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Bradyrhizobia are Gram-negative soil bacteria that regroup a growing number of species. They are widespread in nature and recovered from various biomes that may be explained by a high genetic diversity in this genus. Among the numerous metabolic properties they can harbor, the nitrogen fixation resulting from the association with plants among which important crop legumes (soya bean, peanut, cowpea …) is of great interest, notably in a context of sustainable development. Metabarcoding is widely applied to study biodiversity from complex microbial communities. Here, we demonstrate that using a new species-specific and highly polymorphic 16S-23S rRNA intergenic spacer barcode, we could rapidly estimate the diversity of bradyrhizobial populations that associate with cowpea and peanut plants, two crop legumes of major interest in Senegal. Application of the method on indigenous bradyrhizobia associated with peanut and cowpea grown in soils collected in the center of the peanut basin shows that Bradyrhizobium vignae is a dominant symbiont. We also showed that the two plant species associate with distinct community profiles and that strains introduced by inoculation significantly modified the population structure with these two plants suggesting that application of elite strains as inoculants may well ensure optimized symbiotic performance. This approach may further be used to study the diversity of bradyrhizobia from contrasting agro-eco-climatic zones, to test whether the plant genotype influences the association outputs as well as to estimate the competitiveness for nodule occupancy and the fate of elite strains inoculated in the field. Key points • An amplicon sequencing approach targeting the Bradyrhizobium genus was developed. • Diversity of cowpea and peanut bradyrhizobia from cultivated soils was identified. • The method is well suited to test the competitiveness of defined Bradyrhizobium inoculants.

International audience; Here, we report the complete genome sequence of Bradyrhizobium sp. strain ORS3257, which forms efficient symbioses with cowpea, peanut, or groundnut. These genomic data will be useful to identify genes associated with symbiotic performance and host compatibility on several legumes, including Aeschynomene species , with which a Nod-independent type III secretion system (T3SS)-dependent sym-biosis can be established.

Here, we report the complete genome sequence of Bradyrhizobium sp. strain ORS3257, which forms efficient symbioses with cowpea, peanut, or groundnut., Here, we report the complete genome sequence of Bradyrhizobium sp. strain ORS3257, which forms efficient symbioses with cowpea, peanut, or groundnut. These genomic data will be useful to identify genes associated with symbiotic performance and host compatibility on several legumes, including Aeschynomene species, with which a Nod-independent type III secretion system (T3SS)-dependent symbiosis can be established.

Arbuscular mycorrhizal symbiosis is an important plant root–fungal partnership/interaction that affects the growth response of crops. We have investigated the molecular diversity of arbuscular mycorrhizal fungi (AMF) colonizing cowpea roots and the associated rhizosphere soil to test the hypothesis that community diversity in rhizosphere soil is similar to that in cowpea (Vigna unguiculata) roots. Cowpea plants were grown in farmers’ fields located in seven agro-ecological zones of Benin, and soil and root samples were collected. The molecular diversity of the AMF in these samples was assessed after amplification of the large ribosomal subunit of DNA extracted from the soil and the root samples. At fruition, the frequency of mycorrhizal infection was unaffected by the agro-ecological zone, but there were significant differences in the intensity of AMF colonization among the zones. Multiple regression analysis showed that the main factor affecting mycorrhizal frequency at flowering was available phosphorus. Phylogenetic analysis revealed 25 operational taxonomic units belonging to two fungal families (Glomeraceae and Gigasporaceae). The diversity of AMF colonizing roots of cowpea in Benin was high and fairly similar to that in the rhizosphere soil but with a prevalence of the Glomeraceae. Despite the absence of strict host specificity in mycorrhizal symbiosis, there was a preferential association between some AMF species and cowpea cultivar IT96D-610.

The response of cowpea (Vigna unguiculata (Walp. (L.)) to inoculation with symbiotic microorganisms (arbuscular mycorrhizal fungus and rhizobium) was investigated on cultivated vertisoils (known locally as Dek) and arenosoils (known locally as Dior) in three geographic sites in Senegal. On the basis of soil physico-chemical characteristics, of most probable number (MPN) of indigenous rhizobia and of yields, a hierarchical analysis showed that the sites were grouped in three clusters. Some soils initially considered as Dek were grouped with Dior, so that all of Dior soils were grouped in the same cluster. No significant increasing in shoot biomass or yield was observed when cowpea was inoculated with fungi alone, whatever soil type. However, a significant or noticeable improvement in cowpea seeds production was obtained by rhizobium inoculation. It appeared that cowpea response to inoculation depended on geographical site and were often observed in sandy arenosoils (Dior type) or in loamy sand vertisoils (Dek type) with at least 15mgkg -1 of available

International audience; In a context of global change, alterations in the water cycle may impact the structure and function of terrestrial and aquatic ecosystems. Wetlands are particularly at risk because hydrological regime has a major influence on microbially mediated biogeochemical processes in sediments. While the influence of water availability on wetland biogeochemical processes has been comprehensively studied, the influence of hydrological regime on microbial community structure has been overlooked. We tested for the effect of hydrological regime on the structure and functions of microbial communities by comparing sediments collected at multiple sites in the Ain departement (Eastern France). Each site consisted of two plots, one permanently and one seasonally inundated. At the time of sampling, all plots were continuously inundated for more than 6 months but still harboured distinct bacterial communities. This change in community structure was not associated with marked modifications in the rates of microbial activities involved in the C and N cycles. These results suggest that the observed structural change could be related to bacterial taxa responding to the environmental variations associated with different hydrological regimes, but not strongly associated with the biogeochemical processes monitored here.

Tilemsi rock phosphate (TRP) of Mali is one of the most promising rock phosphate in West Africa for soil fertilization, but it is little used because of its insoluble form. The main objective of this study is to investigate TRP effects on rhizobia associated with the multipurpose leguminous tree Sesbania sesban grown on a sandy soil, poor in phosphorus and not sterilised. The experiment included treatments with and without TRP and was conducted during 105 days. At the end, 114 nodules have been collected and analysed by PCR/RFLP of 16S-23S intergenic spacer. Sixteen different RFLP profiles corresponding to different genomic groups of rhizobia have been detected. Five were dominant and present in both treatments. Five groups appear only in treatments without TRP whereas the six others are only in nodules of plants with TRP, suggesting a different capacity of natural phosphates solubilization by these strains.

The present study aimed at assessing the recovery of phototrophic and heterotrophic biofilm communities after a decrease in copper exposure. An original experiment was designed to evaluate the possible influence of non-exposed (i.e. pristine) communities (e.g. via immigration processes) in recovery dynamics. Laboratory channels were used to study the structural and functional changes in microbial communities after a 4-week Cu exposure period in the presence and absence of pristine biofilms. When pristine biofilms were present, phototrophic communities recovered within 6 weeks, both in terms of biomass, structure and photosynthetic activity. Recovery processes were also detected using the PICT approach. In contrast, in the absence of pristine communities, all of the Cu-induced changes recorded in the phototrophic communities remained throughout the recovery period. Regardless of the presence or absence of pristine biofilms, the decrease in Cu exposure did not abolish Cu-induced changes in bacterial community structure, whereas functional recovery (based on beta-glucosidase activity) was complete in both recovery contexts. These results revealed that microbial community response to a decrease in Cu exposure differs between phototrophic and heterotrophic communities. The presence of pristine communities greatly influences the structural and functional recovery of phototrophic communities, suggesting an important role of microbial immigration processes, but have far less influence on the recovery trajectory of heterotrophic communities.

