Ann Microbiol
DOI 10.1007/s13213-011-0227-4
ORIGINAL ARTICLE
Legume-nodulating bacteria (LNB) from three pasture
legumes (Vicia sativa, Trigonella maritima and Hedysarum
spinosissimum) in Tunisia
Mosbah Mahdhi & Amira Fterich & Mokhtar Rejili &
Ignacio David Rodriguez-Llorente & Mohamed Mars
Received: 21 September 2010 / Accepted: 7 February 2011
# Springer-Verlag and the University of Milan 2011
Abstract Sixty-one bacterial isolates were recovered from
surface-sterilized root nodules of Vicia sativa, Trigonella
maritima and Hedysarum spinosissimum plants growing in
two arid Tunisian soils. The natural nodulation resource of
these legumes, prospected from the two sites, was investi-
gated. The occurrence of nodulation and the morphology of
the nodules were observed. The isolates were examined by
phenotypic characterization and 16S rDNA analysis.
Among the 61 isolates that were screened, the majority
(92%) were fast-growing rhizobia. Twenty-eight strains
tolerated high concentration of salt (3% NaCl) and grew at
temperatures up to 40°C. PCR restriction fragment length
polymorphism (PCR-RFLP) and 16S rRNA gene sequencing
revealed that the majority of the isolates belonged to the
genera Rhizobium (54%) and Sinorhizobium (42%). Five H.
spinosissimum isolates failed to nodulate their host plant, and
were affiliated to Pseudomonas and Kocuria genera. This
study is the first report that describes bacteria of genus
Kocuria occupying root nodules of legumes to the best of
our knowledge.
Keywords Rhizobia . PCR-RFLP . Nodulation .
Gammaproteobacteria . Sequencing
M. Mahdhi (*) : A. Fterich : M. Rejili : M. Mars
Laboratoire de Biotechnologies Végétales Appliquées à
l’Amélioration des Cultures, Université de Gabès,
Faculté des Sciences de Gabès,
Cité Erriadh Zrig,
6072 Gabès, Tunisia
e-mail: mosbahtn@yahoo.fr
I. D. Rodriguez-Llorente
Departamento de Microbiología y Parasitología,
Facultad de Farmacia,
Sevilla, Spain
Introduction
Legume-nodulating bacteria (LNB) are Gram-negative soil
bacteria that fix nitrogen after becoming established inside root
nodules of legumes. In the last few years, a large diversity of
LNB has been revealed, which has caused considerable
changes in the taxonomy of these bacteria. The current
taxonomy of LNB reveals a wide diversity at the genus,
species and intraspecies levels. Most of these bacterial species
are in the Rhizobiaceae family in the α-class of Proteobacteria:
Rhizobium, Azorhizobium, Sinorhizobium (syn. Ensifer),
Mesorhizobium, Bradyrhizobium, Methylobacterium (Jaftha
et al. 2002; Jourand et al. 2004), Devosia (Rivas et al. 2003),
Blastobacter (Van Berkum and Eardly 2002), Ochrobactrum
(Trujillo et al. 2005) and Phyllobacterium (Valverde et al.
2005; Mantelin et al. 2006). Moreover, about eight rhizobial
species within two genera of the β-class of Proteobacteria
(Burkholderia and Ralstonia) have been reported (Moulin et
al. 2001; Chen et al. 2001, 2006, 2008; Leelahawonge et al.
2010; Shiraishi et al. 2010). On the other hand, Agrobacterium
strains have been isolated from nodules of many legume
species (Gurtler et al. 1991; Liu et al. 2005; Mahdhi et al.
2008), but no definitive explanation of the presence of these
bacteria inside nodules could be demonstrated. In addition,
bacteria from the γ-class of Proteobacteria have also been
reported (Benhizia et al. 2004; Muresu et al. 2008;
Leelahawonge et al. 2010; Shiraishi et al. 2010).
Plants of the genus Vicia, Trigonella and Hedysarum are
annuals, which play an important role for forage and
medicinal products. Although nodulation of these legumes
has been reported, a detailed description of the taxonomy of
the rhizobial symbionts has not been made. Legumes from
genera Vicia are commonly nodulated by Rhizobium
leguminosarum bv. viciae (Jordan 1984; Laguerre et al.
2003; Mutch et al. 2003; Slattery et al. 2004; Mutch and

