Chemosphere 296 (2022) 134045

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Phytoremediation potential evaluation of three rhubarb species and

comparative analysis of their rhizosphere characteristics in a Cd- and
Pb-contaminated soil
Jingya Yang a, b, c, 1, Yingqi Huang a, b, c, 1, Gaojuan Zhao a, b, Boqun Li d, Xiangshi Qin e,
Jianchu Xu a, b, **, Xiong Li a, b, e, *
a
Department of Economic Plants and Biotechnology, Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences,
Kunming, 650201, China
b
Honghe Center for Mountain Futures, Kunming Institute of Botany, Chinese Academy of Sciences, Honghe, 654400, China
c
University of Chinese Academy of Sciences, Beijing, 100049, China
d
Science and Technology Information Center, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China
e
Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201, China

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Tolerance indexes of three rhubarbs

were > 60% at 10 mg L− 1 Cd and 100
mg L− 1 Pb.
• Shoot bioconcentration factors of rhu­
barbs were 0.42–0.47 for Cd but < 0.01
for Pb.
• Rhubarbs changed physicochemical
indices of the rhizospheric soils.
• Three rhubarb species recruited differ­
ential plant growth-promoting
rhizobacteria.

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

Handling Editor: T Cutright Screening or breeding exceptional plant species for heavy metal phytoremediation is as important as adopting
feasible measures to enhance phytoremediation efficiency, which are largely based on clarifying the mechanisms
Keywords: of heavy metal tolerance and accumulation by plants. In this study, cadmium (Cd) and lead (Pb) tolerance and
Rheum accumulation characteristics of Rheum officinale, R. palmatum, and R. tanguticum were analysed to assess their
Cd accumulator
phytoremediation potential. The seed germination test indicated that these three rhubarb species could tolerate
Phytoextraction
10 mg L− 1 Cd and 100 mg L− 1 Pb. However, when sown in Cd- and Pb-contaminated soil, all three rhubarb
Rhizosphere effect
Plant growth-promoting rhizobacteria species exhibited a relatively high Cd accumulation capacity but a considerably low Pb accumulation capacity

Abbreviations: ACd, available Cd; AP, available phosphorus; APb, available Pb; BCF, bioconcentration factor; BRS, bare soil; Cd, cadmium; EC, electrical con­
ductivity; HN, hydrolyzable nitrogen; HTS, high-throughput sequencing; ICP–OES, inductively coupled plasma optical emission spectrometer; OTU, operational
taxonomic unit; Pb, lead; PGPR, plant growth-promoting rhizobacteria; RDA, redundancy analysis; ROS, R. officinale rhizosphere soil; RPS, R. palmatum rhizosphere
soil; RSB, root system biomass; RTS, R. tanguticum rhizosphere soil; TF, translocation factor; TI, tolerance index.
* Corresponding author. 132# Lanhei Road, Kunming, 650201, China.
** Corresponding author. 132# Lanhei Road, Kunming, 650201, China.
E-mail addresses: jxu@mail.kib.ac.cn (J. Xu), lixiong@mail.kib.ac.cn (X. Li).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.chemosphere.2022.134045
Received 23 November 2021; Received in revised form 4 January 2022; Accepted 16 February 2022
Available online 17 February 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
J. Yang et al. Chemosphere 296 (2022) 134045

according to the bioconcentration factors of Cd (0.42–0.47 in shoots and 0.11–0.15 in roots) and Pb
(0.004–0.008 in shoots and 0.007–0.013 in roots). The high Cd translocation factors (3.04–4.24) indicated that
these three rhubarb species were suitable for Cd phytoextraction. The changes in rhizospheric physicochemical
indices were generally similar among the three rhubarb plants in comparison with those of the unplanted soil.
However, differential indicator rhizobacteria were identified for the three rhubarb plants, which may be pri­
marily attributed to their different root system characteristics. These enriched rhizobacteria included many plant
growth-promoting bacteria, and several of them were also involved in regulating heavy metal uptake by plants,
indicating that three rhubarb species likely recruit differentially beneficial rhizobacteria to maintain plant
growth and vitality and to regulate heavy metal uptake in the Cd- and Pb-polluted soil. This study identifies new
candidate plant resources for the phytoremediation of Cd-polluted soils and provides novel insights into un­
derstanding the interactions among heavy metals, rhizobacteria, and plants.

1. Introduction systematic and unified understanding of the dynamic regulation process

Toxic metals such as cadmium (Cd) and lead (Pb) in soils are likely
hazardous to organisms at each nutritional level along the food chain
even at low concentrations (Okereafor et al., 2020). In addition to
controlling pollution sources, various techniques have been explored to
remediate heavy metal-contaminated soils (Vareda et al., 2019). Phy­
toremediation, an economic and eco-friendly technology, is promising
and sustainable for soil pollution remediation and has attracted signif­
icant attention (Yan et al., 2020). Phytoextraction and phytostabilisa­
tion, which are the two most commonly used and efficient
phytoremediation techniques, largely depend on the heavy metal
tolerance and accumulation ability of plants (Shen et al., 2021). Over the
past few decades, a great number of plant species and cultivars have
been investigated to determine their suitability for the phytor­
emediation of heavy metal pollution (Reeves et al., 2018).
Although there are considerable potential phytoremediation re­
sources (Reeves et al., 2018), phytoremediation efficiency has the
following bottlenecks: (1) most of the reported heavy metal accumula­
tors possess small biomass and slow growth rates (Shen et al., 2021); (2)
some potentially exceptional phytoremediation resources are prevented
from being popularised because of their regional distribution; and (3)
limited plant species have been reported to be suitable for remediating
soils contaminated with multiple heavy metals (Mahar et al., 2016). In
this context, screening and/or breeding plant resources with the ad­
vantages of strong heavy metal tolerance and accumulation capacities,
large biomass, and fast growth rates for phytoremediation is as impor­
tant as adopting feasible measures that enhance phytoremediation
efficiency.
Clarifying the mechanisms of heavy metal tolerance and accumula­
tion in plants is the basis for developing phytoremediation enhancement
strategies. In addition to the inherent physiological and molecular
mechanisms of plants (Feki et al., 2021), rhizospheric environments play
an important role in regulating the heavy metal tolerance and accu­
mulation of plants (Bian et al., 2021). Many soil factors, such as pH,
oxidation–reduction potential, root exudates, and microorganisms, can
influence the chemical forms and bioavailability of heavy metals (Yuan
et al., 2021), which directly determine the accumulation levels of heavy
metals in plants. The absorption of nutrients and root exudates or resi­
dues during plant growth can greatly influence rhizosphere physico­
chemical properties and nutrient status, leading to significant
differences in microbial community composition and activity between
rhizosphere and non-rhizosphere soils (Liu et al., 2020a). Recruited
rhizospheric microorganisms can activate or immobilise soil heavy
metals to affect their uptake by plants (Wu et al., 2022). Moreover, these
potentially beneficial microorganisms can regulate the geochemical
cycles of nutrients in soil to improve plant nutrient absorption or pro­
mote plant growth through multiple mechanisms (Abhilash et al., 2016).
Although rhizospheric ecological effects of plants under heavy metal
stress have become research hotspots, there are still many undisclosed
problems in related research owing to the dynamics and complexity of
rhizospheric environments, as well as the limitations of detection and
research methods (Yu et al., 2016a). For example, there is a lack of
in the rhizosphere of different accumulators under heavy metal stress.
The effects and mechanisms of microorganisms in rhizosphere ecosys­
tems on heavy metal bioavailability need to be further explored.
Rhubarb (Rheum L.) is an important genus of Polygonaceae and
contains approximately 60 known species worldwide. Most Rheum
species are perennial tall herbs that are primarily distributed in alpine
regions of temperate and subtropical Asia. Rhubarbs (e.g. R. officinale,
R. palmatum, and R. tanguticum) are known as traditional Chinese
medicines (Xiang et al., 2020); however, some rhubarb species (e.g.
R. rhaponticum) are also used as leafy vegetables in some regions
worldwide (van Treuren et al., 2012). Because of these factors, rhubarbs
are typically tested for harmful elements (such as Cd and Pb) to evaluate
their quality and safety (Yang and Zhang, 2021). Moreover, several
studies analysed the effects of heavy metals on seed germination of
rhubarbs (Jiang et al., 2016; Wang et al., 2020b). Although these studies
clarified the effects of heavy metals on the planting and edible safety of
rhubarbs, their potential for the phytoremediation of heavy metal
pollution has rarely been reported. Considering the potential advantage
of the large plant biomass of rhubarbs for phytoremediation, in this
study, we explored the tolerance capacities of three tall rhubarbs (i.e.
R. officinale, R. palmatum, and R. tanguticum) to Cd and Pb via seed
germination experiments. Based on this, these three rhubarbs were
further sown in Cd- and Pb-contaminated soil to evaluate their phytor­
emediation potential. Moreover, the rhizospheric characteristics
(particularly microbial community composition) of the three rhubarbs
were analysed to understand their contributions to the heavy metal
accumulation characteristics of plants. This study will provide funda­
mental information on the heavy metal accumulation characteristics of
rhubarbs and improve our understanding of the effects of the rhizo­
spheric environment on heavy metal transport in the soil–plant system.
2. Materials and methods
2.1. Seed preparation
Mature seeds of R. officinale, R. palmatum, and R. tanguticum,
collected from different areas of China (Supplementary Table S1), were
obtained from the Germplasm Bank of Wild Species (Kunming, China)
and seed companies.
2.2. Seed germination under heavy metal stress
The seeds were sterilised using 1% NaClO solution for 5 min and
subsequently cleaned using deionised water. Five solution gradients
were set for both Cd (0, 5, 10, 20, and 40 mg L− 1 Cd2+ supplied by
CdCl2•2.5H2O) and Pb (0, 50, 100, 200, and 400 mg L− 1 Pb2+ supplied
by PbCl2), according to previous studies (Wei et al., 2016; Wang et al.,
2020b). The germination experiment was conducted in culture dishes as
previously described (Li et al., 2017). Each dish was sown with 10 seeds,
and three replicates were set for each treatment. Seeds with at least 2
mm hypocotyls were defined as germinated seeds. The number of
germinated seeds was recorded after ten days, and the root lengths of

