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Ten mesorhizobial strains isolated from root-nodules of Anthyllis vulneraria by trapping using soils from southern France were studied to resolve their taxonomy. Their 16S rDNA sequences were identical and indicated that they are affiliated to the genus Mesorhizobium within the group M. prunaredense/M. delmotii/M. temperatum/M. mediterraneum/M. wenxiniae and M. robiniae as the closest defined species. Their evolutionary relationships with validated species were further characterized by multilocus sequence analysis (MLSA) using 4 protein-coding housekeeping genes (recA, atpD, glnII and dnaK), that divides the strains in two groups, and suggest that they belong to two distinct species. These results were well-supported by MALDI-TOF mass spectrometry analyses, wet-lab DNA-DNA hybridization (≤58%), and genome-based species delineation methods (ANI 96%, in silico DDH 70%), confirming their affiliation to two novel species. Based on these differences, Mesorhizobium ventifaucium (STM4922

International audience; Two 915-MHz microwave treatments (2 kW-8 min and 4 kW-4 min) were applied to soil and their effects were monitored just after treatments (T0) and 26 days (T26) in soil microcosms. Densities of culturable bacteria, fluorescent pseudomonads and nematodes, hydrolysis activity and soil DNA content declined by over 50% immediately after both microwave treatments (T0), excluding the total fungal 18S rRNA (-13 or -17%) and bacterial 16S rRNA copies (non-significant). A rapid shift in bacterial community composition occurred from T0 towards a large increase in the relative abundance of Firmicutes (+1650%) and a concomitant decrease in various phyla (e.g. Acidobacteria, Actinobacteria, Bacteroidetes, Proteobacteria) from -85 to -61%. At T26 and for both treatments, fluorescein diacetate hydrolysis, density of culturable bacteria, 18S rRNA gene numbers, Simpson diversity, relative abundances of Bacteroidetes and Proteobacteria regained levels similar to controls. Alpha, Delta and Gamma classes Proteobacteria also end up reaching a similar level. In contrast, fluorescent pseudomonad density, nematode diversity and abundance, soil DNA content and relative abundances of some phylum (Acidobacteria, Actinobacteria, Chloroflexi) did not increase in such proportions. Most of the soil biological properties have not been permanently impacted. Nevertheless, the recovery kinetics highly differed according properties, with resilience indices at T26 varying from -87 to +99%.

International audience; To control the soil seed bank of invasive plants with microwaves represents an alternative method to chemical treatments, but it could alter soil quality. Microwave effects were investigated on 17 soil physicochemical and biological properties. Four 915-MHz microwave treatments combining power and duration of exposure were applied on alluvial soil from a grassland using Festuca seeds as an internal standard to evaluate the efficiency of sterilization. Two treatments, 2 kW-8 and 4 kW-4 min, completely inhibited Festuca seed germination and caused the soil temperature to reach at least 80 degrees C. Conversely, the other treatments, 2 kW-4 and 4 kW-2 min, failed to affect seed germination while raising the temperature to 50-60 degrees C. Microbial biomass, the density of culturable heterotrophic bacteria and fluorescent Pseudomonads as well as fluorescein di-acetate hydrolysis decreased significantly by 2.7-, 1.1-, 1.4- and 5.1-fold compared with the control, respectively, following the 2 kW-8 and 4 kW-4 min treatments. These treatments also increased dissolved organic carbon and inorganic phosphorus contents by 1.6- and 1.2-fold, respectively. In contrast, nitrate increased only (about 140%) in the soils under the 2 kW-4 and 4 kW-2 min conditions. Furthermore, the growth of Medicago truncatula Gaertn. seedlings transplanted in microwave-treated soils under the 2 kW-8 and 4 kW-4 min conditions was not impaired despite the reduction in total nitrogen and the microbial population and smaller numbers of nodules. These results suggest that soil seed bank sterilization by microwaves that is 100% effective alters soil microbial indicators more strongly than chemical treatments without compromising the early growth stages of transplanted Medicago plants. Highlights Effects of microwave seed sterilization on soil physicochemical and biological properties. Microbial properties were more sensitive than chemical ones. Microbial biomass, bacterial density, hydrolase activity and root nodulation were reduced. Soluble carbon, nitrate and phosphorus increased depending on treatment effectiveness.

Phytostabilisation can benefit from phytostimulatory rhizobacteria. Forty-three bacterial strains were isolated from the roots of the metallicolous legume Anthyllis vulneraria ssp. carpatica grown in a highly contaminated mine tailing (total Cd, Pb and Zn were up to 1200; 34,000; and 170,000 mg kg(-1), respectively). We aimed at evaluating their phytostimulatory effects on the development of leguminous metallophytes. Strains were screened for fluorescent siderophores and auxin synthesis, inorganic P solubilisation and 1-amino-cyclopropane-1-carboxylate deaminase (ACCd) activity to define a subset of 11 strains that were inoculated on the leguminous metallophytes A. vulneraria and Lotus corniculatus grown in diluted mine spoil (Zn 34,653; Pb 6842; and Cd 242, all in mg kg-1). All strains were affiliated to Pseudomonas spp. (except two), synthetised auxins and siderophores and solubilised P (except three), and seven of them were ACCd positive. The inoculation effects (shoot-root-nodule biomass, chlorophyll content) depended on legume species and bacterial strain genotype. Phytostimulation scores were unrelated to siderophore/auxin synthesis and P solubilisation rates. Inoculations of the strain nos. 17-43 triggered a 1.2-fold significant increase in the chlorophyll content of A. vulneraria. Chlorophyll content and root biomass of L. corniculatus were significantly increased following the inoculations of the strain nos. 17-22 (1.5-1.4-fold, respectively). The strongest positive effects were related to increases in the nodule biomass of L. corniculatus in the presence of three ACCd-positive strains (1.8-fold), one of which was the highest auxin producer. These data suggest to focus on interactions between ACCd activity and auxin synthesis to enhance nodulation of metallicolous legumes.

