Soil & Tillage Research 138 (2014) 35–43
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Soil & Tillage Research
journal homepage: www.elsevier.com/locate/still
Application of 31P NMR spectroscopy in determining phosphatase
activities and P composition in soil aggregates influenced by tillage
and residue management practices
Kai Wei a,b, Zhenhua Chen a, Anning Zhu c, Jiabao Zhang c, Lijun Chen a,*
a
b
c
State Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, China
University of Chinese Academy of Sciences, Beijing 100049, China
State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 8 May 2013
Received in revised form 2 January 2014
Accepted 2 January 2014
Soil phosphorus (P) composition and phosphatase activities in aggregates are essential for agricultural
productivity and remain poorly understood. A field experiment was conducted from 2007 to study the
effect of tillage systems (conventional tillage, T and no tillage, NT) and crop residue management (0, 50%
and 100% crop residue incorporation/coverage) on P composition determined by 31P nuclear magnetic
resonance (NMR) and phosphatase activities in soil aggregates (>2 mm, 0.25–2 mm and 0.053–
0.25 mm). The results showed that crop residue input influenced the concentrations of soil phosphate
monoesters and diesters, alkaline phosphomonoesterase (AlP), acid phosphomonoesterase (AcP),
phosphodiesterase (PD) activities, and soil aggregate stability significantly, and the addition of crop
residue was significantly more effective than tillage. The NT had significantly higher soil phosphatase
activities than tillage treatment but not more soil P content. The 0.25–2 mm aggregates showed higher
total P, organic P, concentrations of monoesters and diesters, and AlP activity. The structure equation
model showed that soil aggregate stability could increase concentrations of monoesters and diesters
indirectly by its direct effects on soil phosphatases. Our results suggest that NT and crop residue input
could increase the P store and sustainable supply in soil aggregates and that the 0.25–2 mm size
aggregates may play an important role in soil organic P maintenance and transformation.
ß 2014 Elsevier B.V. All rights reserved.
Keywords:
Soil P composition
Phosphatase activities
31
P NMR spectroscopy
Aggregate size fractions
Tillage
Crop residue
1. Introduction
Soil phosphorus (P) is one of the major nutrients limiting
agricultural production and consists of organic P and inorganic P.
Organic P accounts for a high proportion of the total P, but most of
it cannot be utilized directly by crops (Schachtman et al., 1998).
Because tillage significantly influences soil P (Selles et al., 1997;
Redel et al., 2007; Zamuner et al., 2008) and long-term tillage
practices have reduced soil P content (Franzluebbers and Hons,
1996; Sisti et al., 2004), it is necessary to study how to increase soil
P storage and the utilization efficiency of organic P by adopting
optimized soil agricultural management. The effect of conservation
tillage on soil nutrients has been widely studied (Franzluebbers
and Hons, 1996; Al-Kaisi et al., 2005; Chen et al., 2009). It has been
confirmed that crop residue inputs and no tillage can enhance soil
organic matter content, improve soil structure and increase the
* Corresponding author at: Institute of Applied Ecology, Chinese Academy of
Sciences, P.O. Box 417, Shenyang 110016, China. Tel.: +86 24 83970355;
fax: +86 24 83970300.
E-mail addresses: [email protected], [email protected] (L. Chen).
0167-1987/$ – see front matter ß 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.still.2014.01.001
accumulation of soil nutrients (Havlin et al., 1990; Malhi and
Lemke, 2007; Wang et al., 2011).
Organic P in soil can be hydrolyzed by phosphatases to release
inorganic orthophosphate for crop uptake. In brief, soil diesters can
be hydrolyzed by phosphodiesterase (PD) to release monoesters,
and then inorganic orthophosphate is released through hydrolysis
of monoesters by alkaline phosphomonoesterase (AlP) and acid
phosphomonoesterase (AcP) (Tabatabai, 1994; Turner and Haygarth, 2005). Traditionally, the determination of soil organic P was
hindered by the difficulties of the extraction, separation and
detection of recalcitrant compounds. However, the adoption of
solution 31P nuclear magnetic resonance (NMR) spectroscopy
eliminated these problems (Turner et al., 2003c). Organic P can be
extracted by using NaOH-EDTA extraction and solution 31P NMR
spectroscopy (Bowman and Moir, 1993), and identified by their
chemical shifts in the spectra (Turner et al., 2003b, 2003c; CadeMenun, 2005). The organic P composition determined by 31P NMR
primarily consists of monoesters and diesters in most soils
(Rheinheimer et al., 2002; Turner et al., 2003a).
Although several investigators have found that residue input
with no tillage can increase soil phosphatase activities (Deng and
Tabatabai, 1997; Wang et al., 2011; Wei et al., 2014) and
36
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
monoester and diester concentrations (Condron et al., 1990; Redel
et al., 2011; Zhang et al., 2012a), little is known about the
relationship between phosphatases and organic P composition of
soils under tillage and crop residue management. Moreover, soil
phosphatase activities in aggregates affected by tillage have been
reported by Gupta and Germida (1988). They found that
macroaggregates in both native and cultivated soils had higher
phosphatase activities than microaggregates. However, the study
of the distribution of P composition and the relationship between
phosphatases and P composition in soil aggregates under tillage
and residue management is limited except for the study conducted
by McDowell et al. (2007) with a pot trail.
The objectives of our study were to investigate the effect of
tillage and crop residue management on soil P composition
determined by 31P NMR and phosphatases activities and to explore
the relationship between phosphatases and organic P composition
in soil aggregates.
