Research Article
Open Access
Characterization of Root-Nodule Bacteria Isolated
from Hedysarum spinosissimum L, Growing in
Mining Sites of Northeastern Region of Morocco
Laila Sbabou1*#, Yassir Idir1#, Odile Bruneel1,2, Antoine Le Quere1,4, Jamal Aurag1, Gilles Bena1,3
and Abdelkarim Filali-Maltouf1
1Laboratoire de Microbiologie et Biologie Moléculaire, Faculté des Sciences, Université Mohammed V, Rabat, Morocco
2Laboratoire Hydro Sciences Montpellier, UMR 5569(IRD, CNRS, UM), Université de Montpellier, CC0057 (MSE), 163 rue Auguste Broussonet, 34090
Montpellier, France
3Laboratoire des Interactions Plante Microorganismes Environnement, UMR 186 (IRD, CIRAD, UM), 911 avenue Agropolis, BP 64501, 34394 Montpellier cedex
5, France
4Institut de Recherche pour le Développement, Laboratoire des Symbioses Tropicales et Méditerranéennes, UMR113 IRD-CIRAD-SupAgro-UM-INRA, Campus
International de Baillarguet, 34 398 Montpellier Cedex
5, France
#Authors are equally contributed
*Corresponding author: Laila Sbabou, Laboratory of Microbiology and molecular Biology, Faculty of Sciences, Mohammed V University, Rabat,
Morocco, E-mail:
@
Received: September 1, 2016; Accepted: October 1, 2016; Published: October 5, 2016
Citation: Sbabou L, Idir Y, Bruneel O, Le Quere A, Aurag J (2016) Characterization of
Root-Nodule Bacteria Isolated from Hedysarum
spinosissimum L, Growing in Mining Sites of Northeastern Region of Morocco. SOJ Microbiol Infect Dis 4(3): 1-8. DOI:
10.15226/sojmid/4/3/00156
The aim of this study was to identify bacteria present in
the nodules of the legume Hedysarum spinosissimum growing in
metal-contaminated soils; and to test whether these root-nodule
bacteria are able to promote host plant growth and enhance their
phytostabilization potential. Seventy-four bacteria were isolated
from nodules of H. spinosissimum growing in 3 different mining sites
in Morocco and were identified by 16S rDNA gene sequencing. They
belonged to 8 genera affiliated to Pseudomonas (49 strains), Pantoea
(11), Rhizobium (6), Herbaspirillum (3), Bacillus (2), and one strain of
Serratia, Agrobacterium and Azospirillum. Seven strains, presenting a
high tolerance to Zn and Pb, exhibited capacity of inorganic phosphate
solubilization and ammonia production from peptone degradation.
The inoculation of H. spinosissimum, growing in 2000 μM of Zn by
Pseudomonas putida and Herbaspirillum huttiense suppresses Zn
toxicity symptoms in plants and also enhances plant growth by
significantly increasing plant shoot and root fresh weights. The
maximal Zn accumulation was observed in roots of plants inoculated
by Pseudomonas putida with a translocation factor of 0.05(± 0.006).
Our results, evidence that the selection of metal-resistant bacteria
is a key step in polluted soils for the use of plants like Hedysarum
spinosissimum for in situ phytoremediation.
Keywords: legumes; Phytoremediation; Nodulation; Mine
tailings; Heavy metal resistance; Phytostabilisation
Introduction
Soil contaminations with heavy metals are principally caused
by mining activities and require strengthening the efforts to
minimize their impact on the environment and human health [1].
Since metals are non biodegradable, they accumulate and persist
in the environment [2]. To cleanup these degraded and polluted
areas, metallic pollutants should be extracted and stored in some
appropriate and secure sites. Conventional techniques, including thermal processes, washing, physical separation, stabilization/
solidification, etc …, are generally very expensive and harmful
to soil integrity and microbial diversity [3,4]. Phytoremediation
appears as a good alternative as plants can indeed bio-concentrate
(phytoextraction) as well as bio-immobilize (phytostabilization)
toxic metals through in situ rhizospheric processes [1,2].
