Definition and evolution of a new symbiovar, sv. rigiduloides, among Ensifer meliloti efficiently nodulating Medicago species

Systematic and Applied Microbiology 36 (2013) 490–496

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Definition and evolution of a new symbiovar, sv. rigiduloides, among Ensifer meliloti efficiently nodulating Medicago species Cécile Gubry-Rangin a,∗ , Gilles Béna b,c , Jean-Claude Cleyet-Marel d , Brigitte Brunel e a

Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen AB24 3UU, United Kingdom IRD, LSTM, UMR 113 IRD SupAgro CIRAD UM2, Montpellier, France LMBM/LMI LMBV, University Mohammed V Agdal, Rabat, Morocco d INRA, USC1242, Laboratoire des Symbioses Tropicales et Méditerranéennes IRD/INRA/CIRAD/Montpellier SupAgro/Université Montpellier II, TA A-82/J, F-34398 Montpellier Cedex 5, France e Montpellier SupAgro, Laboratoire des Symbioses Tropicales et Méditerranéennes, IRD/INRA/CIRAD/Montpellier SupAgro/Université Montpellier II, TA A-82/J, F-34398 Montpellier Cedex 5, France b c

a r t i c l e

i n f o

Article history: Received 16 April 2013 Received in revised form 4 June 2013 Accepted 4 June 2013 Keywords: Species Ecotype Biovar Genetic diversity Symbiosis Nitrogen fixation Nodulation genes

a b s t r a c t Understanding functional diversity is one of the main goals of microbial ecology, and definition of new bacterial ecotypes contributes significantly to this objective. Nitrogen-fixing bacteria provide a good system for investigation of ecotypes/biovars/symbiovars, as they present different specific associations with several host plants. This specific symbiosis is reflected both in the nodulation and fixation efficiency and in genetic characters of the bacteria, and several biovars have already been described in the bacterial species Ensifer meliloti. In the present study, the species affiliation of E. meliloti strains trapped from nodules sampled from Medicago rigiduloïdes roots was analyzed using housekeeping recA genes and DNA–DNA hybridization. The genetic diversity of these isolates was also investigated using several symbiotic markers: nodulation (nodA, nodB, nodC) and nitrogen fixation (nifH) genes, as well as the performance of phenotypic tests of nodulation capacity and nitrogen fixation efficiency. These analyses led to the proposal of a new bacterial symbiovar, E. meliloti sv. rigiduloides, that fixed nitrogen efficiently on M. rigiduloïdes, but not on Medicago truncatula. Using phylogenetic reconstructions, including the different described symbiovars, several hypotheses of lateral gene transfer and gene loss are proposed to explain the emergence of symbiovars within this species. The widespread geographical distribution of this symbiovar around the Mediterranean Basin, in contrast to restriction of M. rigiduloïdes to Eastern European countries, suggests that these isolates might also be associated with other plant species. The description of a new symbiovar within E. meliloti confirms the need for accurate bacterial ecological classification, especially for analysis of bacterial populations. © 2013 Elsevier GmbH. All rights reserved.

Introduction The huge diversity of microorganisms within a specific environment has been extensively reported in different ecosystems [31] and ecotype delineation is now a well-accepted concept. Indeed, the majority of microbial studies identify ecologically distinct populations in order to understand better the repartition, interaction and evolution of the microorganisms of interest. Several microorganisms have been subjected to more detailed study because of their critical importance, especially in relation to pathogenesis in humans (e.g. Legionella pneumophila [8]), or plants (e.g. Pseudomonas syringae and Xanthomonas strains [24]). In addition, the ecotype definition for free-living terrestrial microorganisms is

∗ Corresponding author. Tel.: +44 1224272700. E-mail address: [email protected] (C. Gubry-Rangin). 0723-2020/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.syapm.2013.06.004

often based on the intrinsic properties of associated geographical sites, such as the solar insulation for delineation of Bacillus ecotypes [18]. Host specificity in the legume-nitrogen fixing bacteria interaction has been a focus of interest for ecotype delineation, and several biovars (biological variants) have been described in different rhizobia based on differences in symbiotic specificity within the same species. Such characterizations have been made, particularly, in highly cultivated legumes, such as Phaseolus vulgaris [25] or in plant–bacteria model systems, such as Medicago–Sinorhizobium (reviewed in [32]). The genus Medicago comprises approximately 80 species, 25% of which are perennial. Alfalfa (M. sativa) and several annual species (e.g. barrel medic Medicago truncatula or burr medic Medicago polymorpha) are a biological source of nitrogen that gives them economic significance in cultivation for forage or pasture, as well as environmental value in non-managed ecosystems. Medicago is

