Pseudomonas syringae isolated in lichens for the first time: Unveiling Peltigera genus as the exclusive host.

Pseudomonas syringae is a bacterial complex that is widespread through a range of environments, typically associated with plants where it can be pathogenic, but also found in non-plant environments such as clouds, precipitation, and surface waters. Understanding its distribution within the environment, and the habitats it occupies, is important for examining its evolution and understanding behaviours. After a recent study found P. syringae living among a range of vascular plant species in Iceland, we questioned whether lichens could harbour P. syringae. Sixteen different species of lichens were sampled all over Iceland, but only one lichen genus, Peltigera, was found to consistently harbour P. syringae. Phylogenetic analyses of P. syringae from 10 sampling points where lichen, tracheophyte, and/or moss were simultaneously collected showed significant differences between sampling points, but not between different plants and lichens from the same point. Furthermore, while there were similarities in the P. syringae population in tracheophytes and Peltigera, the densities in Peltigera thalli were lower than in moss and tracheophyte samples. This discovery suggests P. syringae strains can localize and survive in organisms beyond higher plants, and thus reveals opportunities for studying their influence on P. syringae evolution.

Groundwater promotes emergence of asporogenic mutants of emetic Bacillus cereus.

Bacillus cereus is a ubiquitous endospore-forming bacterium, which mainly affects humans as a food-borne pathogen. Bacillus cereus can contaminate groundwater used to irrigate food crops. Here, we examined the ability of the emetic strain B. cereus F4810/72 to survive abiotic conditions encountered in groundwater. Our results showed that vegetative B. cereus cells rapidly evolved in a mixed population composed of endospores and asporogenic variants bearing spo0A mutations. One asporogenic variant, VAR-F48, was isolated and characterized. VAR-F48 can survive in sterilized groundwater over a long period in a vegetative form and has a competitive advantage compared to its parental strain. Proteomics analysis allowed us to quantify changes to cellular and exoproteins after 24 and 72 h incubation in groundwater, for VAR-F48 compared to its parental strain. The results revealed a significant re-routing of the metabolism in the absence of Spo0A. We concluded that VAR-F48 maximizes its energy use to deal with oligotrophy, and the emergence of spo0A-mutated variants may contribute to the persistence of emetic B. cereus in natural oligotrophic environments.

The Pseudomonas viridiflava phylogroups in the P. syringae species complex are characterized by genetic variability and phenotypic plasticity of pathogenicity-related traits.

As a species complex, Pseudomonas syringae exists in both agriculture and natural aquatic habitats. P.viridiflava, a member of this complex, has been reported to be phenotypically largely homogenous. We characterized strains from different habitats, selected based on their genetic similarity to previously described P.viridiflava strains. We revealed two distinct phylogroups and two different kinds of variability in phenotypic traits and genomic content. The strains exhibited phase variation in phenotypes including pathogenicity and soft rot on potato. We showed that the presence of two configurations of the Type III Secretion System [single (S-PAI) and tripartite (T-PAI) pathogenicity islands] are not correlated with pathogenicity or with the capacity to induce soft rot in contrast to previous reports. The presence/absence of the avrE effector gene was the only trait we found to be correlated with pathogenicity of P.viridiflava. Other Type III secretion effector genes were not correlated with pathogenicity. A genomic region resembling an exchangeable effector locus (EEL) was found in S-PAI strains, and a probable recombination between the two PAIs is described. The ensemble of the variability observed in these phylogroups of P.syringae likely contributes to their adaptability to alternating opportunities for pathogenicity or saprophytic survival.

Pseudomonas syringae on Plants in Iceland Has Likely Evolved for Several Million Years Outside the Reach of Processes That Mix This Bacterial Complex across Earth's Temperate Zones.

