Mesorhizobium koreense sp. nov., Isolated from Soil.

An aerobic, Gram-stain-negative, catalase-positive, rod-shaped, and motile bacteria, designated as a strain WR6T was isolated from soil in Republic of Korea. Strain WR6T grew at temperatures of 10-37°C, at pH of 5.0-9.0, and at NaCl concentrations of 0-3.0% (w/v). Phylogenetic and 16S rRNA gene nucleotide sequence analysis confirmed that strain WR6T affiliated to the genus Mesorhizobium, with the nearest relative being Mesorhizobium waimense ICMP 19557T (98.5%). The genome of strain WR6T was 5,035,462 bp with DNA G+C content of 62.6%. In strain WR6T, Q-10 was sole ubiquinone; summed feature 8 (C18:1ω7c and/or C18:1ω6c) and C19:0 cyclo ω8c were predominant fatty acids; and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylcholine, and phosphatidylethanolamine were major polar lipids. Based on these polyphasic taxonomic data, strain WR6T represents a novel species in the genus Mesorhizobium. Accordingly, we propose the name Mesorhizobium koreense sp. nov., with the type strain WR6T (=KCTC 92695T =NBRC 116021T).

O-Methylated Isoflavones Induce nod Genes of Mesorhizobium ciceri and Pratensein Promotes Nodulation in Chickpea.

Legume plants form symbiotic relationships with rhizobia, which allow plants to utilize atmospheric nitrogen as a nutrient. This symbiosis is initiated by secretion of specific signaling metabolites from the roots, which induce the expression of nod genes in rhizobia. These metabolites are called nod gene inducers (NGIs), and various flavonoids have been found to act as NGIs. However, NGIs of chickpea, the second major pulse crop, remain elusive. We conducted untargeted metabolome analysis of chickpea root exudates to explore metabolites with increased secretion under nitrogen deficiency. Principal component (PC) analysis showed a clear difference between nitrogen deficiency and control, with PC1 alone accounting for 37.5% of the variance. The intensity of two features with the highest PC1 loading values significantly increased under nitrogen deficiency; two prominent peaks were identified as O-methylated isoflavones, pratensein and biochanin A. RNA-seq analysis showed that they induce nodABC gene expression in the Mesorhizobium ciceri symbiont, suggesting that pratensein and biochanin A are chickpea NGIs. Pratensein applied concurrently with M. ciceri at sowing promoted chickpea nodulation. These results demonstrate that pratensein and biochanin A are chickpea NGIs, and pratensein can be useful for increasing nodulation efficiency in chickpea production.

Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction.

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.

Periodic cytokinin responses in Lotus japonicus rhizobium infection and nodule development.

Host plants benefit from legume root nodule symbiosis with nitrogen-fixing bacteria under nitrogen-limiting conditions. In this interaction, the hosts must regulate nodule numbers and distribution patterns to control the degree of symbiosis and maintain root growth functions. The host response to symbiotic bacteria occurs discontinuously but repeatedly at the region behind the tip of the growing roots. Here, live-imaging and transcriptome analyses revealed oscillating host gene expression with approximately 6-hour intervals upon bacterial inoculation. Cytokinin response also exhibited a similar oscillation pattern. Cytokinin signaling is crucial to maintaining the periodicity, as observed in cytokinin receptor mutants displaying altered infection foci distribution. This periodic regulation influences the size of the root region responsive to bacteria, as well as the nodulation process progression.

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.

Rhizobial Secretion of Truncated Exopolysaccharides Severely Impairs the Mesorhizobium-Lotus Symbiosis.

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously, we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A nonpolar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here, we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to the secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant, leading to abrogation of the infection process. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phylogenomic analyses and reclassification of the Mesorhizobium complex: proposal for 9 novel genera and reclassification of 15 species.

