Evidence of Horizontal Transfer of Symbiotic Genes from a Bradyrhizobium japonicum Inoculant Strain to Indigenous Diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah Soil

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N₂-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.     

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.     

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of Crotalaria spp., Indigofera spp., Erythrina brucei and Glycine max growing in Ethiopia

Ethiopian Bradyrhizobium strains isolated from root nodules of Crotalaria spp., Indigofera spp., Erythina brucei and soybean (Glycine max) represented genetically diverse phylogenetic groups of the genus Bradyrhizobium. Strains were characterized using the amplified fragment length polymorphism fingerprinting technique (AFLP) and multilocus sequence analysis (MLSA) of core and symbiotic genes. Based on phylogenetic analyses of concatenated recA-glnII-rpoB-16S rRNA genes sequences, Bradyrhizobium strains were distributed into fifteen phylogenetic groups under B. japonicum and B. elkanii super clades. Some of the isolates belonged to the species B. yuanmingense, B. elkanii and B. japonicum type I. However, the majority of the isolates represented unnamed Bradyrhizobium genospecies and of these, two unique lineages that most likely represent novel Bradyrhizobium species were identified among Ethiopian strains. The nodulation nodA gene sequence analysis revealed that all Ethiopian Bradyrhizobium isolates belonged to nodA sub-clade III.3. Strains were further classified into 14 groups together with strains from Africa, as well as some originating from the other tropical and subtropics regions. Strains were also clustered into 14 groups in nodY/K phylogeny similarly to the nodA tree. The nifH phylogenies of the Ethiopian Bradyrhizobium were generally also congruent with the nodA gene phylogeny, supporting the monophyletic origin of the symbiotic genes in Bradyrhizobium. The phylogenies of nodA and nifH genes were also partially congruent with that inferred from the concatenated core genes sequences, reflecting that the strains obtained their symbiotic genes vertically from their ancestor as well as horizontally from more distantly related Bradyrhizobium species.     

Phylogenetic analyses of Bradyrhizobium strains nodulating soybean (Glycine max) in Thailand with reference to the USDA strains of Bradyrhizobium.

To elucidate the phylogenetic relationships between Thai soybean bradyrhizobia and USDA strains of Bradyrhizobium, restriction fragment length polymorphism (RFLP) analysis using the nifDK gene probe and sequencing of the partial 16S rRNA gene were performed. In our previous work, Thai isolates of Bradyrhizobium sp. (Glycine max) were separated clearly from Bradyrhizobium japonicum and Bradyrhizobium elkanii based on the RFLP analysis using the nodDYABC gene probe. RFLP analysis using the nifDK gene probe divided 14 Thai isolates and eight USDA strains of B. japonicum into different groups, respectively, but categorized into the same cluster. All of seven strains within these Thai isolates had the same sequence of the partial 16S rRNA gene, and it was an intermediate sequence between those of B. japonicum USDA 110 and B. elkanii USDA 76T. Furthermore, three USDA strains of B. japonicum, USDA of (B. japonicum ATCC 10324T), USDA 115 and USDA 129, had the same partial 16S rRNA gene sequence that seven Thai isolates had. These results suggest that Thai isolates of Bradyrhizobium sp. (Glycine max) are genetically distinct from USDA strains of B. japonicum and B. elkanii, but also indicate a close relationship between Thai isolates and USDA strains of B. japonicum.     

Biodiversity and biogeography of rhizobia associated with soybean plants grown in the North China Plain

As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.     

Expression and functional roles of Bradyrhizobium japonicum genes involved in the utilization of inorganic and organic sulfur compounds in free-living and symbiotic conditions

Strains of Bradyrhizobium spp. form nitrogen-fixing symbioses with many legumes, including soybean. Although inorganic sulfur is preferred by bacteria in laboratory conditions, sulfur in agricultural soil is mainly present as sulfonates and sulfur esters. Here, we show that Bradyrhizobium japonicum and B. elkanii strains were able to utilize sulfate, cysteine, sulfonates, and sulfur-ester compounds as sole sulfur sources for growth. Expression and functional analysis revealed that two sets of gene clusters (bll6449 to bll6455 or bll7007 to bll7011) are important for utilization of sulfonates sulfur source. The bll6451 or bll7010 genes are also expressed in the symbiotic nodules. However, B. japonicum mutants defective in either of the sulfonate utilization operons were not affected for symbiosis with soybean, indicating the functional redundancy or availability of other sulfur sources in planta. In accordance, B. japonicum bacteroids possessed significant sulfatase activity. These results indicate that strains of Bradyrhizobium spp. likely use organosulfur compounds for growth and survival in soils, as well as for legume nodulation and nitrogen fixation.     

Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.     

Variation in the nod gene RFLPs, nucleotide sequences of 16S rRNA genes, Nod factors, and nodulation abilities of Bradyrhizobium strains isolated from Thai Vigna plants

The analysis of nod genes and 16S rRNA gene regions, Nod factors, and nodulation abilities of Brady rhizobium strains isolated from tropical Thai Vigna species is reported. A total of 55 Bradyrhizobium strains isolated from two cultivated and six wild Vigna species growing in central and northern Thailand were evaluated. Thai Vigna spp. Bradyrhizobium strains showed higher levels of nod gene RFLP diversity compared with Thai soybean Brady rhizobium strains or temperate strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. Analysis of the 16S rRNA gene region using selected strains also suggests a high genetic diversity of the Thai Vigna-Bradyrhizobium association. Based on thin-layer chromatography analysis, Nod factors produced by tropical Thai Vigna spp. Brady rhizobium strains are more diverse than temperate Japanese and US strains of B. japonicum and B. elkanii. Thai Vigna spp. Bradyrhizobium strains showed variation in nodulation ability and affinity, estimated by the number of normal nodules versus green nodules in an inoculation study. There are some Bradyrhizobium-host combinations that could not form any nodules, suggesting that some genetic differentiation has evolved in their host range. However, most of the Thai Vigna spp. Bradyrhizobium strains formed nodules on the cultigens soybean (Glycine max), mungbean (Vigna radiata), azuki bean (Vigna angularis), and cowpea (Vigna unguiculata). This is the first study on Bradyrhizobium strains associated with a range of cultivated and wild Vigna and reveals that these Bradyrhizobium strains are diverse and may provide novel sources of useful variation for the improvement of symbiotic systems.     

Flavonoid-responsive nodY-lacZ expression in three phylogenetically different Bradyrhizobium groups

Previously, restriction fragment length polymorphism analysis using the nodD1YABC gene probe showed the genetic diversity of common nodD1ABC gene regions of Bradyrhizobium japonicum, Bradyrhizobium elkanii, and the Thai soybean Bradyrhizobium. The nodD1 sequences of representative strains of the 3 groups differed phylogenetically, suggesting that responses of NodD1 proteins of the 3 Bradyrhizobium groups to diverse flavonoids may differ. To confirm this hypothesis, 6 representative strains were chosen from the 3 Bradyrhizobium groups. Six reporter strains were constructed, all carrying the pZB32 plasmid, which contains a nod box and the nodY-lacZ fusion of B. japonicum USDA 110. Differences in nodY-lacZ expression among the strains in response to 37 flavonoid compounds at various concentrations were evaluated. Of those compounds, prunetin (4',5-dihydroxy-7-methoxyisoflavone) and esculetin (6,7-dihydroxycoumarin) were identified as Bradyrhizobium group-specific nod gene inducers. Esculetin showed nod gene induction activities unique to Thai Bradyrhizobium strains. The levels of nodY-lacZ induction among B. japonicum and Thai Bradyrhizobium strains increased with increasing concentration of prunetin, whereas, those in B. elkanii strains did not.     

Common soybean inoculant strains in Brazil are members of Bradyrhizobium elkanii.

The Brazilian inoculant strains 29W and 587 were found to be members of Bradyrhizobium elkanii primarily on the basis of 16S rRNA gene sequences identical to that of B. elkanii USDA76 and on the basis of reactivity with antibodies against serogroups 76 and 31, respectively. The agronomic consequences of using strains of B. elkanii as soybean inoculants are discussed.     

Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes

Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.     

Identification of Bradyrhizobium japonicum Nodule Isolates from Wisconsin Soybean Farms

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a more discriminating method than serotyping for identifying strains of Bradyrhizobium japonicum. Analysis of 543 nodule isolates from southeastern Wisconsin soybean farms revealed that none of the isolates were formed by any of the inoculant strains supplied by either of two inoculant companies. Twenty-nine indigenous strains and six inoculant strains were identified. Strain 61A76, the most competitive indigenous strain, formed 21% of the nodules. Indigenous strains 3030, 3058, 0336, and 3052 formed 15, 11, 9, and 9% of the nodules, respectively. These predominant strains were not associated with a particular soybean cultivar, soil type, or farm location.     

