterisation of P forms is limited to a few studies (Smernik and Dougherty 2007; Bunemann et al. 2008b; Doolette et al. 2009), one of which suggested that phytate concentrations can be overestimated in 31p NMR analyses through mis-assignment of phytate peaks (Smernik and Dougherty 2007). This is consistent with the earlier findings of McLaughlin et al. (1988), who suggested that a major portion of organic P that accumulated in cropping soils was of microbial, rather than plant, origin.

In this study we apply solution [sup.31]P NMR spectroscopy to a diverse range of Australian soils with varying chemical and physical properties to provide a detailed characterisation and quantitative assessment of the forms of sodium hydroxide-ethylenediaminetetra-acetic acid (NaOH-EDTA) extractable P.

Materials and methods

Soil chemical and physical analysis

Eighteen topsoils were collected from New South Wales, South Australia, and Tasmania. Eight of these soils (Nowra, Casino, Collector, Coonabarabran, Wollongbar, Tocal, and Somersby A & B) were included in a previous study (Doolette et al. 2009). Of the extra 10 soils included in this study, two are from South Australia and the remaining eight comprise four pairs of soils with contrasting P fertiliser histories from Tasmania. All sites had been under their current management regime for at least 4 years. All soils were sampled, after the removal of the litter layer, to a depth of 0.10 m and air-dried.

Total soil P and inorganic and organic P contents were determined using the acid extraction and ignition methods of Saunders and Williams (1955). Soil pH was determined on 1 : 5 soil : water extracts. Organic C was determined using the method of Walkley (1947). Soils were classified according to the Australian Soil Classification (ASC) (Isbell 1996).

NaOH-EDTA extraction

Samples were ground to pass through a 2-mm sieve before extraction. Soils were extracted in triplicate using methods based on those of Cade-Menun and Preston (1996). For all but three soils, 2.0 g of soil was shaken with 40 mL of 0.25 M NaOH and 0.05 M Na2EDTA for 16 h. For three low-P soils (Somersby A & B and Coonabarabran), 6.0 g rather than 2.0 g of soil was used in the extraction to provide acceptable signal-to-noise ratios in the NMR spectra. All extracts were centrifuged (1400G) for 10 min and filtered using Whatman no. 42 filter paper. A 15-mL aliquot was immediately frozen and freeze-dried for NMR analysis. Triplicate subsamples of the supernatant were also taken to determine the total P concentrations using nitric acid digestion and subsequent analysis by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The determination of inorganic P in the NaOH-EDTA soil extracts was based on colourimetry using the molybdenum blue method of Murphy and Riley (1962). Inorganic P in the NaOH-EDTA extracts was also quantified using deconvolution of 31p NMR spectra (detailed below).

NMR analysis of NaOH-EDTA extracts

Triplicate freeze-dried NaOH-EDTA extracts for each soil were combined for NMR analysis. A 500mg subsample of each composite extract was ground, re-dissolved in 5 mL of deionised water, and centrifuged (1400G) for 20 min. The supernatant solution (3.5mL) and deuterium oxide ([D.sub.2]O, 0.3mL) were placed in a 10-mm-diameter NMR tube. Solution [sup.31]P NMR spectra were acquired at 24[degrees]C on a Varian INOVA400 NMR spectrometer (Varian, Palo Alto, CA) at a [sup.31]P frequency of 161.9 MHz. Recovery delays ranged from 10 to 30s and were set to at least five times the [T.sub.1] value of the orthophosphate resonance determined in preliminary inversion-recovery experiments for each extract (data not presented). We used a 90[degrees] pulse of 32-45 [mu]s, an acquisition time of 1.0 s, and broadband [sup.1]H decoupling. Between 3224 and 49 000 scans were acquired for each sample, depending on the P concentration of the freeze-dried extract. Chemical shifts were referenced to [beta]-glycerophosphate at 4.63 ppm according to Doolette et al. (2009). The spectra presented have a line broadening of 2 Hz.

Quantification of P species from [sup.31]P NMR spectra

The relative concentrations of P species in the NaOH-EDTA extracts were determined from [sup.31]P NMR spectra using a combination of integration and deconvolution. Orthophosphate diester (2.0 to 1.0ppm) and pyrophosphate (-4.5 to -5.5ppm) concentrations were determined using integration alone. Integration was used to determine the combined concentration of inorganic orthophosphate and orthophosphate monoester P (6.5-2.6ppm). The relative concentrations of P species giving rise to the numerous individual peaks in this region of the spectrum were quantified by spectral deconvolution, using a method similar to that of Bunemann et al. (2008b). Each spectrum was fitted with up to 15 sharp peaks as well as a broad signal that spanned most of the monoester region. The sharp peaks included those of orthophosphate, [alpha]- and [beta]-glycerophosphate, phytate, adenosine-5'-monosphosphate (AMP), scyllo-inositol phosphate, five unknown peaks in the monoester region, and two unknown peaks downfield (higher ppm values) of orthophosphate. Each peak was defined by three parameters: the chemical shift (frequency), intensity, and line width. The degrees of freedom for the fit were limited by fixing the line widths of the sharp peaks at 10 Hz (0.06 ppm), and the broad resonance at 200Hz (1.24ppm). The line width of the well-defined inorganic orthophosphate resonance was allowed to vary. The absolute concentration of each P species (including those determined using integration alone and those determined using integration and deconvolution combined) was calculated by multiplying its relative contribution to total NMR signal by the total NaOH-EDTA extractable P concentration determined by ICP-AES.

