Analysis of Rhizobial Strains Nodulating Phaseolus Vulgaris From

Systematic and Applied Microbiology 37 (2014) 149–156

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Systematic and Applied Microbiology

j ournal homepage: www.elsevier.de/syapm

Analysis of rhizobial strains nodulating Phaseolus vulgaris from

Hispaniola Island, a geographic bridge between Meso and South

America and the ﬁrst historical link with Europe

a b,c d

César-Antonio Díaz-Alcántara , Martha-Helena Ramírez-Bahena , Daniel Mulas ,

e b b,c e,∗

Paula García-Fraile , Alicia Gómez-Moriano , Alvaro Peix , Encarna Velázquez , d

Fernando González-Andrés

Facultad de Ciencias Agronómicas y Veterinarias, Universidad Autónoma de Santo Domingo, Dominican Republic

Instituto de Recursos Naturales y Agrobiología, IRNASA (CSIC), Salamanca, Spain

Unidad Asociada Grupo de Interacción Planta-Microorganismo, Universidad de Salamanca-IRNASA (CSIC), Spain

Instituto de Medio Ambiente, Recursos Naturales y Biodiversidad, Universidad de León, Spain

Departamento de Microbiología y Genética, Universidad de Salamanca, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Hispaniola Island was the ﬁrst stopover in the travels of Columbus between America and Spain, and

Received 13 July 2013

played a crucial role in the exchange of Phaseolus vulgaris seeds and their endosymbionts. The analysis of

Received in revised form

recA and atpD genes from strains nodulating this legume in coastal and inner regions of Hispaniola Island

15 September 2013

showed that they were almost identical to those of the American strains CIAT 652, Ch24-10 and CNPAF512,

Accepted 18 September 2013

which were initially named as Rhizobium etli and have been recently reclassiﬁed into Rhizobium phaseoli

after the analysis of their genomes. Therefore, the species R. phaseoli is more abundant in America than

Keywords:

previously thought, and since the proposal of the American origin of R. etli was based on the analysis

Rhizobium

of several strains that are currently known to be R. phaseoli, it can be concluded that both species have

Phaseolus vulgaris

Taxonomy an American origin coevolving with their host in its distribution centres. The analysis of the symbiovar

Phylogeny phaseoli nodC gene alleles carried by different species isolated in American and European countries

Biogeography suggested a Mesoamerican origin of the ␣ allele and an Andean origin of the ␥ allele, which is supported

by the dominance of this latter allele in Europe where mostly Andean cultivars of common beans have

been traditionally cultivated.

Introduction Spain, Portugal and France linked to other chromosomal species

such as Rhizobium leguminosarum, Rhizobium lusitanum, Rhizobium

Phaseolus vulgaris is a legume indigenous to the mountainous gallicum and Rhizobium giardinii [5,11,24,41].

regions of Central America and the Andes that was distributed Most of the studies mentioned focused on strains isolated from

through Spain and Portugal to the rest of Europe after the discov- mainland America and Europe, but no reports are available on

ery of America [14]. Although this legume establishes symbiosis strains nodulating common bean in the American islands located

with different fast-growing rhizobia on mainland America, Rhizo- on the trade routes between these continents. This is the case of

bium etli is its predominant endosymbiont and an American origin Hispaniola Island in the Caribbean Sea, which was the ﬁrst island

has been proposed for this species [1,2,6,7,36,37]. Since it has been discovered by Christopher Columbus on his ﬁrst voyage to America

reported that R. etli is carried on the testa of the bean seeds [25] it and it became an important bridge in the trade routes between

could have been spread with common bean seeds in Europe [15,34]. America and Europe. In this way, P. vulgaris seeds were likely

This fact is supported by several studies given that typical symbi- transported from mainland America to Hispaniola after Columbus’s

otic genes of the American R. etli biovar phaseoli were found in arrival, although it is known that common bean was cultivated on

this island before then [9]. The common bean germplasm culti-

vated on Hispaniola had two different origins, the Mesoamerican

∗ one, with small seeds, and the Andean one, with larger seeds [9].

Corresponding author at: Departamento de Microbiología y Genética, Lab. 209,

After Gepts [13] suggested that the Caribbean was a natural geo-

Ediﬁcio Departamental de Biología, Campus Miguel de Unamuno, 37007 Salamanca,

graphic bridge between the two centres of origin, Duran et al. [9]

Spain. Tel.: +34 923 294532; fax: +34 923 224876.

E-mail address: [email protected] (E. Velázquez). not only found the two types, but also a sort of gradient across the

150 C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156

region related to their prevalence, which was supported by RAPD RAPD ﬁngerprinting

analysis and phaseolin patterns. The small-seed cultivars passed

through the Caribbean islands of Cuba and Hispaniola on their way RAPD patterns were obtained using the primer M13 (5 -

from Mexico and Central America, to the north of South America GAGGGTGGCGGTTCT-3 ), according to Rivas et al. [32], with the

◦

and Brazil [13,14]. In turn, the large-seed cultivars were introduced following PCR conditions: preheating at 95 C for 9 min; 35 cycles

◦ ◦

to Hispaniola Island from the north of South America through the of denaturing at 95 C for 1 min; annealing at 45 C for 1 min and

◦ ◦

“Arawak Arc” of the Leeward Islands and the Great Antilles [9]. extension at 75 C for 2 min, and a ﬁnal extension at 72 C for 7 min.

