1 Introduction

Legumes belong to three subfamilies of the family-Fabaceae, including the Caesalpinioideae, Mimosideae and Papilionoideae. These subfamilies have received great attention because they can establish specific symbioses with rhizobia in the soil. The rhizobia, collectively referred to as the root and stem nodule bacteria of legumes, consist of 238 species in 18 genera and two clades. Under nitrogen (N) deficient conditions these microorganisms fix atmospheric nitrogen and transfer N-containing compounds to the legume plant through the process of symbiotic nitrogen fixation (Sprent 2001). In this biological process leading to symbiosis. The legumes excrete different types of flavonoid molecules, which subsequently induce bacterial strains to produce different types of Nod factors. The later molecules facilitate recognition and penetration of bacteria into root hairs, eventually leading to the production of root/stem nodules. Inside the nodule tissue, the bacteria live in a bacteroid state and are capable of fixing atmospheric dinitrogen. Until the early 980s, all symbiotic nitrogen-fixing bacteria from leguminous plants were classified as belonging to the genus Rhizobium, with six named species: R. leguminosarum, R. meliloti, R. trifolii, R. phaseoli, R. lupine and R. japonicum (Somasegaran and Hoben 1985). This taxonomy, however changed in 1984, and continues to evolve today.

2 Phenotypic and genotypic characteristics used for taxonomy of legume-nodulating bacteria

There are several phenotypic characteristics which have been used to identify and differentiate among bacteria capable of nodulating-legumes (Jordan 1984). Rhizobia were subsequently reclassified into two groups, the fast-growing, acid-producing group (the Rhizobium sp. strains) and a slow-growing, alkaline-producing group (Bradyrhizobium) based on their generation time and pH reaction on yeast extract mannitol medium containing bromophenol blue (Vincent 1970). Nodulation of specific host plants was one of main criteria used to differentiate among the Rhizobium species, as was known as cross inoculation group concept (Somasegaran and Hoben 1985). However, it soon became apparent that this character was not useful to classify species of rhizobia due to the possibility of natural transfer of symbiotic plasmids among bacterial strains in the soil (Mergaert et al. 1997; Finan 2002; Nakatsukasa et al. 2008). The location of symbiotic genes was also used as a genotypic tool to differentiate between the fast and slow-growing legume-nodulating bacteria, they are typically chromosome-located for the slow-growing bradyrhizobia and on plasmids for fast-growing Rhizobium strains. Recently, however a slow growing Bradyrhizobium strain DOA9 was found to carry symbiotic genes on a megaplasmid (Teamtisong et al. 2013).

Subsequent molecular biology tools developed over several decades were used in polyphasic approaches to classify rhizobia and included the mole %G + C content of the bacterial genome, and later the sequencing of 16S rRNA gene. This led to the description of large number of rhizobia in a non-systematic way. In 1991 Graham and colleagues published the minimal standards for the description of rhizobial species (Graham et al. 1991). After this, RFLP analyses of the 16S rRNA gene, the phylogenetic analysis of 16 rRNA gene sequences (Willems and Collins 1993; Yanagi and Yamasato 1993) and DNA-DNA hybridization percentage were being proposed as needed tools for the identification of nitrogen-fixing, legume-nodulating bacteria (Stackebrandt and Gobel 1994). Moreover, strain similarity could be easily assessed by using the REP PCR DNA fingerprinting technique (De Bruijn 1992; Ishii and Sadowsky 2009). Although the sequencing of 16S rRNA gene is still used widely to propose and describe new species of legume-nodulating bacteria; it has some limitation and cannot be solely used to differentiate among the closest Rhizobium species (Ramirez-Bahena et al. 2008). Therefore, researchers suggest using chromosomal housekeeping genes, such as atpD, recA, and glnII to help with speciation of closest-related species of R. leguminosarum sv. trifolii, R. leguminosarum sv. phaseoli, and R. leguminosarum sv. viceae. Multilocus sequence analysis (MLSA) and multilocus sequence typing (MLST) has also been used to differentiate and identify new rhizobial taxa (Ribeiro et al. 2009). Other methods have been used to explain the differences among species, such as extracellular polysaccharide composition (Huber et al. 1984), fatty acid profiles (Tighe et al. 2000). Recently, two new methods have been used for studying the taxonomy of legume-nodulating bacteria: comparative genomics (Ormeno-Orrillo et al. 2015) and average nucleotide identity (ANI) of genome comparisons (Rashid et al. 2015).

