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REGULAR ARTICLE
Rhizobacterial communities associated with the flora of three
serpentine outcrops of the Iberian Peninsula
Vanessa Álvarez-López &Ángeles Prieto-Fernández &
Cristina Becerra-Castro &Carmela Monterroso &Petra S. Kidd
Received: 15 May 2015 /Accepted: 6 August 2015
#Springer International Publishing Switzerland 2015
Abstract
Aim Plant-associated bacteria can improve
phytoextraction by increasing plant growth and/or metal
uptake. This study aimed to characterise the culturable
rhizobacterial community associated with two Ni-
hyperaccumulators and to obtain a collection of isolates
for application in Ni phytomining.
Methods Non-vegetated and rhizosphere soil samples
were collected from the Ni-hyperaccumulator Alyssum
serpyllifolium ssp. lusitanicum (three populations) and
Alyssum serpyllifolium ssp. malacitanum (one popula-
tion), as well as from non-hyperaccumulating plants
(Dactylis glomerata,Santolina semidentata and
Alyssum serpyllifolium ssp. serpyllifolium).
Rhizobacteria were isolated and characterised genotyp-
ically (BOX-PCR, 16S rDNA sequencing) and pheno-
typically (Ni tolerance, plant growth promoting (PGP)
traits, biosurfactant production).
Results Hyperaccumulating Alyssum subspecies hosted
higher densities of bacteria compared to either non-
hyperaccumulators or non-vegetated soil. In some cases
hyperaccumulators showed selective enrichment of Ni-
tolerant bacteria. Most bacterial strains belonged to the
Actinobacteria phylum and presented Ni resistance.
Phosphorus-solubilisers were mostly associated with
the hyperaccumulators, siderophore-producers with
D. glomerata, and IAA-producers with both these
species.
Conclusion Taxonomic diversity and phenotypic char-
acteristics were soil-, plant species- and plant popula-
tion-specific. Moreover, differences were observed be-
tween the two Ni-hyperaccumulating subspecies and
amongst plant populations. Several strains presented
PGP characteristics which could be useful when
selecting microorganisms for bioaugmentation trials.
Keywords Alyssum serpyllifolium .
Hyperaccumulators .Nickel .Plant-associated bacteria .
PGP traits .Bacterial diversity .Phytoextraction
Introduction
Serpentine soils are derived from ultramafic rocks, where
the term ultramafic refers to igneous or metamorphic
rocks containing more than 70 % of ferromagnesium
Plant Soil
DOI 10.1007/s11104-015-2632-0
Responsible Editor: Antony Van der Ent.
Electronic supplementary material The online version of this
article (doi:10.1007/s11104-015-2632-0) contains supplementary
material, which is available to authorized users.
V. Álvarez-López (*):Á. Prieto-Fernández:
C. Becerra-Castro :P. S. Kidd
Instituto de Investigaciones Agrobiológicas de Galicia, CSIC,
Apdo. 122, Santiago de Compostela 15780, Spain
e-mail: vanessa@iiag.csic.es
C. Monterroso
Departamento Edafoloxía e Química Agrícola, Universidade de
Santiago de Compostela, Santiago de Compostela 15706, Spain
Present Address:
C. Becerra-Castro
CBQF –Centro de Biotecnologia e Química Fina –
Laboratório Associado, Escola Superior de Biotecnologia,
Universidade Católica Portuguesa, Rua Arquiteto Lobão
Vital, Apartado 2511, 4202-401 Porto, Portugal
minerals and a low content in silicon (<45 % SiO
2
)
(Brooks 1987). Ultramafic rocks are patchily distributed
throughout the world (occupying approximately 1 % of
the earth’s surface area; (Proctor 1999)) and are well-
known for their physical and chemical anomalies that
present a hostile environment for plant growth. Some
common traits include an elevated concentration of Mg
and Fe, a low availability of Ca relative to Mg
(unfavourable for Ca absorption), a deficiency in essen-
tial nutrients such as N, P and K, and high concentrations
of potentially phytotoxic trace metals such as Ni,
Co and Cr. As a result the plant communities in
these areas often present a high number of endem-
ic species (serpentinophytes), and have evolved
both morphological and physiological adaptations
differentiating them from the flora of adjacent
geological substrates (Brooks 1987). Serpentine
flora includes an unusual plant group, the so-
called hyperaccumulators, which are able to accu-
mulate extremely high concentrations of Ni in their
aerial biomass (>1000 mg kg
−1
dry weight (DW)
matter) (Baker and Brooks 1989;Chaneyetal.
2007). The Iberian Peninsula hosts two subspecies
of Alyssum serpyllifolium Desf. which are both
serpentine-endemic and hyperaccumulators of Ni:
Alyssum serpyllifolium ssp. lusitanicum from
Galicia (NW Spain) and Trás-os-Montes (NE
Portugal), and Alyssum serpyllifolium ssp.
malacitanum from Andalucía (S Spain).
Nickel-hyperaccumulating plants are considered ide-
al candidates for application in phytomining, a non-
destructive approach for the recovery of high value
metals (e.g. Ni) from metal-enriched soils and ores.
Plants are cultivated to accumulate trace metals from
soils and transport them to the shoots which can then be
harvested (Chaney et al. 2007). Bioaugmentation using
bacteria associated with hyperaccumulators can im-
prove the plant’s capacity to phytoextract metals from
soils (Becerra-Castro et al. 2013; Mengoni et al. 2010;
Sessitsch et al. 2013). Plant-associated bacteria can en-
hance plant growth, reduce stress and/or modify soil
metal bioavailability (Becerra-Castro et al. 2013;
Cabello-Conejo et al. 2014; Glick 2014; Kidd et al.
2009; Lebeau et al. 2008; Sessitsch et al. 2013).
Serpentine soils are a potential source of metal-tolerant
(Co, Cr and Ni) bacteria (Pal et al. 2005). Schlegel et al.
(1991) found bacterial strains isolated from serpentine
soils tolerated up to 10–20 mM Ni (in the culture medi-
um), while strains from other soil types tolerated only
1 mM Ni. Pal et al. (2007) found that bacterial strains
isolated from the rhizosphere of the Ni-
hyperaccumulators Rinorea bengalensis and
Dichapetalum gelonoides tolerated up to 28.9 mM of
Ni in the culture medium, while Turgay et al. (2012)
found that, bacterial strains isolated from Turkish ser-
pentine soils, tolerated up to 34 mM Ni in the growth
medium. Furthermore, the rhizosphere bacterial com-
munities associated with Ni-hyperaccumulating plants
have been shown to differ from those of non-
accumulating plants growing at the same site or
of non-vegetated soil, and are also characterised by
a higher number of Ni-tolerant bacteria (Abou-
Shanab et al. 2003;Idrisetal.2004; Mengoni
et al. 2001;Schlegeletal.1991). This selective
enrichment in Ni-tolerant bacteria in the rhizo-
sphere has been correlated with an increase in soil
Ni availability (Becerra-Castro et al. 2009).
Amongst the culturable Ni-tolerant bacterial strains
isolated from serpentine soils and/or associated
with Ni-hyperaccumulating plants, members of
the Actinobacteria,Acidobacteria,Chlorobi,
Firmicutes,Verrucomicrobia and Proteobacteria
have been described (Mengoni et al. 2001;Oline
2006; Pal et al. 2007;Turgayetal.2012).
In this study, the culturable bacterial community
associated with different populations of the two
Ni-hyperaccumulating subspecies of Alyssum
serpyllifolium (subsp. lusitanicum and subsp.
malacitanum) of the Iberian Peninsula were
characterised. The rhizosphere bacterial communi-
ties associated with these plants were compared
with those of non-hyperaccumulating plant species
(the Ni-excluder Dactylis glomerata and the facul-
tative serpentinophyte Santolina semidentata)
growing at the same sites, as well as with the
non-hyperaccumulating Alyssum serpyllifolium
subsp. serpyllifolium growing in calcareous soils
(developed over limestone and dolomite) in Sierra
Nevada (S Spain). A collection of rhizobacterial
isolates was obtained and the strains were
characterised both genotypically (BOX-PCR and
16S rDNA partial sequencing) and phenotypically
(for their resistance to Ni and plant growth pro-
moting (PGP) traits). We focused on the culturable
bacterial community since the global aim is to
obtain potentially useful isolates which can be
used to improve the Ni phytoextraction capacity
of Ni-(hyper)accumulating plant species.
Plant Soil
Materials and methods
Study areas and collection of samples
The study was carried out in five areas of the Iberian
Peninsula in which three subspecies of Alyssum
serpyllifolium Desf. (Brassicaceae) are found growing.
Four of these areas are serpentine outcrops: two sites in
Trás-os-Montes (Samil (S; 41°46′48″N; 6°44′47″W)
and Morais (M; 41°31′21″N; 06°49′20″W), NW
Portugal), one site in Barazón (L) (42°51′09″N; 08°01′
15″W, NW Spain) and one in Sierra Bermeja (SB)
(36°28′48″N; 05°11′52″W, S Spain). These serpentinitic
areas host the two Ni-hyperaccumulating subspecies of
A. serpyllifolium whichareendemictotheIberian
Peninsula: A. serpyllifolium subsp. lusitanicum Dudley
and P. Silva (hereafter referred to as A. pintodasilvae)in
S, M and L, and A. serpyllifolium ssp. malacitanum
Rivas Goday (hereafter referred to as A. malacitanum)
in SB. The fifth sampling site was the calcareous dolo-
mitic area of Sierra Nevada (SN) (37°7′21″N; 03°26′53″
W, S Spain) where the non-hyperaccumulator
A. serpyllifolium subsp. serpyllifolium Desfontaines
grows (hereafter referred to as A. serpyllifolium). Both
Ni-hyperaccumulating subspecies presented leaf Ni
concentrations above 10 g kg
−1
and the highest Ni
accumulation was found in the Spanish population of
L(15.5gkg
−1
; Table 1). For comparative purposes non-
hyperaccumulating plants were also collected in some
study sites: Dactylis glomerata L. was sampled at sites
M, S and L, Santolina semidentata Hoffmanns. & Link.
was sampled at sites M and S.
