Development of a sequence-based in silico OspA typing method for Borrelia burgdorferi sensu lato.

Lyme disease (LD), caused by spirochete bacteria of the genus Borrelia burgdorferi sensu lato, remains the most common vector-borne disease in the northern hemisphere. Borrelia outer surface protein A (OspA) is an integral surface protein expressed during the tick cycle, and a validated vaccine target. There are at least 20 recognized Borrelia genospecies, that vary in OspA serotype. This study presents a new in silico sequence-based method for OspA typing using next-generation sequence data. Using a compiled database of over 400 Borrelia genomes encompassing the 4 most common disease-causing genospecies, we characterized OspA diversity in a manner that can accommodate existing and new OspA types and then defined boundaries for classification and assignment of OspA types based on the sequence similarity. To accommodate potential novel OspA types, we have developed a new nomenclature: OspA in silico type (IST). Beyond the ISTs that corresponded to existing OspA serotypes 1-8, we identified nine additional ISTs that cover new OspA variants in B. bavariensis (IST9-10), B. garinii (IST11-12), and other Borrelia genospecies (IST13-17). The IST typing scheme and associated OspA variants are available as part of the PubMLST Borrelia spp. database. Compared to traditional OspA serotyping methods, this new computational pipeline provides a more comprehensive and broadly applicable approach for characterization of OspA type and Borrelia genospecies to support vaccine development.

Arachis hypogaea L. from Acid Soils of Nanyang (China) Is Frequently Associated with Bradyrhizobium guangdongense and Occasionally with Bradyrhizobium ottawaense or Three Bradyrhizobium Genospecies.

Henan Province is a major area of peanut production in China but the rhizobia nodulating the crop in this region have not been described. A collection of 217 strains of peanut rhizobia was obtained from six field sites across four soil types in Henan Province, North China, by using peanut as a trap host under glasshouse conditions. The 217 strains separated into 8 distinct types on PCR-RFLP analysis of their IGS sequences. Phylogenetic analysis of the 16S rRNA, recA, atpD, and glnII genes of 11 representative strains of the 8 IGS types identified Bradyrhizobium guangdongense, B. ottawaense and three novel Bradyrhizobium genospecies. Bradyrhizobium guangdongense was dominant, accounting for 75.0% of the total isolates across the field sites while B. ottawaense covered 5.1% and the three novel Bradyrhizobium genospecies 4.1 to 8.8% of the total. The symbiosis-related nodA and nifH gene sequences were not congruent with the core genes on phylogenetic analysis and separated into three groups, two of which were similar to sequences of Bradyrhizobium spp. isolated from peanut in south-east China and the third identical to that of B. yuanmingense isolated from Lespedeza cuneata in northern China. A canonical correlation analysis between the distribution of IGS genotypes and soil physicochemical characteristics and climatic factors indicated that the occurrence of IGS types/species was mainly associated with soil pH and available phosphorus.

Phylogenetic Distribution of Ralstonia solanacearum Species Complex Populations in Potato in Kenya.

Ralstonia solanacearum is a pathogen causing bacterial wilt disease of potato, resulting in 70% potato production losses in Kenya. A study was conducted to determine the diversity of R. solanacearum species complex strains within the main potato-growing regions of Kenya. Potato tubers were collected in different potato-growing regions of Kenya from visibly wilted potato plants as well as samples of tomato, irrigation water, and cultures for pathogen isolation. Genomic DNA was isolated from 135 purified cultures of RSSC isolates and PCR-amplified using multiplex and sequevar primers targeting the endoglucanase (egl) partial gene sequences. Pathogenicity tests using R. solanacearum strain (phylotype II sequevar I) were done on the cultivars Kenya Karibu, Shangi, Chulu, Wanjiku, and MoneyMaker. Phylogenetic analysis of the partial egl gene identified two genospecies, R. pseudosolanacearum sp. nov. (1.5%) and R. solanacearum (98.5%). All R. solanacearum strains clustered in sequevar I and were distributed in all the potato-growing regions surveyed. The cultivars were grown in a greenhouse for two cycles in a randomized complete block design and inoculated with R. solanacearum strain. The severity scores were assessed and the area under the disease progress curve (AUDPC) was determined. All the cultivars tested for pathogenicity exhibited wilting symptoms at varying intervals after infection, with none showing complete resistance to R. solanacearum. Cultivar Shangi exhibited minimum disease severity and progression of 41.14% and AUDPC of 1041.7, respectively, while 'Kenya Karibu' was the most susceptible with a high progression rate of 68.24% and AUDPC of 1897.5, respectively. 'MoneyMaker', 'Chulu', and 'Wanjiku' showed no significant difference in disease severity, depicting a simultaneous rate of infection among them. These findings provide valuable information to better understand the pathogen genetic diversity in Kenya and how it spreads.

