Forest Ecology and Management 435 (2019) 170–179

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Forest Ecology and Management

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Phosphorus pool responses under different P inorganic fertilizers for a T

eucalyptus plantation in a loamy Oxisol
Estela Covre Foltrana, , José Henrique Tertulino Rochaa,b, José Henrique Bazanic,
⁎

José Leonardo de Moraes Gonçalvesa, Marcos Rodriguesd, Paulo Pavinatod,

Giancarlo Ribas Valdugae, Javier Errof, Jose M. Garcia-Minaf
a
Forest Science Department, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, SP, Brazil
b
Agriculture and Forest Engineering College, FAEF, Garça, SP, Brazil
c
Geplant Forest Technology, Piracicaba, SP, Brazil
d
Soil Science Department, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Piracicaba, SP, Brazil
e
Timac Agro Brazil, Roullier Group, Porto Alegre, RS, Brazil
f
Department of Chemistry and Soil Chemistry, University of Navarra, Pamplona, Spain

ARTICLE INFO ABSTRACT

Keywords: Phosphate fertilizers play an important role in plant nutrition. Different P fertilizer sources such as high-solu-
Hedley fractionation, Phosphate fertilization bility (simple superphosphate, SSP), low-solubility (rock phosphate, RP) and complex superphosphate (CSP) are
Complex Super Phosphate (CSP) available for plant supplementation. The objective of this study was to investigate the short- and long-term
Organic P redistribution of soil P after application of different P sources at establishment of an Eucalyptus forest stand. We
carried out two experiments to identify the short- and long- term changes in a Brazilian Oxisol. To property
identify the P pools in different times, Hadleýs fractionation methodology was applied to a long-term studies and
citrate and oxalate to short-term. From zero to 180 days, the soluble P fractions were not altered in the non-
fertilized treatment. Under SSP, a slight increase in this P fraction was found until 30 days, followed by a
decrease in later evaluations. During the same period, a slight reduction in Pi extracted by citrate and oxalate
was found under the control and a large reduction (approximately 50%) under the SSP treatment. Intermediate
behavior was observed under the CSP and RP treatments, whereas there was an increase in P-citrate and P-
oxalate until 30 days followed by a reduction afterwards. These results suggest that this pool comprises a po-
tential bioavailability of P to plants. Different fertilizer sources did not increase the most recalcitrant P pool.
However, different sources increased the organic P pool, mainly organic moderately labile P at long-term eva-
luations. Organic labile pools showed a fertilizer-specific response where CSP and RP increased respectively by
43% and 41% during the first year, and decreased to 39% in CSP treatment and 50% in RP treatment during the
third year. Available P pool was highly dependent on inorganic and occluded pools and the organic pool acted
predominantly as a sink of P on available and inorganic pools. The results reinforce the high level of recalci-
trance of the organic pool and the fact that Eucalyptus plants must access pools of residual P in order to maintain
their nutritional demands.

1. Introduction
Total P concentrations in soils range from 101 to 103 mg kg−1, depending
on soil horizon (subsoil < top-soil), substrate (sandy < loamy), pedogen-
esis (older < younger), land-use (forest < pasture < agriculture) and
land-use intensity (extensive < intensive) (Kruse et al., 2015). Soil P exists in
inorganic and organic compounds that range from ions in solution to very
stable inorganic and organic compounds in the solid phase. The inorganic P
(Pi) compounds often bond with amorphous and crystalline forms of Al, Fe,
and Ca in a very stable way, normally called fixation or specific adsorption.
The organic (Po) compounds are associated with rapidly to slowly decom-
posable organic molecules, such as nucleic acids, phospholipids, sugar
phosphates, inositol phosphates, and recalcitrant humic substances (Hedley
et al., 1982; Negassa and Leinweber, 2009).
While the proportion of total fertilizer use in forests and the average
annual application rates are lower than those applied in cereals, ferti-
lizer types are similar to annual crops. In forest systems, P is commonly
applied at rates of 10 to 50 kg ha−1 each rotation (Gonçalves et al.,

⁎
Corresponding author at: USP, ESALQ, 11 Av. Padua Dias, Piracicaba, SP CEP 13418-900, Brazil.
E-mail address: estela.foltran@uni-goettingen.de (E.C. Foltran).

