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

International audience; Crop diseases are responsible for considerable yield losses worldwide and particularly in sub-Saharan Africa. To implement efficient disease control measures, detection of the pathogens and understanding pathogen spatio-temporal dynamics is crucial and requires the use of molecular detection tools, especially to distinguish different pathogens causing more or less similar symptoms. We report here the design a new molecular diagnostic tool able to simultaneously detect five bacterial taxa causing important diseases on rice in Africa: (1) Pseudomonas fuscovaginae, (2) Xanthomonas oryzae, (3) Burkholderia glumae and Burkholderia gladioli, (4) Sphingomonas and (5) Pantoea species. This new detection tool consists of a multiplex PCR, which is cost effective and easily applicable. Validation of the method is presented through its application on a global collection of bacterial strains. Moreover, sensitivity assessment for the detection of all five bacteria is reported to be at 0.5 ng DNA by μl. As a proof of concept, we applied the new molecular detection method to a set of 256 rice leaves collected from 16 fields in two irrigated areas in western Burkina Faso. Our results show high levels of Sphingomonas spp. (up to 100% of tested samples in one field), with significant variation in the incidence between the two sampled sites. Xanthomonas oryzae incidence levels were mostly congruent with bacterial leaf streak (BLS) and bacterial leaf blight (BLB) symptom observations in the field. Low levels of Pantoea spp. were found while none of the 256 analysed samples was positive for Burkholderia or Pseudomonas fuscovaginae. Finally, many samples (up to 37.5% in one studied field) were positive for more than one bacterium (co-infection). Documenting co-infection levels are important because of their drastic consequences on epidemiology, evolution of pathogen populations and yield losses. The newly designed multiplex PCR for multiple bacterial pathogens of rice is a significant improvement for disease monitoring in the field, thus contributing to efficient disease control and food safety.

Using a local Algerian cultivar of chickpea, from 9 sites in Eastern Algeria, along a sub-humid to arid gradient, we isolated 60 nodulating isolates, among which 19 were assigned to Mesorhizobium species and 41 to Ensifer meliloti. Trapping revealed great differences among sites for their ability to induce nodules on plant, but no correlation with chemical and physical characterizations of soil samples could be found. Based on 16S rRNA gene sequences, 16S-23S IGS, recA, nodC and nifH sequences, Mesorhizobium isolates were assigned to at least 5 different species, among which one had never been described as nodulating chickpea before. The symbiotic diversity is conversely low, and shared by several species, reflecting horizontal gene transfer. The ability of Mesorhizobium genus to recombine and exchange symbiotic clusters among different species must play a great role in this ability to be associated with chickpea. The question to what extent the symbiotic species would end in this genus is discussed.

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

The bacterial genus Burkholderia comprises species occupying several habitats, including a group of symbionts of leguminous plants-also called beta-rhizobia-that has been recently ascribed to the new genus Paraburkholderia. We used common bean (Phaseolus vulgaris L.) plants to trap rhizobia from an undisturbed soil of the Brazilian Cerrado under the vegetation type 'Cerradao'. Genetic characterization started with the analyses of 181 isolates by BOX-PCR, where the majority revealed unique profiles, indicating high inter- and intra-species diversity. Restriction fragment length polymorphism-PCR of the 16S rRNA of representative strains of the BOX-PCR groups indicated two main clusters, and gene-sequencing analysis identified the minority (27%) as Rhizobium and the majority (73%) as Paraburkholderia. Phylogenetic analyses of the 16S rRNA and housekeeping (recA and gyrB) genes positioned all strains of the second cluster in the species P. nodosa, and the phylogeny of a symbiotic gene-nodC-was in agreement with the conserved genes. All isolates were stable vis-A -vis nodulating common bean, but, in general, with a low capacity for fixing N-2, although some effective strains were identified. The predominance of P. nodosa might be associated with the edaphic properties of the Cerrado biome, and might represent an important role in terms of maintenance of the ecosystem, which is characterized by acid soils with high saturation of aluminum and low N-2 content.Study of the diversity of nitrogen-fixing symbionts from a 'hot spot area', the Brazilian 'Cerradao', reveals that Paraburkholderia nodosa is the main symbiont trapped by promiscuous common bean (Phaseolus vulgaris L.).Study of the diversity of nitrogen-fixing symbionts from a 'hot spot area', the Brazilian 'Cerradao', reveals that Paraburkholderia nodosa is the main symbiont trapped by promiscuous common bean (Phaseolus vulgaris L.).

