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RESEARCH PAPER
Assessing the role of endophytic bacteria in the halophyte
Arthrocnemum macrostachyum salt tolerance
S. Navarro-Torre
1,†
, J. M. Barcia-Piedras
2,3,†
, E. Mateos-Naranjo
2
, S. Redondo-G
omez
2
,
M. Camacho
3
, M. A. Caviedes
1
, E. Pajuelo
1
& I. D. Rodr
ıguez-Llorente
1
1 Departamento de Microbiolog
ıa y Parasitolog
ıa, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain
2 Departamento de Biolog
ıa Vegetal y Ecolog
ıa, Facultad de Biolog
ıa, Universidad de Sevilla, Sevilla, Spain
3 IFAPA, Centro Las Torres –Tomejil, Sevilla, Spain
Keywords
Arthrocnemum macrostachyum; endophytes;
halophyte; phyllosphere; salt tolerance.
Correspondence
I. D. Rodr
ıguez-Llorente, Departamento de
Microbiolog
ıa y Parasitolog
ıa, Facultad de
Farmacia, Universidad de Sevilla, C/Profesor
Garc
ıa Gonz
alez 2, 41012 Sevilla, Spain.
E-mail: irodri@us.es
†
These authors contributed equal to this work.
Editor
H. Papen
Received: 14 September 2016; Accepted: 18
October 2016
doi:10.1111/plb.12521
ABSTRACT
•There is an increasing interest to use halophytes for revegetation of salt affected eco-
systems, as well as in understanding their mechanisms of salt tolerance. We hypothe-
sized that bacteria from the phyllosphere of these plants might play a key role in its
high tolerance to excessive salinity.
•Eight endophytic bacteria belonging to Bacillus and closely related genera were isolated
from phyllosphere of the halophyte Arthrocnemum macrostachyum growing in salty
agricultural soils. The presence of plant-growth promoting (PGP) properties, enzy-
matic activities and tolerance towards NaCl was determined. Effects of inoculation on
seeds germination and adult plant growth under experimental NaCl treatments (0,
510 and 1030 mM NaCl) were studied.
•Inoculation with a consortium including the best performing bacteria improved con-
siderably the kinetics of germination and the final germination percentage of A.
macrostachyum seeds. At high NaCl concentrations (1030 mM), inoculation of plants
mitigated the effects of high salinity on plant growth and physiological performance
and, in addition, this consortium appears to have increased the potential of A. macro-
stachyum to accumulate Na
+
in its shoots, thus improving sodium phytoextraction
capacity.
•Bacteria isolated from A. macrostachyum phyllosphere seem to play an important role
in plant salt tolerance under stressing salt concentrations. The combined use of A.
macrostachyum and its microbiome can be an adequate tool to enhance plant adapta-
tion and sodium phytoextraction during restoration of salt degraded soils.
INTRODUCTION
Halophytes can be defined as plants that can survive and repro-
duce in environments where the salt concentration exceeds
200 mMNaCl (Flowers & Colmer 2008), or simply plants that
grow exclusively on saline soils (Dansereau 1957). The need for
re-vegetation and remediation of ecosystems affected by salin-
ity, a growing problem and one of the main factors influencing
the reduction in crop yields, has focused research interest on
understanding mechanisms of salt tolerance in halophytes
(Shabala 2013).
Salt tolerance is a complex trait that involves various
biochemical and physiological mechanisms that are also
species-specific (Flowers & Colmer 2008; Shabala 2013). These
mechanisms include increasing the efficiency of external and
internal Na
+
sequestration in trichomes and vacuoles, changes
in trichome shape or regulation of ion channels and trans-
porters, tonoplast antiporters or the development of large vac-
uoles, often with modified lipid composition (reviewed in
Shabala 2013; Hasanuzzaman et al. 2014). Furthermore, halo-
phytes can generate and accumulate compatible osmolytes that
contribute to osmoregulation while preventing prolonged Na
+
transport to shoots (Rozema et al. 1981) and show great stom-
atal control that optimises water use efficiency under extreme
salinity (Redondo-Gomez et al. 2010; Shabala 2013). Despite
extensive knowledge on the mechanisms of tolerance in halo-
phytes, plant tolerance must also be connected with complex
ecological processes, which are highly influenced by microor-
ganisms that colonise the rhizosphere and phyllosphere, the so-
called plant microbiome. Although the halophyte microbiome
may contribute to plant adaptation and salt tolerance, studies
with bacteria inhabiting halophytes are still scarce, and most
are merely descriptive, i.e. tried to classify and count microbial
species (Ruppel et al. 2013). Bacteria collectively known as plant
growth-promoting bacteria (PGPB) have a beneficial effect on
plant growth, health and tolerance to different environmental
stresses, including contamination with organic compounds,
heavy metals or salinity (de Bashan et al. 2012; Glick 2012;
Mateos-Naranjo et al. 2015; Mesa et al. 2015a,b; Ullah et al.
