Geoderma 404 (2021) 115311

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Geoderma
journal homepage: www.elsevier.com/locate/geoderma

Phosphorus deficiency in soils with red color: Insights from the interactions
between minerals and microorganisms
Mu Su a, b, Lingzi Meng a, Lang Zhao a, Yukai Tang a, Jingjing Qiu a, Da Tian c, d, Zhen Li a, b, *
a
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China
b
Jiangsu Provincial Key Lab for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China
c
Anhui Province Key Lab of Farmland Ecological Conservation and Pollution Prevention, School of Resources and Environment, Anhui Agricultural University, Hefei
230036, China
d
Research Centre of Phosphorus Efficient Utilization and Water Environment Protection along the Yangtze River Economic Belt, Anhui Agricultural University, Hefei
230036, China

A R T I C L E I N F O A B S T R A C T

Handling Editor: David Laird Phosphorus (P) deficiency limits agricultural productivity in many tropical and subtropical soils. The aim of this
study was to investigate the influences of typical mineral components and microorganisms on P availability in
Keywords: soil. Three representative soil minerals, i.e., montmorillonite (Mt), calcium carbonate (Cal) and goethite (Gt)
Phosphorous were incubated with phosphate-solubilizing fungus (PSF, Aspergillus niger) and apatite (the dominant P source on
Artificial soil
Earth) in soil. In the control treatment, the fungus promoted phosphate dissolution and P release (from < 1 to
Clay mineral
370.40 mg kg− 1), primarily via secreting oxalic acid. Mt addition promoted fungal respiration and up-regulated
Fe oxide
Calcium carbonate oxaloacetate acetylhydrolase (regulating organic acid secretion, based on RNA sequencing analysis). However,
Phosphate-solubilizing fungi strong adsorption of organic acids by Mt finally caused decline of P availability (from ~ 400 to 24 mg kg− 1). Cal
addition decreased fungal counts (down to < 5% of the value for the control treatment). Additionally, the
consumption of organic acids (by Cal dissolution) further reduced P release. Gt stimulated fungal respiration,
whereas its toxicity significantly decreased fungal abundance. Moreover, Gt demonstrated the maximal P
adsorption capacity, i.e., 1.266 mg g− 1. Therefore, Gt demonstrated evident negativity on P release. Despite the
strong sorption of organic acids, Mt showed high potential to promote P release in the long-term due to the
enhanced fungal bioactivity. Our results hence indicated that different minerals had totally distinct effects on P
release, which should be addressed when investigating biogeochemistry in soil.

1. Introduction
Soils with red color (red soils) are widely distributed in tropical and
subtropical regions, covering 13% of the land area on the Earth (Bir­
keland, 1999; Baligar et al., 2004; Mee et al., 2004) and including oxi­
sols, ultisols, alfisols, mollisols and inceptisols (Yaalon, 1997; Durn
et al., 1999). Abundant rainfall, warm temperature and rapid biological
cycling in red soil regions provide high potential for global agricultural
production. However, many of these soils are characterized by low pH,
low organic matter, and P deficiency (Baligar et al., 2004; Chacon et al.,
2006), which often limit their crop yields.
Intense leaching and immobilization of labile P primarily account for
P deficiency in red soils (Sanchez, 1976; Peretyazhko and Sposito,
2005). In China, approximately 30–50% of fertilized red soils suffer
from P deficiency (Baligar et al., 2004; Zhang et al., 2004). Available P
(AP) in many red soils is even < 5 mg kg− l. Both the secondary clay
minerals (e.g., kaolinite, illite and montmorillonite) and Fe/Al oxides
can reduce AP, mainly via ligand exchange and surface precipitation
(Ler and Stanforth, 2003; Sparks, 2003a; Perassi and Borgnino, 2014;
Wang et al., 2015). In addition, electrostatic interactions would
encourage sorption of negative phosphate anion onto Fe/Al oxides
(Colombo et al., 1994; Fang et al., 2017). Organic matter addition may
further increase P adsorption sites in soil via altering mineral crystalli­
zation (Pizzeghello et al., 2016). However, many physico-chemical
processes in soils may also lead to P desorption. Elevated pH enhances
P desorption via decreasing adsorption sites or increasing the electro­
static repulsion between clays and phosphates. The pH value of red soils
in karst area could be as high as 8 (Liang et al., 2003; Martin, 2017).

* Corresponding author at: College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.
E-mail address: lizhen@njau.edu.cn (Z. Li).

