Effects of vegetative-periodic-induced rhizosphere variationon the uptake and translocation of metals in Phragmites australis(Cav.) Trin ex. Steudel growing in the Sun Island Wetland

Jieting Wu • Li Wang • Fang Ma • Jixian Yang •

Shiyang Li • Zhe Li

Accepted: 9 February 2013 / Published online: 2 March 2013

� Springer Science+Business Media New York 2013

Abstract To evaluate the vegetative periodic effect of

rhizosphere on the patterns of metal bioaccumulation, the

concentrations of Mg, K, Ca, Mn, Zn, Fe, Cu, Cr, Ni, Cd

and Pb in the corresponding rhizosphere soil and tissues of

Phragmites australis growing in the Sun Island wetland

(Harbin, China) were compared. The concentrations of Zn,

Fe, Cu, Cr, Ni, Cd and Pb in roots were higher than in

shoots, suggesting that roots are the primary accumulation

organs for these metals and there exists an exclusion

strategy for metal tolerance. In contrast, the rest of the

metals showed an opposite trend, suggesting that they were

not restricted in roots. Harvesting would particularly be an

effective method to remove Mn from the environment. The

concentrations of metals in shoots were generally higher in

autumn than in summer, suggesting that Ph. australis

possesses an efficient root-to-shoot translocation system,

which is activated at the end of the growing season and

allows more metals into the senescent tissues. Furthermore,

metal bioaccumulation of Ph. australis was affected by

vegetative periodic variation through the changing of

physicochemical and microbial conditions. The rhizo-

spheric microbial characteristics were significantly related

to the concentrations of Mg, K, Zn, Fe and Cu, suggesting

that microbial influence on metal accumulation is specific

and selective, not eurytopic.

Keywords Metals � Phragmites australis � Rhizosphere �Bioaccumulation � Wetland

Introduction

The utilization of wetlands as natural filters for the abate-

ment of metal pollutants transported by rivers or lakes is

considered to be an effective and low-cost option to ame-

liorate the quality of surface waters (Gambrell 1994; Weis

and Weis 2004; Bragato et al. 2006). The vegetation cov-

ering wetlands, especially the macrophytes, may play an

important role in accumulating and sequestering metals by

storing them mainly in their oxygenated rhizosphere and

their roots or shoots (Jana 1988; Ye et al. 2001; Baldantoni

et al. 2004; Bragato et al. 2006). Rhizosphere comprises of

three interacting components—the plant, the soil, and the

microorganisms—and is defined as the volume of soil that

is influenced by the roots of plants (Lynch and Moffat

2005; Kavamura and Esposito 2010). Rhizospheric

microorganisms can improve soil quality and plant per-

formance via certain ecological processes, ultimately

helping plants accumulate more metals either directly or

indirectly (Tinker 1984; Barea et al. 2002; Yang et al.

2009). It makes sense to apply plants combined with some

rhizospheric microorganisms to improve the phytoextrac-

tion and phytoremediation of metal contaminants. Such

technique is called rhizoremediation (Jing et al. 2007).

Wetland macrophytes have higher remediation potential

than other species due to their fast growth, high biomass,

efficient nutrient assimilation, and stimulation of microbial

proliferation (Bragato et al. 2006; Larue et al. 2010).

Phragmites australis, also known as common reed, is one

of the dominant macrophytes that widely distributed in

worldwide wetland ecosystems, spanning temperate and

J. Wu � L. Wang (&) � F. Ma (&) � J. Yang � S. Li � Z. Li

State Key Lab of Urban Water Resource and Environment,

School of Municipal and Environmental Engineering,

Harbin Institute of Technology, Harbin 150090,

People’s Republic of China

e-mail: wanglihit@hotmail.com

F. Ma

e-mail: mafang@hit.edu.cn

123

Ecotoxicology (2013) 22:608–618

DOI 10.1007/s10646-013-1052-2

tropical regions. This macrophyte is a rhizomatous hemi-

cryptophyte and geophyte. It not only form wide stands

known as reed beds that provide microhabitats for birds

and mammals (Bonanno 2011), but also have the capacity

of metal bioaccumulation due to the large intercellular

air space in its cortex parenchyma (Sawidis et al. 1995;

Duman et al. 2007; Bragato et al. 2009). As a hyper-tol-

erant species, Ph. australis has been widely used in the

phytoextraction and phytoremediation of metal pollution in

recent decades, and is characterized as well-established and

eco-friendly (Vymazal et al. 2009; Zabłudowska et al.

2009).

Although there is a great number of plant species

capable of hyper-accumulating various metals using dif-

ferent tissues during certain vegetative periods, their cor-

responding systems of bioaccumulation process could

hardly persist during the whole growth cycle, neither do the

plants sequestrate many metal contaminants simulta-

neously (Wu et al. 2006). In addition, even though rhizo-

remediation has received increased attention, the present

understanding about the roles of various rhizosphere-rela-

ted constituents in metal bioaccumulation is limited,

let alone the information about which constituents are more

closely related to metal bioaccumulation (Choi and Park

2005; Kavamura and Esposito 2010). Consequently, more

research is needed to better understand and demonstrate the

effects of vegetative period change and the resulting vari-

ation in rhizosphere conditions on uptake and translocation

of metals.

The primary objectives of this study were to: (a) explore

the bioaccumulation patterns of Ph. australis to different

metals in various vegetative periods, (b) determine the

potential rhizosphere-related factors influencing the metal

bioaccumulation of Ph. australis and identify the closely

related ones, and (c) discuss the potential use of Ph. aus-

tralis in metals removal from wetlands. The main findings

of this research could be referred to for the utilization of

Ph. australis in the phytoextraction and phytoremediation

of heavy metals in wetland ecosystems.

Materials and methods

Site description and sample collection

The Sun Island Wetland (SIW) is located at 126�310–126�360E, 45�410–45�470N (Harbin, China), on the north

shore of Songhua River. Harbin is in the temperate conti-

nental monsoon climate zone, with an annual rainfall

average of 593.8 mm. The dominant plants of this wetland

are hydrophyte (plants that live in aquatic environments)

and phreatophyte (plants that are supplied with surface

water and often have their roots constantly in touch with

moisture). The Sun Island Wetland is in a triple functional

zone overlapped by urbanization areas, development zones

and scenic spots, making this wetland closely related to

human activities.

For the common reed growing in the northeast China,

the peak and trough of its vegetative periods coincide with

summer and autumn respectively. Based on this reason,

these two seasons which represent the vegetative-periodic-

induced rhizosphere variations warrant observation. During

June (summer) and October (autumn) of both 2010 and

2011, the daily air temperatures and daily relative humidity

at the research plots were recorded. The mean daily air

temperatures ranged from 16.0 to 26.0 �C in June and 2.6

to 11.2 �C in October, while the mean daily relative

humidity ranged from 46.7 to 88.7 % in June and 48.2 to

80.7 % in October. Thirty sampling plots (about

30 9 30 9 30 cm3 in size, 50 m apart) were chosen along

the river bank of the Sun Island Wetland.

After the collection, plant and soil samples were placed

into sterile plastic bags and sealed to avoid extraneous

contamination and then transferred to laboratory in a con-

stant temperature cool box.

