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International audience; The cultivation of grain legumes (e.g., common bean) in sub-Saharan Africa contributes to the provision of food for a growing population and delivers environmental benefits such as inputs of nitrogen (N) to crops and soil via symbiotic nitrogen fixation (SNF). However, the success of SNF is constrained by several factors such as the poor efficiency of native rhizobial strains to fix N, the low availability of phosphorus (P) and the acidity of soils. Two trials have been conducted in low-fertility tropical soils at the smallholder farm scale in Madagascar to assess the effects of Rhizobium inoculation together with inputs of P and lime on the growth of the common bean. We showed that inoculation with native strains of Rhizobium had significant effects on bean root nodulation, which was increased by up to 15-fold on plant growth, which increased by 78% and on bean yield, which increased by 126%. Moreover, we observed positive and significant relationships between inoculation with Rhizobium and P fertilization on nodulation, plant growth and yield. However, the addition of dolomite lime did not show any effect in our study. The addition of P decreased the mycorrhization rate of roots. Additional research is still needed to improve our understanding of soil fertility conditions (mainly on nutrient availability, including micronutrients) allowing better efficiency of legume symbionts (rhizobium and mycorrhiza) in such low-fertility soils.

Malgré des mesures de protection, la forêt dense humide de l'Est malgache tend à se réduire à des fragments forestiers. Le processus de fragmentation n'est pas pris en compte dans les suivis de la déforestation par télédétection qui comparent le couvert forestier entre plusieurs dates ; la question est de savoir s'il est perçu par les populations riveraines au point de les mobiliser dans des actions de conservation ou de restauration. Des enquêtes ethnobotaniques sont menées dans deux communes contrastées de l'Est malgache, la commune de Ranomafana-Est au nord (région Antsiranana) à fragments forestiers et la commune d'Androy au sud (région Haute Matsiatra) à massif forestier, pour recueillir auprès des villageois leurs perceptions de la forêt, son évolution, sa fragmentation, sa biodiversité végétale et animale, ses ressources et les mesures de protection envisagées localement. Les différences de réponses entre les deux sites traduisent les différences de contextes et de niveaux de fragmentation ; elles sont marquées sur la perception des forêts résiduelles et leur dynamique passée, avec une hiérarchie spécifique des usages (la construction de l'habitation venant en premier), une attention particulière à la biodiversité animale et des propositions de conservation surtout issues des interventions d'ONG. Un prolongement serait nécessaire pour co-construire avec les habitants une vision de la fragmentation

Background Among semi-aquatic species of the legume genus Aeschynomene, some have the property of being nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the synthesis of Nod factors. Knowledge of the specificities underlying this Nod-independent symbiosis has been gained from the model legume Aeschynomene evenia but our understanding remains limited due to the lack of comparative genetics with related taxa using a Nod factor-dependent process. To fill this gap, we combined different approaches to perform a thorough comparative analysis in the genus Aeschynomene. Results This study significantly broadened previous taxon sampling, including in allied genera, in order to construct a comprehensive phylogeny. In the phylogenetic tree, five main lineages were delineated, including a novel lineage, the Nod-independent clade and another one containing a polytomy that comprised several Aeschynomene groups and all the allied genera. This phylogeny was matched with data on chromosome number, genome size and low-copy nuclear gene sequences to reveal the diploid species and a polytomy containing mostly polyploid taxa. For these taxa, a single allopolyploid origin was inferred and the putative parental lineages were identified. Finally, nodulation tests with different Bradyrhizobium strains revealed new nodulation behaviours and the diploid species outside of the Nod-independent clade were compared for their experimental tractability and genetic diversity. Conclusions The extended knowledge of the genetics and biology of the different lineages sheds new light of the evolutionary history of the genus Aeschynomene and they provide a solid framework to exploit efficiently the diversity encountered in Aeschynomene legumes. Notably, our backbone tree contains all the species that are diploid and it clarifies the genetic relationships between the Nod-independent clade and the Nod-dependent lineages. This study enabled the identification of A. americana and A. patula as the most suitable species to undertake a comparative genetic study of the Nod-independent and Nod-dependent symbioses. Electronic supplementary material The online version of this article (10.1186/s12870-018-1567-z) contains supplementary material, which is available to authorized users.