However, mining sites in semi-arid areas are characterized by low
vegetation cover due to the unfavorable effects of a combination
of environmental factors, including metals toxicity, nutrient
deficiency, poor soil structure and low water retention [5,6], thus,
the development of a vegetative cover in these environments
is a challenge. Recently, several studies have shown that the
combination of plant-associated bacteria in phytoremediation
may lead to promising results, in particular the group of plant
growth promoting bacteria [7,8]. This approach is furthermore
perceived as cost-effective, eco-friendly, and with good public
acceptance [2]. The choice of bacteria is usually based on their
potential to produce phytohormones such as IAA, gibberellins
and cytokinins that directly promote roots and plant growth [9]. Other bacteria can synthesize organic chelators (siderophores) to
acquire iron [10] that will play a positive role in plant nutrition;
some have great potential for phosphate solubilization or possess
ACC deaminase activity [11] while others are able to fix nitrogen
[1] or provide protection against viral diseases [12]. In addition,
microorganisms can be implicated directly in metal mobilization/
immobilization, by changing the metal bioavailability, solubility
and toxicity by different mechanisms including alteration of
soil pH, releasing of chelators (organic acids, biosurfactants,
siderophores, polymeric substances or glycoprotein, etc),
metal biosorption or by oxidation/ reduction reactions [4,13].
The use of plants for the remediation of contaminated soils by
heavy metals obviously also depends on the plant species, and
on its anatomical, physiological and molecular characteristics [14]. Furthermore, these plants need to be drought-, and metaltolerant
in arid and semi-arid area, and preferentially native [5].
Beside the interest of legumes in soil regeneration, thanks to their
capacity to increase soil nitrogen through atmospheric nitrogen
fixation, many legumes are able to accumulate metals in their
roots, representing good candidates for metal phytostabilization.
They have also been found to be generally the dominant plant
species in metal contaminated environments [15,16]. In this
regard, several legume species have been proposed for metal
phytostabilization [17,18]. Hedysarum spinosissimum is a forage
legume, which is part of the indigenous flora adapted to semiarid
climate of the mining sites located in eastern Morocco. The
soils in these sites are contaminated by metallic pollutants and
constitute a potential source of toxicity for the environment and
peoples in surrounding villages.
The objectives of this study were to isolate and characterize
metal tolerant bacteria from H. spinosissimum's nodules and to
propose a new model for phytoremediation of mining tailing by
using plants and associated symbiotic bacteria.
Material and Methods
Soil collection and characterization
Soil samples were collected, in triplicate, from 12 points
distributed among the 3 mining sites: Oued El heimer smelter
(34°26'38''N/1°53'54''W), Touissit (34°28'06"N/1°46'18"W)
and Sidi Boubker (34°28'23''N/1°42'55''W) mines. Soil debris
was removed by sieving using a steel sieve (2 mm pores
diameter). Each soil was then ground in a mortar until obtaining
a fine powder. Samples were sent for ICP-AES analysis in
Eurofins-France laboratory, under ISO/ IEC 17025:2005 norm
and accreditation by the COFRAC 1-148.
Bacterial trapping and isolation
Nodules were obtained from Hedysarum spinosissimum
plants, aged of 2 months, growing in soils sampled from the
3 mining sites, under growth chamber under day/ night
photoperiod of 16h/ 8h and temperature of 25°C. The nodules
were collected, surface sterilized by soaking in alcohol (ethanol
95%), washed several times in sterile distilled water and crushed.
The suspension obtained was spread on solid YEM medium
(yeast extract mannitol), and incubated for 24-48 hours at 28°C.
Bacterial colonies were purified on the same media by streaking
3 to 4 times on fresh media. Bacterial isolates showing different
morphological appearance on agar media were selected, grown
in liquid YM and stored at -80°C in 20% glycerol.
