Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis

The molecular and physiological mechanisms behind the maturation and maintenance of N₂-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N₂-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N₂ fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C₄ metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules.     

A study of nodulation and nitrogen fixation of alder on the purplish soils in China

Summary The alder has a perennial nodule cluster. The nodule amount on the roots increases with tree age. The N₂-fixing activity of nodules decreases with nodule age. Purple coloured soils with various soil pHs and CaCO₃ contents are, in the main, the ones which influence nodulation and N₂-fixing. Higher N₂-fixing capacity existed in the neutral and low calcium soils. High calcium soils and acid soils can restrain nodulation and the N₂-fixing rate significantly. On the slope, where calcarous light loams are found, the annual nitrogen fixation capacity of alder and cypress mixed plantations, less than 10 years old, is 16 or 17 kg/ha yr, but in the valley, a pure alder plantation can reach 40 kg/ha yr.     

Alder and lupine enhance nitrogen cycling in a degraded forest soil in Northern Sweden

Positive effects of legumes and actinorhizal plants on N-poor soils have been observed in many studies but few have been done at high latitudes, which was the location of our study. We measured N₂ fixation and several indices of soil N at a site near the Arctic Circle in northern Sweden. More than 20 years ago lupine (Lupinus nootkatensis Donn) and gray alder (Alnus incana L. Moench) were planted on this degraded forest site. We measured total soil N, net N mineralization and nitrification with a buried bag technique, and fluxes of NH⁺ ₄ and NO^– ₃ as collected on ion exchange membranes. We also estimated N₂ fixation activity of the N₂-fixing plants by the natural abundance of ¹⁵N of leaves with Betula pendula Roth. as reference species. Foliar nitrogen in the N₂-fixing plants was almost totally derived from N₂ fixation. Plots containing N₂-fixing species generally had significantly higher soil N and N availability than a control plot without N₂-fixing plants. Taken together, all measurements indicated that N₂-fixing plants can be used to effectively improve soil fertility at high latitudes in northern Sweden.     

Assessing the symbiotic dependency of grain and tree legumes on N2 fixation for their N nutrition in five agro-ecological zones of Botswana

To assess the symbiotic dependency of grain and shrub/tree legumes within five agro-ecological zones of Botswana, fully expanded leaves of the test species were sampled from about 26 study sites within Ngwaketse, Gaborone, Central, Ghanzi and Kalahari agro-ecological zones. Isotopic analysis revealed significant differences in δ¹⁵N values of the grain legumes [cowpea (Vigna unguiculata L. Walp), Bambara groundnut (Vigna subterranea L. Verde.), and groundnut (Arachis hypogaea L.)] from the 26 farming areas in both 2005 and 2006. Estimates of %Ndfa of leaves also showed significant differences between farming areas, with cowpea deriving more than 50% of its N nutrition from symbiotic fixation. In terms of distribution, many more symbiotic shrub/tree species were found in the wetter Ngwaketse agro-zone compared to the fewer numbers in the drier Kalahari region. Acacias were the more dominant species at all sites. Leaf δ¹⁵N values of shrub/tree species also varied strongly across Botswana, with 11 out of 18 of these legumes deriving about 50%, or more, of their N from symbiotic N₂ fixation.Acacia caffra, in particular, obtained as much as 93.6% of its N nutrition from symbiotic fixation in the wetter Ngwaketse agro-zone. This study has shown that grain legumes sampled from farmer’s fields in Botswana obtained considerable amounts of their N from symbiotic fixation. We have also shown that shrub and tree legumes probably play an important role in the N economy of the savanna ecosystems in Botswana. However, the decline in the number of functional N₂-fixing shrub/tree legumes along an aridity gradient suggests that soil moisture is a major constraint to N₂ fixation in the tree legumes of Botswana.     