3 Taxonomy of symbiotic, nitrogen-fixing bacteria

Currently the legume-nodulating bacteria belong to three different bacterial classes; the α, β and γ-Proteobacteria. The largest class, the alphaproteobacteria, is composed of six families, including Rhizobiaceae, Phylobactericiae, Bradyrhizobiaceae, Hyphomicrobiaceae, Methylobacteriiaceae and Brucellaceae. The second class comprised of the betaproteobacteria, currently contains one family the Burkholderiales, and contains two genera (Fig. 1).

Fig. 1
figure 1

Schematic diagram to explain the numbers and distribution of legume-nodulating bacterial species in the classes of α- and β-Proteobacteria.

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The classification of symbiotic, legume-nodulating bacteria is in great state of flux, more so than ever before. Zakhia and de Lajudie (2001) subsequently summarized the classification of these bacteria into six Rhizobium genera with 28 recognized species. By 2003 however, Sawada et al. (2003) reported that 44 bacterial species distributed in 12 genera can form nitrogen-fixing symbiosis with legumes. Subsequently, Willems (2006) stated that rhizobia are comprised of 53 bacterial species that are distributed as follows: 16, 11, 11, 7, 5, 2 and 1 species belonging to the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Agrobacterium, Azorhizobium, and Allorhizobium, respectively. The further subdivisions of rhizobia continue and Berrada and Fikri-Benbrahim (2014) recently reported that there are 98 species of legume nodulating bacteria belonging to 14 genera. Here we described about 238 species distributed among 18 genera. Results in Fig. 2 describe 18 genera of root nodulating bacteria with some representative species of each genus. The largest two genera are Rhizobium and Bradyrhizobium and we focus here on species that nodulate edible legumes.

Fig. 2
figure 2

Neighbor-Joining phylogenetic sequence analysis of 16S rRNA (1,450 bp) of for 47 representative species of 18 genera of root-nodulating bacteria. Values of bootstrap probaility greater than 50% are indicated

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4 Whole genome sequence alignments

Ormeno-Orrillo et al. (2015) established a new trend of using whole genome sequence comparisons to define new taxa of rhizobia, which they called genomotaxonomy. Using this approach, they confirmed that the phylogenomic analysis provided support for the revival of Allorhizobium as a bona fide genus within the Rhizobiaceae, the distinctiveness of Agrobacterium and the recently proposed genus Neorhizobium, and suggested that R. giardinii be transferred to a novel genus.

The recent revision of rhizobial taxonomy by Mousavi et al. (2015) led to the description of the novel genus Pararhizobium, comprised of 4 new species combinations and 9 other species combinations belonging to the genus Allorhizobium.

5 Species of rhizobia within the class α-Proteobacteria

Despite these proposed changes, the Rhizobiaceae represent perhaps the most cohesive and preserved among the six families of legume-nodulating bacteria (Fig. 1). The family Rhizobiaceae is a common and widely distributed family containing five genera: Rhizobium, Ensifer (formerly Sinorhizobium), Allorhizobium, Shinella and Pararhizobium. The genus Rhizobium has about 98 species, 69 of which were isolated from various legume hosts around the world and 29 species that are non-symbiotic (Table 1). Recently Shamseldin et al. (2016) identified Rhizobium aegypticaum as a new species which effectively nodulated Egyptian clover (Trifolium alexandrinum L.). The genus Ensifer (formerly Sinorhizobium) has about 18 species (Table 1). Gubry-Rangin et al. (2013) proposed a new bacterial symbiovar Ensifer meliloti sv. rigiduloides, that fixes nitrogen efficiently on Medicago rigiduloides, but not on Medicago truncatula. Each of the other three genera Allorhizobium, Shinella and Pararhizobium contain a single species (Table 1).