Molecular identification
Genomic DNA was extracted from liquid cultures
of bacterial isolates and 16S rDNA was amplified by
Polymerase Chain Reaction (PCR) using the universal
primers, 41F (5'-GCTCAGATTGAACGCTGGCG-3') and 1488R
(5'-CGGTTACCTTGTTACGACTTCACC-3') [19]. The PCR mixture
(25 μl) contained 1 μl of DNA (10 ng/ μl), 5 μl of 5xTaq DNA
polymerase buffer (BIOLINE), 0.5 μl of Taq DNA polymerase (2.5
U), 1.25 μl of 10 pmol primers, 0.125 μl of Taq polymerase and bidistilled water. The PCR was performed in a Veriti® 96-Well
Thermal Cycler (Applied BioSystems®) with a hot starting at
94°C for 5 min, followed by 35 cycles of 94°C for 45s, 64°C for
90s, and 72°C for 90s, followed by a final extension at 72°C for
7 min. The sequencing was performed at Genoscreen Company
(France). The 16S rDNA sequences were compared with those
available in GenBank database using the BLASTN program
through the National Center for Biotechnology Information
server. The 16S rDNA sequences have been deposited in GenBank
under the accession numbers from KP263459 to KP263517 and
KP677886 to KP677900.
Phylogenetic analysis
Multiple alignments of consensus sequences were done with
the program ClustalW [20]. The resulting alignment was used for
construction of phylogenetic trees using MEGA software version
6, using the Neighbor-Joining method [21]. The robustness of
the phylogenetic tree was evaluated according to the bootstrap
analysis based on 1000 re-samplings of the sequences.
Effects of heavy metals on bacterial growth
The tolerance of each bacterial isolate to Zn and Pb of selected
isolates was evaluated on Tryptone-Yeast extract (TY) medium
[22] supplemented with increasing concentrations of both
metals. The choice of concentrations used for tolerance tests was
based on the concentration of these metals in contaminated soils.
ZnSO4 and PbNO3
2- forms were used at concentrations ranged
from 1 to 30 mM. The strains were grown in liquid medium for
24 h at 28°C. At the exponential growth phase of each strain, the
optical density at the wavelength of 600 nm was determined
and standardized at 0.06. Ten μl of the bacterial culture were
deposited onto TY agar plates and incubated at 28°C for 24–72
h. Three replicates were made for each bacterial strain. Strains
tolerance was also tested in liquid medium.
Bioaccumulation of Zn in bacteria
A total of 100 ml of bacterial cultures (in TY medium) were
prepared in 250 ml flasks, incubated at 37°C. After 48h incubation,
the metal (5 mM of ZnSO4) was added to the bacterial cultures,
which were then re-incubated under the same conditions. The cell
mass was collected by centrifugation (8000 rpm for 10 min at 4°C)
and washed twice with Tris-HCl buffer (0.1 M, pH 7.2) to remove
elements adsorbed onto the cell surface [23]. The samples were
then dried at 70°C until a constant dry mass was obtained. The
bacterial pellets were mineralized using the following procedure:
the pellets were introduced into tubes which contained 10 ml of
nitric acid (HNO3) 60%, 0.5 ml of sulfuric acid (H2SO4) and 4 drops
of perchloric acid (HClO4) were added. After incubation at room
temperature for 24 h, the tubes were placed in a heating block at
140°C for 4 h. Finally, the volume was reduced by evaporation for
24 hours by slightly opening the tubes. The concentration of Zn in
the samples was finally determined by ICP-AES method (Ultima
2- Jobin Yvon).
