Soil Biology & Biochemistry 37 (2005) 241–250
www.elsevier.com/locate/soilbio
Cadmium-tolerant plant growth-promoting bacteria associated
with the roots of Indian mustard (Brassica juncea L. Czern.)
A.A. Belimova,*, N. Hontzeasb, V.I. Safronovaa, S.V. Demchinskayaa,
G. Piluzzac, S. Bullittac, B.R. Glickb
a
All-Russia Research Institute for Agricultural Microbiology, Podbelskogo Sh., 3, Pushkin-8, 196608 St-Petersburg, Russian Federation
b
Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1
c
ISPAAM-CNR Sezione Pascoli Mediterranei, 07100 Sassari, Italy
Received 2 December 2003; received in revised form 28 June 2004; accepted 7 July 2004
Abstract
Eleven cadmium-tolerant bacterial strains were isolated from the root zone of Indian mustard (Brassica juncea L. Czern.) seedlings grown
in Cd-supplemented soils as well as sewage sludge and mining waste highly contaminated with Cd. The bacteria also showed increased
tolerance to other metals including Zn, Cu, Ni and Co. The isolated strains included Variovorax paradoxus, Rhodococcus sp. and
Flavobacterium sp., and were capable of stimulating root elongation of B. juncea seedlings either in the presence or absence of toxic Cd
concentrations. Some of the strains produced indoles or siderophores, but none possessed C2H2-reduction activity. All the strains, except
Flavobacterium sp. strain 5P-3, contained the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which hydrolyses ACC
(the immediate precursor of plant hormone ethylene) to NH3 and a-ketobutyrate. V. paradoxus utilized ACC as a sole source of N or energy.
A positive correlation between the in vitro ACC deaminase activity of the bacteria and their stimulating effect on root elongation suggested
that utilization of ACC is an important bacterial trait determining root growth promotion. The isolated bacteria offer promise as inoculants to
improve growth of the metal accumulating plant B. juncea in the presence of toxic Cd concentrations and for the development of plantinoculant systems useful for phytoremediation of polluted soils.
q 2004 Elsevier Ltd. All rights reserved.
Keywords: ACC deaminase; Brassica juncea; Cadmium; Ethylene; Flavobacterium sp.; PGPR; Phytoremediation; Rhizosphere; Rhodococcus sp.; Variovorax
paradoxus
1. Introduction
Among heavy metals, which are widespread pollutants of
the surface soil layer, cadmium is one of the most toxic. In
plants, Cd inhibits root and shoot growth, affects nutrient
uptake and homeostasis, and frequently is accumulated by
agriculturally important crops (Sanita di Toppi and
Gabrielli, 1999). Thus, Cd is consumed by animals and
humans with their diet and can cause diseases. Contamination of soil with Cd also negatively affects biodiversity
and the activity of soil microbial communities (McGrath,
1994).
* Corresponding author. Tel.: C7 812 476 1802; fax: C7 812 470 43 62.
E-mail address: belimov@rambler.ru (A.A. Belimov).
0038-0717/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2004.07.033
Phytoremediation, an emerging low-cost and ecologically benign technology for decontamination of soils, is
defined as the process of utilizing plants to absorb,
accumulate and detoxify contaminants in soil through
physical, chemical and biological processes (Cunningham
and Ow, 1996; Saxena et al., 1999; Wenzel et al., 1999).
Phytoremediation helps to prevent landscape destruction
and enhances activity and diversity of soil microorganisms
to maintain healthy ecosystems. Plants suitable for phytoremediation should have a high biomass production with
enhanced metal tolerance and metal uptake potential. Most
of the commonly known heavy metal accumulators belong
to the Brassicaceae family (Kumar et al., 1995). Although
hyperaccumulator plants have exceptionally high metalaccumulating capacity, most of these have a slow growth
rate and often produce limited amounts of biomass.
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A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
An alternative is to use species with a lower metalaccumulating capacity but higher growth rates, such as
Indian mustard (Brassica juncea L. Czern.), which is
considered to be one of the most promising species for
phytoremediation (Kumar et al., 1995; Saxena et al., 1999).
Along with metal toxicity, there are often additional
factors that limit plant growth in contaminated soils
including arid conditions, a lack of soil structure, low
water supply and nutrient deficiency. Therefore, improvement of plant growth under stressed growth conditions is
critical to the optimum performance of phytoremediation of
soils using both metal hyperaccumulator plant species and
metal accumulating crops like Brassica juncea. The bacteria
associated with plant roots may have profound effects on
plant growth and nutrition through a number of mechanisms
such as N2 fixation, production of phytohormones and
siderophores, and transformation of nutrient elements.
Improvement of the interactions between plants and
beneficial rhizosphere microorganisms can enhance
biomass production and tolerance of the plants to
heavy metals, and are considered is be an important
component of phytoremediation technology (Wenzel
et al., 1999; Glick, 2003).
Although many soil bacteria are tolerant to heavy metals
and play important roles in mobilization or immobilization
of heavy metals (Gadd, 1990), only a few attempts have
been made to study the rhizosphere bacteria of metal
accumulating and hyperaccumulating plants and their role
in the tolerance to and uptake of heavy metals by the plants.
