Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

A Metagenome from a Steam Vent in Los Azufres Geothermal Field Shows an Abundance of Thermoplasmatales archaea and Bacteria from the Phyla Actinomycetota and Pseudomonadota.

Los Azufres National Park is a geothermal field that has a wide number of thermal manifestations; nevertheless, the microbial communities in many of these environments remain unknown. In this study, a metagenome from a sediment sample from Los Azufres National Park was sequenced. In this metagenome, we found that the microbial diversity corresponds to bacteria (Actinomycetota, Pseudomonadota), archaea (Thermoplasmatales and Candidatus Micrarchaeota and Candidatus Parvarchaeota), eukarya (Cyanidiaceae), and viruses (Fussellovirus and Caudoviricetes). The functional annotation showed genes related to the carbon fixation pathway, sulfur metabolism, genes involved in heat and cold shock, and heavy-metal resistance. From the sediment, it was possible to recover two metagenome-assembled genomes from Ferrimicrobium and Cuniculiplasma. Our results showed that there are a large number of microorganisms in Los Azufres that deserve to be studied.

Functional genomics of a Spiroplasma associated with the carmine cochineals Dactylopius coccus and Dactylopius opuntiae.

BACKGROUND: Spiroplasma is a widely distributed endosymbiont of insects, arthropods, and plants. In insects, Spiroplasma colonizes the gut, hemolymph, and reproductive organs of the host. Previous metagenomic surveys of the domesticated carmine cochineal Dactylopius coccus and the wild cochineal D. opuntiae reported sequences of Spiroplasma associated with these insects. However, there is no analysis of the genomic capabilities and the interaction of this Spiroplasma with Dactylopius. RESULTS: Here we present three Spiroplasma genomes independently recovered from metagenomes of adult males and females of D. coccus, from two different populations, as well as from adult females of D. opuntiae. Single-copy gene analysis showed that these genomes were > 92% complete. Phylogenomic analyses classified these genomes as new members of Spiroplasma ixodetis. Comparative genome analysis indicated that they exhibit fewer genes involved in amino acid and carbon catabolism compared to other spiroplasmas. Moreover, virulence factor-encoding genes (i.e., glpO, spaid and rip2) were found incomplete in these S. ixodetis genomes. We also detected an enrichment of genes encoding the type IV secretion system (T4SS) in S. ixodetis genomes of Dactylopius. A metratranscriptomic analysis of D. coccus showed that some of these T4SS genes (i.e., traG, virB4 and virD4) in addition to the superoxide dismutase sodA of S. ixodetis were overexpressed in the ovaries. CONCLUSION: The symbiont S. ixodetis is a new member of the bacterial community of D. coccus and D. opuntiae. The recovery of incomplete virulence factor-encoding genes in S. ixodetis of Dactylopius suggests that this bacterium is a non-pathogenic symbiont. A high number of genes encoding the T4SS, in the S. ixodetis genomes and the overexpression of these genes in the ovary and hemolymph of the host suggest that S. ixodetis use the T4SS to interact with the Dactylopius cells. Moreover, the transcriptional differences of S. ixodetis among the gut, hemolymph and ovary tissues of D. coccus indicate that this bacterium can respond and adapt to the different conditions (e.g., oxidative stress) present within the host. All this evidence proposes that there is a strong interaction and molecular signaling in the symbiosis between S. ixodetis and the carmine cochineal Dactylopius.

Functional characterization of culturable fungi from microbiomes of the "conical cobs" Mexican maize (Zea mays L.) landrace.

Mexican maize landraces, produced for local consumption, are adapted to different environmental conditions, and their yield is affected by abiotic and biotic factors, including the use of agrochemicals. The search for sustainable alternatives to agrochemicals includes the study of the culturable microbial communities. In this study, the fungal communities associated with 2 Mexican maize landraces reddish and bluish "conical cobs" were found to be comprised of Ascomycota fungi, represented by 89 strains within 6 orders (Pleosporales, Hypocreales, Onygenales, Capnodiales, Helotiales, and Eurotiales) and 16 genera. Cellulases and metallophores production were the primary enzymatic products and plant growth-promoting activities were detected among the isolates. Penicillium, Didymella, and Fusarium strains had the most active enzymatic and plant growth promoting activities, however, Aspergillus sp. HES2-2.2, Talaromyces sp. RS1-7, and Penicillium sp. HFS3-3 showed antagonistic activity against the four phytopathogenic Fusarium strains Fusarium oxysporum, Fusarium sambucinum, Fusarium fujikuroi and Fusarium incarnatum-equiseti and also a high and diverse production of enzymatic and plant growth promoting activities; here we identified fungal strains as candidates to promote maize growth.