Phylogeographic distribution of rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia.

Rhizobia are soilborne bacteria that form symbiotic relations with legumes and fix atmospheric nitrogen. The nitrogen fixation potential depends on several factors such as the type of host and symbionts and on environmental factors that affect the distribution of rhizobia. We isolated bacteria nodulating common bean in Southern Ethiopia to evaluate their genetic diversity and phylogeography at nucleotide, locus (gene/haplotype) and species levels of genetic hierarchy. Phylogenetically, eight rhizobial genospecies (including previous collections) were determined that had less genetic diversity than found among reference strains. The limited genetic diversity of the Ethiopian collections was due to absence of many of the Rhizobium lineages known to nodulate beans. Rhizobium etli and Rhizobiumphaseoli were predominant strains of bean-nodulating rhizobia in Ethiopia. We found no evidence for a phylogeographic pattern in strain distribution. However, joint analysis of the current and previous collections revealed differences between the two collections at nucleotide level of genetic hierarchy. The differences were due to genospecies Rhizobium aethiopicum that was only isolated in the earlier collection.

Defining the Rhizobium leguminosarum Species Complex.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Phylogeography and Symbiotic Effectiveness of Rhizobia Nodulating Chickpea (Cicer arietinum L.) in Ethiopia.

Chickpea (Cicer arietinum L.) used to be considered a restrictive host that nodulated and fixed nitrogen only with Mesorhizobium ciceri and M. mediterraneum. Recent analysis revealed that chickpea can also establish effective symbioses with strains of several other Mesorhizobium species such as M. loti, M. haukuii, M. amorphae, M. muleiense, etc. These strains vary in their nitrogen fixation potential inviting further exploration. We characterized newly collected mesorhizobial strains isolated from various locations in Ethiopia to evaluate genetic diversity, biogeographic structure and symbiotic effectiveness. Symbiotic effectiveness was evaluated in Leonard Jars using a locally released chickpea cultivar "Nattoli". Most of the new isolates belonged to a clade related to M. plurifarium, with very few sequence differences, while the total collection of strains contained three additional mesorhizobial genospecies associated with M. ciceri, M. abyssinicae and an unidentified Mesorhizobium species isolated from a wild host in Eritrea. The four genospecies identified represented a subset of the eight major Mesorhizobium clades recently reported for Ethiopia based on metagenomic data. All Ethiopian strains had nearly identical symbiotic genes that grouped them in a single cluster with M. ciceri, M. mediterraneum and M. muleiense, but not with M. plurifarium. Some phylogeographic structure was observed, with elevation and geography explaining some of the genetic differences among strains, but the relation between genetic identity and symbiotic effectiveness was observed to be weak.

Complete Genome Sequences of Trifolium spp. Inoculant Strains Rhizobium leguminosarum sv. trifolii TA1 and CC275e: Resources for Genomic Study of the Rhizobium-Trifolium Symbiosis.

Rhizobium leguminosarum symbiovar trifolii strains TA1 and CC275e are nitrogen-fixing microsymbionts of Trifolium spp. and have been used as commercial inoculant strains for clovers in pastoral agriculture in Australia and New Zealand. Here we present the complete genome sequences of both strains, resolving their multipartite genome structures and allowing for future studies using genomic approaches.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Genetically diverse lentil- and faba bean-nodulating rhizobia are present in soils across Central and Southern Ethiopia.

In total 196 bacterial isolates were obtained from root nodules of lentil (Lens culinaris) and faba bean (Vicia faba) grown on soil samples collected from 10 different sites in central and southern parts of Ethiopia. All isolates were identified as members of the genus Rhizobium by using recA gene sequence analysis. In the recA phylogenetic tree 195 rhizobial strains were classified into nine genospecies. The phylogeny of symbiotic genes nodC and nifH revealed five and six distinct groups respectively, largely dominated by symbiovar viciae. A multivariate analysis showed that environmental variables of the sampling sites considered in this study had more effect on the distribution and composition of the genospecies than the host legumes of the strains. Twenty representative strains, selected based on their isolation site, host plant and nodC group, were able to nodulate all lentil, faba bean, field pea (Pisum abyssinicum) and grass pea (Lathyrus sativus) plants in a greenhouse test in axenic conditions. The majority of the rhizobial strains were effective nitrogen-fixing symbionts for all tested legumes, indicating their potential to serve as broad host-range inoculants in agriculture. The present work suggests the presence of taxonomically and symbiotically diverse rhizobial species for legumes in the Viciae tribe in Ethiopia.

