Members of the Leguminosae form the largest plant family on Earth, with around 18,000 species. The success of legumes can largely be attributed to their ability to form a nitrogen-fixing symbiosis with specific bacteria known as rhizobia, manifested by the development of nodules on the plant roots in which the bacteria fix atmospheric nitrogen, a major contributor to the global nitrogen cycle. Rhizobia described so far belong exclusively to the alpha-subclass of Proteobacteria, where they are distributed in four distinct phylogenetic branches. Although nitrogen-fixing bacteria exist in other proteobacterial subclasses, for example Herbaspirillum and Azoarcus from the phylogenetically distant beta-subclass, none has been found to harbour the nod genes essential for establishing rhizobial symbiosis. Here we report the identification of proteobacteria from the beta-subclass that nodulate legumes. This finding shows that the ability to establish a symbiosis with legumes is more widespread in bacteria than anticipated to date.

Following the initial discovery of two legume-nodulating Burkholderia strains (L. Moulin, A. Munive, B. Dreyfus, and C. Boivin-Masson, Nature 411:948-950, 2001), we identified as nitrogen-fixing legume symbionts at least 50 different strains of Burkholderia caribensis and Ralstonia taiwanensis, all belonging to the ␤-subclass of proteobacteria, thus extending the phylogenetic diversity of the rhizobia. R. taiwanensis was found to represent 93% of the Mimosa isolates in Taiwan, indicating that ␤-proteobacteria can be the specific symbionts of a legume. The nod genes of rhizobial ␤-proteobacteria (␤-rhizobia) are very similar to those of rhizobia from the ␣-subclass (␣-rhizobia), strongly supporting the hypothesis of the unique origin of common nod genes. The ␤-rhizobial nod genes are located on a 0.5-Mb plasmid, together with the nifH gene, in R. taiwanensis and Burkholderia phymatum. Phylogenetic analysis of available nodA gene sequences clustered ␤-rhizobial sequences in two nodA lineages intertwined with ␣-rhizobial sequences. On the other hand, the ␤-rhizobia were grouped with free-living nitrogen-fixing ␤-proteobacteria on the basis of the nifH phylogenetic tree. These findings suggest that ␤-rhizobia evolved from diazotrophs through multiple lateral nod gene transfers.Members of the Leguminosae, comprising about 18,000 species, play an important ecological role, with representatives in nearly every type of plant on Earth. Most species are able to form nitrogen-fixing symbioses with specific bacteria known as rhizobia. The recent identification of two ␤-proteobacterial strains of the genus Burkholderia able to nodulate legumes (10) changed the long-held dogma that only bacteria of the ␣ subdivision are able to nodulate legumes (18, 23). These two strains were subsequently described as Burkholderia tuberum and Burkholderia phymatum (24). In addition, eight strains isolated from root nodules of Mimosa spp. were recently described as Ralstonia taiwanensis, also classified as ␤-proteobacteria (1), although their nodulation capacity was not confirmed. The terms ␣-and ␤-rhizobia were proposed to distinguish the rhizobial ␣-and ␤-proteobacteria, respectively (10). This unexpected discovery raised the question as to whether nodulation by ␤-proteobacteria is an extremely rare phenomenon or whether it had simply been overlooked until now. Moreover, the fact that the first two nodulating Burkholderia strains were isolated from Aspalathus and Machaerium spp., which are known to be associated with Bradyrhizobium (2, 12), may suggest that these ␤-proteobacteria are not the specific partners of the respective host legumes.In this article, we confirm the widespread phylogenetic diversity of nitrogen-fixing legume symbionts by identifying as ␤-rhizobia an additional 2 Burkholderia strains from the species Burkholderia caribensis and a collection of at least 44 R. taiwanensis strains. These data increase to four the number of different ␤-rhizobial species identified so far, originating from three different continents. Moreove...