A minor correction was used in the determination of phytate and orthophosphate concentrations due to the overlap of the phytate C-2 peak with the much larger orthophosphate peak. Total phytate was calculated as 6/5 times the total concentration of the three observable resonances, and 1/5 of this value was subtracted from the total orthophosphate concentration (Doolette et al. 2009).

Spiking experiments

Spiking experiments were carried out on two soil extracts to confirm the accuracy of the spectral deconvolution technique used for quantifying P species. This enabled an independent measure of the concentration of one of the species ([beta]-glycerophosphate) using the method of Smemik and Dougherty (2007). An aqueous solution (0.1 mL) of [beta]-glycerophosphate was spiked into the Parkham +P (0.74 g/L) and Elizabeth Town control (1.4 g/L) redissolved NaOH-EDTA soil extracts (3.5 mL and 0.3 mL [D.sub.2]O). The [sup.31]P NMR spectrum of the unspiked NaOH-EDTA extract was subtracted from the [beta]-glycerophosphate-spiked NaOH-EDTA spectrum, resulting in a spectrum of the [beta]glycerophosphate spike alone. Increasing proportions of the [beta]-glycerophosphate spectrum were then subtracted from the unspiked NaOH-EDTA spectrum until the [beta]-glycerophosphate resonance was null. The lower limit of [beta]-glycerophosphate was defined by the maximum proportion of the [beta]-glycerophosphate spectrum that resulted in the appearance of a positive [beta]-glycerophosphate resonance in the unspiked NaOH-EDTA spectrum. The upper limit was defined by the minimum proportion of the [beta]-glycerophosphate spectrum that resulted in the appearance of a negative [beta]-glycerophosphate resonance in the unspiked NaOH-EDTA spectrum.

Results and discussion

The 18 Australian soils selected for this study span a wide range of soil management, climate, chemical and physical properties, and soil orders (Table 1). Mean annual temperature ranged from 15 to 23[degrees]C and mean annual precipitation from 387 to 1318 mm (www.bom.gov.au). Soil pH([H.sub.2]O) varied from moderately acidic (4.5) to moderately alkaline (8.3). Soil organic carbon ranged from 10 to 95 g/kg. Soils were used for a range of agricultural practices including pasture production (dairy and sheep), horticulture (conventional and organic), cropping, and a cropping/grazing rotation. Four pairs of soils differed in P fertilisation history, with each pair having one unfertilised soil and one with a known history of inorganic P fertilisation (Togari, Parkham, Elliot Research Station, and Elizabeth Town).

Table 2 shows the variation in total P and organic P concentrations of the whole soils. Total soil P concentration across all soils ranged from 239 to 15111mg/kg, of which inorganic P comprised 25-90% (84-12 559 mg/kg) and organic P 10-75% (72-2552mg/kg). Comparisons of the total, inorganic, and organic P fractions of each soil are shown in Fig. 1a.

In all cases, the P-treated soils (+P) had a higher total P concentration, of which the majority of the extra P was inorganic P, except Togari, where the +P treatment was higher in organic P. This is consistent with the results of McLaughlin et al. (1988), which showed, using isotope tracer experiments, that fertiliser was not quickly incorporated into organic P forms.

Extractability of inorganic and organic P

Total NaOH-EDTA extractable P ranged from 149 to 6222 mg/kg (Table 2) representing an extraction efficiency of 21 89%, with an average of 64% (Fig. 1b). Comparable extraction efficiencies have been reported for other [sup.31]P NMR studies. Dou et al. (2009) extracted 67-97% of total P from soils manured for 8-10 years, while Turner et al. (2003a) reported considerably lower extraction efficiencies (12-45%) for a range of cropped soils. Plots of whole soil and NaOH-EDTA extractable inorganic and organic P are shown in Fig. 2a, b. These exclude data for the Nowra sample (total P, 15 111 mg/kg; total extractable P, 6222 mg/kg) as they were several times higher than for any of the other soils presented in Fig. 2a.

The 1 : 1 line shown in Fig. 2a, b indicates the potential maximum (100%) extraction efficiency. It is clear that the average extraction efficiency of inorganic P was greater (82%) than for organic P (47%). This implies that, on average, around half of the organic P in these soils is not extractable and therefore cannot be identified by solution 3~p NMR analysis. However, it is likely that hydrolysis of organic P in the alkaline extract results in an overestimation of the extraction efficiency of inorganic P, consequently underestimating the extraction efficiency of organic P. Indeed, for four soils (Tocal, Wollongbar, Coonabarabran, and Collector) the apparent extraction efficiency of inorganic P was >100%. It should also be noted that the ashing technique used to determine the organic P content of the whole soils can be unreliable (Condron et al. 1990b), and this represents a second possible source of bias in the reported inorganic and organic P extraction efficiencies.

