Setting up Agrobacterium tumefaciens-mediated transformation of the tropical legume Aeschynomene evenia, a powerful tool for studying gene function in Nod Factor-independent symbiosis.

Most legumes are able to develop a root nodule symbiosis in association with proteobacteria collectively called rhizobia. Among them, the tropical species Aeschynomene evenia has the remarkable property of being nodulated by photosynthetic Rhizobia without the intervention of Nod Factors (NodF). Thereby, A. evenia has emerged as a working model for investigating the NodF-independent symbiosis. Despite the availability of numerous resources and tools to study the molecular basis of this atypical symbiosis, the lack of a transformation system based on Agrobacterium tumefaciens significantly limits the range of functional approaches. In this report, we present the development of a stable genetic transformation procedure for A. evenia. We first assessed its regeneration capability and found that a combination of two growth regulators, NAA (= Naphthalene Acetic Acid) and BAP (= 6-BenzylAminoPurine) allows the induction of budding calli from epicotyls, hypocotyls and cotyledons with a high efficiency in media containing 0,5 µM NAA (up to 100% of calli with continuous stem proliferation). To optimize the generation of transgenic lines, we employed A. tumefaciens strain EHA105 harboring a binary vector carrying the hygromycin resistance gene and the mCherry fluorescent marker. Epicotyls and hypocotyls were used as the starting material for this process. We have found that one growth medium containing a combination of NAA (0,5 µM) and BAP (2,2 µM) was sufficient to induce callogenesis and A. tumefaciens strain EHA105 was sufficiently virulent to yield a high number of transformed calli. This simple and efficient method constitutes a valuable tool that will greatly facilitate the functional studies in NodF-independent symbiosis.

Legume rhizodeposition promotes nitrogen fixation by soil microbiota under crop diversification.

Biological nitrogen fixation by free-living bacteria and rhizobial symbiosis with legumes plays a key role in sustainable crop production. Here, we study how different crop combinations influence the interaction between peanut plants and their rhizosphere microbiota via metabolite deposition and functional responses of free-living and symbiotic nitrogen-fixing bacteria. Based on a long-term (8 year) diversified cropping field experiment, we find that peanut co-cultured with maize and oilseed rape lead to specific changes in peanut rhizosphere metabolite profiles and bacterial functions and nodulation. Flavonoids and coumarins accumulate due to the activation of phenylpropanoid biosynthesis pathways in peanuts. These changes enhance the growth and nitrogen fixation activity of free-living bacterial isolates, and root nodulation by symbiotic Bradyrhizobium isolates. Peanut plant root metabolites interact with Bradyrhizobium isolates contributing to initiate nodulation. Our findings demonstrate that tailored intercropping could be used to improve soil nitrogen availability through changes in the rhizosphere microbiome and its functions.

Hopanoid lipids promote soybean-Bradyrhizobium symbiosis.

The symbioses between leguminous plants and nitrogen-fixing bacteria known as rhizobia are well known for promoting plant growth and sustainably increasing soil nitrogen. Recent evidence indicates that hopanoids, a family of steroid-like lipids, promote Bradyrhizobium symbioses with tropical legumes. To characterize hopanoids in Bradyrhizobium symbiosis with soybean, we validated a recently published cumate-inducible hopanoid mutant of Bradyrhizobium diazoefficiens USDA110, Pcu-shc::∆shc. GC-MS analysis showed that this strain does not produce hopanoids without cumate induction, and under this condition, is impaired in growth in rich medium and under osmotic, temperature, and pH stress. In planta, Pcu-shc::∆shc is an inefficient soybean symbiont with significantly lower rates of nitrogen fixation and low survival within the host tissue. RNA-seq revealed that hopanoid loss reduces the expression of flagellar motility and chemotaxis-related genes, further confirmed by swim plate assays, and enhances the expression of genes related to nitrogen metabolism and protein secretion. These results suggest that hopanoids provide a significant fitness advantage to B. diazoefficiens in legume hosts and provide a foundation for future mechanistic studies of hopanoid function in protein secretion and motility.A major problem for global sustainability is feeding our exponentially growing human population while available arable land decreases. Harnessing the power of plant-beneficial microbes is a potential solution, including increasing our reliance on the symbioses of leguminous plants and nitrogen-fixing rhizobia. This study examines the role of hopanoid lipids in the symbiosis between Bradyrhizobium diazoefficiens USDA110, an important commercial inoculant strain, and its economically significant host soybean. Our research extends our knowledge of the functions of bacterial lipids in symbiosis to an agricultural context, which may one day help improve the practical applications of plant-beneficial microbes in agriculture.