Folates in legume root nodules.

Folates are multifunctional metabolites in plants that are essential for cell division, nucleic acids and amino acid synthesis. During symbiotic nitrogen fixation in legumes, these cofactors are needed for de novo purine biosynthesis, meaning that changes in the folate pools could directly affect the flow of fixed nitrogen to the plant. Its role related to symbiotic nitrogen fixation has not been yet explored, but recent data suggest a relevant role during the first steps. Transcriptomic, metabolomic and proteomic analyses indicate that folates are accumulated in symbiotic plant tissue, as they are involved, not only in de novo purines biosynthesis, but in nitrogen translocation, endoreduplication and phytohormones biosynthesis. Understanding the possible implication of folate pools during the nitrogen fixation and assimilation, might aid for new engineering targets, in relation to the two transformylations or the production of glycine by serine hydroxymethyltransferase during the de novo purine biosynthetic pathway. In this review, we intend to deliver and discuss the available evidence that support a relevant role of folates during the symbiotic nitrogen fixation.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Evolution of an Escherichia coli PTS- strain: a study of reproducibility and dynamics of an adaptive evolutive process.

Adaptive laboratory evolution (ALE) has been used to study and solve pressing questions about evolution, especially for the study of the development of mutations that confer increased fitness during evolutionary processes. In this contribution, we investigated how the evolutionary process conducted with the PTS- mutant of Escherichia coli PB11 in three parallel batch cultures allowed the restoration of rapid growth with glucose as the carbon source. The significant findings showed that genomic sequence analysis of a set of newly evolved mutants isolated from ALE experiments 2-3 developed some essential mutations, which efficiently improved the fast-growing phenotypes throughout different fitness landscapes. Regulator galR was the target of several mutations such as SNPs, partial and total deletions, and insertion of an IS1 element and thus indicated the relevance of a null mutation of this gene in the adaptation of the evolving population of PB11 during the parallel ALE experiments. These mutations resulted in the selection of MglB and GalP as the primary glucose transporters by the evolving population, but further selection of at least a second adaptive mutation was also necessary. We found that mutations in the yfeO, rppH, and rng genes improved the fitness advantage of evolving PTS- mutants and resulted in amplification of leaky activity in Glk for glucose phosphorylation and upregulation of glycolytic and other growth-related genes. Notably, we determined that these mutations appeared and were fixed in the evolving populations between 48 and 72 h of cultivation, which resulted in the selection of fast-growing mutants during one ALE experiments in batch cultures of 80 h duration.Key points• ALE experiments selected evolved mutants through different fitness landscapes in which galR was the target of different mutations: SNPs, deletions, and insertion of IS.• Key mutations in evolving mutants appeared and fixed at 48-72 h of cultivation.• ALE experiments led to increased understanding of the genetics of cellular adaptation to carbon source limitation.

Current phylogeny of Rhodospirillaceae: A multi-approach study.

Rhodospirillaceae represents a major family of the class alphaproteobacteria that includes an increasing number of functionally diverse taxa. The aim of this work is to evaluate the present phylogenetic diversity of the Rhodospirillaceae, which includes several metagenome-assembled genomes of uncultivated bacteria, as well as cultivated bacteria that were previously classified in different families. Various methodological approaches have been followed to discern the phylogenetic diversity of the taxa associated with the Rhodospirillaceae, which are grouped in three major sub-divisions and several other taxonomic entities that are currently confined to the genus rank. These genera include Tistrella, Elstera, Dongia and Ferrovibrio among cultivated organisms and alphaproteobacteria bacterium 41-28 among uncultivated bacteria. Overall, this study adds at least 11 genera and over 40 species to the current set of taxa belonging to the Rhodospirillaceae, a taxonomic term that clearly requires amendment. We propose to re-classify all taxa associated with the Rhodospirillaceae family under the new order, Diaforabacterales ord. nov. (from the Greek word for diversity, διάφορα). This study also uncovers the likely root of Rhodospirillaceae among recently reported metagenome-assembled genomes of uncultivated marine and groundwater bacteria.

