Sequential fractionation and characterisation (
31
P-NMR) of
phosphorus-amended soils in
Banksia integrifolia
(L.f.) woodland
and adjacent pasture
M.T. Taranto
a, M.A. Adams
b,*, P.J. Polglase
caSchool of Botany, The University of Melbourne, Parkville, Vic., 3052, Australia
bDepartment of Botany, The University of Western Australia, Nedlands, WA, 6009, Australia
cCSIRO, Division of Forestry, PO Box 4008, Queen Victoria Terrace, Canberra, ACT, 2600, Australia
Accepted 2 August 1999
Abstract
Fractionation and characterisation of soil P by sequential extraction or by 31P-NMR spectroscopy were used to assess a
variety of soil characteristics, including the availability of P to plants, the transformations of native and added phosphorus and the eects of plant growth on pools and distributions of P. We used a modi®cation of the technique of Hedley et al. (1982) and Tiessen et al. (1984) [Hedley, M.J., Stewart, J.W.B. and Chauhan, B.S., 1982. Changes in inorganic and organic soil P fraction induced by cultivation practices and by laboratory incubations. Soil Science Society of America Journal, 46, 970±976 and Tiessen, H., Stewart, J.W.B., Cole, C.V., 1984. Pathways of P transformations in soils of diering pedogenesis. Soil Science Society of America Journal, 48, 854±858] to assess the stability and transformations of sources of P (rock phosphate, Fe-phytate, RNA) added to soil under a stand of Banksia integrifoliaand under an adjacent pasture. Pasture soils contained more P than soils under Banksia, but the distribution of P among fractions was similar for both soils. The dierent sources of P added to soils were recovered in largely discrete fractions. Most of the P in RNA added toBanksiasoil was mineralized and leached, as PO43ÿ, within 2 months, whereas additions of Fe-phytate and rock phosphate produced little PO43ÿ. In the pasture soil, RNA
was mineralized at a slower rate, but root growth (and presumably uptake of P) was rapid and less P was leached. Generally, the amount of P extracted using Chelex 20 cation-exchange resin was less than one third of that extracted using our modi®cation of the methods of Hedley et al.(1982) and Tiessen et al. (1984). Results from31P-NMR spectroscopy showed that most (>090%) of the P compounds extracted by the resin in all treatments were monoesters.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Phosphorus;Banksia; Sequential fractionation;31P-NMR
1. Introduction
Potter et al. (1991) noted the widespread application of methods to fractionate P (e.g. Chang and Jackson, 1957; Bowman and Cole, 1978a; Hedley et al., 1982) in studies of P availability and cycling in soil-plant sys-tems. The principal bene®t of fractionation methods is that they permit ``a complete account or budget of the
P forms present'' (Potter et al., 1991). Furthermore, the growth of plants is often well correlated with con-centrations in soil of the more soluble fractions of or-ganic-P (Po) and inorganic-P (Pi) (Turner and
Lambert, 1985). De®ciencies of fractionation methods are: (i) that they may not be the best possible means with which to estimate some P fractions (e.g. mi-crobial, total, organic) (Potter et al., 1991), (ii) that they may not discriminate between fractions of P that dier in biological importance and (iii) that they may not yield truly discrete fractions, as P can transfer between pools depending on extraction conditions (e.g.
0038-0717/00/$ - see front matter#2000 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 8 - 0 7 1 7 ( 9 9 ) 0 0 1 3 8 - 8
www.elsevier.com/locate/soilbio
* Corresponding author. Fax: +61-8-9380-1001.
Aung Khin and Leeper, 1960) and some extractants may cause partial hydrolysis of Po to Pi (Adams and
Byrne, 1989).
Characterisation of P in soil by31P-NMR is a useful alternative to sequential extraction procedures, but the method also requires that forms of P not be altered greatly by the chemical extractant used (Adams and Byrne, 1989). Identifying various functional groups (e.g. phosphomonoesters versus phosphodiesters) is the principal means by which 31P-NMR characterises dierent fractions of P. In addition to identifying some speci®c classes of organic P in soils [e.g. phosphonates (Newman and Tate, 1980), teichoic acid (Condron et al., 1990)], the results from NMR studies generally complement and support conclusions based on sequen-tial extraction studies. For example, studies using sequential extraction (a series of chemical extractions) and 31P-NMR methodologies both indicate that die-ster-P is more labile than monoedie-ster-P and, hence, more easily mineralized. NMR studies have shown that diester-P was mineralized more readily than monoester-P following changes in land use (Condron et al., 1990) or during incubation of sludge-amended soils (Hinedi et al., 1988) and that much of the `labile' organic P in eucalypt forest soils was in the diester form (Adams, 1990). Bowman and Cole (1978a) had previously demonstrated that diester-P (as RNA) could be recovered in a labile fraction using a chemical extraction (0.5 M NaHCO3, pH 8.5) that is the ®rst
step in the sequential method. There have been few studies that have compared characterisation by 31 P-NMR with chemical fractionation.
