Phylogeny and symbiotic effectiveness of indigenous rhizobial microsymbionts of common bean (Phaseolus vulgaris L.) in Malkerns, Eswatini.

In most legumes, the rhizobial symbionts exhibit diversity across different environments. Although common bean (Phaseolus vulgaris L.) is one of the important legumes in southern Africa, there is no available information on the genetic diversity and N2-fixing effectiveness of its symbionts in Malkerns, Eswatini. In this study, we assessed the phylogenetic positions of rhizobial microsymbionts of common bean from Malkerns in Eswatini. The isolates obtained showed differences in morpho-physiology and N2-fixing efficiency. A dendrogram constructed from the ERIC-PCR banding patterns, grouped a total of 88 tested isolates into 80 ERIC-PCR types if considered at a 70% similarity cut-off point. Multilocus sequence analysis using 16S rRNA, rpoB, dnaK, gyrB, and glnII and symbiotic (nifH and nodC) gene sequences closely aligned the test isolates to the type strains of Rhizobium muluonense, R. paranaense, R. pusense, R. phaseoli and R. etli. Subjecting the isolates in this study to further description can potentially reveal novel species. Most of the isolates tested were efficient in fixing nitrogen and elicited greater stomatal conductance and photosynthetic rates in the common bean. Relative effectiveness (RE) varied from 18 to 433%, with 75 (85%) out of the 88 tested isolates being more effective than the nitrate fed control plants.

Seasonal Effect on Bacterial Communities Associated with the Rhizospheres of Polhillia, Wiborgia and Wiborgiella Species in the Cape Fynbos, South Africa.

The Cape fynbos biome in South Africa is home to highly diverse and endemic shrub legumes, which include species of Aspalathus, Polhillia, Wiborgia and Wiborgiella. These species play a significant role in improving soil fertility due to their ability to fix N2. However, information regarding their microbiome is still unknown. Using the 16S rRNA Miseq illumina sequencing, this study assessed the bacterial community structure associated with the rhizospheres of Polhillia pallens, Polhillia brevicalyx, Wiborgia obcordata, Wiborgia sericea and Wiborgiella sessilifolia growing at different locations during the wet and dry seasons in the Cape fynbos. The results showed that the most dominant bacterial phylum was Actinobacteria during both the dry (56.2-37.2%) and wet (46.3-33.3%) seasons. Unclassified bacterial genera (19.9-27.7%) were the largest inhabitants in the rhizospheres of all five species during the two seasons. The other dominant phyla included Bacteroidetes, Acidobacteria, Proteobacteria and Firmicutes. Mycobacterium and Conexibacter genera were the biggest populations found in the rhizosphere soil of all five test species during both seasons, except for W. obcordata soil sampled during the dry season, which had Dehalogenimonas as the major inhabitant (6.08%). In this study plant species and growth season were the major drivers of microbial community structure, with W. obcordata having the greatest influence on its microbiome than the other test species. The wet season promoted greater microbial diversity than the dry season.

Coinoculation impact on plant growth promotion: a review and meta-analysis on coinoculation of rhizobia and plant growth-promoting bacilli in grain legumes.

Coinoculation of symbiotic N2-fixing rhizobia and plant growth-promoting Bacillus on legume seeds can increase crop productivity. We collected highly resolved data on coinoculation of rhizobia and bacilli on 11 grain legume crops: chickpea, common bean, cowpea, faba bean, groundnut, lentil, mung bean, pea, pigeon pea, soybean, and urad bean to verify the magnitude of additive effects of coinoculation in relation to single inoculation of rhizobia on plant growth and yield of grain legumes. Coinoculation of rhizobia and bacilli on legume seeds and/or soil during sowing significantly increased nodulation, nitrogenase activity, plant N and P contents, and shoot and root biomass, as well as the grain yield of most grain legumes studied. There were however a few instances where coinoculation decreased plant growth parameters. Therefore, coinoculation of rhizobia and Bacillus has the potential to increase the growth and productivity of grain legumes, and can be recommended as an environmental-friendly agricultural practice for increased crop yields.

