agronomy
Article
Molecular-Level Insights into Phosphorus Transformation
Mechanisms in Entisol Soils under Multiple Long-Term
Fertilization Regimes
Jin Liu 1, * , Chaoqun Han 1 , Yuhang Zhao 1 , Dongling Yang 1 , Jianjun Yang 2 , Lei Zheng 3 , Yongfeng Hu 4 ,
Jumei Li 5 , Peng Sui 1 , Yuanquan Chen 1 , Xiaojun Shi 6 and Yibing Ma 7, *
Citation: Liu, J.; Han, C.; Zhao, Y.;
Yang, D.; Yang, J.; Zheng, L.; Hu, Y.;
Li, J.; Sui, P.; Chen, Y.; et al.
Molecular-Level Insights into
Phosphorus Transformation
Mechanisms in Entisol Soils under
Multiple Long-Term Fertilization
Regimes. Agronomy 2022, 12, 2760.
https://doi.org/10.3390/
agronomy12112760
Academic Editor: Wei Zhang
Received: 3 October 2022
Accepted: 3 November 2022
Published: 6 November 2022
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Agronomy 2022, 12, 2760. https://doi.org/10.3390/agronomy12112760
1 College of Agronomy and Biotechnology, China Agricultural University, Beijing 100094, China
2 Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural
Sciences, Beijing 100081, China
3 Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences,
Beijing 100049, China
4 Sinopec Shanghai Research Institute of Petrochemical Technology, Shanghai 201208, China
5 Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences,
Beijing 100081, China
6 College of Resources and Environment, Southwest University, Chongqing 400715, China
7 Macao Environmental Research Institute, Macau University of Science and Technology, Macao 999078, China
* Correspondence: jliu207@cau.edu.cn (J.L.); ybma@must.edu.mo (Y.M.)
Abstract: Improving phosphorus (P) fertilization strategies benefits mitigating future global P short-
age and reducing legacy P loss risk in agricultural lands. In this study, the molecular transformation
mechanisms of P in Entisol soils under multiple long-term fertilization regimes including PK; NK;
NPK; NPK with pig manure (NPKM); and NPK with rice straw return (NPKS) were investigated
by sequential fractionation (SF), synchrotron-based P K-edge X-ray absorption near-edge structure
(P-XANES) and solution 31 P nuclear magnetic resonance (P-NMR) spectroscopy. Compared with
conventional NPK fertilization, a higher accumulation of total P occurred in soils under the PK,
NPKM and NPKS treatments. By SF, there were relatively higher contents of NaHCO3 -extracted
inorganic P (Pi ) fractions for the soils under PK (52.5 mg/kg) and NPKM (35.5 mg/kg) fertilization
relative to the NPK (23.3 mg/kg) treatment. Consistently, P-XANES analysis revealed that there was
a higher proportion of brushite, as a bioavailable P form, in soil under the PK and NPKM treatments
compared with the NPK treatment, indicating higher P availability in the Entisol soils under PK
and NPKM fertilizations. By P-NMR, long-term PK fertilization resulted in relatively a higher ratio
of orthophosphate diesters to orthophosphate monoesters after correction compared with the NPK
fertilization, which strongly suggested that N deficiency enhanced the biodegradability of soil organic
P (Po ) pools, thus providing new molecular-level insights into soil Po transformation. Collectively,
these results, obtained from a long-term experimental study, facilitates the comprehensive under-
standing of P availability and transformation mechanisms in Entisol soils under multiple fertilization
regimes, and thus benefits the improvement of fertilization strategies in agricultural soils.
Keywords: manure; nitrogen deficiency; NMR; XANES; straw return
1. Introduction
Increased concerns about shortage of global phosphorus (P) rocks and risk of P loss
from agricultural lands called for more efficient and sustainable P fertilization strate-
gies [1,2]. Chemical fertilization generally led to the majority of fertilizer P fixed and
accumulated in soils as legacy P, which has low P bioavailability but high environmental
loss risk due to surface runoff [1,3,4]. Amounting evidence indicated that a combination or
partial replacement of chemical P fertilizer with manure and crop straw could increase soil
https://www.mdpi.com/journal/agronomy
Agronomy 2022, 12, 2760 2 of 13

