Complete genomes of two Variovorax endophytes isolated from surface-sterilized alfalfa nodules.

Variovorax species catabolize a wide range of natural and industrial products and have been shown to be integral rhizosphere inhabitants. Here, we report the complete genomes of V. paradoxus 2u118 and V. sp. SPNA7, which were isolated from alfalfa root nodules and possess plant growth-promoting properties.

Chemical Composition of Essential Oils from Eight Tunisian Eucalyptus Species and Their Antifungal and Herbicidal Activities.

Eucalyptus species are known to produce metabolites such as essential oils (EOs) that play an important role in the control of weeds, pests and phytopathogenic fungi. The aims of this study were as follows: (i) to determine the chemical composition of the EOs derived from eight Eucalyptus species growing in Tunisia, and (ii) to study their possible antifungal and herbicidal activities. EOs were obtained by hydrodistillation from the dried leaves of eight Eucalyptus species, namely, E. angulosa, E. cladocalyx, E. diversicolor, E. microcoryx, E. ovata, E. resinifera, E. saligna and E. sargentii, and the determination of their composition was achieved by GC and GC-MS. The EOs' antifungal activities were tested against four Fusarium strains, and the EOs' herbicidal properties were evaluated on the germination and seedling growth of three annual weeds (Trifolium campestre, Lolium rigidum and Sinapis arvensis) and three cultivated crop species (Lepidium sativum, Raphanus sativus and Triticum durum). The EO yields ranged between 0.12 and 1.32%. The most abundant components found were eucalyptol, α-pinene, p-cymene, trans-pinocarveol, α-terpineol and globulol. All EOs showed significant antifungal activity against the four phytopathogenic Fusarium strains. E. cladocalyx EO exhibited the highest level of antifungal activity, and the greatest inhibition of seed germination was obtained even at lowest concentrations used. These findings suggested that E. resinifera, E. ovata and E. cladocalyx EOs could have applications in agriculture as possible biopesticides, as Fusarium antagonists and as bioherbicides.

Reviewing and renewing the use of beneficial root and soil bacteria for plant growth and sustainability in nutrient-poor, arid soils.

A rapidly increasing human population coupled with climate change and several decades of over-reliance on synthetic fertilizers has led to two pressing global challenges: food insecurity and land degradation. Therefore, it is crucial that practices enabling both soil and plant health as well as sustainability be even more actively pursued. Sustainability and soil fertility encompass practices such as improving plant productivity in poor and arid soils, maintaining soil health, and minimizing harmful impacts on ecosystems brought about by poor soil management, including run-off of agricultural chemicals and other contaminants into waterways. Plant growth promoting bacteria (PGPB) can improve food production in numerous ways: by facilitating resource acquisition of macro- and micronutrients (especially N and P), modulating phytohormone levels, antagonizing pathogenic agents and maintaining soil fertility. The PGPB comprise different functional and taxonomic groups of bacteria belonging to multiple phyla, including Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, among others. This review summarizes many of the mechanisms and methods these beneficial soil bacteria use to promote plant health and asks whether they can be further developed into effective, potentially commercially available plant stimulants that substantially reduce or replace various harmful practices involved in food production and ecosystem stability. Our goal is to describe the various mechanisms involved in beneficial plant-microbe interactions and how they can help us attain sustainability.

Comparative Analysis of the Cultured and Total Bacterial Community in the Wheat Rhizosphere Microbiome Using Culture-Dependent and Culture-Independent Approaches.