The Kadina soil had a total P extraction efficiency of only 21% (Fig. 1b), with the extraction efficiency of inorganic P particularly low (16%) compared with the other soils. The low extractability may be due to the calcareous nature of the Kadina soil, as high soil pH and the presence of calcium minerals has potential to complex P, rendering it hard to extract from soil (Cole et al. 1953; Celi et al. 2000). Across all soils, there was no noticeable relationship between P extractability and soil pH. The only other sample with a high soil pH was Nowra, and it too had a comparatively low P extraction efficiency (41%). Differences in extraction efficiency are also likely to have affected inorganic and organic P concentrations of the P-treated and P-untreated paired soil samples. The extraction efficiency of organic P tended to be greater in the treated soils than the untreated soils.

Assessment of the reliability of spectral deconvolution

Overlap of resonances in the 2-7ppm region of [sup.31]P NMR spectra of soil extracts, the region that encompasses the orthophosphate peak and numerous monoester peaks, hampers quantitative assessment of these P types. This problem can be overcome by using spectral deconvolution, which involves a numerical least-squares fit of the spectrum as the sum of multiple peaks of standard shape. Spectral deconvolution has been used in several [sup.31]P NMR soil studies (Turner et al. 2003d; McDowell and Stewart 2005; McDowell et al. 2005). Here, we use a modified version (see Materials and methods for description) that has been used only once before (Bunemann et al. 2008b). The main modification is the inclusion of a broad signal (3.95-5.45 ppm) in the fit. In a study that compared extracts of 'real' soils and 'model' soils (mixtures of pure sand and clay that were incubated with pure organic substrates, e.g. cellulose, starch, and glucose), Bunemann et al. (2008b) observed that the extracts of the model soils contained only sharp resonances. These resonances could be assigned to specific, simple organic P molecules. In contrast, the spectra of the real soils contained a broad signal in addition to the sharp resonances. This modified technique is yet to be applied to a variety of soils with considerably different inorganic and organic P concentrations.

The reliability of our methods for quantifying P species was tested by: (i) comparing orthophosphate concentrations determined using NMR with those determined colourimetrically, and (ii) using the spiking method of Smernik and Dougherty (2007) to provide an independent measure of the concentration of the monoester compound [beta]-glycerophosphate in two soil extracts.

Figure 3 shows the close relationship between NaOH-EDTA extractable orthophosphate concentrations measured using spectral deconvolution and those measured using molybdate colourimetry. Both methods gave very similar values (i.e. the line of regression is close to the 1 : 1 line in Fig. 3), but concentrations determined by colourimetry were generally slightly higher (on average 8%) than those determined using spectral deconvolution.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Spiking experiments were carried out on Elizabeth Town control and Parkham +P extracts. We calculated the [beta]-glycerophosphate concentration in the Elizabeth Town control sample to be 22 [+ or -] 1.9 mg P/kg using the spiking method and 21 mg P/kg using spectral deconvolution. Spiking the Parkham +P NaOH-EDTA soil extract revealed a [beta]-glycerophosphate concentration of 5 [+ or -] 1.8 mg P/kg, which was slightly less than that determined using deconvolution (10 mg P/kg). This suggests that deconvolution may overestimate the concentration of species with low abundances; however, the practical implication of such errors is relatively small.

Quantitative analysis and interpretation of [sup.31]P NMR spectra of NaOH-EDTA soil extracts

Spectra of eight of the soil extracts are not presented here as they have been reported elsewhere (Doolette et al. 2009) but have not been quantitatively analysed. For two of the soils (Kadina and Crystal Brook), we were unable to obtain an adequate [sup.31]P NMR spectrum. As discussed, the extraction efficiency was very low for the Kadina soil and this resulted in a low concentration of P in the NaOH-EDTA extract. This was also the case, but to a lesser extent, for the Crystal Brook soil, which suffered from the combination of a relatively low P concentration and relatively low extraction efficiency. However, it should be noted that we were able to obtain adequate NMR spectra on other soil extracts with similar, or even lower, P contents (e.g. Togari and Collector). This shows that the difficulties encountered in the analysis of these two soil extracts are not entirely explained by their low P content. The Kadina and Crystal Brook soils were also the only alkaline soils analysed, other than the Nowra soil, which had an extremely high P content (Tables 1 and 2). The potential link between difficulties in NMR analysis and high soil pH requires further investigation.