Aims: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro-ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. Methods and Results: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal-nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. Conclusions: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal-nodulating rhizobia could enhance plant growth. Significance and Impact of the Study: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.

In this study, the first experiment analyzed the effects of 0, 85, 171, 256, and 342 mM sodium chloride (NaCl) on the germination of Acacia seyal seeds. In the second in vitro experiment, the effects of inoculation with two rhizobial strains on growth, physiological, and biochemical responses of A. seyal seedlings was evaluated at four levels (0, 85, 171, and 256 mM) of NaCl. Results showed that at 342 mM NaCl, germination rate of A. seyal seeds declined by 56% from the control value, while any germination was recorded for Acacia bivenosa and Acacia sclerosperma seeds at the same salt level. Salt stress gradually decreased the growth, the soluble protein and the leaf-chlorophyll contents of A. seyal seedlings. However, rhizobial inoculation limits these adverse effects of salt on physiological and metabolic processes responses. A. seyal exhibited a moderate halophytic behavior; nodulation was enhanced by moderate salt stress.

Aims: The aim of this study was to optimize DNA extraction from legume nodules to obtain large amounts of high-quality genomic DNA. Methods and Results: Nodules of different legume species were used. Varied concentrations of guanidine thiocyanate (from 6 mol l−1 to 0·05 mmol l−1), a component of DNAzol, were tested. The quality of DNA extract was determined by PCR–RFLP. The best results were obtained with 0·5 mmol l−1 guanidine thiocyanate, which resulted in greater DNA yield than with higher and lower concentrations or with DNAzol. Conclusion: The procedure using 0·5 mmol l−1 guanidine thiocyanate yields the highest DNA amount when compared with previously described protocols and offers a reliable method to isolate DNA from nodules of different origins. Significance and Impact of the Study: Irrespective of nodule origin, DNA yield was increased significantly, by two (e.g., Vigna nodules) to seven (Acacia auricoliformis nodules) times. In addition, the proposed procedure’s costs are lower than those using the DNAzol.

[Departement_IRSTEA]Eaux [TR1_IRSTEA]BELCA; International audience; Acacia senegal (L) Willd. and Acacia seyal Del. are highly nitrogen-fixing and moderately salt tolerant species. In this study we focused on the genetic and genomic diversity of Acacia mesorhizobia symbionts from diverse origins in Senegal and investigated possible correlations between the genetic diversity of the strains, their soil of origin, and their tolerance to salinity. We first performed a multi-locus sequence analysis on five markers gene fragments on a collection of 47 mesorhizobia strains of A. senegal and A. seyal from 8 localities. Most of the strains (60%) clustered with the M. plurifarium type strain ORS 1032T, while the others form four new clades (MSP1 to MSP4). We sequenced and assembled seven draft genomes: four in the M. plurifarium clade (ORS3356, ORS3365, STM8773 and ORS1032T), one in MSP1 (STM8789), MSP2 (ORS3359) and MSP3 (ORS3324). The average nucleotide identities between these genomes together with the MLSA analysis reveal three new species of Mesorhizobium. A great variability of salt tolerance was found among the strains with a lack of correlation between the genetic diversity of mesorhizobia, their salt tolerance and the soils samples characteristics. A putative geographical pattern of A. senegal symbionts between the dryland north part and the center of Senegal was found, reflecting adaptations to specific local conditions such as the water regime. However, the presence of salt does not seem to be an important structuring factor of Mesorhizobium species.

Nitrate in the unsaturated zone between the soil surface and the water table was studied in agroforestry Parklands in north western Senegal by examination of samples obtained by hand auger. Depending on location, water tables existed at depths between 10 and 35m below ground. Previous studies of groundwater in this region had found that large concentrations of nitrate were unconnected with anthropogenic activity. The objective of this study was to determine whether nitrogen fixing vegetation had a role in groundwater nitrate accumulation and whether roots of trees were located deeply enough to access the nitrate. Accordingly, sample profiles were augered close to stems of nitrogen fixing trees, non-nitrogen fixing trees and also in adjacent areas that were unaffected by tree presence. These adjacent areas were typically open pasture or cultivated fields. Tree fine roots were quantified in the samples and examined for the presence of mycorrhizas. Similarly, sand/soil samples were examined and tested for the presence of nitrogen fixing rhizobia that were capable of forming functional nodules on appropriate host plants. Concentrations of nitrate were greatest in soils beneath nitrogen fixing trees and nitrate was more plentiful in profiles augered beneath nitrogen fixing crops than it was elsewhere suggesting that N-fixation was the source of the nitrate. The concentrrations of nitrate that were found in the unsaturated zone were greatly in excess of the WHO recommended limit for nitrate in drinking water. High NO3-N/Cl ratios confirm insitu production of nitrate, and indicate that this is a natural baseline occurrence related to N-fixation. The nitrate is moving down the profile and impacts the groundwater unless it can be intercepted by plant roots. NO3-N amounts in solution in the soil profile varied between 75 and 1000kg ha−1 beneath trees and between 120 and 400kg ha−1 in areas outwith tree crowns. Although these quantities of N occupy the lower end of the range of N values obtained in north American deserts, they comprise a considerable dryland resource where amounts of organic fertilizer are limited and where cost prohibits the use of commercial fertilizers. Roots of both nitrogen fixing and non-nitrogen fixing trees were deep enough to access the nitrate but the small amounts of available water at intermediate depths suggest that large scale uptake of nitrate will only be possible in the wetter zones located close to the water table. Shallow roots tended to be more heavily colonized by mycorrhizas than deeper roots but mycorrhizas were recovered from roots located 22m below ground. Tree roots and rhizobia had similar patterns of distribution. They were commonest close to the soil surface, less frequent at intermediate depths and tended to increase in frequency close to the water table.

Summary Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0–0.25 m and 0.25–0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.

This study was conducted to evaluate the competitiveness and effectiveness of Thelephoroid fungal sp. ORS.XM002 against native ectomycorrhizal fungal species colonizing potted Afzelia africana seedlings during 3 months of growth in different forest soils collected from under mature trees. Using morphotyping and restriction fragment length polymorphism (RFLP) analysis of the nuclear rDNA internal transcribed spacer (ITS), we were able to distinguish the introduced Thelephoroid fungal sp. ORS.XM002 among native ectomycorrhizal fungal species that form ectomycorrhizae in A. africana seedlings. The morphotype (MT) of the introduced fungus showed some color variation, with a shift from light- to dark-brown observed from younger to older mycorrhizal tips. We were able to differentiate the ITS type xm002 of the introduced fungus from the 14 ITS-RFLP types characterizing the 9 native MT that occurred in forest soils. The frequency of ITS type xm002 ranged from 40% to 49% depending on the forest soil used, and was always higher than those of ITS types from native dark-brown MT that occurred in inoculated seedlings 3 months after inoculation. We considered Thelephoroid fungal sp. ORS.XM002 to be responsible for stimulation of mycorrhizal colonization of inoculated A. africana seedlings when compared with control seedlings in forest soils. This fungus appeared to be more effective in increasing the root dry weight of A. africana seedlings. To identify the unknown introduced fungal species and native MT, we sequenced the ML5/ML6 region of the mitochondrial large subunit rRNA. Sequence analysis showed that these fungi belong to three ML5/ML6 groups closely related to the Cortinarioid, Thelephoroid, and Sclerodermataceous taxa. The molecular evidence for the persistence of Thelephoroid fungal sp. ORS.XM002 despite competition from native fungi argues in favor of using this fungus with A. africana in nursery soil conditions in Senegal.