Rhizosphere and root-associated bacteria are key components of crop production and sustainable agriculture. However, utilization of these beneficial bacteria is often limited by conventional culture techniques because a majority of soil microorganisms cannot be cultured using standard laboratory media. Therefore, the purpose of this study was to improve culturability and investigate the diversity of the bacterial communities from the wheat rhizosphere microbiome collected from three locations in Egypt with contrasting soil characteristics by using metagenomic analysis and improved culture-based methods. The improved strategies of the culture-dependent approach included replacing the agar in the medium with gellan gums and modifying its preparation by autoclaving the phosphate and gelling agents separately. Compared to the total operational taxonomic units (OTUs) observed from the metagenomic data sets derived from the three analyzed soils, 1.86 to 2.52% of the bacteria were recovered using the modified cultivation strategies, whereas less than 1% were obtained employing the standard cultivation protocols. Twenty-one percent of the cultivable isolates exhibited multiple plant growth-promoting (PGP) properties, including P solubilization activity and siderophore production. From the metagenomic analysis, the most abundant phyla were Proteobacteria, Actinobacteria, Chloroflexi, Bacteroidetes, and Firmicutes. Moreover, the relative abundance of the specific bacterial taxa was correlated with the soil characteristics, demonstrating the effect of the soil in modulating the plant rhizosphere microbiome. IMPORTANCE Bacteria colonizing the rhizosphere, a narrow zone of soil surrounding the root system, are known to have beneficial effects in improving the growth and stress tolerance of plants. However, most bacteria in natural environments, especially those in rhizosphere soils, are recalcitrant to cultivation using traditional techniques, and thus their roles in soil health and plant growth remain unexplored. Hence, investigating new culture media and culture conditions to bring "not-yet-cultured" species into cultivation and to identify new functions is still an important task for all microbiologists. To this end, we describe improved cultivation protocols that increase the number and diversity of cultured bacteria from the rhizosphere of wheat plants. Using such approaches will lead to new insights into culturing more beneficial bacteria that live in the plant rhizosphere, in so doing creating greater opportunities not only for field application but also for promoting sustainability.

Inoculation With a Microbe Isolated From the Negev Desert Enhances Corn Growth.

Corn (Zea mays L.) is not only an important food source, but also has numerous uses, including for biofuels, fillers for cosmetics, glues, and so on. The amount of corn grown in the U.S. has significantly increased since the 1960's and with it, the demand for synthetic fertilizers and pesticides/fungicides to enhance its production. However, the downside of the continuous use of these products, especially N and P fertilizers, has been an increase in N2O emissions and other greenhouse gases into the atmosphere as well as run-off into waterways that fuel pollution and algal blooms. These approaches to agriculture, especially if exacerbated by climate change, will result in decreased soil health as well as human health. We searched for microbes from arid, native environments that are not being used for agriculture because we reasoned that indigenous microbes from such soils could promote plant growth and help restore degraded soils. Employing cultivation-dependent methods to isolate bacteria from the Negev Desert in Israel, we tested the effects of several microbial isolates on corn in both greenhouse and small field studies. One strain, Dietzia cinnamea 55, originally identified as Planomicrobium chinense, significantly enhanced corn growth over the uninoculated control in both greenhouse and outside garden experiments. We sequenced and analyzed the genome of this bacterial species to elucidate some of the mechanisms whereby D. cinnamea 55 promoted plant growth. In addition, to ensure the biosafety of this previously unknown plant growth promoting bacterial (PGPB) strain as a potential bioinoculant, we tested the survival and growth of Caenorhabditis elegans and Galleria mellonella (two animal virulence tests) as well as plants in response to D. cinnamea 55 inoculation. We also looked for genes for potential virulence determinants as well as for growth promotion.

Exopolysaccharide production in Ensifer meliloti laboratory and native strains and their effects on alfalfa inoculation.

Bacterial surface molecules have an important role in the rhizobia-legume symbiosis. Ensifer meliloti (previously, Sinorhizobium meliloti), a symbiotic Gram-negative rhizobacterium, produces two different exopolysaccharides (EPSs), termed EPS I (succinoglycan) and EPS II (galactoglucan), with different functions in the symbiotic process. Accordingly, we undertook a study comparing the potential differences in alfalfa nodulation by E. meliloti strains with differences in their EPSs production. Strains recommended for inoculation as well as laboratory strains and native strains isolated from alfalfa fields were investigated. This study concentrated on EPS-II production, which results in mucoid colonies that are dependent on the presence of an intact expR gene. The results revealed that although the studied strains exhibited different phenotypes, the differences did not affect alfalfa nodulation itself. However, subtle changes in timing and efficacy to the effects of inoculation with the different strains may result because of other as-yet unknown factors. Thus, additional research is needed to determine the most effective inoculant strains and the best conditions for improving alfalfa production under agricultural conditions.

Trinickia dabaoshanensis sp. nov., a new name for a lost species.