Host-specific competitiveness to form nodules in Rhizobium leguminosarum symbiovar viciae.

Fabeae legumes such as pea and faba bean form symbiotic nodules with a large diversity of soil Rhizobium leguminosarum symbiovar viciae (Rlv) bacteria. However, bacteria competitive to form root nodules (CFN) are generally not the most efficient to fix dinitrogen, resulting in a decrease in legume crop yields. Here, we investigate differential selection by host plants on the diversity of Rlv. A large collection of Rlv was collected by nodule trapping with pea and faba bean from soils at five European sites. Representative genomes were sequenced. In parallel, diversity and abundance of Rlv were estimated directly in these soils using metabarcoding. The CFN of isolates was measured with both legume hosts. Pea/faba bean CFN were associated to Rlv genomic regions. Variations of bacterial pea and/or faba bean CFN explained the differential abundance of Rlv genotypes in pea and faba bean nodules. No evidence was found for genetic association between CFN and variations in the core genome, but variations in specific regions of the nod locus, as well as in other plasmid loci, were associated with differences in CFN. These findings shed light on the genetic control of CFN in Rlv and emphasise the importance of host plants in controlling Rhizobium diversity.

Occurrence of Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti in Ixodes ricinus ticks collected from selected areas of Opolskie Province in south-west Poland.

INTRODUCTION: Ticks (Acari: Ixodida) are vectors and/or reservoirs of many pathogens, i.e. Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Babesia microti. These pathogens are ethiological agents of such diseases as Lyme borreliosis, human granulocytic anaplasmosis and human babesiosis. OBJECTIVE: The aim of the study was to evaluate the role of the Ixodes ricinus in the transmission of Borrelia burgdorferi sensu lato, Borrelia afzelii, Borrelia garinii, Borrelia burgdorferi sensu stricto, Anaplasma phagocytophilum and Babesia microti in Opolskie Province in Poland. MATERIAL AND METHODS: DNA from 222 ticks was isolated by the ammonia method. The pair of primers specific to the flagelline gene was used to detect of B. burgdorferi s. l. To detect of genospecies of this spirochete, three pairs of internal primers were used. In turn, two pairs of primers specific to the 16S rDNA gene and the 18S rRNA were used, respectively, for the detection of A. phagocytophilum and B. microti. Borrelia burgdorferi s. l., A. phagocytophilum, and B. microti were detected in 4.5%, 2.7% and 5.4% of examined ticks, respectively. RESULTS AND CONCLUSIONS: Of the ten ticks infected with B. burgdorferi s. l., B. afzelii was found in seven, undefinied genospecies in two, and mixed infection with B. afzelii and B. burgdorferi s. s. in one. The study demonstrated the potential risk of exposure of humans and animals to infections of B. burgdorferi s. l., A. phagocytophilum and B. microti in the examined area of Poland.

Identification and Characterization of "Haemophilus quentini" Strains Causing Invasive Disease in Ontario, Canada (2016 to 2018).

Haemophilus influenzae is a well-established human pathogen capable of causing a range of respiratory and invasive diseases. Since the 1970s, it has been observed that a nontypeable cryptic genospecies of H. influenzae, most often biotype IV, has been associated with the genitourinary tracts of females and with invasive neonatal infections. This distinct genospecies has been provisionally named "Haemophilus quentini" Here, we report seven cases of invasive H. quentini disease in patients from Ontario, Canada, over a 2-year period. Significantly, while most reports of invasive disease with H. quentini to date have been in neonates, we observed five cases in adults (three in women of childbearing age and two in seniors) as well as two in neonates. Identification of H. quentini is challenging and was not possible for frontline laboratories, requiring work at the reference laboratory level. We describe in detail the biochemical results, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-Tof MS) results, and PCR results with several targets, including the 16S rRNA gene and multilocus sequence typing (MLST) genes, for the seven Ontario H. quentini isolates and several controls. Our data, combined with those of other publications, support the fact that H. quentini is distinct from H. influenzae and Haemophilus haemolyticus This organism is recognized as a pathogen of neonates, but we hypothesize that it may be underrecognized as an important pathogen in adults as well, particularly pregnant women. By sharing the detailed descriptions of these isolates, we hope to enable other laboratories to better identify H. quentini so that the true prevalence of this organism and disease can be explored.