[FIGURE 3 OMITTED]

The [sup.31]P NMR spectra of the other NaOH-EDTA soil extracts, which consist of four pairs of control and P-treated soils, contain signals in four diagnostic chemical shift regions, orthophosphate (6.5-5.3 ppm), orthophosphate monesters (5.3-2.6 ppm), orthophosphate diesters (2.0 to -1.0 ppm), and pyrophosphate (-4.5 to -5.5 ppm) (Fig. 4). An expansion of the orthophosphate and orthophosphate monoester regions is shown in Fig. 5. Equivalent spectra for the other soils can be found in Doolette et al. (2009). The two regions that comprised the majority of signal across all soils were orthophosphate and orthophosphate monoester, together equating to 94-98% of total NaOH-EDTA extractable P. Orthophosphate diesters and pyrophosphate were minor components, comprising only 2-6% of total NaOH-EDTA extractable P. When comparing the control and treated soils, the only noticeable differences were in the orthophosphate region. The differences between the control and treated soils in the orthophosphate monoester, orthophosphate diester, and pyrophosphate chemical shift regions were relatively small. However, it might be expected that with continued P fertiliser application, soil organic P could increase (Condron et al. 1985; McLaughlin et al. 1988).

[FIGURE 4 OMITTED]

Quantitative distributions of P forms determined for all soil extracts are presented in Table 3. The percentage distribution of each P species, excluding orthophosphate in relation to total non-orthophosphate P, is presented in Fig. 1c.

Orthophosphate (5.69-5.78 ppm) produced the most intense resonance in each spectrum and is the most abundant P species detected in every soil extract. Orthophosphate concentrations ranged from 86 to 5622 mg/kg, equivalent to 37-90% of total NaOH-EDTA extractable P (Table 3).

Orthophosphate monoesters represented the next-largest P class and accounted for 7 55% of total NaOH-EDTA extractable P. The majority of the orthophosphate monoester was assigned to a broad signal, centred at 3.95-5.45 ppm. This signal is present in all spectra but is more easily discerned in the spectra that contained less-intense sharp resonances, e.g. Parkham, Elliot, and Elizabeth Town (Fig. 5). The broad monoester signal represented 31-355mgP/kg (equivalent to 2-39% of total extractable P or 32-86% of total orthophosphate monoesters) (Table 3). The absolute P concentration and proportion of organic or total extractable P of the broad resonance did not appear to be associated with any of the climatic, chemical, or physical properties measured, but more soils need to be studied to further examine this possibility.

[FIGURE 5 OMITTED]

Most previous attempts at quantifying monoester P species using deconvolution analysis of [sup.31]P NMR spectra of soil extracts have not included a broad peak in the fit, and consequently, signal from this broad peak is assigned to species that give rise to

the sharper peaks in the spectrum. More specifically, we have previously tested this method of deconvolution and shown that, by not accounting for the broad peak, phytate concentrations were overestimated by 54% (Doolette et al. 2010). Dougherty et al. (2007) suggested that this broad signal is due to large, complex, humic molecules, as opposed to smaller, specific monoester P compounds that produce sharp resonances. This explanation is supported by Bunemann et al. (2008b), who found this broad signal to be absent in model soils that have had insufficient time for extensive humification to have occurred. Earlier evidence for the dominance of humic P can be seen in the work of Anderson and Hance (1963), who found evidence of phytate and other organic phosphate molecules bound in a complex or compound that contained appreciable amounts of carbohydrates and proteins. Omotoso and Wild (1970) also considered that phytate existed in an organic complex combined with other organic P compounds.

The concentrations of humic organic P reported here are consistent with those determined by different methods in previous studies of volcanic and alpine soils. The humic fractionation of volcanic soils showed that P associated with humic and fulvic acids accounted for 32-75% and 51-68% of organic P, respectively (Borie et al. 1989; Escudey et al. 2001; Borie and Rubio 2003). In alpine soils, 52-90% of alkali-extractable organic P was associated with humic acids with phosphate monoesters the dominant P species (Makarov et al. 1997). Furthermore, using [sup.3t]P NMR, He et al. (2006) examined the spectral and chemical characteristics of humic substances and also found phosphate monoesters to be the dominant P species. More importantly, they were unable to identify individual monoester P compounds, as the spectra had no sharp resonances but instead a broad signal in the monoester region. We conclude that failing to include the broad signal in the deconvolution procedure would result in the overestimation of the concentration of species that give rise to the sharp monoester resonances.

The remainder of orthophosphate monoester P was assigned to the [sup.3t]P NMR signal contained within the sharp resonances. Phytate (4.17-4.28, 4.35-4.40, 4.71-4.82ppm), [alpha]-glycerophosphate (4.95-4.98 ppm), and [beta]-glycerophosphate (4.59-4.64ppm) were the most prominent sharp resonances. Phytate was present in all soil extracts except for the Elliot control and +P samples. Concentrations in the other soils ranged from 5 to 86 mg P/kg (equivalent to up to 9% of total NaOH-EDTA extractable P) (Table 3). Phytate concentrations averaged just 3% of total NaOH-EDTA extractable P and 13% of NaOH-EDTA extractable organic P. Both or- and [beta]-glycerophosphate were present in all [sup.3t]P NMR spectra but were only a very minor component in some extracts; [beta]-glycerophosphate comprised <1% of total NaOH-EDTA extractable P in the Wollongbar extract, [alpha]-glycerophosphate comprised <1% in the Coonabarabran extracts, and combined, [alpha]- and [beta]-glycerophosphate comprised <1% in both the Somersby A and B samples. In the remaining spectra, [alpha]- and [beta]-glycerophosphate resonances were more prominent; [alpha]-glycerophopshate concentrations ranged from 3 to 80mgP/kg (1-5% total NaOH-EDTA extracable P) and [beta]-glycerophosphate ranged from 7 to 80 mg P/kg (1-5% total NaOH-EDTA extractable P) (Table 3). In total, [alpha]- and [beta]-glycerophosphate comprised up to a maximum of 10% of extractable P.