2015). General mechanisms underlying these beneficial effects
include, among others, phosphorus solubilisation, nitrogen fix-
ation, iron sequestration, phytohormones (mainly auxins),
ACC (1-aminocyclopropane-1-carboxylic acid) deaminase syn-
thesis and biofilm formation (Ullah et al. 2015). In this context,
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 1
Plant Biology ISSN 1435-8603
several authors reported the use of PGPB to enhance salt toler-
ance and plant productivity under saline conditions (de Bashan
et al. 2012; Ruppel et al. 2013). Nevertheless, there are still few
studies describing PGPB application in halophytes to extract
solid conclusions and, in addition, these are restricted to rhizo-
sphere bacteria in most cases (Ruppel et al. 2013; Jes
us et al.
2015). Therefore, this study was designed and conducted in
order to contribute to filling this gap in knowledge.
Arthrocnemum macrostachyum (Moric) C. Koch is a suitable
model plant to conduct a thorough study of the role of endo-
phytic bacteria on plant performance under salinity stress,
since it is a halophyte that is able to adapt physiologically to a
wide range of salinities and grow under extreme hypersaline
conditions, even with 1030 mMNaCl, and has a high capacity
for accumulation of Na
+
(Redondo-G
omez et al. 2010), which
makes it a suitable candidate for remediation of salt-affected
soils. In the present study we hypothesised that bacteria from
the phyllosphere of A. macrostachyum might play a key role in
the high salinity tolerance of this species. Thus the specific aims
of this study with A. macrostachyum were to: (i) isolate, charac-
terise and select bacterial endophytes with plant growth-pro-
moting (PGP) properties from aerial parts; (ii) investigate how
these bacteria affect seed germination; (iii) explore their contri-
bution togrowth of adult plants in experimental NaCl treat-
ments ranging from 0 to 1030 mMNaCl and ascertain the
extent to which the photosynthetic apparatus determines plant
performance; and (iv) determine bacterial influence on Na
+
accumulation patterns.
MATERIAL AND METHODS
Isolation of endophytic bacteria from A. macrostachyum aerial
parts
Plants of A. macrostachyum were collected from an agricultural
soil in Lebrija (Seville, Spain; 36°540N, 6°120E) in December
2013. Samples of aerial parts from three plants (each sample
containing three fragments of plant material of 2–3 cm length)
were surface disinfected in 50 ml sterile Falcon tubes in aseptic
conditions. First, they were washed with 70% (v/v) ethanol for
1 min by shaking and then submerged in sodium hypochlorite
1:4 in sterile distilled water for 15 min and gently shaken.
Finally, it was washed four times in sterile distilled water. Dis-
infected samples were crushed separately with a sterile mortar
in 1 ml 0.9% sterile saline solution. Aliquots of 100 ll of the
resulting mixtures were plated onto five plates containing tryp-
tic soy agar (TSA) medium (Thompson et al. 1993) and five
containing TSA medium supplemented with 0.3 MNaCl. Petri
dishes were incubated at 28 °C for 72 h and the different colo-
nies based on morphology were isolated. TSA of 0.3 MNaCl
was prepared by adding SW30 solution to the distilled water
(Mesa et al. 2015a).
Isolation of DNA and identification of endophytic bacteria by
16S rRNA amplification
Genomic DNA was extracted using the i-genomic BYF DNA
Extraction kit (Intron Biotechnology, Sangdaewon-Dong,
Korea), according to the manufacturing’ instructions. 16S
rRNA amplification was performed using the 16F27 primer
(50-AGAGTTTGATCM
TGGCTCAG-30) and 16R1488 primer (50-CGGTTACCTTGTT
AGGACTTCACC-30) with the following PCR conditions: ini-
tial denaturation at 95 °C for 2 min, 30 cycles of denaturation
at 95 °C for 45 s, annealing at 58 °C for 45 s, extension at
72 °C for 3 min and a final extension at 72 °C for 5 min. 16S
rRNA sequences were compared with those in the databases
using the EzTaxon server (Chun et al. 2007). 16S rRNA
sequences were deposited in GenBank under the accession
numbers shown in Table 1.