https://doi.org/10.1016/j.foreco.2018.10.053
Received 22 March 2018; Received in revised form 24 October 2018; Accepted 25 October 2018
0378-1127/ © 2018 Published by Elsevier B.V.
E.C. Foltran et al.
2008), but under low soil availability, rates of P exceeding 60 kg ha−1
have doubled in wood volume (Fernandez et al., 2000). There is far less
frequent use of P fertilizers in forest plantations, where the application
is normally just during the first year (Smethurst, 2010). This is possible
because trees are highly efficient in P absorption and retranslocation
(Fife et al., 2008; Saur et al., 2000) and the non-retranslocated P returns
to the soil through litterfall and litter decomposition. In this context,
organic-P forms are essential to maintain forest P nutrition (Vincent
et al., 2010), contributing to the sustainability of commercial planta-
tions (Huang et al., 2017).
In highly weathered acidic soils, rich in Al-and Fe-oxides (e.g.,
Oxisols and Ultisols predominant in tropical regions), Pi is strongly
sorbed on the edges of silicate clay minerals and on pedogenic Al- and
Fe-oxides, decreasing the use efficiency of applied P fertilizer. Tree
response to P-fertilizer application depends on different factors, such as
weather, genetic material, soil P availability, soil P fixation capacity,
fertilizer type, among others. In the field, the type and placement of the
applied fertilizer are very important for use efficiency in soils with high
P-fixation capacity (Fernandez et al., 2000). Proper management of P
application contributes to optimize the costs of fertilization and to
maintain the economic competitiveness of forest plantations.
The usual management of phosphate in commercial plantations in
Brazil is based on soluble and low soluble-P sources, or both. New
technologies of phosphate fertilizers have been developed intending to
increase P use efficiency, one being orthophosphate-humic substances
complexation (P-metal-HS complex). These organic molecules de-
creases the fixation processes in soil and improve the utilization of this
nutrient by plants (Erro et al., 2012).
Despite the relevance of organic P in the nutrition of tropical forest
trees (Huang et al., 2017), this P pool has not been considered in
standard soil tests for P availability, such as Mehlich-III, Bray or Olsen P
(Darch et al., 2016). Different sequential-P-fractionation schemes have
been developed to quantify P in the previously described inorganic and
organic forms, as summarized by Pierzynski et al. (2005). Hedley et al.
(1982) developed a sequential extraction technique for separating soil P
into various inorganic (Pi) and organic (Po) fractions. These schemes
have been widely applied for P dynamics in tropical soils (Gatiboni,
2003; Cherubin et al., 2016; Costa et al., 2016; Rodrigues et al., 2016),
and can be useful to identify the dynamic of P fertilizers poor nutrient
conditions.
In a review on the use of the Hedley method, studying agroeco-
systems of tropical and temperate climates, with different soil and
management use, Negassa and Leinweber (2009) reported that in-
organic and organic fractions of P could act as a source or sink of
Table 1
Soil physical and chemical attributes of the experimental sites.
Soil layer (cm) Clay(B) (g kg−1) pH ΔpH1 Organic C2 (g kg−1)
KCl H2O
0–10 201 3.2 4.5 −1.3 18.0
10–20 176 3.6 4.7 −1.1 10.3
20–30 177 3.6 4.7 −1.1 7.9
30–40 177 3.7 4.7 −1.0 2.9
1
ΔpH – Difference between pH(KCl)e pH(H2O).
2
Dichromate/colorimetric; N sulfuric acid extraction/ Kjeldahl; P, K extrator Mehlich-1; Ca; Mg extractor ammonium acetate 1 mol L−1; Al KCl extractor 1 mol
−1
L (van Raij et al., 2001).
171
Forest Ecology and Management 435 (2019) 170–179
available P to plants, as a result of several factors such as climate, soil
texture, organic matter content and mineral fertilization. Although
some literature supports the potential of the Hedley method in studies
that involve the dynamics of P in agroecosystems soils (Gatiboni, 2003;
Cherubin et al., 2016; Costa et al., 2016; Rodrigues et al., 2016) there
are few studies into tropical soil in Eucalyptus plantations.
In this context, we aimed to investigate the short- and long-term
redistribution of soil P following application of different P sources at
planting of an Eucalyptus forest and to determine the accuracy of some
methods to estimate P availability for forest plantations. The hy-
potheses were that during forest development (1) the organic P pools
are the most responsible for providing P for the plants during the first
year (2) the labile and moderately labile inorganic P pools increase
while occluded P decreases during the long-term Eucalyptus cycle in
non-fertilized plots, (3) P-limited Eucalyptus plants can access stable soil
P pools, and (4) the high-solubility fertilizers promote an increase in
total soil P mainly in the recalcitrant P due to the high affinity of P with
Al-and Fe-oxides.
2. Materials and methods
2.1. Study sites
The experiments were carried out in two Eucalyptus grandis planta-
tions established at Itatinga, São Paulo state (23°02́S, 48°38́W, ∼850 m
elevation), which is managed by the Forest Science Department of the
College of Agriculture “Luiz de Queiroz”, University of São Paulo,
Brazil. This region has a humid subtropical climate (Köppen classifi-
cation, Cfa) characterized by hot and humid summers (Alvares et al.,
2013). The coldest and driest months are June and July (means of 16 °C
and 30 mm, respectively) and the warmest and rainiest months are
December and January (means of 24 °C and 400 mm, respectively). The
mean annual rainfall is 1300 mm over the last four years has been very
irregular, with atypical dry periods during the summer.
The native vegetation of the site was Cerrado, or Brazilian savanna.
The local topography is flat, with deep loamy and dystrophic soils,
classified as Oxisols, developed from Cretaceous sandstones (Table 1)
and their mineralogy is dominated by quartz, kaolinite and oxyhydr-
oxides.
Two experimental sites were established, 300 m apart. The first
experiment was focused at a plant level, using plant as a repetition to
measure the short-term effects, to address the long-term effects, a
second experiment was carried out focusing at stand level using a
randomized block design. Both experiments were established in a
Total N P Exchangeable cation
K Ca Mg Al
−1 −1
mg kg mmolc kg
1792 2.0 0.4 7.2 4.8 15.0
1176 3.0 0.2 5.5 3.4 13.1
952 1.0 0.2 2.7 1.7 9.4
679 1.0 0.1 1.3 0.9 7.0
E.C. Foltran et al. Forest Ecology and Management 435 (2019) 170–179

