The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound "fixation threads" (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-TrnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (a) aided the genus-wide retention of nodulation, and (b) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

Aromatic amino acid biosynthesis impacts root hair development and symbiotic associations in Lotus japonicus.

Legume roots can be symbiotically colonized by arbuscular mycorrhizal (AM) fungi and nitrogen-fixing bacteria. In Lotus japonicus, the latter occurs intracellularly by the cognate rhizobial partner Mesorhizobium loti or intercellularly with the Agrobacterium pusense strain IRBG74. Although these symbiotic programs show distinctive cellular and transcriptome signatures, some molecular components are shared. In this study, we demonstrate that 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 1 (DAHPS1), the first enzyme in the biosynthetic pathway of aromatic amino acids (AAAs), plays a critical role in root hair development and for AM and rhizobial symbioses in Lotus. Two homozygous DAHPS1 mutants (dahps1-1 and dahps1-2) showed drastic alterations in root hair morphology, associated with alterations in cell wall dynamics and a progressive disruption of the actin cytoskeleton. The altered root hair structure was prevented by pharmacological and genetic complementation. dahps1-1 and dahps1-2 showed significant reductions in rhizobial infection (intracellular and intercellular) and nodule organogenesis and a delay in AM colonization. RNAseq analysis of dahps1-2 roots suggested that these phenotypes are associated with downregulation of several cell wall-related genes, and with an attenuated signaling response. Interestingly, the dahps1 mutants showed no detectable pleiotropic effects, suggesting a more selective recruitment of this gene in certain biological processes. This work provides robust evidence linking AAA metabolism to root hair development and successful symbiotic associations.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Evolution of novel strains of Ensifer nodulating the invasive legume Leucaena leucocephala (Lam.) de Wit in different climatic regions of India through lateral gene transfer.

More than 200 root-nodule bacterial strains were isolated from Leucaena leucocephala growing at 42 sampling sites across 12 states and three union territories of India. Genetic diversity was observed among 114 strains from various climatic zones; based on recA, these were identified as strains of Ensifer, Mesorhizobium, Rhizobium, and Bradyrhizobium. In multilocus sequence analysis (MLSA) strains clustered into several novel clades and lineages. Ensifer were predominant nodulating genotype isolated from majority of alkaline soils, while Mesorhizobium and Rhizobium strains were isolated from a limited sampling in North-Eastern states with acidic soils. Positive nodulation assays of selected Ensifer representing different genetic combinations of housekeeping and sym genes suggested their broad host range within the closely related mimosoid genera Vachellia, Senegalia, Mimosa, and Prosopis. Leucaena selected diverse strains of Ensifer and Mesorhizobium as symbionts depending on available soil pH, climatic, and other edaphic conditions in India. Lateral gene transfer seems to play a major role in genetic diversification of Ensifer exhibited in terms of Old World vs. Neotropical genetic make-up and mixed populations at several sites. Although Neotropical Ensifer strains were most symbiotically effective on Leucaena, the native Ensifer are promiscuous and particularly well-adapted to a wide range of sampling sites with varied climates and edaphic factors.

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.

WHIRLY1 functions in the nucleus to regulate barley leaf development and associated metabolite profiles.

The WHIRLY (WHY) DNA/RNA binding proteins fulfil multiple but poorly characterised functions in leaf development. Here, we show that WHY1 transcript levels were highest in the bases of 7-day old barley leaves. Immunogold labelling revealed that the WHY1 protein was more abundant in the nuclei than the proplastids of the leaf bases. To identify transcripts associated with leaf development we conducted hierarchical clustering of differentially abundant transcripts along the developmental gradient of wild-type leaves. Similarly, metabolite profiling was employed to identify metabolites exhibiting a developmental gradient. A comparative analysis of transcripts and metabolites in barley lines (W1-1 and W1-7) lacking WHY1, which show delayed greening compared with the wild type revealed that the transcript profile of leaf development was largely unchanged in W1-1 and W1-7 leaves. However, there were differences in levels of several transcripts encoding transcription factors associated with chloroplast development. These include a barley homologue of the Arabidopsis GATA transcription factor that regulates stomatal development, greening and chloroplast development, NAC1; two transcripts with similarity to Arabidopsis GLK1 and two transcripts encoding ARF transcriptions factors with functions in leaf morphogenesis and development. Chloroplast proteins were less abundant in the W1-1 and W1-7 leaves than the wild type. The levels of tricarboxylic acid cycle metabolites and GABA were significantly lower in WHY1 knockdown leaves than the wild type. This study provides evidence that WHY1 is localised in the nuclei of leaf bases, contributing the regulation of nuclear-encoded transcripts that regulate chloroplast development.

