657

Phaseolus vulgaris is nodulated in northern Spain

by Rhizobium leguminosarum strains harboring
two nodC alleles present in American Rhizobium
etli strains: biogeographical and evolutionary
implications
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Paula Garcı́a-Fraile, Daniel Mulas-Garcı́a, Alvaro Peix, Raúl Rivas,

Fernando González-Andrés, and Encarna Velázquez

Abstract: In this study a collection of rhizobial strains were isolated from effective nodules of Phaseolus vulgaris in a
wide region of northern Spain, which is the major producer region of this legume in Spain. The analysis of their core
genes, rrs, atpD, and recA, and the 16S–23S intergenic spacer showed that all isolates belong to the phylogenetic group of
Rhizobium leguminosarum and some of them were identical to those of strains nodulating Vicia or Trifolium. None of the
isolates was identified as Rhizobium etli; however, all of them carry the nodC alleles a and g harboured by American
strains of this species. These alleles were also found in strains nodulating P. vulgaris in southern Spain identified as R.
etli. These results suggest that R. etli was carried from America to Spain with common bean seeds, but that they could
have found difficulties persisting in the soils of northern Spain, probably because of the climatic conditions. The symbiotic
genes of this species could have been transferred, after the arrival of P. vulgaris, to strains of R. leguminosarum already
For personal use only.

present in northern Spanish soils.

Key words: Rhizobium, Phaseolus, nodC gene.
Résumé : Dans cette étude, une collection de souches de rhizobiums a été isolée de nodules actifs de Phaseolus vulgaris
dans une vaste région du Nord de l’Espagne, principale productrice de cette légumineuse dans cette région. L’analyse des
gènes centraux rrs, atpD et recA, et de l’espaceur intergénique 16S–23S, a démontré que tous les isolats appartenaient au
groupe phylogénique de Rhizobium leguminosarum et que quelques uns d’entre eux étaient identiques aux souches formant
des nodules sur Vicia ou Triflorum. Aucun isolat n’a été identifié à Rhizobium etli, mais tous possédaient les allèles a et g
de nodC retrouvées dans les souches américaines de cette espèce. Ces allèles ont aussi été retrouvés dans les souches for-
mant des nodules sur P. vulgaris du sud de l’Espagne, identifiées à R. etli. Ces résultats suggèrent donc que R. etli a été
apporté de l’Amérique vers l’Espagne par des graines de fèves communes, mais qu’il a eu de la difficulté à persister dans
les sols du nord de l’Espagne, probablement à cause des conditions climatiques. Les gènes symbiotiques de cette espèce
pourraient avoir été transférés après l’arrivée de P. vulgaris vers les souches de R. leguminosarum déjà présentes dans les
sols du nord de l’Espagne.
Mots-clés : Rhizobium, Phaseolus, gène nodC.
[Traduit par la Rédaction]

Introduction et al. 2003). Taxonomically, R. etli originated as a conse-

Phaseolus vulgaris is a legume indigenous to Central and
South American countries that was introduced in Europe
after the 15th century. This legume establishes symbiosis
with different fast-growing rhizobia in American countries,
but the predominant endosymbiont is Rhizobium etli, whose
American origin has been proposed (Aguilar et al. 1998,
2004; Bernal and Graham 2001; Segovia et al. 1993; Silva
quence of the reclassification of Rhizobium leguminosarum
bv. phaseoli type I (Segovia et al. 1993) and is closely
related to Rhizobium phaseoli ATCC 14482T (Ramı́rez-
Bahena et al. 2008). The seeds of P. vulgaris were intro-
duced from America to different continents, namely Europe,
through Spain and Portugal, and Africa, through the Canary
Islands. These seeds could have delivered rhizobia nodulating
P. vulgaris to these continents, since at least R. etli has

Received 21 March 2010. Accepted 14 May 2010. Published on the NRC Research Press Web site at cjm.nrc.ca on 15 July 2010.
P. Garcı́a-Fraile, R. Rivas, and E. Velázquez.1 Departamento de Microbiologı́a y Genética, Universidad de Salamanca, Laboratorio
209, Edificio Departamental de Biologı́a, Campus Miguel de Unamuno, Salamanca 37007, Spain.
D. Mulas-Garcı́a and F. González-Andrés. Departamento de Ingenierı́a y Ciencias Agrarias, Universidad de León, León 24071, Spain.
A. Peix. Instituto de Recursos Naturales y Agrobiologı́a, IRNASA-CSIC, Salamanca 37008, Spain.
1Corresponding author (e-mail: evp@usal.es).