former Eucalyptus saligna (Sm.) plantation, managed by coppice,
without fertilizer application from 1940 to 1998. Stumps from the
previous rotation were suppressed by glyphosate application (4 L ha−1)
and E. grandis seedlings were planted from 1998 to 2011 with low
fertilizer inputs (300 kg ha−1 NPK 10:20:10). Only the bole (wood and
bark) were removed from the plot and harvest residues were spread
uniformly all over the field.
At both sites, E.grandis seedlings were established with genetically
improved (half-sib seeds) in April 2012 for the establishment of the
long-term study and in April 2013 for the short-term study. Seedlings
were planted between stumps of the previous rotation at a planting row
spacing of 2 × 3 m.
2.2. Short-term study
For each treatment, a plot of 612 m2 was established with 102 plants
allocated in six rows of 17 plants with a double-row buffer. Within each
plot, soils were collected at different ages (from 2 to 374 days after
planting). Between each collection, a line of plants was left as a border
to eliminate the sampling influences. In each collection, three trees in
the planting line were selected and used as a replicate. Treatments
tested were as follows: (i) control – without P fertilizer application, (ii)
soluble phosphates (monoammonium phosphate – MAP and single su-
perphosphate – SSP), (iii) low-solubility phosphate (rock phosphate –
RP) and (iv) complexed humic-phosphates (P-metal-HS complex + RP –
CSP).
2.3. Long-term study
A complete randomized block design was established with the same
four treatments as the short-term study considering four replicates with
25 plants on each plot and two buffer rows. Herbicide was applied to
control weeds three times during the first year until canopy closure. In
this study, the soil was sampled before planting or fertilization, and
after one, two, and three years since planting.
2.4. Soil preparation and fertilizer application
At both experimental areas minimal tillage was applied, with soil
preparation just along the planting line (60 cm in depth) and 2 Mg ha−1
of dolomitic lime (not incorporated) was applied before planting. Along
the planting line 60 kg ha−1 of available P2O5 (HCl 2%) was applied at
10 cm depth, 30 kg ha−1 of fritted elements (1.8% B, 0.8% Cu, 2.0%
Mn, 9.0% Zn, 3.0% Fe, 0.1% Mo) and a small amount of N (17 kg ha−1)
and K (36 kg ha−1) to stimulate plant establishment. Highest N and K
doses were divided into two surface applications occurred at 2.7 months
and considered 20 kg ha−1 N as ammonium nitrate, 47 kg ha−1 K2O as
potassium chloride and 5 kg ha−1 B as ulexite; and at 5.7 months with
30 kg ha−1N and 63 kg ha−1of K2O applied with the same previous
fertilizer sources. (Table 2)
3. Soil sampling and P analysis
3.1. Short-term study
Soil samples were collected before planting and at 2, 30, 120, 180
and 360 days after planting. Three subsamples were collected from the
planting row (i.e., 25, 50 and 100 cm from each selected plant) at 0–10,
10–20, 20–30 and 30–40 cm depths, and grouped in one composite
sample per tree and depth. Samples were dried at 45 °C and sieved
through 2 mm mesh.
Field experience shows that soil preparation and fertilizer dis-
tribution are heterogeneous at operational scale. This heterogeneity
generates variation in resource availability, to avoid those errors,
samples were collected every 10 cm as mentioned above and the same
soil volumes were combine into a composite (0–40 cm) sample to soil
analysis.
Soil P fractionation analysis procedure was adapted from Erro et al.
(2011). It provides the P fractions soluble in water, anion-exchange
resin (AMI-7001CRX –2 cm2), sodium citrate (pH 5.0) and sodium ox-
alate (pH 4.0), to evaluate the potential bioavailable P pools. This was
followed by four sequential steps: (1) 1 g soil: 10 mL of deionized water
– 16 h agitation – P-water; (2) anion exchange resin membrane – P-resin
– 16 h agitation; (3) 0.3 M sodium citrate acidified to pH 5.0 with 1 M
HCl– P-citrate – 30 min agitation; and (4) 0.2 M sodium oxalate acid-
ified to pH 4.0 with 1 M HCl –P-oxalate– 4 h agitation (black room).
Each P-extracted fraction was quantified by induced coupled plasma
optical emission spectrometry (ICP-OES).
3.2. Long-term study
Soil was sampled at 0, 12 and 36 months after planting at 0–10 and
10–20 cm depths. At each sampling time 18 subsamples in the planting
row were collected, using only three blocks. Samples were dried at
45 °C and sieved through a 2 mm screen. A proportional soil volume
sample of each depth was collected and combine into a composite
sample 0–20 cm for phosphorus fractionation analysis.
Phosphorus pools were obtained by measuring inorganic P (Pi) and
organic P (Po) following the procedure described by Hedley et al. (1982)
and modified by Condron et al. (1985). P was sequentially extracted from
1.0 g of dried soil sample (Maranguit et al., 2017) in the following order: (1)
anion-exchange resin membrane - Piresin fraction (AMI-7001CRX – 2 cm2);
(2) 0.5 M sodium bicarbonate (NaHCO3at pH 8.5) (Pibic and Pobic fractions);
(3) 0.1 M sodium hydroxide (NaOH) (Pihyd0.1 and Pohyd0.1 fractions); (4)
1.0 M chloridric acid (HCl) (PiHCl fraction); and (5) 0.5 M NaOH (Pihyd0.5
and Pohyd0.5 fractions). The remaining soil was oven dried and digested with
H2SO4 + H2O2 (5) – Presidual fraction to determine residual P. Phosphorus
concentrations in the acid extractants were determined using the Murphy
and Riley (1962) methodology. Inorganic P (Pi) fractions in the alkaline
extractants (NaHCO3 and NaOH) were determined using the Dick and
Tabatabai (1977) methodology. After determining total P in the alkaline

