SeqCode facilitates naming of South African rhizobia left in limbo.

South Africa is well-known for the diversity of its legumes and their nitrogen-fixing bacterial symbionts. However, in contrast to their plant partners, remarkably few of these microbes (collectively referred to as rhizobia) from South Africa have been characterised and formally described. This is because the rules of the International Code of Nomenclature of Prokaryotes (ICNP) are at odds with South Africa's National Environmental Management: Biodiversity Act and its associated regulations. The ICNP requires that a culture of the proposed type strain for a novel bacterial species be deposited in two international culture collections and be made available upon request without restrictions, which is not possible under South Africa's current national regulations. Here, we describe seven new Mesorhizobium species obtained from root nodules of Vachellia karroo, an iconic tree legume distributed across various biomes in southern Africa. For this purpose, 18 rhizobial isolates were delineated into putative species using genealogical concordance, after which their plausibility was explored with phenotypic characters and average genome relatedness. For naming these new species, we employed the rules of the recently published Code of Nomenclature of Prokaryotes described from Sequence Data (SeqCode), which utilizes genome sequences as nomenclatural types. The work presented in this study thus provides an illustrative example of how the SeqCode allows for a standardised approach for naming cultivated organisms for which the deposition of a type strain in international culture collections is currently problematic.

Characterization of the Fusarium circinatum biofilm environmental response role.

The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.

Chromosome-Level Assemblies for the Pine Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome's distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.

Unidirectional mating-type switching is underpinned by a conserved MAT1 locus architecture.

Unidirectional mating-type switching is a form of homothallic reproduction known only in a small number of filamentous ascomycetes. Their ascospores can give rise to either self-sterile isolates that require compatible partners for subsequent sexual reproduction, or self-fertile individuals capable of completing this process in isolation. The limited studies previously conducted in these fungi suggest that the differences in mating specificity are determined by the architecture of the MAT1 locus. In self-fertile isolates that have not undergone unidirectional mating-type switching, the locus contains both MAT1-1 and MAT1-2 mating-type genes, typical of primary homothallism. In the self-sterile isolates produced after a switching event, the MAT1-2 genes are lacking from the locus, likely due to a recombination-mediated deletion of the MAT1-2 gene information. To determine whether these arrangements of the MAT1 locus support unidirectional mating-type switching in the Ceratocystidaceae, the largest known fungal assemblage capable of this reproduction strategy, a combination of genetic and genomic approaches were used. The MAT1 locus was annotated in representative species of Ceratocystis, Endoconidiophora, and Davidsoniella. In all cases, MAT1-2 genes interrupted the MAT1-1-1 gene in self-fertile isolates. The MAT1-2 genes were flanked by two copies of a direct repeat that accurately predicted the boundaries of the deletion event that would yield the MAT1 locus of self-sterile isolates. Although the relative position of the MAT1-2 gene region differed among species, it always disrupted the MAT1-1-1 gene and/or its expression in the self-fertile MAT1 locus. Following switching, this gene and/or its expression was restored in the self-sterile arrangement of the locus. This mirrors what has been reported in other species capable of unidirectional mating-type switching, providing the strongest support for a conserved MAT1 locus structure that is associated with this process. This study contributes to our understanding of the evolution of unidirectional mating-type switching.

Bradyrhizobium xenonodulans sp. nov. isolated from nodules of Australian Acacia species invasive to South Africa.

A genealogical concordance approach was used to delineate strains isolated from Acacia dealbata and Acacia mearnsii root nodules in South Africa. These isolates form part of Bradyrhizobium based on 16S rRNA sequence similarity. Phylogenetic analysis of six housekeeping genes (atpD, dnaK, glnII, gyrB, recA and rpoB) confirmed that these isolates represent a novel species, while pairwise average nucleotide identity (ANIb) calculations with the closest type strains (B. cosmicum 58S1T, B. betae PL7HG1T, B. ganzhouense CCBAU 51670 T, B. cytisi CTAW11T and B. rifense CTAW71T) resulted in values well below 95-96%. We further performed phenotypic tests which revealed that there are high levels of intraspecies variation, while an additional analysis of the nodA and nifD loci indicated that the symbiotic loci of the strains are closely related to those of Bradyrhizobium isolates with an Australian origin. Strain 14ABT (=LMG 31415 T = SARCC-753 T) is designated as the type strain of the novel species for which we propose the name Bradyrhizobium xenonodulans sp. nov.