2. Materials and methods
2.1. Site description
The field experiment was established in September 2007 at the
Fengqiu State Key Agro-Ecological Experimental Station (358010 N,
1148320 E), Henan province, China. The study plots were located in
the Huang-Huai-Hai Plain with a semi-arid and sub-humid warm
temperate monsoon climate. The annual precipitation in this area
ranged from 355 mm to 800 mm, of which two thirds took place
from June to September. The average annual temperature was
13.9 8C, and the lowest and highest mean monthly values were
1.0 8C in January and 27.2 8C in July, respectively (Ding et al.,
2010). The soil type was Ochri-Aquic Cambosol according to the
World Reference Base for Soil Resources (FAO/ISRIC/ISSS, 1998)
with a profile of sandy loam (about 9% clay, 21.8% silt) in the plough
layer (0–20 cm) and 11.13 g kg1 organic matter, total nitrogen
1.39 g kg1, total phosphorus 0.72 g kg1, total potassium
14.53 g kg1, pH (H2O) 8.24 and bulk density 1.16 g cm2 (Cai
and Qin, 2006; Zhu et al., 2009).
2.2. Experimental design
The experiment was set up using a split-plot design with three
replicates and was conducted under field controlled condition.
Tillage treatment was the main plots, and crop residue management was the subplots. The tillage treatments were conventional
tillage (T) and no tillage (NT). Crop residue managements were 0
(no wheat and maize residue incorporation/coverage), 50% and
100% (7.5 t ha1 in the wheat season and 8.12 t ha1 in the maize
season). The tillage treatment was plowed with a moldboard to a
depth of 23 cm, then, the soil was disked twice, with a disk harrow
before seed sowing. The NT treatment was managed similarly to
the tillage treatment, except for tillage which continued to be NT
with a no-tillage planter for seed sowing. The chopped crop
residues were incorporated into the soil for the tillage systems and
covered the soil surface for the no tillage systems after the wheat
and maize had been harvested every year. The average phosphate
content of maize and wheat residues was 21.4 kg hm2 and
6.2 kg hm2, respectively (Gao et al., 2009). The experimental plots
consisted of six sub-plots: T0 (conventional tillage without residue
incorporation/coverage), T50 (conventional tillage with 50%
residue incorporation), T100 (conventional tillage with 100%
residue incorporation), NT0 (no tillage without residue incorporation/coverage), NT50 (no tillage with 50% residue coverage) and
NT100 (no tillage with 100% residue coverage). Each sub-plot was
4 m 100 m and was under a rotation of winter wheat (early
October to mid-May) and summer maize (early June to
mid-September). Two fertilizations were applied in the wheat
and maize seasons for all treatments. One was applied when
sowing in October and June in the amount of 150 kg ha1
(N:P:K = 17:9:5), and the other was applied in March in the wheat
season and in August in the maize season with urea
(120 kg N ha1).
2.3. Soil sampling
Soil was collected on September 9, 2010, before the maize
harvest and has experienced 3 years’ management since the field
experiment was conducted. Three random, undisturbed soil
subsamples were taken from 0 to 20 cm depth of surface soil
(2000 cm3) where tillage mixed soil and the fertilizer and crop
residues well. Then, soil subsamples were put into hard plastic
containers to prevent the disruption of their structure. After being
transported to the laboratory, the three undisturbed subsamples
were combined into one soil sample. After plant materials and
stones were removed, a portion of each soil sample was sieved
(2 mm) and stored at 4 8C until it was analyzed. The remaining
samples were used to fractionate soil aggregates.
2.4. Soil aggregate distribution
Soil aggregate size fractions were obtained by adopting the
methods of Schutter and Dick (2002) and Sainju et al. (2003).
Before soil aggregates were fractionated, large soil clods were
gently broken apart and laid out on brown paper to dry slowly for
several days. This process was conducted at 4 8C to minimize the
impact of air drying on microbial communities and activities
(Schutter and Dick, 2002) until an 80 g kg1 soil gravimetric water
content was reached so that dry sieving method could be
implemented effectively.
Because the disruption of the physical habitat of microbial
communities was less with dry-sieving than with wet-sieving
(Schutter and Dick, 2002), dry-sieving was used to fractionate soil
aggregates. Before sieving, air-dried soils were sieved (5 mm).
Fractionation was achieved by placing 100 g of sieved soils on
nested sieves mounted on a Retsch AS200 Control (Retsch
Technology, Düsseldorf, Germany). Sieves were mechanically
shaken (amplitude 1.5 mm) for 2 min to separate soil into the
following aggregate size fractions: >2 mm (large macroaggregates), 0.25–2 mm (small macroaggregates), 0.053–0.25 mm
(microaggregates) and <0.053 mm (silt + clay size fraction).
Fractionated samples were later combined into one composite
sample for each aggregate size fraction. Aggregate distribution was
obtained by weighing soil from each aggregate size fraction. Bulk
soil and all the soil aggregates were stored at 4 8C until analysis.
The mean weight diameter (MWD) and geometric mean
diameter (GMD) of soil aggregates were calculated as follows
(Kemper and Rosenau, 1986):
MWD ¼
n
X
xi wi
i¼1
where, xi is the mean diameter (mm), and wi is the weight
proportion of each size fraction.
GMD ¼ exp
Pn
i¼1 wi lnxi
P
n
i¼1 wi
where, wi is the weight of aggregates of each size fraction (g), and
ln xi is the natural logarithm of the mean diameter of each size
fraction.