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mainly associated with Ensifer meliloti and Ensifer medicae [33] and the rhizobium-Medicago association is highly specific [5,6]. Regarding the symbiotic specificity of E. meliloti strains, several symbiovars have been described in this species, either associated with medic species (E. meliloti sv. meliloti [37], sv. medicaginis [37]) or with other host plants, such as Acacia tortilis (sv. acaciae [1]), Cicer arietinum (sv. ciceri [23]), Phaseolus vulgaris (sv. mediterranense [26]) and Lotus sp. (sv. lancerrotense [21]). The adaptation of annual Medicago to the dry environments of countries with a Mediterranean climate [14] is of interest, and the presence of adequate rhizobia is essential for the success of their growth. M. rigiduloïdes was first described by Small [34] as a new species segregated from M. rigidula. These two species can scarcely be distinguished morphologically and appear to be very closely related from an evolutionary standpoint [34]. An important characteristic of this speciation relates to the bacterial strains isolated from each of them. The western Mediterranean species (M. rigidula) performs nitrogen fixation with the bacterial species E. meliloti sv. meliloti and E. medicae [4], whereas neither of these species fixes nitrogen with the eastern Mediterranean species (M. rigiduloïdes). The very low morphological divergence and the geographical differentiation are therefore linked to symbiotic specificity divergence [5]. In this study, several rhizobial strains able to nodulate M. rigiduloïdes were trapped and isolated from four soils, and their symbiotic characteristics and genetic diversity were compared by analysis of the chromosome and the nif-nod region located on the symbiotic megaplasmid. A novel symbiovar was then proposed based on its host specificity and genetic distinctiveness. The use of such a symbiovar definition in this well-studied organism is discussed in the context of a different evolutionary hypotheses proposed for rhizobial chromosomal diversification, which will improve our understanding of the mode of adaptation of these organisms and their relationship with their host plant. Materials and methods Origin and isolation of bacterial strains Root nodulating bacteria were trapped using leguminous M. rigiduloïdes cultivar 716 (ICARDA, Aleppo, Syria) at four alkaline soil sites (7.4 ≤ pH ≤ 8.3) situated around the Mediterranean Basin (Lebanon, Bulgaria, Turkey and France), collected between 1990 and 2005 and kept at 4 ◦ C. Trapping was performed on seven plants (as described in [29]), which briefly consisted of growing the plant on a sterile perlite-vermiculite medium topped with a 1-cm layer of selected soil. After growth for 8 weeks, nodules (if any) were collected and kept in glycerol at −80 ◦ C. Bacterial isolation from 3 to 5 nodules per soil was performed after external sterilization of the nodules and plating of the nodule lysate on yeast-extract mannitol (YEM) agar medium. After incubation of plates at 28 ◦ C for 3 days, purification on solid medium was performed twice from single colonies and isolated bacteria were maintained by incubation in liquid YEM medium for 48 h. All isolates were stored at −80 ◦ C in YEM with 25% (v/v) glycerol. DNA extraction, gene sequencing and phylogenetic analyses Genomic DNA extraction was performed using the method of Chen and Kuo [9], as modified in Rangin et al. [29]. Partial nodAB sequences were determined for all isolates [2] using primers XNA2F and XNA4R to amplify 178 bp of nodA and 480 bp of nodB genes. For 13 strains that displayed different nodAB sequences, several other genetic fragments were amplified by end-point PCR targeting the recA gene (primers RAS recAF35: CGGTGGAYAAAAGCAAGGC