In many plant communities (e.g. forests, rangelands, heathlands) to which fertilizers have not been applied, the availability of P depends, in part, on the rate at which P is cycled through plant residues and soil or-ganic matter. A variety of crop (Hoand et al., 1989), pasture (Schwab et al., 1983) and native plants pro-duce root exudates (organic acids, predominantly citric acid; Grierson, 1992) that increase the solubilisation and mineralization of soil P (Marschner et al., 1987). For example, the lateral roots of Banksia integrifolia
dierentiate to produce clusters of rootlets of limited growth (or `proteoid roots') (Purnell, 1960) that exude considerable quantities of citric acid that solubilises soil phosphates (Grierson and Attiwill, 1989; P.F. Grierson, unpublished Ph.D. thesis, University of Melbourne, 1990). The production and function of specialised root structures may be an evolutionary ad-aptation of plants to grow in soils with a low content of P, such as deep coastal sands. Conversely, most grasses in pasture communities do not have such highly specialised root systems. It is, therefore, reason-able to suppose that Banksia and pasture communities dier in how they access various sources of soil P.
Our objective was to compare, in the ®eld, the
eects of roots in two contrasting ecosystems (Banksia
woodland versus a pasture) on transformations and mobility of various P compounds added to soil. Comparisons were made between the eects of the proteoid roots of an Australian native tree, Banksia integrifolia and the roots of exotic species in an adja-cent pasture. A sequential extraction procedure was used to identify temporal changes in various P frac-tions, which were compared with P extracted by a mild chelating resin and characterised by31P-NMR.
2. Materials and methods
2.1. Site description
The study area was at Main Creek, Point Nepean National Park, Victoria, Australia, where two sites were chosen for an intensive study. The ®rst site, a
Banksia woodland, is dominated by Banksia integrifo-lia (var. integrifolia; coast banksia), which grows to a maximum height of about 10 m. B. integrifolia grows on coastal sands of low fertility in south-eastern Australia forming a closed canopy and open unders-tory. B. integrifolia, similar to many other members of
the Proteaceae, Casuarinaceae, Fabaceae and
Mimosaceae, have proteoid roots. The understory of the Banksia woodland includes Leptospermum laeviga-tum, Acacia sophorae and Leucopogon parvi¯orus. The second site was a pasture adjacent to the Banksia
woodland and consists predominantly of exotic species, including Plantago lanceolata, Holcus lanatus, Briza maxima, Anthoxanthum odoratum and Cynosorus echi-natus and fewer native species, such as Themeda trian-dra,Cynoglossum australeandIsolepis nodosa.
Main Creek has a temperate climate with warm summers and wet winters. The mean daily maximum temperature is 178C and the mean daily minimum is 118C; daily maximum temperatures frequently exceed 258C in summer, do not exceed 158C in winter and range between 15 and 208C in autumn. Mean annual rainfall is 750 mm, about half of which falls during winter when the mean monthly rainfall is between 120 and 150 mm. During autumn, rainfall is generally between 40 and 60 mm monthÿ1. The area of Main Creek has developed on stabilized dunes. The soil under both the Banksia woodland and pasture was a `uniform' pro®le of neutral to moderately acidic sand (Northcote, 1979, Uc1.11). A slightly more organic layer at the surface (0±20 cm) grades rapidly into the sand.
2.2. Field experiment
with one of three P-containing compounds, or these soils not amended. Amendments were chosen to rep-resent a few of the P compounds prep-resent in soil (e.g. Dalal, 1977) that vary in form (inorganic or organic) and solubility in water: RNA (prepared from Torula Yeast purchased from Sigma-Aldrich), Fe-phytate [pre-pared in the laboratory by the method of Greaves and Webley (1969)] and rock phosphate (obtained commer-cially as North Carolina rock phosphate, Florida 75% BPI phosphate rock). RNA degrades rapidly to poly-nucleotide diesters that are labile in soil (Bowman and Cole, 1978b; Harrison, 1982), whereas the monoester, Fe-phytate (or Fe-myoinositol hexaphosphate), is re-sistant to microbial hydrolysis in soil (Greaves and Webley, 1969) and rock phosphate (an inorganic P salt of Ca) is insoluble in water.
Soil (0±10 cm) was excavated from each site, sieved (<5 mm) and amended by shaking soil and P sources together in large polypropylene bags. Sources of P were applied at a calculated rate of 100 mg P gÿ1
soil.
Organic sources were contaminated with small
amounts of soluble inorganic P. For calculation of the recovery of added P, the following ®gures derived from digestion of the substrates in H2SO4/H2O2 at
3208C (total P and corrected for soluble inorganic P in the case of organic substrates) were used: RNA, 100
mg P gÿ1soil; Fe-phytate, 82mg P gÿ1soil; rock-phos-phate, 108mg P gÿ1soil.