Can. J. Microbiol. 56: 657–666 (2010) doi:10.1139/W10-048 Published by NRC Research Press
 658
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Table 1. Characteristics of Rhizobium strains isolated in northern Spain and of the type strains of the closest related species also nodulating Phaseolus.

TP-RAPD nodC allele with

Strain Geographical origin Source or reference Host legume pattern respect to R. etli
Rhizobium etli CFN42T Guanajuato (Mexico) Segovia et al. 1993 Phaseolus vulgaris I a
Rhizobium pisi DSM 30132T Ramı́rez-Bahena et al. 2008 Pisum sativum II NA
Rhizobium phaseoli ATCC 14482T Beltsville (USA) Ramı́rez-Bahena et al. 2008 Phaseolus vulgaris III a
Rhizobium leguminosarum USDA 2370T Illinois (USA) Ramı́rez-Bahena et al. 2008 Pisum sativum IV NA
RPVR01 Riego de la Vega (León) This study Phaseolus vulgaris ‘Redondilla’ IV g
RPVF18 Riego de la Vega (León) This study Phaseolus vulgaris ‘Francesa’ IV g
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BAPVPT01, BAPVPT02, BAPVPT03, Barco de Ávila (Ávila) This study Phaseolus vulgaris ‘Pinta’ IV g
BAPVPT05, BAPVPT06, BAPVPT07,
BAPVPT08, BAPVPT09, BAPVPT11
RPVR04 Riego de la Vega (León) This study Phaseolus vulgaris ‘Redondilla’ V a
RPVR06 Riego de la Vega (León) Velázquez et al. 2001 Phaseolus vulgaris ‘Redondilla’ VI g
RPVN04, RPVN06 Riego de la Vega (León) This study Phaseolus vulgaris ‘Negra’ VI g
RPVR24 Riego de la Vega (León) This study Phaseolus vulgaris ‘Redondilla’ VII g
RPVF07 Riego de la Vega (León) This study Phaseolus vulgaris ‘Francesa’ VII g
RPVR32 Riego de la Vega (León) Velázquez et al. 2001 Phaseolus vulgaris ‘Redondilla’ VIII g
LCS0401 Sueros de Cepeda (León) This study Phaseolus vulgaris ‘Riñón’ IX g
LCS0407 Sueros de Cepeda (León) This study Phaseolus vulgaris ‘Riñón’ X g
LCS0303 Sueros de Cepeda (León) This study Phaseolus vulgaris ‘Riñón’ XI g
LPB0111, LPB0114, LPB0201, LPB0205, Bercianos del Páramo (León) This study Phaseolus vulgaris ‘Riñón’ XII g
LPB0209, LPB0211, LPB0212
LCS0305, LCS0403, LCS0404, LCS0408, Sueros de Cepeda (León) This study Phaseolus vulgaris ‘Riñón’ XII g
LCS0411

