Isolation and Assessment of the Symbiotic Efficiency of Faba bean (Vicia faba L.) Nodulating Rhizobial Isolates from Eastern Hararghe Highlands of Ethiopia

This particular work was devoted to isolate and assess the symbiotic efficiency of faba bean (Vicia faba L.)-nodulating rhizobia isolate at few faba bean growing areas of the eastern Hararghe highlands of Ethiopia. Overall 50 rhizobia isolates were obtained from soil samples of three Woredas (districts) of the eastern Hararghe highlands using the host trap method. Out of these 50 isolates, 40 were presumptively identified as rhizobia. Among these 40 rhizobia isolates, only 31 were successful to nodulate faba bean, and authenticated as true faba bean nodulating rhizobia. Concerning the symbiotic efficiency, about 52%, 35%, and 13% of the rhizobial isolates were found to be highly effective, effective, and lowly-effective, respectively. The correlation data on the sand experiment displayed that nodule dry weight was associated positively and significantly (r = 0.494, p<0.05) with shoot dry weight while shoot dry weight was associated positively and significantly (r=0.41, p<0.05) with plant total nitrogen. Positive correlations were also observed concerning shoot dry weight and dry weight of nodules (r = 0.7, p<0.05) on unsterilized soil. Among the observed rhizobium isolates, EHHFR (4A, 6A) showed the highest symbiotic efficiency above 110%, tolerated NaCl concentration ranging from 2% to 6% and 2% to 8%, respectively, and a pH range of 4.5 to 8 and 5 to 8, respectively. Thus, based on their symbiotic efficiency at the greenhouse level and relative tolerance to extreme conditions these faba bean nodulating rhizobia isolates were recommended to be used as nominees for the future development of faba bean rhizobial inoculants after being tested on field conditions.

Increasing the Legume–Rhizobia Symbiotic Efficiency Due to the Synergy between Commercial Strains and Strains Isolated from Relict Symbiotic Systems

The phenomenon of rhizobial synergy was investigated to increase the efficiency of nitrogen-fixing symbiosis of alfalfa (Medicago varia Martyn), common vetch (Vicia sativa L.) or red clover (Trifolium pratense L.). These plants were co-inoculated with the respective commercial strains Sinorhizobium meliloti RCAM1750, Rhizobium leguminosarum RCAM0626 or R. leguminosarum RCAM1365 and with the strains Mesorhizobium japonicum Opo-235, M. japonicum Opo-242, Bradyrhizobium sp. Opo-243 or M. kowhaii Ach-343 isolated from the relict legumes Oxytropis popoviana Peschkova and Astragalus chorinensis Bunge. The isolates mentioned above had additional symbiotic genes (fix, nif, nod, noe and nol) as well as the genes promoting plant growth and symbiosis formation (acdRS, genes associated with the biosynthesis of gibberellins and auxins, genes of T3SS, T4SS and T6SS secretion systems) compared to the commercial strains. Nodulation assays showed that in some variants of co-inoculation the symbiotic parameters of plants such as nodule number, plant biomass or acetylene reduction activity were increased. We assume that the study of microbial synergy using rhizobia of relict legumes will make it possible to carry out targeted selection of co-microsymbionts to increase the efficiency of agricultural legume–rhizobia systems.

Supply of acetyl-CoA to N2-fixing bacteroids: insights from the mutational and proteomic analyses of Sinorhizobium meliloti

Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.