Evidence of Biparental Mitochondrial Inheritance from Self-Fertile Crosses between Closely Related Species of Ceratocystis.

Hybridization is recognized as a notable driver of evolution and adaptation, which closely related species may exploit in the form of incomplete reproductive barriers. Three closely related species of Ceratocystis (i.e., C. fimbriata, C. manginecans and C. eucalypticola) have previously been shown to hybridize. In such studies, naturally occurring self-sterile strains were mated with an unusual laboratory-generated sterile isolate type, which could have impacted conclusions regarding the prevalence of hybridization and inheritance of mitochondria. In the current study, we investigated whether interspecific crosses between fertile isolates of these three species are possible and, if so, how mitochondria are inherited by the progeny. For this purpose, a PCR-RFLP method and a mitochondrial DNA-specific PCR technique were custom-made. These were applied in a novel approach of typing complete ascospore drops collected from the fruiting bodies in each cross to distinguish between self-fertilizations and potential hybridization. These markers showed hybridization between C. fimbriata and C. eucalypticola and between C. fimbriata and C. manginecans, while no hybridization was detected in the crosses involving C. manginecans and C. eucalypticola. In both sets of hybrid progeny, we detected biparental inheritance of mitochondria. This study was the first to successfully produce hybrids from a cross involving self-fertile isolates of Ceratocystis and also provided the first direct evidence of biparental mitochondrial inheritance in the Ceratocystidaceae. This work lays the foundation for further research focused on investigating the role of hybridization in the speciation of Ceratocystis species and if mitochondrial conflict could have influenced the process.

An in Silico Approach to Identifying TF Binding Sites: Analysis of the Regulatory Regions of BUSCO Genes from Fungal Species in the Ceratocystidaceae Family.

Transcriptional regulation controls gene expression through regulatory promoter regions that contain conserved sequence motifs. These motifs, also known as regulatory elements, are critically important to expression, which is driving research efforts to identify and characterize them. Yeasts have been the focus of such studies in fungi, including in several in silico approaches. This study aimed to determine whether in silico approaches could be used to identify motifs in the Ceratocystidaceae family, and if present, to evaluate whether these correspond to known transcription factors. This study targeted the 1000 base-pair region upstream of the start codon of 20 single-copy genes from the BUSCO dataset for motif discovery. Using the MEME and Tomtom analysis tools, conserved motifs at the family level were identified. The results show that such in silico approaches could identify known regulatory motifs in the Ceratocystidaceae and other unrelated species. This study provides support to ongoing efforts to use in silico analyses for motif discovery.

A LAMP Assay for Rapid Detection of the Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen Fusarium circinatum is endemic in the southeastern United States and Central America and represents an invasive threat globally. This ecologically adaptable fungus readily infects all parts of its pine hosts, leading to widespread mortality of nursery seedlings and decline in the health and productivity of forest stands. Because trees infected by F. circinatum can remain asymptomatic for long periods of time, accurate and rapid tools are needed for real-time diagnostics and surveillance at ports, in nurseries, and in plantations. To meet this need and to limit the spread and impact of the pathogen, we developed a molecular test using loop-mediated isothermal amplification (LAMP), a technology that allows for the rapid detection of pathogen DNA on portable, field-capable devices. LAMP primers were designed and validated to amplify a gene region unique to F. circinatum. Using a globally representative collection of F. circinatum isolates and other closely related species, we have demonstrated that the assay can be used to identify F. circinatum across its genetic diversity and that it is sensitive to as few as 10 cells from purified DNA extracts. The assay can also be used with a simple, pipette-free DNA extraction method and is compatible with testing symptomatic pine tissues in the field. This assay has the potential to facilitate diagnostic and surveillance efforts both in the laboratory and in the field and, thus, to reduce the spread and impact of pitch canker worldwide.

Nodulation and Growth Promotion of Chickpea by Mesorhizobium Isolates from Diverse Sources.