Trás-os-Montes has a Mediterranean climate, with a
mean annual temperature of 12.4 °C and annual precip-
itation of 720 mm (Carballeira et al. 1983;Menezesde
Sequeira and Pinto da Silva 1992). Barazón has a
European humid-temperate climate with a mean annual
temperature of 12.9 °C and mean annual precipitation of
1381 mm (Carballeira et al. 1983). Sierra Bermeja and
Sierra Nevada have a Mediterranean oceanic climate
(Rivas-Martínez and Rivas-Saenz 1996–2009): Sierra
Bermeja has a mean annual precipitation between 800
and 1600 mm and mean annual temperature between 14
and 16 °C (Gómez-Zotano et al. 2014) and Sierra
Nevada has a mean temperature of 12 °C and mean
annual precipitation between 450 and 1000 mm
(Castillo-Martín 2000).
At each site, 5 to 7 samples of non-vegetated soil (0–
15 cm) or of rhizosphere soil were collected for the
isolation of bacterial strains. The whole plant and root
system (including root-adhering soil) of each species
were collected at late flowering stage, and after gently
crushing the root ball the tightly held soil (<3mmfrom
the root surface) was considered as rhizosphere soil.
Samples were sieved (<4 mm) and kept at 4 °C until
processing. Soil samples are named as follow: L, M, S,
SB and SN according to the sampling site (Barazón,
Morais, Samil, Sierra Bermeja and Sierra Nevada, re-
spectively); and as A, G and S according to the plant
species (Alyssum,Dactylis and Santolina,respectively).
Rhizosphere soil is indicated by an R and the non-
vegetated soil by NV.
Elemental analysis of field-collected soils and plant
material
Non-vegetated (NV) and rhizosphere samples were air-
dried and sieved through a 2-mm stainless steel sieve.
Soil pH was measured in H
2
OandKClusinga1:2.5
soil:solution ratio. Total C and N were analysed by
combustion with a CHN analyser (Model CHN-1000,
LECO Corp., St Joseph, MI). Exchangeable cations (Ca,
Mg, Al, Na and K) were extracted with 1 M NH
4
Cl and
determined by inductively coupled plasma optical emis-
sion spectrometry (ICP-OES, model Vista-PRO,
Varian). Soils were digested in a 3:1 mixture of concen-
trated HNO
3
:HCl and the total concentrations of Co, Cr
and Ni were analysed by ICP-OES. Soil metal availabil-
ity was evaluated after extraction with water after 24 h
shaking using a 1:2.5 soil:H
2
Oratio.
Plant material collected in the field was separated in
shoots and roots, washed with pressurised tap water
followedbydeionisedwater,oven–driedat45°C,
weighed and ground. Plant shoots (approximately
0.1 g) were digested in a 2:1 concentrated HNO
3
:HCl
mixture on a hot plate at 120 °C, and the concentration
of metals was measured by ICP-OES.
Microbiological analyses
Five grams of fresh rhizosphere soil were suspended in
45 mL sterile sodium hexametaphosphate solution (1 %)
and shaken for 30 min in an end-over-end shaker. Soil
suspensions were diluted in 10-fold series and plated in
duplicate onto 284 agar medium (Schlegel et al. 1991)
supplemented with 100 μgmL
−1
of the fungicide cy-
cloheximide. The 284 medium contains (per litre):
6.06 g Tris–HCl, 4.68 g NaCl, 1.49 g KCl, 1.07 g
Plant Soil
NH
4
Cl, 0.43 g Na
2
SO
4
, 0.2 g MgCl
2
.6H
2
O, 0.03 g
CaCl
2
.2H
2
O, 0.04 g Na
2
HPO
4
.2H
2
O, 10 mL
Fe(III)NH
4
citrate solution (containing 48 mg/100 mL)
plus oligoelements (1.5 mg FeSO
4
.7H
2
O, 0.3 mg
H
3
BO
4
, 0.19 mg CoCl
2
.H
2
O, 0.1 mg MnCl
2
.4H
2
O,
0.08 mg ZnSO
4
.7H
2
O, 0.02 mg CuSO
4
.5H
2
O,
0.036 mg Na
2
MoO
4
.2H
2
O) adjusted to pH 7. The me-
dium was supplemented with a mixture of different
carbon sources: lactate (0.7 g L
−1
), glucose (0.5 g L
−1
),
gluconate (0.7 g L
−1
), fructose (0.5 g L
−1
), and succinate
(0.8 g L
−1
). Densities of metal tolerant bacteria were
determined in 284 agar media supplemented with an
increasing concentration of Ni (0 mM, 0.5 mM,
1.0 mM, 2.0 mM, 3.0 mM; added as NiSO
4
.6H
2
O).
After 7 days incubation at 28 °C, colony forming units
(CFUs) were counted and calculated per gram DW soil.
Distinct colony morphotypes from Ni-enriched media
associated with each plant species and from each site
were sub-cultured at least three times to ensure purity
and cryopreserved at −70 °C in culture medium supple-
mented with 15 % (v/v) glycerol.
Phenotypic characterisation of bacterial isolates
Rhizobacterial strains were screened for Ni resistance,
the ability to produce biosurfactants, and for various
plant growth promoting characteristics: phosphate
solubilisation capacity, siderophore production, organic
acid production, and indoleacetic acid (IAA) produc-
tion. Nickel resistance of the strains was tested using
284 agar medium (see above) supplemented with an
increasing concentration of Ni (0 mM, 1.0 mM,
2.5 mM, 5.0 mM, 10.0 mM; added as NiSO
4
.6H
2
O)
and incubated at 28 °C for 7 days. The Maximal
Tolerable Concentration (MTC) of Ni was recorded for
each isolate, as the highest Ni concentration tested
where the isolate was able to grow. The ability to solu-
bilise inorganic phosphate was assessed in a modified
NBRIP agar medium (1.8 %) supplied with 5 g L
−1
of
hydroxyapatite and incubated at28 °C for 5 days (10.0 g
glucose, 5.0 g MgCl
2
.6H
2
O, 0.25 g MgSO
4
.7H
2
O, 0.2 g
KCl, 0.1 g (NH
4
)
2
SO
4
, 0.1 g yeast extract in 1 L deion-
ized water adjusted to pH 7.0; modified from (Nautiyal
Tabl e 1 Leaf Ni accumulation and soil physicochemical characteristics (mean±SE) at the different study sites. L (Barazón, NW Spain), S
and M (Samil and Morais, NE Portugal), SB and SN (Sierra Bermeja and Sierra Nevada, S Spain)
LSMSBSN
Leaf Ni concentration (g kg
−1
)
Alyssum 15.46±0.72 11.02±0.12 10.88±0.74 10.39±0.77 <LOQ
Dactylis 0.06± 0.01 0.35±0.07 0.14±0.03 - -
Santolina - 0.19±0.04 0.23±0.02 - <LOQ
Soil physicochemical properties
pH (H
2
O) 7.2±0.1 6.9±0.1 6.9 ±0.1 7.4±0.0 8.0±0.0
pH (KCl) 6.0±0.1 6.3±0.1 6.2 ±0.1 5.6±0.0 7.2±0.0
Total C (%) 1.28±0.11 3.05±0.24 3.0±0.27 2.42±0.10 9.73±0.05
Total N (%) 0.10±0.01 0.28±0.02 0.26±0.02 0.21±0.00 0.46±0.01
C/N 12.7±0.6 10.8±0.4 11.5±0.3 11.5± 0.4 21.2±0.2
CEC (cmol
c
kg
−1
) 11.8±0.3 23.2±1.1 22.4±0.5 25.9±0.5 23.9±0.5
Ca/Mg 0.3±0.0 0.6±0.1 0.3 ±0.0 0.5±0.0 2.9±0.0
Total N i (g k g
−1
) 2.20±0.20 3.03±0.12 2.72±0.04 2.10±0.03 0.03±0.0
Water soluble Ni (mg kg
−1
)
Non-vegetated soil 0.97±0.12 1.40±0.23 0.81±0.03 0.33±0.02 <LOQ.