Phytate in soils primarily originates from the decay of crop seeds and fruits (Lott et al. 2000). Relatively high phytate concentrations in soils can result from the addition of animal manures, e.g. poultry and swine manure, as phytate passes though the digestive tracts of these animals since they lack the ability to break it down (Selinger et al. 1996). This may explain the relatively high concentration of phytate in the Tocal soil extract, which had received poultry manure.

The remainder of the soils are predominantly dairy pastures where the strongest monoester resonances have been assigned to [alpha]- and [beta]-glycerophosphate, which are most likely products of the alkaline hydrolysis of phospholipids (Doolette et al. 2009) rather than being actual soil constituents themselves. They are likely to be derived mainly from microbial biomass, as Bunemann et al. (2008a) showed that [alpha]- and [beta]-glycerophosphate were the major forms of monoester P identified in NaOH-EDTA extracts of bacterial and fungal cultures. Microbial biomass is often quite high in dairying soils due to high organic carbon and moisture regimes (Burkitt et al. 2007).

Other orthophosphate monoester compounds present in the soil extracts were detected in much smaller quantities. For example, scyllo-inositol hexakisphosphate (3.85-3.91 ppm), a common stereoisomer of phytate, was present in all soil extracts at concentrations ranging from 2 to 20 mg P/kg or <1-4% of total extractable P (Table 3). Despite the widespread occurrence of scyllo-inositol hexakisphosphate in soils (McKercher and Anderson 1968; Turner et al. 2005; Turner 2007; Murphy et al. 2009) its origins are still unknown. Furthermore, there are conflicting reports regarding the bioavailability or susceptibility of scyllo-inositol hexakisphosphate to hydrolytic attack by phytase (Cosgrove 1966, 1970; Turner et al. 2005).

Trace amounts of AMP ([less than or equal to] 2% extractable P; 4.47-4.52 ppm) were present in all soil extracts except Somersby B. This resonance is also likely to include other ribonucleotides (RNA monomers) and deoxyribonucleotides (DNA monomers), as they resonate at very similar chemical shifts (Turner et al. 2003b; Bunemann et al. 2008a; Doolette et al. 2009). However, due to the instability of RNA it is not possible to conclude whether the nucleotides are present in the soil or are products of alkaline hydrolysis during the sample extraction/preparation stages.

Five other well-resolved orthophosphate monoester P resonances (5.23-5.24, 5.10-5.11, 4.064.17, 3.04ppm) were identified and are labelled as unknown compounds a-e in Table 3. In total, these unknown compounds accounted for [less than or equal to] 3% of total extractable P, but in most cases <1%. Two further unidentified resonances between 6.34 and 6.58ppm (comprising <1% of total extractable P) have previously been assigned as aromatic orthophosphate diesters in the NaOH extracts of Scottish mineral soils by Bedrock et al. (1994). Aromatic orthophosphate diesters are generally thought to be associated with cold and wet environments. They have been identified in grassland soils from Russia and New Zealand (Amelung et al. 2001; McDowell 2003) and pasture soils from New Zealand, England, and Wales (Turner et al. 2003c; McDowell et al. 2005). However, little is known about the possible structures or origins of these compounds, and the chemical shifts of aromatic orthophosphate diesters can vary widely (Turner et al. 2003b; Makarov et al. 2004). It has been proposed that it is more likely that these signals actually represent stereo-isomers of inositol phosphates (Turner et al. 2005).

Orthophosphate diesters represent the next largest class of P compounds and were present in all soil extracts, with concentrations ranging from 4 to 87 mg/kg or 1-5% of total [sup.31]P NMR signal (Table 3). Orthophosphate diesters comprised only DNA with other potential diester P forms such as phospholipids and RNA absent from our [sup.31]P NMR spectra. Phospholipids and RNA are rarely detected in soils (Makarov et al. 2002; Turner et al. 2007), despite their rate of addition to soils being much greater than that of other organic P forms (Anderson 1980). This is likely governed by the low stability of these compounds in the soil and in alkaline solutions (Makarov et al. 2002; Turner et al. 2003b) and the low aqueous solubility of phospholipids' (Doolette et al. 2009).

Low concentrations of diester P reported in some soils have been attributed to the preferential mineralisation of diester P compared with other more stable organic P forms (Hawkes et al. 1984; Condron et al. 1985; Condron et al. 1990a) and are considered to be linked to nutrient turnover, with diester P representing a labile pool of soil organic P (Turner et al. 2003c). In particular, cell-free RNA and DNA are highly susceptible to degradation due to the presence of active nucleases in soils (Paul and Cark 1996). However, extracellular DNA can strongly sorb to sand, clay minerals, and humic substances, providing protection from degradation by nucleases compared with free DNA in solution (Pietramellara et al. 2009). This may be one of the factors contributing to the presence of DNA in preference to other diester P forms.

