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This descriptive study aimed to find out whether instructors practiced self-development activities, whether some factors hindered the practice of self-development activities, and to what extent the instructors implemented self-development activities in their classes to solve a problem. Additionally, it aimed to find out whether the instructors differed in the use of self-development activities in terms of age, gender, experience, education level, and teaching hours. This study was conducted with 348 EFL instructors and four teacher trainers. Three research instruments including a questionnaire, interviews with teacher trainers, and four EFL instructors were used to investigate the self-development activities which were journal writing, self-appraisal, peer-observation, reading, writing a research paper, and action research. The analysis of data revealed that EFL instructors practiced self-development activities on a limited scale except for peer observation, which was carried out as a school policy. The results also showed that the workload was the most important hindrance in practicing self-development activities. Additionally, EFL instructors did not transfer the information gathered from self-development activities in their EFL classes to solve problems and they differed in the use of self-development activities to some extent in terms of age, gender, teaching experience, ELT qualifications like BA, MA, or Ph.D. and teaching hours.

We studied symbiosis of Medicago ciliaris and Medicago polymorpha, two legumes of forage and ecological importance in Algeria, especially in saline soil regions. We report the spatial distribution of the two species and their microsymbionts along salinity gradient transects in the Sebkha of Misserghin (Algeria, North Africa). Seeds and root nodules were sampled from 10 sites. Twenty-seven rhizobial strains were isolated from root nodules and characterized as fast-growers and slime-producers on yeast mannitol agar. By partial sequencing of the gene coding for the 16 S ribosomal RNA, they were found to be affiliated to Rhizobium, Sinorhizobium, and Agrobacterium but several strains had sequences with separate positions.Interestingly one of these was further assigned to Phyllobacterium. Opposite to rhizobia, the distribution of the two Medicago species varied along the salinity gradient, M. ciliaris being dominant in the low NaCl concentration zones and M. polymorpha dominant in the most saline zones. Tolerance to salinity, of both bacterial and plant partners, was studied under laboratory conditions, showing that plants are susceptible to NaCl concentrations of 50 mM, 15-fold lower than that of their associated rhizobia (800 mM). [ABSTRACT FROM AUTHOR]

Phylogenetic studies comparing the Dipterocarpaceae and the Sarcolaenaceae, a tree family endemic to Madagascar, have shown that the Sarcolaenaceae share a common ancestor with Asian dipterocarps. This suggests that Asian dipterocarps drifted away from Madagascar with the India–Seychelles landmass and then dispersed through Asia. Although all dipterocarps examined so far have been found to be ectomycorrhizal, the ectomycorrhizal status of Sarcolaenaceae had not been investigated. Here we establish the ectomycorrhizal status of Sarcolaenaceae using histological and molecular methods. This indicates that the common ancestor of the Sarcolaenaceae and Asian dipterocarps was ectomycorrhizal, at least before the separation of the Madagascar–India landmass, 88 million years ago. [ABSTRACT FROM AUTHOR]

Burkholderia glumae causes bacterial leaf blight in rice, and its global spread has been exacerbated by climate change. To understand the genetic diversity and virulence of B. glumae strains isolated from rice cultivars in Perú, 47 isolates were obtained from infected rice fields, all belonging to B. glumae , and confirmed by rec A and tox B sequences. The BOX-PCR typing group has 38 genomic profiles, and these turn into seven variable number tandem repeats (VNTR) haplotypes. There was no correlation between clustering and geographical origin. Nineteen strains were selected for phenotypic characterization and virulence, using both the maceration level of the onion bulb proxy and inoculation of seeds of two rice cultivars. Several strains produced pigments other than toxoflavin, which correlated with onion bulb maceration. In terms of virulence at the seed level, all strains produced inhibition at the root and coleoptile level, but the severity of symptoms varied significantly between strains, revealing significant differences in pathogenicity. There is no correlation between maceration and virulence scores, probably reflecting different virulence mechanisms depending on the host infection stage. This is the first study to evaluate the VNTR diversity and virulence of Peruvian strains of B. glumae in two commercial cultivars., Competing Interests: The author(s) declare no conflict of interest.

