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

In response to the presence of compatible rhizobium bacteria, legumes form symbiotic organs, called nodules, on their roots. These nodules house nitrogen-fixing bacteroids which are a differentiated form of the rhizobium bacteria. In some legumes, the bacteroid differentiation comprises a dramatic cell enlargement, polyploidization and other morphological changes. Here we demonstrate that a peptidoglycan-modifying enzyme in Bradyrhizobium strains, a DD-carboxypeptidase that contains a peptidoglycan-binding SPOR domain, is essential for normal bacteroid differentiation in Aeschynomene species. The corresponding mutants formed bacteroids that are malformed and hypertrophied. However, in soybean, a plant that does not induce morphological differentiation of its symbiont, the mutation does not affect the bacteroids. Remarkably, the mutation also leads to necrosis in a large fraction of the Aeschynomene nodules indicating that a normally formed peptidoglycan layer is essential for avoiding the induction of pla...

Among semi-aquatic species of the legume genus Aeschynomene, some have the unique property of being root and stem-nodulated by photosynthetic Bradyrhizobium lacking the nodABC genes necessary for the production of Nod factors. These species provide an excellent biological system with which to explore the evolution of nodulation in legumes. Among them, Aeschynomene evenia has emerged as a model legume to undertake the genetic dissection of the so-called Nod-independent symbiosis. In addition to the genetic analysis of nodulation on a reference line, natural variation in a germplasm collection could also be surveyed to uncover genetic determinants of nodulation. To this aim, we investigated the patterns of genetic diversity in a collection of 226 Nod-independent Aeschynomene accessions. A combination of phylogenetic analyses, comprising ITS and low-copy nuclear genes, along with cytogenetic experiments and artificial hybridizations revealed the richness of the Nod-independent Aeschynomene group with the identification of 13 diploid and 6 polyploid well-differentiated taxa. A set of 54 SSRs was used to further delineate taxon boundaries and to identify different genotypes. Patterns of microsatellite diversity also illuminated the genetic basis of the Aeschynomene taxa that were all found to be predominantly autogamous and with a predicted simple disomic inheritance, two attributes favorable for genetics. In addition, taxa displaying a pronounced genetic diversity, notably A. evenia, A. indica and A. sensitiva, were characterized by a clear geographically-based genetic structure and variations in root and stem nodulation. A well-characterized germplasm collection now exists as a major genetic resource to thoroughly explore the natural variation of nodulation in response to different bradyrhizobial strains. Symbiotic polymorphisms are expected to be found notably in the induction of nodulation, in nitrogen fixation and also in stem nodulation. Subsequent genetic analysis and locus mapping will pave the way for the identification of the underlying genes through forward or reverse genetics. Such discoveries will significantly contribute to our understanding of the molecular mechanisms underpinning how some Aeschynomene species can be efficiently nodulated in a Nod-independent fashion.

Rhizobia are soil bacteria that are able to form symbiosis with plant hosts of the legume family. These associations result in the formation of organs, called nodules in which bacteria fix atmospheric nitrogen to the benefit of the plant. Most of our knowledge on the metabolism and the physiology of the bacteria during symbiosis derives from studying roots nodules of terrestrial plants. Here we used a proteomics approach to investigate the bacterial physiology of photosynthetic Bradyrhizobium sp. ORS278 during the symbiotic process with the semi aquatical plant Aeschynomene indica that forms root and stem nodules. We analyzed the proteomes of bacteria extracted from each type of nodule. First, we analyzed the bacteroid proteome at two different time points and found only minor variation between the bacterial proteomes of 2-week- and 3-week-old nodules. High conservation of the bacteroid proteome was also found when comparing stem nodules and root nodules. Among the stem nodule specific proteins were those related to the phototrophic ability of Bradyrhizobium sp. ORS278. Furthermore, we compared our data with those obtained during an extensive genetic screen previously published. The symbiotic role of four candidate genes which corresponding proteins were found massively produced in the nodules but not identified during this screening was examined. Mutant analysis suggested that in addition to the EtfAB system, the fixA locus is required for symbiotic efficiency.