Table 2
Total macro and micro-nutrient applied in each treatment.
Treatment N Total P P P K2O Ca Mg S B Cu Mn Zn Fe Mo
CNA + Water HCi2%
kg ha−1
Control 66 – – – 146 760 240 75 5.5 0.8 2.0 4.1 0.9 0.03
SSP 66 60 60 – 146 772 240 69 5.5 0.8 2.0 4.1 0.9 0.03
CSP 66 71 52 8 146 787 240 63 6.4 0.8 2.0 4.1 0.9 0.03
RP 66 174 – 60 146 976 240 75 5.5 0.8 2.0 4.1 0.9 0.03

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E.C. Foltran et al. Forest Ecology and Management 435 (2019) 170–179

extractants using ammonium persulfate + H2SO4 digestion in an autoclave, 4. Results

organic P (Po) was estimated by the difference between total P and Pi in
each extractant.
Phosphorus fractions were grouped into pools according to their
potential plant availability as “labile P” (Piresin + Pibic + Pobic), “mod-
erately labile P” (Pihyd0.1 + Pohyd0.1 + PiHCl) and “non-labile P”
(Pihyd0.5 + Pohyd0.5 + Presidual) (Rodrigues et al., 2016).
Soil was sampled at Native Cerrado forest (Brazilian Savannah)
nearby from experimental area. Across the native Cerrado forest 4
subsamples were collected, considering 0–10 and 10–20 cm depths, to
use as a baseline.
3.3. Data analysis
The Bartlett test was used to check homoscedasticity and the normal
distribution of the residuals was checked using the Shapiro–Wilk test.
When necessary, the data were log-transformed to compensate for the
heterogeneity of variance, before the analysis of variance. The analysis
of variance (F test) was applied using as variation sources treatment,
time, and interaction treatment x time. When significant (p < 0.05)
means were compared using the LSD test (α = 0.05). Principal com-
ponent analysis (PCA) was performed on soil properties (pH and OM)
and different P pools and fractions by including all sampling points and
time to discern the effect of P fertilization and P-bioavailability at long-
term study site. Soil properties and P pools data were assessed for
normal distribution and transformed accordingly for PCA analysis in R-
Project 3.0.3. For PCA of soil, edaphic vectors with Pearson correlation
greater than 0.5, were overlayed as indicators of the key drivers.
All the data were processed using the software package R-Project
3.0.3 (www.r-project.org) and the graphics were processed using
SigmaPlot software.
4.1. Short-term – labile inorganic P
From zero to 180 days the P-water and P-resin fractions showed no
changes in the control treatment (Table 3). Under SSP, a slight increase
in these two P fractions was found until 30 days, followed by a decrease
for the coming evaluations. During the same period a slight reduction in
P extracted by citrate and oxalate was found under the control and a
large reduction (approximately 50%) under the SSP treatment. Inter-
mediate behavior was observed under the CSP and RP treatments
whereas an increase in P-citrate and P-oxalate until 30 days was found
followed by a reduction afterwards. However, between 180 and
360 days, an atypical decrease in rainfall occurred in the experimental
area, promoting water stress (Fig. 1). Tree growth rate decreased, ob-
viously affecting the P dynamics in the soil. During this dry period the
P-water and P-resin under the control decreased, and the SSP increased.
No change was observed in P-citrate and P-oxalate under the control,
but they increased under SSP and decreased under CSP and RP as
compared to the experimental set up (Table 3).
4.2. Long term - Labile, moderately labile and non-labile P pools
Total soil P concentration increased with P fertilizer application
(Fig. 2). From zero to 12 months after planting, increases of 10% in the
total P under the SSP, 35% under the CSP and 115% under the RP
treatment were found (p > 0.001). From 12 to 36 months after
planting all sources decreased in total P concentration, as expected,
being more intense under soluble P fertilizers, with 31.27 mg kg−1 for
CSP and 28.50 mg kg1 for SSP (Fig. 2).
From zero to 12 months all Po pools decreased under the control,
especially the moderately labile fraction (Fig. 2b). However, at

Table 3
Potential bioavailable P concentrations in soil (0–40 cm) extracted by water and resin (available P), citrate pH 5.0 and oxalate pH 4.0 (potential available P) under
different P fertilizer sources.
Days1 Water Resin Citrate pH 5.0 Oxalate pH 4.0 Total

mg kg−1

0 2.91 ± 0.76 1.19 ± 0.20 29.33 ± 0.68 29.89 ± 1.35 65.0

2
Control
30 14.40 aB3 0.90 aB 27.98 aC 28.73 aC 76.4 aC
120 12.90 abB 1.10 aC 23.78 abB 26.70 aB 66.2 aB
180 14.89 aC 1.03 aC 18.08 bC 17.18 aB 58.5 aC
360 7.13 bC 0.25 aB 18.15 bB 16.45 aC 46.0 aC

SSP
30 21.43 aB 17.38 aA 52.40 aB 85.90 aB 201.9 aB
120 20.77 abA 2.91 cBC 30.08 bA 48.15 bcA 116.2 bA
180 6.46 cD 6.00 cB 26.93 bB 31.88 cB 82.0 cBC
360 26.13 aA 13.60 bA 34.60 bA 55.70 bA 135.4 bA

CSP
30 2.83 bC 5.32 bB 74.10 aA 113.18 aB 203.0 aB
120 18.93 aA 7.95 bB 27.50 bcB 38.05 bAB 101.5 cAB
180 23.49 aB 21.94 aA 41.10 bA 59.78 bAB 158.9 bcB
360 24.02 aAB 4.04 bB 24.65 cAB 29.80 bBC 92.9 cB

