A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus.

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro, the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined ß-1,3/ß-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus. The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

Two members of a Nodule-specific Cysteine-Rich (NCR) peptide gene cluster are required for differentiation of rhizobia in Medicago truncatula nodules.

Legumes have evolved a nitrogen-fixing symbiotic interaction with rhizobia, and this association helps them to cope with the limited nitrogen conditions in soil. The compatible interaction between the host plant and rhizobia leads to the formation of root nodules, wherein internalization and transition of rhizobia into their symbiotic form, termed bacteroids, occur. Rhizobia in the nodules of the Inverted Repeat-Lacking Clade legumes, including Medicago truncatula, undergo terminal differentiation, resulting in elongated and endoreduplicated bacteroids. This transition of endocytosed rhizobia is mediated by a large gene family of host-produced nodule-specific cysteine-rich (NCR) peptides in M. truncatula. Few NCRs have been recently found to be essential for complete differentiation and persistence of bacteroids. Here, we show that a M. truncatula symbiotic mutant FN9285, defective in the complete transition of rhizobia, is deficient in a cluster of NCR genes. More specifically, we show that the loss of the duplicated genes NCR086 and NCR314 in the A17 genotype, found in a single copy in Medicago littoralis R108, is responsible for the ineffective symbiotic phenotype of FN9285. The NCR086 and NCR314 gene pair encodes the same mature peptide but their transcriptional activity varies considerably. Nevertheless, both genes can restore the effective symbiosis in FN9285 indicating that their complementation ability does not depend on the strength of their expression activity. The identification of the NCR086/NCR314 peptide, essential for complete bacteroid differentiation, has extended the list of peptides, from a gene family of several hundred members, that are essential for effective nitrogen-fixing symbiosis in M. truncatula.

Phosphatidylcholine-deficient suppressor mutant of Sinorhizobium meliloti, altered in fatty acid synthesis, partially recovers nodulation ability in symbiosis with alfalfa (Medicago sativa).

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

Aspartate aminotransferase of Rhizobium leguminosarum has extended substrate specificity and metabolizes aspartate to enable N2 fixation in pea nodules.

Rhizobium leguminosarum aspartate aminotransferase (AatA) mutants show drastically reduced symbiotic nitrogen fixation in legume nodules. Whilst AatA reversibly transaminates the two major amino-donor compounds aspartate and glutamate, the reason for the lack of N2 fixation in the mutant has remained unclear. During our investigations into the role of AatA, we found that it catalyses an additional transamination reaction between aspartate and pyruvate, forming alanine. This secondary reaction runs at around 60 % of the canonical aspartate transaminase reaction rate and connects alanine biosynthesis to glutamate via aspartate. This may explain the lack of any glutamate-pyruvate transaminase activity in R. leguminosarum, which is common in eukaryotic and many prokaryotic genomes. However, the aspartate-to-pyruvate transaminase reaction is not needed for N2 fixation in legume nodules. Consequently, we show that aspartate degradation is required for N2 fixation, rather than biosynthetic transamination to form an amino acid. Hence, the enzyme aspartase, which catalyses the breakdown of aspartate to fumarate and ammonia, suppressed an AatA mutant and restored N2 fixation in pea nodules.

The costs and benefits of symbiotic interactions: variable effects of rhizobia and arbuscular mycorrhizae on Vigna radiata accessions.

BACKGROUND: The symbiosis among plants, rhizobia, and arbuscular mycorrhizal fungi (AMF) is one of the most well-known symbiotic relationships in nature. However, it is still unclear how bilateral/tripartite symbiosis works under resource-limited conditions and the diverse genetic backgrounds of the host. RESULTS: Using a full factorial design, we manipulated mungbean accessions/subspecies, rhizobia, and AMF to test their effects on each other. Rhizobia functions as a typical facilitator by increasing plant nitrogen content, plant weight, chlorophyll content, and AMF colonization. In contrast, AMF resulted in a tradeoff in plants (reducing biomass for phosphorus acquisition) and behaved as a competitor in reducing rhizobia fitness (nodule weight). Plant genotype did not have a significant effect on AMF fitness, but different mungbean accessions had distinct rhizobia affinities. In contrast to previous studies, the positive relationship between plant and rhizobia fitness was attenuated in the presence of AMF, with wild mungbean being more responsive to the beneficial effect of rhizobia and attenuation by AMF. CONCLUSIONS: We showed that this complex tripartite relationship does not unconditionally benefit all parties. Moreover, rhizobia species and host genetic background affect the symbiotic relationship significantly. This study provides a new opportunity to re-evaluate the relationships between legume plants and their symbiotic partners.