Amplification of genes related to synthesis of osmoprotectants
The genes ectBC and betB were amplified.For ectBC gene
amplification, Tra3 and CR primers (Reshetnikov et al. 2011)
were used, following the PCR conditions described by the
authors. This amplification gives PCR products of around
900 bp (Reshetnikov et al. 2011). For amplification of betB,
degenerate primers betBF (50-ATYGGYGCMTGGAACTAYCC-
30) and betBR (50-ACCGGBCCRAARATYTCYTC-30) were
designed based on the sequences of several betB genes from dif-
ferent genera deposited in the NCBI Genebank database (se-
quences with accession nos. CP000949.1, CP000749.1, CP00
2447.1, AJ238780.1, JN426961.1, AE014075.1, CP001830.1,
EQ973121.1) using CLUSTAL Omega program (http://www.
ebi.ac.uk/Tools/msa/clustalo/; Figure S1). PCR conditions
were initial denaturation at 93 °C for 2 min, 34 cycles of
denaturation at 93 °C for 45 s, annealing at 55.7 °C for 1 min,
extension at 72 °C for 2 min and a final extension at 72°C for
5 min. The PCR product for this amplification was 700 bp,
approximately.
Enzymatic activities and resistance to NaCl
Pectinase and cellulase activities were examined according to
the method described in Elbeltagy et al. (2000). Amylase activ-
ity was assessed in starch agar plates incubated for 7 days at
28 °C and revealed with 10 ml Lugol. Lipase and protease
activities were observed as the presence of halos around bacte-
ria after incubation in Tween and casein agars, respectively, for
7 days at 28 °C (Prescott 2002). Concerning DNAse activity,
bacteria were incubated 7 days at 28 °C in DNA agar plates.
Plates were then treated with 1 MHCl; halos in dark back-
ground were observed in bacteria with DNAse activity. Chiti-
nase activity was assessed as described in Mesa et al. (2015b).
Plates were supplemented with 0.3 MNaCl.
Table 1. Closest species to the endophytic bacteria isolated from
A. macrostachyum based on 16S rRNA partial sequences.
strain acc. n°related species
sequenced
fragment (bp)
identity
(%)
EA1 KT963445 Bacillus alcalophilus 1417 98.69
EA2 KT963446 Bacillus gibsonii 1440 99.51
EA3 KT963447 Bacillus thuringiensis 1460 99.93
EA4 KT963448 Oceanobacillus picturae 1453 99.65
EA5 KT963449 Bacillus alcalophilus 1417 98.48
EA6 KT963450 Oceanobacillus picturae 1455 99.66
EA7 KT963451 Oceanobacillus kimchii 1455 100
EA8 KT963452 Gracilibacillus saliphilus 1458 98.83
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands2
Role of endophytes in halophyte salt tolerance Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al.
To determinate resistance to NaCl, bacteria were incubated
in TSA plates supplemented with increasing concentrations of
salt (adding SW30 solution) at 28 °C for 72 h.
Plant growth-promoting properties
All the plant growth-promoting traits of isolated bacterial
strains were evaluated in the presence of 0.3 MNaCl. For N fix-
ation, bacteria were plated in NFb (nitrogen fixation bromoth-
ymol blue), a nitrogen-free medium prepared as described in
D€
obereiner (1995), and incubated for 72 h at 28 °C. Sidero-
phore production was assessed in CAS (chrome azurol S) agar
plates (Schwyn & Neilands 1987). Plates were incubated for
7 days at 28 °C in darkness. Bacteria that produced sidero-
phores had an orange halo. Phosphate solubilisation was deter-
minate as the presence of a halo around the bacteria in NBRIP
plates (Nautiyal 1999) after 7 days at 28 °C. IAA (indoleacetic
acid) production was evaluated in nutrient broth media (Bio-
life, Italy) supplemented with L-tryptophan (100 mgl
1
) and
incubated at 28 °C for 72 h with shaking. Salkowsky reagent
(Gordon & Weber 1951) was then added and optical density at
535 nm measured using a spectrophotometer (Lambda25; Per-
kinElmer, Waltham, USA). 1-aminocyclopropane-1-carboxy-
late (ACC) deaminase activity was assessed as describe in
Penrose & Glick (2003). Briefly, isolated strains were incubated
in DF salts minimal medium with (NH
4
)
2
SO
2
as a source of N
for 24 h at 28 °C with shaking. An aliquot was then transferred
to DF salts minimum medium with 3 mMACC and incubated
for a further 24 h at the same temperature. Finally, biofilm for-
mation was assayed following the method described in del Cas-
tillo et al. (2012), starting from a bacterial culture in TSB
supplemented with 0.3 MNaCl.
