Geoderma 337 (2019) 749–757

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

Arbuscular mycorrhizal fungi and the associated bacterial community T

influence the uptake of cadmium in rice
⁎
Xun Wen Chena,b, Li Wua, Na Luoa, Ce Hui Moa, Ming Hung Wonga,b,c, Hui Lia,
a
Guangdong Provincial Research Center for Environment Pollution Control and Remediation Materials, Department of Ecology, College of Life Science and Technology,
Jinan University, Guangzhou 510632, China
b
State Environmental Protection Key Laboratory of Integrated Surface Water-Groundwater Pollution Control, School of Environmental Science and Engineering, Southern
University of Science and Technology, Shenzhen 518055, China
c
Consortium on Health, Environment, Education and Research (CHEER) and Department of Science and Environmental Studies, The Education University of Hong Kong,
Tai Po, Hong Kong, China

A R T I C LE I N FO A B S T R A C T

Handling Editor: Yvan Capowiez Cadmium (Cd)-contaminated rice imposes severe health risks to human. The present study investigated the role
Keywords: of arbuscular mycorrhizal fungi (AMF) in sculpting the rhizospheric bacterial community, and the potential
Symbiosis effects on the Cd uptake by rice. AMF Funneliformis mosseae (Fm) or Rhizophagus intraradices (Ri) were inoculated
Heavy metal to rice grown in soils spiked with 0 or 10 μM Cd. Initial Cd concentration in soil was 0.18 mg/kg. AMF colo-
Oryza sativa nization rate, plant biomass, Cd content in rice, soil properties, rice Cd transporters (Nramp5 and HMA3) and
Microbial community soil bacterial community were analysed. Both AMF decreased (P < 0.05) root and shoot Cd concentrations,
Actinobacteria especially for Ri treatment. The higher relative abundance of Actinobacteria (mostly from genus Arthrobacter)
observed in Ri treatment probably absorbed Cd in soil, and hence decreased the Cd availability for rice.
Expression of genes Nramp5 and HMA3 in root were lower in Ri treatment, but higher in Fm treatment. The gene
expressions were in line with the results of lower root Cd content in Ri treatment, and higher in Fm treatment.
The present study firstly revealed that AMF can reduce rice Cd uptake by changing the expression of Cd
transporters and soil bacterial community in a pot experiment. Effects of plants-bacteria-fungi interaction on
both plant productivity and toxicants uptake deserved further study.

1. Introduction Unfortunately, rice is efficient in taking up Cd. The soil-to-grain bio-

Cadmium (Cd) possesses a higher mobility and toxicity to organisms
than other heavy metals (He et al., 2015; Song et al., 2015). Cadmium
could be detected in crops imposing severe health problems (McBride
et al., 2014; Esposito et al., 2015; Norton et al., 2015). In Japan, in-
gestion rate of Cd can be up to 3–4 mg Cd/week/kg body weight due to
rice consumption (Tsukahara et al., 2003). Accumulating evidence
showed that the increased cancer incidence was related to environ-
mental exposure to Cd (Satarug et al., 2010). The endometrial cancer
risk was significantly associated with Cd intake and the risk was in-
creased in a Swedish cohort who consumed > 15 μg/day of Cd, mainly
from cereals and vegetables (Åkesson et al., 2008). In China, a sig-
nificant amount of arable land (~2.786 × 109 m2) has been con-
taminated by Cd (Liu et al., 2015; Xue et al., 2017), and Cd in cereals
and vegetables were 0.008–0.062 mg/kg and 0.007–0.021 mg/kg, re-
spectively (Song et al., 2017). Rice (Oryza sativa L.) is a major staple
cereal crop throughout the world, especially in Asia (Liu et al., 2014).
concentration factors of rice are 0.300–1.112 (Song et al., 2015). Cd
concentration in rice grain was up to 0.062 ± 0.128 mg/kg (Song
et al., 2017). Rice consumption was the main Cd exposure pathway to
humans (Hu et al., 2009; Li et al., 2017; Song et al., 2017). Health risk
of Chinese people should be of concern due to the high level of rice
intake (218.6 ± 174.5 g/day) (Song et al., 2017).
Cd in rice is mainly taken up by roots in soil. To reach the grain, Cd
in soil has to pass through epidermis, exodermis, cortex and endodermis
before reaching the root xylem (Schreiber and Franke, 2001). Rice takes
up Cd and the necessary manganese (Mn) at the same time via Nramp5
transporter, which is polarly localized at the distal side of both exo-
dermis and endodermis cells (Sasaki et al., 2012). Cd is then translo-
cated from root cortex to xylem due to transpirational pull. During this
step, most Cd is either translocated to the xylem (high Cd-accumulating
cultivar in grain, e.g. Anjana Dhan) or sequestered into the vacuoles
through vacuole membrane transporter HMA3 (low Cd-accumulating
cultivar in grain, e.g. Nipponbare) (Ueno et al., 2009, 2010; Miyadate