Safety and efficacy of bosutinib in fourth-line therapy of chronic myeloid leukemia patients.

Bosutinib is a second-generation tyrosine kinase inhibitor (2GTKI) approved at 400 mg once daily (QD) as first-line therapy in patients with chronic myeloid leukemia (CML) patients and at 500 mg QD in patients who are resistant to or intolerant of prior therapy. In clinical practice, bosutinib is often given to patients who have failed imatinib, nilotinib, and dasatinib (i.e., as fourth-line treatment), despite the limited data on its clinical benefit in this setting. We have retrospectively evaluated the results of bosutinib in a series of 62 CML patients who have failed to prior treatment with all three, imatinib, nilotinib, and dasatinib. Median time on TKI treatment before bosutinib start was 105 (9-163) months, and median duration on bosutinib was 9 months (1-30). Overall, probabilities to achieve complete cytogenetic response (CCyR) and major molecular response (MMR) were 25% and 24% respectively. After a median follow-up period of 14 months, the event-free survival and progression-free survival were 68 and 85%, respectively. Sixty-four percent of patients in CCyR at the time of bosutinib start were able to achieve MMR. In contrast, patients without CCyR, probabilities to obtain CCyR and MMR were 25% and 14%. Bosutinib was well tolerated in this heavily pretreated patients' cohort. Pleural effusions and diarrhea were the most frequent grade II-IV side effects, leading to treatment discontinuation in 16% of patients. Bosutinib is an effective treatment option for patients who have failed previous 2GTKIs due to intolerance. However, efficacy seems to be related to the molecular response that the patient achieved prior to bosutinib.

Minimal standards for the description of new genera and species of rhizobia and agrobacteria.

Herein the members of the Subcommittee on Taxonomy of Rhizobia and Agrobacteria of the International Committee on Systematics of Prokaryotes review recent developments in rhizobial and agrobacterial taxonomy and propose updated minimal standards for the description of new species (and genera) in these groups. The essential requirements (minimal standards) for description of a new species are (1) a genome sequence of at least the proposed type strain and (2) evidence for differentiation from other species based on genome sequence comparisons. It is also recommended that (3) genetic variation within the species is documented with sequence data from several clearly different strains and (4) phenotypic features are described, and their variation documented with data from a relevant set of representative strains. Furthermore, it is encouraged that information is provided on (5) nodulation or pathogenicity phenotypes, as appropriate, with relevant gene sequences. These guidelines supplement the current rules of general bacterial taxonomy, which require (6) a name that conforms to the International Code of Nomenclature of Prokaryotes, (7) validation of the name by publication either directly in the International Journal of Systematic and Evolutionary Microbiology or in a validation list when published elsewhere, and (8) deposition of the type strain in two international culture collections in separate countries.

Klebsiella variicola and Klebsiella quasipneumoniae with capacity to adapt to clinical and plant settings.

OBJECTIVE: To compare the genetic determinants involved in plant colonization or virulence in the reported genomes of K. variicola, K. quasipneumoniae and K. pneumoniae. MATERIALS AND METHODS: In silico comparisons and Jaccard analysis of genomic data were used. Fimbrial genes were detected by PCR. Biological assays were performed with plant and clinical isolates. RESULTS: Plant colonization genes such as cellulases, catalases and hemagglutinins were mainly present in K. variicola genomes. Chromosomal ß-lactamases were characteristic of this species and had been previously misclassified. K. variicola and K. pneumoniae isolates produced plant hormones. CONCLUSIONS: A mosaic distribution of different virulence- and plant-associated genes was found in K. variicola and in K. quasipneumoniae genomes. Some plant colonizing genes were found mainly in K. variicola genomes. The term plantanosis is proposed for plant-borne human infections.

Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants, animals and humans.