For the physicochemical analysis of the plant samples,

the sample-washing procedure to preliminarily remove soil

and dust impurities was performed within 24 h after sam-

pling in a 5 L plastic jar with distilled water in order to

minimize the loss of hairy roots. For the physicochemical

analysis and the microbial community analysis of the rhi-

zospheric soil samples, these samples from all the plots

were carefully collected from the roots. Then all the sam-

ples (including plants and rhizospheric soil) were mixed

into their subsamples before analysis. In addition, the

backup samples were temporarily stored in a refrigerator to

keep them fresh (4 �C within 3 days; -20 �C for a longer

time).

Physicochemical analysis

The organic matter content of the soil was determined by

the wet combustion method (Kandeler 1995). The content

of organic C was determined by the Total Organic Carbon

Analyzer (SSM-5000A, Shimadzu Corp.). The content of

total N and total S in the soil was determined by a CNS

analyzer (Vario EL). The washed plant samples were

separated into aboveground parts (leaves and stems) and

underground parts (roots and rhizomes). All the samples

(soil and plants) were dried at 70 �C until a constant weight

then homogenized and accurately weighed to *0.3 ±

0.0003 g and subsequently mineralized with HCl(36 %)–

HNO3(67 %)–HF(49 %) acids (5:2:2, V/V/V) in a Micro-

wave Accelerated Reaction System (CEM MARS-5). The

mineralized samples were dissolved in 1 mL HNO3 (1 %)

and diluted to 50 mL with ultrapure water, then analyzed

Effects of vegetative-periodic-induced rhizosphere variation 609

123

for total P, Mg, K, Ca, Mn, Zn, Fe and Cu, using induc-

tively coupled plasma-atomic emission spectrometry (ICP-

AES) (Perkin Elmer Optima 5300DV), while Cr, Ni, Cd

and Pb levels were analyzed using inductively coupled

plasma mass spectrometry (ICP-MS) (Agilent 7500a). For

quality control reasons, several blanks, two certified ref-

erence materials, GBW07312 (stream sediments) and

GBW07605 (Tea) were used during the procedure. The

relative standard deviation (RSD) values were mostly

lower than 5 %, except for Fe (7.2 %) and Pb (9.7 %). The

recoveries of the determined elements ranged between 95.5

and 103.5 %. Limits of detection (LOD) were calculated as

3r in over 9 measurements of the chemical blanks

from each procedure. LODs of these metals were Mg

(0.1 lg L-1), K (20 lg L-1), Ca (0.02 lg L-1), Mn (0.4

lg L-1), Zn (1 lg L-1), Fe (2 lg L-1), Cu (0.4 lg L-1),

Cr (0.025 lg L-1), Ni (0.056 lg L-1), Cd (0.015 lg L-1),

and Pb (0.067 lg L-1).

Microbial community analysis

Microbial community structure analysis genetic

characteristics by denaturing gradient gel

electrophoresis (DGGE)

The genomic DNA of rhizospheric soil microbes was

extracted with a FastDNA Spin Kit for Soil (Q-Biogene,

Vista, CA, USA) according to the manufacturer’s instruc-

tions. The extracted DNA was then used as a template for

PCR. The primers of bacteria, actinomycetes and fungi for

the PCR amplification were designed respectively, and the

corresponding thermocycling conditions were set. Partial

16S rRNA genes of bacteria were amplified with primers

GC-341F (50-CCTACGGGAGGCAGCAG-30) and 534R

(50-ATTACCGCGGCTGCTGG-30), and the thermocycling

conditions were(touchdown PCR): 3 min at 95 �C, fol-

lowed by 20 cycles of: 30 s at 95 �C(annealing for 30 s

with a 0.5 �C/cycle decrement until the 56 �C is reached),

1 min at 72 �C, 35 cycles of: 30 s at 95 �C, 30 s at 56 �C

and 1 min at 72 �C, and a final extension for 5 min

at 72 �C (Muyzer et al. 1993; Huws et al. 2007). For

actinomycetes, the genes were amplified with primers

GC-R513 (50-CGGCCGCGGCTGCTGGCACGTA-30) and

F243 (50-GGATGAGCCCGCGGCCTA-30), and the ther-

mocycling conditions were: 5 min at 94 �C, followed by 35

cycles of : 1 min at 95 �C, 1 min at 63 �C, 2 min at 72 �C,

and a final extension for 10 min at 72 �C (Heuer et al.

1997). For fungi, the genes were amplified with primers

GC-FR1(50-AICCATTCAATCGGTAIT-30) and FF390

(50-CGATAACGAACGAGACCT-30). The thermocycling

conditions were: 8 min at 95 �C, followed by 30 cycles of :

30 s at 95 �C, 45 s at 50 �C, 2 min at 72 �C, and a final

extension for 10 min at 72 �C (Vainio and Hantula 2000).

(GC-Clamp50-CGCCCGCCGCGCGCGGCGGGCGGGG

CGGGGGCACGGGGGG-30).Amplicons were loaded onto 6–12 % (w/v) polyacryl-

amide gels with a 40–60 % denaturing gradient of deion-

ised formamide and urea (100 % denaturant consisting of

40 % (v/v) deionised formamide and 7 M urea), and

electrophoresis was performed in a DCode system (BioRad

Co., Ltd., USA). Electrophoresis was conducted at a con-

stant voltage of 140 V for 7 h at 60 �C. Gels were then

stained with silver nitrate (Sanguinetti et al. 1994). The

DGGE profiles were analyzed by software ‘‘Quantity One

version 4.6.2’’ (BIO-RAD Laboratories, Inc. USA). The

gray scales of the fragments were measured.

Metabolic characteristics (BIOLOG)

Community level physiological profiles (CLPPs) were

assessed by the Biolog EcoPlateTM system (Biolog Inc.,

CA, USA) according to previous work (Classen et al. 2003;

Gomez et al. 2006). Each 96-well plate consists of three

replicates with each replicate comprising of 31 sole carbon

sources and a blank of water. Soil suspensions (10 g of soil,

and 100 mL of sterilized distilled water) were shaken for

1 h and then pre-incubated for 18 h before inoculation to

allow microbial utilization of any soluble organic com-

pounds from the soil. The soil suspensions were then

shaken for 30 min on a reciprocal shaker. After settling for

30 min., an 8-mL aliquot of the supernatant was diluted in

792 mL of inoculating solution for a final 1:1,000 dilution.

Inoculations were accomplished by transferring 150 lL of

the soil dilution to each of the 96 wells on the plates using

an 8-channel pipettor. The plates were incubated at 25 �C,

and color development in each well was recorded as optical

density (OD) at 590 and 750 nm with a plate reader at

regular 24 h-intervals.

All work during plate preparation was done under a

laminar-flow hood to minimize the risk of contamination.

All plates were placed in polyethylene bags to reduce des-

iccation while incubating in the dark in growth chambers.

Statistical analysis

All statistical analyses were performed using software

SPSS version17.0 of Statistical Software Package (SPSS

Inc. Chicago, USA). Standard deviation (SD) was used as a

measure of variance. T test and One-Way ANOVA (LSD

test and Duncan test) were used to ascertain whether

parameters were significantly different among samples.

The correlation between parameters was tested by Bivari-

ate Correlations (Pearson Correlation Coefficients).