2
J. Yang et al.
five germinated seeds in each dish were randomly measured. The
tolerance index (TI) was calculated using the root length according to a
previously reported formula (Gaur et al., 2017).
2.3. Pot experiment
The heavy metal-contaminated soils, which were left over from
previous experiments (Li et al., 2017, 2019) were air-dried, sieved, and
mixed to obtain a homogeneous mixture, as previously described (Li
et al., 2021). Basic parameters, including pH, electrical conductivity
(EC), organic matter, and several major elements were analysed in the
homogenised soil prior to the experiment (Supplementary Table S2).
The concentrations of total Cd (21.7 mg kg− 1) and Pb (113 mg kg− 1) in
the soils exceed their safe thresholds of 0.3 and 80 mg kg− 1, respectively,
according to the soil environmental quality ‘Risk control standard for
soil contamination of agricultural land’ in China (GB 15618–2018);
however, the concentrations of other metal elements were within the
statutory limits. Therefore, the soil used in this study was polluted with
Cd and Pb to different degrees.
The homogenizsed soil samples were placed in flowerpots (h = 17.5
cm, d = 18.5 cm) in equal volumes, which were uniformly pressed to a
height of 17 cm. The soils were placed in a greenhouse (light: 12–14 h,
20–25 ◦ C; dark: 10–12 h, 18–20 ◦ C; humidity: 40%–60%) at the
Kunming Institute of Botany to equilibrate for two weeks. The relative
soil water content was maintained at 50%–60%.
The seeds with uniform plumpness and size for each rhubarb species
were first surface-sterilised and cleaned using the aforementioned
method. The sterilised seeds were then seeded in flowerpots (three seeds
per pot), and a group of pots with bare (unplanted) soil was prepared as
control for rhizosphere characteristic analysis. When the seedlings grew
to approximately 2 cm in height, one healthy seedling was left in each
pot. Pots were placed in the same greenhouse for 3 months for growth.
Three biological replicates were prepared for each group.
2.4. Sample collection and biomass measurement
The roots and shoots of the plants were harvested separately and
oven-dried at 80 ◦ C for 48 h to measure the biomass. Root samples were
cleaned according to a previously described method (Wu et al., 2018).
The soil that naturally adhered to the root systems after gentle shaking
were collected as rhizosphere soil (Li et al., 2022) and stored at − 80 ◦ C
to analyse the physicochemical indices and microbial community
composition. For the control group, soil from the centre of the pot (5–10
cm below the surface) was collected.
2.5. Cd and Pb concentration detection
The total Cd and Pb concentrations in dried roots and shoots were
measured via the inductively coupled plasma-mass spectrometry
method, as previously reported (Li et al., 2017). The digestion processes
of both Cd and Pb were identical; however, but they were identified and
quantified according to their respective detection wavelengths and
standard curves. The bioconcentration factors (BCFs), translocation
factors (TFs), and total accumulation contents of both Cd and Pb in the
shoots and roots of the plants were calculated as previously described (Li
and Yang, 2020).
2.6. Determination of soil physicochemical indices
In this study, soil pH, EC, and the concentrations of hydrolysable N
(HN), available P (AP), Cd (ACd), and Pb (APb) were determined using
the methods introduced in the corresponding detection standards in
China (Cha et al., 2020; Li et al., 2020; Zhang and Luo, 2020). Soil pH
was determined via potentiometric method (LY/T 1239–1999), and soil
EC was determined via electrode method (HJ 802–2016); soil HN was
hydrolysed using a 1.8 M NaOH solution, absorbed by a boric acid
3
Chemosphere 296 (2022) 134045
solution, and then titrated using a 0.01 M HCl standard solution (LY/T
1228–2015); soil AP (LY/T 1232–2015) was extracted using HCl (0.05
M)-H2SO4 (0.025 M) extractant and detected via inductively coupled
plasma optical emission spectrometer (ICP–OES); and soil ACd and APb
were extracted using diethylenetriamine pentaacetic acid-calcium
chloride-triethanolamine (DTPA-CaCl2-TEA) buffer solution and then
detected via ICP–OES (HJ 804–2016).
2.7. Soil microbial community composition analysis
Soil microbial DNA extraction, 16S rDNA amplification, sequencing,
and bioinformatics analyses were performed as previously described (Li
et al., 2021). The raw sequencing reads were deposited into the
Sequence Read Archive database of NCBI (Accession Number:
PRJNA772309), which should be released on 2022.7.31or upon publi­
cation, whichever is first.
2.8. Statistical analysis
The differences in indices among different groups were tested by one-
way analysis of variance with Tukey’s test (significance level 0.05). The
networks of correlation coefficients among soil physicochemical indices
and redundancy analysis (RDA) were generated using the Omicsmart
platform (http://www.omicsmart.com).
3. Results and discussion
3.1. Cd and Pb tolerance and accumulation characteristics of three
rhubarbs
Seed germination is a prerequisite for establishing plant seedlings
and is typically used to test the tolerance of plants to heavy metal stress
(Li et al., 2017; Seneviratne et al., 2019; Yao et al., 2021). As shown in
Fig. 1A, the seed germination percentages of each rhubarb species were
similar under different Cd treatment concentrations (0–40 mg L− 1).
Similar results were observed in the 0–400 mg L− 1 Pb treatment, except
that the seed germination percentages of R. tanguticum in the 200 and
400 mg L− 1 Pb treatments significantly decreased (p < 0.05) compared
with that of the control (Fig. 1B). These results correspond with those of
previous studies (Li et al., 2017; Yao et al., 2021), in which the seed
germination percentages were slightly affected by heavy metal stress in
a certain concentration range. This is because the seed cotyledons can
expand and develop normally, thereby breaking the shells. However,
consistent with previous studies (Guleryuz et al., 2016; Li et al., 2017;
Yao et al., 2021), the root lengths of the rhubarbs were significantly
influenced (p < 0.05) with increasing Cd or Pb concentrations (Fig. 1C
and D). Briefly, the root lengths of R. officinale and R. palmatum
decreased significantly (p < 0.05) in the 10–40 mg L− 1 Cd and 100–400
mg L− 1 Pb treatments compared with those of the control, whereas the
root lengths of R. tanguticum were significantly inhibited (p < 0.05) in
the 5 mg L− 1 Cd to 50 mg L− 1 Pb treatments (Fig. 1C and D). The roots
were inhibited, likely because roots produce a large amount of ethylene
after absorbing heavy metals, which can seriously damage plant cells
(Wei et al., 2016). TI was calculated based on root lengths to compare
the tolerance of different species to heavy metals. According to the
formula (Gaur et al., 2017), the TIs exhibited the same trend as the root
length for each species with increasing Cd and Pb concentrations
(Fig. 1E and F). As shown in Fig. 1E and F, the TIs of the rhubarb species
were similar under most Cd and Pb concentrations, indicating that they
have similar Cd and Pb tolerance capacities. The TI results for Pb
treatment in this study were generally consistent with previously re­
ported results for R. tanguticum and R. rhaponticum for a similar Pb
concentration range (Jiang et al., 2016; Wang et al., 2020b). Notably, in
the 10 mg L− 1 Cd and 100 mg L− 1 Pb treatments, the TIs (> 60%) of the
three rhubarbs were relatively high (Fig. 1E and F). Moreover, the Cd TIs
of the three rhubarbs were even higher than that of the potential Cd
J. Yang et al. Chemosphere 296 (2022) 134045