Figure S3. Chromosome numbers in Aeschynomene species. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 135 kb)

Figure S2. Schematic representation of the different steps of the genotyping process from marker selection to data treatment. (PPTX 64 kb)

Figure S3. Detailed NJ trees representing the genetic diversity among the Nod-independent Aeschynomene accessions. The trees were developed separately in DARWIN using the allelic data of 65 SSRs for the 2× (a), 4× (b) and 6× (c) taxa. Well-differentiated taxa are distinctly colored. Identified genotypes are marked with a red dot and numbered. Accessions are designated with their LSTM code mentioned in Additional file 1: Table S1 followed by their geographical origin. Species suspected to be morphological variants are marked with an asterisk. Taxon colours and genotype numbers are the same as in Fig. 3. (PPTX 187 kb)

Figure S1. Chromosome numbers in new Aeschynomene taxa. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome numbers are indicated in brackets. Scale bars: 5 μm. (PPTX 1008 kb)

Table S1. Accessions used for the phylogeny of the genus Aeschynomene and related genera, their origin and characteristics. (PPTX 143 kb)

Figure S4. Chromosome numbers in species of Aeschynomene related genera. Root tip metaphase chromosomes stained in blue with DAPI (4′,6-diamidino-2-phenylindole). Chromosome counts are indicated in brackets. Scale bars: 5 μm. (PPTX 57 kb)

Figure S5. Phylogenetic trees based on nuclear low-copy genes. Bayesian phylogenetic reconstructions obtained for the CYP1, eif1a, SuSy and TIP1;1 genes. Diploid species (2n = 20) are in blue, polyploid species (2n ≥ 28) in black excepted A. afraspera for which the A and B gene copies are distinguished in red and green respectively. -A, −A1, −A2, -B, -B1 and -B2 indicated the different copies found. Putative A and B subgenomes of the polyploid taxa are delineated by red and green boxes in dashed lines, respectively. Numbers at branches represent posterior probability. (PPTX 56 kb)

Table S3. GenBank numbers for the sequences used in the phylogenetic analyses. (PPTX 149 kb)

Table S2. Primers used for gene amplification and sequencing. (PPTX 134 kb)

Figure S6. Ancestral state reconstruction of ploidy levels in the genus Aeschynomene and allied genera. Ancestral state reconstruction was estimated in SIMMAP software using the 50% majority-rule topology obtained by Bayesian analysis of the combined ITS + matK sequences. Ploidy levels are indicated by different colors. Unknown ploidy levels are denoted by a dash. (PPTX 3568 kb)

Doc. S1. ITS sequences obtained for the Nod-independent Aeschynomene accessions. (DOCX 30 kb)

Figure S4. Comparison of the nodulation properties of A. evenia s.s. and A. indica. Different accessions were root inoculated with Bradyrhizobium ORS278 and BTAi1 and analysed at 14dpi. (a) Number of nodules per accession. (b) Acetylene-reducing activity (ARA). A.U. Arbitrary Unit. Error bars represent s.d. (n = 6). (PPTX 179 kb)

International audience; Mycorrhizal symbiosis is extremely important for tree growth, survival and resistance after transplantation particularly in Madagascar where deforestation is a major concern. The importance of mycorrhizal symbiosis is further increased when soil conditions at the planting site are limiting. To identify technical itineraries capable of improving ecological restoration in Madagascar, we needed to obtain native ectomycorrhizal (ECM) saplings with a wide diversity of ECM fungi. To this end, we transplanted ECM seedlings from the wild (wildlings) to a nursery. Using molecular characterisation of internal transcribed spacer (ITS) rDNA, we tested the effect of transplanting Asteropeia mcphersonii wildlings on ECM communities after 8 months of growth in the nursery. With or without the addition of soil from the site where the seedlings were sampled to the nursery substrate, we observed a dramatic change in the composition of fungal communities with a decrease in the ECM infection rate, a tremendous increase in the abundance of an operational taxonomic unit (OTU) taxonomically close to the order Trechisporales and the disappearance of all OTUs of Boletales. Transplanting to the nursery and/or to nursery conditions was shown to be incompatible with the survival and even less with the development in the nursery of most ECM fungi naturally associated with A. mcphersonii wildings.

