Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

A nodule endophytic Bacillus megaterium strain isolated from

Medicago polymorpha enhances growth, promotes nodulation
by Ensifer medicae and alleviates salt stress in alfalfa plants
A. Chinnaswamy1,2,* , T. Coba de la Peña1,3,* , A. Stoll3,4 , D. de la Peña Rojo1 , J. Bravo3,5 , A. Rincón1 ,
M.M. Lucas1,† & J.J. Pueyo1,†
1 Institute of Agricultural Sciences, Spanish National Research Council, ICA-CSIC, Madrid, Spain
2 Present address: Sugarcane Breeding Institute, Division of Crop Improvement, Coimbatore, Tamil Nadu, India
3 Centro de Estudios Avanzados en Zonas Áridas (CEAZA), La Serena, Chile
4 Universidad de La Serena, La Serena, Chile
5 Laboratorio Integral de Investigación en Alimentos-Biotecnología, Instituto Tecnológico de Tepic, Tepic, Mexico

Keywords
Bacillus megaterium; endophyte; Medicago;
nodule; PGPB; salt stress.
Correspondence
J.J. Pueyo, Institute of Agricultural Sciences,
ICA-CSIC, Serrano 115-bis, 28006 Madrid,
Spain. Email: jj.pueyo@csic.es
∗ These authors contributed equally to this study.
† These authors contributed equally to this study.
Received: 22 February 2017; revised version
accepted: 22 December 2017; published online:
19 February 2018.
doi:10.1111/aab.12420
Abstract
A Gram-positive, fast-growing, endophytic bacterium was isolated from root
nodules of Medicago polymorpha and identified as Bacillus megaterium. The isolate,
named NMp082, co-inhabited nodules with the symbiotic rhizobium Ensifer
medicae. B. megaterium NMp082 contained nifH and nodD genes that were 100%
identical to those of Ensifer meliloti, an unusual event that suggested previous
lateral gene transfer from a different rhizobial species. Despite the presence
of nodulation and nitrogen fixation genes, the endophyte was not able to
form effective nodules; however, it induced nodule-like unorganised structures
in alfalfa roots. Axenic inoculation promoted plant growth in M. polymorpha,
Medicago lupulina, Medicago truncatula and Medicago sativa, and co-inoculation
with E. medicae enhanced growth and nodulation of Medicago spp. plants com-
pared with inoculation with either bacterium alone. B. megaterium NMp082 also
induced tolerance to salt stress in alfalfa and Arabidopsis plants. The ability to
produce indole acetic acid (IAA) and the 1-aminocyclopropane-1-carboxylate
(ACC) deaminase activity displayed by the endophyte in vitro might explain the
observed plant growth promotion and salt stress alleviation. The isolate was
also highly tolerant to salt stress, water deficit and to the presence of different
heavy metals. The newly characterised endophytic bacterium possessed specific
characteristics that point at potential applications to sustain plant growth and
nodulation under abiotic stress.

Introduction phytohormones, including auxins and cytokinins, are

Symbioses between legumes and rhizobia are of con-
siderable environmental and agricultural importance
since these associations are the most important ones
for nitrogen fixation (Graham & Vance, 2003). The
establishment of an effective nitrogen-fixing symbiotic
relationship requires sequential signal exchange between
the bacterial microsymbiont and the host plant (Fisher &
Long, 1992; Cooper, 2007). Nodule formation is induced
by rhizobial secreted signal molecules called Nod factors
(controlled by the nod genes), which are recognised by
the host plant (Schultze & Kondorosi, 1998). Several
also involved in the induction of root cortical cell division
leading to the formation of the nodule primordium (Fer-
guson & Mathesius, 2014). The nif genes, codifying for
the nitrogenase enzyme complex and several regulatory
proteins, are essential for nitrogen fixation by bacteria
(Franche et al., 2009).
Many naturally occurring, free-living soil bacteria
that enhance plant health and growth are classified as
plant growth promoting bacteria (PGPB), including plant
growth promoting rhizobacteria (PGPR) and endophytic
PGPB (De Souza et al., 2015; Santoyo et al., 2016). Plant