Transformations of added P compounds were moni-tored in situ by analyzing soil that was contained in corers for various periods. Root ``in-growth'' and root ``exclusion'' corers were used to test for eects of roots and root type on transformations and stability of P. However, dierences between corer type were not sig-ni®cant (P< 0.05) and therefore, data from in-growth and root-exclusion corers were combined. Root-exclu-sion corers were made from polyvinyl chloride (PVC) pipe (10 cm long, 7 cm internal dia) and were perfo-rated with 8 holes, each approximately 7mm dia. Corers of this design have been used in other in situ studies of nutrient transformations in soil (e.g. Adams and Attiwill 1986; Adams et al., 1989) and allow tem-perature and moisture of the enclosed soil to equili-brate with that of the surrounding bulk soil. In-growth corers of the same size were constructed from plastic
garden mesh (10 mm10 mm mesh apertures) to
allow penetration and colonisation of repacked soil by roots.
Corers were inserted into prepared 10 cm deep holes. Polypropylene mesh bags (approx. 80% open area, 7 cm7 cm) containing 4 g of anion exchange
resin (Dowex-1, 80 mesh) were placed at the bottom of corers containing amended or nonamended (control) soil (280 g soil corerÿ1
). The resin bags captured PO43ÿ
leached from the soil core, which was expected to be a signi®cant pathway for the loss of P, as these soils
con-sisted predominantly of coarse sand and, hence, have a small capacity to sorb P.
Two experimental plots (3.3 m3.3 m, 200 m
apart) were established in the pasture adjacent to the
Banksia woodland. Corers were placed randomly at intersecting points of a square grid such that P treat-ments were evenly distributed within the two plots. In the Banksia woodland, soil microclimate, chemical characteristics of soil and root distribution varied with increasing distance from the trees. Consequently, ®ve
B. integrifolia trees were selected, radial grids were constructed around the base of each tree and corers were randomly placed at intersecting points of the grid.
At each site, 180 corers were installed on 13 April
1991. One hundred and twenty corers (4
treatments3 collections10 replicates) were used
for P fractionation studies and 60 in-growth corers were used to assess root growth. Ten replicate corers of each treatment were collected in early June (2-month ®eld exposure), August (4-(2-month ®eld ex-posure) and December (8-month ®eld exex-posure). Five replicate corers were collected for assessment of root growth after the 2- and 4-month ®eld exposures. Soil samples collected in June and August were used for sequential fractionation and those collected in August and December were used for characterisation using
31
P-NMR (replicate samples were combined for Chelex 20 extraction and NMR analysis). At each sampling time, corers and enclosed soil were removed from the plots, placed into sealed plastic bags and stored at 48C before chemical analysis. Resin bags were removed from the base of cores with forceps, brushed to remove excess soil, placed individually in sealed containers and stored at 48C. Before analysis, each resin bag was rinsed with glass-distilled H2O (also used in all other
laboratory procedures) to remove attached soil and root material and shaken intermittently for 1 h in 20 ml of 0.5 M HCl (Gibson et al., 1985). The eluate was centrifuged at 3000 rev minÿ1 for 10 min. Inorganic-P (PO43ÿ) recovered from the resin bags will be referred
to as `leached phosphorus'.
2.3. Laboratory analysis
Root material was sorted from in-growth cores by washing the soil through a 2 mm sieve. The proteoid roots of B. integrifolia were separated from the other root material. Roots were dried at 808C, allowed to cool in a desiccator and weighed.
the soil solution, some Pi held on exchange complexes
and labile (easily mineralized) Po substrates (Bowman
and Cole, 1978b). A separate sample of soil (1 g) was fumigated for 24 h with CHCl3 and then extracted
with NaHCO3, as described above. Extracts from
fumigated and nonfumigated soils were analysed for Pi
and for total P (Pt). The dierence in Pt extracted
from fumigated and nonfumigated soils is commonly described as an index of the P contained in microbial cells (Hedley et al., 1982).
Sequential extractions were continued on the resi-dues of nonfumigated samples only. Moderately labile Po (retained by Fe- and Al-oxides) was extracted with
0.1 M NaOH (1:30 soil:solution, 16 h shaking time) and after centrifugation, the residue was ultrasonicated (10 s at 75 W) in 0.1 M NaOH to remove resistant Po
retained at internal surfaces of soil aggregates (Tiessen et al., 1984). The residue was then digested at 3208C in 2 ml of concentrated H2SO4 catalyzed by 0.5 ml
ad-ditions of 30% H2O2, to extract stable Po as well as
precipitated Pi (Hedley et al., 1982). Each extract was
analyzed for Pi and Pt and the dierence between Pi
and Ptwas assumed to be the amount of Po.
Sequential extraction and fractionation of P using
NaHCO3, with or without CHCl3 and NaOH, are
methods that have been used by numerous workers (e.g. Bowman and Cole, 1978a; Hedley et al., 1982; McLaughlin et al., 1988). The ®nal extraction by oxi-dation and acid digestion has more commonly been separated into two steps: an acid extraction to remove precipitated-P (mainly apatite-type minerals) (e.g. Williams et al., 1967; Potter et al., 1991) and then a complete acid digestion to remove chemically stable forms of P and relatively insoluble Pi (Hedley et al.,
1982). These steps were combined in our study, owing to the small amounts of P in these soils (Grierson, 1990, loc. cit.).