Here we report, for the first time, the occurrence of the bacteria from the species complex Pseudomonas syringae in Iceland. We isolated this bacterium from 35 of the 38 samples of angiosperms, moss, ferns and leaf litter collected across the island from five habitat categories (boreal heath, forest, subalpine and glacial scrub, grazed pasture, lava field). The culturable populations of P. syringae on these plants varied in size across 6 orders of magnitude, were as dense as 107 cfu g-1 and were composed of strains in phylogroups 1, 2, 4, 6, 7, 10 and 13. P. syringae densities were significantly greatest on monocots compared to those on dicots and mosses and were about two orders of magnitude greater in grazed pastures compared to all other habitats. The phylogenetic diversity of 609 strains of P. syringae from Iceland was compared to that of 933 reference strains of P. syringae from crops and environmental reservoirs collected from 27 other countries based on a 343 bp sequence of the citrate synthase (cts) housekeeping gene. Whereas there were examples of identical cts sequences across multiple countries and continents among the reference strains indicating mixing among these countries and continents, the Icelandic strains grouped into monophyletic lineages that were unique compared to all of the reference strains. Based on estimates of the time of divergence of the Icelandic genetic lineages of P. syringae, the geological, botanical and land use history of Iceland, and atmospheric circulation patterns, we propose scenarios whereby it would be feasible for P. syringae to have evolved outside the reach of processes that tend to mix this bacterial complex across the planet elsewhere.

Comparison of Environmental and Culture-Derived Bacterial Communities through 16S Metabarcoding: A Powerful Tool to Assess Media Selectivity and Detect Rare Taxa.

To compare environmental and culture-derived microbial communities, we performed 16S metabarcoding of uncultured samples and their culture-derived bacterial lawns. Microbial communities were obtained from freshwater river samples representative of an anthropization gradient along a river stream. Their culture-derived bacterial lawns were obtained by growing aliquots of the samples on a broad range medium and on two different semi-selective media. The V3-V4 16S rRNA region was amplified and sequenced. The bacterial diversity of water samples decreased from the upper to lower stream sampling sites and, as expected, these differences were mostly suppressed by the culture step. Overall, the diversity of cultured-derived bacterial communities reflected selectivity of each tested medium. Comparison of treatments indicated that the culture selected both detected and rare undetected environmental species. Accurate detection of rare environmental bacteria of the Pectobacterium genus by 16S metabarcoding of the culture lawn was demonstrated. Interestingly, for abundant taxa, such as those of the Pseudomonas genus, the culture/environment ratio varied between sampled sites, indicating the difficulty of comparing cultured-derived taxa abundance between environmental sites. Finally, our study also highlighted media specificity and complementarity: bacterial communities grown on the two selective media, while selecting a small set of specific species, were mostly a subset of the bacterial community observed on the broad range medium.

Diversity of pathogenic Pseudomonas isolated from citrus in Tunisia.

The damages observed in Tunisian citrus orchards have prompted studies on the Pseudomonas spp. responsible for blast and black pit. Prospective orchards between 2015 and 2017 showed that the diseases rapidly spread geographically and to new cultivars. A screening of Pseudomonas spp. isolated from symptomatic trees revealed their wide diversity according to phylogenetic analysis of their housekeeping rpoD and cts genes. The majority of strains were affiliated to Pseudomonas syringae pv. syringae (Phylogroup PG02b), previously described in Tunisia. However, they exhibited various BOX-PCR fingerprints and were not clonal. This work demonstrated, for the first time in Tunisia, the involvement of Pseudomonas cerasi (PG02a) and Pseudomonas congelans (PG02c). The latter did not show significant pathogenicity on citrus, but was pathogenic on cantaloupe and active for ice nucleation that could play a role in the disease. A comparative phylogenetic study of citrus pathogens from Iran, Montenegro and Tunisia revealed that P. syringae (PG02b) strains are closely related but again not clonal. Interestingly P. cerasi (PG02a) was isolated in two countries and seems to outspread. However, its role in the diseases is not fully understood and it should be monitored in future studies. The diversity of pathogenic Pseudomonas spp. and the extension of the diseases highlight that they have become complex and synergistic. It opens questions about which factors favor diseases and how to fight against them efficiently and with sustainable means.

New species of pathogenic Pseudomonas isolated from citrus in Tunisia: Proposal of Pseudomonas kairouanensis sp. nov. and Pseudomonas nabeulensis sp. nov.