https://doi.org/10.1016/j.geoderma.2021.115311
Received 29 March 2021; Received in revised form 14 June 2021; Accepted 15 June 2021
Available online 23 June 2021
0016-7061/© 2021 Elsevier B.V. All rights reserved.
M. Su et al.
However, carbonate weathering in this region usually reduces P avail­
ability by co-precipitation of labile P and Ca (Perassi and Borgnino,
2014; Jiang et al., 2020).
Redox reactions strongly influence P availability. Adsorbed or co-
precipitated P could be released during Fe-oxide reduction (Per­
etyazhko and Sposito, 2005; Chacon et al., 2006; Liptzin and Silver,
2009; Henderson et al., 2012). In particular, the abundant iron in the red
soils intensifies the redox reactions. Moreover, the oxidation–reduction
of Fe-oxides and subsequent P release can be influenced by the labile
carbon (C) addition, mineral surface area, soil water contents and anoxic
condition, etc. (Chacon et al., 2006; Liptzin and Silver, 2009; Hall et al.,
2013; Hall and Huang, 2017). Therefore, the redox reactions are tightly
correlated with mineralogy and environmental factors in soil.
The mineral-microorganism interactions affect many critical envi­
ronmental processes, e.g., soil development, aggregation, and global
cycling of multiple elements (Wei et al., 2014; Pastore et al., 2020;
Zhang et al., 2020). Phosphate-solubilizing microorganisms (PSM) can
dissolve phosphate minerals by secreting organic acids (e.g., oxalic acid,
citric acid, gluconic acid, etc.) and enzymes (e.g., phytases and phos­
phatases) (Plante, 2007; Richardson et al., 2009; Li et al., 2016a, 2019;
Zhang et al., 2019b). Due to the selection pressure, the abundance of
PSM is relatively high in P-limited environments (Mander et al., 2012;
Pastore et al., 2020). For example, Aspergillus niger is a ubiquitous
phosphate-solubilizing fungus (PSF) in highly weathered red soil (Goe­
nadi et al., 2000; Tallapragada and Seshachala, 2012; Islam et al., 2019).
About 80–90% microorganisms are associated with solid mineral sur­
faces in soil (Hattori, 1973). They adhere to mineral surfaces through
secretion of mucus layers, extracellular polymeric substances (EPS),
protein-binding receptors, and hydrophobic/electrostatic interactions
(Cai et al., 2013; Poorni and Natarajan, 2014; Ghashoghchi et al., 2017).
Clay minerals and Fe/Al oxides may inhibit microbial growth by
reducing their mobility, suppressing nutrient acquisition, or releasing
toxic metal cations (e.g., Al3+ and Fe3+) (Lavie and Stotzky, 1986;
Amonette et al., 2003; Wong et al., 2004; Zhao et al., 2014; Imlay, 2019;
Su et al., 2019). Clay mineral can also increase microbial longevity and
metabolic activity by reducing exposure of microorganisms to pollutants
or enhancing aeration (Hwang and Tate, 1997; Wu et al., 2014; Biswas
et al., 2015; Zhang et al., 2019a). However, limited understanding of
interactions among microorganisms and minerals constrains our capa­
bility to elucidate P biogeochemistry in red soils.
To investigate complex interactions among soil minerals, P, and
Fig. 1. Distribution of red soil collection sites in south China (land area).
2
Geoderma 404 (2021) 115311
microorganisms, we conducted a 28-d incubation in an artificial soil
amended with representative minerals in red soils. Because of multiple
confounding factors, such as soil texture, organic matter and soil envi­
ronment in natural soils, we examined the impacts of soil minerals on
PSF and P transformation in this artificial system. We hypothesized that
different minerals would have distinct influences, which are not only
limited to sorption/desorption, but also correlated to microbial physi­
ology. Then, these discrepancies might cause complex P release from
phosphate minerals.
2. Materials and methods
2.1. Field observation
Seven red soils were collected from seven locations in China,
including Chongqing, Zhangjiajie, Nanchang, Zhuzhou, Guilin, Wen­
shan and Chongzuo city (Fig. 1 & S1). The soils from Chongqing, Nan­
chang, Zhuzhou, Guilin, Wenshan and Chongzuo city are Ultisols, and
the soil from Zhangjiajie is belonging to Inceptisol according to U. S. Soil
Taxonomy System.