Lyme neuroborreliosis and bird populations in northern Europe.

Many vector-borne diseases are transmitted through complex pathogen-vector-host networks, which makes it challenging to identify the role of specific host groups in disease emergence. Lyme borreliosis in humans is now the most common vector-borne zoonosis in the Northern Hemisphere. The disease is caused by multiple genospecies of Borrelia burgdorferi sensu lato bacteria transmitted by ixodid (hard) ticks, and the major host groups transmit Borrelia genospecies with different pathogenicity, causing variable clinical symptoms in humans. The health impact of a given host group is a function of the number of ticks it infects as well as the pathogenicity of the genospecies it carries. Borrelia afzelii, with mainly small mammals as reservoirs, is the most common pathogen causing Lyme borreliosis, and it is often responsible for the largest proportion of infected host-seeking tick nymphs in Europe. The bird-borne Borrelia garinii, though less prevalent in nymphal ticks, is more likely to cause Lyme neuroborreliosis, but whether B. garinii causes disseminated disease more frequently has not been documented. Based on extensive data of annual disease incidence across Norway from 1995 to 2017, we show here that 69% of disseminated Lyme borreliosis cases were neuroborreliosis, which is three times higher than predicted from the infection prevalence of B. garinii in host-seeking ticks (21%). The population estimate of migratory birds, mainly of thrushes, explained part of the annual variation in cases of neuroborreliosis, with a one-year time lag. We highlight the important role of the genospecies' pathogenicity and the host associations for understanding the epidemiology of disseminated Lyme borreliosis.

Prevalence of Borrelia Burgdorferi Sensu Lato in Ticks from the Ternopil Region in Ukraine.

INTRODUCTION: Lyme borreliosis/Lyme disease is caused by Borrelia burgdorferi and is one of the most common vector-borne diseases transmitted by ticks. MATERIAL AND METHODS: A total of 136 Ixodes ricinus ticks, collected in the Ternopil (Ukraine) region, including 126 adults (70 females and 56 males), and 10 nymphs were examined. The identification of the species and their developmental form was based on morphological characteristics. RESULTS: PCR with B5S-Bor and 23S-Bor primers resulted in Borrelia burgdorferi sensu lato DNA amplification among six ticks (4.4%). The detailed analysis based on the DNA sequencing showed the presence of DNA of Borrelia afzelii in four samples; the remaining two represented Borrelia burgdorferi sensu lato complex, although their genospecies were not determined. The research confirmed the dominance of Borrelia afzelii genospecies in the ticks from Ukraine. CONCLUSION: It seems reasonable to undertake similar research in ticks from other regions of Ukraine. Knowledge in this field can be useful for public health and planning the prevention of tick-borne diseases.

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia.

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ15N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ15N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ15N=0.73) and GN102 (δ15N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.

Association of Borrelia and Rickettsia spp. and bacterial loads in Ixodes ricinus ticks.

In recent years, awareness of coinfections has increased as synergistic or antagonistic effects on interacting bacteria have been observed. To date, several reports on coinfections of ticks with Rickettsia and Borrelia spp. are available. However, associations are rarely described and studies are based on rather low sample sizes. In the present study, coinfections of Ixodes ricinus with these pathogens were investigated by determining their association in a meta-analysis. A total of 5079 tick samples examined for Rickettsia and Borrelia spp. via probe-based quantitative real-time PCR in previous prevalence studies or as submitted diagnostic material were included. In Borrelia-positive ticks, genospecies were determined by Reverse Line Blot. Determination of bacterial loads resulted in an increase between developmental tick stages with highest mean bacterial loads in female ticks (7.96×104 in Borrelia single-infected, 4.87×105 in Rickettsia single-infected and 3.22×105 in Borrelia-Rickettsia coinfected females). The determined Borrelia-Rickettsia tick coinfection rate was 12.3% (626/5079) with a significant difference to the expected coinfection rate of 9.0% (457/5079). A significant slight association as well as correlation between Borrelia and Rickettsia were determined. In addition, a significant interrelation of the bacterial load in coinfected ticks was shown. At the level of Borrelia genospecies, significant weak associations with Rickettsia spp. were detected for B. afzelii, B. garinii/bavariensis, B. valaisiana and B. lusitaniae. The positive association provides evidence for interactions between Borrelia and Rickettsia spp. in the tick vector, presumably resulting in higher bacterial replication rates in the tick vector and possibly the reservoir host. However, coinfection may impact the vector negatively as indicated by an absent increase in coinfection rates from nymphs to adults. Future studies are needed to investigate the underlying mechanisms of the positive association in ticks and possible associations in the vertebrate host as well as the potential influence of environmental factors.