A collection of Pseudomonas strains was isolated in different regions of Tunisia in the period 2016-2017 from the fruits and leaves of Citrus sinensis cv. 'Valencia Late' and Citrus limon cv. 'Eureka' plants with symptoms of blast and black pit disease. A phylogenetic analysis of the housekeeping gene rpoD was used for strain identification at the species level. The results demonstrated the affiliation of these strains with the genus Pseudomonas and revealed the presence of 11 strains representing two putative new species in two monophyletic branches. These strains were analyzed morphologically and genotypically by multilocus sequence analyses of the rpoD, gyrB and 16S rRNA (rrs) gene sequences, and their phenotypic characteristics by API 20NE and Biolog GEN III. Plant pathogenic properties were confirmed on fruits and detached leaves of C. limon cv. 'Eureka'. Fatty acids and WC MALDI-TOF MS major protein profiles were determined. The genomes of both representatives were sequenced. The average nucleotide index and genome-to-genome distance from KC12T and E10BT are below the cut-off established for a described species. These results support the conclusion that the strains KC12T, KC17, KC20, KC22, KC24A, KC25 and KC26 represent a novel species of Pseudomonas, for which the name of Pseudomonas kairouanensis is proposed. The type strain is KC12T (=CECT9766 and CFBP 8662). The strains E10BT, E10AB, E10CB1 and Iy3BA represent another novel species of Pseudomonas for which the name of Pseudomonas nabeulensis is proposed; the type strain is E10BT (=CECT9765 and CFBP 8661).

Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999.

Research interest in microbial biodiversity over the past 25 years has increased markedly as microbiologists have become interested in the significance of biodiversity for ecological processes and as the industrial, medical, and agricultural applications of this diversity have evolved. One major challenge for studies of microbial habitats is how to account for the diversity of extremely large and heterogeneous populations with samples that represent only a very small fraction of these populations. This review presents an analysis of the way in which the field of microbial biodiversity has exploited sampling, experimental design, and the process of hypothesis testing to meet this challenge. This review is based on a systematic analysis of 753 publications randomly sampled from the primary scientific literature from 1975 to 1999 concerning the microbial biodiversity of eight habitats related to water, soil, plants, and food. These publications illustrate a dominant and growing interest in questions concerning the effect of specific environmental factors on microbial biodiversity, the spatial and temporal heterogeneity of this biodiversity, and quantitative measures of population structure for most of the habitats covered here. Nevertheless, our analysis reveals that descriptions of sampling strategies or other information concerning the representativeness of the sample are often missing from publications, that there is very limited use of statistical tests of hypotheses, and that only a very few publications report the results of multiple independent tests of hypotheses. Examples are cited of different approaches and constraints to experimental design and hypothesis testing in studies of microbial biodiversity. To prompt a more rigorous approach to unambiguous evaluation of the impact of microbial biodiversity on ecological processes, we present guidelines for reporting information about experimental design, sampling strategies, and analyses of results in publications concerning microbial biodiversity.

The exopolysaccharide of Rhizobium sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus roots but contributes to root colonization.

Microbial exopolysaccharides (EPSs) play key roles in plant-microbe interactions, such as biofilm formation on plant roots and legume nodulation by rhizobia. Here, we focused on the function of an EPS produced by Rhizobium sp. YAS34 in the colonization and biofilm formation on non-legume plant roots (Arabidopsis thaliana and Brassica napus). Using random transposon mutagenesis, we isolated an EPS-deficient mutant of strain YAS34 impaired in a glycosyltransferase gene (gta). Wild type and mutant strains were tagged with a plasmid-born GFP and, for the first time, the EPS produced by the wild-type strain was seen in the rhizosphere using selective carbohydrate probing with a fluorescent lectin and confocal laser-scanning microscopy. We show for the fist time that Rhizobium forms biofilms on roots of non-legumes, independently of the EPS synthesis. When produced by strain YAS34 wild type, EPS is targeted at specific parts of the plant root system. Nutrient fluctuations, root exudates and bacterial growth phase can account for such a production pattern. The EPS synthesis in Rhizobium sp. YAS34 is not essential for biofilm formation on roots, but is critical to colonization of the basal part of the root system and increasing the stability of root-adhering soil. Thus, in Rhizobium sp. YAS34 and non-legume interactions, microbial EPS is implicated in root-soil interface, root colonization, but not in biofilm formation.

Frontiers for research on the ecology of plant-pathogenic bacteria: fundamentals for sustainability: Challenges in Bacterial Molecular Plant Pathology.