Anthyllis vulneraria is a legume associated with nitrogen-fixing rhizobia that together offer an adapted biological material for mine-soil phytostabilization by limiting metal pollution. To find rhizobia associated with Anthyllis at a given site, we evaluated the genetic and phenotypic properties of a collection of 137 rhizobia recovered from soils presenting contrasting metal levels. Zn-Pb mine soils largely contained metal-tolerant rhizobia belonging to Mesorhizobium metallidurans or to another sister metal-tolerant species. All of the metal-tolerant isolates harbored the cadA marker gene (encoding a metal-efflux P IB -type ATPase transporter). In contrast, metal-sensitive strains were taxonomically distinct from metal-tolerant populations and consisted of new Mesorhizobium genospecies. Based on the symbiotic nodA marker, the populations comprise two symbiovar assemblages (potentially related to Anthyllis or Lotus host preferences) according to soil geographic locations but independently of metal content. Multivariate analysis showed that soil Pb and Cd concentrations differentially impacted the rhizobial communities and that a rhizobial community found in one geographically distant site was highly divergent from the others. In conclusion, heavy metal levels in soils drive the taxonomic composition of Anthyllis- associated rhizobial populations according to their metal-tolerance phenotype but not their symbiotic nodA diversity. In addition to heavy metals, local soil physicochemical and topoclimatic conditions also impact the rhizobial beta diversity. Mesorhizobium communities were locally adapted and site specific, and their use is recommended for the success of phytostabilization strategies based on Mesorhizobium -legume vegetation. IMPORTANCE Phytostabilization of toxic mine spoils limits heavy metal dispersion and environmental pollution by establishing a sustainable plant cover. This eco-friendly method is facilitated by the use of selected and adapted cover crop legumes living in symbiosis with rhizobia that can stimulate plant growth naturally through biological nitrogen fixation. We studied microsymbiont partners of a metal-tolerant legume, Anthyllis vulneraria , which is tolerant to very highly metal-polluted soils in mining and nonmining sites. Site-specific rhizobial communities were linked to taxonomic composition and metal tolerance capacity. The rhizobial species Mesorhizobium metallidurans was dominant in all Zn-Pb mines but one. It was not detected in unpolluted sites where other distinct Mesorhizobium species occur. Given the different soil conditions at the respective mining sites, including their heavy-metal contamination, revegetation strategies based on rhizobia adapting to local conditions are more likely to succeed over the long term compared to strategies based on introducing less-well-adapted strains.

Genista saharae is an indigenous shrub legume that spontaneously grows in the northeastern Algerian Sahara. It is known for efficient dune fixation and soil preservation against desertification, due to its drought tolerance and its contribution to sustainable nitrogen resources implemented by biological N-2-fixation. In this study, the root nodule bacteria of G. saharae were investigated using phenotypic and phylogenetic characterization. A total of 57 rhizobial strains were isolated from nodules from several sites in the hyper-arid region of Metlili and Taibet (east Septentrional Sahara). They all nodulate G. saharae species but they differed in their symbiotic efficiency and effectiveness. The genetic diversity was assessed by sequencing three housekeeping genes (atpD, recA and 16S rRNA). The majority of isolates (81 %) belonged to the genus Ensifer (previously Sinorhizobium), represented mainly by the species Ensifer meliloti. The next most abundant genera were Neorhizobium (17 %) with 3 different species: N. alkalisoli, N. galegae and N. huautlense and Mesorhizobium (1.75 %) represented by the species M. camelthorni. Most of the isolated strains tolerated up to 4%(w/v) NaCl and grew at 45 degrees C. This study is the first report on the characterization of G. saharae microsymbionts in the Algerian Sahara.

Eight mesorhizobial symbiotic strains isolated from Anthyllis vulneraria root-nodules were studied and compared taxonomically with defined Mesorhizobium species. All strains presented identical 165 rDNA sequences but can be differentiated by multilocus sequence analysis of housekeeping genes (recA, atpD, glnll and dnaK). Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analyses separate these strains in two groups and a separate strain. Levels of DNA-DNA relatedness were less than 55% between representative strains and their closest Mesorhizobium reference relatives. The two groups containing four and three strains, respectively, originating from border mine and non-mining areas in Cevennes, were further phenotypically characterized. Groupings were further supported by average nucleotide identity values based on genome sequencing, which ranged from 80 to 92% with their close relatives and with each other, confirming these groups represent new Mesorhizobium species. Therefore, two novel species Mesorhizobium delmotii sp. nov. (type strain STM4623(T) = LMG 296401(T)= CFBP 84361(T) ) and Mesorhizobium prunaredense sp. nov. (type strain STM4891(T) = LMG 29641(T) = CFBP 8437(T)) are proposed. Type strains of the two proposed species share accessory common nodulation genes within the new symbiovar anthyllidis as found in the Mesorhizobium metallidurans type strain.

International audience; Type three secretion systems (T3SSs) mediate cell-to-cell interactions between Gram-negative bacteria and eukaryotes. We hypothesized that fluorescent pseudomonads harboring T3SS (T3SS+) would be beneficial to arbuscular mycorrhizal symbiosis because non-pathogenic fluorescent pseudomonads have been previously shown to be much more abundant in mycorrhizal than in non-mycorrhizal roots. We tested this hypothesis by comparing mycorrhization and the associated rhizosphere microbial communities of Medicago truncatula grown in a non-sterile soil inoculated with either the T3SS+ mycorrhiza helper bacterium Pseudomonas fluorescens (C7R12) or a T3SS- mutant of the strain. Results showed that the bacterial secretion system was responsible for the promotion of mycorrhization because root colonization by arbuscular mycorrhizal fungi was not promoted by the T3SS- mutant. The observed T3SS-mediated promotion of mycorrhization was associated with changes in the rhizosphere bacterial communities and the increased occurrence of Claroidoglomeraceae within the intraradical arbuscular mycorrhizal fungi. Furthermore, both pseudomonad strains promoted the host-free growth of a model arbuscular mycorrhizal fungus in vitro, suggesting that T3SS-mediated promotion of mycorrhization occurs during plant-fungal interactions rather than during the pre-symbiotic phase of fungal growth. Taken together, these data provide evidence for the involvement of T3SS in promoting arbuscular mycorrhization by a model fluorescent pseudomonad and suggest the implication of interactions between the bacterium and mycorrhizas.