2.2. Experimental procedures
Clay mineral (Ca-montmorillonite, collected from Chifeng, Inner
Mongolia, China), iron oxide (goethite (Sigma-Aldrich®, Germany)),
and calcium carbonate (Xilong Chem. Inc., China) were selected as three
representative soil minerals. Quartz sand was used as a matrix material
to control texture (Wu et al., 2019). The three soil minerals were ground
to < 250 μm. Quartz was prepared with two sizes, i.e., 0.5–1 mm
(coarse) and < 250 μm (fine). Inorganic phosphate was added as
hydroxylapatite (HAp) powder (5–20 μm) (Yuanyeshengwu Inc. China).
All these materials were sterilized before addition.
The Aspergillus niger (CGMCC No.11544, Nanjing Agricultural Uni­
versity) had been studied in our previous studies (Li et al., 2016a,
2016b). A. niger was cultured in a potato dextrose agar medium (PDA)
for 5 d at 28 ◦ C to prepare a spore suspension.
2.3. Artificial soil incubations
Nine treatments were performed, i.e., 5% Mt + 95% quartz (LMt),
10% Mt + 90% quartz (MMt), 20% Mt + 80% quartz (HMt), 2.5% Cal +
M. Su et al. Geoderma 404 (2021) 115311

97.5% quartz (LCal), 5% Cal + 95% quartz (MCal), 10% Cal + 90%
Qm kCf
quartz (HCal), 1% Gt + 99% quartz (LGt), 2% Gt + 98% quartz (MGt), Q= (3)
1 + kCf
and 4% Gt + 96% quartz (HGt) (Table S1). The contents of these soil
minerals were selected on the basis of experimental designs in previous K is a coefficient, which reflects the relative rates of sorption and
studies (Zhang et al., 2011; Steinbach et al., 2015; Wu et al., 2019). In desorption at equilibrium. Qm (mg g− 1) is the maximal sorption
addition, one treatment with 100% quartz was set as the control treat­ capacity.
ment. HAp (0.22 g) was added to 20 g (dry weight) artificial soils in each
treatment. After sterilization, glucose (0.7 g of C per week per kg dry
2.5. RNA-seq analysis
soil) and KNO3 (0.028 g of N per week per kg dry soil) were added, with
a C/N weight ratio of 25 (Kallenbach et al., 2016). Then, 1 mL spore
The RNA-Seq analysis were performed to reveal cellular tran­
suspension of A. niger (1.40 × 107 spores mL− 1) was inoculated into the
scriptome after addition of Mt and Gt. The potato dextrose broth (PDB)
artificial soils. During incubation, the mixed solution of glucose and
medium (100 mL) was transferred to 250 mL conical flasks and was
KNO3 was sprayed evenly to each bottle. The water holding capacity
sterilized by autoclaving at 115 ◦ C for 30 min. Then, 100 mg sterile Mt or
(WHC) of artificial soil was measured by the tube method (Wani et al.,
Gt, and 1 mL spore suspension of A. niger (1.40 × 107 spores mL− 1) were
1994). The artificial soils were incubated in the dark at 25 ◦ C for 28 d,
added to the medium. The samples were incubated in a rotary shaking
with 50% WHC (Table S1). Artificial soils were incubated for 14 d and
incubator at 28 ◦ C with an agitation speed of 180 rpm for 5 d. This
28 d before measuring available P, pH, and organic acids. In addition,
experiment had three treatments, i.e., A. niger (Control), A. niger + Mt
fungal respiration, fungal abundance, and morphological changes were
(Mt-RNA) and A. niger + Gt (Gt-RNA). All the treatments had three
measured after 28-d incubation. All the treatments had four replicates.
replicates.
For respiration measurement, each bottle was circulated with stan­
After 5-d incubation, the fungal pellets were separated and analyzed
dard air for 5 min, and then sealed for 3 h on days 1, 2, 4, 6, 8, 9, 11, 13,
using a commercially available service (BGI, Huada Gene, Wuhan,
16, 19, 23 and 26. Gas samples (10 mL) were collected from the head­
China). Briefly, total RNA was fragmented into short fragments and
space using a polyethylene syringe with a three-way stopcock (Tian
mRNA was enriched using oligo (dT) magnetic beads, followed by cDNA
et al., 2019). Four empty bottles were also incubated under the same
synthesis. Double-stranded cDNA was purified and enriched by PCR
condition as a control (to determine the standard air CO2 concentration).