⁎
Corresponding author.
E-mail address: tlihui@jnu.edu.cn (H. Li).

https://doi.org/10.1016/j.geoderma.2018.10.029
Received 7 May 2018; Received in revised form 21 September 2018; Accepted 15 October 2018
0016-7061/ © 2018 Elsevier B.V. All rights reserved.
X.W. Chen et al.
et al., 2011; Sasaki et al., 2014). After reaching the xylem, Cd is sub-
sequently pulled up by transpirational pull, and distributed to leaves
and grains (Uraguchi et al., 2009; Li et al., 2017). In the first place, Cd
has to pass through the first barrier of root, i.e. epidermis. Here, Cd is
either passively transported through the apoplast pathway or actively
carried in by proteins that mediating transportation of Cd from soil into
root through the symplast pathway (Zhao et al., 2010; Sasaki et al.,
2012). Therefore, the mobility of Cd in soil is essential at the first step
during Cd uptake.
Soil microbes play important roles in affecting soil properties and
plant growth which significantly influence heavy metals uptake.
Arbuscular mycorrhizal (AM) fungi are the most common mycorrhizal
type and form obligately symbiotic association with 80–90% of ter-
restrial plants (Smith and Read, 2008). They play significant roles in
affecting plant growth (Klironomos, 2003; Smith and Read, 2008), not
only by acquiring nutrients (Paszkowski et al., 2002; Karandashov and
Bucher, 2005; Ma et al., 2006; Barrett et al., 2011), but also resisting
biotic and abiotic stresses (e.g. heavy metals) for plants (Wilkinson and
Dickinson, 1995; Ruiz-Lozano, 2003; Hildebrandt et al., 2007; Pozo
et al., 2010). AM fungi have been widely reported for their profound
effects on re-regulating gene expression of the hosts (Paszkowski et al.,
2002; Smith and Read, 2008; Fiorilli et al., 2009; Handa et al., 2015).
Glomalin generated from AM fungi acts as a chelator and can also
immobilize heavy metals, although the amount can be immobilized is
limited (i.e., < 1% of total Cd) (Wu et al., 2014).
For mycorrhizal rice, the arsenite transporters (Chen et al., 2012),
phosphate transporters (Paszkowski et al., 2002; Chen et al., 2013), and
other genes (e.g. encoding a putative peroxidase and a serine/threonine
kinase-like protein) (Angelard et al., 2010) can be significantly altered,
compared with non-mycorrhizal treatment. Mycorrhizal symbiosis can
influence plant growth and acquisition of arsenic and phosphorus.
However, to our knowledge, there is no investigation conducted to
study the effects of AM fungi on changing the expressions of Nramp5
and the Cd-limiting gene HMA3. The mechanisms that AMF can change
the expression of Cd transporters is not clear.
On the other hand, AM fungal symbiosis profoundly influences the
bacterial community in the mycorrhizosphere (Söderberg et al., 2002;
Offre et al., 2007; Lioussanne et al., 2010; Nuccio et al., 2013). Bacteria
can reduce the heavy metal availability in soil by chelation adsorption,
intracellular sequestration or precipitation (White et al., 1997; Gadd,
2004). It has also been shown that bacteria can form siderophores to
alleviate metal-induced oxidative stress (Burd et al., 2000; Dimkpa
et al., 2008) and lower the formation of cell-damaging free radicals
(Dimkpa et al., 2009). Bacterial cells were capable of binding large
quantities of metals including Cd (Mullen et al., 1989). Bacterial com-
munity structure was the key factor in influencing Cd and Zn uptake by
plant Arabidopsis halleri (Muehe et al., 2015). Some bacterial strains, i.e.
Variovorax paradoxus, Rhodococcus sp. and Flavobacterium sp., were
capable of stimulating root elongation of Brassica juncea exposed to Cd
(Belimov et al., 2005). It suggests that bacteria not only can immobilize
heavy metals, but also promote plant growth.
The structure of soil bacterial community depends more on AM
fungi than host plant identity (Vestergård et al., 2008; Bonfante and
Anca, 2009) and also soil pH (Fierer and Jackson, 2006). Plant species
richness has been found to be correlated with AM fungal species rich-
ness (van der Heijden et al., 1998). Taken together, it suggests that
plant and AM fungal association, apart from soil pH, are the two major
factors in determining bacterial communities. The interaction of plants,