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and RASM recARev1006: GGCRATSAGVCCGGCATTCTG) and three other genes involved in nodulation and nitrogen fixation: the full nodA gene (primers nodboxSme/NodB1rSme.Dfr.Rga) [37], internal fragments of nodC (primers f-nodCF21 and r-nodCI21) [37] and nifH (primers nifHF/nifHI) [19]. The recA gene encodes a protein essential for the repair and maintenance of bacterial DNA. The three genes nodABC are involved in Nod factor synthesis, and are reported to play a role in both host specificity and symbiotic effectiveness [3,30]. Symbiotic effectiveness is also controlled, at least partially, by the nitrogenase reductase nifH gene. Sequencing was performed by the High Throughput Genomics Units Company, University of Washington and electropherograms were manually checked using Chromas2 and aligned using ClustalW [20]. Nucleotide sequences were used to construct phylogenetic trees inferred by the maximum likelihood (ML) method using PhyML [16]. The best fitting model of ML phylogenetic reconstruction was inferred from jModelTest 2.1 [11]. Confidence in the nodes was estimated with 100 (ML) bootstrap replications. Reference sequences and closest sequences from a BLASTn search in NCBI were included (Fig. 1). DNA–DNA hybridization assay Bacterial DNA from three M. rigiduloïdes isolates (M29, 6 III 2tg, 6 II 1p) was extracted from 3-day-old 150-ml TY broth cultures following the method described by Villegas et al. [37]. Total DNA from the reference strains E. meliloti ATCC9930T and E. medicae A321T was labeled by random-priming using a Megaprimekit (Amersham). DNA relatedness between the M. rigiduloïdes isolates and the two reference strains was determined using the S1 nuclease/trichloroacetic acid method [15] at the optimal temperature for DNA reassociation (70 ◦ C) for 16 h. Phenotypic characterization for symbiosis Symbiotic properties (bacterial nodulation and nitrogen fixation) were determined by inoculation of 13 isolates (chosen on the basis of the phylogenetic results) on 10 plant replicates of the two plant species M. rigiduloïdes (cultivar 716) and M. truncatula (Jemalong A17), using the nutrient medium described in Rangin et al. [29]. Plant nodulation and fixation phenotypes were recorded after growth for 8 weeks by measuring the dry plant biomass and the presence of nodules, and estimating the colors of the nodules and plants. Dry plant biomass was compared statistically with noninoculated, control plants (data not shown). Nucleotide sequence accession numbers Sequences of the different alleles for each locus were deposited in GenBank under the following accession numbers: recA, KC857453 and KC857454; nodA, KC848517–KC848531; nodC, KC848532–KC848546; nifH, KC848561–KC848575; and nodB, KC848547–KC848560. Results and discussion Thirteen strains isolated from M. rigiduloïdes were compared with respect to both their genetic relatedness for five genetic markers and their symbiotic properties of nodulation and fixation on two plant hosts, M. rigiduloïdes and M. truncatula. A single genetic marker (nodAB) was determined for a further 49 isolated strains that were affiliated to one of the previous 13 strains (Table 1). According to the Committee on the Taxonomy of Plant Pathogenic Bacteria: “The term pathovar is used to refer to a strain or set of strains with the same or similar characteristics, differentiated at the infrasubspecific level from other strains of the same

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species or subspecies on the basis of distinctive pathogenicity to one or more plant hosts”. In 2011, Rogel et al. [32] suggested that symbiovar could similarly be used, based on the symbiotic capabilities in host plants, distinguished by differences in host range and supported by symbiotic gene sequence information. For the majority of bacteria, the notion of biovar is linked to biological variability.

In the case of the symbiovar, we consider that the host plant is the source of variability, inferring that either the term biovar or symbiovar could generally be used, even if the term symbiovar is more precise and accurate since it refers to symbiotic properties. This definition is therefore used here as the starting point for analysis of the status of isolates recovered from Medicago rigiduloïdes.

Fig. 1. Maximum likelihood (ML) phylogenetic trees showing the relationships between M. rigiduloïdes isolates obtained in this study and several other E. meliloti symbiovar sequences retrieved from GenBank. Accession numbers are given beside the strain names, and isolates obtained in this study are in bold. Genus name: E: Ensifer, M: Mesorhizobium, R: Rhizobium. The selection of best-fit models of DNA evolution for each locus was carried out using jModelTest 2.1.1: (a) GTR + I + G model for the 591-bp nodA sequences, (b) GTR + G model for the 709-bp nifH sequences, (c) HKY + I + G model for the 850-bp nodC sequences – the length of the branch between the outgroup R. leguminosarum USDA2071 and other nodC sequences was reduced (real value: 0.1567), and (d) the GTR + G model for the 480-bp nodB sequences. Values along branches are bootstrap values obtained from 100 replicates. ML analyses were performed using PhyML 3.1. Scales bar represent the number of changes per nucleotide position.

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Fig. 1. (Continued.)