Animals continuously detect information via multiple sensory channels, like vision and hearing, and integrate these signals to realise faster and more accurate decisions; a fundamental neural computation known as multisensory integration. A widespread view of this process is that multimodal neurons linearly fuse information across sensory channels. However, does linear fusion generalise beyond the classical tasks used to explore multisensory integration? Here, we develop novel multisensory tasks, which focus on the underlying statistical relationships between channels, and deploy models at three levels of abstraction: from probabilistic ideal observers to artificial and spiking neural networks. Using these models, we demonstrate that when the information provided by different channels is not independent, linear fusion performs sub-optimally and even fails in extreme cases. This leads us to propose a simple nonlinear algorithm for multisensory integration which is compatible with our current knowledge of multimodal circuits, excels in naturalistic settings and is optimal for a wide class of multisensory tasks. Thus, our work emphasises the role of nonlinear fusion in multisensory integration, and provides testable hypotheses for the field to explore at multiple levels: from single neurons to behaviour., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Ghosh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Due to their potential applications for food safety, there is a growing interest in rice root-associated microbial communities, but some systems remain understudied. Here, we compare the assemblage of root-associated microbiota in rice sampled in 19 small farmer's fields from irrigated and rainfed lowlands in Burkina Faso, using an amplicon metabarcoding approach of the 16S rRNA gene (prokaryotes, three plant samples per field) and ITS (fungi, one sample per field). In addition to the expected structure by root compartments (root vs rhizosphere) and geographical zones, we showed that the rice production system is a major driver of microbiome structure. In irrigated systems, we found a higher diversity of prokaryotic communities from the rhizosphere and more complex co-occurrence networks, compared to rainfed lowlands, while fungal communities exhibited an opposite pattern (higher richness in rainfed lowlands). Core taxa were different between the two systems, and indicator species were identified: mostly within Bacillaceae in rainfed lowlands, and within Burkholderiaceae and Moraxellaceae in irrigated areas. Finally, a higher abundance in rainfed lowlands was found for mycorrhizal fungi (both compartments) and rhizobia (rhizosphere only). Our results highlight deep microbiome differences induced by contrasted rice production systems that should consequently be considered for microbial engineering applications., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Burkholderia vietnamiensis LMG10929 and Paraburkholderia kururiensis M130 are bacterial rice growth-promoting models. Besides this common ecological niche, species of the Burkholderia genus are also found as opportunistic human pathogens, while Paraburkholderia species are mostly environmental and plant associated. In this study, we compared the genetic strategies used by B. vietnamiensis and P. kururiensis to colonize two subspecies of their common host, Oryza sativa subsp. japonica (cv. Nipponbare) and O. sativa subsp. indica (cv. IR64). We used high-throughput screening of transposon insertional mutant libraries (Tn-seq) to infer which genetic elements have the highest fitness contribution during root surface colonization at 7 days postinoculation. Overall, we detected twice more genes in B. vietnamiensis involved in rice root colonization than in P. kururiensis , including genes contributing to the tolerance of plant defenses, which suggests a stronger adverse reaction of rice toward B. vietnamiensis than toward P. kururiensis . For both strains, the bacterial fitness depends on a higher number of genes when colonizing indica rice compared to japonica. These divergences in host pressure on bacterial adaptation could be partly linked to the cultivars' differences in nitrogen assimilation. We detected several functions commonly enhancing root colonization in both bacterial strains, e.g., Entner-Doudoroff (ED) glycolysis. Less frequently and more strain specifically, we detected functions limiting root colonization such as biofilm production in B. vietnamiensis and quorum sensing in P. kururiensis . The involvement of genes identified through the Tn-seq procedure as contributing to root colonization, i.e., ED pathway, c-di-GMP cycling, and cobalamin synthesis, was validated by directed mutagenesis and competition with wild-type (WT) strains in rice root colonization assays. IMPORTANCE Burkholderiaceae are frequent and abundant colonizers of the rice rhizosphere and interesting candidates to investigate for growth promotion. Species of Paraburkholderia have repeatedly been described to stimulate plant growth. However, the closely related Burkholderia genus includes both beneficial and phytopathogenic species, as well as species able to colonize animal hosts and cause disease in humans. We need to understand to what extent the bacterial strategies used for the different biotic interactions differ depending on the host and if strains with agricultural potential could also pose a threat toward other plant hosts or humans. To start answering these questions, we used in this study transposon sequencing to identify genetic traits in Burkholderia vietnamiensis and Paraburkholderia kururiensis that contribute to the colonization of two different rice varieties. Our results revealed large differences in the fitness gene sets between the two strains and between the host plants, suggesting a strong specificity in each bacterium-plant interaction.

Multiple constraints affect rice yields in West Africa. Among these constraints are viral, bacterial, and fungal pathogens. We aimed to describe the spatiotemporal patterns of occurrence and incidence of multiple rice diseases in farmers' fields in contrasting rice growing systems in the western Burkina Faso. For this purpose, we selected a set of three pairs of sites, each comprising an irrigated area and a neighboring rainfed lowland, and studied them over four consecutive years. We first performed interviews with the rice farmers to better characterize the management practices at the different sites. This study revealed that the transplanting of rice and the possibility of growing rice twice a year are restricted to irrigated areas, while other practices, such as the use of registered rice cultivars, fertilization, and pesticides, are not specific but differ between the two rice growing systems. Then, we performed symptom observations at these study sites to monitor the following four diseases: yellow mottle disease, Bacterial Leaf Streak (BLS), rice leaf blast, and brown spot. The infection rates were found to be higher in irrigated areas than in rainfed lowlands, both when analyzing all observed symptoms together (any of the four diseases) and when specifically considering each of the two diseases: BLS and rice leaf blast. Brown spot was particularly prevalent in all six study sites, while yellow mottle disease was particularly structured geographically. Various diseases were frequently found together in the same field (co-occurrence) or even on the same plant (coinfection), especially in irrigated areas.