bacteria, AM fungi and soil properties essentially affects the final ac-
cumulation of Cd in plants. However, it is still not clear whether the
reduced Cd uptake is caused by changing Cd transporters in mycor-
rhizal rice, or by reducing the availability of soil Cd due to AM fungi-
sculpted bacterial community. It is hypothesized that AMF can change
soil bacterial community, but not the Cd transporters. Thus, the ob-
jectives of this study are to investigate whether the inoculation of AM
fungi can change the expression of Cd transporters and alter soil
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Geoderma 337 (2019) 749–757
bacterial community, which would possibly influence Cd uptake in rice.
2. Materials and methods
2.1. Plant cultivation
Rice cultivar Hanyou 3 was employed since it takes up less Cd when
inoculated with Rhizophagus intraradices, based on our previous study
(Luo et al., 2017). The seeds of Hanyou 3, obtained from Shanghai
Academy of Agricultural Sciences, China, were disinfected using 10%
H2O2 for 15 min, and washed with sterile distilled water three times.
The seeds were then germinated and cultured hydroponically with
Hoagland solution (Hoagland and Arnon, 1950) in a greenhouse, under
day/night temperatures of 28/22 °C and relative humidity of 80–85%.
2.2. Soil preparation and pot experiment
The soil for the pot experiment was collected in a flooded paddy
field, Guangzhou, China. Soil properties were 81 g/kg organic matter,
1.3 g/kg total N, 1.1 g/kg total P, 0.61 g/kg total K, 0.18 mg/kg total
Cd, and pH 5.8 (determined in Li et al., 2016). The soil was air-dried for
two weeks and sieved (2 mm). Subsequently, cadmium chloride (fol-
lowing our previous study (Li et al., 2016)) was spiked to the soil and
incubated for two months. Total Cd concentration was determined
(10.02 mg/kg) before use.
Two seedlings (height 15 cm) were chosen and transplanted into
each pot containing 1.5 kg soil. The plants were inoculated with AM
fungi R. intraradices (Ri), F. mosseae (Fm), or sterilized inoculum (un-
inoculated, UI, as a control) and grown in soil with or without spiked
Cd. The two AM fungal strains were chosen since we have demonstrated
that they could reduce Cd uptake in Hanyou 3 (Li et al., 2016; Luo et al.,
2017). The amount of inoculum for each pot was 50 g. The inoculum
was a mix of root pieces, mycelium pieces and spores (> 10 infectious
propagules/g). There were three replicates for each treatment using a
random block design. The plants were irrigated daily. Hoagland solu-
tion was applied every week.
2.3. Determination of AMF colonization rate and Cd concentration in plant
After 50 d, the plants were harvested and washed with deionized
water. The fresh weights were measured. Subsamples of the roots were
stained following Phillips and Hayman protocol. Briefly, roots were
heated at 90 °C in 10% KOH for 2 h, washed with fresh KOH, and im-
mersed in an alkaline solution of hydrogen peroxide until bleached.
Roots were then rinsed by water, acidified in dilute HCl and stained
using 0.05% trypan blue in lactophenol. Excess stain was removed
using clear lactophenol (Phillips and Hayman, 1970). Subsequently,
sixty roots were randomly collected for each subsample to determine
the colonization rate using the gridline intersection method
(Giovannetti and Mosse, 1980).
Shoots and roots were dried in an oven at 65 °C and the dry weight
(DW) were measured. Grinded samples were digested with con-
centrated nitric acid (Li et al., 2016). Cd concentrations were de-
termined using atomic absorption spectrometry (PinAAcle 900 T, Per-
kinElmer, USA). Blanks and standard reference material [GBW07602
(GSV-1)] (China Standard Materials Research Center, Beijing, China)
were used for QA/QC purposes. Recovery rates of Cd were within
90 ± 10%. The translocation factor (TF) was calculated as shoot Cd
concentration divided by root Cd concentration.
2.4. Determination of the soil physicochemical properties
Mobile proportion of Cd in soil was extracted by agitation with a
solution containing 0.005 M diethylenetriaminepentaacetic acid
(DTPA), 0.1 M triethanolamine (TEA) and 0.01 M CaCl2 (pH 7.3, solu-
tion: soil = 2:1) for 2 h. The Cd concentrations were determined using
X.W. Chen et al. Geoderma 337 (2019) 749–757