Inoculation of A. macrostachyum seeds and plant growth
conditions
Preparation of bacterial inoculum
The three strains that showed the best plant beneficial traits
were selected for plant inoculation. They were incubated
separately in TSB medium supplemented with 0.3 MNaCl for
18–24 h at 28 °C with continuous shaking to reach an optical
density at 600 nm around 0.9–1.0, corresponding to 10
8
cellsml
1
. Cultures were then centrifuged in 50-ml Falcon
tubes at 8000 rpm for 10 min. Pellets were washed with 0.9%
saline solution, centrifuged for 10 min in the same conditions
and then re-suspended in 5 ml 0.9% saline solution. Finally,
cultures were mixed in a 50-ml tube.
Seed germination experiment
Seeds of A. macrostachyum were collected in March 2015 in
Lebrija (Seville, Spain; 36°540N, 6°120E) from 20 different
plants chosen randomly from an anthropic marsh with no tidal
influence (mean sea level +2.7 m relative to SHZ) and stored in
the dark for 2 months at 4 °C until the start of the experiments.
In May 2015 a germination experiment was performed to
assess the effect of bacterial inoculation on seed germination
capacity of A. macrostachyum in presence of 0.9% NaCl. Thus,
prior to bacterial inoculation, seeds were surface disinfected
with 10% sodium hypochlorite for 10 min and then six times
washed with sterile distilled water. Seeds were then divided in
two blocks, one was inoculated with the selected bacterial
consortium by submerging seeds in 15 ml culture (containing
5 ml of each strain EA1, EA3 and EA8, prepared as described
above) for 1 h with shaking, while the second block was not
inoculated (submerged in the same culture media without bac-
teria). Five 25-seed replicates were then placed on 9% agar
plates containing 0.9% NaCl (n =250, five plates with 25 seeds
each 92 inoculation treatments) and placed in a growth
chamber (AGP-700-HR ESP; Radiber, Barcelona, Spain) with a
regime of 10 h light (20 °C, 50% RH and 35 lmolm
2
s
1
,
400–700 nm) and 14 h dark (5 °C and 50% RH). Seeds were
inspected daily for 20 days and seeds were considered to have
germinated after radicle appearance (Redondo-G
omez et al.
2004). Germination kinetics and the final germination percent-
age were determined.
Plant growth experiment
Another set of inoculated and non-inoculated seeds (see above)
were germinated on 9% agar plates with regime of 10 h light
(20 °C, 50% RH and 35 lmolm
2
s
1
, 400–700 nm) and 14 h
dark (5 °C and 50% RH). Seedlings were then transferred to
pots with perlite and separated into different trays depending
of the inoculation treatment. Pots were maintained at con-
trolled temperature between 21 and 25 °C, 40–60% RH, natu-
ral daylight of 250 lmolm
2
s
1
as minimum and
1000 lmolm
2
s
1
as maximum light and supplemented with
20% Hoagland solution (Hoagland & Arnon 1938) without
NaCl supplementation.
In September 2015, pots with 4-month-old plants were
placed in individual trays and randomly assigned to three salin-
ity treatments (0, 510, 1030 mMNaCl), maintaining the inocu-
lation treatment under which they had grown, i.e. with or
without bacterial inoculation (n =54, nine plants 9three NaCl
concentrations 92 inoculation treatments). Thus each plant
derived from inoculated seeds was re-inoculated with 200 llof
a culture with the bacterial consortium (prepared as described
above), every week for the first 3 weeks and once a month dur-
ing the experimental period. Saline solutions were established
by combining 20% Hoagland solution with the appropriate
amount of NaCl. Thus, at the beginning, 3 l of each solution
(0, 510, 1030 mMNaCl) were placed in each tray to a depth of
1 cm, and during the experiment the levels of the trays were
monitored and topped up to the marked level with 20% Hoag-
land solution (without additional NaCl) to limit changes in
NaCl concentration caused by water evaporation from the
nutrient solution. In addition, the entire solution (including
the NaCl) was changed every week.
At the end of the experiment, after 3 months growing with dif-
ferent NaCl concentrations (7-month-old plants), in order to
assess the effect of bacterial inoculation on A. macrostachyum
response to salinity in terms of growth, photosynthetic response
and Na
+
accumulation capacity, the following measurements
were performed. For growth analysis, at the beginning and the
end of the experiment, three and six plants per treatment were
harvested and dried at 60 °C for 48 h to estimate total shoot and
root dry weights and calculate the relative growth rate (RGR;
Redondo-G
omez et al. 2010). For photosynthetic response
assessment, instantaneous gas exchange and fluorescence mea-
surements were taken on random primary branches of each
plant (n =6) using an infrared gas analyser in an open system
(LI-6400-XT; Li-Cor, Lincoln, NE, USA) and a modulated fluo-
rimeter (FMS-2; Hansatech Instruments, UK), respectively.
