Improving fertilizer response of crop yield through liming and targeting to landscape positions in tropical agricultural soils.

Nutrient management research was conducted across locations to investigate the influence of landscape position (hill, mid-, and foot slope) in teff (Eragrostis tef) and wheat (Triticum aestivum) yield response to fertilizer application and liming in the 2018 and 2019 cropping seasons. The treatments included 1) NPS fertilizer as a control treatment (42 N + 10P + 4.2S kg ha-1 for teff and 65 N + 20P + 8.5S kg ha-1 for wheat); 2) NPS and potassium (73 N + 17P + 7.2S + 24 K kg ha-1 for teff and 103 N + 30P + 12.7S + 24 K kg ha-1 for wheat) and 3) NPSK and zinc (73 N + 17P + 7.2S + 24K + 5.3Zn kg ha-1 for teff and 103 N + 30P + 12.7S + 24K + 5,3Zn kg ha-1 for wheat) in acid soils with and without liming. Results showed that the highest teff and wheat grain yields of 1512 and 4252 kg ha-1 were obtained at the foot slope position, with the respective yield increments of 71% and 57% over the hillslope position. Yield response to fertilizer application significantly decreased with increasing slope owing to the decrease in soil organic carbon and soil water content and the increase in soil acidity. The application of lime with NPSK and NPSKZn fertilizer increased teff and wheat yields by 43-54% and 32-35%, respectively compared to the application of NPS fertilizer without liming where yield increments were associated with the application of N and P nutrients. Orthogonal contrasts revealed that landscape position, fertilizer application, and their interaction effects were significant on teff and wheat yields. Soil properties including soil pH, organic carbon, total N, and soil water content were increased down the slope, which might be attributed to sedimentation down the slope. However, available P is yet very low both in acidic and non-acidic soils. We conclude that crop response to applied nutrients could be enhanced by targeting nutrient management practices to agricultural landscape features and addressing other yield-limiting factors such as soil acidity and nutrient availability by conducting further research.

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.

Affordable and robust phenotyping framework to analyse root system architecture of soil-grown plants.

The phenotypic analysis of root system growth is important to inform efforts to enhance plant resource acquisition from soils; however, root phenotyping remains challenging because of the opacity of soil, requiring systems that facilitate root system visibility and image acquisition. Previously reported systems require costly or bespoke materials not available in most countries, where breeders need tools to select varieties best adapted to local soils and field conditions. Here, we report an affordable soil-based growth (rhizobox) and imaging system to phenotype root development in glasshouses or shelters. All components of the system are made from locally available commodity components, facilitating the adoption of this affordable technology in low-income countries. The rhizobox is large enough (approximately 6000 cm2 of visible soil) to avoid restricting vertical root system growth for most if not all of the life cycle, yet light enough (approximately 21 kg when filled with soil) for routine handling. Support structures and an imaging station, with five cameras covering the whole soil surface, complement the rhizoboxes. Images are acquired via the Phenotiki sensor interface, collected, stitched and analysed. Root system architecture (RSA) parameters are quantified without intervention. The RSAs of a dicot species (Cicer arietinum, chickpea) and a monocot species (Hordeum vulgare, barley), exhibiting contrasting root systems, were analysed. Insights into root system dynamics during vegetative and reproductive stages of the chickpea life cycle were obtained. This affordable system is relevant for efforts in Ethiopia and other low- and middle-income countries to enhance crop yields and climate resilience sustainably.

A common mechanism for efficient N2 O reduction in diverse isolates of nodule-forming bradyrhizobia.

Bradyrhizobia are abundant soil bacteria, which can form nitrogen-fixing symbioses with leguminous plants, including important crops such as soybean, cowpea and peanut. Many bradyrhizobia can denitrify, but studies have hitherto focused on a few model organisms. We screened 39 diverse Bradyrhizobium strains, isolated from legume nodules. Half of them were unable to reduce N2 O, making them sources of this greenhouse gas. Most others could denitrify NO3 - to N2 . Time-resolved gas kinetics and transcription analyses during transition to anaerobic respiration revealed a common regulation of nirK, norCB and nosZ (encoding NO2 - , NO and N2 O reductases), and differing regulation of napAB (encoding periplasmic NO3 - reductase). A prominent feature in all N2 -producing strains was a virtually complete hampering of NO3 - reduction in the presence of N2 O. In-depth analyses suggest that this was due to a competition between electron transport pathways, strongly favouring N2 O over NO3 - reduction. In a natural context, bacteria with this feature would preferentially reduce available N2 O, produced by themselves or other soil bacteria, making them powerful sinks for this greenhouse gas. One way to augment such populations in agricultural soils is to develop inoculants for legume crops with dual capabilities of efficient N2 -fixation and efficient N2 O reduction.

Host Range and Symbiotic Effectiveness of N2O Reducing Bradyrhizobium Strains.

Emissions of the potent greenhouse gas N2O is one of the environmental problems associated with intensive use of synthetic N fertilizers, and novel N2O mitigation strategies are needed to minimize fertilizer applications and N2O release without affecting agricultural efficiencies. Increased incorporation of legume crops in agricultural practices offers a sustainable alternative. Legumes, in their symbiosis with nitrogen fixing bacteria, rhizobia, reduce the need for fertilizers and also respond to the need for increased production of plant-based proteins. Not all combinations of rhizobia and legumes result in efficient nitrogen fixation, and legume crops therefore often need to be inoculated with compatible rhizobial strains. Recent research has demonstrated that some rhizobia are also very efficient N2O reducers. Several nutritionally and economically important legumes form root nodules in symbiosis with bacteria belonging to Bradyrhizobium. Here, the host-ranges of fourteen N2O reducing Bradyrhizobium strains were tested on six legume hosts; cowpea, groundnut, mung bean, haricot bean, soybean, and alfalfa. The plants were grown for 35 days in pots in sterile sand supplemented with N-free nutrient solution. Cowpea was the most promiscuous host nodulated by all test strains, followed by groundnut (11 strains) and mungbean (4 strains). Three test strains were able to nodulate all these three legumes, while none nodulated the other three hosts. For cowpea, five strains increased the shoot dry weight and ten strains the shoot nitrogen content (pairwise comparison; p < 0.05). For groundnut the corresponding results were three and nine strains. The symbiotic effectiveness for the different strains ranged from 45 to 98% in cowpea and 34 to 95% in groundnut, relative to fertilized controls. The N2O reduction capacity of detached nodules from cowpea plants inoculated with one of these strains confirmed active N2O reduction inside the nodules. When released from senescent nodules such strains are expected to also act as sinks for N2O produced by denitrifying organisms in the soil microbial community. Our strategy to search among known N2O-reducing Bradyrhizobium strains for their N2-fixation effectiveness successfully identified several strains which can potentially be used for the production of legume inoculants with the dual capacities of efficacious N2-fixation and N2O reduction.

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.

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.

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.

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.

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.

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.

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.