Sequential fractionation and characterisation (31P-NMR) of phosphorus-amended soils in Banksia integrifolia (L.f.) woodland and adjacent pasture

Soil Biology & Biochemistry 32 (2000) 169±177

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Sequential fractionation and characterisation (31P-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 c a School of Botany, The University of Melbourne, Parkville, Vic., 3052, Australia Department of Botany, The University of Western Australia, Nedlands, WA, 6009, Australia c CSIRO, Division of Forestry, PO Box 4008, Queen Victoria Terrace, Canberra, ACT, 2600, Australia b

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 e€ects 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 di€ering 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 integrifolia and 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 di€erent sources of P added to soils were recovered in largely discrete fractions. Most of the P in RNA added to Banksia soil was mineralized and leached, as 3ÿ PO3ÿ 4 , within 2 months, whereas additions of Fe-phytate and rock phosphate produced little PO4 . 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 from 31P-NMR spectroscopy showed that most (> 0 90%) 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;

31

P-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 systems. The principal bene®t of fractionation methods is that they permit ``a complete account or budget of the

* Corresponding author. Fax: +61-8-9380-1001. E-mail address: [email protected] (M.A. Adams).

P forms present'' (Potter et al., 1991). Furthermore, the growth of plants is often well correlated with concentrations in soil of the more soluble fractions of organic-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. microbial, total, organic) (Potter et al., 1991), (ii) that they may not discriminate between fractions of P that di€er 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

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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 by 31P-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 di€erent 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 sequential extraction studies. For example, studies using sequential extraction (a series of chemical extractions) and 31P-NMR methodologies both indicate that diester-P is more labile than monoester-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 31PNMR 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 organic matter. A variety of crop (Ho‚and et al., 1989), pasture (Schwab et al., 1983) and native plants produce 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 di€erentiate 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 adaptation 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, reasonable to suppose that Banksia and pasture communities di€er in how they access various sources of soil P. Our objective was to compare, in the ®eld, the

e€ects 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 e€ects of the proteoid roots of an Australian native tree, Banksia integrifolia and the roots of exotic species in an adjacent pasture. A sequential extraction procedure was used to identify temporal changes in various P fractions, which were compared with P extracted by a mild chelating resin and characterised by 31P-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 integrifolia (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 understory. 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 laevigatum, 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 echinatus and fewer native species, such as Themeda triandra, Cynoglossum australe and Isolepis 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 In the in situ study we examined the transformations and mobility of P in Banksia and pasture soil amended

M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177

with one of three P-containing compounds, or these soils not amended. Amendments were chosen to represent a few of the P compounds present 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 [prepared in the laboratory by the method of Greaves and Webley (1969)] and rock phosphate (obtained commercially as North Carolina rock phosphate, Florida 75% BPI phosphate rock). RNA degrades rapidly to polynucleotide diesters that are labile in soil (Bowman and Cole, 1978b; Harrison, 1982), whereas the monoester, Fe-phytate (or Fe-myoinositol hexaphosphate), is resistant 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 gÿ1 soil; Fe-phytate, 82 mg P gÿ1 soil; rock-phosphate, 108 mg P gÿ1 soil. Transformations of added P compounds were monitored 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 e€ects of roots and root type on transformations and stability of P. However, di€erences between corer type were not signi®cant (P < 0.05) and therefore, data from in-growth and root-exclusion corers were combined. Root-exclusion corers were made from polyvinyl chloride (PVC) pipe (10 cm long, 7 cm internal dia) and were perforated 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 temperature and moisture of the enclosed soil to equilibrate with that of the surrounding bulk soil. In-growth corers of the same size were constructed from plastic garden mesh (10 mm  10 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 cm  7 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 PO3ÿ 4 leached from the soil core, which was expected to be a signi®cant pathway for the loss of P, as these soils con-