A high proportion of metal resistant bacteria persist in the
rhizosphere of the hyperaccumulators Thalaspi caerulescens (Delorme et al., 2001) and Alyssum bertolonii
(Mengoni et al., 2001) or Alyssum murale (Abou-Shanab
et al., 2003a) grown in soil contaminated with Zn and Ni or
Ni, respectively. The presence of rhizosphere bacteria
increased concentrations of Zn (Whiting et al., 2001), Ni
(Abou-Shanab et al., 2003b) and Se (De Souza et al., 1999)
in T. caerulescens, A. murale and B. juncea, respectively.
Inoculation of Indian mustard and canola (Brassica
campestris) seeds with the plant growth-promoting rhizobacteria (PGPR) strain Kluyvera ascorbata SUD165, which
produces siderophores and contains the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, protected
the plants against Ni, Pb and Zn toxicity (Burd et al., 1998).
Inoculation of rape (canola; Brassica napus) with
metal-resistant PGPR containing ACC deaminase stimulated growth of plants cultivated in Cd contaminated soil
(Belimov et al., 2001). In addition, various N2-fixing and
auxin-producing PGPR immobilized Cd and promoted
growth and nutrient uptake by barley plants in the presence
of toxic Cd concentrations (Belimov and Dietz, 2000;
Pishchik et al., 2002).
Our aim was to isolate and characterize Cd-tolerant
bacteria associated with the roots of the metal accumulating
plant Brassica juncea L. Czern. grown in heavy metal
contaminated soils, and to select PGPR strains which might
be useful to increase plant biomass production under
unfavourable environmental conditions. The creation of
such metal tolerant plant-microbe associations is aimed at
improving the efficiency of phytoremediation of heavy
metal polluted soils.
2. Materials and methods
2.1. Characterisation, sampling and treatment of soils
Characteristics of the soils used are described in Table 1.
The soils were classified using the FAO-UNESCO Soil
Taxonomic System and soil samples were conventionally
labelled 1–5. Sod-podzolic soils (samples 1 and 2) were
sampled to a depth of 0–10 cm from agricultural fields in the
Saint-Petersburg region (30837 0 N, 59847 0 E) in June 2001,
supplemented with 50 mg Cd kgK1, as CdCl2 dissolved in
water, immediately after sampling and incubated in
sterile enameled pots in the dark at room temperature
(20 8CG2 8C) for 3 months before use. Soil moisture was
maintained at 60% of water holding capacity via addition of
sterile tap water. Sewage sludge pits (samples 3 and 4),
which had been left to stand for 13 y, were sampled to a
depth of 0–10 cm in the Gatchina sewage treatment works in
the Saint-Petersburg region (29812 0 N, 58833 0 E) in September 2001 and immediately used. Mining waste (sample 5)
was sampled to a depth of 0–10 cm at the Campo Pisano
mine in the Iglesiente area in southwest Sardinia, Italy
(3854 0 N, 39817 0 E) in March 2001 and stored moist in sterile
plastic bags at 4 8C for 6 months before use.
Total C in soil samples 1–4 and 5 was determined as
described by Arinushkina (1970) and by the Walkey-Black
method (Violante, 2000), respectively. The total N content
Table 1
Characteristics of the soils used for isolation of rhizobacteria
Designation and soil
type
1 Sod-podzolic
2 Sod-podzolic
3 Sewage sludge
4 Sewage sludge
5 Mining waste
a
pHKCl
Total content of elements (mg kgK1)
C
4.7
5.2
6.9
6.6
6.9
12,300
24,800
36,100
109,000
18,600
N
1240
1830
2200
85,800
700
P
350
410
360
640
400
K
280
360
1730
1890
1540
Mn
240
340
714
738
2409
Zn
32
28
826
1250
5961
Cu
22
15
662
875
39
Ni
24
23
63
87
30
Cr
32
31
201
540
19
The data are given as Cd concentrations at the time of sampling before addition of 50 mg Cd kgK1, as CdCl2 dissolved in water.
Cd
Pb
a
0.1
0.2a
43
112
36
18
25
84
106
2827
A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
was determined by the Kjehldhal method. The total content
of P, K, Mn, Zn, Cu, Ni, Cr, Cd, and Pb in soil samples 1–4
was determined using an AI1029 X-Ray fluorescence
analyser (BALTIETS, Narva, Estonia) equipped with a
BDRC-1425 detector (RRIEI, Riga, Latvia). The total
content of P and K in soil sample 5 was determined by
Olsen’s method and the Barium Chloride–Ethanolamine
method, respectively (Violante, 2000). The content of heavy
metals in soil sample 5 was determined using a Perkin
Elmer 372 atomic absorption spectrophotometer (USA).
2.2. Isolation of cadmium tolerant rhizobacteria
The bacteria were isolated from the root zone of Indian
mustard (Brassica juncea L. Czern.) variety VIR-3129
cultivated in the above-mentioned soils and sewage sludge.
Seeds of Indian mustard were surface-sterilised by treatment with a mixture of ethanol and 30% H2O2 (1:1) for
20 min. The treated seeds were tested for surface sterility
via incubation on solid Bacto-Pseudomonas F (BPF)
medium (Belimov et al., 2001) for 3 days (d) at 28 8C.