Rhizosphere soil
Alyssum 0.74± 0.17 1.09±0.30 2.52±0.58 0.69± 0.12 <LOQ
Dactylis 1.42±0.31 1.18±0.21 2.12±1.63 - <LOQ
Santolina - 1.03±0.09 1.13±0.18 - -
LOQ limit of quantification; 0.025 mg kg
−1
for water soluble Ni and 0.005 g kg
−1
for leaf Ni concentration
Plant Soil
1999)). A clear halo around the bacterial colony indi-
cated solubilisation of mineral phosphate. Yeast extract
was added since some strains were unable to grow in
yeast-freeNBRIP medium. Siderophore production was
detected in a modified 284 liquid medium (without Fe)
using the Chrome Azurol S (CAS) method described by
Schwyn and Neilands (1987). All glassware used in this
assay was previously cleaned with 30 % HNO
3
follow-
ed by washing in distilled water (Cox 1994). Each
isolate wasscreened for acid production. Single colonies
were plated on agar medium containing 0.002 %
bromocresol purple (per litre medium): 10.0 g glucose,
1.0 g tryptone, 0.5 g yeast extract, 0.5 g NaCl, 0.03 g
CaCl
2
.2H
2
O. Colonies forming a yellow halo after
1 day of growth at 28 °C indicated a pH change in the
medium and were considered acid producers. The abil-
ity of isolated strains to produce IAA was evaluated in
liquid medium (5.0 g glucose, 1.0 g (NH
4
)
2
SO
4
,2.0g
K
2
HPO
4
,0.5gCaCO
3
,0.5gMgSO
4
.7H
2
O, 0.1 g NaCl,
0.1 g yeast extract adjusted to pH 7; modified from
Sheng et al. (2008) supplemented with 0.5 mg mL
−1
of tryptophan). After 5 days incubation at 28 °C, cul-
tures were centrifuged and the supernatant was incubat-
ed with Salkowski reagent for 25 min. The production
of IAAwas recognized by the presence of red colouring
and isolates were considered IAA-producers when the
concentration of IAA determined was more than
4mgL
−1
culture. Strains were screened for potential
biosurfactant production using the qualitative method of
Chen et al. (2007). The strains were inoculated in 284
liquid medium and cultured overnight at 28 °C, at
150 rpm on a rotary shaker. A 100 μL sample was taken
from the supernatant of each strain and added to a
microwell of a 96-microwell plate. The plate was then
viewed using a backing sheet of paper with a black and
white grid. The optical distortion of the grid provided a
qualitative assay for the presence of surfactants.
Isolates were classified in different phenotype groups,
according to their metal resistance (low Ni tolerance (LT):
≤2.5 mM Ni MTC, or high Ni tolerance (HT): 5–10 mM
Ni MTC) and PGP traits (N: non PGP traits producers; A:
organic acid-producers; P: P-solubilisers; S: Siderophore-
producers; T: biosurfactants-producers; H: indoleacetic
acid-producers; and their possible combinations).
Eighty four out of the 550 rhizobacterial isolates
obtained were already phenotypically characterised
in a prior study and used to evaluate the potential
of cell-free cultures to mobilise soil Ni under
in vitro conditions (Becerra-Castro et al. 2011).
This previous study targeted only rhizobacterial
strains associated with Alyssum sp. and their capac-
ity for soil Ni mobilisation.
Genotypic characterisation of bacterial isolates
BOX-PCR genomic DNA profiling
BOX-PCR fingerprinting was used to genotype and
group bacterial strains within each isolate collection (L,
S, M, SB, SN). Crude cell lysates (colonies suspended in
100μL and heated to 100 °C for 5 min) were used as
DNA templates for BOX-PCR reactions. Box reactions
were performed in a total volume of 20 μL containing: 1x
Taq buffer (Invitrogen), 1.5 mM MgCl
2
, 0.1 mM of each
dNTP, 0.5U Taq polymerase (Invitrogen), 2 μMofBOX
A1R primer (5′- CTACGGCAAGGCGACGCTGACG-
3′) (Versalovic et al. 1994), and 2 μLofcelllysate.
Thermocycling conditions were: 1 cycle of 94 °C for
5min;35cyclesof1minat94°C;1.5minat50°C
and 8 min at 68 °C; and 1 cycle of 8 min at 68 °C. The
obtained PCR products were separated by gel electropho-
resisin1.8%agaroserunfor3hat3.3Vcm
−1
gel. Gel
images were analysed, using the Pearson correlation
coefficient and UPGMA clustering algorithm of the Gel
Compar Bionumerics program (Bionumerics Version
6.6, Applied Maths, Belgium). Rhizobacterial isolates
weregroupedaccordingtotheirBOX-PCRprofilesata
similarity level of 82 %. BOX-PCR fingerprints were
analysed for each sampling site (L, S, M, SB and SN)
and moreover, bacterial strains associated with the Ni-
hyperaccumulator Alyssum subspecies were analysed
separately (LA, SA, MA and SBA). Genetic diversity
of isolates was assessed for each plant species and pop-
ulation using the Shannon diversity index (H′=−∑(x
i
/
x
0
)ln(x
i
/x
0
)) where x
i
is the number of strains per BOX-
PCR group for each level of metal tolerance (LT or HT),
and x
0
is the total number of strains in either LT or HT.
Isolates within each BOX-PCR group were also classi-
fied according to the phenotypes (PGP traits and Ni
resistance) described above.
DNA extraction and 16S rRNA gene amplification
For DNA extraction, purified strains were grown in 1/10
strength 869 liquid medium (1.0 g tryptone, 0.5 g yeast
extract,0.5gNaCl,0.1gglucose,0.035gCaCl
2
.2H
2
O
in 1 L deionised water (Mergeay et al. 1985)) and
genomic DNAwas extracted from bacterial cell pellets.
Plant Soil
Briefly, the method consists of alkaline cell lysis follow-
ed by phenol/chloroform/isopropanol alcohol purifica-
tion. DNA quality was checked by gel electrophoresis
on a 0.8 % agarose gel. PCR amplification targeting the
16S rRNA gene was carried out using the primers 16S-
27F (5′-AGAGTTTGATCMTGGCTCAG-3′)and16S-
1492R (5′-TACGGYTACCTTGTTA CGACTT-3′)
(Lane 1991). PCR reactions were performed in a total
volume of 50 μL containing: 1x Taq buffer (Invitrogen),
2.5 mM MgCl
2
, 0.1 mM of each dNTP, 1.75U Taq
polymerase (Invitrogen), 0.4 μM of each primer, and
1μL of extracted DNA. Thermocycling conditions
were: 2 min at 94 °C; 30 cycles of 1 min at 94 °C;
1 min at 55 °C and 2 min at72 °C; and 1 cycle of 10 min
at 72 °C. PCR products were partially sequenced (be-
tween 750 and 1000 bases) using the primer 16S-27F
(Lane 1991). Sequence data were checked using the
Chromas v. 1.45 software (Technelysium Pty. Ltd.,
Australia), uploaded and aligned in the Ribosomal
Database Project (RDP; Cole et al. (2009)) and assessed
for similarity with sequences in the RDP. The sequences
used for identification of the culturable strains are avail-
able in the EMBL database (www.ebi.ac.uk)under
accession numbers HG941722 - HG942010,
HE646570 - HE646571, and FN908759 - FN908797
(the latter group were identified and described for Ni
mobilisation capacity by Becerra-Castro et al. 2011).
Genetic diversity of isolates was assessed for each
plant species and population using the Shannon diversity
index (H′=−∑(x
i
/x
0
)ln(x
i
/x
0
)) where x
i
is the number of
strains per genera, and x
0
is the total number of strains.
Statistical analyses
Analyses of variance (ANOVA) were used to detect
statistically significant differences between bacterial
counts. Comparisons of prevalence of PGP phenotypes
(Plant Growth Promoting traits) between plant species
were achieved through a chi-squared test or Fisher’s
exact test when necessary. Statistical analyses were per-
formed using SPSS v.22.0, SPSS Inc., Chicago, IL.
Results
General soil physicochemical properties
Some general physicochemical properties of the soils at
each site are given in Table 1, as well as the total
concentration of Ni in aboveground shootsof each plant
species studied. All four serpentine soils were
characterised by pH values close to neutrality, a low C
and N content, a predominance of Mg in the exchange
complex, and Ca/Mg quotients of <1. In contrast, SN
soil presented alkaline pH values and Ca dominating the
cation exchange complex, higher organic matter content
and C/N ratio. Total Ni concentrations were higher in
serpentine soils than the calcareous SN soil. Values were
similar in the Portuguese sites (S and M; 3.03 and
2.72 g kg
−1
, respectively) and lower in the L and SB
soils (2.20 and 2.10 g kg
−1
, respectively). In contrast,
total Ni in SN calcareous soil was only 0.03 mg kg
−1
.In
accordance, labile Ni (water-soluble) was highest in the
S site. Water-soluble Ni concentrations were higher in
the rhizosphere soil of the M population of the Ni-
hyperaccumulator compared to the non-
hyperaccumulators D. glomerata (1.2-fold lower) and
S. semidentata (2.2-fold lower) or the non-vegetated soil
(3.1-fold lower). Also the rhizosphere soil of the Ni-
hyperaccumulator in SB presented a higher concentra-
tion of water-soluble Ni than the non-vegetated soil
(2.1-fold lower).
Culturable bacterial densities and the rhizosphere effect
The densities of culturable bacteria in non-vegetated and
rhizosphere soils of the different plant species and sites
are presented in Table 2. Bacterial densities in non-
vegetated serpentine soils did not differ significantly,
and ranged from 3.7×10
7
CFU g
−1
DW soil in L
to 7.9×10
7
CFU g
−1
DW soil in SB (Tukey post-
hoc test showed no differences between NV serpen-
tine soils; p>0.05). On the other hand, bacterial
densities in non-vegetated soils of SN were at least
10-fold higher than those of the serpentine sites
(5.2×10
8
CFU g
−1
DW soil).