Acacia holosericea seedlings were planted in 1-l pots filled with a soil collected from an Australian Acacia plantation in Southern Senegal. After 6 months of culture, mycorrhizosphere soil, roots, galls induced by root-knot nematodes and Rhizobium nodules were sampled from each pot. The diversity of this bacterial group was characterized by siderotyping (pyoverdine IsoElectric Focusing (IEF) analysis) and by restriction fragment length polymorphism (RFLP). The effect of these isolates on the establishment of the ectomycorrhizal symbiosis between an Australian Acacia (A. holosericea) and Pisolithus sp. strain IR100 was studied. In the mycorrhizosphere soil, the population of fluorescent pseudomonads was represented by strains of two different siderovars (groups of bacterial strains presenting an identical pyoverdine-IEF pattern): siderovar 1 (74%) and siderovar 2 (26%). The siderotyping of the isolates around galls of the root-knot nematodes revealed three siderovars (40% from siderovar 1, 40% from siderovar 2 and about 15% from siderovar 3). RFLP of 16S rDNA divided the isolates into four different groups with MspI, two with HhaI and two with HaeIII endonucleases. The establishment of the ectomycorrhizal symbiosis with A. holosericea was promoted by 14 bacterial strains isolated from the mycorrhizosphere soil, three isolates from the roots and four from the galls. Shoot biomass of A. holosericea seedlings was stimulated by eight bacterial isolates from soil, six isolates from galls and seven from roots. These mycorrhiza helper bacteria could have a great ecological importance in tropical areas through the reforestation programs.

Summary A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus erinaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S–23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S–23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorhizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrhizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrhizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.

Intact soil cores were sampled from around three leguminous tree species (Acacia nilotica, Acacia tortilis and Prosopis juliflora) in 10-year-old alley-cropping plots at Thienaba, Senegal. Cores were removed from two depths (0–25 and 25–50 cm) and at two distances from the trunk (1 and 5 m). Duplicate soil cores were taken for assessment of root concentration (cm/100 cm3 soil), mycorrhizal infection (% of infected root length) and spore concentration in the soils. In order to determine the mycorrhizal inoculum potential (MIP) of the soils, a mycorrhizal bioassay of the soil cores was conducted in the greenhouse using millet seedlings. For all plots, seedlings grown in soils from the surface layer (0–25 cm) were larger and formed higher levels of infection than those grown in soils from 25 to 50 cm depth. Mycorrhizal infection of the seedlings was greatest in soil from the A. tortilis plots and, unlike the other tree species, also the greatest in soil collected near the tree. Positive relationships were found between the growth and infection of the bioassay seedlings and the root and spore concentrations in the field soils. Seedling growth and infection may also have been related to higher levels of carbon in the field soils. The results indicated that root and spore concentrations in the field soils were good indicators of MIP, but that levels of root infection were not. The results also indicated the potential benefit to crop yield of maintaining high levels of mycorrhizal propagules in alley-cropping soils and a possible role of the trees in maintaining these sources of inoculum.

Nous avons étudié une collection de 76 nouveaux isolats bactériens de nodules obtenus par piégeage sur plantes d'Acacia tortilis subsp. raddiana in vitro, inoculées avec des suspensions de sols prélevés dans divers sites au nord (Tunisie) et au sud (Sénégal, Mauritanie) du Sahara, en comparaison avec 54 souches de références. L'analyse des nouveaux isolats par RAPD et SDS-PAGE a montré une grande diversité taxonomique parmi les isolats d'A. tortilis subsp. raddiana. En effet, bien que toutes les souches puissent être qualifiées de « rhizobium à croissance rapide », elles se répartissent en au moins 9 groupes différents, dont plusieurs ne correspondent à aucun des groupes de rhizobia déjà décrits. Aucune relation entre la position taxonomique des souches, leur origine géographique, les conditions édaphiques ou la profondeur d'isolement n'a pu être établie. La caractérisation symbiotique des souches d'A. tortilis subsp. raddiana a, au contraire, montré une grande homogénéité des caractéristiques symbiotiques majeures. Toutes les souches nodulent également A. Senegal, Prosopis julifora et Leucaena leucocephala, qui constituent avec A. tortilis subsp. raddiana un groupe d'inoculation. L'analyse des profils chromatographiques des facteurs Nod (FN) d'une quinzaine de souches réparties dans les différents groupes taxonomiques montre qu'ils sont très similaires. L'homogénéité structurale des FN des souches d'A. tortilis subsp. raddiana a été confirmée par la détermination de la structure des FN de quelques souches représentatives. Cette étroite relation entre FN et plante d'isolement/spectre d'hôte indépendamment de la taxonomie des souches suggère l'existence d'une forte pression de sélection de la part de la plante hôte et confirme nos observations précédentes, à savoir que les facteurs Nod sont de bons indicateurs du potentiel de nodulation d'un rhizobium donné. We studied a collection of 76 new bacterial isolates from nodules developed on Acacia tortilis subsp. raddiana plants inoculated in vitro with soil suspensions harvested in different sites in North (Tunisia) and South (Senegal, Mauritania) parts of Sahara. RAPD and SDS-PAGE analyses in comparison with 54 reference strains from diverse origins showed great taxonomie diversity among the new isolates. All of them are fast-growing and form at least 9 different groups, several of which could correspond to undescribed groups. No relationship could be established between taxonomie groups, geographical origin (North/South of Sahara) of the strains, edaphic conditions, or depth of soil sampling (down to - 32 m). Opposite to their taxonomie diversity, the new isolates share identical main symbiotic properties. All strains nodulating A. tortilis subsp. raddiana can also nodulate Prosopis juliflora, Acacia Senegal and Leucaena leucocephala, these four plants then constituting a cross-inoculation group. By thin-layer chromatography, we showed that 20 A. tortilis subsp. raddiana strains, representative of the different taxonomie groups, share very similar Nod factor (NF) profiles, independently from their geographical origins. NF structure homogeneity was confirmed by NF structure determination of some representative strains. The consistency between NF and plant of isolation/nodulation spectrum, independently of the taxonomie position of the strains suggests a high selection pressure from the plant, and confirms our previous observations that NF are good markers for predicting the nodulation potential of a given rhizobial strain.