BACKGROUD: The genus Mesorhizobium is shown by phylogenomics to be paraphyletic and forms part of a complex that includes the genera Aminobacter, Aquamicrobium, Pseudaminobacter and Tianweitania. The relationships for type strains belong to these genera need to be carefully re-evaluated. RESULTS: The relationships of Mesorhizobium complex are evaluated based on phylogenomic analyses and overall genome relatedness indices (OGRIs) of 61 type strains. According to the maximum likelihood phylogenetic tree based on concatenated sequences of 539 core proteins and the tree constructed using the bac120 bacterial marker set from Genome Taxonomy Database, 65 type strains were grouped into 9 clusters. Moreover, 10 subclusters were identified based on the OGRIs including average nucleotide identity (ANI), average amino acid identity (AAI) and core-proteome average amino acid identity (cAAI), with AAI and cAAI showing a clear intra- and inter-(sub)cluster gaps of 77.40-80.91% and 83.98-86.16%, respectively. Combined with the phylogenetic trees and OGRIs, the type strains were reclassified into 15 genera. This list includes five defined genera Mesorhizobium, Aquamicrobium, Pseudaminobacter, Aminobacterand Tianweitania, among which 40/41 Mesorhizobium species and one Aminobacter species are canonical legume microsymbionts. The other nine (sub)clusters are classified as novel genera. Cluster III, comprising symbiotic M. alhagi and M. camelthorni, is classified as Allomesorhizobium gen. nov. Cluster VI harbored a single symbiotic species M. albiziae and is classified as Neomesorhizobium gen. nov. The remaining seven non-symbiotic members were proposed as: Neoaquamicrobium gen. nov., Manganibacter gen. nov., Ollibium gen. nov., Terribium gen. nov., Kumtagia gen. nov., Borborobacter gen. nov., Aerobium gen. nov.. Furthermore, the genus Corticibacterium is restored and two species in Subcluster IX-1 are reclassified as the member of this genus. CONCLUSION: The Mesorhizobium complex are classified into 15 genera based on phylogenomic analyses and OGRIs of 65 type strains. This study resolved previously non-monophyletic genera in the Mesorhizobium complex.

SeqCode facilitates naming of South African rhizobia left in limbo.

South Africa is well-known for the diversity of its legumes and their nitrogen-fixing bacterial symbionts. However, in contrast to their plant partners, remarkably few of these microbes (collectively referred to as rhizobia) from South Africa have been characterised and formally described. This is because the rules of the International Code of Nomenclature of Prokaryotes (ICNP) are at odds with South Africa's National Environmental Management: Biodiversity Act and its associated regulations. The ICNP requires that a culture of the proposed type strain for a novel bacterial species be deposited in two international culture collections and be made available upon request without restrictions, which is not possible under South Africa's current national regulations. Here, we describe seven new Mesorhizobium species obtained from root nodules of Vachellia karroo, an iconic tree legume distributed across various biomes in southern Africa. For this purpose, 18 rhizobial isolates were delineated into putative species using genealogical concordance, after which their plausibility was explored with phenotypic characters and average genome relatedness. For naming these new species, we employed the rules of the recently published Code of Nomenclature of Prokaryotes described from Sequence Data (SeqCode), which utilizes genome sequences as nomenclatural types. The work presented in this study thus provides an illustrative example of how the SeqCode allows for a standardised approach for naming cultivated organisms for which the deposition of a type strain in international culture collections is currently problematic.

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

Lineage-specific evolution of Aquibium, a close relative of Mesorhizobium, during habitat adaptation.

The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes.

Proximity-based defensive mutualism between Streptomyces and Mesorhizobium by sharing and sequestering iron.

Microorganisms living in soil maintain intricate interactions among themselves, forming the soil microbiota that influences the rhizosphere microbiome and plant growth. However, the mechanisms underlying the soil microbial interactions remain unclear. Streptomyces and Mesorhizobium are commonly found in soil and serve as plant growth-promoting rhizobacteria (PGPR). Here, we identified an unprecedented interaction between the colonies of red-soil-derived Streptomyces sp. FXJ1.4098 and Mesorhizobium sp. BAC0120 and referred to it as "proximity-based defensive mutualism (PBDM)." We found that metabolite-mediated iron competition and sharing between the two microorganisms were responsible for PBDM. Streptomyces sp. FXJ1.4098 produced a highly diffusible siderophore, desferrioxamine, which made iron unavailable to co-cultured Mesorhizobium sp. BAC0120, thereby inhibiting its growth. Streptomyces sp. FXJ1.4098 also released poorly diffusible iron-porphyrin complexes, which could be utilized by Mesorhizobium sp. BAC0120, thereby restoring the growth of nearby Mesorhizobium sp. BAC0120. Furthermore, in ternary interactions, the PBDM strategy contributed to the protection of Mesorhizobium sp. BAC0120 close to Streptomyces sp. FXJ1.4098 from other microbial competitors, resulting in the coexistence of these two PGPR. A scale-up pairwise interaction screening suggested that the PBDM strategy may be common between Mesorhizobium and red-soil-derived Streptomyces. These results demonstrate the key role of iron in complex microbial interactions and provide novel insights into the coexistence of PGPR in soil.