Reducing the use of chemical inputs (fertilizers, pesticides) in agriculture while maintaining crop productivity is the main challenge facing sub-Saharan African family farming systems. The use of effective microorganisms (EM) is among the various innovative approaches for minimizing chemical inputs and the environmental impact of agricultural production and protecting soil health while enhancing crop yields and improving food security. This study sought to characterize the microbial biodiversity of local beneficial microorganisms (BMs) products from locally fermented forest litter and investigate their ability to enhance tomato plant growth and development. Beneficial microorganisms (BMs) were obtained by anaerobic fermentation of forest litter collected in four agroecological regions of Senegal mixed with sugarcane molasses and various types of carbon sources (groundnut shells, millet stovers, and rice bran in different proportions). The microbial community composition was analyzed using next-generation rDNA sequencing, and their effects on tomato growth traits were tested in greenhouse experiments. Results show that regardless of the litter geographical collection site, the dominant bacterial taxa in the BMs belonged to the phyla Firmicutes (27.75–97.06%) and Proteobacteria (2.93–72.24%). Within these groups, the most prevalent classes were Bacilli (14.41–89.82%), α-proteobacteria (2.83–72.09%), and Clostridia (0.024–13.34%). Key genera included Lactobacillus (13–65.83%), Acetobacter (8.91–72.09%), Sporolactobacillus (1.40–43.35%), and Clostridium (0.08–13.34%). Fungal taxa were dominated by the classes Leotiomycetes and Sordariomycetes, with a prevalence of the acidophilic genus Acidea. Although microbial diversity is relatively uniform across samples, the relative abundance of microbial taxa is influenced by the litter’s origin. This is illustrated by the PCoA analysis, which clusters microbial communities based on their litter source. Greenhouse experiments revealed that five BMs (DK-M, DK-G, DK-GM, NB-R, and NB-M) significantly (p < 0.05) enhanced tomato growth traits, including plant height (+10.75% for DK-G and +9.44% for NB-R), root length (+56.84–62.20%), root volume (+84.32–97.35%), root surface area (+53.16–56.72%), and both fresh and dry shoot biomass when compared to untreated controls. This study revealed that forest-fermented litter products (BMs), produced using litter collected from various regions in Senegal, contain beneficial microorganisms known as plant growth-promoting microorganisms (PGPMs), which enhanced tomato growth. These findings highlight the potential of locally produced BMs as an agroecological alternative to inorganic inputs, particularly within Senegal’s family farming systems.

Soybean (Glycine max) is a protein-rich legume crop that plays an important role in achieving food security. The aim of this study was to isolate soybean-nodulating rhizobia from Côte d’Ivoire soils and evaluate their potential as efficient strains in order to develop local bioinoculants. For this objective, 38 composite soil samples were collected from Côte d’Ivoire’s five major climatic zones. These soils were used as substrate to trap the nodulating rhizobia using the promiscuous soybean variety R2-231. A total of 110 bacterial strains were isolated and subsequently identified. The analysis of ITS (rDNA16S-23S), glnII and recA sequences revealed a relatively low genetic diversity of these native rhizobia. Moreover, the ITS phylogeny showed that these were scattered into two Bradyrhizobium clades dominated by the B. elkanii supergroup, with ca. 75% of all isolates. Concatenated glnII-recA sequence phylogeny confirmed that the isolates belong in the majority to ‘B. brasilense’, together with B. vignae and some putative genospecies of Bradyrhizobium that needs further elucidation. The core gene phylogeny was found to be incongruent with nodC and nifH phylogenies, probably due to lateral gene transfer influence on the symbiotic genes. The diversity and composition of the Bradyrhizobium species varied significantly among different sampling sites, and the key explanatory variables identified were carbon (C), magnesium (Mg), nitrogen (N), pH, and annual precipitation. Based on both shoot biomass and leaf relative chlorophyll content, three isolates consistently showed a higher symbiotic effectiveness than the exotic inoculant strain Bradyrhizobium IRAT-FA3, demonstrating their potential to serve as indigenous elite strains as bioinoculants.