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A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
growth promoting bacteria inoculants are able to increase
plant growth, improving seed germination, seedling
emergence and plant responses to external stress factors,
and thus protect plants from disease (Bhattacharyya &
Jha, 2012; De Souza et al., 2015). Plant growth promoting
bacteria belong to diverse genera, such as Pseudomonas,
Streptomyces, Azospirillum, Agrobacterium, Rhizobium, Bacil-
lus and others, and they have been isolated from a wide
range of plant species (Bhattacharyya & Jha, 2012).
Plant growth promoting bacteria are of great interest as
biofertilizers in agriculture and can play an important
role in phytoremediation (Vessey, 2003; Glick, 2010).
The direct mechanisms of plant growth stimula-
tion by PGPB include solubilisation and mobilisation
of nutrients, thus improving plant nutritional status
(Glick, 1995; Srivastwa et al., 2014), nitrogen fixa-
tion (Castellano-Hinojosa et al., 2016; Kifle & Laing,
2016), siderophore production that increases iron
availability in the rhizosphere (Kloepper et al., 1980;
Sharma et al., 2016), production of phytohormones
(such as IAA, cytokinins and gibberellins) and other
growth regulators (Cassán et al., 2014) and production of
1-aminocyclopropane-1-carboxylate (ACC) deaminase,
that cleaves the intermediate ethylene precursor ACC,
reducing plant ethylene levels, thus promoting growth
and protecting against abiotic stresses (Glick, 2014;
Gamalero & Glick, 2015). Indirect mechanisms include
production of antibiotics and lytic enzymes, competitive
exclusion of soil-borne plant pathogens, induction of
systemic resistance (phenotypically similar to systemic
acquired resistance) or removal of phytotoxic substances
(Bhattacharyya & Jha, 2012; Paul & Lade, 2014).
There is an increasing interest in the identification and
characterisation of PGPB associated with legumes because
of the positive effects on growth, nitrogen fixation and
tolerance to biotic and abiotic stresses (Grover et al.,
2011; Dudeja et al., 2012; Taurian et al., 2012), including
salt stress (Paul & Lade, 2014). Nodule endophytes dis-
playing PGPB properties have been isolated and charac-
terised in different legumes and include species from the
genera Agrobacterium, Bacillus, Pseudomonas, Salmonella,
Klebsiella, Burkholderia, Enterobacter, Pantoea, Phyllobac-
terium and others (Bai et al., 2002; Rajendran et al., 2008;
Stajković et al., 2009; Dudeja et al., 2012). Moreover,
it has been reported that co-inoculation of symbiotic
rhizobia with nodule endophytic PGPB or PGPR can
enhance legume plant growth, nodulation and nitrogen
fixation, compared to inoculation with rhizobia alone
(Bai et al., 2002; Stajković et al., 2009; Masciarelli et al.,
2014). Some legume nodule endophytes contain either
nifH-like genes and/or nod genes in the genome, acquired
through horizontal gene transfer (Zakhia et al., 2006;
Li et al., 2008).
296
A. Chinnaswamy et al.
Here, we report the isolation and characterisation of an
endophytic Bacillus megaterium present in naturally occur-
ring root nodules of Medicago polymorpha plants growing
in a heavy metal polluted area. This bacterial strain was
highly tolerant to salinity and heavy metals, and displayed
plant growth promoting activities in vitro. It also promoted
plant growth in several Medicago species and in the model
plant Arabidopsis thaliana L., and improved salt tolerance
in M. sativa and A. thaliana. Moreover, co-inoculation with
Ensifer medicae enhanced nodulation and plant growth
in several Medicago species (M. sativa, M. polymorpha, M.
lupulina and M. truncatula) to a higher extent than inocu-
lation with either of the two bacteria alone. nifH and nodD
genes within this B. megaterium strain were identified and
characterised. This bacterium also induced the develop-
ment of nodule-like structures in roots of M. sativa in the
absence of rhizobial bacteria. We discuss here the possible
mechanisms involved in the promotion of plant growth
upon inoculation with this B. megaterium strain and its
potential application in the field.
Materials and methods
Isolation of nodule bacteria
Healthy-nodulated M. polymorpha plants were collected in
the Almadén mining district (Ciudad Real, Spain) in order
to isolate rhizobia and endophytic bacteria, as previously
described (Nonnoi et al., 2012). Root nodule sampling and
soil characteristics are described in Nonnoi et al. (2012).
Nodules were surface sterilised with 96% ethanol for 30 s
and with 0.1% HgCl2 for 30 s, and exhaustively washed in
sterile distilled water. Isolation of rhizobia from nodules
was performed as previously described by Nonnoi et al.
(2012). Colony morphology, growth time and Gram and
Congo red staining were examined.
Isolation of genomic DNA, polymerase chain reaction
amplification and gene sequencing
Genomic DNA isolation was performed according to the
procedure described previously (Nonnoi et al., 2012). The
16S rRNA gene was amplified by PCR as described pre-
viously (Weisburg et al., 1991). The nodAB genes were
amplified following the method of Moulin et al. (2001).
The nodC and nifH genes were amplified as described by
Laguerre et al. (2001). The nodD gene was amplified as
reported by Rivas et al. (2002). Polymerase chain reaction
products were purified using the GFX™ PCR Gel Band
Purification Kit (Amersham Pharmacia Biotech Inc., -
Buckinghamshire, UK). Gene sequencing was performed
as described in El-Akhal et al. (2008). Sequences were
analysed using the BLASTN tool from the NCBI website.
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A. Chinnaswamy et al. A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
Plant growth promoting activities in vitro
One of the bacterial isolates obtained, B. megaterium
NMp082, was selected for testing its plant growth promot-
ing capacities, in different completely randomised tests
(Table S1), as detailed below. In all cases, an initial bac-
terial inoculum of OD600nm = 0.8 was used.
Phosphate solubilisation activity was verified by plac-
ing four separated 5 μL drops of bacterial inoculum on
Pikovskaya medium-PVK plates (Vazquez et al., 2000).
Plates (n = 3) were incubated at 28∘ C, and the solubilisa-
tion efficiency E ((halo diameter/colony diameter) × 100)
was evaluated after 72 h (Nguyen et al., 1992).
Siderophore production was assessed by placing four
separated 5 μL drops of bacterial inoculum on plates with
agar-CAS (Alexander & Zuberer, 1991). Plates (n = 3)
were incubated at 28∘ C for 72 h, and siderophore pro-
duction was revealed by the formation of an orange halo
around bacterial colonies.
In order to test whether the bacterial isolate was able to
use ACC as a sole nitrogen source, serial dilutions (10−2 ,
10−3 and 10−4 ) of the initial bacterial inoculum were
plated onto solid Dworkin and Foster (DF) salt minimal
medium supplemented with ACC, and plates (n = 3 per
dilution) were incubated at 28∘ C for 3 days (Penrose &
Glick, 2003). The ACC deaminase activity was quantified
by measuring the amount of 𝛼-ketobutyrate produced
when the enzyme ACC deaminase cleaves ACC (Honma
& Shimomura, 1978; Penrose & Glick, 2003).
For indolic compounds determination, the bacte-
rial isolate was incubated in liquid Luria-Bertani (LB)
medium at 150 rpm and 28∘ C, for 66 h. Auxin production
was estimated by assay with Salkowski reagent (Glick-
mann & Dessaux, 1995). Flasks were used as biological
replicates (n = 3), and each one was measured three times
(i.e. technical replicates).
For detection of nitrogen fixation activity, the bacterial
strain was plated on Nfb medium (Kirchhof et al., 1997;
n = 3 plates). After incubation for 24 h at 28∘ C, the colour
change from green to blue was indicative of bacterial N
fixation activity.
Tolerance to sodium chloride, water deficit and heavy
metals
Sensitivity to salt stress, water deficit and heavy metals
were determined as previously described (Nonnoi et al.,
2012), for which different randomised experiments with