The cultivation of chickpea (Cicer arietinum L.) in South Africa is dependent on the application of suitable Mesorhizobium inoculants. Therefore, we evaluated the symbiotic effectiveness of several Mesorhizobium strains with different chickpea genotypes under controlled conditions. The tested parameters included shoot dry weight (SDW), nodule fresh weight (NFW), plant height, relative symbiotic effectiveness (RSE) on the plant as well as indole acetic acid (IAA) production and phosphate solubilization on the rhizobia. Twenty-one Mesorhizobium strains and six desi chickpea genotypes were laid out in a completely randomized design (CRD) with three replicates in a glasshouse pot experiment. The factors, chickpea genotype and Mesorhizobium strain, had significant effects on the measured parameters (p < 0.001) but lacked significant interactions based on the analysis of variance (ANOVA). The light variety desi genotype outperformed the other chickpea genotypes on all tested parameters. In general, inoculation with strains LMG15046, CC1192, XAP4, XAP10, and LMG14989 performed best for all the tested parameters. All the strains were able to produce IAA and solubilize phosphate except the South African field isolates, which could not solubilize phosphate. Taken together, inoculation with compatible Mesorhizobium promoted chickpea growth. This is the first study to report on chickpea-compatible Mesorhizobium strains isolated from uninoculated South African soils with no history of chickpea production; although, their plant growth promotion ability was poorer compared to some of the globally sourced strains. Since this study was conducted under controlled conditions, we recommend field studies to assess the performance of the five highlighted strains under environmental conditions in South Africa.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

Characterization of Host-Specific Genes from Pine- and Grass-Associated Species of the Fusarium fujikuroi Species Complex.

The Fusarium fujikuroi species complex (FFSC) includes socioeconomically important pathogens that cause disease for numerous crops and synthesize a variety of secondary metabolites that can contaminate feedstocks and food. Here, we used comparative genomics to elucidate processes underlying the ability of pine-associated and grass-associated FFSC species to colonize tissues of their respective plant hosts. We characterized the identity, possible functions, evolutionary origins, and chromosomal positions of the host-range-associated genes encoded by the two groups of fungi. The 72 and 47 genes identified as unique to the respective genome groups were potentially involved in diverse processes, ranging from transcription, regulation, and substrate transport through to virulence/pathogenicity. Most genes arose early during the evolution of Fusarium/FFSC and were only subsequently retained in some lineages, while some had origins outside Fusarium. Although differences in the densities of these genes were especially noticeable on the conditionally dispensable chromosome of F. temperatum (representing the grass-associates) and F. circinatum (representing the pine-associates), the host-range-associated genes tended to be located towards the subtelomeric regions of chromosomes. Taken together, these results demonstrate that multiple mechanisms drive the emergence of genes in the grass- and pine-associated FFSC taxa examined. It also highlighted the diversity of the molecular processes potentially underlying niche-specificity in these and other Fusarium species.

Intra-Species Genomic Variation in the Pine Pathogen Fusarium circinatum.

Fusarium circinatum is an important global pathogen of pine trees. Genome plasticity has been observed in different isolates of the fungus, but no genome comparisons are available. To address this gap, we sequenced and assembled to chromosome level five isolates of F. circinatum. These genomes were analysed together with previously published genomes of F. circinatum isolates, FSP34 and KS17. Multi-sample variant calling identified a total of 461,683 micro variants (SNPs and small indels) and a total of 1828 macro structural variants of which 1717 were copy number variants and 111 were inversions. The variant density was higher on the sub-telomeric regions of chromosomes. Variant annotation revealed that genes involved in transcription, transport, metabolism and transmembrane proteins were overrepresented in gene sets that were affected by high impact variants. A core genome representing genomic elements that were conserved in all the isolates and a non-redundant pangenome representing all genomic elements is presented. Whole genome alignments showed that an average of 93% of the genomic elements were present in all isolates. The results of this study reveal that some genomic elements are not conserved within the isolates and some variants are high impact. The described genome-scale variations will help to inform novel disease management strategies against the pathogen.

Novel mating-type-associated genes and gene fragments in the genomes of Mycosphaerellaceae and Teratosphaeriaceae fungi.