This work was designed to study the antimicrobial activity of Crotalaria bernieri Baill. (Fabaceae). Extracts from leaf, root, pod and seed using hexane, ethyl acetate and methanol were tested in vitro for their activity against 17 bacteria, 5 fungi (3 yeasts and 2 molds) using disc diffusion and micro dilution methods. At the concentration of 1 mg/disc, all the extracts exhibited antimicrobial activity depending on the plant part and the extraction method used. The most sensitive germs were Salmonella enteridis, Streptococcus pyogenes and Candida guilliermondii with inhibition zone diameter (IZD) of 11 mm, 15 mm and 13 mm respectively. Most of extracts showed, broad activity spectrum varying from one extract to another. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and minimum fungicidal concentration (MFC) of all extracts were recorded. Ten extracts displayed an excellent effect (MIC 1000 µg/ml). Leaf methanol extracts were the most efficient and Gram positive bacteria the most sensitive. All extracts had bactericidal (MBC/MIC ≤ 4) or fungicidal action (MFC/MIC ≤ 4) in certain microorganisms and bacteriostatic (MBC/MIC > 4) or fungistatic action (MFC/MIC > 4) in others. Antimicrobial activity might be due to tannins, polyphenols, steroids, triterpenes and flavonoids that were present in most of the plant organs, but alkaloids in leaf and pod and saponosides in root might also be involved. C. bernieri with the effectiveness of all its parts, the variety of its secondary metabolites, the great number of sensitive pathogen microorganisms and its ubiquity make this plant species an interesting source of antimicrobial agents. Key words: Crotalaria bernieri, antimicrobial activity, disc diffusion method, microdilution method, minimum inhibitory concentration, minimum bactericidal concentration, minimum fungicidal concentration.

Forest ecosystem disturbances generate changes including soil changes, generally with loss of topsoil and loss of much of the diversity of microorganisms, which are usually replaced by others that are more adapted to the new conditions. We describe the effect of rainforest clear-cutting in Madagascar, with stripping of surface soil horizons among ectomycorrhizal fungal communities associated with natural Asteropeia mcphersonii seedlings. An experimental device was placed on four sites, each including an undisturbed plot with litter and a plot in clear-cut areas on a soil composed of a homogeneous mineral substrate. On each of these eight plots the ectomycorrhizal infection rate was determined and root tips were collected to sequence the internal transcribed spacer. 219 sequences grouped into 59 operational taxonomic units (OTUs) were obtained. The main taxonomic groups were Russulaceae, Boletales and Thelephoraceae. Marked spatial aggregation was observed for OTUs. The disturbance significantly affected the ectomycorrhizal infection rate of A. mcphersonii seedlings and the OTU composition of mycorrhizal communities. However, some OTUs were found in both types of environments. We discuss the effect of the disturbance on mycorrhizal communities associated with natural ectomycorrhizal A. mcphersonii seedlings, particularly in view of its application to ecological restoration of disturbed forest environments.