Can. J. Microbiol. Vol. 56, 2010

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 Garcı́a-Fraile et al.
Fig. 1. Different TP-RAPD patterns found in this study: Rhizobium
etli CFN42T (lane 1), Rhizobium pisi DSM 30132T (lane 2), Rhizo-
bium phaseoli ATCC 14482T (lane 3), Rhizobium leguminosarum
USDA 2370T (lane 4), RPVR04 (lane 5), RPVN06 (lane 6), RPVR24
(lane 7), RPVR32 (lane 8), LCS0401 (lane 9), LCS0407 (lane 10),
LCS0303 (lane 11), and LPB0205 (lane 12). MW, molecular
weight standard VI (Roche, Mannheim, Germany).
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been reported to be carried by bean grains, on their testa
(Pérez-Ramı́rez et al. 1998). The presence of this species
has been detected in Africa (Mhamdi et al. 1999; Beyene et
al. 2004) and Europe (southern Spain), as it is the most
common endosymbiont of Phaseolus in some of these soils
(Herrera-Cervera et al. 1999; Rodrı́guez-Navarro et al.
2000). In southern Spain, 2 nodC alleles (a and g), com-
monly harbored in R. etli strains, have been found (Aguilar
et al. 2004). However, in Africa and the Canary Islands, P.
vulgaris may be nodulated by Sinorhizobium meliloti strains
that carry a different nodC gene (Mnasri et al. 2007; Zurdo-
Piñeiro et al. 2009). The dominance of R. etli (73% of
strains) in southern Spain and the presence of the 2 nodC
alleles found in this species in America supports the hypoth-
esis that this species was introduced in Spain by associating
with bean seeds. Nevertheless, the data obtained in southern
Spain correspond to soils without a cultivation history of the
common bean, as northern Spain is the main producer re-
gion with the longest history of cultivation of this legume.
Since only 6 strains isolated in a single soil of this region
had been previously analysed (Velázquez et al. 2001), and
none of them were identified as R. etli, we performed a
wider study of strains nodulating common bean in different
soils from northern Spain. The phylogenetic relationships of
these strains were analysed on the basis of several chromo-
659
somal loci (rrs, atpD, and recA genes and 16S–23S inter-
genic spacer) and a symbiotic locus (nodC), and a
comparative study was carried out with respect to strains
that have been isolated from other geographical locations
and whose genes are available in databases.
Material and methods
Isolation of rhizobial strains and nodulation tests
A total of 43 rhizobial isolates (Table 1) able to form ef-
fective nodules on P. vulgaris were isolated according to
Vincent (1970) on medium containing yeast extract –
mannitol – agar, from common bean plants growing in 4 dif-
ferent locations in northern Spain, in which different vari-
eties of this legume are commonly cultivated in rotation
with maize, potatoes, or sugar beet. The locations Riego de
la Vega (42823’N, 5858’W), Sueros de Cepeda (42836’N,
6802’W), and Bercianos del Páramo (42822’N, 5842’W) be-
long to León province, and Barco de Ávila to Ávila prov-
ince (40821’N, 5831’W). The nodulation experiments were
carried out on Phaseolus vulgaris ‘Riñón’, Pisum sativum
‘Frisson’, Trifolium repens ‘Huia’, and a local variety of Vi-
cia ervilia, as previously described (Ramı́rez-Bahena et al.
2009).
Two primer randomly amplified polymorphic DNA (TP-
RAPD) pattern analysis
DNA extraction and TP-RAPD analysis were performed
as reported in Rivas et al. (2002), using the primers pair
879F–1522R at 2 mmol/L final concentration and an Ampli-
Taq Gold reagent kit (Applied Biosystems Inc., Foster City,
Calif., USA). PCR conditions were as follows: preheating at
95 8C for 9 min; 35 cycles of denaturing at 95 8C for 1 min;
annealing at 50 8C for 1 min and extension at 72 8C for
2 min; and a final extension at 72 8C for 7 min. An 8 mL
aliquot of loading solution (40% sucrose and 0.25% bromo-
phenol blue) was added to 25 mL of PCR product. The elec-
trophoresis was carried out on a 1.5% agarose gel in TBE
buffer (100 mmol/L Tris, 83 mmol/L boric acid, 1 mmol/L
EDTA, pH 8.5) at 6 V/cm. The gels were stained in a solu-
tion containing 0.5 mg/mL ethidium bromide and photo-
graphed under ultraviolet light.
Sequence analysis of 16S rRNA, recA, atpD, and nodC
genes and the 16S–23S intergenic spacer
The amplification and sequencing of 16S rRNA gene was
carried out according to Rivas et al. (2007a), internal tran-
scribed spacer (ITS) according to Kwon et al. (2005), recA
and atpD according to Gaunt et al. (2001), and nodC as de-
scribed by Laguerre et al. (2001). PCR amplification and
gene sequencing were performed as was recently described
by Ramı́rez-Bahena et al. (2009). The sequences obtained
were compared with those from GenBank using the
BLASTN program (Altschul et al. 1990) and aligned using
the Clustal W software (Thompson et al. 1997). The distan-
ces were calculated according to Kimura’s 2-parameter
model (Kimura 1980). Phylogenetic trees were inferred us-
ing the neighbour-joining method (Saitou and Nei 1987).
Bootstrap analysis was based on 1000 resamplings. The
MEGA 4 package (Tamura et al. 2007) was used for all
analyses.
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 660 Can. J. Microbiol. Vol. 56, 2010

