Consistency, variability, and predictability of on-farm nutrient responses in four grain legumes across East and West Africa.

Grain legumes are key components of sustainable production systems in sub-Saharan Africa, but wide-spread nutrient deficiencies severely restrict yields. Whereas legumes can meet a large part of their nitrogen (N) requirement through symbiosis with N2-fixing bacteria, elements such as phosphorus (P), potassium (K) and secondary and micronutrients may still be limiting and require supplementation. Responses to P are generally strong but variable, while evidence for other nutrients tends to show weak or highly localised effects. Here we present the results of a joint statistical analysis of a series of on-farm nutrient addition trials, implemented across four legumes in four countries over two years. Linear mixed models were used to quantify both mean nutrient responses and their variability, followed by a random forest analysis to determine the extent to which such variability can be explained or predicted by geographic, environmental or farm survey data. Legume response to P was indeed variable, but consistently positive and we predicted application to be profitable for 67% of farms in any given year, based on prevailing input costs and grain prices. Other nutrients did not show significant mean effects, but considerable response variation was found. This response heterogeneity was mostly associated with local or temporary factors and could not be explained or predicted by spatial, biophysical or management factors. An exception was K response, which displayed appreciable spatial variation that could be partly accounted for by spatial and environmental covariables. While of apparent relevance for targeted recommendations, the minor amplitude of expected response, the large proportion of unexplained variation and the unreliability of the predicted spatial patterns suggests that such data-driven targeting is unlikely to be effective with current data.

Inoculation and phosphorus fertilizer improve food-feed traits of grain legumes in mixed crop-livestock systems of Ethiopia.

Grain legumes play an important role as source of food and feed in smallholder mixed systems. They also contribute to soil fertility improvement through biological nitrogen fixation. Although rhizobium inoculation and phosphorus fertilizer are known to improve grain yield of legumes, information is limited on the effect of this practice on the yield and fodder quality of the haulm. This study was conducted to evaluate the effects of rhizobium inoculation (I) and phosphorus fertilizer (P) on yield and nutritional quality of grains and haulms of grain legumes (faba bean, chickpea, common bean and soybean) on farm across diverse agroecological locations in the Ethiopian highlands. The crops were subjected to four treatments [+I, +P, -I + P and a negative control (-P-I)] at multiple locations on farm during the main cropping season in 2016. Yield data was recorded during grain harvesting, and subsequently representative samples of grains and haulms were collected and analyzed for quality variables. Effects of the treatments were significant (P < 0.05) with 30% increase on grain yield for all studied crops and 28% increase on haulm dry matter yield for faba bean, common bean and soybean. Crude protein (CP) and in vitro organic matter digestibility (IVOMD) values of faba bean, common bean and soybean haulms were higher (P < 0.05); and neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were lower (P < 0.05) for the treatments than the control. The haulm CP content and IVOMD of chickpea also responded positively (P < 0.05) to the treatments. The current results demonstrated the possibility of improving both yield and quality of grains and haulms of grain legumes with the application of efficient rhizobium inocula and P fertilization. This practice offers an opportunity for smallholders in the crop-livestock system to improve the food-feed traits of grain legumes with minimal input and environmental footprint.

Genetic diversity and symbiotic effectiveness of Bradyrhizobium strains nodulating selected annual grain legumes growing in Ethiopia.