Fig. 1. Seed germination characteristics of three rhubarb species under different Cd and Pb treatment concentrations. (A) Seed germination percentages under Cd
treatment. (B) Root length under Cd treatment. (C) Tolerance index under Cd treatment. (D) Seed germination percentages under Pb treatment. (E) Root length
under Pb treatment. (F) Tolerance index under Pb treatment. Data represent the means ± standard deviations (n = 3 or 15); the similar coloured bars labelled with
different lowercase letters (A–F) and bars at the same Cd or Pb treatment concentration labelled with different uppercase letters (E and F) indicate significant
differences among three species at the p < 0.05 level according to Tukey’s test.

hyperaccumulator Amaranthus caudatus under 10 mg L− 1 Cd treatment
(Peng et al., 2018), while the Pb TIs of three rhubarbs were close to those
of the Pb hyperaccumulator Pogonatherum crinitum under low Pb treat­
ment concentrations (e.g. 50 and 100 mg L− 1) (Hou, 2013). These results
suggest that the three rhubarb species have a relatively high Cd and Pb
tolerance.
Based on the seed germination test results, the heavy metal accu­
mulation characteristics of the three rhubarb species were further
explored in Cd- and Pb-contaminated soil. All three rhubarb species
survived normally and grew rapidly in the Cd- and Pb-polluted soils,
which further confirms that they have relatively high Cd and Pb toler­
ance capacities. However, Fig. 2A and B indicate that the biomass yields
and shoot/root ratios of the three rhubarb species were markedly
different. After three months of growth, the average shoot biomass was
in the order R. tanguticum (10.44 g) > R. officinale (7.46 g) > R. palmatum
(4.96 g); however, R. tanguticum (6.30 g) had a significantly stronger (p
< 0.05) root system than R. officinale (1.51 g) and R. palmatum (1.19 g)
(Fig. 2B). These differential growth characteristics of the three rhubarbs,
which are inherent genetic characteristics of these plants, indicate that
they may have different heavy metal accumulation characteristics.
Notably, their root systems likely had differential interactions with the
rhizosphere environment.
In this study, the average Cd concentration, whether in shoots
(9.00–10.30 mg kg− 1 DW) or in roots (2.32–3.18 mg kg− 1 DW),
exhibited no significant difference among the three rhubarb species
(Fig. 2C), indicating a similarity in the average Cd BCFs for both shoots
(0.42–0.47) and roots (0.11–0.15) (Fig. 2D). Moreover, the average Cd
TFs (3.04–4.24) were similar among the three rhubarb species (Fig. 2E).
These results suggest that the three rhubarb species have similar Cd
accumulation characteristics. The values of shoot Cd BCFs and TFs
indicate that the Cd accumulation capacities of these rhubarbs are high.
The shoot BCF and TF values > 1 are defined as the necessary conditions
for hyperaccumulators (Zhang et al., 2021). In this study, the shoot Cd
BCFs of the three rhubarb species were not far from 1 (Fig. 2D).
Furthermore, the Cd TFs were even greater than those of the Cd
hyperaccumulator Arabidopsis halleri (Dietrich et al., 2021), indicating
strong capacities to transport Cd from roots to aerial parts in the three
species. These results suggest that the three rhubarb species can be
designed as Cd accumulators. The total Cd accumulation in the rhubarb
plants was calculated to assess the potential phytoextraction efficiency
of Cd. Approximately 86.0%–94.6% of the total accumulated Cd was
transported into the plant shoots (Supplementary Fig. 1), which facili­
tates the removal of Cd by harvesting aerial parts. These findings suggest
that R. officinale, R. palmatum, and R. tanguticum have the potential for
the phytoextraction of Cd from polluted soils. In this study, R. tanguticum
exhibited a higher Cd phytoextraction efficiency (Supplementary Fig. 1)
than R. officinale and R. palmatum because of its larger biomass yield
(Fig. 2B).
Pb accumulation characteristics were also similar among the three
rhubarb species (Fig. 2F–H). However, unlike Cd, Pb BCFs, whether in
shoots (0.004–0.008) or in roots (0.007–0.013), were very low in the
three rhubarb species (Fig. 2G), indicating that these species are not
suitable for Pb phytoextraction or Pb phytostabilisation. However,
further confirmation is required to clarify whether the low Pb absorption
and accumulation in these three rhubarb species is an inherent trait or
hindered by environmental conditions (e.g. high Cd concentrations).
As mentioned earlier, these rhubarb species are used in traditional
Chinese medicine, and their roots are consumed orally (Xiang et al.,
2020). Therefore, the risk of heavy metal intake from these plants should
be considered when consuming them. According to the international

4
J. Yang et al. Chemosphere 296 (2022) 134045

Fig. 2. Plant growth and heavy metal accumulation characteristics of three rhubarb species growing in a Cd- and Pb-polluted soil. (A) Representative plant
morphology. (B) Shoot and root biomasses (dry weight). (C) Cd concentrations. (D) Cd bioconcentration factors (BCFs). (E) Cd translocation factor (TFs). (F) Pb
concentrations. (G) Pb BCFs. (H) Pb TFs. Data represent the means ± standard deviations (n = 3); the similar coloured bars labelled with different letters indicate
significant differences among three species at the p < 0.05 level according to Tukey’s test (B–H).

standard (BS ISO 18664:2015), the limits of Cd and Pb in natural
traditional Chinese medicines for oral use are 0.3 and 5.0 mg kg− 1,
respectively, in China. Based on the root BCFs of Cd and Pb for the three
rhubarb species in this study, when planted in soils (pH ≤ 6.5) with Cd
and/or Pb concentrations below their pollution risk screening values
(GB15618—2018: 0.3 mg kg− 1 Cd and 70–90 mg kg− 1 Pb; China) (Wang
et al., 2019), there is a low risk of Cd and Pb exceeding the standard for
these herbs.
In summary, this study revealed similarities in Cd and Pb tolerance
and accumulation characteristics among R. officinale, R. palmatum, and
R. tanguticum. The high Cd tolerance and accumulation characteristics
enable them to be potentially used for phytoextraction in Cd-polluted
soils. The results also suggest that the accumulation characteristics of
rhubarbs for other heavy metals with chemical properties similar to Cd
are also noteworthy. In addition, the mechanisms underlying the low Pb
accumulation characteristics would be potentially helpful for producing
Pb-safe crops and vegetables in Pb-contaminated soils.
3.2. Rhizospheric characteristics of the three rhubarbs in Cd- and Pb-
polluted soil
As mentioned earlier, the accumulation characteristics of heavy
metals in plants are not only regulated by genetic mechanisms but also
largely affected by the rhizospheric environment. Rhizospheric physi­
cochemical indices and microorganisms can affect plant growth and
heavy metal uptake (Abhilash et al., 2016; Yuan et al., 2021; Liu et al.,
2020b), which thereby regulates the phytoextraction efficiency of heavy
metals. Revealing the relationship between rhizospheric characteristics
and Cd and Pb accumulation characteristics of the three rhubarb species
will help to further improve their phytoremediation efficiency. For this,
changes in the physicochemical indices and microbial community
composition in rhizospheric soils compared with bare (unplanted) soil
(BRS) were explored in this study.
Table 1 presents the variations in physicochemical indices (i.e. soil
pH, EC, HN, AP, ACd, APb) in the rhizospheric soils of R. officinale