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.

Nodulation Experiment by Cross-Inoculation of Nitrogen-Fixing Bacteria Isolated from Root Nodules of Several Leguminous Plants.

Root-nodule nitrogen-fixing bacteria are known for being specific to particular legumes. This study isolated the endophytic root-nodule bacteria from the nodules of legumes and examined them to determine whether they could be used to promote the formation of nodules in other legumes. Forty-six isolates were collected from five leguminous plants and screened for housekeeping (16S rRNA), nitrogen fixation (nifH), and nodulation (nodC) genes. Based on the 16S rRNA gene sequencing and phylogenetic analysis, the bacterial isolates WC15, WC16, WC24, and GM5 were identified as Rhizobium, Sphingomonas, Methylobacterium, and Bradyrhizobium, respectively. The four isolates were found to have the nifH gene, and the study confirmed that one isolate (GM5) had both the nifH and nodC genes. The Salkowski method was used to measure the isolated bacteria for their capacity to produce phytohormone indole acetic acid (IAA). Additional experiments were performed to examine the effect of the isolated bacteria on root morphology and nodulation. Among the four tested isolates, both WC24 and GM5 induced nodulation in Glycine max. The gene expression studies revealed that GM5 had a higher expression of the nifH gene. The existence and expression of the nitrogen-fixing genes implied that the tested strain had the ability to fix the atmospheric nitrogen. These findings demonstrated that a nitrogen-fixing bacterium, Methylobacterium (WC24), isolated from a Trifolium repens, induced the formation of root nodules in non-host leguminous plants (Glycine max). This suggested the potential application of these rhizobia as biofertilizer. Further studies are required to verify the N2-fixing efficiency of the isolates.

Impact of antagonistic endophytic bacteria on productivity of some economically important legumes.

Understanding the interactions within and between endophytes and their hosts is still obscure. Investigating endophytic bacterial plant growth-promoting (PGP) traits and co-inoculation effects on legumes' performance is a candidate. Endophytic bacteria were isolated from Vicia sativa root nodules. Such endophytes were screened for their PGP traits, hydrolytic enzymes, and antifungal activities. Sterilized Vicia faba and Pisum sativum seedlings were co-inoculated separately with seven different endophytic bacterial combinations before being planted under sterilized conditions. Later on, several growth-related traits were measured. Eleven endophytes (six rhizobia, two non-rhizobia, and three actinomycetes) could be isolated, and all of them were indole-acetic-acid (IAA) producers, while seven isolates could solubilize phosphorus, whereas three, five, five, and four isolates could produce protease, cellulase, amylase, and chitinase, respectively. Besides, some of these isolates possessed powerful antifungal abilities against six soil-borne pathogenic fungi. Co-inoculation of tested plants with endophytic bacterial mixes (Rhizobiamix+Actinomix+non-Rhizobiamix), (Rhizobiamix+Actinomix), or (Rhizobiamix+non-Rhizobiamix) significantly improved the studied growth parameters (shoot, root fresh and dry weights, length and yield traits) compared to controls, whereas co-inoculated plants with (Rhizobiaalone), (non-Rhizobiamix), or (Actinomix) significantly recorded lower growth parameters. Five efficient endophytes were identified: Rhizobium leguminosarum bv. Viciae, Rhizobium pusense, Brevibacterium frigoritolerans, Streptomyces variabilis, and Streptomyces tendae. Such results suggested that these isolates could be utilized as biocontrols and biofertilizers to improve legumes productivity. Also, co-inoculation with different endophytic mixes is better than single inoculation, a strategy that should be commercially exploited.

Unraveling the rhizobial infection thread.

Most legumes can form an endosymbiotic association with soil bacteria called rhizobia, which colonize specialized root structures called nodules where they fix nitrogen. To colonize nodule cells, rhizobia must first traverse the epidermis and outer cortical cell layers of the root. In most legumes, this involves formation of the infection thread, an intracellular structure that becomes colonized by rhizobia, guiding their passage through the outer cell layers of the root and into the newly formed nodule cells. In this brief review, we recount the early research milestones relating to the rhizobial infection thread and highlight two relatively recent advances in the symbiotic infection mechanism, the eukaryotically conserved 'MYB-AUR1-MAP' mitotic module, which links cytokinesis mechanisms to intracellular infection, and the discovery of the 'infectosome' complex, which guides infection thread growth. We also discuss the potential intertwining of the two modules and the hypothesis that cytokinesis served as a foundation for intracellular infection of symbiotic microbes.

Phyllobacteriaceae: a family of ecologically and metabolically diverse bacteria with the potential for different applications.

