World Journal of Microbiology and Biotechnology (2020) 36:63

https://doi.org/10.1007/s11274-020-02839-w

REVIEW

The promiscuity of Phaseolus vulgaris L. (common bean)

for nodulation with rhizobia: a review
Abdelaal Shamseldin1 · Encarna Velázquez2,3

Received: 16 January 2020 / Accepted: 9 April 2020

© Springer Nature B.V. 2020

Abstract
Phaseolus vulgaris L. (common bean) is a legume indigenous to American countries currently cultivated in all continents,
which is nodulated by different rhizobial species and symbiovars. Most of species able to nodulate this legume worldwide
belong to the genus Rhizobium, followed by those belonging to the genera Ensifer (formerly Sinorhizobium) and Pararhizo-
bium (formerly Rhizobium) and minority by species of the genus Bradyrhizobium. All these genera belong to the phylum
alpha-Proteobacteria, but the nodulation of P. vulgaris has also been reported for some species belonging to Paraburkholderia
and Cupriavidus from the beta-Proteobacteria. Several species nodulating P. vulgaris were originally isolated from nodules
of this legume in American countries and are linked to the symbiovars phaseoli and tropici, which are currently present in
other continents probably because they were spread in their soils together with the P. vulgaris seeds. In addition, this legume
can be nodulated by species and symbiovars originally isolated from nodules of other legumes due its high promiscuity, a
concept currently related with the ability of a legume to be nodulated by several symbiovars rather than by several species.
In this article we review the species and symbiovars able to nodulate P. vulgaris in different countries and continents and
the challenges on the study of the P. vulgaris endosymbionts diversity in those countries where they have not been studied
yet, that will allow to select highly effective rhizobial strains in order to guarantee the success of P. vulgaris inoculation.

Keywords P. vulgaris (common bean) · Promiscuity · Nitrogen-fixation · Rhizobial species · Symbiovars

Origin and promiscuity of P. vulgaris
Phaseolus vulgaris L. (common bean) is a legume indig-
enous to Middle- and South-America currently cultivated in
countries of all continents by small farmers and large pro-
ducers being commercialized for both the green pods and the
Electronic supplementary material The online version of this
article (https​://doi.org/10.1007/s1127​4-020-02839​-w) contains
supplementary material, which is available to authorized users.
* Abdelaal Shamseldin
yabdelall@yahoo.com
1
Environmental Biotechnology Department, Genetic
Engineering and Biotechnology Research Institute (GEBRI),
City of Scientific Research and Technological Applications,
New Borg El‑Arab, Alexandria, Egypt
2
Departamento de Microbiología Y Genética and CIALE,
Universidad de Salamanca, Salamanca, Spain
3
Unidad Asociada Grupo de Interacción
Planta-Microorganismo (Universidad de
Salamanca-IRNASA-CSIC), Salamanca, Spain
dry seeds. This legume is among the three most important
cultivated crops worldwide together with soybean (Glycine
max L.) and peanut (Arachis hypogaea L.). P. vulgaris can
grow in different edafo-climatic conditions and there are
many varieties with different maturation times and seeds
with great variety of sizes, shapes and colors (de Ron et al.
2015). They are a source of protein, dietary fiber and starch
particularly in developing countries (Gomes Basso Los et al.
2018).
From the American diversification centers, the seeds of
P. vulgaris contributed to spread their microsymbionts into
other continents worldwide. This is supported by the finding
in the testa of P. vulgaris seeds of Rhizobium etli (Pérez-
Ramírez et al. 1998), which is the main microsymbiont of
this legume in its American diversification centers (Aguilar
et al. 2004) but it has also been found in Europe (Herrera-
Cervera et al. 1999), Africa (Diouf et al. 2000; Mhamdi et al.
2002; Shamseldin et al. 2005; Aserse et al. 2012; Faghire
et al. 2012; Zinga et al. 2017) and Asia (Gurkanli et al. 2013;
Adhikari et al. 2013; Cao et al. 2014). The same can be hap-
pened with other American species nodulating P. vulgaris,

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such as Rhizobium phaseoli and Rhizobium tropici which
have been found in Asia (Wang et al. 2016; Gurkanli et al.
2013; Adhikari et al. 2013; Rouhrazi et al. 2016), Rhizobium
leucaenae and Rhizobium hidalgonense in Europe (Valverde
et al. 2011; Rajnovic et al. 2019), and R. phaseoli, R. tropici
and R. leucaenae in Africa (Diouf et al. 2000; Aserse et al.
2012; Faghire et al. 2012; Zinga et al. 2017; Kawaka et al.
2018; Mwenda et al. 2018).
