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Due to the wide cultivation of bean (Phaseolus vulgaris L.), rhizobia associated with this plant have been isolated from many different geographical regions. In order to investigate the species diversity of bean rhizobia, comparative genome sequence analysis was performed in the present study for 69 Rhizobium strains mainly isolated from root nodules of bean and clover (Trifolium spp.). Based on genome average nucleotide identity, digital DNA:DNA hybridization, and phylogenetic analysis of 1,458 single-copy core genes, these strains were classified into 28 clusters, consistent with their species definition based on multilocus sequence analysis (MLSA) of atpD, glnII, and recA. The bean rhizobia were found in 16 defined species and nine putative novel species; in addition, 35 strains previously described as Rhizobium etli, Rhizobium phaseoli, Rhizobium vallis, Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium spp. should be renamed. The phylogenetic patterns of symbiotic genes nodC and nifH were highly host-specific and inconsistent with the genomic phylogeny. Multiple symbiovars (sv.) within the Rhizobium species were found as a common feature: sv. phaseoli, sv. trifolii and sv. viciae in Rhizobium anhuiense; sv. phaseoli and sv. mimosae in Rhizobium sophoriradicis/R. etli/Rhizobium sp. III; sv. phaseoli and sv. trifolii in Rhizobium hidalgonense/Rhizobium acidisoli; sv. phaseoli and sv. viciae in R. leguminosarum/Rhizobium sp. IX; sv. trifolii and sv. viciae in Rhizobium laguerreae. Thus, genomic comparison revealed great species diversity in bean rhizobia, corrected the species definition of some previously misnamed strains, and demonstrated the MLSA a valuable and simple method for defining Rhizobium species., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Phaseolus vulgaris (common bean) was introduced to Kenya several centuries ago but the rhizobia that nodulate it in the country remain poorly characterised. To address this gap in knowledge, 178 isolates recovered from the root nodules of P. vulgaris cultivated in Kenya were genotyped stepwise by the analysis of genomic DNA fingerprints, PCR-RFLP and 16S rRNA, atpD, recA and nodC gene sequences. Results indicated that P. vulgaris in Kenya is nodulated by at least six Rhizobium genospecies, with most of the isolates belonging to Rhizobium phaseoli and a possibly novel Rhizobium species. Infrequently, isolates belonged to Rhizobium paranaense, Rhizobium leucaenae, Rhizobium sophoriradicis and Rhizobium aegyptiacum. Despite considerable core-gene heterogeneity among the isolates, only four nodC gene alleles were observed indicating conservation within this gene. Testing of the capacity of the isolates to fix nitrogen (N 2 ) in symbiosis with P. vulgaris revealed wide variations in effectiveness, with ten isolates comparable to Rhizobium tropici CIAT 899, a commercial inoculant strain for P. vulgaris. In addition to unveiling effective native rhizobial strains with potential as inoculants in Kenya, this study demonstrated that Kenyan soils harbour diverse P. vulgaris-nodulating rhizobia, some of which formed phylogenetic clusters distinct from known lineages. The native rhizobia differed by site, suggesting that field inoculation of P. vulgaris may need to be locally optimised., (Copyright © 2018 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Analysis of spoilage-associated microbiota of modified-atmosphere packaged poultry meat revealed four different bacterial isolates that could not be assigned to known species. They showed a Gram-negative staining behavior, were facultatively aerobic, non-motile with variable cell morphology. Phylogenetic analysis of 16S rDNA and gyrB, rpoD and recA revealed a distinct lineage within the genus Photobacterium with Photobacterium (P.) iliopiscarium DSM 9896 T , P. phosphoreum DSM 15556 T , P. kishitanii DSM 19954 T , P. piscicola LMG 27681 T and P. aquimaris DSM 23343 T as closest relatives. The designated type strain TMW 2.2021 T is non-luminous and grew at 0-20°C (optimum 10-15°C), within pH 5.0-8.5 (optimum 6-8) and in the presence of 0.5-3% (w/v) NaCl (optimum 1%). Major cellular fatty acids of TMW 2.2021 T were summed feature 3 (C 16:1 ω7c/iso-C 15 3-OH), C 16:0 , C 18:1 ω7c and summed feature 2 (C 12:0 aldehyde and C 10.928 unknown). Quinone analysis revealed Q-8 as sole respiratory ubiquinone. The genome of TMW 2.2021 T has a size of 4.56Mb and a G+C content of 38.49mol%. The ANI value between TMW 2.2021 T and the type strain of closest relative P. iliopiscarium DSM 9896 T was 91.43%. Fingerprinting on the base of M13-RAPD-PCR band pattern and MALDI-TOF MS profiles allowed intraspecies differentiation between our isolates but also supported their distinct lineage to a novel species. Based on phylogenetic, genomic, phenotypic and chemotaxonomic data, strain TMW 2.2021 T and further strains represent a novel species of the genus Photobacterium, for which the name Photobacterium carnosum sp. nov. is proposed. The type strain is TMW 2.2021 T (=DSM 105454 T =CECT 9394 T )., (Copyright © 2017. Published by Elsevier GmbH.)

The genera Microbispora and Sphaerimonospora were examined for GyrB and RecA amino-acid signatures to determine whether molecular-signature analyses support the recent establishment of the genus Sphaerimonospora. The creation of Sphaerimonospora was based mainly upon morphological differences between Microbispora and Sphaerimonospora and the clustering of the type strains of the two genera in phylogenetic trees based on a multilocus sequence analysis. The molecular-signature analyses showed that all members of Sphaerimonospora can be distinguished from all members of Microbispora at 14 amino acid positions in the GyrB protein and at four positions in the shorter RecA protein. These amino acid differences can be used as signatures to differentiate the members of these genera from each other and thus provide support for the establishment of the genus Sphaerimonospora. This is the first demonstration of the use of molecular signatures to support the establishment of a new genus in the family Streptosporangiaceae. Following the transfer of Microbispora mesophila and Microbispora thailandensis from Microbispora to Sphaerimonospora, all species in the genus Microbispora are characterised by the insertion of a small, hydrophobic amino acid after position 208 in the GyrB protein. This insertion is absent from the GyrB protein of members of the genus Sphaerimonospora., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

An assessment was made of the evolutionary relationships of soybean nodulating bacteria associated with legumes native to eastern Canada to identify potential new sources of soybean inoculant strains. Short season soybeans were used to selectively trap bacteria from root zone soils of four native legume species. Screening of more than 800 bacterial isolates from soybean root nodules by analysis of recA gene sequences followed by analyses of selected genotypes using six core and two symbiosis (nodC and nifH) gene sequences permitted identification of diverse taxa that included eight novel and four named Bradyrhizobium species as well as lineages attributed to the genera Afipia and Tardiphaga. Plant tests showed that symbionts related to four named species as well as a novel Bradyrhizobium lineage were highly efficient with regard to nitrogen fixation on soybeans relative to an inoculant strain. A new symbiovar (sv. septentrionalis) is proposed based on a group of four novel Bradyrhizobium spp. that possess distinctive nodC and nifH gene sequences and symbiotic characteristics. Evidence is provided for horizontal transfer of sv. septentrionalis symbiosis genes between novel Bradyrhizobium spp., a process that rendered recipient bacteria ineffective on soybeans. Diverse lineages of non-symbiotic and symbiotic Bradyrhizobium spp. co-occured within monophyletic clusters in a phylogenetic tree of concatenated core genes, suggesting that loss and/or gain of symbiosis genes has occurred in the evolutionary history of the bacterial genus. Our data suggest that symbiont populations associated with legumes native to eastern Canada harbour elite strains of Bradyrhizobium for soybean inoculation., (Crown Copyright © 2017. Published by Elsevier GmbH. All rights reserved.)