P availability and P uptake by crops [5–7], which not only reduced the amounts of chem-
ical P fertilizer applied in agricultural lands but also enhanced P fertilizer use efficiency.
For example, Liu et al. [5] reported that a combined application of chemical fertilizers
with manure significantly enhanced the accumulation of total soil P and soil P availability
(i.e., Olsen-P). Qin et al. [8] found that the application of chemical nitrogen (N), P and
potassium (K) fertilizers with manure increased the content of orthophosphate, which
resulted from the orthophosphate present in manure and the transformation of organic P
(Po ) into inorganic P (Pi ) in soil. Jiang et al. [7] reported that the combination of chemical fer-
tilizers with crop straw significantly increased soil P availability and use efficiency, but the
fertilization of chemical fertilizers with manure exhibited an opposite impact. Moreover, in
P-enriched agricultural lands, crops could reuse soil legacy P without further P fertilization,
but maintained a stable crop yield production [1]. Such fertilization strategies could dig soil
legacy P resources and reduce P application if crop yield production was maintained at a
level equal or acceptable to that with continuous P fertilization when taking environmental
benefits into consideration. Recently, the transformation of soil Po was found to be linked
to soil N deficiency, which could stimulate ectomycorrhizas to release exoenzymes and
enhance formation of orthophosphate diesters (Di) [9]. Therefore, long-term PK fertilization
without N addition probably causes different Po transformation compared with conven-
tional NPK fertilization. Previous studies indicated that soil P availability and speciation
depended critically on fertilization treatments and soil properties [1,2,10]. However, the
transformation mechanisms of P in Entisol soils under multiple fertilization regimes remain
largely unknown at the molecular level, particular for long-term fertilization experimental
sites, thus deserving further study.
Sequential fractionation (SF) was widely applied to split soil P fractions, but it is
sometimes insufficient to clarify accurately P availability in soils based on the operationally
defined P fractions [11–13]. Therefore, state-of-art techniques including synchrotron-based
X-ray absorption near-edge structure (P-XANES) and liquid-state 31 P NMR spectroscopy
(P-NMR) were combined for the characterization of soil P speciation at the molecular
level [11]. Generally, P-XANES is sensitive to probe soil Pi speciation, including calcium-
associated P (Ca-P), aluminum-associated P (Al-P), and iron-associated P (Fe-P), while
P-NMR is capable of performing Po speciation, including various Po species, such as
orthophosphate monoesters (Mono), and Di, etc., and biogenic Pi species such as polyphos-
phate, and pyrophosphate [1,2,11]. These two techniques combined greatly facilitate our
investigation on the availability, speciation and transformation of soil P under various
fertilization treatments. For examples, Weyers et al. [14] reported that manure application
significantly increased P availability in the soils but did not change P species among all
amendments in calcareous soil using P K-edge XANES and solution P-NMR spectroscopy.
Using the same two techniques, Liu et al. [2] reported the increase of soil P availability
in a desert soil (Haplic Calcisol) under NPK plus manure fertilization compared with
NPK fertilization as a result of newberyite (magnesium (Mg)-P compound) formation and
deoxyribonucleic acid accumulation. These inconsistent results on the impact of manure
on soil P availability probably resulted from different soil properties and P species in exotic
fertilizers as well as their varied influences on soil P speciation and transformation, and
highlighted the need of further investigation.
Purple soil, belonging to the Entisols in USDA Taxonomy [15], is the most impor-
tant agricultural soil type in the Sichuan basin of southwestern China, with an area of
160,000 km2 [16], which is regarded as the ‘grain barn’ in China with a long-term history.
However, nutrient loss of purple soils due to surface runoff caused eutrophication in some
tributaries of the Three Gorges Reservoir, which is one of the largest reservoirs in the
world [4]. Therefore, an effective fertilization strategy in agricultural lands enriched with
purple soils was of significant importance to improve agricultural sustainability and water
quality. Since 1990, a long-term fertilization experiment has been established in this region,
with replicated plots under various fertilizer treatments, which provide an indispensable
platform to investigate the impact of various fertilization treatments on soil P availability
Agronomy 2022, 12, 2760 3 of 13

and transformation. To date, few studies have been conducted to investigate the impact of
multiple fertilizer treatments on the transformation of P in Entisol soils at the molecular
level. Therefore, the aims of this study were: (1) to identify P pools in the Entisol soils
under various long-term (25 years) fertilization treatments using sequential fractionation,
and (2) to clarify the transformation mechanisms of specific P forms using a combination of
synchrotron-based P-XANES and P-NMR spectroscopy.

2. Materials and Methods

2.1. Experimental Sites and Soil Sampling
This field experiment, belonging to one of the National Monitoring Stations of Soil Fer-
tility and Fertilizer Efficiency, is located on the campus of Southwest University, Chongqing,
China (30◦ 600 N, 10◦ 260 E). This region has a subtropical monsoon climate with an annual
mean temperature of 18.3 ◦ C, precipitation of 1106 mm, and sunshine of 1294 h, respec-
tively [17]. The detailed experimental design was reported previously [17]. In brief, the
long-term field experiment, consisting of 10 m × 12 m plots, was arranged in a com-
pletely randomized design with a rice–rice–winter wheat rotation system under various
fertilization treatments including PK (application of chemical P and K fertilizers), NK
(application of chemical N and K fertilizers), NPK (application of chemical N, P and K
fertilizers), NPKM (application of chemical P and K fertilizers with pig manure), and NPKS
(application of chemical P and K fertilizers with rice straw). For each crop growth season,
chemical fertilizers of N 150 kg/hm2 , P2 O5 75 kg/hm2 , and K2 O 75 kg/hm2 were applied.
Pig manure (M), containing total N 1.34%, total P 1.30% and total K 0.8%, was applied
at 22,500 kg/hm2 per year; while crop straw, with percentages of N 0.49%, P2 O5 0.18%
and K2 O 0.75%, was returned to agricultural fields at 7500 kg/hm2 per year. Soil samples
(sandy loam), collected from three subplots (top 0–20 cm) in each plot in 2015 were used
for this study. The reference soil, collected in 1991 before starting the long-term experiment,
was also included in this study for comparison. All the soils were air dried and sieved
to <2 mm before analysis. Soil pH was measured in water (1:2.5 w/v), total phosphorus
(TP) was digested with H2 SO4 -HClO4 , and Po was determined by the ignition method,
according to the recommended methods [18]. Total carbon, inorganic carbon and total N of
each soil was determined by an element analyzer (Elementar EA1112, Germany), and the
organic carbon content of each soil was obtained by the subtraction of inorganic carbon
from total carbon. Three replicates were measured for these selected properties of each soil
under different fertilization regimes.

