Bradyrhizobium ivorense sp. nov. as a potential local bioinoculant for Cajanus cajan cultures in Côte d'Ivoire.

For many smallholder farmers of Sub-Saharan Africa, pigeonpea (Cajanus cajan) is an important crop to make ends meet. To ascertain the taxonomic status of pigeonpea isolates of Côte d'Ivoire previously identified as bradyrhizobia, a polyphasic approach was applied to strains CI-1BT, CI-14A, CI-19D and CI-41S. Phylogeny of 16S ribosomal RNA (rRNA) genes placed these nodule isolates in a separate lineage from current species of the B. elkanii super clade. In phylogenetic analyses of single and concatenated partial dnaK, glnII, gyrB, recA and rpoB sequences, the C. cajan isolates again formed a separate lineage, with strain CI-1BT sharing the highest sequence similarity (95.2 %) with B. tropiciagri SEMIA 6148T. Comparative genomic analyses corroborated the novel species status, with 86 % ANIb and 89 % ANIm as the highest average nucleotide identity (ANI) values with B. elkanii USDA 76T. Although CI-1BT, CI-14A, CI-19D and CI-41S shared similar phenotypic and metabolic properties, growth of CI-41S was slower in/on various media. Symbiotic efficacy varied significantly between isolates, with CI-1BT and CI-41S scoring on the C. cajan 'Light-Brown' landrace as the most and least proficient bacteria, respectively. Also proficient on Vigna radiata (mung bean), Vigna unguiculata (cowpea, niébé) and additional C. cajan cultivars, CI-1BT represents a potential bioinoculant adapted to local soil conditions and capable of fostering the growth of diverse legume crops in Côte d'Ivoire. Given the data presented here, we propose the 19 C. cajan isolates to belong to a novel species called Bradyrhizobium ivorense sp. nov., with CI-1BT (=CCOS 1862T=CCMM B1296T) as a type strain.

Loss of NifQ Leads to Accumulation of Porphyrins and Altered Metal-Homeostasis in Nitrogen-Fixing Symbioses.

Symbiotic nitrogen fixation between legumes and rhizobia involves a coordinated expression of many plant and bacterial genes as well as finely tuned metabolic activities of micro- and macrosymbionts. In spite of such complex interactions, symbiotic proficiency remains a resilient process, with host plants apparently capable of compensating for some deficiencies in rhizobia. What controls nodule homeostasis is still poorly understood and probably varies between plant species. In this respect, the promiscuous Sinorhizobium (Ensifer) fredii strain NGR234 has become a model to assess the relative contribution of single gene products to many symbioses. Here, we describe how a deletion in nifQ of NGR234 (strain NGRΔnifQ) makes nodules of Vigna unguiculata, V. radiata, and Macroptilium atropurpureum but not of the mimisoid tree Leucaena leucocephala, purple-red. This peculiar dark-nodule phenotype did not necessarily correlate with a decreased proficiency of NGRΔnifQ but coincided with a 20-fold or more accumulation of coproporphyrin III and uroporphyrin III in V. unguiculata nodules. Porphyrin accumulation was not restricted to plant cells infected with bacteroids but also extended to the nodule cortex. Nodule metal-homeostasis was altered but not sufficiently to prevent assembly and functioning of nitrogenase. Although the role of NifQ in donating molybdenum during assembly of nitrogenase cofactor FeMo-co makes it essential in free-living diazotrophs, our results highlight the dispensability of NifQ in many legume species.

Ribosomal protein biomarkers provide root nodule bacterial identification by MALDI-TOF MS.

Accurate identification of soil bacteria that form nitrogen-fixing associations with legume crops is challenging given the phylogenetic diversity of root nodule bacteria (RNB). The labor-intensive and time-consuming 16S ribosomal RNA (rRNA) sequencing and/or multilocus sequence analysis (MLSA) of conserved genes so far remain the favored molecular tools to characterize symbiotic bacteria. With the development of mass spectrometry (MS) as an alternative method to rapidly identify bacterial isolates, we recently showed that matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) can accurately characterize RNB found inside plant nodules or grown in cultures. Here, we report on the development of a MALDI-TOF RNB-specific spectral database built on whole cell MS fingerprints of 116 strains representing the major rhizobial genera. In addition to this RNB-specific module, which was successfully tested on unknown field isolates, a subset of 13 ribosomal proteins extracted from genome data was found to be sufficient for the reliable identification of nodule isolates to rhizobial species as shown in the putatively ascribed ribosomal protein masses (PARPM) database. These results reveal that data gathered from genome sequences can be used to expand spectral libraries to aid the accurate identification of bacterial species by MALDI-TOF MS.

The core bacteriobiome of Côte d'Ivoire soils across three vegetation zones.

