Chemosphere 85 (2011) 11301138

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Chemosphere
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Analysis and characterization of cultivable heavy metal-resistant bacterial

endophytes isolated from Cd-hyperaccumulator Solanum nigrum L. and their
potential use for phytoremediation
Sheng-lian Luo a,b,c,d,, Liang Chen a, Jue-liang Chen b,c, Xiao Xiao b,c, Tao-ying Xu b,c, Yong Wan b,c,
Chan Rao b,c, Cheng-bin Liu a, Yu-tang Liu a, Cui Lai b,c, Guang-ming Zeng b,c
a

State Key Laboratory of Chemo/Biosensing and Chemometrics, Hunan University, Changsha 410082, Peoples Republic of China
College of Environmental Science and Engineering, Hunan University, Changsha 410082, Peoples Republic of China
c
Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Ministry of Education, Changsha 410082, Peoples Republic of China
d
Key Laboratory of Jiangxi Province for Ecological Diagnosis-Remediation and Pollution Control, Nanchang Hangkong University, Nanchang 330063, Peoples Republic of China
b

a r t i c l e

i n f o

Article history:
Received 24 February 2011
Received in revised form 24 July 2011
Accepted 25 July 2011
Available online 24 August 2011
Keywords:
Heavy metal-resistant bacterial endophytes
Solanum nigrum L.
Phylogenetic analysis
Phytoremediation

a b s t r a c t
This study investigates the heavy metal-resistant bacterial endophytes of Cd-hyperaccumulator Solanum
nigrum L. grown on a mine tailing by using cultivation-dependent technique. Thirty Cd-tolerant bacterial
endophytes were isolated from roots, stems, and leaves of S. nigrum L. and classied by amplied ribosomal DNA-restriction analysis into 18 different types. Phylogenetic analysis based on 16S rDNA
sequences showed that these isolates belonged to four groups: Actinobacteria (43%), Proteobacteria
(23%), Bacteroidetes (27%) and Firmicutes (7%). All the isolates were then characterized for their plant
growth promoting traits as well as their resistances to different heavy metals; and the actual plant
growth promotion and colonization ability were also assessed. Four isolates were re-introduced into S.
nigrum L. under Cd stress and resulted in Cd phytotoxicity decrease, as dry weights of roots increased
from 55% to 143% and dry weights of above-ground from 64% to 100% compared to the uninoculated
ones. The total Cd accumulation of inoculated plants increased from 66% to 135% (roots) and from 22%
to 64% (above-ground) compared to the uninoculated ones. Our research suggests that bacterial endophytes are a most promising resource and may be the excellent candidates of bio-inoculants for enhancing the phytoremediation efciency.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Bacterial endophytes are dened as those bacteria that colonize
the inner parts of their host plants without causing disease symptoms and can be isolated from surface-disinfected plants or extracted from within plant (Hallmann et al., 1997; Schulz and
Boyle, 2006). They have been isolated from both monocotyledonous and dicotyledonous plants, ranging from woody tree species
to herbaceous crop plants, suggesting a ubiquitous existence in
nearly all higher plants. On the other hand, the composition of bacterial endophytes in various plants was different, with many
strains closely related to common soil bacteria representative of
genera such as Enterobacter, Pseudomonas, Bacillus, Arthrobacter,
Burkholderia and Methylobacterium (Lodewyckx et al., 2002). Bacterial endophytes are widely applied in the area of sustainable
Corresponding author at: State Key Laboratory of Chemo/Biosensing and
Chemometrics, Hunan University, Changsha 410082, Peoples Republic of China.
Tel./fax: +86 731 88823805.
E-mail address: sllou@hnu.cn (S.-l. Luo).
0045-6535/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.chemosphere.2011.07.053

agriculture. Numerous reports have shown that bacterial endophytes can promote their host establishment as well as hasten
plant development under adverse conditions (Bent and Chanway,
1998; Sturz et al., 2000). Moreover, they have the capacity to control plant pathogens, insects and nematodes, which make them
suitable as bio-control agents (Berg and Hallmann, 2006). Recent
research suggests that benecial bacterial endophytes may also
play an important role in remediation of contaminated soils and
water (Mastretta et al., 2006; Guo et al., 2010; Xiao et al., 2010).
Release of heavy metals from various industrial sources, agrochemicals and sewage sludge presents a major threat to the soil
environment. Conventional remediation methods such as soil
excavation followed by coagulation-ltration or ion exchange are
expensive and disruptive to the sites. In situ phytoremediation is
a low-cost and effective method, but this technique is timeconsuming (Lebeau et al., 2008). Therefore, the development of
phytoremediation strategies for heavy metal contaminated soils
is necessary. Symbiosis between plants and microbes in the rhizosphere has long been studied by microbial ecologists (Kuiper et al.,
2004). Colonizing the internal tissues of plants, endophytes are