Phylogenetic relationships among Bradyrhizobium species nodulating groundnut (Arachis hypogea L.), jack bean (Canavalia ensiformis L.) and soybean (Glycine max Merr.) in Eswatini.

This study assessed the genetic diversity and phylogenetic relationships of rhizobial isolates obtained from root nodules of groundnut, jack bean and soybean planted in different locations within Eswatini. Seventy-six rhizobial isolates were studied using ERIC-PCR (enterobacterial repetitive intergenic consensus) fingerprinting and PCR amplification of 16S rRNA, housekeeping genes (atpD, dnaK, glnll and rpoB) and symbiotic genes (nifH and nodC). The dendrogram generated from the ERIC-PCR banding patterns grouped the test rhizobial isolates into 16 major clusters (Cluster I-XVI), with three isolates, namely TUTAHeS60, TUTGMeS3 and TUTAHeS127, forming outgroups of Clusters IV, VI and IX, respectively. Furthermore, the 76 test isolates were grouped into 56 ERIC-PCR types at 70% similarity level. The phylogenetic analysis of the 16S rRNA gene and multilocus sequence analysis of four housekeeping (atpD, dnaK, glnII and rpoB) and two symbiotic (nifH and nodC) genes showed that all three legumes (groundnut, jack bean and soybean) were nodulated by bacterial symbionts belonging to the genus Bradyrhizobium, with some isolates exhibiting high divergence from the known reference type strains. The results also showed that B. arachidis, B. iriomotense and B. canariense were the closest type strains to the groundnut isolates, while B. pachyrhizi and B. elkanii were the closest relatives to the bacterial symbionts associated with the nodulation of both jack bean and soybean. This study is the first report to describe of the bacterial symbionts nodulating jack bean in African soils.

Ecological adaptation and phylogenetic analysis of microsymbionts nodulating Polhillia, Wiborgia and Wiborgiella species in the Cape fynbos, South Africa.

Polhillia, Wiborgia and Wiborgiella species are shrub legumes endemic to the Cape fynbos of South Africa. They have the ability to fix atmospheric N2 when in symbiosis with soil bacteria called 'rhizobia'. The aim of this study was to assess the morpho-physiological and phylogenetic characteristics of rhizobia associated with the nodulation of Polhillia, Wiborgia and Wiborgiella species growing in the Cape fynbos. The bacterial isolates from root nodules consisted of a mixture of fast and intermediate growers that differed in colony shape and size. The isolates exhibited tolerance to salinity (0.5-3% NaCl) and pH (pH 5-10) and different antibiotic concentrations, and could produce 0.51 to 51.23 µg mL-1 of indole-3-acetic acid (IAA), as well as solubilize tri-calcium phosphate. The ERIC-PCR results showed high genomic diversity in the rhizobial population and grouped them into two major clusters. Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, nifH and nodC gene sequences revealed distinct and novel evolutionary lineages related to the genus Rhizobium and Mesorhizobium, with some of them being very close to Mesorhizobium australicum. However, the phylogenetic analysis of glnII and nifH genes of some isolates showed incongruency.

Adaptability to local conditions and phylogenetic differentiation of microsymbionts of TGx soybean genotypes in the semi-arid environments of Ghana and South Africa.

The study of the nitrogen fixation and phylogenetic diversity of nodule microsymbionts of grain legumes in many parts of the globe is often carried out in order to identify legume-rhizobia combinations for agricultural sustainability. Several reports have therefore found that rhizobial species diversity is shaped by edapho-climatic conditions that characterize different geographic locations, suggesting that rhizobial communities often possess traits that aid their adaptation to their habitat. In this study, the soybean-nodulating rhizobia from semi-arid savannahs of Ghana and South Africa were evaluated. The authenticated rhizobial isolates were highly diverse based on their colony characteristics, as well as their BOX-PCR profiles and gene sequences. In the 16S rRNA phylogeny, the isolates were placed in the different clades Bradyrhizobium iriomotense and Bradyrhizobium jicamae together with two superclades Bradyrhizobium japonicum and Bradyrhizobium elkanii. The multilocus (atpD, glnII, gyrB, recA) phylogenetic analyses indicated the dominance of Bradyrhizobium diazoefficiens and putative new Bradyrhizobium species in the semi-arid Ghanaian region. The phylogenetic analyses based on the symbiotic genes (nifH and nodC) clustered the test isolates into different symbiovars (sv. glycinearum, sv. retame and sv. sojae). Principal component analysis (PCA) showed that soil factors played a significant role in favoring the occurrence of soybean-nodulating microsymbionts in the tested local conditions. The results suggested that isolates had marked local adaptation to the prevailing conditions in semi-arid regions but further studies are needed to confirm new Bradyrhizobium species.