The growing understanding that soil bacteria play a critical role in ecosystem servicing has led to a number of large-scale biogeographical surveys of soil microbial diversity. However, most of such studies have focused on northern hemisphere regions and little is known of either the detailed structure or function of soil microbiomes of sub-Saharan African countries. In this paper, we report the use of high-throughput amplicon sequencing analyses to investigate the biogeography of soil bacteria in soils of Côte d'Ivoire. 45 surface soil samples were collected from Côte d'Ivoire, representing all major biomes, and bacterial community composition was assessed by targeting the V4-V5 hypervariable region of the 16S ribosomal RNA gene. Causative relationships of both soil physicochemical properties and climatic data on bacterial community structure were infered. 48 phyla, 92 classes, 152 orders, 356 families, and 1,234 genera of bacteria were identified. The core bacteriobiome consisted of 10 genera ranked in the following order of total abundance: Gp6, Gaiella, Spartobacteria_genera_incertae_sedis, WPS-1_genera_incertae_sedis, Gp4, Rhodoplanes, Pseudorhodoplanes, Bradyrhizobium, Subdivision3_genera_incertae_sedis, and Gp3. Some of these genera, including Gp4 and WPS-1_genera_incertae_sedis, were unequally distributed between forest and savannah areas while other taxa (Bradyrhizobium and Rhodoplanes) were consistently found in all biomes. The distribution of the core genera, together with the 10 major phyla, was influenced by several environmental factors, including latitude, pH, Al and K. The main pattern of distribution that was observed for the core bacteriobiome was the vegetation-independent distribution scheme. In terms of predicted functions, all core bacterial taxa were involved in assimilatory sulfate reduction, while atmospheric dinitrogen (N2) reduction was only associated with the genus Bradyrhizobium. This work, which is one of the first such study to be undertaken at this scale in Côte d'Ivoire, provides insights into the distribution of bacterial taxa in Côte d'Ivoire soils, and the findings may serve as biological indicator for land management in Côte d'Ivoire.

The Rhizobial Microbiome from the Tropical Savannah Zones in Northern Côte d'Ivoire.

Over the past decade, many projects have been initiated worldwide to decipher the composition and function of the soil microbiome, including the African Soil Microbiome (AfSM) project that aims at providing new insights into the presence and distribution of key groups of soil bacteria from across the African continent. In this national study, carried out under the auspices of the AfSM project, we assessed the taxonomy, diversity and distribution of rhizobial genera in soils from the tropical savannah zones in Northern Côte d'Ivoire. Genomic DNA extracted from seven sampled soils was analyzed by sequencing the V4-V5 variable region of the 16S rDNA using Illumina's MiSeq platform. Subsequent bioinformatic and phylogenetic analyses showed that these soils harbored 12 out of 18 genera of Proteobacteria harboring rhizobia species validly published to date and revealed for the first time that the Bradyrhizobium genus dominates in tropical savannah soils, together with Microvirga and Paraburkholderia. In silico comparisons of different 16S rRNA gene variable regions suggested that the V5-V7 region could be suitable for differentiating rhizobia at the genus level, possibly replacing the use of the V4-V5 region. These data could serve as indicators for future rhizobial microbiome explorations and for land-use decision-making.

Sinorhizobium fredii Strains HH103 and NGR234 Form Nitrogen Fixing Nodules With Diverse Wild Soybeans (Glycine soja) From Central China but Are Ineffective on Northern China Accessions.

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals.

Two Major Clades of Bradyrhizobia Dominate Symbiotic Interactions with Pigeonpea in Fields of Côte d'Ivoire.

In smallholder farms of Côte d'Ivoire, particularly in the northeast of the country, Cajanus cajan (pigeonpea) has become an important crop because of its multiple beneficial facets. Pigeonpea seeds provide food to make ends meet, are sold on local markets, and aerial parts serve as forage for animals. Since it fixes atmospheric nitrogen in symbiosis with soil bacteria collectively known as rhizobia, C. cajan also improves soil fertility and reduces fallow time. Yet, seed yields remain low mostly because farmers cannot afford chemical fertilizers. To identify local rhizobial strains susceptible to be used as bio-inoculants to foster pigeonpea growth, root nodules were collected in six fields of three geographically distant regions of Côte d'Ivoire. Nodule bacteria were isolated and characterized using various molecular techniques including matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) and DNA sequencing. These molecular analyses showed that 63 out of 85 nodule isolates belonged to two major clades of bradyrhizobia, one of which is known as the Bradyrhizobium elkanii super clade. Phylogenies of housekeeping (16S-ITS-23S, rpoB) and symbiotic (nifH) genes were not always congruent suggesting that lateral transfer of nitrogen fixation genes also contributed to define the genome of these bradyrhizobial isolates. Interestingly, no field-, plant-, or cultivar-specific effect was found to shape the profiles of symbiotic strains. In addition, nodule isolates CI-1B, CI-36E, and CI-41A that belong to distinct species, showed similar symbiotic efficiencies suggesting that any of these strains might serve as a proficient inoculant for C. cajan.