Development of an Illumina-based analysis method to study bradyrhizobial population structure-case study on nitrogen-fixing rhizobia associating with cowpea or peanut.

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.

Complete Genome Sequence of Bradyrhizobium sp. Strain ORS3257, an Efficient Nitrogen-Fixing Bacterium Isolated from Cowpea in Senegal.

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.

Genetic and genomic diversity studies of Acacia symbionts in Senegal reveal new species of Mesorhizobium with a putative geographical pattern.

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.

Eco-geographical diversity of cowpea bradyrhizobia in Senegal is marked by dominance of two genetic types.

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.

Long-term impact of hydrological regime on structure and functions of microbial communities in riverine wetland sediments.

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 département (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.

Effects of the fungicide tebuconazole on microbial capacities for litter breakdown in streams.

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 ß-glucosidase, ß-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.

Structural and functional recovery of microbial biofilms after a decrease in copper exposure: influence of the presence of pristine communities.

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.

Effects of a rock phosphate on indigenous rhizobia associated with Sesbania sesban.

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.

Genetic diversity of Acacia seyal Del. rhizobial populations indigenous to Senegalese soils in relation to salinity and pH of the sampling sites.

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.

Symbiosis of Acacia auriculiformis and Acacia mangium with mycorrhizal fungi and Bradyrhizobium spp. improves salt tolerance in greenhouse conditions.

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.

Rhizobial populations in soils from natural Acacia senegal and Acacia nilotica forests in Mauritania and the Senegal river valley.

Eighty-two strains of rhizobia were isolated from soils taken from several sites in Mauritania and Senegal. These soil samples were collected from natural stands of Acacia nilotica and Acacia senegal. The soils from Mauritania were less rich in native rhizobia than the soils from Senegal. The strains were characterized using polymerase chain reaction-restriction fragment length polymorphism and by sequencing the rDNA 16S-23S intergenic spacer region (IGS). They were sorted into seven IGS groups. These groups were not associated with the geographical origin of the strains or with the host-plant species at the site where the soils were collected. Most of the strains were in three of the IGS groups (I, IV, and V). One representative strain from each IGS group was sequenced and showed that the strains were from the genus Mesorhizobium. IGS groups I, IV, and VI were close to the species M. plurifarium (AF34563), IGS groups IIand III were close to the species Mesorhizobium sp. (AF510360), IGS group V was close to the species Mesorhizobium sp. (AF510366), and IGS group VII was close to Mesorhizobium sp. (AF510346).

Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal.

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.

Phylogenetic analysis of partial bacterial 16S rDNA sequences of tropical grass pasture soil under Acacia tortilis subsp. raddiana in Senegal.

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 25th 1999 (dry season) and August 28th 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.

Symbiotic and taxonomic diversity of rhizobia isolated from Acacia tortilis subsp. raddiana in Africa.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana from various sites in the North and South of Sahara was analyzed for their diversity at both taxonomic and symbiotic levels. On the basis of whole cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most of the strains were found to belong to the Sinorhizobium and Mesorhizobium genera where they may represent several different genospecies. Despite their chromosomal diversity, most A. tortilis Mesorhizobium and Sinorhizobium symbionts exhibited very similar symbiotic characters. Nodulation tests showed that the strains belong to the Acacia-Leucaena-Prosopis nodulation group, although mainly forming non-fixing nodules on species other than A. tortilis. Most of the strains tested responded similarly to flavonoid nod gene inducers, as estimated by using heterologous nodA-lacZ fusions. Thin layer chromatography analysis of the Nod factors synthesized by overproducing strains showed that most of the strains exhibited similar profiles. The structures of Nod factors produced by four different Sinorhizobium sp. strains were determined and found to be similar to other Acacia-Prosopis-Leucaena nodulating rhizobia of the Sinorhizobium-Mesorhizobium-Rhizobium branch. They are chitopentamers, N-methylated and N-acylated by common fatty acids at the terminal non reducing sugar. The molecules can also be 6-O sulfated at the reducing end and carbamoylated at the non reducing end. The phylogenetic analysis of available NodA sequences, including new sequences from A. tortilis strains, confirmed the clustering of the NodA sequences of members of the Acacia-Prosopis-Leucaena nodulation group.

Phylogenetic relationships in Termitomyces (Family Agaricaceae) based on the nucleotide sequence of ITS: a first approach to elucidate the evolutionary history of the symbiosis between fungus-growing termites and their fungi.

Termitomyces constitutes a very poorly known genus of fungi whose essential characteristic is that all representatives of the genus are cultivated by termites (Macrotermitinae) in their nest and that all the fungi cultivated by termites belong to this genus. For the first time, the phylogenetic relationships of several African Termitomyces species was studied by the sequencing of their internal transcriber spacer region (ITS1--5.8S--ITS2). It appeared that this group is clearly monophyletic and belongs to the Tricholomataceae family. The total homology of the ITS zone of several Termitomyces symbionts of different termite genera indicated that the specific diversity of this group is in fact less important than previously supposed. Finally, the comparison between the Termitomyces phylogenetic tree and the taxonomic tree of Macrotermitinae showed that if for certain genera the hypothesis of termite/fungus coevolution is acceptable, it should not be applied for all symbiosis.

Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal.

A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus 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 Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium 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.

Interactions between ectomycorrhizal symbiosis and fluorescent pseudomonads on Acacia holosericea: isolation of mycorrhiza helper bacteria (MHB) from a Soudano-Sahelian soil.

Abstract 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.