The main goal of this work was to describe the mycorrhizal status, AM or ECM or dual AM and ECM or nonmycorrhizal (NM), of different trees species in five coastal humid forests located along the eastern coast of Madagascar. Identification of the plants was carried out by carefully tracing the root system in the field. Several field collection surveys were under-taken and the biological material was examined in laboratory conditions to check the mycorrhizal status of roots and to identify sporophores. (Resume d'auteur)

International audience; Ecological restoration in severely disturbed environments can fail because of lack of knowledge of the functioning of the original ecosystem. Nevertheless, facilitating establishment between plant species can help accelerate ecological succession, especially in stressful environments. Mycorrhizal symbiosis plays a key role in plant growth, particularly in harsh environments, and could also play a role in facilitation between plants, as mycorrhizal fungi can form a mycelial network that simultaneously interacts with the root systems of several plant species. In a high-elevation Malagasy tropical rainforest on acidic and iron-rich soil surrounding an active mining site, four genera of ectomycorrhizal plants are locally abundant: Leptolaena, Sarcolaena, Uapaca and Asteropeia. A floristic survey showed that only Asteropeia seedlings can grow on bare soil. Molecular analysis of ectomycorrhizal fungi ITS rDNA enabled us to describe ectomycorrhizal communities and their distribution among these four plant genera. Russulaceae, Boletales, Cortinariaceae and Thelephoraceae are abundant in these forests. There is extensive sharing between ectomycorrhizal communities associated with Asteropeia mcphersonii and other ectomycorrhizal plants. There are also many mycorrhizal fungi species which are common to ectomycorrhizal communities of seedlings and adult trees. This abundance of generalist fungi allows us to envisage the use of A. mcphersonii in the ecological restoration of the mine site. Planting ectomycorrhizal fungi in the bare soil at the beginning of ecological restoration could allow them to grow, thereby establishing a source of inoculum to colonize other ectomycorrhizal plants and consequently facilitate their establishment.

It is well known that the over exploitation of soil resources decreases the density and diversity of arbuscular mycorrhizal (AM) fungal propagules. It has been postulated that agricultural practices could promote the occurrence and functioning of remaining and resilient AM fungal ecotypes in order to benefit from AM associations. Using C. ochroleuca, a highly mycotrophic plant species, the aims of this study were to determine in controlled conditions, the impact of this legume species on: (i) the mycorrhizal soil infectivity (MSI), (ii) the Rock Phosphate (RP) solubilising activity and (iii) the soil microbial functions. The expected benefits of C. ochroleuca cultivation have been evaluated by comparing its biological influences with those recorded with a non-mycorrhizal plant species, Brassica oleracea in order to attest of the importance of the AM potential in RP weathering and soil functioning. The results show that: C. ochroleuca was effective in improving the mycorrhizal soil potential, (ii) functional abilities of soil microflora were influenced by the cultured plant species and the mycorrhizal soil potential and (iii) the efficiency of rock phosphate amendment on plant growth depended on the level of mycorrhizal soil potential and was linkedto the extent of the mycorrhizal network.The C. ochroleuca impact was significantly reinforced with KRP amendment (i.e. +29.3% for the Catabolic Eveness, +45.7% for the hyphal length). According to these results, it could be concluded that agricultural strategies based on the management of local biodiversity (i.e. the use of native legume species) could be considered as a sustainable practice to optimally manage AM fungi and to ensure their functionalities in order to maintain a sustainable production of food crops in the tropics.