Burkholderia glumae is responsible for the panicle blight disease of rice. This disease is present worldwide and can result in significant drop in yields. To estimate the genetic diversity of the bacterial strains present in a rice paddy field in Colombia, we sampled 109 strains from infected panicles. To detect fine genetic relationships among related haplotypes, and to overcome a very low nucleotide diversity detected in previous studies, we designed primers to amplify and sequence several highly variable minisatellite loci, or variable number tandem repeats (VNTRs), as well as part of the Toxoflavin toxA gene in all strains. Results show that the toxA nucleotide diversity defined four lineages and was similar to that detected in several fields in Japan; data suggest that B . glumae has spread from Asia to America without major loss of genetic diversity, and that five VNTR loci discriminated the strains within the field revealing single and multi-infections of the rice panicles with a wide distribution of the haplotypes among the different plots. Even though disease levels vary considerably from year to year, the bacterial genetic diversity is maintained within a field. We do not detect any geographical structuring within the field, nor any effect of the rice cultivar on the observed diversity. The consequences on the origin and evolution of the bacteria are discussed.

Burkholderia sensu lato species are prominent for their diversity of hosts. The type 3 secretion system (T3SS) is a major mechanism impacting the interactions between bacteria and eukaryotic hosts. Besides the human pathogenic species Burkholderia pseudomallei and closely affiliated species, the T3SS has received little attention in this genus as in taxonomically and evolutionary close genera Paraburkholderia , Caballeronia , Trinickia , and Mycetohabitans . We proceeded to identify and characterize the diversity of T3SS types using the genomic data from a subset of 145 strains representative of the species diversity found in the Burkholderia s.l. group. Through an analysis of their phylogenetic distribution, we identified two new T3SS types with an atypical chromosomal organization and which we propose to name BCI ( Burkholderia cepacia complex Injectisome) and PSI ( Paraburkholderia Short Injectisome). BCI is the dominant T3SS type found in Burkholderia sensu stricto ( s.s. ) species and PSI is mostly restricted to the Paraburkholderia genus. By correlating their distribution with the ecology of their strains of origin, we propose a role in plant interaction for these T3SS types. Experimentally, we demonstrated that a BCI deficient B. vietnamiensis LMG10929 mutant was strongly affected in its rice colonization capacity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Wallner, Moulin, Busset, Rimbault and Béna.)

Here, we assessed the relative influence of wheat genotype, agricultural practices (conventional vs organic) and soil type on the rhizosphere microbiome. We characterized the prokaryotic (archaea and bacteria) and eukaryotic (fungi and protists) communities in soils from four different countries (Cameroon, France, Italy, Senegal) and determined if a rhizosphere core microbiome existed across these different countries. The wheat genotype had a limited effect on the rhizosphere microbiome (2% of variance) as the majority of the microbial taxa were consistently associated to multiple wheat genotypes grown in the same soil. Large differences in taxa richness and in community structure were observed between the eight soils studied (57% variance) and the two agricultural practices (10% variance). Despite these differences between soils, we observed that 177 taxa (2 archaea, 103 bacteria, 41 fungi and 31 protists) were consistently detected in the rhizosphere, constituting a core microbiome. In addition to being prevalent, these core taxa were highly abundant and collectively represented 50% of the reads in our data set. Based on these results, we identify a list of key taxa as future targets of culturomics, metagenomics and wheat synthetic microbiomes. Additionally, we show that protists are an integral part of the wheat holobiont that is currently overlooked., (© FEMS 2020.)

Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by μl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety., Competing Interests: The authors have declared that no competing interests exist.

Background: Burkholderia cenocepacia is a human opportunistic pathogen causing devastating symptoms in patients suffering from immunodeficiency and cystic fibrosis. Out of the 303 B. cenocepacia strains with available genomes, the large majority were isolated from a clinical context. However, several isolates originate from other environmental sources ranging from aerosols to plant endosphere. Plants can represent reservoirs for human infections as some pathogens can survive and sometimes proliferate in the rhizosphere. We therefore investigated if B. cenocepacia had the same potential., Results: We selected genome sequences from 31 different strains, representative of the diversity of ecological niches of B. cenocepacia, and conducted comparative genomic analyses in the aim of finding specific niche or host-related genetic determinants. Phylogenetic analyses and whole genome average nucleotide identity suggest that strains, registered as B. cenocepacia, belong to at least two different species. Core-genome analyses show that the clade enriched in environmental isolates lacks multiple key virulence factors, which are conserved in the sister clade where most clinical isolates fall, including the highly virulent ET12 lineage. Similarly, several plant associated genes display an opposite distribution between the two clades. Finally, we suggest that B. cenocepacia underwent a host jump from plants/environment to animals, as supported by the phylogenetic analysis. We eventually propose a name for the new species that lacks several genetic traits involved in human virulence., Conclusion: Regardless of the method used, our studies resulted in a disunited perspective of the B. cenocepacia species. Strains currently affiliated to this taxon belong to at least two distinct species, one having lost several determining animal virulence factors.