Since antiquity the main port in the trade routes between

America and Europe within Hispaniola Island was located on the Sequence analysis of rrs, recA, atpD and nodC genes

Caribbean coast of the oriental part of the island, where at the end

of the 15th century the city of Santo Domingo de Guzmán was The ampliﬁcation and sequencing of the rrs gene was performed

founded, which is now the capital city of the Dominican Republic. according to Rivas et al. [30], whereas recA and atpD genes were

This country occupies 60% of the territory of Hispaniola Island. according to Gaunt et al. [12] and the nodC gene as described

Therefore, the port of Santo Domingo played a crucial role in the by Laguerre et al. [19]. The sequences obtained were compared

distribution of common bean seeds to Spain after the ﬁrst Colum- with those from GenBank using the BLASTN programme [3], and

bus voyage. As the large grains were preferred to the small ones were aligned using the Clustal W software [40]. The distances were

for human consumption, the large-seeded cultivars progressively calculated according to Kimura’s two-parameter model [18]. Phy-

replaced the small-seeded ones in the region near Santo Domingo, logenetic trees were inferred using the neighbour-joining method

although the latter remained longer in the inner and isolated moun- [35]. Bootstrap analysis was based on 1000 resamplings. The MEGA

tainous regions of the island [9], distant from the commercial trade 5 package [39] was used for all analyses.

routes. As a consequence, the Andean germplasm was mostly trans-

ported to Spain and Portugal and from here to the rest of Europe Results and discussion

and Africa, and for this reason the Andean germplasm is predomi-

nant in these areas [14]. Therefore, Hispaniola played an essential Isolation of rhizobia and nodulation tests

role in the exchange of common bean germplasm between Amer-

ica and Europe, and the analysis of rhizobia nodulating this legume Twenty ﬁve isolates were obtained from nodules that pre-

on the island is therefore particularly interesting for biographical sented the typical morphology of effective common bean nodules

studies. (pink internal colour due to leghaemoglobin). All strains were able

In this study, effective rhizobial strains nodulating common to induce effective nodules in P. vulgaris. The inoculated plants

bean were isolated in the Santo Domingo region near the coast, had shoot weights ranging from 475 mg/plant to 932 mg/plant

as well as from inner mountainous regions of Hispaniola Island in and plant nitrogen contents ranging from 11 to 37 mg/plant.

the Dominican Republic zone in order to analyze their phyloge- The uninoculated controls (without added nitrogen) had aver-

netic relationships with rhizobia isolated from the American and age shoot weights of 468 mg/plant and contained 8 mg/plant of

European countries linked by trade routes since the discovery of nitrogen.

America. Several strains of Rhizobium phaseoli carrying identical

core genes to those isolated in mainland America and novel Rhi- RAPD ﬁngerprinting

zobium chromosomal lineages not previously reported in America

or Europe to date were isolated on Hispaniola Island. This ﬁnd- RAPD ﬁngerprinting allows differentiation between strains of

ing, together with the fact that several American strains initially rhizobial species nodulating common bean and provides an esti-

named R. etli have been reclassiﬁed into R. phaseoli, showed that mation of their genetic diversity [24,41,42]. In our case, the strains

this species is as abundant in America as R. etli. Moreover, all strains presented 14 RAPD proﬁles, which indicated high genetic variabil-

from symbiovar phaseoli carried one of the two American nodC ity even in isolates originating from the same zone (Fig. 1 and

gene alleles, indicating a common phylogenetic origin of this gene Table 1). A representative strain from each RAPD proﬁle was sub-

in strains nodulating P. vulgaris on mainland America, Hispaniola jected to gene sequence analyses.

Island and Europe.

Analysis of chromosomal genes

The rrs gene analysis showed that strains from RAPD groups

Materials and methods

II, VI, VII, VIII, IX, X and XI had identical sequences and therefore

only a representative strain, VPA1100, was included in the phy-

Isolation of rhizobial strains and nodulation tests

logenetic tree (Fig. S1). This strain clustered with the type strain

of the species R. phaseoli ATCC 14482 , whereas the remaining

Rhizobial strains (Table 1) were isolated according to Vin-

strains were located in different branches of the tree related to R.