atomic absorption spectrometry (Lindsay and Norvell, 1978). Soil pH

was determined (soil: water =1: 2.5, w/v) using a pH meter (Fangzhou
pHS-4C+, China).

2.5. Analyses of Cd transporter (Nramp5) and vacuole sequestration gene

(HMA3)

Subsamples of fresh roots were collected and frozen in liquid ni-

trogen. Total RNA was extracted using an RNA extraction kit (RNeasy
Plant Mini Kit, Qiagen, Germany). 250 ng RNA was used for first-strand
cDNA synthesis using Reverse Transcriptase M-MLV (RNase H-)
(TaKaRa, Japan). Nramp5, HMA3 and GAPDH (Glyceraldehyde 3-
phosphate dehydrogenase, as internal control) were amplified by using
SYBR Premix Ex Taq (Tli RNase H Plus) (TaKaRa, Japan) for real-time
polymerase chain reaction (PCR) with the designed primer pairs:
5′-GCCTTGGTGCTATCGAGGAA-3′ and 5′-TACAGGAAGAACCTGCA
CCC-3′ for Nramp5; 5′-CTGGCTCTGGTGATGCTTGT-3′ and 5′-TGAAG
ATCCCCATCCTCGCA-3′ for HMA3; 5′-CCTTTTGTAAGGAGAAAGGAG
Fig. 1. Plant dry masses derived from different treatments. UI, uninoculated;
CAAC-3′ and 5′-ATGGCTCCTCCCAAGCAATC-3′ for GAPDH. Relative Fm, F. mosseae; Ri, R. intraradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and
expression of Nramp5 and HMA3 were determined from the Ct values Ri treatments spiked with 10 mg/kg of Cd in soil, respectively. NS, not sig-
obtained from the detection system ABI 7500 (Applied Biosystems, MA, nificant (P > 0.05); *P < 0.05; **P < 0.01. Data are mean ± S.D. (n = 3).
USA) and the Pfaffl's method (Pfaffl, 2001).
3. Results
2.6. Analysis of bacterial community
3.1. Plant biomass and AM fungal colonization rate
For each pot, three soil subsamples closely attached to the roots
were sampled, put in three different centrifuge tubes respectively and Without Cd addition (i.e. treatments UI, Fm and Ri), Fm enhanced
submerged in liquid nitrogen. Total DNA in soil was extracted using both root and shoot dry mass (P < 0.05), compared with UI and Ri
MOBIO PowerSoil® DNA Isolation Kit (MOBIO, CA, USA). Primers 515F (Fig. 1). Ri did not influence the root and shoot dry mass, compared
(5′-GTGCCAGCMGCCGCGGTAA-3′) (Walters et al., 2016) and 907R with UI (Fig. 1). Cd addition decreased (P < 0.05) root biomass of
(5′-CCGTCAATTCMTTTRAGTTT-3′) (Tischer et al., 2015) were used to UI·Cd and Fm·Cd treatments, but not Ri·Cd treatment, compared with
amplify the V4–V5 region of the 16S rRNA gene. Barcodes for sample their corresponding non-Cd treatments, respectively. The shoot biomass
multiplexing were added to the 5′ terminus of the forward primer. Dual- of all treatments was decreased (P < 0.05) due to Cd addition. Com-
index barcodes and sequencing adapters were attached to the amplicon pared to UI, the decrement of shoot biomass was the largest for UI·Cd,
using NEBNext® Multiplex Oligos for Illumina® (NEB, CA, USA). followed by Fm·Cd and Ri·Cd. A similar trend was observed for root
The PCR was conducted in a 50-μL system using a thermal cycler biomass (Fig. 1). For inoculated treatments, AM fungal colonization
(T100 Gradient Cycler, Bio-Rad, USA) Subsequently, the PCR products rates ranged from 32 to 43% (Fig. 2). A significantly higher coloniza-
were purified using OMEGA Gel Extraction Kit (Omega Bio-tek, tion rate was observed in plants inoculated with Ri (> 40%), while
Norcross, GA) and quantified using a Qubit®2.0 fluorometer (Life 32–35% colonization rate was observed in those inoculated with Fm.
Technologies, Shanghai, China). Addition of Cd did not notably change the colonization rate of Fm and
PCR products were then sequenced using the MiSeq sequencing Ri (Fig. 2).
platform (PE300, Illumina, CA, USA) according to manufacturer's in-
struction. The final contigs were analysed using QIIME (Quantitative
Insights Into Microbial Ecology) v1.9.1 (Caporaso et al., 2010). The
obtained sequences were classified into different operational taxonomic
units (OTUs) using open reference OTUs picking method against
Greengenes 13.8 database with a similarity of 97% (Desantis et al.,
2006; McDonald et al., 2012). The most abundant sequence for each
OTU was selected as the representative sequence for further analyses.
Taxonomy information was assigned to each OTU using QIIME.

2.7. Data analysis

Statistical analyses were conducted using SPSS v22. Student's t-test

was used to compare the means derived from different treatments. One-
sample t-test was used to compare Nramp5 and HMA3 with the re-
ference gene (test value = 1). For all tests, 95% confidence level was
used.
For bacterial community, a table of OTU counts was generated.
Together with the phylogenetic tree, the OTU table was used to cal-
culate the alpha and beta diversities using QIIME. The relative abun-
dance (%) of each OTU was calculated. Principal coordinates analysis Fig. 2. AM fungal colonization rates derived from different treatments. UI,
(PCoA) was carried out using matrix of unweighted UniFrac distance uninoculated; Fm, F. mosseae; Ri, R. intraradices; UI·Cd, Fm·Cd and Ri·Cd in-
which considers the phylogenetic distance between sets of taxa dicate UI, Fm and Ri treatments spiked with 10 mg/kg of Cd in soil, respec-
(Lozupone and Knight, 2005). tively. *P < 0.05. Data are mean ± S.D. (n = 3).