In-silico Taxonomic Classification of 373 Genomes Reveals Species Misidentification and New Genospecies within the Genus Pseudomonas.

The genus Pseudomonas has one of the largest diversity of species within the Bacteria kingdom. To date, its taxonomy is still being revised and updated. Due to the non-standardized procedure and ambiguous thresholds at species level, largely based on 16S rRNA gene or conventional biochemical assay, species identification of publicly available Pseudomonas genomes remains questionable. In this study, we performed a large-scale analysis of all Pseudomonas genomes with species designation (excluding the well-defined P. aeruginosa) and re-evaluated their taxonomic assignment via in silico genome-genome hybridization and/or genetic comparison with valid type species. Three-hundred and seventy-three pseudomonad genomes were analyzed and subsequently clustered into 145 distinct genospecies. We detected 207 erroneous labels and corrected 43 to the proper species based on Average Nucleotide Identity Multilocus Sequence Typing (MLST) sequence similarity to the type strain. Surprisingly, more than half of the genomes initially designated as Pseudomonas syringae and Pseudomonas fluorescens should be classified either to a previously described species or to a new genospecies. Notably, high pairwise average nucleotide identity (>95%) indicating species-level similarity was observed between P. synxantha-P. libanensis, P. psychrotolerans-P. oryzihabitans, and P. kilonensis- P. brassicacearum, that were previously differentiated based on conventional biochemical tests and/or genome-genome hybridization techniques.

Europe-Wide Meta-Analysis of Borrelia burgdorferi Sensu Lato Prevalence in Questing Ixodes ricinus Ticks.

Lyme borreliosis is the most common zoonotic disease transmitted by ticks in Europe and North America. Despite having multiple tick vectors, the causative agent, Borrelia burgdorferisensu lato, is vectored mainly by Ixodes ricinus in Europe. In the present study, we aimed to review and summarize the existing data published from 2010 to 2016 concerning the prevalence of B. burgdorferi sensu lato spirochetes in questing I. ricinus ticks. The primary focus was to evaluate the infection rate of these bacteria in ticks, accounting for tick stage, adult tick gender, region, and detection method, as well as to investigate any changes in prevalence over time. The data obtained were compared to the findings of a previous metastudy. The literature search identified data from 23 countries, with 115,028 ticks, in total, inspected for infection with B. burgdorferi sensu lato We showed that the infection rate was significantly higher in adults than in nymphs and in females than in males. We found significant differences between European regions, with the highest infection rates in Central Europe. The most common genospecies were B. afzelii and B. garinii, despite a negative correlation of their prevalence rates. No statistically significant differences were found among the prevalence rates determined by conventional PCR, nested PCR, and real-time PCR.IMPORTANCEBorrelia burgdorferisensu lato is a pathogenic bacterium whose clinical manifestations are associated with Lyme borreliosis. This vector-borne disease is a major public health concern in Europe and North America and may lead to severe arthritic, cardiovascular, and neurological complications if left untreated. Although pathogen prevalence is considered an important predictor of infection risk, solitary isolated data have only limited value. Here we provide summarized information about the prevalence of B. burgdorferi sensu lato spirochetes among host-seeking Ixodes ricinus ticks, the principal tick vector of borreliae in Europe. We compare the new results with previously published data in order to evaluate any changing trends in tick infection.

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of tree and annual legume species growing in Ethiopia.

Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating from root nodules of the leguminous trees and crops Acacia saligna, Faidherbia albida, Erythrina brucei, Albizia gummifera, Millettia ferruginea, Cajanus cajan, Vigna unguiculata and Phaseolus vulgaris, growing in southern Ethiopia. Multilocus sequence analyses (MLSA) of the 16S rRNA, glnII, recA, gyrB and dnaK genes and the ITS region grouped the test strains into seven well-supported genospecies (I-VII), six of which occupied distinct positions excluding all hitherto defined Bradyrhizobium species. Analyses of the nodA, nodC and nifH genes suggested different evolutionary history of the chromosomal and symbiosis-related genes. Our study corroborates earlier findings that Ethiopia is a hotspot for rhizobial biodiversity, justifying further search for novel strains from this region and calling for intensified research on the ecology and biochemistry of these organisms.