S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

likely to interact more closely with their host compared with rhizosphere and phyllosphere microbes (Weyens et al., 2009). For
studying the composition of bacterial communities living on a
niche of heavy metals stress and possible biotechnological application in bioremediation, the interactions between endophytes and
hyperaccumulator have been attracted much attention (Idris
et al., 2004; Barzanti et al., 2007; Mengoni et al., 2009; Luo et al.,
2010). Bacterial endophytes have been isolated from many different plant species and they may promote plant growth or confer
higher tolerance to plant grown in heavy metal contaminated soil
(Lodewyckx et al., 2001; Madhaiyan et al., 2007; Sheng et al., 2008;
Chen et al., 2010). The mechanisms of bacterial endophytes benecial to their host plant may include the production of phytohormones, enzymes involved in growth regulator metabolism, such
as ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, indole-3-acetic acid (IAA) and production of a siderophore
(Glick et al., 1998; Hardoim et al., 2008; Rajkumar et al., 2010).
Moreover, they can also improve plant growth via the xation of
nitrogen (Triplett, 1996) and enhance phosphate availability during initial colonization (Kuklinsky-Sobral et al., 2004).
Therefore, searching for new and benecial endophytic microorganisms among the different hyperaccumulating plants in metalpolluted environment then becomes meaningful. Furthermore, an
analysis of genotypic and phenotypic characteristics of endophytes
may help to clarify the mechanism related to plant-endophyte
interaction. Consequently, the objectives of this study are to (a)
analyze the diversity of the cultivable metal-resistant bacterial
endophytes isolated from Cd-hyperaccumulator plant Solanum nigrum L. in heavy metal mine tailings, (b) characterize these cultivable endophytic bacterial isolates, and (c) select the plant growthpromoting (PGP) endophytes which might be useful for increasing
plant biomass production and Cd uptake of S. nigrum L. to accelerate
the efciency of phytoremediation of Cd-polluted soils.

2. Materials and methods

2.1. Isolation of Cd-resistant endophytic bacteria
The Cd-hyperaccumulator of S. nigrum L. was obtained from
Shuikoushan Mine tailings located in Hengyang, Hunan province,
China. Roots, stems and leaves from plant were separated and
washed extensively in order to remove any nonspecically bound
metals and dried to constant weight. Dried plant tissues were
ground and then digested in a mixture of concentrated HNO3 and
HClO4 (4:1, v/v). Rhizoshpere soil was also dried, crushed and
sieved for metal and physicochemical analysis. Metal concentrations of all samples were determined using ame atomic absorption spectrometer (Z-2000, Hitachi, Japan).
Endophytic bacteria were isolated from surface-sterilized tissues of S. nigrum L. The protocol of surface-sterilized is as follows:
rst immersion in 70% ethanol for 40 s, then 2.5% sodium hypochlorite for 30 min, and nally 3 rinses in sterile distilled water. To conrm that the surface disinfection process was successful, 100 lL of
the last washing water were plated onto solid tryptone soya broth
(TSB) agar medium, and then the TSB plate was incubated at 28 C
for 3 d. The leaf, stem and root tissue were then macerated respectively using a sterile mortar and pestle in a small volume of sterile
phosphate buffered saline (PBS, pH 7.4), with sterile quartz sand
being added to improve the wall disruption. Samples (100 lL) of
tissue extracts and their different dilutions were plated onto TSB
agar medium supplemented with 0.5 mM of CdCl2. After incubation
at 28 C for 4 d, colonies of varying morphology were picked and
repeatedly re-streaked on TSB agar medium until the colony morphology of each isolate reached homogenous. Thirty Cd-resistant
isolates were nally selected and stored on slants for further study.

