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Plasmid analysis of B. japonicum NK5 and nod geneacquired B. elkanii USDA94DNOD. (A) Plasmid profiles of B. japonicum USDA110 and NK5. Strain NK5 possessed two plasmids 240 kb and 180 kb in size. E. coli HB101 harboring pRjUT10 was analyzed as a positive control of nod hybridization . Rhizobium leguminosarum bv. phaseoli NOKO-311 and Rhizobium etli NOKO-312 were used as plasmid size markers (Tabel 1). (B) Southern hybridization of the agarose gel (A) with B. japonicum nodD 1 YABC from pRjUT10. (C) Plasmid analysis of B. elkanii USDA94DNOD derivatives, Sir3, Sir12 and Sir27, that acquired nod genes.
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We investigated the horizontal transfer of nodulation (nod) genes to a Bradyrhizobium elkanii strain, lacking common nod genes as a recipient, in soils and microcosms using selection systems of antibiotic resistance and legume nodulation. We observed the horizontal transfer of nod genes at 4°C in Nakazawa soil where pecu-liar strains (HRS strains)...
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Bacterial death can release eARGs into the environment. Agricultural soils can contain upwards of 10 ⁹ ARGs g ⁻¹ soil, which may facilitate the movement of eARGs from dead to live bacteria through a mechanism of horizontal gene transfer called natural transformation.
Citations
... While the 31 housekeeping genes used by AMPHORA2 indicated a wide variety of possible origins of the RH 3a 1 strain, the nod-based trees point to a more recent acquisition of these genes upon association to local plant hosts, adapted to a Mediterranean and semi-arid climate. Thus, these nod genes were likely acquired by RH 3a 1 via horizontal gene transfer from local nodulating bacteria, a phenomenon known to occur in Bradyrhizobium (Minamisawa et al., 2002;Moulin et al., 2004). ...
Plant–microbe mutualisms, such as the legume‐rhizobium symbiosis, are influenced by the geographical distributions of both partners. However, limitations on the native range of legumes, resulting from the absence of a compatible mutualist, have rarely been explored.
We used a combination of a large‐scale field survey and controlled experiments to determine the realized niche of Calicotome villosa , an abundant and widespread legume shrub.
Soil type was a major factor affecting the distribution and abundance of C. villosa . In addition, we found a large region within its range in which neither C. villosa nor Bradyrhizobium , the bacterial genus that associates with it, were present. Seedlings grown in soil from this region failed to nodulate and were deficient in nitrogen. Inoculation of this soil with Bradyrhizobium isolated from root nodules of C. villosa resulted in the formation of nodules and higher growth rate, leaf N and shoot biomass compared with un‐inoculated plants.
We present evidence for the exclusion of a legume from parts of its native range by the absence of a compatible mutualist. This result highlights the importance of the co‐distribution of both the host plant and its mutualist when attempting to understand present and future geographical distributions of legumes.
... These mobile elements, in addition to HGT, are involved in recombination, rearrangements, and streamlining of bacterial genomes (30). Such IS-mediated HGT and genomic rearrangements have been observed in soybean bradyrhizobia (31)(32)(33) and work in tandem with large, self-replicating, and conjugable plasmids of rhizobia and bradyrhizobia (34). ...
... All observed IS families were evenly distributed within the S06B-Bj and USDA 76-Be genomes (Fig. 5), and the number of IS elements from each IS family ranged from 1 to 40 per genome. The high number of IS elements in the S06B-Bj and USDA 76-Be genomes also suggested elevated levels of gene transfer and rearrangement activities; indeed, previous studies have shown that HRS strains transfer nod genes from B. japonicum to B. elkanii via HGT (32). The potential impacts of IS elements on soybean bradyrhizobia diversity vary. ...
... While further studies are needed to confirm specialized transduction, mapping analysis of the SPP ppUSDA76Be-2 and ppS10JBj-1 suggested that headful packaging terminases could be involved in specialized transduction in these SPP. IS and plasmids have been shown to transfer symbiotic genes between different strains of bradyrhizobia (31,32). While such transfers can have a significant impact on their gene pool, they require direct contact between two bradyrhizobia cells. ...
The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation (nod) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.
... inoculants ( Andrews et al., 2018). Horizontal gene transfer from B. japonicum to B. elkanii was already observed by Minamisawa et al. (2002), and later Silva- Batista et al. (2007) obtained evidence that this phenomenon occurred in soils of Brazil. However, the identity of nodC and nifH sequences from R. radiobacter SNAP-isolates with those from R. radiobacter MQ-110s and gx-178, respectively, and their lower relatedness with Bradyrhizobium spp. ...
