Soybeans inoculated with root zone soils of Canadian native legumes harbour diverse and novel Bradyrhizobium spp. that possess agricultural potential

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains.Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga.Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain.A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics.Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans.Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus.Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation.     

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.     

Genetic diversity of root nodulating bacteria associated with Retama sphaerocarpa in sites with different soil and environmental conditions

The genetic diversity of root nodulating bacteria isolated from Retama sphaerocarpa was studied using BOX-A1R PCR and phylogenetic analysis of the 16S rRNA region, as well as the housekeeping genes atpD, glnII and recA. A total of 193 isolates were obtained from eight different sites with different soil and environmental conditions in the Iberian Peninsula. These isolates corresponded to 31 different strains that successfully nodulated R. sphaerocarpa seedlings in reinoculation trials. About one-third of the strains clustered with B. canariense or B. cytisi within Bradyrhizobium group I. The remaining strains clustered with B. elkanii/B. pachyrhizi within Bradyrhizobium group II or in separate clades that could represent new lineages. Based on the 16S rRNA and combined atpD + glnII + recA sequences, two to three lineages of root nodulating bacteria were found at each sampling site, except for Collado Garcia where five species were detected. B. canariense and B. elkanii/B. pachyrhizi were the most abundant species, whereas the least abundant were those related to B. retamae and a putative new lineage. B. canariense was found only in soils with neutral and acid pH, whereas B. retamae was the dominant species in alkaline soils.     

Molecular characterization of novel Bradyrhizobium strains nodulating Eriosema chinense and Flemingia vestita,important unexplored native legumes of the sub-Himalayan region (Meghalaya) of India

Root nodule bacterial strains were isolated from the little-studied legumes Eriosema chinense and Flemingia vestita (both in tribe Phaseoleae, Papilionoideae) growing in acidic soil of the sub-Himalayan region of the Indian state of Meghalaya (ME), and were identified as novel strains of Bradyrhizobium on the basis of their 16S rRNA sequences. Seven isolates selected on the basis of phenotypic characters and assessment of ARDRA and RAPD patterns were subjected to multilocus sequence analysis (MLSA) using four protein-coding housekeeping genes (glnII, recA, dnaK and gyrB). On the basis of 16S rRNA phylogeny as well as a concatenated MLSA five strains clustered in a single separate clade and two strains formed novel lineages within the genus Bradyrhizobium. The phylogenies of the symbiotic genes (nodA and nifH) were in agreement with the core gene phylogenies. It appears that genetically diverse Bradyrhizobium strains are the principal microsymbionts of these two important native legumes. The novel genotypes of Bradyrhizobium strains isolated in the present study efficiently nodulate the Phaseoloid crop species Glycine max, Vigna radiata and Vigna umbellata. These strains are genetically different from strains of Bradyrhizobium isolated earlier from a different agro-climatic region of India suggesting that the acidic nature of the soil, high precipitation and other local environmental conditions are responsible for the evolution of these newly-described Bradyrhizobium strains. In global terms, the sub-Himalayan region of India is geographically and climatically distinct and the Bradyrhizobium strains nodulating its legumes appear to be novel and potentially unique to the region.     

Rhizobial hitchhikers from Down Under: invasional meltdown in a plant–bacteria mutualism?

Aim This study analysed the diversity and identity of the rhizobial symbionts of co‐existing exotic and native legumes in a coastal dune ecosystem invaded by Acacia longifolia. Location An invaded coastal dune ecosystem in Portugal and reference bradyrhizobial strains from the Iberian Peninsula and other locations. Methods Symbiotic nitrogen‐fixing bacteria were isolated from root nodules of plants of the Australian invasive Acacia longifolia and the European natives Cytisus grandiflorus, Cytisus scoparius and Ulex europaeus. Total DNA of each isolate was amplified by polymerase chain reaction (PCR) with the primer BOX A1R. Subsequent PCR‐sequencing and phylogenetic analyses of the internal transcribed spacer region and the nifD and nodA genes were performed for all different strains. Results The four plant species analysed were nodulated by bacteria from three different Bradyrhizobium lineages, although most of the isolates belonged to the Bradyrhizobium japonicum lineage sensu lato. Ninety‐five per cent of the bradyrhizobia isolated from A. longifolia, C. grandiflorus and U. europaeus in the invaded ecosystem had nifD and nodA genes of Australian origin. Seven isolates obtained in this study define a new distinctive nifD group of Bradyrhizobium from western and Mediterranean Europe. Main conclusions These results reveal the introduction of exotic bacteria with the invasive plant species, their persistence in the new geographical area and the nodulation of native legumes by rhizobia containing exotic symbiotic genes. The disruption of native mutualisms and the mutual facilitation of the invasive spread of the introduced plant and bradyrhizobia could constitute the first report of an invasional meltdown documented for a plant–bacteria mutualism.     