It should be noted that the concentrations of diester P in this study (as in other 31p NMR studies) are likely to be underestimated due to its degradation in alkaline solution during extraction. Turner et al. (2003b) showed RNA to be highly unstable in NaOH-EDTA, completely degrading to orthophosphate monoesters within 24 h, and this is consistent with the findings of Makarov et al. (2002).

Phospholipids are also susceptible to hydrolysis, although the rate of degradation varies between phospholipids. One of the most common phospholipids, phosphatidyl choline (lecithin), completely degrades in NaOH-EDTA within 24 h (Turner et al. 2003b) to [alpha]- and [beta]-glycerophosphate (Doolette et al. 2009). In contrast, DNA is more stable (Turner et al. 2003b) and can remain intact in alkaline solutions for at least 24 h (Makarov et al. 2002). Low concentrations of diester P may also be due to a combination of the low aqueous solubility of phospholipids in NaOH-EDTA (Doolette et al. 2009) and low organic P extractability. On average only 47% of total organic P was extracted, which suggests some forms of diester P that are not extracted from the soil.

Pyrophosphate was present in all soil extracts at concentrations ranging from 1 to 62 mg/kg (up to 5% of total NaOH-EDTA extractable P). No higher polyphosphates, which produce resonances in the range -18 to -22 ppm, were detected in any of the soil extracts. Pyrophosphate concentrations reported here are similar to those reported elsewhere: 1-7% of total extractable P in pasture soils (Turner et al. 2003c), 0.5-4.3% in cropping soils (Turner et al. 2003a), <1-4.85% in manure-treated soils (Dou et al. 2009), and on average 3% in grassland soils (Murphy et al. 2009). Inorganic polyphosphates, of which pyrophosphate is the simplest form, are believed to accumulate in microbial biomass as a P storage form. The factors controlling their abundance remain unclear (Blanchar and Hossner 1969; Turner et al. 2003a).

The results presented here suggest that the largest portion of organic P in some Australian soils is bound in complex humic P molecules and not in smaller specific compounds such as phytate. This not only challenges our understanding of soil organic P cycling but has implications for the way in which methods are developed to increase P availability in the soil profile. In terms of sustainable P management, it is likely that a slightly new approach is required, especially in low-input and organic agricultural systems where recalcitrant or poorly available forms of P such as phytate are seen as potentially available forms of organic P (Guppy and McLaughlin 2009). There is likely to be great promise for plant-based approaches to increase P efficiency in agricultural systems (Richardson et al. 2009). However, some of the most effective strategies for increased P availability may not be through techniques that target specific soil organic P compounds, such as the genetic modification of plants with increased phytase activity (George et al. 2004, 2005) or the direct application of manufactured extracellular enzymes to increase mineralisation of inositol P (Conyers and Moody 2009). Instead, greater success could come from techniques isolating genes in plants that aid or alter root development (Grierson et al. 2001; Williamson et al. 2001), thereby increasing the chances of P uptake, or focusing on the exudation of organic anions with a known capability of mobilising P bound in humic-metal complexes (Gerke 1993). It is doubtful that a single strategy that increases the availability of organic P would be applicable to all soils. However, we suggest that the development of strategies to increase uptake of P by plants and crops should not be focused only on increasing the availability of P contained in small, supposedly recalcitrant compounds, such as phytate. Consideration should also be given to the availability of P in larger, complex molecules.

Conclusions

We have used an improved method for assessing the speciation of soil P using solution [sup.31]P NMR and spectral deconvolution. This method not only provided a more detailed characterisation of organic P forms in Australian soils than has hitherto been undertaken, but also provided a more accurate estimation of the quantity of these forms by minimising the potential for overestimation of the P concentrations of simple orthophosphate monoesters. The [sup.31]P NMR spectra of 18 NaOH-EDTA soil extracts revealed that a large proportion of organic P is present in large, complex molecules such as humic acids. Phytate was the most abundant identifiable organic P compound, but only comprised a maximum of 10% of total extractable P. In most cases, humic organic P represented more P than all of the small discrete P molecules combined. These findings have implications for attempts to increase the availability of stabilised forms of organic P. Consideration should be given to further investigation of the presence and availability of P in large, complex molecules, rather than solely developing methods to increase the availability of individual P compounds (such as phytate) that are present in the soils at much lower concentrations.