The effect of the plant on bacterial densities (rhizo-
sphere effect) was assessed using the R / NV ratio
(CFUs g
−1
rhizosphere soil / CFUs g
−1
non-vegetated
soil) (Table 2). Bacterial densities were higher (albeit not
always significantly) in the rhizosphere of all the plant
species compared to non-vegetated soil, with the excep-
tion of D. glomerata in M (R / NV of 0.9). The highest
densities were always associated with the Alyssum sub-
species, and this was the case for all five populations.
The increase in bacterial densities in the rhizosphere
ranged from 1.3-fold in the S population of
D. glomerata to 4.0-fold in the L population of
Plant Soil
A. pintodasilvae. The most pronounced rhizosphere ef-
fect was observed in the L population of
A. pintodasilvae (R / NV ratio of 4; p<0.001), where
densities were up to one order of magnitude higher in
the rhizosphere soil compared to non-vegetated soil. On
the other hand, the biggest differences in rhizosphere
bacterial densities between different plant species grow-
ing at the same site were observed in M. In this case the
bacterial densities associated with A. pintodasilvae were
1.7× 10
8
CFU g
−1
soil, which was 3.4- and 2.3-fold
higher than those found in the rhizosphere soil of
D. glomerata (7.5 × 10
7
CFU g
−1
soil; p<0.05) and
S. semidentata (5.0×10
7
CFU g
−1
soil; p<0.001), re-
spectively. In fact, the rhizosphere bacterial densities
associated with S. semidentata from this site (5.0×
10
7
CFU g
−1
soil) were similar to those determined in
non-vegetated soil (5.5× 10
7
CFU g
−1
soil).
Nickel resistance of the culturable bacterial community
Bacterial densities of soils (NVand rhizospheric) plated
in culture media supplemented with Ni decreased with
an increase in the medium Ni concentration, and this
was observed for all plant species and populations
(Table 2). Compared to the total bacterial population,
densities of Ni-resistant bacteria in serpentine soils were
reduced by at most one order of magnitude at the highest
metal concentration tested. In contrast, in SN, densities
were reduced by up to three orders of magnitude: in
non-vegetated soil, densities decreased from 5.2×
10
8
CFU g
−1
soil (0 mM Ni) to 6.0×10
5
CFU g
−1
soil
at the highest Ni concentration (3 mM) and in
A. serpyllifolium rhizosphere soil they fell from 1.1 ×
10
9
CFU g
−1
soil (0 mM Ni) to 1.6×10
6
CFU g
−1
soil
(3 mM Ni).
Tabl e 2 Colony forming units per gram dry weight soil (CFUs *
10
6
g
−1
±SE) in non-vegetated and rhizosphere soils, and the ratio
rhizosphere (R): non-vegetated soil (NV), of the serpentine
(Barazón, Samil, Morais, S. Bermeja) and the calcareous soils (S.
Nevada). Different letters indicate significant differences between
plant species and non-vegetated soil at the same site (p<0.05). NV
(non-vegetated soil), LA (A. pintodasilvae from Barazón), LG
(D. glomerata from Barazón), SA (A. pintodasilvae from Samil),
SG (D. glomerata from Samil), SS (S. semidentata from Samil),
MA (A. pintodasilvae from Morais), MG (D. glomerata from
Morais), MS (S. semidentata from Morais), SBA (A. malacitanum
from S. Bermeja) and SNA (A. serpyllifolium from S. Nevada)
Control Ni 0.5 mM Ni 1 mM Ni 2 mM Ni 3 mM
CFUs * 10
6
R/NV CFUs * 10
6
R/NV CFUs * 10
6
R/NV CFUs * 10
6
R/NV CFUs * 10
6
R/NV
Barazón
NV 37.4±28.3 a20.6±16.1 a10.6± 10.3 a5.2±6.5 a2.7±3.3 a
LA 149.5±20.1 b4.0 68.1±14.3 b3.3 40.3±10.4 b3.8 15.6±6.3 a37.3±3.1a2.7
LG 115.2±20.3 b3.1 44.4±7.8 ab 2.2 30.8±6.5 ab 2.9 17.1± 3.4 a3.3 9.1±2.1 a3.3
Samil
NV 50.9±6.4 a47.0±6.3 a28.3±2.3 a15.1± 1.1 a7.6± 1.3 a
SA 98.8±10.0 b1.9 49.1±9.2 a1 36.8±6.3 a1.2 23.4±6.1 a1.6 15.1± 3.2 b2.2
SG 75.7±13.4 ab 1.3 42.8±5.1 a0.9 25.6±1.8 a0.9 16.2±3.8 a1.1 10.9±0.6 ab 1.4
SS 67.5±5.3 ab 1.5 47.8±6.0 a1 35.0±5.8 a1.2 21.6±2.9 a1.4 11.8 ±1.7 ab 1.6
Morais
NV 54.6±9.6 a30.2±6.0 a22.9±2.8 a10.2± 1.1 a5.4± 1.4 a
MA 171.6±19.7 b3.1 120.1± 14.5 b4 97.6 ±15.9 b4.3 62.0±6.7 b6.1 20.3±4.3 b3.7
MG 74.8±7.0 a0.9 24.8± 3.3 a0.8 18.2±1.4 a0.8 10.0±2.4 a15.5±1.4a1
MS 50.0±4.8 a1.4 23.7± 0.4 a0.8 29.6± 2.5 a1.3 14.9±5.3 a1.5 9.3±4.7 a1.7
S. Bermeja
NV 79.4±7.8 a25.8±1.0 a15.3±1.8 a5.0±0.4 a2.3± 0.1 a
SBA 237.8 ±40.6 b3.0 96.1±9.0 b3.7 63.6±14.2 b3.7 24.6±4.7 b5 11.2±1.6 b4.9
S. Nevada
NV 520.0±33.4 a 16.1±3.0 a4.6±1.4 a1.8±0.6 a0.6±0.2 a
SNA 1083.0±179.9 b2.1 41.0±9.5 a2.5 12.7±2.4 b2.8 2.0± 0.7 a1.2 1.6±0.6 a2.6
Plant Soil
As observed for the total culturable bacterial popula-
tion, the Ni-resistant population (CFU g
−1
soil) was
highest in the rhizosphere of the Ni-hyperaccumulator
compared to non-vegetated soil, these plants also tended
to present higher R / NV ratios compared to non-accu-
mulators. A strong rhizosphere effect (R / NV ratios >2)
was not observed for any population of S. semidentata
or D. glomerata (except for the L population of
D. glomerata;Table2). The M population of
A. pintodasilvae hosted up to 6-fold higher densities of
Ni-resistant bacteria in culture medium with 2 mM Ni
compared to non-vegetated soil (R / NV of 6.1), while
the R / NV ratios for the same population of
D. glomerata and S. semidentata were at most 1.5. At
the same site the abundance of Ni tolerant bacteria was
also significantly higher in the rhizosphere of the
hyperaccumulator than in the rhizosphere of
S.semidentata or D. glomerata. In the SB population
of A. malacitanum Ni-resistant bacterial densities were
5-fold higher than those of non-vegetated soil in media
supplemented with either 2 or 3 mM Ni (Table 2).
In most cases the presence of the plant did not affect
bacterial Ni tolerance. For example, in the case of L no
differences were observed between the % Ni-resistant
bacteria associated with the different plant species and
the non-vegetated soil. In S, at the two lower concentra-
tions of Ni (0.5 and 1 mM) the rhizosphere of all three
plant species harboured a lower % of Ni-tolerant bacte-
ria comparedto non-vegetated soil. However, in the case
of the Ni-hyperaccumulators A. pintodasilvae (M pop-
ulation) and A. malacitanum (SB population), these
plants harboured a higher proportion of Ni-tolerant bac-
teria than their corresponding non-vegetated soils: in-
creasing from 19.6 % in non-vegetated soil to 36.8 % in
the rhizosphere of the M population of A. pintodasilvae
(at 2 mM Ni) and from 19.5 to 27 % in the SB popula-
tion of A. malacitanum (at 1 mM Ni) (Table 2).
Phenotypic characteristics of the bacterial isolates
A total of 550 different colony morphotypes (74 asso-
ciated to LA, 64 to SA, 54 to MA, 95 to SBA, 48 to LG,
41 to SG, 44 to MG, 43 to SS, 45 to MS, and 42 to SNA)
were isolated and analysed for their plant growth pro-
moting traits and Ni tolerance (MTC). Figure 1a sum-
marises the frequency of PGP traits found amongst the
rhizobacterial isolates. All of the plant species harboured
rhizobacteria which were capable of producing organic
acids. This was in fact the most common trait amongst
the collection of rhizobacteria: 29.3 % of the isolated
strains were characterised as organic acid-producers.
Depending on the plant species or population between
12 and 61 % of the isolates were organic acid-producers.
The highest numbers of organic acid-producers were
associated with S. semidentata (51.2 to 62.2 % of the
isolates from this plant species showed this trait),
followed by the Alyssum subspecies (24.3 to 37.5 %)
and finally, D. glomerata (12.2 to 15.9 %). The ability to
produce IAA was also present in all plant species and
represented 23.1 % of the total of number of isolates.