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 3
Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al. Role of endophytes in halophyte salt tolerance
Thus net photosynthesis rate (A
N
), stomatal conductance (g
s
)
and intercellular CO
2
concentration (C
i
) were obtained at a light
intensity of 1000 lmolm
2
s
1
, atmospheric CO
2
of 400 ppm,
temperature of 25 °C and RH of 50 5%. Maximum quantum
efficiency of PSII photochemistry (F
v
/F
m
) and quantum effi-
ciency of PSII (Φ
PSII
) were recorded in light and dark-adapted
shoots at midday (1400 lmolm
2
s
1
) according to the proto-
col described in Redondo-G
omez et al. (2010). Finally, total
concentration of Na
+
in dried shoot and root samples of the six
replicates plants of each treatment were measured with induc-
tively coupled plasma (ICP) spectroscopy, as previously
described in Redondo-G
omez et al. (2010).
Statistical analysis
Statistical analysis was carried out using Statistica version 6.0
(Tulsa, OK, USA). Comparisons between means of the differ-
ent salinity and inoculation treatments at the end of the experi-
ment were assessed using two-way ANOVA (F-test).
RESULTS
Isolation and identification of endophytic bacteria from
A. macrostachyum
From the morphologies of colonies grown in TSA medium and
TSA supplemented with 0.3 MNaCl, eight different bacterial
strains were isolated from the aerial part of A. macrostachyum;
all were Gram-positive, spore-forming and rod-shaped. 16S
rRNA sequences indicated that isolates belonged to Bacillus,
Oceanobacillus and Gracilibacillus genera (Table 1). Although
EA1 and EA5, as well as EA4 and EA6, showed the highest
identity with the same species, they had different properties
and were considered different isolates.
Bacterial resistance to NaCl and presence of genes related to
salt tolerance
Bacteria were incubated from TSA plates containing increasing
NaCl concentrations. Resistance to NaCl ranged from 0.75 to
2.5 M(Table S1). Most isolates could be considered halotoler-
ant; however EA8 did not grow in TSA plates without NaCl,
starting to grow when plates were supplemented with at least,
0.3 MNaCl, hence it was considered halophilic. Due to this
NaCl resistance, the presence of genes related to osmoprotec-
tant synthesis, ectBC genes (for ectoine synthesis) and betB
gene (betaine aldehyde dehydrogenase for betaine synthesis)
was investigated with PCR using degenerate primers, either
described in the literature or designed in this work. For betB
amplification, betBF and betBR degenerate primers were
designed (Figure S1), as described in Material and Methods.
After PCR, all the isolates showed the presence of bands of the
expected sizes (Figure S2). The sequences of these bands
showed high identity with either ectBC or betB previously
described (data not shown).
Strains show enzymatic activities and PGP properties in the
presence of NaCl
Since some bacteria did not grow at all or poorly in media
without salt, assays for enzymatic activities and PGP properties
were performed in the presence of 0.3 MNaCl. Pectinase activ-
ity was present only in strain EA1 and chitinase in EA3,
whereas four isolates, including EA3, showed cellulase activity
(Table S1). In terms of other activities, amylase and protease
appeared in three strains, while only EA7 had DNAse. Finally,
lipase activity could not be observed in any strain (Table S1).
Concerning motility, half of the isolates were mobile, which is
an interesting feature of endophytic bacteria to reach their
appropriate location inside the plant.
For PGP properties, all strains had at least one plant-benefi-
cial property. Seven out of the eight isolates were auxin pro-
ducers, with amounts of IAA from 0.64 mgl
1
(EA2) to
4.17 mgl
1
(EA8; Table S2). Four strains were able to solu-
bilise phosphate: EA1, EA2 and EA5 were best solubilisers
(Table S2). In the case of siderophores, only EA3 and EA8 pro-
duced these compounds, EA3 being the best producer
(Table S2). Finally, half of the isolated bacteria were able to
form biofilms. Nitrogen fixation and ACC deaminase activity
were also investigated, but these properties could not be found
in any of the isolated bacteria.
In terms of PGP properties, enzymatic activities and resis-
tance to NaCl, a bacterial consortium containing strains Bacil-
lus alcalophilus EA1, B. thuringiensis EA3 and Gracilibacillus
saliphilus EA8 was selected for further experiments. Strain EA8
showed the highest auxin production (and also produced side-
rophores) and strains EA1 and EA3 had three PGP properties
each. Combining EA1, EA3 and EA8, each PGP property was
represented in two of the selected strains. In addition, EA1 had
pectinase and EA3 cellulase and chitinase activity.