-1
soil h )
Samples of bulk soil and aggregates were sieved through
0.15 mm mesh for total P analysis and through 2 mm for
phosphatases and other P analyses. Total P was determined by
the perchloric acid (HClO4) digestion method by putting 1.000 g of
soil and 15 ml of HClO4 into a 100 ml digestion tube and digesting
until dense white fumes appeared (Kuo, 1996). Inorganic P was
determined by the method of Saunders and Williams (1955) with
extraction in 0.5 M H2SO4 (1:25 soil-to-solution ratio for 16 h)
(Kuo, 1996). Available P was extracted with 0.5 M NaHCO3 (Olsen
et al., 1954). All the resulting PO43 was detected according to the
method described by Murphy and Riley (1962). Organic P was
calculated as the difference between total P and inorganic P (Gupta
and Germida, 1988). The soil samples for determine P should be
air-dried prior to analysis, while the samples for analyze
phosphatase activities should be stored at 4 8C to retain the field
moisture. The activities of alkaline phosphomonoesterase (AlP),
acid phosphomonoesterase (AcP) and phosphodiesterase (PD)
were determined as described by Tabatabai (1994). Briefly, AlP and
AcP were assayed using p-nitrophenyl phosphate as the substrate
with the modified universal buffer of pH 11.0 and 6.5, respectively.
PD was assayed using bis-p-nitrophenyl phosphate as the substrate
with a buffer of pH 8.0. Phosphatase activities were expressed as
mg p-nitrophenol kg1 soil h1.
P NMR spectroscopy
Phosphorus was extracted by shaking soil (2.5 g) with 50 ml of a
solution containing 0.25 M NaOH and 0.05 M Na2EDTA for 16 h at
20 8C (Cade-Menun and Preston, 1996). Each extract was
centrifuged at 10,000 g for 25 min, the pH of the extract was
adjusted to pH 6.6–7.0 by adding 5 ml of 2 mol L1 HCl solution
rapidly to reduce the hydrolysis of organic P during the following
freeze-dry process (Cade-Menun et al., 2006). The extract was
immediately frozen at 40 8C and subsequently freeze-dried over
2 days. Before total P concentrations were determined by ICP-OES,
the extracts were diluted 25-fold to prevent interference from
EDTA during the analysis (Turner et al., 2003c).
After the NaOH-EDTA extracts were freeze-dried into powder
form, they were transported to the laboratory for analysis
subsequently. Freeze-dried NaOH-EDTA extracts (200 mg) were
re-dissolved in 0.1 ml D2O (for signal lock) and 0.9 ml of a solution
containing 1.0 M NaOH and 100 mM Na2EDTA and then transferred to 5 mm diameter NMR tubes (Turner and Engelbrecht,
2011). Solution 31P NMR spectra were obtained using a JOEL ECA
600 spectrometer operating at 243 MHz, and samples were
analyzed using a 458 pulse, 1.077-s acquisition time, and 1.0-s
delay time. Approximately 26,000 scans were performed for all
samples, and the P composition was identified by the chemical
shifts (ppm) as described by Turner et al. (2003b,c) and CadeMenun (2005). The spectra were plotted using a line broadening of
16 Hz and processed using NMR Utility Transform Software (NUTS)
for Windows (Acorn NMR, Livermore, CA). An example spectrum
was shown in Fig. 2.
2.7. Statistical analysis
Bc
C
BC
300
250
Bb
Bb
Ab
200
C
B
Bb
AB
C
Ba
Ba
Ba
C
ABb
ABc
B
A
Ba
Ba
Aa
150
100
50
0
T0
T50
T100
NT0
NT50
NT100
180
160
Cb
140
Bb
BCb
140
120
C
C
A
ABb
BCb
BCb
BC Cb
AB
Cb
ABb
AB ABab
ABCa
Ab
Ca
BCa
Ab
BCa
100
ABa
80
Aa
60
40
20
0
T0
T50
T100
NT0
B
NT50
100
Cb
BCDb
A
A
A
B
ABCb
AB
Aa
A
CD
ABC
A
BC
DA
B
BCb
Aa
NT100
B
B
B
120
A
80
60
40
20
0
T0
T50
bulk soil
All results were calculated based on oven-dried (105 8C) weight.
Soil data are presented as the arithmetic mean value of three
replicates with their standard deviation. The Duncan test at the
P = 0.05 level under one-way ANOVA was used to analyze the effect
of tillage and residue management on soil parameters. The effects
of tillage systems, crop residue input amounts and aggregate size
on soil characteristics in the aggregates were tested by three-way
ANOVA. The correlation between soil properties was based on
ABc
-1
-1
soil h )
37
Ac
Acid phosphomonoesterase activities (mg para-nitrophenol kg
31
350
-1
-1
phosphodiesterase activities (mg para-nitrophenol kg soil h )
2.6. Soil P composition analysis by
Alkaline phosphomonoesterase activities (mg para-nitrophenol kg
2.5. Analysis of soil P concentrations and phosphatase activities
-1
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
T100
>2mm
NT0
0.25-2mm
NT50
NT100
0.053-0.25mm
Fig. 1. Phosphatase activities in bulk soil and soil aggregates under tillage and
residue management. Values are means, and error bars represent standard
deviation. Bars followed by different uppercase letters indicate differences
(P < 0.05) among tillage systems. Bars followed by different lowercase letters
indicate differences (P < 0.05) in aggregate size fractions in each tillage system. Bars
without letters indicate no significant difference (P > 0.05).
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
38
Fig. 2. Example of a solution 31P NMR spectrum of an NaOH-EDTA extract of the 0.25–2 mm aggregate size fraction under NT0 treatment showing the location of different P
compositions in the spectrum.