Before measurement of Pi, soil extracts were
deco-lorized with activated charcoal (pre-washed Darco G-60) that removes only background color and no P. The Pi in all solutions was measured by either
auto-mated colorimetry (Technicon Instruments, 1977) or manual colorimetric methods (Murphy and Riley (1962) as modi®ed by Watanabe and Olsen (1965)). The Pt in soil extracts was measured by evaporating
(1208C) an aliquot (10 ml) to dryness and digesting the residual crust in concentrated H2SO4 (as described
above) after adding 0.5 ml of saturated MgCl2(1 g
dis-solved in 0.6 ml H2O) to prevent losses of P by
volatil-isation (Brookes and Powlson, 1981). The Pi in the
supernatant was measured with a Technicon Auto-Analyzer II (Technicon Instruments, 1977).
Soil extracts for 31P-NMR analysis were prepared using Chelex 20 cation-exchange resin, as described by Adams and Byrne (1989) but omitting the dilute acid pre-extraction step. In brief, 6 samples of 10 g (fresh weight) of soil were each shaken for 2 h with 60 g of resin and 200 ml of distilled H2O after which the
`Chelex extracts' were separated by centrifugation. The pH of the extracts ranged between 11.2 and 11.5. The extracts were combined and lyophilised. After being redissolved in 50 ml of H2O, the extract was
centri-fuged (18,000 rev minÿ1, 45 min) and concentrated by rotary evaporation at 40±458C to a volume of 6 to 10 ml (e.g. Newman and Tate, 1980). This procedure extracts labile forms of P, which are of interest in stu-dies of cycling and availability to plants of P and which might be expected to be similar to those forms
extracted by NaHCO3 or low concentrations of
NaOH.
The recording of spectra, the identi®cation of peaks and the quanti®cation of fractions were as described by Adams and Byrne (1989); Adams (1990). Brie¯y, to samples consisting of approximately 2 ml of
concen-Table 1
Summary of analysis of variance (ANOVA) of data from 2- and 4-month ®eld exposures. The signi®cancea of phosphorus (P) amendments
(RNA, Fe-phytate, rock phosphate) and corer type (root in-growth or root-exclusion) on P fractionsbwere tested by 2-way ANOVA Site Field exposure period (months) P fractions of soil
leached NaHCO3 NaOH NaOH/ultrasonicated residual sum
Phosphorus amendment
Banksia 2
4
Pasture 2
4 ns
Corer type
Banksia 2 ns ns ns ns
4 ns ns ns ns ns
Pasture 2 ns ns ns ns ns ns
4 ns ns ns ns ns ns
aLevels for signi®cance:P< 0.05,P< 0.01,P< 0.001, ns not signi®cant.
b
trated Chelex extract 0.5 ml of D2O was added to
pro-vide the ®eld-frequency lock signal. 31P-NMR spectra were recorded on a Bruker AM-300 instrument with an aquisition time of 0.82 s and a pulse angle of 318. Dierent P compounds were identi®ed by their chemi-cal shift relative to external orthophosphoric acid (85%). Chemical shift assignments were con®rmed by the addition of known P compounds to the concen-trated extracts.
Moist soil samples were used in all sequential and
31
P-NMR fractionations and the results were verted to an oven-dried weight basis. The water con-tent of soils was determined gravimetrically after drying the soils at 1058C.
The results from the sequential fractionation of soil are presented as unadjusted means. A Tukey-Kramer test was used to evaluate the ranges of Pi and Po in
each fraction. For each of the 2- and 4-month ®eld
ex-posures, 2-way analysis of variance (ANOVA) was used to test for the main eects of treatment (RNA, Fe-phytate, rock phosphate) and type of corer (root in-growth, root-exclusion) on concentrations of Pt in
each of the sequential fractions. The main eects of community (Banksia, pasture), root type (proteoid, nonproteoid in Banksia only) and treatment (RNA, Fe-phytate, rock phosphate) on growth of roots (mass inside in-growth corers) were tested by ANOVA fol-lowed by separation of treatment means (within root types) with the Tukey-Kramer test.
