We investigated the diversity of a collection of 76 Xenorhabdus strains, isolated from at least 27 species of Steinernema nematodes and collected in 32 countries, using three complementary approaches: 16S rRNA gene sequencing, molecular typing and phenotypic characterization. The 16S rRNA gene sequences of the Xenorhabdus strains were highly conserved (similarity coefficient >95 %), suggesting that the common ancestor of the genus probably emerged between 250 and 500 million years ago. Based on comparisons of the 16S rRNA gene sequences, we identified 13 groups and seven unique sequences. This classification was confirmed by analysis of molecular typing profiles of the strains, leading to the classification of new isolates into the Xenorhabdus species described previously and the description of ten novel Xenorhabdus species: Xenorhabdus cabanillasii sp. nov. (type strain USTX62 T =CIP 109066 T =DSM 17905 T

Steinernema species are entomopathogenic nematodes. They are symbiotically associated with Enterobacteriaceae of the genus Xenorhabdus. These nematode-bacteria symbioses are extremely diversified and constitute an important new model in ecology and evolution to investigate symbioses between microbes and invertebrates. However, no study has so far adequately evaluated either the outcome of the interactions or the obligate nature of interactions in different Steinernema species in the same way. Studying three different species of Steinernema, we showed that symbiotic nematodes are always fitter than aposymbiotic ones. Nevertheless, we revealed contrasting types of interaction in terms of outcome and obligate nature of the interaction. Bacterial analyses showed that nematode species differed dramatically in the number of symbiotic Xenorhabdus they carried. We suggested that when the interaction appeared more facultative for a nematode species, the nematodes carried fewer Xenorhabdus cells than strongly dependent worm species. Thus, the symbiont transmission appeared to become more efficient as the relationship between the nematode and the bacteria became tighter.

Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

We used the information from a set of concatenated sequences from four genes (recA, gyrB, dnaN and gltX) to investigate the phylogeny of the genera Photorhabdus and Xenorhabdus (entomopathogenic bacteria associated with nematodes of the genera Heterorhabditis and Steinernema, respectively). The robustness of the phylogenetic tree obtained by this multigene approach was significantly better than that of the tree obtained by a single gene approach. The comparison of the topologies of single gene phylogenetic trees highlighted discrepancies which have implications for the classification of strains and new isolates; in particular, we propose the transfer of Photorhabdus luminescens subsp. thracensis to Photorhabdus temperata subsp. thracensis comb. nov. (type strain CIP 108426T =DSM 15199T). We found that, within the genus Xenorhabdus, strains or isolates that shared less than 97 % nucleotide identity (NI), calculated on the concatenated sequences of the four gene fragments (recA, gyrB, dnaN and gltX) encompassing 3395 nucleotides, did not belong to the same species. Thus, at the 97 % NI cutoff, we confirm the current 20 species of the genus Xenorhabdus and propose the description of a novel species, Xenorhabdus vietnamensis sp. nov. (type strain VN01T = CIP 109945T =DSM 22392T). Within each of the three current species of the genus Photorhabdus, P. asymbiotica, P. luminescens and P. temperata, strains or isolates which shared less than 97 % NI did not belong to the same subspecies. Comparisons of the four gene fragments plus the rplB gene fragment analysed separately led us to propose four novel subspecies: Photorhabdus luminescens subsp. caribbeanensis subsp. nov. (type strain HG29T =CIP 109949T =DSM 22391T), P. luminescens subsp. hainanensis subsp. nov. (type strain C8404T = CIP 109946T =DSM 22397T), P. temperata subsp. khanii subsp. nov. (type strain C1T =NC19T =CIP 109947T =DSM 3369T), and P. temperata subsp. tasmaniensis subsp. nov. (type strain T327T = CIP 109948T =DSM 22387T).