Fifty-eight new isolates were obtained from root nodules of common bean (#Phaseolus vulgaris$) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 #Rhizobium$ isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to #Rhizobium tropici$ type B and #Rhizobium etli$, respectively. By MLEE #R. etli$ and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains). (Résumé d'auteur)

Thirty isolates of root-nodulating bacteria obtained from Acacia cyanophylla, A. karroo, A. cyclops, A. tortilis (subsp.raddiana), Faidherbia albida and Acacia sp., grown in different regions of Libya, were studied by performing numerical analysis of 104 characteristics. Three fast- and one slow-growing reference strains from herbaceous and woody legumes were included. Five distinct clusters were formed. The fast-growing reference strains were separated from the isolates whereas the slow-growing was included in cluster 4. With the exception of one cluster, the majority of clusters were formed regardless of the host plant or site of origin. Based on plant tests, generation times, acid production and carbon utilization the isolates were diverse (fast and slow-growing isolates). Like slow-growing isolates, most of the fast-growing isolates appeared to be non-specific, nodulated many species from the same genus notably F. albida, known to nodulate only with slow-growing strains. Most clusters grew at temperatures 35 °C and 37 °C; some grew at temperatures above 40 °C. The majority of isolates grew at acid and alkaline pH and only one isolate grew below pH 4. Most isolates were able to utilize many amino acids as nitrogen sources and to reduce nitrate. Urea was hydrolysed by all clusters. Monosaccharides and polyols were used by slow and fast-growing isolates as the only carbon sources whereas assimilation of disaccharides varied: Some isolates, like slow-growing isolates, failed to utilize these carbon sources. Most isolates were unable to utilize polysaccharides. Regarding tolerance to NaCl on agar medium, the majority of isolates were unable to grow at a concentration of 2% NaCl, but some were highly resistant and there was one isolate which grew at 8% NaCl. Most isolates were resistant to heavy metals and to antibiotics.

Twenty-eight reference strains, 79 rhizobia isolated from Trigonella foenum graecum (fenugreek), 26 strains isolated from root nodules of Ceratonia siliqua (carob tree), 30 strains isolated from nodules of Adenocarpus decorticans and five isolated from Cytisus arboreus root nodules, were screened for their tolerance to increased concentrations of NaCl. Nine isolates of fenugreek were able to grow on medium containing 10% NaCl and one strain still grew at 14%. The effect of a range of salts at 2·5 and 5% (w/v) on the growth of rhizobia was assessed and it was shown that this effect depended on the ion form and the strains used. In general, NaCl appeared to be a good indicator of the tolerance of the strains to salts. The concentrations of the salts used were high and even at 5%, growth was not completely arrested in the less tolerant strains. Some substances, such as glutamate, proline, glycine betaine and CaCl2, were tested as osmotica. The effect of the two amino acids and betaine was confirmed with all the strains used whereas the alleviating effect of CaCl2 was not observed in all strains. This salt had different effects on two isolates of fenugreek. These results revealed a great diversity in salt tolerance, correlated with different responses to other stress conditions, which may be due to diversity in microbial ecology.

Several strains of Bradyrhizobium, Mesorhizobium and Sinorhizobium strains were tested for their compatibility with an Australian acacia: Acacia holosericea. All bacterial strains induced some nodule formation on roots. However, the rhizobia which greatly increased the development of seedlings belonged to the Bradyrhizobium genus. The root-knot nematode Meloidogyne javanica, strongly inhibited the symbiosis with most of the rhizobial strains. The rhizobia had no effect on nematode multiplication except for bacterial strain ORS 1020 where the final nematode population was higher than in the control treatment. The hypothesis concerning the interactions between nematodes and the nitrogen fixative process are discussed. Moreover, this investigation underlines the decreasing effect of nematodes on the potential benefits that may result from growing A. holosericea inoculated with selected rhizobial strains.

The genetic diversity of native cowpea rhizobia originating from 60 sites across four eco-geographic zones in Senegal Was studied. More than 300 cowpea nodules were analyzed by PCR-RFLP of the 16S-23S rDNA InterGenic Spacer region (IGS). Alignments of IGS sequences indicated that all genotypes were grouping within the Bradyrhizobium genus. The geographical distribution showed that apart from five IGS types, the others were specifically found in only one region. The diversity was significantly higher in the Senegal River valley zone, which presents lower mean annual rainfalls and slightly alkaline soils. Interestingly, two IGS types dominated the Senegalese rhizobial collection, one IGS type (VI) was found on more than half of the nodules collected in the northern Senegal River valley while another IGS type (I) was recovered from the great majority of nodules in the three other regions sampled. Two representative strains from each of these two dominant types were isolated and further analyzed. Multi Locus Sequence Analyses using 6 housekeeping genes indicate that they belong to a new Bradyrhizobium species closely related to B. yuanmingense. Phylogenetic analyses of 2 symbiotic genes nodC and nifH show that they are clustered with B. arachidis. Physiological tests on these strains have shown that under laboratory conditions, the growth of the IGS type VI strains was slightly less affected by a higher osmotic strength in the medium and to alkaline pH, which corroborates the soil physico-chemical parameters. (C) 2013 Elsevier GmbH. All rights reserved.

La culture du Niebe [Vigna unguiculata (L.) Walp.] en Afrique de l’Ouest est marquee par l’instabilite de ses rendements et sa faible production liee a la pauvrete des sols, a la faiblesse de la pluviometrie et aux contraintes parasitaires. Dans cette etude, nous nous sommes interesses a la reponse a l’inoculation mycorhizienne de quatre varietes de niebe dans une perspective de selection de souches efficaces de champignons endomycorhiziens pour l’amelioration de sa productivite au champ. Quatre varietes de niebe dont deux du Burkina Faso [Gorom Local (GO) et KVX 396-4-5-2D (KVX)] et deux du Senegal [Melakh (Mel) et Yacine (Yac)] ont ete inoculees en pepiniere avec trois (3) inoculums mixtes du Burkina Faso [Yakouta (Ya), Tale Mossi (Ta) et Soumousso (Sou)] et trois inoculums monospecifiques de la collection du Laboratoire Commun de Microbiologie de Dakar (LCM) [Glomus mossea (Gm), Glomus fasciculatum (Gf) et Glomus intraradices (Gi)]. Les effets des differents inoculums sur la croissance des plants ont ete evalues par la mesure de la hauteur des plants, des biomasses aerienne et racinaire et de la frequence et intensite de mycorhization a la floraison et a la fructification. Les resultats obtenus montrent que l’inoculation n’a pas eu d’effets significatifs sur les differents parametres mesures pour les varietes du Senegal malgre des taux de mycorhization de 6% a 92% a la floraison et de 44% a 100% a la fructification. Pour les varietes du Burkina Faso, a la floraison, la croissance en hauteur et la production de biomasse aerienne de la variete KVX ont ete ameliorees par toutes les souches, exceptee Gm. Pour la variete GO, les inoculums mixtes indigenes Ya et Ta ont significativement ameliore la croissance en hauteur des plants. En general, les frequences et les intensites de mycorhization sont elevees pour les souches monospecifiques, mais ceci ne se traduit pas par un effet significatif sur la croissance des plants inocules. L’inoculum mixte indigene Ya apparait plus performant pour la croissance en hauteur et la production de biomasse de la variete la plus sensible a l’inoculation (KVX). Cette etude a montre que l’inoculation endomycorhizienne a un effet benefique sur la croissance du Niebe, particulierement la variete KVX ; et que l’utilisation de souches indigenes conviendrait mieux que les souches monospecifiques. Il s’avere donc interessant de poursuivre la selection de souches performantes pour la productivite du Niebe sur les souches indigenes. Mots cles : champignons mycorhiziens, croissance du Niebe, pepiniere, Afrique de l’Ouest