Functional and genetic diversity of native rhizobial isolates nodulating cowpea (Vigna unguiculata L. Walp.) in Mozambican soils.

Identification and symbiotic characterization of indigenous rhizobial isolates are the basis for inoculant formulations needed for sustainable grain legume production. This study screened for morpho-genetic diversity of indigenous cowpea nodulating rhizobia in farmers' fields across two contrasting agroecological zones of Northern Mozambique. The photosynthetic function induced by the isolates in their homologous cowpea was assessed. The results showed high genetic variability among the isolates based on morphology and ERIC-PCR fingerprinting. The trap cowpea genotype did not influence the diversity of isolates collected from the two different agroecologies, suggesting that the cowpea-rhizobia compatibility may be conserved at species level. Phylogenetic analysis of the 16S rRNA gene assigned representative rhizobial isolates to species in the Bradyrhizobium and Rhizobium genera, with some isolates showing high divergence from the known reference type strains. The isolates from both agroecologies highly varied in the number and biomass of nodules induced in the homologous cowpea, resulting in variable plant growth and photosynthetic activities. A total of 72% and 83% of the isolates collected from the agroecological zones 7 and 8 were respectively classified as highly effective candidates with > 80% relative effectiveness compared to plants fertilized with nitrate, indicating that elite native strains populated the studied soils. Moreover, the top 25% of high N2-fixing isolates from the two agroecologies recorded relative effectiveness ranging from 115 to 154%, values higher than the effectiveness induced by the commercial Bradyrhizobium sp. strain CB756. These strains are considered as having potential for use in inoculant formulations. However, future studies should be done to assess the ecologically adaptive traits and symbiotic performance under field conditions.

Diverse symbiovars nodulating cowpea (Vigna unguiculata L. Walp.) in highly adaptable agro-ecological zones in Mozambique.

The presence of effective microsymbionts in the soil and their compatibility with the host plant are the key determinants to the N2 fixation process. In Sub-Saharan Africa, nitrogen fixation in locally adapted cowpea and the distribution of their symbiovars are not well understood. The Aim of the study was to assess the distribution and symbiotic phylogenetic position of cowpea microsymbionts. Root nodules were sampled from various cowpea genotypes planted in Agro-Ecological Zone 7 and 8 (AEZ 7 and AEZ 8). Root-nodule bacteria were isolated and their molecular characterization was conducted. Physicochemical properties of soil were recorded. Enterobacterial Repetitive Intergenic Consensus (ERIC) distribution patterns in rhizobial genomes resulted in genetically diverse rhizobial population in Northern Mozambique. Principal component analysis showed that location-specific soil environment determined the presence of particular microsymbionts. Based on 16S rRNA and symbiotic gene analysis many diverse symbiovars were found in Mozambican soils. With few discrepancies, the results further confirmed the coevolution of the nifH, nodD, nodC and nodY/K genes, which was indicative of natural events such as vertical/horizontal gene transfer. The results suggested that ecological and phylogenetic studies of the microsymbionts are necessary to better reflect symbiovar identification and the ecological adaptation of the cowpea-nodulating rhizobial community.

Studies of Phylogeny, Symbiotic Functioning and Ecological Traits of Indigenous Microsymbionts Nodulating Bambara Groundnut (Vigna subterranea L. Verdc) in Eswatini.