"Burkholderia dabaoshanensis" was described in 2012. Although the name was effectively published, it could not be validly published, because the description provided in the original paper did not comply with the Rule 27 (2) (c) of the Bacterial Code. The Code requiresthat the properties of the taxon form part of the protologue. As the name of this species does not have standing in nomenclature, the recently published new combination Trinickia dabaoshanensis could also not be validly published. The current proposal attempts to rectify the situation by providing the information required to meet the criteria stipulated in Rule 27 for valid publication.

Draft Genome of Burkholderia cenocepacia TAtl-371, a Strain from the Burkholderia cepacia Complex Retains Antagonism in Different Carbon and Nitrogen Sources.

Burkholderia cenocepacia TAtl-371 was isolated from the rhizosphere of a tomato plant growing in Atlatlahucan, Morelos, Mexico. This strain exhibited a broad antimicrobial spectrum against bacteria, yeast, and fungi. Here, we report and describe the improved, high-quality permanent draft genome of B. cenocepacia TAtl-371, which was sequenced using a combination of PacBio RS and PacBio RS II sequencing methods. The 7,496,106 bp genome of the TAtl-371 strain is arranged in three scaffolds, contains 6722 protein-coding genes, and 99 RNA only-encoding genes. Genome analysis revealed genes related to biosynthesis of antimicrobials such as non-ribosomal peptides, siderophores, chitinases, and bacteriocins. Moreover, analysis of bacterial growth on different carbon and nitrogen sources shows that the strain retains its antimicrobial ability.

Engineering root microbiomes for healthier crops and soils using beneficial, environmentally safe bacteria.

The Green Revolution developed new crop varieties, which greatly improved food security worldwide. However, the growth of these plants relied heavily on chemical fertilizers and pesticides, which have led to an overuse of synthetic fertilizers, insecticides, and herbicides with serious environmental consequences and negative effects on human health. Environmentally friendly plant-growth-promoting methods to replace our current reliance on synthetic chemicals and to develop more sustainable agricultural practices to offset the damage caused by many agrochemicals are proposed herein. The increased use of bioinoculants, which consist of microorganisms that establish synergies with target crops and influence production and yield by enhancing plant growth, controlling disease, and providing critical mineral nutrients, is a potential solution. The microorganisms found in bioinoculants are often bacteria or fungi that reside within either external or internal plant microbiomes. However, before they can be used routinely in agriculture, these microbes must be confirmed as nonpathogenic strains that promote plant growth and survival. In this article, besides describing approaches for discovering plant-growth-promoting bacteria in various environments, including phytomicrobiomes and soils, we also discuss methods to evaluate their safety for the environment and for human health.

Antifungal Activity of Bacillus Species Against Fusarium and Analysis of the Potential Mechanisms Used in Biocontrol.

Fusarium is a complex genus of ascomycete fungi that consists of plant pathogens of agricultural relevance. Controlling Fusarium infection in crops that leads to substantial yield losses is challenging. These economic losses along with environmental and human health concerns over the usage of chemicals in attaining disease control are shifting focus toward the use of biocontrol agents for effective control of phytopathogenic Fusarium spp. In the present study, an analysis of the plant-growth promoting (PGP) and biocontrol attributes of four bacilli (Bacillus simplex 30N-5, B. simplex 11, B. simplex 237, and B. subtilis 30VD-1) has been conducted. The production of cellulase, xylanase, pectinase, and chitinase in functional assays was studied, followed by in silico gene analysis of the PGP-related and biocontrol-associated genes. Of all the bacilli included in this study, B. subtilis 30VD-1 (30VD-1) demonstrated the most effective antagonism against Fusarium spp. under in vitro conditions. Additionally, 100 µg/ml of the crude 1-butanol extract of 30VD-1's cell-free culture filtrate caused about 40% inhibition in radial growth of Fusarium spp. Pea seed bacterization with 30VD-1 led to considerable reduction in wilt severity in plants with about 35% increase in dry plant biomass over uninoculated plants growing in Fusarium-infested soil. Phase contrast microscopy demonstrated distortions and abnormal swellings in F. oxysporum hyphae on co-culturing with 30VD-1. The results suggest a multivariate mode of antagonism of 30VD-1 against phytopathogenic Fusarium spp., by producing chitinase, volatiles, and other antifungal molecules, the characterization of which is underway.