Polymeric Formulations of Liquid Inoculants with Rhizobia Exopolysaccharides Increase Survival and Symbiotic Efficiency of Bradyrhizobium Strains with Cowpea and Soybean

We studied the survival of four elite strains of Bradyrhizobium in liquid inoculants with three formulations with EPS extracted from other rhizobia genera, and their symbiotic efficiency, with soybean and cowpea, in a greenhouse. For this purpose, we verified the utility of formulations for maintaining the cell viability of strains by counting the colony forming units (CFU) per milliliter of the liquid inoculants with formulations over 90 days. Survival of the soybean inoculant strains, 29W and CPAC15, in the PEPS formulation had the largest number of CFU (> 1010 mL− 1) after 90 days. For the cowpea inoculant strains, INPA3-1B and UFLA3-84, the formulations REPS1 had the largest number of CFU (> 1010 mL− 1) after 90 days. Symbiotic efficiency in soybean of the formulations PEPS and REPS2 was higher than that shown by the commercial inoculant. For cowpea, the three formulations with EPS showed symbiotic efficiency bigger than that of the commercial inoculant.

Multiple Domains in the Rhizobial Type III Effector Bel2-5 Determine Symbiotic Efficiency With Soybean

Bradyrhizobium elkanii utilizes the type III effector Bel2-5 for nodulation in host plants in the absence of Nod factors (NFs). In soybean plants carrying the Rj4 allele, however, Bel2-5 causes restriction of nodulation by triggering immune responses. Bel2-5 shows similarity with XopD of the phytopathogen Xanthomonas campestris pv. vesicatoria and possesses two internal repeat sequences, two ethylene (ET)-responsive element-binding factor-associated amphiphilic repression (EAR) motifs, a nuclear localization signal (NLS), and a ubiquitin-like protease (ULP) domain, which are all conserved in XopD except for the repeat domains. By mutational analysis, we revealed that most of the putative domains/motifs in Bel2-5 were essential for both NF-independent nodulation and nodulation restriction in Rj4 soybean. The expression of soybean symbiosis- and defense-related genes was also significantly altered by inoculation with the bel2-5 domain/motif mutants compared with the expression upon inoculation with wild-type B. elkanii, which was mostly consistent with the phenotypic changes of nodulation in host plants. Notably, the functionality of Bel2-5 was mostly correlated with the growth inhibition effect of Bel2-5 expressed in yeast cells. The nodulation phenotypes of the domain-swapped mutants of Bel2-5 and XopD indicated that both the C-terminal ULP domain and upstream region are required for the Bel2-5-dependent nodulation phenotypes. These results suggest that Bel2-5 interacts with and modifies host targets via these multiple domains to execute both NF-independent symbiosis and nodulation restriction in Rj4 soybean.

Symbiotic efficiency of pigeonpea (Cajanus cajan) with different sources of nitrogen

Pigeonpea is an important grain legume. It contributes to the improvement of soil fertility through biological nitrogen (N) fixation. However, the symbiotic efficiency of pigeonpea with native soil rhizobia has not been determined adequately. This study was designed to determine the variation in the N fixation ability of pigeonpea inoculated with the native rhizobia. Forty soil samples were collected from diverse locations across South Africa and used for inoculating pigeonpea seed. Each pigeonpea genotype was inoculated separately with each soil sample and raised in a nitrogen-depleted growth medium in the greenhouse. A split-plot experimental design was used in the study. Several N fixation variables of pigeonpea were measured. There was >40.0% difference in the number of nodules between genotypes ‘Ex-PP-MD-321’ and ‘Mpuma-B-Spot’ but the nodule dry weight between the two genotypes was >80.0%. In contrast, the heaviest dry shoots (0.4513 g), weighed 52.0% heavier than those that were observed for ‘Mpuma-B-Spot’. Pigeonpea showed differential N fixation ability with the nodules, suggesting that there was potential to select for optimum host × rhizobial isolate combinations for the process and to expand the production area of the crop.