amplification, after which the library products were sequenced using
The CO2 efflux of soil was calculated using the equation proposed by Liu
BGIseq-500. Genes with a fold change (FC) greater than a pre-defined
et al. (2009). In addition, the cumulative CO2 efflux during incubation
threshold and a p-value less than a pre-defined threshold were consid­
was calculated as:
ered as differentially expressed genes (DEGs). In this study, Log2 Fold
∑
n Change (LogFC) values were generated for RNA-Seq samples by
Cumulative respiration = Ri ti (1) comparing the average Fragments Per Kilobase per Million mapped
fragments (FPKM) values of genes for the Mt-RNA/Gt-RNA treatment vs.
i=0

Where n is the incubation day, Ri is the mean rate of CO2 efflux be­ the control treatment. All DEGs were selected with the average FPKM >
tween two successive CO2 efflux measurements, ti is the time (h) be­ 1, false discovery rate (FDR) < 0.05 and log2 fold change ≥ 1 or ≤ − 1.
tween two successive CO2 efflux measurements. All gene assignments were analyzed based on automatic annotation of
Each artificial soil sample was divided into two subsamples. One part A. niger CBS 513.88 (ascomycetes) gene models.
was analyzed by SEM after air drying (for mineralogical investigation).
Another was fixed by 2.5% (v) distilled glutaraldehyde (pH = 7.2) for
2.6. Instrumentation
12 h at room-temperature, and then rinsed. Then, the fixed samples were
dehydrated through a graded ethanol series (50–100% (v)) (for micro­
Soil total carbon (TC) and nitrogen (TN) were analyzed using a CN
bial investigation). All the samples were subsequently mounted onto
analyzer (Elementar Vario Micro cube, Germany). Soil carbonate was
aluminum stubs using carbon adhesive tape and stored in a desiccator at
determined by measurement of CO2 by gas chromatography (GC)
room temperature for SEM analysis.
equipped with flame ionization detector (GC-7890B, Agilent, Santa
Clara, CA, USA) following sample acidification (hydrochloric acid)
2.4. Phosphorus adsorption on soil minerals
(Amundson et al., 1988). Soil pH was measured in a 1:2.5 ratio of soil to
CO2-free water by SG98 InLab pH meter (Mettler Toledo Int. Inc.) with
Phosphate (added as KH2PO4) solution was prepared with a series of
an Expert Pro-ISM probe. Soil total P was measured after microwave
P concentrations (0.0, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0 mg L− 1) in 50 mL
acid-digest (perchloric acid and sulfuric acid) and measured by induc­
polyethylene centrifuge tubes. Meanwhile, 500 mg montmorillonite
tively coupled plasma optic emission spectrometry (ICP-OES, Agilent
(Mt), goethite (Gt), or calcium carbonate (Cal) were mixed with 0.1 M
710). Available P in neutral and alkaline soils was extracted by 0.5 M
NaNO3 (as background electrolyte) in solution. Then, the pH was
NaHCO3, while the AP in acid soils was extracted by 0.05 M HCl-0.025
adjusted to 9.0 ± 0.5 based on the results from soil incubation. After
M H2SO4. The molybdenum blue method was used to determine avail­
shaken with an oscillation rate of 180 rpm at 25 ◦ C for 24 h, the solutions
able P (AP) concentrations (Watanabe and Olsen, 1965). Organic acids
were centrifuged. The supernatant solutions were extracted and filtered
extracted in 10 mM NaH2PO4 were analyzed by high performance liquid
through 0.22 μm membrane for P analysis. The amount of P adsorbed by
chromatography (HPLC) (Agilent 1200) with column temperature of
the soil mineral was calculated using the following Eq. (2):
30 ◦ C and detection wavelength of 210 nm (Mimmo et al., 2008). The
V(Ci − Cf ) conidia concentration was determined by haemacytometer under
Q= (2)
M Olympus Inverted Microscope BX53 (Olympus, Japan). The fungal
abundance in the artificial soils was counted by dilution coating method
Q is sorption capacity (mg g− 1). V is the volume of solution (L). M is
(Dickinson et al., 1975; Martin et al., 2012). Information about the XRF,
the weight of the Mt (g). Ci and Cf are the initial concentration and
XRD, and SEM can be referred to Supplementary Materials.
equilibrium concentration of P (mg L− 1), respectively.
The corresponding sorption isotherms for P was quantitatively
described by fitting the experimental data to Langmuir isotherm equa­ 2.7. Statistical analysis
tions according to Eq. (3):
Effects of Mt, Cal and Gt addition on CO2 emissions, fungal abun­
dance, organic acids and AP were evaluated by one-way analysis of

3
M. Su et al.
variance (ANOVA). We used least significant difference (LSD) test at the
significance level of p < 0.05 to further analyze differences among
different mineral content levels. All statistical analyses were performed
using SPSS 16.0. The residuals from the models were examined for
normality and homoscedasticity using graphical diagnostic plots (i.e.