The composition of BPF medium was as follows (g lK1):
peptone, 10; casein hydrolysate, 10; glycerol, 12.5;
K2HPO4, 1.5; MgSO4, 1.5; agar, 15. Twenty aseptic seeds
were sown in each enameled pot containing 2 kg of soil,
sewage sludge or mining waste (1 pot was prepared for each
substrate). The pots were held for 15 d in a growth chamber
at 25 8C, lit by OSRAM L36W/77 FLUORA metal halide
lamps with a 16 h photoperiod and a photosynthetic photon
flux density at the plant surface of 500 mmol mK2 sK1. The
whole roots of 20 seedlings (about 70 mg fresh weight) were
removed from the soils, carefully washed in sterile tap
water, and homogenised in 2 ml of sterile tap water using a
sterile mortar and pestle. To select for the dominant species
of the culturable component of bacterial community, the
root homogenates were diluted 1000-fold with sterile tap
water. Aliquots of diluted homogenates (50 ml) were plated
onto Petri dishes (3 replicates) with 20 ml of solid BPF,
SMN or SMC media supplemented with 400, 200 and
200 mM of CdCl2, respectively. The appropriate Cd
concentrations resulting in complete growth inhibition of
Cd-sensitive bacteria were established in preliminary
experiments (data not shown). To prepare SMN medium,
a salts minimal (SM) medium (containing (lK1): KH2PO4,
0.4 g; K2HPO4, 2 g; MgSO4, 0.2 g; CaCl2, 0.1 g; FeSO4,
5 mg; H3BO3, 2 mg; ZnSO4, 5 mg; Na2MoO4, 1 mg;
MnSO4, 3 mg; CoSO4, 1 mg; CuSO4, 1 mg; NiSO4, 1 mg;
pH 6.4) was supplemented with (g lK1): ACC, 0.5; glucose,
1; sucrose, 1; Na-acetate, 1; Na-citrate, 1; malic acid, 1; and
mannitol, 1. To prepare SMC medium, the SM medium was
supplemented with (g lK1): ACC, 0.5; and NH4NO3, 0.3. In
many instances a-ketobutyrate may act as a precursor of
branched chain amino acids such as leucine, isoleucine and
valine. Thus, we proposed that some bacteria should be
capable of utilizing ACC as an energy source and therefore
tried to isolate such bacteria using selective SMC medium
243
supplemented with ACC as a sole source of carbon. To
prevent the growth of soil fungi, the media were
supplemented with 10 mg cycloheximide lK1 after autoclaving. SMN and SMC media were additionally supplemented with 10 ml lK1 of the stock solution containing
the vitamins (mg lK1): biotin, 2; pyridoxine, 10; thiamine,
2; pantothenic acid, 5; folic acid; 2; riboflavin, 5; nicotinic
acid, 5; cyanocobalamine, 0.1. After incubation of Petri
dishes for 4 d at 28 8C, colonies varying in morphology were
picked and repeatedly re-streaked on BPF medium supplemented with 200 mM of CdCl2 until the colony
morphology of each isolate was homogenous.
2.3. Identification of bacteria
The strains were identified by determination of 16S
rRNA gene sequences. Colony PCR was performed from
live cells cultured on solid BPF medium. The PCR
mixture (25 ml) contained 1.5 mM MgCl2, 1.5 units of Taq
DNA polymerase (MBI Fermentas), 10 mM Tris–HCl,
50 mM KCl, 200 mM of each dNTP (MBI Fermentas).
Approximately 500 bp of the 16S rDNA were amplified by
PCR using the following primers 8f (5 0 -AGAGTTTGAT
CCTGGCTCAG-3 0 ) and 519r (5 0 -GWATTACCGCGG
CKGCTG-3 0 where W indicates A/T) as described by
Preisfeld et al. (2000). Aliquots of PCR reaction products
were electrophoresed in 1% agarose containing 10 mg
ethidium bromide mlK1. The PCR product was purified
using a QIEAX II DNA gel purification kit (Qiagen). The
small subunit rRNA gene was sequenced using the dye
termination method (Long-Read Towere System, Visible
Genetics Inc. Toronto, Canada). The sequences were then
compared to similar sequences in the databases using
BLAST analysis (Basic logical alignment search tool,
BLAST at NCBI). The 16S rRNA gene sequences of
the studied strains have been submitted to the GenBank/
DDBJ/EMBL databases under the accession numbers given
in Table 2.
Table 2
Distribution of the isolated bacterial strains by their effect on root
elongation of B. juncea variety VIR3129
Origin of strain isolation
1
2
3
4
5
Sod-podzolic
Sod-podzolic
Sewage sludge
Sewage sludge
Mining waste
Number of strains with:
Stimulating
effecta
No
effectb
Inhibiting
effectc
0
3
4
1
3
3
2
2
3
1
2
0
4
5
9
The data are means of two experiments with 80 seedlings each.
a
Details on the stimulating effect of the bacteria on root elongation of Cd
untreated seedlings are given in Table 4.
b
Statistically insignificant effect varied from C11 to K11% (PO0.05;
Fisher’s LSD test).
c
Inhibiting effect varied from K13 to K39% depending on the strain
(P!0.05; Fisher’s LSD test).