Methods to ensure the health of crops owe their efficacy to the extent to which we understand the ecology and biology of environmental microorganisms and the conditions under which their interactions with plants lead to losses in crop quality or yield. However, in the pursuit of this knowledge, notions of the ecology of plant-pathogenic microorganisms have been reduced to a plant-centric and agro-centric focus. With increasing global change, i.e. changes that encompass not only climate, but also biodiversity, the geographical distribution of biomes, human demographic and socio-economic adaptations and land use, new plant health problems will emerge via a range of processes influenced by these changes. Hence, knowledge of the ecology of plant pathogens will play an increasingly important role in the anticipation and response to disease emergence. Here, we present our opinion on the major challenges facing the study of the ecology of plant-pathogenic bacteria. We argue that the discovery of markedly novel insights into the ecology of plant-pathogenic bacteria is most likely to happen within a framework of more extensive scales of space, time and biotic interactions than those that currently guide much of the research on these bacteria. This will set a context that is more propitious for the discovery of unsuspected drivers of the survival and diversification of plant-pathogenic bacteria and of the factors most critical for disease emergence, and will set the foundation for new approaches to the sustainable management of plant health. We describe the contextual background of, justification for and specific research questions with regard to the following challenges: Development of terminology to describe plant-bacterial relationships in terms of bacterial fitness. Definition of the full scope of the environments in which plant-pathogenic bacteria reside or survive. Delineation of pertinent phylogenetic contours of plant-pathogenic bacteria and naming of strains independent of their presumed life style. Assessment of how traits of plant-pathogenic bacteria evolve within the overall framework of their life history. Exploration of possible beneficial ecosystem services contributed to by plant-pathogenic bacteria.

Toxin gene profiling of enterotoxic and emetic Bacillus cereus.

Very different toxins are responsible for the two types of gastrointestinal diseases caused by Bacillus cereus: the diarrhoeal syndrome is linked to nonhemolytic enterotoxin NHE, hemolytic enterotoxin HBL, and cytotoxin K, whereas emesis is caused by the action of the depsipeptide toxin cereulide. The recently identified cereulide synthetase genes permitted development of a molecular assay that targets all toxins known to be involved in food poisoning in a single reaction, using only four different sets of primers. The enterotoxin genes of 49 strains, belonging to different phylogenetic branches of the B. cereus group, were partially sequenced to encompass the molecular diversity of these genes. The sequence alignments illustrated the high molecular polymorphism of B. cereus enterotoxin genes, which is necessary to consider when establishing PCR systems. Primers directed towards the enterotoxin complex genes were located in different CDSs of the corresponding operons to target two toxin genes with one single set of primers. The specificity of the assay was assessed using a panel of B. cereus strains with known toxin profiles and was successfully applied to characterize strains from food and clinical diagnostic labs as well as for the toxin gene profiling of B. cereus isolated from silo tank populations.

Isolation and identification of Pseudomonas syringae facilitated by a PCR targeting the whole P. syringae group.

We present a reliable PCR-based method to avoid the biases related to identification based on the conventional phenotypes currently used in the identification of Pseudomonas syringae sensu lato, a ubiquitous environmental bacterium including plant pathogens. We identified a DNA target suitable for this purpose by applying a comparative genomic pipeline to Pseudomonas genomes. We designed primers and developed PCR conditions that led to a clean and strong PCR product from 97% of the 185 strains of P. syringae strains tested and gave a clear negative result for the 31 non-P. syringae strains tested. The sensitivity of standard PCR was determined with pure strains to be 10(6) bacteria mL(-1) or 0.4 ng of DNA µL(-1). Sensitivity could be improved with the touchdown method. The new PCR-assisted isolation of P. syringae was efficient when deployed on an environmental sample of river water as compared to the isolation based on phenotypes. This innovation eliminates the need for extensive expertise in isolating P. syringae colonies, was simpler, faster and very reliable. It will facilitate discovery of more diversity of P. syringae and research on emergence, dispersion and evolution to understand the varied functions of this environmental bacterium.

Agrifood systems and the microbial safety of fresh produce: Trade-offs in the wake of increased sustainability.