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X.W. Chen et al.
Fig. 3. Cd concentrations in roots (a), shoots (b) and extractable Cd in soil (c)
derived from different treatments. UI, uninoculated; Fm, F. mosseae; Ri, R. in-
traradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri treatments spiked
with 10 mg/kg of Cd in soil, respectively. NS, not significant (P > 0.05);
*P < 0.05; **P < 0.01. Data are mean ± S.D. (n = 3).
3.2. Cd in plant and soil
For treatments without Cd addition (UI, Fm and Ri), Cd
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Geoderma 337 (2019) 749–757
Fig. 4. Cd content accumulated in root and shoot. UI, uninoculated; Fm, F.
mosseae; Ri, R. intraradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri
treatments spiked with 10 mg/kg of Cd in soil, respectively. The values were
calculated by multiplying the dry biomass (g) with the corresponding Cd con-
centrations (μg/g). *P < 0.05. Data are mean ± S.D. (n = 3).
concentrations ranged from 0.28 to 0.35 mg/kg in roots, while ranged
from 0.02 to 0.04 mg/kg in shoots (Fig. 3a). For treatments with Cd
addition, the Cd concentrations in root and shoot (UI·Cd treatment)
were 27.6 and 8.3 mg/kg, respectively. AM fungi Fm and Ri sig-
nificantly decreased (P < 0.05) Cd concentration in root by ~18 and
~38%, respectively, compared with UI·Cd (Fig. 3a). For shoot, Fm and
Ri significantly decreased (P < 0.05) the Cd concentrations by ~20
and ~23%, respectively (Fig. 3b).
Extractable Cd in soil of Ri·Cd treatment was significantly lower
(P < 0.05) than that of UI·Cd (Fig. 3c). As a consequence, the amount
of Cd content accumulated in root of treatment Ri·Cd was the lowest,
compared with UI·Cd and Fm·Cd (Fig. 4). The treatment Ri·Cd with
lower extractable Cd in soil (Fig. 3c) has lower Cd content in root
(Fig. 4). However, the amount of Cd content accumulated in shoot of
Ri·Cd treatment was not significantly lower than those of UI·Cd and
Fm·Cd (Fig. 4). This was caused by moderately more shoot mass yielded
in Ri·Cd treatment leading to more accumulated Cd content, compared
with UI·Cd and Fm·Cd (Fig. 1). For Fm·Cd, the TF was unchanged, while
for Ri·Cd, the TF was increased significantly, compared with UI·Cd
(Fig. 5).
3.3. Relative expression of Cd transporters (Nramp5 and HMA3)
Relative expression of Nramp5 in root was not significantly changed
in UI·Cd, but it was significantly decreased in Ri·Cd (Fig. 6a). This co-
incided with the findings that a lower Cd concentration in root of Ri·Cd
treatment was recorded compared with UI·Cd (Fig. 3a). However, the
significantly higher level of Nramp5 in Fm·Cd (Fig. 6a) contradicted
with the results that a lower Cd concentration was observed in Fm·Cd,
compared with UI·Cd (Fig. 3a). Since the changes of plant biomass (due
to the effects from both AM fungi and Cd) influenced the concentration
in the long run (50 d of growing), the temporary gene expression level
was not correlated with the Cd concentration, but rather, the total Cd
content. In this case, expression levels of Nramp5 were in line with the
total Cd contents in roots which is shown in Fig. 4. The greatest amount
of Cd content in roots was found in Fm·Cd, followed by UI·Cd and Ri·Cd
(Fig. 4). This observation was in line with the results that the highest
Nramp5 expression was found in Fm·Cd treatment, followed by UI·Cd
and Ri·Cd (Fig. 6a). A similar trend was observed for HMA3 gene
(Fig. 6b).
X.W. Chen et al. Geoderma 337 (2019) 749–757

a 4.0

**
3.0

(relative abundance)
Nramp5 / GAPDH
2.0

1.0

** **
**
0.0
Fm Ri UI.Cd Fm.Cd Ri.Cd

Treatment
b 4.0
Fig. 5. Comparison of translocation factors (Cd concentration in shoot/Cd
concentration in root) of Cd in rice plant. UI, uninoculated; Fm, F. mosseae; Ri,
R. intraradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri treatments spiked
with 10 mg/kg of Cd in soil, respectively. Student's t-test at a probability level of 3.0 **