Influence of cyclic di-GMP metabolism to T3SS expression, biofilm formation and symbiosis efficiency in Mesorhizobium japonicum MAFF303099.

A link between the T3SS and inhibition of swimming motility by the transcriptional regulator TtsI in Mesorhizobium japonicum MAFF303099 has been previously reported. Here, we show that mutants in T3SS components display impaired biofilm formation capacity, indicating that a functional T3SS, or at least pili formation, is required for this process. As a first approach to the cdiG regulation network in this bacterium, we started a study of the second messenger cdiG by overexpressing or by deleting some genes encoding cdiG metabolizing enzymes. Overexpression of two putative PDEs as well as deletion of various DGCs led to reduced biofilm formation on glass tubes. Mutation of dgc9509 also affected negatively the nodulation and symbiosis efficiency on Lotus plants, which can be related to the observed reduction in adhesion to plant roots. Results from transcriptional nopX- and ttsI-promoter-lacZ fusions suggested that cdiG negatively regulates T3SS expression in M. japonicum MAFF303099.

A Lipopolysaccharide O-Antigen Synthesis Gene in Mesorhizobium huakuii Plays Differentiated Roles in Root Nodule Symbiotic Compatibility with Astragalus sinicus.

Lipopolysaccharide (LPS) is a ubiquitous microbial-associated molecular pattern. Plants can sense the three components of LPS, including core polysaccharide, lipid A, and O-antigen. LPS biosynthesis is an essential factor for the successful establishment of symbiosis in the rhizobium-legume plant system. The MCHK_1752 gene (Mesorhizobium huakuii 7653R gene) encodes O-antigen polymerase and affects the synthesis of O-antigen. Here, we investigated the symbiotic phenotypes of six Astragalus sinicus accessions inoculated with the MCHK_1752 deletion mutant strain. The results revealed that the MCHK_1752 deletion mutant strain had a suppressing effect on the symbiotic nitrogen fixation of two A. sinicus accessions, a promoting effect in three A. sinicus accessions, and no significant effect in one A. sinicus accessions. In addition, the effect of MCHK_1752 on the phenotype was confirmed by its complementary strains and LPS exogenous application. Deletion of MCHK_1752 showed no effect on the growth of a strain, but affected biofilm formation and led to higher susceptibility to stress in a strain. At the early symbiotic stage, Xinzi formed more infection threads and nodule primordia than Shengzhong under inoculation with the mutant, which might be an important reason for the final symbiotic phenotype. A comparison of early transcriptome data between Xinzi and Shengzhong also confirmed the phenotype at the early symbiotic stage. Our results suggest that O-antigen synthesis genes influence symbiotic compatibility during symbiotic nitrogen fixation. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

A Mesorhizobium japonicum quorum sensing circuit that involves three linked genes and an unusual acyl-homoserine lactone signal.