Rhizobial microsymbionts of grain legumes are ubiquitous in soils and exhibit a wide range of diversity with respect to colony morphology, genetic variability, biochemical characteristics, and phylogenetic relationships. This study assessed the phylogenetic positions of rhizobial microsymbionts of Bambara groundnut from Eswatini exhibiting variations in morpho-physiology, adaptive characteristics, and N2-fixing efficiency. The isolates' ERIC-PCR profiles revealed the presence of high genetic variation among them. These test isolates also exhibited differences in pH tolerance and IAA production. Multilocus sequence analysis based on the 16S rRNA, atpD, glnII, gyrB, and recA gene sequences of representative test isolates closely aligned them to the type strains of Bradyrhizobium arachidis, B. manausense, B. guangdongense, B. elkanii, and B. pachyrhizi. However, some isolates showed a high divergence from the known reference type strains, indicating that they may represent species yet to be properly characterized and described. Functional characterization in the glasshouse revealed that most of the isolates from the contrasting Agro-ecologies of Eswatini were efficient in N2 fixation, and therefore elicited greater stomatal conductance and photosynthetic rates in the homologous Bambara groundnut. Of the 75 isolates tested, 51% were more effective than the commercial Bradyrhizobium sp. strain CB756, with relative symbiotic effectiveness ranging from 138 to 308%. The findings of this study indicated that the analysis of housekeeping genes and functional traits of Bambara-nodulating microsymbionts can provide a clear view for understanding and predicting rhizobial community structure across environmental gradients.

Insights into nitrogen fixing traits and population structure analyses in cowpea (Vigna unguiculata L. Walp) accessions grown in Ghana.

With legumes, symbiotic N2 fixation can meet the species N demand and reduce the over-reliance on chemical fertilizers in tropical regions where N deficiency is a major factor limiting crop yields and increased agricultural sustainability. Therefore, to optimize the use of cowpea (Vigna unguiculata L. Walp) germplasm in effective breeding, evaluation of genetic diversity and quantification of N2 fixation are essential prerequisites. The aim of this study was to explore the level of diversity using SSR markers and N2-fixing traits in a set of cowpea germplasm grown in Ghana. We analysed 49 cowpea accessions collected from Northern Ghana using qualitative vegetative and N2 fixation traits, and simple sequence repeat (SSR) markers. Experimental field results revealed considerable morpho-physiological variation for plant growth habits, grain yield and symbiotic performance between and among the cowpea accessions. Results from both the 15N natural abundance and ureides in the xylem sap were able to descriminate between high and low levels of N2 fixation in cowpea accessions. Five subpopulations were identified within accessions inferred from STRUCTURE 2.3.4. A general linear model was used to assess the association of SSR markers with N2-fixing traits. There were significant (p ≤ 0.05) links between SSR markers and symbiosis-related traits such as nodule number, nodule dry weight, shoot dry weight, N-fixed, N derived from air (Ndfa), and relative uried-N (RU-N).

Symbiotic effectiveness and ecologically adaptive traits of native rhizobial symbionts of Bambara groundnut (Vigna subterranea L. Verdc.) in Africa and their relationship with phylogeny.

Bambara groundnut (Vigna subterranea L. Verdc.) is an indigenous, drought-tolerant, underutilized African food legume, with the ability to fix atmospheric N2 in symbiosis with soil bacteria called rhizobia. The aim of this study was to assess the morpho-physiological, symbiotic and phylogenetic characteristics of rhizobia nodulating Bambara groundnut in Ghana, Mali and South Africa. The morpho-physiologically diverse isolates tested were also found to exhibit differences in functional efficiency and phylogenetic positions. Based on Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR banding patterns, the isolates were grouped into eight major clusters. The concentrations of Ca, Na and K in soils had a significant (p ≤ 0.01) effect on the distribution of rhizobia. Though many isolates were symbiotically very effective, the effectiveness index varied markedly (p ≤ 0.05) among them. Moreover, the isolates also exhibited tolerance to a wide range of NaCl (0.5-7%), streptomycin (50-500 µg.ml-1), and kanamycin (25-150 µg.ml-1) concentrations. Additionally, these isolates could produce 0.02 to 69.71 µg.ml-1 of indole-3-acetic acid (IAA) in tryptophan-supplemented medium, as well as solubilize tri-calcium phosphate. Phylogenetic analysis of these rhizobial isolates using 16S rRNA, atpD, glnII, gyrB, recA and symbiotic (nifH and nodC) gene sequences revealed distinct and novel evolutionary lineages related to the genus Bradyrhizobium, with some of them being very close to Bradyrhizobium vignae, B. kavangense, B. subterraneum, B. elkanii and B. pachyrhizi.