cent [43] on YMA medium, from effective nodules of P. vulgaris T T

etli CFN42 or R. vallis CCBAU 65647 (Fig. S1). Nevertheless, sev-

var. “José Beta” plants grown in four provinces of the Dominican

eral species of the genus Rhizobium are not easily distinguishable

Republic in the oriental part of Hispaniola Island, in which this

through rrs gene analysis, as occurs with some species nodulat-

local variety is commonly cultivated (Table 1). In each ﬁeld, 15

ing P. vulgaris (Fig. S1). To distinguish these species the analysis

plants at least 1 m apart were randomly taken, and the most effec-

of the housekeeping recA and atpD genes is much more reliable

tive nodules (pink-red coloured) were selected from each plant

[27,41]. In fast growing rhizobia nodulating P. vulgaris, the species

for rhizobial isolation. The sampling was carried out in two dif-

delineation limit for housekeeping gene identities has been estab-

ferent agrosystems. The ﬁrst was a lowland area near sea level

lished as 95–96% when compared with DNA-DNA hybridization

represented by Santo Domingo Province, and the second ones

data [27,41].

were isolated highlands areas, represented by the rest of the

The analysis of recA and atpD genes (Figs. 2 and 3) showed that

sampling sites (Table 1). The nodulation experiments were car-

Dominican strains occupied different branches within the genus

ried out on P. vulgaris var. “José Beta”, as previously described

Rhizobium. Strains showing the proﬁles II, VI, VII, VIII, IX, X and

[24].

XI, represented by strains VPA1100, OLQ1051, OLQ1052, VIAD5,

C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156 151

Table 1

Characteristics of strains isolated in different geographic locations from Dominican Republic at Hispaniola Island.

Strains Isolation site RAPD pattern nodC allele Species

ESC1020 Sabana Chen, Hondo Valle; Elías Pina˜ I ␥ Rhizobium sp.

ESC1060 Sabana Chen, Hondo Valle; Elías Pina˜ II ␥ Rhizobium phaseoli

ESC1110 Sabana Chen, Hondo Valle; Elías Pina˜ III ␥ Rhizobium sp.

␥

ESC1120 Sabana Chen, Hondo Valle; Elías Pina˜ III Rhizobium sp.

␥

ESC1129 Sabana Chen, Hondo Valle; Elías Pina˜ III Rhizobium sp.

OAC1020 Arroyo Cana;˜ San José de Ocoa IV ␣ Rhizobium sp.

OAC1031 Arroyo Cana;˜ San José de Ocoa IV ␣ Rhizobium sp.

OAC1140 Arroyo Cana;˜ San José de Ocoa IV ␣ Rhizobium sp.

OAC1150 Arroyo Cana;˜ San José de Ocoa V ␥ Rhizobium sp.

OLQ1051 Los Quemaos; San José de Ocoa VI ␣ Rhizobium phaseoli

OLQ1052 Los Quemaos; San José de Ocoa VII ␥ Rhizobium phaseoli

␣

VIAD3 Santo Domingo VIII Rhizobium phaseoli

VIAD5 Santo Domingo VIII ␣ Rhizobium phaseoli

␣

VIAD8G Santo Domingo IX Rhizobium phaseoli

VIAD10 Santo Domingo IX ␣ Rhizobium sp.

VIAD11 Santo Domingo X ␣ Rhizobium phaseoli

VIAD12G Santo Domingo XI ␣ Rhizobium phaseoli

VIAD13G Santo Domingo XII ␥ Rhizobium sp.

VIAD14G Santo Domingo XI ␣ Rhizobium phaseoli

VCS3041 Canada˜ Seca, Constanza; la Vega IV ␣ Rhizobium sp.

VLP1091 Las Palmas, Constanza; la Vega XIII ␣ Rhizobium sp.

VPA1010 Palero, Constanza; la Vega II ␥ Rhizobium phaseoli

VPA1100 Palero, Constanza; la Vega II ␥ Rhizobium phaseoli

VRI2100 El Río, Constanza, la Vega IV ␣ Rhizobium sp.

VRI2090 El Río, Constanza, la Vega XIV ␣ Rhizobium sp.

VIAD8G, VIAD11 and VIAD12G, respectively, were unambiguously initially named as R. etli but have recently been included in the

identiﬁed as R. phaseoli because they had recA and atpD gene species R. phaseoli [20].

sequence identities higher than 99.9% with respect to R. phaseoli Strain ESC1110, representing RAPD proﬁle III, had identity val-

ATCC 14482 . Other P. vulgaris-nodulating strains isolated in Amer- ues for the recA and atpD genes of lower than 95% with respect

ica, such as the Mexican ch24-10, the Colombian CIAT 652 and the to the remaining strains isolated in this study and to the cur-

Brazilian CNPAF 512, were also located in this cluster with almost rently described species of Rhizobium. The strain representing RAPD

100% identity for both recA and atpD genes. These strains were pattern XII, VIAD13G, was related to R. etli in the recA phylogenetic

Fig. 1. RAPD patterns of strains isolated in this study: ESC1020 (lane 1), ESC1060 (lane 2), ESC1110 (lane 3), ESC1120 (lane 4), ESC1129 (lane 5), OAC1020 (lane 6), OAC1031

(lane 7), OAC1140 (lane 8), OAC1150 (lane 9), OLQ1051 (lane 10), OLQ1052 (lane 11), VIAD3 (lane 12), VIAD5 (lane 13), VIAD8G (lane 14), VIAD10 (lane 15), VIAD11 (lane

16), VIAD12G (lane 17), VIAD13G (lane 18), VIAD14G (lane 19), VCS3041 (lane 20), VLP1091 (lane 21), VPA1010 (lane 22), VPA1100 (lane 23), VRI2100 (lane 24) and VRI2090

(lane 25). The different RAPD groups are indicated by Roman numerals.