The symbiovars phaseoli and tropici linked to the Ameri-
can species nodulating P. vulgaris could be also spread in
other continents together with the seeds of this legume,
and their symbiotic genes could be transferred to local
strains. This occurred with the symbiovar phaseoli, which
has been found in strains of Rhizobium leguminosarum
in Asia, Europe and Africa (Mhamdi et al. 2002; García-
Fraile et al. 2010; Mulas et al. 2011; Gurkanli et al. 2013;
Adhikari et al. 2013; Cao et al. 2014; Rajnovic et al. 2019),
Rhizobium lusitanum and Rhizobium pisi in Europe (Val-
verde et al. 2011; Rajnovic et al. 2019), Rhizobium vallis
and Rhizobium chutanense in Asia (Wang et al. 2011; Huo
et al. 2019) and Rhizobium sophoriradicis in Africa (Zinga
et al. 2017; Mwenda et al. 2018). However the symbiovar
tropici, which is also present in Europe, Asia and Africa,
has not been found to date in local strains of other species
and it is always linked to the American species R. tropici
or R. leucaenae (Valverde et al. 2011; Rouhrazi et al. 2016;
Mwenda et al. 2018).
The species reported to date nodulating P. vulgaris mostly
belong to the alpha-Proteobacteria and concretely to the gen-
era Rhizobium, Pararhizobium (formerly Rhizobium), Ensi-
fer (formerly Sinorhizobium) and Bradyrhizobium. Several
species of genera Rhizobium and Pararhizobium were origi-
nally isolated from P. vulgaris nodules in different continents
and carry different symbiovars (Table 1). In addition, P. vul-
garis can be nodulated some beta-Proteobacteria, concretely
by some strains belonging to species from genera Burk-
holderia (currently Paraburkholderia) (Talbi et al. 2010;
Martínez-Aguilar et al. 2013; Dall’Agnol et al. 2017) and
Cupriavidus (da Silva et al. 2012; Dall’Agnol et al. 2017).
Due to the ability of P. vulgaris to nodulate with different
rhizobial genera and species it has been traditionally consid-
ered as a promiscuous host (Michiels et al. 1998). However,
the promiscuity degree of a legume is not currently related
with the concept of species but with that of symbiovar and
then, the high promiscuity degree of P. vulgaris is not due
to its ability to nodulate with several rhizobial species, but
with several symbiovars (Peix et al. 2015). In addition to
the symbiovars phaseoli and tropici firstly found in Ameri-
can countries, P. vulgaris is nodulated by symbiovars found
outside America, such as gallicum and giardinii linked to
Rhizobium and Pararhizobium, respectively, that firstly
found in Europe (Amarger et al. 1997), and mediterranense
linked to Ensifer and firstly found in Africa (Mnasri et al.
2007; Zurdo-Piñeiro et al. 2009). From them, the symbiovars

Table 1  Species of rhizobia originally isolated from Phaseolus vulgaris nodules and symbiovars of these species nodulating this legume
Species Country (continent) (for Symbiovar (for the References
the type strain) type strain)

Rhizobium aethiopicum Ethiopia Unnamed Aserse et al. (2017)

Rhizobium acidisoli Mexico (America) Phaseoli Román-Ponce et al. (2016)
Rhizobium azibense Tunisia (Africa) Gallicum Mnsari et al. (2014)
Rhizobium chutanense China (Asia) Phaseoli Huo et al. (2019)
Rhizobium ecuadorense Ecuador (America) Phaseoli Ribeiro et al. (2015)
Rhizobium etli Mexico (America) Phaseoli Segovia et al. (1993)
Rhizobium esperanzae Mexico (America) Phaseoli Cordeiro et al. (2017)
Rhizobium freirei Brazil (America) Tropici Dall’Agnol et al. (2013), Ormeño-Orrillo et al. (2012)
Rhizobium gallicum France (Europe) Gallicum Amarger et al. (1997)
Phaseoli
Rhizobium hidalgonense Mexico (America) Phaseoli Yan et al. (2017)
Rhizobium leucaenae Brazil (America) Tropici Ribeiro et al. (2012), Ormeño-Orrillo et al. (2012)
Rhizobium lusitanum Portugal (Europe) Phaseoli Valverde et al. (2006), Ormeño-Orrillo et al. (2012)
Tropici
Rhizobium mesoamericanum Mexico (America) Unnamed López-López et al. (2012)
Rhizobium paranaense Brazil (America) Unknown Dall’Agnol et al. (2014)
Rhizobium phaseoli USA (America) Phaseoli Dangeard (1926), Ramírez-Bahena et al. (2008)
Rhizobium tropici Colombia (America) Tropici Martínez-Romero et al. (1991), Ormeño-Orrillo et al. (2012)
Rhizobium vallis China (Asia) Phaseoli Wang et al. (2011)
Pararhizobium giardini France (Europe) Giardinii Amarger et al. (1997), Mousavi et al. (2015)
Phaseoli

13
World Journal of Microbiology and Biotechnology (2020) 36:63
gallicum and mediterranense were later found in America
(Silva et al. 2003; Verástegui-Valdés et al. 2014) where some
new symbiovars linked to species originally isolated from P.
vulgaris nodules remain unnamed (Table 1).