5
J. Yang et al. Chemosphere 296 (2022) 134045

Table 1 their role in contributing to the plant traits based on 16S

Physicochemical indices in the bare soil (BRS) and rhizosphere soils (dry weight) high-throughput sequencing (HTS).
of Rheum officinale (ROS), R. palmatum (RPS), and R. tanguticum (RTS) growing
in soils polluted by Cd and Pb. 3.3.1. Richness, diversity, and composition of bacterial communities in
Soil index BRS ROS RPS RTS different soils
pH 5.21 ± 0.05 b 5.55 ± 0.02 a 5.46 ± 0.03 a 5.48 ± 0.04 a To date, there have been very few studies on the rhizospheric mi­
EC (μS 547.00 ± 289.33 ± 7.32 367.33 ± 329.67 ± crobial community composition of rhubarbs, particularly in heavy
cm− 1) 30.34 a c 15.92 b 12.82 bc metal-contaminated soils. This study provides original rhizobacterial
HN (mg 846.67 ± 747.33 ± 731.33 ± 756.00 ± information to understand plant growth in rhubarbs and their response
kg− 1) 36.75 a 28.99 ab 52.16 b 10.03 ab
AP (mg 34.73 ± 0.87 24.80 ± 1.56 c 29.43 ± 0.62 24.37 ± 1.97 c
to heavy metals. The HTS results showed that 120,539–136,575
kg− 1) a b sequencing reads were obtained from different samples (Supplementary
ACd (mg 15.03 ± 0.33 19.80 ± 0.51 21.00 ± 0.50 19.63 ± 0.50 Table S3), with 97.92%–98.37% library coverage (Supplementary
kg− 1) b a a a Table S4). All rarefaction curves of alpha indices, including Sobs,
APb (mg 24.77 ± 2.76 42.27 ± 1.39 37.17 ± 1.70 39.40 ± 4.95
Shannon, Simpson, Chao1, and Ace, approached the saturation plateau
kg− 1) b a a a
(Supplementary Fig. S3), indicating that the sequencing depth was
Data represent the means ± standard deviations (n = 3); data in the same row reasonable in this study. These alpha indices, which reflect the richness
labelled with different letters indicate significant differences among different and diversity of the bacterial community, exhibited no significant dif­
soils at the p < 0.05 level according to Tukey’s test. ACd: available Cd; AP:
ferences among the soils (Supplementary Table S4). The results can be
available P; APb: available Pb; EC: electrical conductivity; HN: hydrolyzable N.
attributed to the decreased or increased abundance of some rhizobac­
teria taxa when plants were sown, consistent with the reason mentioned
(ROS), R. palmatum (RPS), and R. tanguticum (RTS) compared with the in a previous study (Li et al., 2021).
BRS soil after 3 months of plant growth. Each of these soil indices The average bacterial OTU numbers were 5,157, 5,169, 5,141, and
showed a significant change in the rhizosphere of the three rhubarbs 5,108 in the BRS, ROS, RPS, and RTS soils respectively, which were
compared with the BRS soil, while most of them (excluding EC and AP) similar among the different soils (Supplementary Table S3). The soils
were similar among the rhizospheric soils of the three rhubarb species shared 3163 core bacterial OTUs (Fig. 3A), which accounted for 61.3%,
(Table 1). The average pH of the ROS (5.55), RPS (5.46), and RTS (5.48) 61.2%, 61.5%, and 61.9% of the total OTUs of BRS, ROS, RPS, and RTS
soils showed an increasing trend (p < 0.05) compared with that of the soils, respectively. The unique bacterial OTU number exhibited the
BRS soil (5.21), whereas the average EC values showed a decreasing following trend: RPS (478) > BRS (426) > RTS (388) > ROS (377)
trend (p < 0.05) (Table 1). The increase in rhizospheric pH is likely (Fig. 3A). The highly similar levels of OTUs in different soils conformed
attributed to the accumulation of some cations (e.g., Ca2+ and Mg2+) to the undifferentiated bacterial diversity and richness.
across the rhizosphere, as previously reported (Youssef and Chino, Consistent with the OTU results, the four soils shared most of the
1989). In contrast, the rhizospheric EC may decrease owing to the ab­ bacterial phyla and genera (Fig. 3B and C). No specific bacterial phyla
sorption of soluble nutrients by plants, as soil EC is an important indi­ were detected in each soil (Fig. 3B), whereas 16–23 specific bacterial
cator of soil water-soluble salts (Yao et al., 2020). The deficit (p < 0.01) genera were identified in the different soils (Fig. 3C). A total of 15
in the concentrations of HN and AP in the rhizospheric soils (i.e. ROS, bacterial phyla (Supplementary Table S5) and 85 bacterial genera
RPS, and RTS) conformed to the variation in EC (Table 1), and the up­ (Supplementary Table S6), with a relative abundance > 0.1% in at least
take of N and P by plants and microorganisms may be primarily one sample, were identified in the soils. The abundance of these 15
responsible for this deficit. Interestingly, the average concentrations of bacterial phyla among the four soils showed a complex correlation
both ACd (19.63–21.00 mg kg− 1) and APb (37.17–42.27 mg kg− 1) in the network (Fig. 3D). The phyla Patescibacteria, Firmicutes, and Cyano­
rhizospheric soils were significantly higher (p < 0.05) than those in the bacteria exhibited the highest connectivity (Fig. 3D), indicating that
BRS soil (ACd: 15.03 mg kg− 1, APb: 24.77 mg kg− 1), as shown in they may affect a large number of other bacterial taxa. The top 10
Table 1, which may be partially attributed to the adsorption and inter­ bacterial phyla followed the order of relative abundance: Proteobacteria
ception of heavy metals by plant roots. (39.4%–42.7%) > Actinobacteria (9.8%–13.2%) > Acidobacteria
Complex interactions among various soil indices have been observed (9.7%–11.7%) > Chloroflexi (6.1%–7.7%) > Gemmatimonadetes
in soils (Centenaro et al., 2018). In the present study, different soil (6.4%–8.0%) > Bacteroidetes (4.8%–7.8%) > Patescibacteria (4.1%–
indices were divided into two clusters in the network of Pearson cor­ 8.1%) > Planctomycetes (3.1%–7.8%) > Verrucomicrobia (0.4%–1.2%)
relation coefficients (Supplementary Fig. S2). The first cluster included > Armatimonadetes (0.4%–0.9%). These accounted for more than 95%
pH, APb, and ACd, whereas the second cluster included EC, AP, and HN of the total bacterial community (Fig. 3E). Most of these dominant phyla
(Supplementary Fig. S2). The soil indices exhibited significantly positive (e.g. Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, Gem­
correlations (p < 0.01 or p < 0.001) with each other within the same matimonadetes, and Verrucomicrobia) have been identified in heavy
cluster but significantly negative correlations (p < 0.05, p < 0.01, or p < metal-polluted soils (Jiang et al., 2019; Li et al., 2021). Several of these
0.001) with those in the other cluster (Supplementary Fig. S2). These bacterial phyla, such as Proteobacteria, Acidobacteria, and Bacter­
correlations indicate the complex interrelations among the soil physi­ oidetes, are generally tolerant to metal toxicity because of their
cochemical indices after planting the rhubarb plants. Of these, the var­ complexation and adsorption capacities (Guo et al., 2017; Kasemodel
iations in the concentrations of ACd and APb were associated with the et al., 2019). Consistent with many reports (Shao et al., 2016; Naren­
dynamics of soil pH, EC, and concentrations of HN and AP (Supple­ drula-Kotha and Nkongolo, 2017; Li et al., 2021), Proteobacteria, the
mentary Fig. S2), which partially conformed to previous studies (Quan most dominant phylum in this study (Fig. 3E), likely plays an important
et al., 2011; Yu et al., 2016b; Yang et al., 2021). role in carbon and nitrogen cycles to maintain a stable nutritional level
for the growth of rhubarb plants (Narendrula-Kotha and Nkongolo,
3.3. Variations in bacterial community composition in the rhizosphere of 2017).
the three rhubarbs
3.3.2. Variations in bacterial taxa and functions in different soils
As variations in soil physicochemical indices mould the rhizospheric LEfSe analysis was used to identify the differential bacterial taxa
microbiome (Liu et al., 2020a, 2020b), which can affect the growth and (LDA > 2) in ROS, RPS, and RTS soils compared to the BRS soil
fitness of plants (Hakim et al., 2021), the differences in rhizobacterial (Fig. 4A–C; Supplementary Tables S7–S9). The number of differential
communities of the three rhubarb species were compared to understand bacterial taxa in different rhizospheric soils are shown in Fig. 4D–F.

6
J. Yang et al. Chemosphere 296 (2022) 134045

Fig. 3. Bacterial community composition between the bare soil (BRS) and rhizosphere soils of Rheum officinale (ROS), R. palmatum (RPS), and R. tanguticum (RTS)
growing in a Cd- and Pb-polluted soil. (A) Venn diagram of operational taxonomic units among different soils. (B) Venn diagram of the identified bacterial phyla
among different soils. (C) Venn diagram of the identified bacterial genera among different soils. (D) Pearson correlation network among the top 15 bacterial phyla.
The solid and dotted lines represent positive and negative correlation, respectively. (F) Circos diagram showing the relative abundance of the top 10 bacterial phyla
identified in different soils.