Whole Genome Analyses Suggests that Burkholderia sensu lato Contains Two Additional Novel Genera (Mycetohabitans gen. nov., and Trinickia gen. nov.): Implications for the Evolution of Diazotrophy and Nodulation in the Burkholderiaceae.

Burkholderia sensu lato is a large and complex group, containing pathogenic, phytopathogenic, symbiotic and non-symbiotic strains from a very wide range of environmental (soil, water, plants, fungi) and clinical (animal, human) habitats. Its taxonomy has been evaluated several times through the analysis of 16S rRNA sequences, concantenated 4⁻7 housekeeping gene sequences, and lately by genome sequences. Currently, the division of this group into Burkholderia, Caballeronia, Paraburkholderia, and Robbsia is strongly supported by genome analysis. These new genera broadly correspond to the various habitats/lifestyles of Burkholderia s.l., e.g., all the plant beneficial and environmental (PBE) strains are included in Paraburkholderia (which also includes all the N2-fixing legume symbionts) and Caballeronia, while most of the human and animal pathogens are retained in Burkholderia sensu stricto. However, none of these genera can accommodate two important groups of species. One of these includes the closely related Paraburkholderia rhizoxinica and Paraburkholderia endofungorum, which are both symbionts of the fungal phytopathogen Rhizopus microsporus. The second group comprises the Mimosa-nodulating bacterium Paraburkholderia symbiotica, the phytopathogen Paraburkholderia caryophylli, and the soil bacteria Burkholderia dabaoshanensis and Paraburkholderia soli. In order to clarify their positions within Burkholderia sensu lato, a phylogenomic approach based on a maximum likelihood analysis of conserved genes from more than 100 Burkholderia sensu lato species was carried out. Additionally, the average nucleotide identity (ANI) and amino acid identity (AAI) were calculated. The data strongly supported the existence of two distinct and unique clades, which in fact sustain the description of two novel genera Mycetohabitans gen. nov. and Trinickia gen. nov. The newly proposed combinations are Mycetohabitans endofungorum comb. nov., Mycetohabitansrhizoxinica comb. nov., Trinickia caryophylli comb. nov., Trinickiadabaoshanensis comb. nov., Trinickia soli comb. nov., and Trinickiasymbiotica comb. nov. Given that the division between the genera that comprise Burkholderia s.l. in terms of their lifestyles is often complex, differential characteristics of the genomes of these new combinations were investigated. In addition, two important lifestyle-determining traits-diazotrophy and/or symbiotic nodulation, and pathogenesis-were analyzed in depth i.e., the phylogenetic positions of nitrogen fixation and nodulation genes in Trinickia via-à-vis other Burkholderiaceae were determined, and the possibility of pathogenesis in Mycetohabitans and Trinickia was tested by performing infection experiments on plants and the nematode Caenorhabditis elegans. It is concluded that (1) T. symbiotica nif and nod genes fit within the wider Mimosa-nodulating Burkholderiaceae but appear in separate clades and that T. caryophyllinif genes are basal to the free-living Burkholderia s.l. strains, while with regard to pathogenesis (2) none of the Mycetohabitans and Trinickia strains tested are likely to be pathogenic, except for the known phytopathogen T. caryophylli.

Cell Autoaggregation, Biofilm Formation, and Plant Attachment in a Sinorhizobium meliloti lpsB Mutant.

Bacterial surface molecules are crucial for the establishment of a successful rhizobia-legume symbiosis, and, in most bacteria, are also critical for adherence properties, surface colonization, and as a barrier for defense. Rhizobial mutants defective in the production of exopolysaccharides (EPSs), lipopolysaccharides (LPSs), or capsular polysaccharides are usually affected in symbiosis with their plant hosts. In the present study, we evaluated the role of the combined effects of LPS and EPS II in cell-to-cell and cell-to-surface interactions in Sinorhizobium meliloti by studying planktonic cell autoaggregation, biofilm formation, and symbiosis with the host plant Medicago sativa. The lpsB mutant, which has a defective core portion of LPS, exhibited a reduction in biofilm formation on abiotic surfaces as well as altered biofilm architecture compared with the wild-type Rm8530 strain. Atomic force microscopy and confocal laser microscopy revealed an increase in polar cell-to-cell interactions in the lpsB mutant, which might account for the biofilm deficiency. However, a certain level of biofilm development was observed in the lpsB strain compared with the EPS II-defective mutant strains. Autoaggregation experiments carried out with LPS and EPS mutant strains showed that both polysaccharides have an impact on the cell-to-cell adhesive interactions of planktonic bacteria. Although the lpsB mutation and the loss of EPS II production strongly stimulated early attachment to alfalfa roots, the number of nodules induced in M. sativa was not increased. Taken together, this work demonstrates that S. meliloti interactions with biotic and abiotic surfaces depend on the interplay between LPS and EPS II.