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Manuscript received 21 April 2010, accepted 2 September 2010

A. L. Doolette (A,C), R.J. Smernik (A), and W. J. Dougherty (B)

(A) School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, SA 5064, Australia. (B) Department of Primary Industries, Industry and Investment New South Wales, Locked Bag 4, Richmond, NSW 2753, Australia. (C) Corresponding author. Email: ashlea.doolette@adelaide.edu.au

10.1071/SR10092
Table 1. Selected physical, climatic, and chemical properties of the
18 Australian soils used in this study

MAT, Mean annual temperature; MAP, mean annual precipitation; OC,
organic carbon, pH in water (1:5 soil/water ratio)

Soil                     Land use/treatment

Nowra (A)                Dairy pasture + effluent
Casino (A)               Dairy pasture
Collector (A)            Sheep pasture
Coonabarabran (A)        Horticulture
Wollongbar (A)           Dairy pasture
Tocal (A)                Dairy pasture + poultry litter
Somersby A (A)           Organic vegetable
Somersby B (A)           Conventional vegetable
Kadina                   Cropping
Crystal Brook            Cropping/grazing rotation
Togari control           Dairy pasture
Togari +P                Dairy pasture
Parkham control          Dairy pasture
Parkham +P               Dairy pasture
Elliot control           Dairy pasture
Elliot +P                Dairy pasture
Elizabeth Town control   Dairy pasture
Elizabeth Town +P        Dairy pasture

Soil                     MAT ([degrees]C)   MAP (mm)   pH    OC (g/kg)

Nowra (A)                       16            1110     8.1      38
Casino (A)                      20            1096     6.5      42
Collector (A)                   14            644      5.0      27
Coonabarabran (A)               16            748      4.5      16
Wollongbar (A)                  19            1793     5.2      38
Tocal (A)                       18            922      6.1      43
Somersby A (A)                  17            1318     6.7      11
Somersby B (A)                  17            1318     5.9      10
Kadina                          23            387      8.3      19
Crystal Brook                   23            471      7.9      17
Togari control                  15            1243     5.5      87
Togari +P                       15            1243     5.3      87
Parkham control                 16            947      5.7      28
Parkham +P                      16            947      5.9      28
Elliot control                  16            1188     5.2      95
Elliot +P                       16            1188     5.3      95
Elizabeth Town control          16            947      5.4      60
Elizabeth Town +P               16            947      5.3      60

Soil                     Soil texture      Soil Classif. (B)

Nowra (A)                Clay loam         Chromosol
Casino (A)               Silty clay        Vertosol
Collector (A)            Loam              Chromosol
Coonabarabran (A)        Loamy sand        Podosol
Wollongbar (A)           Clay              Ferrosol
Tocal (A)                Loamy sand        Chromosol
Somersby A (A)           Sandy loam        Tenosol
Somersby B (A)           Loamy sand        Tenosol
Kadina                   Loamy sand        Calcarosol
Crystal Brook            Sandy loam        Calcarosol
Togari control           Loamy sand        Hydrosol
Togari +P                Loamy sand        Hydrosol
Parkham control          Clay loam/loam    Vertosol
Parkham +P               Clay loam/loam    Vertosol
Elliot control           Silty loam        Ferrosol
Elliot +P                Silty loam        Ferrosol
Elizabeth Town control   Silty clay loam   Ferrosol
Elizabeth Town +P        Silty clay loam   Ferrosol

(A) Data reproduced from Doolette et al. (2009).

(B) Australian Soil Classification (Isbell 1996).

Table 2. Phosphorus characteristics (mg/kg) of the whole soil and
NaOH-EDTA extractable P fractions

Values in parentheses are percentages of total NaOH-EDTA extractable P

Soil                     Whole soil P
                                             NaOH-EDTA
                         Total   Organic   extractable P

Nowra (A)                15111      2552     6222 (41)
Casino (A)                1576       423     1184 (75)
Collector (A)              257       172      166 (64)
Coonabarabran (A)          239       144      149 (63)
Wollongbar (A)            1113       650      882 (79)
Tocal (A)                 1214       564      887 (73)
Somersby A (A)             743        90      542 (73)
Somersby B (A)             702        72      627 (89)
Kadina                     844       200      178 (21)
Crystal Brook              571       214      335 (59)
Togari control             465       220      340 (73)
Togari +P                  665       490      336 (51)
Parkham control            627       470      355 (60)
Parkham +P                1386       465      950 (69)
Elliot control            1106       816      677 (61)
Elliot +P                 2526       776     1912 (76)
Elizabeth Town control    1910      1094     1066 (56)
Elizabeth Town + P        2293       963     1443 (63)

(A) Data reproduced from Doolette et al. (2009).

Table 3. Concentrations (mg-kg) of phosphorus forms and their
percentages (in parentheses) of NaOH-EDTA extractable total P as
detected by 31P NMR and quantified using spectral deconvolution

Ortho-P, Orthophosphate; GP, glycerophosphate; AMP,
adenosine-5'-monophosphate; s-IHP, scyllo-inositol hexakisphosphate;
unknown monoesters (a-e) are the sum of five individual resonances
with the following chemical shift ranges (ppm): (a) 5.23-5.24, (b)
5.10-5.11, (c) 4.06-4.17, (d) 3.04, (e) 2.89; Pyro-P, pyrophosphate;
detected n.d., not

Soil                     Unknown P    Ortho-P    [alpha]-GP
Chemical shift           6.53-6.58   5.69-5.78   4.95-4.98
ranges (ppm):            6.34-6.36