Most of the differential bacterial taxa (i.e. phyla and genera) were spe­
cific to the rhizospheric soils of different rhubarbs (Supplementary
Figs. S4 and S5). Notably, there was a high overlap in the upregulated
bacterial taxa between ROS and RTS soils and the downregulated bac­
terial taxa between RPS and RTS soils (Fig. 3D and F). These results
suggest that the three rhubarb species differentially remodelled the
rhizobacterial composition in Cd- and Pb-contaminated soil. Similar
results have typically been detected in different plant homologous spe­
cies, ecotypes, and cultivars that have differential heavy metal accu­
mulation capacities (Hanaka et al., 2019; Wang et al., 2020a; Chen et al.,
2021; Dong et al., 2021). Wang et al. (2020a) found distinct functional
assemblies of rhizobacteria between the Pb-accumulating ecotype and
non-accumulating ecotypes of Sedum alfredii under Pb stress. Chen et al.
(2021) also found certain variations in rhizobacterial communities in
four Miscanthus cultivars in Cd-polluted soils. These studies demonstrate
that plants typically recruit various rhizobacteria to regulate plant
growth and heavy metal uptake, indicating that the differential rhizo­
bacteria of the three rhubarb species are likely responsible for the plant
growth and heavy metal uptake characteristics in heavy
metal-contaminated soils.
The rhizobacteria in the soils were divided into 34 functional cate­
gories (level 2) (Supplementary Table S10) and involved in 186 KEGG
pathways (level 3) (Supplementary Table S11). The dynamics of bac­
terial taxa led to variations in the abundance of several bacterial func­
tional categories in the rhizospheric soils compared with the BRS soil
(Supplementary Fig. S6). The rhizobacteria involved in sesquiterpenoid
and triterpenoid biosynthesis were significantly enriched (p < 0.05) in
the ROS and RTS soils than in the BRS soil (Supplementary Figs. S6A and
C). The rhizobacteria involved in salivary secretion were specifically
identified in the ROS and RPS soils (Supplementary Figs. S6A and B).
The rhizobacteria involved in the proteasome, biosynthesis of type II
polyketide backbone, and photosynthesis-antenna proteins showed a
significant enrichment (p < 0.05) in the RTS soil compared with the BRS
soil (Supplementary Fig. S6C). These altered bacterial KEGG pathways
in the rhizosphere of rhubarbs were possibly influenced by multiple soil
factors. Because changes in these bacterial KEGG pathways are rarely
reported in other studies, they are likely related to the special traits of
rhubarb plants, such as root exudates. However, further studies are
required to establish whether the rhizobacteria enriched in these KEGG
pathways are associated with growth or heavy metal uptake in plants.
3.3.3. Correlations between soil physicochemical indices and bacterial
community composition
RDA was conducted to evaluate the correlations among soil factors
for the abundance and diversity of bacterial communities in soils. The
top 15 and 20 bacterial phyla and genera in the soils (Supplementary
Tables S5 and S6) were used as response variables. In addition to the
physiochemical indices, the different root system biomasses (RSBs) of
the three rhubarb species (Fig. 1B), which indicate diverse root char­
acteristics, such as root surface area, number of lateral roots, and root
exudates, were also used as explanatory variables.
The results showed that the four soils could be evidently separated
(Fig. 5A and B), suggesting that planting of different rhubarb plants
greatly significantly the rhizosphere environment. The seven soil indices
accounted for 75.56% (first axis: 51.97%, second axis: 23.59%) of the
total variation in bacterial phyla (Fig. 5A). The main soil indices
affecting the bacterial phylum variation were RSB (R2 = 0.8435, p =
0.001) and AP (R2 = 0.4653, p = 0.052) (Fig. 5A; Supplementary
Table S12). Approximately 77.27% (first axis: 61.72%, second axis:
15.55%) of the total variation in bacterial genera could be explained by
the seven selected variables (Fig. 5B). Notably, the main soil indices
affecting the bacterial genus variation were AP (R2 = 0.6458, p =

7
J. Yang et al. Chemosphere 296 (2022) 134045

Fig. 4. LEfSe analysis results with linear discriminate analysis (LDA) scores > 2 between the bare soil (BRS) and rhizosphere soils of Rheum officinale (ROS),
R. palmatum (RPS), and R. tanguticum (RTS) growing in a Cd- and Pb-polluted soil. (A) Cladogram showing indicator bacteria between BRS and ROS. The identifiers
labelled on the cladogram correspond to those in Supplementary Table S7. (B) Cladogram showing indicator bacteria between BRS and RPS. The identifiers labelled
on the cladogram correspond to those in Supplementary Table S8. (C) Cladogram showing indicator bacteria between BRS and RTS. The identifiers labelled on the
cladogram correspond to those in Supplementary Table S9. Cladogram circles indicate phylogenetic taxa from phylum to species. The diameter of each circle is
proportional to the abundance of the group; only the dominant groups are exhibited in the cladograms (A–C). (D) Number of indicator bacteria at different clas­
sification levels between ROS and BRS soils. (E) Number of indicator bacteria at different classification levels between RPS and BRS soils. (F) Number of indicator
bacteria at different classification levels between RTS and BRS soils.

0.004), RSB (R2 = 0.6918, p = 0.012), and ACd (R2 = 0.4796, p = 0.053)
(Fig. 5B; Supplementary Table S13). These results suggest that the dy­
namics of rhizobacteria in the experiment are closely associated with the
root systems of different rhubarb species, which agrees with the
conclusion that there are complex interactions between plant root sys­
tems and rhizobacteria (Marasco et al., 2012; Chen et al., 2016; Lund­
berg and Teixeira, 2018; Lima et al., 2021). Marasco et al. (2012) found
that the plant root system was able to act as a ‘‘resource island’’ to
attract and select microbial communities containing multiple plant
growth-promoting traits that sustain plant development under
water-limiting conditions. Lundberg and Teixeira (2018) also reported
that root-exuded coumarins could shape the root microbiome. In
contrast, plant growth-promoting rhizobacteria (PGPR) have been
shown to modulate root-system architecture in many studies (Chen
et al., 2016; Lima et al., 2021). These studies indicate that the different
root systems of the three rhubarb species recruit different rhizobacteria,
which may in turn produce a micromodification of root architecture that
affects the absorption of nutrients and heavy metals. In addition, the AP
and ACd levels may have two-way interactions with rhizobacteria as
previously reported (Cosme and Wurst, 2013; Sharma and Archana,
2016).
3.4. Effects of rhizobacteria on heavy metal uptake by rhubarbs
Rhizospheric microbes such as bacteria, fungi, protozoa, and algae
typically exhibit various associations with plants (Zubair et al., 2016).
Microbes can generate quorum sensing between the soil environment
and plants, which activates the microbial population to form a biofilm
around the root surface and trigger a correlative response (Daniels et al.,
2004). A number of plant-associated bacteria are beneficial for
enhancing plant growth and mitigating the negative effects of heavy
metals on plants (Manoj et al., 2020). In addition, some bacteria can
minimise the limiting factors associated with phytoremediation
including soil chemistry and metal solubility (Tangahu et al., 2011).
The bacterial genera enriched in the rhizosphere of the three rhu­
barbs in this study are shown in Fig. 6. Notably, many bacterial genera
have been shown to promote plant growth and/or affect heavy metal
uptake by plants. Bacterial genera, including Cupriavidus, Pseudomonas,
Ralstonia, Rhizobium, Bryobacter, Streptomyces, Burkholderia, Nocar­
dioides, Mesorhizobium, and Flavisolibacter (Fig. 6), are important PGPR
(Kruasuwan and Thamchaipenet, 2016; Alka et al., 2020; Efe, 2020;
Chen et al., 2021) and can enhance plant growth through various
mechanisms (Manoj et al., 2020). The presence of heavy metal-tolerant
PGPR in the plant rhizosphere can enhance plant health and vitality,