Microbial community structure in the rhizosphere of the orphan legume Kersting's groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet].

The presence of microbial communities in the rhizosphere of plants is an important determinant of plant health and soil organic matter composition. Plant species play significant roles in selecting the specific microbial communities that inhabit the root zone. However, till now, there is no solid information regarding the presence of specific plant-microbiome in the rhizosphere of many plants, especially under-exploited and under-researched species such as Kersting's groundnut. This study assessed the effect of five Kersting's groundnut landraces on the structure of microbial communities in rhizosphere of field-grown plants. The five tested Kersting's groundnut landraces (Belane Mottled, Boli, Funsi, Puffeun and Heng Red Mottled) were found to exert a marked selective influence on bacteria associated with their rhizospheres, measured using 16S rDNA MiSeq illumina sequencing. Community differences in microbial composition and relative abundance were both significant. Numerous phyla in the rhizosphere were affected by the test landraces. Except for Belane mottled whose rhizospheres were dominated by Proteobacteria, the rhizosphere soils of the other landraces were dominated by Bacteroidetes. With the exception of landrace Puffeun which showed only Mesorhizobium in its rhizosphere, all the other test landraces revealed the presence of Bradyrhizobium and Rhizobium species of alpha Proteobacteria. Furthermore, the rhizosphere of all landraces were abundant in species of the indole-3-acetic-acid producing Sphingomonas and cellulose-degrading Fibrobacteres. The results of this study suggest that Kersting's groundnut landraces can shape bacterial community composition in the rhizosphere via plant-related changes in the rhizosphere soil.

Phylogenetic evidence of allopatric speciation of bradyrhizobia nodulating cowpea (Vigna unguiculata L. walp) in South African and Mozambican soils.

The legume host and soil environment play a major role in establishing effective symbiosis with diverse rhizobia for plant growth promotion and nodule formation. The aim of this study was to assess the morpho-physiology, distribution and phylogenetic position of rhizobia nodulating cowpea from South Africa and Mozambique. The results showed that the isolates were highly diverse in their appearance on yeast mannitol agar plates. The isolates tested also showed an ability to produce IAA at concentrations ranging from 0.64 to 56.46 µg.ml-1 and to solubilise phosphorus at levels from 0 to 3.55 index. Canonical correspondence analysis showed that soil pH and mineral nutrients significantly influenced bradyrhizobial distribution. Analysis of BOX-PCR placed the isolates in eight major clusters with 0.01 to 1.00 similarity coefficient which resulted in 45 unique BOX-types. Phylogenetic analyses based on 16S rRNA, atpD, glnII, gyrB and recA gene sequences showed distinct novel evolutionary lineages within the genus Bradyrhizobium, with some of them being closely related to Bradyrhizobium kavangense, B. subterraneum and B. pachyrhizi. Furthermore, symbiotic gene phylogenies suggested that the isolates' sym loci probably relates to the isolates' geographical origin. The results indicated that geographical origin did affect the isolates' phylogenetic placement and could be the basis for allopatric speciation.

Insights into the Phylogeny, Nodule Function, and Biogeographic Distribution of Microsymbionts Nodulating the Orphan Kersting's Groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] in African Soils.