RP
30 24.35 bcA 26.63 aA 49.28 aB 147.98 aA 261.3 aA
120 26.86 bA 15.45 bA 33.00 bA 60.38 bA 141.9 cA
180 37.29 aA 20.06 bA 37.25 abA 87.30 bA 200.7 bA
360 17.45 cB 2.50 cB 20.48 bAB 41.63 bAB 87.2 dB

1
0 – prior to planting, 30, 120, 180 and 360 days.
2
Treatments: Control – without P fertilizer, Rock phosphate (RP), Complex super phosphate (CSP) and simple superphosphate (SSP).
3
Means with the same lowercase letter do not differ among age in the same treatment at 5% significance by LSD test and mean with the same capital letters do not
differ among treatment in the same age.

173
E.C. Foltran et al.
Fig. 1. Potential bioavailable P (P-water + P-resin + P-citrate + P-oxalate) concentration in the 0–40 cm soil layer in a Eucalyptus grandis plantation with different P
fertilizer sources prior to the experimental set up (0) and 30, 120,180 and 360 days. Water balance by Thornthwaite and Mather (1955) method (WAC = 280 mm).
Bars indicate the standard error of the mean (n = 3).
36 months after planting, the replacement of this Po pool was observed,
diminishing in this instance for non-labile P (113.9 mg kg−1 to
93.4 mg kg−1). This non-labile P reduction was observed under all
treatments except RP (Fig. 2c).
All sources of P enhanced the labile inorganic P pools (PAER and
PiBIC), in response to fertilizer application, as expected. However,
the P-resin levels, commonly used as P indicators for crops and trees in
Brazil, were very similar among the sources, being approximately
14 mg kg−1at the end of the first year (t12). During the same period, the
PiBIC pool decreased in the control and increased under P sources,
especially CSP (8.8 mg kg−1). After the third year (t36), the P con-
centration as PiBIC decreased 25% under SSP and 19% under CSP
compared to t12 but it was not significant (p > 0.10). The organic
labile pools (PoBIC) reported a fertilizer-specific response, CSP and RP
increased 43% and 41% during the first year, with a large decrease
during the third year (39% and 50%, respectively) (Table 4).
Inorganic moderately labile P pools (PiHID0.1 + PiHCl) had a small
contribution to total P, except for the RP treatment. However, the or-
ganic moderately labile pool was affected by the P sources, being the
most important Po stock in the soil, responsible for approximately 40 to
60% of total Po. CSP had a greater contribution to the total Po stock,
with 60% during the first year, and remaining constant at that value
over the third year. SSP presented a high contribution during the first
year (60%), decreasing to 45% during the third year (Table 4).
The RP fertilizer application increased the PiHCl during the first year
due to calcium-phosphate presence in the fertilizer, resulting from the
slow solubility of this fertilizer. This pool was still high after 3 years,
but decreased approximately 23% compared to t12. No changes were
found in this pool to the other treatments (Table 4).
174
Forest Ecology and Management 435 (2019) 170–179
Different phosphate sources did not increase the non-labile pool.
Control treatment showed a decrease in residual P pool only during the
first to third year, decreasing 13%. The opposite behavior was observed
under the RP treatment, which decreased only during the first year, 8%,
and remaining constant until the third year. Phosphate fertilization
with highly solubility sources showed a continuous decrease over the
years, 18% under the SSP treatment and 17% under CSP.
Principal component analysis (PCA) was used to determine the ef-
fect of the solubility of fertilizers and plant age on soil P fraction, OM
content and pH (Fig. 3). According to the parameters of Kaiser (1958),
the data variance can be represented using two main components. The
first two components explain 71% of the data variance in the 0–20 cm
soil layer (Fig. 3a). The first factor, which explains 38% of the varia-
bility of the data, is composed mainly of the OM and non-labile P. It
affects the first year of all treatments. These data present an inverse
relation to pH, which influenced expressively during the third year. The
second factor is composed mainly of the labile and moderately labile P
fractions and represents 33% of the data variance.
After the first 12 months after planting, the OM and non-labile P
influenced all treatments, especially the control and RP treatments. For
these treatments the OM and non-labile P were an important pool P for
plant uptake. This interpretation is supported by Maranguit et al.
(2017) in agroforestry plantations. Otherwise, the labile and moder-
ately labile P contents did not change over the time. At 36 months after
planting, the control and source SSP and CSP influenced only the soil
pH. The pH decreased due to a possible P solubilization under a higher
concentration of CO2 in the rhizosphere originating from plant roots
and microbial respiration (Bünemann et al., 2011).
The fertilizer solubilization intensified over time and increased the
E.C. Foltran et al. Forest Ecology and Management 435 (2019) 170–179

Fig. 2. Labile (a), moderately labile (b) and non-labile P (c) concentrations of inorganic and organic P in the 0–20 cm soil layer in a Eucalyptus grandis plantation
under different P fertilizer sources. Means within each P pool followed by the same letter do not differ themselves among treatments in the same age according to
LSD́s test (p < 0.05). Treatments: Control – without P fertilizer, Rock phosphate (RP), Complex super phosphate (CSP) and simple superphosphate (SSP).