Fresh produce has been a growing cause of food borne outbreaks world-wide prompting the need for safer production practices. Yet fresh produce agrifood systems are diverse and under constraints for more sustainability. We analyze how measures taken to guarantee safety interact with other objectives for sustainability, in light of the diversity of fresh produce agrifood systems. The review is based on the publications at the interface between fresh produce safety and sustainability, with sustainability defined by low environmental impacts, food and nutrition security and healthy life. The paths for more sustainable fresh produce are diverse. They include an increased use of ecosystem services to e.g. favor predators of pests, or to reduce impact of floods, to reduce soil erosion, or to purify run-off waters. In contrast, they also include production systems isolated from the environment. From a socio-economical view, sustainability may imply maintaining small tenures with a higher risk of pathogen contamination. We analyzed the consequences for produce safety by focusing on risks of contamination by water, soil, environment and live stocks. Climate change may increase the constraints and recent knowledge on interactions between produce and human pathogens may bring new solutions. Existing technologies may suffice to resolve some conflicts between ensuring safety of fresh produce and moving towards more sustainability. However, socio-economic constraints of some agri-food systems may prevent their implementation. In addition, current strategies to preserve produce safety are not adapted to systems relying on ecological principles and knowledge is lacking to develop the new risk management approaches that would be needed.

Mutability in Pseudomonas viridiflava as a programmed balance between antibiotic resistance and pathogenicity.

Mutable bacterial cells are defective in their DNA repair system and often have a phenotype different from that of their wild-type counterparts. In human bacterial pathogens, the mutable and hypermutable phenotypes are often associated with general antibiotic resistance. Here, we quantified the occurrence of mutable cells in Pseudomonas viridiflava, a phytopathogenic bacterium in the P. syringae complex with a broad host range and capacity to live as a saprophyte. Two phenotypic variants (transparent and mucoid) were produced by this bacterium. The transparent variant had a mutator phenotype, showed general antibiotic resistance and could not induce disease on the plant species tested (bean). In contrast, the mucoid variant did not display mutability or resistance to antibiotics and was capable of inducing disease on bean. Both the transparent and mucoid variants were less fit when grown in vitro, whereas, in planta, both of the variants and wild-types attained similar population densities. Given the importance of the methyl-directed mismatch repair system (MMR) in the occurrence of mutable and hypermutable cells in human bacterial pathogens, we investigated whether mutations in mut genes were associated with mutator transparent cells in P. viridiflava. Our results showed no mutations in MMR genes in any of the P. viridiflava cells tested. Here, we report that a high mutation rate and antibiotic resistance are inversely correlated with pathogenicity in P. viridiflava, but are not associated with mutations in MMR. In addition, P. viridiflava variants differ from variants produced by other phytopathogenic bacteria in the absence of reversion to the wild-type phenotype.

The Water Cycle, a Potential Source of the Bacterial Pathogen Bacillus cereus.

The behaviour of the sporulating soil-dwelling Bacillus cereus sensu lato (B. cereus sl) which includes foodborne pathogenic strains has been extensively studied in relation to its various animal hosts. The aim of this environmental study was to investigate the water compartments (rain and soil water, as well as groundwater) closely linked to the primary B. cereus sl reservoir, for which available data are limited. B. cereus sl was present, primarily as spores, in all of the tested compartments of an agricultural site, including water from rain to groundwater through soil. During rain events, leachates collected after transfer through the soil eventually reached the groundwater and were loaded with B. cereus sl. In groundwater samples, newly introduced spores of a B. cereus model strain were able to germinate, and vegetative cells arising from this event were detected for up to 50 days. This first B. cereus sl investigation in the various types of interrelated environments suggests that the consideration of the aquatic compartment linked to soil and to climatic events should provide a better understanding of B. cereus sl ecology and thus be relevant for a more accurate risk assessment of food poisoning caused by B. cereus sl pathogenic strains.

A framework to gauge the epidemic potential of plant pathogens in environmental reservoirs: the example of kiwifruit canker.