Kersting's groundnut [Macrotyloma geocarpum (Harms) Marechal & Baudet] is a neglected indigenous African legume adapted to growth in N-deficient soils due to its ability to fix atmospheric N2 via symbiosis with rhizobia. Despite its nutritional and medicinal uses, to date there is little information on the phylogeny and functional traits of its microsymbionts, aspects that are much needed for its conservation and improvement. This study explored the morphogenetic diversity, phylogenetic relationships, and N2-fixing efficiency of Kersting's groundnut rhizobial isolates from contrasting environments in Ghana, South Africa, and Mozambique. BOX-PCR fingerprinting revealed high diversity among the rhizobial populations, which was influenced by geographic origin. Of the 164 isolates evaluated, 130 BOX-PCR types were identified at a 70% similarity coefficient, indicating that they were not clones. Soil pH and mineral concentrations were found to influence the distribution of bradyrhizobial populations in African soils. Phylogenetic analysis of 16S rRNA genes and multilocus sequence analysis of protein-coding genes (atpD, glnII, gyrB, and rpoB) and symbiotic genes (nifH and nodC) showed that Kersting's groundnut is primarily nodulated by members of the genus Bradyrhizobium, which are closely related to Bradyrhizobium vignae 7-2T, Bradyrhizobium kavangense 14-3T, Bradyrhizobium subterraneum 58-2-1T, Bradyrhizobium pachyrhizi PAC48T, the type strain of Bradyrhizobium elkanii, and novel groups of Bradyrhizobium species. The bradyrhizobial populations identified exhibited high N2 fixation and induced greater nodulation, leaf chlorophyll concentration, and photosynthetic rates in their homologous host than did the 5 mM KNO3-fed plants and/or the commercial Bradyrhizobium sp. strain CB756, suggesting that they could be good candidates for inoculant formulations upon field testing.IMPORTANCE Rhizobia play important roles in agroecosystems, where they contribute to improving overall soil health through their symbiotic relationship with legumes. This study explored the microsymbionts nodulating Kersting's groundnut, a neglected orphan legume. The results revealed the presence of different bradyrhizobial populations with high N2-fixing efficiencies as the dominant symbionts of this legume across diverse agroecologies in Africa. Our findings represent a useful contribution to the literature in terms of the community of microsymbionts nodulating a neglected cultivated legume and its potential for elevation as a major food crop. The presence of potentially novel bradyrhizobial symbionts of Kersting's groundnut found in this study offers an opportunity for future studies to properly describe, characterize, and delineate these isolates functionally and phylogenetically for use in inoculant production to enhance food/nutritional security.

Widespread Distribution of Highly Adapted Bradyrhizobium Species Nodulating Diverse Legumes in Africa.

Bradyrhizobium is one of the most cosmopolitan and diverse bacterial group nodulating a variety of host legumes in Africa, however, the diversity and distribution of bradyrhizobial symbionts nodulating indigenous African legumes are not well understood, though needed for increased food legume production. In this review, we have shown that many African food legumes are nodulated by bradyrhizobia, with greater diversity in Southern Africa compared to other parts of Africa. From a few studies done in Africa, the known bradyrhizobia (i.e., Bradyrhizobium elkanii, B. yuanmingense) along with many novel Bradyrhizobium species are the most dominant in African soils. This could be attributed to the unique edapho-climatic conditions of the contrasting environments in the continent. More studies are needed to identify the many novel bradyrhizobia resident in African soils in order to better understand the biogeography of bradyrhizobia and their potential for inoculant production.

Phylogeny and distribution of Bradyrhizobium symbionts nodulating cowpea (Vigna unguiculata L. Walp) and their association with the physicochemical properties of acidic African soils.

In the N2-fixing symbiosis, the choice of a symbiotic partner is largely influenced by the host plant, the rhizobial symbiont, as well as soil factors. Understanding the soil environment conducive for the survival and multiplication of root-nodule bacteria is critical for microbial ecology. In this study, we collected cowpea-nodules from acidic soils in Ghana and South Africa, and nodule DNA isolates were characterized using 16S-23S rRNA-RFLP, phylogenetic analysis of housekeeping and symbiotic genes, and bradyrhizobial community structure through canonical correspondence analysis (CCA). The CCA ordination plot results showed that arrow of soil pH was overlapping on CCA2 axis and was the most important to the ordination. The test nodule DNA isolates from Ghana were positively influenced by soil Zn, Na and K while nodule DNA isolates from South Africa were influenced by P. The amplified 16S-23S rRNA region yielded single polymorphic bands of varying lengths (573-1298bp) that were grouped into 28 ITS types. The constructed ITS-dendrogram placed all the nodule DNA isolates in five major clusters at low cut-off of approx. 0.1 Jaccard's similarity coefficient. The phylogenetic analysis of 16S rRNA and housekeeping genes (glnII, gyrB, and atpD) formed distinct Bradyrhizobium groups in the phylogenetic trees. It revealed the presence of highly diverse bradyrhizobia (i.e. Bradyrhizobium vignae, Bradyrhizobium elkanii, Bradyrhizobium iriomotense, Bradyrhizobium pachyrhizi, and Bradyrhizobium yuanmingense) together with novel/unidentified bradyrhizobia in the acidic soils from Ghana and South Africa. Discrepancies noted in the phylogenies of some nodule DNA isolates could be attributed to horizontal gene transfer or recombination.