152 C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156

Rh izobium etli IE4804 (AY907457)

65 Rh izobium etli IE4876 (AY907455)

94 Rh izobium etli IE2730 (AY907456)

Rh izobium etli IE4810 (AY907453)

83 Rh izobium etli IE4837 (AY907463)

Rh izobium etli IE2704 (AY907461)

66 Rhizobium etli KIM5s (AY907360)

63 Rh izobium etli IE4794 (AY907454)

Rh izobium etli GR12 (AY907361)

97 95 Rh izobium etli GR56 (NZ ABRB01002262)

Rh izobium etli CNPSO679 (JN129355)

65 Rh izobium etli (formerly Rhizobium etli) CNPAF512 (AEYZ01000264)

Rhizob ium phaseoli OLQ1052 (KF638391)

Rhizob ium phaseoli OLQ1051 (KF638390)

100 Rhizob ium phaseoli (formerly Rhizobium etli) Ch24-10 (NZ_AHJU01000019)

Rhizob ium phaseoli ATCC 14482T (EF113136)

89 Rhizob ium phaseoli (formerly Rhizobium etli) CIAT 652 (NC_010994)

Rhizob ium phaseoli VPA1100 (KF638385)

66 Rhizob ium phaseoli VIAD5 (KF638393)

Rhizob ium phaseoli VIAD8G (KF638394)

Rhizob ium phaseoli VIAD12G (KF638395)

Rhizob ium phaseoli VIAD11 (KF638396)

Rhiz obium sp. ESC1110 (KF638397)

100 Rhizob ium sp. BLR160 (JN649055)

72 Rhiz obium sp. VLP1091 (KF638386)

100

Rhizobium sp. VRI2090 (KF638389)

Rhiz obium sp. OAC1150 (KF638388)

Rhiz obium sp. ESC1020 (KF638398)

Rhiz obium sp. OAC1020 (KF638387)

Rh izobium vallis CCBAU 65647T (GU211770)

85 Rhiz obium sp. VIAD13G (KF638392)

99 100 Rhiz obium sp. BLR62 (JN649042)

Rh izobium etli CFN42T (NC 007761)

Rh izobium etli CNPSO666 (JN129350)

99 Rhizob ium sp. RPVR24 (GQ863533)

100 Rhizob ium sp. LBM1123 (JF792209)

Rhizob ium sp. RPVN06 (GQ863531)

Rhizob ium sp. RPVR32 (GQ863534)

92 Rhizob ium sp. RPVR04 (GQ863532)

64 Rhizob ium fabae CCBAU 33202T (EF579941)

97 Rhizob ium pisi DSM 30132T (EF113134)

Rhizob ium indigoferae CCBAU71042T (EF027965)

Rhizob ium leguminosarum USDA 2370T (AJ294376)

100 Rhizob ium leguminosarum RPVF18 (GQ863530)

53 64 Rhizob ium leguminosarum LCS0313 (JF792208)

Rhizob ium leguminosarum BAPVPT02 (GQ863529)

Rhizob ium oryzae Alt505T (FJ712274)

Rhiz obium tibeticum LMG 24453T (HQ735075)

Rhiz obium mesoamericanum CCGE 501T (JF424620)

83 97 Rhizobium grahamii CCGE 502 (JF424622)

97 Rhizob ium gallicum R602spT (AY907357)

Rhizob ium gallicum PhD12 (HQ394250)

99 Rhizob ium yanglingense SH22623T (AY907359)

Rhizob ium loessense LMG21975T (HQ735076)

Rhizob ium mongolense USDA 1844T (AY907358)

100 Rhizob ium alkalisoli CCBAU01393T (EU672490)

Rhizob ium lusitanum P1-7T (DQ431674)

Rhiz obium lusitanum p3-13 (DQ431675)

Rhizob ium giardinii H152T (EU488819)

Rhizob ium leucaenae USDA 9039T (AJ294372)

Rhizob ium multihospitium CCBAU 838401T (EF490029)

Rhizob ium rhizogenes NCPPB 2991T (AJ294374)

Rh izobium freirei PRF 81T (EU488827)

Rhizob ium tropici USDA9030T (AJ294373)

Rhizob ium miluonense CCBAU 41251T (HM047131)