Species affiliation of isolates fixing nitrogen with M. rigiduloïdes The recA phylogeny, now often used instead of 16S rRNA gene sequences in bacterial taxonomy [7], unambiguously clustered all 12 strains (one missing sequence) together with E. meliloti (Fig. S1), and 11 of them had an identical sequence. Among these strains, three (6II1p, 6III2tg and M29) were chosen for the DNA–DNA hybridization assay, which is (still) the accepted methodology

for defining bacterial species (even if whole-genome sequencing provides highly valuable information about the overall nucleotide similarity and the extent of the core genome). Two of these three strains (6II1p and 6III2tg) had an identical nodA sequence, the third (M29) being slightly different (two single nucleotide mutations over the 591-bp gene). High DNA relatedness (85–88%) was found between these three M. rigiduloïdes isolates and E. meliloti strain RCR2011, whereas a low value of relatedness (37%) was estimated

1 1 1 2 5 5 5 5 8 9 10 10

1 2 6

1 1 1 1 1 1 3 2 1 2 2 2 3 4 1 3 5 6

1 1 1 4 1 2

nodC

nifH

recA

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with the type strain of E. medicae. This data indicated that these bacterial strains isolated from M. rigiduloïdes belonged to the species E. meliloti. Previous studies [5,36] also positioned isolates sampled from M. rigiduloïdes (or alternative Medicago species that displayed the same symbiotic specificity characteristics) in the E. meliloti species, based on several chromosomal sequences. Both phylogenetic and hybridization analyses confirmed that these isolates were true Ensifer meliloti. Supplementary material related to this article can be found, in the online version, at doi:10.1016/j.syapm.2013.06.004.

8 9 10 10 STM4099, E7 n2 E1 p1 n2, E2 p1 n2

6 6 8 7 STM4089, STM4093, STM4064, STM4057

Fix− Fix− Fix− Fix− Bulgaria Bulgaria Bulgaria France

Fix+ Fix− Fix+ Fix+

7 Fix+

STM4103 S. meliloti clade V STM3283 STM4054 STM4069 E4 n5

Turkey

Fix−

STM4076, STM4113, STM4067, STM3173, STM4095, STM4055, STM3133, STM4046, STM3549, STM4058, STM4070, STM4101, STM4098, STM4090 STM4073, STM4096, STM4060

Fix+ Fix+ Fix+ Fix+ Fix+ Fix+ 6 II 1 p STM4065 STM4082 STM4084 M29 [12] STM3164

France Lebanon Lebanon Lebanon Syria Turkey

Fix− Fix− Fix− Fix− Fix− Fix−

STM4056

4

3 4 5 6 – 3 3 5 1 3 4 4

1 2 1 2 Bulgaria Bulgaria

Fix+ Fix+

Fix− Fix−

STM4052, STM3224, STM4088, STM4092 STM3244, STM3255, STM4066, STM4048, STM4102, STM4094, STM4053, STM4059, STM4061, STM4047, STM4062 6 II tg I 2, 6 III 2 tg, 6 III 3 g, E7 n1, STM4079 STM4100, STM4085, STM4081

nodA

S. meliloti sv. rigiduloides STM3226 STM4097

M. rigiduloïdes

Origin

N2 fixation tests

M. truncatula

Identical nodAB strains

Genotypes

nodB

The isolates displayed atypical nodulation gene content divergent from known E. meliloti symbiovars

Strain

Table 1 Bacterial strains isolated in this study from M. rigiduloïdes. Their geographical origin and their phenotype of nitrogen fixation on the two plant hosts are indicated. Other strains with identical nodAB genotypes are presented.