The mating-type (MAT1) locus encodes transcription factors essential for the onset of the sexual cycle in ascomycete fungi. This locus has been characterised in only a few heterothallic, plant pathogenic Mycosphaerellaceae and Teratosphaeriaceae. We used available genome sequences for Mycosphaerellales species to investigate the presence of two unique mating-type-associated features. The accessory MAT1 genes, MAT1-1-10 (MATORF2) and MAT1-2-12 (MATORF1), typically occurred in both MAT idiomorphs of Mycosphaerellaceae species. In contrast, they were associated with only one idiomorph in Teratosphaeriaceae species. In Pseudocercospora, phylogenetic analyses showed that homologs present in different idiomorphs were paralogous and subject to different selective pressures, indicating that their evolution is linked to mating type. In almost half of the investigated Mycosphaerellales genomes, numerous short fragment sequences, almost identical to portions of the MAT1-1-1 and MAT1-2-1 genes, were present in multiple areas outside of the MAT1 locus. Aligned to the MAT1 genes, these sequences resembled an mRNA transcript. Fragment sequences were similar among species groups and occurred at the same genomic positions, implying that monophyletic groups have the same origins of these sequences. Although the functions of the MAT fragment sequences and accessory MAT1 genes remain unknown, both were expressed in the representative Mycosphaerellaceae and Teratosphaeriaceae species that were investigated.

Delineation of Paraburkholderia tuberum sensu stricto and description of Paraburkholderia podalyriae sp. nov. nodulating the South African legume Podalyria calyptrata.

Since the discovery of Paraburkholderia tuberum, an indigenous South African species and one of the first beta-rhizobia described, several other South African rhizobial Paraburkholderia species have been recognized. Here, we investigate the taxonomic status of 31 rhizobial isolates from the root nodules of diverse South African legume hosts in the Core Cape Subregion, which were initially identified as P. tuberum. These isolates originate from the root nodules of genera in the Papilionoideae as well as Vachellia karroo, from the subfamily Caesalpinioideae. Genealogical concordance analysis of five loci allowed delineation of the isolates into two putative species clusters (A and B). Cluster A included P. tuberum STM678T, suggesting that this monophyletic group represents P. tuberum sensu stricto. Cluster B grouped sister to P. tuberum and included isolates from the Paarl Rock Nature Reserve in the Western Cape Province. Average Nucleotide Identity (ANI) analysis further confirmed that isolates of Cluster A shared high genome similarity with P. tuberum STM678T compared to Cluster B and other Paraburkholderia species. The members of Cluster B associated with a single species of Podalyria, P. calyptrata. For this new taxon we accordingly propose the name Paraburkholderia podalyriae sp. nov., with the type strain WC7.3bT (= LMG 31413T; SARCC 750T). Based on our nodA and nifH phylogenies, P. podalyriae sp. nov. and strains of P. tuberum sensu stricto (including one from V. karroo) belong to symbiovar africana, the symbiotic loci of which have a separate evolutionary origin to those of Central and South American Paraburkholderia strains.

Rhizosphere Diazotrophs and Other Bacteria Associated with Native and Encroaching Legumes in the Succulent Karoo Biome in South Africa.

Total and diazotrophic bacteria were assessed in the rhizosphere soils of native and encroaching legumes growing in the Succulent Karoo Biome (SKB), South Africa. These were Calobota sericea, Lessertia diffusa, Vachellia karroo, and Wiborgia monoptera, of Fabaceae family near Springbok (Northern Cape Province) and neighboring refugia of the Fynbos biome for C. sericea for comparison purposes. Metabarcoding approach using 16S rRNA gene revealed Actinobacteria (26.7%), Proteobacteria (23.6%), Planctomycetes, and Acidobacteria (10%), while the nifH gene revealed Proteobacteria (70.3%) and Cyanobacteria (29.5%) of the total sequences recovered as the dominant phyla. Some of the diazotrophs measured were assigned to families; Phyllobacteriaceae (39%) and Nostocaceae (24.4%) (all legumes), Rhodospirillaceae (7.9%), Bradyrhizobiaceae (4.6%) and Methylobacteriaceae (3%) (C. sericea, V. karroo, W. monoptera), Rhizobiaceae (4.2%; C. sericea, L. diffusa, V. Karroo), Microchaetaceae (4%; W. monoptera, V. karroo), Scytonemataceae (3.1%; L. diffusa, W. monoptera), and Pseudomonadaceae (2.7%; V. karroo) of the total sequences recovered. These families have the potential to fix the atmospheric nitrogen. While some diazotrophs were specific or shared across several legumes, a member of Mesorhizobium species was common in all rhizosphere soils considered. V. karroo had statistically significantly higher Alpha and distinct Beta-diversity values, than other legumes, supporting its influence on soil microbes. Overall, this work showed diverse bacteria that support plant life in harsh environments such as the SKB, and shows how they are influenced by legumes.