Vigna unguiculata, Vigna radiata and Arachis hypogaea growing in Ethiopia are nodulated by a genetically diverse group of Bradyrhizobium strains. To determine the genetic identity and symbiotic effectiveness of these bacteria, a collection of 36 test strains originating from the root nodules of the three hosts was investigated using multilocus sequence analyses (MLSA) of core genes including 16S rRNA, recA, glnII, gyrB, atpD and dnaK. Sequence analysis of nodA and nifH genes along with tests for symbiotic effectiveness using δ15N analysis were also carried out. The phylogenetic trees derived from the MLSA grouped most test strains into four well-supported distinct positions designated as genospecies I-IV. The maximum likelihood (ML) tree that was constructed based on the nodA gene sequences separated the entire test strains into two lineages, where the majority of the test strains were clustered on one of a well-supported large branch that comprise Bradyrhizobium species from the tropics. This clearly suggested the monophyletic origin of the nodA genes within the bradyrhizobia of tropical origin. The δ15N-based symbiotic effectiveness test of seven selected strains revealed that strains GN100 (δ15N=0.73) and GN102 (δ15N=0.79) were highly effective nitrogen fixers when inoculated to cowpea, thus can be considered as inoculants in cowpea production. It was concluded that Ethiopian soils are a hotspot for rhizobial diversity. This calls for further research to unravel as yet unknown bradyrhizobia nodulating legume host species growing in the country. In this respect, prospective research should also address the mechanisms of symbiotic specificity that could lead to high nitrogen fixation in target legumes.

Additive yield response of chickpea (Cicer arietinum L.) to rhizobium inoculation and phosphorus fertilizer across smallholder farms in Ethiopia.

The impacts of rhizobium inoculation on growth and yield of chickpea have mainly been tested in experiments conducted in greenhouses or on research stations. We report the response of the crop to inoculation (I) and phosphorus fertilizer (P) application across a large number of smallholder's farms over four regions of Ethiopia, covering diverse soil fertility and agro-ecological conditions. Increased grain yields due to the soil fertility treatments was evident for 99% target farmers. On average, I and P increased grain yield by 21% and 25% respectively, while the combined application of I and P resulted in a 38% increase. However, observed grain yields on control plots and responses to the treatments on individual farms varied greatly, and relative yield responses (%; yield of P and/I minus control yield, divided by control yield) ranged from 3% to 138%. With the exception of a few extremely poorly yielding locations, average responses to P and I were high across a wide range of control yields, indicating the possibility of boosting chickpea productivity for smallholders with P fertilizer and inoculant technology. Variation in response to rhizobium inoculation was mostly independent of agro-ecology and soil type although it was found to be low on a number of farms with extremely high N contents (%). Assuming that a relative yield increase of 10% due to treatment effects is required to be visible, 71%, 73% and 92% of the farmers observed a yield benefit by applying P, I, and P + I, respectively. The results are discussed with respect to the additive benefits of P fertilizers and rhizobial inoculation and their implications for wide scale promotion of inoculant technology to smallholders.

Soyabean response to rhizobium inoculation across sub-Saharan Africa: Patterns of variation and the role of promiscuity.

Improving bacterial nitrogen fixation in grain legumes is central to sustainable intensification of agriculture in sub-Saharan Africa. In the case of soyabean, two main approaches have been pursued: first, promiscuous varieties were developed to form effective symbiosis with locally abundant nitrogen fixing bacteria. Second, inoculation with elite bacterial strains is being promoted. Analyses of the success of these approaches in tropical smallholder systems are scarce. It is unclear how current promiscuous and non-promiscuous soyabean varieties perform in inoculated and uninoculated fields, and the extent of variation in inoculation response across regions and environmental conditions remains to be determined. We present an analysis of on-farm yields and inoculation responses across ten countries in Sub Saharan Africa, including both promiscuous and non-promiscuous varieties. By combining data from a core set of replicated on-farm trials with that from a large number of farmer-managed try-outs, we study the potential for inoculation to increase yields in both variety types and evaluate the magnitude and variability of response. Average yields were estimated to be 1343 and 1227 kg/ha with and without inoculation respectively. Inoculation response varied widely between trials and locations, with no clear spatial patterns at larger scales and without evidence that this variation could be explained by yield constraints or environmental conditions. On average, specific varieties had similar uninoculated yields, while responding more strongly to inoculation. Side-by side comparisons revealed that stronger responses were observed at sites where promiscuous varieties had superior uninoculated yields, suggesting the availability of compatible, effective bacteria as a yield limiting factor and as a determinant of the magnitude of inoculation response.