Fig. 2. Neighbour-joining phylogenetic rooted tree based on rrs gene sequences (1475 nt) showing the taxonomic location of representative
strains from each TP-RAPD group. Bootstrap values calculated for 1000 replications are indicated. Scale bar = 1 nt substitution per 100 nt.
A single strain from each soil has been included in the phylogenetic tree, since the remaining strains have an identical rrs gene: LCS0303
(GQ863501), LCS0401 (GQ863502), LCS0411 (GQ863504), RPVN06 (GQ863497), RPVR04 (GQ863498), RPVR24 (GQ863499), and
RPVR32 (GQ863500). Accession numbers from GenBank are given in parentheses.
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Results and discussion TP-RAPD pattern analysis

Isolation of rhizobia and nodulation tests
Effective nodules were found in all P. vulgaris plants har-
vested in all the cultivated soils analysed, and we selected
those presenting morphology typical of effective common
bean nodules (pink internal colour due to leghaemoglobin).
All the strains were able to reinfect P. vulgaris; however,
they did not form nodules on Vicia, Pisum, or Trifolium.
The inoculated bean plants had shoot masses ranging from
833 to 1203 mg/plant and plant nitrogen contents ranging
from 23 to 42 mg/plant. The uninoculated controls (without
added nitrogen) had a mean of 670 mg/plant and contained
7.4 mg/plant of nitrogen. These results showed that all strains
were able to form an effective symbiosis with P. vulgaris.
TP-RAPD pattern analysis is a useful technique for group-
ing rhizobial isolates before gene sequencing, because rhizo-
bial strains with the same TP-RAPD patterns have identical
rrs gene and internal variations in atpD and recA gene se-
quences lower than 2% (Valverde et al. 2006). The results
are summarized in Fig. 1 and Table 1, showing the existence
of 9 different patterns, from which only pattern IV was
found in 3 soils: those from Riego de la Vega and Barco de
Ávila (Spain) and Illinois (USA). The strains presenting this
pattern were isolated from different hosts; Phaseolus in the
case of Spanish strains and Pisum in the case of strain
USDA 2307T, isolated in America. These patterns were dif-
ferent from those of reference strains included in this study,
which as expected were also different among them. These