IAA-producers were most common in the L, S and M
serpentine sites. Similar proportions of IAA-producers
were found for isolates of A. pintodasilvae and
D. glomerata in L (31.1–39.6 %) and S (21.9–24.4 %)
populations. In M a higher % of IAA-producers were
found amongst isolates of A. pintodasilvae (44.4 %),
and a similar % was obtained from isolates of either
D. glomerata (20.5 %) or S. semidentata (17.8 %). IAA-
producers were less frequently associated with either
A. malacitanum from SB (9.5 % of isolates) or
A. serpyllifolium from SN (14 %). The mean production
of IAA (mg L
−1
) by strains varied from 5.3 in SA to 48.0
in LG, however, a high variability was found between
isolates from each site. The maximum IAA production
was recorded in the population of D. glomerata,from
Barazón, where three isolates showed IAA production
higher than 200 mg L
−1
(247.9 mg L
−1
for strain LG105;
315.9 mg L
−1
for strain LG120 and 211.4 mg L
−1
for
strain LG121). In addition, one strain associated with
the grass species in Samil also showed IAA production
(up to 118.2 mg L
−1
; strain SG5), while in the case of the
hyperaccumulator, the maximum IAA production was
recorded in A. pintodasilvae from Barazón
(136.2 mg L
−1
;strainLA24).
Siderophore-producing strains were found in the rhi-
zosphere of all the Alyssum populations studied except
in A. pintodasilvae from M. The frequency of isolates
showing this characteristic varied between 6.8 % in L
and 33.3 % in SN. The siderophore-producing ability
was abundant in isolates from the rhizosphere of
D. glomerata collected in M (51.2 %) and S (21.7 %).
In contrast, in the rhizosphere of S. semidentata these
were much less frequent (only 2.3 % of isolates in S).
The abilities to solubilise mineral phosphate or to
produce biosurfactants were relatively rare traits
amongst the bacterial isolates, representing 4.5 and
0.5 % of the total number of the isolates, respectively.
P solubilisers were not found in isolates from
Plant Soil
D. glomerata but were isolated from the rhizosphere of
the Alyssum and S. semidentata populations (except in
L). In Alyssum between 4.8 and 11.6 % of the isolates
were able to solubilise P while in S. semidentata this
characteristic represented 2.2 and 4.6 % of the isolates.
Biosurfactant production was only detected amongst 3
rhizobacteria isolated from A. malacitanum (SB),
S. semidentata (of M) and D. glomerata (of S), and the
percentage was never greater than 3 %.
Figure 1b shows the frequency of isolates (as a % of
the total number of isolates in each group) for each
MTC. All of the serpentine plant species harboured
rhizobacteria capable of growing in all the tested Ni
concentrations. However, the level of Ni tolerance
depended on both the soil type and the plant species.
About 90 % of rhizobacterial isolates associated with the
non-hyperaccumulator (SN) were only able to grow in
medium without Ni (MTC<1 mM), while for the plant
species growing in the serpentine sites from 4.2 to
18.5 % of rhizobacteria presented a MTC of 10 mM.
Of the rhizobacterial isolates associated with non-
hyperaccumulators species growing on serpentine sites
the majority presented MTC values < than 1 mM Ni. For
instance, most of the isolates associated with the Ni-
excluder D. glomerata (66–73 %) did not tolerate Ni
concentrations ≥2.5 mM, while in the rhizosphere of the
Fig. 1 a Frequency of plant growth promoting (PGP) traits pre-
sented by isolates obtained from the different populations of each
plant species (presented as a % of the total number of isolates per
group). bMaximal Tolerable Concentration (MTC) of bacteria
isolated from the different rhizosphere soils (presented as a per-
centage of the total number of isolates in each group). The metal
resistance was tested using 284 agar medium supplemented with 1,
2.5, 5 or 10 mM Ni added as NiSO
4
and incubated at 28 °C for
7 days. LA (A. pintodasilvae from Barazon), LG (D. glomerata
from Barazon), SA (A. pintodasilvae from Samil), SG
(D. glomerata from Samil), SS (S. semidentata from Samil), MA
(A. pintodasilvae from Morais), MG (D. glomerata from Morais),
MS (S. semidentata from Morais), SBA (A. malacitanum from S.
Bermeja) and SNA (A. serpyllifolium from S. Nevada)
Plant Soil
Ni-hyperaccumulators this percentage ranged from 36
to 51 %.
Within the Ni-hyperaccumulators, only 9.4 % of
strains associated with the S population of
A. pintodasilvae were unable to grow in the presence
of Ni in the growth medium. The same population
presented the highest percentage (45.3 %) of associated
microorganisms with a MTC of 2.5 mMNi. While the L
population presented the highest number of isolates with
an MTC of 5 mM (30 %) and the M population of
10 mM (18.5 %). Similarly, it was the Ni-
hyperaccumulators which presented the highest num-
bers of associated bacteria with an MTC of 10 mM Ni
(6.8, 10.9 and 18.5 % in the L, S and M sites, respec-
tively). Nonetheless, a high percentage (at times similar
or even higher than the Ni-hyperaccumulators) of iso-
lates associated with the non-hyperaccumulator
S. semidentata also presented elevated Ni MTC values
(up to 5 or 10 mM).
Online resource 1summarises the frequency of each
phenotype within the collection of isolates. The most
frequent phenotype was LT-N (47 % in L, 25 % in M,
28%inS,31%inSBand38%inSN),which
represents low Ni tolerance (up to 2.5 mM Ni MTC)
and no PGP trait. This was followed in the serpentine
sites, by HT-N (high Ni tolerance but no PGP trait)
(18 % in L, 16 % in M, 12 % in S, 23 % in SB) and
by LT-A in the SN site (17 %). Phenotypes A and H
(which correspond with organic acid- and IAA-
producers respectively), were also frequently found in
LT strains (up to 18 % in L) but were less often repre-
sented by HT strains (up to 10 % in S). Phenotypes
presenting three PGP traits (phenotypes APH, ASH
and PSH) were rarely found (at most 2 % of strains).
In general, all phenotypes were represented in both
ranges of metal tolerance (LT and HT), except pheno-
types T and APH which were exclusive to HT isolates
and phenotypes AP, PH, ST and PSH which were only
present in LT isolates. Strains isolated from L showed a
maximum of two PGP traits (HT-SH, siderophore- and
IAA-producers; Online resource 1). Whereas in S, M
and SB several strains with more than two PGP traits
were found (phenotypes APH, ASH and PSH); and
these were mainly associated with the Ni-
hyperaccumulator. Rhizobacterial strains associated
with Alyssum ssp. generally presented a higher diversity
in phenotypes than the other plant species. For example,
in the L population A. pintodasilvae strains were allo-
cated into 12 different phenotypes, while only five
phenotypes were found in rhizobacteria from the
gramineae growing in the same site, and none of these
were specific to the grass species. In the Portuguese (M
and S) populations, phenotype distribution depended on
the plant species. In M, phenotypes LT-AP and HT-APH
were associated with A. pintodasilvae, while HT-S, LT-
AS and LT-SH (all of which include siderophore-
producers) with D. glomerata, and finally, HT-T with
S. semidentata. In S, the 18 phenotypes were distributed
among the plant species as follows: A. pintodasilvae (15
groups) > D. glomerata (10) > S. semidentata (9); four
of the groups were specific to the hyperaccumulator (LT-
PH, LT-PSH, HT-AS, and HT-ASH), two to
D. glomerata (LT-ST and LT-ASH) and only one (HT-
APH) was specific to S. semidentata.InSBandSN,
strains associated with Alyssum sp. were represented by
ASH and PH phenotypes, respectively.
Identification and genotypic diversity of culturable
rhizobacteria
BOX-PCR profiles
A BOX-PCR profile was obtained for 538 out of the 550
isolated bacterial strains. Profiles were analysed on a
site-by-site basis (for each plant species present) and for
all the Ni-hyperaccumulating Alyssum species together
(Online Resource 2- Online Resource 6and Online
Resource 7). Table 3presents a summary of the total
number of BOX-PCR groups obtained in each of these
analyses and the distribution of bacterial isolates
amongst these groups for each site (Table 3a) and plant
species (Table 3b). In the case of L the BOX-PCR
profiles of isolates were distributed amongst 19 groups,
strains associated with the Ni-hyperaccumulator
A. pintodasilvae were distributed amongst 13 groups
and those of D. glomerata in 13 groups. Twelve of these
BOX-PCR groups are represented exclusively by strains
of either A. pindoasilvae or D. glomerata (groups L01-
L02, L04, L07-L08, L11-L12, L14 and L16-L19), while
seven groups are made-up of isolates from both species
(Online Resource 2). In the case of S, the BOX-PCR
profiles of isolates from A. pintodasilvae and
S. semidentata were distributed amongst 19 and 9
groups each, while for D. glomerata isolates were dis-
tributed amongst only 4 groups. Twelve groups were
exclusively made up of isolates associated with
A. pintodasilvae (S01, S02, S07, S09, S11, S12, S15,
S16, S17, S18, S20 and S21), three groups with isolates
Plant Soil
of S. semidentata (S10, S19 and S22), and some groups
(S04, S08 and S13) were composed of isolates from
both the Ni-hyperaccumulator and S. semidentata.
There were no groups solely represented by isolates of
D. glomerata (Online Resource 3). In the case of plant
populations of M, isolates were distributed in 24 BOX-
PCR groups; A. pintodasilvae isolates were distributed
amongst 17 groups, S. semidentata amongst 17 groups
and D. glomerata amongst 8 groups. The groups are
generally represented by isolates from all three plant
species, with some exceptions being group M01 (solely
represented by isolates of D. glomerata), M02, M10,
M19, M23-M24 (each represented by a single isolate of
A. pintodasilvae), M03, M14, M16-18 and M22 (iso-
lates of S. semidentata) (Online Resource 4).