Young 2004; Moschetti et al. 2005). Although, Trigonella
plants mainly nodulate with Sinorhizobium meliloti
(Roumiantseva et al. 2002; You et al. 2008), strains
belonging to the genera Bradyrhizobium (Pandey et al.
2004) and Rhizobium (Wang et al. 2006; Hou et al. 2009)
have also been isolated from two Trigonella species in
China. The genus Hedysarum L. comprises about 100
species, of herbaceous legumes, widely distributed in the
Mediterranean, temperate Europe, North and South Africa,
Asia Minor, Siberia, North America from Arizona into
Alaska and the Arctic regions of Canada. Only 19 species
of Hedysarum are recorded as being nodulated and root-
nodule bacteria have only been purified and authenticated
from a few of these. Since previous studies on biological
nitrogen fixation in the genus Hedysarum focused mainly
on H. coronarium, there is little information regarding
root-nodulating bacteria associated with other Hedysarum
species such as H. spinosissimum. Previous researches
showed that Hedysarum species have been reported to be
nodulated by Mesorhizobium (Kishinevsky et al. 2003;
Safronova et al. 2004), Rhizobium (Squartini et al. 2002;
Hung et al. 2005), Bradyrhizobium (Hung et al. 2005) and
Sinorhizobium (Zakhia et al. 2004). In addition, strain
SH199 isolated from Hedysarum scoparium in China was
grouped as Agrobacterium tumefaciens (Wei et al. 2008).
Another study showed that some isolates from the
nodules of Hedysarum species belong to the γ-class of
Proteobacteria (Benhizia et al. 2004; Muresu et al. 2008).
In Tunisia, there are no reports about rhizobial symbionts
from Hedysarum species, and only one Sinorhizobium
strain has been isolated from Hedysarum carnosum
(Zakhia et al. 2004).
Considering the importance of Vicia sativa, Trigonella
maritima and Hedysarum spinosissimum in forage produc-
tion and the insufficient study on the diversity of rhizobia
associated with these three legumes, the aim of this study is
to analyse, using both phenotypic and genotypic methods,
the taxonomic diversity of 61 nodule isolates from these
legumes grown in two arid areas of Tunisia. Natural
nodulation of these three legumes is also investigated and
is reported for the first time.
Materials and methods
Occurrence of nodulation
Spontaneous nodulation of the three plant species pro-
spected in this study is reported from their sites of origin.
The intensity of nodulation (number of nodules per plant)
and the morphology of nodules (shape and colour) were
investigated visually. Twelve plants were considered at
each site.
Ann Microbiol
Bacterial isolates and reference strains
Sixty-one isolates and three reference strains (Table 1),
representing different rhizobial species belonging to
Rhizobium and Sinorhizobium genera, were used. Rhizo-
bial bacteria were isolated from naturally occurring root
nodules collected in two arid soils of Tunisia: National
park of Bouhedma (34°42′47″N, 9°28′27″E) and Matmata
33°41′N, 10°23′E). For rhizobia isolation, nodules were
rehydrated in sterile water and surface sterilised by
immersion in 95% ethanol for 30 s and 0.1% mercuric
chloride for 2 min. Each nodule was rinsed ten times in
sterile water. A 100-μl aliquot of the last washing solution
was checked for sterility by inoculation on YMA agar
plates and incubation. Only nodules resulting in a sterile
final washing liquid were further considered for bacterial
isolation. Nodules were then individually squashed and
streaked on plates containing YMA agar (Vincent 1970).
After incubation for 5 days at 28°C, single colonies were
selected and transferred on to YEMA plates to ascertain
purity. Pure cultures of the isolates or strains were
maintained on YEM agar slants at 4°C or in 25% glycerol
at −80°C.
Phenotypic and nodulation tests
All isolates were observed for colour and colonoy mor-
phology after growth on YMA plates for 48 h at 28°C.
Generation time was determined by inoculation in 50 ml of
YM broth into 250-ml Erlenmeyer flasks and incubation in
a gyratory shaker at 180g and 28°C. Growth was checked
by measuring the optical density at 600 nm every 2 h. The
generation time was deduced from the exponential growth
phase. The growth temperature ranges (15, 28, 35, 37, 40,
42°C), the ability to grow in the presence of NaCl (1, 2, 3,
4%) and at different pH (4, 5, 6, 7, 9, 10, 12) were tested on
YMA plates as described by Mohamed et al. (2000).
Each plate was divided into 12 equal sectors, and each
sector was then inoculated with 10 μl of exponential phase
growth cultures of the test strain. Acid and alkali
production were determined in YEM agar medium with
bromothymol blue indicator (0.0025%, w/v). All pheno-
typic tests were done in triplicate.
All isolates were tested for their ability to re-nodulate
their host plant. Inoculation and seed treatment were
performed as described by Mahdhi et al. (2008). Seeds
were scarified with 95% sulphuric acid for 10 min.
Germinated seedlings were aseptically transferred to indi-
vidual pots of about 200 ml containing vermiculite and
sterilised nitrogen-free nutrient solution (Vincent 1970).
The pots were placed in a growth chamber at 25°C during
the day and 18°C at night, with a 16-h photoperiod and 60–
70% relative humidity. Two days later, each seedling was
Ann Microbiol