2.2. Sequential Fractionation

The soil P fractions from different fertilization treatments were investigated using the
method recommended by Jiang and Gu [19], which defined P fractions as: Ca2 -P, Ca8 -P,
Al-P, Fe-P, occluded (O)-P and Ca10 -P, extracted by 0.25 M NaHCO3 , 0.5 M NH4 Ac, 0.5 M
NH4 F, 0.1 mol L−1 NaOH-Na2 CO3 , 0.3 M citrate-dithionite and 0.25 M H2 SO4 , respectively.
In brief, the soil was sequentially extracted by the aforementioned chemical solutions
in three replicates using a soil–water ratio of 1: 50. All fractions were centrifuged, and
the supernatants were analyzed for TP by inductively coupled plasma-optical emission
spectroscopy (ICP-OES, Agilent 5110, USA) and molybdate-reactive P (MRP, hereby referred
to as Pi ) by the Murphy and Riley method [20], respectively. The Po concentration of each
fraction was calculated as the corresponding difference between TP and Pi .

2.3. Synchrotron-Based XANES Spectroscopy

Phosphorus K-edge XANES spectra of all the soil samples were collected at the Beam-
line 4B7A of the Beijing Synchrotron Radiation Facility (Beijing, China). Detailed informa-
tion on sample preparation and XANES measurement was described previously [11]. In
brief, powder soil samples from each treatment were attached on a P-free tape and mounted
on a sample holder. After transferring the samples into a chamber with a high vacuum,
partial fluorescence yield mode was used to collect XANES spectra of all soil samples using
Agronomy 2022, 12, 2760 4 of 13

a silicon drifted detector. Due to the limited beamtime, only one mixed soil sample for
each treatment was used to collected the P K-edge XANES spectra as before [11]. Beamline
energy was calibrated by the first derivative peak of the hydroxyapatite (HAP) spectrum to
2151.4 eV (E0). All the data were processed and normalized using Ifeffit softwares including
Athena and SixPack [21]. Library P reference spectra, which were reported previously [11],
were used for fingerprint and linear combination fitting analysis. These P reference spectra
included berlinite (AlPO4 ), hydroxyapatite [Ca5 (PO4 )3 OH, HAP], dicalcium phosphate
dihydrate (brushite, CaHPO4 ·2H2 O), monocalcium phosphate [Ca(H2 PO4 )2 , MCP], iron
phosphate (FePO4 ), and myo-inositol hexakisphosphate (myo-IHP). R factor was used to
judge the goodness of LCF.

2.4. P-NMR Analysis

All the soil samples were analyzed by liquid-state P-NMR spectroscopy after ex-
tractions using 0.25 M NaOH plus 0.05 M Na2 EDTA solution (NaOH-Na2 EDTA). The
detailed information on the NaOH-Na2 EDTA extraction and the parameters for P-NMR
measurements were described in a previous study [2]. A 10 mm probe was used for NMR
measurements. For each treatment, soils from replicate plots were composited to one sam-
ple for the NaOH-Na2 EDTA extraction. Signals were assigned to individual P compounds
or compound classes based on publications after standardizing the peak of orthophosphate
at 6 ppm. Selected sample spectra were spiked with β-glycerophosphate, myo-IHP, and
choline phosphate to confirm peak identifications. Signal areas were processed using
NUTS (Acorn NMR). Results were corrected for diester degradation by including α- and
β-glycerophosphate within the orthophosphate diesters instead of the monoesters [22,23].
Other unidentified orthophosphate monoesters and diesters were grouped into general
categories (Mono1, Mono2, Mono3, Di1, Di2; Table S1).

2.5. Statistic Analysis

One-way analysis of variance (ANOVA) was conducted for the data of soil properties
and P fractionation using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA), using a least significant
difference (LSD) test with α = 0.05.