Rhizobacterial strains isolated from A. malacitanum
(from SB) and from A. serpyllifolium (from SN) were
allocated into 18 and 10 BOX-PCR groups, respectively
(Online resource 5and 6).
BOX-PCR profiles of strains associated with the two
Alyssum subspecies (from L, M, S, SB) were allocated
into 40 groups (Online resource 7). Approximately half
of these (19 groups) were made up of strains from
various populations. On the other hand, some groups
were predominantly represented by isolates from a sin-
gle population. This was the case for groups A36 or A37
which were principally composed of strains isolated
from the SB population of A. malacitanum,orfor
groups A08, A14, A20, A23 and A38 which were made
up of strains almost exclusively isolated from the
Portuguese serpentine populations (M or S) of
A. pintodasilvae. BOX-PCR group A18 represents iso-
lates associated with A. pintodasilvae from both the
Spanish population (L) and Portuguese populations (M
and S) (Online Resource 7). In general no correlation
was found between BOX- and phenotypic-groups, how-
ever some phenotypes were shown to be specific to a
particular BOX-group. Phenotype SH was specific to
the L05 BOX-group in Barazón (Online Resource 2),
phenotype APH was specific to group M08 in Morais
and to S06 in Samil (Online Resource 4and 3), and
finally, phenotype ASH was specific to group S05 in
Samil (Online Resource 3). In SB, phenotype PSH was
associated with SB09 and in SN phenotype ASH was
specific to SN07 (Online Resource 5and 6,
respectively). Regarding the Ni-hyperaccumulators, on-
ly phenotype ASH (organic acid-, siderophore- and
IAA-producer) was found exclusively in the BOX
group A36 (Online Resource 7).
The Shannon diversity index (H′) is presented in
Online resource 8. Diversity indices were calculated
separately for isolates presenting low (LT) and high
(HT) Ni tolerance. No correlation was found between
plant species and bacterial diversity of isolates with low
Tabl e 3 (a) Total numbers of
BOX-PCR groups obtained for
each site and the number associ-
ated with each plant species. (b)
Total n umber o f BOX-PCR
groups obtained for all the Ni-
hyperaccumulators and the num-
ber associated with each
population
LBarazón; SSamil; MMorais; SB
Sierra Bermeja; SN Sierra Nevada
A, Alyssum;G,Dactylis;Sa,
Santolina
(a) Number of BOX-PCR groups
Site (total no. of BOX profiles) Total Host plant
L(n= 119) 19 A 13
G13
S(n=144) 22 A 19
G4
Sa 9
M(n=139) 24 A 17
G8
Sa 17
SB (n=94) 18 A 18
SN (n=42) 10 A 18
(b) Number of BOX-PCR groups
Plant type (total no. of BOX profiles) Total Site
Ni-hyperaccumulator (n=280) 40 L 17
S20
M22
SB 19
Plant Soil
Ni tolerance (LT), the highest H′values were recorded in
the bacterial community associated with the M popula-
tion of S. semidentata. (H′=1.04) and the S population
of A. pintodasilvae (H′=0.95). However, bacterial di-
versity of isolates with high Ni tolerance (HT),
tended to be higher in the rhizosphere of the Ni-
hyperaccumulating species than the non-accumula-
tors. The highest index was recorded in the bacte-
rial population of A. pintodasilvae growing in
Morais (H′=0.96) while that of the excluder
D. glomerata growing at the same site presented
the lowest bacterial diversity (H′=0.58).
16S rDNA identif ication
A total of 329 isolates were identified through compar-
ative sequence analysis of 16S rDNA sequences, and
isolates wereaffiliated with a total of 24 different genera
belonging to four phyla. The Shannon diversity (H′)
index was also calculated on the basis of the 16S
rDNA sequencing. The highest value was found in the
SN population of A. serpyllifolium (1.9), while for the
serpentine populations, the highest diversity was asso-
ciated with the Ni-hyperaccumulators, and particularly
the S population of A.pintodasilvae (1.7) (Online
resource 8).
Figure 2presents the taxonomic breakdown for each
plant species and population. Four different phyla were
represented in the bacterial collection, two phyla corre-
spond with Gram-positive bacteria (Actinobacteria and
Firmicutes) and two with Gram-negative (Bacteroidetes
and Proteobacteria). The distribution of isolates within
these phyla differed according to the soil type, 94 % of
the isolates obtained from serpentine soils (L, S, M, SB)
were Gram-positive bacteria and 6 % were Gram-
negative (data not shown). In contrast, in the calcareous
soil (SN) Gram-positive bacteria represented 77 % of
the total community and Gram-negative 23 %.
Proteobacteria or Actinobacteria were the most diverse
taxonomic classes (including 9 and 11 different genera,
respectively).
Differences in bacterial diversity were observed re-
garding the distribution of phyla between plant species
and populations. For the M population of S. semidentata
(MS) and the S population of D. glomerata (SG) 100 %
of the isolates were affiliated with the phylum
Actinobacteria (Fig. 2e and g). For the Ni-
hyperaccumulators, the proportion of isolates belonging
to the Actinobacteria decreased in the order SB (95 %)
(Fig. 2i), S (90 %) (Fig. 2f), L (88.6 %) (Fig. 2a)andM
(74.3 %) (Fig. 2c). Actinobacteria was least represented
in the SN population of A. serpyllifolium,although63%
of isolates were still affiliated with this phylum (Fig. 2j).
The second most important phylum was Proteobacteria
(Alpha-andGamma-proteobacteria), particularly in the
SN population of A. serpyllifolium (25.9 % of isolates
belonged to this phylum) (Fig. 2j). The phylum
Firmicutes was primarily associated with the Alyssum
subspecies (11.4 % of isolates in M, 10.0% in S, 5 % in
SB and 11.1 % in SN) (Fig. 2c, f, i and j), while isolates
belonging to the Bacteroidetes were only found in the L
population of A. pintodasilvae (5.7 % of isolates)
(Fig. 2a).
Regarding the distribution of genera amongst the
bacterial isolates associated with each plant species
(considering all their populations together), the Ni-
hyperaccumulators showed a higher diversity and
hosted members of 19 different genera, while only 9
genera were associated with the non-hyperaccumulating
A. serpyllifolium. The rhizosphere community of
D. glomerata and S. semidentata were represented by
members of 10 and 6 genera, respectively (data not
shown). Some bacterial genera, such as Arthrobacter,
Streptomyces and Rhodococcus (all members of the
Actinobacteria), were found in the rhizosphere of all
the plant species (Fig. 2a to j). In contrast, some genera
were only isolated in specific plant species: members of
the genera Stenotrophomonas (Gammaproteobacteria),
Methylobacterium (Alphaproteobacteria),
Staphylococcus (Firmicutes), Amycolatopsis and
Mycobacterium (both Actinobacteria) and the phylum
Bacteroidetes (exclusively represented by the genera
Olivibacter and Chitinophaga) were only found in as-
sociation with the Ni-hyperaccumulators (either
A. pintodasilvae or A. malacitanum)(Fig.2a, c, f and
i). On the other hand, members of the genera Janibacter
(Actinobacteria)andMesorhizobium and Aminobacter
(Alphaproteobacteria) were exclusively isolated
from the rhizosphere of D. glomerata (specifically
its L population, Fig. 2b), and Enterobacter
(Gammaproteobacteria) from S. semidentata (S
population, Fig. 2h).Finally, members of two gen-
era, Pseudomonas and Rhizobium (Gamma-and
Alpha-proteobacteria,respectively),wereonlyfound
in the rhizosphere of the non-hyperaccumulator
A. serpyllifolium (Fig. 2j).
Amongst the Ni hyperaccumulators the predominant
genera represented by isolates associated with the L
Plant Soil
population of A. pintodasivae (Fig. 2a)were
Arthrobacter (48.6 %) and Amycolatopsis (25.7 %).
While the main genera associated with the two
Portuguese populations of A. pintodasilvae were
(Fig. 2c and f): Arthrobacter (37.1 % in M and 20 % in
S) and Streptomyces (37.1 % in M and 32.0 % in S).
Similarly, the rhizosphere community of A. malacitanum
(SB) (Fig. 2i) was predominantly represented by
Arthrobacter (31.0 %) and Streptomyces (40.5 %). The
latter two genera were also important components of the
rhizobacterial community of the non-hyperaccumulator
A. serpyllifolium, but in this case 18.5 % of isolates were
Fig. 2 Taxonomic breakdown of 16 s rDNA sequences a35
sequences from Alyssum pintodasilvae (Barazón), b25 from
Dactylis glomerata (Barazón), c35 from Alyssum pintodasilvae
(Morais), d26 from Dactylis glomerata (Morais), e28 from San-
tolina semidentata (Morais), f50 from Alyssum pintodasilvae
(Samil), g30 from Dactylis glomerata (Samil), h30 from Santolina
semidentata (Samil), i42 from Alyssum malacitanum (S. Bermeja),
j26 from Alyssum serpyllifolium (S. Nevada). The central pie
shows percentages by phyla; each outer ring progressively breaks
these down to finer taxonomic levels (phyla, class, family, genera)
Plant Soil
identified as Pseudomonas. As observed for the
hyperaccumulators, members of the genera
Arthrobacter and Streptomyces were predominant in
the rhizosphere of both D. glomerata and
S. semidentata: 60 % of isolates of the L population of
D. glomerata were affiliated with Arthrobacter,while
53 % and 75 % of isolates from S and M populations of
D. glomerata and S. semidentata, respectively, were
affiliated with Streptomyces. Members of the genera
Nocardia were generally found in association with
D. glomerata from Portuguese sites (M and S),
representing 19.2 % and 16.7 % of isolates, respectively.