An assessment of plant growth and N2 fixation in soybean genotypes grown in uninoculated soils collected from different locations in Ethiopia.

Achieving food and nutritional security is a major challenge in Ethiopia, especially with increasing human population and low crop productivity. Legumes offer an alternative choice to chemical fertilizers for increasing crop yields. The aim of this study was to assess, under glasshouse conditions, plant growth and symbiotic performance of uninoculated soybean genotypes planted in soils collected from different locations in Ethiopia. The results showed significant differences in plant growth and symbiotic performance among the soybean genotypes planted in different soils. There was a location-specific effect of soil on plant growth and symbiotic N nutrition of soybean. Whole-plant biomass was highest in soil from Amaro, followed by Boricha, Dorebafano, Pawe, and Mambuk. The δ15N values ranged from +0.82‰ for Pawe to +5.11‰ at Dorebafano. However, %Ndfa of soybean was greater in plants grown in Mambuk soil, followed by Pawe with the lowest %Ndfa being in Amaro soil. The amount of N-fixed followed similar pattern as %Ndfa. The significant interaction found between soil type and soybean genotype for plant DM, shoot N concentration, δ15N, %Ndfa, N-fixed and soil N-uptake clearly indicated the effect of soil factors. This study revealed the presence of native rhizobia in Ethiopian soils that are compatible with soybean. The N contribution of the soybean genotypes was variable, and strongly influenced by the soil factors.

Genetic and phenotypic diversity of rhizobia nodulating chickpea (Cicer arietinum L.) in soils from southern and central Ethiopia.

Forty-two chickpea-nodulating rhizobia were isolated from soil samples collected from diverse agro-ecological locations of Ethiopia and were characterized on the basis of 76 phenotypic traits. Furthermore, 18 representative strains were selected and characterized using multilocus sequence analyses of core and symbiotic gene loci. Numerical analysis of the phenotypic characteristics grouped the 42 strains into 4 distinct clusters. The analysis of the 16S rRNA gene of the 18 strains showed that they belong to the Mesorhizobium genus. On the basis of the phylogenetic tree constructed from the combined genes sequences (recA, atpD, glnII, and gyrB), the test strains were distributed into 4 genospecies (designated as genospecies I-IV). Genospecies I, II, and III could be classified with Mesorhizobium ciceri, Mesorhizobium abyssinicae, and Mesorhizobium shonense, respectively, while genospecies IV might represent an unnamed Mesorhizobium genospecies. Phylogenetic reconstruction based on the symbiosis-related (nifH and nodA) genes supported a single cluster together with a previously described symbiont of chickpea (M. ciceri and Mesorhizobium mediterraneum). Overall, our results corroborate earlier findings that Ethiopian soils harbor phylogenetically diverse Mesorhizobium species, justifying further explorative studies. The observed differences in symbiotic effectiveness indicated the potential to select effective strains for use as inoculants and to improve the productivity of chickpea in the country.

Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov., isolated from root nodules of different agroforestry legume trees.

A total of 18 strains, representing members of the genus Mesorhizobium, obtained from root nodules of woody legumes growing in Ethiopia, have been previously shown, by multilocus sequence analysis (MLSA) of five housekeeping genes, to form three novel genospecies. In the present study, the phylogenetic relationship between representative strains of these three genospecies and the type strains of their closest phylogenetic neighbours Mesorhizobium plurifarium, Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium huakuii was further evaluated using a polyphasic taxonomic approach. In line with our earlier MLSA of other housekeeping genes, the phylogenetic trees derived from the atpD and glnII genes grouped the test strains into three well-supported, distinct lineages that exclude all defined species of the genus Mesorhizobium. The DNA-DNA relatedness between the representative strains of genospecies I-III and the type strains of their closest phylogenetic neighbours was low (≤59 %). They differed from each other and from their closest phylogenetic neighbours by the presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon and nitrogen sources. The strains belonging to genospecies I, II and III therefore represent novel species for which we propose the names Mesorhizobium shonense sp. nov., Mesorhizobium hawassense sp. nov. and Mesorhizobium abyssinicae sp. nov. The isolates AC39a(T) ( = LMG 26966(T) = HAMBI 3295(T)), AC99b(T) ( = LMG 26968(T) = HAMBI 3301(T)) and AC98c(T) ( = LMG 26967(T) = HAMBI 3306(T)) are proposed as type strains for the respective novel species.