Distribution and correlation between phylogeny and functional traits of cowpea (Vigna unguiculata L. Walp.)-nodulating microsymbionts from Ghana and South Africa.

Cowpea (Vigna unguiculata L. Walp.) is indigenous to Africa, and highly valued for its N2-fixing trait and the nutritional attributes of its grain and leaves. The species' ability to establish effective symbiosis with diverse rhizobial populations gives it survival and growth advantage in N-limited environments. To explore the functional diversity and phylogenetic positions of rhizobia nodulating cowpea in Africa, nodules were collected from various cowpea varieties grown in soils from the Guinea savanna and Sudano-sahelian agroecologies of Northern Ghana, and from the lowveld and middleveld areas of Mpumalanga Province in South Africa. Box-PCR profiling and multilocus sequence analysis revealed the presence of diverse microsymbionts responsible for cowpea nodulation across the study sites. BOX-PCR amplifications yielded variable band sizes, ranging from 618 bp to 5354 bp, which placed the isolates in six major clusters (Cluster A-F). Phylogenetic analysis based on 16S rRNA, atpD, glnII, gyrB, rpoB, nifH and nodC genes revealed the presence of diverse Bradyrhizobium sp. closely related to Bradyrhizobium daqingense, Bradyrhizobium subterraneum, Bradyrhizobium yuanmingense, Bradyrhizobium embrapense, Bradyrhizobium pachyrhizi, Bradyrhizobium elkanii and novel Bradyrhizobium species in the soils studied, a finding that could be attributed to the unique edapho-climatic conditions of the contrasting environments. The test isolates exhibited distinct symbiotic efficiencies, and also induced variable (p ≤ 0.001) photosynthetic rates, leaf transpiration, total chlorophyll and shoot biomass accumulation on cowpea (their homologous host). Canonical correspondence analysis showed that the distribution of these microsymbionts was influenced by the concentrations of macro- and micronutrients in soils. The pairwise genetic distances derived from phylogenies and nodule functioning showed significant (p < 0.05) correlation, which suggests that local environmental factors played a major role in the cowpea-Bradyrhizobium symbiosis.

Symbiotic N2 Fixation and Grain Yield of Endangered Kersting's Groundnut Landraces in Response to Soil and Plant Associated Bradyrhizobium Inoculation to Promote Ecological Resource-Use Efficiency.

Kersting's groundnut (Macrotyloma geocarpum Harms) is a neglected, endangered food and medicinal legume in Africa. Efforts to harness the benefits of the legume-rhizobia symbiosis have focused on few major legumes to the neglect of underutilized ones such as Kersting's groundnut. This study assessed plant growth, N-fixed and grain yield of five Kersting's groundnut landraces in response to inoculation with Bradyrhizobium strain CB756 at two locations in the Northern Region of Ghana. The transferability of cowpea-derived Simple Sequence Repeat (SSR) markers to Kersting's groundnut was also assessed. The symbiotic results revealed significant variation in nodulation, shoot biomass, δ15N, percent N derived from fixation, amount of N-fixed and soil N uptake. The cross-taxa SSR primers revealed monomorphic bands with sizes within the expected range in all the Kersting's groundnut landraces. The results of the aligned nucleotide sequences revealed marked genetic variability among the landraces. Kersting's groundnut was found to be a low N2-fixer, with 28-45% of its N derived from fixation at Nyankpala and 15-29% at Savelugu. Nitrogen contribution was 28-50 kg N-fixed·ha-1 at Nyankpala, and 12-32 kg N-fixed·ha-1 at Savelugu. Uninoculated plants of the Kersting's groundnut landraces Puffeun, Dowie, Sigiri and Boli, respectively, contributed 22, 16, 13, and 15 kg N-fixed·ha-1 from symbiosis at Savelugu as opposed to 89, 82, 69, and 89 kg N·ha-1 from soil. Landrace Puffeun was highly compatible with the introduced strain CB756 if based on δ15N and %Ndfa values, while Dowie, Funsi and Boli showed greater compatibility with native rhizobia in Ghanaian soils. The unimproved Kersting's groundnut in association with soil microsymbionts could produce grain yield of 1,137-1,556 kg ha-1 at Nyankpala, and 921-1,192 kg ha-1 at Savelugu. These findings suggest the need for further work to improve the efficiency of the Kersting's groundnut-rhizobia symbiosis for increased grain yield and resource-use efficiency in cropping systems.