Motility Assay
The procedure described by Murray, et al. [24] was followed
with slight modifications. Bacterial motility was evaluated on a semi-solid TY (0.3%) medium. Liquid cultures were standardized
to a bacterial concentration of 105 to 106 cells/ ml. Five μl of
each bacterial suspension were inoculated in TY semi-solid
agar medium by gently pressing the tip into the medium. Three
repetitions were performed for each strain for both treatments:
metal stress treatment (5 mM of Zn) and a control treatment (no
metal added). Petri dishes were incubated at 30°C for 24-48h
depending on the strain. The bacterial motility was evaluated by
determination of the diameter of the halo formed.
Mineral phosphate solubilizing activity and ammonia
production
Phosphate solubilization was first roughly estimated on
modified solid PVK medium [25]. 10μl of each strain were
deposited on the media in triplicates and incubated at 28°C for
5 days. The solubilization was evaluated by the halo diameter
obtained. To precisely quantify the solubilized phosphate, flasks
containing 100 ml of PVK medium were inoculated with 1 ml of
pre-cultures and incubated at 28°C for 5 days with stirring at 150
rpm. The determination of soluble phosphate was done according
to the phosphomolybdate method [26].
Bacterial strains were also tested for the production of NH3
in peptone water [27] using Nessler's reagent (0.5 ml/ tube).
The appearance of a faint yellow color indicates production of a
small amount of NH3 whereas a deep yellow to brownish color
indicates high NH3 production.
Influence of selected strains on plant growth and Zn
uptake
For inoculation, two strains (LMR23 and LMR51) were
grown at 28°C for 24h in TY medium on a shaker at 150 rpm.
The bacterial suspensions were washed twice and resuspended
in sterile distillated water after determination of optical density
(i.e. roughly 106 bacteria ml-1). After inoculum preparation, the
experiment was conducted with 3 replicates for each of the
3 following treatments: (1) plants grown in nutrient solution
without metal and inoculum, (2) plants grown in nutrient
solution supplemented with the Zn MIC (Zn Minimal Inhibitory
Concentration) of plants growth and (3) plants grown in nutrient
solution with different combinations of strains with Zn at the MIC.
Before the start of the experiment, plants were grown in normal
conditions for 15 days. After treatment, plants were grown under
the different conditions for 15 days and their roots and shoots
were separated and rinsed several times in 0.2 mM CaSO4 and
then with distilled water. Samples were dried at 70°C for 48h and
then treated according to the acid hydrolysis protocol described
by Temminghoff, et al. [28]. Zn concentration was determined by
ICP-AES method. The Translocation Factor (TF) for metals within
a plant was expressed by the ratio of concentration of metal
(shoot)/ metal (root), to estimate metal translocation properties
from roots to shoots [29].
Statistical analysis
The mean and standard deviation of the 3 replicates for each
treatment were calculated. Tukey's test was conducted to assess
determine significant differences. Statistical analyses were
performed with STATISTICA version 6.
Results and Discussion
Isolation and identification of bacteria
Overall, 74 endophytic bacteria were isolated from Hedysarum
spinosissimum nodules. These isolates were selected based on
their morphological differences observed between colonies on
YEM solid medium and were identified using 16S rRNA gene
sequencing. Most of the strains were isolated from Oued El
heimer site (37 isolates) and Touissit (34 isolates), unlike Sidi
Boubker where the number of isolates was limited to 3 (Table
1). The analysis of the 16S rRNA gene sequences revealed that
the isolates belonged to 8 different taxonomic genera, including
Pseudomonas genus (largest number of representatives, 49),
Pantoea (11), Rhizobium (6), Herbaspirillum (3), Bacillus (2) and
one representative of Serratia, Agrobacterium and Azospirillum
(Table 1). The phylogenetic tree shows the relationships between
the bacterial strains and related reference species (Figure 1).
Phylogenetic analysis indicated that the bacterial strains isolated
from the nodules of H. spinosissimum belong to 2 phyla: the
Firmicutes represented by Bacillus sp. (2%) and Proteobacteria
represented by the rest of the collection (98%), including the
classes of α-, β- and γ-Proteobacteria (Table 1).