Relationships of Bradyrhizobia from the Legumes Apios americana and Desmodium glutinosum

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.     

Genetic diversity of native soybean bradyrhizobia from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal

Soybean-nodulating bradyrhizobia are genetically diverse and are classified into different species. In this study, the genetic diversity of native soybean bradyrhizobia isolated from different topographical regions along the southern slopes of the Himalayan Mountains in Nepal was explored. Soil samples were collected from three different topographical regions with contrasting climates. A local soybean cultivar, Cobb, was used as a trap plant to isolate bradyrhizobia. A total of 24 isolates selected on the basis of their colony morphology were genetically characterized. For each isolate, the full nucleotide sequence of the 16S rRNA gene and ITS region, and partial sequences of the nifD and nodD1 genes were determined. Two lineages were evident in the conserved gene phylogeny; one representing Bradyrhizobium elkanii (71% of isolates), and the other representing Bradyrhizobium japonicum (21%) and Bradyrhizobium yuanmingense (8%). Phylogenetic analyses revealed three novel lineages in the Bradyrhizobium elkanii clade, indicating high levels of genetic diversity among Bradyrhizobium isolates in Nepal. B. japonicum and B. yuanmingense strains were distributed in areas from 2420 to 2660 m above sea level (asl), which were mountain regions with a temperate climate. The B. elkanii clade was distributed in two regions; hill regions ranging from 1512 to 1935 m asl, and mountain regions ranging from 2420 to 2660 m asl. Ten multi-locus genotypes were detected; seven among B. elkanii, two among B. japonicum, and one among B. yuanmingense-related isolates. The results indicated that there was higher species-level diversity of Bradyrhizobium in the temperate region than in the sub-tropical region along the southern slopes of the Himalayan Mountains in Nepal.     

NONRANDOM GENOTYPIC ASSOCIATIONS IN A LEGUME—BRADYRHIZOBIUM MUTUALISM

Genetically divergent lineages often coexist within populations of the annual legume Amphicarpaea bracteata. At one site dominated by two such lineages (termed biotypes “C” and “S”), isolates of root-nodule bacteria (Bradyrhizobium sp.) were sampled from both hosts and analyzed by enzyme electrophoresis. Symbiont populations on the two plant biotypes were highly distinct. Out of 15 bacterial multilocus genotypes detected (among 51 isolates analyzed), only one was shared in common by the two plant biotypes. Cluster analysis revealed three bacterial lineages (designated I, II, and III), with lineage I found exclusively on biotype C plants, and the two other lineages almost completely restricted to biotype S hosts. Laboratory inoculation tests indicated that lineage I bacteria were strictly specialized on biotype C hosts, forming few or no nodules on plants of the other host biotype. Bacterial lineages II and III were capable of forming nodules on both kinds of plants, but nodule numbers were often significantly higher on biotype S hosts. The nonrandom association between plant and bacterial lineages at this site implies that genetic diversity of hosts is an important factor in the maintenance of polymorphism within the symbiont population.     

Phenotypic and phylogenetic characterization of native peanut Bradyrhizobium isolates obtained from Córdoba, Argentina

Peanut is an economically important legume nodulated by slow-growing bacteria of the genus Bradyrhizobium. In this study, a collection of native slow-growing peanut rhizobial isolates from Argentina was obtained and characterized. The phenotypical characterization included the determination of the symbiotic properties, whereas the genetic and phylogenetic diversity was assessed through ERIC-PCR and sequencing of the internal transcribed spacer (ITS) region, as well as the dnaK and nodA genes. The results obtained indicated that peanut nodulating bradyrhizobia were phenotypically and genotypically diverse, and included locally adapted variants of B. yuanmingense and B. iriomotense carrying novel nodA alleles.     

