Proof that Burkholderia strains form effective symbioses with legumes: a study of novel Mimosa-nodulating strains from South America

Twenty Mimosa-nodulating bacterial strains from Brazil and Venezuela, together with eight reference Mimosa-nodulating rhizobial strains and two other beta-rhizobial strains, were examined by amplified rRNA gene restriction analysis. They fell into 16 patterns and formed a single cluster together with the known beta-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 symbiosis-related 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 N2-fixing symbioses. Finally, both wild-type and green fluorescent protein-expressing transconjugant strains of Br3461 and MAP3-5 produced N2-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.     

Burkholderia phymatum Strains Capable of Nodulating Phaseolus vulgaris Are Present in Moroccan Soils

Phylogenetic analysis of 16S rRNA, nodC, and nifH genes of four bacterial strains isolated from root nodules of Phaseolus vulgaris grown in Morocco soils were identified as Burkholderia phymatum. All four strains formed N₂-fixing nodules on P. vulgaris and Mimosa, Acacia, and Prosopis species and reduced acetylene to ethylene when cultured ex planta.Until 2001 all known bacteria involved in root nodule symbioses with leguminous plants were classified as members of the order Rhizobiales of the Alphaproteobacteria, including Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium (28, 36, 38). Moulin et al. (21), however, described two Burkholderia nodule-forming strains isolated from Machaerium lunatum in French Guiana and Aspalathus carnosa in South Africa, respectively, this being the first report on the presence of a betaproteobacterium within root nodules of legumes. Later, the strains were formally classified as Burkholderia phymatum STM815^T and Burkholderia tuberum STM678^T, respectively (33). Burkholderia species are the predominant isolates from nodules of mimosoid legumes from Panama (2), Costa Rica (3), Taiwan (4, 6), Brazil (5, 7), Venezuela (5), and Madagascar (24), which indicates a high affinity of Burkholderia for forming effective symbioses with Mimosa. Diazotrophy is well represented in Burkholderia; among the more than 55 species presently classified as Burkholderia, 9 have been shown to fix N₂ ex planta by using either the acetylene reduction activity (ARA) assay or the presence of nifH genes encoding nitrogenase reductase (3, 5, 11, 24) and more recently by ¹⁵N₂ isotopic dilution experiments (17).Common bean (Phaseolus vulgaris) is an herbaceous leguminous plant which establishes N₂-fixing symbiosis with at least 5 species of the genus Rhizobium. Rhizobium etli is the predominant species in America (29) and is also detected in Europe and Africa (13, 20). Rhizobium leguminosarum bv. phaseoli is commonly found in Europe (13) and has also been reported to be present in Tunisia (20) and Colombia (10). Rhizobium tropici is found in acid soils of South America and is also present in Europe and several African countries (18). Rhizobium giardinii has been detected only in European and Tunisian soils (1, 20), and Rhizobium gallicum has been found nodulating beans in Europe, North Africa, and Mexico (1, 13). In this study we report on the isolation and characterization of B. phymatum from root nodules of P. vulgaris grown in alkaline soils from Morocco. Our results show that strains formed effective nodules on species of Mimosa, Acacia, and Prosopis and fixed atmospheric N₂ under free-living conditions.Soil was taken from a field near Oulade Mansour (34°47′N, 2°15′W, Oujda province, Morocco) where maize and common bean have traditionally been grown as rotational crops without N fertilization. Soil had a sandy-clay texture and the following characteristics: pH (in water), 8.1; 55.18% sand; 17.17% silt; 27.65% clay; 6.1% carbonates; 7.69% organic carbon; 0.069 total nitrogen. Seeds of P. vulgaris cv. Flamingo were surface sterilized (96% ethanol for 30 s followed by immersion in 15% [vol/vol] H₂O₂ for 8 min), washed several times with sterile water, germinated in the dark, and planted in 1-kg pots containing soil and sterile sand (1:1, vol/vol). Plants were grown for 30 days in controlled environmental chambers under conditions previously described (9). Nodules were collected, pooled together, surface sterilized with 2.5% HgCl₂ for 5 min, and rinsed thoroughly with sterile distilled water. Then, 12 nodules were placed independently on petri dishes and crushed in a drop of sterile water with a sterile glass rod. The resulting suspension was streaked onto petri dishes containing either yeast extract-mannitol (YEM) medium (35) or peptone-mineral salts-yeast extract (PSY) medium (25). After incubation of the plates at 30°C for 7 days, CFU which represented all of the colony types that could be distinguished by microscopic observation of living cells were chosen. After identification, Burkholderia strains were routinely grown in BAc medium (12).For DNA extraction and PCR amplifications, genomic DNA was isolated from bacterial cells using the RealPure genomic DNA extraction kit (Durviz, Spain) according to the manufacturer''s instructions. Repetitive extragenic palindromic (REP) fingerprinting was performed using primers REPIR-I and REP2-I according to the method of de Bruijn (8). PCR amplifications of the 16S rRNA gene fragments were done with the Bphym-F and Bphym-R species-specific primer pair (37).After isolation from root nodules, 52 strains forming morphologically different colonies were obtained and grouped in two main clusters after REP-PCR fingerprinting (data not shown), a technique that is extensively used to group bacteria at subspecies or strain level (8, 34) and has proven to be a powerful tool for studies of microbial ecology and evolution (14). The nearly complete sequence of the 16S rRNA gene from a representative strain of each REP-PCR group was obtained and compared with those held in GenBank. Forty-six strains in cluster I were members of the family Rhizobiaceae from the Alphaproteobacteria. Another 4 strains, here referred to as GR strains (GR01 to GR04), grouped in cluster II and were classified into the family Burkholderiaceae within the Betaproteobacteria. The remaining two strains have not been clearly classified as yet. The four GR strains have almost identical 16 rRNA gene sequences, and BLAST searches showed that they were phylogenetically close (99% identity) to B. phymatum STM815^T, a strain originally isolated from the papilionoid legume Machaerium lunatum (21, 33). A phylogenetic analysis including 30 Burkholderia reference strains showed that strains from root nodules of P. vulgaris form a tight cluster with B. phymatum STM815^T (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Phylogenetic tree showing the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia based on 16S rRNA gene sequence comparisons. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses. The bar represents one nucleotide substitution per 1,000 nucleotides. The multiple alignments of the sequences were performed with CLUSTAL W software (30). The tree topology was inferred by the neighbor-joining method (27), based on 1,310 DNA sites, and the distance matrix method was performed according to the method of Jukes and Cantor (15) using the program MEGA version 2.1 (16).The nodC gene was amplified with the primer pairs and conditions previously described (11). Amplification of the nodC gene from each GR strain yielded a DNA fragment of about 0.4 kb (data not shown) whose nucleotide sequences were identical for all four strains and showed 99% identity to those of B. phymatum strains STM815^T and NGR195A (11). A phylogenetic tree inferred from NodC sequences from B. phymatum strains and members of the order Rhizobiales is shown in Fig. ​Fig.2.2. Primers IGK (23) and NDR-1 (31) were used for amplification of the nifH genes as indicated earlier (22). PCR amplifications of the nifH gene and further sequencing from each GR strain revealed that they all had almost identical DNA sequences, which were 99% identical to those of B. phymatum STM815^T and NGR195A (11). A phylogenetic tree based on NifH sequences showing the relationships between B. phymatum and other Burkholderia and rhizobial species is shown in Fig. ​Fig.33.Open in a separate windowFIG. 2.Phylogenetic tree inferred from NodC sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.Open in a separate windowFIG. 3.Phylogenetic tree inferred from NifH sequences shows the positions of four P. vulgaris-isolated strains, GR01, GR03, GR05, and GR06, within the genus Burkholderia. The tree topology was inferred by the neighbor-joining method (27) based on 195 sites. The bar represents the number of amino acid substitutions per site. One thousand bootstrap samplings were performed. The NCBI GenBank accession number for each strain is shown in parentheses.For nodulation tests, seeds of Glycine max, Cicer arietinum, Pisum sativum, Lens culinaris, Lotus corniculatus, and Medicago sativa were surface sterilized as described above for common beans. Seeds of Mimosa, Leucaena, Prosopis, and Acacia were surface sterilized with concentrated sulfuric acid for 10 min followed by 3% sodium hypochlorite for 10 min and then washed thoroughly with sterile water. Plant