Seed germination response to bacterial inoculation
Seed inoculation considerably improved the kinetics of germi-
nation and final germination percentage of A. macrostachyum
(Fig. 1). Thus, both velocity of germination and number of ger-
minated seeds were higher in inoculated seeds than in non-
inoculated control seeds, reaching 50% germination in 5 days,
while non-inoculated seeds needed 1 week to reach this per-
centage germination. The highest germination rate after
Fig. 1. Kinetics of germination rate of A. macrostachyum seeds without
and with bacterial pre-inoculation, treated with 0.9% saline solution for
20 days. Values represent mean of five replicates.
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands4
Role of endophytes in halophyte salt tolerance Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al.
10 days was 80% for inoculated seeds versus 57% for non-
inoculated seeds (Fig. 1).
Plant response to combine effect of salinity and bacterial
inoculation
The effects of salinity and bacterial inoculation on plant growth
were significant after 3 months of treatment (two-way ANOVA:
P<0.05). Thus total shoot and root dry mass was optimal at
510 mMNaCl external salinity with respect to control treat-
ment (i.e. zero NaCl), and did not show differences between
inoculation treatments at both salinity levels. Furthermore,
both parameters decreased considerably in plants grown in
1030 mMNaCl external salinity, however, this reduction was
mitigated in plants inoculated with the bacterial consortium
(Fig. 2A and B). Similar trends were reported in mean RGR,
with 45% and 72% reduction in plants grown at 1030 mMNaCl
with and without bacterial inoculation, respectively (Fig. 2C).
With respect to photosynthetic responses, results indicated
that A
N
,g
s
and C
i
were significantly influenced by salinity con-
centration (two-way ANOVA:P<0.05), with maximum values
in plants grown in 510 mMNaCl external salinity, while the
inoculation treatment had no statistically significant effect in
these parameters in either of the salinity concentrations
(Table 2). In addition, maximum quantum efficiency of PSII
photochemistry (F
v
/F
m
) and Φ
PSII
at midday did not vary with
salinity and inoculation treatments, with values around 0.78
and 0.70 in all inoculation and salinity treatments, respectively
(Table 2).
Finally, the concentrations of Na
+
in tissues of
A. macrostachym increased markedly in plants growth in the
presence of NaCl in the growth medium with respect to control
plants, with mean values ca.70mgg
1
and 20 mgg
1
for
shoots and roots, respectively, for both salinity levels and inoc-
ulation treatments (Fig. 3A and B), except in inoculated plants
grown at 1030 mMNaCl, which showed a significant increment
in shoot Na
+
concentration up to 103 mgg
1
(Fig. 3A).
DISCUSSION
In this study we attempted to elucidate the role of a select
group of endophytic bacteria isolated from the halophyte
A. machrostachyum and its tolerance to salinity stress in two
phases of its life cycle (germination and plant development).
For this purpose, eight endophytic strains of Bacillus and clo-
sely related genera were isolated from A. macrostachyum
growing in salt-affected soil. These genera are frequently iso-
lated from degraded soils due to their capacity to resist con-
taminants and NaCl (Abou-Shanab et al. 2007; Mesa et al.
2015b). Recently, Navarro-Torre et al. (2016) reported isola-
tion of 11 strains from the phyllosphere of A. macrostachyum
growing in the Odiel marshes (SW Spain). Although these
strains were completely different to those reported in this
work, the vast majority also belonged to a couple of genera.
The high level of NaCl in the phyllosphere of this halophyte
could explain both the small number of isolates and the
reduced biodiversity found. Strains B. alcalophilus EA1,
B. thuringiensis EA3 and Gracilibacillus saliphilus EA8 were
selected based on their PGP properties and enzymatic activi-
ties in order to assess their role in seed germination and
plant development under salt stress. Usually, the use of a
consortium for plant inoculants guarantees the highest plant
growth promotion with regard to single bacterium inocula-
tion, due to the synergistic effect of the PGP properties pre-
sent in the different strains (for recent examples see, Kumar
et al. 2014, 2016).
Inhibition of seed germination by salinity is not unusual for
halophytes (Ungar 1978; Woodell 1985; Baskin & Baskin
1998), and may be attributed to reduced osmotic potential of
the solution caused by salinity (Beadle 1952; Osmond et al.
1980; Ungar & Khan 2001), which hinders seed hydration
(Ramoliya & Pandey 2002). Concerning A. macrostachyum,
several studies have indicated that its seeds are able to
Fig. 2. Growth analysis of A. macrostachyum in response to a range of
NaCl concentrations and absence (non-inoculated) or presence (inoculated)
of bacteria after 3 months. A: Shoot dry mass; B: root dry mass, C: RGR.
Values represent mean SE, n =6. Different letters indicate means that are
significantly different from each other (P<0.05).