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 Garcı́a-Fraile et al.
Fig. 3. Neighbour-joining phylogenetic tree based on 16S–23S rDNA intergenic sequences (1108 nt) showing the position of representative
strains from each TP-RAPD group. Bootstrap values calculated for 1000 replications are indicated. Scale bar = 2 nt substitutions per 100 nt.
Accession numbers from GenBank are given in parentheses.
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results show a high genetic diversity of strains isolated from
P. vulgaris in the soils analysed, and representative strains
were selected for gene sequencing.
Analysis of rrs gene
The results of rrs gene phylogenetic anaysis are shown in
Fig. 2. In spite of the genetic variability found with TP-
RAPD pattern analysis, the rrs gene was identical in all our
strains. The high similarity of rrs genes is common among
different species of the genus Rhizobium; nevertheless, we
have found a high variability in ITS and housekeeping genes
even when the rrs genes were identical (Valverde et al.
2006; Ramı́rez-Bahena et al. 2008; Santillana et al. 2008;
Álvarez-Martı́nez et al. 2009).
ITS sequence analysis
The results of the ITS analysis showed that all Phaseolus
isolates belong to the phylogenetic group of R. leguminosa-
rum (Fig. 3), as they show identity values ranging from 93%
to 100% with respect to R. leguminosarum bv. viciae USDA
2370T. With the exception of the strains presenting TP-
RAPD pattern IV, which showed 100% identity with respect
661
to the type strain of R. leguminosarum, the remaining strains
clustered in 5 different branches of the tree. In some cases,
the identity values of these strains with respect to the strain
USDA 2370T were similar to that found between this last
strain and Rhizobium pisi DSM 30132T, such as especially
occurs in the case of the strain RPVR04. Therefore, these
strains could possibly be classified into different species of
the genus Rhizobium; nevertheless, the ITS region is not the
most suitable phylogenetic marker for species delineation in
this genus because of its high variability in size and se-
quence. However, it is very useful to recognize strains with
a common phylogenetic origin; for example, RPVF18, iso-
lated from Phaseolus in Riego de la Vega; RVS03 and
RVS11, isolated from Vicia in the same soil (Álvarez-Martı́nez
et al. 2009); BAPVPT2, isolated in Barco de Avila; PEVF01
and PEVF02, isolated from Vicia faba in Peru (Santillana et
al. 2008); and USDA 2370T and ATCC 14482, isolated from
Pisum and Trifolium, respectively, in the United States.
These results confirmed a worldwide distribution of this
chromosomal background, as we pointed out already in a pre-
vious study on Vicia isolates, suggesting a possible European
origin of the species R. leguminosarum (Álvarez-Martı́nez et
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 662
Fig. 4. Neighbour-joining phylogenetic tree based on recA gene sequences (520 nt) showing the position of representative strains from each
TP-RAPD group. Bootstrap values calculated for 1000 replications are indicated. Scale bar = 2 nt substitutions per 100 nt. Accession num-
bers from GenBank are given in parentheses.
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al. 2009). However, the results of the present work offer
new perspectives in the analysis of the biogeography of
these strains, since strains with the same chromosomal back-
ground but different host range (RPVF18, BAPVPT02,
RVS03, and RVS11) exist in the same soil because of the
acquisition of different nodulation genes during the course
of evolution (see below).
Analysis of atpD and recA genes
The phylogenetic analyses of recA and atpD genes
(Figs. 4 and 5, respectively) are congruent with the results
of the ITS analysis, showing again that our Phaseolus strains
clustered in 5 different branches related to R. leguminosa-
rum. Strains with total identity in their rrs genes can have
highly variable housekeeping genes (Valverde et al. 2006;
Ramı́rez-Bahena et al. 2008; Santillana et al. 2008;
Álvarez-Martı́nez et al. 2009). As occurred in the case of
ITS sequences, RPVR04 was the most divergent strain with
respect to R. leguminosarum (92% identity in both recA and
Can. J. Microbiol. Vol. 56, 2010
atpD). The new Phaseolus isolates from different soils in
León (represented by RPVF18, LCS0407, and LPB0205)
and Barco de Ávila (represented by strain BAPVPT02),
those isolated from Vicia in Peru (South America), and
strains USDA 2370T and ATCC 14480, isolated in Illinois