(relative abundance)
5% was used to separate the differences between treatments. NS, not significant
(P > 0.05); *P < 0.05. Data are mean ± S.D. (n = 3). HMA3 / GAPDH
2.0
3.4. Soil bacterial community *
Ten phyla of bacteria (relative abundance > 1%), i.e. Acidobacteria,
1.0
Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes,
Gemmatimonadetes, OD1, Planctomycetes, Proteobacteria and
*
**
Verrucomicrobia, were observed in different soil samples. Other phyla
with relative abundance < 1% were clustered as “Others” (Fig. 7). The 0.0
most abundant phylum was Proteobacteria (~50%), followed by Fir- Fm Ri UI.Cd Fm.Cd Ri.Cd

micutes (~20%), Acidobacteria (~15%) and Chloroflexi (~5%). The Treatment

relative abundances of these four phyla were similar across all treat-
ments. However, Ri·Cd treatment increased the relative abundances of
Actinobacteria to 15%, compared to other treatments which had only
1–2% (Fig. 7). Data points representing the structure of bacterial
community were clustered to the negative side of PC1 due to Cd ex-
posure, and data points of Fm·Cd treatment were separated from those
of Ri·Cd treatment (Fig. 8). Such a pattern was not observed in non-Cd
treatment. Although the PCoA explains only 18.12% of the variance, it
seems Cd and AM fungi exerted a combined effect on changing the
structure of soil bacterial community.
4. Discussion
4.1. Protective effects of AM fungi on rice plant subjected to Cd stress
Fig. 6. The relative abundances of Nramp5 and HMA3 genes in roots of dif-
ferent treatments, compared to uninoculated treatment (UI). The abundances of
Nramp5 and HMA3 genes for UI are presented as the dotted lines (value = 1).
Gene expressions were normalized using the ΔΔCt method (Pfaffl, 2001) with
the housekeeping gene GAPDH. Gene expressions of other treatments, i.e., Fm,
Ri, UI·Cd, Fm·Cd and Ri·Cd, were compared with 1 to study the differences
between treatments and control (i.e., UI). Values higher than 1 indicate the
gene expressions were up-regulated compared to control, while values lower
than 1 indicate gene expressions were down-regulated. Fm, F. mosseae; Ri, R.
intraradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri treatments spiked
with 10 mg/kg of Cd in soil, respectively. * and ** indicate significant differ-
ences (P < 0.05 and < 0.01, respectively) between the level of gene expres-
sion and the control (UI) using one-sample t-test (test value = 1). Data are
mean ± S.D. (n = 3).

and Ri·Cd were not enhanced. Instead, the accumulated Cd content in

In the present pot experiment, for non-Cd treatments, Fm enhanced
the root and shoot biomass significantly, while Ri had no such an effect
(Fig. 1). For Cd treatments, AM fungus Fm significantly increased root
biomass, while Ri significantly increased both root and shoot biomass
(Fig. 1). This may suggest that the AM fungi (especially Ri) exerted
protective effects on plants against Cd exposure. In other pot studies,
similar protective effects of AM fungi on their hosts were observed in
carrot and pea subjected to Cd stress (Janoušková and Vosátka, 2005;
Rivera-Becerril et al., 2005). Enhanced biomass in Cd treatments could
lead to a dilution effect which has been observed in other pot experi-
ments (Liu et al., 2005; Ahmed et al., 2006; Chen et al., 2007, 2013). In
the present study, dilution effect was also observed. With a similar
accumulated Cd content in shoot (Fig. 4), the enhanced shoot biomass
(Fm·Cd and Ri·Cd, Fig. 1) led to a lower Cd concentration in shoot
(Fig. 3a, b). Even root biomass increased in Fm·Cd and Ri·Cd compared
with UI·Cd (Fig. 1), the accumulated Cd contents in roots from Fm·Cd
roots was even decreased in Ri·Cd treatment (Fig. 4). The decrease in
root Cd content could be possibly caused by the changes of Cd trans-
porters (Fig. 6) and the lower level of soil Cd availability (Fig. 3c).
4.2. AM fungi influenced expression of Cd transporters
Fig. 6a shows the relative abundance of Nramp5 gene expression,
which is responsible for the transport of Mn and Cd from the external
solution into root cells (Sasaki et al., 2012). On the other hand, HMA3 is
a Cd-limiting gene in root that sequesters Cd into the vacuoles of root
cells thus decreases the translocation rate of Cd from root to shoot
(Ueno et al., 2010; Miyadate et al., 2011). It would be expected that
when a higher level of HMA3 expression is observed in root, more Cd
content in root and less in shoot would be found (Ueno et al., 2009,
2010; Miyadate et al., 2011; Sasaki et al., 2014). In the present study,