New economically important diseases on crops and forest trees emerge recurrently. An understanding of where new pathogenic lines come from and how they evolve is fundamental for the deployment of accurate surveillance methods. We used kiwifruit bacterial canker as a model to assess the importance of potential reservoirs of new pathogenic lineages. The current kiwifruit canker epidemic is at least the fourth outbreak of the disease on kiwifruit caused by Pseudomonas syringae in the mere 50 years in which this crop has been cultivated worldwide, with each outbreak being caused by different genetic lines of the bacterium. Here, we ask whether strains in natural (non-agricultural) environments could cause future epidemics of canker on kiwifruit. To answer this question, we evaluated the pathogenicity, endophytic colonization capacity and competitiveness on kiwifruit of P. syringae strains genetically similar to epidemic strains and originally isolated from aquatic and subalpine habitats. All environmental strains possessing an operon involved in the degradation of aromatic compounds via the catechol pathway grew endophytically and caused symptoms in kiwifruit vascular tissue. Environmental and epidemic strains showed a wide host range, revealing their potential as future pathogens of a variety of hosts. Environmental strains co-existed endophytically with CFBP 7286, an epidemic strain, and shared about 20 virulence genes, but were missing six virulence genes found in all epidemic strains. By identifying the specific gene content in genetic backgrounds similar to known epidemic strains, we developed criteria to assess the epidemic potential and to survey for such strains as a means of forecasting and managing disease emergence.

A user's guide to a data base of the diversity of Pseudomonas syringae and its application to classifying strains in this phylogenetic complex.

The Pseudomonas syringae complex is composed of numerous genetic lineages of strains from both agricultural and environmental habitats including habitats closely linked to the water cycle. The new insights from the discovery of this bacterial species in habitats outside of agricultural contexts per se have led to the revelation of a wide diversity of strains in this complex beyond what was known from agricultural contexts. Here, through Multi Locus Sequence Typing (MLST) of 216 strains, we identified 23 clades within 13 phylogroups among which the seven previously described P. syringae phylogroups were included. The phylogeny of the core genome of 29 strains representing nine phylogroups was similar to the phylogeny obtained with MLST thereby confirming the robustness of MLST-phylogroups. We show that phenotypic traits rarely provide a satisfactory means for classification of strains even if some combinations are highly probable in some phylogroups. We demonstrate that the citrate synthase (cts) housekeeping gene can accurately predict the phylogenetic affiliation for more than 97% of strains tested. We propose a list of cts sequences to be used as a simple tool for quickly and precisely classifying new strains. Finally, our analysis leads to predictions about the diversity of P. syringae that is yet to be discovered. We present here an expandable framework mainly based on cts genetic analysis into which more diversity can be integrated.

The life history of Pseudomonas syringae: linking agriculture to earth system processes.

The description of the ecology of Pseudomonas syringae is moving away from that of a ubiquitous epiphytic plant pathogen to one of a multifaceted bacterium sans frontières in fresh water and other ecosystems linked to the water cycle. Discovery of the aquatic facet of its ecology has led to a vision of its life history that integrates spatial and temporal scales spanning billions of years and traversing catchment basins, continents, and the planet and that confronts the implication of roles that are potentially conflicting for agriculture (as a plant pathogen and as an actor in processes leading to rain and snowfall). This new ecological perspective has also yielded insight into epidemiological phenomena linked to disease emergence. Overall, it sets the stage for the integration of more comprehensive contexts of ecology and evolutionary history into comparative genomic analyses to elucidate how P. syringae subverts the attack and defense responses of the cohabitants of the diverse environments it occupies.

Rhizobium alamii sp. nov., an exopolysaccharide-producing species isolated from legume and non-legume rhizospheres.

A group of exopolysaccharide-producing bacteria was isolated from the root environment of Arabidopsis thaliana. The genetic diversity revealed by REP-PCR fingerprinting indicated that the isolates correspond to different strains. 16S rRNA gene sequence analysis showed that the isolates are closely related to the strains Rhizobium sp. YAS34 and USDA 1920, respectively isolated from sunflower roots and Medicago ruthenica nodules. These bacteria belong to the Rhizobium lineage of the Alphaproteobacteria, and the closest known species was Rhizobium sullae. DNA-DNA hybridization experiments and biochemical analysis demonstrated that the nine strains isolated from A. thaliana and Rhizobium strains YAS34 and USDA 1920 constitute a novel species within the genus Rhizobium, for which the name Rhizobium alamii sp. nov. is proposed. The type strain is GBV016(T) (=CFBP 7146(T) =LMG 24466(T)).

Mesorhizobium metallidurans sp. nov., a metal-resistant symbiont of Anthyllis vulneraria growing on metallicolous soil in Languedoc, France.

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)).