(USA) from Pisum and Trifolium, respectively, grouped to-
gether with identity values higher than 99%. Therefore,
these strains are unambiguously identified as having a com-
mon phylogenetic origin in R. leguminosarum, even though
they were isolated from different and distant soils. The re-
maining strains cluster into 4 groups, some of them probably
corresponding to different species from those currently rec-
ognized within the phylogenetic group of R. leguminosarum,
since they are more distant from R. leguminosarum than R.
pisi or R. fabae. All strains isolated in this study were phy-
logenetically distant from R. etli and R. phaseoli as deduced
from the ITS and housekeeping gene analysis. These results
contrast with those found in southern Spain, in which up to
73% of strains were identified as R. etli (Herrera-Cervera et
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 Garcı́a-Fraile et al.
Fig. 5. Neighbour-joining phylogenetic tree based on atpD gene sequences (500 nt) showing the position of representative strains from each
TP-RAPD group. Bootstrap values calculated for 1000 replications are indicated. Scale bar = 1 nt substitution per 100 nt. Accession num-
bers from GenBank are given in parentheses.
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al. 1999). The fact that we did not find R. etli in northern
Spain suggests a lesser occurrence or even a possible ab-
sence of this species, which could be due to a poor adapta-
tion to the climate or edaphic and chemical characteristics of
northern Spanish soils.
Analysis of nodC gene
The species P. vulgaris was reported as a nonselective
host by Michiels et al. (1998), since it was nodulated by a
wide variety of rhizobial species. Nevertheless, it is cur-
rently known that the promiscuity of a legume cannot be
based on the number of chromosomal bacterial species able
to nodulate it, but rather on the variety of symbiotic genes
able to induce the nodulation process (Rivas et al. 2007b).
From the symbiotic gene pool, nodC has been widely ana-
lysed in rhizobia, as the gene is related to the host range
and the promiscuity degree of the hosts (Roche et al. 1996;
Perret et al. 2000; Laguerre et al. 2001; Rivas et al. 2007b;
Iglesias et al. 2007; Álvarez-Martı́nez et al. 2009; Zurdo-
Piñeiro et al. 2009). Laguerre et al. (2001) pointed out that
nodC genes of various species nodulating P. vulgaris were
phylogenetically close even if they were included in several
biovars (phaseoli, gallicum, and giardinii). Later, Mnasri et
663
al. (2007) described a new biovar named mediterranense in
the species S. meliloti and Sinorhizobium fredii that includes
strains able to nodulate Phaseolus but not alfalfa and carry-
ing nodC genes relatively divergent to those of biovar pha-
seoli (Mnasri et al. 2007). Recently, we found strains of S.
meliloti from this biovar in the Canary Islands, but we also
found that P. vulgaris can be nodulated by strains of S. me-
liloti bv. meliloti carrying a highly divergent nodC, leading
us to conclude that P. vulgaris is a very promiscuous host
(Zurdo-Piñeiro et al. 2009). Although the convenience to as-
sign biovar status to different rhizobia nodulating a too-
promiscuous host can be discussed, it may be reasonable to
maintain the biovar phaseoli at least in R. leguminosarum.
This species includes strains nodulating a promiscuous host
such as Phaseolus and restrictive hosts such as Vicia and
Trifolium (Álvarez-Martı́nez et al. 2009; Ramı́rez-Bahena et
al. 2009). Although most strains nodulating Vicia or Trifo-
lium were also able to nodulate Phaseolus, the strains iso-
lated from Vicia were not able to nodulate Trifolium, and
only a few strains isolated from Trifolium were able to nod-
ulate Pisum (Álvarez-Martı́nez et al. 2009; Ramı́rez-Bahena
et al. 2009). These results support the convenience of main-
taining the biovars viciae, trifolii, and phaseoli, which can
be differentiated by the type of nodC gene.
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 664
Fig. 6. Neighbour-joining phylogenetic tree based on nodC gene sequences (840 nt) showing the position of representative strains from each
TP-RAPD group. Bootstrap values calculated for 1000 replications are indicated. Scale bar = 2 nt substitutions per 100 nt. Accession num-
bers from GenBank are given in parentheses.
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According to the nodC gene analysis (Fig. 6), our Phaseolus
strains belong to biovar phaseoli of R. leguminosarum and
grouped with some strains of R. etli, Rhizobium gallicum,