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Not all of the sampled strains could efficiently fix nitrogen with M. rigiduloïdes, even if they had originally been isolated from nodules collected on this species. This Fix−/Fix+ phenotype could also be observed on strains obtained from root nodules of M. rigiduloïdes directly cultivated at the French site (data not shown). The symbiotic associations of combinations of nodulating bacteria on one legume species ranged from no effect to efficient nitrogen fixation, but with an intermediate state (nodulation but no fixation). Strains that fixed nitrogen with M. rigiduloïdes could nodulate M. truncatula, but only inefficiently (Table 1; quantitative data not shown). This symbiotic specificity corresponded to part of the symbiovar definition, with respect to host range specificity. Genetic analysis of accessory genes involved in symbiosis (nodulation and nitrogen fixation) was also performed, in order to clarify genetic differentiation in terms of symbiotic genes and relative phylogenetic and evolutionary positions among all E. meliloti symbiovars defined so far. Phylogenetic analyses confirmed the distinction between the two groups of strains (Fix+ vs Fix− ) irrespective of which locus was analyzed. The group that did not fix nitrogen with M. rigiduloïdes clustered with E. meliloti clade V isolates (following the Mnasri et al. [27] subdivision). For this bacterial cluster, a symbiotic polymorphism was shown on several host plants as these strains performed efficient symbiosis on other hosts not tested in this study, such as Argyrolobium uniflorum [27]. The symbiotic status of these isolates remains to be studied. All three nodulation genes studied, nodA, nodB and nodC, placed E. meliloti isolates that fixed nitrogen with M. rigiduloïdes in divergent clades, but with slight differences between them. In the nodA phylogeny, the nine isolates (plus three previously published sequences) formed a monophyletic group, diverging at the base of a clade that included E. medicae and all biovars of E. meliloti strains sequenced so far at this locus. In the nodB and nodC phylogenies, these isolates split into two (and potentially three) closely related groups. One group clustered as a sister clade of E. meliloti sv. medicaginis isolates. A previous study [2] additionally determined the intergenic sequences between nodE and nodG for three E. meliloti isolates, and the resultant nodE-nodG phylogenetic tree also positioned these strains in an equivalent diverging sister clade of E. meliloti sv. medicaginis. The split of all these isolates into two clades was also present, although not well supported, in the nodA phylogeny. Chinese strains isolated from Trigonella [17] falling into a putative third clade on the nodC phylogeny could not be tested for nodulation and fixation on M. rigiduloïdes. However, the close relationships of these strains to our isolates fixing nitrogen on M. rigiduloïdes, based on the nifH and nodC phylogenies, suggested that they may belong to the same symbiovar. Taken together, these data confirmed the symbiovar status of these strains, belonging to the E. meliloti species but with a specific host range correlated to specific symbiotic gene pool content.

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Due to its host specificity and its genetic distinctiveness, it is therefore proposed that the strains belonging to this new symbiovar be referred to as E. meliloti sv. rigiduloides. According to the symbiotic classification proposed for Medicago species [5], we inferred that these strains also performed efficient nitrogen fixation with several other Medicago species, such as M. noeana, M. monantha or M. radiata. Supporting this view, the two strains USDA1614 and USDA1613, originally isolated from M. radiata and M. noeana, respectively, fell within the sv. rigiduloides cluster for each nod locus and should be considered as members of this symbiovar. Following this view, the Chinese strains potentially belonging to the E. meliloti sv. rigiduloides clade 3 (nodC phylogeny) were isolated from Trigonella arcuata, strongly suggesting that this biovar might not be restricted to M. rigiduloïdes but has rather a wider host spectrum.

Emergence of a new symbiovar and evolutionary relationships among Ensifer meliloti symbiovars The spread of symbiotic genes among various genetic backgrounds, via horizontal transfer, has been documented for a long time [28,35]. The acquisition of novel symbiotic abilities via lateral transfer of symbiotic mobile elements has usually been suggested in order to explain the incongruence between chromosomal and symbiotic gene phylogenies. Six symbiovars have previously been described or suggested for Ensifer meliloti, including the newly described sv. rigiduloides. However, a single major transfer of the entire symbiotic cluster does not explain the modification of the host plant range, and the emergence of several symbiovars may have resulted from several different genomic modifications. In the nodC phylogeny, for which most sequences from all biovars are available in the databases, the three symbiovars sv. lancerottense, sv. mediterranense and sv. ciceri fell into the same clade as E. fredii, diverging from the three other E. meliloti symbiovars (sv. rigiduloides, sv. medicaginis, sv. meliloti) that clustered in a well supported clade together with E. medicae. Members of this last clade shared the ability to nodulate Medicago species. Conversely, in the nodA phylogeny, sv. mediterranense (efficient on Phaseolus vulgaris) fell within the same clade as these Medicago symbiovars, far away from E. fredii. Therefore, the discrepancy between the symbiotic phylogenies suggested that, rather than transfer of the entire symbiotic cluster, the different symbiovars have emerged following the acquisition of a subset of nodulation genes, made possible by non-obligate physical linkage in a symbiotic cluster of the nodulation genes. Either a single gene transfer or transfer of the entire symbiotic cluster, followed by the loss of several transferred genes to the benefit of others present in the acceptor strain, might thus explain the emergence of different biovars in E. meliloti. Reinforcing this view, it has been shown that modification of a single locus can control a drastic change in symbiotic specificity, since the bacterial specificity of E. meliloti sv. medicaginis in association with Medicago laciniata relies on a single nodC gene polymorphism [3]. The hypothesis of a “patchwork” of alleles (donor and receptor) was also supported by the nifH phylogeny. The nitrogenase is coded by a set of genes that are co-localized with nodulation genes. However, lateral gene transfer of nodulation genes might not have involved nitrogen fixation genes, especially when these genes are already present in the receptor strains and might then be adapted to their genetic background. The nifH phylogeny obtained did follow this suggestion and displayed contrasting topology from nodulation genes. For instance, sv. ciceri strains that clustered with E. fredii and the other symbiovars in the nodC tree fell within the E. meliloti main clade in the nifH phylogeny.