The family Phyllobacteriaceae is a heterogeneous assemblage of more than 146 species of bacteria assigned to its existing 18 genera. Phylogenetic analyses have shown great phylogenetic diversity and also suggested about incorrect classification of several species that need to be reassessed for their proper phylogenetic classification. However, almost 50% of the family members belong to the genus Mesorhizobium only, of which the majority are symbiotic nitrogen fixers associated with different legumes. Other major genera are Phyllobacterium, Nitratireductor, Aquamicrobium, and Aminobacter. Nitrogen-fixing, legume nodulating members are present in Aminobacter and Phyllobacterium as well. Aquamicrobium spp. can degrade environmental pollutants, like 2,4-dichlorophenol, 4-chloro-2-methylphenol, and 4-chlorophenol. Chelativorans, Pseudaminobacter, Aquibium, and Oricola are the other genera that contain multiple species having diverse metabolic capacities, the rest being single-membered genera isolated from varied environments. In addition, heavy metal and antibiotic resistance, chemolithoautotrophy, poly-ß-hydroxybutyrate storage, cellulase production, etc., are the other notable characteristics of some of the family members. In this report, we have comprehensively reviewed each of the species of the family Phyllobacteriaceae in their eco-physiological aspects and found that the family is rich with ecologically and metabolically highly diverse bacteria having great potential for human welfare and environmental clean-up.

Comparative phylogenomics and phylotranscriptomics provide insights into the genetic complexity of nitrogen-fixing root-nodule symbiosis.

Plant root-nodule symbiosis (RNS) with mutualistic nitrogen-fixing bacteria is restricted to a single clade of angiosperms, the Nitrogen-Fixing Nodulation Clade (NFNC), and is best understood in the legume family. Nodulating species share many commonalities, explained either by divergence from a common ancestor over 100 million years ago or by convergence following independent origins over that same time period. Regardless, comparative analyses of diverse nodulation syndromes can provide insights into constraints on nodulation-what must be acquired or cannot be lost for a functional symbiosis-and the latitude for variation in the symbiosis. However, much remains to be learned about nodulation, especially outside of legumes. Here, we employed a large-scale phylogenomic analysis across 88 species, complemented by 151 RNA-seq libraries, to elucidate the evolution of RNS. Our phylogenomic analyses further emphasize the uniqueness of the transcription factor NIN as a master regulator of nodulation and identify key mutations that affect its function across the NFNC. Comparative transcriptomic assessment revealed nodule-specific upregulated genes across diverse nodulating plants, while also identifying nodule-specific and nitrogen-response genes. Approximately 70% of symbiosis-related genes are highly conserved in the four representative species, whereas defense-related and host-range restriction genes tend to be lineage specific. Our study also identified over 900 000 conserved non-coding elements (CNEs), over 300 000 of which are unique to sampled NFNC species. NFNC-specific CNEs are enriched with the active H3K9ac mark and are correlated with accessible chromatin regions, thus representing a pool of candidate regulatory elements for genes involved in RNS. Collectively, our results provide novel insights into the evolution of nodulation and lay a foundation for engineering of RNS traits in agriculturally important crops.

Diversity and pathogenic characteristics of the Fusarium species isolated from minor legumes in Korea.

Legumes are primarily grown agriculturally for human consumption, livestock forage, silage, and as green manure. However, production has declined primarily due to fungal pathogens. Among them, this study focused on Fusarium spp. that cause Fusarium wilt in minor legumes in Korea. Diseased legume plants were collected from 2020 to 2021, and diverse fungal genera were isolated from the internal tissues of the plant roots and stems. Fusarium spp. were the most dominant, accounting for 71% of the isolates. They were identified via morphological characteristics and molecular identification. In the pathogenicity test, Fusarium oxysporum and Fusarium fujikuroi generally exhibited high virulence. The host range investigation revealed that the NC20-738, NC20-739, and NC21-950 isolates infected all nine crops, demonstrating the widest host range. In previous studies, the focus was solely on Fusarium wilt disease in soybeans. Therefore, in this study, we aimed to investigate Fusarium wilt occurred in minor legumes, which are consumed as extensively as soybeans, due to the scarcity of data on the diversity and characteristics of Fusarium spp. existing in Korea. The diverse information obtained in this study will serve as a foundation for implementing effective management strategies against Fusarium-induced plant diseases.

Comparative Analysis of Secondary Metabolites Produced by Ascochyta fabae under In Vitro Conditions and Their Phytotoxicity on the Primary Host, Vicia faba, and Related Legume Crops.