The ability of P. vulgaris to be nodulated by different
symbiovars linked to species primarily isolated from nod-
ules of this legume and also by symbiovars linked to species
initially isolated from nodules of other legumes allows P.
vulgaris to establish nitrogen-fixing symbiosis in very differ-
ent ecosystems around the world. Several reviews have been
published along the time about the legume symbiosis, but to
date only one review has been published in the present cen-
tury about the diversity of rhizobia establishing symbiosis
with P. vulgaris (Martinez-Romero 2003). Since the publi-
cation of this review, multiple new species and symbiovars
have been identified from P. vulgaris nodules. In this article
we review the species and symbiovars of Rhizobium reported
to date, which are able to nodulate this legume in countries
of different continents, and the challenges on studying P.
vulgaris and their endosymbionts diversity in those countries
where they have not been studied yet, that will help to select
the more effective rhizobial strains in order to guarantee the
success of P. vulgaris inoculation allowing the substitution
of chemical fertilizers by rhizobial-based inoculants (Fig. 1).
Species and symbiovars of rhizobia
nodulating P. vulgaris around the world
P. vulgaris is the most widely cultivated species of genus
Phaseolus around the world, although other species such
as Phaseolus coccienus, Phaseolus dumosus and Phaseolus
Fig. 1  Neighbour-joining phylo-
genetic rooted tree based on 16S
rRNA gene sequences (1410
nt) showing the phylogenetic
relationships among the type
strains of species which have
been originally isolated from P.
vulgaris nodules. Bradyrhizo-
bium japonicum USDA ­6T was
used as out-group. Bootstrap
values calculated for 1000
replications are indicated. Bar,
1 nt substitution per 1000 nt.
Accession numbers from Gene
59
Bank are given in brackets.
Only bootstrap values > 50% are
shown
0.01
Page 3 of 12 63
lunatus are also cultivated mainly in American countries.
It has been reported that the nodulation of P. coccinus in
America by strains of genus Rhizobium belonged to R. etli
sv. phaseoli (Souza et al. 1994) and by strains closely related
to R. leguminosarum, R. ecuadorense and R. tropici in the
case of P. dumosus (Ramírez-Puebla et al. 2019). The spe-
cies Phaseolus lunatus, although it is nodulated by strains
of different rhizobial genera (Matsubara and Zuniga-Davila
2015; Oliveira-Santos et al. 2011), most of strains nodulating
this legume belong to the genus Bradyrhizobium (Ormeño-
Orrillo et al. 2006; Oliveira-Santos et al. 2011; Lopez-Lopez
et al. 2013; Matsubara and Zuniga-Davila 2015). Neverthe-
less, the main studied endosymbionts are those of P. vulgaris
and most of them are indigenous to American diversification
centers of this legume, which migrated to other continents
through several ways. Perez-Ramirez et al. (1998) reported
the presence of R. etli viable on the surface of the P. vulgaris
seeds for several years at room temperature suggesting that
this may a way to move rhizobial strains among countries.
The rhizobial cells can also survive in water environments,
as evidenced the nodulation of aquatic legumes (de Lajudie
et al. 1998; Wang and Martinez-Romero 2000; Rivas et al.
2002, 2003; Rahman et al. 2018) and therefore water may
have also contributed to the dispersion of rhizobia, including
those nodulating P. vulgaris, such as R. tropici which is also
able to nodulate aquatic legumes (Zurdo-Piñeiro et al. 2004).
In addition, the inoculation allochthonous rhizobial strains
becoming from producing countries, such as Colombia can
contribute to the establishment of Rhizobium strains in new
regions of importing countries where these strains are not
previously found (Wang and Chen 2004).
87 Rhizobium chutanense C5 (NWSV01000078)
T
Rhizobium phaseoli ATCC14482T (EF141340)
Rhizobium hidalgonense FH14T (|KJ921034)
63
100 Rhizobium acidisoli FH13T (KJ921033)
99 Rhizobium ecuadorense CNPSO 671T (LFIO01000095)
66 Rhizobium esperanzae CNPSo 668T (NZ_MXPU01000055)
Rhizobium etli CFN42T (CP000133)
Rhizobium azibense 23C2T (JN624691)
63
99 Rhizobium gallicum R602spT (U86343)
Rhizobium vallis CCBAU65647T (FJ839677)
Rhizobium paranaense PRF35T (EU488753)
97
Rhizobium lusitanum p1-7T (AY738130)
82
Rhizobium tropici CIAT 899T (CP004015)
63
89 Rhizobium leucaenae LMG 9517T (NR_118993)
73 Rhizobium freirei PRF 81T (EU488742)
Rhizobium mesoamericanum STM3625T (CANI01000030)
Pararhizobium giardinii H152T (ARBG01000149)
Bradyrhizobium japonicum USDA 6T (AP012206)
13
 63 Page 4 of 12
Therefore, several species and symbiovars initially found
in P. vulgaris nodules in America have been later identified
in countries from other continents and also some symbiovars
probably indigenous to Africa have been later found linked
to strains nodulating P. vulgaris in America. These findings
come from several studies about the species and symbiovars
nodulating common bean, which have been carried out in
the countries from different continents and are shown below.