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A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
Conventional tests were performed in accordance with
Bergey’s Manual of Determinative Bacteriology (1989) and
Willems et al. (1991) to establish the compatibility of the
morphological and biochemical properties of the studied
strains with their respective closest phylogenetic
relatives found by BLAST analysis. Cell form and size,
Gram-staining, motility, colony pigmentation, production of
UV-fluorescent pigments, the presence of oxidase, catalase
and urease, hydrolysis of gelatin and starch, denitrification,
nitrite and nitrate reduction, growth in the presence of 8%
NaCl, utilization of acetamide, acetate, adipinate,
adonitol, b-alanine, L-arabinose, L-arginine, L-asparagine,
benzoic acid, butane-2-3-diol, citrate, D-fructose, L-fucose,
D-galactose, geraniol, D-gluconate, D-glucose, D-glucosamine, L-histidine, meso-inositol, 2-ketoglutarate, 5-keto-Dgluconate, lactose, L-leucine, L-lisin, D-maltose, D-mannitol,
D -mannose, D -melibiose, D -melezitose, DL -norleucine,
DL-ornithine, L-phenylalanine, pimelic acid, D-raffinose,
D-ribose, sebacic acid, D-sorbitol, D-sucrose, D-tartrate,
meso-tartrate, D -trehalose, L -tryptophan, DL -tyrosine,
L -valine, D-xylose were determined as described by
Zvyagintsev (1991).
2.4. Characteristics of bacteria
Tolerance to Cd was estimated visually after incubation
of the bacteria for 5 d at 28 8C on solid BPF medium
containing Cd at concentrations from 0 to 5000 mM CdCl2.
A threshold growth-inhibitory concentration and a minimum lethal concentration of Cd were determined for each
strain. Tolerance of bacteria to other heavy metals was
monitored in the same manner via incubation on BPF
medium in the presence of 1 mM and 2 mM ZnCl2, CuCl2,
NiCl2 or CoCl2. Bacterial siderophore production was
determined using a chrome azurol S (CAS) shuttle solution
as described by Schwyn and Neilands (1987). The assay was
calibrated by generating a standard curve for samples
containing 1–100 mM deferoxamine mesylate (DFM).
Production of indoles was measured spectrophotometrically
using Salkowsky’s reagent (Ehman, 1977) after incubation
of the bacteria for 5 d at 28 8C on liquid SMN medium
without ACC and supplemented with 0.5 g tryptophan lK1
and 0.3 g NH4NO3 lK1. The assay was calibrated by
generating a standard curve for samples containing
indoleacetic acid (IAA). Acetylene-reduction (N2-fixing)
activity was determined by gas chromatography (Rennie,
1981). For this assay, the bacteria were incubated for 5 d at
28 8C in flasks containing 5 ml of a liquid SMN
medium without ACC and supplemented with 10 mg
(NH4)2SO4 lK1. All tests were conducted twice with two
replicates for each strain.
(aKB) generated by the enzymatic hydrolysis of ACC (Saleh
and Glick, 2001). The bacteria were grown in test tubes
containing 10 ml of a liquid BPF medium for 24 h at 30 8C
and harvested by centrifugation at 9000g for 10 min at room
temperature. Cell pellets were washed twice with 5 ml of
0.1 M Tris–HCl buffer (pH 7.5), resuspended in 1 ml of SM
medium and then 0.5 ml of each suspension was added to
2.5 ml of liquid SMN medium containing 5 mM ACC or
2 mM (NH4)2SO4 as a sole source of N. Bacteria were
incubated for 24 h at 30 8C, centrifuged as indicated above,
resuspended in 1 ml of 0.1 M Tris–HCl buffer (pH 7.5) and
centrifuged at 9000g for 10 min. The pellets were resuspended in 600 ml of 0.1 M Tris–HCl buffer (pH 8.5) and cells
were disrupted by the addition of 30 ml of toluene and
vigorous vortexing. After reaction of mixtures, containing
100 ml of cell suspension, 10 ml of 0.5 M ACC and 100 ml of
0.1 M Tris–HCl buffer (pH 8.5), for 30 min at 30 8C, 1 ml of
0.56 N HCl was added, and the mixtures were centrifuged at
14,000g for 5 min. The mixtures containing no cell
suspension or no ACC were used as controls. Then, 400 ml
of 0.56 N HCl and 150 ml of 0.2% 2,4-dinitrophenylhydrazine in 2 N HCl were added to 500 ml of the supernatant. The
mixtures were reacted for 30 min at 30 8C, supplemented
with 1 ml of 2 N NaOH and assayed for a-ketobutyrate via
determination of the optical density at 540 nm.
2.6. Protein determination
The protein concentration of cell suspensions and in cells
disrupted by toluene was determined by the method of
Bradford (1976). For this assay, 100 ml of 0.1 N NaOH was
added to 100 ml of sample, heated in boiling water for
10 min, and assayed for protein concentration using the
Bio-Rad protein reagent (Bio-Rad Lab., USA). Bovine
serum albumin was used to establish a standard curve.
2.7. Utilization of ACC in batch culture
The strains V. paradoxus 2C-1 and 5C-2 were incubated
for 25 d at 25 8C without shaking in flasks containing 50 ml
of a liquid SM medium containing 50 mg yeast extract lK1
as a source of vitamins and as an initial source of N (SMY
medium). The SMY medium was supplemented with
different C and N sources such as 6.7 mM glucose,
10 mM ACC or 5 mM (NH4)2SO4 such that the amounts
of C and N were equal. Initial bacterial concentration in
SMY medium was 105 cells mlK1. Bacterial growth was
monitored daily by measuring the optical density (OD) at
540 nm. The inoculated SMY medium without supplements
was used as a blank.