levels of soil labile and moderately labile P, but did not change the non-
labile fraction (Fig. 3). Grouping the levels of P in organic and inorganic
pools (Fig. 3b), the first factor by PCA analysis can explain 46% of the
data variability and is composed mainly of inorganic P. The second
factor explains 28% and is composed mainly of soil pH. The inorganic P
content in the soil explains a large part of the variation observed in the
CSP and RP treatments during the first year, due to the high mineral P
input. At 36 months, biochemical processes such as exudation of low
molecular weight organic acids (LMWOA) by the root system and ac-
tivity of microorganisms in the soil increased (Hinsinger et al., 2011),
contributing to the soil pH change. Rock phosphate was an exception,
where the inorganic P- and total P-content of the soil explain the var-
iation of the data at this age.
In tropical ecosystems, the turnover of OM is an important source of

Table 4
Soil P fractions extracted sequentially by anion exchange resin (PAER), bicarbonate in inorganic (PiBIC) and organic (PoBIC) forms, NaOH 0.1 mol L−1 (PiHID0.1 and
PoHID0.1), HCl 1 mol L−1 (PHCl) NaOH 0.5 mol L−1 (PiHID0.5 and PoHID0.5) and residual P (PResid), in Eucalyptus grandis plantations under different P fertilizer sources
over time.
Treatment1 PAER PiBIC PoBIC PiHID0.1 PoHID0.1 PHCl PiHID0.5 PoHID0.5 PResid

mg kg−1

Prior to planting
3.3 1.6 9.5 2.3 24.3 0.7 14.1 16.5 108.7

One year after planting

Control 3.6 b2 1.0 c 7.8 c 4.1 19.0 C 2.6 b 10.5 b 14.1 b 114.0 aA
SSP 14.9 a 5.8 b 9.3 bc 4.4 38.9 bA 2.6 b 9.7 b 16.4 b 100.4 bA
CSP 14.6 a 8.8 a 16.5 aA 6.0 61.1 A 4.0 b 10.4 b 20.0 ab 103.9 ab
RP 12.2 aB 3.5 bcB 16.0 abA 3.8 32.4 B 181.4 aA 12.6 a 22.5 a 99.9 b

Three years after planting

Control 5.8 c 1.1 c 9.5 5.6 25.1 B 3.6 b 10.1 b 17.8 b 93.4 B
SSP 11.5 b 4.3 b 11.7 3.0 25.2 bB 1.6 b 9.3 b 18.5 ab 88.8 B
CSP 18.8 a 7.1 ab 10.1 B 3.2 49.8 A 3.2 b 9.5 b 19.4 ab 93.3
RP 22.7 aA 7.9 aA 8.0 B 2.2 40.4 A 147.1 aB 13.8 a 23.8 a 98.3

1
Treatments applied: Control – without P fertilizer, Simple superphosphate (SSP), Complex superphosphate (CSP), Rock phosphate (RP).
2
Means followed by the same letter or not followed by any letter do not differ by LSD test at 5% of probability. Lower case letter show the difference among
treatment to the same age and capital letter show the difference among ages for each treatment.

175
E.C. Foltran et al. Forest Ecology and Management 435 (2019) 170–179

Fig. 3. Principal Component Analysis of pH, OM content and distinct soil P pools of the 0–20 cm soil layers with different P sources. The P pools were grouped in
labile, moderately labile and non-labile (a) and in organic and inorganic (b). The black-filled symbols represent the first year, and the unfilled symbols represent the
third year after planting. Only vectors with Pearson correlations > 0.5 are presented.

Po, being fundamental in maintaining the plant P supply (Condron and
Tiessen, 2015; Maranguit et al., 2017). The high accumulation of lit-
terfall in forest conditions could results in high and easily mineralized
Po. The total litterfall over three years was about 20 Mg ha-1 year-1
(data not shown). The high litter depositions in this system promoted a
strong contribution to a moderate Po pool. This fraction involves a long-
term soil transformation in natural forests and acts as a buffer for labile
Pi in highly weathered soils (Guo et al., 2000). Additionally, the high
microbiological communities contribute to fungal symbiosis and thus to
Po mineralization (Plassard and Dell, 2010).
5. Discussion
5.1. Effect of phosphate fertilizers on soil P
Soluble P fertilizers provided more P for the Eucalyptus during the
beginning of the cycle; however, due to high Al and Fe content, this
high P availability can result in high P fixation (Bol et al., 2016; Fox
et al., 2011). Under soluble sources, such as SSP, the non-labile P
fraction did not change (Fig. 2c). Otherwise, a small and temporary
increase in the moderately labile P fraction was observed, but it