Ascochyta blight, caused by Ascochyta fabae, poses a significant threat to faba bean and other legumes worldwide. Necrotic lesions on stems, leaves, and pods characterize the disease. Given the economic impact of this pathogen and the potential involvement of secondary metabolites in symptom development, a study was conducted to investigate the fungus's ability to produce bioactive metabolites that might contribute to its pathogenicity. For this investigation, the fungus was cultured in three substrates (Czapek-Dox, PDB, and rice). The produced metabolites were analyzed by NMR and LC-HRMS methods, resulting in the dereplication of seven metabolites, which varied with the cultural substrates. Ascochlorin, ascofuranol, and (R)-mevalonolactone were isolated from the Czapek-Dox extract; ascosalipyrone, benzoic acid, and tyrosol from the PDB extract; and ascosalitoxin and ascosalipyrone from the rice extract. The phytotoxicity of the pure metabolites was assessed at different concentrations on their primary hosts and related legumes. The fungal exudates displayed varying degrees of phytotoxicity, with the Czapek-Dox medium's exudate exhibiting the highest activity across almost all legumes tested. The species belonging to the genus Vicia spp. were the most susceptible, with faba bean being susceptible to all metabolites, at least at the highest concentration tested, as expected. In particular, ascosalitoxin and benzoic acid were the most phytotoxic in the tested condition and, as a consequence, expected to play an important role on necrosis's appearance.

Plant growth promoting activities of Pseudomonas sp. and Enterobacter sp. isolated from the rhizosphere of Vachellia gummifera in Morocco.

The Moroccan endemic Vachellia gummifera grows wild under extreme desert conditions. This plant could be used as an alternative fodder for goats, and camels, in order to protect the Argan forests against overgrazing in Central and Southwestern Moroccan semiarid areas. With the aim to improve the V. gummifera population's density in semiarid areas, we proposed its inoculation with performing plant growth-promoting bacteria. Hence, 500 bacteria were isolated from the plant rhizosphere. From these, 291 isolates were retained for plant growth-promoting (PGP) activities assessment. A total of 44 isolates showed the best phosphates solubilization potential, as well as siderophore and auxin production. The combination of REP-PCR (repetitive extragenic palindromic-polymerase chain reaction) fingerprinting, PGP activities, and phenotypic properties, allowed the selection of three strains for the inoculation experiments. The three selected strains' 16S rRNA sequencing showed that they are members of the Enterobacter and Pseudomonas genera. The inoculation with three strains had diverse effects on V. gummifera growth parameters. All single and combined inoculations improved the plant shoot weight by more than 200%, and the root length by up to 139%, while some combinations further improved protein and chlorophyll content, thereby improving the plant's forage value. The three selected strains constitute an effective inoculum for use in the arid and semiarid zones of southern Morocco.

Pioneering Desmodium spp. are nodulated by natural populations of stress-tolerant alpha- and beta-rhizobia.

The rhizobia-Desmodium (Leguminosae, Papilionoideae) symbiosis is generally described by its specificity with alpha-rhizobia, especially with Bradyrhizobium. Our study aimed to isolate rhizobia from root nodules of native D. barbatum, D. incanum, and D. discolor, collected in remnants of the biomes of Atlantic Forest and Cerrado in protected areas of the Paraná State, southern Brazil. Based on the 16S rRNA phylogeny, 18 out of 29 isolates were classified as Alphaproteobacteria (Bradyrhizobium and Allorhizobium/Rhizobium) and 11 as Betaproteobacteria (Paraburkholderia). Phylogeny of the recA gene of the alpha-rhizobia resulted in ten main clades, of which two did not group with any described rhizobial species. In the 16S rRNA phylogeny of the beta-rhizobia, Paraburkholderia strains from the same host and conservation unity occupied the same clade. Phenotypic characterization of representative strains revealed the ability of Desmodium rhizobia to grow under stressful conditions such as high temperature, salinity, low pH conditions, and tolerance of heavy metals and xenobiotic compounds. Contrasting with previous reports, our results revealed that Brazilian native Desmodium can exploit symbiotic interactions with stress-tolerant strains of alpha- and beta-rhizobia. Stress tolerance can highly contribute to the ecological success of Desmodium in this phytogeographic region, possibly relating to its pioneering ability in Brazil. We propose Desmodium as a promising model for studies of plant-rhizobia interactions.

Both incompatible and compatible rhizobia inhabit the intercellular spaces of leguminous root nodules.