Phylogenetic multilocus sequence analysis identifies seven novel Ensifer genospecies isolated from a less-well-explored biogeographical region in East Africa.

The diversity of 71 rhizobial strains belonging to the genus Ensifer, isolated from root nodules of woody legumes growing in southern Ethiopia, was studied using multilocus sequence analysis (MLSA) and phenotypic approaches. Phylogenetic analyses based on core genes revealed that 43 strains were clustered in seven distinct and consistent positions (genospecies I-VII), while another 25 strains were also distinct but were discrepant in their placement on the different gene trees. The remaining three strains occupied the same phylogenetic branches as defined Ensifer species and thus were not distinct. Irrespective of their chromosomal background, the majority of the test strains were highly related with respect to their nifH and nodC gene sequences, suggesting that these symbionts might have acquired these genes recently from a common origin. On the nifH phylogenetic tree, the branch containing the test strains and reference species isolated from woody legumes in Africa was clearly separate from those isolated outside the continent, suggesting that these symbionts have a long history of separate evolution within Ensifer for this gene. A cross-inoculation study showed that our strains were capable of eliciting effective nodulation on the homologous host and on other host species. This suggests a potential to improve nitrogen fixation by selecting for broad-host-range inoculants. Our study confirms the presence of a wide diversity of Ensifer in East Africa and, while contributing to the general knowledge of the biodiversity within the genus, also highlights the need to focus on previously less-well-explored biogeographical regions to unravel as-yet-unidentified rhizobial resources.

Phylogeographic distribution of rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia.

Rhizobia are soilborne bacteria that form symbiotic relations with legumes and fix atmospheric nitrogen. The nitrogen fixation potential depends on several factors such as the type of host and symbionts and on environmental factors that affect the distribution of rhizobia. We isolated bacteria nodulating common bean in Southern Ethiopia to evaluate their genetic diversity and phylogeography at nucleotide, locus (gene/haplotype) and species levels of genetic hierarchy. Phylogenetically, eight rhizobial genospecies (including previous collections) were determined that had less genetic diversity than found among reference strains. The limited genetic diversity of the Ethiopian collections was due to absence of many of the Rhizobium lineages known to nodulate beans. Rhizobium etli and Rhizobiumphaseoli were predominant strains of bean-nodulating rhizobia in Ethiopia. We found no evidence for a phylogeographic pattern in strain distribution. However, joint analysis of the current and previous collections revealed differences between the two collections at nucleotide level of genetic hierarchy. The differences were due to genospecies Rhizobium aethiopicum that was only isolated in the earlier collection.

Phylogenetically diverse groups of Bradyrhizobium isolated from nodules of tree and annual legume species growing in Ethiopia.

Bacteria belonging to the genus Bradyrhizobium nodulate various leguminous woody plants and herbs, including economically important crops such as soybean, peanut and cowpea. Here we analysed 39 Bradyrhizobium strains originating from root nodules of the leguminous trees and crops Acacia saligna, Faidherbia albida, Erythrina brucei, Albizia gummifera, Millettia ferruginea, Cajanus cajan, Vigna unguiculata and Phaseolus vulgaris, growing in southern Ethiopia. Multilocus sequence analyses (MLSA) of the 16S rRNA, glnII, recA, gyrB and dnaK genes and the ITS region grouped the test strains into seven well-supported genospecies (I-VII), six of which occupied distinct positions excluding all hitherto defined Bradyrhizobium species. Analyses of the nodA, nodC and nifH genes suggested different evolutionary history of the chromosomal and symbiosis-related genes. Our study corroborates earlier findings that Ethiopia is a hotspot for rhizobial biodiversity, justifying further search for novel strains from this region and calling for intensified research on the ecology and biochemistry of these organisms.