Genetic diversity and population structure of 268 Lens culinaris symbiotic rhizobia collected from 40 cultivated fields in the main lentil production regions in Morocco were estimated. Three chromosomal housekeeping genes (recA, glnII and atpD) and one common symbiotic gene (nodC) were sequenced and analyzed in order to identify the local symbionts of lentil. The molecular phylogeny of the concatenated housekeeping genes clustered more than 95% of the isolates in one main clade together with Rhizobium laguerreae species. R. laguerreae represents the main symbiont of cultivated lentil in Morocco and, for the first time, a large sample of individuals is obtained for this species. There is a significant and high genetic differentiation of bacterial populations among the four regions for their symbiotic gene, and much lower for their housekeeping genes. The reasons why R. laguerreae is so frequently recovered in our study is discussed., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Some species of the genus Paraburkholderia that are able to nodulate and fix nitrogen in symbiosis with legumes are called β-rhizobia and represent a group of ecological and biotechnological importance. We used Mimosa pudica and Phaseolus vulgaris to trap 427 rhizobial isolates from rhizospheric soil of Mimoseae trees in the Brazilian Atlantic Forest. Eighty-four representative strains were selected according to the 16S rRNA haplotypes and taxonomically characterized using a concatenated 16S rRNA-recA phylogeny. Most strains were assembled in the genus Paraburkholderia, including Paraburkholderia sabiae and Pa. nodosa. Mesorhizobium (α-rhizobia) and Cupriavidus (β-rhizobia) were also isolated, but in smaller proportions. Multilocus sequence analysis and BOX-PCR analyses indicated that six clusters of Paraburkholderia represent potential new species. In the phylogenetic analysis of the nodC gene, the majority of the strains were positioned in the same groups as in the 16S rRNA-recA tree, indicative of stability and vertical inheritance, but we also identified horizontal transfer of nodC in Pa. sabiae. All α- and β-rhizobial species were trapped by both legumes, although preferences of the host plants for specific rhizobial species have been observed., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 bacterial genus Burkholderia comprises species occupying several habitats, including a group of symbionts of leguminous plants-also called beta-rhizobia-that has been recently ascribed to the new genus Paraburkholderia We used common bean (Phaseolus vulgaris L.) plants to trap rhizobia from an undisturbed soil of the Brazilian Cerrado under the vegetation type 'Cerradão'. Genetic characterization started with the analyses of 181 isolates by BOX-PCR, where the majority revealed unique profiles, indicating high inter- and intra-species diversity. Restriction fragment length polymorphism-PCR of the 16S rRNA of representative strains of the BOX-PCR groups indicated two main clusters, and gene-sequencing analysis identified the minority (27%) as Rhizobium and the majority (73%) as Paraburkholderia Phylogenetic analyses of the 16S rRNA and housekeeping (recA and gyrB) genes positioned all strains of the second cluster in the species P. nodosa, and the phylogeny of a symbiotic gene-nodC-was in agreement with the conserved genes. All isolates were stable vis-à-vis nodulating common bean, but, in general, with a low capacity for fixing N2, although some effective strains were identified. The predominance of P. nodosa might be associated with the edaphic properties of the Cerrado biome, and might represent an important role in terms of maintenance of the ecosystem, which is characterized by acid soils with high saturation of aluminum and low N2 content., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hirschfeldia incana, a pseudometallophyte belonging to the Brassicaceae family and widespread in the Mediterranean region, was selected for its ability to grow on soils contaminated by lead (Pb). The global comparison of gene expression using microarrays between a plant susceptible to Pb (Arabidopsis thaliana) and a Pb tolerant plant (H. incana) enabled the identification of a set of specific genes expressed in response to lead exposure. Three groups of genes were particularly over-represented by the Pb exposure in the biological processes categorized as photosynthesis, cell wall, and metal handling. Each of these gene groups was shown to be directly involved in tolerance or in protection mechanisms to the phytotoxicity associated with Pb. Among these genes, we demonstrated that MT2b, a metallothionein gene, was involved in lead accumulation, confirming the important role of metallothioneins in the accumulation and the distribution of Pb in leaves. On the other hand, several genes involved in biosynthesis of ABA were shown to be up-regulated in the roots and shoots of H. incana treated with Pb, suggesting that ABA-mediated signaling is a possible mechanism in response to Pb treatment in H. incana. This latest finding is an important research direction for future studies.

Pterocarpus officinalis Jacq. is a legume tree native to the Caribbean islands and South America growing as a dominant species in swamp forests. To analyze (i) the genetic diversity and (ii) the symbiotic properties of its associated nitrogen-fixing soil bacteria, root nodules were collected from P. officinalis distributed in 16 forest sites of the Caribbean islands and French Guiana. The sequencing of the 16S-23S ribosomal RNA intergenic spacer region (ITS) showed that all bacteria belonged to the Bradyrhizobium genus. Bacteria isolated from insular zones showed very close sequence homologies with Bradyrhizobium genospecies V belonging to the Bradyrhizobium japonicum super-clade. By contrast, bacteria isolated from continental region displayed a larger genetic diversity and belonged to B. elkanii super-clade. Two strains from Puerto Rico and one from French Guiana were not related to any known sequence and could be defined as a new genospecies. Inoculation experiments did not show any host specificity of the Bradyrhizobium strains tested in terms of infectivity. However, homologous Bradyrhizobium sp. strain-P. officinalis provenance associations were more efficient in terms of nodule production, N acquisition, and growth than heterologous ones. The dominant status of P. officinalis in the islands may explain the lower bacterial diversity compared to that found in the continent where P. officinalis is associated with other leguminous tree species. The specificity in efficiency found between Bradyrhizobium strains and host tree provenances could be due to a coevolution process between both partners and needs to be taken in consideration in the framework of rehabilitation plantation programs.