2.8. Root elongation assay on filter paper culture
2.5. ACC deaminase assay
The ACC deaminase activity of cell-free extracts was
determined by monitoring the amount of a-ketobutyrate
The plant root elongation promoting (PREP) activity of
the isolated bacteria was determined using the modified root
elongation assay of Belimov et al. (2001). Bacteria were
A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
grown on solid BPF medium for 48 h at 28 8C and
resuspended to 5!107 cells mlK1 in sterile tap water
filtered through an AQUAPHOR (Electrophor Inc., Dobbs
Ferry, NY, USA). Six ml of the bacterial suspensions or
sterile water (uninoculated control) were added to glass
Petri dishes with filter paper. Bacterial suspensions and
water were supplemented or not with 8 mM CdCl2
(final concentration). The seeds of Brassica juncea variety
VIR-3129 were surface-sterilised with a mixture of ethanol
and 30% H2O2 (1:1) for 20 min, washed with sterile water
and placed on wetted filter paper. Root length of seedlings
was measured after incubation of closed Petri dishes for 6 d
at 28 8C in the dark. The assay was repeated two times with
four dishes (with 20 seeds per dish) for each treatment.
2.9. Statistical analysis
The data were processed by variance and correlation
analysis using the software STATISTICA version 5.5
(StatSoft, Inc. 1999, USA). LSD stands for Fisher’s least
significant difference.
3. Results
3.1. Isolation and identification of bacteria
Forty-two Cd-tolerant bacterial strains were isolated
from the rhizoplane of plants grown in the soils, sewage
245
sludge and mining waste studied. All strains were
repeatedly screened for their effect on root elongation of
B. juncea VIR-3129 without the addition of Cd. The
results showed that the response of seedlings to inoculation significantly varied from inhibition to stimulation of
root elongation depending on the strain (Table 2). All of
the strains having a stimulatory effect, seven strains
having no effect and one strain having an inhibitory effect
on root elongation were characterized by determination of
16S rRNA gene sequences and then appropriate conventional tests were made to compare the biochemical
properties of the isolated strains with their closest genetic
relatives found in the NCBI database using BLAST
analysis (Table 3). Nine strains having PREP activity
were assigned to the species Variovorax paradoxus,
while two others were characterized as Rhodococcus
sp. strain 4N-4 and Flavobacterium sp. strain 5P-4. The
isolated V. paradoxus strains had relatively high degree of
16S rRNA gene sequence similarity (98–99%) and few
differences between strains were observed in their
biochemical properties such as utilization of citrate,
D-raffinose or D-trehalose (data not shown). The root
length-promoting bacteria were isolated from all of the
soils, except sample 1 of sod-podzolic soil, using BPF and
SMC media (Table 3). The only Rhodococcus sp. 4N-4
was isolated using SMN medium. The strains without
root length-promoting activity were assigned to
different genera and their taxonomic positions are shown
in Table 3.
Table 3
Identification and characteristics of the isolated Cd-tolerant bacterial strains
Straina
Taxon
Accession
no.
ACC deaminase activity
(mM aKB mgK1 hK1)
Strains with plant root elongation promoting activity of Cd untreated seedlings
2C-1
Variovorax paradoxus AY196950
6.2G0.1
2P-1
V. paradoxus
AY196997
6.4G0.1
2P-4
V. paradoxus
AY196998
5.5G0.2
3C-2
V. paradoxus
AY196999
1.3G0.4
3C-3
V. paradoxus
AY197000
9.0G0.8
3C-5
V. paradoxus
AY197001
7.3G0.5
3P-3
V. paradoxus
AY197002
1.2G0.1
4N-4
Rhodococcus sp.
AY197005
10.5G0.2
5C-2
V. paradoxus
AY197003
9.3G0.8
5P-3
V. paradoxus
AY197004
1.6G0.1
5P-4
Flavobacterium sp.
AY197006
0
Strains without plant root elongation promoting activity of Cd untreated seedlings
1P-5
Rhodococcus sp.
AY197007
0
2P-2
Ralstonia sp.
AY323204
0
4N-3
Arthrobacter sp.
AY323203
0
4P-6
Acidovorax facilis
AY197008
0.7G0.1
4P-7
Stenotrophomonas sp.
AY197011
0
5N-1
Flavobacterium sp.
AY197009
0
5P-1
Cytophagales
AY323202
0
5P-2
Pseudomonas sp.
AY197010
0
Indole production
(mg IAA mgK1)
Siderophore production
(mM DFM mgK1)
Cadmium toleranceb (mM CdCl2)
0.4G0.1
0
6G0.1
43G0.5
24G0.8
73G2.1
41G0.4
34G0.6
29G0.5
4G0.1
0
29G0.8
10G0.8
27G0.6
0
0
0
0
0
0
0
8G0.8
0.2/0.6
0.2/0.6
1.0/1.8
0.8/2.3
1.0/3.2
1.0/2.2
1.0/2.3
0.1/0.3
1.4/3.5
2.2/3.5
0.2/1.2
35G0.4
4.1G0.1
38G2.3
4G0.1
0
0
0
3G0.1
25G4.2
24G0.5
15G0.8
0
11G2.2
29G5.9
0
90G1.2
0.1/2.6
1.0/4.0
0.1/3.0
0.4/2.2
1.2/2.8
0.1/2.0
1.0/3.0
0.4/2.3
Gshows standard deviation.
a
The strains are designated as follows: the number on the left stands for the soil used for isolation of the strain (see Table 1); the capital letter identifies the
medium used for isolation of the strain (C, N and P are SMC, SMN and BPF media, respectively).
b
The data are reported as threshold growth-inhibitory/- minimum lethal concentrations of CdCl2 in the nutrient medium.