Rhizobium giardinii, and the type strain R. phaseoli ATCC
14482T (Ramı́rez-Bahena et al. 2008). This group is divided
into 2 subgroups coincident with the previously described
alleles a and g of R. etli (Aguilar et al. 2004). Northern
Spain isolates mainly harboured the g allele of nodC, also
present in the strain R. etli Viking I, and this was also the
allele found in R. leguminosarum H132, R. gallicum bv.
phaseoli PhD12, and R. giardinii bv. phaseoli H251, all of
them isolated in Europe. Only a strain isolated in northern
Spain, RPVR04, harboured the nodC allele named a, also
found in R. etli CFN42T and type strain of R. phaseoli
ATCC 14482T. These results suggest that whatever R. etli
strains survived or disappeared in northern Spanish soils, its
symbiotic genes have been maintained in other related spe-
cies able to nodulate Phaseolus.
Can. J. Microbiol. Vol. 56, 2010
Biogeographic and evolutionary remarks
Rhizobium etli is the predominant species associated with
both the wild and cultivated common bean in different
American countries such as Mexico, Argentina, Ecuador,
and the United States (Aguilar et al. 1998, 2004; Bernal et
al. 2004; Silva et al. 2003), and the coevolution of this spe-
cies in centres of host diversification has been proposed
(Aguilar et al. 2004). This species could have been dissemi-
nated together with bean seeds, since it has been found on
their testa (Pérez-Ramı́rez et al. 1998), explaining its pres-
ence in African (Mhamdi et al. 1999; Beyene et al. 2004)
and southern Spanish soils (Herrera-Cervera et al. 1999;
Rodrı́guez-Navarro et al. 2000), in which R. etli is the major
species nodulating P. vulgaris. Although R. etli has not been
found in northern Spain, the R. leguminosarum strains iso-
lated from bean nodules in this region harbored the 2 major
R. etli nodC alleles (a and g) found in both America and
southern Spain (Aguilar et al. 2004). Therefore, the nodC
genes carried by the strains nodulating bean in northern
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 Garcı́a-Fraile et al.
Spain seem to come from R. etli, probably by horizontal
gene transfer in the rhizosphere. In a previous work, we pro-
posed the European origin of R. leguminosarum, because R.
leguminosarum strains nodulating Vicia in different conti-
nents carry a close nodC gene that was highly divergent
from those of biovar phaseoli, suggesting a coevolution be-
tween R. leguminosarum and Vicia in Europe (Álvarez-
Martı́nez et al. 2009). This proposal could be supported by
the results of the present work showing the existence, in the
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same soil (Riego de la Vega), of R. leguminosarum strains
with the same chromosomal background but nodC genes
with different phylogenetic origins (see Fig. 6). Therefore,
the more plausible hypothesis is that when P. vulgaris was
introduced into northern Spanish soils, R. leguminosarum
strains were already present and horizontally acquired the
nodulation genes carried by the seed-borne R. etli. Although
R. leguminosarum strains nodulating Phaseolus are also
present in North America, the available data for these strains
are not enough to support an American origin of R. legumi-
nosarum, since only chromosomal genes were analyzed
(Bernal et al. 2004). Moreover, the presence in South Amer-
ican R. leguminosarum strains of a nodC allele different
from a or g (Aguilar et al. 2004) that has not been found in
northern Spanish strains seems also to exclude the American
origin of this species. The comparative analysis of chromo-
somes and symbiotic genes of strains isolated in northern
For personal use only.
Spain soils could help us to draw a picture about their ori-
gin, but it is necessary to complete the study with Trifolium
strains isolated in the same soil. Finding unambiguous data
about donors and acceptors of nodulation genes in R. legu-
minosarum that confirm the geographical origin of this spe-
cies and its nodC genes is a hard task, but further studies
about strains isolated from soils in which Phaseolus has not
been cultivated in Spain could provide interesting data for
evolutionary and biogeographical studies.
Acknowledgements
This work was supported by the Ministerio de Ciencia e
Innovación (MICINN; Spanish central government) and
Junta de Castilla y León (JCyL; Spanish regional govern-
ment). This work was supported by MICINN funding to E.
Velázquez and JCyL funding to E. Velázquez and F. Gonzá-
lez-André.
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