n = 3 replicates each, were performed (Table S1).
Salt tolerance was tested in Tryptone Yeast (TY) agar
medium supplemented with increasing NaCl concentra-
tions (0–2000 mM,) in 100 mM steps (n = 3 plates per
concentration).
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Water deficit was tested in TY agar medium sup-
plemented with increasing PEG200 concentrations
(0–2000 mM) in 100 mM steps (n = 3 plates per
concentration).
Metal tolerance was tested with the same metals
detailed in Nonnoi et al. (2012). All metal concentrations
ranged from 0.01 to 10 mM, in 25 μM steps for Hg and Ag,
100 μM steps for Ni, Pb, Cd, Zn, Cr, Cu and Co, and 1 mM
steps for As (n = 3 plates per concentration), depending
on the metal, and controls consisting of medium without
metal were included in all tests. The minimum inhibitory
concentrations (MICs), defined as the lowest concentra-
tion of a chemical that prevents visible growth of a bac-
terium, were determined.
In vitro antifungal activity
Bacillus megaterium NMp082 was screened for in vitro
antagonism against the plant pathogenic fungi Sclero-
tinia sp. and Botrytis sp. obtained from lettuce. The test
consisted of a randomised design with three replicates,
in which each fungal pathogen was grown in presence
or absence of the bacterial isolate (n = three plates per
fungus in each case). Fungi and bacteria were grown
on Potato Dextrose Agar (PDA) plates for 4 days at
room temperature. Halo formation and growth inhibi-
tion of the pathogen were evaluated, using the equation
I = ((C-T)/C) × 100, where I is the percentage of inhibi-
tion, C is the diameter of fungi growth in control (i.e.
without bacteria), and T is the diameter of fungi growth in
dual culture with bacteria (Odebode et al., 2004; Simon-
etti et al., 2011).
Bacterial inoculation and co-inoculation of Medicago
spp. plants in low nitrogen conditions
Seeds of Medicago sativa cv. Aragon R-1 (Rocalba, Girona,
Spain), M. polymorpha cv. Scimitar, Medicago lupulina cv.
Virgo Pajbjerg and Medicago truncatula cv. Parabinga,
(Semillas Silvestres S.L., Córdoba, Spain) were scarified
with sandpaper and surface sterilised in 70% ethanol
(1 min) and 50% sodium hypochlorite (10 min). Seeds
were placed on 1% water agar on Petri dishes and incu-
bated for 24–36 h at 25 ∘ C. Germinated seedlings (root
length 1–1.5 cm) were transferred to growth pouches
(CYG Seed Germination Pouches, Mega International,
Minneapolis) (five seedlings, i.e. technical replicates, per
pouch).
To test the response of the different Medicago species
to inoculation with the selected bacterial strains, a
three-factorial design experiment with the factors
(a) ‘plant species’ with four treatments: M. sativa,
M. polymorpha, M. lupulina, M. truncatula, (b) ‘inoculation
297
A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
with B. megaterium’ with two treatments (+/−) and
(c) ‘inoculation with E. medicae’ with two treat-
ments (+/−), was performed (n = 12 pouches) per
plant species and bacterial inoculation treatments
(Table S1).
Bacteria were grown to the exponential phase and
inoculations were performed 3 days after seedling trans-
plantation to pouches. The pouches were watered with
low-nitrogen (12.6 mg L−1 KNO3 ) Hoagland’s nutrient
solution and placed in a growth chamber under controlled
conditions (24∘ C, 180 μmol photon m−2 s−1 , 16/8 h pho-
toperiod). After 30 days, different morphometric param-
eters of plants (nodule number, root fresh weight, root
length, shoot fresh weight, shoot length and number of
trifoliate leaves) were recorded per pouch.
Light microscopy
Sample preparation and light microscopy were per-
formed as explained elsewhere (Redondo et al., 2009).
Briefly, M. sativa nodule-like root outgrowths were col-
lected 20 days after inoculation with B. megaterium and
immediately fixed for 2 h at 4 ∘ C in 5% glutaraldehyde
and 4% paraformaldehyde in 100 mM Na-cacodylate
buffer (pH 7.4) containing sucrose (25 mg mL−1 ). After
a second fixation stage (1.5 h at 4∘ C), specimens were
washed in the sucrose-cacodylate buffer three times (1 h
each, 4∘ C). Tissues were post-fixed with a solution of
1% osmium tetroxide in the same buffer (16 h, 4∘ C).
After two washes with the same buffer (5 min each,
4∘ C), specimens were dehydrated in an ethanol series in
water (30%, 50%, 70%; 10 min each, 4∘ C). Dehydra-
tion continued with 1% uranyl acetate in 70% ethanol
(24 h, 4∘ C), 90% ethanol (10 min, 4∘ C), 96% ethanol
(30 min, 4∘ C) and 100% ethanol (2 h, 4∘ C). Samples
were embedded in London Resin White (London Resin
Co., London, UK) and polymerised into gelatin capsules
for 24 h at 60∘ C. Semithin (1 mm) sections were cut with
a Reichert Ultracut S ultramicrotome (Leica, Vienna, Aus-
tria) fitted with a diamond knife. Semithin sections were
stained with 1% (w/v) toluidine blue in aqueous 1%
sodium borate.
Direct observation of sections was performed under a
Zeiss Axiophot photomicroscope (Carl Zeiss, Oberkochen,
Germany) with an attached digital camera (Leica DFC
420C, Heerbrugg, Switzerland).
Plant growth promoting bacteria effect on alfalfa seed
germination under salt stress
The effect of B. megaterium on seed germination was
tested in an in vitro test, considering the factors (a) ‘inoc-
ulation’ with two treatments: control without bacteria,
298
A. Chinnaswamy et al.
and inoculated with bacteria and (b) ‘salt stress’ with
two treatments: control without NaCl, and 100 mM
NaCl stress, with a total of 12 plates (n = three plates per
treatment) (Table S1).
Medicago sativa cv. Aragon R-1 (Rocalba, Girona, Spain)
seeds were sterilised in 70% ethanol for 10 min, washed
in sterile distilled water and incubated in 0.1% HgCl2 for
2 min and washed again in sterile water.
Colonies of B. megaterium were firstly grown on LB
agar medium, and then transferred to 0.5× Murashige
and Skoog (MS; Murashige & Skoog, 1962) agar supple-
mented with 10 g L−1 sucrose (and 0 or 100 mM NaCl
depending on the treatment) in 120 mm × 120 mm square
Petri dishes. Bacteria were streaked in straight lines over
the agar surface, dividing the agar medium surface in four
identical squares (i.e. technical replicates), and plates (i.e.
experimental unit) were incubated at 28∘ C during 48 h
in the dark. Right after, M. sativa seeds were placed on
the Petri dishes (25 seeds per quadrant, i.e. 100 seeds
per plate). Plates were placed upside down in a growth
chamber at 24∘ C in the dark, and germination rates were
recorded after 18, 42 and 66 h.
Plant growth promoting bacteria effect on alfalfa plant
growth under salt stress
The effect of B. megaterium on plant growth was tested
following the same two-factorial design explained above
(i.e. ± bacteria and ± salt stress), in an experiment using
growth pouches as the experimental unit (n = 11 per
treatment) (Table S1).
Sterile seeds of M. sativa were placed on 1% water
agar on Petri dishes and incubated for 24–36 h at 24∘ C in
the dark. Germinated seedlings (root length 1–1.5 cm)
were incubated for 30 min in B. megaterium liquid inocu-
lum grown to exponential phase in LB (OD600nm 0.8).
Then, seedlings were transferred to growth pouches
(six seedlings, i.e. technical replicates, per pouch)
with Hoagland’s nutrient solution containing nitro-
gen (1000 mg L−1 KNO3 ) (Hoagland & Arnon, 1950),
and 1.5 mL of B. megaterium inoculum at exponential
phase was added, or not, to each pouch, depending on
the treatment. Plants were grown in a growth cham-
ber under controlled conditions (24∘ C, 180 μmol photon
m−2 s−1 , 16/8 h photoperiod). After 5 days of acclimation,
the nutrient solution was removed, new nutrient solu-
tion supplemented or not with NaCl (0 mM or 100 mM
depending on the treatment), and pouches were main-
tained in the growth chamber for other 9 days. After this
time, the morphometric parameters of plants (root length
and fresh weight, number and length of secondary roots,
shoot length and fresh weight, and number of leaves)
were recorded per pouch.
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A. Chinnaswamy et al. A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa