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A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
Table 4
Root length of B.juncea variety VIR3129 seedlings inoculated with the isolated strains and grown in the absence or presence of Cd in nutrient solution (the data
are means of two experiments with 80 seedlings each)
Strain
Uninoculated control
V. paradoxus 2C-1
V. paradoxus 2P-1
V. paradoxus 2P-4
V. paradoxus 3C-2
V. paradoxus 3C-3
V. paradoxus 3C-5
V. paradoxus 3P-3
Rhodococcus sp. 4N-4
V. paradoxus 5C-2
V. paradoxus 5P-3
Flavobacterium sp. 5P-4
Rhodococcus sp. 1P-5
Ralstonia sp. 2P-2
Arthrobacter sp. 4N-3
A. facilis 4P-6
Stenotrophomonas sp. 4P-7
Flavobacterium sp. 5N-1
Cytophagales 5P-1
Pseudomonas sp. 5P-2
Treated with 8 mM CdCl2
Untreated seedlings
Root length (mm)
Bacterial effect (%)
Root length (mm)
Bacterial effect (%)
90
113 c
106 b
113 c
108 b
117 c
112 c
112 c
104 b
127 c
113 c
102 a
100
87
83
89
80
95
98
71 c
C26
C18
C26
C20
C30
C24
C24
C15
C41
C25
C13
C11
K3
K8
K1
K11
C5
C9
K21
65
101 c
98 c
100 c
95 c
86 c
103 c
98 c
83 b
100 c
96 c
86 c
74
84c
64
71
74
66
71
62
C55
C51
C54
C46
C32
C63
C51
C28
C54
C48
C32
C14
C29
K2
C9
C14
C1
C9
K5
Letters a, b and c indicate a statistically significant difference between inoculated seedlings and the uninoculated controls, according to Fisher’s LSD test at
P%0.05, P%0.01 and P%0.001, respectively.
3.2. Root length promotion
The effects of each identified strain on root elongation of
B. juncea VIR-3129 in the absence of Cd is shown in
Table 4. Addition of 8 mM Cd to the filter paper culture
inhibited root elongation of uninoculated seedlings by 30%
(Fisher’s LSD test, P%0.001). Inoculations with strains
having PREP activity in the absence of Cd also significantly
increased the root length of Cd-treated seedlings. The
maximum root length-promoting effect on Cd-treated plants
was observed after inoculation with V. paradoxus strains
2C-1, 2P-4, 3C-5 and 5C-2. Stimulation of root elongation
by the bacteria was more pronounced with Cd-treated plants
as compared to Cd-untreated plants. The root elongation of
Cd-treated seedlings was not significantly affected by the
other strains tested with the exception of Ralstonia sp. 2P-2,
which increased root elongation. There was a positive
correlation (rZC0.84, P!0.001, nZ19) between bacterial
effects on root length of Cd treated and untreated plants.
Such a correlation was also significant when only the strains
having PREP activity, including Ralstonia sp. 2P-2, were
analysed (rZC0.60, PZ0.04, nZ12).
3.3. Characteristics of the bacteria
The isolated bacteria had a high tolerance to Cd
(Table 3). The most Cd-tolerant strains that also had a
stimulating effect on root elongation were V. paradoxus
5C-2 and 5P-3, both isolated from mining waste (sample 5),
whereas the least Cd-tolerant strains were Rhodococcus sp.
4N-4 and V. paradoxus 2C-1 and 2P-1 isolated from soil
samples 2 and 4, respectively. Bacterial tolerance to Cd did
not correlate with their PREP activity. The bacteria were
also tolerant to other heavy metals and grew on BPF
medium supplemented with 2 mM ZnCl2, or CuCl2, or
NiCl2, or with 1 mM CoCl2 (data not shown). The only
exception was that the growth of Flavobacterium sp. 5P-4
was completely inhibited in the presence of 1 mM CoCl2.
The strains V. paradoxus 3C-2, 3C-3, 3C-5, 3P-3 and 5C-2,
as well as Rhodococcus sp. 4N-4, were the most Co-tolerant
bacteria and grew in the presence of 2 mM CoCl2.
The strains without PREP activity were also tolerant to
these heavy metals, except for A. facilis 4P-6 and
Flavobacterium sp. 5N-1, which were both sensitive to Co
and Cu (data not shown).
Many of the strains were capable of producing
indoleacetic acid (IAA) or siderophores, and significant
differences between strains were also observed in amount of
IAA and siderophores produced (Table 3). There was no
correlation between IAA or siderophore production and
bacterial effect on root elongation. Among the root lengthpromoting strains, only V. paradoxus 2C-1, 2P-1 and 2P-4,
Flavobacterium sp. 5P-4 and Ralstonia sp. 2P-2 produced
siderophores. None of the strains possessed C2H2-reduction
(N2-fixing) activity.