Strains nodulating Lupinus albus on different continents belong to several new chromosomal and symbiotic lineages within Bradyrhizobium

In this work we analysed different chromosomal and symbiotic markers in rhizobial strains nodulating Lupinus albus (white lupin) in several continents. Collectively the analysis of their rrs and atpD genes, and 16S-23S intergenic spacers (ITS), showed that they belong to at least four chromosomal lineages within the genus Bradyrhizobium. Most isolates from the Canary Islands (near to the African continent) grouped with some strains isolated on mainland Spain and were identified as Bradyrhizobium canariense. These strains are divided into two ITS subgroups coincident with those previously described from isolates nodulating Ornithopus. The remaining strains isolated on mainland Spain grouped with most isolates from Chile (American continent) forming a new lineage related to Bradyrhizobium japonicum. The strains BLUT2 and ISLU207 isolated from the Canary Islands and Chile, respectively, formed two new lineages phylogenetically close to different species of Bradyrhizobium depending on the marker analyzed. The analysis of the nodC gene showed that all strains nodulating L. albus belong to the biovar genistearum; nevertheless they form four different nodC lineages of which lineage C is at present exclusively formed by L. albus endosymbionts isolated from different continents.     

Soybean biofertilization in sandy soils of Egypt using15N tracer technique

The effect of inoculating soybean plants withBradyrhizobium japonicum andAzospirillum brasilense either solely or in mixture, and of using different N fertilizer levels was studied in pot experiments. The nodulation of soybean grown in a sandy soil was enhanced by the inoculation while the highest nodule numbers and fresh mass, recorded at a N-fertilizer dose of 20 kg N/hm², decreased when the fertilizer quantity increased to 40 kg N/hm². By contrast, the dry mass of above-ground parts and the N uptake was increased with increasing N fertilizer level. A similar effect was observed for inoculation as compared with the uninoculated variant. Although the nodulating and non-nodulating soybeans has nearly the same dry mass, the nodulating isoline accumulated more N than the non-nodulating. The percentage of nitrogen derived from air (%, Ndfa) and estimated by isotope dilution (ID) or N difference method (DM) dropped with increasing N level from 10 to 40 kg N/hm². Dual inoculation resulted in a high per cent of fixed N₂ (42.5%) at 10 kg N/hm². Correlation between the ID and DM methods was found to be dependent on inoculation treatments. The amount of nitrogen utilized by nodulating soybean (FUE %) was enhanced as a function of inoculation withB. japonicum.     

Native bradyrhizobia from Los Tuxtlas in Mexico are symbionts of Phaseolus lunatus (Lima bean)

Los Tuxtlas is the northernmost rain forest in North America and is rich in Bradyrhizobium with an unprecedented number of novel lineages. ITS sequence analysis of legumes in polycultures from Los Tuxtlas led to the identification of Phaseolus lunatus and Vigna unguiculata in addition to Phaseolus vulgaris as legumes associated with maize in crops. Bacterial diversity of isolates from nitrogen-fixing nodules of P. lunatus and V. unguiculata was revealed using ERIC-PCR and PCR-RFLP of rpoB genes, and sequencing of recA, nodZ and nifH genes. P. lunatus and V. unguiculata nodule bacteria corresponded to bradyrhizobia closely related to certain native bradyrhizobia from the Los Tuxtlas forest and novel groups were found. This is the first report of nodule bacteria from P. lunatus in its Mesoamerican site of origin and domestication.     

Symbiotic Relationships of Legumes and Nodule Bacteria on Barro Colorado Island,Panama: A Review

New data on 129 bacterial isolates were analyzed together with prior samples to characterize community-level patterns of legume–rhizobial symbiosis on Barro Colorado Island (BCI), Panama. Nodules have been sampled from 24 BCI legume species in 18 genera, representing about one quarter of the legume species and one half of the genera on the island. Most BCI legumes associated exclusively with nodule symbionts in the genus Bradyrhizobium, which comprised 86.3% of all isolates (315 of 365). Most of the remaining isolates (44 of 365) belonged to the β-proteobacterial genus Burkholderia; these were restricted to two genera in the legume subfamily Mimosoideae. Multilocus sequence analysis indicated that BCI Bradyrhizobium strains were differentiated into at least eight lineages with deoxyribonucleic acid divergence of the same magnitude as found among currently recognized species in this bacterial genus. Two of these lineages were widely distributed across BCI legumes. One lineage was utilized by 15 host species of diverse life form (herbs, lianas, and trees) in 12 genera spanning two legume subfamilies. A second common lineage closely related to the taxon B. elkanii was associated with at least five legume genera in four separate tribes. Thus, BCI legume species from diverse clades within the family frequently share interaction with a few common lineages of nodule symbionts. However, certain host species were associated with unique symbiont lineages that have not been found on other coexisting BCI legumes. More comprehensive sampling of host taxa will be needed to characterize the overall diversity of nodule bacteria and the patterns of symbiont sharing among legumes in this community.     

Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils

Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia.     

The interaction of rockphosphate,Bradyrhizobium, vesicular-arbuscular mycorrhizae and phosphate-solubilizing microbes on soybean grown in a sub-Himalayan mollisol

A factorial design 2³ × 4 with two levels of Mussorie rockphosphate (RP) with or without vesicular-arbuscular mycorrhizal (VAM) fungi and Bradyrhizobium japonicum, and four treatments of phosphate-solubilizing microbes (PSM) Pseudomonas striata, Bacillus polymyxa, Aspergillus awamori was employed using Patharchatta sandy loam soil (Typic Hapludoll). The observations included mycorrhization, nodulation, grain and straw yield, N and P uptake, available soil P and the PSM population in the soil after crop harvest. Inoculation with endophytes alone caused about 70% root colonization. Addition of rockphosphate or inoculation with PSM, except B. polymyxa, stimulated root infection of native as well as introduced VAM endophytes. Application of RP or inoculation with Bradyrhizobium japonicum, mycorrhizal fungi or phosphate-solubilizing microorganisms significantly increased nodulation, N uptake, available soil P and the PSM population in the soil after the crop harvest. The grain and straw yields did not increase following RP addition or mycorrhizal inoculation but increased significantly after inoculation wit Bradyrhizobium or PSM. In general, the application of RP, Bradyrhizobium, VAM and PSM in combinations of any two or three resulted in significant increases in nodulation, plant growth, grain yield and uptake of N and P. Among the four factor interactions, rockphosphate, Bradyrhizobium and P. striata in the absence of VAM resulted in maximal nodulation, grain and straw yields and N uptake by soybean. The highest P uptake by soybean grain was recorded with Bradyrhizobium and A. awamori in the absence of rockphosphate and VAM. Generally, available soil P and PSM population after crop harvest were not significantly increased by the treatment combinations giving the maximal uptake of nutrients. However, they increased significantly in response to PSM, which produced no significant increase in total uptake of nutrients.Research paper no. 7498     

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznań University of Life Sciences Experiment Station in Gorzyń, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.     

Geographic structure of lineage associations in a plant-bacterial mutualism

Two species linked by a mutualistic relationship may evolve correlated population differentiation if there is long-term continuity of interactions between specific partners. This phenomenon was analyzed by multilocus enzyme electrophoresis on the annual legume Amphicarpaea bracteata and its nitrogen-fixing bacterial symbionts (Bradyrhizobium sp.) sampled from >20 sites over a 1000 km area. Three analyses indicated that genetic differentiation was correlated in the two organisms. First, the genetic distance between bacterial populations at each pair of sites was significantly positively related to the genetic distance between their host plant populations, as evaluated by the Mantel test. Second, a cluster analysis revealed that several divergent lineages were present both among plants and among bacteria. Bacterial lineages showed a highly nonrandom distribution across plant lineages that was consistent in each of two regions sampled. Finally, there were numerous cases where populations of the same plant lineage 1000 km apart harbored bacterial isolates with an identical multilocus genotype. Thus, despite recurrent opportunities for partner switching, particular genotypes of these two organisms associate consistently across multiple habitats throughout their geographic range.     

Variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> and <Emphasis Type="Italic">B. elkanii</Emphasis> Seven Years after Introduction of both the Exotic Microsymbiont and the Soybean Host in a Cerrados Soil

The plasticity of rhizobial genomes is far greater than previously thought, with complex genomic recombination events that may be accelerated by the often stressful environmental conditions of the tropics. This study aimed at evaluating changes in soybean rhizobia due to adaptation to inhospitable environmental conditions (high temperatures, drought, and acid soils) in the Brazilian Cerrados. Both the host plant and combinations of four strains of soybean Bradyrhizobium were introduced in an uncropped soil devoid of rhizobia capable of nodulating soybean. After the third year, seeds were not reinoculated. Two hundred and sixty-three isolates were obtained from nodules of field-grown soybean after the seventh year, and their morphological, physiological, serological, and symbiotic properties determined, followed by genetic analysis of conserved and symbiotic genes. B. japonicum strain CPAC 15 (same serogroup as USDA 123) was characterized as having high saprophytic capacity and competitiveness and by the seventh year represented up to 70% of the cultivable population, in contrast to the poor survival and competitiveness of B. japonicum strain CPAC 7 (same serogroup as CB 1809). In general, adapted strains had increased mucoidy, and up to 43% of the isolates showed no serological reaction. High variability, presumably resulting from the adaptation to the harsh environmental conditions, was verified in rep-PCR (polymerase chain reaction) profiles, being lower in strain CPAC 15, intermediate in B. elkanii, and higher in CPAC 7. Restriction fragment length polymorphism (RFLP)-PCR types of the 16S rDNA corresponded to the following: one type for B. elkanii species, two for B. japonicum, associated to CPAC 15 and CPAC 7, and unknown combinations of profiles. However, when nodC sequences and RFLP-PCR of the nifH region data were considered, only two clusters were observed having full congruence with B. japonicum and B. elkanii species. Combining the results, variability was such that even within a genetically more stable group (such as that of CPAC 15), only 6.4% of the isolates showed high similarity to the inoculant strain, whereas none was similar to CPAC 7. The genetic variability in our study seems to result from a variety and combination of events including strain dispersion, genomic recombination, and horizontal gene transfer. Furthermore, the genetic variability appears to be mainly associated with adaptation, saprophytic capacity, and competitiveness, and not with symbiotic effectiveness, as the similarity of symbiotic genes was higher than that of conserved regions of the DNA.     

DIVERGENCE IN SYMBIOTIC COMPATIBILITY IN A LEGUME-BRADYRHIZOBIUM MUTUALISM

Geographic variation in the mutualism between the legume Amphicarpaea bracteata and its nitrogen-fixing root nodule bacteria (Bradyrhizobium sp.) was analyzed by sampling genotypes from 11 sites separated by distances ranging from 0.6 km to more than 1000 km. Cross inoculation experiments revealed that plants were genetically differentiated in traits determining compatibility with mutualist partners from different sites. Combinations of plant and bacterial genotypes native to the same local habitat yielded 26% higher plant growth relative to non-native combinations (range across 4 experiments; 9% to 48%). Among non-native symbioses, plant growth was unrelated to the geographic distance between sites of plant and bacterial origin. However, compatibility varied significantly with the genetic distance among host populations (inferred by multilocus enzyme electrophoresis): genetically similar plants from separate sites showed superior growth with each other's mutualist partners. Nevertheless, the tree structure of population genetic similarity was not congruent in plants versus bacteria. This implies that adaptive variation in symbiotic compatibility has evolved without strictly parallel divergence in the two species.     

Rhizobitoxine: A phytotoxin of unknown function which is commonly produced by bradyrhizobia

Summary Fifty-six percent of 93 strains ofBradyrhizobium japonicum andBradyrhizobium sp. (various hosts) from diverse geographical areas were found to produce a chlorosis-inducing toxin. Toxin production was common among bradyrhizobia originating from the USA, Africa, Central America, and South America. Toxin produced by West African strains was compared with rhizobitoxine by cation exchange chromatography, paper chromatography, and soybean (Glycine max (L.) Merr.) bioassay. The comparison suggested that the chlorosis-inducing toxin produced by West African bradyrhizobia is rhizobitoxine. Purified toxin from a West AfricanBradyrhizobium sp. (Vigna) strain inhibited the growth ofBacillus subtilis on minimal medium. The growth inhibition was reduced by addition of yeast-extract or casamino acids but not by any of 21 individual amino acids, including methionine. The same toxin did not inhibit the growth of 14 Bradyrhizobium strains, including eight strains that did not produce toxin. Mixed inoculum experiments revealed that a toxin-producing West African strain could not assist toxin non-producingB. japonicum strains in nodulating non-nodulating (rj₁ rj₁) soybeans.