Nowra                      0 (0)     5622 (90)     80 (1)
Casino                     0 (0)      904 (76)     22 (2)
Collector                  0 (0)       86 (52)      8 (5)
Coonabarabran              1 (<1)      94 (63)      3 (2)
Wollongbar                 0 (0)      455 (52)      6 (<1)
Tocal                      2 (<1)     595 (67)     17 (2)
Somersby A                 0 (0)      460 (85)      2 (<1)
Somersby B                 0 (0)      509 (81)      1 (<1)
Kadina                     n.d.       104 (58)      n.d.
Crystal Brook              n.d.       249 (74)      n.d.
Togari control             3 (<1)     134 (39)     16 (5)
Togari +P                  0 (0)      176 (52)     13 (4)
Parkham control            4 (1)      132 (37)     12 (4)
Parkham +P                 4 (<1)     739 (78)     14 (2)
Elliot control             6 (<1)     310 (46)     17 (3)
Elliot +P                  4 (<1)    1574 (82)     18 (1)
Elizabeth Town control     9 (1)      520 (49)     37 (4)
Elizabeth Town +P          6 (<1)     939 (65)     34 (2)

Soil                      Humic P     Phytate    [beta]-GP      AMP
Chemical shift           3.95-5.45   4.17-4.28   4.59-4.64   4.47-4.52
ranges (ppm):                        4.35-4.40
                                     4.71-4.82

Nowra                    143 (2)      86 (1)      80 (1)      41 (Q)
Casino                   143 (12)     26 (2)      23 (2)      15 (1)
Collector                 31 (19)     14 (8)       8 (5)       3 (2)
Coonabarabran             37 (24)      5 (3)       1 (<1)      1 (<1)
Wollongbar               274 (31)     55 (6)      10 (1)       7 (<1)
Tocal                    131 (15)     65 (7)      15 (2)      11 (1)
Somersby A                43 (8)      15 (3)       3 (<1)      2 (<1)
Somersby B                77 (12)     19 (3)       4 (<1)      0 (0)
Kadina                     n.d.        n.d.        n.d.        n.d.
Crystal Brook              n.d.        n.d.        n.d.        n.d.
Togari control           108 (32)     29 (9)      12 (4)       8 (2)
Togari +P                 90 (27)     13 (4)      12 (4)       4 (1)
Parkham control          135 (38)     10 (3)       7 (2)       7 (2)
Parkham +P               130 (14)     14 (1)      10 (1)      10 (1)
Elliot control           263 (39)      0 (0)      10 (2)      10 (2)
Elliot +P                210 (11)      0 (0)      18 (1)      19 (1)
Elizabeth Town control   355 (33)     23 (2)      21 (2)      19 (2)
Elizabeth Town +P        323 (22)     24 (2)      17 (1)      13 (1)

Soil                       s-IHP      Unknown     Diester
Chemical shift           3.85-3.91   monoesters   2 to -1
ranges (ppm):                           a-e

Nowra                      5 (<1)      11 (<1)     87 (1)
Casino                    11 (1)        6 (<1)     14 (l)
Collector                  5 (3)        2 (1)       8 (1)
Coonabarabran              2 (1)      0.4 (<1)      5 (1)
Wollongbar                14 (2)        6 (<1)     39 (2)
Tocal                     20 (2)        6 (<1)     14 (1)
Somersby A                 4 (<1)       0 (0)       8 (1)
Somersby B                 5 (<1)       0 (0)       8 (1)
Kadina                     n.d.         n.d.       n.d.
Crystal Brook              n.d.         n.d.       n.d.
Togari control             12(4)        0 (0)       6 (3)
Togari +P                  7 (2)        0 (0)       4 (5)
Parkham control            8 (2)        9 (3)      16 (4)
Parkham +P                 9 (1)        2 (<1)      5 (1)
Elliot control             5 (<1)       0 (0)      31 (4)
Elliot +P                  9 (<1)       0 (0)      28 (2)
Elizabeth Town control    14 (1)       27 (3)      19 (2)
Elizabeth Town +P         13 (1)       22 (2)      28 (2)

Soil                        Pyro-P
Chemical shift           -4.5 to -5.5
ranges (ppm):

Nowra                       62 (1)
Casino                      11 (1)
Collector                    1 (1)
Coonabarabran                2 (1)
Wollongbar                  14 (2)
Tocal                       10 (1)
Somersby A                   5 (1)
Somersby B                   5 (1)
Kadina                       n.d.
Crystal Brook                n.d.
Togari control              12 (3)
Togari +P                   17 (5)
Parkham control             16 (4)
Parkham +P                  13 (1)
Elliot control              24 (4)
Elliot +P                   33 (2)
Elizabeth Town control      20 (2)
Elizabeth Town +P           25 (2)
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Author:Doolette, A.L.; Smernik, R.J.; Dougherty, W.J.
Publication:Soil Research
Article Type:Report
Geographic Code:8AUST
Date:Mar 1, 2011
Words:9511
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