Abandoned tailing basins and waste heaps of orphan mining sites are of great concern since extreme metal contamination makes soil improper for any human activity and is a permanent threat for nearby surroundings. Although spontaneous revegetation can occur, the process is slow or unsuccessful and rhizostabilisation strategies to reduce dispersal of contaminated dust represent an option to rehabilitate such sites. This requires selection of plants tolerant to such conditions, and optimization of their fitness and growth. Arbuscular mycorrhizal fungi (AMF) can enhance metal tolerance in moderately polluted soils, but their ability to survive extreme levels of metal contamination has not been reported. This question was addressed in the tailing basin and nearby waste heaps of an orphan mining site in southern France, reaching in the tailing basin exceptionally high contents of zinc (ppm: 97,333 total) and lead (ppm: 31,333 total). In order to contribute to a better understanding of AMF ecology under severe abiotic stress and to identify AMF associated with plants growing under such conditions, that may be considered in future revegetation and rhizostabilisation of highly polluted areas, nine plant species were sampled at different growing seasons and AMF root colonization was determined. Glomeromycota diversity was monitored in mycorrhizal roots by sequencing of the ribosomal LSU. This first survey of AMF in such highly contaminated soils revealed the presence of several AMF ribotypes, belonging mainly to the Glomerales, with some examples from the Paraglomerales and Diversisporales. AMF diversity and root colonization in the tailing basin were lower than in the less-contaminated waste heaps. A Paraglomus species previously identified in a polish mining site was common in roots of different plants. Presence of active AMF in such an environment is an outstanding finding, which should be clearly considered for the design of efficient rhizostabilisation processes.

Restoration of metalliferous mine soils requires using plant species tolerant to high metal concentrations and adapted to nutrient-poor soil. Legumes can increase plant productivity through N2-fixation, but they are often scarce in metalliferous sites. We examined survival, growth, and tolerance of four populations of a legume, Anthyllis vulneraria, from two metalliferous (MET) Zn-Pb mine sites, Avinieres (AV) ([Zn-EDTA] = 26,000 mg/kg) and Eylie (EY) ([Zn-EDTA] = 4,632 mg/kg), and two non-metalliferous (NMET) sites located in the south of France with the aim to select the most appropriate populations for restoration of mined soils. In a common garden experiment, plants from each population were reciprocally grown in soil from the provenance of each population. The two NMET populations exhibited high mortality and low growth rates in soil from the mined sites. The AV MET exhibited a high growth rate in metalliferous soils, but showed high mortality in non-metalliferous soils. The growth of the EY MET was very low in the AV-contaminated soil, but was the highest of all populations in moderately and non-metalliferous soils. Plants from the AV MET population showed a high growth and survival in metalliferous soil and would be appropriate in the restoration of metal-contaminated sites (>30,000 mg Zn kg−1). The EY MET population would be adapted to the restoration of moderate metal-contaminated soils (

International audience; Abandoned mines are a recurrent problem for nearby communities in Mediterranean regions because mine tailings represent a major source of polymetallic contamination. Metal contaminants are emitted in mining areas and dispersed by wind and water erosion in the surroundings. The goal of this literature review was to identify the specific features of polymetallic contamination arising from abandoned mines in the Mediterranean regions. Mediterranean climate conditions and local geochemical context are the most important factors that control the metal-bearing particle dispersion toward the different compartments of ecosystems. Acid mine drainage, as an important source of damage to the environment, is limited to a certain extent by the predominance of carbonate rocks in the Mediterranean regions. In opposite, aeolian contamination is specific to the semiarid conditions of the Mediterranean climate. In this context, impacts on different compartments such as agricultural soils and edible plants or human populations were underlined. The analysis of environmental laws and regulations of North and South Mediterranean countries shows that one of the main differences is the lack of identification and definition of mining waste as a public concern in the latter countries. In order to limit the transfer of contaminants from mining waste to the different components of the environment, phytostabilization of mine tailings was considered as the more adapted green technology even in the Mediterranean region where water access is limited. Finally, this review of polymetallic pollution from abandoned mines in Mediterranean regions enabled to identify priority actions for future research.

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.

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. Tolera...

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.

Nous avons effectue des inoculations ponctuelles sur le systeme racinaire de Medicago laciniata, cultivee in vitro La bacterie symbiotique Sinorhizobium meliloti et le champignon mycorhizien arbusculaire Glomus sp. S043 ont ete utilises. Nous avons etudie la reponse chimique du tissu racinaire en presence et en l'absence de la bacterie ou du champignon par observation en microscopie visible et a epi-fluorescence. L'inoculation ponctuelle de Sinorhizobium meliloti ou Glomus sp. S043 induit une accumulation de flavonoides localisee autour du point d'inoculation. Les autres zones de la racine ne presentent aucune accumulation de flavonoides. Il n'a ete observe d'accumulation de tannins ni chez le controle ni dans les racines inoculees. Le role de ces flavonoides est discute. (Resume d'auteur)

International audience; Plant growth‐promoting bacteria (PGPB) have been reported to stimulate root morphogenesis. To improve our knowledge of the PGPB effect, the early modifications of Brassica napus root system architecture induced by the PGPB Phyllobacterium sp. (29‐15) were analysed. Plants were grown in Petri dishes on a vertical medium supplemented with variable doses of Phyllobacterium sp. in gnotobiotic conditions. Root system elementary variables were measured in a nondestructive manner and the distribution of the bacteria throughout the primary root was quantified. Phyllobacterium sp. in doses from 3 × 107 to 3 × 108 colony‐forming units ml−1 significantly promoted B. napus total root length up to 50% by increasing both lateral root density throughout the primary root and growth rate of mature lateral roots. The primary root was progressively colonized by the bacteria from the tip to the base and the number of colonizing cells was positively correlated with the inoculum density. • Relationships between inoculum density, root colonization and root system architecture emphasized the relevance of this approach to specify PGPB effects on plants.