cultivation was carried out as indicated above. Acetylene reduction activity (ARA) by nodulated plants was assayed on detached root systems excised at the cotyledonary node as previously described (19). The GR strains are true symbionts of P. vulgaris as, after nodule isolation, they were able to establish new effective symbiosis with common beans, with values of ARA ranging from 492 to 525 μmol ethylene/plant/h. B. phymatum STM815^T also infected P. vulgaris, but the efficiency of the symbiosis, determined as plant dry weight (1.06 ± 0.18 g/plant/h), was half of that found in plants nodulated by the GR strains. These strains also nodulated Mimosa pigra, Acacia cochliacantha, Acacia bilimeki, Leucaena glauca, and Prosopis laevigata but were unable to form nodules on P. sativum, L. culinaris, L. corniculatus, M. sativa, G. max, and C. arietinum.Diazotrophy is common among Burkholderia species, as shown recently by N₂ isotopic dilution studies (17). Under free-living conditions, ARA by the GR strains was tested in semisolid JMV medium as indicated earlier (26). At the end of the experiments, the culture purity was routinely checked by plating to verify uniform colony morphology. Like strain STM815^T, strains isolated from P. vulgaris also had nitrogenase activity when grown ex planta. Values of activity, however, were about half of that detected in strain STM815^T (83 ± 15 nmol C₂H₄/h).Preparation of whole-cell proteins and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) assays were performed as described previously (12). Protein profiles were compared with type and reference strains of legume-nodulating Burkholderia species. P. vulgaris-isolated strains showed SDS-PAGE protein profiles (evaluated by visual comparison) almost identical to those from the STM815^T type strain of B. phymatum but clearly different from those of other legume-nodulating Burkholderia species (Fig. ​(Fig.4).4). No differences were found when API 20 NE and API 50 CH strips were used to check for differences in nitrogen and carbon sources between B. phymatum STM815^T and the GR strains (data not shown).Open in a separate windowFIG. 4.Protein electrophoregrams (SDS-PAGE) of P. vulgaris-isolated strains and type strains of known legume-nodulating Burkholderia species. Lane 1, B. tuberum STM678^T; lane 2, B. phymatum STM815^T; lanes 3 to 6, B. phymatum GR01, GR03, GR04, and GR06, respectively; lane 7, B. mimosarum PAS44^T; lane 8, B. nodosa Br3437^T.Based on 16S rRNA gene sequences and protein profiles, which provide strong evidence for the delineation of bacterial species (32), the Phaseolus-isolated strains could be assigned to the species B. phymatum. Moreover, analysis of the phylogenetic relationships of such sequences and other Burkholderia species showed that they formed a robust clade with B. phymatum STM815^T. In addition, sequencing of the nodC and nifH genes revealed that the phylogenetically closest bacterial species was B. phymatum STM815^T. All these results support the affiliation of Phaseolus-isolated strains as B. phymatum. Since genomic DNAs from the GR strains had the same DNA band pattern after REP-PCR fingerprinting and extremely similar 16S rRNA and nifH gene sequences, as well as identical nodC sequences, the four strains could be derived from a single clone. Our results also suggest that strains of B. phymatum isolated from Mimosa and Phaseolus have acquired their symbiosis genes either from a common ancestor or by lateral transfer between them, the direction of transfer being unknown. Although limited to three isolates, strains NGR114 and NGR195A from Mimosa invisa and Mimosa pudica in Papua New Guinea, respectively (11), and STM815^T from M. lunatum in French Guiana (21, 33), and four strains from P. vulgaris in Morocco, our results raise questions concerning the biogeographical, environmental, and host taxon distribution of B. phymatum nodule symbionts. B. phymatum was originally discovered in nodules from M. lunatum in French Guiana, and most other strains in this lineage have been found associated with host legumes in the genus Mimosa, primarily in the Neotropics. Thus, the current results extend both the known host distribution and geographic range of this group of nodule symbionts. Whether B. phymatum is prevalent on rhizobial species within nodules of P. vulgaris in the geographic site where soil samples were taken cannot be elicited from the present results. Accordingly, it will be important in future work to survey additional sites both within the native geographic range and elsewhere to understand the consistency of the association between Phaseolus and Burkholderia.     