OBJECTIVE: Due to the fact that K. variicola, K. quasipneumoniae and K. pneumoniae are closely related bacterial species, misclassification can occur due to mistakes either in normal biochemical tests or during submission to public databases. The objective of this work was to identify K. variicola and K. quasipneumoniae genomes misclassified in GenBank database. MATERIALS AND METHODS: Both rpoB phylogenies and average nucleotide identity (ANI) were used to identify a significant number of misclassified Klebsiella spp. genomes. RESULTS: Here we report an update of K. variicola and K. Quasipneumoniae genomes correctly classified and a list of isolated genomes obtained from humans, plants, animals and insects, described originally as K. pneumoniae or K. variicola, but known now to be misclassified. CONCLUSIONS: This work contributes to recognize the extensive presence of K. variicola and K. quasipneumoniae isolates in diverse sites and samples.

Species-specific diversity of novel bacterial lineages and differential abundance of predicted pathways for toxic compound degradation in scorpion gut microbiota.

Scorpions are considered 'living fossils' that have conserved ancestral anatomical features and have adapted to numerous habitats. However, their gut microbiota diversity has not been studied. Here, we characterized the gut microbiota of two scorpion species, Vaejovis smithi and Centruroides limpidus. Our results indicate that scorpion gut microbiota is species-specific and that food deprivation reduces bacterial diversity. 16S rRNA gene phylogenetic analysis revealed novel bacterial lineages showing a low level of sequence identity to any known bacteria. Furthermore, these novel bacterial lineages were each restricted to a different scorpion species. Additionally, our results of the predicted metagenomic profiles revealed a core set of pathways that were highly abundant in both species, and mostly related to amino acid, carbohydrate, vitamin and cofactor metabolism. Notably, the food-deprived V. smithi shotgun metagenome matched almost completely the metabolic features of the prediction. Finally, comparisons among predicted metagenomic profiles showed that toxic compound degradation pathways were more abundant in recently captured C. limpidus scorpions. This study gives a first insight into the scorpion gut microbiota and provides a reference for future studies on the gut microbiota from other arachnid species.

Endemic Mimosa species from Mexico prefer alphaproteobacterial rhizobial symbionts.

The legume genus Mimosa has > 500 species, with two major centres of diversity, Brazil (c. 350 spp.) and Mexico (c. 100 spp.). In Brazil most species are nodulated by Burkholderia. Here we asked whether this is also true of native and endemic Mexican species. We have tested this apparent affinity for betaproteobacteria by examining the symbionts of native and endemic species of Mimosa in Mexico, especially from the central highlands where Mimosa spp. have diversified. Nodules were tested for betaproteobacteria using in situ immunolocalization. Rhizobia isolated from the nodules were genetically characterized and tested for their ability to nodulate Mimosa spp. Immunological analysis of 25 host taxa suggested that most (including all the highland endemics) were not nodulated by betaproteobacteria. Phylogenetic analyses of 16S rRNA, recA, nodA, nodC and nifH genes from 87 strains isolated from 20 taxa confirmed that the endemic Mexican Mimosa species favoured alphaproteobacteria in the genera Rhizobium and Ensifer: this was confirmed by nodulation tests. Host phylogeny, geographic isolation and coevolution with symbionts derived from very different soils have potentially contributed to the striking difference in the choice of symbiotic partners by Mexican and Brazilian Mimosa species.

Rhizobium favelukesii sp. nov., isolated from the root nodules of alfalfa (Medicago sativa L).

Strains LPU83T and Or191 of the genus Rhizobium were isolated from the root nodules of alfalfa, grown in acid soils from Argentina and the USA. These two strains, which shared the same plasmid pattern, lipopolysaccharide profile, insertion-sequence fingerprint, 16S rRNA gene sequence and PCR-fingerprinting pattern, were different from reference strains representing species of the genus Rhizobium with validly published names. On the basis of previously reported data and from new DNA-DNA hybridization results, phenotypic characterization and phylogenetic analyses, strains LPU83T and Or191 can be considered to be representatives of a novel species of the genus Rhizobium, for which the name Rhizobium favelukesii sp. nov. is proposed. The type strain of this species is LPU83T (=CECT 9014T=LMG 29160T), for which an improved draft-genome sequence is available.

Microbiota from Rhabditis regina may alter nematode entomopathogenicity.