Plant growth promoting bacteria effect on growth district (Ciudad Real, Spain) were examined in order to
and survival of Arabidopsis thaliana under salt stress isolate rhizobia and endophytic bacteria. As described in
Nonnoi et al. (2012), bacterial isolates were classified as
The effect of B. megaterium isolate on the growth of the
Gram-negative. In one of the analysed nodules, besides
model plant A. thaliana was tested in a two-factorial in
the described Gram-negative bacteria, a different isolate
vitro test, considering the factors (a) ‘inoculation’ with two
was identified as a fast growing bacterium, which formed
treatments: control without bacteria, and inoculated with
colonies that were evident on TY agar plates after less
bacteria and (b) ‘salt stress’ with three treatments: control
than 24 h at 28∘ C. This bacterial strain named NMp082
without NaCl, and with 50 and 100 mM NaCl, with a total
produced white colonies with serrated margins, and it
of 18 plates (n = 3 plates per treatment) (Table S1).
was positive in Gram and Congo red staining.
Sterile and hibernated seeds of A. thaliana were placed
Polymerase chain reaction amplification of the 16S
on 0.4× MS agar plates and incubated for 4–5 days in
rRNA gene produced a single 1357 bp amplicon for
a growth chamber at 20∘ C (16/8 h photoperiod). When
all bacterial isolates (Nonnoi et al., 2012), except for
seedling roots reached 0.5 cm length, 10 seedlings (i.e.
NMp082, which yielded a 1432 bp PCR product. 16S
technical replicates) were transferred on a new 0.4× MS
rRNA gene sequencing and data base analysis identified
agar plate enriched with 10 g L−1 sucrose and adjusted for
isolate NMp082 as B. megaterium (99% sequence iden-
salt concentration depending on the treatment (0, 50 and
tity). The rest of the isolates were classified as E. medicae
100 mM NaCl). B. megaterium was streaked on the surface
(Nonnoi et al., 2012).
of the agar in a straight line, at a distance of approximately
Polymerase chain reaction amplifications of the B.
5 cm below lined up seedlings. Control plates without
megaterium genomic DNA were performed using specific
bacteria were also established. The plates were placed
primers to identify symbiosis related genes. Sequence
vertically and incubated for 20 days in a growth chamber
analysis of a 780 bp PCR product revealed 100% sequence
at 20∘ C (16/8 h photoperiod). After this time, root length
identity with the nifH gene of E. meliloti. A 501 bp-long
and number of secondary roots were measured with
PCR product was 100% identical to the nodD gene of
WinRHIZO 2013a software (Regent Instruments Inc.,
E. meliloti. Polymerase chain reaction amplification with
Quebec, Canada), and root and leaf areas were measured
nodC specific primers produced a 503 bp-long PCR prod-
using the ImageJ software (NIH, Bethesda, Maryland,
uct, which had no sequence similarity to any known nodC
USA). Plant survival was also assessed.
gene. Similarly, PCR amplification using nodAB primers
produced a 1100 bp PCR product; however, sequence
Statistical analyses analysis did not identify any putative nodAB genes.
Due to the provenance of B. megaterium NMp082, iso-
Data were analysed with the SPSS v.23 software (IBM
lated from M. polymorpha nodules growing in a heavy
Corporation Software Group, Somers, NY, USA). Prior to
metal-polluted area, its tolerance to several metals was
analysis, all variables were tested for a Normal distribution
tested. The MICs for each metal are shown in Table 1.
and sqrt, log or logit (for percentages) transformed to meet
variance homoscedasticity, when required.
Seed germination was analysed by repeated measures Table 1 Tolerance to metals, salt stress and water deficit of Bacillus mega-
terium NMp082.
of analysis of variance (ANOVA) (P < 0.05; F-test). Facto-
rial experiments with pouches and plates, in which the MIC (mM)
effect of inoculation and plant species or inoculation and
Ag (AgNO3 ) 0.15
salt stress was tested, were analysed by three or two-factor
Zn (ZnSO4 .7H2 O) 1
ANOVA (P < 0.05; F-test). The standard error of difference
Pb (C4 H6 O4 Pb.3H2 O) 2
(SED) and the least significant difference (LSD) at 5% Cu (CuSO4 .5H2 O) 2
significance were calculated to detect differences among Cr (KCr(SO4 )2 .12H2 O) 3
means. Hg (HgCl2 ) 0.075
Cd (CdCl2 ) 1
Co (CoCl2 ) 3
Results Ni (NiSO4 .6H2 O) 2
As (NaAsO2 ) 7
Isolation and characterisation of nodule endophytic NaCl 1700
plant growth promoting bacteria B. megaterium Polyethylene Glycol 200 1000
NMp082
Metal salts are indicated in parenthesis. MIC is the lowest concentration of a
Nodules of M. polymorpha plants growing in chemical that prevents visible growth of a bacterium.
mercury-contaminated soils in the Almadén mining MIC, minimum inhibitory concentration.