0.01

Fig. 2. Neighbour-joining phylogenetic tree based on partial recA gene sequences showing the position of representative strains from each RAPD group. Bootstrap values

calculated for 1000 replications are indicated. Bar, 2 nt substitutions per 100 nt. Accession numbers from GenBank are given in brackets.

tree and had 97% identity with respect to strain CFN42 , how- that it did not belong to the same species, since the recA gene

ever, the atpD genes of both strains were divergent, and showed had an identity of lower than 92%. The strains with proﬁles V, XIII

less than 94% identity. The two mentioned strains, ESC1110 and and XIV, represented by strains OAC1150, VLP1091 and VRI2090,

VIAD13G, were related to strains BLR160 and BRL62 nodulating respectively, were closely related and probably belong to the same

Vicia in Asia [28], and could represent undescribed species of Rhizo- species, since they had more than 98% identity in both recA and

bium, although in the latter case of VIAD13G, the identity of the recA atpD genes (Fig. 2). These strains were phylogenetically divergent

gene with R. etli CFN42 was at the limit of species differentiation. to all described species from the genus Rhizobium (Fig. 2) and

The remaining Rhizobium strains isolated from the Dominican therefore they probably belong to an undescribed species from this

Republic were not identiﬁed as described species of this genus genus.

according to the recA and atpD gene sequence analyses. Moreover, These results showed that, in addition to R. phaseoli, several

these strains were not related to any rhizobial strains whose recA putative new species in the genus Rhizobium were endosymbionts

and atpD genes are currently available in the public databases. of P. vulgaris from Hispaniola Island and conﬁrmed the greater use-

ESC1020, representing RAPD proﬁle I, occupied a phylogenetically fulness of housekeeping genes compared to the rrs gene for species

divergent branch with identities lower than 95% for the recA and delineation within the genus.

atpD genes, respectively, with respect to the remaining Dominican

strains (Figs. 2 and 3), as well as to the current species of Rhizobium Analysis of the nodC gene

(Figs. 2 and 3). Strain OAC1020, displaying RAPD type IV, formed

an independent branch related to the previous strain in the case The nodC gene has been widely analyzed in rhizobia, and is

of the atpD gene, but an identity of lower than 96% suggested related to their host range and the degree of host promiscuity

C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156 153

89 T (OAC1150, ESC1020, ESC1110 and VIAD13G) that belonged to chro-

96 Rhizobium gallicum R602sp (AY907371)

Rhizob ium gallicum PhD12 (AY929482)

mosomal lineages divergent to R. phaseoli had the ␥ allele. However,

Rhizobium yanglingense SH22623T (AY907373)

55 Rhizobium mongolense USDA 1844 (AY907372) two strains, VLP1091 and VRI2090, isolated at two sites from the

68 68 Rhizob ium loessense CCBAU 7190BT (HM142761)

inner La Vega region (Table 1) carried a slightly divergent nodC gene

Rhizobium mesoamericanum CCGE 501T (JF424611)

Rhizobium tibeticum LMG 24453 (HQ735093) phylogenetically related to the ␣ allele (Fig. 4), suggesting a recent

Rhizob ium alkalisoli 01393T (EU672461)

diversiﬁcation of this gene in some local ecosystems, since this vari-

64 Rhizobium grahamii CCGE 502T (JF424612)

Rhizobium lusitanum P1-7 (DQ431671) ation has not been found in the remaining strains analyzed to date

100 Rhizob ium lusitanum p3-13 (DQ431672)

Rhizob ium leucaenae USDA 9039T (AJ294396) from different continents.

54 Rh izobium freirei PRF 81T (NZ AQHN01000005)

Rhizob ium miluonense CCBAU 41251T (HM047116)

T Taxonomic, evolutionary and biogeography remarks

52 Rhizobium multihospitium CCBAU 83401 (EF490019)

Rhizobium tropici USDA 9030T (AJ294397)

Rhizob ium giardinii H152T (HQ394216)

Rhizobium sp. ESC1110 (KF638364) For several decades, the species R. etli was considered to be the

Rhizob ium sp. BLR160 (JN648965)

Rhiz obium sp. VIAD13G (KF638365) major endosymbiont of P. vulgaris in the distribution centres of

100 Rhizobium sp. BLR62 (JN648952) common bean in America [1,2,6,36,37]. However, the taxonomy

Rhiz obium sp. OAC1020 (KF638359)

Rhiz obium sp. OAC1150 (KF638360) of this species was not clearly established, mainly because rhizo-

70 Rhiz obium sp. VLP1091 (KF638358)

97 biologists considered R. phaseoli as an invalid species after Jordan’s

51 Rhiz obium sp. VRI2090 (KF638361)

Rh izobium etli CFN42T (NC_007761) reclassiﬁcation [17]. At the beginning of rhizobiology, this species

Rhiz obium sp. ESC1020 (KF638370)

was deﬁned as an endosymbiont of P. vulgaris and was included in

Rhizob ium phaseoli OLQ1051 (KF638362)