Dickeya dadantii (Erwinia chrysanthemi) is a phytopathogenic bacterium causing soft rot diseases on many crops. The sequencing of its genome identified four genes encoding homologues of the Cyt family of insecticidal toxins from Bacillus thuringiensis, which are not present in the close relative Pectobacterium carotovorum subsp. atrosepticum. The pathogenicity of D. dadantii was tested on the pea aphid Acyrthosiphon pisum, and the bacterium was shown to be highly virulent for this insect, either by septic injury or by oral infection. The lethal inoculum dose was calculated to be as low as 10 ingested bacterial cells. A D. dadantii mutant with the four cytotoxin genes deleted showed a reduced per os virulence for A. pisum, highlighting the potential role of at least one of these genes in pathogenicity. Since only one bacterial pathogen of aphids has been previously described (Erwinia aphidicola), other species from the same bacterial group were tested. The pathogenic trait for aphids was shown to be widespread, albeit variable, within the phytopathogens, with no link to phylogenetic positioning in the Enterobacteriaceae. Previously characterized gut symbionts from thrips (Erwinia/Pantoea group) were also highly pathogenic to the aphid, whereas the potent entomopathogen Photorhabdus luminescens was not. D. dadantii is not a generalist insect pathogen, since it has low pathogenicity for three other insect species (Drosophila melanogaster, Sitophilus oryzae, and Spodoptera littoralis). D. dadantii was one of the most virulent aphid pathogens in our screening, and it was active on most aphid instars, except for the first one, probably due to anatomical filtering. The observed difference in virulence toward apterous and winged aphids may have an ecological impact, and this deserves specific attention in future research.Dickeya dadantii (syn. Erwinia chrysanthemi, Pectobacterium chrysanthemi) (53), as well as many other erwinia species, is one of the phytopathogenic Enterobacteriaceae that can cause soft rot diseases in a wide range of economically important crops. These bacteria can survive in soils, from where they are transmitted to plants by water, miscellaneous insects, or cultural techniques. There is no recorded specific vector organism for such pathogens, although Drosophila melanogaster was claimed to be a potential vector for some Erwinia species (3). The common symptoms for D. dadantii infection, as well as other so-called pectinolytic soft rot Erwinia, are characterized by the rapid disorganization of parenchymatous tissues, mainly driven by pectic enzymes (38). Nevertheless, plant colonization by soft rot Erwinia is a multifactorial process requiring numerous additional factors, such as cellulases, iron assimilation, an Hrp type III secretion system, exopolysaccharides, motility, and proteins involved in resistance against plant defense mechanisms (58). The recent deciphering of the complete genome sequence of D. dadantii, strain 3937 (28), after that of Pectobacterium carotovorum subsp. atrosepticum (syn....

Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg 2؉ -dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg 2؉ -regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.

In this paper, we investigate the level of specialization of the symbiotic association between an entomopathogenic nematode (Steinernema carpocapsae) and its mutualistic native bacterium (Xenorhabdus nematophila). We made experimental combinations on an insect host where nematodes were associated with non-native symbionts belonging to the same species as the native symbiont, to the same genus or even to a different genus of bacteria. All non-native strains are mutualistically associated with congeneric entomopathogenic nematode species in nature. We show that some of the non-native bacterial strains are pathogenic for S. carpocapsae. When the phylogenetic relationships between the bacterial strains was evaluated, we found a clear negative correlation between the effect a bacterium has on nematode fitness and its phylogenetic distance to the native bacteria of this nematode. Moreover, only symbionts that were phylogenetically closely related to the native bacterial strain were transmitted. These results suggest that co-evolution between the partners has led to a high level of specialization in this mutualism, which effectively prevents horizontal transmission. The pathogenicity of some non-native bacterial strains against S. carpocapsae could result from the incapacity of the nematode to resist specific virulence factors produced by these bacteria.

Background: The holistic view of bacterial symbiosis, incorporating both host and microbial environment, constitutes a major conceptual shift in studies deciphering host-microbe interactions. Interactions between Steinernema entomopathogenic nematodes and their bacterial symbionts, Xenorhabdus, have long been considered monoxenic two partner associations responsible for the killing of the insects and therefore widely used in insect pest biocontrol. We investigated this "monoxenic paradigm" by profiling the microbiota of infective juveniles (IJs), the soil-dwelling form responsible for transmitting Steinernema-Xenorhabdus between insect hosts in the parasitic lifecycle. Results: Multigenic metabarcoding (16S and rpoB markers) showed that the bacterial community associated with laboratory-reared IJs from Steinernema carpocapsae, S. feltiae, S. glaseri and S. weiseri species consisted of several Proteobacteria. The association with Xenorhabdus was never monoxenic. We showed that the laboratory-reared IJs of S. carpocapsae bore a bacterial community composed of the core symbiont (Xenorhabdus nematophila) together with a frequently associated microbiota (FAM) consisting of about a dozen of Proteobacteria (Pseudomonas, Stenotrophomonas, Alcaligenes, Achromobacter, Pseudochrobactrum, Ochrobactrum, Brevundimonas, Deftia, etc.). We validated this set of bacteria by metabarcoding analysis on freshly sampled IJs from natural conditions. We isolated diverse bacterial taxa, validating the profile of the Steinernema FAM. We explored the functions of the FAM members potentially involved in the parasitic lifecycle of Steinernema. Two species, Pseudomonas protegens and P. chlororaphis, displayed entomopathogenic properties suggestive of a role in Steinernema virulence and membership of the Steinernema pathobiome. Conclusions: Our study validates a shift from monoxenic paradigm to pathobiome view in the case of the Steinernema ecology. The microbial communities of low complexity associated with EPNs will permit future microbiota manipulation experiments to decipher overall microbiota functioning in the infectious process triggered by EPN in insects and, more generally, in EPN ecology.