The overexploitation of natural resources, resulting in an increased need for arable lands by local populations, causes a serious dysfunction in the soil's biological functioning (mineral deficiency, salt stress, etc.). This dysfunction, worsened by the climatic conditions (drought), requires the implementation of ecological engineering strategies allowing the rehabilitation of degraded areas through the restoration of essential ecological services. The first symptoms of weathering processes of soil quality in tropical and Mediterranean environments result in an alteration of the plant cover structure with, in particular, the pauperization of plant species diversity and abundance. This degradation is accompanied by a weakening of soils and an increase of the impact of erosion on the surface layer resulting in reduced fertility of soils in terms of their physicochemical characteristics as well as their biological ones (e.g., soil microbes). Among the microbial components particularly sensitive to erosion, symbiotic microorganisms (rhizobia, Frankia, mycorrhizal fungi) are known to be key components in the main terrestrial biogeochemical cycles (C, N and P). Many studies have shown the importance of the management of these symbiotic microorganisms in rehabilitation and revegetation strategies of degraded environments, but also in improving the productivity of agrosystems. In particular, the selection of symbionts and their inoculation into the soil were strongly encouraged in recent decades. These inoculants were selected not only for their impact on the plant, but also for their ability to persist in the soil at the expense of the residual native microflora. The performance of this technique was thus evaluated on the plant cover, but its impact on soil microbial characteristics was totally ignored. The role of microbial diversity on productivity and stability (resistance, resilience, etc.) of eco- and agrosystems has been identified relatively recently and has led to a questioning of the conceptual bases of controlled inoculation in sustainable land management. It has been suggested that the environmental characteristics of the area to rehabilitate should be taken into account, and more particularly its degradation level in relation to the threshold of ecological resilience. This consideration should lead to the optimization of the cultural practices to either (i) restore the original properties of an ecosystem in case of slightly degraded environments or (ii) transform an ecosystem in case of highly degraded soils (e.g., mine soils). In this chapter, we discuss, through various examples of experiments conducted in tropical and Mediterranean areas, the performance of different strategies to manage the microbial potential in soils (inoculation of exotic vs. native species, inoculation or controlled management potential microbial stratum via aboveground vegetation, etc.) based on the level of environmental degradation.

La surexploitation des ressources naturelles, consécutive à un besoin accru en terres arables des populations locales, entraîne de graves dysfonctionnements dans le bio-fonctionnement du sol (carences minérales, stress salins, etc.). Ce dysfonctionnement, aggravé par les aléas climatiques (sécheresse), nécessite la mise en oeuvre de stratégies d'ingénierie écologique permettant une réhabilitation des milieux dégradés via la restauration de services écologiques essentiels. Les premiers symptômes du processus d'altération de la qualité des sols des milieux tropicaux et méditerranéens se traduisent par une altération de la structure de la strate épigée avec, en particulier, une paupérisation de la diversité et de l'abondance des espèces végétales. Cette dégradation est accompagnée par une fragilisation des sols et une augmentation de l'impact de l'érosion sur la couche superficielle, entraînant une baisse de la fertilité des sols au niveau de leurs caractéristiques physico-chimiques, mais aussi biologiques (par exemple, la microflore tellurique). Parmi les composantes microbiennes particulièrement sensibles à ces phénomènes d'érosion, les microorganismes symbiotiques (rhizobia, champignons mycorhiziens) sont connus pour être des composantes clés dans le fonctionnement des principaux cycles biogéochimiques telluriques (C, N et P). De nombreux travaux ont montré l'importance de la gestion de ces microorganismes symbiotiques dans les stratégies de réhabilitation et de revégétalisation des milieux dégradés, mais également pour améliorer la productivité des agro-systèmes. En particulier, la sélection de symbiotes et leur inoculation en masse dans le sol de plantation ou de culture ont été fortement encouragées au cours de ces dernières décennies. Ces inoculants étaient sélectionnés, non seulement pour leur impact sur la plante, mais également pour leur capacité à persister dans le sol, au détriment de la microflore résiduelle native. La performance de cette technique était donc évaluée au niveau du couvert végétal, mais les répercussions au niveau des caractéristiques de la microflore tellurique étaient généralement ignorées. Le rôle de la diversité microbienne sur la productivité et la stabilité (résistance, capacité de résilience, etc.) des éco- et agro-systèmes a été mis en évidence relativement récemment et a conduit à une remise en cause des bases conceptuelles de l'inoculation contrôlée dans la gestion durable des terres. Il a été notamment suggéré que les caractéristiques du milieu à réhabiliter devaient être prises en compte, et plus particulièrement son niveau de dégradation par rapport au seuil de résilience écologique. Cette prise en compte devrait permettre d'optimiser les pratiques culturales pour soit (i) redonner à l'écosystème ses propriétés originelles dans le cas de milieux peu dégradés ou (ii) transformer un écosystème dans le cas de sols hautement dégradés (par exemple, les sols miniers). Dans ce chapitre, nous évoquerons, à travers différents exemples d'expérimentations réalisées en milieu tropical et méditerranéen, les performances des différentes stratégies de gestion du potentiel microbien des sols (inoculation d'espèces exotiques vs des espèces natives, inoculation contrôlée vs la gestion du potentiel microbien via la strate végétale épigée, etc.) suivant le niveau de dégradation du milieu.