Few studies have been made on wild legume Hedysarum
spinosissimum nodule bacterial populations. Wei, et al. [30]
suggested a high level of genetic diversity among isolates
of Hedysarum. Benhizia, et al. [31] reported the presence of
isolates belonging to bacterial genera retrieved in our study,
Pseudomonas and Pantoea agglomerans, in addition to three other
Table 1: Identification, using 16S rDNA gene, of the bacterial isolates
present in the nodules of the plant Hedysarum spinosissimum.
Bacterial species |
Taxonomical division |
Number of representatives |
Sampling sites |
Pseudomonas sp. |
γ-Proteobacteria |
36 |
Touissit / Oued El heimer |
Pseudomonas putida |
γ-Proteobacteria |
8 |
Touissit |
Pseudomonas brassicacearum |
γ-Proteobacteria |
1 |
Oued El heimer |
Pseudomonas frederiksbergensis |
γ-Proteobacteria |
4 |
Touissit/ Sidi Boubker |
Pantoea agglomerans |
γ-Proteobacteria |
11 |
Touissit/ Sidi Boubker/Oued El heimer |
Serratia proteamaculans |
γ-Proteobacteria |
1 |
Oued El heimer |
Herbaspirillum huttiense |
β -Proteobacteria |
3 |
Touissit/ Oued El heimer |
Rhizobium leguminosarum |
α -Proteobacteria |
4 |
Oued El heimer / Touissit |
Rhizobium galegae |
α -Proteobacteria |
2 |
Oued El heimer |
Agrobacterium tumefaciens |
α -Proteobacteria |
1 |
Oued El heimer |
Azospirillum lipoferum |
α -Proteobacteria |
1 |
Oued El heimer |
Bacillus sp. |
Firmicutes |
2 |
Oued El heimer |
genera (Enterobacter, Leclercia and Escherichia), while Zakhia,
et al. [32] only isolated one strain related to Sinorhizobium.
Symbiotic Rhizobium species have been already isolated from
nodules of another leguminous plant, Anthyllis vulneraria,
growing in contaminated soils from a different mining district
in Morocco [18]. The presence of Agrobacterium in nodules of
plants in polluted soils was also previously described [33,34].
Herbaspirillum and Azospirillum are 2 root associated nitrogen
fixing bacterium [9,35,36]. Species belonging to Herbaspirillum
genus are known root endophytic Plant Growth Promoting
Bacteria (PGPB) especially in cereals. In addition, Herbaspirillum
strains have a considerable potential to produce auxin and
siderophores, and to solubilize inorganic phosphates [37]. In a
bioremediation frame, Herbaspirillum strains showed also an
important resistance to As, Zn, Cu and Pb, and some good abilities
for the leaching of Cu from contaminated soil [38]. Plant-growth promoting
Bacillus strains were also isolated from soybean root
nodules [39]. This genus was also able to accelerate root and shoot
growth by increasing chlorophyll content [40]. Different strains
of Bacillus species such as B. methylotrophicus, B. aryabhattai,
and B. licheniformis isolated from Spartina maritime rhizosphere
exhibited multiple plant growth promoting properties and were
selected as performing strains for restoration programs [41]. The
presence of Serratia sp. in legume nodules of Lupinus luteus was
previously reported [17]. This genus has also been shown to have
some PGPR properties like ACC deaminase activity [42].
The combination of both harsh conditions encountered by
the roots in metal polluted soils combined with the tolerance of
Hedysarum to co-infestation of nodules by rhizospheric bacteria
should explain the large taxonomic diversity we recovered in
this study. Such diversity gives the opportunity for the selection
of various strains, with different properties, that could be used
either alone or in combination for inoculation.
Effect of Pb and Zn on bacterial growth
To select strains tolerant to heavy metals, we conducted
Figure 1: Phylogenetic tree showing the relative position of isolates
based on 16S rDNA partial sequences, using the neighbor-joining method.