En 1966, l'aire de répartition d'Acacia mangium était encore restreinte à quelques ilots dans le Northern Queensland Australien et la Papouasie Nouvelle Guinée. En 2010, cette espèce représente un quart des surfaces sylvicoles plantées au monde, en proportion similaire au teck, avec plus de 2 millions d'Ha cultivés. A. mangium est une légumineuse s'associant à des bactéries du genre Bradyrhizobium. Dans le cadre d'une étude de diversité, des graines et des nodules (et les bactéries symbiotiques se cachant à l'intérieur..) avaient été récoltés en 1986 dans plusieurs sites australiens. En 2007 et 2009, sur approximativement les mêmes sites, nous avons procédé à de nouveaux échantillonnages et isolements bactériens. De plus, A. mangium ayant été introduit notamment en Malaisie et au Brésil, nous avons caractérisé les symbiotes spontanément retrouvés dans les plantations non inoculées artificiellement. Nous obtenons ainsi une image de l'évolution de la diversité des bactéries symbiotiques s'associant avec A. mangium au niveau temporel mais aussi spatiale. Nos résultats montrent que sur 20 ans, plusieurs allèles recA et nodA se retrouvent, avec un niveau de recouvrement plus élevé pour les prélèvements situés à deux ans d'intervalle. De plus, un haplotype multilocus est retrouvé à 20 ans d'intervalle, suggérant une très faible évolution des populations bactériennes, malgré des indices de recombinaison évidents. La similitude des fréquences sur deux ans renforce cette image de forte stabilité des populations. Inversement, la dispersion des isolats et des gènes apparait extrêmement limitée, avec une absence totale d'haplotype commun avec le Brésil, et deux entre la Malaisie et le Brésil. Les gènes de ménage et symbiotiques ne présentent cependant pas les mêmes patrons de diversification, le Brésil possédant une diversité symbiotique propre par rapport aux autres sites. Une possible histoire globale des Bradyrhizobium peut alors être proposée pour comprendre comment ce genre a évolué et évoluera encore.

Insularity, geological history and biogeography have made from New-Caledonia a hot spot of biodiversity where extremely diversified ecosystems occupies ultramafic terrains with drastic edaphic conditions in terms of fertility and metallic toxicity. In the framework of the mine project of the Koniambo Massif, a large nickel deposit, we tried to explore the diversity of ectomycorrhizal symbioses within these poorly explored natural ultramafic ecosystems. Floristic inventories along an altitudinal gradient ranging from 700 to 900 m evidenced 4 different plant communities. The 2 lower plant communities, 3 and 4, were dominated by 2 endemic tree genera, Tristaniopsis (Leptospermoideae) and Nothofagus (Nothofagaceae) respectively, whose ectomycorrhizal (ECM) status was shown and explored through molecular methods on sporocarps, mycorrhizae and soil mycelium. We evidenced a diversified fungal community in the basal plant community dominated by two tree species of the genus Nothofagus. The molecular characterization of these ECM fungi was established on the total ribosomal inter transcribed spacer (ITS) by PCR-sequencing and BLASTn analysis, revealing the relative abundance of the Cortinariaceae among our samples. Samples belonging to this fungal family were phylogenetically analyzed on the same ITS, in reference to sequences of samples with geographically different origins, including countries derived from the Gondwanaland fragmentation. If no clear phylogenetical relationships were evidenced, our study confirmed the same relative dominance of ECM Nothofagaceae, as well as the relative abundance of associated Cortinariaceae, in New Caledonia as in several of the Gondwanaland-originating countries.