3. Results and Discussion

3.1. Changes in Soil Properties
Different long-term fertilization treatments significantly changed the properties of
the investigated soils. Compared with the reference soil, all the investigated soils af-
ter long-term fertilization with urea had relatively low pH values, particularly for the
2015-NPK soil (Table 1). This agreed with previous reports that long-term chemical N
fertilization generally resulted in the decrease in soil pH [24,25]. Under P fertilization, the
TP contents significantly increased relative to the reference soil (~451.2 mg kg−1 , Table 1),
which suggested the accumulation of soil P after fertilization. Obviously, the amount of
P fertilization generally exceeded crop needs, which resulted in a substantial enrichment
of soil TP as a legacy P resource during the long-term experiments [26]. Furthermore, P
was relatively enriched in the PK- and NPKM-treated soils compared with the NPK- and
NPKS-treated soils after long-term fertilization. However, Po showed a relatively higher
accumulation in the NPKS- and NPKM-treated soils than in the PK- and NPK-treated soils.
The accumulation of Po was probably derived from the long-term input of manure and
crop straw [27,28]. Correspondingly, the total organic C and total N contents in the soil
after long-term NPKS treatment were relatively higher than those with other fertilization
treatments, which agreed with the previous report on organic C accumulation with long-
term residual return [29]. Lu et al. [6] found that the application of fertilizer with straw or
manure might enhance the Pi and Po pools in a Mollisol in Northeast China. Wei et al. [30]
also reported an increase of Po in the NPKS-treated soil but a slight decrease of Po in the
NPKM-treated soil. Though having a low Po content, the soil receiving long-term NK
fertilization had the highest proportion of Po , which was probably due to the depletion
Agronomy 2022, 12, 2760 5 of 13

of Pi contents in the NK-treated soils by crop uptake and enhanced Po proportion in TP

accordingly [5].

Table 1. Selected properties of the studied soil samples.

pH Organic C Total N Total P Organic P

Year Treatment
g kg−1 g kg−1 g kg−1 mg/kg %
1991 Reference 8.3 ± 0.1 a 12.23 ± 0.08 d 1.13 ± 0.02 e 451.2 ± 3.3 e 138.3 ± 24.6 e 30.7
NK 8.0 ± 0.1 a 13.57 ± 0.25 bc 1.32 ± 0.02 cd 472.8 ± 7.6 de 195.7 ± 6.1 cd 41.4
PK 7.3 ± 0.2 b 12.91 ± 0.11 cd 1.25 ± 0.02 d 1014.0 ± 14.7 a 221.3 ± 16.7 bc 21.8
2015 NPK 6.9 ± 0.2 b 13.74 ± 0.44 bc 1.28 ± 0.01 d 811 ± 22.0 c 232.3 ± 1.8 bc 28.6
NPKM 7.0 ± 0.1 b 13.94 ± 0.03 b 1.40 ± 0.02 b 1008.6 ± 7.0 a 258.7 ± 14.2 ab 25.6
NPKS 7.2 ± 0.1 b 15.86 ± 0.35 a 1.58 ± 0.02 a 883.6 ± 9.1 b 282.6 ± 12.3 a 32.0
Values in each column followed by the same letters are not significantly different (p < 0.05).

3.2. Soil P Transformation by Chemical Extractions

Sequential fractionation revealed varied P fractions in the soils under different long-term
fertilization treatments. The majority of Pi was predominate as Ca10 -Pi in the soils regardless
of P fertilization or not (Table 2), which agreed with the fact that P tended to form Ca-P
precipitates in the soils with relatively high pH [2,31]. The second major Pi fraction was Fe-Pi
for all the investigated soils (Table 2), although purple soil generally had a low content of Fe
oxides but a high clay content [32]. One possible reason is because Fe oxides were usually
coated on clay mineral surfaces and could serve as the binding sites of Pi [33]. Other Pi frac-
tions with fewer contents were arranged in an order of Al-Pi > Ca8 -Pi /Ca2 -Pi > O-Pi (Table 2).
Among the four P fertilization treatments, Ca2 -Pi fraction, as a bioavailable P pool, showed
a stronger accumulation trend in the soils under PK and NPKM fertilization relative to
the conventional NPK treatment (Table 2). These results strongly suggested that the com-
bination of manure with conventional NPK fertilization facilitated the improved soil P
availability. Oliveira et al. [34] also reported that long-term manure application significantly
increased the content of the labile Ca2 -Pi fraction in soils. The presence of Pi source in the
manure could directly contribute to the Ca2 -Pi fraction [35]. Additionally, manure induced
the release of dissolved organic carbon and its subsequent complexation of P-binding
cations (Fe3+ , Ca2+ etc.) [6], and competitive sorption with PO4 3− on mineral surfaces
was the major driving force in maintaining high availability of P [36]. Furthermore, there
were significant increases in the contents of Ca8 -Pi , Fe-Pi , O-Pi , but no significant differ-
ences for Ca2 -Pi , Al-Pi and Ca10 -Pi occurred in the soil under NPKS fertilization compared
with NPK fertilization (Table 2). Generally, crop straw could readily release PO4 3− after
degradation, which could be easily transformed into Ca8 -Pi and Fe-Pi . This is because Fe
oxides generally have relatively lower points of zero charge than Al oxides [37], and thus
have a higher binding affinity to PO4 3− under alkaline conditions. Meanwhile, crop straw
released dissolved organic carbon, which could prohibit the formation of more crystalline
Ca10 -Pi [35], thus helpful to form Ca8 -Pi .
The Po fraction extracted by the NaOH-Na2 CO3 solution was the most abundant,
accounting for more than 60% of the total Po in the investigated soils, and exhibited insignif-
icant differences among the four different P fertilization treatments (Table 2). Obviously,
Fe oxides served as the major binding phase of Po species in the investigated purple soil,
which agreed with the generally high association of organic moiety including Po to Fe
oxides in soils. The Po fraction extracted by NaHCO3 was generally considered as the
bioavailable form [38], and showed insignificant changes in the soils under PK, NPK, NPKS
and NPKM treatments (Table 2). Therefore, there was little contribution of long-term
manure and crop straw return to the enrichment of bioavailable Po in the purple soils
compared with chemical fertilization including PK and NPK treatments. Generally, crop
residue, containing various Po species [29], could directly contribute to soil Po pools after
returning into agricultural lands. Li et al. [39] reported that a two-year straw return could
significantly increase Po in paddy soil, which also facilitated the formation of labile P
 Agronomy 2022, 12, 2760 6 of 13