3. Results
The eect of treatment (form of added P) and corer type (root in-growth or root-exclusion) on the concen-tration of P in discrete fractions was generally
signi®-Table 2
Mass of root tissue (mg coreÿ1) from cores exposed in situ for 2 or 4 months. Values are means of ®ve replicates and means in the same column
followed by the same letter or by no letter are not signi®cantly dierent (P< 0.05). Values in parentheses are the standard deviation of the mean
Phosphorus amendment Community
Banksia woodland Pasture
nonproteoid proteoid total total
2 4 2 4 2 4 2 4
Control 40 (40) 60 (20) 10 (0) 70 (30) 50 (40) 130 (40) 250 (70) 330a,b(110)
RNA 90 (90) 130 (20) 20 (20) 60 (30) 110 (90) 190 (60) 360 (200) 460b(210)
Fe-phytate 50 (40) 130 (50) 20 (10) 50 (40) 70 (40) 180 (40) 300 (80) 210a(70)
Rock phosphate 40 (20) 150 (120) 20 (20) 30 (20) 60 (40) 180 (40) 310 (50) 600c(150)
Table 3
P concentration (mg gÿ1
dry weight soil) in dierent fractions of soil from theBanksiacommunity. Within each column, means followed by the same letter are not signi®cantly dierent (P< 0.05)
Treatment Leached Soil P fraction Suma NaHCO3 NaOH NaOH/ultrasonicated Residue Pi Pt
PO43ÿ Pi Po Pi Po Pi Po Piand Po 2 monthsb
Control 1.8a 2.2a 13.8a 8.4a 13.3a 3.7b 2.9a 11.5a 14.3a 55.7a
RNA 86.4b 19.4c 20.1b 24.8b 7.6a 2.0a 4.9a 13.3a 44.1c 91.8b
Fe-phytate 0.3a 2.7a 24.2c 8.4a 60.0b 2.0a 9.4b 12.8a 13.0a 119.4c
Rock phosphate 3.8a 6.6b 16.1a 14.3a 17.3a 3.2ab 4.6a 98.7b 24.1b 160.8d 4 months
Control 0.3a 0.8a 13.9a 1.8a 20.8a 0.0a 7.5a 12.1a 2.6a 56.9a
RNA 71.5b 19.6b 21.6b 3.2a 25.6a 0.3a 16.2b 12.6a 23.0b 99.0b
Fe-phytate 0.7a 0.0a 27.9c 0.0a 65.1b 0.2a 9.9ab 14.3a 0.2a 117.4c Rock phosphate 6.3a 6.1a 16.5ab 1.7a 26.2a 0.0a 13.3ab 85.1b 7.8a 148.8d
a
Sum of extractable Pi=sum of Piin fractions excluding leached P (PO4
3ÿ). Sum of extractable P
t=sum of Pi and Po in fractions excluding
lea-ched P (PO4 3ÿ).
b
cant (P< 0.05) for both soil type and ®eld exposure period (Table 1). The eect of fumigation on the pool of P extractable from soils with NaHCO3 (also:
Grierson, P.F., 1990. Ph.D. thesis, Unversity of Melbourne) resulted in no signi®cant (P< 0.05) dier-ences and hence, these data are not presented.
Root growth (mass) in the pasture community was greater than that in theBanksiacommunity during the 2- and 4-month in situ studies. During the ®rst 2 months, root growth in the pasture was between 2-and 5-fold greater than in the Banksia woodland for all treatments (Table 2). In the following 2 months, root growth in the pasture was up to 3-fold greater for all treatments except in soil amended with Fe-phytate. Signi®cant dierences were only evident between treat-ments (P amendtreat-ments) after 4 months in the pasture soil. Root growth (proteoid and non-proteoid) in the
Banksia woodland during the ®rst 2 months was between 50 and 110 mg coreÿ1
, similar to that in the following 2 months (Table 2). Proteoid roots com-prised 18 to 33% of total root mass after 2 months and this proportion increased 2.7-fold in the control soils and 1.7-fold in the RNA-treated soils in the fol-lowing 2 months. After 8 months, root growth in both
Banksia and pasture sites followed the same trends as after 2 and 4 months (data not presented).
Summation of the individual P fractions shows that the nonamended (control) pasture soil contained about 20 mg gÿ1
dry weight of soil more P than the nona-mended Banksia soil (Tables 3 and 4). However, irre-spective of vegetation type or ®eld exposure period, there was a similar trend in the distribution of P among fractions in nonamended soil. Moderately labile
P compounds (NaOH fraction) constituted the largest fraction (37 to 40% of the total P extracted from con-trol soils), followed by labile P (NaHCO3; 23 to 29%),
stable and precipitated P (residual fraction soluble only in H2SO4/H2O2 digestion; 20 to 22%) and
resist-ant P (NaOH/ultrasonication; 12 to 19%).
Table 4
P concentration (mg gÿ1dry weight soil) in dierent fractions of soil from the Pasture community. Within each column, means followed by the
same letter are not signi®cantly dierent (P< 0.05)
Treatment Leached Soil P fraction Suma NaHCO3 NaOH NaOH/ultrasonicated Residue Pi Pt
PO43ÿ Pi Po Pi Po Pi Po Piand Po 2 monthsb
Control 0.0a 1.4a 15.1a 11.4a 15.1a 4.4a 9.0a 15.9a 17.2ab 72.4a
RNA 0.1b 7.5b 62.3b 10.3a 22.6b 3.8a 8.7a 16.4a 21.5b 136.5b
Fe-phytate 0.02a 1.4a 21.1a 10.1a 65.3c 4.4a 12.4b 17.2a 15.9a 131.9b
Rock phosphate 0.02a 3.0a 17.5a 11.3a 15.8a 4.1a 6.9a 81.5b 18.3ab 140.1b 4 months
Control 5.0a 1.5a 18.4a 2.5a 28.5a 4.9b 11.1a 16.2a 8.8a 83.0a
RNA 25.1c 8.0b 46.3b 2.7a 32.6a 0.0a 15.8b 17.4a 10.7d 122.9b
Fe-phytate 8.1a 0.9a 28.7a 3.3a 58.3b 0.0a 16.5b 17.4a 4.2b 125.1b
Rock phosphate 13.0b 4.4a 16.9a 2.3a 27.3a 0.0a 13.8b 67.0b 6.7c 131.6b a
Sum of extractable Pi=sum of Piin fractions excluding leached-P (PO4
3ÿ). Sum of extractable P
t=sum of Pi and Po in fractions excluding
leached-P (PO4 3ÿ).
b
Values for 2-months ®eld exposures are means of 10 replicates, values for 4-months ®eld exposures are means of 6 replicates.