Variations in the patterns of diversity of symbionts have been described worldwide on Mimosa pudica, a pan-tropical invasive species that interacts with both α and β-rhizobia. In this study, we investigated if symbiont competitiveness can explain these variations and the apparent prevalence of β- over α-rhizobia. We developed an indirect method to measure the proportion of nodulation against a GFP reference strain and tested its reproducibility and efficiency. We estimated the competitiveness of 54 strains belonging to four species of β-rhizobia and four of α-rhizobia, and the influence of the host genotype on their competitiveness. Our results were compared with biogeographical patterns of symbionts and host varieties. We found: (i) a strong strain effect on competitiveness largely explained by the rhizobial species, with Burkholderia phymatum being the most competitive species, followed by B. tuberum, whereas all other species shared similar and reduced levels of competitiveness; (ii) plant genotype can increase the competitiveness of Cupriavidus taiwanensis. The latter data support the likelihood of the strong adaptation of C. taiwanensis with the M. pudica var. unijuga and help explain its prevalence as a symbiont of this variety over Burkholderia species in some environments, most notably in Taiwan., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The genetic structure of rhizobia nodulating pea and lentil in Algeria, Northern Africa was determined. A total of 237 isolates were obtained from root nodules collected on lentil (Lens culinaris), proteaginous and forage pea (Pisum sativum) growing in two eco-climatic zones, sub-humid and semi-arid, in Eastern Algeria. They were characterised by PCR-restriction fragment length polymorphism (RFLP) of the 16S-23S rRNA intergenic region (IGS), and the nodD-F symbiotic region. The combination of these haplotypes allowed the isolates to be clustered into 26 distinct genotypes, and all isolates were classified as Rhizobium leguminosarum. Symbiotic marker variation (nodD-F) was low but with the predominance of one nod haplotype (g), which had been recovered previously at a high frequency in Europe. Sequence analysis of the IGS further confirmed its high variability in the studied strains. An AMOVA analysis showed highly significant differentiation in the IGS haplotype distribution between populations from both eco-climatic zones. This differentiation was reflected by differences in dominant genotype frequencies. Conversely, no host plant effect was detected. The nodD gene sequence-based phylogeny suggested that symbiotic gene diversity in pea and lentil nodulating rhizobial populations in Algeria was low compared to that reported elsewhere in the world., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Legumes in the genus Aeschynomene form nitrogen-fixing root nodules in association with Bradyrhizobium strains. Several aquatic and subaquatic species have the additional capacity to form stem nodules, and some of them can symbiotically interact with specific strains that do not produce the common Nod factors synthesized by all other rhizobia. The question of the emergence and evolution of these nodulation characters has been the subject of recent debate. We conducted a molecular phylogenetic analysis of 38 different Aeschynomene species. The phylogeny was reconstructed with both the chloroplast DNA trnL intron and the nuclear ribosomal DNA ITS/5.8S region. We also tested 28 Aeschynomene species for their capacity to form root and stem nodules by inoculating different rhizobial strains, including nodABC-containing strains (ORS285, USDA110) and a nodABC-lacking strain (ORS278). Maximum likelihood analyses resolved four distinct phylogenetic groups of Aeschynomene. We found that stem nodulation may have evolved several times in the genus, and that all Aeschynomene species using a Nod-independent symbiotic process clustered in the same clade. The phylogenetic approach suggested that Nod-independent nodulation has evolved once in this genus, and should be considered as a derived character, and this result is discussed with regard to previous experimental studies., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Understanding functional diversity is one of the main goals of microbial ecology, and definition of new bacterial ecotypes contributes significantly to this objective. Nitrogen-fixing bacteria provide a good system for investigation of ecotypes/biovars/symbiovars, as they present different specific associations with several host plants. This specific symbiosis is reflected both in the nodulation and fixation efficiency and in genetic characters of the bacteria, and several biovars have already been described in the bacterial species Ensifer meliloti. In the present study, the species affiliation of E. meliloti strains trapped from nodules sampled from Medicago rigiduloïdes roots was analyzed using housekeeping recA genes and DNA-DNA hybridization. The genetic diversity of these isolates was also investigated using several symbiotic markers: nodulation (nodA, nodB, nodC) and nitrogen fixation (nifH) genes, as well as the performance of phenotypic tests of nodulation capacity and nitrogen fixation efficiency. These analyses led to the proposal of a new bacterial symbiovar, E. meliloti sv. rigiduloides, that fixed nitrogen efficiently on M. rigiduloïdes, but not on Medicago truncatula. Using phylogenetic reconstructions, including the different described symbiovars, several hypotheses of lateral gene transfer and gene loss are proposed to explain the emergence of symbiovars within this species. The widespread geographical distribution of this symbiovar around the Mediterranean Basin, in contrast to restriction of M. rigiduloïdes to Eastern European countries, suggests that these isolates might also be associated with other plant species. The description of a new symbiovar within E. meliloti confirms the need for accurate bacterial ecological classification, especially for analysis of bacterial populations., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