1131

2.2. DNA extraction, PCR amplication and amplied ribosomal DNA

restriction analysis (ARDRA)
DNA was extracted from each bacterial isolate after cultured
20 h in Luria-Berani (LB) broth with the protocol described by
Arajo et al. (2002). Amplication of 16S rDNA was performed in
a total volume of 50 lL containing 1.5 mM MgCl2, 5 lL of 10
reaction buffer, 200 lM of each dNTPs, 10 pmol of each primer
[27f, 50 -GAGAGTTTGATCCTGGCTCAG, and 1495r, 50 -CTACGGCTACCTTGTTACGA], 1 lL of template DNA, 2 U of Taq DNA polymerase. PCR reaction condition was initial denaturing step of 5 min at
94 C followed by 30 cycles of 50 s at 94 C, 50 s of annealing at
52 C, and a 65 s extension at 72 C, and a nal polymerization step
of 72 C for 10 min. PCR products were examined electrophoretically in a 1% (w/v) agarose gel. The length of the amplicon was
about 1400 bp.
After the amplication of 16S rDNA fragments, 10 lL of the PCR
products were digested with the restriction endonuclease MspI
(10 U) for 12 h at 37 C with buffer supplied. The restriction fragments were resolved by horizontal gel electrophoresis of 10 lL of
each restriction mixture at 7 V cm1 for 3 h in 2.5% (w/v) agarose
in 0.5 TBE (Tris/Borate/EDTA) buffer, pH 8.0. After which the gel
was soaked for 1 h in GelRed nucleic acid stain (1:3300 dilution,
biotium, American) and immediately photographed under UV
light. ARDRA patterns were used in cluster analysis with the unweighted pair grouping with mathematic method in the MVSP3.1
software package.
2.3. Sequencing and phylogenetic analysis
Puried PCR products of one isolate representative of each
ARDRA group were selected for 16S rDNA sequencing (Invitrogen,
Shanghai). Partial 16S rDNA sequences obtained were matched
against nucleotide sequences present in GenBank using the BLAST
tool in the NCBI website. The multiple sequence alignment program Clustal x1.8 was used to align the sequences retrieved from
databases. Neighbor joining phylogenetic trees were constructed
after calculation of a Maximum Composite Likelihood distance matrix with the software MEGA4.0 (Tamura et al., 2007). The robustness of the phylogeny was evaluated by bootstrap analysis with
1000 replicates. The sequences used for identication of the cultivable endophytes are available in the GenBank database under
accession numbers HQ331124 through HQ331141.
2.4. Heavy metal resistance of the isolates
The level of heavy metals tolerance was tested on Tris-buffered
low-phosphate (TLP) medium (Madhaiyan et al., 2007). Isolates
were streaked on TLP agar plates containing Zn, Cd, Pb and Cu
respectively, in different concentrations. Metals used were Zn as
ZnSO4, Cd as CdCl2, Pb as PbNO3 and Cu as CuSO4. For each isolates
and each metal the lowest concentration that inhibited visible
growth at 28 C within 5 d was determined.
2.5. Screening for PGP properties
Metal resistant isolates were tested for their ability to grow on
ACC as the sole N source, to produce siderophores and IAA, and to
solubilize phosphate. For the ACC deaminase activity detected, the
isolates were cultured rst in LB medium and then transferred into
tubes with Dworkin and Foster (DF) mineral medium (Dworkin
and Foster, 1958) containing 3.0 mM ACC instead of (NH4)2SO4 as
N source (Penrose and Glick, 2003). Solution of ACC (0.5 M) was lter through a 0.22-lm pore size membrane and store at 20 C.
Prior to inoculation, the ACC solution was thawn and appropriately
added to sterile DF medium. The DF medium without any N source

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S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

was used as control. Following inoculation with the appropriate

stain, the cultures were incubated at 28 C for 48 h. Growth was
positive when the cultures developed turbidity. The production
of IAA by strains was determined according to the methods of
Sheng et al. (2008). Briey, 1 mL cell suspension obtained from
bacterial culture grown in sucrose-minimal salts (SMS) medium
with L-tryptophan (0.5 mg mL1) was transferred into a tube and
mixed vigorously with 2 mL of Salkowski0 s reagent (Gordon and
Weber, 1951). A pink color developed after 20 min incubation at
room temperature, and the absorbance was obtained at 530 nm.
The IAA concentration was determined using a calibration curve
of pure IAA as a standard following the linear regression analysis.
The production of siderophores by strains was determined according to the chrome azurol-S (CAS) analytical method (Schwyn and
Neilands, 1987). 1.0 mL of cell free culture supernatant obtained
from bacterial culture grown in King B medium (King et al.,
1954) without phosphate was mixed with 1.0 mL of CAS assay
solution. The non-inoculated supernatant used as reference. After
3 h of incubation in the dark at room temperature, the decrease
in absorbance at 630 nm was recorded and the values were compared with the OD630 of the reference (k/k0). The phosphate solubilizing activity of the isolates was analyzed in NBRIP medium
(Nautiyal, 1999) amended with tricalcium phosphate. After cultured at 30 C for 192 h at 150 rpm, the solubilized phosphate in
the culture supernatant was quantied by Mo-blue method
(Watanabe and Olsen, 1965).
2.6. Inoculation of S. nigrum L. with endophytic bacteria and analysis
of seedling growth
Isolates selected for inoculation were grown overnight in
100 mL LB medium at 30 C on a rotary shaker. Cells were collected
by centrifugation, washed twice with PBS, and suspended in biological saline (0.85% KCl) to obtain the nal inoculum density of
108 CFU mL1.
Seeds of S. nigrum L. were surface-sterilized with sodium hypochlorite solution (1% available Cl and 1 droplet Tween 80 per
100 mL solution) for 30 min and washed with sterile water. Seed
sterility was veried by incubating several seeds on LB agar plate
at 30 C for 4 d. Seeds were considered sterile when no bacterial
grown was observed. About 20 seeds were soaked in the bacterial
suspension or sterile water for 2 h, and then were sowed in a sterile
pot in which lled with sterilized vermiculite and saturated with a
half-strength sterile Hoaglands nutrient solution (Barac et al.,
2004). After the seeds had germinated, plantlets were allowed to
grow for 20 d in a growth chamber with constant temperature of
25 C, relative humidity of 65%, and a 16/8 h photoperiod. Five
plantlets were harvested and growth parameters such as root
length, shoot length and fresh weight of each plantlet were
measured. Subsequently, fresh whole plant tissues were surfacesterilized for 5 min in a solution containing 1% active chloride supplemented with 1 droplet Tween 80 per 100 mL solution and rinsed
three times in sterile water. A 100 lL sample of the third rinsing
water was plated on TSB agar plate to verify the efciency of sterilization. After sterilization, the plantlets were macerated in a sterilized mortar, diluted, and plated as described above. The total
number of bacterial colonies was counted after incubation for 3 d
at 30 C. The identity of re-isolates was checked by morphological
characteristics and 16S rDNA sequencing.
2.7. Plant growth and Cd uptake of S. nigrum L. by PGP endophytes
Based on the PGP traits, the strains LKR01, LSE02, LSE03 and
LSE04 were selected for exploring the effects of plant-endophyte
partnership in heavy metal remediation. Consequently, plantlets
inoculated with these bacterial strains and uninoculated control