American countries
Different Rhizobium species are able to nodulate P. vulgaris
in America where the distribution centers of this legume
are located (Gepts and Debouck 1991). The first described
species nodulating P. vulgaris was isolated in this continent
and it was named R. phaseoli, which was later considered a
biovar (currently re-named symbiovar) of R. leguminosarum
(Jordan 1984) and later partially transferred to a new species
named R. etli (Segovia et al. 1993). Already in the present
century, Ramírez-Bahena et al. (2008) showed that both spe-
cies of R. phaseoli and R. etli are distinguished between
them on the basis of their housekeeping genes. In a later
study it was showed that several American strains initially
identified as R. etli really belong to the species R. phaseoli
(López-Guerrero et al. 2012).
The type strains of both R. phaseoli and R. etli belong to
the symbiovar phaseoli, which is also linked to other species
mainly isolated in America from P. vulgaris nodules, such
as Rhizobium acidisoli, R. ecuadorense, Rhizobium esper-
anzae and Rhizobium hidalgonense (Table 1). This sym-
biovar together with the symbiovar tropici of the species R.
tropici, R. leucaenae and Rhizobium freirei is considered to
be indigenous to the American continent (Martínez-Romero
et al. 1991; Silva et al. 2003; Aguilar et al. 2004; Valverde
et al. 2011; Ormeño-Orrillo et al. 2012; Díaz-Alcántara
et al. 2014; Verástegui-Valdés et al. 2014; Román-Ponce
et al. 2016; Cordeiro et al. 2017; Yan et al. 2017). How-
ever, the origin of other species and symbiovars nodulat-
ing P.vulgaris is not clear because they were firstly isolated
in other continents and later in American countries. This
occurs, for example, with Rhizobium gallicum sv. gallicum
firstly located in France (Amarger et al. 1997) and later in
Mexico (Silva et al. 2003) and with Ensifer americanus sv.
mediterranense isolated firstly in Africa (Mnasri et al. 2007)
and later also in Mexico (Verástegui-Valdés et al. 2014).
The species R. etli and/or R. phaseoli are dominant in
P. vulgaris nodules in American countries, such as Chile
(Junier et al. 2014; Baginsky et al. 2015), Brazil (Grange
and Hungria 2004; Costa et al. 2018), Colombia (López-
Guerrero et al. 2012), Dominican Republic (Diaz-Alcantara
et al. 2014) and Mexico, Bolivia and Argentina (Pereira et al.
2002; Aguilar et al. 2004), supporting the hypothesis of the
13
World Journal of Microbiology and Biotechnology (2020) 36:63
existence of a co-evolution of P. vulgaris and its microsym-
bionts in the centers of host genetic diversification (Aguilar
et al. 2004; Díaz-Alcántara et al. 2014). This hypothesis
is also supported for the species R. tropici and R. leucae-
nae which are found in P. vulgaris nodules in American
countries such as Colombia (Martinez-Romero et al. 1991),
Argentina (Aguilar et al. 2001), Chile (Baginsky et al. 2015)
and Brazil (Grange and Hungria 2004; Costa et al. 2018).
P. vulgaris is also nodulated by strains that do not belong to
any of the currently described species in several American
countries indicating that several new rhizobial species will
be described in the future (Díaz-Alcántara et al. 2014; Costa
et al. 2018). In addition to the American species originally
isolated from nodules of P. vulgaris, this legume is nodu-
lated in American countries by strains of species originally
isolated from nodules of other legumes, some of them con-
taining strains from the symbiovar phaseoli. For example, R.
leguminosarum sv. phaseoli nodulates P. vulgaris in Mexico
and Brazil (Grange and Hungria 2004; Verástegui-Valdés
et al. 2014; Baginsky et al. 2015), Rhizobium grahamii in
Mexico (López-López et al. 2012), and E. americanus sv.
mediterranense in Mexico (Verástegui-Valdés et al. 2014).
From the species belonging to the beta-Proteobacteria
able to nodulate P. vulgaris, two of them were also isolated
from American countries. The species Paraburkholderia
caballeronis was isolated from rhizosphere of tomato plants
in Mexico being able to nodulate P. vulgaris (Martínez-Agu-
ilar et al. 2013) and the strains from Cupriavidus necator
were isolated from P. vulgaris nodules in Brazil (da Silva
et al. 2012). Also, more recently several strains belonging
to these two genera have been found in Brazil and isolated
from nodulated roots of P. vulgaris (Dall’Agnol et al. 2017).
European countries
Europe has been considered a secondary center of diver-
sification of P. vulgaris with both Andean and Mesoa-
merican gene pools present in this continent where a high
proportion of the bean germplasm was derived from the
hybridization of these two gene pools, being the hybrids
very frequent in the Central Europe and less in countries
as Spain and Italy (Angioi et al. 2010). However a wide
area is cultivated with beans in Europe leading to a high
productivity that reached to 1500.000 Tons in year 2018,
including dry and green beans, most of them belonging
to the species P. vulgaris (https​://fao.org/faost​at/en/#data/
QC). Due to the importance of this legume in this conti-
nent, several researchers have been focused on the analysis
of the microsymbionts of this legume in different Euro-
pean countries. The first report was carried out in France
in the last decade of the past century and they identified
the microsymbionts of P. vulgaris as R. leguminosarum sv.