Bradyrhizobium comprises most tropical symbiotic nitrogen-fixing strains, but the correlation between symbiotic and core genes with host specificity is still unclear. In this study, the phylogenies of the nodY/K and nifH genes of 45 Bradyrhizobium strains isolated from legumes of economic and environmental importance in Brazil (Arachis hypogaea, Acacia auriculiformis, Glycine max, Lespedeza striata, Lupinus albus, Stylosanthes sp. and Vigna unguiculata) were compared to 16S rRNA gene phylogeny and genetic diversity by rep-PCR. In the 16S rRNA tree, strains were distributed into two superclades-B. japonicum and B. elkanii-with several strains being very similar within each clade. The rep-PCR analysis also revealed high intra-species diversity. Clustering of strains in the nodY/K and nifH trees was identical: 39 strains isolated from soybean grouped with Bradyrhizobium type species symbionts of soybean, whereas five others occupied isolated positions. Only one strain isolated from Stylosanthes sp. showed similar nodY/K and nifH sequences to soybean strains, and it also nodulated soybean. Twenty-one representative strains of the 16S rRNA phylogram were selected and taxonomically classified using a concatenated glnII-recA phylogeny; nodC sequences were also compared and revealed the same clusters as observed in the nodY/K and nifH phylograms. The analyses of symbiotic genes indicated that a large group of strains from the B. elkanii superclade comprised the novel symbiovar sojae, whereas for another group, including B. pachyrhizi, the symbiovar pachyrhizi could be proposed. Other potential new symbiovars were also detected. The co-evolution hypotheses is discussed and it is suggested that nodY/K analysis would be useful for investigating the symbiotic diversity of the genus Bradyrhizobium., (Copyright © 2017 Elsevier GmbH. All rights reserved.)

Rhizobia nodulating native Astragalus and Oxytropis spp. in Northern Europe are not well-studied. In this study, we isolated bacteria from nodules of four Astragalus spp. and two Oxytropis spp. from the arctic and subarctic regions of Sweden and Russia. The phylogenetic analyses were performed by using sequences of three housekeeping genes (16S rRNA, rpoB and recA) and two accessory genes (nodC and nifH). The results of our multilocus sequence analysis (MLSA) of the three housekeeping genes tree showed that all the 13 isolates belonged to the genus Mesorhizobium and were positioned in six clades. Our concatenated housekeeping gene tree also suggested that the isolates nodulating Astragalus inopinatus, Astragalus frigidus, Astragalus alpinus ssp. alpinus and Oxytropis revoluta might be designated as four new Mesorhizobium species. The 13 isolates were grouped in three clades in the nodC and nifH trees. 15 N analysis suggested that the legumes in association with these isolates were actively fixing nitrogen., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The present study was aimed to evaluate the diversity and probiotic properties of Lactobacillus plantarum-group cultures from vegetable origin. First, genotypic diversity of L. plantarum (n=34) was achieved by PCR of Random Amplified Polymorphic DNA and recA gene-specific multiplex PCR. The isolates were segregated into five groups namely, Lactobacillus pentosus, Lactobacillus paraplantarum, Lactobacillus arizonensis, Lactobacillus plantarum subsp. plantarum and argentoratensis. Further discrimination was achieved by restriction fragment length polymorphism of probiotic adhesion genes viz.fbp, mub and msa gene. As determined by nucleotide sequence analysis and bioinformatics Pfam database, the putative Fbp protein had only one FBP domain, whereas Mub protein had 8-10 MUB domain repeats. However, L. pentosus (except CFR MFT9), L. plantarum subsp. argentoratensis (except CFR MFT5) and L. arizonensis (except CFR MFT2) isolates gave no amplicon for the tested marker genes. Selected cultures (n=15) showed tolerance to simulated digestive fluids (20-85%), exhibited auto-aggregation (10-77%), cellular hydrophobicity (12-78%), and broad spectrum of anti-microbial activity. Concurrently, high adherence capacity to mucin was achieved for L. plantarum subsp. plantarum (MCC 2974 and CFR MFT1) and L. paraplantarum (MTCC 9483, MCC 2977, MCC 2978), which had an additional MUB domain repeat., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In this study four Mesorhizobium strains isolated from Lotus corniculatus nodules in Granada (Spain) were characterized. Their 16S rRNA gene sequences were closely related to those of M. albiziae LMG 23507 T and M. chacoense Pr5 T showing 99.4 and 99.2% similarity values, respectively. The analysis of concatenated rpoB, recA, atpD and glnII genes showed they formed a cluster with internal similarities higher than 97%. The closest species also were M. albiziae LMG 23507 T and M. chacoense Pr5 T showing similarity values lower than 92% in rpoB, recA and glnII genes and lower than 96.5% in the atpD gene. These results indicated that the L. corniculatus strains belong to a new species of genus Mesorhizobium which was confirmed by DNA-DNA hybridization and phenotypic characterization. Therefore a new species with the name Mesorhizobium olivaresii sp. nov. is proposed, and the type strain is CPS13 T (LMG 29295 T =CECT 9099 T )., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Phylogenetically diverse Ensifer strains associated with five species of Tephrosia growing in alkaline soils of semi-arid regions of the Thar Desert were characterized using multi locus sequence analysis. Based on 16S rRNA and four protein-coding housekeeping gene (recA, atpD, glnII and dnaK) sequences, the Tephrosia-Ensifer strains were genetically different from the type strains of Ensifer saheli, Ensifer kostiensis, Ensifer terangae (African origin) and Ensifer psoraleae (Asiatic origin). One strain, Ensifer sp. TL4, showed maximum similarity (99%) to Ensifer adhaerens LMG 20216 T and formed a separate lineage close to it. Phylogenetic incongruence between sym and housekeeping genes was observed. The monophyletic origin of symbiotic genes from Asia in the Tephrosia-Ensifer strains from the Thar Desert suggests that they might have