171

sisted predominantly of coarse sand and, hence, have a small capacity to sorb P. Two experimental plots (3.3 m  3.3 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 treatments 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 treatments  3 collections  10 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 (2month ®eld exposure), August (4-month ®eld exposure) and December (8-month ®eld exposure). 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 (PO3ÿ 4 ) 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 sequential fractionation procedure used was adapted from that described by Grierson (1990, loc.cit.) and is a modi®cation of that originally described by Hedley et al. (1982); Tiessen et al. (1984). A subsample (1 g) of fresh soil was shaken with 30 ml of 0.5 M NaHCO3 for 16 h to extract a combination of Pi in

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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 fractionsb were tested by 2-way ANOVA Site

Phosphorus amendment Banksia Pasture Corer type Banksia Pasture a b

Field exposure period (months)

P fractions of soil leached

NaHCO3

NaOH

NaOH/ultrasonicated

residual

sum

























2 4 2 4













ns











2 4 2 4



ns ns ns ns

ns ns ns ns

ns ns ns ns

ns



ns ns ns



ns ns

ns ns ns

Levels for signi®cance:  P < 0.05,  P < 0.01,  P < 0.001, ns not signi®cant. Results are presented for Pi and Po of each fraction and for the sum of Pi and Po of all fractions (sum).

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 di€erence 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 residues 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 additions 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 di€erence between Pi and Pt was 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 oxidation 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 decolorized with activated charcoal (pre-washed Darco G60) that removes only background color and no P. The Pi in all solutions was measured by either automated 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 dissolved in 0.6 ml H2O) to prevent losses of P by volatilisation (Brookes and Powlson, 1981). The Pi in the supernatant was measured with a Technicon AutoAnalyzer 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 centrifuged (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 studies 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-

M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177

173

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 di€erent (P < 0.05). Values in parentheses are the standard deviation of the mean Phosphorus amendment

Community Banksia woodland nonproteoid 2

Control RNA Fe-phytate Rock phosphate

40 90 50 40

proteoid 4

(40) (90) (40) (20)

Pasture total

2

60 130 130 150

(20) (20) (50) (120)

10 20 20 20

4 (0) (20) (10) (20)

2

70 60 50 30

(30) (30) (40) (20)

total 4

50 110 70 60

(40) (90) (40) (40)

130 190 180 180

2 (40) (60) (40) (40)

250 360 300 310

4 330a,b (110) 460b (210) 210a (70) 600c (150)

(70) (200) (80) (50)

posures, 2-way analysis of variance (ANOVA) was used to test for the main e€ects 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 e€ects 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 followed by separation of treatment means (within root types) with the Tukey-Kramer test.

trated Chelex extract 0.5 ml of D2O was added to provide 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. Di€erent P compounds were identi®ed by their chemical shift relative to external orthophosphoric acid (85%). Chemical shift assignments were con®rmed by the addition of known P compounds to the concentrated extracts. Moist soil samples were used in all sequential and 31 P-NMR fractionations and the results were converted to an oven-dried weight basis. The water content 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-

3. Results The e€ect of treatment (form of added P) and corer type (root in-growth or root-exclusion) on the concentration of P in discrete fractions was generally signi®-

Table 3 P concentration (mg gÿ1 dry weight soil) in di€erent fractions of soil from the Banksia community. Within each column, means followed by the same letter are not signi®cantly di€erent (P < 0.05) Treatment

Leached

NaHCO3

2 monthsb Control RNA Fe-phytate Rock phosphate 4 months Control RNA Fe-phytate Rock phosphate

