Burkholderia has only recently been recognized as a potential nitrogen-fixing symbiont of legumes, but we find that the origins of symbiosis in Burkholderia are much deeper than previously suspected. We sampled 143 symbionts from 47 native species of Mimosa across 1800 km in central Brazil and found that 98% were Burkholderia. Gene sequences defined seven distinct and divergent species complexes within the genus Burkholderia. The symbiosis-related genes formed deep Burkholderia-specific clades, each specific to a species complex, implying that these genes diverged over a long period within Burkholderia without substantial horizontal gene transfer between species complexes.

Summary• The ability of Burkholderia phymatum STM815 to effectively nodulate Mimosa spp., and to fix nitrogen ex planta , was compared with that of the known Mimosa symbiont Cupriavidus taiwanensis LMG19424.• Both strains were equally effective symbionts of M. pudica , but nodules formed by STM815 had greater nitrogenase activity. STM815 was shown to have a broader host range across the genus Mimosa than LMG19424, nodulating 30 out of 31 species, 21 of these effectively. LMG19424 effectively nodulated only nine species. GFP-marked variants were used to visualise symbiont presence within nodules.• STM815 gave significant acetylene reduction assay (ARA) activity in semisolid JMV medium ex planta , but no ARA activity was detected with LMG19424. 16S rDNA sequences of two isolates originally from Mimosa nodules in Papua New Guinea (NGR114 and NGR195A) identified them as Burkholderia phymatum also, with nodA , nodC and nifH genes of NGR195A identical to those of STM815.• B. phymatum is therefore an effective Mimosa symbiont with a broad host range, and is the first reported beta-rhizobial strain to fix nitrogen in free-living culture.

Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other ␤-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known ␤-rhizobia, Burkholderia caribensis, Burkholderia phymatum, and Burkholderia tuberum. The 16S rRNA gene sequences of 15 of the 20 strains were determined, and all were shown to belong to the genus Burkholderia; four distinct clusters could be discerned, with strains isolated from the same host species usually clustering very closely. Five of the strains (MAP3-5, Br3407, Br3454, Br3461, and Br3469) were selected for further studies of the symbiosisrelated genes nodA, the NodD-dependent regulatory consensus sequences (nod box), and nifH. The nodA and nifH sequences were very close to each other and to those of B. phymatum STM815, B. caribensis TJ182, and Cupriavidus taiwanensis LMG19424 but were relatively distant from those of B. tuberum STM678. In addition to nodulating their original hosts, all five strains could also nodulate other Mimosa spp., and all produced nodules on Mimosa pudica that had nitrogenase (acetylene reduction) activities and structures typical of effective N 2 -fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N 2 -fixing nodules on their original hosts, Mimosa bimucronata (Br3461) and Mimosa pigra (MAP3-5), and hence this confirms strongly that Burkholderia strains can form effective symbioses with legumes.Although it was generally accepted for many years that legumes (and the nonleguminous plant Parasponia) were nodulated exclusively by members of the Rhizobiaceae in the ␣-Proteobacteria (including the genera Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium) (30, 34), recently there has been an increasing number of reports of members of the ␤-Proteobacteria being isolated from nodules. So far, these include Burkholderia tuberum strain STM678 and Burkholderia phymatum strain STM815 (originally isolated from Aspalathus carnosa in South Africa and Machaerium lunatum in French Guiana, respectively [26,39]), Ralstonia taiwanensis strains (isolated from Mimosa pudica in Taiwan and India and Mimosa diplotricha in Taiwan [8,41] and now renamed Cupriavidus taiwanensis [38]), and several Burkholderia strains isolated from Mimosa casta, Mimosa pigra (synonym, Mimosa pellita), M. pudica, and another mimosoid legume, Abarema macradenia, in Panama (3). Although symbiotic genes (nifH and nodA) have been identified in the Burkholderia strains STM678 and STM815, so far there is very little physiological and structural evidence of their symbiotic nature, and they have been shown to form only ineffective nodules on the promiscuous legume Macroptilium atropurpureum (26). More convincingly, not only have some of the Panamanian Burkholderia strains been shown to possess symbiosis-relat...

Repoils of i-iodiihition in tbt-LcKLiminosae an-c\aniiiK-il in tbe li.ulit ol curri-nt views on tlu-taxonomy of tbe family. In tile subfamilv-C'ac-salpinioideac, nodulation is largely restricted to the tribe Caesalpmieae and the Ljeiuis ('/tctiiittccristit lrom tlu-C'assieae. .All nodules studied have rhizobiii retained within infection threads diirinu the nitrogen lixin^ period. In tbe .Mimosoideae, nodulation is general, except lor 4 groups w ithin the tribe Mimoseae. and a verv-few species of Acacia. The onlv' tribe from tbe Papilionoideae which appears not to ncHlulate is the Diptery^eae, altbouyh the monogeneric luichresteae has not been examined. .\ tiumbei-of genera in the Swartzieae ilo not nodulate. Takint; the family as a whole, nodulation appears to be very iinitorm certain sections niHhilate, otbers do not.

No abstract