species. Lu et al. [6] found that the combination of NPK with crop straw significantly
increased Po fractions in the top soil of a Mollisol in Northeast China after long-term fertil-
ization. However, crop straw return, although enhancing soil Po contents, was reported to
contribute little to soil P availability [40]. Generally, the operationally defined soil P pools
by sequential fractionation may not accurately reflected soil P speciation and availability.
Therefore, synchrotron-based XANES and liquid-state 31 P-NMR spectroscopy were applied
to probe P speciation of the investigated soils under various fertilization treatments at the
molecular level.

3.3. Soil P Transformation by P K-Edge XANES

In the P K-edge XANES spectra of all the P references, different peak features could be
observed for the spectra of Ca-P, Al-P, Fe-P and IHP (Figure 1a). The Fe-P spectrum was char-
acterized by a pre-edge peak at 2148 eV, which was absent from all other spectra [1,41–43].
All the Ca-P spectra were featured by two peaks at 2155 eV and 2162.5 eV, respectively,
particularly for HAP with more prominent features [2,5,44]. Compared with the Fe-P and
Agronomy 2022, 12, 2760 7 of 13
Ca-P spectra, the Al-P spectrum had a sharper main peak at slightly higher energy and
the IHP spectrum was relatively featureless [11,41]. For the reference soil and NK-treated
soil, the resolved peak at 2162.5 eV in their P XANES spectra (Figure 1b) were well aligned
pHtotended
that of HAP
to form(Figure 1a), which
the stable Ca-Pindicated
as HAP the presence
[2,5]. of HAP
The results in these
agreed soils.
with theThis was
broadened
feature around 2155 eV in their spectra, indicating the presence of Ca-P species.toCon-
probably due to the relatively high soil pH, since P in the soil with high pH tended
form theLCF
sistently, stable Ca-P asindicated
analysis HAP [2,5].PThe
in results agreed with
the reference soil the
wasbroadened
present asfeature
HAParound
and Fe-P
2155 eV in their spectra, indicating the presence of Ca-P species. Consistently, LCF analysis
species, having a proportion of 78% and 21% of total P, respectively (Table 3). For the
indicated P in the reference soil was present as HAP and Fe-P species, having a proportion
NK-treated soil, the predominance of HAP (61%) over Fe-P (24%) and IHP (15%) oc-
of 78% and 21% of total P, respectively (Table 3). For the NK-treated soil, the predominance
curred (Table
of HAP (61%) 3).over Fe-P (24%) and IHP (15%) occurred (Table 3).

Figure 1. P K-edge XANES spectra of reference (a) and soils under different fertilization treat-
Figure 1. (b).
ments P K-edge
Dash XANES spectra ofdata;
line, experimental reference
solid (a) and
line, soilsdata.
fitting underMCP,
different fertilization
monocalcium treatments
phosphate.
(b).HAP,
Dashhydroxyapatite.
line, experimental data; solid line, fitting data. MCP, monocalcium phosphate. HAP, hy-
droxyapatite.

Table 3. Linear combination fitting of P K-edge XANES spectra of soils under different fertilization
treatments.

Year Linear Combination Fitting Goodness of Fit

Treatment HAP FePO4 Brushite IHP R Factor
Agronomy 2022, 12, 2760 7 of 13

Table 2. Sequential fractionation results of P in the soils under various fertilization treatments a .