The regulation of symbiotic N2 fixation: a conceptual model of N feedback from the ecosystem to the gene expression level

Symbiotic nitrogen (N₂) fixation in legumes may give the host plant a distinct competitive advantage; at the same time it is mainly responsible for introducing N into terrestrial ecosystems which may ultimately benefit all organisms. Depending on environmental conditions, symbiotic N₂ fixation may be tuned to the plant's N demand or specifically inhibited (a disadvantage for plants which depend mainly on symbiotic N₂ fixation), or even prevented. Thus, the ecological range for symbiotic N₂ fixation can be narrower than that of the host plants. A shortage of mineral N is the only case in which adverse environmental conditions clearly favour symbiotic N₂ fixation. Variations in number or mass of nodules or nodule morphology are persistent features, that may represent one kind of regulation of N₂ fixation. In addition, varying O₂ permeability of nodules functions as a rapid and reversible control of N₂ fixation which may compensate partially or fully for poor nodulation. The plant's demand for symbiotically fixed N is thought to play a central role in modulating both nodulation and N₂ fixation activity; an N feedback mechanism is assumed. The control of symbiotic N₂ fixation operates through a series of ecophysiological triggers which are also influenced by complex interactions between legume plants and other organisms in the ecosystem. The proportion of legume biomass and the performance of symbiotic N₂ fixation in each individual legume are the main parameters which determine the amount of symbiotically fixed N introduced into a terrestrial ecosystem. The various triggers and N feedback mechanisms from the whole ecosystem to the gene expression level which regulate symbiotic N₂ fixation in terrestrial ecosystems are reviewed and discussed in terms of a conceptual model. Although the presented model is based primarily on our knowledge about the physiology of a few leguminous crop species and of ecosystem processes in managed, perennial grassland in temperate climatic conditions, it may stimulate thinking about functional relationships between symbiotic N₂ fixation and terrestrial ecosystems at various system levels.     

Nodules from Fynbos legume Virgilia divaricata have high functional plasticity under variable P supply levels

Legumes have the unique ability to fix atmospheric nitrogen (N₂) via symbiotic bacteria in their nodules but depend heavily on phosphorus (P), which affects nodulation, and the carbon costs and energy costs of N₂ fixation. Consequently, legumes growing in nutrient-poor ecosystems (e.g., sandstone-derived soils) have to enhance P recycling and/or acquisition in order to maintain N₂ fixation. In this study, we investigated the flexibility of P recycling and distribution within the nodules and their effect on N nutrition in Virgilia divaricata Adamson, Fabaceae, an indigenous legume in the Cape Floristic Region of South Africa. Specifically, we assessed tissue elemental localization using micro-particle-induced X-ray emission (PIXE), measured N fixation using nutrient concentrations derived from inductively coupled mass-spectrometry (ICP-MS), calculated nutrient costs, and determined P recycling from enzyme activity assays. Morphological and physiological features characteristic of adaptation to P deprivation were observed for V. divaricata. Decreased plant growth and nodule production with parallel increased root:shoot ratios are some of the plastic features exhibited in response to P deficiency. Plants resupplied with P resembled those supplied with optimal P levels in terms of growth and nutrient acquisition. Under low P conditions, plants maintained an increase in N₂-fixing efficiency despite lower levels of orthophosphate (Pi) in the nodules. This can be attributed to two factors: (i) an increase in Fe concentration under low P, and (ii) greater APase activity in both the roots and nodules under low P. These findings suggest that V. divaricata is well adapted to acquire N under P deficiency, owing to the plasticity of its nodule physiology     

On the efficiency of legume supernodulating mutants

The supernodulating mutants of legumes lack the internal regulation of the number of symbiotic root nodules that harbour N₂-fixing nodule bacteria. On one hand, these mutants represent an efficient tool for dramatic increase in the degree of rhizobial symbiosis development. The trait of released nodulation is often associated with the desirable resistance of nodule initiation and functioning to the inhibition by ambient nitrate. On the other hand, the more intense and stable atmospheric nitrogen fixation of supernodulated plants is devalued by plant growth depression that results from the disproportion between the photosynthetic capacity of the shoot and the catabolic demands of symbiotic nodules. The deleterious effects of excessive nodulation can be neutralised or alleviated by a breeding strategy aimed at creating an ideotype of N₂-fixing legume. The growth depression can be diminished by the reduction in the nodule number typical for supernodulators, that is, 6–10-fold of the wild type, to the level found permissive for the particular crop. This shift should be accompanied with breeding aimed at the increased photosynthetic capacity of the shoot. Forage varieties of legumes represent a reserve of high photosynthetic and shoot growth capacity, thanks to a long-term breeding history for green biomass accumulation. Moreover, the deleterious effects of supernodulation are less perceived after introgression into the background of forage varieties in view of different criteria in their evaluation, such as nitrogen accumulation and biomass production per crop area unit. The growth of supernodulators can be further corrected by breeding for auxiliary traits such as long-vine shoot architecture, a longer vegetation period and late flowering. The same strategy is applicable to the compensation for inherent pleiotropic changes in plant development, which are often associated with primarily symbiotic mutations. Supporting evidence for the efficiency of the described approach has already been reported.     

