Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

BACKGROUND AND AIMS: Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). METHODS: Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. KEY RESULTS: Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a beta-rhizobial strain can effectively nodulate papilioinoid legumes. CONCLUSIONS: Papilionoid legumes from widely different tribes can be nodulated by beta-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.     

Beta-rhizobia from Mimosa pigra, a newly discovered invasive plant in Taiwan

A total of 191 rhizobial isolates from the root nodules of three geographically separate populations of the invasive plant Mimosa pigra in Taiwan were examined using amplified rDNA restriction analysis, 16S rDNA sequences, protein profiles and ELISA. Of these, 96% were identified as Burkholderia and 4% as Cupriavidus taiwanensis. The symbiosis-essential genes nodA and nifH were present in two strains of Burkholderia (PAS44 and PTK47), and in one of C. taiwanensis (PAS15). All three could nodulate M. pigra. Light and electron microscopy studies with a green fluorescent protein transconjugant variant of strain PAS44 showed the presence of fluorescent bacteroids in M. pigra nodules. These bacteroids expressed the nifH protein, hence this is the first confirmation that Burkholderia is a genuine symbiont of legume nodules. The predominance of Burkholderia in Taiwanese M. pigra suggests that this species may have brought its symbionts from its native South America, rather than entering into association with the Taiwanese Mimosa symbiont C. taiwanensis which so successfully nodulates Mimosa pudica and Mimosa diplotricha.