NaHCO3 -P (Ca2 -P) NH4 Ac-P(Ca8 -P) NH4 F-P (Al-P) NaOH + Na2 CO3 -P (Fe-P) CBD-P (O-P) d H2 SO4 -P (Ca10 -P)
Treatment Recovery b c
Year Pi Po Pi Po Pi Po Pi Po Pi Po Pi Po
mg/kg
1991 Reference 123.9 1.6 ± 0.6 d 9.9 ± 0.6 ab 23.2 ± 0.3 e 11.2 ± 0.6 c 22.8 ± 1.1 c 2.7 ± 0.8 b 34.7 ± 0.6 e 54.2 ± 1.0 c 5.4 ± 0.1 c 3.1 ± 0.4 bc 299.5 ± 2.5 c 6.7 ± 1.5 c
NK 126.3 0.5 ± 0.1 d 5.4 ± 0.3 b 3.4 ± 0.5 f 2.7 ± 0.5 d 12.9 ± 0.8 d 2.3 ± 0.1 b 25.5 ± 0.9 f 86.3 ± 0.9 b 1.3 ± 0.3 d 2.5 ± 0.5 c 306.5 ± 8.1 c 4.4 ± 1.1 c
PK 106.2 52.5 ± 2.3 a 10.4 ± 1.4 a 42.2 ± 0.4 b 18.5 ± 1.1 b 93.8 ± 2.6 a 5.1 ± 1.6 ab 187.8 ± 2.4 a 105.7 ± 2.1 a 18.6 ± 0.1 a 5.2 ± 0.4 a 446.8 ± 11.8 a 8.5 ± 2.0 c
2015 NPK 111.6 23.3 ± 1.4 c 11.0 ± 2.1 a 27.2 ± 0.5 d 13.3 ± 0.6 c 64.4 ± 3.2 b 3.1 ± 1.5 b 117.6 ± 1.5 d 97.5 ± 4.9 ab 14.2 ± 0.7 b 5.0 ± 1.2 ab 399.0 ± 12.3 b 26.7 ± 0.4 a
NPKM 104.5 35.5 ± 1.7 b 11.1 ± 1.3 a 46.4 ± 0.6 a 22.7 ± 1.6 a 87.5 ± 2.9 a 6.5 ± 1.3 a 171.5 ± 4.8 b 109.4 ± 7.3 a 18.9 ± 0.4 a 4.6 ± 0.5 ab 426.1 ± 6.0 a 20.5 ± 1.4 b
NPKS 113.3 26.7 ± 2.0 c 14.1 ± 2.1 a 34.8 ± 0.4 c 17.0 ± 0.7 b 64.9 ± 2.2 b 3.7 ± 0.8 ab 139.6 ± 2.0 c 110.2 ± 4.6 a 18.4 ± 1.6 a 3.3 ± 0.1 abc 396.2 ± 7.4 b 27.2 ± 0.2 a
a–fValues in each column followed by the same letters are not significantly different (p < 0.05). Pi , inorganic phosphorus. Po , organic phosphorus. CBD-P, citrate-bicarbonate-dithionite-
extractable phosphorus.
Agronomy 2022, 12, 2760 8 of 13

Table 3. Linear combination fitting of P K-edge XANES spectra of soils under different fertiliza-
tion treatments.

Goodness
Year Linear Combination Fitting
of Fit
Treatment
HAP FePO4 Brushite IHP R Factor
Proportion (%)
1991 Reference 72 ± 1 28 ± 1 0.0028
NK 61 ± 1 24 ± 1 15 ± 1 0.0024
PK 30 ± 1 35 ± 1 35 ± 1 0.0035
2015 NPK 37 ± 1 32 ± 1 31 ± 2 0.0035
NPKM 30 ± 1 29 ± 1 41 ± 1 0.0051
NPKS 36 ± 1 34 ± 1 30 ± 2 0.003

Under long-term P fertilization, the formation of new P species as brushite, besides

HAP and Fe-P, occurred for all the soils under various P treatments, including PK, NPK,
NPKM and NPKS, according to the LCF analysis (Table 3). The formation of brushite in
the alkaline soils after long-term fertilization using calcium superphosphate was reported
previously [2,45,46]. For all the long-term fertilization treatments, the proportions of
brushite ranged from 30 to 41% of the total P in the investigated soils (Table 3). Generally,
brushite were regarded to be available, and could assigned as NaHCO3 -P fraction. The
relatively higher percentage of brushite by the LCF analysis over NaHCO3 -P fraction by SF
for each soil probably resulted from the inaccuracy in the characterization of P fractions by
SF [11], as well as the limited P standards for the LCF analysis [35]. For the soil under the
NPKM treatment, there was relatively higher brushite proportion of the TP than that under
the NPK treatment. This result agreed with the higher proportion of Ca2 -P in the NPKM-
treated soil compared with the NPK-treated soil by SF (Table 2). Considering the high
availability of brushite in the soil, long-term NPKM fertilization facilitated the retention
of available P pools in the soil for crop uptake, which benefited enhancing P utilization
efficiency. Conventional application of chemical NPK fertilizer resulted in the formation
of brushite, with a percentage of 31% of the total P in this soil (Table 3). This could help
to supply nutrients for crop uptake, but also lead to the relatively high accumulation of
residual fertilizer P in the soil as legacy P. Consequently, a higher cost of fertilization was
expected due to its relatively low P utilization efficiency. Replacing manure with crop
straw for long-term NPK fertilization showed insignificant changes in the proportion of P
speciation in this soil compared with conventional NPK fertilization (Table 3). However, the
total Po content in this soil significantly increased after long-term NPKS fertilization relative
to the NPK fertilization (Table 1). Therefore, crop straw probably mainly contributed to
Po accumulation in this soil. Compared with the long-term NPK treatment, the soil under
long-term PK fertilization maintained relatively high proportion of brushite (Table 3). This
agreed with the high content of the Ca2 -P fraction in this soil by SF (Table 2). These results
were probably caused by low crop biomass under N deficiency, which led to less P uptake
by crops. The lack of IHP as probed by P-XANES in these soils was probably due to the
limited sensitivity of P-XANES to Po . Generally, the proportion of IHP greater than 15%
could be probed by P-XANES in soils [41]. As indicated by NMR analysis (shown later
in Table 4), the percentages of IHP ranged from 4.0 to approx. 12.1% in the investigated
soils under different fertilization treatments.