The Defective in Autoregulation (DAR) gene of Medicago truncatula encodes a protein involved in regulating nodulation and arbuscular mycorrhiza.

BACKGROUND: Legumes utilize a long-distance signaling feedback pathway, termed Autoregulation of Nodulation (AON), to regulate the establishment and maintenance of their symbiosis with rhizobia. Several proteins key to this pathway have been discovered, but the AON pathway is not completely understood. RESULTS: We report a new hypernodulating mutant, defective in autoregulation, with disruption of a gene, DAR (Medtr2g450550/MtrunA17_Chr2g0304631), previously unknown to play a role in AON. The dar-1 mutant produces ten-fold more nodules than wild type, similar to AON mutants with disrupted SUNN gene function. As in sunn mutants, suppression of nodulation by CLE peptides MtCLE12 and MtCLE13 is abolished in dar. Furthermore, dar-1 also shows increased root length colonization by an arbuscular mycorrhizal fungus, suggesting a role for DAR in autoregulation of mycorrhizal symbiosis (AOM). However, unlike SUNN which functions in the shoot to control nodulation, DAR functions in the root. CONCLUSIONS: DAR encodes a membrane protein that is a member of a small protein family in M. truncatula. Our results suggest that DAR could be involved in the subcellular transport of signals involved in symbiosis regulation, but it is not upregulated during symbiosis. DAR gene family members are also present in Arabidopsis, lycophytes, mosses, and microalgae, suggesting the AON and AOM may use pathway components common to other plants, even those that do not undergo either symbiosis.

Timely symbiosis: circadian control of legume-rhizobia symbiosis.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialised polyploid cells within root nodules. This results in a mutualistic relationship whereby the plant host receives fixed nitrogen from the bacteria in exchange for dicarboxylic acids. This plant-microbe interaction requires the regulation of multiple metabolic and physiological processes in both the host and symbiont in order to achieve highly efficient symbiosis. Recent studies have showed that the success of symbiosis is influenced by the circadian clock of the plant host. Medicago and soybean plants with altered clock mechanisms showed compromised nodulation and reduced plant growth. Furthermore, transcriptomic analyses revealed that multiple genes with key roles in recruitment of rhizobia to plant roots, infection and nodule development were under circadian control, suggesting that appropriate timing of expression of these genes may be important for nodulation. There is also evidence for rhythmic gene expression of key nitrogen fixation genes in the rhizobium symbiont, and temporal coordination between nitrogen fixation in the bacterial symbiont and nitrogen assimilation in the plant host may be important for successful symbiosis. Understanding of how circadian regulation impacts on nodule establishment and function will identify key plant-rhizobial connections and regulators that could be targeted to increase the efficiency of this relationship.

An overview of Parafrankia (Nod+/Fix+) and Pseudofrankia (Nod+/Fix-) interactions through genome mining and experimental modeling in co-culture and co-inoculation of Elaeagnus angustifolia.