Nodulation of native woody legumes in Hong Kong, China

Woody legumes can play an important role in forest restoration on degraded land but the knowledge of woody legumes has lagged behind their uses. This study is a pioneer investigation to explore the ability of native woody legumes to form root nodules and fix nitrogen in Hong Kong. Nine sites of different habitat types were surveyed during both wet and dry seasons for two years. Young plants of woody legumes along studied transects were excavated. The patterns of nodulation and nodule morphology were recorded and the nitrogen fixing ability was tested by acetylene-reduction-assay. Twenty-eight species in 16 genera were examined, of which 20 species were nodulating and eight non-nodulating, including all six species in the Caesalpinioideae. Five species were new records to the world’s nodulation inventory. Bowringia callicarpa was a new species and genus examined, which was non-nodulating. The overall nodulation pattern was consistent with previous studies. Nodulation was more profuse in some shrub species while inconsistent in most tree species. Species with higher proportion of nodulated individual plants also tended to have more nodules in each plant. Spherical nodules were common in shrub and woody climber species whilst tree species usually had woody indeterminate nodules. Seasonal difference in the amount of senescent nodules was noted in most species. All the nodules tested by acetylene-reduction-assay were effectively nitrogen-fixing, with nitrogenase activity ranging from 4 μmol C₂H₄ g^?1 h^?1 to 20 μmol C₂H₄ g^?1 h^?1, which was comparable to other tropical tree species. The findings in nodulation pattern and nitrogen fixing ability of these species are essential in their application in forest restoration on degraded lands.     

Facultative nitrogen fixation by canopy legumes in a lowland tropical forest

Symbiotic dinitrogen (N₂) fixation is often invoked to explain the N richness of tropical forests as ostensibly N₂-fixing trees can be a major component of the community. Such arguments assume N₂ fixers are fixing N when present. However, in laboratory experiments, legumes consistently reduce N₂ fixation in response to increased soil N availability. These contrasting views of N₂ fixation as either obligate or facultative have drastically different implications for the N cycle of tropical forests. We tested these models by directly measuring N₂-fixing root nodules and nitrogenase activity of individual canopy-dominant legume trees (Inga sp.) across several lowland forest types. Fixation was substantial in disturbed forests and some gaps but near zero in the high N soils of mature forest. Our findings suggest that canopy legumes closely regulate N₂ fixation, leading to large variations in N inputs across the landscape, and low symbiotic fixation in mature forests despite abundant legumes.     

Natural 15N abundance of presumed N2-fixing and non-N2-fixing plants from selected ecosystems

Summary The ¹⁵N/¹⁴N ratios of plant and soil samples from Northern California ecosystems were determined by mass spectrometry. The ¹⁵N abundance of 176 plant foliar samples averaged 0.0008 atom % ¹⁵N excess relative to atmospheric N₂ and ranged from-0.0028 to 0.0064 atom % ¹⁵N excess relative to atmospheric N₂. Foliage from reported N₂-fixing species had significantly lower mean ¹⁵N abundance (relative to atmospheric N₂ and total soil N) and significantly higher N concentration (% N dry wt.) than did presumed non-N₂-fixing plants growing on the same sites. The mean difference between N₂-fixing species and other plants was 0.0007 atom % ¹⁵N. N₂-fixing species had lower ¹⁵N abundance than the other plants on most sites examined despite large differences between sites in vegetation, soil, and climate. The mean ¹⁵N abundance of N₂-fixing plants varied little between sites and was close to that of atmospheric N₂. The ¹⁵N abundance of presumed non-N₂-fixing species was highest at coastal sites and may reflect an input of marine spray N having relatively high ¹⁵N abundance. The ¹⁵N abundance of N₂-fixing species was not related to growth form but was for other plants. Annual herbaceous plants had highest ¹⁵N abundance followed in decreasing order by perennial herbs, shrubs, and trees. Several terrestrial ferns (Pteridaceae) had ¹⁵N abundances comparable to N₂-fixing legumes suggesting N₂-fixation by these ferns. On sites where the ¹⁵N abundance of soil N differs from that of the atmosphere, N₂-fixing plants can be identified by the natural ¹⁵N abundance of their foliage. This approach can be useful in detecting and perhaps measuring N₂-fixation on sites where direct recovery of nodules is not possible.     

Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N₂-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191.     

Significance of nitrogen-fixing actinorhizal symbioses for restoration of depleted,degraded, and contaminated soil

Atmospheric nitrogen (N₂)-fixing legume trees are frequently used for the restoration of depleted, degraded, and contaminated soils. However, biological N₂ fixation (BNF) can also be performed by so-called actinorhizal plants. Actinorhizal plants include a high diversity of woody species and therefore can be applied in a broad spectrum of environments. In contrast to N₂-fixing legumes, the potential of actinorhizal plants for soil restoration remains largely unexplored. In this Opinion, we propose related basic research requirements for the characterization of environmental stress responses that determine the restoration potential of actinorhizal plants for depleted, degraded, and contaminated soils. We identify advantages and unexplored processes of actinorhizal plants and describe a mainly uncharted avenue of future research for this important group of plant species.     

Improvement of symbiotic nitrogen fixation in plants: molecular-genetic approaches and evolutionary models

Efficiency of symbiotic nitrogen fixation in legumes depends on bringing together the processes of N₂ fixation, assimilation of its products, supply of nitrogenase with energy, and development of nodule tissue and cellular structures. Coordination of these processes could arise from the evolutionary old functions of the nodules associated with deposition of the products of photosynthesis governed by systemic signals traveling between the above-ground organs and the roots. Further increase in symbiotic efficiency was associated with a pronounced ability to fix N₂ by intracellular bacteroids that lost capability to propagate (as observed in galegoid legumes from the tribes Viciae, Trifolieae, and Galegae producing indeterminate nodules). However, efficiency of these symbioses is restricted by a slow removal from the nodules of the products of N₂ fixation, which are assimilated along the same amide pathway as nitrogen compounds arriving from the soil. In legumes from the tribe Phaseoleae, such a restriction was overcome owing to a particular way of nitrogen assimilation via its incorporation into ureides (in determinate nodules). Development of symbioses where specialization of bacteroids in symbiotic fixation of atmospheric nitrogen is combined with its ureide assimilation will make it possible to produce new forms of plants highly efficient in symbiotic nitrogen fixation.     

新疆干旱区豆科植物结瘤的固氮特性

调查了新疆干旱区72种豆科植物的结瘤固氮活性,其中33种尚未见报道。这些植物所结根瘤在外形上多数不规则,以皮层厚和白色、棕色者居多,与非干旱区的根瘤形态显著不同。根瘤固氮活力相差较大,但比一般豆科植物根瘤活性高,最高者可达当地大豆根瘤的42倍。根瘤活性与宿主的抗逆境能力有关。此外,从11种豆科植物根瘤观察到10种具有吸氢活性。对干旱区豆科共生固氮生理生态的特性进行了讨论。     

Recent development and new insight of diversification and symbiosis specificity of legume rhizobia: mechanism and application

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N₂) into ammonia (NH₃) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a ‘1+n−N’ model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where ‘1’ refers to appreciate rhizobium; ‘+n’ means the addition of multiple trace elements and PGPR bacteria; and ‘−N’ implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches.     

N2-Fixation, nodule efficiency and biomass accumulation after two years in three Chilean legume trees and Tagasaste Chamaecytisus proliferus subsp. palmensis

Initial results of a long-term field experiment are presented for the above and below-ground biomass accumulation after two years, as well as root nodulation, nodule efficiency (g N fixed/g nodules) and biological N₂ fixation (using the ¹⁵N isotope dilution method) of four N₂-fixing tree species (NFTs) grown in the subhumid mediterranean-climate zone of central Chile. Two non-legume tree species, Fraxinus excelsior and Schinus polygamus, were used as reference plants for the isotope dilution calculations.Over two years, Tagasaste (Chamaecytisus proliferus subsp. palmensis, a Papilionoideae from the Canary Islands), produced 10 to 20 times more biomass than the other three NFTS (Acacia caven, Prosopis alba and P. chilensis); all Mimosoideae native to Chile, and nodulation and nitrogen fixed were an order of magnitude higher as well. At the end of the second year, the percentage of N derived from N₂ fixation (%Ndfa) in Tagasate averaged 85.6, equivalent to ca. 49.1 g N fixed per tree. For all four NFTs, however, %Ndfa, nodule efficiency, and total N accumulation varied from one year to the next; caution is thus required in interpreting or predicting NFT performance over the long term, even if Tagasaste can already be considered a highly promising NFT for central Chile.     