Suma

Soil P fraction NaOH

NaOH/ultrasonicated

Residue Pi and Po

Pi

Pt

PO3ÿ 4

Pi

Po

Pi

Po

Pi

1.8a 86.4b 0.3a 3.8a

2.2a 19.4c 2.7a 6.6b

13.8a 20.1b 24.2c 16.1a

8.4a 24.8b 8.4a 14.3a

13.3a 7.6a 60.0b 17.3a

3.7b 2.0a 2.0a 3.2ab

2.9a 4.9a 9.4b 4.6a

11.5a 13.3a 12.8a 98.7b

14.3a 44.1c 13.0a 24.1b

55.7a 91.8b 119.4c 160.8d

0.3a 71.5b 0.7a 6.3a

0.8a 19.6b 0.0a 6.1a

13.9a 21.6b 27.9c 16.5ab

1.8a 3.2a 0.0a 1.7a

20.8a 25.6a 65.1b 26.2a

0.0a 0.3a 0.2a 0.0a

7.5a 16.2b 9.9ab 13.3ab

12.1a 12.6a 14.3a 85.1b

2.6a 23.0b 0.2a 7.8a

56.9a 99.0b 117.4c 148.8d

Po

a Sum of extractable Pi=sum of Pi in fractions excluding leached P (PO3ÿ 4 ). Sum of extractable Pt=sum of Pi and Po in fractions excluding leached P (PO3ÿ 4 ). b Values for 2-months ®eld exposures are means of 10 replicates; values for 4-months ®eld exposures are means of 6 replicates.

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M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177

Table 4 P concentration (mg gÿ1 dry weight soil) in di€erent fractions of soil from the Pasture community. Within each column, means followed by the same letter are not signi®cantly di€erent (P < 0.05) Treatment

Leached

NaHCO3 PO3ÿ 4 2 monthsb Control RNA Fe-phytate Rock phosphate 4 months Control RNA Fe-phytate Rock phosphate

0.0a 0.1b 0.02a 0.02a 5.0a 25.1c 8.1a 13.0b

Suma

Soil P fraction NaOH

NaOH/ultrasonicated

Residue

Pi

Pt

Pi

Po

Pi

Po

Pi

Po

Pi and Po

1.4a 7.5b 1.4a 3.0a

15.1a 62.3b 21.1a 17.5a

11.4a 10.3a 10.1a 11.3a

15.1a 22.6b 65.3c 15.8a

4.4a 3.8a 4.4a 4.1a

9.0a 8.7a 12.4b 6.9a

15.9a 16.4a 17.2a 81.5b

17.2ab 21.5b 15.9a 18.3ab

72.4a 136.5b 131.9b 140.1b

1.5a 8.0b 0.9a 4.4a

18.4a 46.3b 28.7a 16.9a

2.5a 2.7a 3.3a 2.3a

28.5a 32.6a 58.3b 27.3a

4.9b 0.0a 0.0a 0.0a

11.1a 15.8b 16.5b 13.8b

16.2a 17.4a 17.4a 67.0b

8.8a 10.7d 4.2b 6.7c

83.0a 122.9b 125.1b 131.6b

a Sum of extractable Pi=sum of Pi in fractions excluding leached-P (PO3ÿ 4 ). Sum of extractable Pt=sum of Pi and Po in fractions excluding leached-P (PO3ÿ 4 ). b Values for 2-months ®eld exposures are means of 10 replicates, values for 4-months ®eld exposures are means of 6 replicates.

cant (P < 0.05) for both soil type and ®eld exposure period (Table 1). The e€ect 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) di€erences and hence, these data are not presented. Root growth (mass) in the pasture community was greater than that in the Banksia community during the 2- and 4-month in situ studies. During the ®rst 2 months, root growth in the pasture was between 2and 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 di€erences were only evident between treatments (P amendments) 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 comprised 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 following 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 nonamended Banksia soil (Tables 3 and 4). However, irrespective 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 control 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 resistant P (NaOH/ultrasonication; 12 to 19%).

Fig. 1. Recovery (%) of added phosphorus (RNA, 100 mg P gÿ1 soil; Fe-phytate, 82 mg P gÿ1 soil; rock-phosphate, 108 mg P gÿ1 soil) in di€erent 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 Po in each fraction (Tables 3 and 4) after subtracting amounts in each fraction in the respective (Banksia and Pasture) control treatment.