Phylogenetic diversity and plant growth-promoting activities of rhizobia nodulating fenugreek (Trigonella foenum-graecum Linn.) cultivated in different agroclimatic regions of India.

Fenugreek (Trigonella foenum-graecum Linn.), is an extensively cultivated legume crop used as a herb, spice, and traditional medicine in India. The symbiotic efficiency and plant growth-promoting potential of fenugreek rhizobia depend on the symbiont strain and environmental factors. We isolated 176 root-nodulating bacteria from fenugreek cultivated in different agroclimatic regions of India. MALDI-TOF MS-based identification and phylogenetic analyses based on 16S rRNA and five housekeeping genes classified the fenugreek-rhizobia as Ensifer (Sinorhizobium) meliloti. However, the strains represent separate sub-lineages of E. meliloti, distinct from all reported sub-lineages across the globe. We also observed the spatial distribution of fenugreek rhizobia, as the three sub-lineages of E. meliloti recorded during this study were specific to their respective agroclimatic regions. According to the symbiotic gene (nodC and nifH) phylogenies, all three sub-lineages of E. meliloti harboured symbiotic genes similar to symbiovar meliloti; as with the housekeeping genes, these also revealed a spatial distribution for different clades of sv. meliloti. The strains could nodulate fenugreek plants and they showed plant growth-promoting potential. Significant differences were found in the plant growth parameters in response to inoculation with the various strains, suggesting strain-level differences. This study demonstrates that fenugreek rhizobia in India are diverse and spatially distributed in different agro-climatic regions.

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.

Role and Regulation of Poly-3-Hydroxybutyrate in Nitrogen Fixation in Azorhizobium caulinodans.

An Azorhizobium caulinodans phaC mutant (OPS0865) unable to make poly-3-hydroxybutyrate (PHB), grows poorly on many carbon sources and cannot fix nitrogen in laboratory culture. However, when inoculated onto its host plant, Sesbania rostrata, the phaC mutant consistently fixed nitrogen. Upon reisolation from S. rostrata root nodules, a suppressor strain (OPS0921) was isolated that has significantly improved growth on a variety of carbon sources and also fixes nitrogen in laboratory culture. The suppressor retains the original mutation and is unable to synthesize PHB. Genome sequencing revealed a suppressor transition mutation, G to A (position 357,354), 13 bases upstream of the ATG start codon of phaR in its putative ribosome binding site (RBS). PhaR is the global regulator of PHB synthesis but also has other roles in regulation within the cell. In comparison with the wild type, translation from the phaR native RBS is increased approximately sixfold in the phaC mutant background, suggesting that the level of PhaR is controlled by PHB. Translation from the phaR mutated RBS (RBS*) of the suppressor mutant strain (OPS0921) is locked at a low basal rate and unaffected by the phaC mutation, suggesting that RBS* renders the level of PhaR insensitive to regulation by PHB. In the original phaC mutant (OPS0865), the lack of nitrogen fixation and poor growth on many carbon sources is likely to be due to increased levels of PhaR causing dysregulation of its complex regulon, because PHB formation, per se, is not required for effective nitrogen fixation in A. caulinodans.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Deciphering Molecular Host-Pathogen Interactions During Ramularia Collo-Cygni Infection on Barley.

Ramularia collo-cygni is the causal agent of Ramularia leaf spot disease (RLS) on barley and became, during the recent decades, an increasing threat for farmers across the world. Here, we analyze morphological, transcriptional, and metabolic responses of two barley cultivars having contrasting tolerance to RLS, when infected by an aggressive or mild R. collo-cygni isolate. We found that fungal biomass in leaves of the two cultivars does not correlate with their tolerance to RLS, and both cultivars displayed cell wall reinforcement at the point of contact with the fungal hyphae. Comparative transcriptome analysis identified that the largest transcriptional differences between cultivars are at the early stages of fungal colonization with differential expression of kinases, calmodulins, and defense proteins. Weighted gene co-expression network analysis identified modules of co-expressed genes, and hub genes important for cultivar responses to the two R. collo-cygni isolates. Metabolite analyses of the same leaves identified defense compounds such as p-CHDA and serotonin, correlating with responses observed at transcriptome and morphological level. Together these all-round responses of barley to R. collo-cygni provide molecular tools for further development of genetic and physiological markers that may be tested for improving tolerance of barley to this fungal pathogen.