This work studied the effects of P fertilization on nodulation of field-grown soybean by two Bradyrhizobium strains (SMGS1 and THA7), and checked if differences between strains were consistent with bacterial growth and growth pouch nodulation ability in response to P availability. In the field, nodule dry weight and nitrogen fixation activity of inoculated soybean were studied on typical acid soils of Thailand at the flowering (R1) stage and at the end of grain filling. Grain yield, growth and phosphorus content were recorded. The bradyrhizobial strains were cultivated in culture medium, and growth parameters recorded. Nodulation patterns were observed during growth pouch experiments: infective root cells were inoculated with strains cultivated at two P concentrations in their culture media, namely 1 μM and 1 mM. Ten days after inoculation, the position of each nodule was measured relative to the root tip (RT) mark, expressed relative to the smallest emerging root hairs-RT distance in the nodulation frequency profile, and the consistency of responses was tested. In the field, on P deficient soils, dry weight of nodules was higher with Bradyrhizobium japonicum strain SMGS1 than with strain THA7. P supply increased the number and dry weight of nodules for both strains, with a higher dry weight response for THA7 than for SMGS1. It also had a positive effect on tissue phosphorus status and grain yield at R8 stage. In growth media, significant differences were recorded between strains under P-limiting conditions: The growth rate was higher for strain SMGS1, as well as the maximal number of bacterial cells supported. With growth pouch, inoculating plants with bacteria grown in P-deficient medium resulted in a less intense nodulation of roots by THA7, and with nodules appearing earlier on roots than in the case of SMGS1. At 1 mM P, there was no significant difference between strains. Thus, strain THA7 is more affected by P deficiency than strain SMGS1. Although P was not supplied in the same way in the soil and in the growth pouch experiments, this consistency of behaviour between work scales indicates that phosphorus availability is a key component for a successful inoculation. Furthermore, the study of bacterial growth rates and nodulation profile represents an interesting step for bacterial screening for low P soils. [-11pt]

Le sol, theâtre de nombreuses transformations initiees par des organismes vivants, voit son fonctionnement perturbe par les pratiques de l’agriculture intensive. C’est le cas notamment du cycle de l’azote qui est fortement modifie par les apports massifs de fertilisants azotes de synthese et ceci au detriment de la fixation biologique dont le potentiel est de moins en moins valorise. Globalement, l’utilisation de fertilisants chimiques et de pesticides a conduit a minimiser et a negliger le potentiel biologique des sols. L’evaluation de la qualite des sols devra desormais integrer la composante biotique et les mesures biologiques devront passer dans la pratique courante pour completer les analyses physico-chimiques traditionnelles. Une meilleure valorisation du potentiel naturel des sols passera tres certainement par une meilleure maitrise des interactions dans la rhizosphere, lieu privilegie de rencontre entre la plante et les micro-organismes du sol.

Mesorhizobium metallidurans STM 2683 T is a nitrogen-fixing bacterium that nodulates Anthyllis vulneraria in mine tailings highly contaminated in zinc, lead and cadmium. To study the mechanisms whereby this bacterium copes with metals, we functionally screened a cosmid genomic library of M. metallidurans for zinc or cadmium tolerance. A cosmid clone harbored a gene encoding P IB -type ATPase homologous to CadA that leads to cadmium and zinc resistance in Escherichia coli . The CadA protein structure presents one duplication of the two N-terminal metal binding domains (i.e. a heavy metal-associated domain followed by a histidine-rich domain) which allows specific binding to zinc and cadmium cations. A cadA -deleted strain of M. metallidurans failed to grow at high zinc concentrations (2 mM) and its growth was delayed at lower zinc concentrations. Expression studies using a transcriptional fusion of cadA promoter to gfp showed that cadA is specifically induced in a dose-dependent manner by zinc and cadmium in M. metallidurans in vitro conditions and into A. vulneraria nodules after Zn stress. Metal induction sensitivity was increased in the strain where cadA gene was deleted. This study identified cadA as a first mesorhizobial resistance determinant involved in detoxification of cadmium and zinc and which confers upon M. metallidurans greater capacity for coping with high zinc concentrations. This improves the knowledge of this bacterium for potential use as a symbiotic inoculant of Anthyllis in phytostabilization strategies of metal-rich sites.

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca. Methods Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using 15?N isotopic methods. Results Starter N fertilization (28 kg N ha?1) improved symbiotic N2 fixation and the growth of both species. Belowground N balance (N rhizodeposition - soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence. Conclusions i) soil N enrichment by the N2-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.