Here we report the presence of the entomopathogenic nematode Rhabditis (Rhabditoides) regina affecting white grubs (Phyllophaga sp. and Anomala sp.) in Mexico and R. regina-associated bacteria. Bioassays were performed to test the entomopathogenic capacity of dauer and L2 and L3 (combined) larval stages. Furthermore, we determined the diversity of bacteria from laboratory nematodes cultivated for 2 years (dauer and L2-L3 larvae) and from field nematodes (dauer and L2-L3 larvae) in addition to the virulence in Galleria mellonella larvae of some bacterial species from both laboratory and field nematodes. Dauer and non-dauer larvae of R. regina killed G. mellonella. Bacteria such as Serratia sp. (isolated from field nematodes) and Klebsiella sp. (isolated from larvae of laboratory and field nematodes) may explain R. regina entomopathogenic capabilities. Different bacteria were found in nematodes after subculturing in the laboratory suggesting that R. regina may acquire bacteria in different environments. However, there were some consistently found bacteria from laboratory and field nematodes such as Pseudochrobactrum sp., Comamonas sp., Alcaligenes sp., Klebsiella sp., Acinetobacter sp., and Leucobacter sp. that may constitute the nematode microbiome. Results showed that some bacteria contributing to entomopathogenicity may be lost in the laboratory representing a disadvantage when nematodes are cultivated to be used for biological control.

Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola.

BACKGROUND: Klebsiella variicola was very recently described as a new bacterial species and is very closely related to Klebsiella pneumoniae; in fact, K. variicola isolates were first identified as K. pneumoniae. Therefore, it might be the case that some isolates, which were initially classified as K. pneumoniae, are actually K. variicola. The aim of this study was to devise a multiplex-PCR probe that can differentiate isolates from these sister species. RESULT: This work describes the development of a multiplex-PCR method to identify K. variicola. This development was based on sequencing a K. variicola clinical isolate (801) and comparing it to other K. variicola and K. pneumoniae genomes. The phylogenetic analysis showed that K. variicola isolates form a monophyletic group that is well differentiated from K. pneumoniae. Notably, the isolate K. pneumoniae 342 and K. pneumoniae KP5-1 might have been misclassified because in our analysis, both clustered with K. variicola isolates rather than with K. pneumoniae. The multiplex-PCR (M-PCR-1 to 3) probe system could identify K. variicola with high accuracy using the shared unique genes of K. variicola and K. pneumoniae genomes, respectively. M-PCR-1 was used to assay a collection of multidrug-resistant (503) and antimicrobial-sensitive (557) K. pneumoniae clinical isolates. We found K. variicola with a prevalence of 2.1% (23/1,060), of them a 56.5% (13/23) of the isolates were multidrug resistant, and 43.5% (10/23) of the isolates were antimicrobial sensitive. The phylogenetic analysis of rpoB of K. variicola-positive isolates identified by multiplex-PCR support the correct identification and differentiation of K. variicola from K. pneumoniae clinical isolates. CONCLUSIONS: This multiplex-PCR provides the means to reliably identify and genotype K. variicola. This tool could be very helpful for clinical, epidemiological, and population genetics studies of this species. A low but significant prevalence of K. variicola isolates was found, implying that misclassification had occurred previously. We believe that our multiplex-PCR assay could be of paramount importance to understand the population dynamics of K. variicola in both clinical and environmental settings.

Bradyrhizobium viridifuturi sp. nov., encompassing nitrogen-fixing symbionts of legumes used for green manure and environmental services.

Symbiotic nitrogen-fixing bacteria, commonly called rhizobia, are agronomically important because they can provide significant amounts of nitrogen to plants and help in recovery of impoverished soils and improvement of degraded environments. In recent years, with advances in molecular techniques, several studies have shown that these bacteria have high levels of genetic diversity, resulting in taxonomic reclassifications and descriptions of new species. However, despite the advances achieved, highly conserved 16S ribosomal genes (16S rRNA) do not elucidate differences between species of several genera, including the genus Bradyrhizobium. Other methodologies, such as multilocus sequence analysis (MLSA), have been used in such cases, with good results. In this study, three strains (SEMIAs 690T, 6387 and 6428) of the genus Bradyrhizobium, isolated from nitrogen-fixing nodules of Centrosema and Acacia species, without clear taxonomic positions, were studied. These strains differed from genetically closely related species according to the results of MLSA of four housekeeping genes (dnaK, glnII, gyrB and recA) and nucleotide identities of the concatenated genes with those of related species ranged from 87.8 % to 95.7 %, being highest with Bradyrhizobium elkanii. DNA-DNA hybridization (less than 32 % DNA relatedness) and average nucleotide identity values of the whole genomes (less than 90.5 %) indicated that these strains represented a novel species, and phenotypic traits were determined. Our data supported the description of the SEMIA strains as Bradyrhizobium viridifuturi sp. nov., and SEMIA 690T ( = CNPSo 991T = C 100aT = BR 1804T = LMG 28866T), isolated from Centrosema pubescens, was chosen as type strain.