The genetic characteristics of the rhizospheric microbial

community were distinguishable in terms of the Shannon–

Weaver diversity index (H) and richness index (S), which

610 J. Wu et al.

123

were determined as the following equation as described by

Yang et al. (2000):

H ¼ �X

pi lnpið Þ ¼ �X

Ni=Nð Þln Ni=Nð Þ

where H is the Shannon–Weaver diversity index, pi is the

percentage of the DGGE band gray degree to each DNA

sample, Ni is the net gray degree quantity (subtracted by

the background gray degree quantity of a gel) of the DGGE

band to each DNA sample. N is the total net gray degree

quantity of all DGGE bands examined in each DNA

sample. The range of H is between 0 and ln(S), and S is the

number of DGGE bands to each DNA sample (richness

index).

The metabolic characteristics of the rhizospheric

microbial community were distinguishable in terms of the

Shannon–Weaver diversity index (H), richness index (S)

(Garland 1997; Derry et al. 1999), and the microbial met-

abolic activity was estimated by the average well-color

development (AWCD) (Gomez et al. 2006), which was

determined as follows:

AWCD ¼X

ODi=31

where ODi is the optical density value from each well and

31 is the number of the sole carbon sources in the EcoPlate

(Classen et al. 2003).

The ability of Ph. australis to take up metals from the

soil and translocate them within itself was evaluated by the

following indices: Translocation factor (TF) = shoot/root

ratio, the shoot-to-root quotient of metal concentrations (or

translocation ratio) typically is [1 in hyperaccumulators

(Macnair 2003; Van der Ent et al. 2013). Another criterion

sometimes proposed for defining hyperaccumulation is the

bioaccumulation factor (BAF); bioaccumulation factor

(BAF) = shoot/soil ratio. BAF[1 means that a given plant

is an accumulator of this metal while BAF \1 means that

it is an excluder of the given metal (Tu and Ma 2002;

Kovacik et al. 2012).

Results

The main physicochemical properties of rhizosphere soils

were shown in Table 1. The summer samples (S) and the

autumn samples (A) were significantly different in terms of

pH and Total P. The soils in summer had the higher pH,

organic matter, organic C, total N, total S and total P than

in autumn.

Based on the DGGE banding patterns, the genetic Shan-

non–Weaver indices of the rhizospheric microorganisms

(bacteria, actinomycetes and fungi) are presented in Fig. 1.

Generally, the microbial community of the rhizosphere of

Ph. australis had higher genetic diversity and richness in

summer than in autumn. Nevertheless, the significant dif-

ference in diversity between summer and autumn only

occurred in fungi, while significant differences in richness

were observed in bacteria and fungi. The community level

physiological profiles (CLPPs) of microorganisms are

shown in Fig. 2. The AWCD and the metabolic Shannon–

Weaver indices increased along with the incubation time. In

addition, the microbial community of the Ph. australis rhi-

zosphere had higher diversity and richness in summer than in

autumn. Though the microbial metabolic activity, expressed

as AWCD, was not significantly different between two sea-

sons, the rhizospheric microbial community in summer still

showed higher metabolic activity.

Concentrations of the metals in soil and plant organs are

reported in Table 2. Within Ph. australis, significant dif-

ferences in the concentration of metals between roots and

shoots were noticed. The underground tissues (roots) and

the aboveground tissues (shoots) are significantly different

in terms of the metal concentrations. Zn (only significant in

summer), Fe, Cu, Cr, Ni, Cd (only significant in summer)

and Pb showed higher concentrations in roots. Mg (only

significant in autumn), K, Ca and Mn showed higher

concentrations in shoots. In addition, the metal concentra-

tions in rhizosphere soils and Ph. australis tissues showed

significant differences between the two vegetative periods.

The soil samples of summer showed higher concentrations

of Ca and Zn, while the samples of autumn showed higher

concentrations of Mg, Mn, Fe, Cr, Ni and Cd. The root

samples of summer showed higher concentrations of Zn

and Cu, while the samples of autumn showed higher con-

centrations of Ni and Pb. The shoot samples of summer

only showed a higher concentration of K, while the sam-

ples of autumn showed higher concentrations of Mn, Ni

and Pb. However, K, Cu and Pb in soils, Mg, K, Ca, Mn,

Fe, Cr and Cd in roots and Mg, Ca, Zn, Fe, Cu, Cr and Cd

in shoots didn’t show significantly different concentrations

between the two vegetative periods. Besides, in terms of

Table 1 Physicochemical properties of soil from Phragmites aus-tralis rhizosphere

S A

pH 8.26 ± 0.03b 7.72 ± 0.04a

Organic matter (%) 14.9 ± 2.34a 13.0 ± 1.03a

Organic C (mg kg-1) 15711 ± 2375.93a 12648 ± 1729.06a

Total N (mg kg-1) 1072 ± 118.14a 909 ± 81.64a

Total P (mg kg-1) 442 ± 35.06b 331 ± 66.91a

Total S (mg kg-1) 319 ± 68.18a 269 ± 76.55a

Different letters indicate significant differences between two seasons

per physicochemical property (p \ 0.05) after T test. Data are

mean ± SD (n = 30)

S summer, A autumn

Effects of vegetative-periodic-induced rhizosphere variation 611

123

the correlations between metal concentrations in soil and

Ph. australis tissues, Zn in soil only correlated with its

concentration in roots, while Mn, Cr and Cd in soil only

correlated with their concentrations in shoots. Fe and Ni in

soils correlated with their concentration in both shoots and

roots. However, the rest of the metals in soil didn’t have

significant correlations with their concentrations in any

plant tissues. Although different samples followed different

decreasing trends of metal concentration in different veg-

etative periods, K generally showed the highest concen-

tration in both underground and aboveground issues of

Ph. australis, whereas Cd showed the lowest concentration.

The translocation factor (TF) and bioaccumulation fac-

tor (BAF) of the 11 metals considered in Ph. australis are

shown in Fig. 3. The mobility of a given metal, expressed

as TF, varied according to the vegetative periods. Summer

only showed a higher value of K, whereas, autumn showed

higher values in 7 cases, namely, Mn, Zn, Cu, Cr, Ni, Cd

and Pb. Regarding the BAF, summer showed a higher

value only in one case—K, while autumn showed a higher

value only in Zn. The TF and BAF values of the rest of the

metals didn’t show significant differences between the two

vegetative periods. In particular, Ca and Fe respectively

showed the highest and lowest values of TF, whereas Mg

and Fe respectively showed the highest and lowest values

of BAF. It also indicated that, as a whole, TF was signif-

icant higher (0.17–3.89) than BAF (0.01–0.76).