Pearson correlation coefficients. All statistical analyses were
conducted with the software SPSS 16.0 (SPSS, Chicago, IL, USA).
Structural equation modeling (SEM) was performed using
AMOS 7.0 software to explore the causal relationship between soil
phosphatases and soil P composition determined by 31P NMR. We
used the x2 test to judge whether the covariance structures
implied by the model adequately fit the actual covariance
structures of the data. A non-significant chi-squared test
(P > 0.05) indicated adequate model fit. As the calculated
standardized coefficients, the path coefficients were determined
using the correlation matrices analysis.
macroaggregate proportions were higher in the T50 and T100
treatments compared to the T0 treatment (Table 1). Microaggregate (<0.053 mm and 0.053–0.25 mm) proportions, however,
were higher in the T0 treatment. Moreover, the MWD and GMD of
aggregates were also affected by crop residue management
(Table 2). The no tillage, T50 and T100 treatments showed higher
MWD and GMD than the T0 treatment. However, no significant
differences in MWD and GMD were found among the no tillage
systems.
3. Results
The soil samples collected in September 2010 were used to
analyze P concentrations, and since the field experiment was
conducted under controlled condition, P concentrations was
independent of variation of season, crop and crop residue type.
In our study, available P concentration was affected by tillage and
crop residue management in bulk soil and in aggregates (Tables 2
3.1. Soil aggregate distribution
Crop residue management significantly affected soil aggregate
distribution (Table 1). Small and large (0.25–2 mm and >2 mm)
3.2. Soil P concentrations
Table 1
Aggregate size distribution in g aggregate kg1 soil and mean weight diameter (MWD) and geometric mean diameter (GMD) under tillage (T and NT) and residue
management (0, 50 and 100) at the depth of 0–20 cm.
Aggregates (mm)
T0
T50
T100
NT0
NT50
NT100
<0.053
0.053–0.25
0.25–2
>2
MWD
GMD
8.1 6.5Ba
258.3 95.0Bb
474.0 121.8Ac
259.7 26.4Ab
1.48 0.08A
0.89 0.18A
2.2 1.6Aa
158.8 50.0Ab
541.4 60.3ABd
297.6 29.1Bc
1.67 0.08B
1.14 0.12B
0.7 0.4Aa
135.8 39.6Ab
601.6 40.2Bd.
262.0 19.4Ac
1.61 0.07B
1.15 0.10B
2.4 1.0Aa
198.3 73.7ABb
521.2 68.5ABd
278.1 19.9ABc
1.59 0.11B
1.04 0.17AB
1.7 1.6Aa
172.2 45.6Ab
537.3 53.7ABd
288.8 36.3ABc
1.64 0.10B
1.10 0.13B
1.2 1.2Aa
158.5 33.8Ab
546.1 35.7ABd
294.2 24.4ABc
1.67 0.08B
1.14 0.10B
T0: conventional tillage without residue incorporation/coverage; T50: conventional tillage with 50% residue incorporation: T100: conventional tillage with 100% residue
incorporation: NT0: no tillage without residue incorporation/coverage: NT50: no tillage with 50% residue coverage; NT100: no tillage with 100% residue coverage; Values
followed by different uppercase letters within a row indicate difference (P < 0.05) among tillage systems. Values followed by different lowercase letters within a column
indicate difference (P < 0.05) in aggregate size fractions in each tillage system.
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
39
Table 2
Significant levels (ANOVA) of the effects of tillage treatment (T and NT), residue management (0, 50 and 100), aggregate size fractions (>2, 0.25–2 and 0.053–025 mm) and
their interaction on soil chemical and biological characteristics in aggregates.
Factors
TP
OP
AP
AlP
AcP
PD
Monoesters
Diesters
MWD
GMD
Tillage (T)
Residues (R)
Aggregate
(A)
sizes
TR
TA
RA
TRA
F value
P value
F value
P value
F value
P value
F value
P value
F value
P value
F value
P value
F value
P value
0.03
0.02
43.40
25.89
14.17
3.87
0.21
54.94
2.18
0.54
0.870
0.880
0.000
0.000
0.001
0.057
0.648
0.000
0.150
0.467
1.35
1.74
7.80
37.14
21.93
11.46
19.52
73.00
6.98
6.72
0.273
0.191
0.002
0.000
0.000
0.000
0.000
0.000
0.003
0.004
49.26
31.30
2.98
170.84
80.00
6.73
15.64
15.89
–
–
0.000
0.000
0.063
0.000
0.000
0.003
0.000
0.000
–
–
2.24
1.59
1.08
14.21
6.86
0.17
0.24
7.93
2.02
1.76
0.121
0.218
0.495
0.000
0.003
0.847
0.788
0.001
0.150
0.189
1.52
2.37
0.92
9.77
1.09
0.62
1.36
6.42
–
–
0.233
0.108
0.408
0.000
0.346
0.542
0.270
0.004
–
–
1.69
0.70
0.86
4.60
0.98
1.31
4.67
28.34
–
–
0.173
0.596
0.500
0.004
0.432
0.286
0.004
0.000
–
–
0.90
1.57
1.28
1.26
0.48
0.72
0.92
4.62
–
–
0.476
0.204
0.297
0.304
0.753
0.585
0.464
0.004
–
–
TP: total P; OP: organic P; AP: available P; AlP: alkaline phosphomonoesterase; AcP: acid phosphomonoesterase; PD: phosphodiesterase; MWD: mean weight diameter;
GMD: geometric mean diameter.
and 3), whereas total P and organic P concentrations were affected
by aggregate size. The T0 and T100 samples showed significantly
higher available P concentrations than the NT0 and NT100 samples
in the 0.053–0.25 mm and >2 mm fractions (Table 3). When crop
residues were applied, the available P concentration significantly
decreased in bulk soil, in the >2 mm and 0.053–0.25 mm sizes in
tillage systems, and in the >2 mm size in the no-tillage systems
(Table 3). Small macroaggregates (0.25–2 mm) showed significantly higher total P and organic P concentrations than the other
size fractions in all treatments (Table 3), and the proportion of
organic P in the total P was also higher in the 0.25–2 mm size
fraction.