Tropical aquatic species of the legume genus Aeschynomene are stem- and root-nodulated by bradyrhizobia strains that exhibit atypical features such as photosynthetic capacities or the use of a nod gene-dependent (ND) or a nod gene-independent (NI) pathway to enter into symbiosis with legumes. In this study we used a comparative genomics approach on nine Aeschynomene symbionts representative of their phylogenetic diversity. We produced draft genomes of bradyrhizobial strains representing different phenotypes: five NI photosynthetic strains (STM3809, ORS375, STM3847, STM4509 and STM4523) in addition to the previously sequenced ORS278 and BTAi1 genomes, one photosynthetic strain ORS285 hosting both ND and NI symbiotic systems, and one NI non-photosynthetic strain (STM3843). Comparative genomics allowed us to infer the core, pan and dispensable genomes of Aeschynomene bradyrhizobia, and to detect specific genes and their location in Genomic Islands (GI). Specific gene sets linked to photosynthetic and NI/ND abilities were identified, and are currently being studied in functional analyses.

Tropical aquatic legumes of the genus Aeschynomene are unique in that they can be stem-nodulated by photosynthetic bradyrhizobia. Moreover, a recent study demonstrated that two Aeschynomene indica symbionts lack canonical nod genes, thereby raising questions about the distribution of such atypical symbioses among rhizobial-legume interactions. Population structure and genomic diversity were compared among stem-nodulating bradyrhizobia isolated from various Aeschynomene species of Central America and Tropical Africa. Phylogenetic analyses based on the recA gene and whole-genome amplified fragment length polymorphism (AFLP) fingerprints on 110 bacterial strains highlighted that all the photosynthetic strains form a separate cluster among bradyrhizobia, with no obvious structuring according to their geographical or plant origins. Nod-independent symbiosis was present in all sampling areas and seemed to be linked to Aeschynomene host species. However, it was not strictly dependent on photosynthetic ability, as exemplified by a newly identified cluster of strains that lacked canonical nod genes and efficiently stem-nodulated A. indica, but were not photosynthetic. Interestingly, the phenotypic properties of this new cluster of bacteria were reflected by their phylogenetical position, as being intermediate in distance between classical root-nodulatingBradyrhizobium spp. and photosynthetic ones. This result opens new prospects about stem-nodulating bradyrhizobial evolution., (© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The diversity of ectomycorrhizal (ECM) fungi on adult trees and seedlings of five species, Anthonotha fragrans, Anthonotha macrophylla, Cryptosepalum tetraphyllum, Paramacrolobium coeruleum and Uapaca esculenta, was determined in a tropical rain forest of Guinea. Ectomycorrhizae were sampled within a surface area of 1600 m(2), and fungal taxa were identified by sequencing the rDNA Internal Transcribed Spacer region. Thirty-nine ECM fungal taxa were determined, of which 19 multi-hosts, 9 single-hosts and 11 singletons. The multi-host fungi represented 92% (89% when including the singletons in the analysis) of the total abundance. Except for A. fragrans, the adults of the host species displayed significant differentiation for their fungal communities, but their seedlings harboured a similar fungal community. These findings suggest that there was a potential for the formation of common mycorrhizal networks in close vicinity. However, no significant difference was detected for the δ(13)C and δ(15)N values between seedlings and adults of each ECM plant, and no ECM species exhibited signatures of mixotrophy. Our results revealed (i) variation in ECM fungal diversity according to the seedling versus adult development stage of trees and (ii) low host specificity of ECM fungi, and indicated that multi-host fungi are more abundant than single-host fungi in this forest stand., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula-Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.

A polyphasic taxonomic approach was used to characterize 31 rhizobial isolates obtained from Anthyllis vulneraria, a metallicolous legume species, growing close to a zinc mine in the south of France (Saint Laurent le Minier). Comparative analysis of nearly full-length 16S rRNA gene sequences showed that these Gram-negative bacteria belonged to the genus Mesorhizobium and that they were related most closely to Mesorhizobium tianshanense ORS 2640(T). The phylogenetic relationships of these isolates with other Mesorhizobium species were confirmed by sequencing and analysis of the recA and atpD genes, which were used as alternative chromosomal markers. These novel mesorhizobial strains tolerated high concentrations of heavy metals: 16-32 mM Zn and 0.3-0.5 mM Cd. DNA-DNA hybridizations revealed >73 % relatedness between the strains isolated from A. vulneraria, but only 19-33 % relatedness between these and the type strains of M. tianshanense and Mesorhizobium mediterraneum. These results, together with other phenotypic characteristics, support the conclusion that these isolates represent a single, novel species of the genus Mesorhizobium, for which the name Mesorhizobium metallidurans sp. nov. is proposed. The type strain is STM 2683(T) (=CFBP 7147(T)=LMG 24485(T)).