Table 1 Isolates and reference strains used in this study and their relevant characteristics

Isolates and Other Host plant Site of origin 16S Isolates and Host plant Site of origin 16S
reference strains designation rDNA reference rDNA
type strains type

VB1 V. sativa Bouhedma Park, 1 TB6 T. maritima Bouhedma Park, 2

Tunisia Tunisia
VB2 V. sativa Bouhedma Park 1 TB7 T. maritima Bouhedma Park 2
VB3 V. sativa Bouhedma Park 1 TB8 T. maritima Bouhedma Park 2
VB4 V. sativa Bouhedma Park 1 TB9 T. maritima Bouhedma Park 2
VB5 V. sativa Bouhedma Park 1 TB10 T. maritima Bouhedma Park 2
VB6 V. sativa Bouhedma Park 1 TB11 T. maritima Bouhedma Park 2
VB7 V. sativa Bouhedma Park 1 TB12 T. maritima Bouhedma Park 2
VB8 V. sativa Bouhedma Park 1 TB13 T. maritima Bouhedma Park 2
VB9 V. sativa Bouhedma Park 1 TB14 T. maritima Bouhedma Park 2
VB10 V. sativa Bouhedma Park 1 TB15 T. maritima Bouhedma Park 2
VB11 V. sativa Bouhedma Park 1 TB16 T. maritima Bouhedma Park 2
VB12 V. sativa Bouhedma Park 1 TB17 T. maritima Bouhedma Park 2
VB13 V. sativa Bouhedma Park 1 TB18 T. maritima Bouhedma Park 2
VB14 V. sativa Bouhedma Park 1 TB19 T. maritima Bouhedma Park 2
VB15 V. sativa Bouhedma Park 1 TB20 T. maritima Bouhedma Park 2
VB16 V. sativa Bouhedma Park 1 TB21 T. maritima Bouhedma Park 2
VB17 V. sativa Bouhedma Park 1 TB22 T. maritima Bouhedma Park 2
VB18 V. sativa Bouhedma Park 1 TB23 T. maritima Bouhedma Park 2
VB19 V. sativa Bouhedma Park 1 TB24 T. maritima Bouhedma Park 2
VB20 V. sativa Bouhedma Park 1 TB25 T. maritima Bouhedma Park 2
VB21 V. sativa Bouhedma Park 1 TB26 T. maritima Bouhedma Park 2
VB22 V. sativa Bouhedma Park 1 TB27 T. maritima Bouhedma Park 2
VB23 V. sativa Bouhedma Park 1 TB28 T. maritima Bouhedma Park 2
VB24 V. sativa Bouhedma Park 1 TB29 T. maritima Bouhedma Park 2
TB1 T. maritima Bouhedma Park 2 TB30 T. maritima Matmara, Tunisia 2
TB2 T. maritima Bouhedma Park 2 HM1 H. spinosissimum Matmara 2
TB3 T. maritima Bouhedma Park 2 HM2 H. spinosissimum Matmara 4
TB4 T. maritima Bouhedma Park 2 HM3 H. spinosissimum Matmara 4
TB5 T. maritima Bouhedma Park 2 HM4 H. spinosissimum Matmara 4
S. meliloti NZP4027T, LMG6133T Medicago sativa Virginia, USA 2 HM5 H. spinosissimum Matmara 4
ORS665T
Sinorhizobium medicae LMG16580, Medicago. Syria 3 HM6 H. spinosissimum Matmara 5
M102, ORS504 HAMBI1809 truncatula
R. leguminosarum bv. VF39SM 1 HM7 H. spinosissimum Matmara 2
viciae ORS639