Table 1 Species within the family Rhizobiaceae
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The second family Phylobactericiae contains three genera: Mesorhizobium, Phyllobacterium and Aminobacter. The genus Mesorhizobium contains about 40 species, while the genus Phyllobacterium has 8 species, and the genus Aminobacter only one species (Table 2).

Table 2 Species within the family Phylobactericiae
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The third family, the Bradyrhizobiaceae, contains three genera Bradyrhizobium, Blastobacter and Photorhizobium. The genus Bradyrhizobium includes 36 species, while both Blastobacter and Photorhizobium contain only a single species (Table 3). Guerrouj et al. (2013) proposed a novel symbiovar named Bradyrhizobium retamae sp. nov., nodulating Retama sphaerocarpa and Retama monosperma.

Table 3 Species within the family Bradyrhizobiaceae
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The fourth family is the Hyphomicrobiaceae and includes Devosia and Azorhizobium as the two known genera. The genus Devosia has only one species, while genus Azorhizobium has three species (Table 4).

Table 4 Species within the family Hyphomicrobiaceae
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The fifth family, Methylobacteriiaceae, is comprised of genera Methylobacterium and Microvirga. The genus Methylobacterium includes 3 species and Microvirga includes 4 species (Table 5). Lastly, the family Brucellaceae contains only a single genus, Ochrobacterium, which has 2 species (Table 6).

Table 5 Species within the family Methylbacteriiaceae
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Table 6 Species within the family Brucellaceae
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6 Species of legume-nodulating bacteria within the β- and γ-Proteobacteria classes

While most rhizobia were originally placed in the α-Proteobacteria, more recent studies showed that the legume-nodulating bacteria belong to a much wider group (Gyaneshwar et al. 2011; Shiraishi et al. 2010). There are about 18 species of rhizobia belonging to two genera of the β-Proteobacteria, Burkholderia with 17 species and Ralstonia (former Cupriavidus) with 2 species (Table 7). More recently, Shiraishi et al. (2010) noted that there is a Pseudomonas sp. belonging to the γ-Proteobacteria that can nodulate Robinia pseudoacacia.

Table 7 Species of symbiotic strains of β-Proteobacteria
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7 Non-symbiotic bacteria

There are about 33 non-symbiotic bacterial species reported here that are related to the nodule bacteria and are distributed in different genera. The majority of them belong to the genus Rhizobium (29), although non-nodulating Bradyrhizobium have been isolated (Pongsilp et al. 2002). Each of the genera Pararhizobium, Mesorhizobium, Azorhizobium and Burkholderia has one species. Kimes et al. (2015) identified a novel genus, Pseudorhizobium pelagicum which had 95.6 to 97 % a sequence similarity to members of genera Neorhizobium and Rhizobium, but this new species lacked symbiosis-related genes.

8 Conclusions

The considerable and continued development of molecular biology tools over the last 20 years has facilitated the identification of new legume-nodulating bacteria and resulted in considerable changes in the classification and proposal of new and different species. Although in this review we described about 238 species distributed in 18 genera, larger efforts from researchers around the world are needed. This is, in large part, due to our lack of understanding of legume-Rhizobium interactions. For example, the description of rhizobia species above comprises only about 23 % of legumes, and it has been estimated that there are roughly 19,000 legume species. Hopefully, the discovery and designation of new different species of bacteria-nodulating edible legumes can contribute to improve productivity, especially in developing countries which suffer from a lack of protein.