3.4. Soil P Transformation by P-NMR

The extracted Pi by NaOH-EDTA solution accounted for 73.7–84.4% of the total P in
the long-term P-fertilized soils, but had a percentage of 29.1% of the total P in the soil under
long-term NK fertilization (Table 4). Obviously, the majority of P in the soil after long-term
NK fertilization was Po , while long-term P fertilization maintained high amounts of Pi in
the investigated soils. The low proportions of Po in soils under long-term PK and NPK
Agronomy 2022, 12, 2760 9 of 13

treatments agreed with previous reports that chemical P fertilization had little impact on Po
variation in soils [47,48]. The relatively high proportion of Po extracted by NaOH–EDTA
solution in the NPKM- and NPKS-treated soils indicated the accumulation of Po in the soils,
which was consistent with higher amounts of Po in NPKM- and NPKS-treated soils relative
to the PK- and NPK-treated soils by the ignition method (Table 1).

Table 4. Phosphorus form classes or ratios of form classes in NaOH-EDTA extractions of the studied
soils in 2015 determined by P-NMR spectroscopy.

NaOH-EDTA Extraction
Extracted
Total
Treatment Total P Pi Po Mono Di Cmono Cdi D:M cD:M
IHP
—————————————————–Proportion (%) —————————————–
NK 19.4 29.1 70.9 12.1 59.4 10.4 43.4 26.4 0.2 0.6
PK 22.9 84.4 15.6 4.4 13.3 1.9 8.5 6.7 0.1 0.8
NPK 32.2 82.0 18.0 4.0 16.1 1.5 12.1 5.5 0.1 0.5
NPKM 16.9 73.7 26.3 5.1 23.3 2.6 16.8 9.2 0.1 0.5
NPKS 33.2 76.1 23.9 5.1 21.3 2.2 14.7 8.9 0.1 0.6
Pi , inorganic P; Po , organic P; total IHP, the total of inositol hexakisphosphate; Mono, orthophosphate monoester;
Di, orthophosphate diesters; D:M, the ratio of orthophosphate diesters to orthophosphate monoester; C denotes a
correction for diester degradation products.

Multiple biogenic P species were characterized by liquid-state P-NMR, which could

be grouped into Mono, Di, phosphonates and complex Pi (Figure 2). The orthophosphate
monoesters consisted of stereoisomers of inositol hexakisphosphate (myo- and scyllo-IHP),
choline phosphate, glucose 6-phosphate, and degradation products of orthophosphate
diesters (α- and β-glycerophosphate). Changes in the proportion of individual biogenic
P species reflected varied P biochemistry among the treatments. Compared with the
P-fertilized soils, there were much higher proportions of Mono and total IHP in the soil after
long-term NK fertilization with or without correction for degradation products (Table 4).
Previous studies also indicated there were relatively high proportions of Mono and IHP in
the P-depleted soils without long-term P fertilization [49]. Moreover, the soils receiving
NPKM and NPKS fertilization exhibited a higher accumulation trend of Mono and IHP
relative to the conventional NPK-treated soil (Table 4). The contribution of manure and crop
straw to the accumulation of Mono and IHP had been widely reported, because Mono and
IHP in the manure and crop straw had relatively high resistances to biodegradation and
transformation mediated by soil microbes, and thus tended to accumulate in soils [50,51].
Among stereoisomers of inositol hexakisphosphate, myo-IHP was predominant in all the
investigated soils where the distribution of myo-IHP was relatively higher in the soils under
NPKM and NPKS treatments than under NPK and PK treatments (Table S1). Generally,
myo-IHP was widely reported to be present in crop straw and manure [1,2,51], which
had relatively high stability after being sorbed on soil clay minerals [11]. This probably
accounted for the higher accumulation of myo-IHP in the soils after long-term NPKM and
NPKS fertilization, since purple soils generally were enriched with clay minerals [32].
Furthermore, orthophosphate diesters, as a readily bioavailable Po category, were
mainly DNA in these soils (Figure 2 and Table S1). Similar to Mono, the distribution of Di
in these soils followed in the order of NK > NPKM ≈ NPKS > PK ≈ NPK (Table 4). The
highest proportion of Di in the NK-treated soil resulted from the highest Po proportion
in this soil. For the NPKM and NPKS treatments, the relatively higher proportion of Di
could probably be due to the contribution of manure and crop straw during the long-term
fertilization. Previous studies indicated that manure and crop straw both contained Di
species [27,28], and thus long-term fertilization of manure and crop straw might facilitate
the accumulation of Di species. Moreover, DNA was the most predominant Di species
in each of the soils, and the addition of manure and crop straw probably stimulated the
growth of microbes in soils. This also probably accounted for the relatively high proportion
Agronomy 2022, 12, 2760 10 of 13