World Journal of Microbiology and Biotechnology (2020) 36:63
phaseoli (Geniaux et al. 1993; Laguerre et al. 1993a, b). In
a later study Amarger et al. (1997) isolated by first time in
France from P. vulgaris nodules two species which were
named R. gallicum and R. giardinii (currently Pararhizo-
bium giardinii), each one harboring two symbiovars. One
of them was the symbiovar phaseoli, which is considered
to be indigenous to America, but the geographical origins
of the other two symbiovars, gallicum and giardinii, are
not clear because the symbiovar gallicum has been found
in P. vulgaris nodules in Europe, America and Africa
(Amarger et al. 1997; Silva et al. 2003; Shamseldin and
Werner 2007; Faghire et al. 2012), and the symbiovar
giardinii in Europe, Africa and Asia (Amarger et al. 1997;
Aserse et al. 2012; Rouhrazi et al. 2016).
Despite of that, the first studies about the rhizobia nodu-
lating P. vulgaris were carried out in France, the two entry
doors of this legume in Europe were Spain and Portugal.
In Spain different studies showed that in South Spain this
legume is nodulated by strains of R. etli, R. gallicum, P.
giardinii, R. leguminosarum and Ensifer fredii (Herrera-Cer-
vera et al. 1999) and in North Spain by R. leguminosarum
(Velázquez et al. 2001; García-Fraile et al. 2010; Mulas
et al. 2011). The strains of R. leguminosarum belonged to
the symbiovar phaseoli and carry nodC gene alleles typi-
cal of American endosymbionts of P. vulgaris showing that
these genes probably were transferred to local strains of R.
leguminosarum whose European origin has been suggested
(Álvarez-Martínez et al. 2009). More recently, it has been
shown that R. laguerreae sv. viciae is able to nodulate P.
vulgaris in Spanish soils where this legume has never been
cultivated (Flores-Félix et al. 2019).
In Portugal a new species named R. lusitanum was firstly
isolated from P. vulgaris nodules (Valverde et al. 2006)
together with the species R. leucaenae (Valverde et al.
2011). In the case of R. leucaenae, all strains belonged to the
symbiovar tropici indicating the spread of R. leucaenae sv.
tropici together with P. vulgaris seeds in Portugal (Valverde
et al. 2011). However, the strains of R. lusitanum carried
the nodC genes typical of the American symbiovars pha-
seoli or tropici, suggesting a lateral transfer of nodC genes
to Portuguese strains of the species R. lusitanum, whose high
abundance in Portugal suggest that it is indigenous to Europe
(Valverde et al. 2011).
Recently, in Croatian soils strains belonging to R. legumi-
nosarum, R. hidalgonense and R. pisi were identified from
P. vulgaris nodules and all of them carry the nodC genes
typical of the symbiovar phaseoli being the first report about
the presence of R. hidalgonense in European soils, as well as
about the existence of the symbiovar phaseoli in the species
R. pisi (Rajnovic et al. 2019). In this country strains belong-
ing to the symbiovar phaseoli of two putative new species
related to R. ecuadorense and R. sophoriradicis were also
found in nodules of P. vulgaris (Rajnovic et al. 2019).
Page 5 of 12 63
Asian countries
Phaseolus vulgaris was also directly introduced from Amer-
ican countries into China where many local landraces of
Andean and Mesoamerican origins are cultivated, suggest-
ing that China may also be considered a secondary center of
diversification of this legume (Zhang et al. 2008). Neverthe-
less, compared with other legumes, the species and symbio-
vars nodulating P. vulgaris in China have been studied in few
articles to date, despite some new species of Rhizobium have
been originally isolated in this country, such as Rhizobium
vallis and R. chutanense, whose type strains belong to the
symbiovar phaseoli (Wang et al. 2011; Huo et al. 2019).
Moreover, in China the symbiovar phaseoli has been
identified in strains of R. etli, R. vallis, R. phaseoli and R.
leguminosarum and of an undescribed species nodulating
P. vulgaris, suggesting that the symbiotic genes typical of
the symbiovar phaseoli were transferred to local strains of
R. leguminosarum from strains of R. etli, which should have
been introduced in Asia through P. vulgaris seeds (Cao et al.
2014). This is supported also by the finding of R. phaseoli
sv. phaseoli in the Shaanxi province from nodules of P. vul-
garis (Wang et al. 2016). Due to the high promiscuity of this
legume, it was also able to nodulate in different Chinesse
regions with strains of E. fredii, P. giardinii and Bradyrhizo-
bium liaoningense (Wang et al. 2016) and with Bradyrhizo-
bium diazoefficiens and Bradyrhizobium strains belonging
to different undescribed species and symbiovars (Cao et al.
2014; Lopez-Lopez et al. 2013).