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands 5
Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al. Role of endophytes in halophyte salt tolerance
germinate well in the presence of NaCl (Pujol et al. 2001;
Rubio-Casal et al. 2003; Vicente et al. 2004). Our results
showed that both velocity of germination and number of ger-
minated seeds improved with bacterial inoculation. This bene-
ficial effect could be explaining by the presence of enzymatic
activities in the isolates employed for inoculation, such as
pectinase, cellulase and chitinase, which are important to break
the seed cell wall and could contribute to improve hydration of
seeds under saline conditions and, as consequence, could facili-
tate colonisation of saline soils by A. macrostachyum through
seeds.
Regarding plant growth, our results showed that
A. macrostachyum is extremely well adapted to salinity, with
optimal growth at 510 mMNaCl and without differences
between inoculation treatments, since the largest values of
shoot and root dry mass and RGR were recorded at this salin-
ity level. This optimal growth at 510 mMNaCl was consistent
with salt stimulation of dry mass production with 170–
510 mMNaCl reported previously for A. macrostachyum
(Khan et al. 2005; Redondo-G
omez et al. 2010). However,
growth decreased considerably in plants grown with 1030 mM
NaCl, although this reduction was lower in plants inoculated
with the bacterial consortium. Thus mechanisms underlying
the bacterial effect on salt tolerance of growth are evident
from our experiments. Salinity effects on growth were highly
supported by gas exchange measurements, which showed
responses to salinity that generally correspond well with RGR,
as previously reported (Redondo-G
omez et al. 2010). Thus A
N
was stimulated at salinity up to 510 mMNaCl, before a drastic
reduction at 1030 mMNaCl, which could be ascribed to direct
stomatal limitation of the CO
2
diffusion pathway, as indicated
in lower g
s
and C
i
at both extremes of salinity (i.e. zero and
1030 mMNaCl). However, contrary to expectations, mitiga-
tion of the deleterious effect of NaCl excess on growth of
A. macrostachyum did not mirror any positive effect on A
N
or
g
s
or in terms of functionality of PSII, since ours results
showed that F
v
/F
m
and Φ
PSII
did not vary between inoculation
or salinity treatments. Therefore, the bacterial inoculation
effects at high salinity can be attributed to an indirect benefi-
cial effect on global plant photosynthetic assimilation due to
the differences in development of the photosynthetic area
rather than to variations in A
N
. Hence, similar A
N
could be
more than compensated for with a larger photosynthetic area
at 1030 mMNaCl, as a result of shoot biomass stimulation
induced by the PGPB employed in plant inoculation. These
PGPB might improve plant growth, increasing accessibility or
supply of nutrients under this stressing condition (Bashan
1998). The production of auxins by strains EA1 and EA8
could help plant development through an increase in root sur-
face (Brader et al. 2014). In addition, auxins might contribute
to plant salt tolerance, acting as antagonists of ethylene pro-
duced by plants in stressing conditions. Also, EA1 increased
phosphorus availability to the plant through solubilising phos-
phate, and the capacity of EA3 and EA8 to produce sidero-
phores might improve iron solubilisation and uptake, thus
ameliorating the nutritional status of salinised plants (Burd
et al. 2000; Kpomblekou & Tabatabai 2003). This response
might provide positive feedback, since a larger photosynthetic
Table 2. Net photosynthesis rate, A
N
=stomatal conductance, g
s
=intercelular CO
2
concentration, C
i
=maximum quantum efficiency of PSII photochemistry,
F
v
/F
m
and quantum efficiency of PSII, Φ
PSII
in randomly selected primary branches of A. macrostachyum in response to treatment with a range of NaCl concen-
trations without and with bacterial inoculation for 3 months. Values represent mean SE, n =6.
Parameters
0m
MNACl 510 mMNaCl 1030 mMNaCl
non inoculated inoculated non inoculated inoculated non inoculated inoculated
A
N
(lmolm
2
s
1
) 4.2 0.7 4.8 0.5 5.7 0.5 6.0 0.2 3.3 0.3 3.8 0.3
g
s
(mmolm
2
s
1
) 38.2 2.6 43.7 4.9 62.9 9.9 67.3 4.3 29.4 2.7 32.1 5.9
C
i
(lmolmol
1
) 190 7 218 10 244 6 246 5 181 5 170 15
F
v
/F
m
0.76 0.01 0.77 0.01 0.78 0.01 0.79 0.01 0.78 0.01 0.78 0.01
Φ
PSII
0.68 0.01 0.67 0.02 0.73 0.02 0.72 0.01 0.71 0.01 0.70 0.02
Fig. 3. Total sodium (Na
+
) concentration for A: shoots and B: roots of
A. macrostachyum in response to a range of NaCl concentrations and
absence (non-inoculated) or presence (inoculated) of bacteria after
3 months. Values represent mean SE, n =6. Different letters indicate
means that are significantly different from each other (P<0.05).