were allowed to continue grow (each pot contained ve plantlets

and three replicates were used for each treatment). The plantlets
were watered with 1/4 Hoaglands nutrient solution every 3 d
and 40 lmol CdCl2 were added as solution. After 35 d, plants were
carefully removed from the pots, the roots and shoots were separated, roots were immersed in 10 mM EDTA for 30 min, and then
rinsed thoroughly with deionized water to remove surface adsorbed metal. Subsequently, plant tissues were oven-dried at
80 C to constant weight for determining dry weight. The accumulation of total Cd in root and shoot tissue of plant were quantied
following the method of DellAmico et al. (2008).
2.8. Statistical analysis
Statistical analysis was done with SPSS 11.0. ANOVA followed
by post hoc Fisher Least Signicant Difference (LSD) test was used
to compare treatment means. All analyses were performed at the
p 6 0.05 level.
3. Results
3.1. Isolation of endophytic bacteria from S. nigrum L
In Table 1, heavy metal concentrations in rhizosphere soil and in
S. nigrum L. tissues collected from the mine waste tailings were
reported. The concentrations of Pb (4410 mg kg1) and Zn
(3310 mg kg1) in the soil were especially high, and concentration
of Cd (32 mg kg1) also overcame the soil baseline. Moreover, the
metal concentrations in leaves and stem were higher than those in
roots. Based on morphological characteristics, a total of 30 heavy
metal-resistant cultivable endophytic bacteria were isolated from
plant tissues. Bacterial titers and the number of isolates in each tissue are also shown in Table 1. The highest bacterial titers were found
in the roots and decreased progressively from stem to leaves.
3.2. Analysis of genotypic of cultivable endophytes bacterial isolates
After digestion of amplied 16S rDNA with MspI restriction enzyme, the 30 isolates were classied into 18 different ARDRA types.
As shown in Fig. 1, the distribution of ARDRA types in the isolates
from the different plant portions was reported. Seven ARDRA types
were exclusively recovered from roots, while 4 (IX, XI, XII, XIII) and
3 (XVI, XVII, XVIII) were recovered only from leaves and stems,
respectively. Partial sequencing of 16S rDNA of isolates belonging
to the 18 ARDRA types allowed us to phylogenetically assign the
ARDRA types to distinct bacterial taxonomic groups (Fig. 2). Among
the 30 Cd-resistant isolates, 6 could be ascribed to the Microbacterium, 2 to Bacillus, 3 to Arthrobacter, 5 to Flavobacterium, 3 to
Chryseobacterium, 1 to Agrobacterium, 1 to Sphingomonas, 2 to Pseudomonas, 3 to Serratia, and 4 to Curtobacterium.
3.3. Heavy metal-resistance and PGP properties
Most of the tested isolates were resistant to various metals
simultaneously (Table 2). Among all strains, 60% were tolerant to
1 mM Cd. Some isolates were able to resist to 5 mM Zn and
5 mM Pb. Moreover, most of them could tolerate to 2 mM Cu.
Bacterial endophytes with PGP properties may play an important role on plant growth in the stressed environment. Table 2
showed that almost all strains had at least one PGP trait except
LKR02 and LKR09. Two strains, LKR01 and LSE06 had all four traits.
A majority of strains (70%) had the capacity to produce IAA at levels of 1.6122 mg L1. About 50% and 56% of the tested isolates had
the ability to produce siderophores and to grow on ACC, respectively. In particular, strains LSE06 had the highest siderophores

S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

1133

Table 1
Metal concentration and number of endophytic bacteria in S. nigrum L. plant tissue and the properties of rhizoshpere soil.
Items

Roots

Stems

Leaves

Rhizoshpere soil

56 0.1
43 0.1
16 0.3
17 0.1
80 0.2
3.0 0.2  105
11

118 0.1
185 3.5
6.6 0.0
55 1.2
265 3.7
2.8 0.4  104
9

142 0.5
164 4.4
6.8 0.4
36 0.4
278 3.2
7.0 0.3  102
10

32 0.7
3340 6.8
1962 4.2
4410 8.2
2052 1.4

Total metal concentration

Cadmium (Cd)
Zinc (Zn)
Copper (Cu)
Lead (Pb)
Manganese (Mn)
Total bacteriab
No. of isolated bacteriac
a
b
c

Physicochemical properties/value
pH (1:2.5 w/v water)/6.6
Organic matter/3.2%
Total phosphor/952 mg kg1
Total nitrogen/2.2 g kg1
Dry mass/88.7%
Cation exchange capacity/3.2 cmol kg1

Metal concentrations are expressed in mg kg1 dry weight standard deviation.