3.3. Soil phosphatase activities
The AlP, AcP and PD activities in bulk soil and in aggregates
were significantly affected by tillage and crop residue management
(Table 2, Fig. 1). Compared to the T0 and T100 treatments, the NT0
and NT100 samples showed significantly higher AlP activity in bulk
soil and in the >2 mm and 0.053–0.25 mm size fractions. The AcP
activity in the 0.25–2 mm and 0.053–0.25 mm size fractions and
the PD activity in bulk soil and in the 0.25–2 mm size fractions
were also significantly higher in the NT0 and NT100 samples than
in the T0 and T100 samples, respectively. With increasing crop
residue input amounts, the AlP, AcP and PD activities in the tillage
systems increased in bulk soil and in all size fractions. In contrast,
the AlP and AcP activities in the 0.25–2 mm size fraction and the PD
activity in bulk soil, >2 mm and 0.053–2 mm size fractions showed
no significant change in any of the tillage systems (Fig. 1).
Moreover, aggregate size also affected AlP, AcP and PD activities
(Table 2). The AlP activity was significantly higher in small
macroaggregates (0.25–2 mm), whereas AcP and PD showed
higher activities in large and small macroaggregates (>2 mm
and 0.25–2 mm) (Fig. 1).
Table 3
Soil chemical characteristics in bulk soil and soil aggregates under tillage (T and NT) and residue management (0, 50 and 100).
Tillage systems
Aggregate sizes (mm)
Total P
Organic P
Available P
OP/TP (%)
(mg kg1)
T0
BS
>2
0.25–2
0.053–0.25
609.2 48.7
631.8 23.6a
688.8 20.7b
610.4 28.0ABa
224.1 44.4
260.9 14.5a
307.8 16.4b
256.0 24.1Aa
26.3 5.0D
23.1 4.7 C
28.3 8.0B
27.1 3.9 C
36.6 4.9
41.3 0.9
44.7 1.5
41.9 2.7A
T50
BS
>2
0.25–2
0.053–0.25
626.4 9.0
631.5 13.5a
671.9 5.2b
649.4 27.6Bab
249.2 10.4
263.0 31.8
327.9 39.4
307.1 20.6B
19.3 0.4BC
14.9 2.5AB
23.5 9.3AB
18.5 3.8AB
39.8 1.5
41.6 4.6
48.8 6.0
47.3 1.6B
T100
BS
>2
0.25–2
0.053–0.25
619.2 22.4
658.0 23.3
691.3 10.7
614.2 23.9AB
224.5 39.3
262.7 39.3a
323.4 19.0b
248.3 13.7Aa
23.0 4.0CD
21.2 2.3 C
22.6 2.5AB
22.7 3.2BC
36.1 5.2
40.0 6.8
46.8 2.2
40.5 2.5A
NT0
BS
>2
0.25–2
0.053–0.25
647.4 25.6
653.7 19.7a
702.0 12.0b
623.1 18.8ABa
249.9 26.8
271.7 20.2a
317.3 8.1b
262.2 10.3Aa
14.8 1.3AB
18.7 3.5BC
18.6 3.0AB
13.9 3.9A
38.5 2.9
41.5 1.9a
45.2 1.8b
42.1 1.4Aab
NT50
BS
>2
0.25–2
0.053–0.25
639.9 10.3
629.0 10.9a
681.0 19.0b
599.5 24.4Aa
266.0 11.8
282.0 7.7b
320.0 16.6c
253.5 13.9Aa
13.4 2.1AB
11.1 1.4A
13.7 1.3A
16.5 5.4AB
41.6 1.2
44.9 2.0ab
47.0 1.2b
42.3 0.9Aa
NT100
BS
>2
0.25–2
0.053–0.25
638.4 21.5
661.8 43.3ab
694.8 22.2b
611.1 17.0ABa
250.1 5.7
289.0 34.4ab
309.6 11.7b
259.8 8.4Aa
12.9 4.1A
13.2 1.1Aa
14.9 0.1Aab
16.4 1.3ABb
39.2 2.1
43.6 2.5
44.6 0.3
42.5 1.7A
TP: total P; OP: organic P; values followed by different uppercase letters within a column indicate difference (P < 0.05) among tillage systems. Values followed by different
lowercase letters within a column indicate difference (P < 0.05) in aggregate size fractions in each tillage system. Columns without letters indicate no significant difference
(P > 0.05).
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
40
Table 4
Mean concentration (mg kg1) and the percentage of extracted total P (in parentheses) of P composition determined by 31P NMR spectroscopy in bulk soil and soil aggregates
under tillage (T and NT) and residue management (0, 50 and 100).