been acquired from a common ancestor and horizontally transferred. These novel strains are promiscuous, cross-nodulating some papilionoid crop species, mimosoid trees and the caesalpinioid Chamaecrista pumila. This study improves understanding of the distribution of Ensifer in unexplored and threatened alkaline arid regions of the Thar Desert and how this relates to other similar regions in the world., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Seven isolates were obtained from different culture stages of carpet shell clam (Ruditapes decussatus) reared in a bivalve hatchery (Galicia, NW Spain). Three groups were differentiated by genotyping techniques and phenotypic profiles and representative trains were selected to further taxonomic studies. These strains were studied by a polyphasic approach and in basis of the phylogenetic analysis based on concatenated sequences of the five housekeeping genes ftsZ, gyrB, pyrH, recA and rpoA formed a tight group into the Mediterranei clade of the genus Vibrio. Percentages of genomic resemblance, including average nucleotide identity, in silico genome-to-genome comparison and wet DNA-DNA hybridization between the type strain and the closest relatives Vibrio mediterranei and Vibrio thalassae were below of the proposed boundaries for the definition of species. The novel isolates could be also differentiated from the related taxa on the basis of several phenotypic traits and fatty acid profiles. Results obtained support the description of a novel species into the Mediterranei clade, for which the name Vibrio barjaei sp. nov. is proposed, with strain 3062 T (=CECT 9090 T =CAIM 1921T T =LMG 29358 T ) as the type strain., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In this work we analyze through a polyphasic approach several Bradyrhizobium strains isolated in Venezuela from root nodules of Centrosema species. The analysis of the 16S rRNA gene showed that the strains belong to three clusters within genus Bradyrhizobium which have 100% similarity with Bradyrhizobium daqingense CCBAU 15774(T)Bradyrhizobium guangxiense CCBAU 53363(T) and Bradyrhizobium viridifuturi SEMIA 690(T). The results of recA and glnII gene analysis confirmed the identification of the strains CMVU02 and CMVU30 as Bradyrhizobium viridifuturi but the nodC gene analysis showed that they belong to a new symbiovar for which we propose the name tropici. Nevertheless, the concatenated recA and glnII gene phylogenetic analysis, DNA-DNA hybridization and phenotypic characterization showed that the strains A9(T), CMVU44(T) and CMVU04 belong to two novel Bradyrhizobium species. The analysis of the nodC gene showed that these strains also represent two new symbiovars. Based on these results we propose the classification of the strain A9(T) isolated from Centrosema molle into the novel species Bradyrhizobium centrosemae (sv. centrosemae) sp. nov. (type strain A9(T)=LMG 29515(T)=CECT 9095(T)). and the classification of the strains CMVU44(T) and CMVU04 isolated from C. macrocarpum into the novel species Bradyrhizobium americanum (sv. phaseolarum) sp. nov. (type strain CMVU44(T)=LMG 29514(T)=CECT 9096(T))., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A multilocus sequence analysis was undertaken in order to redefine the Splendidus clade of the genus Vibrio, a large group of species containing several pathogenic members that affect fish and shellfish, and are difficult to identify through both phenotypic and genotypic approaches. The study included analysis of partial sequences of recA, gyrB, mreB, rpoD and pyrH genes, as well as the 16S rRNA gene. Seventeen type strain species were included that were complemented with other reference strains and a collection of isolates tentatively identified as members of this clade, as well as a set of other Vibrio species. The clade was well defined and stable in all analyses, and was confirmed to contain V. celticus, V. atlanticus, V. artabrorum, V. toranzoniae and V. hemicentroti, in addition to the twelve previously recognized species. While some species were well-defined members (e.g. Vibrio cyclitrophicus, V. chagasii) others formed tight groups that were related by sequence similarities and lineage topology, which suggested a synonymy among their members, particularly the V. splendidus-V. hemicentroti pair. Most of the isolates were related to two major groups: the V. celticus-V. crassostreae-V. gigantis subclade that contained all isolates from oysters sampled in the cold season, and V. chagasii that included oyster isolates from warm months. This suggested a sharp seasonal occurrence for these species. None of the single genes were able to mimic the resolving power of the five-gene MLSA and none worked well for the identification of the whole group of species in the clade., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

To reveal the genetic diversity and geographic distribution of peanut (Arachis hypogaea L.) rhizobia in Guangdong Province, one of the main peanut producing regions in China, 216 bradyrhizobial isolates were trapped by peanut plants inoculated with soil samples (pH 4.7-7.4) collected from ten sites in Guangdong. Based on BOX-PCR fingerprinting analysis, 71 representative isolates were selected for sequence analyses of ribosomal IGS, recA, atpD and symbiotic gene nodA. As a result, 22 genospecies were detected in the peanut rhizobia, including eight minor groups or single strains corresponding to Bradyrhizobium diazoefficiens, B. japonicum, B. yuanmingense, B. arachidis, B. guangdongense, B. guangxiense, B. iriomotense and B. liaoningense, as well as 14 novel Bradyrhizobium genospecies covering the majority of isolates. Five symbiotic clusters were obtained based on the phylogenetic relationships of nodA genes, related to the soybean-nodulating or peanut-nodulating reference strains. Biogeographic patterns, which were mainly correlated with potassium content and pH, were detected in the peanut bradyrhizobial community in Guangdong Province. These findings enriched the diversity of peanut rhizobia, and added the K content as a special determinant for peanut rhizobial distribution in acid soils., (Copyright © 2016. Published by Elsevier GmbH.)