67 Rhizobium phaseoli (formerly Rhizobium etli) CNPAF512 (AEYZ01000362) the validation list of Skerman et al. [38]. The discovery of the exist-

Rhizob ium phaseoli OLQ1052 (KF638363)

ence of interchangeable symbiotic plasmids in some Rhizobium

99 Rhizob ium phaseoli VIAD5 (KF638366)

T species led Jordan [17] to reclassify the species R. phaseoli and R. tri-

79 Rhizobium phaseoli ATCC 14482 (EF113151)

Rhizob ium phaseoli (formerly Rhizobium etli) CIAT 652 (NC_010994)

folii as R. leguminosarum. From this date, in the description of new

Rhizob ium phaseoli VIAD8G (KF638367)

64 Rhizobium phaseoli VIAD12G (KF638368) rhizobial species, such as R. etli [36], R. phaseoli was not considered,

Rhizob ium phaseoli VIAD11 (KF638369)

Rhizob ium phaseoli VPA1100 (KF638357) although Jordan’s reclassiﬁcation was never ofﬁcially validated. As

Rhizobium phaseoli (formerly Rhizobium etli) Ch24-10 (NZ_AHJU01000173) a consequence, many strains isolated from P. vulgaris in America

100 Rhizob ium fabae CCBAU 33202T (EF579929)

Rhizob ium pisi DSM 30132T (EF113149) were classiﬁed as R. etli and the American origin of this species was

100

Rhizobium sp. RPVR24 (GQ863522) proposed [36]. However, in 2008, the validity of the old species R.

Rhizobium sp. LBM1123 (JF792200)

Rhizobium sp. RPVN06 (GQ863520) phaseoli was recognized again, since it could be differentiated from

71 Rhizob ium sp. RPVR32 (GQ863523)

R. etli on the basis of housekeeping gene analysis [27] and, recently,

Rhizob ium sp. RPVR04 (GQ863521)

98 Rhizobium vallis CCBAU 65647T (GU211768) the American P. vulgaris-nodulating strains CIAT 652, CNPAF 512

53 Rh izobium etli GR12 (AY907375)

and ch24-10, whose complete genomes are available, have been

61 Rhizob ium leguminosarum USDA 2370T (AJ294405)

Rhizobium leguminosarum RPVF18 (GQ863519) reported to be R. phaseoli rather than R. etli [20]. Also, some strains

100 Rhizob ium indigoferae CCBAU71042T (GU552925)

isolated in Mexico (IE6823, IE6862 and IE6760) and classiﬁed as

88 Rhizobium leguminosarum LCS0313 (JF792195)

96Rhizobium leguminosarum BAPVPT02 (GQ863518) R. etli clearly belong to the species R. phaseoli since they have been

Rhizob ium oryzae Alt505T (FJ712275) T

Rh izobium etli KIM5s (AY907374) shown to cluster with strain CIAT 652 and not with strain CFN42

88 Rhizobium etli IE4794 (AY907435) in the study of Lozano et al. [21]. Strain CIAT 151, which is used as

100 Rh izobium etli IE2737 (AY907439)

Rhizobium etli IE2704 (AY907442) an inoculant for common bean, and strain IPR-Pv589 [8] clustered

Rh izobium etli IE2730 (AY907436)

88 with R. phaseoli instead of R. etli (Fig. S1). On the other hand, some

Rh izobium etli IE4804 (AY907437)

Rhizobium etli IE4837 (AY907444) strains named R. etli isolated from America, such as IE2737 and

IE4804 [2,21,37], IE4837 [21], IE2704, IE2730, IE4794, IE4876 and

0.01

IE4810 [37], CNPSO679 and CNPSO666 [29] from Mexico and KIM5s

Fig. 3. Neighbour-joining phylogenetic tree based on partial atpD gene sequences from Idaho (USA) [37], as well as some Spanish strains initially

showing the position of representative strains from each RAPD group. Bootstrap named as R. etli [15,34], such as G12 and GR56 [15], do not belong

values calculated for 1000 replications are indicated. Bar, 1 nt substitution per 100 nt.

to this species or to R. phaseoli (Figs. 2 and 3). The same occurred

Accession numbers from GenBank are given in brackets.

with several Spanish strains initially named as R. etli [15,34] that

did not cluster with the type strain of R. etli CFN42 in the study of

[4,10,11,16,19,23,26,31,33]. P. vulgaris is a promiscuous host [22] Lozano et al. [21]. This means that the taxonomic position of strains

that is nodulated by Rhizobium strains carrying very divergent nodC named as R. etli isolated to date should be revised, particularly

and it belongs to different symbiovars [11,19,42]. Amongst these, when it has only been based on partial sequences of the 16S rRNA

the biovar phaseoli is widely distributed in the world and two dif- gene.