8
J. Yang et al.
Fig. 5. Redundancy analysis (RDA) between bacterial communities and soil physicochemical indices. (A) RDA results showing the influence of soil indices on the
variation in the bacterial community at the phylum level. (B) RDA results showing the influence of soil indices on the variation in the bacterial community at the
genus level. ACd: available Cd; AP: available P; APb: available Pb; AZn: available Zn; BRS: bare soil; EC: electrical conductivity; HN: hydrolysable N; ROS: Rheum
officinale rhizosphere soil; RPS: R. palmatum rhizosphere soil; RTS: R. tanguticum rhizosphere soil.
thereby enabling plants to tolerate heavy metal stress in soils (Ma et al.,
2016). Among these, the genera Cupriavidus, Pseudomonas, Rhizobium,
Streptomyces, Burkholderia, and Mesorhizobium (Fig. 6) have also been
reported to affect heavy metal uptake by plants through multiple
mechanisms (Rajkumar et al., 2010; Hao et al., 2015; Ali et al., 2017;
Guo et al., 2020; Shi et al., 2020; Wu et al., 2020). For example, the
Cd-tolerant Cupriavidus sp. WS2 has the ability to immobilise Cd by
biosynthesising intracellular Cd-containing nanoparticle inclusions,
thereby leading to a significant decrease in Cd accumulation in rice
seedlings (Shi et al., 2020). Pseudomonas has proven to be effective in
influencing the efficiency of phytoextraction or phytostabilisation in
metal-contaminated soils. Many Pseudomonas species/stains have been
shown to enhance heavy metal uptake by plants (Babu et al., 2015; Wu
et al., 2020; Wang et al., 2021), and several species and stains have been
reported to immobilise heavy metals (Shahid et al., 2017). Consistently,
the rhizobacteria Rhizobium (Rajkumar et al., 2010), Streptomyces pac­
tum (Ali et al., 2017), Burkholderia sp. D54 (Guo et al., 2020), and
Mesorhizobium amorphae (Hao et al., 2015) could modulate the uptake of
various heavy metals by different plants.
Fig. 6 illustrates that most of the PGPR (i.e. Ralstonia, Rhizobium,
Bryobacter, Streptomyces, Burkholderia, and Mesorhizobium) were signif­
icantly enriched in the rhizosphere of both R. officinale and R. tanguticum
(Fig. 6), whereas two genera (Cupriavidus and Pseudomonas) and other
two genera (Nocardioides and Flavisolibacter) were specifically enriched
in the rhizosphere of R. officinale and R. palmatum, respectively (Fig. 6).
These results suggest that the growth and heavy metal accumulation
characteristics of the three rhubarb plants were likely assisted by these
PGPR. The differential rhizobacteria recruited by the three rhubarb
species may be closely related to their different root features as dis­
cussed earlier. These potential PGPR need to be screened and identified
to explore their explicit functions, which are helpful for establishing
combined plant-microorganism bioremediation techniques when these
rhubarb plants are used for phytorextraction.
4. Conclusions
The present study explored the tolerance and accumulation charac­
teristics of heavy metals (i.e., Cd and Pb) in R. officinale, R. palmatum,
and R. tanguticum. Seed germination results showed that these three
9
Chemosphere 296 (2022) 134045
rhubarb species had relatively high tolerance to Cd and Pb. Pot experi­
ments performed in a Cd- and Pb-polluted soil found that the three
rhubarb species had a relatively high Cd-accumulating capacity (root Cd
BCFs: 0.11–0.15; shoot Cd BCFs: 0.42–0.47) but a very low Pb-
accumulating capacity (root Pb BCFs: 0.007–0.013; shoot Pb BCFs:
0.004–0.008). These results indicate that these three rhubarb species are
potentially suitable for Cd phytoextraction. The changes in rhizospheric
physicochemical indices and bacterial diversity and richness were
similar among the three rhubarb plants in comparison with those of the
bare (unplanted) soil. However, differential indicator rhizobacteria
were identified for three rhubarb plants, which was likely attributed to
their different root system characteristics. The differential PGPR
enriched in the rhizosphere of the three rhubarb plants might improve
plant growth and regulate heavy metal uptake by plants in Cd- and Pb-
contaminated soil. This study identifies new candidate plant resources
for the phytoremediation of Cd-polluted soils and provides novel in­
sights into the interactions among heavy metals, rhizobacteria, and
plants. However, the heavy metal accumulation characteristics of the
three rhubarb plants in different soil types should be considered in the
future. Moreover, the definite effects of rhizobacteria on the absorption
of heavy metals by rhubarb plants also require further study.
Author statememt
Jingya Yang: Methodology, Formal analysis, Investigation, Writing -
Review & Editing, Visualization. Yingqi Huang: Methodology, Formal
analysis, Investigation, Writing - Review & Editing, Visualization.
Gaojuan Zhao: Investigation, Writing - Review & Editing, Visualization.
Boqun Li: Investigation, Writing - Review & Editing. Xiangshi Qin:
Investigation. Jianchu Xu: Conceptualization, Methodology, Writing -
Review & Editing, Supervision. Xiong Li: Conceptualization, Method­
ology, Validation, Formal analysis, Investigation, Writing - Original
Draft, Writing - Review & Editing, Visualization, Supervision, Funding
acquisition.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
J. Yang et al. Chemosphere 296 (2022) 134045

Fig. 6. Bacterial genera enriched in rhizosphere soils

of Rheum officinale (ROS), R. palmatum (RPS), and
R. tanguticum (RTS) growing in a Cd- and Pb-polluted
soil in comparison with bare soil (BRS). Plus (+) signs
indicate the bacteria enriched in the rhizospheric
soils of plants. Bacterial genera in red font indicate
plant growth-promoting rhizobacteria, and bacterial
genera with blue rectangular boxes indicate bacteria
involved in affecting heavy metal uptake by plants.
(For interpretation of the references to colour in this
figure legend, the reader is referred to the Web
version of this article.)

the work reported in this paper. Ali, A., Guo, D., Mahar, A., Ma, F., Li, R.H., Shen, F., Wang, P., Zhang, Z.Q., 2017.
Streptomyces pactum assisted phytoremediation in Zn/Pb smelter contaminated soil
of Feng County and its impact on enzymatic activities. Sci. Rep. 7, 46087.
Acknowledgements Alka, S., Shahir, S., Ibrahim, N., Chai, T.T., Bahari, Z.M., Abd Manan, F., 2020. The role
of plant growth promoting bacteria on arsenic removal: a review of existing
We thank the Germplasm Bank of Wild Species for providing the perspectives. Environ. Technol. Innovat. 17, 100602.
Babu, A.G., Shea, P.J., Sudhakar, D., Jung, I.B., Oh, B.T., 2015. Potential use of
seeds. This work was financially supported by the Youth Innovation Pseudomonas koreensis AGB-1 in association with Miscanthus sinensis to remediate
Promotion Association CAS (2020387) and the National Natural Science heavy metal(loid)-contaminated mining site soil. J. Environ. Manag. 151, 160–166.
Foundation of China (31800226). Bian, F., Zhong, Z., Li, C., Zhang, X., Gu, L., Huang, Z., Gai, X., Huang, Z., 2021.
Intercropping improves heavy metal phytoremediation efficiency through changing
properties of rhizosphere soil in bamboo plantation. J. Hazard Mater. 416, 125898.
Appendix A. Supplementary data Centenaro, G., Hudek, C., Zanella, A., Crivellaro, A., 2018. Root-soil physical and biotic
interactions with a focus on tree root systems: a review. Appl. Soil Ecol. 123,
318–327.
Supplementary data to this article can be found online at https://doi. Cha, L.J., Zhao, S.Y., Feng, H.J., Zhou, D.D., 2020. Study on influencing factors of heavy
org/10.1016/j.chemosphere.2022.134045. metal forms in wild fungi growth soil. Ecol. Environ. Sci. 29, 2457–2464.
Chen, W.L., Li, J., Zhu, H.H., Chen, J.Z., Yao, Q., 2016. A review of the regulation of
plant root system architecture by rhizosphere microorganisms. Acta Ecol. Sin. 36,
References 5285–5297.
Chen, Z.J., Tian, W., Li, Y.J., Sun, L.N., Chen, Y., Zhang, H., Li, Y.Y., Han, H., 2021.
Abhilash, P.C., Dubey, R.K., Tripathi, V., Gupta, V.K., Singh, H.B., 2016. Plant growth- Responses of rhizosphere bacterial communities, their functions and their network
promoting microorganisms for environmental sustainability. Trends Biotechnol. 34, interactions to Cd stress under phytostabilization by Miscanthus spp. Environ. Pollut.
847–850. 287, 117663.