International audience; Increasing interest has been given so far to the role of mycorrhizal symbiosis on plant diversity and ecosystem productivity. However much remains unknown about the effect of ectomycorrhizal fungal diversity on plant growth and rhizobial symbiosis. The purpose of this study is to investigate the influence of ectomycorrhizal diversity on root nodulation and plant nutrient uptake during plant growth. A gradient of ectomycorrhizal diversity was created (ranged from one to six fungal isolates) on Australian Acacia (Acacia mangium) seedlings grown on a phosphate-deficient soil in controlled conditions. The results dearly show (i) an enhancement of A. mangium growth due to ectomycorrhizal fungal inoculation, (ii) as well as significant effects of increasing ectomycorhizal diversity and soil ectomycorrhizal mycelium density on whole plant biomass and plant nutrient uptake. They also reveal an effect of ectomycorrhizal diversity effect on the functioning of the nitrogen-fixing symbiosis. These results emphasize the need to manage soil ectomycorrhizal potential (abundance and diversity of ectomycorrhizal mycelium) in forest management practices in order to optimize N-2 fixation and seedling growth, leading thus to dramatic improvements in outplanting performances with fast-growing tree legumes on various reafforestation sites, more particularly in tropical and Mediterranean areas. (C) 2012 Elsevier Ltd. All rights reserved.

Assessment of diversity and understanding factors underlying species distribution are fundamental themes in ecology. However, the diversity of native arbuscular mycorrhizal fungi (AMF) species in African tropical agro-ecosystems remains weakly known. This research was carried out to assess the morphological diversity of indigenous AMF species associated with rhizosphere of cowpea (Vigna unguiculata (L.) Walp.) in different agro-ecological zones (AEZ) of Benin and to examine the effects of soil chemical properties, climatic factors and agricultural practices on this diversity. Results showed that, in Benin, cowpea was grown by farmers in very exhausted soils, where available phosphorus and potassium were deficient. The indigenous AMF spore density was on average 202 spores per 100 g dry soil and there was no difference in the density among the agro-ecological zones (P = 0.56). Fifteen AMF morphospecies belonging to eight genera (Gigaspora, Scutellospora, Racocetra, Acaulospora, Funneliformis, Rhizophagus, Glomus and Claroideoglomus) were detected. The computed species richness estimators indicated that a limited number of additional undetected morphospecies are probably present in cowpea fields. According to analysis of similarity (ANOSIM), the arbuscular mycorrhizas community composition of the Sudanian zone and Guinean zone were not significantly different (r = −0.01; P = 0.517). The diversity of AMF morphospecies in the fields was weak with prevalence of Glomeraceae (92%). Furthermore, AMF diversity and evenness indices were negatively correlated with annual rainfall (P < 0.01) and with available phosphorus (P < 0.05). However, no significant correlation was observed between AMF diversity indices and soil organic carbon. Ultimately, this study tends to confirm that soil management practices (tillage, soil disturbance) have negative effect on AMF diversity.

Streams draining agricultural basins are subjected to the input of fungicides which can affect aquatic microbial communities. We analyzed the effect of the fungicide tebuconazole (TBZ) on Alnus glutinosa and Populus nigra litter breakdown by aquatic microorganisms. For six weeks, fungal and bacterial responses were analyzed in indoor stream channels subjected to TBZ-contaminated (33.1±12.4 μg L(-1)) and uncontaminated conditions. Litter breakdown rates decreased in presence of TBZ. The decrease was explained by reductions in microbial biomass development and shifts in community structure. At the same time, TBZ modified the kinetics of β-glucosidase, β-xylosidase and cellobiohydrolase enzymes resulting in lower affinities for cellulose and hemicellulose decomposition in leaves. These alterations were modulated by the litter quality; the greatest structural impairment was observed in Populus whereas Alnus were more affected in terms of leaf breakdown rate. Our results suggest that chronic exposure to TBZ can affect aquatic microbial communities and their capacity to break down leaf litter in streams.

Présentation du contexte Les systèmes écologiques des zones africaines sont soumis depuis des décennies à d’importantes perturbations climatiques (effets de la sécheresse et des fortes températures) et anthropiques (surexploitation des terres, modification des pratiques culturales). Ainsi, on assiste généralement à un accroissement de l’aridité d’origine édaphique et à une modification du couvert végétal et des paysages, qui affectent la productivité des systèmes de production et les conditio...

Many cowpea varieties (Vigna unguiculataL. Walp) have been proposed by research in Mali. None of these varieties were investigated for their symbiotic potential in terms of root nodulation and mycorrhizal infection. An experiment was conducted at Cinzana Research Station, from 2007 to 2009 with an objective to identify a cowpea variety with high symbiotic potential which may improve millet/cowpea cropping global production. Randomized complete block (RCBD) design with a factorial combination of 3 cowpea varieties (IT89KD-374, CZ1-94-23-1, and CZ11-94-5C) and 2 cropping systems (millet/cowpea intercropping and cowpea-millet rotation) was used. On farm test was conducted to evaluate CZ11-94-5C and IT89KD-374 nodulation performance. Cowpea variety CZ11-94-5-C had the highest nodule number and nodule weight. Millet/cowpea alternate rows intercropping (1/1), only, had a significant influence on cowpea root infection rates by mycorrhizae, on the 45th day after emergence. IT89KD-374 gave the best cowpea grain yield (1540 kg ha−1) in sole crop. The highest millet grain yield (1650 kg ha−1) was obtained under CZ11-94-5C-millet rotation. Farmers' fields assessments results confirmed CZ11-94-5C performance on research station. The CZ11-94-5C cowpea variety needs to be more characterized.

Il est désormais fréquent que des chercheurs en sciences sociales soient sollicités dans des programmes de recherche sur l'environnement et la biodiversité. Ce type de collaboration mobilise une attention croissante à l'hétérogénéité des formes de production et de légitimation des savoirs, d'autant plus forte lorsqu'il s'agit de programmes qui impliquent des experts non-scientifiques. Ce contexte génère des tensions, entre d'une part, la rationalisation gestionnaire des formes du travail scientifique et notamment de son évaluation standardisée, et d'autre part, l'ouverture épistémologique à la diversité des modes de légitimation des savoirs et par conséquent à une créativité dans les formes du travail qui contrecarre les tendances à la standardisation. C'est pour rendre compte de l'attention à la pluralité des savoirs et de ce qu'elle implique et qu'elle crée précisément dans le courant d'une recherche collective, que nous revenons sur une démarche photo-ethnographique menée dans le cadre d'un programme de recherche participatif et interdisciplinaire au Sénégal. Cette démarche a permis de : (i) recueillir des données sur les savoirs locaux et les savoirs scientifiques ; (ii) participer au partage des savoirs propres aux différents acteurs impliqués et (iii) restituer et diffuser des résultats de la recherche partenariale. Mais elle a surtout constitué une forme d'expérimentation collective de formes de communication, de mise en discussion et en circulation des savoirs singuliers mobilisés et produits dans le cadre du projet. Au travers de cette démarche photo-ethnographique est révélée l'importance de la construction d'une sémiotique pratique commune à l'ensemble des acteurs pour le partage effectif de savoirs multiples dans le cadre de recherche collaborative. Cette étude souligne la manière dont toute construction et toute expression d'un savoir quel qu'il soit est liée à des situations de communication. Une démarche réflexive sur les modes de co-construction de connaissance implique donc nécessairement un travail d'analyse et d'expérimentation sur les pratiques de communication dans des recherches qui associent des collectifs différents.