Broad-spectrum antimicrobial activity by Burkholderia cenocepacia TAtl-371, a strain isolated from the tomato rhizosphere.

The Burkholderia cepacia complex (Bcc) comprises a group of 24 species, many of which are opportunistic pathogens of immunocompromised patients and also are widely distributed in agricultural soils. Several Bcc strains synthesize strain-specific antagonistic compounds. In this study, the broad killing activity of B. cenocepacia TAtl-371, a Bcc strain isolated from the tomato rhizosphere, was characterized. This strain exhibits a remarkable antagonism against bacteria, yeast and fungi including other Bcc strains, multidrug-resistant human pathogens and plant pathogens. Genome analysis of strain TAtl-371 revealed several genes involved in the production of antagonistic compounds: siderophores, bacteriocins and hydrolytic enzymes. In pursuit of these activities, we observed growth inhibition of Candida glabrata and Paraburkholderia phenazinium that was dependent on the iron concentration in the medium, suggesting the involvement of siderophores. This strain also produces a previously described lectin-like bacteriocin (LlpA88) and here this was shown to inhibit only Bcc strains but no other bacteria. Moreover, a compound with an m/z 391.2845 with antagonistic activity against Tatumella terrea SHS 2008T was isolated from the TAtl-371 culture supernatant. This strain also contains a phage-tail-like bacteriocin (tailocin) and two chitinases, but the activity of these compounds was not detected. Nevertheless, the previous activities are not responsible for the whole antimicrobial spectrum of TAtl-371 seen on agar plates, suggesting the presence of other compounds yet to be found. In summary, we observed a diversified antimicrobial activity for strain TAtl-371 and believe it supports the biotechnological potential of this Bcc strain as a source of new antimicrobials.

Draft genome of Paraburkholderia caballeronis TNe-841T, a free-living, nitrogen-fixing, tomato plant-associated bacterium.

10.1601/nm.26956 caballeronis is a plant-associated bacterium. Strain TNe-841T was isolated from the rhizosphere of tomato (Solanum lycopersicum L. var. lycopersicum) growing in Nepantla Mexico State. Initially this bacterium was found to effectively nodulate Phaseolus vulgaris L. However, from an analysis of the genome of strain TNe-841T and from repeat inoculation experiments, we found that this strain did not nodulate bean and also lacked nodulation genes, suggesting that the genes were lost. The genome consists of 7,115,141 bp with a G + C content of 67.01%. The sequence includes 6251 protein-coding genes and 87 RNA genes.

Combating Fusarium Infection Using Bacillus-Based Antimicrobials.

Despite efforts to control toxigenic Fusarium species, wilt and head-blight infections are destructive and economically damaging diseases that have global effects. The utilization of biological control agents in disease management programs has provided an effective, safe, and sustainable means to control Fusarium-induced plant diseases. Among the most widely used microbes for biocontrol agents are members of the genus Bacillus. These species influence plant and fungal pathogen interactions by a number of mechanisms such as competing for essential nutrients, antagonizing pathogens by producing fungitoxic metabolites, or inducing systemic resistance in plants. The multivariate interactions among plant-biocontrol agent-pathogen are the subject of this study, in which we survey the advances made regarding the research on the Bacillus-Fusarium interaction and focus on the principles and mechanisms of action among plant-growth promoting Bacillus species. In particular, we highlight their use in limiting and controlling Fusarium spread and infestations of economically important crops. This knowledge will be useful to define strategies for exploiting this group of beneficial bacteria for use as inoculants by themselves or in combination with other microbes for enhanced crop protection.

Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.

Chitinase-producing bacteria and their role in biocontrol.