In many frankia, the ability to nodulate host plants (Nod+) and fix nitrogen (Fix+) is a common strategy. However, some frankia within the Pseudofrankia genus lack one or two of these traits. This phenomenon has been consistently observed across various actinorhizal nodule isolates, displaying Nod- and/or Fix- phenotypes. Yet, the mechanisms supporting the colonization and persistence of these inefficient frankia within nodules, both with and without symbiotic strains (Nod+/Fix+), remain unclear. It is also uncertain whether these associations burden or benefit host plants. This study delves into the ecological interactions between Parafrankia EUN1f and Pseudofrankia inefficax EuI1c, isolated from Elaeagnus umbellata nodules. EUN1f (Nod+/Fix+) and EuI1c (Nod+/Fix-) display contrasting symbiotic traits. While the prediction suggests a competitive scenario, the absence of direct interaction evidence implies that the competitive advantage of EUN1f and EuI1c is likely contingent on contextual factors such as substrate availability and the specific nature of stressors in their respective habitats. In co-culture, EUN1f outperforms EuI1c, especially under specific conditions, driven by its nitrogenase activity. Iron-depleted conditions favor EUN1f, emphasizing iron's role in microbial competition. Both strains benefit from host root exudates in pure culture, but EUN1f dominates in co-culture, enhancing its competitive traits. Nodulation experiments show that host plant preferences align with inoculum strain abundance under nitrogen-depleted conditions, while consistently favoring EUN1f in nitrogen-supplied media. This study unveils competitive dynamics and niche exclusion between EUN1f and EuI1c, suggesting that host plant may penalize less effective strains and even all strains. These findings highlight the complex interplay between strain competition and host selective pressure, warranting further research into the underlying mechanisms shaping plant-microbe-microbe interactions in diverse ecosystems. IMPORTANCE: While Pseudofrankia strains typically lack the common traits of ability to nodulate the host plant (Nod-) and/or fix nitrogen (Fix-), they are still recovered from actinorhizal nodules. The enigmatic question of how and why these unconventional strains establish themselves within nodule tissue, thriving either alongside symbiotic strains (Nod+/Fix+) or independently, while considering potential metabolic costs to the host plant, remains a perplexing puzzle. This study endeavors to unravel the competitive dynamics between Pseudofrankia inefficax strain EuI1c (Nod+/Fix-) and Parafrankia strain EU1Nf (Nod+/Fix+) through a comprehensive exploration of genomic data and empirical modeling, conducted both in controlled laboratory settings and within the host plant environment.

Cell-layer specific roles for gibberellins in nodulation and root development.

Gibberellins (GA) have a profound influence on the formation of lateral root organs. However, the precise role this hormone plays in the cell-specific events during lateral root formation, rhizobial infection and nodule organogenesis, including interactions with auxin and cytokinin (CK), is not clear. We performed epidermal- and endodermal-specific complementation of the severely GA-deficient na pea (Pisum sativum) mutant with Agrobacterium rhizogenes. Gibberellin mutants were used to examine the spatial expression pattern of CK (TCSn)- and auxin (DR5)-responsive promoters and hormone levels. We found that GA produced in the endodermis promote lateral root and nodule organogenesis and can induce a mobile signal(s) that suppresses rhizobial infection. By contrast, epidermal-derived GA suppress infection but have little influence on root or nodule development. GA suppress the CK-responsive TCSn promoter in the cortex and are required for normal auxin activation during nodule primordia formation. Our findings indicate that GA regulate the checkpoints between infection thread (IT) penetration of the cortex and invasion of nodule primordial cells and promote the subsequent progression of nodule development. It appears that GA limit the progression and branching of IT in the cortex by restricting CK response and activate auxin response to promote nodule primordia development.

The native distribution of a common legume shrub is limited by the range of its nitrogen-fixing mutualist.

Plant-microbe mutualisms, such as the legume-rhizobium symbiosis, are influenced by the geographical distributions of both partners. However, limitations on the native range of legumes, resulting from the absence of a compatible mutualist, have rarely been explored. We used a combination of a large-scale field survey and controlled experiments to determine the realized niche of Calicotome villosa, an abundant and widespread legume shrub. Soil type was a major factor affecting the distribution and abundance of C. villosa. In addition, we found a large region within its range in which neither C. villosa nor Bradyrhizobium, the bacterial genus that associates with it, were present. Seedlings grown in soil from this region failed to nodulate and were deficient in nitrogen. Inoculation of this soil with Bradyrhizobium isolated from root nodules of C. villosa resulted in the formation of nodules and higher growth rate, leaf N and shoot biomass compared with un-inoculated plants. We present evidence for the exclusion of a legume from parts of its native range by the absence of a compatible mutualist. This result highlights the importance of the co-distribution of both the host plant and its mutualist when attempting to understand present and future geographical distributions of legumes.