Names of strains reflect their host plant and site of isolation: TB Trigonella maritima isolates from Bouhedma Park, Tunisia; HM Hedysarum
spinosissimum isolates from Matmata, Tunisia; VB Vicia sativa isolates from Bouhedma Park, Tunisia; HAMBI Culture Collection of the
Department of Microbiology, University of Helsinki, Helsinki, Finland; LMG Collection of Bacteria of the Laboratorium voor Microbiologie,
Universiteit Ghent, Belgium; NZP Culture Collection of the Department for Scientific and Industrial Research, Biochemistry Division, Palmerston
North, New Zealand

inoculated with 1 ml of culture of the appropriate rhizobial
strain (approximately 109 cells). Uninoculated plants served
as control treatment. Three replicates were maintained for
each treatment. Thirty days later, plants were examined for
root nodulation.
PCR-RFLP of 16S rRNA gene
Bacterial genomic DNA from each strain was extracted
according to the methodology described by Mhamdi et al.
(2002). Bacteria were grown on TY agar slopes at 28°C for
48 h and suspended in sterile water. A volume,
corresponding to 200 μl matching a DO620 nm=1, was
centrifuged. The cell pellet was resuspended in Tris-HCl
(10 mM, pH 8.3) and 20 μl of proteinase K (1 mg/ml).
Cells were incubated overnight at 55°C and then 10 min
in boiling water. Bacterial Cell preparations were stored
at −20°C until use and a volume of 5 μl was used for PCR
amplification as emplate DNA. Two primers FGPS 6 (5′-
GGAGAGTTAGATCTTGGCTCAG-3′) and FGPS 1509