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X.W. Chen et al.
Fig. 8. PCoA of unweighted UniFrac distances for the rhizospheric microbiota
of different treatments. UI, uninoculated; Fm, F. mosseae; Ri, R. intraradices;
UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri treatments spiked with 10 mg/kg
of Cd in soil, respectively. Number 1, 2 and 3 indicate replicates. PC, principal
coordinate. UniFrac distance reveals the similarity between sequences
(Lozupone and Knight, 2005).
Nramp5 expression was up-regulated in Fm·Cd treatment compared
with UI·Cd, indicating that Cd content accumulated in both root and
shoot should be higher versus UI·Cd. On the other hand, HMA3 ex-
pression was also up-regulated in Fm·Cd treatment compared with
UI·Cd. Based on the function of HMA3 gene, it suggests that Cd content
in root should be higher while that in shoot should be lower, compared
with UI·Cd. Taken together, it would be expected that higher root Cd
contents and relatively stable shoot Cd contents could be observed.
Indeed, our results are in line with the expectation (Fig. 4). For Ri·Cd,
based on a similar interpretation, it would be anticipated that less root
Cd content and unchanged shoot Cd content could be recorded. Fig. 4
shows significantly less root Cd content and unchanged shoot Cd con-
tent in Ri·Cd treatment versus UI·Cd. The TF of Ri·Cd treatment was
increased compared with UI·Cd (Fig. 5). This is also in line with the
result that a lower HMA3 expression led to more shoot Cd. To our
754
Geoderma 337 (2019) 749–757
Fig. 7. Comparison of relative abundance (> 1%) of dif-
ferent bacterial phyla detected in soils under different
treatments. UI, uninoculated; Fm, F. mosseae; Ri, R. in-
traradices; UI·Cd, Fm·Cd and Ri·Cd indicate UI, Fm and Ri
treatments spiked with 10 mg/kg of Cd in soil, respec-
tively. Asterisk indicates significantly higher relative
abundance observed (P < 0.05). Data are mean ± S.D.
(n = 3).
knowledge, the re-regulation of Nramp5 and HMA3 genes expression
upon mycorrhizal symbiosis in rice was firstly reported in the present
study.
4.3. Effects of soil bacterial community on Cd accumulation
The relative abundance of Actinobacteria was increased in Ri·Cd
treatment (Fig. 7). Increased abundance of Actinobacteria was com-
pensated mostly by the decrease of Firmicutes. It has been reported that
Actinobacteria could absorb Cd and decrease Cd availability for plant
(Tsuruta et al., 2014). It has been found that the bacterium A. nicotianae
(among 51 microorganism strains tested) was highly capable in ab-
sorbing Cd from an aqueous solution (Tsuruta et al., 2014). Such a
feature probably contributed to the lower extractable Cd in soil
(Fig. 3c), significantly lower Cd concentration in plant (Fig. 3a, b), and
lower Cd content in plant (Fig. 4) in Ri·Cd treatment.
On the other hand, relative abundance of Actinobacteria was en-
hanced only in Ri treatments under Cd stress, not in Fm treatments
(either with or without Cd) (Fig. 7). Here, the PCoA showed that the
structure of bacterial communities in soils were influenced by Cd rather
than AM fungal species (PC1) (Fig. 8). For non-Cd treatments plus AM
fungi, soil bacterial communities were more heterogeneous (Fig. 8,
positive side of PC1). With Cd, the bacterial communities were mod-
erately altered due to AM fungal treatments, i.e. Ri·Cd vs. Fm·Cd (Fig. 8,
PC2). Cd had influenced the bacterial community and deviated it away
from that of the non-Cd treatments (Fig. 8, PC1). Similar findings that
Cd influences the structure of bacterial community were observed in
other studies (Ranjard et al., 2006; Lazzaro et al., 2006). It also has
been pointed out that not only Cd, but also pH play a role (coupling
effects) in changing soil bacterial community (Lazzaro et al., 2006).
From our results, soil pH, one of the most important factors influencing
plant Cd uptake (Yu et al., 2016), was not significantly affected by
mycorrhizal treatment (Fig. A1).
Plant species has greater effects on the bacterial community in the
rhizosphere than AM colonization (Söderberg et al., 2002), while plant
species influences the AM fungal community (Torrecillas et al., 2012).
Mycorrhizal fungi also shape the bacterial community in the mycor-
rhizosphere (Uroz et al., 2007). At the same time, fungi-bacteria in-
teraction in the mycorrhizosphere relies on soil properties (Toljander
et al., 2008). Bacteria Comamonadaceae was more abundant in my-
corrhizal treatment than non-mycorrhizal treatment (host: Medicago
truncatula) (Offre et al., 2007). Firmicutes responded positively, while
Actinobacteria and Comamonadaceae responded negatively to AMF
Glomus hoi (Nuccio et al., 2013) (host: Plantago lanceolata). The results
of these two studies showed that the phylum Comamonadaceae re-
sponded differently to AM fungal association. Plant species (Söderberg
X.W. Chen et al.
et al., 2002) and soil properties (Fierer and Jackson, 2006) have a more
profound influence, while AM fungi have less on the structure of bac-