An invasive Mimosa in India does not adopt the symbionts of its native relatives

Background and Aims

The large monophyletic genus Mimosa comprises approx. 500 species, most of which are native to the New World, with Central Brazil being the main centre of radiation. All Brazilian Mimosa spp. so far examined are nodulated by rhizobia in the betaproteobacterial genus Burkholderia. Approximately 10 Mya, transoceanic dispersal resulted in the Indian subcontinent hosting up to six endemic Mimosa spp. The nodulation ability and rhizobial symbionts of two of these, M. hamata and M. himalayana, both from north-west India, are here examined, and compared with those of M. pudica, an invasive species.

Methods

Nodules were collected from several locations, and examined by light and electron microscopy. Rhizobia isolated from them were characterized in terms of their abilities to nodulate the three Mimosa hosts. The molecular phylogenetic relationships of the rhizobia were determined by analysis of 16S rRNA, nifH and nodA gene sequences.

Key Results

Both native Indian Mimosa spp. nodulated effectively in their respective rhizosphere soils. Based on 16S rRNA, nifH and nodA sequences, their symbionts were identified as belonging to the alphaproteobacterial genus Ensifer, and were closest to the ‘Old World’ Ensifer saheli, E. kostiensis and E. arboris. In contrast, the invasive M. pudica was predominantly nodulated by Betaproteobacteria in the genera Cupriavidus and Burkholderia. All rhizobial strains tested effectively nodulated their original hosts, but the symbionts of the native species could not nodulate M. pudica.

Conclusions

The native Mimosa spp. in India are not nodulated by the Burkholderia symbionts of their South American relatives, but by a unique group of alpha-rhizobial microsymbionts that are closely related to the ‘local’ Old World Ensifer symbionts of other mimosoid legumes in north-west India. They appear not to share symbionts with the invasive M. pudica, symbionts of which are mostly beta-rhizobial.     

Rapid identification of nitrogen-fixing and legume-nodulating Burkholderia species based on PCR 16S rRNA species-specific oligonucleotides

Several novel N₂-fixing Burkholderia species associated with plants, including legume-nodulating species, have recently been discovered. Presently, considerable interest exists in studying the diazotrophic Burkholderia species, both for their ecology and their great potential for agro-biotechnological applications. However, the available methods used in the identification of these Burkholderia species are time-consuming and expensive. In this study, PCR species-specific primers based on the 16S rRNA gene were designed, which allowed rapid, easy, and correct identification of most known N₂-fixing Burkholderia. With this approach, type and reference strains of Burkholderia kururiensis, B. unamae, B. xenovorans, B. tropica, and B. silvatlantica, as well as the legume-nodulating B. phymatum, B. tuberum, B. mimosarum, and B. nodosa, were unambiguously identified. In addition, the PCR species-specific primers allowed the diversity of the diazotrophic Burkholderia associated with field-grown tomato and sorghum plants to be determined. B. tropica and B. xenovorans were the predominant species found in association with tomato, but the occurrence of B. tropica with sorghum plants was practically exclusive. The efficiency of the species-specific primers was validated with the detection of B. tropica and B. xenovorans from DNA directly recovered from tomato rhizosphere soil samples. Additionally, using PCR species-specific primers, all of the legume-nodulating Burkholderia were correctly identified, even from single nodules collected from inoculated common bean plants. These primers could contribute to rapid identification of the diazotrophic and nodulating Burkholderia species associated with important crop plants and legumes, as well as revealing their environmental distribution.     

Burkholderia sp. Induces Functional Nodules on the South African Invasive Legume Dipogon lignosus (Phaseoleae) in New Zealand Soils