Bacterial number are expressed in CFU g1 plant fresh weight.
Number of bacteria selected for further characterizations.

Fig. 1. Distribution of 30 different bacteria isolates among 18 ARDRA types in the different plant portions. Serratia: I, XV; Arthrobacter: II, III, IX; Microbacterium: IV, VI, XIII,
XVI; Agrobacterium: V; Bacillus: VII; Sphingomonas: VIII; Flavobacterium: X; Chryseobacterium: XI, XVII, XVIII; Pseudomonas: XII; Curtobacterium: XIV.

production among all the isolates. In addition, only eight strains

were able to solubilize phosphate (Table 2).
3.4. Seeds germination and colonization ability
Eighteen representative isolates from diverse ARDRA type infected sterile S. nigrum L. seeds, and results showed that bacterial
inoculation had no obvious inhibition or stimulation effects on
seeds germination. After 20 d of seed growth, the growth parameters such as fresh weight, root length and shoot length of the seedlings were measured. As shown in Table 3, seeds inoculated with
different isolates were all increased in root length compared with
non-inoculated seeds. The maximum effect on root elongation
was found for LKR08, which increased the root length by 88%.
However, the shoot length and fresh weight were not always increased as the increase of root length. For instance, the shoot
length and fresh weight of the seeds inoculated with LKL04 were
lower than the control, with a 39% reduction in shoot length and
11% reduction in fresh weight. About 61% and 66% of the total inoculants had positive effects on shoot length and fresh weight,
respectively.
Seedlings were surface sterilized after 20 d of growth and endophytic bacteria were recovered. Bacterial titers (CFU g1 fresh

weight) in interior tissue of seedling are reported in Table 3. Plant

colonization ability of the isolates was different. Most of the tested
bacteria were successfully recovered. However, no colonies were
recovered after infection of LKR07, LKR09, LKS07, LKL04 and LKL06.
3.5. Improve plant biomass production and Cd-uptake of S. nigrum L.
The effects of the selected bacterial isolates on plant biomass,
Cd-uptake, bio-concentration factor (BCF) and translocation factor
(TF) of S. nigrum L. are shown in Fig. 3. The BCF is dened as the ratio between the metal concentrations in the above-ground and the
metal concentration in the soils. The TF is dened as the ratio between the metal concentrations in the above-ground and the metal
concentration in the roots. All the tested strains were able to significant increase (p 6 0.05) the plant biomass and total Cd uptake of S.
nigrum L. Compared to the uninoculated control, roots and shoots
dry weights of bacterial inoculated plants increased from 55% to
143% and from 64% to 100%, respectively. As the plant biomass increased, the total Cd-uptake per pot plant signicantly increased as
well. For bacterial inoculated plants, roots and shoots Cd-uptake
were increased from 66% to 135% and from 22% to 64% compared
to the uninoculated control, respectively. Furthermore, the BCF
and TF of plants inoculated by bacteria were also increased com-

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S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

Serratia sp. [GU120657]

LKR01 [HQ331124]
Serratia marcescens [EF114344]
95
88
LKR09 [HQ331131]
Pseudomonas oryzihabitans [GQ250598]
66
100
LKS06 [HQ331135]
99 Sphingomonas sp. [AB495350]
LKS09 [HQ331138]
96
Agrobacterium tumefaciens [GQ140317]
99
LKR05 [HQ331128]
Flavobacterium sp. [AY230767]
LKL06 [HQ331140]
78
100
41 LKS03 [HQ331133]
42 Chryseobacterium sp. [DQ981464]
LKS04 [HQ331134]
78
LKL10 [HQ331141]
Bacillus subtilis [EU373393]
99
LKR08 [HQ331130]
84 LKS08 [HQ331137]
55
83 99 Microbacterium foliorum [EU714341]
93 LKR04 [HQ331127]
Microbacterium hydrocarbonoxydans [AJ698726]
99 LKR07 [HQ331129]
100 Microbacterium oleivorans [AJ698725]
100 99 LKS07 [HQ331136]
LKL04 [HQ331139]
Arthrobacter sp. [EF550164]
LKR02 [HQ331125]
LKR03 [HQ331126]
100
LKS02 [HQ331132]
Arthrobacter oxydans [EU086782]
Curtobacterium sp. [AM396911]
100

Proteobacteria

Bacteroidetes

Firmicutes

Actinobacteria

0.1
Fig. 2. Neighbor-joining phylogenetic tree of partial 16S rDNA sequences of 18 representative isolates. Scale bars represent the Maximum Composite Likelihood distance.
Bootstrap values are indicated at the node.

pared with the uninoculated control (Fig. 3). It is suggested that

more of Cd was stored in the above-ground tissues.