␣ ␥

ferent nodC alleles named and have been found in American However, based on the available data, it can be concluded that

distribution centres [2]. The phylogenetic analysis of the nodC gene R. phaseoli is also very abundant in American countries, and it was

showed that the two types of alleles were clearly separated (about found in both coastal and mountainous regions of Hispaniola Island.

97% identity), and proved that strains from the same species, such as All these strains belonged to the symbiovar phaseoli according to

R. phaseoli ATCC 14482 and CIAT 652, carry different nodC alleles, the nodC gene analysis, and this gene seems to be exclusive for

␣

␥

and , respectively. Also, strains from different species carried common bean nodulation [5,42]. Therefore, the plasmids carried by

the same nodC allele, as occurs with the type strains of R. phaseoli the symbiovar phaseoli should have evolved with native common

T T

ATCC 14482 and R. etli CFN42 . bean plants in America [2] and R. etli sv. phaseoli is considered to

All Dominican strains belonged to the biovar phaseoli and were be the predominant endosymbiont of wild and cultivated common

␣ ␥

distributed in the groups corresponding to and nodC alleles, bean in different American countries, such as Mexico, Argentina,

␣

although the allele was slightly more abundant (Fig. 4 and Ecuador and USA [1,2,6,7,36,37]. In accordance with these results,

Table 1). Most strains identiﬁed as R. phaseoli (OLQ1051, VIAD5, coevolution of R. etli with P. vulgaris has been proposed in centres

␣

VIAD8G, VIAD11 and VIAD12G) had the allele that was carried of host diversiﬁcation [2]. Nevertheless, this proposal was based

by the type strain of this species (ATCC 14482 ), as well as by on some strains that have been reclassiﬁed recently as R. phaseoli

the type strain of R. etli CFN42 , and only two strains, VPA1100 and, therefore, we propose that this species also coevolved with P.

␥

and OLQ1052, showed the allele. Most of the remaining strains vulgaris in its diversiﬁcation centres and was disseminated in other

154 C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156

Rhizob ium giardinii sv. phaseoli H251 (AF217264) France

Rhizobium phaseoli sv. phaseoli VPA1100 (KF638371) Dominican Republic

Rhizob ium leguminosarum sv. phaseoli H132 (AF217263) France

Rhizob ium sp. sv. phaseoli RPVR32 (GQ863545) North Spain

Rh izobium etli sv. phaseoli CNPAF512 (AEYZ01000361) Brazil

Rhiz obium sp. sv. phaseoli ESC1110 (KF638373) Dominican Republic

Rhiz obium sp. sv. phaseoli OAC1150 (KF638375) Dominican Republic

Rhiz obium sp. sv. phaseoli VIAD13G (KF638377) Dominican Republic

99 Rhiz obium sp. sv. phaseoli ESC1020 (KF638382) Dominican Republic allele Rhizobium phaseoli sv. phaseoli OLQ1052 (KF638384)Dominican Republic

Rhizobium etli sv. phaseoli CIAT652 (NC_010996) Colombia

Rh izobium etli sv. phaseoli IE6823 (GU084774) Mexico Rhizobium lusitanum P3-13 (HM852099) Portugal Rhizobium etli sv. phaseoli GR10 (GU084788) South Spain

Rhizobium etli sv. phaseoli 21PR-1 (GU084781) South Spain

Rhizob ium leguminosarum sv. phaseoli BAPVPT02 (GQ863540) Central Spain

100 Rh izobium etli sv. phaseoli GR56 (GU084770) South Spain

Rhizob ium leguminosarum sv. phaseoli LBM1123 (JF792200) North Spain

Rhizob ium gallicum sv. phaseoli PhD12 (AF217265) France

96 Rhizobium sp. sv. phaseoli VLP1091 (KF638372) Dominican Republic

Rhiz obium sp. sv. phaseoli VRI2090 (KF638376) Dominican Republic

Rhizobium sp. sv. phaseoli RPVR04 (GQ863543) North Spain

Rh izobium etli sv. phaseoli CFN42T (NC_004041) Mexico

Rhizob ium phaseoli sv. phaseoli ATCC 14482T (HM441255) USA

Rhiz obium sp. sv. phaseoli OAC1020 (KF638374) Dominican Republic

92 Rhizobium phaseoli sv. phaseoli VIAD5 (KF638378)Dominican Republic

Rhizob ium phaseoli sv. phaseoli VIAD8G (KF638379) Dominican Republic

Rhizob ium phaseoli sv. phaseoli VIAD12G (KF638380) Dominican Republic

allele

Rhizob ium phaseoli sv. phaseoli VIAD11 (KF638381) Dominican Republic

87 Rhizob ium phaseoli sv. phaseoli OLQ1051 (KF638383) Dominican Republic

Rhizobium etli sv. phaseoli Ch24-10 (NZ_AHJU01000233) Mexico

Rh izobium etli sv. phaseoli IE2730 (GU084771) Mexico

Rh izobium etli sv. phaseoli GR14 (GU084756) South Spain

92 Rh izobium etli sv. phaseoli 21NJ-2 (GU084792) South Spain

Rh izobium etli sv. phaseoli IE4810 (GU084778) Mexico

Rh izobium etli sv. phaseoli IE4837 (GU084764) Mexico

Rhizob ium leguminosarum sv. phaseoli LCS0313 (JF792202) North Spain

En sifer fredii sv. mediterranense 16b1 (AF481764)