Correlation analyses were performed between each of

the metal’s concentration in Ph. australis roots versus the

physicochemical properties of the soil (pH, organic matter,

a a ba a a

0

1

2

3

4

5

6D

iver

sity

inde

x(a)

S A

b

a

baa

a

0

5

10

15

20

25

30

35

40

45

50

Bacteria Actinomycetes Fungi

Ric

hnes

s in

dex

(b)

Fig. 1 Shannon–Weaver indices at DNA level of microorganisms

(bacteria, actinomycetes and fungi) derived from Phragmites aus-tralis rhizosphere. S summer, A autumn. a Shannon–Weaver diversity

index (H). b Shannon–Weaver richness index (S). Different letters

indicate significant differences of Shannon–Weaver indices

(p \ 0.05) between two seasons after One-Way ANOVA (Duncan

test). Data are mean ± SD (n = 3)

0

0.2

0.4

0.6

0.8

1

1.2

AW

CD

(a)

S A

0

0.5

1

1.5

2

2.5

3

3.5

Div

ersi

ty in

dex

(b)

0

5

10

15

20

25

30

0 24 48 72 96 120 144 168 192

Ric

hnes

s in

dex

Incubation time (h)

(c)

Fig. 2 Community level physiological profiles (CLPPs) of microor-

ganisms derived from Phragmites australis rhizosphere. S summer,

A autumn. a Average well color development (AWCD). b Shannon–

Weaver diversity index (H). c Shannon–Weaver richness index (S).

Data are mean ± SD (n = 9)

612 J. Wu et al.

123

organic C, total N, total S, total P) and the characteristics of

rhizospheric microbial community (Tables 3, 4). Note that

pH had significant relationships with K, Zn and Cu con-

tents. Regarding the nutrient variables, organic C and total

N had a similar effect on Mg, K, Zn, Fe, Cu. However,

none of the physicochemical properties of the soil showed

significant effects on Cd and Pb. Regarding rhizospheric

microorganisms, significant correlations between the

microbial characteristics and metal content were found in

Mg, K, Zn, Fe and Cu. In addition, compared to the met-

abolic characteristics, the genetic characteristics of the

rhizospheric microbial community have relationships with

more metals.

Discussion

In line with previous findings, Ph. australis roots accu-

mulated greater concentrations of Zn, Fe, Cu, Cr, Ni, Cd

and Pb than shoots in this research (Table 2), which indi-

cated that a certain fraction of these metals were available

to the underground tissues of Ph. australis and were sub-

sequently accumulated by them. However, once inside the

plant, these metals’ mobility was limited. Previous data

also indicated that Ph. australis appears to concentrate

more metals in roots and translocate smaller amounts to

shoots, which suggested that it is able to restrict the amount

of metals translocated to aerial tissues (Peverly et al. 1995;

Weis et al. 2003). This is probably due to the need of plants

to prevent toxicity to their photosynthetic apparatus (Stoltz

and Greger 2002). To Ph. australis, its roots acted as a kind

of filter to certain metals, and this filter exclusion effect is

an effective tolerance strategy to protect its shoots from

metal induced injuries (Furtig et al. 1999). Besides, the

elevated metal concentrations in the underground tissues

indicated the capability of rather well-balanced uptake and

translocation of metals in Ph. australis. This largely inde-

pendent ion budget is typical of many grass-like species,

which is supposed to partly contribute to their wide eco-

logical amplitude (Gries and Garbe 1989; Deng et al.

2004). Nevertheless, compared to roots, shoots accumu-

lated greater concentrations of Mg, K, Ca and Mn in this

research, which indicated that this rooted species can

absorb metals through their underground tissues as well as

aboveground tissues. This is because the latter can provide

an expanded area to trap particulate matter, absorb metal

ions, and accumulate and sequester pollutants (Welsh and

Denny 1980; Levine et al. 1990). However, as previously

noted by Macnair (2003) and Faucon et al. (2007), some-

times the phenomenon of aboveground tissues’ accumula-

tion has been over-reported, because it appeared to be the

result of airborne contamination of the leaf surface, rather

than root uptake and translocation. Although in our

research the plant samples were washed before the metal

content analysis, the direct absorption of airborne con-

tamination could not be totally eliminated.

The observed metal mobility and bioaccumulation

capability of Ph. australis (expressed as TF and BAF)

might depend on the vegetative period. The concentrations

of metals in roots were generally higher in summer, while

Table 2 Metal element concentrations (mg metal element kg-1 dry weight) in soil and Phragmites australis tissues

Soil S A

Root Shoot Soil Root Shoot

Mg 1098 ± 94.13A 691 ± 43.15a 834 ± 130.54ab 1320 ± 42.6B 667 ± 13.11a 965 ± 184.53b

K 22156 ± 1538.85A 7769 ± 299.25a 11683 ± 573.52c 21085 ± 2126.55A 6938 ± 355.58a 9633 ± 656.2b

Ca 13314 ± 1018B 817 ± 115.89a 2896 ± 383.13b 9811 ± 1719.19A 689 ± 63a 2649 ± 462.16b

Mn 456 ± 64.94A 57.5 ± 10.06a 84.1 ± 13.91b* 645 ± 83.59B 46.3 ± 9.41a 107 ± 11c*

Zn 75.3 ± 1.73B 11.9 ± 0.3b** 10.0 ± 0.5a 63.5 ± 6.59A 10.7 ± 0.47a** 10.2 ± 0.5a

Fe 19950 ± 2079.02A 1844 ± 109.44b** 314 ± 36.17a* 22921 ± 2231.26B 1910 ± 87.23b** 420 ± 109.2a*

Cu 25.9 ± 5.97A 3.6 ± 0.42c 1.5 ± 0.15a 30.1 ± 2.97A 2.8 ± 0.26b 1.7 ± 0.2a

Cr 33.9 ± 3.8A 12.8 ± 2.53b 2.8 ± 0.55a* 46.1 ± 8.83B 10.8 ± 2.85b 6.4 ± 0.76a*

Ni 18.5 ± 1.24A 3.2 ± 0.36b* 1.3 ± 0.21a** 26.9 ± 0.7B 4 ± 0.31c* 2.7 ± 0.53b**

Cd 0.71 ± 0.03A 0.2 ± 0.01b 0.12 ± 0.01a** 0.78 ± 0.03B 0.19 ± 0.03b 0.16 ± 0.03ab**

Pb 9 ± 1.12A 0.36 ± 0.01b 0.13 ± 0.01a 8 ± 1.47A 0.45 ± 0.07c 0.33 ± 0.03b

Different letters in uppercase indicate significant differences of rhizosphere soil between two seasons per element (p \ 0.05) after One-Way

ANOVA (Duncan test). Different letters in lowercase indicate significant differences among Phragmites australis tissues between two seasons

per element (p \ 0.05) after One-Way ANOVA (Duncan test). Data are mean ± standard deviation (n = 3)

S summer, A autumn

* Correlations between concentrations of (Mn, Fe, Cr etc.) in soil and Phragmites australis tissues are significant at the 0.05 level

** Correlations between concentrations of (Zn, Fe and Ni) in soil and Phragmites australis tissues are significant at the 0.01 level