In a natural environment, major disturbances cause the destruction of the vegetation as well as of soil microorganisms such as mycorrhizal fungi. Plant and mycorrhizal fungus communities are independent and so it is important to understand the dynamics of mycorrhizal communities during the stages in plant colonisation successions that generate a forest canopy. These successions go along with an increase in the wealth of species and the diversity of the mycorrhizal community through the gradual establishment of late-stage fungi and the decrease in the number of multi-stage fungi. Plant-fungus interactions become specialised as the plant succession runs its course or as the population ages. These dynamics of mycorrhizal communities can be explained by differences in the life history traits between species of fungi, but also by a change in the characteristics of the plant community and the soil over time

The dynamics of mycorrhizal communities during the stages of the plant colonisation successions leads to an increase in the wealth of species and the diversity of the mycorrhizal community through the gradual establishment of late-stage fungi and the decrease in the number of multi-stage fungi. This brings about, amongst other things, a modification in the structure of the mycorrhizal networks, which has a direct impact on the stability of the plant community and on forest stands. Due to the specialisation of some interactions, the recruitment of new fungi has either direct or feedback effects on the diversity and productivity of the plant community. By taking into account the natural dynamics of the mycorrhizal community, the process of ecological remediation can be speeded up by inoculating the appropriate fungi or introducing suitable mycorrhizal communities by means of nurse plants.

Forest ecosystem disturbances generate changes including soil changes, generally with loss of topsoil and loss of much of the diversity of microorganisms, which are usually replaced by others that are more adapted to the new conditions. We describe the effect of rainforest clear-cutting in Madagascar, with stripping of surface soil horizons among ectomycorrhizal fungal communities associated with natural Asteropeia mcphersonii seedlings. An experimental device was placed on four sites, each including an undisturbed plot with litter and a plot in clear-cut areas on a soil composed of a homogeneous mineral substrate. On each of these eight plots the ectomycorrhizal infection rate was determined and root tips were collected to sequence the internal transcribed spacer. 219 sequences grouped into 59 operational taxonomic units (OTUs) were obtained. The main taxonomic groups were Russulaceae, Boletales and Thelephoraceae. Marked spatial aggregation was observed for OTUs. The disturbance significantly affected the ectomycorrhizal infection rate of A. mcphersonii seedlings and the OTU composition of mycorrhizal communities. However, some OTUs were found in both types of environments. We discuss the effect of the disturbance on mycorrhizal communities associated with natural ectomycorrhizal A. mcphersonii seedlings, particularly in view of its application to ecological restoration of disturbed forest environments.

Forest ecosystem disturbances generate changes including soil changes, generally with loss of topsoil and loss of much of the diversity of microorganisms, which are usually replaced by others that are more adapted to the new conditions. We describe the effect of rainforest clear-cutting in Madagascar, with stripping of surface soil horizons among ectomycorrhizal fungal communities associated with natural Asteropeia mcphersonii seedlings. An experimental device was placed on four sites, each including an undisturbed plot with litter and a plot in clear-cut areas on a soil composed of a homogeneous mineral substrate. On each of these eight plots the ectomycorrhizal infection rate was determined and root tips were collected to sequence the internal transcribed spacer. 219 sequences grouped into 59 operational taxonomic units (OTUs) were obtained. The main taxonomic groups were Russulaceae, Boletales and Thelephoraceae. Marked spatial aggregation was observed for OTUs. The disturbance significantly affected the ectomycorrhizal infection rate of A. mcphersonii seedlings and the OTU composition of mycorrhizal communities. However, some OTUs were found in both types of environments. We discuss the effect of the disturbance on mycorrhizal communities associated with natural ectomycorrhizal A. mcphersonii seedlings, particularly in view of its application to ecological restoration of disturbed forest environments.