Members of the genus Mesorhizobium, which are core components of the rhizosphere and specific symbionts of legume plants, possess genes for acyl-homoserine lactone (AHL) quorum sensing (QS). Here we show Mesorhizobium japonicum MAFF 303099 (formerly M. loti) synthesizes and responds to N-[(2E, 4E)-2,4-dodecadienoyl] homoserine lactone (2E, 4E-C12:2-HSL). We show that the 2E, 4E-C12:2-HSL QS circuit involves one of four luxR-luxI-type genes found in the sequenced genome of MAFF 303099. We refer to this circuit, which appears to be conserved among Mesorhizobium species, as R1-I1. We show that two other Mesorhizobium strains also produce 2E, 4E-C12:2-HSL. The 2E, 4E-C12:2-HSL is unique among known AHLs in its arrangement of two trans double bonds. The R1 response to 2E, 4E-C12:2-HSL is extremely selective in comparison with other LuxR homologs, and the trans double bonds appear critical for R1 signal recognition. Most well-studied LuxI-like proteins use S-adenosylmethionine and an acyl-acyl carrier protein as substrates for synthesis of AHLs. Others that form a subgroup of LuxI-type proteins use acyl-coenzyme A substrates rather than acyl-acyl carrier proteins. I1 clusters with the acyl-coenzyme A-type AHL synthases. We show that a gene linked to the I1 AHL synthase is involved in the production of the QS signal. The discovery of the unique I1 product enforces the view that further study of acyl-coenzyme A-dependent LuxI homologs will expand our knowledge of AHL diversity. The involvement of an additional enzyme in AHL generation leads us to consider this system a three-component QS circuit. IMPORTANCE We report a Mesorhizobium japonicum quorum sensing (QS) system involving a novel acyl-homoserine lactone (AHL) signal. This system is known to be involved in root nodule symbiosis with host plants. The chemistry of the newly described QS signal indicated that there may be a dedicated cellular enzyme involved in its synthesis in addition to the types known for production of other AHLs. Indeed, we report that an additional gene is required for synthesis of the unique signal, and we propose that this is a three-component QS circuit as opposed to the canonical two-component AHL QS circuits. The signaling system is exquisitely selective. The selectivity may be important when this species resides in the complex microbial communities around host plants and may make this system useful in various synthetic biology applications of QS circuits.

Population genomics of Australian indigenous Mesorhizobium reveals diverse nonsymbiotic genospecies capable of nitrogen-fixing symbioses following horizontal gene transfer.

Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

Two New Rhizobiales Species Isolated from Root Nodules of Common Sainfoin (Onobrychis viciifolia) Show Different Plant Colonization Strategies.

Root nodules of legume plants are primarily inhabited by rhizobial nitrogen-fixing bacteria. Here, we propose two new Rhizobiales species isolated from root nodules of common sainfoin (Onobrychis viciifolia), as shown by core-gene phylogeny, overall genome relatedness indices, and pan-genome analysis. Mesorhizobium onobrychidis sp. nov. actively induces nodules and achieves atmospheric nitrogen and carbon dioxide fixation. This species appears to be depleted in motility genes and is enriched in genes for direct effects on plant growth performance. Its genome reveals functional and plant growth-promoting signatures, like a large unique chromosomal genomic island with high density of symbiotic genetic traits. Onobrychidicola muellerharveyae gen. nov. sp. nov. is described as a type species of the new genus Onobrychidicola in Rhizobiaceae. This species comprises unique genetic features and plant growth-promoting traits (PGPTs), which strongly indicate its function in biotic stress reduction and motility. We applied a newly developed bioinformatics approach for in silico prediction of PGPTs (PGPT-Pred), which supports the different lifestyles of the two new species and the plant growth-promoting performance of M. onobrychidis in the greenhouse trial. IMPORTANCE The intensive use of chemical fertilizers has a variety of negative effects on the environment. Increased utilization of biological nitrogen fixation (BNF) is one way to mitigate those negative impacts. In order to optimize BNF, suitable candidates for different legume species are required. Despite intensive search for new rhizobial bacteria associated with legumes, no new rhizobia have recently been identified from sainfoin (Onobrychis viciifolia). Here, we report on the discovery of two new rhizobial species associated with sainfoin, which are of high importance for the host and may help to increase sustainability in agricultural practices. We employed the combination of in silico prediction and in planta experiments, which is an effective way to detect promising plant growth-promoting bacteria.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Chickpea (Cicer arietinum L.) Seeds as a Reservoir of Endophytic Plant Growth-Promoting Bacteria.