The South African invasive legume Dipogon lignosus (Phaseoleae) produces nodules with both determinate and indeterminate characteristics in New Zealand (NZ) soils. Ten bacterial isolates produced functional nodules on D. lignosus. The 16S ribosomal RNA (rRNA) gene sequences identified one isolate as Bradyrhizobium sp., one isolate as Rhizobium sp. and eight isolates as Burkholderia sp. The Bradyrhizobium sp. and Rhizobium sp. 16S rRNA sequences were identical to those of strains previously isolated from crop plants and may have originated from inocula used on crops. Both 16S rRNA and DNA recombinase A (recA) gene sequences placed the eight Burkholderia isolates separate from previously described Burkholderia rhizobial species. However, the isolates showed a very close relationship to Burkholderia rhizobial strains isolated from South African plants with respect to their nitrogenase iron protein (nifH), N-acyltransferase nodulation protein A (nodA) and N-acetylglucosaminyl transferase nodulation protein C (nodC) gene sequences. Gene sequences and enterobacterial repetitive intergenic consensus (ERIC) PCR and repetitive element palindromic PCR (rep-PCR) banding patterns indicated that the eight Burkholderia isolates separated into five clones of one strain and three of another. One strain was tested and shown to produce functional nodules on a range of South African plants previously reported to be nodulated by Burkholderia tuberum STM678^T which was isolated from the Cape Region. Thus, evidence is strong that the Burkholderia strains isolated here originated in South Africa and were somehow transported with the plants from their native habitat to NZ. It is possible that the strains are of a new species capable of nodulating legumes.     

Complete Genome sequence of Burkholderia phymatum STM815T,a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815^T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).     

Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp. Nodule Bacteria on two Mimosa spp. in Costa Rica

rRNA gene sequencing and PCR assays indicated that 215 isolates of root nodule bacteria from two Mimosa species at three sites in Costa Rica belonged to the genera Burkholderia, Cupriavidus, and Rhizobium. This is the first report of Cupriavidus sp. nodule symbionts for Mimosa populations within their native geographic range in the neotropics. Burkholderia spp. predominated among samples from Mimosa pigra (86% of isolates), while there was a more even distribution of Cupriavidus, Burkholderia, and Rhizobium spp. on Mimosa pudica (38, 37, and 25% of isolates, respectively). All Cupriavidus and Burkholderia genotypes tested formed root nodules and fixed nitrogen on both M. pigra and M. pudica, and sequencing of rRNA genes in strains reisolated from nodules verified identity with inoculant strains. Inoculation tests further indicated that both Cupriavidus and Burkholderia spp. resulted in significantly higher plant growth and nodule nitrogenase activity (as measured by acetylene reduction assays) relative to plant performance with strains of Rhizobium. Given the prevalence of Burkholderia and Cupriavidus spp. on these Mimosa legumes and the widespread distribution of these plants both within and outside the neotropics, it is likely that both β-proteobacterial genera are more ubiquitous as root nodule symbionts than previously believed.     

Nodulation and effective nitrogen fixation of Macroptilium atropurpureum (siratro) by Burkholderia tuberum, a nodulating and plant growth promoting beta-proteobacterium, are influenced by environmental factors

Background and aims

Burkholderia tuberum STM678^T was isolated from a South African legume, but did not renodulate this plant. Until a reliable host is found, studies on this and other interesting beta-rhizobia cannot advance. We investigated B. tuberum STM678^T’s ability to induce Fix⁺ nodules on a small-seeded, easy-to-propagate legume (Macroptilium atropurpureum). Previous studies demonstrated that B. tuberum elicited either Fix^- or Fix⁺ nodules on siratro, but the reasons for this difference were unexplored.

Methods

Experiments to promote effective siratro nodule formation under different environmental conditions were performed. B. tuberum STM678^T’s ability to withstand high temperatures and desiccation was checked as well as its potential for promoting plant growth via mechanisms in addition to nitrogen fixation, e.g., phosphate solubilization and siderophore production. Potential genes for these activities were found in the sequenced genomes.

Results

Higher temperatures and reduced watering resulted in reliable, effective nodulation on siratro. Burkholderia spp. solubilize phosphate and produce siderophores. Genes encoding proteins potentially involved in these growth-promoting activities were detected and are described.

Conclusions

Siratro is an excellent model plant for B. tuberum STM678^T. We identified genes that might be involved in the ability of diazotrophic Burkholderia species to survive harsh conditions, solubilize phosphate, and produce siderophores.     

Monophyly of nodA and nifH Genes across Texan and Costa Rican Populations of Cupriavidus Nodule Symbionts

nodA and nifH phylogenies for Cupriavidus nodule bacteria from native legumes in Texas and Costa Rica grouped all strains into a single clade nested among neotropical Burkholderia strains. Thus, Cupriavidus symbiotic genes were not acquired independently in different regions and are derived from other Betaproteobacteria rather than from α-rhizobial donors.     