Studies on Genetic Male-Sterile Soybeans : IV. Effect of Male Sterility and Source of Nitrogen Nutrition on Accumulation, Partitioning, and Transport of Nitrogen

Soybean (Glycine max [L.] Merr.) germplasm, isogenic except for loci controlling male sterility (ms₁) and nodulation (rj₁), was used to investigate the effects of reproductive tissue development and source of nitrogen nutrition on accumulation, transport, and partitioning of nitrogen in a greenhouse experiment. Nodulated plants were supplied nitrogen-free nutrient solution, and nonnodulated plants were supplied nutrient solution containing 20 millimolar KNO₃. Plants were sampled from flowering until maturity (77 to 147 days after transplanting).

Accumulation rates of nitrogen in whole plants during reproductive growth were not significantly different among the four plant types. Nitrogen accumulation in the sterile, nonnodulated plants, however, ceased 2 weeks earlier than in fertile, nonnodulated or fertile and sterile, nodulated plants. This early cessation in nitrogen accumulation resulted in sterile, nonnodulated plants accumulating significantly less whole plant nitrogen by 133 days after transplanting (DAT) than fertile, nonnodulated plants. Thus, changing the site of nitrogen assimilation from nodules (N₂-fixing plants) to roots and leaves (NO₃-fed plants) resulted in similar whole-plant nitrogen accumulation rates in fertile and sterile plants, despite the absence of seed in the latter.

Leaflet and stem plus petiole tissues of both types of sterile plants had significantly higher nitrogen concentrations after 119 DAT than both types of fertile plants. Significantly higher concentrations and exudation rates of nonureide, reduced-nitrogen in xylem sap of sterile than of fertile plants after 105 DAT were observed. These latter results indicated possible cycling of nonureide, reduced-nitrogen from the downward phloem translocation stream to the upward xylem translocation stream in roots of sterile plants. Collectively, these results suggest a lack of sinks for nitrogen utilization in the shoots of sterile plants. Hence, comparison of nitrogen accumulation rates for sterile and fertile plants does not provide a definitive test of the hypothesis that reproductive tissue development limits photosynthate availability for support of N₂ fixation and nitrate assimilation in determinate soybeans.

Nitrogen assimilation during reproductive growth met a larger proportion of the reproductive-tissue nitrogen requirement of nitrate-dependent plants (73%) than of N₂-fixing plants (63%). Hence, vegetative-tissue nitrogen mobilization to reproductive tissue was a more prominent process in N₂-fixing than in nitrate-dependent plants. N₂-fixing plants partitioned nitrogen to reproductive tissue more efficiently than nitrate-dependent plants as the reproductive tissues of the former and latter contained 65 and 55%, respectively, of the whole-plant nitrogen at the time that nitrogen accumulation in reproductive parts had ceased (133 DAT).

     

Nodulation in Dimorphandra wilsonii Rizz. (Caesalpinioideae), a Threatened Species Native to the Brazilian Cerrado

The threatened caesalpinioid legume Dimorphandra wilsonii, which is native to the Cerrado biome in Brazil, was examined for its nodulation and N₂-fixing ability, and was compared with another, less-threatened species, D. jorgei. Nodulation and potential N₂ fixation was shown on seedlings that had been inoculated singly with five bradyrhizobial isolates from mature D. wilsonii nodules. The infection of D. wilsonii by two of these strains (Dw10.1, Dw12.5) was followed in detail using light and transmission electron microscopy, and was compared with that of D. jorgei by Bradyrhizobium strain SEMIA6099. The roots of D. wilsonii were infected via small transient root hairs at 42 d after inoculation (dai), and nodules were sufficiently mature at 63 dai to express nitrogenase protein. Similar infection and nodule developmental processes were observed in D. jorgei. The bacteroids in mature Dimorphandra nodules were enclosed in plant cell wall material containing a homogalacturonan (pectic) epitope that was recognized by the monoclonal antibody JIM5. Analysis of sequences of their rrs (16S rRNA) genes and their ITS regions showed that the five D. wilsonii strains, although related to SEMIA6099, may constitute five undescribed species of genus Bradyrhizobium, whilst their nodD and nifH gene sequences showed that they formed clearly separated branches from other rhizobial strains. This is the first study to describe in full the N₂-fixing symbiotic interaction between defined rhizobial strains and legumes in the sub-family Caesalpinioideae. This information will hopefully assist in the conservation of the threatened species D. wilsonii.