Beach pea [Lathyrus maritimus Bigelow, or Lathyrus japonicus subsp. maritimus (L.) P.W. Ball] is a wild legume distributed on the seashore line, and the rhizobia nodulating with this plant have been reported only rarely. In order to reveal the diversity of beach pea rhizobia on the seashore line of Shandong Peninsula, China, a total of 124 bacterial strains were isolated from the root nodules of beach pea plants collected from five sites. All the isolates were divided into five recA types after screening by recA gene sequence analysis and they consisted of Rhizobium anhuiense covering 122 symbiotic isolates in three recA types, as well as two single isolates Rhizobium sp. and Rhizobium lusitanum representing distinct recA types. The recA genotype III of R. anhuiense (103 isolates) represented by strain YIC11270 was dominant at all five sampling sites. Identical symbiotic genes (nodC and nifH) were detected in the three recA genotypes of R. anhuiense isolates that were closely related to those of the pea and faba rhizobia. This study clarified that R. anhuiense was the main symbiont for beach pea rhizobia on the seashore line of Shandong Peninsula. The low level genetic diversity of beach pea rhizobia revealed by both MLSA and the symbiotic genes might be related to the strong selection pressure produced by the saline-alkaline environment and the host plants., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

A total of 212 symbiotic bacteria were isolated from nodules of Vicia ervilia, a traditional crop cultivated in Northern Morocco. The isolates were recovered from 10 different sites, trapped each time with the local cultivar grown in the same field. Four loci were sequenced in order to characterize the isolates, including two housekeeping genes (recA and glnII), one plasmidic symbiotic gene (nodC) and one locus from another plasmid (prL11). In several isolates, two different copies of glnII were detected and sequenced, suggesting a unique duplication event, which has never been reported previously. There was no correlation between the genetic differentiation among cultivars and among bacteria, showing that the evolution of the bacterial population was independent, at least partially, from the host plant. By placing the haplotypes in a wide-ranging phylogenetic reconstruction, it was shown that the diversity detected in Morocco was spread throughout the different clades detected worldwide. The differentiation between areas relied on frequency variations of haplotypes rather than a presence/absence pattern. This finding raises new questions concerning bacterial genetic resource preservation, and confirms the old tenet "everything is everywhere but the environment selects"., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The symbiosis between leguminous plants and symbiotic nitrogen-fixing bacteria is a key component of terrestrial ecosystems. Woody legumes are well represented in tropical African forests but despite their ecological and socio-economic importance, they have been little studied for this symbiosis. In this study, we examined the identity and diversity of symbiotic-nitrogen fixing bacteria associated with Acacia xanthophloea, Faidherbia albida and Albizia versicolor in the Gorongosa National Park (GNP) in Mozambique. To the best of our knowledge, this is the first report on the identity of symbiotic-nitrogen fixing bacteria in this region. 166 isolates were obtained and subjected to molecular identification. BOX-A1R PCR was used to discriminate different bacterial isolates and PCR-sequencing of 16S rDNA, and two housekeeping genes, glnII and recA, was used to identify the obtained bacteria. The gene nifH was also analyzed to assess the symbiotic capacity of the obtained bacteria. All isolates from F. albida and Al. versicolor belonged to the Bradyrhizobium genus whereas isolates from Ac. xanthophloea clustered with Mesorhizobium, Rhizobium or Ensifer strains. Soil chemical analysis revealed significant differences between the soils occupied by the three studied species. Thus, we found a clear delimitation in the rhizobial communities and soils associated with Ac. xanthophloea, F. albida and Al. versicolor, and higher rhizobial diversity for Ac. xanthophloea than previously reported., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

It is known that the genetic diversity of conspecific rhizobia present in root nodules differs greatly among populations of a legume species, which has led to the suggestion that both dispersal limitation and the local environment affect rhizobial genotypic composition. However, it remains unclear whether rhizobial genotypes residing in root nodules are representative of the entire population of compatible symbiotic rhizobia. Since symbiotic preferences differ among legume populations, the genetic composition of rhizobia found within nodules may reflect the preferences of the local hosts, rather than the full diversity of potential nodulating rhizobia present in the soil. Here, we assessed whether Vicia cracca legume hosts of different provenances select different Rhizobium leguminosarum genotypes than sympatric V. cracca hosts, when presented a natural soil rhizobial population. Through combining V. cracca plants and rhizobia from adjacent and more distant populations, we found that V. cracca hosts are relatively randomly associated with rhizobial genotypes. This indicates that pre-infection partner choice is relatively weak in certain legume hosts when faced with a natural population of rhizobia., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Lupinus micranthus is a lupine distributed in the Mediterranean basin whose nitrogen fixing symbiosis has not been described in detail. In this study, 101 slow-growing nodule isolates were obtained from L. micranthus thriving in soils on both sides of the Western Mediterranean. The diversity of the isolates, 60 from Algeria and 41 from Spain, was addressed by multilocus sequence analysis of housekeeping genes (16S rRNA, atpD, glnII and recA) and one symbiotic gene (nodC). Using genomic fingerprints from BOX elements, 37 different profiles were obtained (22 from Algeria and 15 from Spain). Phylogenetic analysis based on 16S rRNA and concatenated atpD, glnII and recA sequences of a representative isolate of each BOX profile displayed a homogeneous distribution of profiles in six different phylogenetic clusters. All isolates were taxonomically ascribed to the genus Bradyrhizobium. Three clusters comprising 24, 6, and 4 isolates, respectively, accounted for most of the profiles. The largest cluster was close to the Bradyrhizobium canariense lineage, while the other two were related to B. cytisi/B. rifense. The three remaining clusters included only one isolate each, and were close to B. canariense, B. japonicum and B. elkanii species, respectively. In contrast, phylogenetic clustering of BOX profiles based on nodC sequences yielded only two phylogenetic groups. One of them included all the profiles except one, and belonged to symbiovar genistearum. The remaining profile, constituted by a strain related to B. elkanii, was not related to any well-defined symbiotic lineage, and may constitute both a new symbiovar and a new genospecies., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Three novel actinobacterial strains, GUI2(T), GUI42 and CR21 isolated from nodular tissues and the rhizosphere of a sweet pea plant collected in Cañizal, Spain were identified according to their 16S rRNA gene sequences as new members of the genus Micromonospora. The closest phylogenetic members were found to be Micromonospora saelicesensis (99.2%) "Micromonospora zeae" (99.1%), "Micromonospora jinlongensis" (99%), Micromonospora lupini (98.9%) and Micromonospora zamorensis (98.8%). To resolve their full taxonomic position, four additional genes (atpD, gyrB, recA, rpoB) were partially sequenced and compared to available Micromonospora type strain sequences. DNA-DNA hybridization, BOX-PCR and ARDRA profiles confirmed that these strains represent a novel genomic species. All strains contained meso-diaminopimelic and hydroxy-diaminopimelic acids in their cell wall. Their fatty acid profiles comprised iso-C15:0, iso-C16:0 and anteiso-C15:0 as major components. The polar lipids diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylinositol were found in the type strain GUI2(T) which also contained MK-10(H4) as the major menaquinone. Physiological and biochemical characteristics also differentiated the new isolates. Based on the integration of the above studies, strains GUI2(T), GUI42 and CR21 represent a novel Micromonospora species and we propose the name