Mixotrophic metabolism in Burkholderia kururiensis subsp. thiooxydans subsp. nov., a facultative chemolithoautotrophic thiosulfate oxidizing bacterium isolated from rhizosphere soil and proposal for classification of the type strain of Burkholderia kururiensis as Burkholderia kururiensis subsp. kururiensis subsp. nov.

A thiosulfate-oxidizing facultative chemolithoautotrophic Burkholderia sp. strain ATSB13^T was previously isolated from rhizosphere soil of tobacco plant. Strain ATSB13^T was aerobic, Gram-staining-negative, rod shaped and motile by means of sub-terminal flagellum. Strain ATSB13^T exhibited mixotrophic growth in a medium containing thiosulfate plus acetate. A phylogenetic study based on 16S rRNA gene sequence analysis indicated that strain ATSB13^T was most closely related to Burkholderia kururiensis KP23^T (98.7%), Burkholderia tuberum STM678^T (96.5%) and Burkholderia phymatum STM815^T (96.4%). Chemotaxonomic data [G+C 64.0 mol%, major fatty acids, C_18:1 ω7c (28.22%), C_16:1 ω7c/15 iso 2OH (15.15%), and C_16:0 (14.91%) and Q-8 as predominant respiratory ubiquinone] supported the affiliation of the strain ATSB13^T within the genus Burkholderia. Though the strain ATSB13^T shared high 16S rRNA gene sequence similarity with the type strain of B. kururiensis but considerably distant from the latter in terms of several phenotypic and chemotaxonomic characteristics. DNA–DNA hybridization between strain ATSB13^T and B. kururiensis KP23^T was 100%, and hence, it is inferred that strain ATSB13^T is a member of B. kururiensis. On the basis of data obtained from this study, we propose that B. kururiensis be subdivided into B. kururiensis subsp. kururiensis subsp. nov. (type strain KP23^T = JCM 10599^T = DSM 13646^T) and B. kururiensis subsp. thiooxydans subsp. nov. (type strain ATSB13^T = KACC 12758^T).     

Delineation of Paraburkholderia tuberum sensu stricto and description of Paraburkholderia podalyriae sp. nov. nodulating the South African legume Podalyria calyptrata

Since the discovery of Paraburkholderia tuberum, an indigenous South African species and one of the first beta-rhizobia described, several other South African rhizobial Paraburkholderia species have been recognized. Here, we investigate the taxonomic status of 31 rhizobial isolates from the root nodules of diverse South African legume hosts in the Core Cape Subregion, which were initially identified as P. tuberum. These isolates originate from the root nodules of genera in the Papilionoideae as well as Vachellia karroo, from the subfamily Caesalpinioideae. Genealogical concordance analysis of five loci allowed delineation of the isolates into two putative species clusters (A and B). Cluster A included P. tuberum STM678^T, suggesting that this monophyletic group represents P. tuberum sensu stricto. Cluster B grouped sister to P. tuberum and included isolates from the Paarl Rock Nature Reserve in the Western Cape Province. Average Nucleotide Identity (ANI) analysis further confirmed that isolates of Cluster A shared high genome similarity with P. tuberum STM678^T compared to Cluster B and other Paraburkholderia species. The members of Cluster B associated with a single species of Podalyria, P. calyptrata. For this new taxon we accordingly propose the name Paraburkholderia podalyriae sp. nov., with the type strain WC7.3b^T (= LMG 31413^T; SARCC 750^T). Based on our nodA and nifH phylogenies, P. podalyriae sp. nov. and strains of P. tuberum sensu stricto (including one from V. karroo) belong to symbiovar africana, the symbiotic loci of which have a separate evolutionary origin to those of Central and South American Paraburkholderia strains.     