4. Discussion
In the present study we used cultivation-based methods to analyze the genotypic and phenotypic diversity of the heavy metalresistant bacterial endophytes of Cd-hyperaccumulator S. nigrum
L. grown on a mine tailings. The mine was rich in heavy non-ferrous
metals, which have been exploited for 100 years. Meanwhile, it has
been leading to serious heavy metal contamination for the soil
around. Heavy metal pollution cannot only cause adverse effects
on various parameters relating to plant quality and yield but also
bring about changes in the size, composition, and activity of plantassociated microbial community (Giller et al., 1998). Although only
30 Cd-resistant isolates were obtained from the plants, the metalresistant bacterial endophytes were diversiform. The total isolates
were classied by ARDRA, and 18 strains representing different
types were subject to 16S rDNA sequence phylogenetic analysis
and found to be afliate with 10 different genera (Fig. 2). Some genera have already been reported to be bacterial endophytes. Barzanti
et al. (2007) isolated 83 bacterial endophytes from roots, stems, and
leaves of a Ni-hyperaccumulator Alyssum bertolonii and classied by
ARDRA and partial 16S rDNA sequencing in 23 different taxonomic
groups. In particular, the genera represented were Bacillus, Microbacterium, Leifsonia, Curtobacterium, Paenibacillus, and Staphyloccoccus. Idris et al. (2004) also reported that a high number of different
divisions, genera, and species were colonized Ni-hyperaccumulator
Thlaspi goesingense. Among the 62 endophytic isolates, the dominant
species were Methylobacterium (42%) and Sphingomonas (37%), and

remaining endophytes showed high homology to the genera Rhodococcus, Curtobacterium, and Plantibacter. Moreover, these genera
identied in our studies or other study were commonly found in soil
bacteria suggested that majority of the endophytic bacteria may origin from soil bacteria through root colonization. The highest bacterial densities are commonly observed in root tissues, which also
explained the close relationship between endophytic bacteria and
soil bacteria.
Hyperaccumulator accumulated high concentration of heavy
metals which provided a specic environment (the constant metal
stress) for bacterial endophytes (Idris et al., 2004). Numerous studies have demonstrated that the bacteria isolated from polluted
environment are tolerance to higher concentrations of metals than
those isolated from unpolluted areas. In our study, the result of
heavy metal resistance analysis of the isolates conrmed that heavy metal stress had an acclimation role on microorganisms survive
in the adverse environment. Sun et al. (2009) also reported that the
proportion of copper-resistant-endophytic bacteria in Commelina
coommunis was higher than that of in Elsholtzia splendens, which
may be due to the higher copper concentration in C. coommunis tissues. It is widely believed that endophytes are ltered by plants
from soil microbe, and plants may select those specically attractive microorganisms for their own ecological and evolutionary
benet (Hardoim et al., 2008). These bacterial endophytes were detected for some properties which were thought to be important for
PGP activities (Table 2). Benecial bacteria can accelerate seedling
emergence, promote plant root elongation particularly under adverse conditions (DellAmico et al., 2008). To investigate the actual
role of the bacterial isolates in plant growth and health, re-infection experiments was conducted in laboratory condition. The effects of inoculants on seedling depended on various factors due

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S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

Table 2
Characteristics of metal-resistant plant growth promotion of bacterial endophytes.
Endophytic
bacteria

ARDRA types

IAA synthesis
(mg L1)