of DNA in the soils after long-term NPKM and NPKS treatments compared with the NPK
treatment (Table S1) [25,52]. Additionally, the ratio of Di to Mono was generally regarded
as an indicator of the biodegradability of Po in soils. There was very limited variation in
the corrected ratio of Di to Mono among the treatments of NK, NPK, NPKM and NPKS,
but it was relatively lower than that of the PK treatment (Table 4). These results indicated
that N deficiency might increase the biodegradability of Po in soils, which was probably
linked to microbe-mediated degradation of soil organic matter for N nutrition and thus
enhanced the Di contents in soils with long-term PK treatments. Such molecular-level
evidence on the enhanced biodegradability of Po in soils by N deficiency has been never
reported at long-term experimental sites in previous studies [1,2,49,53]. Tian et al. [54]
reported there was a relatively high proportion of Di but a low proportion of Mono in the
soils without N addition compared with the soils with N addition. Meeds et al. [9] also
reported that soil N deficiency facilitated the release of exoenzymes by ectomycorrhizal
fungi and enhanced Di in soils. Therefore, this study strongly suggested that the reduction
of N fertilization facilitated enhancing the biodegradability of Po in the investigated soils,
although the reasonable amounts of N reduction for the increase of soil Po availability needs
further study. Consistently, there were relatively higher proportions of pyrophosphate and
Agronomy 2022, 12, 2760 polyphosphate in the NK-treated soil relative to other P-fertilized soils (Table10 S1),
of 13which
also indicated stronger microbial involvements on P transformation in this soil [49,55].

Figure 2.
Figure 2.Solution
SolutionP-NMR
P-NMRspectra of of
spectra thethe
investigated soilssoils
investigated under different
under fertilization
different treatments.
fertilization treatments.

4. Conclusions
This study enabled us to understand comprehensively the impact of multiple
long-term fertilization regimes on the transformation mechanisms of P in Entisol soils at
the molecular level. It indicated that HAP, as a stable P form, had the highest proportion
in the soil with NK fertilization, but significantly reduced under long-term P fertilization
Agronomy 2022, 12, 2760 11 of 13

4. Conclusions
This study enabled us to understand comprehensively the impact of multiple long-
term fertilization regimes on the transformation mechanisms of P in Entisol soils at the
molecular level. It indicated that HAP, as a stable P form, had the highest proportion in
the soil with NK fertilization, but significantly reduced under long-term P fertilization
including PK, NPK, NPKM and NPKS, thus increasing soil P bioavailability. Moreover,
long-term withdrawing of N application under a PK-fertilization regime could facilitate the
increase of soil Po bioavailability. Given the large stock of Po in soils, the attempt to reduce
N fertilization may be helpful to dig legacy Po pools, particular in Southwest China, rich in
Entisol (purple) soils, which could help us to mitigate the shortage of chemical P fertilizers
in future. Moreover, the NPKM could maintain a relatively high P availability by mainly
forming an available Pi form as brushite in this soil, although manure input contributed
little to the available Po . Additional attention should be paid to P loss during the rice
growth season due to high P availability after NPKM fertilization and the subsequent
potential P loss through surface runoff. Additionally, the NPKS could significantly increase
Po accumulation in this soil, though having limited impact on the increase of soil available
P pools. This study has provided new molecular-level insights into P speciation and
transformation under multiple fertilization treatments, which benefits improving future
fertilization strategies and protection of surface water quality in Southwest China.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/agronomy12112760/s1, Table S1: Phosphorus forms in the NaOH-
EDTA extractions as determined by integration of P-NMR signals from the studied soils.
Author Contributions: Conceptualization, J.L. (Jin Liu) and Y.M.; supervision, funding acquisition,
project administration, writing—original draft preparation, J.L. (Jin Liu); investigation, C.H., Y.Z.
and D.Y.; methodology, J.L. (Jin Liu), J.Y., L.Z. and Y.H.; resources, J.L. (Jumei Li) and X.S.; writing—
review and editing, J.Y., P.S. and Y.C. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the National Natural Science Foundation of China (No. 41977091).
Data Availability Statement: Not applicable.
Acknowledgments: Thanks to the staff for assistance with P-NMR measurements in the Agro-
Environmental NMR Microstructure Lab, Institute of Environment and Sustainable Development
in Agriculture, Chinese Academy of Agricultural Sciences. We also thanks the staff at the Beamline
4B7A of the Beijing Synchrotron Radiation Facility for their help on P K-edge XANES measurements.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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