All the strains with PREP activity, except Flavobacterium sp. 5P-4 and Ralstonia sp. 2P-2, contained ACC
deaminase (Table 3). On the other hand, all other strains,
except A. facilis 4P-6, possessed no ACC deaminase
activity. The strains differed in their amount of ACC
A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
deaminase activity by a factor of 15. ACC deaminase
activity was not detected in cells of any V. paradoxus strains
or A. facilis 4P-6 incubated in the presence of excess NHC
4
ion (2 mM (NH4)2SO4), which was added to the nutrient
medium as a source of N. Only Rhodococcus sp. 4N-4
possessed very high ACC deaminase activity (4.9G0.3 mM
aKB mgK1 hK1) in the presence of (NH4)2SO4.
The fact that all but one of the root length-promoting
strains possessed ACC deaminase suggests the importance
of this enzyme for bacterial root length promotion.
Surprisingly, correlation between ACC deaminase activity
in toluenized cells (in vitro) and PREP activity on Cd
untreated seedlings was not significant (rZC0.43,
PZ0.18, nZ11). However, this correlation became significant after excluding Rhodococcus sp. 4N-4, which exhibited
a unique induction of this enzyme in the presence of NHC
4 ,
from the analysis (rZC0.65, PZ0.04, nZ10). A scatter
plot showing this relationship between ACC deaminase
activity and the stimulation of root elongation on Cd
untreated seedlings is shown in Fig. 1. However, no
correlation was found between ACC deaminase activity in
vitro and the bacterial effect on root elongation of Cd treated
seedlings (rZC0.39, PZ0.27, nZ10).
Five of nine strains with PREP activity belonging to
V. paradoxus were isolated using SMC medium containing
ACC as a sole source of C. Therefore, the ability of
V. paradoxus to utilize ACC as a C source was studied in
batch culture using strains 2C-1 and 5C-2. Both strains were
capable of utilizing ACC as a sole source of C, both in the
presence and absence of NHC
4 (Fig. 2). Strain 2C-1 was
characterized by a prolonged lag phase in the medium
supplemented with ACC, whereas strain 5C-2 had a short
lag phase and its growth rate on ACC was comparable
with its growth rate on glucose. The growth curve of
bacteria in the presence of both glucose and ACC had
two peaks, suggesting successive utilization of these
potential C sources.
Fig. 1. Linear regression curve showing the relationship between ACC
deaminase activity (mM aKB mgK1 hK1) of bacterial strains in vitro and
their effect on root elongation of B. juncea variety VIR3129 in the absence
of Cd. Names of strains are shown in the plot according with Table 2.
Asterisk shows position of strain Rhodococcus sp. 4N-4, which is not
included in the analysis. Dotted lines show regression confidence area at
PZ0.05.
247
Fig. 2. Growth of V. paradoxus strains 2C-1 and 5C-2 on a liquid SMY
medium supplemented with different C and N compounds. Supplements:
:, 6.7 mM glucose; &, 6.7 mM glucose and 10 mM ACC; ,, 6.7 mM
glucose and 5 mM (NH4)2SO4; B, 10 mM ACC; C, 10 mM ACC and
5 mM (NH4)2SO4. OD stands for optical density of bacterial suspensions at
540 nm. In all instances the standard errors of the means are smaller than
the symbol size.
4. Discussion
In the present study, 1000-fold diluted homogenates of
washed roots of young Indian mustard seedlings were
used for the isolation of bacteria. The approach used here
allowed the selection of those strains, which can be
considered as the dominant culturable bacteria on the
roots of plant seedlings (Belimov et al., 1999). This is
especially important since active root colonization is
necessary for successful application of PGPR as inoculants. Several Cd-tolerant PGPR strains containing ACC
deaminase have been also isolated from the rhizoplane of
Indian mustard and pea seedlings (Belimov et al., 2001),
however that isolation procedure significantly differed
from the method used here since previously: (i) the root
samples were first serially incubated on BPF medium to
enrich the culture for pseudomonads; (ii) the isolation
media were not supplemented with Cd; (iii) a mixture of
roots of two plant species (pea and Indian mustard) was
used making it difficult to recognise which strain was
isolated from which plant; and (iv) the concentrations of
Cd in soils used for seedling germination were relatively
low. Here, priorincubation of root samples was eliminated
to avoid changes in the composition of the bacterial
248
A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
community inhabiting the roots, and to isolate those
bacteria which are abundant in the root zone in situ. In
addition, in the present study the seedlings were grown in
soils containing high Cd concentrations and Cd-tolerant
strains were selected through the supplementation of Cd
to the nutrient media at the first step of the isolation
procedure. In fact, the strains isolated in our study are
more tolerant to Cd as compared with those isolated by
Belimov et al. (2001). Interestingly, Cd-tolerant
V. paradoxus were present in soil sample 2, which was
artificially contaminated with Cd before the experiment,
and were found in only one of two heavily contaminated
samples that originated from the Gatchina sewage
treatment works.