Three Phylogenetic Groups of nodA and nifH Genes in Sinorhizobium and Mesorhizobium Isolates from Leguminous Trees Growing in Africa and Latin America

The diversity and phylogeny of nodA and nifH genes were studied by using 52 rhizobial isolates from Acacia senegal, Prosopis chilensis, and related leguminous trees growing in Africa and Latin America. All of the strains had similar host ranges and belonged to the genera Sinorhizobium and Mesorhizobium, as previously determined by 16S rRNA gene sequence analysis. The restriction patterns and a sequence analysis of the nodA and nifH genes divided the strains into the following three distinct groups: sinorhizobia from Africa, sinorhizobia from Latin America, and mesorhizobia from both regions. In a phylogenetic tree also containing previously published sequences, the nodA genes of our rhizobia formed a branch of their own, but within the branch no correlation between symbiotic genes and host trees was apparent. Within the large group of African sinorhizobia, similar symbiotic gene types were found in different chromosomal backgrounds, suggesting that transfer of symbiotic genes has occurred across species boundaries. Most strains had plasmids, and the presence of plasmid-borne nifH was demonstrated by hybridization for some examples. The nodA and nifH genes of Sinorhizobium teranga ORS1009^T grouped with the nodA and nifH genes of the other African sinorhizobia, but Sinorhizobium saheli ORS609^T had a totally different nodA sequence, although it was closely related based on the 16S rRNA gene and nifH data. This might be because this S. saheli strain was originally isolated from Sesbania sp., which belongs to a different cross-nodulation group than Acacia and Prosopis spp. The factors that appear to have influenced the evolution of rhizobial symbiotic genes vary in importance at different taxonomic levels.     

The Tomato Rhizosphere, an Environment Rich in Nitrogen-Fixing Burkholderia Species with Capabilities of Interest for Agriculture and Bioremediation

Burkholderia strains are promising candidates for biotechnological applications. Unfortunately, most of these strains belong to species of the Burkholderia cepacia complex (Bcc) involved in human infections, hampering potential applications. Novel diazotrophic Burkholderia species, phylogenetically distant from the Bcc species, have been discovered recently, but their environmental distribution and relevant features for agro-biotechnological applications are little known. In this work, the occurrence of N₂-fixing Burkholderia species in the rhizospheres and rhizoplanes of tomato plants field grown in Mexico was assessed. The results revealed a high level of diversity of diazotrophic Burkholderia species, including B. unamae, B. xenovorans, B. tropica, and two other unknown species, one of them phylogenetically closely related to B. kururiensis. These N₂-fixing Burkholderia species exhibited activities involved in bioremediation, plant growth promotion, or biological control in vitro. Remarkably, B. unamae and B. kururiensis grew with aromatic compounds (phenol and benzene) as carbon sources, and the presence of aromatic oxygenase genes was confirmed in both species. The rhizospheric and endophyte nature of B. unamae and its ability to degrade aromatic compounds suggest that it could be used in rhizoremediation and for improvement of phytoremediation. B. kururiensis and other Burkholderia sp. strains grew with toluene. B. unamae and B. xenovorans exhibited ACC (1-aminocyclopropane-1-carboxylic acid) deaminase activity, and the occurrence of acdS genes encoding ACC deaminase was confirmed. Mineral phosphate solubilization through organic acid production appears to be the mechanism used by most diazotrophic Burkholderia species, but in B. tropica, there presumably exists an additional unknown mechanism. Most of the diazotrophic Burkholderia species produced hydroxamate-type siderophores. Certainly, the N₂-fixing Burkholderia species associated with plants have great potential for agro-biotechnological applications.     

Nodule Symbiosis of Invasive <Emphasis Type="Italic">Mimosa pigra</Emphasis> in Australia and in Ancestral Habitats: A Comparative Analysis

Ninety isolates of root nodule bacteria from an invasive Mimosa pigra population in Australia were characterized by PCR assays and by sequencing of ribosomal genes. All isolates belonged to the same bacterial genus (Burkholderia) that predominates on M. pigra in its native geographic range in tropical America. However, the Australian Burkholderia strains represented several divergent lineages, none of which had a close relationship to currently known Burkholderia strains in American M. pigra populations. Inoculation of M. pigra with Australian strains resulted in equal or higher plant growth and nodule nitrogenase activity (measured by acetylene reduction assays) relative to outcomes with bacteria from M. pigra’s native geographic region. The main difference in symbiotic phenotype for bacteria from the two regions involved responses to an alternate Mimosa host species: Central American strains failed to fix nitrogen in association with Mimosa pudica, while most Australian Burkholderia isolates tested had high nodule nitrogenase activity in association with both Mimosa species. Invasive M. pigra populations in Australia have therefore acquired a diverse assemblage of nodule bacteria that are effective nitrogen-fixing symbionts, despite having a broader host range and a distant genetic relationship to bacterial strains found in the plant’s ancestral region.     