Tillage systems
T0
T50
T100
NT0
NT50
NT100
NaOH-EDTA extracted total Pa
Aggreagte sizes (mm)
BS
>2
0.25–2
0.053–0.25
BS
>2
0.25–2
0.053–0.25
BS
>2
0.25–2
0.053–0.25
BS
>2
0.25–2
0.053–0.25
BS
>2
0.25–2
0.053–0.25
BS
>2
0.25–2
0.053–0.25
196(32.2)
166(26.3)
191(27.7)
141(23.1)
156(24.9)
151(23.9)
182(27.1)
131(20.2)
179(28.9)
189(28.7)
217(31.3)
158(25.7)
196(30.2)
169(25.9)
223(31.8)
157(25.2)
162(25.3)
169(26.9)
152(22.4)
130(21.7)
189(29.6)
176(26.5)
192(27.6)
146(23.9)
Inorganic P
Organic P
Orthophosphate
Pyrophosphate
Monoesters
Diesters
154.7(78.9)
137.3(82.7)
159.9(83.7)
111.5(79.1)
122.5(78.5)
117.2(77.6)
146.5(80.5)
98.8(75.4)
135.3(75.6)
153.1(81.0)
164.3(75.7)
120.9(76.5)
155.8(79.5)
136.6(80.8)
191.8(86.0)
127.0(80.9)
122.0(75.3)
132.0(78.1)
108.2(71.2)
93.7(72.1)
140.4(74.3)
140.4(79.8)
126.0(65.6)
106.3(72.8)
6.0(3.1)
2.5(1.5)
1.9(1.0)
2.0(1.4)
4.1(2.6)
0.9(0.6)
2.9(1.6)
1.8(1.4)
3.9(2.2)
1.2(0.6)
1.1(0.5)
1.1(0.7)
7.1(3.6)
4.1(2.4)
3.1(1.4)
2.8(1.8)
4.1(2.5)
3.0(1.8)
0.6(0.4)
1.7(1.3)
4.5(2.4)
2.6(1.5)
1.2(0.6)
2.0(1.4)
26.2(13.4)
20.8(12.5)
25.4(13.3)
24.1(17.1)
21.1(13.5)
28.2(18.7)
28.2(15.5)
24.4(18.6)
29.4(16.4)
27.0(14.3)
39.7(18.3)
29.9(18.9)
25.8(13.1)
22.5(13.3)
23.4(10.5)
22.6(14.4)
26.9(16.6)
25.0(14.8)
33.9(22.3)
24.6(18.8)
32.5(17.2)
26.9(15.3)
46.5(24.2)
27.6(18.9)
9.1(4.6)
5.5(3.3)
3.8(2.0)
3.4(2.4)
8.4(5.4)
4.7(3.1)
4.4(2.4)
6.2(4.7)
10.4(5.8)
7.7(4.1)
11.9(5.5)
6.2(3.9)
7.3(3.7)
5.9(3.5)
4.7(2.1)
4.6(2.9)
9.1(5.6)
9.0(5.3)
9.3(6.1)
10.1(7.8)
11.5(6.1)
6.0(3.4)
18.6(9.7)
10.1(6.9)
a
Values in the column represent mean concentration of NaOH-EDTA extracted P and the proportion of total P (in parentheses) recovered by NaOH-EDTA in bulk soil and
soil aggregates.
3.4. Soil P composition determined by
31
P NMR spectroscopy
Total P concentration extracted by NaOH-EDTA in bulk soil
ranged from 156 to 196 mg kg1, equivalent to 24.9 to 32.2% of the
total P in soils (Table 4). Organic P composition determined by 31P
NMR consisted primarily of monoesters and diesters (Table 4). In
our study, tillage had a significant effect on diester concentrations
(Table 2). The no tillage system showed higher diester concentrations than the corresponding tillage system. Moreover, the input of
crop residue affected the concentrations of monoester and diester
significantly (Table 2), and with increasing crop residue input
amounts, monoester and diester concentrations also increased
(Table 4). Small macroaggregates (0.25–2 mm) showed higher
monoester and diester concentrations than other sizes of
aggregates (Table 4). The inorganic P composition determined
by 31P NMR in our study mainly consisted of inorganic
orthophosphate and pyrophosphate (Table 4). In contrast to
monoesters and diesters, orthophosphate and pyrophosphate
concentrations decreased with increasing crop residue input
amounts.
and AcP) on monoesters, AlP showed a significantly greater effect
(path coefficient = 0.25, P < 0.05) than AcP (not significant).
Moreover, the effect of PD on diesters was also significant (path
coefficient = 0.36, P < 0.01). It is worth mentioning that soil MWD
had a strong, direct effect on the AlP, AcP and PD activities (all
P < 0.001), but it had no significant effect on monoesters and
diesters. Soil MWD influenced monoesters and diesters indirectly
mainly by its effect on phosphatases (Fig. 3).
4. Discussion
4.1. Soil aggregate distribution
In our study, the NT, T100 and T50 treatments resulted in more
small and large macroaggregates (0.25–2 mm and >2 mm) than
3.5. Correlations between soil P characteristics and phosphatase
activities
Monoesters were significantly positively correlated with AlP
(P < 0.01) and AcP (P < 0.05), whereas diesters had significant
positive correlations with all phosphatases (P < 0.01) (Table 5). All
parameters were significantly positively correlated with soil
aggregate MWD and GMD (Table 5).
3.6. Structure equation model
The model explained 61% of the variation in monoesters
determined by 31P NMR, and provided the best fit to our data
according to the three indices of model fit (x2 = 9.27; df = 5;
P > 0.05) (Fig. 3). This model revealed that diesters had the
strongest direct effect on monoesters (path coefficient = 0.75,
P < 0.001). Compared to the effect of phosphomonoesterases (AlP
Fig. 3. Structural equation model results for effects of soil MWD and phosphatases
on organic P composition determined by 31P NRM under tillage and residue
management. AlP, alkaline phosphomonoesterase; AcP, acid phosphomonoesterase;
PD, phosphodiesterase; MWD, mean weight diameter. Numbers on arrows are
standardized path coefficients. Numbers in bold indicate the variance explained by the
model (R2). Line thickness represents the magnitude of the path coefficient. Nonsignificant pathways are dashed and removed in the final model. Significance levels are
as follows: *P < 0.05, **P < 0.01 and ***P < 0.001.