The large mimosoid genus Inga Mill. (tribe Ingeae, Caesalpinioideae) is nodulated by diverse Bradyrhizobium strains in its main centers of diversity in Brazil.

Inga (Caesalpinioideae) is the type genus of the Ingeae tribe in the mimosoid clade. It comprises about 300 species, all trees or treelets, and has an exclusively neotropical distribution, with Brazil as its main center of diversity. In this study, we analyzed the diversity of 40 strains of rhizobia isolated from root nodules collected from ten species of Inga belonging to different types of vegetation in Brazil. Sequences of their housekeeping genes (dnaK, recA, rpoB, gyrB and glnII), 16S rRNA genes, internal transcribed spacer (ITS) regions, as well as their symbiosis-essential genes (nodC and nifH) were used to characterize them genetically. The ability of the rhizobia to form nodules on Inga spp., and on the promiscuous legume siratro (Macroptilium atropurpureum) was also evaluated. A multilocus sequence analysis (MLSA) combined with an analysis of the ITS region showed that the isolates were distributed into four main groups (A-D) within the large genus Bradyrhizobium. Analysis of the nodC and nifH genes showed that the isolates formed a separate branch from all described species of Bradyrhizobium, except for B. ingae. Most of the tested isolates formed nodules on siratro and all isolates tested nodulated Inga spp. Our results suggest a unique co-evolutionary history of Bradyrhizobium and Inga and demonstrate the existence of potential new species of microsymbionts nodulating this important and representative genus of leguminous tree from the Caesalpinioideae mimosoid clade.

Genomic Diversity of Pigeon Pea (Cajanus cajan L. Millsp.) Endosymbionts in India and Selection of Potential Strains for Use as Agricultural Inoculants.

Pigeon pea (Cajanus cajan L. Millsp. ) is a legume crop resilient to climate change due to its tolerance to drought. It is grown by millions of resource-poor farmers in semiarid and tropical subregions of Asia and Africa and is a major contributor to their nutritional food security. Pigeon pea is the sixth most important legume in the world, with India contributing more than 70% of the total production and harbouring a wide variety of cultivars. Nevertheless, the low yield of pigeon pea grown under dry land conditions and its yield instability need to be improved. This may be done by enhancing crop nodulation and, hence, biological nitrogen fixation (BNF) by supplying effective symbiotic rhizobia through the application of "elite" inoculants. Therefore, the main aim in this study was the isolation and genomic analysis of effective rhizobial strains potentially adapted to drought conditions. Accordingly, pigeon pea endosymbionts were isolated from different soil types in Southern, Central, and Northern India. After functional characterisation of the isolated strains in terms of their ability to nodulate and promote the growth of pigeon pea, 19 were selected for full genome sequencing, along with eight commercial inoculant strains obtained from the ICRISAT culture collection. The phylogenomic analysis [Average nucleotide identity MUMmer (ANIm)] revealed that the pigeon pea endosymbionts were members of the genera Bradyrhizobium and Ensifer. Based on nodC phylogeny and nod cluster synteny, Bradyrhizobium yuanmingense was revealed as the most common endosymbiont, harbouring nod genes similar to those of Bradyrhizobium cajani and Bradyrhizobium zhanjiangense. This symbiont type (e.g., strain BRP05 from Madhya Pradesh) also outperformed all other strains tested on pigeon pea, with the notable exception of an Ensifer alkalisoli strain from North India (NBAIM29). The results provide the basis for the development of pigeon pea inoculants to increase the yield of this legume through the use of effective nitrogen-fixing rhizobia, tailored for the different agroclimatic regions of India.

Conditional sanctioning in a legume-Rhizobium mutualism.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.

Distinct signaling routes mediate intercellular and intracellular rhizobial infection in Lotus japonicus.

