Directory UMM :Data Elmu:jurnal:S:Soil Biology And Chemistry:Vol32.Issue2.2000:
Soil Biology & Biochemistry 32 (2000) 169±177
www.elsevier.com/locate/soilbio
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 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 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 dierent 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
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
170
M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177
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
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 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 (Hoand 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
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 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
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 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
(
www.elsevier.com/locate/soilbio
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 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 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 dierent 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
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
170
M.T. Taranto et al. / Soil Biology & Biochemistry 32 (2000) 169±177
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
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 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 (Hoand 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
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 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
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 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
(