Rhizobium symbiotic efficiency meets CEP signaling peptides.

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation.

Is there a latitudinal diversity gradient for symbiotic microbes? A case study with sensitive partridge peas.

Mutualism is thought to be more prevalent in the tropics than temperate zones and may therefore play an important role in generating and maintaining high species richness found at lower latitudes. However, results on the impact of mutualism on latitudinal diversity gradients are mixed, and few empirical studies sample both temperate and tropical regions. We investigated whether a latitudinal diversity gradient exists in the symbiotic microbial community associated with the legume Chamaecrista nictitans. We sampled bacteria DNA from nodules and the surrounding soil of plant roots across a latitudinal gradient (38.64-8.68 °N). Using 16S rRNA sequence data, we identified many non-rhizobial species within C. nictitans nodules that cannot form nodules or fix nitrogen. Species richness increased towards lower latitudes in the non-rhizobial portion of the nodule community but not in the rhizobial community. The microbe community in the soil did not effectively predict the non-rhizobia community inside nodules, indicating that host selection is important for structuring non-rhizobia communities in nodules. We next factorially manipulated the presence of three non-rhizobia strains in greenhouse experiments and found that co-inoculations of non-rhizobia strains with rhizobia had a marginal effect on nodule number and no effect on plant growth. Our results suggest that these non-rhizobia bacteria are likely commensals-species that benefit from associating with a host but are neutral for host fitness. Overall, our study suggests that temperate C. nictitans plants are more selective in their associations with the non-rhizobia community, potentially due to differences in soil nitrogen across latitude.

Soluble humic acid suppresses plant immunity and ethylene to promote soybean nodulation.

Symbiotic nitrogen fixation (SNF) is a crucial process for nitrogen geochemical cycling and plant-microbe interactions. Water-soluble humic acid (WSHM), an active component of soil humus, has been shown to promote SNF in the legume-rhizobial symbiosis, but its molecular mechanism remains largely unknown. To reveal the SNF-promoting mechanism, we conducted transcriptomic analysis on soybean treated with WSHM. Our findings revealed that up- and downregulated differentially expressed genes (DEGs) were mainly involved in plant cell-wall/membrane formation and plant defence/immunity in the early stage, while the late stage was marked by the flavonoid synthesis and ethylene biosynthetic process. Further study on representative DEGs showed that WSHM could inhibit GmBAK1d-mediated immunity and BR signalling, thereby promoting rhizobial colonisation, infection, and nodulation, while not favoring pathogenic bacteria colonisation on the host plant. Additionally, we also found that the ethylene pathway is necessary for promoting the soybean nodulation by WSHM. This study not only provides a significant advance in our understanding of the molecular mechanism of WSHM in promoting SNF, but also provides evidence of the beneficial interactions among the biostimulator, host plant, and soil microbes, which have not been previously reported.

Nodulating another way: what can we learn from lateral root base nodulation in legumes?

Certain legumes provide a special pathway for rhizobia to invade the root and develop nitrogen-fixing nodules, a process known as lateral root base (LRB) nodulation. This pathway involves intercellular infection at the junction of the lateral roots with the taproot, leading to nodule formation in the lateral root cortex. Remarkably, this LRB pathway serves as a backbone for various adaptative symbiotic processes. Here, we describe different aspects of LRB nodulation and highlight directions for future research to elucidate the mechanisms of this as yet little known but original pathway that will help in broadening our knowledge on the rhizobium-legume symbiosis.

A pathogenesis-related protein, PRP1, negatively regulates root nodule symbiosis in Lotus japonicus.