We investigated the genetic diversity and symbiotic efficiency of 223 Sinorhizobium sp. isolates sampled from a single Mediterranean soil and trapped with four Medicago truncatula lines. DNA molecular polymorphism was estimated by capillary electrophoresis-single-stranded conformation polymorphism and restriction fragment length polymorphism on five loci (IGS(NOD), typA, virB11, avhB11, and the 16S rRNA gene). More than 90% of the rhizobia isolated belonged to the Sinorhizobium medicae species (others belonged to Sinorhizobium meliloti), with different proportions of the two species among the four M. truncatula lines. The S. meliloti population was more diverse than that of S. medicae, and significant genetic differentiation among bacterial populations was detected. Single inoculations performed in tubes with each bacterial genotype and each plant line showed significant bacterium-plant line interactions for nodulation and N(2) fixation levels. Competition experiments within each species highlighted either strong or weak competition among genotypes within S. medicae and S. meliloti, respectively. Interspecies competition experiments showed S. meliloti to be more competitive than S. medicae for nodulation. Although not highly divergent at a nucleotide level, isolates collected from this single soil sample displayed wide polymorphism for both nodulation and N(2) fixation. Each M. truncatula line might influence Sinorhizobium soil population diversity differently via its symbiotic preferences. Our data suggested that the two species did not evolve similarly, with S. meliloti showing polymorphism and variable selective pressures and S. medicae showing traces of a recent demographic expansion. Strain effectiveness might have played a role in the species and genotype proportions, but in conjunction with strain adaptation to environmental factors.

Despite the great popularity and potential of microarrays, their use for research and clinical applications is still hampered by lengthy and costly design and optimization processes, mainly because the technology relies on the end point measurement of hybridization. Thus, the ability to monitor many hybridization events on a standard microarray slide in real time would greatly expand the use and benefit of this technology, as it would give access to better prediction of probe performance and improved optimization of hybridization parameters. Although real-time hybridization and thermal denaturation measurements have been reported, a complete walk-away system compatible with the standard format of microarrays is still unavailable. To address this issue, we have designed a biochip tool that combines a hybridization station with active mixing capability and temperature control together with a fluorescence reader in a single compact benchtop instrument. This integrated live hybridization machine (LHM) allows measuring in real time the hybridization of target DNA to thousands of probes simultaneously and provides excellent levels of detection and superior sequence discrimination. Here we show on an environmental single nucleotide polymorphism (SNP) model system that the LHM enables a variety of experiments unachievable with conventional biochip tools.

Using nitrogen-fixing Sinorhizobium species that interact with Medicago plants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genus Medicago on the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact with Medicago species, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, using Medicago truncatula and Medicago laciniata as host plants, to study the differentiation between populations of Sinorhizobium medicae, Sinorhizobium meliloti bv. meliloti, and S. meliloti bv. medicaginis. The former two can be associated with M. truncatula (among other species of Medicago), whereas the last organism is the specific symbiont of M. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids of S. meliloti. The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification of Medicago symbionts. Within S. meliloti, the analyses show that nod genes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars of S. meliloti, whereas reproductive isolation between S. meliloti bv. meliloti and S. medicae is maintained even though these bacteria can cooccur in sympatry on the same individual host plants.

The use of DNA microarrays for detection and identification of bacteria and genes of interest from various environments (e.g. soil, sediment, water column...) is a major challenge for microbiologists working on functional diversity. So far, most of the genomic methods that have been described rely on the use of taxonomic markers (such as 16S rRNA) that can be easily amplified by PCR prior to hybridization on microarrays. However, taxonomical markers are not always informative on the functions present in these bacteria. Moreover, genes for which sequence database is limited or that lack any conserved regions will be difficult to amplify and thus to detect in unknown samples. Furthermore, PCR amplification often introduces biases that lead to inaccurate analysis of microbial communities. An alternative solution to overcome these strong limitations is to use genomic DNA (gDNA) as target for hybridisation, without prior PCR amplification. Though hybridization of gDNA is already used for comparative genome hybridization or sequencing by hybridization, yet to the high cost of tiling strategies and important data filtering, its adaptation for use in environmental research poses great challenges in terms of specificity, sensitivity and reproducibility of hybridization. Considering the very faint number of publications that have described hybridization of gDNA to microarrays for environmental applications, we confront in this review the different approaches that have been developed so far, and propose alternative strategies that may contribute to improve the development of microarrays for studying the microbial genetic structure and composition of samples of high environmental and ecological value.

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.

We investigate the genetic structure and molecular selection pattern of a sympatric population of Sinorhizobium meliloti and Sinorhizobium medicae. These bacteria fix nitrogen in association with plants of the genus Medicago. A set of 116 isolates were obtained from a soil sample, from root nodules of three groups of plants representing among-species, within-species and intraline diversity in the Medicago genus. Bacteria were characterized by sequencing at seven loci evenly distributed along the genome of both Sinorhizobium species, covering the chromosome and the two megaplasmids. We first test whether the diversity of host plants influence the bacterial diversity recovered. Using the same data set, we then analyse the selective pattern at each locus. There was no relationship between the diversity of Medicago plants that were used for sampling and the diversity of their symbionts. However, we found evidence of selection within each of the two main symbiotic regions, located on the two different megaplasmids. Purifying selection or a selective sweep was found to occur in the nod genomic region, which includes genes involved in nodulation specificity, whereas balancing selection was detected in the exo region, close to genes involved in exopolysaccharide production. Such pattern likely reflects the interaction between host plants and bacterial symbionts, with a possible conflict of interest between plants and cheater bacterial genotypes. Recombination appears to occur preferentially within and among loci located on megaplasmids, rather than within the chromosome. Thus, recombination may play an important role in resolving this conflict by allowing different selection patterns at different loci.