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E. meliloti sv. rigiduloides geographical distribution and the ecotype concept The new symbiovar sv. rigiduloides appeared to be geographically widespread around the Mediterranean Basin, as it was recovered from five different sites from different countries (Turkey, Syria, Lebanon, Bulgaria and France). Sequences of this new symbiovar are only weakly represented in the nucleotide database for the four symbiotic genes used in this study, but several strains supposedly belonging to this biovar have been trapped in a large sampling E. meliloti dataset [12] using diverse plant species in different countries (Pakistan, Syria, Turkey and Jordan). All these strains were inefficient on M. sativa [12,36]. Béna et al. [5] previously showed that strains belonging to this symbiovar could form nodules and efficiently fix nitrogen with several Medicago species: M. radiata, M. monantha, M. brachycarpa, M. orthoceras, M. rigiduloïdes and M. noeana. According to Lesins and Lesins [22] all these Medicago species grow in the eastern part of the Mediterranean Basin and in Asia, contributing to an apparent trend for an eastern distribution of these plant species and their symbiotic bacteria. Therefore, it was quite surprising to detect this symbiovar in western European soil (France). Moreover, no evidence of any genetic divergence or isolation was detected between these isolates and eastern ones, even if the genetic typing in our study might not have been sufficiently powerful or deep to detect traces of geographic differentiation. Such wide dispersion may result from several (non-exclusive) parameters, such as high rates of migration and homologous recombination between strains. The maintenance of these strains in western soils also implies the presence of associated plant cover. In these areas, E. meliloti sv. rigiduloides might be associated with non-Medicago host species and closely related genera including Trigonella and Melilotus, as found for the Chinese strains isolated from Trigonella acuarta [17]. Finally, it is important to determine whether one or several distinct symbiovars/ecotypes should be defined for sv. rigiduloides, since the discrimination of specific ecotypes is fundamental for an understanding of recombination patterns within a species [2]. Indeed, among-ecotype recombination is expected to be lower than within-ecotype recombination, due to adaptation to specific niches and/or geographical specialization. Such findings suggest that the conclusions inferred from extensive recombination analyses, performed in the absence of ecotype classification, should be revisited by considering the existence of different E. meliloti symbiovars [13,36] in order to estimate this parameter better within each species. Furthermore, this among-ecotype recombination is expected to decrease over time, potentially leading to different chromosomal genetic divergence due to cohesive forces constraining each ecotype [10]. Defining this new symbiovar rigiduloides will help to clarify bacterial ecological symbiotic classification and should enhance the accuracy of population analysis methods.

Acknowledgements We would like to thank Lucette Mauré and Odile Domergue for help in plant inoculation and molecular sequencing, respectively.

References [1] Ba, S., Willems, A., de Lajudie, P., Roche, P., Jeder, H., Quatrini, P., Neyra, M., Ferro, M., Prome, J.-C., Gillis, M., Boivin-Masson, C., Lorquin, J. (2002) Symbiotic and taxonomic diversity of rhizobia isolated from Acacia tortilis subsp. raddiana in Africa. Syst. Appl. Microbiol. 25, 130–145. [2] Bailly, X., Olivieri, I., Brunel, B., Cleyet-Marel, J.-C., Béna, G. (2007) Horizontal gene transfer and homologous recombination drive the evolution of the nitrogen-fixing symbionts of Medicago species. Appl. Environ. Microbiol. 189, 5223–5236.