Assessing host range, symbiotic effectiveness, and photosynthetic rates induced by native soybean rhizobia isolated from Mozambican and South African soils.

Host range and cross-infectivity studies are important for identifying rhizobial strains with potential for use as inoculants. In this study, 10 native soybean rhizobia isolated from Mozambican and South African soils were evaluated for host range, symbiotic effectiveness and ability to induce high rates of photosynthesis leading to enhanced plant growth in cowpea (Vigna unguiculata L. Walp.), Bambara groundnut (Vigna subterranean L. Verdc.), Kersting's groundnut (Macrotyloma geocarpum Harm) and soybean (Glycine max L. Merr). The test isolates had different growth rates and colony sizes. Molecular analysis based on enterobacterial repetitive intergenic consensus (ERIC)-PCR revealed high genetic diversity among the test isolates. The results further showed that isolate TUTLBC2B failed to elicit nodulation in all test plants, just as TUTNSN2A and TUTDAIAP3B were also unable to nodulate cowpea, Kersting's bean and Bambara groundnut. Although the remaining strains formed ineffective nodules on cowpea and Kersting's bean, they induced effective nodules on Bambara groundnut and the two soybean genotypes. Bacterial stimulation of nodule numbers, nodule dry weights and photosynthetic rates was generally greater with isolates TUTRSRH3A, TUTM19373A, TUTMCJ7B, TUTRLR3B and TUTRJN5A. As a result, these isolates elicited significantly increased accumulation of biomass in shoots and whole plants of Bambara groundnut and the two soybean genotypes. Whole-plant symbiotic nitrogen (N) of soybean and Bambara groundnut was highest for the commercial strains CB756 and WB74, as well as for TUTRLR3B, TUTMCJ7B and TUTRSRH3A, suggesting that the three native rhizobial isolates have potential for use as inoculants.

Identification and distribution of microsymbionts associated with soybean nodulation in Mozambican soils.

Indigenous soybean rhizobial strains were isolated from root nodules sampled from farmers' fields in Mozambique to determine their identity, distribution and symbiotic relationships. Plant infection assays revealed variable nodulation and symbiotic effectiveness among the 43 bacterial isolates tested. Strains from Ruace generally promoted greater whole-plant growth than the others. 16S rRNA-RFLP analysis of genomic DNA extracted from the rhizobial isolates produced different banding patterns, a clear indication of high bacterial diversity. However, the multilocus sequence analysis (MLSA) data showed alignment of the isolates with B. elkanii species. The 16S rRNA sequences of representative soybean isolates selected from each 16S rRNA-RFLP cluster showed their relatedness to B. elkanii, as well as to other Bradyrhizobium species. But a concatenated phylogeny of two housekeeping genes (glnII and gyrB) identified the soybean nodulating isolates as Bradyrhizobium, with very close relatedness to B. elkanii. The nifH and nodC sequences also showed that the majority of the test soybean isolates were closely related to B. elkanii, albeit the inconsistency with some isolates. Taken together, these findings suggest that the B. elkanii group are the preferred dominant microsymbiont of soybean grown in Mozambican soils. Furthermore, the distribution of soybean rhizobia in the agricultural soils of Mozambique was found to be markedly influenced by soil pH, followed by the concentrations of plant-available P and Mn. This study suggested that the identified isolates TUTMJM5, TUTMIITA5A and TUTLBC2B can be used as inoculants for increased soybean production in Mozambique.