Effects of vegetative-periodic-induced rhizosphere variation 613

123

the concentrations in shoots were generally higher in

autumn, suggesting an increase of metals in aboveground

tissues over the growing season, until the leaves die and

begin to decay. Previous publications have also reported

the highest concentrations of metals during autumn com-

pared to relatively low levels during the summer. The

senescent leaves had greater metal concentrations than

green leaves, which indicated that plants may use leaf fall

as a detoxification mechanism (Matthews and Thornton

1982; Dahmani-Muller et al. 2000; Weis et al. 2003). It

means that plants possess an efficient root-to-shoot trans-

location system, which is activated at the end of the

growing season and allows the concentration of toxic ele-

ments in the senescent tissues, so they can eliminate metals

through litter fall in this way (Bragato et al. 2009). This

strategy has been suggested for metal hyperaccumulator

plants (Baker and Brooks 1989). The older leaves fall off

the plant, which results in increased metal concentrations

in the soil, thus, metals may be released back to the envi-

ronment, either directly by excretion or indirectly when

dead leaves become detritus, finally being available for

circulation. This can also explain why the concentrations of

most of the metals in the soil were generally higher in

autumn. The concentrations of certain metals in soil

decreased from summer to autumn. These metals were

probably removed through root uptake or formed iron

plaque in parts of the roots, which made them unavailable

for analysis (Duman et al. 2007). Besides the tolerance and

protective mechanisms mentioned above, that the concen-

trations of metals in shoots increased from summer to

autumn can also be referred to as a dilution effect (slower

uptake than growth), since summer coincides with the peak

of the vegetative periods of Ph. australis. The dilution

effect also indicated that an increase in biomass is not

necessarily linked to an increase in metal bioaccumulation

(Duman et al. 2007). Another possible explanation is that

the leaf transpiration rates are higher in summer than in

autumn. A small fraction of metals may be released into the

environment through living aerial leaf tissues. Although the

exact nature is uncertain, it may be the result of leaching

from the leaf surfaces accompanying transpirational water

loss (Burke et al. 2000).

The movement of certain metals into the plants appeared

to be concentration dependent (Table 2). Metal ions gen-

erally settle and accumulate in the soil, which in turn

became important sinks and sources of metals (Wang et al.

2009). However, some researchers consider that the uptake

of metals had no linear correlation with the external con-

tent. They attribute it to the variations of other environmen-

tal factors, which are more important in determining metal

uptake compared with labile total metal concentrations in

Ab

Bb

Ab

Ab

Ab

AbAb

AbAb

AbAb

Ab Ab

Ab

Bb

Bb

Ab

Bb Bb BbBb

Bb

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

Tra

nslo

cati

on f

acto

r (T

F)

(a)

Aa

Ba

AaAa

Aa

Aa

AaAa Aa

Aa

Aa

Aa

Aa

Aa

Aa Ba

AaAa

AaAa

Aa

Aa

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Mg K Ca Mn Zn Fe Cu Cr Ni Cd Pb

Bio

accu

mul

atio

n fa

ctor

(B

AF

)

(b)

S A

Fig. 3 Translocation factor (TF) and bioaccumulation factor (BAF)

of metal elements in Phragmites australis. S summer, A autumn. a TF

(shoot/root), b BAF (shoot/soil). Different letters in uppercase

indicate significant differences between two seasons per element

(p \ 0.05) after One-Way ANOVA (LSD test and Duncan test).

Different letters in lowercase indicate significant differences between

TF and BAF per element (p \ 0.05) after One-Way ANOVA (LSD

test and Duncan test). Data are mean ± SD (n = 3)

Table 3 Coefficients of Pearson’s correlations between the soil

physicochemical properties and metal elements in roots of Phrag-mites australis

pH Organic

matter

Organic

C

Total N Total S Total P

Mg ns ns 0.824* 0.843* ns ns

K 0.886* ns 0.953** 0.860** ns 0.886**

Ca ns 0.812* ns ns 0.834* ns

Mn ns ns ns ns 0.868** ns

Zn 0.912* ns 0.900* 0.913* ns 0.875**

Fe ns ns -0.878* -

0.881*

ns ns

Cu 0.869* ns 0.966** 0.872** ns 0.832**

Cr ns ns ns ns 0.918** ns

Ni ns -0.903* ns ns ns ns

Cd ns ns ns ns ns ns

Pb ns ns ns ns ns ns

ns no significant correlation at the 0.05 level (2-tailed)

* Correlation is significant at the 0.05 level (2-tailed)

** Correlation is significant at the 0.01 level (2-tailed)

614 J. Wu et al.

123

soil (Keller et al. 1998). Besides, the aboveground tissues

are likely to be affected by external sorption due to

atmospheric deposition, which also affects the metal con-

centration in soil. This may be one of the reasons of rather

good correlations between concentrations of some met-

als in soil and shoots. In addition, the various physiological

functions of metals might provide another reason for their

different behaviors. Certain metals are required for the

normal growth and healthy functioning of plants, pos-

ing toxicity only when their concentration limits are

exceeded, while certain non-essential metals are extremely

toxic to plants even at low concentrations (Rengel 2004;

Bragato et al. 2009). For the essential nutrients, plants

may actively promote these metals’ release from soil

via the excretion of root exudates and accumulate them

even if their concentrations in the soil are relatively low

(Lasat 2002).

The metals’ uptake and translocation by plants rely

on numerous external factors, which control the balance

of metal exchange between soil, water and plants

(Sundareshwar et al. 2003; Bragato et al. 2009). The

variations were induced by the changing of seasons, mainly

with the accompanying changes in temperature. Average

air temperatures were used to evaluate the effect of temper-

ature in this research, which can reflect the diurnal changes.

A number of researchers indicated that temperature has a

positive effect on metal uptake and accumulation in various

species (Fritioff et al. 2005; Brunham and Bendell 2011).

The reason is probably that increasing temperatures coin-

cide with an increase in ambient carbon dioxide (CO2)

level, and their combined effects would increase certain

metal accumulation of wetland plants (Reddy et al. 2010;

Brunham and Bendell 2011). By contrast, temperature did

not affect metal uptake in some cases (Brunham and

Bendell 2011). Regarding pH, it may have a positive effect

on the concentrations of K, Zn, Cu in the root tissues. Batty

et al. (2000) found that the uptake of Mn and Cu in

P. australis was lower at pH 3.5 than at 6.0. Similar rela-

tionships have also been observed in the concentrations of

Pb, Cu and Cd in J. effuses (Deng et al. 2004). Van der

Merwe et al. (1990) also found that Zn, Mn and Ni uptakes

by Typha capensis and Arundo donax were pH dependent,

and this dependence under alkaline conditions became

prominent. This effect could be interpreted in terms of the

competition between metal ions and protons at the plant–

soil–water interface in the waterlogged soil/sediment

(Deng et al. 2004). As well, almost all the metals uptake

seemed to be positively affected by soil nutrients (organic

matter, organic C, total N, P and S contents) in this

research. In many areas characterized by alkaline soil like

the presently studied area, low organic matter content may

constrain the availability of certain metals to plants (Rashid

and Ryan 2004; de Santiago et al. 2011). However, some

researchers found that the plants might accumulate par-

ticular metals if organic matter contents are low, because

high organic matters might cause certain metals to pre-

cipitate as sulphides and thus reducing their availability

(Du Laing et al. 2007; Du Laing et al. 2009). Regarding P

and N, their concentrations were related to Mg, K, Zn, Fe

and Cu uptake, which was similar with previous findings

Table 4 Coefficients of Pearson’s correlations between the characteristics of rhizospheric microbial community and metal elements in roots of

Phragmites australis

Genetic characteristics Metabolic characteristics

(H)-B (H)-A (H)-F (S)-B (S)-A (S)-F AWCD (H) (S)