(5′-AAGGAGGGGATCCAGCCGCA-3′) (Weisburg et al.
1991) were used for PCR amplification of 16S rRNA
gene. PCR reaction was carried out in a final volume of
25 μl containing: 1× PCR buffer, 2.5 mM of MgCl2,
0.02 mM of each dNTP, 1 U of Taq DNA polymerase,
0.1 μM of each primer, 6 μl of H2O and 5 μl of template
DNA (100 ng). The amplifications were performed in a
Thermocycler (Gene Amp PCR System 9700; Applied
Biosystems) and programmed for an initial denaturation of
3 min at 95°C, followed by 35 cycles of 1 min at 94°C,
1 min at 55°C and 2 min at 72°C, and a final extension of
2 min at 72°C. PCR products were checked by electro-
phoresis on 1% agarose gel. Aliquots (7 μl) of PCR
products were digested with each of the following
enzymes: RsaI, NdeII, AluI, HinfI, MspI and TaqI.
Restriction DNA was analysed by horizontal electropho-
resis in 4% agarose gel at 4 V cm−1. Each restriction band
was considered as a unit feature and was scored as 1
(present) or 0 (absent) for all strains. The similarity
coefficient (Ssm) was calculated as Ssm ¼ m=ðm þ nÞ,
where m is the number of matched bands and n is the
number of mismatched bands. A dendrogram was con-
structed from the similarity matrix using the unweighted
pair group method with averages (UPGMA) (Sneath and
Sokal 1973).
Sequencing of 16S rDNA
The 16S rRNA genes of three H. spinosissimum
isolates, chosen as representatives for each 16S rDNA
pattern, were amplified under the conditions described
above. PCR products were run on a 1% agarose gel, band
was excised and DNA purified using CTB DNA Extrac-
tion Mini Kit (iNtRON Biotechnology, Korea). Two
forward primers (FGPS6 and 16S-370f) and one reverse
primer (FGPS1509) were used to obtain the complete
gene sequence. 16S rRNA gene cycle sequencing was
performed using the ABI PRISM BigDye Terminator
cycle sequencing kit according to the manufacturer’s
protocol and analysed on an ABI PRISM 310 Genetic
Analyzer (Applied Biosystems). The sequences obtained
were deposited in the GenBank database and were
compared with related sequences from the database. The
sequences were aligned using the programs in the
package of Clustal X and Genedoc software. The
phylogenetic analyses were performed using mega 3.1
software (Kumar et al. 2001). A neighbour-joining tree
was reconstructed and bootstrapped with 1000 replica-
tions of each sequence using Kimura two-parameter
model (Kimura 1980) of evolution. The GenBank acces-
sion numbers for the 16S rRNA gene sequences reported
in this paper are HM2 (GU384322), HM6 (GU384323)
and HM1 (GU384324).
Ann Microbiol
Results
Occurrence of nodulation and nodule morphology
Natural nodulation of the three plant species prospected in
this study is reported. Results show that V. sativa was
strongly nodulated (52 nodules/plant). The lowest number
of nodules (12 nodules/plant) was observed for H.
spinosissimum. The external colour of the nodules is white
or brown. The size of the nodules with globular shapes
varied from 0.2 to 0.3 cm, and could exceed the diameter of
the root of their host plant.
Phenotypic properties
Sixty-one isolates investigated in this study (Table 1)
were purified from root nodules of three forage legumes
(V. sativa, T. maritima and H. spinosissimum) growing on
two arid soils of Tunisia. A nodulation test was performed
for all the isolates. Results showed that only five H.
spinosissimum isolates, which were classified as γ-class of
Proteobacteria by 16S rRNA gene sequencing analyses
(see below) failed to nodulate their host plant of origin.
Phenotypically, the majority of the isolates were acid
producers (colonies change colour after 72 h at 28°C to
yellow) and fast growing with average mean generation
time of 3 h. Most of the isolates were able to grow at pH
between 6 and 9, but only two isolates (VB15 and VB13)
grow at pH 4 (Table 2). The tolerance to NaCl and
resistance to temperature were variable among the studied
isolates: most isolates were able to grow only at 2% NaCl
and at 37°C while the majority of T. maritima isolates
grew well in YEM broth with 3% NaCl and at 40°C but
not with a 4% NaCl concentration and at 42°C. A summary of
some phenotypic properties is described in Table 2.
16S-RFLP analysis
The 16S rDNA of all the isolates and reference strains was
amplified, resulting in a characteristic single band of
1500 bp. Restriction analysis was performed by using six
endonucleases. The analysis revealed three to five restric-
tion patterns per enzyme. Five 16S rDNA types were
distinguished among the 61 isolates and the reference
strains. Each 16S rDNA type comprising 1–33 isolates
(Table 1). All V. sativa isolates had 16S rDNA type
identical to that of the reference R. leguminosarum bv.
viciae ORS639 (16S rDNA type 1). 16S rDNA type 2
included 32 new isolates, originating from T. maritima (30
isolates) and H. spinosissimum (2 isolates). These isolates
share identical PCR- 16S rDNA RFLP pattern with
Sinorhizobium meliloti type strain (ORS665T). Four H.
spinosissimum isolates (HM2, HM3, HM4, and HM5) have
Ann Microbiol

Table 2 Phenotypic characteris-

tics of the isolates Characteristics V. sativa isolates T. maritima isolates H. spinosissimum isolates

Number of isolates 24 30 7
Generation time in YEM medium
GT≤3 h + (20) + (20) + (2)
3<GT≤6 h + (4) + (4) -
GT≥6 h - - + (5)
NaCl tolerance
2% + (22) + (28) +
3% + (3) + (25) -
4% - - -
Growth at pH
4 + (2) - -
9 + + +
12 - - -
Growth at temperature
20°C + (4) + (5) + (22)
37°C + (5) + + (5)
+ Positive growth/present, - no
growth/absent. Numbers in 40°C + (3) + (25) -
parentheses indicate the number 42°C - - -
of positive isolates of the total Nodulation tests + + + (2)
number of isolates tested