Q–Q plots and residuals plots) (Quinn and Keough, 2002). Different
letters indicate significant differences between treatments.
3. Results
3.1. Chemical properties of natural red soils
Comparing with acid soils, the neutral and alkaline red soils showed
higher total P and AP contents (Table S2). The AP values were very low
in six soils, i.e., 2.14 (Chongqing), 3.84 (Zhangjiajie), 1.28 (Zhuzhou),
4.04 (Guilin), 1.90 (Wenshan) and 0.53 (Chongzuo) mg kg− 1, and also
low in the Nanchang soil (7.83 mg kg− 1). There are abundant Fe and Al
in all the soils. Fe and Al (denoted by oxide) contents in Yunnan soil
were the highest, i.e., 27.91% and 13.49%, respectively (Table S3).
These results confirmed the P deficiency and Fe/Al abundance in the red
soils.
The soil pH in Chongqing, Zhangjiajie, Nanchang and Wenshan soils
were 7.48, 8.28, 7.87 and 6.93, and 4.58, 4.39 and 4.95 in Zhuzhou,
Guilin and Chongzuo soils (Table S2). These soils all demonstrated low
organic C, with the range from 1 to 8 g kg− 1. There are abundant Ca
cations in the red soils that located in Chongqing and Zhangjiajie. i.e., 1
and 1.85% (denoted as CaO), respectively (Table S3). XRD results
confirmed that quartz, calcium carbonate, clays (montmorillonite and
kaolinite), and Fe oxides (hematite and goethite) were the four repre­
sentative minerals (Fig. S2). Then, these minerals were added to the
artificial soils.
3.2. The fungal respiration and abundance
Microbial activities can be tracked by their respiration (Su et al.,
2019; Tian et al., 2019). In the control treatment, the cumulative fungal
CO2 emission during 28-d incubation was 715.51 mg C kg− 1 soil (Fig. 2).
Fig. 2. The cumulative CO2 emission (A-C) and fungal counts (D-F) after incubation with montmorillonite (Mt), calcium carbonate (Cal), and goethite (Gt) addition.
Values with different lowercase letters indicated statistically significantly different at p＜0.05 level.
4
Geoderma 404 (2021) 115311
Mt and Gt addition significantly increased cumulative CO2 emission to
~ 1400 and 1230 mg C kg− 1 soil, respectively (Fig. 2A, C). However, Cal
addition had no effect on CO2 emission (693 mg C Kg− 1 soil) (Fig. 2B). In
addition, there was no significant difference among the treatments with
different mineral contents.
In the control treatment, the fungal count after 28-d incubation was
4.47 × 106 cfu g− 1 soil (Fig. 2). It was significantly decreased to1.14 ×
106 cfu g− 1 soil after low Mt addition (p < 0.05), while remained high
(4.69 × 106 and 4.40 × 106 cfu g− 1 soil) in the MMt and HMt treatments
(Fig. 2D). After addition of Cal and Gt, the fungal abundance signifi­
cantly decreased to 2.01 × 105 and 1.45 × 106 cfu g− 1 soil, respectively
(p < 0.05) (Fig. 2E, F), which indicated that Cal and Gt inhibited the
anabolism of the fungus. Similar to the respiration, there was no sig­
nificant change of fungal abundance among the treatments regarding
Cal and Gt addition.
3.3. Chemical properties in the artificial soils
The initial pH in the control treatment was 7.28 (Fig. S3). Addition of
Mt, Cal and Gt changed the initial pH to 6.77, 8.64 and 7.57, respec­
tively. After incubation, the pH decreased to 6.73 at 14 d and 5.57 at 28
d in the control treatment. In contrast, the pH increased to approxi­
mately 8.94, 9.15 and 9.26 after addition of Mt, Gt and Cal, respectively.
In the control treatment, organic acids (oxalic and citric acids) after
14-d and 28-d incubation were 180.40 and 201.98 mg kg− 1. Mt, Gt and
Cal addition lowered organic acid contents (Fig. 3A, B and C). The oxalic
acid in the control treatment was 15.01 mg kg− 1 after 14-d incubation,
and under the detection limit after 28-d incubation (Fig. 3D, E and F).