A total of 80 bacterial strains isolated from different Sesbania and Acacia species growing in various sites in Senegal (West Africa) were compared with 35 reference strains of Rhizobium, Bradyrhizobium, Azorhizobium, and Agrobacterium species and with 33 representative strains of the different groups of Brazilian isolates described on the basis of the results of a numerical analysis of the whole-cell protein patterns obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fifty-two strains could be placed in three protein electrophoretic clusters, two of which were different from the clusters containing various reference or representative strains, while 30 other strains could not be placed in any group. The strains belonging to the three clusters were studied by determining their nodulation host ranges and their morphological, physiological, and auxanographic characteristics. Representative strains of the three clusters were also genotypically characterized by determining their DNA base compositions, by performing DNA-DNA and DNA-rRNA hybridization experiments, and by determining their 16S rRNA gene sequences. Our results showed that two of the clusters identified on the basis of SDS-PAGE data are genotypically and phenotypically distinct groups that belong on the Rhizobium meliloti-Rhizobium fredii rRNA branch. The third cluster is localized on the Rhizobium loti rRNA branch in the vicinity of Rhizobium huakuii and contains strains isolated in Africa, in Brazil, and in New Zealand from different leguminous species. On the basis of the results of the present study, we propose to emend the genus Sinorhizobium and to reclassify R. meliloti as Sinorhizobium meliloti comb. nov. In addition, two new species, Sinorhizobium saheli and Sinorhizobium teranga, are proposed for isolates from Senegal.

Many fast growing tree species have been introduced to promote biodiversity rehabilitation on degraded tropical lands. Although it has been shown that plant productivity and stability are dependent on the composition and functionalities of soil microbial communities, more particularly on the abundance and diversity of soil symbiotic micro-organisms (mycorrhizal fungi and rhizobia), the impact of tree introduction on soil microbiota has been scarcely studied. This research has been carried in a field plantation of Acacia holosericea (Australian Acacia species) inoculated or not with an ectomycorrhizal fungus isolate, Pisolithus albus IR100. After 7 year's plantation, the diversity and the symbiotic properties of Bradyrhizobia isolated from the plantation soil or from the surrounding area ( Faidherbia albida (Del.) a. Chev. parkland) and able to nodulate F. albida , a native Sahelian Acacia species, have been studied. Results clearly showed that A. holosericea modified the structure of Bradyrhizobia populations and their effectiveness on F. albida growth. This negative effect was counterbalanced by the introduction of an ectomycorrhizal fungus, P. albus , on A. holosericea root systems. In conclusion, this study shows that exotic plant species can drastically affect genotypic and symbiotic effectiveness of native Bradyrhizobia populations that could limit the natural regeneration of endemic plant species such as F. albida . This effect could be counterbalanced by controlled ectomycorrhization with P. albus . These results have to be considered when exotic tree species are used in afforestation programs that target preservation of native plants and soil ecosystem rehabilitation.

Acacia nilotica (var. adansonii and var. tomentosa) is a tree species belonging to the family Leguminosae, subfamily Fabaceae (Allen and Allen, 1981). They are endemic to the sahelian arid and Sudanean semi-arid tropical regions, respectively. They have high socio-economic and ecological importance as sources of forage and wood for local populations and might improve the fixed-N status of soil due to their nitrogen-fixing

Aims: We characterized phenotypically and genotypically root-nodulating bacteria associated with Acacia senegal (L.) Willd. isolated from the soils surrounding A. senegal trees in the dry land area of Senegal. Methods and Results: The phenotypical and genotypical characterizations we carried out showed a high diversity of A. senegal root-nodulating bacteria. Phenotypic patterns showed adaptations of the rhizobial strains to many environmental stresses such as heat, drought, and salinity. Twelve molecular groups were distinguished by profiles obtained using polymerase chain reaction/restriction fragment length polymorphism techniques from intergenic spacer region rDNA. The highest genetic diversity was found around the A. senegal rhizosphere. Therefore, A. senegal seemed to have a positive influence on occurrence and genotypical diversity of rhizobial populations. Rhizobial isolates obtained in this study belonged phylogenetically to the genera Mesorhizobium and Rhizobium. Conclusions: Our results provided information about the genetic diversity of the rhizobial strains associated with A. senegal and suggested the adaptability of natural rhizobial populations to major ecological environmental stress within these soil environments. Significance and Impact of the Study: These results suggested a potential selection of compatible and well adapted strains under stress conditions as inoculants for successful A. senegal growth in arid lands.

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N(2)-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.

Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) of the InterGenic Spacer (IGS) between rDNA 16S and 23S was used to identify indigenous strains nodulating four clones of Acacia mangium–Acacia auriculiformis hybrids cultivated in non-sterilized sandy soil from Sangalkam (Senegal) under greenhouse conditions. The experiment was for 4 months. The analysis of restriction fragment length polymorphism obtained with MspI and HaeIII restriction enzymes allowed the identification of 15 different IGS Groups with a distribution which significantly differed according to the clone of the hybrid (strains of one clone can belong to three and five different IGS Groups). Three large multi-lobed nodules were obtained on the root system of clone 3.26 within 5 months. Also, the nature of the rhizobia contained in each lobe was determined. The results showed that the lobes of large nodules can be occupied by one or two strains and the nodules analysed were mainly occupied by those belonging to IGS Group 12.

Aims: In order to understand the genetic diversity of Acacia tortilis ssp. raddiana-rhizobia in Tunisia, isolates from nine geographical locations were obtained and analysed. Methods and Results: Characterization using restriction fragment length polymorphism analysis (RFLP) of PCR-amplified 16S rRNA gene and the intergenic spacer (IGS) between the 16S and 23S rRNA genes was undertaken. Symbiotic efficiency of the strains was also estimated. Analysis of the 16S rRNA by PCR-RFLP showed that the isolates were phylogenetically related to Ensifer ssp., Rhizobium tropicii-IIA, and Rhizobium tumefaciens species. Analysis of 16S-23S spacer by PCR-RFLP showed a high diversity of these rhizobia and revealed eleven additional groups, which indicates that these strains are genetically very diverse. Full 16S rRNA gene-sequencing showed that the majority of strains form a new subdivion inside the genera Ensifer, with Ensifer meliloti being its nearest neighbour. Nodulation test performed on the plant host demonstrated differences in the infectivity among the strains. Conclusion: Rhizobial populations that nodulate specifically and efficiently Acacia tortilis ssp. raddiana in representative soils of Tunisia is dominated by E. meliloti-like genomospecies. Significance and Impact of the Study: This paper provides the first clear characterization and symbiotic efficiency data of rhizobia strains nodulating A. tortilis in Tunisia.