Gram-negative bacteria were isolated from the rhizoplane of Brassica napus in France and from root nodules of Argyrolobium uniflorum, Astragalus algerianus and Lathyrus numidicus growing in the infra-arid zone of southern Tunisia. Based on phylogenetic analysis of the 16S rRNA gene sequences, the seven isolates belong to the Alphaproteobacteria and are related to Phyllobacterium myrsinacearum strains. The isolates formed three clusters; clusters A and C consist of Tunisian strains, whereas cluster B consists of two strains from Brassica napus from France. Phylogenetic reconstruction based on the atpD gene strongly supports their affiliation to the genus Phyllobacterium. DNA-DNA hybridizations revealed that (i) none of the isolates belong to the species P. myrsinacearum, (ii) clusters A and C represent two distinct genomospecies and (iii) the two strains of cluster B represent two separate genomospecies. Distinctive phenotypic features were deduced from numerical analysis of phenotypic data. Based on this polyphasic approach, four novel species are proposed: Phyllobacterium leguminum sp. nov. (type strain ORS 1419T = CFBP 6745T = LMG 22833T), Phyllobacterium ifriqiyense sp. nov. (type strain STM 370T = CFBP 6742T = LMG 22831T), Phyllobacterium brassicacearum sp. nov. (type strain STM 196T = CFBP 5551T = LMG 22836T) and Phyllobacterium bourgognense sp. nov. (type strain STM 201T = CFBP 5553T = LMG 22837T). The description of the genus Phyllobacterium is emended accordingly.

Although legume biodiversity is concentrated in tropical regions, the majority of studies on legume nodulating bacteria (LNB) are focused on cultivated leguminous plants from temperate regions. However, recent works on tropical regions tend to indicate that the actual diversity of LNB is largely underestimated. In this study, we report the isolation and characterization of 68 nitrogen-fixing root nodule bacteria collected from eight endemic tree species of Dalbergia in Madagascar. The isolates were characterized by (i) restriction fragment length polymorphism (RFLP) analysis of 16S-IGS rDNA, (ii) 16S rDNA gene sequencing and (iii) nodulation tests. Results revealed a wide diversity of bacteria present in the nodules of Dalbergia. Among the 68 isolated bacteria, 65 belonged to Bradyrhizobium, Mesorhizobium, Rhizobium, Azorhizobium and Phyllobacterium from the alpha-class of Proteobacteria, and three isolates belonged to Burkholderia and Ralstonia from the beta-class of Proteobacteria. Our results also show for the first time that a strain belonging to the Burkholderia cepacia complex is able to induce efficient nodules on a legume plant.

S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions. Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms. The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene. This topology suggests the occurrence of lateral transfer of this gene between the domains. Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an "early" acquisition by some bacterial and eukaryotic phyla. Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins. SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi. On the other hand, the lack of affinity to the sequences from the alpha-Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses. Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine. However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high. This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism. On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase. This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb-million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase. This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity. The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes. The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the beta- or gamma-Proteobacteria.

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C(1) compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060(T)=CNCM I 2342(T)=LMG 21967(T)) is proposed for this group of root-nodule-forming bacteria.

Symbiotic nitrogen fixing bacteria-known as rhizobia-harbour a set of nodulation (nod) genes that control the synthesis of modified lipo-chitooligosaccharides, called Nod factors that are required for legume nodulation. The nodA gene, which is essential for symbiosis, is responsible for the attachment of the fatty acid group to the oligosaccharide backbone. The nodZ, nolL, and noeI genes are involved in specific modifications of Nod factors common to bradyrhizobia, i.e., the transfer of a fucosyl group on the Nod factor core, fucose acetylation and fucose methylation, respectively. PCR amplification, sequencing and phylogenetic analysis of nodA gene sequences from a collection of diverse Bradyrhizobium strains revealed the monophyletic character with the possible exception of photosynthetic Bradyrhizobium, despite high sequence diversity. The distribution of the nodZ, nolL, and noeI genes in the studied strains, as assessed by gene amplification, hybridization or sequencing, was found to correlate with the nodA tree topology. Moreover, the nodA, nodZ, and noeI phylogenies were largely congruent, but did not closely follow the taxonomy of the strains shown by the housekeeping 16S rRNA and dnaK genes. Additionally, the distribution of nodZ, noeI, and nolL genes suggested that their presence may be related to the requirements of their legume hosts. These data indicated that the spread and maintenance of nodulation genes within the Bradyrhizobium genus occurred through vertical transmission, although lateral gene transfer also played a significant role.