Genetic Characterization of Cylindrospermopsis raciborskii (Cyanobacteria) Isolates from Diverse Geographic Origins Based on nifH and cpcBA-IGS Nucleotide Sequence Analysis

Isolates of the toxic, N₂-fixing species Cylindrospermopsis raciborskii from various geographic locations were analyzed with respect to their genetic diversity based on the nifH and cpcBA-IGS genes. Gene sequences clustered according to their geographic origin, with the nifH sequences separating into European, Australian, and American groups and the cpcBA-IGS sequences separating into American and European or Australian groups. PCR primers for both genes were designed to exclusively amplify DNA from Cylindrospermopsis species, and an additional primer set for cpcBA-IGS was designed to specifically amplify the American C. raciborskii strains.     

Brazilian species of Calliandra Benth. (tribe Ingeae) are nodulated by diverse strains of Paraburkholderia

The Chapada Diamantina in NE of Brazil is a biodiversity hotspot and a center of radiation for many Neotropical legume genera, such as Calliandra and Mimosa. The present study aimed to evaluate nodulation in Calliandra species endemic to various environments, and to characterize the diversity of their symbiotic rhizobia using housekeeping (16S rRNA, recA) and plasmid-borne, symbiosis-related (nifH and nodC) genes. The nodulation ability of selected isolates was assessed. All of the 126 bacterial isolates from 18 Calliandra species collected in six different vegetation types were identified as Paraburkholderia according to their housekeeping and symbiosis gene phylogenies. They were grouped in seven clades in relation to the dominant vegetation type in their native environments. The majority, particularly those from highland “campo rupestre” vegetation, were similar to Paraburkholderia nodosa, but had nodC genes identical to the Mimosa symbiont Paraburkholderia tuberum sv. mimosae. The other smaller groups were related to Paraburkholderia diazotrophica and Paraburkholderia sabiae, and some single strains were not close to any known species. The symbionts of Calliandra spp. in NE Brazil are Paraburkholderia strains closely-related to Mimosa symbionts from the same region. NE Brazil is a reservoir of symbiotic Paraburkholderia that have an affinity for genera in the Mimosoid clade.     

Burkholderia, a Genus Rich in Plant-Associated Nitrogen Fixers with Wide Environmental and Geographic Distribution

The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N₂-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N₂-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75^T. These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75^T. Although the ability to fix N₂ is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N₂-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N₂-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N₂-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.     

Nodulation in black locust by the Gammaproteobacteria Pseudomonas sp. and the Betaproteobacteria Burkholderia sp.

Nodulation abilities of bacteria in the subclasses Gammaproteobacteria and Betaproteobacteria on black locust (Robinia pseudoacacia) were tested. Pseudomonas sp., Burkholderia sp., Klebsiella sp., and Paenibacillus sp. were isolated from surface-sterilized black locust nodules, but their nodulation ability is unknown. The aims of this study were to determine if these bacteria are symbiotic. The species and genera of the strains were determined by RFLP analysis and DNA sequencing of 16S rRNA gene. Inoculation tests and histological studies revealed that Pseudomonas sp. and Burkholderia sp. formed nodules on black locust and also developed differentiated nodule tissue. Furthermore, a phylogenetic analysis of nodA and a BLASTN analysis of the nodC, nifH, and nifHD genes revealed that these symbiotic genes of Pseudomonas sp. and Burkholderia sp. have high similarities with those of rhizobial species, indicating that the strains acquired the symbiotic genes from rhizobial species in the soil. Therefore, in an actual rhizosphere, bacterial diversity of nodulating legumes may be broader than expected in the Alpha-, Beta-, and Gammaproteobacteria subclasses. The results indicate the importance of horizontal gene transfer for establishing symbiotic interactions in the rhizosphere.