Bradyrhizobium tropiciagri sp. nov. and Bradyrhizobium embrapense sp. nov., nitrogen-fixing symbionts of tropical forage legumes.

Biological nitrogen fixation is a key process for agricultural production and environmental sustainability, but there are comparatively few studies of symbionts of tropical pasture legumes, as well as few described species of the genus Bradyrhizobium, although it is the predominant rhizobial genus in the tropics. A detailed polyphasic study was conducted with two strains of the genus Bradyrhizobium used in commercial inoculants for tropical pastures in Brazil, CNPSo 1112T, isolated from perennial soybean (Neonotonia wightii), and CNPSo 2833T, from desmodium (Desmodium heterocarpon). Based on 16S-rRNA gene phylogeny, both strains were grouped in the Bradyrhizobium elkanii superclade, but were not clearly clustered with any known species. Multilocus sequence analysis of three (glnII, gyrB and recA) and five (plus atpD and dnaK) housekeeping genes confirmed that the strains are positioned in two distinct clades. Comparison with intergenic transcribed spacer sequences of type strains of described species of the genus Bradyrhizobium showed similarity lower than 93.1 %, and differences were confirmed by BOX-PCR analysis. Nucleotide identity of three housekeeping genes with type strains of described species ranged from 88.1 to 96.2 %. Average nucleotide identity of genome sequences showed values below the threshold for distinct species of the genus Bradyrhizobium ( < 90.6 %), and the value between the two strains was also below this threshold (91.2 %). Analysis of nifH and nodC gene sequences positioned the two strains in a clade distinct from other species of the genus Bradyrhizobium. Morphophysiological, genotypic and genomic data supported the description of two novel species in the genus Bradyrhizobium, Bradyrhizobium tropiciagri sp. nov. (type strain CNPSo 1112T = SMS 303T = BR 1009T = SEMIA 6148T = LMG 28867T) and Bradyrhizobium embrapense sp. nov. (type strain CNPSo 2833T = CIAT 2372T = BR 2212T = SEMIA 6208T = U674T = LMG 2987).

Rhizobium ecuadorense sp. nov., an indigenous N2-fixing symbiont of the Ecuadorian common bean (Phaseolus vulgaris L.) genetic pool.

There are two major centres of genetic diversification of common bean (Phaseolus vilgaris L.), the Mesoamerican and the Andean, and the legume is capable of establishing nitrogen-fixing symbioses with several rhizobia; Rhizobium etli seems to be the dominant species in both centres. Another genetic pool of common bean, in Peru and Ecuador, is receiving increasing attention, and studies of microsymbionts from the region can help to increase our knowledge about coevolution of this symbiosis. We have previously reported several putative new lineages from this region and here present data indicating that strains belonging to one of them, PEL4, represent a novel species. Based on 16S rRNA gene sequence phylogeny, PEL4 strains are positioned in the Rhizobium phaseoli/R. etli/Rhizobium leguminosarum clade, but show unique properties in several morphological, physiological and biochemical analyses, as well as in BOX-PCR profiles ( < 75% similarity with related species). PEL4 strains also differed from related species based on multilocus sequence analysis of three housekeeping genes (glnII, gyrB and recA). Nucleotide identities of the three concatenated genes between PEL4 strains and related species ranged from 91.8 to 94.2%, being highest with Rhizobium fabae. DNA-DNA hybridization ( < 47% DNA relatedness) and average nucleotide identity values of the whole genomes ( < 90.2%) also supported the novel species status. The PEL4 strains were effective in nodulating and fixing N2 with common beans. The data supported the view that PEL4 strains represent a novel species, Rhizobium ecuadorense sp. nov. The type strain is CNPSo 671(T) ( = UMR 1450(T) = PIMAMPIRS I 5(T) = LMG 27578(T)).