Bradyrhizobium diazoefficiens USDA110 Nodulation of Aeschynomene afraspera Is Associated with Atypical Terminal Bacteroid Differentiation and Suboptimal Symbiotic Efficiency

ABSTRACT Legume plants can form root organs called nodules where they house intracellular symbiotic rhizobium bacteria. Within nodule cells, rhizobia differentiate into bacteroids, which fix nitrogen for the benefit of the plant. Depending on the combination of host plants and rhizobial strains, the output of rhizobium-legume interactions varies from nonfixing associations to symbioses that are highly beneficial for the plant. Bradyrhizobium diazoefficiens USDA110 was isolated as a soybean symbiont, but it can also establish a functional symbiotic interaction with Aeschynomene afraspera. In contrast to soybean, A. afraspera triggers terminal bacteroid differentiation, a process involving bacterial cell elongation, polyploidy, and increased membrane permeability, leading to a loss of bacterial viability while plants increase their symbiotic benefit. A combination of plant metabolomics, bacterial proteomics, and transcriptomics along with cytological analyses were used to study the physiology of USDA110 bacteroids in these two host plants. We show that USDA110 establishes a poorly efficient symbiosis with A. afraspera despite the full activation of the bacterial symbiotic program. We found molecular signatures of high levels of stress in A. afraspera bacteroids, whereas those of terminal bacteroid differentiation were only partially activated. Finally, we show that in A. afraspera, USDA110 bacteroids undergo atypical terminal differentiation hallmarked by the disconnection of the canonical features of this process. This study pinpoints how a rhizobium strain can adapt its physiology to a new host and cope with terminal differentiation when it did not coevolve with such a host. IMPORTANCE Legume-rhizobium symbiosis is a major ecological process in the nitrogen cycle, responsible for the main input of fixed nitrogen into the biosphere. The efficiency of this symbiosis relies on the coevolution of the partners. Some, but not all, legume plants optimize their return on investment in the symbiosis by imposing on their microsymbionts a terminal differentiation program that increases their symbiotic efficiency but imposes a high level of stress and drastically reduces their viability. We combined multi-omics with physiological analyses to show that the symbiotic couple formed by Bradyrhizobium diazoefficiens USDA110 and Aeschynomene afraspera, in which the host and symbiont did not evolve together, is functional but displays a low symbiotic efficiency associated with a disconnection of terminal bacteroid differentiation features.

Lineage-Specific Rewiring of Core Pathways Predating Innovation of Legume Nodules Shapes Symbiotic Efficiency

ABSTRACT The interkingdom coevolution innovated the rhizobium-legume symbiosis. The application of this nitrogen-fixing system in sustainable agriculture is usually impeded by incompatible interactions between partners. However, the progressive evolution of rhizobium-legume compatibility remains elusive. In this work, deletions of rhcV encoding a structural component of the type three secretion system allow related Sinorhizobium strains to nodulate a previously incompatible soybean cultivar (Glycine max). These rhcV mutants show low to medium to high symbiotic efficiency on the same cultivated soybean while being indistinguishable on wild soybean plants (Glycine soja). The dual pantranscriptomics reveals nodule-specific activation of core symbiosis genes of Sinorhizobium and Glycine genes associated with genome duplication events along the chronogram. Unexpectedly, symbiotic efficiency is in line with lineage-dependent transcriptional profiles of core pathways which predate the diversification of Fabaceae and Sinorhizobium. This is supported by further physiological and biochemical experiments. Particularly, low-efficiency nodules show disordered antioxidant activity and low-energy status, which restrict nitrogen fixation activity. Collectively, the ancient core pathways play a crucial role in optimizing the function of later-evolved mutualistic arsenals in the rhizobium-legume coevolution. IMPORTANCE Significant roles of complex extracellular microbiota in environmental adaptation of eukaryotes in ever-changing circumstances have been revealed. Given the intracellular infection ability, facultative endosymbionts can be considered pioneers within complex extracellular microbiota and are ideal organisms for understanding the early stage of interkingdom adaptation. This work reveals that the later innovation of key symbiotic arsenals and the lineage-specific network rewiring in ancient core pathways, predating the divergence of legumes and rhizobia, underline the progressive evolution of rhizobium-legume compatibility. This insight not only is significant for improving the application benefits of rhizobial inoculants in sustainable agriculture but also advances our general understanding of the interkingdom coevolution which is theoretically explored by all host-microbiota interactions.