Bradyrhizobium altum sp. nov., Bradyrhizobium oropedii sp. nov. and Bradyrhizobium acaciae sp. nov. from South Africa show locally restricted and pantropical nodA phylogeographic patterns.

Africa is known for its rich legume diversity with a significant number of endemic species originating in South Africa. Many of these legumes associate with rhizobial symbionts of the genus Bradyrhizobium, of which most represent new species. Yet, none of the Bradyrhizobium species from South Africa have been described. In this study, phylogenetic analysis of 16S rRNA gene sequences of fourteen strains isolated in southern Africa from root nodules of diverse legumes (i.e., from the tribes Crotalarieae, Acacieae, Genisteae, Phaseoleae and Cassieae) revealed that they belong to the Bradyrhizobium elkanii supergroup. The taxonomic position and possible novelty of these strains were further interrogated using genealogical concordance of five housekeeping genes (atpD, dnaK, glnII, gyrB and rpoB). These phylogenies consistently recovered four monophyletic groups and one singleton within Bradyrhizobium. Of these groups, two were conspecific with Bradyrhizobium brasilense UFLA 03-321T and Bradyrhizobium ivorense CI-1BT, while the remaining three represented novel taxa. Their existence was further supported with genome data, as well as metabolic and physiological traits. Analysis of nodA gene sequences further showed that the evolution of these bacteria likely involved adapting to local legume hosts and environmental conditions through the acquisition, via horizontal gene transfer, of optimal symbiotic loci. We accordingly propose the following names Bradyrhizobium acaciae sp. nov. 10BBT (SARCC 730T = LMG 31409T), Bradyrhizobium oropedii sp. nov. Pear76T (SARCC 731T = LMG 31408T), and Bradyrhizobium altum sp. nov. Pear77T (SARCC 754T = LMG 31407T) to accommodate three novel species, all of which are symbionts of legumes in South Africa.

Extracellular Vesicles in Teasing Apart Complex Plant-Microbiota Links: Implications for Microbiome-Based Biotechnology.

Extracellular vesicles (EVs) are subcellular carriers of bioactive compounds with a complex array of functional effects on target cells. In mammals, circulating bodily fluid microbiota EVs (mbEVs) deliver cargo from source cells and adversely or favorably alter the physiology of the same source, neighboring, and distant recipient cells in an autocrine, paracrine, or endocrine fashion, respectively. Plant mbEVs may similarly mediate these interactive effects within the holobiont framework. However, the majority of plant EV research has focused on a small number of individual microbes, thus failing to reflect the importance of EVs in a community and consequently leaving a wide gap in scientific knowledge. Addressing this gap should entail a systems-level approach that combines vesicle characterization with microbiome analyses. This would certainly usher in a new age in microbial biotechnology entailing EVs as a microbiome manipulation strategy, a biomarker for stable microbiomes, and a diagnostic tool for plant infectious diseases.

Culture-independent assessment of the diazotrophic Bradyrhizobium communities in the Pampa and Atlantic Forest Biomes localities in southern Brazil.

The isolation of rhizobial strains from the root and stem nodules remains a commonly used method despite its limitations as it enables the identification of mainly dominant symbiotic groups within rhizobial communities. To overcome these limitations, we used genus-specific nifD primers in a culture-independent assessment of Bradyrhizobium communities inhabiting soils in southern Brazil. The majority of nifD sequences were generated from DNA isolated from tropical-lowland pasture soils, although some soil samples originated from the Campos de Cima da Serra volcanic plateau. In the nifD tree, all the bradyrhizobial sequences comprised 38 clades, including 18 new clades. The sequences generated in this study were resolved into 22 clades and 21 singletons. The nifD bradyrhizobial assemblage contained Azorhizobium and α-proteobacterial methylotrophic genera, suggesting that these genera may have acquired their nif loci from Bradyrhizobium donors. The most common in the lowland pasture soils subclade III.3D branch comprises the isolates of mainly an American origin. On the other hand, subclade III.4, which was earlier detected in Brazil among Bradyrhizobium isolates nodulating native lupins, appears more common in the Campos de Cima da Serra soils. The second-largest group, Clade XXXVIII, has not yet been reported in culture-dependent studies, while another common group called Clade I represents a symbiovar predominating in Australia. The identification of the diverse nifD Clade I haplotypes in the tropical-lowland pastures infested by Australian Acacia spp implies that the introduction of these legumes to southern Brazil has resulted in the dissemination of their bradyrhizobial symbionts.