Lentil (Lens culinaris Medik.) nodulates with genotypically and phenotypically diverse rhizobia in Ethiopian soils.

Forty-eight lentil-nodulating rhizobia were isolated from soil samples collected from diverse agro-ecological locations in Ethiopia, and characterized based on 76 phenotypic traits. Furthermore, 26 representative strains were selected and characterized using multilocus sequence analyses (MLSA) of core (16S rRNA, recA, atpD, glnII and gyrB) and symbiotic (nodA and nifH) genes. Numerical analysis of phenotypic characteristics showed that the 48 test strains fell into three major distinct clusters. The phylogenetic trees based on 16S rRNA genes showed that they belong to the Rhizobium genus. Our phylogenetic reconstruction based on combined gene trees (recA, atpD and glnII) supported three distinct sub-lineages (Clades I-III). While genospecies I and II could be classified with Rhizobium etli and Rhizobium leguminosarum, respectively, genospecies III, might be an unnamed genospecies within the genus Rhizobium. Phylogenetic reconstruction based on the symbiosis-related genes supported a single cluster, indicating differences in the evolutionary histories between chromosomal and symbiotic genes. Overall, these results confirmed the presence of a great diversity of lentil-nodulating Rhizobium species in Ethiopia, inviting further exploration. Moreover, the differences in symbiotic effectiveness of the test strains indicated the potential for selecting and using them as inoculants to improve the productivity of lentil in the country.

Metabolic and genomic diversity of rhizobia isolated from field standing native and exotic woody legumes in southern Ethiopia.

Eighty-seven rhizobial strains isolated from root nodules of field standing native and exotic woody legumes in southern Ethiopia were characterized using the Biolog method and AFLP fingerprinting technique. Cluster analysis of the metabolic and genomic fingerprints revealed 18 and 25 groups, respectively, demonstrating considerable diversity in rhizobial population indigenous to Ethiopian soils. While 25 strains (29%) were linked to members of Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium or Sinorhizobium, the bulk of the strains formed several distinct groups in both methods and did not relate to reference species included in the study. In contrast to exotic species which formed symbiosis with strains of only one specific genomic group, indigenous host species nodulated by metabolically and genomically diverse groups. The results in this study support the view, that long-term association between the symbionts allows gradual differentiation and diversity in compatible rhizobial population resident in native soils. Lack of significant metabolic and genomic relatedness to the reference strains in our results suggested that test strains in our collection probably included 'unique' types, which belong to several yet undefined rhizobial species.

Phylogenetic diversity of Rhizobium strains nodulating diverse legume species growing in Ethiopia.

The taxonomic diversity of thirty-seven Rhizobium strains, isolated from nodules of leguminous trees and herbs growing in Ethiopia, was studied using multilocus sequence analyses (MLSA) of six core and two symbiosis-related genes. Phylogenetic analysis based on the 16S rRNA gene grouped them into five clusters related to nine Rhizobium reference species (99-100% sequence similarity). In addition, two test strains occupied their own independent branches on the phylogenetic tree (AC86a2 along with R. tibeticum; 99.1% similarity and AC100b along with R. multihospitium; 99.5% similarity). One strain from Milletia ferruginea was closely related (>99%) to the genus Shinella, further corroborating earlier findings that nitrogen-fixing bacteria are distributed among phylogenetically unrelated taxa. Sequence analyses of five housekeeping genes also separated the strains into five well-supported clusters, three of which grouped with previously studied Ethiopian common bean rhizobia. Three of the five clusters could potentially be described into new species. Based on the nifH genes, most of the test strains from crop legumes were closely related to several strains of Ethiopian common bean rhizobia and other symbionts of bean plants (R. etli and R. gallicum sv. phaseoli). The grouping of the test strains based on the symbiosis-related genes was not in agreement with the housekeeping genes, signifying differences in their evolutionary history. Our earlier studies revealing a large diversity of Mesorhizobium and Ensifer microsymbionts isolated from Ethiopian legumes, together with the results from the present analysis of Rhizobium strains, suggest that this region might be a potential hotspot for rhizobial biodiversity.