A number of PGPR, which stimulate root growth of
different plant species including Indian mustard (Burd et al.,
1998; Belimov et al., 2001), contain the enzyme ACC
deaminase, which hydrolyses ACC (the immediate precursor of the plant hormone ethylene). Some of the plant
ACC is exuded from roots or seeds and cleaved by ACC
deaminase to NH3 and a-ketobutyrate (Penrose and Glick,
2001). The bacteria utilize the NH3 evolved from ACC as a
source of N and thereby decrease ACC within the plant
(Penrose et al., 2001) with the concomitant reduction of
plant ethylene (Burd et al., 1998; Mayak et al., 1999;
Grichko and Glick, 2001; Belimov et al., 2002). In our
study, 10 of 11 newly-selected root length-promoting strains
assigned to V. paradoxus and Rhodococcus sp. contained
ACC deaminase. Only one strain A. facilis 4P-6, with low
ACC deaminase activity, had an insignificant effect on root
elongation. ACC-utilizing PGPR belonging to V. paradoxus
and Rhodococcus sp. have been described by Belimov et al.
(2001), but this is the first report that bacteria of the genus
Acidovorax contain ACC deaminase, and have a high
tolerance to heavy metals present in the root zone of metal
accumulating plants.
The isolated PGPR differed significantly in ACC
deaminase activity and a positive correlation was observed
between the activity of this enzyme in vitro and the bacterial
effect on plant root elongation. However, such a correlation
was significant only when Rhodococcus sp. 4N-4 was
excluded from the analysis. Two observations provide
reasons to exclude this strain. First, Rhodococcus sp. 4N-4
differed from the other strains in that it has the unique ability
to induce ACC deaminase in the presence of a high NHC
4
concentration and absence of ACC. Second, to obtain this
sort of correlation it is also important that all of the strains
beings compared should belong to the same or closely
related species. In this respect, the genus Acidovorax is
phylogenetically much closer to the genus Variovarax
compared to Rhodococcus. Previously, Belimov et al.
(2001) did not find such a correlation when 15 ACCutilizing strains belonging to various genera and species
were analysed. It was assumed that a number of bacterial
properties associated with their taxonomic position could
affect the interaction of rhizobacteria with plant roots,
and there was no reason to expect a strong correlation
between a particular bacterial property and its quantitative
effect on plant growth. At the same time, a clear quantitative
relationship between ACC deaminase activity and plant
root length promotion was established using ACC
deaminase deficient mutants of Pseudomonas putida
GR12-2 (Glick et al., 1994) and Enterobacter cloacae
UW4 (Li et al., 2000).
The results obtained here confirm previous conclusions
about the importance of bacterial ACC deaminase in plant
growth promotion (Glick et al., 1998), and expanded our
knowledge of bacterial metabolism of ACC, since this is the
first report showing utilization of ACC by bacteria as an
energy source. The ability of V. paradoxus to use ACC as
both N and C sources gives these bacteria an additional
competitive advantage in rhizosphere colonization. Taking
into account that Cd induces plant stress ethylene biosynthesis (Pennasio and Roggero, 1992) and probably contributes to accumulation of ACC in roots, it is not surprising that
a number of V. paradoxus strains were isolated among
bacterial communities dominating the root zone of B. juncea
grown in contaminated soils in the present study.
The mechanisms of root length promotion by the strains
Flavobacterium sp. 5P-4 and Ralstonia sp. 2P-2 which do
not have ACC deaminase activity need further investigation.
One possibility is that Flavobacterium sp. 5P-4 contains
ACC deaminase, but the enzyme was not induced when the
bacteria were cultivated in vitro. Alternatively, the root
length-promoting effect of Ralstonia sp. 2P-2 in the
presence of Cd could be associated with decreased Cd
toxicity for the plants through production of some metalimmobilizing substances by the bacteria. The ability of
several PGPR strains to immobilize Cd in nutrient media
(Belimov et al., 1998) and in soil (Pishchik et al., 2002) has
been described previously. In addition, depending on the
conditions, plant root growth may also be stimulated by
indoleacetic acid produced by PGPR bound to the seeds or
roots (Patten and Glick, 2002).
In conclusion, the results show that Cd-tolerant PGPR
are present in the root zone of metal accumulating plant
B. juncea grown in soils contaminated with heavy metals
and originating from different geographical regions. The
isolated strains belonging to V. paradoxus are of particular
interest being dominant PGPR representatives of a
culturable bacterial community associated with the roots
of young seedlings. The root length-promoting effect of
the selected V. paradoxus strains is most probably due to
their ability to act as a sink for ACC, the immediate
precursor of the plant hormone ethylene. The results
suggest that V. paradoxus, particularly strain 5C-2, offers
promise as a bacterial inoculant for improvement of root
growth of B. juncea plants in the presence of toxic metal
concentrations. Further work aims to study the effects of
these bacteria on tolerance to, and uptake of, heavy metals
by plants, for development of plant-microbe systems
useful for phytoremediation.
A.A. Belimov et al. / Soil Biology & Biochemistry 37 (2005) 241–250
Acknowledgements
The authors are very grateful to Dr O.O. Dzyuba
for kindly supplying us with Indian mustard seeds, to
Dr Y.V. Alekseyev for soil analyses, to Dr I.C. Dodd for
valuable assistance in experiments on ACC utilization by
the bacteria and preparation of the manuscript, and to
Professor W.J. Davies for valuable discussions. Special
thanks from Dr A.A. Belimov are to Dr D.M. Penrose for
her valuable guidance and support during his tenure at
Waterloo. This work was supported by NATO, the Royal
Society (IES grant for Ex-Agreement Visits), the ItalyRussia Commission for Science and Technology
Cooperation, the Russian Foundation of Basic Research,
and the Natural Sciences and Engineering Research Council
of Canada.
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