Bootstrap values for 1000 replicates are shown.
tests on solid and liquid TY media supplemented with ascending
concentrations of Pb and Zn (from 1 to 30 mM) that were the
most abundant metals present in the mining site of Oujda. The
experiment on solid TY medium demonstrated contrasted
responses of the isolates to the different amounts of heavy metals
tested. Seven highly tolerant bacteria were selected for further
studies. In liquid media, the effect of both metals on the growth of
these strains was estimated by measuring the optical density at
600 nm. As seen in figure 2, the 7 strains showed the same level
of resistance to Pb (25 mM) and Zn (5 mM). However, metals
affected negatively the growth of these bacteria by reducing
the number of bacterial cells (Figure 2). Compared with control
treatment the reduction of optical density was more pronounced
in Zn treatment (approximately 2/ 3 of reduction) than in Pb
treatment with 1/ 3 of OD decrease.
Determination of Zn uptake of the most tolerant
isolates
A deeper study has been done for these 7 resistant strains
in order to evaluate their metal accumulation capacity. Zn
was chosen for further investigations based on its important
concentrations in the 3 sampling stations and also its standing as
critical metal for the growth of living organisms, especially plants
and bacteria.
As shown in Table 2, the Rhizobium galegae strain LMR64
or the Herbaspirillum huttiense strain LMR51 showed a very
high level of zinc accumulation inside the cells (representing
respectively 58 and 31 ppm), while other isolates showed a
lower accumulation. The different levels of Zn accumulation in
the strains were not correlated with the level of inhibition of their
detected on TY medium containing high Zn concentration.
However, bioaccumulation of metals, inside the cell or on
cell surface can lead to some applications in remediation by
metal adsorption in soils [43-45] and could decrease the metal
bioavailability around the plant rhizosphere and thereby reduce
plant stress and metal accumulation [1,15,18,46].
Motility assay
In their environment, bacteria are either mobile or bound to a
substrate. In the present work, we were interested in swimming
bacteria, because it is a physiological strategy developed by
bacteria against stress [47]. The swimming was assessed by
comparing the halo of motility between strains living under Zn
stress and normal conditions (Figure 3). We found that motility
was significantly reduced (p < 0.05) under Zn treatment for
some strains (23% for LMR27, 40% for LMR51 and 42% for
LMR64), or completely inhibited for other strains (LMR80 and
LMR54). Conversely, motility was unaffected for LMR23 and
LMR79 with no significant differences (p < 0.05). Under adverse
environmental conditions, the maintaining of motility is useful to
bacteria to find nutrients and to survive, and the movement via
flagella appears to be important for the plant root colonization
[48,49]. However, it has been reported that heavy metals could
affect the motility [47]. In Pseudomonas putida, some proteins
are involved in regulating motility reducing the migration of bacteria and facilitating Pseudomonas putida biofilm formation,
an important trait to protect plants against pathogens [48].
Plant growth promoting properties
The determination of free phosphorus resulting from the
activity of microbial solubilization of mineral phosphate was
performed by the vanadate-molybdate method. The results are
shown in Table 3. All strains tested showed a great potential of
phosphate solubilization (with co-relation with pH decrease)
from 27.25 ± 5.49 ppm to 90.91 ± 7.95 ppm depending on
the strains and was maximum for Pseudomonas putida. The
solubilization of insoluble phosphate into available form is a
mechanism commonly observed in most metals resistant PGPB.
This process is realized by means of organic acids secretion out
of the cell, acidification, chelation and exchange reactions [1].
Pandey, et al [50] also showed the importance of the phosphate
solubilizing activity of a strain of Pseudomonas putida isolated
in Indian Central Himalaya associated with antifungal activity
resulting in significant improvement of the plant biomass.
Several other studies have also demonstrated the importance of
phosphates solubilizing bacteria and their effects on plants by
increasing the bioavailability of this macronutrient [1,51].