The legume-rhizobium symbiosis represents a unique model within the realm of plant-microbe interactions. Unlike typical cases of pathogenic invasion, the infection of rhizobia and their residence within symbiotic cells do not elicit a noticeable immune response in plants. Nevertheless, there is still much to uncover regarding the mechanisms through which plant immunity influences rhizobial symbiosis. In this study, we identify an important player in this intricate interplay: Lotus japonicus PRP1, which serves as a positive regulator of plant immunity but also exhibits the capacity to decrease rhizobial colonization and nitrogen fixation within nodules. The PRP1 gene encodes an uncharacterized protein and is named Pathogenesis-Related Protein1, owing to its orthologue in Arabidopsis thaliana, a pathogenesis-related family protein (At1g78780). The PRP1 gene displays high expression levels in nodules compared to other tissues. We observed an increase in rhizobium infection in the L. japonicus prp1 mutants, whereas PRP1-overexpressing plants exhibited a reduction in rhizobium infection compared to control plants. Intriguingly, L. japonicus prp1 mutants produced nodules with a pinker colour compared to wild-type controls, accompanied by elevated levels of leghaemoglobin and an increased proportion of infected cells within the prp1 nodules. The transcription factor Nodule Inception (NIN) can directly bind to the PRP1 promoter, activating PRP1 gene expression. Furthermore, we found that PRP1 is a positive mediator of innate immunity in plants. In summary, our study provides clear evidence of the intricate relationship between plant immunity and symbiosis. PRP1, acting as a positive regulator of plant immunity, simultaneously exerts suppressive effects on rhizobial infection and colonization within nodules.

Rhizobium hidalgonense and Rhizobium redzepovicii as faba bean (Vicia faba L.) microsymbionts in Mexican soils.

As a legume crop widely cultured in the world, faba bean (Vicia faba L.) forms root nodules with diverse Rhizobium species in different regions. However, the symbionts associated with this plant in Mexico have not been studied. To investigate the diversity and species/symbiovar affiliations of rhizobia associated with faba bean in Mexico, rhizobia were isolated from this plant grown in two Mexican sites in the present study. Based upon the analysis of recA gene phylogeny, two genotypes were distinguished among a total of 35 isolates, and they were identified as Rhizobium hidalgonense and Rhizobium redzepovicii, respectively, by the whole genomic sequence analysis. Both the species harbored identical nod gene cluster and the same phylogenetic positions of nodC and nifH. So, all of them were identified into the symbiovar viciae. As a minor group, R. hidalgonense was only isolated from slightly acid soil and R. redzepovicii was the dominant group in both the acid and neutral soils. In addition, several genes related to resistance to metals (zinc, copper etc.) and metalloids (arsenic) were detected in genomes of the reference isolates, which might offer them some adaptation benefits. As conclusion, the community composition of faba bean rhizobia in Mexico was different from those reported in other regions. Furthermore, our study identified sv. viciae as the second symbiovar in the species R. redzepovicii. These results added novel evidence about the co-evolution, diversification and biogeographic patterns of rhizobia in association with their host legumes in distinct geographic regions.

Exploring the role of symbiotic modifier peptidases in the legume - rhizobium symbiosis.

Legumes can establish a mutual association with soil-derived nitrogen-fixing bacteria called 'rhizobia' forming lateral root organs called root nodules. Rhizobia inside the root nodules get transformed into 'bacteroids' that can fix atmospheric nitrogen to ammonia for host plants in return for nutrients and shelter. A substantial 200 million tons of nitrogen is fixed annually through biological nitrogen fixation. Consequently, the symbiotic mechanism of nitrogen fixation is utilized worldwide for sustainable agriculture and plays a crucial role in the Earth's ecosystem. The development of effective nitrogen-fixing symbiosis between legumes and rhizobia is very specialized and requires coordinated signaling. A plethora of plant-derived nodule-specific cysteine-rich (NCR or NCR-like) peptides get actively involved in this complex and tightly regulated signaling process of symbiosis between some legumes of the IRLC (Inverted Repeat-Lacking Clade) and Dalbergioid clades and nitrogen-fixing rhizobia. Recent progress has been made in identifying two such peptidases that actively prevent bacterial differentiation, leading to symbiotic incompatibility. In this review, we outlined the functions of NCRs and two nitrogen-fixing blocking peptidases: HrrP (host range restriction peptidase) and SapA (symbiosis-associated peptidase A). SapA was identified through an overexpression screen from the Sinorhizobium meliloti 1021 core genome, whereas HrrP is inherited extra-chromosomally. Interestingly, both peptidases affect the symbiotic outcome by degrading the NCR peptides generated from the host plants. These NCR-degrading peptidases can shed light on symbiotic incompatibility, helping to elucidate the reasons behind the inefficiency of nitrogen fixation observed in certain groups of rhizobia with specific legumes.