Symbiotic bacteria associated with the Medicago genus are separated into two closely related species named Sinorhizobium meliloti and Sinorhizobium medicae. To discriminate rapidly between these two bacterial species, two 15-base DNA probes, 16Smfs and 16Smed, were designed from the alignment of 16S rDNA sequences to differentiate S. meliloti from S. medicae. Their specificities were evaluated by dot-blot hybridization experiments on 25 reference strains representing 13 species of Rhizobium and Sinorhizobium, and by comparison with all 16S rDNA sequences available in the GenBank data base. No cross-reaction was found with 16Smed, which was thus considered species specific for S. medicae. By contrast, as expected according to the 16S rDNA sequence alignment, the labeled 16Smfs probe cross-hybridized with the DNAs of S. meliloti, Sinorhizobium fredii, and Sinorhizobium saheli but not with the DNA of S. medicae. Since S. saheli and S. fredii do not nodulate Medicago, 16Smed and 16Smfs can be routinely used to characterize the two Sinorhizobium species nodulating Medicago from pure cultures or from Medicago root nodules. Fifty strains isolated from eight annual Medicago species were then characterized by using colony hybridizations. Sinorhizobium meliloti was more frequently obtained (>80% isolates) than was S. medicae. Both Sinorhizobium species seemed to be trapped by annual Medicago and no plant-host specificity was detected.Key words: Sinorhizobium meliloti, Sinorhizobium medicae, Medicago, oligonucleotide probe, 16S rDNA gene.

The intraspecific genetic variability of Tuber aestivum was studied using molecular markers at various geographical scales. We used the polymerase chain reaction (PCR) coupled with restriction fragment length polymorphism (RFLP) analysis to examine the variation of the nuclear and mitochondrial ribosomal DNA (rDNA). RFLPs were found in the nuclear internal transcribed spacer (ITS) and three alleles were detected in the six populations analysed. No variability was found in mitochondrial rDNA. We found, in a very few cases, that truffles sharing different ITS genotypes could be present within a single symbiotic tree.

Summary Induction by Rhizobium bacteria of symbiotic nitrogen-fixing root nodules on legume plants is a host-specific process. The use of a reporter gene, whose transcription can be detected easily by assaying for s-galactosidase activity allowed quickly description of inducers in seed extracts and root exudates of various legumes. The transcription of nodulation (nod) genes in the symbiotic bacteria is induced by particular flavonoid signals, mainly flavones and isoflavones. The amounts, the diversity and the activity of the flavonoids interacting with products from nodD genes determine specificity and intensity of the nodulation. Flavonoids are molecules active at low concentrations and their technological use open new field to control plant-microbe interactions in the rhizosphere.

This work examines Zn accumulation in four Anthyllis vulneraria subspecies supplemented with mineral nitrogen or grown in the presence of their symbiotic bacteria. Anthyllis vulneraria subspecies were grown hydroponically in the presence of high levels of ZnSO4. The plants were either grown in symbiosis with one of two non-metallicolous or metallicolous Mesorhizobium inoculants or in the presence of KNO3. When exposed to 1,000 mu M Zn, shoot and root biomass of three out of our four Anthyllis subspecies cultivated with NO3 dropped significantly by about 24-28 %; carpatica, the fourth subspecies, was not affected. Subspecies carpatica Zn tolerance was confirmed when in symbiosis with the metallicolous strain. In the presence of 1,000 mu M Zn, the different Anthyllis subspecies concentrated more Zn in their roots than in their shoots and only subsp. carpatica accumulated a significant amount of Zn in its shoots. The most remarkable feature was the drastic decrease in Zn concentration in both roots (up to 2.5-3 fold) and shoots (2.6-fold) of subsp. carpatica exposed to 1,000 mu M Zn and nodulated whatever the Mesorhizobium strain used, compared to the N-grown plants. Our results bring new perspectives as regards phytostabilization, with the potential use of a rhizobium-inoculated leguminous subspecies displaying unusual Zn tolerance.

Background Mesorhizobium metallidurans STM 2683T and Mesorhizobium sp. strain STM 4661 were isolated from nodules of the metallicolous legume Anthyllis vulneraria from distant mining spoils. They tolerate unusually high Zinc and Cadmium concentrations as compared to other mesorhizobia. This work aims to study the gene expression profiles associated with Zinc or Cadmium exposure and to identify genes involved in metal tolerance in these two metallicolous Mesorhizobium strains of interest for mine phytostabilization purposes. Results The draft genomes of the two Mezorhizobium strains were sequenced and used to map RNAseq data obtained after Zinc or Cadmium stresses. Comparative genomics and transcriptomics allowed the rapid discovery of metal-specific or/and strain-specific genes. Respectively 1.05% (72/6,844) and 0.97% (68/6,994) predicted Coding DNA Sequences (CDS) for STM 2683 and STM 4661 were significantly differentially expressed upon metal exposure. Among these, a significant number of CDS involved in transport (13/72 and 13/68 for STM 2683 and STM 4661, respectively) and sequestration (15/72 and 16/68 for STM 2683 and STM 4661, respectively) were identified. Thirteen CDS presented homologs in both strains and were differentially regulated by Zinc and/or Cadmium. For instance, several PIB-type ATPases and genes likely to participate in metal sequestration were identified. Among the conserved CDS that showed differential regulation in the two isolates, we also found znuABC homologs encoding for a high affinity ABC-type Zinc import system probably involved in Zinc homeostasis. Additionally, global analyses suggested that both metals also repressed significantly the translational machinery. Conclusions The comparative RNAseq-based approach revealed a relatively low number of genes significantly regulated in the two Mesorhizobium strains. Very few of them were involved in the non-specific metal response, indicating that the approach was well suited for identifying genes that specifically respond to Zinc and Cadmium. Among significantly up-regulated genes, several encode metal efflux and sequestration systems which can be considered as the most widely represented mechanisms of rhizobial metal tolerance. Downstream functional studies will increase successful phytostabilization strategies by selecting appropriate metallicolous rhizobial partners.

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 rigiduloides 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. rigiduloides, 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. rigiduloides 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.

The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range. The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis. The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA. We identified a total of five genomic species, including the previously described species Rhizobium ciceri. All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R. ciceri. Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles. The complete 16S rRNA sequences of strains representing the two major groups, R. ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches. Both of these strains belonged to the Rhizobium loti-Rhizobium huakuii branch; R. ciceri UPM-Ca7T was closely related to R. loti, and strain UPM-Ca36T was clearly separated from R. ciceri and closely related to R. huakuii. Thus, genomic species 2 could be distinguished from R. ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics. Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain.