Siderophore
(k/k0)a

ACC deaminae

Phosphate
solubilization (mg L1)b

Metal tolerance (mM)c

Cd

Zn

Pb

Cu

Root
LKR01
LKR02
LKR03
LKR04
LKR05
LKR06
LKR07
LKR08
LKR09
LKR10
LKR11

I
II
III
IV
V
XIV
VI
VII
VIII
XII
IV

49.5 0.3

8.8 0.1
18.3 0.2
91.5 2.4

0.6 0.2
13.8 0.3

4.6 0.2
15.2 0.2

0.79

0.84

0.36
0.88

0.53

+
+

+
+

400 5

64 2
22 3

2
0.5
0.5
1
1
0.5
1
0.5
0.5
1
0.5

5
3
3
3
3
3
1.5
2
3
3
2

2
5
1.5
2
2
1
2
2
2
1
1.5

2
2
2
2
2
1.5
2
0.5
2
1.5
2

Stem
LKS01
LKS02
LKS03
LKS04
LKS05
LKS06
LKS07
LKS08
LKS09

XIII
IX
X
XI
X
XII
XIII
XIV
XV

1.6 0.2

34.9 0.2
4.8 0.1

15.1 0.1
12.4 0.3
34.2 0.5
122 0.1

0.81

0.89
0.75
0.06

+
+
+

+
+

28 1

392 2

0.5
1.5
2
1.5
2
0.5
0.5
0.5
1

1
3
5
5
2.5
2.5
1.5
5
3

1
5
5
2
1
2
2
2
2

2
5
1
2
2
2
2
1.5
2

Leaf
LKL01
LKL02
LKL03
LKL04
LKL05
LKL06
LKL07
LKL08
LKL09
LKL10

X
VII
X
XVI
XV
XVII
X
XIV
XIV
XVIII

21.4 1.2
11.3 0.6

30.6 0.9

3.5 0.3
5.6 0.3

4.7 0.4

0.85

0.81
0.86
0.93

0.75
0.87

+
+

+
+

33 2

255 4

100 3

0.5
1.5
1
0.5
1.5
1.5
1
0.5
1
1

2
1
2
1.5
1
5
3
2
1
2

1
1.5
2
2
1
1
1.5
1.5
2
1

1.5
1
2
1
2
2
1
1.5
2
2

, Standard deviation; +, positive; , negative.

a
Siderophore production: little, 0.81.0; low, 0.60.8; moderate, 0.40.6; high, 0.20.4; very high, 00.2.
Concentration of phosphorus.
c
Indicates the strain can tolerant the indicated metal concentration contained in TLP plates after culture 4 d.

to the complex external environment. As shown in Table 3, no clear

connection was found between PGP traits in vitro and promoting
activities of seedling growth. Pearson correlation analysis revealed
that single PGP trait did not signicantly affect the growth parameters such as root length, shoot length and fresh weight (data not
shown). Moreover, the IAA synthesis is negative correlation with
growth parameters. Xie et al. (1996) indicated that a low level of
IAA promotes primary root elongation whereas a high level of
IAA stimulates lateral and adventitious root formation but inhibits
the primary root growth. However, the data show that in most
cases there is signicant growth promotion when both IAA and
ACC deaminase are present. For example, in the case of almost
the same IAA level, the plant growth promotion of LKR08 was better than that of LKR03, and LKS03 was better than LSE08. Although
bacteria synthetic IAA can stimulate plant cell proliferation, plant
cell elongation may induce the transcription of ACC synthase
which is the enzyme that catalyzes the formation of ACC. In this
case, bacteria with ACC deaminase activity can act as a sink for
plant ACC and as a result, lower either the endogenous or
IAA-stimulated ACC level, and then reduce the amount of ethylene
and facilitate the plant growth at last (Glick et al., 2007). Bacterial
siderophore may also stimulate the growth of plant because it can
improve the iron translocation from roots to shoots in the seedlings. Tripathi et al. (2005) reported that Phaseolus vulgaris inoculated with siderophore producing strain KNP9 signicantly
increased plant growth compared with the controls. This effect
was attributed to the increased level of siderophores produced

by the KNP9 that were able to provide iron to the plant. However,
siderophore productions of the isolates in this study were commonly low which may plays less important role in the process of
promoting plant growth. The presence of more than one PGP traits
in these endophytic isolates can facilitate plant growth by utilizing
one or more of those mechanisms at various times during their life
cycle. The endophytes used in growth promotion or plant-assisted
remediation should not only possess various PGP traits but also require excellent re-colonization ability (Rajkumar et al., 2009). The
majority of inoculants were successfully recovered with the protocol of endophytes isolation, which indicated the efcient colonization of these inoculants. In addition, the method of bacterial
endophytes introduced to the host plant by infusion of bacterial
suspension into imbibed seeds was turned out to be successful
(Table 3). However, three fourths of strains belong to Microbacterium sp. could not be recovered from the seedlings suggested that
this genus may be not suitable for this method of inoculation.
Under conditions of metal stress, most of the commonly known
hyperaccumulators have a slow growth rates and low biomass
which will limit the efciency of heavy metal phytoremediation
(Raskin et al., 1997). In addition, the low availability of heavy metals in soils was another main constraint for phytoremediation. Besides PGP potential, metal-resistant PGP bacteria inoculated to
seeds can affect the metal mobility and availability to the plant,
through acidication, chelation, immobilization, and oxidation
reduction reactions in the rhizosphere (Zhuang et al., 2007). Several studies have evidenced that bacterial endophytes equipped

1136

S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

Table 3
Growth parameters of S. nigrum L. seedlings after infection of bacterial endophytes in sterile culture.
Strain

Root length (cm)

Shoot length (cm)

Fresh weight (mg)a

Vigor indexb

Bacterial titers

Uninoculated control
Serratia marcescens LKR01
Arthrobacter oxydans LKR02
Arthrobacter sp. LKR03
Microbacterium foliorum LKR04
Agrobacterium tumefaciens LKR05
Microbacterium hydrocarbonoxydans LKR07
Bacillus sp. LKR08
Serratia sp. LKR09
Arthrobacter sp. LKS02
Flavobacterium sp. LKS03
Chryseobacterium sp. LKS04
Pseudomonas oryzihabitants LKS06
Microbacterium sp. LKS07
Curtobacterium sp. LKS08
Sphingomonas sp. LKS09
Microbacterium sp. LKL04
Chryseobacterium sp. LKL06
Chryseobacterium sp. LKL10