Mg 0.818* 0.862* ns 0.830* 0.873* ns ns ns ns

K 0.936** 0.866* 0.836** 0.920** 0.853* 0.868** ns 0.831* ns

Ca ns ns ns ns ns Ns ns ns ns

Mn ns ns ns ns ns Ns ns ns ns

Zn 0.946** 0.904* 0.935** 0.930** 0.887* 0.945** 0.869* 0.905* 0.863*

Fe ns -0.849* ns ns -0.834* ns ns ns ns

Cu 0.921** 0.816* 0.876* 0.913* 0.819* 0.914* ns ns ns

Cr ns ns ns ns ns ns ns ns ns

Ni ns ns ns ns ns ns ns ns ns

Cd ns ns ns ns ns ns ns ns ns

Pb ns ns ns ns ns ns ns ns ns

B bacteria, A actinomycetes, F fungi, (H) Shannon–Weaver diversity index, (S) Shannon–Weaver richness index

ns no significant correlation at the 0.05 level (2-tailed)

* Correlation is significant at the 0.05 level (2-tailed)

** Correlation is significant at the 0.01 level (2-tailed)

Effects of vegetative-periodic-induced rhizosphere variation 615

123

(Deng et al. 2004). In addition, the negative effect of soil

nutrients on certain metals may result from the ‘‘dilution

effect’’ via improving the biomass of plants under rich

nutrient conditions (Deng et al. 2004). Another possible

factor was the seasonal fluvial inputs, which might have

affected the metal concentrations in soil, subsequently

affecting the metal concentrations in plant indirectly.

The content of certain metals in roots were significantly

related to the characteristics of the rhizospheric microbial

community, which suggested that the microbial effect on

metal uptake exists. Metals can affect microbial communi-

ties by changing the total viable counts, shifting the popu-

lation distribution, or altering the metabolic characteristics

of the community (Frostegard et al. 1996; Chen and Cutright

2003). Vice versa, metal tolerance is probably the result of

changes in the rhizospheric microbial communities change

(Bennisse et al. 2004). Microorganisms have developed

various complex resistance mechanisms to minimize the

adverse effects posed by metals, which can potentially

improve phytoextraction (Bruins et al. 2000; Chen and

Cutright 2003; Ma et al. 2011). Soil microorganisms are

known to play a key role in the mobilization and immobi-

lization of metals in plants via changing their bioavailability

(Bennisse et al. 2004). Actually, only a limited fraction of

the total amount of certain metals is available for plants

(Madejon et al. 2007). Previous studies also showed that

microbial exudates can even supply additional Fe, Zn and Cu

to plants (Howell 2003; de Santiago et al. 2011). Further-

more, the results also indicated that the effects of rhizo-

spheric microorganisms on metal uptake are selective and

specific, not eurytopic. Similarly, the resistance mechanisms

mentioned above are plasmid mediated and are highly spe-

cific to a particular metal, for example, the cop plasmid

facilitates the sequestration of Cu2? ions outside of the

cytoplasmic membrane (Chen and Cutright 2003).

It is well documented that aquatic plants are being used

successfully for the phytoremediation of metals (Gupta and

Chandra 1998; Rajkumar et al. 2009). However, the main

disadvantages of most aquatic flowering plants which can

remove various metals from environment are that their

metal removal efficiency is low. This is because their sizes

are relatively small and roots are slow-growing (Rajkumar

et al. 2009). Since BAF [1 means that a given plant is an

accumulator of a given metal (Tu and Ma 2002; Kovacik

et al. 2012), we know that Ph. australis is not a typical

hyperaccumulator. However, compared to the rest of the

metals, a relatively high TF of Mn ([1 in hyperaccumu-

lators) (Macnair 2003; Van der Ent et al. 2013) was also

detected in this wetland plant. According to Marchand

et al. (2010), a removal rate of 99 % for Mn by Ph. aus-

tralis in the summer was obtained, and this rate remains at

99 % for Mn in the winter. In addition, Ph. australis is fast-

growing and perennial which can produce a relatively large

biomass. Besides, it also has deep root apparatuses and can

tolerate and/or accumulate a range of metals in its aerial

portion. In addition, the possibility of using this plant

species is attractive due to their high ecological adapt-

ability (Rossato et al. 2012). According to Han et al.

(2007), the dual-function of landscaping and environmental

remediation is also an important consideration when

selecting a plant species for phytoremediation. Given these

points, Ph. australis should be considered to remove the

metal contaminants during the phytoremediation of both

natural and constructed wetlands. However, it’s noteworthy

in that the consumption of metal-laden detritus may bring

deleterious effects on consumers as discussed above.

Conclusion

Phragmites australis is one of the hyper-tolerant plants that

has extensive and well-developed root system. In the

present research, since the translocation of Zn, Fe, Cu, Cr,

Ni, Cd and Pb have been reduced from the roots to the

shoots, Ph. australis is suitable as a phytostabilizer for

revegetation. Moreover, in the view of toxicology, this

could be a desirable property, as these metals would not

pass into the food chain via defoliation if they are harvested

and treated, avoiding potential risk to the environment. By

contrast, although the level of Mn concentration in the

sampling soil is rather low, Mn appeared to be mainly

accumulated in the aboveground tissues of Ph. australis, so

harvesting would be a potentially effective method for

phytoextraction. In addition, the uptake and translocation

of metals in Ph. australis are significantly related to certain

vegetative-periodic-induced factors of the rhizosphere. The

identification of these factors and the vegetative period that

coincides with the maximum metal accumulation in the

tissues of Ph. australis are important in optimizing its

potential for metal removal, for example, Ni and Pb in both

shoots and roots of Ph. australis significantly increased

from summer to autumn. However, the lack of correlation

in some cases might imply that the corresponding variation

was not dominant, or was obscured by its concurrence with

other variations. As a result, complementary experiments

should be performed to better assess the interactions

between Ph. australis and its rhizospheric constituents, to

develop methods that aim at enhancing the phytoextraction

and phytoremediation of fragile wetland ecosystems.

Acknowledgments This work was supported by National Natural

Science Foundation of China (51179041), the Major Science and

Technology Program for Water Pollution Control and Treatment

(2012ZX07201003), the National Creative Research Group from the

National Natural Science Foundation of China (51121062), and the

State Key Lab of Urban Water Resource and Environment, Harbin

Institute of Technology, China (HIT) (2011TS07).

616 J. Wu et al.

123

Conflict of interest The authors declare that they have no conflict

of interest.

References

Baker AJM, Brooks RR (1989) Terrestrial higher plants which

hyperaccumulate metallic elements—a review of their distribu-

tion, ecology and phytochemistry. Biorecovery 1:81–126

Baldantoni D, Alfani A, Di Tommasi P, Bartoli G, De Virzo Santo A

(2004) Assessment of macro and microelement accumulation

capability of two aquatic plants. Environ Pollut 130:149–156

Barea JM, Azcon R, Azcon-Aguilar C (2002) Mycorrhizosphere

interactions to improve plant fitness and soil quality. Antonie

Van Leeuwenhoek Int J G 81:343–351

Batty LC, Baker AJM, Wheeler BD, Curtis CD (2000) The effect of

pH and plaque on the uptake of Cu and Mn in Phragmitesaustralis (Cav.) Trin ex Steudel. Ann Bot Lond 86:647–653