International audience; 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)). (C) 2011 Elsevier GmbH. All rights reserved.

International audience; Responses of the Medicago truncatula-Sinorhizobium interaction to variation in N₂-fixation of the bacterial partner were investigated. * Split-root systems were used to discriminate between local responses, at the site of interaction with bacteria, and systemic responses related to the whole plant N status. * The lack of N acquisition by a half-root system nodulated with a nonfixing rhizobium triggers a compensatory response enabling the other half-root system nodulated with N₂-fixing partners to compensate the local N limitation. This response is mediated by a stimulation of nodule development (number and size) and involves a systemic signaling mechanism related to the plant N demand. In roots co-infected with poorly and highly efficient strains, partner choice for nodule formation was not modulated by the plant N status. However, the plant N demand induced preferential expansion of nodules formed with the most efficient partners when the symbiotic organs were functional. The response of nodule expansion was associated with the stimulation of symbiotic plant cell multiplication and of bacteroid differentiation. * A general model where local and systemic N signaling mechanisms modulate interactions between Medicago truncatula and its Sinorhizobium partners is proposed.

The excessive concentrations of toxic heavy metals in mine tailings and their very low N content make soil reclamation strategies by phytostabilization difficult. Our objective was to test if the symbiotic association between the legume Anthyllis vulneraria subsp. carpatica and the bacteria Mesorhizobium metallidurans originating from highly polluted mine tailings is able to increase N concentration in soils with contrasting Zn, Pb and Cd contents. Plants of A. vulneraria subsp. carpatica from a mine site and of a non-metallicolous subsp. praeopera from non-polluted soil were inoculated with a metallicolous or a non-metallicolous compatible Mesorhizobium spp. and grown on low and high heavy metal-contaminated soils. In contaminated soil, many nodules were observed when the metallicolous A. vulneraria was inoculated with its rhizobium species M. metallidurans, whereas the non-metallicolous A. vulneraria died after a few weeks regardless of the rhizobium inoculant. Eighty percent of the total nitrogen was derived from biological nitrogen fixation through the association between metallicolous A. vulneraria and the rhizobium grown on metal-enriched soil. The ability of the metallicolous A. vulneraria to develop a high nitrogen fixing potential opens new possibilities for promoting a low-maintenance plant cover and for stabilizing the vegetation in high heavy metal-contaminated soils.

Mining activities generate spoils and effluents with extremely high metal concentrations of heavy metals that might have adverse effects on ecosystems and human health. Therefore, information on soil and plant metal concentrations is needed to assess the severity of the pollution and develop a strategy for soil reclamation such as phytoremediation. Here, we studied soils and vegetation in three heavily contaminated sites with potential toxic metals and metalloids (Zn, Pb, Cd, As, TI) in the mining district of Les Malines in the Languedoc region (southern France). Extremely high concentrations were found at different places such as the Les Avini,res tailing basins (up to 160,000 mg kg(-1) Zn, 90,000 mg kg(-1) Pb, 9,700 mg kg(-1) of As and 245 mg kg(-1) of Tl) near a former furnace. Metal contamination extended several kilometres away from the mine sites probably because of the transport of toxic mining residues by wind and water. Spontaneous vegetation growing on the three mine sites was highly diversified and included 116 plant species. The vegetation cover consisted of species also found in non-contaminated soils, some of which have been shown to be metal-tolerant ecotypes (Festuca arvernensis, Koeleria vallesiana and Armeria arenaria) and several Zn, Cd and Tl hyperaccumulators such as Anthyllis vulneraria, Thlaspi caerulescens, Iberis intermedia and Silene latifolia. This latter species was highlighted as a new thallium hyperaccumulator, accumulating nearly 1,500 mg kg(-1). These species represent a patrimonial interest for their potential use for the phytoremediation of toxic metal-polluted areas., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; 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 2640T. 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 2683T (=CFBP 7147T=LMG 24485T).

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.

International audience; Phyllobacterium brassicacearum is a plant growth promoting bacterium (PGPB) that stimulates Brassica napus root morphogenesis in absence of bacterial competition. We evaluated in which extent indigenous bacteria associated with B. napus roots and rhizosphere may interfere with the plant growth promoting ability of P. brassicacearum. Thus, bacteria from B. napus roots and rhizosphere were isolated and co-inoculated with the PGPB at different proportions. The root growth promoting ability of P. brassicacearum was impaired only when it was co-inoculated in low proportion together with the indigenous bacteria. This was not linked to a decrease in its population size on roots. Hence, this study highlights that the efficacy of a PGPB is not primarily dependent on the extent of its root colonization but also depends on its initial size compared to the indigenous bacteria.

National audience; Plant symbioses are fundamental for ecosystem stability and sustainability. Introduction of legumes associated with microorganisms efficient in nitrogen fixation and promoting nutrient biodisponibility in limited-nutrient degraded sites is a key step for a successful plant establishment in stress situations such as those encountered in Mediterranean soils. A vegetation strategy based on microbial engineering of locally-adapted plants was investigated and tested in field conditions within the framework of calcareous quarry rehabilitation. Choice of symbiotic plants requires, first, a prospection of native legume species growing in relatively degraded sites in Mediterranean areas and easy to multiply, and second, an identification and production of efficient rhizobial partners. Plants produced in nursery and then transplanted into the quarry showed a good capacity for establishment after three years. Shrub species performed best with spontaneous mycorrhizal association and ability to enrich topsoil with organic matter; Les symbioses végétales sont une composante fondamentale de la stabilité et de la durabilité des écosystèmes. L’introduction de légumineuses associées à leurs auxiliaires microbiens fixateurs d’azote ou améliorant la biodisponibilité d’éléments nutritifs, dans des sites dégradés fortement appauvris en éléments nutritifs, est une condition primordiale pour réussir l’installation de plants en milieu particulièrement contraignant, comme le sont les sites de carrière de calcaire après exploitation sous climat méditerranéen. Une stratégie de végétalisation basée sur l’ingénierie microbiologique de plants adaptés aux conditions locales, a été élaborée puis testée sur le terrain en grandeur nature dans le cas particulier de carrières de granulats calcaires. Le choix des plants symbiotiques utilisables pour la végétalisation repose, d’une part, sur la prospection d’espèces de légumineuses repérées sur des sites assez dégradés en milieu méditerranéen, faciles à multiplier et, d’autre part, sur l’identification et la production de rhizobiums partenaires efficaces. Ces plants produits en pépinière puis transférés sur le terrain présentent après trois années une bonne capacité de croissance. Les espèces arbustives sont plus particulièrement performantes avec une capacité spontanée à s’associer aux mycorhizes et à favoriser l’enrichissement en matière organique de la surface du substrat.