Two strains of Bradyrhizobium sp., Aust 13C and Aust 11C, were dually or singly inoculated with an ectomycorrhizal fungus, Pisolithus albus to assess the interactions between ectomycorrhizal symbiosis and the nodulation process in glasshouse conditions. Sequencing of strains Aust 13C and Aust 11C confirmed their previous placement in the genus Bradyrhizobium. After 4 months' culture, the ectomycorrhizal symbiosis promoted plant growth and the nodulation process of both Bradyrhizobium strains, singly or dually inoculated. PCRIRFLP analysis of the nodules randomly collected in each treatment with Aust 13C and/or Aust 11C: (1) showed that all the nodules exhibited the same patterns as those of the Bradyrhizobium strains, and (2) did not detect contaminant rhizobia. When both Bradyrhizobium isolates were inoculated together, but without P. a/bus IR100, Aust 11C was recorded in 13% of the treated nodules compared to 87% for Aust 13C, where-as Aust 11C and Aust 13C were represented in 20 and 80% of the treated nodules, respectively, in the ectomycorrhizal treatment. Therefore Aust 13C had a high competitive ability and a great persistence in soil. The presence of the fungus did not significantly influence the frequencies of each Bradyrhizobium sp. root nodules. Although the mechanisms remain unknown, these results showed that the ectomycorrhizal and biological nitrogen-fixing symbioses were very dependent on each other. From a practical point of view, the role of ectomycorrhizal symbiosis is of great importance to N2 fixation and, consequently, these kinds of symbiosis must be associated in any controlled inoculation.

The purpose of this work was to determine the most efficient methods of inoculation to significantly improve nodulation and growth of Acacia senegal and Acacia nilotica, grown under greenhouse conditions. Our results showed that inoculation using dissolved alginate beads containing rhizobia significantly improved the growth of both acacia species better than the growth of plants in other treatments. The experiment with A. nilotica was conducted in two unsterilised soils from different areas. Plants grown in soil from Bel Air were well-nodulated and showed better growth than plants grown in soil from Sangalkam. However, no difference between these soils was shown between the several methods of inoculation and their effect on the nodulation and growth of plants. An interaction between A. senegal and A. nilotica provenances and the effect of inoculation with rhizobia was also demonstrated. Bel Air provenance of A. senegal, Dahra and RIM provenances of A. nilotica grew best of several provenances tested. These results suggest that (1) it may be possible to improve growth and yield of A. senegal and A. nilotica by careful selection of each symbiotic partner; and (2) nursery-grown seedlings of A. senegal and A. nilotica should be inoculated, just after sowing, with dissolved alginate beads containing a mixture of selected rhizobia. Resume. L’object de notre travail etait de determiner la methodologie d’inoculation la plus efficace pour ameliorer significativement la nodulation et la croissance d’Acacia senegal et d’Acacia nilotica poussant dans des conditions de serre. Nos resultats ont montre que l’inoculation utilisant des billes d’alginate dissoutes ameliorait significativement la croissance des deux especes d’Acacia par rapport a celle des plantes des autres traitements. L’experience avec A. nilotica a ete faite dans deux types de sol non steriles. Nous avons observe que les plants poussant dans le sol de Bel Air etaient plus nodules et mieux developpes que ceux poussant dans le sol de Sangalkam. Cependant, aucune difference significative entre les deux sols n’a ete mise en evidence en termes d’effet des differentes methodologies d’inoculation sur la nodulation et la croissance des plantes. Il a aussi ete demontre l’existence d’une interaction entre les provenances d’A. senegal et d’A. nilotica et l’effet de l’inoculation avec rhizobium. C’est ainsi que les provenances Bel Air d’A. senegal, Dahra et RIM d’A. nilotica ont le mieux pousse parmi les differentes provenances testees. L’ensemble de ces resultats suggerent: (1) il devrait etre possible d’ameliorer la croissance et la production d’A. senegal et d’A. nilotica par une selection des deux partenaires symbiotiques; (2) Les jeunes plantules d’A. senegal et d’A. nilotica produites en pepiniere devraient etre inoculees juste apres le semis avec des billes d’alginate dissoutes contenant un cocktail de souches selectionnees.

The aim of our work was to assess the growth and mineral nutrition of salt stressed Acacia auriculiformis A. Cunn. ex Benth. and Acacia mangium Willd. seedlings inoculated with a combination of selected microsymbionts (bradyrhizobia and mycorrhizal fungi). Plants were grown in greenhouse conditions in non-sterile soil, irrigated with a saline nutrient solution (0, 50 and 100 mm NaCl). The inoculation combinations consisted of the Bradyrhizobium strain Aust 13c for A. mangium and Aust 11c for A. auriculiformis, an arbuscular mycorrhizal fungus (Glomus intraradices, DAOM 181602) and an ectomycorrhizal fungus (Pisolithus albus, strain COI 007). The inoculation treatments were designed to identify the symbionts that might improve the salt tolerance of both Acacia species. The main effect of salinity was reduced tree growth in both acacias. However, it appeared that, compared with controls, both rhizobial and mycorrhizal inoculation improved the growth of the salt-stressed plants, while inoculation with the ectomycorrhizal fungus strain appeared to have a small effect on their growth and mineral nutrition levels. Endomycorrhizal inoculation combined with rhizobial inoculation usually gave good results. Analysis of foliar proline accumulation confirmed that dual inoculation gave the trees better tolerance to salt stress and suggested that the use of this dual inoculum might be beneficial for inoculation of both Acacia species in soils with moderate salt constraints.

Summary We used direct recovery of bacterial 16S rRNA gene sequences to investigate the bacterial diversity under Acacia tortilis subsp. raddiana , a legume tree naturally growing in the dry land part of Senegal (West Africa). Microbial DNA was purified directly from soil samples and subjected to PCR with primers specific for bacterial 16S rRNA gene sequences. 16S rDNA clone libraries were constructed from two soil samples taken at two dates, i.e. June 25 th 1999 (dry season) and August 28 th 1999 (rainy season) at depths of 0.25–0.50 m and at 3 m distance from the stem. The 16S rDNA of 117 clones was partially sequenced. Phylogenetic analysis of these sequences revealed extensive diversity (100 phylotypes). Comparative sequence analysis of these clones identified members of the Gammaproteobacteria (35% of the phylotypes) as the most important group, followed by the Firmicutes division with 24%. Alphaproteobacteria , Betaproteobacteria , Acidobacteria and Actinobacteria were found to be less represented. Our data suggest that bacterial communities under Acacia tortilis subsp. raddiana might differ according to the season. The relative compositions of the populations is different in both samples: the Acidobacteria are present in a much higher percentage in the dry season than in the rainy season sample while the inverse effect is observed for the members of the other groups. Within the Gammaproteobacteria we found a shift between the dry season and the rainy season from pseudomonads to Acinetobacter and Escherichia related organisms.

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.

The aim of this study was to use several approaches to assess the diversity occuring in a collection of rhizobial strains that had been isolated from Gliricidia sepium (Jacq.) Walp. originating from Kenya, Reunion Island and New Caledonia. Results showed that G. sepium establishes an effective symbiosis only with fast-growing rhizobia. This suggests that this legume nodulates preferentially with this type of rhizobia, although ineffective nodules were observed on root systems of plants inoculated with slow-growing rhizobia. It was possible to distinguish several groups of strains which have the same behaviour in terms of utilization of amino acids and sources of carbon and intrinsic resistance to antibiotics and salinity. Six molecular groups were distinguished by profiles obtained using PCR/RFLP techniques. No relationship was demonstrated between phenotypic groups and molecular groups. Neither was it possible to find a relationship between the geographic origins of the strains and their distribution in the several phenotypic and/or molecular groups. In an experiment carried out under glasshouse conditions, we observed that all strains from the collection significantly improved the growth of G. sepium cultivated in a substrate totally devoid of nitrogen. The strain GsK5 induced the greatest stimulation of plant growth.