The detection of ammonia production was done using a
qualitative method. As shown in table 3, all the 7 strains tested
had a positive NH3 production but this production was more
important for Pseudomonas putida and Herbaspirillum huttiense.
Effects of the inoculation of selected strains on H.
spinosissimum plants growth and Zn tolerance
The effects of the isolates LMR23 (Pseudomonas putida)
and LMR51 (Herbaspirillum huttiense) on the growth of H.
spinosissimum grown in control condition and in nutrient solution
containing 2000 μM of Zn (the minimal inhibitory concentration
of Zn for H. spinosissimum plants) are shown in Table 4. The
plants inoculated with the selected isolates showed a significant
increase in plant fresh weight accompanied by the suppression of
Zn toxicity symptoms and a high level of Zn accumulation in roots
and aerial parts compared with the uninoculated control. The 2
endophytes used separately or together significantly (P < 0.05)
enhanced plants fresh weight as compared to control plants.
Figure 2: Growth of different bacterial strains in TY liquid medium
under Pb and Zn treatments Asterisks indicate significant differences
compared to the control (p < 0.05).
Figure 3: Impact of Zn treatment on the motility of strains estimated by
measuring the halo diameter (mm). Asterisks indicate significant differences
compared to the control (p < 0.05).
Table 2: Amounts of Zn accumulated in bacteria
Strain ID |
Closest relatives |
Zn concentration in cells (ppm) |
|
|
Control |
Zn (5mM) |
LMR23 |
Pseudomonas putida |
0.28 ± 0.14 |
8.07 ± 2.16* |
LMR27 |
Pseudomonas putida |
0.11 ± 0.04 |
14.54 ± 3.24* |
LMR51 |
Herbaspirillum huttiense |
0.24 ± 0.04 |
31.19 ± 17.54* |
LMR54 |
Bacillus sp. |
0.51 ± 0.14 |
4.24 ± 0.57* |
LMR64 |
Rhizobium galegae |
0.51 ± 0.23 |
58.51 ± 9.92* |
LMR79 |
Rhizobium leguminosarum |
0.26 ± 0.07 |
2.2 ± 1.12* |
LMR80 |
Serratia proteamaculans |
0.42 ± 0.34 |
11.05 ± 5.65* |
Results were expressed as means ± Standard Deviation (SD) (n = 3).
Asterisks indicate significant differences compared to the control (p <
0.05).
Table 3: In vitro screening for NH3 production and phosphate
solubilization abilities
Strain |
Ammonia production |
Phosphate solubilization |
|
|
Soluble phosphate (ppm) |
pH |
LMR23 |
+++ |
90.91 ± 7.95 |
4.4 ± 0.21 |
LMR27 |
+++ |
54.16 ± 4.75 |
4.5 ± 0.2 |
LMR51 |
+++ |
55.95 ± 2.98 |
4.7 ± 0.41 |
LMR54 |
++ |
51.79 ± 11.18 |
4.4 ± 0.61 |
LMR64 |
++ |
27.25 ± 5.49 |
4.5 ± 0.1 |
LMR79 |
++ |
54.54 ± 2.35 |
4.7 ± 0.17 |
LMR80 |
++ |
64.12 ± 2.71 |
5.09 ± 0.27 |
Results were expressed as means ± Standard Deviation (SD) (n = 3).