Both root architecture and plant N nutrition are altered by inoculation with the plant growth-promoting rhizobacteria (PGPR) Phyllobacterium strain STM196. It is known that NO 3 − and N metabolites can act as regulatory signals on root development and N transporters. In this study, we investigate the possible interrelated effects on root development and N transport. We show that the inhibition of Arabidopsis lateral root growth by high external NO 3 − is overridden by Phyllobacterium inoculation. However, the leaf NO 3 − pool remained unchanged in inoculated plants. By contrast, the Gln root pool was reduced in inoculated plants. Unexpectedly, NO 3 − influx and the expression levels of AtNRT1.1 and AtNRT2.1 genes coding for root NO 3 − transporters were also decreased after 8 days of Phyllobacterium inoculation. Although the mechanisms by which PGPR exert their positive effects remain unknown, our data show that they can optimize plant development independently from N supply, thus alleviating the regulatory mechanisms that operate in axenic conditions. In addition, we found that Phyllobacterium sp. elicited a very strong induction of AtNRT2.5 and AtNRT2.6, both genes preferentially expressed in the shoots whose functions are unknown.

International audience; We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega of- ficinalis and G. orientalis in northern regions so far

International audience; Thirteen of the most abundant Gram-negative bacteria which are able to grow in N-depleted culture conditions were isolated from the rhizoplane and endorhizosphere of canola (Brassica napus) and identified by 16S rDNA sequence analysis. Eight of these bacteria induced a significant increase in root dry weight ranging from 11 to 52%. Phylogenetic positioning based on 16S rDNA sequences indicated that at least four genera are represented, Pseudomonas, Variovorax, Agrobacterium and Phyllobacterium. The most important direct plant growth-promoting-rhizobacteria effect was found with both isolates belonging to the Phyllobacterium.

International audience

International audience; Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization.

summary Isozyme variation and somatic incompatibility were investigated in order to study intraspecific genetic variation and to identify genets in natural populations of the ectomycorrhizal fungus Suillus collinitus (Fr.) O. Kuntze, Forty-three isolates, obtained from basidiocarps collected in 11 forest sites under Pinus trees, were examined. Isozyme analysis indicated high intraspecific variation between isolates from the same location and from different locations. Phenetic analysis based on calculated dissimilarity values derived from isozyme banding patterns separated the isolates into two main groups (I and II) with 35% dissimilarity. A specific and highly active acid phosphatase isoform (ACP-2) was detected in most group II isolates. Dikaryotic isolates displaying isozyme similarity coefficient of 100 % were found to be somatically compatible and were considered to originate from the same genet. The same genet was never found at more than two forest sites. This study showed a high correlation between the results of electrophoretic isozyme analysis and those of somatic incompatibility reactions. The results were discussed in terms of population biology.

The taxonomic position of isolates of a new genomic species (designated genomic species 2) obtained from several annual Medicago species and originating from different geographical locations was established through the results of phenotypic tests (including the results of auxanographic and biochemical tests and symbiotic properties) and 16S rRNA phylogenetic inferences. A comparison of the complete 16S rRNA sequence of a representative of genomic species 2 (strain A 321T [T = type strain]) with the 16S rRNA sequences of other members of the Rhizobiaceae and closely related taxa showed that genomic species 2 was phylogenetically related to Sinorhizobium meliloti, Sinorhizobium fredii, Sinorhizobium saheli, and Sinorhizobium teranga. The levels of sequence similarity and observed numbers of nucleotide substitutions in Sinorhizobium strains indicated that A 321T and S. meliloti exhibited the highest level of sequence similarity (99.7%), with four nucleotide substitutions and one deletion. The results of a numerical analysis based on data from 63 auxanographic and biochemical tests clearly separated genomic species 2 isolates from S. meliloti. Genomic species 2 isolates nodulated and fixed nitrogen with Medicago polymorpha, whereas S. meliloti isolates were ineffective and formed rudimentary nodules on this host plant. On the basis of phenotypic and 16S sequence analysis data, genomic species 2 isolates cannot be assigned to a previously described species. We propose that these isolates belong to a new species, Sinorhizobium medicae.

The ureide assay method has been found to be suitable for soybean (Glycine max) to assess nitrogen ixation in a wide range of genotypes and Bradyrhizobium strains. The relationships between the relative abundance of ureide-N in xylem-sap and the proportion of plant N derived from nitrogen fixation were studied for early maturing soybeans grown under greenhouse and field conditions. Plants grown under greenhouse conditions were either supplemented with N-free nutrients or supplied with different levels of 15N-nitrate. In the field, soybeans were grown with or without inoculation, in the presence or absence of nitrogen fertilizer. Treatments were applied in order to obtain a wide range of nitrogen nutrition levels. The effect of the source and level of nitrogen nutrition on growth and nitrogen accumulation in plants, proportion of fixed nitrogen in the xylem-sap and in the shoots, and the proportions of N-compounds in the xylem-sap was determined. The greenhouse study demonstrated that the relative...