The diversity of rhizobia nodulating P. vulgaris in
other Asian countries has been poorly studied, although
it has been reported the nodulation of this legume by the
symbiovar phaseoli of R. leguminosarum, R. etli and R.
phaseoli in Nepal, a border country with China (Adhi-
kari et al. 2013). In other countries located in Western
Asia, such as Jordan P. vulgaris is nodulated by R. etli and
strains phylogenetically related to R. tropici (Tamimi and
Young 2004), and in Turkey, this legume nodulated by R.
etli sv. phaseoli, R. phaseoli and R. leguminosarum (Gur-
kanli et al. 2013). More diversity has been found in Iran
where P. vulgaris is nodulated by the symbiovar phaseoli
of species phylogenetically related to R. sophoriradicis, R.
leguminosarum, R. vallis, and by R. tropici sv. tropici and
P. giardinii sv. giardinii (Rouhrazi et al. 2016).
African countries
Phaseolus vulgaris arrived to Africa from diverse Euro-
pean and American countries (Gepts and Bliss 1988)
being and it is currently cultivated in several regions of
13
 63 Page 6 of 12
this continent where this legume has evolving from sub-
sistence crop to a market-oriented crop (Buruchara et al.
2011). The diversity of the rhizobia nodulating P. vulgaris
has been analyzed in many African countries and two new
species of genus Rhizobium have been described in the
present decade. The species named Rhizobium azibense,
whose strains belong to the symbiovar gallicum, was iso-
lated from the Azib region in North Tunisia (Mnsari et al.
2014), and the species Rhizobium aethiopicum isolated
from Ethiopian soils whose type strains belonged to the
symbiovar phaseoli (Aserse et al. 2017).
In Tunisia several studies had been carried out since
the end of the past century showing the presence of strains
related to R. etli, R. leguminosarum and R. gallicum in nod-
ules of P. vulgaris (Mhamdi et al. 1999). In a later study, R.
gallicum sv. gallicum, R. leguminosarum sv. phaseoli, R.
leguminosarum sv. viciae, R. etli sv. phaseoli, P. giardinii
sv. giardinii (previously R. giardinii), E. meliloti, E. medicae
and E. fredii were identified among 160 rhizobial isolates
that have the ability to nodulate and fix nitrogen with P.
vulgaris in Tunisia (Mhamdi et al. 2002).
Another Tunisian research group identified by first time
the symbiovar mediterranense linked to different Ensifer
species (Mnasri et al. 2007). The symbiovar mediterran-
ense of E. meliloti, E. fredii and E. americanum seems to
be indigenous to the African continent (Mnasri et al. 2007,
2012; Zurdo-Piñeiro et al. 2009), although it has been found
linked to the species E. americanus in both Tunisia and
Mexico (Mnasri et al. 2012; Verástegui-Valdés et al. 2014).
This symbiovar was also found in Canary Islands, geograph-
ically near to African coasts, particularly in the Lanzarote
Island where P. vulgaris is nodulated by E. meliloti strains
unable to nodulate the original host Medicago sativa (Zurdo-
Piñeiro et al. 2009).
In Ethiopia too, Beyene et al. (2004) identified the species
of R. leguminosarum and R. etli as microsymbionts of P. vul-
garis, and more recently it has been reported the nodulation
of this legume by the new species R. aethiopicum (Aserse
et al. 2017) and by the symbiovar phaseoli of R. phaseoli,
R. etli and two putative new species related to these species,
by the symbiovar giardinii of P. giardinii and by the sym-
biovar tropici of R. leucaenae (Aserse et al. 2012). In other
African countries whose soils are also affected by salinity,
such as Morocco, R. gallicum sv. gallicum, R. etli sv. pha-
seoli and R. phaseoli sv. phaseoli were found in P. vulgaris
nodules (Faghire et al. 2012). In this country the nodulation
of this legume by the beta- Proteobacteria Paraburkholderia
phymatum was also reported (Talbi et al. 2010). In Senegal
and Gambia, strains closely related to R. etli and R. tropici
were identified in P. vulgaris nodules collected from several
agroecological areas (Diouf et al. 2000).
In Egypt, Shamseldin et al. (2005) identified R. etli and R.
gallicum in P. vulgaris nodules and Shamseldin and Werner
13
World Journal of Microbiology and Biotechnology (2020) 36:63
(2007) reported that Rhizobium etli sv. phaseoli, a species
indigenous to American countries, was the dominant species
nodulating this legume in Egypt being R. gallicum sv. galli-
cum minority in the nodules of P. vulgaris in this country. In
a later study, Elbanna et al. (2009) also found that the more
effective strains nodulating P. vulgaris in different regions
of Egypt belong to the phylogenetic group of R. legumino-
sarum and R. etli.
In Mozambique, the symbiovar phaseoli of R. etli was
identified in nodules of P. vulgaris, and in some South Afri-
can soils this symbiovar was also identified in nodules of
this legume linked to, R. phaseoli, R. sophoriradicis and a
putative new species of Rhizobium together with strains that
belong to the symbiovar tropici of R. leucaenae (Zinga et al.
2017). These two symbiovars were also found in a recent
work in Kenya, where P. vulgaris was mostly nodulated by
the symbiovar phaseoli of R. phaseoli and a putative new
species of genus Rhizobium, although some strains were
identified as R. leucaenae sv. tropici, R. sophoriradicis sv.
phaseoli and Rhizobium aegyptiacum sv. phaseoli (Mwenda
et al. 2018). In the same country, several strains nodulating
P. vulgaris were identified as R. leguminosarum, R. tropici
and Rhizobium sp. although the symbiovars to which strains
belong were not analyzed (Kawaka et al. 2018).