Guidelines for the description of rhizobial symbiovars.

Rhizobia are bacteria that form nitrogen-fixing nodules in legume plants. The sets of genes responsible for both nodulation and nitrogen fixation are carried in plasmids or genomic islands that are often mobile. Different strains within a species sometimes have different host specificities, while very similar symbiosis genes may be found in strains of different species. These specificity variants are known as symbiovars, and many of them have been given names, but there are no established guidelines for defining or naming them. Here, we discuss the requirements for guidelines to describe symbiovars, propose a set of guidelines, provide a list of all symbiovars for which descriptions have been published so far, and offer a mechanism to maintain a list in the future.

Reclassification of type strains of Rhizobium indigoferae and Sinorhizobium kummerowiae into the species Rhizobium leguminosarum and Sinorhizobium meliloti, respectively.

The species Rhizobium indigoferae and Sinorhizobium kummerowiae were isolated from legume nodules and the 16S rRNA sequences of their respective type strains, CCBAU 71042T and CCBAU 71714T, were highly divergent from those of the other species of the genera Rhizobium and Sinorhizobium, respectively. However, the 16S rRNA gene sequences obtained for strains CCBAU 71042T and CCBAU 71714T several years after description, were different from the original ones, showing 100 % similarity to the type strains of Rhizobium leguminosarum and Sinorhizobium meliloti, respectively. Phylogenetic analyses of two housekeeping genes, recA and atpD, confirmed the high phylogenetic closeness of strains CCBAU 71042T and CCBAU 71714T to the respective type strains of R. leguminosarum and S. meliloti. In the present work, we compared the genomes of the type strains of R. indigoferae and S. kummerowiae available in several culture collections with those of the respective type strains of R. leguminosarum and S. meliloti, some of them obtained in this study. The calculated average nucleotide identity-blast and digital DNA-DNA hybridization values in both cases were higher than those recommended for species differentiation, supporting the proposal for the reclassification of the type strains of R. indigoferae and S. kummerowiae into the species R. leguminosarum and S. meliloti, respectively.

Mesorhizobium retamae sp. nov., a novel non-nodulating and non-nitrogen-fixing species isolated from the root nodules of Retama raetam sampled in Tunisia.

A comprehensive polyphasic taxonomic investigation integrating taxongenomic criteria was conducted on strain IRAMC:0171T isolated from the root nodules of Retama raetam in Tunisia. This Gram-stain-negative and aerobic bacterium thrived within a temperature range of 5-45 °C, optimal at 28 °C, and tolerated salt concentrations from 0-6 % NaCl, with an optimal range of 0-3 %. It displayed pH tolerance from pH 4 to 10, thriving best at pH 6.8-7.5. Chemotaxonomically, strain IRAMC:0171T was characterized by diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine, and phosphatidylethanolamine as polar lipids. Its predominant fatty acid composition was C18 : 1 ω7c (61.2 %), and the primary ubiquinone was Q10 (97 %). Analysis of the 16S rRNA gene of strain IRAMC:0171T showed 99.08 % similarity to Mesorhizobium waimense ICMP 19557T, Mesorhizobium amorphae ACCC 19665T, and Mesorhizobium huakuii IAM 14158. However, digital DNA-DNA hybridization and average nucleotide identity analyses revealed values ranging from 21.1 to 25.2 % and 77.05 to 82.24 %, respectively, signifying significant deviation from established species demarcation thresholds. Phylogenetic studies, encompassing 16S rRNA, whole-genome-based tree reconstruction, and core protein analysis, positioned strain IRAMC:0171T closest to Mesorhizobium terrae KCTC 72278T and 'Mesorhizobium hungaricum' UASWS1009T, forming together a distinct branch within the genus Mesorhizobium. In consideration of this comprehensive data, we propose strain IRAMC:0171T (=DSM 112841T=CECT 30767T) as the type strain of a new species named Mesorhizobium retamae sp. nov.