98 En sifer meliloti sv. mediterranense GVPV12 (FJ462796)

93 En sifer fredii sv. mediterranense GR-06 (AF217269)

Rhizob ium gallicum sv. gallicum R602spT (AF217266)

94 Rhizob ium giardinii sv. giardinii H152T (AF217267)

Rhizob ium yanglingense CCBAU 71623T (JQ308167)

100 Rhizob ium mongolense USDA1844T (GQ507367)

100 Rhizob ium leguminosarum sv. trifolii USDA 2071 (AF217271)

100

Rhizob ium leguminosarum sv. trifolii ATCC14480 (FJ895269)

78 Rhizob ium leguminosarum sv. trifolii WSM1325 (CP001623)

Rhizob ium leguminosarum sv. trifolii WSM2304 (CP001192)

99 Rhizob ium leguminosarum sv. viciae 248 (Y00548)

Rhizob ium leguminosarum sv. viciae 3841 (NC_008381)

100 Rhizob ium sp. sv. viciae BLR160 (JN649009)

67 Rhizobium pisi DSM 30132 (HQ394248)

60 Rhizob ium leguminosarum sv. viciae USDA 2370T (FJ596038)

Rhizob ium grahamii CCGE 502T (JN021932)

100 Rhiz obium mesoamericanum CCGE 501T (JN021931)

Rhizob ium leucaenae CFN299T (N98514)

Rh izobium freirei PRF 81T (NZ AQHN01000086)

100

Rhizobium lusitanum p1-7T (HM852098)

69 Rhizob ium tropici CIAT 899T (HQ824719)

0.02

Fig. 4. Neighbour-joining phylogenetic tree based on partial nodC gene sequences showing the position of representative strains from each RAPD group. Bootstrap values

calculated for 1000 replications are indicated. Bar, 2 nt substitutions per 100 nt. Accession numbers from GenBank are given in brackets.

geographical sites with common bean seeds. This can explain the genes by horizontal transfer from the mainland America strains,

presence of R. phaseoli on Hispaniola Island that was an impor- since they carry the typical nodC gene alleles (␣ and ␥) of these

tant trade bridge for bean seeds and their endosymbionts between strains.

the countries of North and South America. In this way, R. phaseoli The proportion of the ␣ and ␥ nodC alleles in strains isolated

was found not only in Santo Domingo, nearest to South America, from Hispaniola Island was similar to that found in mainland Amer-

but also in inland regions of Hispaniola on the central border with ican countries, although with a slight dominance of the ␣ allele [2]

Haiti, which could have received bean seeds from North American that is characteristic of the type strains of R. phaseoli and R. etli, and

mainland countries. From the new chromosomal lineages found on the Mexican strains ch24-10, IE4810 and IE2730 (Fig. 4). The cur-

Hispaniola Island, two of them, represented by strains ESC1110 and rently available data showed a clear prevalence of the nodC ␣ allele

VIAD13G, have been found recently in Asia in nodules of Vicia [28] in the strains from North America, including the type strains of R.

T T

and the remaining ones, represented by strains OAC1020, OAC1150, phaseoli ATCC 14482 (isolated in USA) and R. etli CFN42 (isolated

VLP1091 and VRI2090, have not been found to date in any other in Mexico), as well as many strains isolated from P. vulgaris nodules

countries and they do not cluster with the American species R. etli in different geographical locations in Mexico [2,21,29]. Therefore,

and R. phaseoli or with the strains isolated in Mexico by Silva et al. these ﬁndings suggested that this allele could have originated in

[37]. These strains were isolated from remote locations of Hispan- North America and was distributed via Caribbean islands to South

iola and it is possible that they were present on this island prior to America with the small-seeded Mesoamerican cultivars of common

the arrival of common bean seeds, and they acquired the symbiotic bean. This proposal agrees with the results of Aguilar et al. [2] who

C.-A. Díaz-Alcántara et al. / Systematic and Applied Microbiology 37 (2014) 149–156 155

found that Mesoamerican cultivars of common beans are mostly [5] Amarger, N., Macheret, V., Laguerre, G. (1997) Rhizobium gallicum sp. nov. and

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␥

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Characteristics of rhizobia nodulating beans in the central region of Minnesota.

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Brazil. Therefore, strains carrying the allele could have been dis- Can. J. Microbiol. 50, 1023–1031.

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M.H., Velázquez, E., Peix, A. (2012) Identiﬁcation at the species and symbiovar

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and A/030020/10 to C-AD-A and FG-A, a MEC project to EVP and by

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the Castilla y León Regional Government to AP.

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