Discussion
This study describes the culturable rhizosphere bacterial
community associated with different populations of the
only two Ni-hyperaccumulating subspecies of Alyssum
serpyllifolium in the Iberian Peninsula. The Ni tolerance,
PGP traits and diversity of these communities were
compared with those of non-hyperaccumulating plants
growing at the same sites and with a close relative and
non-hyperaccumulator, A. serpyllifolium, growing in
calcareous soils. Trace metals are well known to affect
the soil bacterial density and activity, as well as the
community structure and diversity (Bååth 1989). A high
soil metal content has been suggested to explain the
lower bacterial densities observed in serpentine soils
compared to nearby non-serpentine soils (Amir and
Pineau 1998; Pal et al. 2005). Higher bacterial densities
in the non-serpentine (SN) soil were therefore to be
expected. Toxic concentrations of trace metals were
not detected in this calcareous soil and the higher organ-
ic matter content should favour microbial development
(Acea and Carballas 1986). In accordance, bacteria from
the serpentine soils (L, M, S, SB) showed a higher
resistance to Ni than those from the calcareous (SN)
soil. An increase in the Ni concentration of the culture
medium led to a significant decrease in bacterial densi-
ties of all soils. However, this decrease was less pro-
nounced in the serpentine soils, indicating their adapta-
tion to the phytotoxic levels of Ni to which they are
normally exposed. Microorganisms from serpentine
Fig. 2 (continued)
Plant Soil
soils have previously been shown to present a high level
of tolerance to trace metals, such as Ni, Co and Cr
(Becerra-Castro et al. 2009; Pal et al. 2005; Schlegel
et al. 1991; Turgay et al. 2012).
Plant root exudates, secretions and lysates contain
labile C sources and growth factors which are well
known to stimulate microbial growth and metabolic
activity (Delorme et al. 2001;Graystonetal.1998).
Bacterial densities were therefore unsurprisingly higher
in the rhizosphere soils of all the plant species studied
compared to corresponding non-vegetated soils (R / NV
ratios>1). This rhizosphere effect was most pronounced
in the case of Alyssum (in both the Ni-hyperaccumulators
and non-hyperaccumulator). The Ni-hyperaccumulating
A. pintodasilvae and A. malacitanum also hosted a
higher density of Ni-resistant bacteria in their rhizo-
sphere than either the non-accumulating plants from
serpentine sites or non-vegetated soil. This effect was
not observed in the case of the non-hyperaccumulator
A. serpyllifolium (SN). Population-specific differences
were observed in the case of A. pintodasilvae, and the
highest densities of Ni-resistant bacteria were associated
with the M population. In addition, both the M popula-
tion of A. pintodasilvae and the SB population of
A. malacitanum showed a selective enrichment of Ni-
resistant bacteria in the rhizosphere. This has been ob-
served for other metal-hyperaccumulating plants,
such as Alyssum bertolonii,Noccaea goesingense
and Sebertia acuminata (Lodewyckx et al. 2002;
Mengoni et al. 2001,2010;Schlegeletal.1991).
Likewise, Zn tolerance has often been described in
bacterial communities exposed to this metal:
Delorme et al. (2001) found higher densities of
Zn-tolerant bacteria in the rhizosphere of the Zn-
hyperaccumulator N. caerulescens compared to the
non-accumulator Trifolium pratense or to non-
vegetated soil. Moreover, bacteria isolated from
serpentine soils have been shown to present co-
resistance to various metals, including Cr, Co and
Zn (Mengoni et al. 2010). It would also have been
interesting to study root architecture of the different
plant species and in particular to compare those of
the different populations of Alyssum. Root architec-
ture and proliferation would influence exudation
and therefore also the rhizosphere effect.
Trace metal uptake by hyperaccumulators has been
found to be associated with partial depletion of labile,
easily bioaccessible trace metal pools in the rhizosphere
(e.g. Fitz et al. 2003; Puschenreiter et al. 2005;
Whiting et al. 2001) and active root proliferation to-
wards soil patches rich in metals (Schwartz et al.
1999). Such depletion of labile metal pools has often
been associated with sustained or even enhanced metal
solubility(i.e. soil solution concentration) due to a more
intense weathering of Ni-bearing silicates in the rhizo-
sphere (Kidd et al. 2009;Puschenreiter et al. 2005). In
agreement, water-soluble Ni concentrations in the rhi-
zosphere soils of the Ni-hyperaccumulating Alyssum
from M and SB were significantly higher than those
detected in the rhizosphere soil of non-accumulators
(e.g. D. glomerata or S. semidentata)orofnon-
vegetated soil. In addition, previous studies have also
shown a higher labile Ni fraction (assessed via sequen-
tial extraction procedures or Sr(NO
3
)
2
-extractable con-
centrations) in the rhizosphere soil of these Alyssum
populations compared to non-vegetated soils or non-
accumulators (Becerra-Castro 2006; Cabello-Conejo
2015). This tendency towards a higher Ni mobility in
the rhizosphere of the hyperaccumulator could be attrib-
uted to the activity of the Ni-tolerant bacteria and at the
same time lead to their further enrichment in the rhizo-
sphere. Of the 508 isolates obtained from serpentine
soils the vast majority (65 %) were able to growth with
the presenceof Ni in the medium (with concentrations>
1 mM Ni). In contrast, 90 % of isolates from the close
relative of the Ni-hyperaccumulator, A. serpyllifolium
were affected by the presence of the minimal concen-
tration of Ni tested (1 mM) and none of them were able
to tolerate more than 2.5 mM. Our results are in agree-
ment with previous studies: Abou-Shanab et al. (2003)
found the majority of rhizobacteria isolated from ser-
pentine soils in Oregon (USA) were able to grow in the
presence of 8 mM Ni, and Mengoni et al. (2001)found
bacterial isolates from serpentine soils in Tuscany (Italy)
resisted between 7 and 10 mM Ni in their growth
medium. These authors did not study the Ni resistance
of bacterial isolates associated with non-accumulators
growing at the same site. Here the MTC for isolates
associated with the hyperaccumulators was higher than
that of the Ni-excluder D. glomerata, which could rein-
force the idea that the activity of these hyperaccumulator
plants leads toan increase in labile Ni and hence enrich-
ment in Ni-tolerant bacteria or that this plant group
selects for Ni-tolerant bacteria which in turn modify soil
Ni availability. However, Ni-resistance was an equally
important phenotype of the bacterial isolates associated
with the serpentinophyte S. semidentata (a similar % of
isolates showed Ni MTC of 10 mM Ni), indicating that
Plant Soil
this may not be a phenomenon specific to the
hyperaccumulators.
The potential application of these Ni-tolerant plant-
associated bacteria in phytoextraction (or phytomining)
processes is based on their ability to improve plant
growth and biomass production and/or modify soil met-
al availability and plant uptake. Bacterial strains for
bioaugmentation trials are frequently selected on the
presence of PGP traits (such as the production of IAA,
ACC deaminase or siderophore production) or their
ability to release compounds which could potentially
modify metal bioavailability (such as the production of
biosurfactants, siderophores or organic acids) (Abou-
Shanab et al. 2003; Braud et al. 2006; Cabello-Conejo
et al. 2014; Idris et al. 2004). Half of the isolates obtain-
ed in this study showed at least one PGP trait (54 % of
isolates). The rarest phenotypes were the biosurfactant-
producers and the ability to solubilise inorganic phos-
phate. Biosurfactant-producing strains were onlydetect-
ed amongst isolates from A. malacitanum,D. glomerata
or S. semidentata (and were identified as members of the
genus Bacillus (Firmicutes)andArthrobacter
(Actinobacteria)). Some authors have shown that micro-
bial produced surfactants can increase the mobility of
Cd, Cu, Pb and Zn in soil, and in some cases, increase
plant metal uptake (Venkatesh and Vedaraman 2012). It
would be interesting to further characterise the
biosurfactant-producing bacterial strains identified in
this study and particularly their Ni-solubilising capaci-
ties. Isolates which were able to solubilise inorganic
phosphate were primarily associated with the Ni-
hyperaccumulators (at most 12 % isolates). Most of
these were Actinobacteria and identified as members
of the genera Arthrobacter (strains SA37, SA40,
SBA82, SNA110), Streptomyces (strains SA46,
SBA57, SBA89) or Rhodococcus (strains SBA86 and
SBA70). One P-solubilising isolate from the M popula-
tion of A. pintodasilvae was identified as
Methylobacterium (Alphaproteobacteria). Phosphate-
solubilising bacteria are effective in promoting plant
growth and biomass by releasing P from inorganic and
organic P pools through solubilisation and
mineralisation (Rodrı́guez and Fraga 1999). These bac-
terial associations may play an important role in P
mobilisation for hyperaccumulating species of
Alyssum, which seems to form non or weak mycorrhizal
associations since reports of arbuscular mycorrhizal
symbiosis with hyperaccumulating Alyssum sp. cannot
be found in the literature.