176
E.C. Foltran et al.
returned to the initial levels 36 months after fertilizer application
(Fig. 2b). This fact indicates that in forest environments, the soluble
phosphate is adsorbed as a moderately labile P pool but is reversible to
a labile P over a long time frame, with the tree’s root system accessing it
later. This temporary P adsorption for Eucalyptus probably is promoted
by recalcitrant OM, especially when such plantations are managed by
minimum tillage at establishment (Gonçalves et al., 2013; Rocha et al.,
2016).
The use of humic complexed simple superphosphate (CSP) resulted
in more labile P in the soil over both the short (t12) and long term (t36)
compared to that of SSP (Figs. 1 and 2), due to lower P adsorption. The
CSP technology consists of the protection of P by humic substances (P-
Ca-HS) that decreases the soil adsorption process (Urrutia et al., 2013,
García-Mina et al., 2004). To carry out the “breaking” of the metal
bridge between P-HS, the plant needs to exude organic acids to promote
this P dissociation. Thus, the absorption occurs in an active way and,
therefore, can achieve a greater efficiency in P use over a long-term
plant cycle (Erro et al., 2007).
Rock phosphate promoted the same increase in labile P in the short
term compared to that of SSP. Moreover, in the long term (t36) the RP
resulted in the highest level of labile P (Figs. 1 and 2a), due to the great
amount of calcium phosphate (Ca-P) in RP, which was solubilizing over
a long time frame as soon as the soil pH dropped. In this way, a high
amount of P was detected by HCl extractor, meaning that a Ca-P pro-
portion remained in the soil and had not solubilized yet. Overall, the
HCl P fraction generated a significant moderately labile P stock in the
soil which improved the labile P pool after 36 months, indicating that
this low solubility P source can supply the plant demand adequately
and maintain soil P availability in the strongly P-fixing soil.
5.2. Soil P pools and water effect on plant absorption
Eucalyptus shows fast development during the early stages, conse-
quently, high nutrient uptake occurs during the initial phases (du Toit
and Dovey, 2005). The labile inorganic pool was the primary P source
for plant growth (Table 3), but over the long term this pool represented
less than 10% of the total P in the soil (Table 4) which probably was not
sufficient for adequate plant development. A similar proportion was
found by Cherubin et al. (2016) and Rodrigues et al. (2016) in annual
crop systems and Costa et al. (2016) in Eucalyptus forests.
High water availability was detected during the first 120 days
(Fig. 1). When soil water content is favorable, root production increase
(Aoki et al., 2012; Laclau et al., 2013; Pinheiro et al., 2016) promoting
a higher tree growth rate and consequently decreasing the soil labile Pi
content (Table 3) by plant absorption and microbial immobilization.
Between 120 and 180 days water stress conditions (50.6 mm water
deficit) decreased trees P uptake, as a consequence the lower solubility
of the CSP and RP fertilizers resulted in an increase in total Pi in the
soil; otherwise, SSP decreased the size of this pool because it is highly
soluble. Indicating that P fixation is intensified by water stress, prob-
ably increasing organic and/or occlude P pools. The Hedley fractiona-
tion during the long-term soil analysis showed a high contribution to
the organic moderate pool during the third year after planting.
Under nutrient stress, forest trees have evolved strategies to opti-
mize acquisition and use of P, such as internal adaptation (nutrients
remobilization between tissues) (Smith et al., 2003) and external ac-
quisition strategies, including organic acid exudations in the rhizo-
sphere by plants (Maillard et al., 2015) and mycorrhizae. Among the
exuded organic acids, citrate is recognized as the most effective to in-
crease P availability in soil (Oburger et al., 2011). Citrate, as many
other forms of dissolved carbon compounds, is an important source of
energy for most microorganisms (Henintsoa et al., 2017), and microbial
P inmobilization is usually linked to a rapid decrease in soil solution P
concentration (Oehl et al., 2001).
The release of adsorbed organic P for microorganisms can be higher
with increasing surface coverage in the soil (Shang et al., 1996; Olsson
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Forest Ecology and Management 435 (2019) 170–179
et al., 2012) as well as with exudation of low molecular weight organic
acids (LMWOA) (Giles et al., 2012) and enzymes (Hinsinger et al.,
2011). Although the excretion of LMWOA was not assessed in our
study, therefore a study conducted by Foltran (2017) in the same ex-
perimental founds increases of 26% in manganese (Mn) concentration
during the autumn on senescent leaves under the control treatment
compared to the SSP. This higher Mn concentration is an indirect in-
dication of carboxylate exudation by the root system, as mentioned by
Lambers et al. (2015), who demonstrated the strong relationship be-
tween Mn content in senescent leaves and organic anion exudation in
the rhizosphere. The soil Mn availability normally increases with pH
depletion until approximately 5.0, at which point and below the
availability declines again (Lambers et al., 2008). However, Mn avail-
ability is also increased by root carboxylate exudation. Within a plant
community of low soil P availability, leaf Mn concentration also pro-
vides a strong indication of specific P-mobilizing carboxylate releasing
strategies by plants.
Considering the soil P fractions in isolation, CSP presented the
highest increase in moderately labile organic P (PoHYD0.1) at the be-
ginning of the experiment. This P pool extracted by NaOH 0.1 mol L−1
comprises inositol phosphates, which are usually the largest proportion
of Po present in the soil and are highly complex forms associated with
clay minerals and fulvic and humic acids (Quiquampoix and Mousain
2005). The humic acid presented in CSP can increase this interaction,
thus increasing the moderately labile organic P pool.
Reduction of Presidual fraction was observed in all treatments over
the trees’ cycles, being more expressive for the SSP and control, which
decreased 22 and 15% compared to the initial levels (Fig. 3). This P
pool (Presidual) decreased because of the depletion of the labile fractions
by plant absorption and consequently remobilization of P from more
stable forms. The soil solution removal of P as well as its replenishment
is a result of a combination of physicochemical (adsorption/desorption
and precipitation/dissolution) and biological/biochemical (miner-