Rhizobial infection of legume roots during the development of nitrogen-fixing root nodules can occur intracellularly, through plant-derived infection threads traversing cells, or intercellularly, via bacterial entry between epidermal plant cells. Although it is estimated that around 25% of all legume genera are intercellularly infected, the pathways and mechanisms supporting this process have remained virtually unexplored due to a lack of genetically amenable legumes that exhibit this form of infection. In this study, we report that the model legume Lotus japonicus is infected intercellularly by the IRBG74 strain, recently proposed to belong to the Agrobacterium clade of the Rhizobiaceae. We demonstrate that the resources available for L. japonicus enable insight into the genetic requirements and fine-tuning of the pathway governing intercellular infection in this species. Inoculation of L. japonicus mutants shows that Ethylene-responsive factor required for nodulation 1 (Ern1) and Leu-rich Repeat Receptor-Like Kinase (RinRK1) are dispensable for intercellular infection in contrast to intracellular infection. Other symbiotic genes, including nod factor receptor 5 (NFR5), symbiosis receptor-like kinase (SymRK), Ca2+/calmodulin dependent kinase (CCaMK), exopolysaccharide receptor 3 (Epr3), Cyclops, nodule inception (Nin), nodulation signaling pathway 1 (Nsp1), nodulation signaling pathway 2 (Nsp2), cystathionine-ß-synthase (Cbs), and Vapyrin are equally important for both entry modes. Comparative RNAseq analysis of roots inoculated with IRBG74 revealed a distinctive transcriptome response compared with intracellular colonization. In particular, several cytokinin-related genes were differentially regulated. Corroborating this observation, cyp735A and ipt4 cytokinin biosynthesis mutants were significantly affected in their nodulation with IRBG74, whereas lhk1 cytokinin receptor mutants formed no nodules. These results indicate a differential requirement for cytokinin signaling during intercellular rhizobial entry and highlight distinct modalities of inter- and intracellular infection mechanisms in L. japonicus.

Comparative Genomics Provides Insights into the Taxonomy of Azoarcus and Reveals Separate Origins of Nif Genes in the Proposed Azoarcus and Aromatoleum Genera.

Among other attributes, the Betaproteobacterial genus Azoarcus has biotechnological importance for plant growth-promotion and remediation of petroleum waste-polluted water and soils. It comprises at least two phylogenetically distinct groups. The "plant-associated" group includes strains that are isolated from the rhizosphere or root interior of the C4 plant Kallar Grass, but also strains from soil and/or water; all are considered to be obligate aerobes and all are diazotrophic. The other group (now partly incorporated into the new genus Aromatoleum) comprises a diverse range of species and strains that live in water or soil that is contaminated with petroleum and/or aromatic compounds; all are facultative or obligate anaerobes. Some are diazotrophs. A comparative genome analysis of 32 genomes from 30 Azoarcus-Aromatoleum strains was performed in order to delineate generic boundaries more precisely than the single gene, 16S rRNA, that has been commonly used in bacterial taxonomy. The origin of diazotrophy in Azoarcus-Aromatoleum was also investigated by comparing full-length sequences of nif genes, and by physiological measurements of nitrogenase activity using the acetylene reduction assay. Based on average nucleotide identity (ANI) and whole genome analyses, three major groups could be discerned: (i) Azoarcus comprising Az. communis, Az. indigens and Az. olearius, and two unnamed species complexes, (ii) Aromatoleum Group 1 comprising Ar. anaerobium, Ar. aromaticum, Ar. bremense, and Ar. buckelii, and (iii) Aromatoleum Group 2 comprising Ar. diolicum, Ar. evansii, Ar. petrolei, Ar. toluclasticum, Ar. tolulyticum, Ar. toluolicum, and Ar. toluvorans. Single strain lineages such as Azoarcus sp. KH32C, Az. pumilus, and Az. taiwanensis were also revealed. Full length sequences of nif-cluster genes revealed two groups of diazotrophs in Azoarcus-Aromatoleum with nif being derived from Dechloromonas in Azoarcus sensu stricto (and two Thauera strains) and from Azospira in Aromatoleum Group 2. Diazotrophy was confirmed in several strains, and for the first time in Az. communis LMG5514, Azoarcus sp. TTM-91 and Ar. toluolicum TT. In terms of ecology, with the exception of a few plant-associated strains in Azoarcus (s.s.), across the group, most strains/species are found in soil and water (often contaminated with petroleum or related aromatic compounds), sewage sludge, and seawater. The possession of nar, nap, nir, nor, and nos genes by most Azoarcus-Aromatoleum strains suggests that they have the potential to derive energy through anaerobic nitrate respiration, so this ability cannot be usefully used as a phenotypic marker to distinguish genera. However, the possession of bzd genes indicating the ability to degrade benzoate anaerobically plus the type of diazotrophy (aerobic vs. anaerobic) could, after confirmation of their functionality, be considered as distinguishing phenotypes in any new generic delineations. The taxonomy of the Azoarcus-Aromatoleum group should be revisited; retaining the generic name Azoarcus for its entirety, or creating additional genera are both possible outcomes.

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.