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
41
Table 5
Correlations between soil phosphatases, organic composition and aggregates stability parameters.
AlP
AcP
PD
Monoesters
Diesters
MWD
GMD
AlP
AcP
PD
Monoesters
Diesters
MWD
GMD
1
0.754**
0.645**
0.402**
0.383**
0.482**
0.467**
1
0.616**
0.277*
0.350**
0.401**
0.420**
1
ns
0.364**
0.540**
0.509**
1
0.773**
0.276**
0.347**
1
0.362**
0.398**
1
0.937**
1
AlP: alkaline phosphomonoesterase; AcP: acid phosphomonoesterase; PD: phosphodiesterase; MWD: mean weight diameter; GMD: geometric mean diameter;
*
Correlation is significant at the 0.05 level.
**
Correlation is significant at the 0.01 level.
the T0 treatment, indicating that crop residues and no tillage had a
positive impact on soil aggregation. It could be concluded that
more binding agents were generated in soils as a result of the
decomposition of the crop residues (Six et al., 2002; Abiven et al.,
2007) or of the microbial activities under no tillage (Zhang et al.,
2012b), thus causing more microaggregates to be bound together
into macroaggregates (Tisdall and Oades, 1982; Zhang et al.,
2012b). Our results suggest that no tillage and the input of crop
residues facilitated the stability of soil aggregates. Similar results
have also been reported by other investigators (Yang and Wander,
1998; Malhi and Lemke, 2007; Chung et al., 2008).
However, the absence of significant differences among the no
tillage systems in the 0–20 cm soil layer for MWD and GMD could
be attributed to two factors: (1) crop residues were not mixed
effectively into the soil under the no tillage systems during the
three year field experiment; and (2) the dilution effect of deeper
soil layers during sampling because the no tillage systems
increased soil surface stability (Abid and Lal, 2008; Chen et al.,
2009). Moreover, when crop residues were applied, the no tillage
systems did not show significantly greater stability of soil
aggregates compared to the corresponding tillage systems. This
could be ascribed to crop residues and their decomposition
products in the tillage systems being incorporated and mixed into
the soil more fully during the three year field experiment,
promoting soil aggregation (Olchin et al., 2008) and thus offsetting
the effect of tillage on soil aggregates.
4.2. Soil P concentrations
Available P was lower in the no tillage systems than in the
tillage systems, which was inconsistent with previous studies
(Ismail et al., 1994; Franzluebbers and Hons, 1996; Sisti et al.,
2004), which found that no tillage increased available P
concentration. The authors attributed this to enhanced P binding
resulting from an increase in the contact between solution P and
soil particles during the tillage treatment. Nevertheless, the lower
available P concentration observed here could be due to increased
microbial immobilization of P in the no tillage systems where
microbial activity increased because of more C sources. Similar
results were also reported by Omidi et al. (2008).
Our results showed that the available P concentration
decreased with increasing crop residue input amounts. This result
was in contrast to the finding of Alamgir et al. (2012) who reported
that the application of crop residues increased available P
concentration. The conflicting results could be due to differences
in the type and quality of the residues (Schomberg and Steiner,
1999; Salas et al., 2003). Nziguheba et al. (2000) reported that
residues containing high P concentrations benefited P mineralization and thus increased available P, whereas residues with low P
would result in the reduction in available P by microbial
immobilization. Moreover, Lupwayi et al. (2007) also found
that phosphorus release was positively correlated with the
P concentration in residues. In our study, maize and wheat
residues containing low P concentrations (Lqbal, 2009) might
increase the microbial immobilization of P, thereby resulting in the
reduction of available P concentrations. Similar results were also
found by Singh and Jones (1976) and Nziguheba et al. (1998).
Due to soil P primarily accumulating in the soil surface layers
under the no tillage and crop residue management (Cade-Menun
et al., 2010), no significant effect of tillage and crop residue
management on the total P and organic P concentrations could be
attributed to the dilution of deeper soil layers during sampling. In
contrast, total P and organic P concentrations were affected by
aggregate size (Elliott, 1986; He et al., 1995; Green et al., 2005). In
our study, small macroaggregates (0.25–2 mm) showed significantly higher total P and organic P concentrations than other size
fractions, indicating that the 0.25–2 mm size was of great
importance for maintaining and sustaining the supply of soil P.
4.3. Soil phosphatase activities
Soil enzymes have been regarded as very good indicators of the
changes in the biological properties of soil because of their
sensitivity to soil management practices (Roldan et al., 2005). Our
results showed that the no tillage systems had higher AlP, AcP and
PD activities compared to the tillage systems, a finding in accord
with those of other researchers (Doran, 1980; Dick, 1984; Wang
et al., 2011). The AlP, AcP and PD studied here were also affected by
crop residue management, and their activities increased with
increasing residue input amounts. This result could be attributed
to two factors: (1) increased substrates of soil phosphatases due to
the application of crop residues, and (2) increased microbial
activities because phosphatases in soils are believed to be derived
primarily from microorganisms (Browman and Tabatabai, 1978;
Tabatabai, 1994; Turner and Haygarth, 2005). Microbial activity
might be increased with increasing C sources resulting from
residue inputs. Deng and Tabatabai (1997) found that the different
effects of tillage and crop residue management on phosphatase
activities might be due to differences in the origin, states, and/or
persistence of different groups of enzymes. Moreover, soil MWD
showed significant positive effects on AlP, AcP and PD (all
P < 0.001, Fig. 3), suggesting that the improvement of soil structure
was conducive to improving the activities of phosphatases. This
could be a result of increased microbial populations and their
activities because of the improvement of the soil structure.