International audience; The competitiveness of two Bradyrhizobium japonicum strains was determined under controlled conditions. The relationship between the ratio of the nodules formed by the two bradyrhizobial strains and the ratio of the two strains in the inoculum was established and used as an internal standard to evaluate the equilibrium ratio between populations of the two strains in the soil. The fluorescent antibody (FA) technique was also used for counting the bradyrhizobial populations directly in the soil. Results with the FA technique were analysed and compared with the estimate obtained through nodule occupancy. The initial ratio of the two bradyrhizobial strains in populations introduced simultaneously into the soil changed during incubation time and the two strains reached the same equilibrium levels after 53 days. Never-theless, the strain introduced into the soil as an overpopulation remained clearly dominant for the nodule occupancy. We conclude that direct counts with the FA approach are selective and these counts could be mainly of the unattached bacteria.

National audience

The taxonomic status of 16 collection strains of chickpea (Cicer arietinum L.) rhizobia which were previously determined to belong to two groups (groups A and B) were compared with reference strains belonging to different genera and species of the family Rhizobiaceae. We used the following taxonomic, phylogenetic, and phenotypic characteristics and approaches to study these organisms: DNA homology, guanine-plus-cytosine content, restriction fragment length polymorphism of the amplified 16S-intergenic spacer rRNA gene, partial 16S rRNA sequencing, and auxanographic tests performed with 147 carbon sources. Similar groups of chickpea strains were identified by the different approaches. The chickpea strains were found to belong to the genus Rhizobium regardless of the phylogenetic group to which they belonged (group A or B). All strains fell into a tight cluster which included Rhizobium loti and Rhizobium galegae, and the group B strains were closely related to R. loti. An analysis of partial 16S ribosomal DNA sequences revealed identical nucleotide sequences for the slowly growing strains and fast-growing strains that were used as representatives of groups A and B, respectively, and these organisms fell into the Rhizobium-Agrobacterium lineage. When the sequences of these organisms were compared with the partial sequences of Rhizobium huakuii and R. loti, one- and two-nucleotide mismatches were observed, respectively, indicating that the chickpea rhizobia are closely related to these two species. The DNA-DNA hybridization data revealed that the chickpea rhizobia exhibited low levels of homology (less than 17%) to previously described Rhizobium and Bradyrhizobium species. Moreover, when we compared chickpea strains to R. loti and R. huakuii, the most closely related species as determined by the partial 16S rRNA sequence analysis, the homology values ranged from 21 to 52% and the delta Tm values were greater than 5 degrees C (delta Tm is the difference between the denaturation temperatures of the heterologous and homologous duplexes). These results confirmed that the rhizobia that nodulate chickpeas cannot be assigned to a previously described species. Within the chickpea rhizobia, the DNA homology values obtained when members of groups A and B were compared were less than 38%, indicating that the group A and group B organisms belong to different species. Furthermore, these organisms can be distinguished from each other by the results of phenotypic tests. We propose that the group B chickpea rhizobia should be assigned to a new species, Rhizobium ciceri; UPM-Ca7 is the type strain of R. ciceri.

Summary In the mediterranean region, the strong edaphic, climatic and economic constraints lead to the increase of the transplantation crisis of young trees. The use of controlled mycorrhizal infection is one of the means open to reduce the effects of this crisis by improving the quality of the forest trees grown in nursery, and the reforestation. To achieve this objective, a research/development programme was started and preliminary results are presented: screening of mycorrhizal isolates using physiological criteria; controlled mycorrhizal infection including the study of enzymic polymorphism in mycorrhizae as a tool for characterization of these organs; measurement of the effects of mycorrhizal infection on the nutrition of trees in nursery; ecophysiological study of experimental wood lots.

An active proteolysed isoform of apple pulp polyphenoloxidase (PPO) was purified by a very quick three-step method based on its resistance to further sodium dodecyl sulphate-proteinase K digestion. After extraction from a thylakoid membrane pellet and pre-purification by temperature-induced phase partitioning, PPO was subjected to sodium dodecyl sulphate-proteinase K digestion, and then 388-fold to homogeneity purified by DEAE-cellulose column chromatography with a yield greater than 40%. This purified, enzymatically active PPO isoform was used to raise polyclonal antibodies. High titred specific serum was obtained and immunoblots were performed to detect active and latent forms of the enzyme.

The diversity of 16 strains of chickpea-infective rhizobia from various geographical origins was analysed using genotypic and phenotypic approaches. Multilocus enzyme electrophoresis was performed, and restriction fragment length polymorphisms of the amplified 16S+IGS (intergenic spacer) rRNA gene, assimilation of 147 carbon sources, antibiotic resistance, and tolerance to NaCl and extreme pH values and temperatures were tested. These approaches had different discriminating powers. Esterase polymorphisms gave a unique pattern for each strain, allowing this method to be used for strain fingerprinting. Genetic distances between strains were estimated. The three approaches used in this study yielded consistent results. They evidenced high heterogeneity among the strains, and made it possible to classify the strains into two clusters. Isozyme patterns for superoxide dismutase were particularly interesting, since they delineated the same two groups. The phenotypic tests clearly confirmed the existence of two genetic groups on the basis of 11 phenotypic characters. Owing to the large phylogenetic distance between the two groups of strains, the taxonomic status of chickpea-infective strains is discussed.Key words: Rhizobium sp. (Cicer arietinum L.), genetic diversity, multilocus enzyme electrophoresis, restriction fragment length polymorphisms, phenotypic diversity.

ACC oxidase from Golden delicious apple fruits has been purified 170-fold to homogeneity in a 5-step procedure and some of its kinetic parameters have been determined. Antibodies raised against a recombinant ACC oxidase from pTOM13 cDNA recognise enzyme of apple fruit with high specificity. The enzyme appears to be non glycosylated. Evidence is given by various methods, including immunocytology, that most of ACC oxidase of ripening fruits was located in the aploplasm.