10
J. Yang et al.
Cosme, M., Wurst, S., 2013. Interactions between arbuscular mycorrhizal fungi,
rhizobacteria, soil phosphorus and plant cytokinin deficiency change the root
morphology, yield and quality of tobacco. Soil Biol. Biochem. 57, 436–443.
Daniels, R., Vanderleyden, J., Michiels, J., 2004. Quorum sensing and swarming
migration in bacteria. FEMS Microbiol. Rev. 28, 261–289.
Dietrich, C.C., Tandy, S., Murawska-Wlodarczyk, K., Banas, A., Korzeniak, U., Seget, B.,
Babst-Kostecka, A., 2021. Phytoextraction efficiency of Arabidopsis halleri is driven
by the plant and not by soil metal concentration. Chemosphere 285, 131437.
Dong, Z.Y., Rao, M.P.N., Liao, T.J., Li, L., Liu, Y.H., Xiao, M., Mohamad, O.A.A., Tian, Y.
Y., Li, W.J., 2021. Diversity and function of rhizosphere microorganisms between
wild and cultivated medicinal plant Glycyrrhiza uralensis Fisch under different soil
conditions. Arch. Microbiol. 203, 3657–3665.
Efe, D., 2020. Potential plant growth-promoting bacteria with heavy metal resistance.
Curr. Microbiol. 77, 3861–3868.
Feki, K., Tounsi, S., Mrabet, M., Mhadhbi, H., Brini, F., 2021. Recent advances in
physiological and molecular mechanisms of heavy metal accumulation in plants.
Environ. Sci. Pollut. Res. Int. 28, 64967–64986.
Gaur, N., Kukreja, A., Yadav, M., Tiwari, A., 2017. Assessment of phytoremediation
ability of Coriander sativum for soil and water co-contaminated with lead and arsenic:
a small-scale study. 3 Biotech 7, 196.
Guleryuz, G., Kirmizi, S., Arslan, H., Derya, S., 2016. The effects of heavy metals on the
seed germination and seedling growth of two endemic Verbascum species. Fresenius
Environ. Bull. 25, 1134–1142.
Guo, H.H., Nasir, M., Lv, J.L., Dai, Y.C., Gao, J.K., 2017. Understanding the variation of
microbial community in heavy metals contaminated soil using high throughput
sequencing. Ecotoxicol. Environ. Saf. 144, 300–306.
Guo, J.K., Zhao, J., Ren, X.H., Jia, H.L., Muhammad, H., Lv, X., Wei, T., Hua, L., 2020.
Effects of Burkholderia sp. D54 on growth and cadmium uptake of tomato, ryegrass
and soybean plants. Int. J. Environ. Sci. Technol. 17, 1149–1158.
Hakim, S., Naqqash, T., Nawaz, M.S., Laraib, I., Siddique, M.J., Zia, R., Mirza, M.S.,
Imran, A., 2021. Rhizosphere engineering with plant growth-promoting
microorganisms for agriculture and ecological sustainability. Front. Sustain. Food
Syst. 5, 617157.
Hanaka, A., Plak, A., Zagorski, P., Ozimek, E., Rysiak, A., Majewska, M., Jaroszuk-
Scisel, J., 2019. Relationships between the properties of Spitsbergen soil, number
and biodiversity of rhizosphere microorganisms, and heavy metal concentration in
selected plant species. Plant Soil 436, 49–69.
Hao, X.L., Xie, P., Zhu, Y.G., Taghavi, S., Wei, G.H., Rensing, C., 2015. Copper tolerance
mechanisms of Mesorhizobium amorphae and its role in aiding phytostabilization by
Robinia pseudoacacia in copper contaminated soil. Environ. Sci. Technol. 49,
2328–2340.
Hou, X.L., 2013. Response Mechanism of Pb Hyperaccumulator Pogonatherum Crinitum to
Pb Stress. Fujian Agriculture and Forestry University: Doctoral Dissertation.
Jiang, B., Adebayo, A., Jia, J.L., Xing, Y., Deng, S.Q., Guo, L.M., Liang, Y.T., Zhang, D.Y.,
2019. Impacts of heavy metals and soil properties at a Nigerian e-waste site on soil
microbial community. J. Hazard Mater. 362, 187–195.
Jiang, L.N., SHao, Z.T., Song, Z.W., Cai, Z.G., 2016. Effects of lead treatment on seed
germination and seedling growth of vegetable rhubarb. Jiangsu Agric. Sci. 44,
223–225.
Kasemodel, M.C., Sakamoto, I.K., Varesche, M.B.A., Rodrigues, V.G.S., 2019. Potentially
toxic metal contamination and microbial community analysis in an abandoned Pb
and Zn mining waste deposit. Sci. Total Environ. 675, 367–379.
Kruasuwan, W., Thamchaipenet, A., 2016. Diversity of culturable plant growth-
promoting bacterial endophytes associated with sugarcane roots and their effect of
growth by co-inoculation of Diazotrophs and Actinomycetes. J. Plant Growth Regul.
35, 1074–1087.
Li, B.Q., Chen, D., Yang, Y.P., Li, X., 2019. Effects of soil properties on accumulation
characteristics of copper, manganese, zinc, and cadmium in Chinese turnip. Plant
Divers 41, 340–346.
Li, X., Chen, D., Li, B.Q., Yang, Y.P., 2021. Cd accumulation characteristics of Salvia
tiliifolia and changes of rhizospheric soil enzyme activities and bacterial communities
under a Cd concentration gradient. Plant Soil 463, 225–247.
Li, X., Yang, Y.P., 2020. Preliminary study on Cd accumulation characteristics in
Sansevieria trifasciata Prain. Plant Divers 42, 351–355.
Li, X., Zhang, X.M., Li, B.Q., Wu, Y.S., Sun, H., Yang, Y.P., 2017. Cadmium
phytoremediation potential of turnip compared with three common high Cd-
accumulating plants. Environ. Sci. Pollut. Res. Int. 24, 21660–21670.
Li, Y.J., Ma, J.W., Li, Y.Q., Xiao, C., Shen, X.Y., Chen, J.J., Xia, X.H., 2022. Nitrogen
addition facilitates phytoremediation of PAH-Cd cocontaminated dumpsite soil by
altering alfalfa growth and rhizosphere communities. Sci. Total Environ. 806,
150610.
Li, Z.J., Shen, Y.X., Zhao, G.J., Yu, Z.F., Chen, F.J., Xiao, G.Y., 2020. Changes of soil fungi
and bacteria after forest mother soil transplantation. Microbiol. China 47,
3196–3205.
Lima, J.V., Tinoco, R.S., Olivares, F.L., Chia, G.S., de Melo, J.A.G., da Silva, G.B., 2021.
Rhizobacteria modify root architecture and improve nutrient uptake in oil palm
seedlings despite reduced fertilizer. Rhizosphere 19, 100420.
Liu, F., Mo, X., Kong, W., Song, Y., 2020a. Soil bacterial diversity, structure, and function
of Suaeda salsa in rhizosphere and non-rhizosphere soils in various habitats in the
Yellow River Delta, China. Sci. Total Environ. 740, 140144.
Liu, H.K., Wang, C., Xie, Y.L., Luo, Y., Sheng, M.P., Xu, F., Xu, H., 2020b. Ecological
responses of soil microbial abundance and diversity to cadmium and soil properties
in farmland around an enterprise-intensive region. J. Hazard Mater. 392, 122478.
Lundberg, D.S., Teixeira, P.J.P.L., 2018. Root-exuded coumarin shapes the root
microbiome. Proc. Natl. Acad. Sci. U. S. A. 115, 5629–5631.
11
Chemosphere 296 (2022) 134045
Ma, Y., Rajkumar, M., Zhang, C., Freitas, H., 2016. Beneficial role of bacterial
endophytes in heavy metal phytoremediation. J. Environ. Manag. 174, 14–25.
Mahar, A., Wang, P., Ali, A., Awasthi, M.K., Lahori, A.H., Wang, Q., Li, R., Zhang, Z.,
2016. Challenges and opportunities in the phytoremediation of heavy metals
contaminated soils: a review. Ecotoxicol. Environ. Saf. 126, 111–121.
Manoj, S.R., Karthik, C., Kadirvelu, K., Arulselvi, P.I., Shanmugasundaram, T., Bruno, B.,
Rajkumar, M., 2020. Understanding the molecular mechanisms for the enhanced
phytoremediation of heavy metals through plant growth promoting rhizobacteria: a
review. J. Environ. Manag. 254, 109779.
Marasco, R., Rolli, E., Ettoumi, B., Vigani, G., Mapelli, F., Borin, S., Abou-Hadid, A.F., El-
Behairy, U.A., Sorlini, C., Cherif, A., Zocchi, G., Daffonchio, D., 2012. A drought
resistance-promoting microbiome is selected by root system under desert farming.
PLoS One 7, e48479.
Narendrula-Kotha, R., Nkongolo, K.K., 2017. Bacterial and fungal community structure
and diversity in a mining region under long-term metal exposure revealed by
metagenomics sequencing. Ecol. Gen. Genom. 2, 13–24.
Okereafor, U., Makhatha, M., Mekuto, L., Uche-Okereafor, N., Sebola, T.,