terial community. Soil bacterial communities are mediated through the
effects of plant-derived resources on antagonistic soil microbes. The
interaction among the four components (i.e., fungi, bacteria, plants and
soil) is complex. Results from different studies are contradicting and
fluctuating. The findings are not conclusive using different plants, soils,
fungi and bacteria.
According to these observations, the lowest Cd concentration in root
and shoot for Ri·Cd treatment (Fig. 3a, b) could be probably caused by
the higher relative abundance of Actinobacteria (Fig. 7), which poten-
tially caused by the coupling effects of Ri and Cd. The Actinobacteria in
the present study, mostly contained the species from genus Arthrobacter.
The ability of Arthrobacter sp. in absorbing Cd has been demonstrated
(Grappelli et al., 1989; Pagnanelli et al., 2000; Tsuruta et al., 2014). In
addition, lower Cd content was observed in roots inoculated with Ri
(Fig. 4). This led to the conclusion that AM fungus Ri promoted the
abundance of Arthrobacter sp., which immobilized Cd and subsequently
reduced the Cd availability (Fig. 3c) for the rice plant.
It should be noted that AM fungal colonization is host-specific. Host
plant species influences AM fungal community structure (Eom et al.,
2000; Davison et al., 2011). In rice field, different AM fungal species
may co-exist in soil and those can associate with rice would be selected
by the rice genotype. A dialog between AM fungi and the host is re-
quired prior to the symbiosis (Maillet et al., 2011; Sun et al., 2015;
Gutjahr et al., 2015). Inoculation of mixed AM fungal species could
alleviate environmental stresses resulting in better plant performance
compared with inoculation of a single species (Schreiner and
Bethlenfalvay, 1997; Labidi et al., 2015). It is not clear what AM fungal
species would colonize the host. However, it is known that different AM
fungi generally restricted the Cd uptake in plants (Li et al., 2016; Luo
et al., 2017). Dual inoculation of AM fungi Glomus geosporum and G.
mosseae decreased total arsenic uptake in upland rice (Chan et al.,
2013). The present study also shows that different AM fungi exerted
different influences on Cd transporters and soil bacterial community
(Figs. 6 and 7). Generally, it is expected that the assemblage of AM
fungi could limit Cd uptake in rice no matter what AM fungal species
are associated with the rice. However, the actual effects should be
further investigated due to the species-dependent issue. The present
study implies that different combinations of soil properties (e.g. Cd
concentration in the present study) and mycorrhizal fungal species
could exert different effects on the bacterial community, and hence the
uptake of Cd in rice. The present study investigated the fundamentals of
AM fungi in affecting Cd uptake in rice in controlled environment be-
fore field application. The roles of multiple AM fungal species in af-
fecting Cd uptake need to be further investigated.
5. Conclusions
In this pot experiment, inoculation of Ri decreased (P < 0.05) Cd
concentration in both root and shoot of rice plant. Soil pH has not been
significantly altered upon mycorrhizal and Cd treatments. The de-
creases in Cd concentrations, therefore, was probably caused by lower
extractable Cd concentration in soil. Less phytoavailable Cd presented
in soil could be caused by the higher relative abundance of
Actinobacteria (mostly from genus Arthrobacter) that can probably ad-
sorb or chelate Cd. Inoculation of AMF also influenced the expression of
rice Cd transporters in a species-dependent manner. The present study
firstly revealed that, AMF can indirectly and directly influence Cd up-
take through shaping the structure of rhizospheric soil bacterial com-
munity and changing the abundance of Cd transporters. It should be
noted that the present findings could be species- and soil-dependents.
Further experiments under field conditions (anthropogenic and geo-
genic contaminations) are also needed. This study indicates the im-
portance of plants-bacteria-fungi interaction in soil on both plant pro-
ductivity and toxicants uptake.
755
Geoderma 337 (2019) 749–757
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.geoderma.2018.10.029.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The present project was supported by the National Key R&D
Program of China (2018YFD0800701), National Natural Science
Foundation of China (41877350), NSFC-Guangdong Joint Fund
(U1501233), the Research Team Project of the Natural Science
Foundation of Guangdong Province (2016A030312009), and the
Fundamental Research Funds for the Central Universities of China. This
work was also partially sponsored by Guangdong Provincial Key
Laboratory of Soil and Groundwater Pollution Control (No.
2017B030301012), and State Environmental Protection Key Laboratory
of Integrated Surface Water-Groundwater Pollution Control.
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