the same 16S rDNA type (16S rDNA type 4). Only isolate
HM6 has a 16S rDNA type different from those of all
isolates investigated in this study.
16S rDNA sequencing
Sequence analysis of 16S rRNA genes of three H.
spinosissimum isolates representing each 16S rDNA types
was performed to confirm and clarify the phylogenetic
position estimated by the RFLP analysis. Sequences were
compared with the 16S rDNA sequences available in
GenBank. Figure 1 show a phylogenetic tree based on the
similarity of 16S rRNA sequences of the selected isolates
and reference strains. Isolate HM1 (16S rDNA type 2) was
found in the branch of Sinorhizobium, closely related to S.
meliloti Lse-2 (99.97%). The alignments of 16S rDNA
sequences of the two other isolates (HM2 and HM6)
indicated that all of them were highly similar (>99%
identity) to those of 16S ribosomal genes belonging to
members of the γ-class of Proteobacteria, Pseudomonas
(for isolate HM2) and Kocuria (for isolate HM6) genera.
Discussion
The N2-fixing leguminous plants are key components of the
natural succession in semi-arid Mediterranean ecosystems
because these plant species, upon establishing rhizobial
symbioses, constitute a fundamental source of N input to
the ecosystem (Zahran 2001). Spontaneous nodulation of
the three legumes was investigated in natural conditions.
Variation in the number of nodules per plant was shown.
Similar results were reported by Zahran (1998) for
Trigonella hamosa nodules collected from cultivated lands
of Egypt. This result seems to be in relation to the
adaptative character of different species. A collection of
61 isolates was obtained from nodules of V. sativa, T.
maritima and H. spinosissimum growing in two arid areas
of Tunisia (National parks of Bouhedma and Matmata) and
characterized by a polyphasic approach including pheno-
typic and genotypic analyses (Vandamme et al. 1996). All
isolates, except HM2, HM3, HM4, HM5 and HM6, induce
nodules in their host plant. These five isolates can be
considered as opportunistic as already proposed (Zakhia et
al. 2006; Mahdhi et al. 2007, 2008).
Phenotypically, our study indicates that microsymbionts
demonstrated a high heterogeneity of several phenotypic
characters. The diversity within the rhizobial population
could offer them advantages to adapt to different environ-
ments for survival and nodulation (Wei et al. 2008). Our
results reported that isolates showed a high resistance to
salinity (2%) and most of them possessed optimum growth
at 37°C and grew well at pH 9. Similar results were
observed by Kishinevsky et al. (2003), Pandey et al. (2004)
and Wei et al. (2008) with strains isolated from three
Hedysarum species, Trigonella foenum-graecum and Vicia
angustifolia. Moschetti et al. (2005) reported that 19
isolates from the nodules of several Vicia species (V.
hybrida, V. sativa, V. faba and V. villosa) in central and
southern Italy were not capable of tolerating salt concen-