For citric acid, it increased from 165.39 to 202.00 mg kg− 1 during in­
cubation (Fig. 3G, H and I). Both oxalic and citric acids were not
detected in the treatments with the Mt addition, possibly due to the
strong adsorption by Mt (Zhang et al., 2018).
3.4. P availability in soil
Comparing with the low release rate of P from apatite, approximately
19% P from apatite was activated into AP in the control treatment. AP
M. Su et al. Geoderma 404 (2021) 115311

Fig. 3. The oxalic and citric acids secreted by PSF after 14-d and 28-d incubation. The dotted line represented the organic acids in the control treatment.

values were 394.08 (14 d) and 370.40 (28 d) mg kg− 1 (Fig. 4). AP
significantly decreased with addition of the three minerals (p < 0.05).
However, mineral contents did not significantly alter AP concentrations.
The AP concentrations decreased to 51.63, 62.82 and 127.01 mg kg− 1
after 14-d incubation and 24.28, 34.60 and 79.19 mg kg− 1 after 28-d in­
cubation in the treatments after addition of Mt, Cal and Gt, respectively
(p < 0.05).
The ideal adsorption of PO4 on Ca-montmorillonite could be divided
into two stages (Fig. 5A, B), i.e., P with low concentration (0–5 mg L− 1)
due to surficial adsorption or precipitation (> 5 mg L− 1). The maximal P
adsorption capacity of Mt was 0.149 mg g− 1 based on Langmuir model
(Fig. 5B). Similarly, Cal could only adsorb a few P (as P < 6 mg L− 1), and
the precipitation will dominate P fate with increasing P concentration
(Fig. 5C). However, Gt displayed a strong P adsorption capacity, i.e.,
1.266 mg g− 1 (Fig. 5D).
3.5. SEM analysis
Abundant fungal mycelia and spores were observed on the soil
samples (Fig. 6A). Calcium oxalate (CaC2O4) was observed on mycelia
surface, which were attributed to the reaction between oxalic acid and
phosphate (HAp) (Fig. 6B) (Michael et al., 2014). Abundant Mt particles

Fig. 4. Available P contents after incubation with montmorillonite (Mt), calcium carbonate (Cal), and goethite (Gt). Values with different lowercase letters indicated
statistically significantly different at p＜0.05 level.

5
M. Su et al.
Fig. 5. Adsorption of AP on the three minerals. B and D showed the parameters of Langmuir equation based on the sorption of AP on montmorillonite and goethite
(N = 3). Qm is the P sorption capacity of mineral.
were also observed on the hyphae (Fig. 6C and 6D). However, CaCO3
particles did not show tight sorption/precipitation on hypha (Fig. 6E, F,
G) in the treatment of MCal. Additionally, Ca-P complex was observed
around the CaCO3 particles (Fig. 6H). Abundant Gt particles were
observed on the surface of fungus in the MGt treatment (Fig. 6I),
showing relatively high P sorption based on the EDS result (see Fig. 6J).
3.6. RNA sequencing analysis
The DEGs were selected by applying cutoffs of the t-test padjgt <
0.05. Comparing with the control treatment, only the RNA gene
encoding oxaloacetate acetylhydrolase was significantly up-regulated in
both the Mt-RNA and Gt-RNA treatments (Fig. 7A). Other RNA expression
related to oxalic and citrate acids secretion, i.e., pyruvate dehydroge­
nase, and citrate synthase, showed no significant changes.
For the organophosphorus conversion, Mt-RNA and Gt-RNA treatments
showed evident contrasts (Fig. 7B). The genes encoding 3-phytase B1,
acid phosphatase 1, 2, 4 and 2-deoxyglucose-6-phosphate phosphatase
were significantly up-regulated. Furthermore, LogFC values of these
genes were higher in the treatment of Gt-RNA than Mt-RNA. Many other P-
related genes also showed evident changes in the Gt-RNA treatment. For
example, the genes encoding protein-tyrosine phosphatase 2, acyl­
phosphatase, trehalose-phosphatase, and NADH pyrophosphatase were
up-regulated, while encoding 3-phytase B1, acid phosphatase 3 and
histidine acid phosphatase 1 were down-regulated. Therefore, addition
of Gt caused more intense dual effects on fungal physiology.