Multilocus sequence analyses reveal several unnamed Mesorhizobium genospecies nodulating Acacia species and Sesbania sesban trees in Southern regions of Ethiopia.

Leguminous trees play an important role in agroforestry in Ethiopia, but studies of their rhizobial symbionts are scarce. In earlier studies, we surveyed natural nodulation of native leguminous trees growing in different agro-ecological zones in Southern Ethiopia, isolated 400 rhizobia, and characterized them based on different phenotypic and genotypic methods. In the present study we characterized 18 strains belonging to the genus Mesorhizobium, isolated from nodules of Acacia abyssinica, A. senegal, A. tortilis and Sesbania sesban. Phylogenetic analysis of nearly full-length 16S rRNA gene grouped the test strains into three distinct clades separated from all currently recognized Mesorhizobium species. Three divergent strains formed separate branches while the other 15 strains formed three distinct groups, genospecies I-III. Grouping of the isolates under study based on the house-keeping genes recA, gyrB, rpoB and gltA were consistent and in agreement with that of 16S rRNA. Similarly phylogenetic relationships based on the symbiosis-related genes nodC, nodA and nifH were generally similar to those shown by the core genes, suggesting that these Acacia and Sesbania symbionts have a long history of separate evolution within Mesorhizobium. Cross inoculation experiments demonstrated a large variation in the ability of the test strains to elicit effective nodules. The Sesbania isolates, occupying a distinct clade in the nodC phylogenetic tree, formed effective nodules only with this host legume. The study strongly suggests that this collection of Mesorhizobium strains comprises several new species, and also indicates the role of the symbiotic genes in determining the host range of these bacteria.

Genetic diversity and phylogeny of rhizobia isolated from agroforestry legume species in southern Ethiopia.

The genetic diversity within 195 rhizobial strains isolated from root nodules of 18 agroforestry species (15 woody and three herbaceous legumes) growing in diverse ecoclimatic zones in southern Ethiopia was investigated by using PCR-RFLP of the ribosomal operon [16S rRNA gene, 23S rRNA gene and the internal transcribed spacer (ITS) region between the 16S rRNA and 23S rRNA genes] and 16S rRNA gene partial sequence (800 and 1350 bp) analyses. All of the isolates and the 28 reference strains could be differentiated by using these methods. The size of the ITS varied among test strains (500-1300 bp), and 58 strains contained double copies. UPGMA dendrograms generated from cluster analyses of the 16S and 23S rRNA gene PCR-RFLP data were in good agreement, and the combined distance matrices delineated 87 genotypes, indicating considerable genetic diversity among the isolates. Furthermore, partial sequence analysis of 67 representative strains revealed 46 16S rRNA gene sequence types, among which 12 were 100% similar to those of previously described species and 34 were novel sequences with 94-99% similarity to those of recognized species. The phylogenetic analyses suggested that strains indigenous to Ethiopia belonged to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium and Sinorhizobium. Many of the rhizobia isolated from previously uninvestigated indigenous woody legumes had novel 16S rRNA gene sequences and were phylogenetically diverse. This study clearly shows that the characterization of symbionts of unexplored legumes growing in previously unexplored biogeographical areas will reveal additional diversity.