Fig. 1. Recovery (%) of added phosphorus (RNA, 100mg P gÿ1soil;
Fe-phytate, 82mg P gÿ1soil; rock-phosphate, 108
mg P gÿ1soil) in
dierent sequential fractions from (top) Banksia soil and (bottom) Pasture soil, exposed in situ for 4 months. Recovery was calculated from amounts of P added (see above) and amounts of Pi plus Poin
At the end of the ®rst 2 months, the amount of P (as PO43ÿ) leached from Banksia soils amended with
RNA (86.4mg P gÿ1
dry weight of soil) was more than one order of magnitude greater than that leached from soil amended with other P sources (up to 4 mg P gÿ1
, Table 3). Negligible amounts of P were leached from pasture soils (a maximum of 0.1mg P gÿ1, Table 4). A further 2 months ®eld exposure produced no signi®-cant increase in the amount of P leached from the
Banksia soil but increased leaching from the pasture soil. More P was leached from pasture soil amended with RNA (25 mg P gÿ1, Table 4) than with other forms of P.
The recovery (in sequential fractions and from resin bags) of added P varied signi®cantly (P< 0.05) between sites (Banksia versus pasture) and among treatments (P< 0.05) and with duration of ®eld ex-posure. Fig. 1 illustrates the general trends in recovery using the 4-month data. As with other samplings, less P was recovered from the pasture soil (53±60% of that added) than from the Banksia soil (74±113 %). In
Banksia soil amended with rock phosphate, by far the largest portion of the recovered P was in the residual fraction (Fig. 1, top) as it was in the corresponding pasture soil (Fig. 1, bottom). In contrast, in both
Banksia and pasture soil amended with RNA, most of the recovered P was in the NaHCO3 fraction or was
leached and collected in the resin bags (Fig. 1). P in Fe-phytate was recovered mostly in the NaOH fraction in both soils.
The ratio of Pi-to-Po changed between 2 and 4
months. First, the ratio of the sum of all fraction Pi:
sum of all fraction Ptwas less (by 45 to 98%) after 4
months than after 2 months for all treatments in both
Banksia woodland and pasture sites (Tables 3 and 4). Secondly, the reduction in the proportion of Pi was
always greater in the Banksia woodland than in the pasture for all treatments.
Control soils and soils amended with Fe-phytate or RNA were extracted using Chelex 20 resin for 31 P-NMR analysis. The classes of P compounds present were the same as those noted in previous NMR studies and a maximum of less than 50mg of P gÿ1dry weight soil was extracted (results not presented). Monoester-P was predominant in all extracts, even in those from soils amended with RNA. Concentrations of Chelex-extractable monoester P were about 9mg gÿ1soil after 4 months ®eld exposure of control soils from either the
Banksia woodland or the pasture. At the same sampling, concentrations inBanksia soil amended with either Fe-phytate or RNA were about 45mg P gÿ1
and then decreased to about 39 mg P gÿ1
after 8 months. Concentrations of Chelex-extractable monoester P in pasture soil after 4 months were 37mg gÿ1
for the Fe-phytate treatment and 30 mg gÿ1 for the RNA treat-ment. After 8 months, these concentrations increased
to 43mg gÿ1
for the Fe-phytate treatment and to 40mg gÿ1
for the RNA treatment. Concentrations of PO43ÿ
in Chelex extracts were generally R1 mg P gÿ1
, except in soils amended with Fe-phytate where they reached about 6 mg P gÿ1
after 8 months. Compared with sequential extraction, Chelex extraction was inecient; little or no inorganic P was extracted by the Chelex resin and extraction of organic P was generally between 20 and 50% of that extracted by the sequen-tial procedure. Only in the Banksia soil amended with RNA did extraction with Chelex resin account for most (95%) of the P extracted by the ®rst two steps of the sequential procedure.