4. Discussion
4.1. Enhancement of P release by PSF
Almost > 90% P on Earth is stored as inorganic phosphates, pri­
marily as apatite. A. niger can secrete oxalic acid and subsequently
accelerate dissolution of inorganic phosphate via acidolysis and
complexation (see Eq. (4)) (Jones and Oburger, 2011; Lazo et al., 2017;
Mendes et al., 2020). Among the various low molecular weight organic
acids, oxalic acid has a greater influence on soil acidity due to their
higher acidity constant (Kα1 = 6.5 × 10− 2) (Bolan et al., 1994; Li et al.,
2016a). According to Fig. 6B, oxalic acid was consumed by phosphate
dissolution and subsequent calcium oxalate formation (Eq. (5)).
6
Geoderma 404 (2021) 115311
Moreover, filamentous microorganisms can enhance the weathering of
minerals/rocks by biomechanical deterioration (e.g., by hypha pene­
tration), in addition to the above biochemical dissolution (Sterflinger,
2000; Gadd, 2007; Tian et al., 2020). Therefore, comparing with the low
release rate (~0.015%) of P from apatite (in water), 19% P in apatite
was activated to AP after involvement by A. niger (Fig. 5).
Ca5(PO4)3OH + 7H+ = 5Ca2+ + 3H2PO−4 + H2O (4)
Ca2+
+ C2O2−
4 = CaC2O4 (5)
4.2. Negative function of calcium carbonate on P availability
Calcium carbonate addition significantly decreased P release from
apatite (Fig. 4). Calcium carbonate is able to adsorb phosphate via
chemisorption when phosphate concentration is low (Karageorgiou
et al., 2007). At high concentration, this adsorption is performed as the
precipitation of Ca-compounds (Yagi and Fukushi, 2012). The low
adsorption of AP on Cal surface in this study proved that the decrease of
AP may not be attributed to sorption by calcium carbonate. Cal can
indirectly suppress fungal growth via elevating soil pH (Fig. S3). Portion
of conjugated bases of either oxalic acid or oxalate could also be
consumed during CaCO3 dissolution (Tian et al., 2020). Thus, both a
decrease in fungal abundance and suppression of organic acids (see
Fig. 2E and 3B) may contribute to the low P release (Arvieu et al., 2003).
There is also a possibility that microbial respiration could accelerate
phosphate dissolution via forming carbonic acid, especially in alkaline
soils (Emmerich, 2003; Filippelli, 2008). The potential reactions are
shown in Eqs. (6) and (7) (Filippelli, 2008). However, this potential
enhancement should be at a low degree based on our chemical analysis.
CaCO3 + CO2 + H2O → Ca2+ + 2HCO−3 (6)
Ca5(PO4)3OH + 4CO2 + 3H2O → 5Ca 2+
+ 3HPO2−
4 + 4HCO (7)
4.3. Complex function of clay minerals on P release
Fungal respiration (CO2 emission) increased 95.69% after Mt
M. Su et al.
addition. Meanwhile, the fungal abundance remained stable. Mt addi­
tion was able to stimulate PSF respiration via enhancing aeration (Zhang
et al., 2019a). In addition, clay minerals are able to create many pores,
and thus provided a suitable habitat for many microorganisms (Fatemeh
et al., 2020). Clay minerals also supplied nutrients for fungal growth,
especially in soils with limited nutrients (Biswas et al., 2015). Mean­
while, the low sorption of PO4 by Mt allowed abundant P released to the
medium (Fig. 7B). Moreover, the genes encoding oxaloacetate ace­
tylhydrolase was significantly up-regulated (Fig. 7A) after Mt addition.
Oxalic acid can be synthesized through the cytoplasmic pathway, the
tricarboxylic acid (TCA) pathway and glyoxylate (GLOX) pathway
(Palmieri et al., 2019). Oxaloacetate acetylhydrolase is one of the pri­
mary enzymes in these processes (Kubicek et al., 1988; Han et al., 2007).
Therefore, the positive function of Mt on P release can be confirmed,
either from chemical or genetic view.
Mt can also inhibit fungal growth via increasing viscosity (impeding
7
Geoderma 404 (2021) 115311
Fig. 6. SEM imaging on the soil samples
collected from the control (A, B), MMt (C, D),
MCal (E-H), and MGt treatments (I, J). The
samples in A, C, E and I were air dried for
observation. The samples in B, D, F, G, H and
J were fixed by glutaraldehyde for observa­
tion. A. A. niger grown on the surface of
quartz. B. Formation of calcium oxalate. C.
Mycelium of A. niger on montmorillonite. D.