Soybean is the most important oilseed in the world, cropped in 120–130 million hectares each year. The three most important soybean producers are Argentina, Brazil, and United States, where soybean crops are routinely inoculated with symbiotic N2-fixing Bradyrhizobium spp. This extended inoculation gave rise to soybean-nodulating allochthonous populations (SNAPs) that compete against new inoculant for nodulation, thus impairing yield responses. Competitiveness depends on intrinsic factors contributed by genotype, extrinsic ones determined by growth and environmental conditions, and strain persistence in the soil. To assess these factors in Argentinean SNAPs, we studied 58 isolates from five sites of the main soybean cropping area. BOX-A1R DNA fingerprint distributed these isolates in 10 clades that paralleled the pHs of their original soils. By contrast, reference Bradyrhizobium spp. strains, including those used as soybean-inoculants, were confined to a single clade. More detailed characterization of a subset of 11 SNAP-isolates revealed that five were Bradyrhizobium japonicum, two Bradyrhizobium elkanii, two Rhizobium radiobacter (formerly Agrobacterium tumefaciens), one Bradyrhizobium diazoefficiens, and one Paenibacillus glycanilyticus-which did not nodulate when inoculated alone, and therefore was excluded from further characterization. The remaining subset of 10 SNAP-isolates was used for deeper characterization. All SNAP-isolates were aluminum- and heat-tolerant, and most of them were glyphosate-tolerant. Meanwhile, inoculant strains tested were sensitive to aluminum and glyphosate. In addition, all SNAP-isolates were motile to different degrees. Only three SNAP-isolates were deficient for N2-fixation, and none was intrinsically more competitive than the inoculant strain. These results are in contrast to the general belief that rhizobia from soil populations evolved as intrinsically more competitive for nodulation and less N2-fixing effective than inoculants strains. Shoot:root ratios, both as dry biomass and as total N, were highly correlated with leaf ureide contents, and therefore may be easy indicators of N2-fixing performance, suggesting that highly effective N2-fixing and well-adapted strains may be readily selected from SNAPs. In addition, intrinsic competitiveness of the inoculants strains seems already optimized against SNAP strains, and therefore our efforts to improve nodules occupation by inoculated strains should focus on the optimization of extrinsic competitiveness factors, such as inoculant formulation and inoculation technology.
... To our knowledge, this is the first report of Pseudomonas with high SE nodulating herbaceous legumes, such as C. mucunoides. Some authors have suggested that endophytic bacteria may acquire genes related to nodulation via horizontal gene transfer, which may be the case for the presently sampled strains (Moulin et al. 2001;Minamisawa et al. 2002). ...
Aims
The objective of this work was to isolate and characterize indigenous rhizobia from coal‐mining areas able to efficiently nodulate and fix nitrogen in association with Calopogonium mucunoides (calopo).
Methods and Results
Isolation, authentication and morphological, biochemical and molecular characterization of the autochthonous rhizobia were performed and their symbiotic efficiency evaluated. Efficient rhizobial isolates suitable for the inoculation of calopo in coal‐mining regions were obtained. A total of thirty isolates were obtained after nodulation authentication, of which five presented high symbiotic efficiency with plant‐growth promoting traits such as indole‐3‐acetic acid production, phosphate solubilization, and biofilm formation. These isolates were identified as belonging to Bradyrhizobium, Pseudomonas and Rhizobium.
Conclusions
Bradyrhizobium sp. A2‐10 and Pseudomonas sp. A6‐05 were able to promote calopo plant growth using soil obtained from coal‐mining degraded areas, thus indicating their potential as inoculants aiming at land reclamation.
Significance and Impact of the Study
To our knowledge, this is the first report of Pseudomonas nodule formation in calopo. Furthermore, the results demonstrated that autochthonous rhizobia obtained from degraded soils presented high symbiotic efficiency in calopo and possess a wide range of plant‐growth promoting traits. Ultimately, they may all contribute to an increased leguminous plant growth under stress conditions. The selected rhizobia strains may be used as inoculants and present a valuable role in the development of strategies aiming to recover coal‐mining degraded areas. Bacterial inoculants would greatly reduce the use of often harmful nitrogen fertilizers vastly employed in revegetation programs of degraded areas.
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... Burkholderia species isolated from Papilionoideae species are distributed in tropical regions of Africa and South America (51), and isolates of Cupriavidus (formerly Ralstonia) have also been identified as root nodule bacteria (2,12,13,75). These findings support the theory of symbiotic lateral gene transfer from rhizobia to other bacteria (17,18,49,59,75). Chen et al. (13) also reported that Cupriavidus taiwanensis was the predominant symbiont of Mimosa pudica and M. diplotricha in Taiwan, and several isolates of Burkholderia phymatum and other species were confirmed as root nodule symbionts of Mimosa species (12,21,50,75). ...