Mavumengwana, V., 2020. Toxic metal implications on agricultural soils, plants,
animals, aquatic life and human health. Int. J. Environ. Res. Publ. Health 17, 2204.
Peng, C.Q., Chen, X.Y., Yang, P., Mu, S.H., Guan, P., 2018. Effects of cadmium stress on
seed germination of Amaranthus caudatus and physiological characteristics of
seedlings. Seed 37, 43–48.
Quan, Z., Wu, J.S., Wei, W.X., Qin, H.L., Zhu, Y.J., Liu, X.L., Shu, R.B., Li, M.D., 2011.
Change characteristics of soil available nitrogen and phosphorus and heavy metal
contents after long-term cultivation of vegetables. Chin. J. Appl. Ecol. 22,
2919–2929.
Rajkumar, M., Ae, N., Prasad, M.N., Freitas, H., 2010. Potential of siderophore-producing
bacteria for improving heavy metal phytoextraction. Trends Biotechnol. 28,
142–149.
Reeves, R.D., Baker, A.J.M., Jaffre, T., Erskine, P.D., Echevarria, G., van der Ent, A.,
2018. A global database for plants that hyperaccumulate metal and metalloid trace
elements. New Phytol. 218, 407–411.
Seneviratne, M., Rajakaruna, N., Rizwan, M., Madawala, H.M.S.P., Ok, Y.S.,
Vithanage, M., 2019. Heavy metal-induced oxidative stress on seed germination and
seedling development: a critical review. Environ. Geochem. Health 41, 1813–1831.
Shahid, M., Dumat, C., Khalid, S., Niazi, N.K., Antunes, P.M.C., 2017. Cadmium
bioavailability, uptake, toxicity and detoxification in soil-plant system. In: Reviews
of Environmental Contamination and Toxicology. Springer, New York, pp. 73–137.
Shao, J.H., He, Y.X., Zhang, H.L., Chen, A.W., Lei, M., Chen, J.F., Peng, L., Gu, J.D., 2016.
Silica fertilization and nano-MnO2 amendment on bacterial community composition
in high arsenic paddy soils. Appl. Microbiol. Biotechnol. 100, 2429–2437.
Sharma, R.K., Archana, G., 2016. Cadmium minimization in food crops by cadmium
resistant plant growth promoting rhizobacteria. Appl. Soil Ecol. 107, 66–78.
Shen, X., Dai, M., Yang, J., Sun, L., Tan, X., Peng, C., Ali, I., Naz, I., 2021. A critical
review on the phytoremediation of heavy metals from environment: performance
and challenges. Chemosphere 132979.
Shi, Z., Zhang, Z., Yuan, M., Wang, S., Yang, M., Yao, Q., Ba, W., Zhao, J., Xie, B., 2020.
Characterization of a high cadmium accumulating soil bacterium, Cupriavidus sp.
WS2. Chemosphere 247, 125834.
Tangahu, B.V., Abdullah, S.R.S., Basri, H., Idris, M., Anuar, N., Mukhlisin, M., 2011.
A review on heavy metals (As, Pb, and Hg) uptake by plants through
phytoremediation. Int. J. Chem. Eng. 2011, 939161.
van Treuren, R., Coquin, P., Lohwasser, U., 2012. Genetic resources collections of leafy
vegetables (lettuce, spinach, chicory, artichoke, asparagus, lamb’s lettuce, rhubarb
and rocket salad): composition and gaps. Genet. Resour. Crop Evol. 59, 981–997.
Vareda, J.P., Valente, A.J.M., Duraes, L., 2019. Assessment of heavy metal pollution from
anthropogenic activities and remediation strategies: a review. J. Environ. Manag.
246, 101–118.
Wang, R., Zhang, F.L., Xu, S.S., Zhang, Y.W., 2019. Method of dividing the value of soil
heavy metal pollution risk screening: using Cd as an example. Environ. Sci. 40,
5082–5089.
Wang, R.Z., Hou, D.D., Chen, J.Z., Li, J.H., Fu, Y.Y., Wang, S., Zheng, W., Lu, L.L., Tian, S.
K., 2020a. Distinct rhizobacterial functional assemblies assist two Sedum alfredii
ecotypes to adopt different survival strategies under lead stress. Environ. Int. 143,
105912.
Wang, Y., Yang, R., Hao, J.J., Sun, M.Q., Wang, H.Y., Ren, H.J., 2021. The impact of
Pseudomonas monteilii PN1 on enhancing the alfalfa phytoextraction and responses of
rhizosphere soil bacterial communities in cadmium-contaminated soil. J. Environ.
Chem. Eng. 9, 106533.
Wang, Y.N., Ru, G., Wu, G.F., 2020b. Effects of exogenous NO on seed germination and
seedling physiological characteristics of Rheum tanguticum Maxim under Pb stress.
Seed 39, 47–52.
Wei, X.D., Huang, Y.G., Wang, Y., 2016. Effect of heavy metal cadmium and lead stress
on seed germination and seedlings growth of the Trifolium Repens. N. Hortic 71–74.
Wu, M.X., Luo, Q., Liu, S.L., Zhao, Y., Long, Y., Pan, Y.Z., 2018. Screening ornamental
plants to identify potential Cd hyperaccumulators for bioremediation. Ecotoxicol.
Environ. Saf. 162, 35–41.
Wu, Y.J., Ma, L.Y., Liu, Q.Z., Sikder, M.M., Vestergard, M., Zhou, K.Y., Wang, Q.,
Yang, X., Feng, Y., 2020. Pseudomonas fluorescens promote photosynthesis, carbon
fixation and cadmium phytoremediation of hyperaccumulator Sedum alfredii. Sci.
Total Environ. 726, 138554.
Wu, Y.J., Santos, S.S., Vestergard, M., Gonzalez, A.M.M., Ma, L.Y., Feng, Y., Yang, X.E.,
2022. A field study reveals links between hyperaccumulating Sedum plants-
associated bacterial communities and Cd/Zn uptake and translocation. Sci. Total
Environ. 805, 150400.
J. Yang et al.
Xiang, H., Zuo, J.X., Guo, F.Y., Dong, D.S., 2020. What we already know about rhubarb: a
comprehensive review. Chin. Med. 15, 88.
Yan, A., Wang, Y., Tan, S.N., Yusof, M.L.M., Ghosh, S., Chen, Z., 2020. Phytoremediation:
a promising approach for revegetation of heavy metal-polluted land. Front. Plant Sci.
11, 359.
Yang, H.Y., Zhang, M.J., 2021. Determination of seven harmful elements in Rhei Radix et
Rhizoma by ICP-MS with ultra-high pressure microwave digestion and preliminary
risk assessment. Chin. J. Pharm. Anal. 41, 852–860.
Yang, Y., Li, Y., Wang, M., Chen, W., Dai, Y., 2021. Limestone dosage response of
cadmium phytoavailability minimization in rice: a trade-off relationship between
soil pH and amorphous manganese content. J. Hazard Mater. 403, 123664.
Yao, L.R., Wang, J.C., Li, B.C., Meng, Y.X., Ma, X.L., Si, E.J., Yang, K., Shang, X.W.,
Wang, H.J., 2021. Influences of heavy metals and salt on seed germination and
seedling characteristics of halophyte Halogeton glomeratus. Bull. Environ. Contam.
Toxicol. 106, 545–556.
Yao, S.T., Lu, G.X., Wang, J.B., Huang, C.X., Wang, Z.H., Zhao, L.R., 2020. Effect of
simulated warming on soil conductivity. Arid Zone Res. 37, 598–606.
Youssef, R.A., Chino, M., 1989. Root-induced changes in the rhizosphere of plants. I. pH
changes in relation to the bulk soil. Soil Sci. Plant Nutr. 35, 461–468.
12
Chemosphere 296 (2022) 134045
Yu, H., Xiang, Y.C., Zou, D.S., 2016a. Rhizosphere ecological effect of cultivars differing
in heavy metal accumulation. Ecol. Sci. 35, 199–204.
Yu, H.Y., Liu, C.P., Zhu, J.S., Li, F.B., Deng, D.M., Wang, Q., Liu, C.S., 2016b. Cadmium
availability in rice paddy fields from a mining area: the effects of soil properties
highlighting iron fractions and pH value. Environ. Pollut. 209, 38–45.
Yuan, C., Gao, B., Peng, Y., Gao, X., Fan, B., Chen, Q., 2021. A meta-analysis of heavy
metal bioavailability response to biochar aging: importance of soil and biochar
properties. Sci. Total Environ. 756, 144058.
Zhang, D.M., Dyck, M., Filipovic, L., Filipovic, V., Lv, J.L., He, H.L., 2021.
Hyperaccumulators for potentially toxic elements: a scientometric analysis. Agron.
Basel 11, 1729.
Zhang, K.X., Luo, Z.J., 2020. Migration characteristics of typical anions in the process of
electrokinetic remediating Cr(Ⅵ)-contaminated soil. Chin. J. Environ. Eng. 14,
3515–3526.
Zubair, M., Shakir, M., Ali, Q., Rani, N., Fatima, N., Farooq, S., Shafiq, S., Kanwal, N.,
Ali, F., Nasir, I.A., 2016. Rhizobacteria and phytoremediation of heavy metals.
Environ. Technol. Rev. 5, 112–119.