Micromonospora luteifusca sp. nov. The type strain is GUI2(T) (=CECT 8846(T); =DSM 100204(T))., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Previously, 159 bacterial strains were isolated from the root nodules of wild perennial Glycyrrhiza legume species grown on 40 sites in central and north-western China, in which 57 strains were classified as "true symbionts" belonging to the genus Mesorhizobium based on amplified fragment length polymorphism (AFLP) genomic fingerprinting and partial sequences of the 16S rRNA gene [20]. In the present work, the phylogeny of Glycyrrhiza nodulating mesorhizobia was further examined by multilocus sequence analysis (MLSA). The concatenated gene tree of three housekeeping genes (16S rRNA, recA, and rpoB) of 59 strains including the 29 mesorhizobial test strains and 30 type mesorhizobial species, was constructed applying the maximum likelihood method and Bayesian inference. In the concatenated gene tree, the 29 test strains were distributed in seven separate clades. Seventeen test strains clustered with Mesorhizobium tianshanense, Mesorhizobium temperatum, Mesorhizobium muleiense, and Mesorhizobium alhagi with high bootstrap support (BS>85%). Eight test strains did not cluster with any of the described Mesorhizobium species. Based on the results, we proposed these eight test strains might belong to a putative new species of the genus Mesorhizobium. The sequences of three accessory genes (nodA, nodC, and nifH) of the test strains were also analyzed and were compared with those of representatives of the 30 described mesorhizobial species. The results showed that mesorhizobia involved in symbiosis with Glycyrrhiza plants probably have acquired some genetic material from other rhizobia in co-evolution with Glycyrrhiza and other legume species., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

In the present work we analyzed the taxonomic status of several Rhizobium strains isolated from Trifolium alexandrinum L. nodules in Egypt. The 16S rRNA genes of these strains were identical to those of Rhizobium bangladeshense BLR175(T) and Rhizobium binae BLR195(T). However, the analyses of recA and atpD genes split the strains into two clusters. Cluster II strains are identified as R. bangladeshense with >98% similarity values in both genes. The cluster I strains are phylogenetically related to Rhizobium etli CFN42(T) and R. bangladeshense BLR175(T), but with less than 94% similarity values in recA and atpD genes. DNA-DNA hybridization analysis showed 42% and 48% average relatedness between the strain 1010(T) from cluster I with respect to R. bangladeshense BLR175(T) and R. etli CFN42(T), respectively. Phenotypic characteristics of cluster I strains also differed from those of their closest related Rhizobium species. Analysis of the nodC gene showed that the strains belong to two groups within the symbiovar trifolii which was identified in Egypt linked to the species R. bangladeshense. Based on the genotypic and phenotypic characteristics, the group I strains belong to a new species for which the name Rhizobium aegyptiacum sp. nov. (sv. trifolii) is proposed, with strain 1010(T) being designated as the type strain (= USDA 7124(T)=LMG 29296(T)=CECT 9098(T))., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The biodiversity and biogeography of rhizobia associated with bean in Shaanxi Province were investigated. A total of 194 bacterial isolates from bean nodules collected from 13 sampling sites were characterized based on phylogenetic analyses of the 16S rRNA gene, the housekeeping genes recA, glnII and atpD, and the symbiotic genes nodC and nifH. Fifteen genospecies belonging to the genera Rhizobium, Agrobacterium, Ensifer, Bradyrhizobium and Ochrobactrum were defined among the isolates, with Rhizobium sp. II, Agrobacterium sp. II, E. fredii and R. phaseoli being the dominant groups. Four symbiotic gene lineages corresponding to Rhizobium sp. I, Rhizobium sp. II, R. phaseoli and B. liaoningense were detected in the nodC and nifH sequence analyses, indicating different origins for the symbiotic genes and their co-evolution with the chromosome of the bacteria. Moreover, the Ensifer isolates harbored symbiotic genes closely related to bean-nodulating Pararhizobium giardinii, indicating possible lateral gene transfer from Rhizobium to Ensifer. Correlation of rhizobial community composition with moisture, temperature, intercropping, soil features and nutrients were detected. All the results demonstrated a great diversity of bean rhizobia in Shaanxi that might be due to the adaptable evolution of the bean-nodulating rhizobia subjected to the diverse ecological conditions in the area., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The taxonomic status of eleven rhizospheric bacterial strains belonging to the genus Burkholderia and isolated from roots of Costularia (Cyperaceae), tropical herbaceous pioneer plants growing on ultramafic soils in New Caledonia, was investigated using a polyphasic taxonomic approach. The genetic analyses (16S rRNA genes, gyrB, recA, nreB and cnr) confirmed that all strains are Burkholderia and cluster into two separated groups. The DNA hybridization results showed low relatedness values to the closest relatives Burkholderia species. The phenotypic analyses confirmed that the two groups of strains could be differentiated from each other and from other known Burkholderia species. This polyphasic study revealed that these two groups of strains represent each a novel species of Burkholderia, for which the names Burkholderia novacaledonica sp. nov. (type strain STM10272(T)=LMG28615(T)=CIP110887(T)) and B. ultramafica sp. nov. (type strain STM10279(T)=LMG28614(T)=CIP110886(T)) are proposed, respectively. These strains of Burkholderia presented specific ecological traits such as the tolerance to the extreme edaphic constraints of ultramafic soils: they grew at pH between 4 and 8 and tolerate the strong unbalanced Ca/Mg ratio (1/19) and the high concentrations of heavy metals i.e. Co, Cr, Mn and Ni. Noteworthy B. ultramafica tolerated nickel until 10mM and B. novacaledonica up to 5mM. The presence of the nickel (nreB) and cobalt/nickel (cnr) resistance determinants encoding for protein involved in metal tolerance was found in all strains of both groups. Moreover, most of the strains were able to produce plant growth promoting molecules (ACC, IAA, NH3 and siderophores). Such ecological traits suggest that these new species of Burkholderia might be environmentally adaptable plant-associated bacteria and beneficial to plants., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Sesbania cannabina is a plant that grows naturally along the seashores in Rudong County, China (RDC) and it has been introduced into the Yellow River Delta (YRD) as a pioneer plant to improve the saline-alkaline soils. In order to investigate the diversity of S. cannabina rhizobia in these soils, a total of 198 rhizobial isolates were characterized and phylogenetic trees were constructed based on data from multilocus sequence analysis (MLSA) of the housekeeping genes recA, atpD and glnII, as well as 16S rRNA. Symbiotic features were also studied by establishing the phylogeny of the symbiotic genes nodA and nifH, and by performing nodulation assays. The isolates had highly conserved symbiotic genes and were classified into nine genospecies belonging to the genera Ensifer, Agrobacterium, Neorhizobium and Rhizobium. A unique community structure was detected in the rhizobia associated with S. cannabina in the saline-alkaline soils that was characterized by five novel genospecies and four defined species. In addition, Ensifer sp. I was the predominant rhizobia in YRD, whereas Ensifer meliloti and Neorhizobium huautlense were the dominant species in RDC. Therefore, the study demonstrated for the first time that this plant strongly selected the symbiotic gene background but not the genomic background of its microsymbionts. In addition, biogeographic patterns existed in the rhizobial populations associated with S. cannabina, which were mainly correlated with pH and salinity, as well as the mineral nutrient contents. This study provided novel information concerning the interaction between soil conditions, host plant and rhizobia, in addition to revealing the diversity of S. cannabina rhizobia in saline-alkaline soils., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Earthworms carry species-specific Verminephrobacter symbionts in their nephridia (excretory organs). The symbionts are vertically transmitted via the cocoon, can only colonize the host during early embryonic development, and have co-speciated with their host for about 