Diversity among Bradyrhizobium isolates nodulating yardlong bean and sunnhemp in Guam

AIMS: To isolate and characterize bradyrhizobia that nodulate yardlong bean and sunnhemp in Guam. METHODS AND RESULTS: Bradyrhizobia populations that nodulate yardlong bean and sunnhemp in Guam were examined for genetic diversity and their relatedness to Bradyrhizobium japonicum and B. elkanii reference strains. Genomic DNA of 58 isolates of Bradyrhizobium spp. was hybridized with B. japonicum nodY and B. elkanii nodK genes. Based on the hybridization patterns, the isolates were classified into three nodY-nodK hybridizing groups. Group I comprised the majority of the isolates and hybridized with nodY whereas group II isolates hybridized with nodK. The group III isolates, that did not hybridize with either nodY or nodK, formed nitrogen-fixing nodules on cowpea but did not nodulate soybean. DNA sequence analysis of a 280-bp fragment of the variable region of the 16S rRNA gene of a few group III isolates showed that these isolates were more similar to Bradyrhizobium spp. than to B. japonicum or B. elkanii. CONCLUSIONS: The majority of the isolates nodulating yardlong bean and sunnhemp in Guam are similar to B. japonicum, although some isolates are similar to Bradyrhizobium spp. that nodulate a miscellaneous group of legumes including cowpea. SIGNIFICANCE AND IMPACT OF THE STUDY: Since both yardlong bean and sunnhemp are nodulated by a range of bradyrhizobia, selection of superior strains may be based on nodulation effectiveness on both legumes.     

Diverse rhizobia that nodulate two species of Kummerowia in China

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.     

Conservation of type III secretion system genes in Bradyrhizobium isolated from soybean

The distribution of rhcRST genes encoding the type III secretion system (T3SS) in a collection of Bradyrhizobium strains was characterized by PCR and Southern blot hybridization. The polymorphism of the corresponding sequences amplified by PCR was characterized by RFLP and sequencing together with those available in the databank. Genomic group I is characterized by the presence of Bradyrhizobium elkanii strains and group II by the presence of B. japonicum and B. liaoningense strains. Highly conserved T3SS-like genes were detected by PCR in all Bradyrhizobium strains isolated from soybean belonging to genomic group II, and in none of the strains belonging to genomic group I. These data were confirmed by Southern blot hybridization that further indicated the presence of sequences showing similarity to the rhcRST sequence in B. elkanii strains. The high level of conservation of rhcRST among Bradyrhizobia of genomic group II and sharing the same host-plant suggests that T3SS-like genes might have undergone horizontal genetic transfer within this genomic group. When considering the three Rhizobiaceae genera, a clear congruence was recorded between the rhcRST, rRNA gene and ITS sequences in bacteria harbouring sequences encoding T3SS, suggesting a relatively ancient emergence of the T3SS in these genera.     

Methods To Alter the Recovery and Nodule Location of Bradyrhizobium japonicum Inoculant Strains on Field-Grown Soybeans

Three strains of Bradyrhizobium japonicum, I17, 110, and 61A76, were evaluated for their ability to form nodules on field-grown soybeans in soil with a highly competitive indigenous B. japonicum population. The predominant indigenous strain, 0336, in the field site used was unlike the more common isolates from Midwestern soils which belong to the 123 or 138 serogroups. This strain persisted in the soil for at least 30 years without any soybean crops. The three inoculant strains differed in their ability to compete with indigenous strains for nodule formation. Four different inoculation treatments were tested in three adjacent fields. When the amount of inoculum was increased, a higher proportion of nodules contained the inoculant strain. The most competitive inoculant strain was I17, a recent field isolate. Strain 61A76 was better than 110. There was no difference in recovery of the inoculant strains on the Hodgson or Corsoy soybean cultivars, nor was there a difference in recovery of the inoculant strains during the growing season. The vertical distribution of nodules containing the inoculant strains was affected by the method of adding the inoculant to the soil. Inoculant added to the seed furrow produced nodules mainly in the top region of the soybean root. Inoculant tilled into the soil produced nodules primarily in the bottom part of the root. The nodules that were produced in the bottom part of the root are younger and may contribute significant amounts of fixed nitrogen to the soybean during seed formation.     

Diversity and relationships of Bradyrhizobia from Amphicarpaea bracteata based on partial nod and ribosomal sequences.