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[3] Barran, L.R., Bromfield, E.S.P., Brown, D.C.W. (2002) Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti–Medicago laciniata symbiosis. Can. J. Microbiol. 48, 765–771. [4] Béna, G., Jubier, M.-F., Olivieri, I., Lejeune, B. (1998) Ribosomal external and internal transcribed spacers: combined use in the phylogenetic analysis of Medicago (Leguminosae). J. Mol. Evol. 46, 299–306. [5] Béna, G., Lyet, A., Huguet, T., Olivieri, I. (2005) Medicago-Sinorhizobium symbiotic specificity evolution and the geographic expansion of Medicago. J. Evol. Biol. 18, 1547–1558. [6] Brockwell, J. (1985) The role of Rhizobium meliloti in annual medic ley pastures in central western New South Wales: a pragmatic appraisal. In: Hochman, Z. (Ed.), The ecology and agronomy of annual medics. Proc. Worshop, Condolin, NSW Dep. Agric. Tech. Bull., vol. 34, pp. 39–42. [7] Bull, M.J., Marchesi, J.R., Vandamme, P., Plummer, S., Mahenthiralingam, E. (2012) Minimum taxonomic criteria for bacterial genome sequence depositions and announcements. J. Microbiol. Methods 89, 18–21. [8] Cazalet, C., Jarraud, S., Ghavi-Helm, Y., Kunst, F., Glaser, P., Etienne, J., Buchrieser, C. (2008) Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species. Genome Res. 18, 431–441. [9] Chen, W.P., Kuo, T.T. (1993) A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Res. 21, 2260. [10] Cohan, F.M. (2006) Towards a conceptual and operational union of bacterial systematic, ecology, and evolution. Phil. Trans. R. Soc. B 361, 1985–1996. [11] Darriba, D., Taboada, G.L., Doallo, R., Posada, D. (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat. Method 9, 772. [12] Eardly, B.D., Materon, L.A., Smith, N.H., Johnson, D.A., Rumbaugh, M.D., Selander, R.K. (1990) Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti. Appl. Environ. Microbiol. 56, 187–194. [13] Epstein, B., Branca, A., Mudge, J., Bharti, A.K., Briskine, R., Farmer, A.D., Sugawara, M., Young, N.D., Sadowsky, M.J., Tiffin, P. (2012) Population genomics of the facultatively mutualistic bacteria Sinorhizobium meliloti and S. medicae. PLoS Genet. 8, e1002868. [14] Gintzburger, G., Rochon, J.J., Conesa, A.P. (1990) The French Mediterranean zones: sheep rearing systems and the present and potential role of pasture legumes. In: Osman, A.E. (Ed.), The role of legumes in the farming systems of the mediterranean areas, ICARDA, The Netherlands, pp. 174–194. [15] Grimont, P.A.D., Popoff, M.Y., Grimont, F., Coynault, C., Lemelin, M. (1980) Reproducibility and correlation study of three deoxynucleic acid hybridization procedures. Curr. Microbiol. 4, 325–330. [16] Guindon, S., Dufayard, J.F., Lefort, V., Anisimova, M., Hordijk, W., Gascuel, O. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321. [17] He, Y.R., Wang, J.Y., Wang, E.T., Feng, G., Chang, Y.L., Sui, X.H., Chen, W.X. (2011) Trigonella arcuata-associated rhizobia—an Ensifer (Sinorhizobium) meliloti population adapted to a desert environment. Plant Soil 345, 89–102. [18] Koeppel, A., Perry, E.B., Sikorski, J., Krizanc, D., Warner, A., Ward, D.M., Rooney, A.P., Brambilla, E., Connor, N., Ratcliff, R.M., Nevo, E., Cohan, F.M. (2008) Identifying the fundamental units of bacterial diversity: a paradigm shift to incorporate ecology into bacterial systematics. Proc. Natl. Acad. Sci. U.S.A. 105, 2504–2509. [19] Laguerre, G., Nour, S.M., Macheret, V., Sanjuan, J., Drouin, P., Amarger, N. (2001) Classification of rhizobia based on nodC and nifH gene analysis reveals a close phylogenetic relationship among Phaseolus vulgaris symbionts. Microbiology 147, 981–993. [20] Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin, F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D.,

[21]

[22] [23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

[32] [33]