Acacia mangium, légumineuse originaire d'Australie, a été introduit dans de nombreux pays tropicaux en particulier dans le cadre de plantations industrielles et de phytostabilisation. Sa productivité et son adaptabilité sont attribuées à sa capacité, assez exceptionnelle, d'établir des symbioses avec des bactéries fixatrices d'azote ainsi qu'avec des champignons mycorhiziens. Dans ce cadre, de nombreux essais de plantation ont été mis en place, en inoculation contrôlée avec des bactéries symbiotiques sélectionnées. En zone d'introduction, A. mangium s'associe spontanément à des souches locales souvent peu efficaces. En inoculation contrôlée, la persistance des inoculants n'est pas connue ainsi que leur impact sur la biodiversité locale, voire de possibles recombinaisons génétiques entre inoculées et locales. Afin de répondre à ces questions, au Brésil, une pépinière industrielle d'A. mangium et un site minier ont été étudiés, chacun avec et sans inoculation artificielle. Ainsi la diversité des souches spontanément nodulantes a été étudiée. Le Brésil interdisant l'utilisation d'inoculants étrangers, les souches sont donc originaires de légumineuses locales. Leur persistance sur A. mangium a été étudiée afin d'évaluer l'intérêt de ces pratiques. Pour cela, 66 souches renodulants A. mangium ont été séquencées sur trois gènes de ménage: glnB, recA et dnaK et un gène symbiotique: nodA. Ces analyses montrent qu'au Brésil, A. mangium est nodulé par des bactéries du genre Bradyrhizobium et Mesorhizobium. On observe également une nette séparation entre les souches issues des deux sites mais aussi entre parcelles inoculés et non-inoculés. Curieusement, les deux souches brésiliennes sélectionnées et inoculées ne semblent pas se maintenir quatre mois après inoculation, remettant en cause les pratiques locales d'inoculation. Enfin, la majorité des Mesorhizobium ont leur séquence du gène nodA, identique à 100%, ce qui pourrait correspondre au transfert d'un îlot symbiotique. (Texte intégral)

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

Acacia mangium is a legume tree native of Australasia. Tt has been introduced in many tropical countries especially in the context of industrial plantations. Tt has the exceptional capacity to establish symbioses with three types of microorganisms: nitrogen-fixing bacteria, arbuscular and ecto-mycorrhizal fungi, enabling a great productivity and adaptability. In this context, many field trials were established to test the effects of controlled inoculation with selected symbiotic bacteria versus natural colonization with indigenous strains. In introduction areas, A. mangium spontaneously nodulate with local and often ineffective bacteria. When inoculated, the persistence of inoculants, their impact on local biodiversity and possible genetic recombination with local strains have to be explored. The aim ofthis study was to describe the genetic diversity ofbacteria spontaneously nodulating A. mangium in Brazil and to evaluate, by molecular tracing, the durability ofselected strains used as inoculants. Three different sites, several hundred kilometres distant, were studied, each with inoculated and non inoculated plots. About one hundred strains were isolated and sequenced on three housekeeping (g/nB, dnaK and recA) and one symbiotic (nodA) genes. We showed that, whatever the site and the treatment, A. mangium is nodulated by bacteria of the genera Bradyrhizobium and Mesorhizobium. We observed a significant genetic differentiation not only among the three sites, but also between inoculated and non-inoculated plots in each site. The two inoculated strains were never detected, even few months after inoculation, be they in nursery or in plantation, raising the question ofthe usefulness of such artificial inoculation. At last, Mesorhizobium strains, display a genetic diversity within the housekeeping genes, shared exactly the same symbiotic sequence gene, opening the door to the possible transfer of the symbiotic island from unknown symbiotic strain. (Texte integral)