Plant Biology ©2016 German Botanical Society and The Royal Botanical Society of the Netherlands6
Role of endophytes in halophyte salt tolerance Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al.
area would induce higher growth rates, which in turn produce
more photosynthetic area, amplifying the difference between
inoculated and non-inoculated plants at 1030 mMNaCl over
time. Although the increased salt tolerance could be attributed
to the presence of PGP properties in the selected isolates,
other well-known plant mechanisms are subject to osmotic
stress, e.g. accumulation of organic solutes or reduced levels
of antioxidant enzymes, which could also be produced by
these bacteria. Future research should detail the concrete
mechanisms leading to this enhanced salt tolerance and the
basis for the selection of the most efficient PGPB.
In terms of Na
+
accumulation, our study showed markedly
accumulation of Na
+
on a dry mass basis in root and shoots
subject to increasing external salinity, which relates to salt
uptake for osmoregulation, as suggested by Redondo-G
omez
et al. (2006, 2010). In addition, the biomass development after
bacterial inoculation at 1030 mMNaCl was also accompanied
by a higher shoot Na
+
concentration, indicating that the bacte-
rial consortium increased the potential of A. macrostachyum to
accumulate Na
+
in shoots. Osmotic adjustment, stomatal regu-
lation and enhanced uptake of minerals are beneficial effects
attributed to endophytes (Compant et al. 2005; Rajkumar et al.
2012). Enhanced uptake of Na
+
by the endophytic consortium
at high salt concentrations could explain the increased Na
+
accumulation in inoculated plants. In addition, Na
+
accumula-
tion into endophytes that colonise aerial parts of the plant
could also help increase salt phytoextraction. Several studies
have documented that PGPB can be used reliably to promote
growth of halophytic and non-halophytic plants under saline
conditions (Rueda-Puente et al. 2007; Gamalero et al. 2009; de
Bashan et al. 2012). Although the increase in plant yield
induced by PGPB may lead to enhanced salt phytoextraction
(Chang et al. 2014), there are no studies in the literature show-
ing increased salt phytoextraction on a per mass basis as a
result of PGPB addition (Jes
us et al. 2015). To our knowledge,
this is the first study reporting increased salt phytoextraction
on a per mass basis as a result of PGPB addition.
In conclusion, endophytes selected from the phyllosphere of
A. macrostachyum seem to play an important role in its toler-
ance to stressful concentrations of NaCl. Bacterial inoculation
improved seed germination rate, enhanced plant growth at
high NaCl concentrations and, what is more interesting and
novel, favour Na
+
phytoextraction capacity in such situations.
Thus, the combined use of A. macrostachyum and its micro-
biome can provide an adequate tool to enhance plant adapta-
tion and Na
+
phytoextraction during restoration of salt-
degraded soils.
ACKNOWLEDGEMENTS
This work was funded by Junta de Andaluc
ıa (P11-RNM-
7274MO project), INIA (RTA 2012-0006-C03-03 project) and
University of Sevilla (VPPI-US Project). S.N-T thanks Junta de
Andaluc
ıa and J.M.B-P thanks INIA for personal financial sup-
port. The authors are grateful to University of Seville Green-
house General Services and Microscopy Service (CITIUS) for
their collaboration.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in the
supporting information tab for this article:
Figure S1. Alignment of betaine aldehyde dehydrogenase
(betB) sequences from different bacterial genera showing the
conserved regions chosen to design the degenerate primers,
betBF (red box) and betBR (black box).
Figure S2. Presence of genes for synthesis of osmoprotectans
in bacterial strains EA1 to EA8. (A) Amplification of ectBC
genes. 1-kb DNA ladder was used to estimate band size. c:
negative control (without ADN); c+: positive control (amplifi-
cation with DNA from Chromohalobacter salixigens). Bands of
around the expected 0.9 kb are observed in all strains. (B)
Amplification of betB gene. 100-bp DNA ladder was used to
estimate band size. c: negative control (without ADN). Bands
of around the expected 0.7 kb are observed in all strains.
Table S1. Enzymatic activities, motility and NaCl tolerance
of the endophytic strains. (+) presence of enzymatic activity or
motility. () absence of enzymatic activity or motility.
Table S2. PGP properties of the endophytes isolated.
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Role of endophytes in halophyte salt tolerance Navarro-Torre, Barcia-Piedras, Mateos-Naranjo et al.