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Table 2 Effects of inoculation of different Medicago species with Bacillus megaterium NMp082 (−/+) and/or with Ensifer medicae (−/+) on growth and nodulation
of plants, analysed by three or two-factora ANOVA (P < 0.05; F-test). Non-inoculated plants were used as controls. Plants were grown in hydroponic pouches
with N-free nutrient solution and harvested 30 days after inoculation. For each plant species and inoculation treatment, values are means of n = 12 pouches (with
five plants per pouch). Nodule-like outgrowths elicited by B. megaterium in M. sativa plants were not counted as nodules.

Bacillus Ensifer No Trifoliate leaves1 Shoot length (cm) Shoot weight (g) Root weight (g) No. nodules2 a

M. sativa − − 2.1 (1.449) 4.695 0.060 0.037

− + 4.1 (2.028) 9.335 0.110 0.059 7.3 (−1.101)
+ − 4.2 (2.056) 9.245 0.101 0.060
+ + 4.3 (2.077) 9.823 0.141 0.098 11.2 (−0.899)
M. polymorpha − − 2.0 (1.414) 4.050 0.045 0.030
− + 3.0 (1.726) 5.423 0.082 0.059 4.5 (−1.325)
+ − 3.1 (1.760) 5.682 0.090 0.069
+ + 3.6 (1.910) 5.780 0.122 0.089 7.7 (−1.080)
M. lupulina − − 1.8 (1.341) 4.250 0.037 0.030
− + 2.7 (1.648) 5.210 0.071 0.043 3.2 (−1.481)
+ − 2.6 (1.622) 5.183 0.070 0.048
+ + 2.9 (1.702) 5.450 0.103 0.076 5.8 (−1.210)
M. truncatula − − 1.7 (1.303) 4.100 0.040 0.030
− + 3.6 (1.897) 6.517 0.091 0.058 5.4 (−1.241)
+ − 3.5 (1.870) 6.660 0.090 0.050
+ + 4.1 (1.991) 7.517 0.131 0.080 9.8 (−0.962)
SED (0.003) 0.007 0.0001 0.0001 (0.002)
LSD 5% (0.007) 0.013 0.0001 0.0001 (0.013)
df 7 7 7 7 3

a Two-wayANOVA performed for the factors ‘Plant species’ and ‘Inoculation with Bacillus’.
1 sqrt
and 2 logit (transformed).
ANOVA, analysis of variance; LSD, least significant difference; SED, standard error of difference.

According to Nieto et al. (1989), strains that are not inhib-
ited by 10 mM As (i.e. MIC >10 mM), 1 mM Ag, Cd, Co,
Cr, Cu, Ni, Pb and Zn (MIC >1 mM) and 0.1 mM Hg (MIC
>100 μM) are considered tolerant. According to these cri-
teria, the B. megaterium strain could be considered tolerant
to Pb, Cu, Cr, Co and Ni. With respect to salt and water
stress, it was observed that this strain tolerated high lev-
els of salinity (MIC = 1.7 M NaCl) and of water deficit
(MIC = 1 M PEG) (Table 1).
Concerning the plant growth promoting ability of B.
megaterium NMp082, it displayed ACC deaminase activity
(7.35 ± 0.31 nmol 𝛼-ketobutyrate mg−1 h−1 ) and produc-
tion of auxins (3.28 ± 0.21 μg mL−1 in the absence of tryp-
tophan and 8.82 ± 0.57 μg mL−1 in the presence of 1 mM
tryptophan). Neither phosphate solubilisation activity nor
siderophore production was detected. B. megaterium was
not able to grow in N-free medium, and no antifungal
activity against the plant pathogenic fungi Sclerotina spp.
or Botrytis spp. was detected.
Bacillus megaterium NMp082 promoted the growth
of Medicago spp. plants in low-nitrogen conditions
and had a synergistic effect when co-inoculated
with Ensifer medicae
The nodule endophytic origin of B. megaterium NMp082
and the presence of nifH and nodD genes suggested the
possibility that it might be able to form nodules and/or
provide fixed nitrogen to the plant when inoculated
alone. A positive effect on plant growth due to its PGPB
traits could also be expected when inoculated alone or in
co-inoculated with E. medicae.
The growth and nodulation of plants were significantly
influenced by the factors studied, that is, ‘inoculation
or not with B. megaterium’, ‘inoculation or not with E.
medicae’ and ‘plant species’, and significant interaction
between factors was observed for all variables (Table S2).
With respect to the factor plant species, significant
differences (P < 0.05, LSD), in growth and nodulation
were observed among the different Medicago spp. in all
cases, with higher values observed in general for M. sativa
(Table 2).
With respect to the factor inoculation, the application
of B. megaterium NMp082 to the Medicago species, under
limiting nitrogen conditions, produced in all cases a sig-
nificant improvement (P < 0.05, LSD) of the shoot length
and plant biomass (root and shoot fresh weight), and of
the number of trifololiate leaves in the case of M. sativa
and M. truncatula, compared to the control non-inoculated
treatment (Table 2). Similarly, the inoculation of all Med-
icago species with the microsymbiont E. medicae improved
all plant growth parameters with respect to the control
non-inoculated plants (P < 0.05, LSD) (Table 2).