Chitin is an important component of the exteriors of insects and fungi. Upon degradation of chitin by a number of organisms, severe damage and even death may occur in pathogens and pests whose external surfaces contain this polymer. Currently, chemical fungicides and insecticides are the major means of controlling these disease-causing agents. However, due to the potential harm that these chemicals cause to the environment and to human and animal health, new strategies are being developed to replace or reduce the use of fungal- and pest-killing compounds in agriculture. In this context, chitinolytic microorganisms are likely to play an important role as biocontrol agents and pathogen antagonists and may also function in the control of postharvest rot. In this review, we discuss the literature concerning chitin and the basic knowledge of chitin-degrading enzymes, and also describe the biocontrol effects of chitinolytic microorganisms and their potential use as more sustainable pesticides and fungicides in the field.

Blue light does not inhibit nodulation in Sesbania rostrata.

Earlier, we reported that root nodulation was inhibited by blue light irradiation of Lotus japonicus. Because some legumes do not establish nodules exclusively on underground roots, we investigated whether nodule formation in Sesbania rostrata, which forms both root and "stem" nodules following inoculation with Azorhizobium caulinodans, is inhibited by blue light as are L. japonicus nodules. We found that neither S. rostrata nodulation nor nitrogen fixation was inhibited by blue light exposure. Moreover, although A. caulinodans proliferation was not affected by blue light irradiation, bacterial survival was decreased. Therefore, blue light appears to impose different responses depending on the legume-rhizobial symbiosis.

Blue Light Perception by Both Roots and Rhizobia Inhibits Nodule Formation in Lotus japonicus.

In many legumes, roots that are exposed to light do not form nodules. Here, we report that blue light inhibits nodulation in Lotus japonicus roots inoculated with Mesorhizobium loti. Using RNA interference, we suppressed the expression of the phototropin and cryptochrome genes in L. japonicus hairy roots. Under blue light, plants transformed with an empty vector did not develop nodules, whereas plants exhibiting suppressed expression of cry1 and cry2 genes formed nodules. We also measured rhizobial growth to investigate whether the inhibition of nodulation could be caused by a reduced population of rhizobia in response to light. Although red light had no effect on rhizobial growth, blue light had a strong inhibitory effect. Rhizobial growth under blue light was partially restored in signature-tagged mutagenesis (STM) strains in which LOV-HK/PAS- and photolyase-related genes were disrupted. Moreover, when Ljcry1A and Ljcry2B-silenced plants were inoculated with the STM strains, nodulation was additively increased. Our data show that blue light receptors in both the host plant and the symbiont have a profound effect on nodule development. The exact mechanism by which these photomorphogenetic responses function in the symbiosis needs further study, but they are clearly involved in optimizing legume nodulation.

Symbiotic Burkholderia Species Show Diverse Arrangements of nif/fix and nod Genes and Lack Typical High-Affinity Cytochrome cbb3 Oxidase Genes.

Genome analysis of fourteen mimosoid and four papilionoid beta-rhizobia together with fourteen reference alpha-rhizobia for both nodulation (nod) and nitrogen-fixing (nif/fix) genes has shown phylogenetic congruence between 16S rRNA/MLSA (combined 16S rRNA gene sequencing and multilocus sequence analysis) and nif/fix genes, indicating a free-living diazotrophic ancestry of the beta-rhizobia. However, deeper genomic analysis revealed a complex symbiosis acquisition history in the beta-rhizobia that clearly separates the mimosoid and papilionoid nodulating groups. Mimosoid-nodulating beta-rhizobia have nod genes tightly clustered in the nodBCIJHASU operon, whereas papilionoid-nodulating Burkholderia have nodUSDABC and nodIJ genes, although their arrangement is not canonical because the nod genes are subdivided by the insertion of nif and other genes. Furthermore, the papilionoid Burkholderia spp. contain duplications of several nod and nif genes. The Burkholderia nifHDKEN and fixABC genes are very closely related to those found in free-living diazotrophs. In contrast, nifA is highly divergent between both groups, but the papilionoid species nifA is more similar to alpha-rhizobia nifA than to other groups. Surprisingly, for all Burkholderia, the fixNOQP and fixGHIS genes required for cbb3 cytochrome oxidase production and assembly are missing. In contrast, symbiotic Cupriavidus strains have fixNOQPGHIS genes, revealing a divergence in the evolution of two distinct electron transport chains required for nitrogen fixation within the beta-rhizobia.