100 million years. Although several studies have addressed Verminephrobacter diversity between worm species, the intra-species diversity of the symbiont population has never been investigated. In this study, symbiont population structure was examined by using a multi-locus sequence typing (MLST) approach on Verminephrobacter isolated from two contrasting ecological types of earthworm hosts: the high population density, fast reproducing compost worms, Eisenia andrei and Eisenia fetida, and the low-density, slow reproducing Aporrectodea tuberculata, commonly found in garden soils. Three distinct populations were investigated for both types and, according to MLST analysis of 193 Verminephrobacter isolates, the symbiont community in each worm individual was very homogeneous. The more solitary A. tuberculata carried unique symbiont populations in 9 out of 10 host individuals, whereas the symbiont populations in the social compost worms were homogeneous across host individuals from the same population. These data suggested that host ecology shaped the population structure of Verminephrobacter symbionts. The homogeneous symbiont populations in the compost worms led to the hypothesis that Verminephrobacter could be transferred bi-parentally or via leaky horizontal transmission in high-density, frequently mating worm populations., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different stone fruit rootstocks in Poland: strains F5.1(T) and F5.3 from Prunus avium F12/1, strains CP3.5 and CP17.2.1 from Prunus avium and strain AL5.1.8 from Prunus cerasifera. On the basis of 16S rDNA phylogeny, the strains cluster together and belong to the genus Pararhizobium with type strain of Pararhizobium herbae (99.6-99.8%) as their closest relative. Phylogenetic analysis of the novel strains using housekeeping genes atpD, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to P. herbae. DNA-DNA hybridization of strains F5.1(T), with the type strain of P. herbae LMG 25718(T) and Pararhizobium giardinii R-4385(T) revealed 28.3% and 27.9% of DNA-DNA relatedness, respectively. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium polonicum sp. nov. (type strain F5.1(T)=LMG 28610(T)=CFBP 8359(T)) is proposed., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Two hundred and twenty-one strains representative of all Aeromonas species were characterized using the recA gene sequence, assessing its potential as a molecular marker for the genus Aeromonas. The inter-species distance values obtained demonstrated that recA has a high discriminatory power. Phylogenetic analysis, based on full-length gene nucleotide sequences, revealed a robust topology with clearly separated clusters for each species. The maximum likelihood tree showed the Aeromonas bestiarum strains in a well-defined cluster, containing a subset of four strains of different geographical origins in a deep internal branch. Data analysis provided strong evidence of recombination at the end of the recA sequences in these four strains. Intergenomic recombination corresponding to partial regions of the two adjacent genes recA and recX (248 bp) was identified between A. bestiarum (major parent) and Aeromonas eucrenophila (minor parent). The low number of recombinant strains detected (1.8%) suggests that horizontal flow between recA sequences is relatively uncommon in this genus. Moreover, only a few nucleotide differences were detected among these fragments, indicating that recombination has occurred recently. Finally, we also determined if the recombinant fragment could have influenced the structure and basic functions of the RecA protein, comparing models reconstructed from the translated amino acid sequences of our A. bestiarum strains with known Escherichia coli RecA structures., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Three isolates were obtained from cultures of carpet shell clam (Ruditapes decussatus) reared in a bivalve hatchery (Galicia, NW Spain) from different sources: healthy broodstock, moribund larvae and the seawater corresponding to the larval tank. All isolates were studied by a polyphasic approach, including a phylogenetic analysis based on concatenated sequences of the five housekeeping genes ftsZ, gyrB, pyrH, recA and rpoA. The analysis supported their inclusion in the Orientalis clade of the genus Vibrio, and they formed a tight group separated from the closest relatives: Vibrio tubiashii subsp. europaensis, Vibrio tubiashii subsp. tubiashii and Vibrio orientalis. The percentages of genomic resemblance, including average nucleotide identity, DNA-DNA hybridization and in silico genome-to-genome comparison, between the type strain and the closest relatives were below values for species delineation and confirmed the taxonomic position of the new species, which could be differentiated from the related taxa on the basis of several phenotypic and chemotaxonomic features, including FAME and MALDI-TOF-MS. The pathogenicity of the new species was demonstrated in larvae of R. decussatus, Ruditapes philippinarum, Ostrea edulis and Donax trunculus. The results demonstrated that the strains analyzed represented a novel species in the Orientalis clade of the genus Vibrio, for which the name Vibrio bivalvicida sp. nov. is proposed, with 605(T) (= CECT 8855(T)=CAIM 1904(T)) designated as the type strain., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The South African Fynbos soils are renowned for nitrogen-fixing Burkholderia associated with diverse papilionoid legumes of the tribes Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae. However, despite numerous rhizobial studies in the region, the symbiotic diversity of Burkholderia has not been investigated in relation to a specific host legume and its geographical provenance. This study analyzed the diversity of nodulating strains of Burkholderia from the legume species Podalyria calyptrata. Diverse lineages were detected that proved to be closely related to Burkholderia taxa, originating from hosts in other legume tribes. By analyzing the genetic variation of chromosomal (recA) and nodulation (nodA) sequence data in relation to the sampling sites we assessed the geographical distribution patterns of the P. calyptrata symbionts. Although we found a degree of genetically differentiated rhizobial populations, a correlation between genetic (recA and nodA) and geographic distances among populations was not observed, suggesting high rates of dispersal and rhizobial colonization within Fynbos soils., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The family Streptosporangiaceae (suborder Streptosporangineae) comprises 13 genera and 100 species with validly published names. In a recent study, gyrB gene sequences were obtained for members of the family Streptosporangiaceae and the GyrB amino acid sequences were analysed for molecular signatures. In this study, recA gene sequences (895nt) were determined for the type strains of members of the family Streptosporangiaceae. The sequences used represent 81% of the full-length recA gene of Streptosporangium roseum DSM 43021(T). The recA gene sequences were used for phylogenetic analyses and the trees were compared to the corresponding 16S-rRNA and gyrB gene trees. RecA amino acid alignments (298 amino acids) were generated and inspected for unique amino acid signatures to distinguish the genera in the family from each other. As was observed for the gyrB gene trees, the recA gene trees generally supported the division of the members of the family Streptosporangiaceae into 13 genera. The genus Nonomuraea was not monophyletic in any of the recA gene trees, while the genera Planomonospora and Streptosporangium were not monophyletic in the maximum likelihood and maximum parsimony trees. The gyrB-recA concatenated-gene tree was more robust than the recA gene tree, with 63 nodes in the gyrB-recA tree having bootstrap values ≥95%. The only insertions in the recA gene sequences were inteins identified in the type strains of Acrocarpospora phusangensis, Acrocarpospora pleiomorpha and Microbispora mesophila. Examination of the RecA sequence alignments for genus-specific amino acid sequences showed that the genera Herbidospora, Planobispora, Planomonospora and Streptosporangium contain unique amino acid sequences that distinguish these genera from all other genera in the family Streptosporangiaceae. The results of this investigation extend the results of the GyrB study and will be useful in future taxonomic studies in the family Streptosporangiaceae by providing additional genus-specific molecular signatures., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Despite the diversity of Burkholderia