300 Ann Appl Biol 172 (2018) 295–308

© 2018 Association of Applied Biologists
A. Chinnaswamy et al.
A B
Figure 1 Pseudonodules formed by Bacillus megaterium NMp08 on alfalfa roots (A). Micrograph of a pseudonodule section showing a cortex and an internal
zone. Notice the presence of cells with different sizes, very small freshly divided cells (white arrowheads) and larger cells in the internal zone (B). Detail of a
pseudonodule with cells containing abundant starch granules (white arrows) (C). Bars: 50 μm.
Ensifer medicae-inoculated plants and B. mega-
terium-inoculated plants presented significant (P < 0.05,
LSD) differences for some morphometric parameters
depending on the Medicago species (Table 2). However,
when inoculated alone, B. megaterium did not support
plant growth beyond the 30 days of the experiment,
leaves turned yellow and plants eventually died, suggest-
ing that B. megaterium NMp082 failed to provide nitrogen
to the plants, and that the positive effects occurred as
a result of the PGPB traits of the strain. Interestingly
though, B. megaterium NMp082 induced nodule-like root
outgrowth structures that became visible about 20 days
after inoculation in the roots of approximately half of
the M. sativa plants; none of the other Medicago spp.
exhibited such formations. The pseudonodules appeared
on the main root associated with the emergence of
lateral roots (Fig. 1A). They did not present the typical
elongated shape of alfalfa nodules, they were round and
whitish in colour, and lacked the pink colour due to the
leghaemoglobin that characterises active nitrogen-fixing
nodules. No nitrogenase activity could be detected. They
presented a cortex composed by 3–4 layers of cells and
an internal zone formed by uninfected cells (Fig. 1B and
Fig. 1C) of different sizes, denoting areas with different
rate of cell division and a diffuse meristem. Big starch
granules were observed in many cells in the internal
zone (Fig. 1C). We did not observe differentiated vascular
tissue in the nodule-like structures.
Co-inoculation of Medicago spp. plants with both B.
megaterium and E. medicae produced a significant (P < 0.05,
LSD) enhancement of all plant growth parameters in
comparison with the non-inoculated treatment and with
treatments of individual inoculations (i.e. either B. mega-
terium or E. medicae) (Table 2). Nodulation of all plant
Ann Appl Biol 172 (2018) 295–308
© 2018 Association of Applied Biologists
A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
C
species by E. medicae was also significantly (P < 0.05,
LSD) improved by co-inoculation with B. megaterium. The
co-existence of the endophytic B. megaterium inside the
nodules of all Medicago spp. along with E. medicae was fur-
ther confirmed by re-isolation from surface-sterilised root
nodules after co-inoculation.
Bacillus megaterium NMp082 enhanced seed
germination and growth of Medicago sativa under
non-stress and salinity conditions
The elevated salt tolerance of the endophytic bacterium
and its notable levels of ACC deaminase activity led us to
test its effects on alfalfa under salinity stress conditions.
The factors ‘inoculation’ and ‘salt stress’ were considered
in order to check whether the germination of M. sativa
seeds was affected by compounds secreted by the bac-
terium. Both factors significantly affected seed germina-
tion, and their interaction was also significant (Table S3;
Fig. 2).
Inoculation with B. megaterium improved early seed
germination (8.0% and 15.8% increase, in absence or
presence of salt, respectively; Fig. 2). Additionally, seeds
inoculated with B. megaterium overcame the negative
effect of salt stress, and germinated significantly (P < 0.05,
LSD) better than control seeds without bacteria (Fig. 2).
Over time, germination significantly improved after 18 h
and similar germination rates were observed after 42 and
66 h in all cases (P < 0.05, LSD; Fig. 2).
Similarly, inoculated and uninoculated M. sativa plants
grown in hydroponic pouches were subjected to salin-
ity (100 mM NaCl) conditions for 9 days. The factor salt
stress had a main effect on all seedlings growth vari-
ables, interacting with inoculation for the shoot fresh
weight of plants (Table S4; Fig. 3). Inoculation had a
301
A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
90 1.4
80
1.2
70
1 secondary roots than non-inoculated plants (Fig. 4B).
Germination (logit)
Germination (%)
60
50 0.8
40
0.6
Control
30 Control + 100 mM NaCl
0.4
Bacillus
20
Bacillus +100 mM NaCl
0.2
10 Average (Control)
Average (Bacillus)
0 0
18h 42h 66h
Figure 2 Germination rate of Medicago sativa seeds after 18, 42 and 66 h
in absence (circles) or presence (triangles) of Bacillus megaterium NMp08 at
two salt concentrations (0 and 100 mM NaCl, continuous or dotted lines,
respectively). Factors were analysed by repeated measures analysis of variance
(ANOVA) (P < 0.05; F-test) (see Table S3). Percentages and logit-transformed
values are means of three plates per treatment (n = 3 plates, with 100 seeds
each). Vertical bars show the least significant difference (LSD) at 5% for
comparison of treatments at each time point. Means (logit) of Bacillus × salt
interaction: control/0 mM NaCl = 72.1 (1.028); control/100 mM NaCl 60.9
(0.900) Bacillus/0 mM NaCl 73.6 (1.040); Bacillus/100 mM NaCl 71.9 (1.023);
df = 24; standard error of difference (SED) = 0.014; LSD (5%) = 0.03.
significant effect on the number of trifoliate leaves, inde-
pendently of the salt stress (1.3-fold and 1.6-fold increase
at 0 and 100 mM NaCl, respectively; Table S4; Fig. 3A).
Compared with non-inoculated plants, those inoculated
with B. megaterium showed a significant improvement of
shoot fresh weight under salt stress conditions (1.6-fold;
Fig. 3B), and a positive (1.4-fold), although not signifi-
cant, effect on root fresh weight.
In order to test whether B. megaterium NMp082 could
also promote growth in non-legume plant species, the
effects of its secreted compounds on the model plant A.
thaliana in the absence or presence of salt stress were anal-
ysed. Both factors, inoculation and salt stress, significantly
affected seedling growth (Table S5; Fig. 4), and significant
interactions were observed for all parameters except the
root area (Table S5).
Concerning the effect of inoculation, in absence and
low NaCl, the primary root length of uninoculated plants
was significantly (P < 0.05; LSD) higher than that of plants
inoculated with B. megaterium, while under high salt
stress (100 nM NaCl) the effect was inverted (Fig. 4A).
Concerning the salt effect, the primary root length of
non-inoculated plants were severely affected by high
salinity, whereas inoculated plants showed even higher
values under salt conditions (Fig. 4A).
The inoculation with B. megaterium significantly
(P < 0.05; LSD) improved the other morphometric
parameters, compared with non-inoculated plants (Fig. 4
302
A. Chinnaswamy et al.
B-E) at all salt concentrations. For example, in the
absence of NaCl, inoculated plants had a significantly
(P < 0.05; LSD) higher number (about threefold) of
The presence of salt (50 or 100 mM) induced a strong
reduction in the number of secondary roots, but signifi-
cant (P < 0.05; LSD) differences between inoculated and
uninoculated plants were still observed (Fig. 4B). Total
root length and root area were significantly (P < 0.05;
LSD) higher in A. thaliana plants inoculated with B. mega-
terium than in non-inoculated ones (Fig. 4C and Fig. 4D).
Regarding total root length, inoculated plants were sig-
nificantly (P < 0.05; LSD) less affected than uninoculated
plants in the presence of 50 mM and 100 mM NaCl
(Fig. 4C). Leaf area of inoculated plants was also signifi-
cantly (P < 0.05; LSD) higher than that of non-inoculated
plants in all salt treatments (Fig. 4E). The inoculation
with B. megaterium significantly (P < 0.05; LSD) improved
the survival of plants under high salt concentration
(Fig. 4F).
Discussion
A Gram-positive, fast-growing, non-rhizobial endophytic
bacterium was isolated from symbiotic nodules of M.
polymorpha, where E. medicae was also present. It was
identified as a B. megaterium strain, and called NMp082.
Bacillus megaterium NMp082 harboured nifH and nodD
genes 100% identical to those of E. meliloti. This data
suggest that lateral gene transfer of nifH and nodD genes
might have happened between the symbiotic and endo-
phytic bacteria. It has been reported that different rhi-
zobia species have symbiosis islands, that is, a type of
mobile genetic elements harbouring gene clusters that
can include nodulation genes and other symbiosis-related
genes (Syvanen, 1994; Sullivan & Ronson, 1998). Hori-
zontal transfer of symbiotic genes from E. meliloti to other
symbiotic and non-symbiotic nodule bacteria has been
previously described (Zakhia et al., 2006; Muresu et al.,
2008), also horizontal transfer of nifH from Bradyrhizo-
bium japonicum to endophytic Bacillus has been reported
(Li et al., 2008). The horizontal transfer of symbiotic
genes from symbiotic to endophytic bacteria is ecolog-
ically important because it offers a mechanism for the
emergence of new symbiotic bacteria by one-step evolu-
tion, as well a way to avoid the environmental stresses
(Barcellos et al., 2007). Interestingly, the endophytic bac-
terium that we describe here obtained its symbiotic genes
from a rhizobial species that was not the one found in the
sampled nodules. Both E. meliloti and E. medicae are able to
form nodules with Medicago species. Gene transfer prob-
ably happened between E. meliloti and B. megaterium in
a previous interaction and then, B. megaterium associated
Ann Appl Biol 172 (2018) 295–308
© 2018 Association of Applied Biologists
A. Chinnaswamy et al. A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa

A B
Salt Inoculation 50 Control Bacillus
No. Trifoliate Leaves 2 40

Shoot Fresh Weight

1.55 30

(mg)
1 20

0.55 10

0 0

C D
30 3.6
Root Fresh Weight (mg)

3.2
25
2.8
Increment (cm)
Root Length
20 2.4
2
15
1.6
10 1.2
0.8
5
0.4
0 0
0 mM NaCl 100 mM NaCl 0 mM NaCl 100 mM NaCl

E 6 F 7
No. Secondary Roots

Length Secondary

5 6
Roots (cm)

4 5
4
3
3
2
2
1 1
0 0
0 mM NaCl 100 mM NaCl 0 mM NaCl 100 mM NaCl

Figure 3 Effect of the factors ‘inoculation’ (control non-inoculated, or inoculated with Bacillus megaterium NMp08), and ‘salt stress’ (0 or 100 mM NaCl)
on morphometric parameters of Medicago sativa plants grown in hydroponic pouches during 9 days, analysed by two-factorial analysis of variance (ANOVA)
(P < 0.05; F-test) (see Table S4). Values are means of 11 pouches (with six seedlings each). Vertical bars show the least significant difference (LSD) at 5% for
comparison of treatments. A) Independent significant effect of each factor; (B) significant interaction of factors; (C–F) significant salt stress effect (Table S4).

and colonised the nodule with E. medicae, which is the pre-
dominant rhizobial species that nodulates Medicago spp. in
the sampling site (Nonnoi et al., 2012), but not necessarily
the only one.
Despite the presence of symbiotic genes, B. mega-
terium NMp082 was not able to induce efficient
nodules in its original host M. polymorpha or in
other Medicago spp., including alfalfa, when grown in
nitrogen-limiting medium. We failed to detect nodABC
genes in B. megaterium using primers specific for E. meliloti.
Thus, it is probable that B. megaterium has not acquired
a whole symbiosis island as results of horizontal gene
transfer events. The bacteria induced the formation
of root nodule-like outgrowths in alfalfa plants. These
outgrowths lacked the typical structure of M. sativa
nitrogen-fixing nodules. Production of IAA by B. mega-
terium could be involved in the generation of these
nodule like-structures. It was observed that formation

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© 2018 Association of Applied Biologists
A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa A. Chinnaswamy et al.

A B

Main root length (cm)

No. Secondary Roots

50 90
40 70
30 50
20
30
10
10
0
0 mM 50 mM100 mM 0 mM 50 mM 100 mM 0 mM 50 mM100 mM 0 mM 50 mM 100 mM
Control Bacillus Control Bacillus

C D
Total Root Length (cm)

275 32

Root Area (cm2)

225
24
175
125 16
75 8
25
0 mM 50 mM100 mM 0 mM 50 mM 100 mM
0
Control Bacillus 0 mM 50 mM 100 mM
Control Bacillus

E F
32
Leaf Area (cm2)

100
Survival (%)

24 75
16 50
8 25
0 0 mM 50 mM100 mM 0 mM 50 mM 100 mM 0
0 mM 50 mM 100 mM 0 mM 50 mM 100 mM
Control Bacillus
Control Bacillus

Figure 4 Effects of the factors ‘inoculation’ (control non-inoculated, or inoculated with Bacillus megaterium NMp08), and ‘salt stress’ (0, 50 or 100 mM NaCl),
on growth and survival of Arabidopsis thaliana, growing in plates during 9 days, analysed by two-factorial analysis of variance (ANOVA) (P < 0.05; F-test) (Table
S5). Values are means of three plates (with 10 seedlings per plate). Vertical bars show the least significant difference (LSD) at 5% for comparison of treatments.
(A–C, E–F) significant interaction of factors; (D) independent significant effect of each factor (see Table S5).

of pseudonodules in plants are linked to application
of IAA and synthetic auxins (Baca & Elmerich, 2003)
and that auxin-transport inhibitors induce nodule-like
structure in legumes (Rightmyer & Long, 2011; Spaepen
& Vanderleyden, 2011; Nagata & Suzuki, 2014). It cannot
be discarded that this B. megaterium strain could also
produce and secrete cytokinins, which play an important
role in nodule organogenesis. Exogenous application
of cytokinins also induces formation of pseudonodule
structures in legumes (Suzaki et al., 2013).
Inoculation with NMp082 remarkably enhanced plant
growth in all Medicago species tested. For 30 days after
inoculation, enhanced growth parameters were even
similar to those observed in E. medicae-inoculated plants.
B. megaterium NMp082 displayed in vitro IAA produc-
tion (even without addition of tryptophan). This is an
important bacterial characteristic for plant growth promo-
tion (De Souza et al., 2015). The endogenous pool of plant
IAA may be altered by the acquisition of IAA secreted by
PGPB. Bacterial IAA can increase root surface area and
length, secondary root formation, number of root hairs
and root tip number, increasing plant nutrient uptake and
growth (Ribaudo et al., 2006; Glick, 2012).
Co-inoculation of E. medicae with B. megaterium sig-
nificantly enhanced growth and nodulation of Medicago
spp. plants compared with inoculation with E. medicae