Jeong et al. (2013)inoculatedBrassica juncea with
strains of phosphate-solubilising Bacillus sp. According
to these authors the release of organic acids and drop in
soil pH led to a mobilisation of Cd and consequent
increase in Cd uptake as well as an enhanced plant
biomass. An increase in plant growth and P uptake
were reported by Ma et al. (2010) after inoculating
Ricinus communis and Helianthus annuus with the
rhizobacterial strain Psychrobacter sp. SRS8. Since
metal-enriched soils are characteristically deficient in
essential nutrients such as P, the identification of bacte-
rial inoculants which could potentially improve the nu-
tritive state of the phytoextracting plant are of interest.
More than 50 % of the hyperacccumulator associated P-
solubilisers also showed Ni tolerance (tolerating Ni
concentrations in the growth medium of ≥2.5 mM).
One of these strains (Arthrobacter nicotinovorans
SA40) was shown to significantly improve the biomass
production of A. pintodasilvae when grown in contrast-
ing Ni-rich soils (serpentine soils and sewage sludge-
amended agricultural soils) (Cabello-Conejo et al.
2014). Strain SA40 was also characterised as a
siderophore- and IAA-producer. The fact that this strain
has a beneficial effect on plant growth in both naturally-
rich and anthropogenic-contaminated soils is notewor-
thy since it could be applied to a wider range of soils
with contrasting edaphic properties.
A substantial number of isolates were able to produce
organic acids, siderophores or IAA, although the frequen-
cy of these traits varied according to the host plant species
or population. Plant-associated bacteria have been shown
to modify soil trace metal availability through the release
of organic acids, such as citric, oxalic or acetic acid,
which in turn can lead to an increase in plant metal uptake
(Sessitsch et al. 2013). More than 50 % of organic acid-
producers isolated from the hyperaccumulators or
S. semidentata tolerated > 2.5 mM Ni and some of them
showed additional PGP characteristics. In a previous
study, Becerra-Castro et al. (2011) showed that by using
the cell-free culture of 13 of the strains associated with
the Alyssum sp. (included in this study) the extractable Ni
concentration from serpentine soils was increased.
However, these authors found no relation between the
capacity for Ni mobilisation and the phenotypic traits of
the strains. In a more detailed study, strain LA44 (iden-
tified as Arthrobacter nitroguajacolicus) was shown to
be an efficient mobiliser of Ni from ultramafic rocks
under in vitro conditions, and principally liberated Ni
associated with Mn oxides through the exudation of
Plant Soil
oxalate (Becerra-Castro et al. 2013). This strain also
shows intense IAA-production, is highly Ni-resistant,
and in a previous study was able to increase shoot Ni
concentrations in A. pintodasilvae growing in serpentine
soil (likely due to an enhanced Ni phytoavailability
and hence plant uptake). The strains identified in
this study as P-solubilisers or organic acid-pro-
ducers, and particularly those which show high Ni
tolerance, would be good candidates for further
studies related to Ni mobilisation.
Zaidi et al. (2006)reportedthatanIAA-producing
Bacillus subtilis strain was able to promote the growth
of Brassica juncea and thereby increased Ni extraction.
In this study, IAA-producers were particularly associat-
ed with the Ni-hyperaccumulator A. pintodasilvae,
while the production of siderophores was associated
with strains isolated from D. glomerata. The fact that
siderophore-producing bacteria were found in associa-
tion with the grass species is interesting since this type
of plant is known to be a highly efficient
phytosiderophore producer. The presence of associated
siderophore-producing bacteria could be related to com-
petition between the bacteria and the plant for obtaining
Fe. Siderophore-producing bacterial strains were mainly
affiliated with the genera Streptomyces and
Arthrobacter, although one isolate associated with
D. glomerata was identified as Microbacterium sp.
(strain SG22) and one isolate associated with
A. malacitanum was identified as Mycobacterium sp.
(strain SBA60). Abou-Shanab et al. (2003) found that
inoculating ultramafic soils with the actinobacterial
Microbacterium arabinogalactanolyticum AY509224
increased soil Ni extractability and uptake by Alyssum
murale. The predominance of siderophore-producers
within the isolate collection obtained from the non-
hyperaccumulator (A. serpyllifolium) could be associat-
ed with the limited availability of Fe in calcareous soils
(due to the pH-dependent low solubility of Fe). The
appearanceof a specific phenotype linked to a particular
plant species also suggests a plant-driving effect in
microorganism selection, while specific phenotypes as-
sociated with sites indicates that the soil conditions are
important factors shaping the bacterial community.
Bacterial diversity was assessedusing the BOX-PCR
technique and 16S rDNA sequencing. Although the
majority of BOX-PCR groups were affiliated with iso-
lates from two or three different plant species, or from
different populations of the same species, some were
specific to a certain plant population or plant species.
The phenotypes associated with the different BOX-PCR
groups, shows that there is no correlation between
BOX-patterns and phenotype, probably because of the
high intra-BOX phenotypic variability. These results are
in agreements with Mengoni et al. (2001), who found no
correlation between OTUs obtained by ARDRA (re-
striction analysis of 16S) and heavy-metal tolerant phe-
notype in bacterial strains isolated from the rhizosphere
of A. bertolonii.
To our knowledge differences in the bacterial commu-
nities associated with different populations of the same
Ni-hyperaccumulating species have not previously been
shown. The phyla Proteobacteria and, in particular,
Actinobacteria, dominated the culturable rhizobacterial
community. Isolates were affiliated with members of
genera, such as Arthrobacter, Streptomyces,
Rhodococcus or Microbacterium, which have been fre-
quently described amongst soil bacteria. Bacteria of the
phyla Proteobacteria (particularly Alphaproteobacteria)
and Actinobacteria were also the most numerous within
the rhizosphere of the Ni-hyperaccumulator Alyssum
murale (Abou-Shanab et al. 2010). In the rhizosphere
of Alyssum bertolonii the culturable bacterial population
was dominated by Ni-resistant Pseudomonas strains
(Gammaproteobacteria) (Mengoni et al. 2001). In this
study, strains identified as Pseudomonas sp. were exclu-
sively found in the rhizosphere of A. serpyllifolium grow-
ing in the calcareous soil. Culturable rhizobacteria asso-
ciated with the Ni-hyperaccumulator N. goesingense
were mainly represented by Methylobacterium spp., an
alphaproteobacterial genus, as well as Rhodococcus spp.
and Okibacterium spp., belonging to the Actinobacteria
(Idris et al. 2004). Gremion et al. (2003) constructed
clone libraries based on the 16S rRNA and 16S rDNA
and found Actinobacteria to be a dominant part of the
metabolically active bacteria in the rhizosphere of the Cd/
Zn-hyperaccumulator N. caerulescens. The predomi-
nance of actinobacterial strains in serpentine soils has
been related to the high adaptability of such gram-
positive bacteria to toxic concentrations of trace metals
(DeGrood et al. 2005). Metal toxicity or nutrient defi-
ciency has been shown to lower the frequency of r-
strategists (bacteria capable of rapid growth and utiliza-
tion of resources), such as Bacillus or Pseudomonas,as
these are more sensitive to toxic substances (Kozdrój
1995;Kunitoetal.2001). This could partly explain the
lower frequency of r-strategists in the serpentine soils. On
the other hand, despite a reduced growth and metabolic
activity, k-strategists (such as the Actinomycetales)
Plant Soil
present stable populations over a long time period (mak-
ing them recommended candidates for bioaugmentation
purposes) (Lebeau et al. 2008). The major differences in
taxonomic diversity were therefore found between ser-
pentine and non-serpentine soils.
Since the effects of the same bacterial inoculum on
plant growth and metal bioaccumulation can be both
plant- (Becerra-Castro et al. 2012) and soil-specific
(Cabello-Conejo et al. 2014), the selection of microor-
ganisms adapted to the soil characteristics and an ability
to colonize the rhizosphere will be a pre-requisite for
successful bioaugmentation. This study generated a
large number of plant-associated rhizobacterial strains
with potential application in these techniques. Many
were affiliated with genera which have previously been
shown to be beneficial for soil metal removal processes.
Although plant population-specific differences in the
diversity of associated rhizobacteria were not large and
the Ni-hyperaccumulators did not seem to harbour a
characteristic bacterial taxonomic diversity; a small
number of strains were only found in association with
the Alyssum hyperaccumulators and would be interest-
ing candidates for further studies related to the applica-
tion of Ni hyperaccumulating species in phytomining.
For example, strains MA98 and MA106 both identified
as Stenotrophomonas sp. and IAA- and organic
acid-producers, respectively; MA91 which is a P-
solubiliser (identified as Methylobacterium sp.); LA24,
LA22, and LA23, all of them IAA-producers (identified
as Amycolatpsis sp.) or SBA60 identified as
Mycobacterium sp. and siderophore-producers.
Moreover, the beneficial effects of stains could also be
more pronounced when these are used as a mixed inoc-
ulum. These strains will be used in future bioaugmenta-
tion trials (individually or as mixed inoculum) to evaluate
their effects on the Ni phytoextraction capacity of Ni-
hyperaccumulating plant species.
Acknowledgments This research was supported by the Spanish
Ministerio de Economía y Competitividad (CTM2012-39904-
C02-01) and FEDER, and by the 7th Framework Program of the
European Commission (FP7-KBBE-266124, GREENLAND).
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