species known to nodulate legumes in introduced and native regions, relatively few taxa have been formally described. For example, the Cape Floristic Region of South Africa is thought to represent one of the major centres of diversity for the rhizobial members of Burkholderia, yet only five species have been described from legumes occurring in this region and numerous are still awaiting taxonomic treatment. Here, we investigated the taxonomic status of 12 South African root-nodulating Burkholderia isolates from native papilionoid legumes (Hypocalyptus coluteoides, H. oxalidifolius, H. sophoroides and Virgilia oroboides). Analysis of four gene regions (16S rRNA, recA, atpD and rpoB) revealed that the isolates represent a genealogically unique and exclusive assemblage within the genus. Its distinctness was supported by all other aspects of the polyphasic approach utilized, including the genome-based criteria DNA-DNA hybridization (≥70.9%) and average nucleotide identities (≥96%). We accordingly propose the name B. kirstenboschensis sp. nov. for this taxon with isolate Kb15(T) (=LMG 28727(T); =SARC 695(T)) as its type strain. Our data showed that intraspecific genome size differences (≥0.81 Mb) and the occurrence of large DNA regions that are apparently unique to single individuals (16-23% of an isolate's genome) can significantly limit the value of data obtained from DNA-DNA hybridization experiments. Substitution of DNA-DNA hybridization with whole genome sequencing as a prerequisite for the description of Burkholderia species will undoubtedly speed up the pace at which their diversity are documented, especially in hyperdiverse regions such as the Cape Floristic Region., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Cicer canariense is a threatened endemic legume from the Canary Islands where it can be nodulated by mesorhizobial strains from the symbiovar ciceri, which is the common worldwide endosymbiont of Cicer arietinum linked to the genus Mesorhizobium. However, when C. canariense was cultivated in a soil from mainland Spain, where the symbiovar ciceri is present, only fast-growing rhizobial strains were unexpectedly isolated from its nodules. These strains were classified into the genus Rhizobium by analysis of the recA and atpD genes, and they were phylogenetically related to Rhizobium leguminosarum. The analysis of the nodC gene showed that the isolated strains belonged to the symbiovar trifolii that harbored a nodC allele (β allele) different to that harbored by other strains from this symbiovar. Nodulation experiments carried out with the lacZ-labeled strain RCCHU01, representative of the β nodC allele, showed that it induced curling of root hairs, infected them through infection threads, and formed typical indeterminate nodules where nitrogen fixation took place. This represents a case of exceptional performance between the symbiovar trifolii and a legume from the tribe Cicereae that opens up new possibilities and provides new insights into the study of rhizobia-legume symbiosis., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Two novel Alphaproteobacteria strains, R1-200B4(T) and R2-400B4, were isolated from the Mediterranean Sea off the coast of Alicante in Spain. The phylogenetic analysis of the 16S rRNA gene showed that they are related to members of Family Rhizobiaceae. The 16S rRNA gene sequence of strain R1-200B4(T) presents 97.0% and 95.6% similarity with respect to the type strains of the type species from genera Neorhizobium and Rhizobium, Neorhizobium galegae HAMBI 540(T) and Rhizobium leguminosarum USDA 2370(T), respectively. The remaining genera of family Rhizobiaceae showed similarities lower than 95%. The recA and atpD gene sequences of strain R1-200B4(T) showed, respectively, 90% and 88.6% similarity with respect to N. galegae HAMBI 540(T) and 87% and 86% with respect to R. leguminosarum USDA 2370(T). The calculated ANI values between the genomes of the strain R1-200B4(T) and those of N. galegae HAMBI 540(T) and R. leguminosarum 3841 are 75.9% and 74.0%, respectively. The major fatty acids are those from summed feature 8 (C18:1 ω6c/C18:1 ω7c) and the C16:0. Catalase and oxidase were positive. Nitrate reduction and aesculin hydrolysis were positive. Production of β-galactosidase and urease was positive. The production of indol, arginine dehydrolase or gelatinase was negative. Growth was observed in presence of 7% NaCl. Therefore, based on the phylogenetic, chemotaxonomic and phenotypic data obtained in this study, we propose to classify the strains isolated in this study in a new genus named Pseudorhizobium gen. nov. and a new species named Pseudorhizobium pelagicum sp. nov. with the type strain R1-200B4(T) (=LMG 28314(T)=CECT 8629(T))., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Forty eight rhizobial isolates from New Zealand (NZ) native Sophora spp. growing in natural ecosystems were characterised. Thirty eight isolates across five groups showed greatest similarity to Mesorhizobium ciceri LMG 14989(T) with respect to their 16S rRNA and concatenated recA, glnll and rpoB sequences. Seven isolates had a 16S rRNA sequence identical to M. amorphae ATCC 19665(T) but showed greatest similarity to M. septentrionale LMG 23930(T) on their concatenated recA, glnll and rpoB sequences. All isolates grouped closely together for their nifH, nodA and nodC sequences, clearly separate from all other rhizobia in the GenBank database. None of the type strains closest to the Sophora isolates based on 16S rRNA sequence similarity nodulated Sophora microphylla but they all nodulated their original host. Twenty one Sophora isolates selected from the different 16S rRNA groupings produced N2-fixing nodules on three Sophora spp. but none nodulated any host of the type strains for the related species. DNA hybridisations indicated that these isolates belong to novel Mesorhizobium spp. that nodulate NZ native Sophora species., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Eleven Burkholderia cepacia-like isolates of human clinical and environmental origin were examined by a polyphasic approach including recA and 16S rRNA sequence analysis, multilocus sequence analysis (MLSA), DNA base content determination, fatty acid methyl ester analysis, and biochemical characterization. The results of this study demonstrate that these isolates represent a novel species within the B. cepacia complex (Bcc) for which we propose the name Burkholderia pseudomultivorans. The type strain is strain LMG 26883(T) (=CCUG 62895(T)). B. pseudomultivorans can be differentiated from other Bcc species by recA gene sequence analysis, MLSA, and several biochemical tests including growth at 42°C, acidification of sucrose and adonitol, lysine decarboxylase and β-galactosidase activity, and esculin hydrolysis., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Forty-eight Burkholderia isolates from different land use systems in the Amazon region were compared to type strains of Burkholderia species for phenotypic and functional characteristics that can be used to promote plant growth. Most of these isolates (n=46) were obtained by using siratro (Macroptilium atropurpureum - 44) and common bean (Phaseolus vulgaris - 2) as the trap plant species; two isolates were obtained from nodules collected in the field from Indigofera suffruticosa and Pithecellobium sp. The evaluated characteristics were the following: colony characterisation on "79" medium, assimilation of different carbon sources, enzymatic activities, solubilisation of phosphates, nitrogenase activity and antifungal activity against Fusarium oxysporium f. sp. phaseoli. Whole cell protein profiles, 16S rRNA, gyrB, and recA gene sequencing and multilocus sequence typing were used to identify the isolates. The isolates showed different cultural and biochemical characteristics depending on the legume species from which they were obtained. Except for one isolate from I. suffruticosa, all isolates were able to solubilise calcium phosphate and present nitrogenase activity under free-living conditions. Only one isolate from common beans, showed antifungal activity. The forty four isolates from siratro nodules were identified as B. fungorum; isolates UFLA02-27 and UFLA02-28, obtained from common bean plants, were identified as B. contaminans; isolate INPA89A, isolated from Indigofera suffruticosa, was a close relative of B. caribensis but could not be assigned to an established species; isolate INPA42B, isolated from Pithecellobium sp., was identified as B. lata. This is the first report of nitrogenase activity in B. fungorum, B. lata and B. contaminans., (Copyright © 2012. Published by Elsevier GmbH.)