The climate, topography, fauna, and flora of Venezuela are highly diverse. However, limited information is currently available on the characterization of soybean rhizobia in Venezuela. To clarify the physiological and genetic diversities of soybean rhizobia in Venezuela, soybean root nodules were collected from 11 soil types located in different topographical regions. A total of 395 root nodules were collected and 120 isolates were obtained. All isolates were classified in terms of stress tolerance under different concentrations of NaCl and Al³⁺. The tolerance levels of isolates to NaCl and Al³⁺ varied. Based on sampling origins and stress tolerance levels, 44 isolates were selected for further characterization. An inoculation test indicated that all isolates showed the capacity for root nodulation on soybean. Based on multilocus sequence typing (MLST), 20 isolates were classified into the genera Rhizobium and Bradyrhizobium. The remaining 24 isolates were classified into the genus Burkholderia or Paraburkholderia. There is currently no evidence to demonstrate that the genera Burkholderia and Paraburkholderia are the predominant soybean rhizobia in agricultural fields. Of the 24 isolates classified in (Para) Burkholderia, the nodD–nodB intergenic spacer regions of 10 isolates and the nifH gene sequences of 17 isolates were closely related to the genera Rhizobium and Bradyrhizobium, respectively. The root nodulation numbers of five (Para) Burkholderia isolates were higher than those of the 20 α-rhizobia. Furthermore, among the 44 isolates tested, one Paraburkholderia isolate exhibited the highest nitrogen-fixation activity in root nodules.
... Thus, sequence analysis of symbiotic genes nifD (encoded the α subunit of dinitrogenase) and nodD1 (nodulation regulation protein) was also conducted. Several studies reported that genes located in symbiosis island might not show diversity even among related species in rhizobial genera commonly due to horizontal gene transfer as directed by their location (Minamisawa et al. 2002;Barcellos et al. 2007;Ramirez-Bahena et al. 2009;Ling et al. 2016). But the role of NodD regulator proteins (including nodD1) in activating the transcription of nod genes is known to be a key factor that influences the competitiveness of rhizobia (Maj et al. 2010) due to its assumed specific interaction with flavonoids (Redmond et al. 1986;Zaat et al. 1989). ...
... The very high similarity (99-100%) observed between the isolates and B. elkanii strains for both nifD and nodD1 and its congruence with 16S rRNA gene phylogeny might be an indication that the evolution of symbiotic genes from the isolates of Kumamoto and Okinawa, Japan and Nueva Ecija, Philippines have progressed similarly with their conserved genes. Previous studies (Minamisawa et al. 2002;Barcellos et al. 2007;Ling et al. 2016) stated that horizontal gene transfer seldom occur for symbiotic genes in rhizobial genera that commonly causes the conformity in phylogenetic analyses. Although we cannot say that this is the same case with our study because we did not perform an analysis that will support this. ...
AimsUnderstanding the factors that influence the diversity of soybean-nodulating rhizobia is important before doing inoculation. Since studies about this topic in tropical regions are limited, this could lay the groundwork for related research particularly on Bradyrhizobium elkanii. Methods
To determine the genetic diversity of B. elkanii in different regions, we conducted Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and sequence analysis of 16S rRNA gene, internal transcribed spacer (ITS) region and rpoB gene. Also, sequence analysis of symbiotic nifD and nodD1 genes was conducted. ResultsAnalysis of the rpoB gene revealed a higher genetic diversity than the ITS region, and possible endemic B. elkanii strains were observed. Meanwhile, no variation was detected among the strains in both nifD and nodD1 phylogenies. Through rpoB gene analysis, variations in the ITS-rpoB type of B. elkanii strains were distinguished and differentiated with that of the closest reference strains. We identified potential soybean inoculants which possess symbiotic efficiency regardless of the Rj genotypes used, suggesting broad host-range of the strains. Conclusions
We show how the genetic diversity of soybean-nodulating B. elkanii strains in subtropical and tropical regions might be influenced by temperature and soil pH and, provided some insights between the symbiotic genes and Rj genotypes.
... No difference between the symbiotic abilities of HRS and non-HRS strains has been observed so far (12)(13)(14). The HRS strain NK5 has the potential to transfer the nod genes to nod-minus mutants of Bradyrhizobium elknaii in soil microcosms (17). ...
... Surface-sterilized soybean seeds (Glycine max cv. Enrei) were inoculated with NK6 and USDA110 as described previously (17). Plants were cultivated in a greenhouse and were repeatedly supplied with nitrogen-free nutrient solution (17). ...
... Enrei) were inoculated with NK6 and USDA110 as described previously (17). Plants were cultivated in a greenhouse and were repeatedly supplied with nitrogen-free nutrient solution (17). Nitrogen fixation and nodulation were analyzed 50 days after germination. ...
Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07-0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7-10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136-212 kb) and a large chromosome (9780 kb). Strong co-linearity was found between 7.4-Mb core regions of NK6 and USDA110 chromosomes. USDA110 symbiosis islands mainly corresponded to five small regions (S1-S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. USDA110 nif gene cluster (nifDKENXSBZHQW/fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.
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... This difference between the two strains did not result from a higher genetic divergence of one inoculant from local strains. Minamisawa et al. (33) detected nod gene transfers among bradyrhizobia and other bacterial populations in soil and microcosms and showed that B. japonicum isolates harboring a high copy number of insertion sequences in their genome might make them potentially more efficient as donors. The different patterns detected between the two strains might be explained by such genomic particularity, probably coupled with a higher selection pressure promoting the stability of the local core genome diversity and the spread of the efficient symbiotic cluster. ...
Introducing nitrogen-fixing bacteria as inoculum, in association with legume crops, is a common practice in agriculture. However, the question of the evolution of these introduced microorganisms remains crucial, both in terms of microbial ecology and agronomy. We explored this question by analyzing the genetic and symbiotic evolution of two Bradyrhizobium strains inoculated on Acacia mangium in Malaysia and Senegal, 15 and 5 years respectively after their introduction. Based on several locus typing, we showed that these two strains, although closely related and originally sampled in Australia, evolved differently. One strain was recovered in soil with the same five loci than the original isolate, whereas the symbiotic cluster of the other strain was detected, with no trace of the three housekeeping genes of the original inoculum. Moreover, the nitrogen fixation efficiency was variable among these isolates (either recombinant or not), with significant higher, lower, or similar efficiency compared to the two original strains, and no significant difference between recombinant and not-recombinant isolates. These data suggested that 15 years after their introduction, nitrogen-fixing bacteria remain in the soil, but that closely related inoculant strains may not evolve in the same way, both genetically and symbiotically. In a context of an increasing agronomical use of microbial inoculants (for biological control, nitrogen fixation or plant growth promotion), this result feeds the debate on the consequences associated to such practices.
... The seeds were then immersed in 0.5% (vol/vol) sodium hypochlorite for 1 min and washed 10 times with sterile distilled water. Finally, the seeds were placed in sterilized plates containing tissue paper wetted with sterile distilled water for 2 days at 30°C in the dark and then transplanted to a Leonard jar (CUL-JAR300; Asahi Glass Co., Ltd., Tokyo, Japan) containing sterile vermiculite and nitrogen-free nutrient solution (28). After transplantation, the seedlings were inoculated with bacteria at 1 ϫ 10 8 cells per plant. ...
Agromonas oligotrophica (Bradyrhizobium oligotrophicum) S58T is a nitrogen-fixing oligotrophic bacterium isolated from paddy field soil that is able to grow in extra-low-nutrient environments.
Here, the complete genome sequence of S58 was determined. The S58 genome was found to comprise a circular chromosome of 8,264,165
bp with an average GC content of 65.1% lacking nodABC genes and the typical symbiosis island. The genome showed a high level of similarity to the genomes of Bradyrhizobium sp. ORS278 and Bradyrhizobium sp. BTAi1, including nitrogen fixation and photosynthesis gene clusters, which nodulate an aquatic legume plant, Aeschynomene indica, in a Nod factor-independent manner. Although nonsymbiotic (brady)rhizobia are significant components of rhizobial populations
in soil, we found that most genes important for nodule development (ndv) and symbiotic nitrogen fixation (nif and fix) with A. indica were well conserved between the ORS278 and S58 genomes. Therefore, we performed inoculation experiments with five A. oligotrophica strains (S58, S42, S55, S72, and S80). Surprisingly, all five strains of A. oligotrophica formed effective nitrogen-fixing nodules on the roots and/or stems of A. indica, with differentiated bacteroids. Nonsymbiotic (brady)rhizobia are known to be significant components of rhizobial populations
without a symbiosis island or symbiotic plasmids in soil, but the present results indicate that soil-dwelling A. oligotrophica generally possesses the ability to establish symbiosis with A. indica. Phylogenetic analyses suggest that Nod factor-independent symbiosis with A. indica is a common trait of nodABC- and symbiosis island-lacking strains within the members of the photosynthetic Bradyrhizobium clade, including A. oligotrophica.
... The seeds were then washed 10 times with sterile distilled water. The seeds were germinated in sterile vermiculite for 2 d at 25°C, and then transplanted into a Leonard jar (41,72) containing sterile vermiculite and nitrogen-free nutrient solution (47). The seedlings were inoculated at 1×10 7 cells per seed with either S23321 or USDA110. ...
Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.