4. Discussion
Growth of Banksia roots is seasonal, often with a pronounced winter±spring peak and proteoid roots can provide a large proportion of the total mass of ®ne roots. These specialized roots produce exudates with enzymic (e.g. phosphatase) and chelating (e.g. or-ganic acids) properties (Grierson and Attiwill, 1989; Grierson, 1990 (loc.cit.), 1992). Few cluster roots grew in the in-growth cores amended with P Ð a result that was in contrast to numerous reports of enhanced growth of cluster roots in zones of greater P avail-ability (e.g. Lamont, 1972; 1973) and may be a conse-quence of the generally slow root growth of Banksia. In comparison, growth of pasture roots was rapid (Table 2). The greater rate of mineralization of RNA in soil underBanksia compared with soil under pasture (Tables 3 and 4) cannot be attributed solely to root exudates. It is more likely a result of greater concen-trations of organic acids and phosphatase activity and possibly greater microbial activity in the litter and soil under Banksia (Grierson, 1990, loc.cit.). The low rate of root development inBanksiaapparently reduced the potential for plant uptake and, hence, most (090%) of the RNA-P was leached within the ®rst 2-months (Fig. 1, Table 3). In contrast, RNA-P added to pasture soils was leached less rapidly (Fig. 1, Table 4), root devel-opment and growth were rapid (Table 2) and little P was leached within the ®rst 2 months. Although con-siderable P (25mg P gÿ1) was leached from the pasture soil between 2 and 4 months, the amount was about 30% of that leached underBanksiain the same period. The poorer recovery of added P from the pasture soil (050±60% of that added, Fig. 1) may be partly attrib-uted to root uptake (not measured) of P.
or humic acids (Crecchio and Stotzky, 1998) and are among the more soluble fractions of Po in soil
(Adams, 1990). For example, Bowman and Cole (1978b) found that RNA was completely mineralized within 18 d after being added to a sandy-loam soil. After 2 months in the pasture soil in our study, it appeared that RNA was somewhat slower to minera-lize, as 47% of the added P was recovered in the Po
fraction of the NaHCO3 extract. However, much of
the RNA was probably degraded, but without conco-mitant mineralization of the P, as 31P-NMR analysis revealed that most of the compounds extracted by the Chelex 20 resin were monoester-P. Extraction of P using Chelex 20 resin is a mild procedure that, in con-trast to more severe extractants, has a limited eect on the inherent composition of organic compounds of P (Adams and Byrne, 1989).
In comparison with diester-P, monoester com-pounds, such as phytic acid, are more stable and ac-cumulate in soil (Dalal, 1977). More than 57% of the Fe-phytate in our study was recovered in the ®rst extraction with 0.1 M NaOH (not ultrasonicated) and 23% was extracted by 0.5 M NaHCO3. In comparison,
Bowman and Cole (1978a) were unable to recover phy-tates in 0.5 M NaOH, NaHCO3,or 1.0 M H2SO4. The
texture of the soils in the study by Bowman and Cole (1978a) ranged from a silty clay to a silty loam and the authors suggested that phytates were rendered re-sistant through cationic bonds to clay minerals. The soils in our study were sands to loamy sands (1.4% C, 0 to 10 cm depth) where organic matter dominates ionic sorption and exchange. Organic matter strongly binds organic P, particularly phytates (McKercher and Anderson, 1989), but not as strongly as clays. Added Fe-phytate was, by comparison with other P com-pounds, relatively stable in the sandy soils of our study and changed little in concentration between 4 and 8 months.
The data presented here provide some con®rmation of the capacity of sequential fractionation procedures to discriminate among dierent forms of P (Bowman and Cole 1978a, 1978b) and illustrate the eects of vegetation type on mobilisation and immobilisation of sources of P. Some native plant communities (e.g. those dominated byBanksia spp.) apparently have the capacity to mobilize a variety of added sources of P in excess of their requirements for growth.
Acknowledgements
We thank all members of the BioEcosystems
Analytical Unit (BEAUT) group for their help, par-ticularly Joanne Iser and Dr Pauline Grierson. MAA acknowledges support from the Australian Research Council.
References
Adams, M.A., 1990. 31P-NMR identi®cation of phosphorus
com-pounds in neutral extracts of Mountain ash (Eucalyptus regnans
F. Muell.) soils. Soil Biology & Biochemistry 22, 419±421. Adams, M.A., Attiwill, P.M., 1986. Nutrient cycling and nitrogen
mineralization in eucalypt forests of south-eastern Australia. II. Indices of nitrogen mineralization. Plant and Soil 92, 341±362. Adams, M.A., Byrne, L.T., 1989.31P-NMR analysis of P compounds
in extracts of surface soils from selected karri (Eucalyptus diversi-colorF. Muell.) forests. Soil Biology & Biochemistry 21, 523±528. Adams, M.A., Polglase, P.J., Attiwill, P.M., Weston, C.J., 1989. In situ studies of nitrogen mineralization and uptake in forest soils; some comments on methodology. Soil Biology & Biochemistry 21, 423±429.
Aung Khin, Leeper, G.W., 1960. Modi®cations in Chang and Jackson's procedure for fractionating soil phosphorus. Agrochimica 4, 246±254.
Beever, R.E., Burns, D.J.W., 1980. Phosphorus uptake, storage and utilisation by fungi. Advances in Botanical Research 8, 127±219. Bowman, R.A., Cole, C.V., 1978a. An exploratory method for
frac-tionation of organic P from grassland soils. Soil Science 125, 95± 101.
Bowman, R.A., Cole, C.V., 1978b. Transformations of organic sub-strates in soils evaluated by NaHCO3 extraction. Soil Science
125, 49±54.