L'association d'Acacia tortilis (Forssk.) Hayne subsp. raddiana (Savi) Brenan avec les bactéries du sol à croissance rapide de la famille des rhizobiacées joue un rôle important dans l'amélioration de la nutrition minérale de cette plante. Cependant, les caractéristiques de cette symbiose ont été très peu étudiées en conditions naturelles. Ce travail a été conduit au Sénégal sur des peuplements naturels d'Acacia raddiana de deux sites du Ferlo sénégalais : Souilène et Dahra-Djoloff. La répartition dans l'espace et les variations saisonnières des nodules présents sur le système racinaire de surface d'Acacia raddiana (0-1 m) ont été étudiées en relation avec les conditions hydriques du sol. À cet effet, des prélèvements de sol et des extractions ont été effectués à différentes périodes de l'année. Le maximum de nodules a été rencontré entre 2 et 3 mètres du tronc, et à une profondeur comprise entre 25 et 75 cm. Une biomasse importante de nodules apparemment très fixateurs d'azote a été produite pendant la saison pluvieuse. Ces nodules ont une durée de vie courte, et apparaissent desséchés pendant la majeure partie de l'année. La densité des nodules apparaît corrélée à la biomasse racinaire, mais également à l'humidité du sol de la rhizosphère. The association between Acacia tortilis (Forssk.) Hayne subsp. raddiana (Savi) Brenan and soil bacteria with rapid growth belonging to the Family Rhizobiaceae plays an important role in the improvement of the mineral nutrition of this plant. However, the characteristics of this symbiotic relationship have been very little studied under natural conditions. This work has been conducted in Senegalese natural populations of Acacia raddiana in two sites: Souilène and Dahra-Djoloff (Ferlo). The spatial distribution around tree and the seasonal variations of nodules present on the root system of Acacia raddiana (0-1 m) were studied under different soil moisture conditions. For that, extraction of soil samples was made at the different periods of the year. The maximum number of nodules was obtained between 2 and 3 m far from the tree trunk, and between 25 and 75 cm soil depth. A significant nodule biomass apparently more efficient in N fixation was obtained during the rainy season. These nodules have short life span, and appeared dried during the greater part of the year. The density of the nodules correlated significantly with the root biomass, and also with the soil moisture content of the rhizosphere. nodulation, root, natural population, soil water budget, acacia tortilis (forssk.), hayne subsp. raddiana (savi), brenan

The diversity of Bradyrhizobium strains nodulating three cowpea (Vigna unguiculata L. Walp.) cultivars in favorable and water-limited conditions occuring at flowering was analysed. PCR- Restriction Fragment Length Polymorphism (PCR-RFLP) analysis of 16S-23S rDNA intergenic spacer region (IGS) directly applied on 85 crushed nodules distinguished four genetic profiles, IGS types I, II, III and IV. The distribution of these IGS types according to water conditions and cowpea cultivars (B-21, TN 88-63 and Mouride) showed that nodulating strains appeared more diverse in water-limited condition. More than three quarters of prospected nodules presented the IGS type I. They were formed on all three cultivars and in both water conditions. Only a small part of nodules was distributed between the IGS type II, III and IV. Nodules showing the IGS types II and III were found mainly in limited conditions on TN 88-63 and Mouride cultivars, whereas nodules presenting the IGS type IV were collected only from cultivars B-21 and Mouride, in both water conditions. Strains corresponding to the different profiles were isolated. The phylogenetic analysis of 16S rRNA gene sequences showed that they belong to the genus, Bradyrhizobium. The sequence analysis of 16S-23S rDNA IGS revealed that the strains exhibiting IGS types II, III and IV were closely related to some Faidherbia albida isolates from Senegal. IGS type II can be assigned with at least 98% similarity to Bradyrhizobium genospecies IV. IGS types III and IV showed more than 96% similarity with genospecies VII and could belong to the same genospecies. IGS type I, the most frequent, exhibits low IGS similarity with reported sequences in the databases, and could represent a new genospecies. (African Journal of Biotechnology: 2003 2(1): 13-23)

International audience; Our work concerned the optimization of inoculation of two agroforestry species of woody leguminous plants: Leucaena leucocephala and Acacia mangium with various strains of rhizobium. First, we showed that the physiological stage of the bacterial culture had no effect on nodulation and growth of the seedlings of Acacia mangium inoculated and cultivated in vitro for four months. For Leucaena leucocephala, the number of nodules was significantly higher when the seedlings were inoculated with a bacterial culture in stationary phase. On the other hand, whatever the species, no significant difference was noted with regards to the dry weight of the shoots. The effect of the size of inoculum on the nodulation and growth of the seedlings was studied in L. leucocephala after five months in a greenhouse. Our result show that an inoculation with bacterial cultures containing 10$^9$ to 10$^{10}$ bacteria per milliliter are the optimal conditions to have a maximum nodulation and growth of the seedlings. The two legume plant species showed significant differences with regard to the effect of the method of inoculation on nodulation and growth of the seedlings. For Acacia mangium, inoculation with a liquid culture one week after sowing was more favourable for the growth of the seedlings. On the other hand, for Leucaena leucocephala, this method of inoculation and the coating of seeds with a bacterial culture mixed with arabic gum improved significantly the growth of the seedlings. Results obtained in our study can be useful for the partners from developing countries involved in the large scale production of tree seedlings.; Optimisation de l'inoculation de Leucaena leucocephala et de Acacia mangium avec rhizobium en conditions de serre. Nos travaux ont porté sur l'optimisation de l'inoculation de 2 espèces agroforestières de légumineuses ligneuses : Leucaena leucocephala et Acacia mangium avec différentes souches de rhizobium. Tout d'abord, nous avons montré que le stade physiologique de la culture bactérienne n'a aucune influence sur la nodulation et la croissance des plants d'Acacia mangium inoculés et cultivés in vitro pendant 4 mois. Pour Leucaena leucocephala, le nombre de nodules est significativement supérieur pour les plants inoculés avec une culture bactérienne en phase plateau. En revanche, quelle que soit l'espèce, aucune différence significative n'est notée en ce qui concerne le poids sec des parties aériennes. L'effet de la taille de l'inoculum sur la nodulation et la croissance des plants de L. leucocephala a été étudié après 5 mois de culture en serre. Nos résultats ont montré qu'une inoculation avec des cutures bactériennes contenant 10$^{10}$ à 10$^9$ bactéries par millilitre sont les conditions optimales pour avoir une nodulation et une croissance maximale des plants. Les 2 espèces de légumineuses montrent des différences significatives en ce qui concerne l'effet de la méthode d'inoculation sur la nodulation et la croissance des plants. Pour Acacia mangium, l'inoculation avec une culture liquide une semaine après les semis est plus favorable pour la croissance de la plante. En revanche, pour Leucaena leucocephala, cette méthode classique d'inoculation de même que l'enrobage des semis avec une culture bactérienne mélangée avec de la gomme arabique améliorent significativement la croissance de la plante. Nos travaux pourront servir aux partenaires du développement pour la production en régie de plants forestiers de ces 2 espèces.