2.2 0.2a
3.3 0.5b
2.8 0.2ab
3.0 0.3ab
3.4 0.4b
2.5 0.6a
3.3 0.3b
4.2 0.2c
2.8 0.4ab
3.0 0.2ab
3.5 0.2b
2.9 0.2ab
3.2 0.8ab
3.2 0.5ab
3.0 0.2ab
2.9 0.6ab
2.7 0.5a
3.4 0.6b
3.7 0.6b

4.1 0.3d
5.3 0.5 g
4.7 0.3ef
4.2 0.4de
4.1 0.3de
3.4 0.3bc
4.2 0.5de
4.7 0.2ef
3.7 0.5c
4.5 0.1ef
5.0 0.2f
4.9 0.4f
4.4 0.2e
3.3 0.2ab
3.1 0.2ab
3.5 0.4bc
2.9 0.2a
3.9 0.2 cd
4.9 0.3ef

48.9 3.8bc
67.0 6.5e
63.2 9.5d
49.1 5.8bc
49.9 8.7bc
49.9 3.7bc
47.0 6.8bc
64.6 4.8e
60.1 4.5d
56.7 9.0d
70.2 4.5e
63.3 5.6d
54.1 6.4 cd
45.4 5.3b
38.9 7.5ab
35.9 7.2a
43.9 8.0b
46.7 3.2bc
78.5 8.9f

5.7b
7.7de
7.2d
6.5c
6.1b
5.0a
6.4c
8.0e
6.2bc
6.4c
8.5f
7.3d
7.5d
6.1bc
5.2ab
5.8b
4.8a
6.2bc
7.5d

n.d.
1.1 0.2  105
8.5 0.5  105
6.5 0.1  104
5.6 0.3  104
4.4 0.2  104
n.d.
1.4 0.2  105
n.d.
6.2 0.4  104
1.2 0.1  105
1.2 0.3  105
1.1 0.1  105
n.d.
7.7 0.1  104
3.3 0.2  104
n.d.
n.d.
1.1 0.2  105

Average standard deviation from ve samples. Dates of columns indexed by the same letter are not signicantly different between bacterial endophytes treatments
according to Fishers protected LSD test (p 6 0.05).
n.d.: Not detected.
a
Fresh weight of seedling.
b
Vigor index = germination (%)  seedling length (root length + shoot length).
c
Bacterial titers are in CFU g1 plant tissue.

Fig. 3. The inuence of the tested isolates on plant biomass, total Cd uptake, bioconcentration factor (BCF) and translocation factor (TF) of S. nigrum L. Values followed by the
same letter are not signicantly different according to Fishers protected LSD test (p 6 0.05).

S.-l. Luo et al. / Chemosphere 85 (2011) 11301138

with PGP traits could support heavy metal uptake and reduce metal stress symptoms in plants (Sheng et al., 2008; Sun et al., 2009;
Chen et al., 2010). Study by Mastretta et al. (2009) showed that the
inoculation of Nicotiana tabacum with Cd-resistant endophytic bacteria Sanguibacter sp. increased the Cd concentration in shoot tissue by approximately 3-fold compared with control. In addition,
the plants seem, due to the inoculants, to store more toxic metal
into their aerial parts. In our study, four selected strains with
PGP abilities were used as inoculants to explore the effects of
plant-endophyte symbiosis in heavy metal remediation. The increased biomass and total Cd uptake of the plants compared to
the uninoculated control revealed that the important role of the
benecial endophytes in heavy metal phytoremediation. Furthermore, these inoculants were found to inuence the translocation
and distribution of metal in their host plants that result in increases in BCF and TF which was good for increasing phytoremediation efciency. However, we obtained these results just in
gnotobiotic system and, therefore, these inoculants should be
tested in a eld trial.
5. Conclusions
Our research reported that a variety of heavy metal-resistant bacterial endophytes lived in Cd-hyperaccumulator S. nigrum L. in heavily metal-polluted mine tailing, and Actinobacteria was predominant
among these isolates. Most of isolates showed co-resistance to more
than one heavy metal and multiple PGP traits, and four of which
were successfully used for microbe-assisted metal-contaminated
phytoremediation. More studies should be addressed to the interactive mechanisms between the bacteria and plants. Moreover, a little
information is available on the functional activities or the effects on
plant performance of these uncultivated bacteria. Therefore, the use
of cultivation-independent techniques and metagenomic approaches to analyze the bacterial endophytes is necessary in the
future.
Acknowledgements
This work was nancially supported by a grant from National
Science Fund for Distinguished Young Scholars (No. 50725825),
the Key Program of National Natural Science Foundation of China
(No. 50830301), and the Hunan Provincial Natural Science Foundation of China (No. 10JJ5051).
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