Montmorillonite particles adsorbed on the
mycelium of A. niger. E. A. niger grown on the
surface of quartz. F-G. CaCO3 particles
adsorbed on the surface of hypha. H. P-Ca
complexes were detected together with
CaCO3. EDS (see the inserts) showed distri­
bution of C, P and Ca. I-J. Goethite particles
adsorbed on A. niger surface, accompanying
with adsorbed P. EDS (see the inserts)
showed the distribution of Fe, Ca and P.
the transport of nutrients, metabolites, and gases across the mycelial
wall) (Stotzky and Rem, 1968; Lavie and Stotzky, 1986). Therefore, soils
with abundant clays may suppress microbial growth (Hassink et al.,
1993; Arndt et al., 2013; Shi and Marschner, 2013). In addition,
although oxaloacetate acetylhydrolase gene was up-regulated with Mt
addition, organic acids were actually under the detection limit (see
Fig. 3A, D, G). Previous studies showed that Mt can adsorb organic acids
on its surface (Zhang et al., 2018), mainly via cation bridging (Mikutta
et al., 2007; Iskrenova-Tchoukova et al., 2010; Saidy et al., 2012).
Positively charged cations can act as binding agents between negatively
charged clay surfaces and organic compounds (Mikutta et al., 2007).
Moreover, hyphae were tightly bound to the Mt particles (Fig. 6C, D),
limiting the dispersion of fungal secretion. Furthermore, the uptake of
NO−3 and transformation of organic nitrogen to ammonium by organisms
can induce soil alkalinity (Marschner, 1995; Letoffe et al., 2014).
Therefore, the strong adsorption of organic acids by Mt might cause a
M. Su et al.
Fig. 7. Expression patterns (P-related) based on RNA sequencing (RNA-Seq). A: Gene related to organic acid secretion gene; B: Gene related to organic P decom­
position. Log2 Fold Change (LogFC) values were generated for RNA-Seq samples by comparing the average FPKM values of genes for the Mt-RNA/Gt-RNA treatment
vs. the control treatment. Effective DEGs were demonstrated by applying the cutoffs of the t-test padjgt < 0.05, average FPKM > 1 and log2 fold change ≥ 1 or ≤ − 1.
Dotted line indicates log2 fold-change threshold of ± 1.0.
decline of AP.
4.4. Negative role of goethite on P availability in soil
Gt addition significantly stimulated fungal respiration (Fig. 2C),
while decreased the fungal abundance (Fig. 2F). This was consistent
with previous studies regarding microbial growth under various severe
stresses (Schimel et al., 2007; Ye et al., 2019). Tightly adsorbed iron
oxide on cell surface may increase intracellular Fe and induced subse­
quent Fenton reaction (reactive oxygen). Then, it could enhance cell
permeabilization (Imlay et al., 1988; Schoonen et al., 2010; Williams
et al., 2011; Qu et al., 2019). Under the stresses, microorganisms may
allocate more C to metabolism, rather than to microbial biomass
(Schimel et al., 2007). In addition, metal oxides can adsorb abundant
organic matter via electrostatic attraction or ligand-exchange reactions
(Mikutta et al., 2014), which further decreased nutrient availability for
microorganisms.
Gt showed high adsorption capability for AP (Fig. 5D). Phosphates
adsorbed on Gt usually appear as stable inner-sphere bidentate and
monodentate complexes (Sparks, 2003b). This adsorption might
contribute to the decreasing P (Fig. 4). Meanwhile, the formation of
ferric citrate also reduce the acidolysis for inorganic phosphates.
Therefore, although Gt significantly altered the expression of genes
related to phosphatase and phytase (for the living cells), the declined
fungal counts and its strong sorption should eventually degrade P
availability.
5. Conclusions
PSF promoted P release, mainly through secreting oxalic acids to
dissolve phosphates. Based on the biochemical and genetical analysis,
we finally confirmed that different soil minerals influenced P release via
distinct pathways. Calcium carbonate addition inhibited the fungal
growth via pH buffering, showing negative role on P release. Montmo­
rillonite addition promoted the fungal respiration and up-regulated gene
encoding oxaloacetate acetylhydrolase, whereas, it could also decrease
AP via adsorption of organic acids. Therefore, Mt showed complex role
8
Geoderma 404 (2021) 115311
in regulating P biogeochemistry. Decline of fungal abundance and high
sorption capability of Gt may result in low P availability.
Declaration of Competing 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.
Acknowledgements
This work was supported by National Key R&D Program of China
(2020YFC1808000) and National Natural Science Foundation of China
(NO. 42007030). This work was also partially supported by Program for
Student Innovation Through Research and Training (S20190010 &
201910307090P).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.geoderma.2021.115311.
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