Bennisse R, Labat M, El Asli A, Brhada F, Chandad F, Lorquin J,

Liegbott PP, Hibti M, Qatibi AI (2004) Rhizosphere bacterial

populations of metallophyte plants in heavy metal-contaminated

soils from mining areas in semiarid climate. World J Microbiol

Biotechnol 20:759–766

Bonanno G (2011) Trace element accumulation and distribution in the

organs of Phragmites australis (common reed) and biomonitor-

ing applications. Ecotoxicol Environ Safe 74:1057–1064

Bragato C, Brix H, Malagoli M (2006) Accumulation of nutrients and

heavy metals in Phragmites australis (Cav.) Trin. ex Steudel and

Bolboschoenus maritimus (L.) Palla in a constructed wetland of

the Venice lagoon watershed. Environ Pollut 144:967–975

Bragato C, Schiavon M, Polese R, Ertani A, Pittarello M, Malagoli M

(2009) Seasonal variations of Cu, Zn, Ni and Cr concentration in

Phragmites australis (Cav.) Trin. Ex Steud. in a constructed

wetland of North Italy. Desalination 246:35–44

Bruins MR, Kapil S, Oehme FW (2000) Microbial resistance to

metals in the environment. Ecotoxicol Environ Saf 45:198–207

Brunham W, Bendell LI (2011) The effect of temperature on the

accumulation of cadmium, copper, zinc, and lead by Scirpusacutus and Typha latifolia: a comparative analysis. Water Air

Soil Pollut 219:417–428

Burke DJ, Weis JS, Weis P (2000) Release of metals by the leaves of

salt marsh grasses Spartina alterniflora and Phragmites austral-is. Estuar Coast Shelf S 51:153–159

Chen H, Cutright TJ (2003) Preliminary evaluation of microbially

mediated precipitation of cadmium, chromium, and nickel by

rhizosphere consortium. J Environ Eng 129:4–9

Choi J, Park JW (2005) Competitive adsorption of heavy metals and

uranium on soil constituents and microorganism. Geosci J

9:53–61

Classen AT, Boyle SI, Haskins KE, Overby ST, Hart SC (2003)

Community-level physiological profiles of bacteria and fungi:

plate type and incubation temperature influences on contrasting

soils. FEMS Microbiol Ecol 44:319–328

Dahmani-Muller H, van Oort F, Gelie B, Balabane M (2000)

Strategies of heavy metal uptake by three plant species growing

near a metal smelter. Environ Pollut 109:231–238

de Santiago A, Quintero JM, Aviles M, Delgado A (2011) Effect of

Trichoderma asperellum strain T34 on iron, copper, manganese,

and zinc uptake by wheat grown on a calcareous medium. Plant

Soil 342:97–104

Deng H, Ye ZH, Wong MH (2004) Accumulation of lead, zinc,

copper and cadmium by 12 wetland plant species thriving in

metal-contaminated sites in China. Environ Pollut 132:29–40

Derry AM, Staddon WJ, Kevan PG, Trevors JT (1999) Functional

diversity and community structure of microorganisms in three

arctic soils as determined by sole-carbon-source-utilization.

Biodivers Conserv 8:205–221

Du Laing G, Vanthuyne DRJ, Vandecasteele B, Tack FMG, Verloo

MG (2007) Influence of hydrological regime on pore water metal

concentrations in a contaminated sediment-derived soil. Environ

Pollut 147:615–625

Du Laing G, Van de Moortel AMK, Moors W, De Grauwe P, Meers E,

Tack FMG, Verloo MG (2009) Factors affecting metal concen-

trations in reed plants (Phragmites australis) of intertidal marshes

in the Scheldt estuary. Ecol Eng 35:310–318

Duman F, Cicek M, Sezen G (2007) Seasonal changes of metal

accumulation and distribution in common club rush (Schoeno-plectus lacustris) and common reed (Phragmites australis).

Ecotoxicology 16:457–463

Faucon MP, Shutcha N, Meerts P (2007) Revisiting copper and cobalt

concentrations in supposed hyperaccumulators from SC Africa:

influence of washing and metal concentrations in soil. Plant Soil

301:29–36

Fritioff A, Kautsky L, Greger M (2005) Influence of temperature and

salinity on heavy metal uptake by submersed plants. Environ

Pollut 133:265–274

Frostegard A, Tunlid A, Baath E (1996) Changes in microbial

community structure during long-term incubation in two soils

experimentally contaminated with metals. Soil Biol Biochem

28:55–63

Furtig K, Pavelic D, Brunold C, Brandle R (1999) Copper-and-iron

induced injuries in roots and rhizomes of reed (Phragmitesaustralis). Limnologica 29:60–63

Gambrell RP (1994) Trace and toxic metals in wetlands—a review.

J Environ Qual 23:883–891

Garland JL (1997) Analysis and interpretation of community-level

physiological profiles in microbial ecology. FEMS Microbiol

Ecol 24:289–300

Gomez E, Ferreras L, Toresani S (2006) Soil bacterial functional

diversity as influenced by organic amendment application.

Bioresour Technol 97:1484–1489

Gries C, Garbe D (1989) Bioamss and nitrogen, phosphorus and

heavy metal content of Phragmites australis during the third

growing season in a root zone wastewater treatment. Archiv fur

Hydrobiologie 117:97–105

Gupta M, Chandra P (1998) Bioaccumulation and toxicity of mercury

in rooted-submerged macrophyte Vallisneria spiralis. Environ

Pollut 103:327–332

Han YL, Yuan HY, Huang SZ, Guo Z, Xia B, Gu JG (2007)

Cadmium tolerance and accumulation by two species of Iris.

Ecotoxicology 16:557–563

Heuer H, Krsek M, Baker P, Smalla K, Wellington EMH (1997)

Analysis of actinomycete communities by specific amplification

of genes encoding 16S rRNA and gel-electrophoretic separation

in denaturing gradients. Appl Environ Microbiol 63:3233–3241

Howell CR (2003) Mechanism employed by Trichoderma species in

the biological control of plant diseases: the history and evolution

of current concepts. Plant Dis 87:4–10

Huws SA, Edwards JE, Kim EJ, Scollan ND (2007) Specificity and

sensitivity of eubacterial primers utilized for molecular profiling

of bacteria within complex microbial ecosystems. J Microbiol

Methods 70:565–569

Jana S (1988) Accumulation of Hg and Cr by three aquatic species

and subsequent changes in several physiological and biochem-

ical plant parameters. Water Air Soil Pollut 38:105–109

Jing Y, He Z, Yang X (2007) Role of soil rhizobacteria in

phytoremediation of heavy metal contaminated soils. J Zhejiang

Univ Sci B 8:192–207

Kandeler E (1995) Organic matter by wet combustion. In: Schinner F,

Ohlinger R, Kandeler E, Margesin R (eds) Methods in soil

biology, 1st edn. Springer, Heidelberg, pp 397–398

Effects of vegetative-periodic-induced rhizosphere variation 617

123

Kavamura VN, Esposito E (2010) Biotechnological strategies applied

to the decontamination of soils polluted with heavy metals.

Biotechnol Adv 28:61–69

Keller BEM, Lajtha K, Cristofor S (1998) Trace metal concentration

in the sediments and plants of the Danube delta, Romania.