[34] [35]

[36]

[37]

Gibson, T.J., Higgins, D.G. (2007) Clustal W and clustal X version 2.0. Bioinformatics 23, 2947–2948. Leon-Barrios, M., Lorite, M.J., Donate-Correa, J., Sanjuan, J. (2009) Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range. Syst. Appl. Microbiol. 32, 413–420. Lesins, K.A., Lesins, I. (1979) Genus Medicago (Leguminosae), a taxogenetic study, Junk, The Hague. Maatallah, J., Berraho, E., Munoz, S., Sanjuan, J., Lluch, C. (2002) Phenotypic and molecular characterization of chickpea rhizobia isolated from different areas of Morocco. J. Appl. Microbiol. 93, 531–540. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S.V., Machado, M.A., Toth, I., Salmond, G., Foster, G.D. (2012) Top 10 plant pathogenic bacteria in molecular plant pathology. Mol. Plant Pathol. 13, 614–629. Mhamdi, R., Laguerre, G., Aouani, M.E., Mars, M., Amarger, N. (2002) Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils. FEMS Microbiol. Ecol. 41, 77–84. Mnasri, B., Mrabet, M., Laguerre, G., Aouani, M.E., Mhamdi, R. (2007) Salttolerant rhizobia isolated from a Tunisian oasis that are highly effective for symbiotic N2 -fixation with Phaseolus vulgaris constitute a novel biovar (bv. mediterranense) of Sinorhizobium meliloti. Arch. Microbiol. 187, 79–85. Mnasri, B., Badri, Y., Saïdi, S., de Lajudie, P., Mhamdi, R. (2009) Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia. Syst. Appl. Microbiol. 32, 583–592. ˜ Pérez-Mendoza, D., Sepúlveda, E., Pando, V., Munoz, S., Nogales, J., Olivares, J., Soto, M.J., Herrera-Cervera, J.A., Romero, D., Brom, S., Sanjuán, J. (2005) Identification of the rctA gene, which is required for repression of conjugative transfer of rhizobial symbiotic megaplasmids. J. Bacteriol. 187, 7341–7350. Rangin, C., Brunel, B., Cleyet-Marel, J.C., Perrineau, M.M., Béna, G. (2009) Effects of Medicago truncatula genetic diversity, rhizobial competition, and strain effectiveness on the diversity of a natural Sinorhizobium species community. Appl. Environ. Microbiol. 74, 5653–5661. Roche, P., Maillet, F., Plazanet, C., Debellé, F., Ferro, M., Truchet, G., Promé, J.C., Dénarié, J. (1996) The common nodABC genes of Rhizobium meliloti are hostrange determinants. Proc. Natl. Acad. Sci. U.S.A. 93, 15305–15310. Roesch, L.F., Fulthorpe, R.R., Riva, A., Casella, G., Hadwin, A.K., Kent, A.D., Daroub, S.H., Camargo, F.A., Farmerie, W.G., Triplett, E.W. (2007) Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J. 1, 283–290. ˜ Rogel, M.A., Ormeno-Orrillo, E., Martinez Romero, E. (2011) Symbiovars in rhizobia reflect bacterial adaptation to legumes. Syst. Appl. Microbiol. 34, 96–104. Rome, S., Fernandez, M.P., Brunel, B., Normand, P., Cleyet-Marel, J.C. (1996) Sinorhizobium medicae sp. nov., isolated from annual Medicago spp. Int. J. Syst. Bacteriol. 46, 972–980. Small, E. (1990) Medicago rigiduloïdes, a new species segregated from M. rigidula. Can. J. Bot. 68, 2614–2617. Sullivan, J.T., Ronson, C.W. (1998) Evolution of rhizobia by acquisition of a 500kb symbiosis island that integrates into a phe-tRNA gene. Proc. Natl. Acad. Sci. U.S.A. 95, 5145–5149. van Berkum, P., Elia, P., Eardly, B.D. (2010) Application of multilocus sequence typing to study the genetic structure of megaplasmids in Medicago-nodulating rhizobia. Appl. Environ. Microbiol. 76, 3967–3977. Villegas, M.C., Rome, S., Maure, L., Domergue, O., Gardan, L., Bailly, X., CleyetMarel, J.-C., Brunel, B. (2006) Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv medicaginis) of E. meliloti. Syst. Appl. Microbiol. 29, 526–538.