The AlP activity studied here was significantly higher in small
macroaggregates (0.25–2 mm), whereas AcP showed higher
activity in large and small macroaggregates (>2 mm and 0.25–
2 mm). According to the structure equation model, we compared
the effect of phosphomonoesterases (AlP and AcP) on monoesters
determined by 31P NMR and found that AlP had a significantly
greater effect (path coefficient = 0.25, P < 0.05). This result could
be attributed to the predominance of AlP activity in the slightly
alkaline soil with average pH of 7.44 (Wang et al., 2011) studied
K. Wei et al. / Soil & Tillage Research 138 (2014) 35–43
42
here (Juma and Tabatabai, 1977). Although AcP activity showed
significant correlation with monoesters (P < 0.05), its direct effect
on monoesters was not significant, suggesting that AlP not only
dominated but also had a significantly stronger effect on
monoesters than AcP in the slightly alkaline soil. Therefore, small
macroaggregates (0.25–2 mm) that contained higher AlP activity
may play a greater role in soil P transformation. The effect of PD on
diesters determined by 31P NRM was also significant (path
coefficient = 0.36, P < 0.01), but it only partly explained the
variation in diesters, implying that there are other factors in soils
that influence diesters that were not included in the model.
4.4. Soil P composition determined by
31
P NMR spectroscopy
In our study, NaOH-EDTA only extracted 24.9–32.2% of the total
P in bulk soil. The relatively low proportion extracted could be
related to the calcareous soils studied here (Wang et al., 2011)
because NaOH-EDTA is regarded as an inefficient extractor for total
P in calcareous soils (Turner et al., 2003d). The P composition
determined by 31P NMR showed that organic P mainly consisted of
monoesters and diesters, and that the proportion of monoesters
was higher than that of diesters (Table 4). This result is consistent
with the results reported by other scientists (Rheinheimer et al.,
2002; Cardoso et al., 2003; Bunemann et al., 2008). We observed
that only the diester concentrations were affected by tillage,
possibly due to diesters being more liable to be degraded in soils
than monoesters (Condron et al., 1990; Turner et al., 2003a).
Moreover, the no tillage systems showed higher diester concentrations than the tillage systems, which could be attributed to the
increased mineralization of diesters under tillage treatment
(Condron et al., 1990; Solomon et al., 2002; Zhang et al., 2012a).
Crop residue management also affected monoesters and
diesters significantly, and their concentrations increased with
increasing amounts of crop residue inputs (Table 4). This could be
the result of increased substrates for phosphatases and C sources
for soil microbes because diesters mainly consist of phospholipids
and nucleic acids (Tate and Newman, 1982), and most of these
compounds in soils are derived from microorganisms and enter the
soil in the form of crop residues (Turner and Newman, 2005; Noack
et al., 2012). Monoesters could be released by the hydrolysis of
diesters by phosphodiesterase (Turner and Newman, 2005). The
similar distributions of these compounds in tillage systems could
be partly explained by the significant positive correlation between
monoesters and diesters (P < 0.01) and the significant direct effect
of diesters on monoesters (P < 0.001). According to the structure
equation model, we found that the improvement of soil structure
could increase monoester and diester concentrations indirectly by
effects on phosphatases. Diesters were the most important factors
for monoesters in the tillage systems. This observation indicates
that monoester concentrations could be increased effectively by
adopting proper agricultural measures that increase the amounts
of diesters, such as adding residues or increasing the microbial
population, thus providing more P for soil. Inorganic P composition, however, showed a trend opposite that of monoesters and
diesters, a trend that might be attributed to increased microbial
immobilization due to the application of residues.
Monoesters and diesters in soils were also affected by aggregate
size. In our study, small macroaggregates (0.25–2 mm) contained
relatively high monoester and diester concentrations, which may
reflect the influence of small macroaggregates (0.25–2 mm) on soil
organic P storage.
5. Conclusions
In our study, the no tillage and crop residue management
increased soil phosphate monoester and diester concentrations,
AlP, AcP and PD activities, and enhanced the soil aggregate
stability. Crop residue input played a greater role than tillage in
these effects. Moreover, NT showed significantly higher soil
phosphatase activities but not soil P content than CT. According
to the structure equation model, we found that AlP not only had
higher activity but also had a significantly stronger effect on
phosphate monoesters determined by 31P NMR than AcP in the
alkaline soil. The improvement of soil structure could improve soil
phosphatase activities and increase concentrations of phosphate
monoesters and diesters indirectly by its effects on phosphatases.
Agricultural management practices that increased the amounts of
phosphate diesters could increase phosphate monoester concentrations effectively. In addition, small macroaggregates (0.25–
2 mm) showed high concentrations of phosphate monoesters and
diesters, and high AlP activity. In conclusion, our results suggest
that NT and crop residue input could increase P stores and the
capacity for a sustainable supply in soil aggregates. The 0.25–2 mm
size may play an important role in the maintenance and
transformation of soil organic P.
Acknowledgements
This study was supported by the National Basic Research
Program of China (973 Program) (2011CB100506, 2011CB100504),
the National Natural Science Foundation of China (41171241)
and the National Key Technology R&D Program of China
(2012BAD14B04).
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