Brookes, P.C., Powlson, D.S., 1981. Preventing P losses during per-chloric acid digestion of sodium bicarbonate soil extracts. Journal of the Science of Food and Agriculture 32, 671±674.
Chang, S.C., Jackson, M.L., 1957. Fractionation of soil phosphorus. Soil Science 84, 133±144.
Condron, L.M., Frossard, E., Tiessen, H., Newman, R.H., Stewart, J.W.B., 1990. Chemical nature of organic P in cultivated and uncultivated soils under dierent environmental conditions. Journal of Soil Science 41, 41±50.
Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: eect on transformation of Bacillus subtilis and resistance to DNase. Soil Biology & Biochemistry 30, 1061±1067.
Dalal, R.C., 1977. Soil organic phosphorus. Advances in Agronomy 29, 83±117.
Gibson, D.J., Colquhoun, I.A., Greig-Smith, P., 1985. A new method for measuring nutrient supply rates in soils using ion-exchange resins. In: Fitter, A.H., Atkinson, D., Read, D.J., Usher, M.B. (Eds.), Ecological Interaction in Soil. Blackwell Scienti®c Publications, Oxford.
Greaves, M.P., Webley, D.M., 1969. The hydrolysis of myoinositol hexaphosphate by soil microorganisms. Soil Biology & Biochemistry 1, 37±43.
Grierson, P.F., 1992. Organic acids in the rhizosphere of Banksia integrifoliaL.f. Plant and Soil 144, 259±265.
Grierson, P.F., Attiwill, P.M., 1989. Chemical characteristics of the proteoid root mat ofBanksia integrifoliaL.f. Australian Journal of Botany 37, 137±143.
Harrison, A.F., 1982.32P-method to compare rates of mineralisation of labile organic P to woodland soils. Soil Biology & Biochemistry 14, 337±341.
Hedley, M.J., Stewart, J.W.B., Chauhan, B.S., 1982. Changes in inorganic and organic soil P fraction induced by cultivation prac-tices and by laboratory incubations. Soil Science Society of America Journal 46, 970±976.
Hinedi, Z.R., Chang, A.C., Lee, R.W.K., 1988. Mineralisation of P in sludge-amended soils monitored by phosphorus-31-nuclear magnetic resonance spectroscopy. Soil Science Society of America Journal 52, 1593±1596.
organic acids as a response to P-starvation. Plant and Soil 113, 161±165.
Lamont, B.B., 1972. The eect of soil nutrients on the production of proteoid roots by Hakea species. Australian Journal of Botany 20, 27±40.
Lamont, B.B., 1973. Factors aecting the distribution of proteoid roots within the root systems of two Hakea species. Australian Journal of Botany 21, 165±187.
Marschner, H., Romheld, V., Cakmak, I., 1987. Root-induced changes of nutrient availability in the rhizosphere. Journal of Plant Nutrition 10, 1175±1184.
McKercher, R.B., Anderson, G., 1989. Organic phosphate sorption by neutral and basic soils. Communications in Soil Science and Plant Analysis 20, 723±732.
McLaughlin, M.J., Alston, A.M., Martin, J.K., 1988. Phosphorus cycling in wheat-pasture rotations. I. The source of phosphorus taken up by wheat. Australian Journal of Soil Research 26, 323± 331.
Murphy, J., Riley, J.P., 1962. A modi®ed single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31±36.
Newman, R.H., Tate, K.R., 1980. Soil P characterisation by 31P nuclear magnetic resonance. Communications in Soil Science and Plant Analysis 11, 835±842.
Northcote, K.H., 1979. A Factual Key for the Recognition of Australian Soils, fourth ed. Rellim Technical Publications, Adelaide.
Potter, R.L., Jordan, C.F., Guedes, R.M., Batmanian, G.J., Han,
X.G., 1991. Assessment of a P fractionation method for soils: problems for further investigation. Agricultural, Ecosystems and Environment 34, 453±463.
Purnell, H.M., 1960. Studies of the family Proteaceae. I. Anatomy and morphology of the roots of some Victorian species. Australian Journal of Botany 8, 38±50.
Schwab, S.M., Menge, J.A., Leonard, R.T., 1983. Quantitative and qualitative eects of P on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation. Plant Physiology 73, 761±765.
Technicon Instruments, 1977. Individual/simultaneous determination of nitrogen and/or phosphorus in BD acid digests. Industrial Method No. 329-74. W./B. Technicon Industrial System, Tarry town, NY.
Tiessen, H., Stewart, J.W.B., Cole, C.V., 1984. Pathways of P trans-formations in soils of diering pedogenesis. Soil Science Society of America Journal 48, 854±858.
Turner, J., Lambert, M.J., 1985. Soil P forms and related tree growth in a long-term Pinus radiata phosphate fertilizer trial. Communications in Soil Science and Plant Analysis 16, 275± 288.
Watanabe, F.S., Olsen, S.R., 1965. Test of an ascorbic acid method for determining P in water and NaHCO3 extracts. Soil Science
Society of America Proceedings 29, 677±678.