M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177

At the end of the ®rst 2 months, the amount of P (as PO3ÿ 4 ) leached from Banksia soils amended with RNA (86.4 mg 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 0.1 mg 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 exposure. 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 Pt was 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 31PNMR analysis. The classes of P compounds present were the same as those noted in previous NMR studies and a maximum of less than 50 mg of P gÿ1 dry weight soil was extracted (results not presented). Monoester-P was predominant in all extracts, even in those from soils amended with RNA. Concentrations of Chelexextractable monoester P were about 9 mg gÿ1 soil after 4 months ®eld exposure of control soils from either the Banksia woodland or the pasture. At the same sampling, concentrations in Banksia soil amended with either Fe-phytate or RNA were about 45 mg 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 37 mg gÿ1 for the Fephytate treatment and 30 mg gÿ1 for the RNA treatment. After 8 months, these concentrations increased

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to 43 mg gÿ1 for the Fe-phytate treatment and to 40 mg gÿ1 for the RNA treatment. Concentrations of PO3ÿ 4 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 inecient; 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 sequential 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. organic 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 availability (e.g. Lamont, 1972; 1973) and may be a consequence 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 under Banksia compared with soil under pasture (Tables 3 and 4) cannot be attributed solely to root exudates. It is more likely a result of greater concentrations 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 in Banksia apparently 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 development and growth were rapid (Table 2) and little P was leached within the ®rst 2 months. Although considerable P (25 mg P gÿ1) was leached from the pasture soil between 2 and 4 months, the amount was about 30% of that leached under Banksia in the same period. The poorer recovery of added P from the pasture soil (050±60% of that added, Fig. 1) may be partly attributed to root uptake (not measured) of P. Nucleic acids and their constituent nucleotides can be signi®cant stores of P in microbial cells (Beever and Burns, 1980) and are concentrated in spores or cysts in which other organic P compounds involved in cellular metabolism are minimal. Nucleic acids are readily mineralized in soils, unless stabilized by clay minerals

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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 mineralize, 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 concomitant 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 contrast to more severe extractants, has a limited e€ect on the inherent composition of organic compounds of P (Adams and Byrne, 1989). In comparison with diester-P, monoester compounds, such as phytic acid, are more stable and accumulate 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 phytates 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 resistant 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 compounds, 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 di€erent forms of P (Bowman and Cole 1978a, 1978b) and illustrate the e€ects of vegetation type on mobilisation and immobilisation of sources of P. Some native plant communities (e.g. those dominated by Banksia 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, particularly 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 compounds 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 diversicolor F. 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 fractionation of organic P from grassland soils. Soil Science 125, 95± 101. Bowman, R.A., Cole, C.V., 1978b. Transformations of organic substrates in soils evaluated by NaHCO3 extraction. Soil Science 125, 49±54. Brookes, P.C., Powlson, D.S., 1981. Preventing P losses during perchloric 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 di€erent environmental conditions. Journal of Soil Science 41, 41±50. Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: e€ect 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 ionexchange 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 integrifolia L.f. Plant and Soil 144, 259±265. Grierson, P.F., Attiwill, P.M., 1989. Chemical characteristics of the proteoid root mat of Banksia integrifolia L.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 practices 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. Ho‚and, E., Findenegg, G.R., Nelemans, J.A., 1989. Solubilisation of rock phosphate by rape. II. Local root exudation of

M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177 organic acids as a response to P-starvation. Plant and Soil 113, 161±165. Lamont, B.B., 1972. The e€ect of soil nutrients on the production of proteoid roots by Hakea species. Australian Journal of Botany 20, 27±40. Lamont, B.B., 1973. Factors a€ecting 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,

177

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 e€ects 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 transformations in soils of di€ering 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. Williams, J.D.H., Syers, J.K., Walker, T.W., 1967. Fractionation of soil inorganic phosphate by a modi®cation of Chang and Jackson's procedure. Soil Science Society of America Proceedings 31, 736±739.