Challenges in the study of P. vulgaris
endosymbionts
In Figure S1 we show the current distribution of P. vulagris
nodulating rhizobial species in 28 countries from America,
Africa, Asia and Europe. It also shows that, to date, most
of the studies about the identity of rhizobia nodulating P.
vulagris have been carried out in America, Africa, and
Asia, being Brazil, Mexico, Tunisia and China the coun-
tries with the higher number of reported species, whereas
in Europe the studies are limited to Portugal, Spain, France
and Croatia, but more reported Rhizobium species nodulat-
ing this legume crop was from Spain. The American species
R. phaseoli and R. etli are widely distributed in the world
as they are present at least in 17 and 15 countries, respec-
tively, which represent in both cases more than 50% of the
28 analyzed ones. However, the American species R. tropici
and R. leucaenae has been found in less countries, 7 and 3
respectively, representing in both cases less than 25% of the
analyzed ones (Figure S1).
Figure S2 shows the distribution of symbiovars nodulat-
ing P. vulgaris in different countries and continents to date.
As expected, the symbiovar phaseoli is the most frequently
isolated from the nodules of this legume worldwide followed
by the symbiovar tropici, since these two symbiovars are
linked to the most widely distributed American Rhizobium
species worldwide. These American species carry nodC
World Journal of Microbiology and Biotechnology (2020) 36:63
genes typical of two symbiovars phaseoli and tropici, and
since highly similar nodC genes have been found in strains
nodulating P. vulgaris outside America. It is accepted by the
scientific community that this is an indication of a horizontal
transfer of symbiotic genes from American species to local
rhizobial strains nodulating P. vulgaris (Rouhrazi et al. 2016;
Andrews et al. 2018; Li et al. 2018; Mwenda et al. 2018;
Rajnovic et al. 2019; Tong et al. 2020). In fact, the number
of different symbiovars found worldwide is lower than the
number of species while it is true that some of them are cur-
rently unknown or unnamed (Table 1). Both Figures S1 and
S2 are evidence on the absence of data about the rhizobial
species and symbiovars nodulating P. vulgaris in the vast
majority of countries of the world indicating on the need
of performing further studies about the identity of rhizo-
bia nodulating this legume in these countries in order to
increase the knowledge of the diversity and biogeography of
this symbiosis. The results of these future studies probably
also will lead to increase the number of species and sym-
biovars able to nodulate this legume in different parts of the
world helping us to the selection of legume inoculants better
adapted to environmental conditions of the regions where
inoculants will be used.
The knowledge of the rhizobial strains nodulating P.
vulgaris present in different countries and their symbiotic
effectiveness is essential to have success with the use of
rhizobia-based inoculants, because it has been reported that
a correct selection of local strains can replace chemical fer-
tilization (Mulas et al 2011; Morad et al 2013; koskey et al.
2017; Samago et al. 2018). In this regard, it is necessary to
know the rhizobial species and symbiovars able nodulate
P. vulgaris present in a soil before to apply an inoculant,
since the competition between native and inoculated strains
is one of the major biotic factors affecting the nodulation
(Gwyn and Handelsman 1993; Shamseldin and Werner
2004; Mrabet et al. 2005; Koskey et al. 2017; Pastor-Bueis
et al. 2019). Moreover not all symbiovars nodulating this
legume, are equally effective in nodulation and nitrogen
fixation, as occurs for example, with the symbiovars gal-
licum and giardinii which have different effectiveness on
P. vulgaris (Amarger et al. 1997). Also it has been reported
that strains from the symbiovar gallicum form lower number
of nodules than the symbiovar phaseoli (Silva et al. 2003).
However, the number of nodules and the effectiveness of the
symbiovar mediterranense is similar to those found with the
symbiovar tropici (Mnasri et al. 2007) and phaseoli (Zurdo-
Piñeiro et al. 2009). Concerning to the strains from symbio-
vars originally isolated from nodules of other legumes (not
from P. vulgaris), it has been reported that strains from the
symbiovar meliloti and viciae formed a lower number of
nodules than those from the symbiovar phaseoli, although
the percentage of fixed nitrogen was similar (Zurdo-Piñeiro
et al. 2009; Flores-Félix et al. 2019).
Page 7 of 12 63
Other relevant biotic factor needed to be consider when
designing a rhizobial inoculants is the macrosymbiont itself
because the legumes control the nodulation process (Fer-
guson et al. 2019) and recent studies reported several genes
of P. vulgaris involved in the regulation of the nodulation
process (Barraza et al. 2018; Carrasco-Castilla et al. 2018;
del Cerro et al. 2019; Ren et al. 2019), and in the selection
of concrete strains within the same species (Rípodas et al.