The seed microbiome, the primary source of inoculum for plants, may play an important role in plant growth, health and productivity. However, the structure and function of chickpea seed endophytes are poorly characterized. Bacteria with beneficial characteristics can be selected by the plant and transmitted vertically via the seed to benefit the next generation. Studying the diversity and multifunctionality of seed microbial communities can provide innovative opportunities in the field of plant-microbe interaction. This study aimed to isolate, identify and characterize culturable endophytic bacteria from chickpea (Cicer arietinum L.) seeds. Phylogenetic analysis based on 16S rDNA showed that the endophytic bacteria belong to the genera Mesorhizobium, Burkholderia, Bacillus, Priestia, Paenibacillus, Alcaligenes, Acinetobacter, Rahnella, Enterobacter, Tsukamurella, and Microbacterium. The most frequently observed genus was Bacillus; however, rhizobia typically associated with chickpea roots were also found, which is a novel finding of this study. Siderophore production and phosphorus solubilization were the most widespread plant growth-promoting features, while hydrogen cyanide production was relatively rare among the isolates. Most of the isolates possess two or more plant growth-promoting features; however, only Bacillus thuringiensis Y2B, a well-known entomopathogenic bacteria, exhibited the presence of all plant growth-promoting traits evaluated. Results suggest that endophytic bacteria such as Bacillus, Mesorhizobium, and Burkholderia may be vertically transferred from inoculated plants to seeds to benefit the next generation.

The introduced strain Mesorhizobium ciceri USDA 3378 is more competitive than an indigenous strain in nodulation of chickpea in newly introduced areas of China.

The present study aimed to compare the competitive advantage of two chickpea nodulating rhizobia strains (an indigenous strain Mesorhizobium muleiense CCBAU 83963T and an introduced strain Mesorhizobium ciceri USDA 3378) in different soils originated from new chickpea cultivation areas of China. The results showed that USDA 3378 had a significant competitive advantage in nodulation, with nodulation occupation rates ranging from 84·6% to 100% in all the sampled soils. According to the efficiency of symbiosis under single inoculation, chickpea plants inoculated with USDA 3378 showed better symbiotic performance based on the plant dry weight, leaf chlorophyll content and nodule numbers. The chickpea plants inoculated with USDA 3378 formed nodules about 2 days earlier than those inoculated with CCBAU 83963T . The higher growth in media and the stronger adsorption on chickpea roots of USDA 3378 when mixed with CCBAU 83963T may explain why USDA 3378 shows a competitive advantage. The results from this study will contribute towards the development of effective chickpea rhizobial inoculants for soil conditioning and more environmentally friendly production of chickpeas in China.

Phenolic Acids Induce Nod Factor Production in Lotus japonicus-Mesorhizobium Symbiosis.

In legume-rhizobia symbiosis, partner recognition and the initiation of symbiosis processes require the mutual exchange of chemical signals. Chemicals, generally (iso)flavonoids, in the root exudates of the host plant induce the expression of nod genes in rhizobia, and, thus, are called nod gene inducers. The expression of nod genes leads to the production of lipochitooligosaccharides (LCOs) called Nod factors. Natural nod gene inducer(s) in Lotus japonicus-Mesorhizobium symbiosis remain unknown. Therefore, we developed an LCO detection method based on ultra-high-performance liquid chromatography-tandem-quadrupole mass spectrometry (UPLC-TQMS) to identify these inducers and used it herein to screen 40 phenolic compounds and aldonic acids for their ability to induce LCOs in Mesorhizobium japonicum MAFF303099. We identified five phenolic acids with LCO-inducing activities, including p-coumaric, caffeic, and ferulic acids. The induced LCOs caused root hair deformation, and nodule numbers in L. japonicus inoculated with M. japonicum were increased by these phenolic acids. The three phenolic acids listed above induced the expression of the nodA, nodB, and ttsI genes in a strain harboring a multicopy plasmid encoding NodD1, but not that encoding NodD2. The presence of p-coumaric and ferulic acids in the root exudates of L. japonicus was confirmed by UPLC-TQMS, and the induction of ttsI::lacZ in the strain harboring the nodD1 plasmid was detected in the rhizosphere of L. japonicus. Based on these results, we propose that phenolic acids are a novel type of nod gene inducer in L. japonicus-Mesorhizobium symbiosis.