Wetlands 40:42–50

Kovacik J, Gruz J, Klejdus B, Stork F, Hedbavny J (2012)

Accumulation of metals and selected nutritional parameters in

the field-grown chamomile anthodia. Food Chem 131:55–62

Larue C, Korboulewsky N, Wang R, Mevy J (2010) Depollution

potential of three macrophytes: exudated, wall-bound and

intracellular peroxidase activities plus intracellular phenol con-

centrations. Bioresour Technol 101:7951–7957

Lasat MM (2002) Phytoextraction of toxic metals: a review of

biological mechanisms. J Environ Qual 31:109–120

Levine SN, Rudnick DT, Kelly JR, Morton RD, Buttel LA (1990)

Pollutant dynamics as influenced by seagrass beds: experiments

with tributyltin in Thalassia microcosms. Mar Environ Res

30:297–322

Lynch JM, Moffat AJ (2005) Bioremediation—prospects for the

future application of innovative applied biological research. Ann

Appl Biol 146:217–221

Ma Y, Prasad MNV, Rajkumar M, Freitas H (2011) Plant growth

promoting rhizobacteria and endophytes accelerate phytoreme-

diation of metalliferous soils. Biotechnol Adv 29:248–258

Macnair MR (2003) The hyperaccumulation of metals by plants. Adv

Bot Res 40:63–105

Madejon P, Murillo JM, Maranon T, Lepp NW (2007) Factors

affecting accumulation of thallium and other trace elements in

two wild Brassicaceae spontaneously growing on soils contam-

inated by tailings dam waste. Chemosphere 67:20–28

Marchand L, Mench M, Jacob DL, Otte ML (2010) Metal and

metalloid removal in constructed wetlands, with emphasis on the

importance of plants and standardized measurements: a review.

Environ Pollut 158:3447–3461

Matthews H, Thornton I (1982) Seasonal and species variation in the

content of cadmium and associated metals in pasture plants at

Shipham. Plant Soil 66:181–193

Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex

microbial populations by denaturing gradient gel electrophoresis

analysis of polymerase chain reaction-amplified genes coding for

16S rRNA. Appl Environ Microbiol 59:695–700

Peverly JH, Surface JM, Wang T (1995) Growth and trace metal

absorption by Phragmites australis in wetlands constructed for

landfill leachate treatment. Ecol Eng 5:21–35

Rajkumar K, Sivakumar S, Senthilkumar P, Prabha D, Subbhuraam

CV, Song YC (2009) Effects of selected heavy metals (Pb, Cu,

Ni, and Cd) in the aquatic medium on the restoration potential

and accumulation in the stem cuttings of the terrestrial plant,

Talinum triangulare Linn. Ecotoxicology 18:952–960

Rashid A, Ryan J (2004) Micronutrient constraints to crop production

in soils with Mediterranean-type characteristics: a review. J Plant

Nutr 27:959–975

Reddy AR, Rasineni GK, Raghavendra AS (2010) The impact of

global elevated CO2 concentrations on photosynthesis and plant

productivity. Curr Sci India 99:46–57

Rengel Z (2004) Heavy metals as essential nutrients. In: Prasad MNV

(ed) Heavy metal stress in plants: from biomolecules to

ecosystems, 2nd edn. Springer, New York, pp 271–285

Rossato LV, Nicoloso FT, Farias JG, Cargnelluti D, Tabaldi LA, Antes

FG, Dressler VL, Morsch VM, Schetinger MRC (2012) Effects of

lead on the growth, lead accumulation and physiological responses

of Pluchea sagittalis. Ecotoxicology 21:111–123

Sanguinetti CJ, Dias Neto E, Simpson AJ (1994) Rapid silver staining

and recovery of PCR products separated on polyacrylamide gels.

Biotechniques 17:914–921

Sawidis T, Chettri MK, Zachariadis GA, Stratis JA (1995) Heavy

metals in aquatic plants and sediments from water systems in

Macedonia, Greece. Ecotoxicol Environ Safe 32:73–80

Stoltz E, Greger M (2002) Accumulation properties of As, Cd, Cu, Pb

and Zn by four wetland plant species growing on submerged

mine tailings. Environ Exp Bot 47:271–280

Sundareshwar PV, Morris JT, Koepfler EK, Fornwalt B (2003)

Phosphorous limitation of coastal ecosystem processes. Science

299:563–565

Tinker PB (1984) The role of microorganisms in mediating and

facilitating the uptake of plant nutrients from soil. Plant Soil

76:77–91

Tu C, Ma LG (2002) Effects of arsenic concentrations and forms on

arsenic uptake by the hyperaccumulator ladder brake. J Environ

Qual 31:641–647

Vainio EJ, Hantula J (2000) Direct analysis of wood-inhabiting fungi

using denaturing gradient gel electrophoresis of amplified

ribosomal DNA. Mycol Res 104:927–936

Van der Ent A, Baker AJM, Reeves RD, Pollard AJ, Schat H (2013)

Hyperaccumulators of metal and metalloid trace elements: facts

and fiction. Plant Soil 362:319–334

Van der Merwe CG, Schoonbee HJ, Pretorius J (1990) Observations

on concentrations of the heavy metals zinc, manganese, nickel

and iron in the water, in the sediments and in two aquatic

macrophytes, Typha capensis (Rohrb.) N.E. Br. and Arundodonax L., of a stream affected by goldmine and industrial

effluents. Water SA 16:119–124

Vymazal J, Kropfelova L, Svehla J, Chrastny V, Stıchova J (2009)

Trace elements in Phragmites australis growing in constructed

wetlands for treatment of municipal wastewater. Ecol Eng

35:303–309

Wang H, Jia Y, Wang S, Zhu H, Wu X (2009) Bioavailability of

cadmium adsorbed on various oxides minerals to wetland plant

species Phragmites australis. J Hazard Mater 167:641–646

Weis JS, Weis P (2004) Metal uptake, transport and release by

wetland plants: implications for phytoremediation and restora-

tion. Environ Int 30:685–700

Weis JS, Windham L, Weis P (2003) Patterns of metal accumulation

in leaves of the tidal marsh plants Spartina alterniflora Loisel

and Phragmites australis Cav. Trin Ex Steud. over the growing

season. Wetlands 23:459–465

Welsh RPH, Denny P (1980) The uptake of lead and copper by

submerged aquatic macrophytes in two English lakes. J Ecol

68:443–455

Wu CH, Wood TK, Mulchandani A, Chen W (2006) Engineering

plant-microbe symbiosis for rhizoremediation of heavy metals.

Appl Environ Microbiol 72:1129–1134

Yang YH, Yao J, Hu S, Qi Y (2000) Effects of agricultural chemicals

on dna sequence diversity of soil microbial community: a study

with RAPD marker. Microbiol Ecol 39:72–79

Yang J, Kloepper JW, Ryu CM (2009) Rhizosphere bacteria help

plants tolerate abiotic stress. Trends Plant Sci 14:1–4

Ye ZH, Whiting SN, Lin ZQ, Lytle CM, Qian JH, Terry N (2001)

Removal and distribution of iron, manganese, cobalt and nickel

within a Pennsylvania constructed wetland treating coal com-

bustion by-product leachate. J Environ Qual 30:1464–1473

Zabłudowska E, Kowalska J, Jedynak Ł, Wojas S, Skłodowska A,

Antosiewicz DM (2009) Search for a plant for phytoremedia-

tion—what can we learn from field and hydroponic studies?

Chemosphere 77:301–307

618 J. Wu et al.

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