Partial sequences of three nod genes (nodC, nodD1, and nodA 5' flanking region) and of 16S and 23S rDNA were obtained from isolates of Bradyrhizobium sp. associated with the native North American legume Amphicarpaea bracteata. Isolates from Amphicarpaea had identical sequences in the three nod gene regions, but differed from all other Bradyrhizobium taxa at > 10% of nucleotide sites. Parsimony analysis of all nod gene segments indicated a phylogenetic relationship of these bacteria to B. elkanii, with B. japonicum diverging prior to the diversification of these taxa. All Bradyrhizobium isolates from Amphicarpaea were also identical to B. elkanii in the size of the intervening sequence (IVS) in the 5' region of the 23S rRNA gene, while B. japonicum had an IVS length variant with 29 additional nucleotides. Parsimony analysis of both 16S and 23S partial rDNA sequences grouped Bradyrhizobium sp. isolates from Amphicarpaea into a clade together with B. elkanii, consistent with the relationships inferred from nod sequences.     

Genetic diversity of indigenous Bradyrhizobium nodulating promiscuous soybean [Glycine max (L) Merr.] varieties in Kenya: Impact of phosphorus and lime fertilization in two contrasting sites

While soybean is an exotic crop introduced in Kenya early last century, promiscuous (TGx) varieties which nodulate with indigenous rhizobia have only recently been introduced. Since farmers in Kenya generally cannot afford or access fertilizer or inoculants, the identification of effective indigenous Bradyrhizobium strains which nodulate promiscuous soybean could be useful in the development of inoculant strains. Genetic diversity and phylogeny of indigenous Bradyrhizobium strains nodulating seven introduced promiscuous soybean varieties grown in two different sites in Kenya was assayed using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 16S-23S rDNA intergenic spacer region and 16S rRNA gene sequencing. PCR-RFLP analysis directly applied on 289 nodules using Msp I distinguished 18 intergenic spacer groups (IGS) I–XVIII. Predominant IGS groups were I, III, II, IV and VI which constituted 43.9%, 24.6%, 8.3% 7.6% and 6.9% respectively of all the analyzed nodules from the two sites while IGS group VII, IX, X, XI, XII, XIV, XVI, XVII, XVIII each constituted 1% or less. The IGS groups were specific to sites and treatments but not varieties. Phylogenetic analysis of the 16S rRNA gene sequences showed that all indigenous strains belong to the genus Bradyrhizobium. Bradyrhizobium elkanii, Bradyrhizobium spp and Bradyrhizobium japonicum related strains were the most predominant and accounted for 37.9%, 34.5%, and 20.7% respectively while B. yuanmigense related accounted for 6.9% of all strains identified in the two combined sites. The diversity identified in Bradyrhizobium populations in the two sites represent a valuable genetic resource that has potential utility for the selection of more competitive and effective strains to improve biological nitrogen fixation and thus increase soybean yields at low cost.     

Genetic diversity of Bradyrhizobium japonicum field population revealed by RAPD fingerprinting

RAPD fingerprinting was used for strain identification and the assessment of genetic diversity within a field population of Bradyrhizobium japonicum . Total genomic DNAs from 13 field isolates and two inoculant strains were amplified using six different 10-mer primers. Different and informative band patterns were obtained for all strains analysed. Cluster analysis unexpectedly revealed that none of the field isolates was identical to inoculant strains which were regularly used for soybean inoculation. Among field isolates two highly divergent groups were determined. The results indicate that RAPD is a very discriminative and efficient method for differentiating and studying genetic diversity of B. japonicum strains.     

Bradyrhizobium iriomotense sp. nov., isolated from a tumor-like root of the legume Entada koshunensis from Iriomote Island in Japan

A polyphasic study was performed to determine the taxonomic position of strain EK05(T) isolated from a root-outgrowth of Entada koshunensis, a legume available in Okinawa, Japan. Phylogenetic analysis of the 16S rRNA gene showed that the strain belongs to the genus Bradyrhizobium. Subsequent multilocus sequence analysis with ITS, glnII, recA, gyrB, and atpD sequences revealed that the isolate represents a distinct evolutionary lineage within the genus Bradyrhizobium. DNA-DNA hybridization indicated that strain EK05(T) shares <61% DNA relatedness with the type strains of all six recognized species of Bradyrhizobium, confirming that this strain is a novel species within the genus. Phylogenetic trees based on symbiotic loci, nifH and nodC, also placed strain EK05(T) clearly in a novel branch. On the basis of its phylogenetic distinctiveness, we propose Bradyrhizobium iriomotense sp. nov. for strain EK05(T). The type strain is EK05(T) (= NBRC 102520(T) = LMG 24129(T)).