trations above 0.1%, and only two of them tolerated 2% (w/
w) NaCl. These results prove that the phenotypic properties
of rhizobial strains, such as temperature resistance and
NaCl tolerance, can be influenced by climatic and edaphic
conditions. Many T. maritima isolates identified as S.
meliloti tolerate high temperatures (40°C) and NaCl
concentrations (up 3%, w/v). These isolates may be
candidates for Trigonella species inoculation in arid soils.
These appropriate native rhizobia resistant to temperature
and salinity would guarantee root nodulation and enhance
plant performance. However, selection of these rhizobia
would need to take into consideration not only their N2-
fixing capacity but also their competitive ability against
native rhizobia. Superior N2-fixing strains have to outcom-
pete native rhizobia and occupy a significant proportion of
the nodules (Rengel 1992).
It has been previously reported that rhizobial strains
from several Vicia sp. were grouped with R. leguminosarum
bv. viciae (Laguerre et al. 2003; Slattery et al. 2004; Mutch
and Young 2004; Moschetti et al. 2005). Alvarez-Martinez
et al. (2009) suggest a world distribution of strains from R.
leguminosarum together with V. sativa and V. faba seeds.
Similar results were found for all V. sativa isolates
investigated in our study.
PCR-RFLP analysis showed that all T. maritima isolates
have their 16S rDNA type identical to S. meliloti LMG6133T
(Table 1). This finding confirmed recent reports on root
nodule bacteria of other Trigonella sp. (You et al. 2008).
However, strains belonging to the genera Bradyrhizobium
(Pandey et al. 2004) and Rhizobium (Hou et al. 2009) have
also been isolated from T. foenum-graecum and Trigonella
archiducis-nicolai, respectively.
Full-length 16S rDNA sequencing grouped one H.
spinosissimum (HM6) to the genus Kocuria. Micro-
organisms of the genus Kocuria are Gram-positive, belong-
ing to the family Micrococcaceae, Their normal habitats
include mammalian skin (Savini et al. 2010; Tsai et al.
2010), soil (Li et al 2006), the rhizosphere (Takarada et al.
2008), fermented foods, and freshwater and marine sedi-
Fig. 1 Phylogenetic tree of 16S
rDNA showing the relationships
among the isolates and the related
bacterial species. Significant
bootstraps (>80%) are indicated
as percentages (1000 replica-
tions). Sequence accession
numbers are listed in parentheses
Ann Microbiol
ments. No reports have been published relating to Kocuria–
plant interactions or to their nitrogen fixing ability
In our collection, four H. spinosissimum isolates are
identified as the genus Pseudomonas by PCR-RFLP and
16S rRNA gene sequencing. Pseudomonas is a genus of
gamma Proteobacteria. Pseudomonas species are a group
of bacteria found commonly in soil and other natural
environments. Some Pseudomonas species have been
reported to be endophytic of legume (Elvira-Recuenco
and Van Vuurde 2000) and also non-legume plants (Kovacs
et al. 1999; Lodewyckx et al. 2002). Previous studies
(Benhizia et al. 2004; Muresu et al. 2008) showed that
Pseudomonas strains could be isolated from root nodules of
several Hedysarum species. On the other hand, nitrogen
fixation has been reported for some species of Pseudomonas
(Young 1992; Desnoues et al. 2003). Shiraishi et al. (2010)
reported that Pseudomonas strains formed nodules on black
locust and also developed differentiated nodule tissue.
Leelahawonge et al. (2010) proposed a bacterium related to
Pseudoalteromonas as a new symbiont of the medicinal
legume Indigofera tinctoria. These two Gammaproteobacteria
(Pseudoalteromonas and Pseudomonas) are found to be true
symbionts of their legumes as they harbour nifH and nodC
genes. These two major symbiotic genes have high similarities
with those of rhizobial species. Lateral gene transfer from
Rhizobiales is suggested as one of the mechanisms explaining
the acquisition of endophytic interactivity with leguminous
plants. In our study, we could not find evidence of nodulation
capacity among Pseudomonas and Kocuria isolates, so these
isolates can be considered as opportunistic endophytic
bacteria as already reported by Zakhia et al. (2006) and
Mahdhi et al. (2007, 2008), and this explanation confirms the
nodulation failures of these isolates. Here, we did not screen
these isolates for symbiotic genes. Therefore, further inves-
tigations on the nodulation gene properties of Pseudomonas
and Kocuria would be helpful to understand the nature of
bacterial interaction. Such investigations are necessary before
the status of these isolates as nodules symbionts could be
considered. The non-nodulation in vitro of H. spinosissimum
Ann Microbiol
by some isolates may also indicate that these legumes are not
the natural hosts of these strains. It would now be interesting
to demonstrate their capacities to induce nodule development
on other wild legumes grown on the same Tunisian soils.
Using two phylogenetic methods (16S rDNA PCR-RFLP
and sequencing), two H. spinosissimum isolates (HM1 and
HM7) were grouped with Sinorhizobium. The HM1 isolate
was similar to the Sinorhizobium meliloti Lse-2 strain
originating from Lotus sp. in the Canary Islands (Fig. 1).
This finding supported results reported by Zakhia et al.
(2004) with Sinorhizobium strain isolated from H. carnosum
in Tunisia. However, Kishinevsky et al. (2003) found that
H. spinosissimum isolates were closely related to both
Mesorhizobium loti and M. ciceri, sharing 97% identity
with each species.
In summary, this study is the first report on the
characterisation of H. spinosissimum microsymbionts in
Tunisia. Our results support the presence of the γ-class of
Proteobacteria in root-nodules of Hedysarum species. Two
Sinorhizobium isolates were also isolated from this legume.
We also confirmed that the majority of the rhizobia
originating from nodules of V. sativa and T. maritima
growing in arid Tunisian soils are grouped with R.
leguminosarum and S. meliloti, respectively. However, soils
from other locations that were not covered in this study
should be investigated in order to provide further informa-
tion about the diversity of rhizobia, especially those that
nodulate Trigonella and Hedysarum species in Tunisia.
Acknowledgements This work was supported by the Ministry of
High Education and Research Development-Tunisia and partly
supported by AECI-Spain project (No. A/018017/08). The authors
thank Dr. Philippe de Lajudie, who kindly provided the references
strains and for his helpful discussion.
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