2019). Also the plant genotype can influence the nodulation
as occurs in the case of P. vulgaris since it has been reported
that R. etli and R. tropici strains showed different efficiency
on different plant genotypes, which affect the stability of the
performance of inoculants (Gunnabo et al. 2019). Therefore,
as it was indicated by Gunnabo et al. (2019), the selection
of legume genotypes on the basis of their response to dif-
ferent rhizobial inoculants is also an important challenge to
improve the nitrogen fixation (Gunnabo et al. 2019).
It is also necessary to take into account that several a
biotic factors can influence the efficacy of rhizobial inocu-
lants, such as the edapho-climatic conditions of the regions
where they will be applied, since in several of them, the
crops are subjected to diverse type of stresses, such as
drought, pH, temperature and soil salinity. The Salinization
is one of the most crucial factors threatening agricultural
lands worldwide with a predicted impact of 50% of all arable
land by 2050 (Wang et al. 2003; Butcher et al. 2016). The
use of plants has been proposed to alleviate this problem
(Litalien and Zeeb 2020), including some none cultivated
species of genus Phaseolus (Bruning and Rozema 2013;
Abiala et al. 2018). Concerning to the symbionts, several
works have been showed the osmotolerance of Rhizobium
strains from some species (Shamseldin and Werner 2005;
Bouhmouch et al. 2001; Mnasri et al. 2007; Faghire et al.
2012) and commonly they have higher tolerance to salinity
than their hosts (Abiala et al. 2018). For example, in Egypt,
Shamseldin and Werner (2005) isolated R. etli strains able
to grow up to 3% NaCl, however their symbiotic perfor-
mance could only be examined at 0.4% NaCl because the
host plant was unable to grow on higher salt concentrations.
This fact has limited the studies in field conditions, although
some local salt tolerant strains were effective as inoculants
on Moroccoeon soils (Bouhmouch et al. 2001) and Mnasri
et al. (2007) isolated from Tunisian saline soils a salt tolerant
strain with high effectiveness on P. vulgaris recommending
it as a good inoculants for this legume in salt-affected soils.
Consequently, it is necessary to develop breeding or selec-
tion programs to select the best cultivars of P. vulgaris able
to grow in presence of high salinity levels in parallel with the
selection of the best salt tolerant microsymbionts to improve
the symbiotic nitrogen fixation in salt affected ecosystems.
Also, it has been reported that the inoculation with Rhizo-
bium strains improves the drought tolerance and the yield
of P. vulgaris (Aserse et al. 2020) and concerning to the
13
 63 Page 8 of 12
tolerance at high temperatures, although some rhizobia
nodulating P. vulgaris can survive at 45 °C, their symbi-
otic effectiveness is negatively affected at this tempera-
ture (Karanja and Wood 1988). Also, Shamseldin (2007)
reported that rhizobial strains formed very small nodules on
P. vulgaris in experiments which performed at 35 °C indicat-
ing a lower efficiency of these strains at temperatures higher
than the optimal values, which ranged from 28 to 30 °C.
Although most of studies focusing on the isolation and
symbiotic effectiveness of strains with tolerance to dif-
ferent stresses have been performed in African countries,
the genomic basis of this tolerance has been analyzed in
some American strains nodulating P. vulgaris, finding in
their genomes genes linked to several metabolic pathways
involved in salt, pH and heat tolerance (Ormeño-Orrillo
et al. 2012). Nevertheless, it is necessary to expand to other
countries and continents the works focusing on the selection
of strains resistant to different stresses in order to design
inoculants for P. vulgaris suitable for the soils affected by
these stresses considering that the substitution of chemical
fertilizers by biofertilizers is a priority objective to face the
scenario of climatic change worldwide.
Conclusion
This article reviews the diverse rhizobial genera, species and
symbiovars that can originate nodules on Phaseolus vul-
garis L., a legume known as a promiscuous host for nodu-
lation. The data currently available showed that this leg-
ume is mostly nodulated by strains from species and genera
belonging to the alpha-Proteobacteria, such as Rhizobium,
Pararhizobium, Ensifer and Bradyrhizobium and minority
by species from Paraburkholderia and Cupriavidus within
the beta-Proteobacteria. Most of these strains belong to sym-
biovars indigenous to America, such as phaseoli and tropici,
where this legume has its distribution centers. Nevertheless,
due to the promiscuity of this legume, it can establish sym-
biosis with species and symbiovars in different geographi-
cal locations most of them still undescribed. Therefore it
is necessary to carry out further studies about the diversity
and the efficiency of rhizobia nodulating P. vulgaris in dif-
ferent soils worldwide, including those subjected to differ-
ent stresses, in order to select strains with a good perfor-
mance on the plant genotypes cultivated in each country to
can achieve a reduction of chemical nitrogen fertilization in
legume crops, which is priority to mitigate the effects of the
climatic changes.
Acknowledgements This review article was funded under the finan-
cial project STDF1268 from the Academy of Scientific Research and
Technology Applications, Cairo, Egypt. Special thanks should be given
by authors to the reviewers who gave important notes that contributed
significantly to strength our manuscript.
13
World Journal of Microbiology and Biotechnology (2020) 36:63
Author contributions AS and EV are having equal contribution for
writing this article and approving it.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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