304 Ann Appl Biol 172 (2018) 295–308

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A. Chinnaswamy et al. A nodule endophyte enhances growth, nodulation and salt tolerance in alfalfa
or B. megaterium alone. Co-inoculation of legumes with
isolated PGPB, including nodule endophytes, and the
corresponding nodulating symbiotic rhizobia induced
enhanced plant growth and/or nodulation (Bai et al.,
2002; Bullied et al., 2002; Shaharoona et al., 2006; Tilak
et al., 2006; Rajendran et al., 2008; Fox et al., 2011). Sta-
jković et al. (2009) observed the same phenomena upon
co-inoculation of alfalfa with E. meliloti and different
B. megaterium strains in gnotobiotic culture. The actual
mechanisms involved remain unknown. One mechanism
that may play a role is phytohormone production. As
stated above, IAA promotes formation of lateral roots
and root hairs. It can be speculated that co-inoculated
plants might have more sites for rhizobial infection,
either whether rhizobia invade the legume at the sites of
lateral root emergence (Ibáñez et al., 2009) or through
root hairs (Srinivasan et al., 1996). Root nodulation is
affected by IAA and increases in auxin levels in the host
legume are necessary for nodule formation and develop-
ment (Mathesius et al., 1998; Fedorova et al., 2005; Glick,
2012). Nevertheless, a clear correspondence between
IAA production by PGPB and enhanced plant growth and
nodulation upon inoculation by IAA-producing bacteria
is not always clear, and other direct or indirect effects
on either the rhizobia or the plant cannot be discarded
(Srinivasan et al., 1996; Dudeja et al., 2012). In fact,
ethylene is a potent negative regulator of nodulation
(Ligero et al., 1999), and it has been proposed that the
presence of a PGPR with ACC-deaminase activity on the
roots of legumes promoted nodulation by decreasing the
synthesis of ethylene (Shaharoona et al., 2006).
The fact that B. megaterium NMp082 displayed high tol-
erance to NaCl, water deficit and several heavy metals,
besides ACC deaminase activity, suggested that it could
have positive effects on plant growth of alfalfa (and other
plant species) under stress conditions. We observed that
compounds secreted by B. megaterium NMp082 induced
an increase in the percentage of alfalfa seed germina-
tion in the absence and in the presence of salt stress.
Enhanced seed germination by IAA-producing bacteria
has been observed in other plants (Martínez-Viveros et al.,
2010), and it has been postulated that IAA stimulates
seed germination, and that IAA produced and secreted by
PGPB likely interferes with different plant physiological
processes by changing the plant auxin pool (Ahemad &
Kibret, 2014). However, auxin by itself is not considered
a necessary hormone for seed germination (Miransari &
Smith, 2014). The possibility cannot be excluded that
NMp082 could also secrete other compounds, such as
the plant hormones gibberellins, which are known to
promote seed germination, and/or cytokinins, which are
able to enhance seed germination in salinity conditions
(Miransari & Smith, 2014).
Ann Appl Biol 172 (2018) 295–308
© 2018 Association of Applied Biologists
Bacillus megaterium NMp082 enhanced alfalfa shoot
and primary root growth in the presence of NaCl stress.
In Arabidopsis, it promoted shoot and secondary root
growth, and enhanced plant survival under salinity stress.
1-Aminocyclopropane-1-carboxylate deaminase activity
can reduce the stress-induced ethylene levels in plants,
inducing tolerance, root elongation and growth pro-
motion in plants subjected to salt, drought and heavy
metal-induced stresses (Ali et al., 2014; Glick, 2014). It is
admitted that the ethylene precursor ACC may be exuded
from plant roots to the rhizosphere, where it will be
taken in by the rhizobacteria and subsequently hydrol-
ysed (Glick et al., 1998). However, effects of PGPB by
secretion of other phytohormones or compounds, such as
volatile organic compounds, on plant abiotic stress toler-
ance cannot be discarded (Liu & Zhang, 2015).
Among different bacteria, various Bacillus species have
been encountered as endophytes in symbiotic nodules
of legumes, and some of them displayed plant growth
promotion activity (Bai et al., 2002; Li et al., 2008; Sta-
jković et al., 2009; Khalifa & Almalki, 2015). B. mega-
terium NMp082 presented specific characteristics that
confer stress tolerance to abiotic stress in legume and
non-legume plants. Further work aims to assess the suit-
ability of B. megaterium to sustain plant growth and nodu-
lation under heavy metal stress and its potential applica-
tion in phytoremediation enhancement.
Acknowledgements
This work was supported by grants from MINECO
(AGL2013-40758-R), Comunidad de Madrid (S2009/
AMB-1511), MEC (SB2009-0075) and CONICYT Chile
(R16A10003). The authors have no conflict of interest to
declare.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. List of assays and experimental designs car-
ried out in this work. PGPB, plant growth promoting bac-
teria.
Table S2. Effects of the factors ‘plant species’ (Medicago
sativa, Medicago polymorpha, Medicago lupulina, Medicago
truncatula), ‘inoculation with Bacillus megaterium’ (+/−),
‘inoculation with Ensifer medicae’ (+/−), and their interac-
tions on the growth and nodulation of plants, analysed
by three-way analysis of variance (ANOVA) (P < 0.05,
F-test). Data = F (P values) (see means in Table 2).
Table S3. Effects of the factors ‘inoculation’ (control
non-inoculated, or inoculated with Bacillus megaterium)
and ‘salt stress’ (0 and 100 mM NaCl), and their interac-
tion on germination of Medicago sativa seeds at 18, 42 and
66 h, analysed by repeated measures analysis of variance
(ANOVA) (P < 0.05, F-test). Data = F (P values) (n = 3
plates; 100 seeds per plate). Significant values are high-
lighted in bold. 1 logit transformed.
Table S4. Effects of the factors ‘inoculation’ (control
non-inoculated, or inoculated with Bacillus megaterium)
and ‘salt stress’ (0 or 100 mM NaCl) and their interaction
on Medicago sativa growth, analysed by two-factor analysis
of variance (ANOVA) (P < 0.05; F-test). Data = F (P val-
ues) (n = 11 pouches; 6 seedlings per pouch). Significant
values are highlighted in bold.
Table S5. Effects of the factors ‘inoculation’ (control
non-inoculated, or inoculated with Bacillus megaterium)
and ‘salt stress’ (0, 50, or 100 mM NaCl), and their inter-
action on growth and survival of Arabidopsis thaliana,
analysed by two-factor analysis of variance (ANOVA)
(P < 0.05, F-test). Data = F (P values) (n = 3 plates; 10
seedlings per plate). Significant values are highlighted
in bold.
Ann Appl Biol 172 (2018) 295–308
© 2018 Association of Applied Biologists