It was recently reported that Micromonospora inhabits the intracellular tissues of nitrogen fixing nodules of the wild legume Lupinus angustifolius. To determine if Micromonospora populations are also present in nitrogen fixing nodules of cultivated legumes such as Pisum sativum, we carried out the isolation of this actinobacterium from P. sativum plants collected in two man-managed fields in the region of Castilla and León (Spain). In this work, we describe the isolation of 93 Micromonospora strains recovered from nitrogen fixing nodules and the rhizosphere of P. sativum. The genomic diversity of the strains was analyzed by amplified ribosomal DNA restriction analysis (ARDRA). Forty-six isolates and 34 reference strains were further analyzed using a multilocus sequence analysis scheme developed to address the phylogeny of the genus Micromonospora and to evaluate the species distribution in the two studied habitats. The MLSA results were evaluated by DNA-DNA hybridization to determine their usefulness for the delineation of Micromonospora at the species level. In most cases, DDH values below 70% were obtained with strains that shared a sequence similarity of 98.5% or less. Thus, MLSA studies clearly supported the established taxonomy of the genus Micromonospora and indicated that genomic species could be delineated as groups of strains that share > 98.5% sequence similarity based on the 5 genes selected. The species diversity of the strains isolated from both the rhizosphere and nodules was very high and in many cases the new strains could not be related to any of the currently described species., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T))., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Two Gram negative, facultatively anaerobic, halophilic, motile, slightly curved rod-shaped bacterial strains MSSRF60(T) and MSSRF64 were isolated from the roots of a mangrove-associated wild rice collected in the Pichavaram mangroves, India. These strains possess the key functional nitrogenase gene nifH. Phylogenetic analysis based on the 16S rRNA, recA, gapA, mreB, gyrB and pyrH, gene sequences revealed that strains MSSRF60(T) and MSSRF64 belong to the genus Vibrio, and had the highest sequence similarity with the type strains of Vibrio diazotrophicus LMG 7893(T) (99.7, 94.8, 98.5, 97.9, 94.0 and 90.7%, respectively), Vibrio areninigrae J74(T) (98.2, 87.5, 91.5, 88.9, 86.5 and 84.6% respectively) and Vibrio hispanicus LMG 13240(T) (97.8, 87.1, 91.7, 89.8, 84.1 and 81.9%, respectively). The fatty acid composition too confirmed the affiliation of strains MSSRF60(T) and MSSRF64 to the genus Vibrio. These strains can be differentiated from the most closely related Vibrio species by several phenotypic traits. The DNA G+C content of strain MSSRF60(T) was 41.8mol%. Based on phenotypic, chemotaxonomic, genotypic (multilocus sequence analysis using five genes and genomic fingerprinting using BOX-PCR) and DNA-DNA hybridization analyses, strains MSSRF60(T) and MSSRF64 represent a novel species of the genus Vibrio, for which the name Vibrio plantipsonsor sp. nov. is proposed. The type strain is MSSRF60(T) (=DSM 21026(T)=LMG 24470(T)=CAIM 1392(T))., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

The aim of this work is the taxonomic characterization of three biosurfactant-producing bacterial isolates from the Churince system at Cuatro Ciénegas Basin (CCB) in the Mexican State of Coahuila, and the study of the possible role of biosurfactant production in their ecology and evolution. We determined that these isolates belong to a Pseudomonas koreensis lineage endemic to CCB, using standard taxonomical techniques, phylogenetic analysis of three chromosomal loci and phenotypic characterization. This new lineage has the distinct capacity to produce a biosurfactant when compared with previously reported P. koreensis isolates recovered from agricultural soils in Korea. We present evidence suggesting that the biosurfactant secreted by CCB P. koreensis strains is involved in their ability to compete with a CCB Exiguobacterium aurantiacum strain (m5-66) used as a model organism in competition experiments. Furthermore, the ethyl acetate extract of culture supernatant of CCB P. koreensis strains results in growth inhibition not only of E. aurantiacum m5-66, but also of a Bacillus subtilis type strain (ATCC6633). Based on these results we propose that the production of biosurfactant could be of ecological importance and could play a role in the separation of the P. koreensis CCB lineage., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

This study represents the first report describing the genetic diversity of nodule-endophytic agrobacteria isolated from diverse legumes and their phylogenetic relationships with the valid species of agrobacteria, as well as the non-recognized genomospecies of the former Agrobacterium tumefaciens (Rhizobium radiobacter). The genetic diversity of a collection of 18 non-nodulating agrobacteria-like strains, previously isolated from root nodules of Vicia faba, Cicer arietinum and Phaseolus vulgaris from different geographical regions of Tunisia, was studied by REP-PCR and PCR-RFLP of the 16S-23S rDNA IGS, as well as by sequence analysis of the 16S rDNA and the housekeeping genes recA and atpD. The aim of the work was to study the genetic diversity of the different isolates and to check for any host-specificity. The results from the different techniques were congruent and suggested a specific interaction for P. vulgaris, whereas no specific endophytic interaction was observed for V. faba and C. arietinum. The phylogenetic analysis clearly indicated that some isolates were affiliated to R. radiobacter or to its non-recognized genomic species (genomovars G2, G4 and G9). However, the other isolates probably constitute new species within Rhizobium (Agrobacterium) and Shinella., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Forty three Bradyrhizobium strains isolated in Poland from root nodules of lupin species (Lupinus albus, L. angustifolius and L. luteus), and pink serradella (Ornithopus sativus) were examined based on phylogenetic analyses of three housekeeping (atpD, glnII and recA) and nodulation (nodA) gene sequences. Additionally, seven strains originating from root-nodules of yellow serradella (O. compressus) from Asinara Island (Italy) were included in this study. Phylogenetic trees revealed that 15 serradella strains, including all yellow serradella isolates, and six lupin strains grouped in Bradyrhizobium canariense (BC) clade, whereas eight strains from pink serradella and 15 lupin strains were assigned to Bradyrhizobium japonicum (BJ1). Apparently, these species are the two dominant groups in soils of central Europe, in the nodules of lupin and serradella plants. Only three strains belonged to other chromosomal lineages: one formed a cluster that was sister to B. canariense, one strain grouped outside the branch formed by B. japonicum super-group, and one strain occupied a distant position in the genus Bradyrhizobium, clustering with strains of the Rhodopseudomonas genus. All strains in nodulation nodA gene tree grouped in a cluster referred to as Clade II, which is in line with earlier data on this clade dominance among Bradyrhizobium strains in Europe. The nodA tree revealed four well-supported subgroups within Clade II (II.1-II.4). Interestingly, all B. canariense strains clustered in subgroup II.1 whereas B. japonicum strains dominated subgroups II.2-II.4., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality., (Copyright © 2011 Elsevier GmbH. All rights reserved.)