(++) intermediate, (+++) strong production
Fresh weights were increased by 44% (LMR23), 41% (LMR51)
and 43% (LMR23/51) for these shoots and 59% (LMR23), 52%
(LMR51) and 57% (LMR23/51) for the roots. Bacteria belonging
to Pseudomonas genus are known for their beneficial effects that
may ameliorate heavy metals phytoextraction [52]. It has been
also shown that Pseudomonas fluorescens strain MH15 was able
to increase the accumulation of Zn, Cd and Cu in the tissues of
Table 4: Effect of inoculation of Hedysarum spinosissimum with 2 bacterial isolates on the plants fresh weight and Zn uptake
|
|
Plant fresh weight(mg) |
Zn concentration (ppm) |
Added Zn
in plant medium (µM) |
Treatment |
Shoots |
Roots |
Shoots |
Roots |
Translocation factor (Tf) |
2000 |
Inoculated
with LMR23 |
47.96 ± 22.1 |
219.9 ± 62.06* |
1093.76 ± 143.84* |
19695 ± 3677.73* |
0.05 ± 0.006 |
2000 |
Inoculated
with LMR51 |
51.83 ± 3.8* |
251.76 ± 32.15* |
1860.54 ± 405.06* |
7100.03 ± 2735.83* |
0.27 ± 0.08* |
2000 |
Inoculated with LMR23/51 |
49.93 ± 9.15* |
228.5 ± 43.41* |
699.58 ±150.38 |
5136.51 ± 1807.66 |
0.15 ± 0.08 |
0 |
Uninoculated |
110.36 ± 39.57 |
606.93 ± 154.78 |
41.06 ± 4.65 |
98.26 ± 16.03 |
0.42 ± 0.04 |
2000 |
Uninoculated |
21.23 ± 5.22 |
129.8 ± 20.13 |
397.47 ± 82.36 |
3590.12 ± 541.37 |
0.11 ± 0.01 |
Results were expressed as means ± Standard Deviation (SD) of three independent experiments. Asterisks indicate significant differences compared
to the control (uninoculated treatments) at p < 0.05
Sinapis alba, which that is consistent with our results. Many
rhizobacteria and endophytic bacteria, including Pseudomonas,
associated with yellow lupine, are also known to promote the
growth of this plant and to increase its resistance and extraction
capacity of Cd [53]. However, in our study, the accumulation
of Zn was higher at the root level compared to the aerial part
thus reducing translocation factor. Zn maximum accumulation
was observed when inoculating the Pseudomonas putida strain
alone, remarkably in the root level, which indicates a very low
Zn translocation to aboveground biomass (Tf = 0.05 ± 0.006),
suggesting an important phytostabilisation potential. In a recent
study, the inoculation of poplar roots with a Pseudomonas
fluorescens strain resulted in improved Cd absorption in
the roots, showing the interest to use such bacteria in a Cd
phytostabilisation program [54]. The use of several bacterial
strains presenting different beneficial features is also often
described as a very effective strategy to improve plant growth
and activity of phytoremediation [55].
Conclusions
The metal contaminated soils harbor a diverse group
of microorganisms [56] among which some are capable of
enhancing the effectiveness of phytoremediation [8,57]. This
study analyzed nodule bacteria population of the legume
Hedysarum spinosissimum and describes the potential of
different bacterial strains to promote plant growth and enhance
Zn phytostabilization potential. Growing in heavy metalscontaminated
soils, this plant hosted a high diversity of endophytic
bacteria inside their root nodules. These endophytes, that belong
to 8 different genera, are resistant to high concentrations of
Pb and Zn, produced multiple PGP traits such as, phosphate
solubilization or ammonia production with a great potential
of Zn bioaccumulation. Inoculation of Pseudomonas putida
LMR23 and Herbaspirillum huttiense LMR51 of H. spinosissimum
seedlings, grown in a nutrient solution supplemented with the
minimal inhibitory concentration of Zn enabled plant survival
and growth. By accumulating Zn mainly in the roots, these
associations may further be tested for their phytostabilisation
potential. The bacteria identified here may thus be used as bioinoculants in situ and may constitute a biological alternative to
improve phytostabilization efficiency in the contaminated sites
from where they originate. However, the molecular mechanisms
whereby these bacteria help the plant to tolerate metals remain
to be elucidated [58].
Conflict of Interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors warmly thanks GEODERIS the organization in
charge of technical expertise in post- mining risk assessment in
France for its collaboration and helping analyzing soil samples.
This study was financed, in part, by the EC2CO program (INEE,
CNRS).
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