alization/immobilization) processes (Bünemann 2015). These processes
were also detected in annual crop production in Brazil (Pavinato et al.,
2010) and natural forests in Central America (Dieter et al., 2010).
5.3. Role of organic P in plant nutrition
Soil management in Eucalyptus stands in Brazil is based on minimal
tillage (Gonçalves et al. 2013). The maintenance of the forest residues
on the soil surface increases the soil OM content in the top layer (Rocha
et al., 2016). As a consequence, it results in a strong soil P buffer ca-
pacity, allowing the maintenance of adequate Pi levels in soil solution.
Our results suggest that the increase in labile P pools was followed by
decreases in recalcitrant or occluded P pools in order to maintain mass
balance. Both organic pools, Po BIC and Po HYD0.1, decreased during
the first year, when the highest uptake rate occurs (Fig. 2a and 2b).
Thus, although P inputs by OM do not represent a “new” P addition to
the ecosystem per se, organic P recycling is a crucial mechanism for
maintaining P stocks in tropical rain forests (Bünemann et al., 2011),
becoming a main source of P under stress nutritional conditions (Fig. 2).
In native Cerrado forests (Brazilian Savanna), the moderately labile
Po pool represents a large proportion of soil total P (Fig. 4). The es-
tablishment of the cultivated forest, even with low input of fertilizers,
contributes to an increase in the total P content in the soil, especially
the occluded P pool (Fig. 4). The inorganic P input from mineral sources
increases the labile and moderately labile P and does not change the
non-labile P in the forest system. In general, the organic pools showed
strong contribution to the plants as a result of biogeochemical cycling,
especially at 12 months but also at 36 months.
5.4. Potential P availability indicators for forest plantations
Ion exchange resin as an indicator of the available P content for
plants is widely used for forest soils in tropical regions. However, resin
E.C. Foltran et al.
Fig. 4. Phosphorus content in each soil fraction, under the effect of eucalyptus management and P fertilizer sources over time. Native Cerrado; eucalyptus stand (-P);
eucalyptus stand low P input: Eucalyptus grandis (13 years) without nutrient input. Treatments applied: Control – without P fertilizer, Rock phosphate (RP), Simple
superphosphate (SSP), Complex superphosphate (CSP) + RP. Bars colors (in order) – Yellow (Labile P): P-resin, Pi and Po NaHCO3; Green (Moderately labile P): Pi and
Po NaOH0.1, P-HCl; Gray (non-labile P): Pi and Po NaOH0.5, P-residual.
extractant did not show a significant difference for the control treat-
ment during the first 12 months (Table 3 and 4). This fact indicates that
resin pool is replenished from other pools over time and the plants can
accesses P pools not extracted by resin. On other hand, at 120 days
there was a reduction of 17 mg kg−1 of Pi extracted by citrate (Pi-ci-
trate-pH 5.0) under SSP and 2.2 mg kg−1 for the control. The use of
citrate at pH 5.0 as an extractor of the labile inorganic P seems to ap-
proach a real condition that occurs in the rhizosphere of acidic soils.
Giles et al. (2017), studying the accumulated P content in the above-
ground of Nicotiana tabacum using citrate as soil P extractant, found a
positive and highly relevant relationship between plant absorption and
soil P levels (p < 0.05).
Recent research has been conducted to detect actual eucalyptus
scavenger capacity in accessing nutrients such as K (Pradier et al.,
2017) and P (Hinsingeret al., 2011). The great ability to promote
symbiosis with mycorrhizal fungi and exude compounds, e.g., carbox-
ylates and phosphatases, makes this species highly efficient in accessing
recalcitrant soil P pools (Fox et al., 2011). Our results suggest that the
content of Pohyd0.1 decreased 28%, Pihyd0.5 decreased 40% and Pohyd0.5
decreased 16% during the first year after planting in the control, re-
sulting from solubilization processes. The P-Ca pool determined by the
HCl extractant decreased 23% under the RP treatment due its solubi-
lization. At the same time, the organic moderately labile P pool in-
creased, acting as a buffer for the easily available P. Because of this ion
exchange resin does not seem to be a good indicator of soil P avail-
ability in eucalyptus plantations, especially in Oxisols.
Our results suggest that Eucalyptus plantations can access the labile
and moderately P pools using the Hedley procedure. Despite being a
good indicator of the actual amount of P that the trees can uptake, the
Hedley procedure is laborious and expensive. However, the extraction
of soil P using citrate and oxalate (potentially available P) can also be a
good indicator of the amount of P available.
6. Conclusions
The forms and distribution of soil P in various pools are determined
by fertilizer source and solubility. The proportion of Po was higher than
Pi in the original soil, and Pi was mainly composed of stable P (residual
P). In the short term the P-fertilized treatments increased the labile P
pool. The P extracted by water and resin showed uncertain changes due
to the high buffer power in the forest’s soils. The procedure we pro-
posed can better determine potential bioavailable P for forest plants.
178
Forest Ecology and Management 435 (2019) 170–179
The soluble phosphate fertilizer improved the labile P during the
short term, but this P is shortly fixed and converted into a moderately
labile fraction. However, P fixation is reversible and the fixed P could
be taken up later by plant. On the other hand, the low soluble RP fer-
tilizer enhanced the labile P form during the long term, due to slow
solubilization of P-Ca. The P fertilizer complexed by humic substances
improved the labile P fraction during both the short and long term.
Hedley fractionation was a good indicator of the available P for the
plant, as well as the citrate + oxalate method. However, the laboratory
and cost make citrate + oxalate more interesting to predict the P
available to plants, specifically during the beginning of their develop-
ment.
Acknowledgments
The authors would like to thank FAPESP (2012/18.234-5) and
CAPES for the scholarship provided. To the Forestry Science and
Research Institute (IPEF) which coordinate the Silviculture and
Management Thematic Programme (PTSM-IPEF). We also thank to
Itatinga Forest station staff for their technical support and Antonio
Carlos Gama-Rodrigues for valuable comments and suggestions on a
prior version of the manuscript.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.foreco.2018.10.053.
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