In addition to rhizobia, many types of co-existent bacteria are found in leguminous root nodules, but their habitats are unclear. To investigate this phenomenon, we labeled Bradyrhizobium diazoefficiens USDA122 and Bradyrhizobium sp. SSBR45 with Discosoma sp. red fluorescent protein (DsRed) or enhanced green fluorescent protein (eGFP). USDA122 enhances soybean growth by forming effective root nodules, but SSBR45 does not form any nodules. Using low-magnification laser scanning confocal microscopy, we found that infected cells in the central zone of soybean nodules appeared to be occupied by USDA122. Notably, high-magnification microscopy after co-inoculation of non-fluorescent USDA122 and fluorescence-labeled SSBR45 also revealed that SSBR45 inhabits the intercellular spaces of healthy nodules. More unexpectedly, co-inoculation of eGFP-labeled USDA122 and DsRed-labeled SSBR45 (and vice versa) revealed the presence of USDA122 bacteria in both the symbiosomes of infected cells and in the apoplasts of healthy nodules. We then next inspected nodules formed after a mixed inoculation of differently-labeled USDA122, without SSBR45, and confirmed the inhabitation of the both populations of USDA122 in the intercellular spaces. In contrast, infected cells were occupied by single-labeled USDA122. We also observed Mesorhizobium loti in the intercellular spaces of active wild-type nodules of Lotus japonicus using transmission electron microscopy. Compatible intercellular rhizobia have been described during nodule formation of several legume species and in some mutants, but our evidence suggests that this type of colonization may occur much more commonly in leguminous root nodules.

What determines symbiotic nitrogen fixation efficiency in rhizobium: recent insights into Rhizobium leguminosarum.

Symbiotic nitrogen fixation (SNF) by rhizobium, a Gram-negative soil bacterium, is an essential component in the nitrogen cycle and is a sustainable green way to maintain soil fertility without chemical energy consumption. SNF, which results from the processes of nodulation, rhizobial infection, bacteroid differentiation and nitrogen-fixing reaction, requires the expression of various genes from both symbionts with adaptation to the changing environment. To achieve successful nitrogen fixation, rhizobia and their hosts cooperate closely for precise regulation of symbiotic genes, metabolic processes and internal environment homeostasis. Many researches have progressed to reveal the ample information about regulatory aspects of SNF during recent decades, but the major bottlenecks regarding improvement of nitrogen-fixing efficiency has proven to be complex. In this mini-review, we summarize recent advances that have contributed to understanding the rhizobial regulatory aspects that determine SNF efficiency, focusing on the coordinated regulatory mechanism of symbiotic genes, oxygen, carbon metabolism, amino acid metabolism, combined nitrogen, non-coding RNAs and internal environment homeostasis. Unraveling regulatory determinants of SNF in the nitrogen-fixing protagonist rhizobium is expected to promote an improvement of nitrogen-fixing efficiency in crop production.

Promoting the growth of Sulla flexuosa L. by endophytic root nodule bacteria authors and affiliations.

Legume plants rely upon multipartite interactions between rhizobia and bacterial endophytes within root nodules to facilitate plant growth. This study aimed to isolate and identify indigenous endophytic bacteria from root nodules of Sulla aculeolata L. in Northeast Morocco. Based on their tri-calcium phosphate (TCP) solubilization capacity, five endophytes were chosen for further evaluation of their plant growth traits. All isolates were hydrogen cyanide (HCN) and siderophore producers, while only BCH24 tested positive for ACC deaminase activity. Indole-3-acetic acid (IAA) synthesis ranged from 1.27 mgL- 1 to 2.89 mgL- 1, while soluble phosphate concentrations was between 7.99 mg L- 1 and 110.58 mg L- 1. Additionally, all the endophytes were able to produce more than two lytic enzymes. Based on the analysis of 16 S rRNA gene sequences five isolates were identified as Enterobacter sp (BCH13, BCH2), Pseudomonas sp (BCH16, BCH24), and Serratia sp (BCH10). The strains inhibited the growth of three phytopathogenic fungi, with BCH13 exhibiting the highest rate against Aspergillus ochraceus (45%), followed by BCH24 against Fusarium oxysporum (40%) and Botrytis cinerea (35%), respectively. In vivo inoculation of halotolerant strains Enterobacter hormaechei (BCH13) and Pseudomonas moraviensis (BCH16) under gnotobiotic conditions revealed that co-inoculation with Rhizobium sullae KS6 improved plant development compared to single inoculation, making it a promising eco-friendly bio-inoculant for legume Sulla flexuosa L. production.

Pseudomonas quebecensis sp. nov., a bacterium isolated from root-zone soil of a native legume, Amphicarpaea bracteata (L.) Fernald, in Quebec, Canada.

Four bacterial strains (S1Bt3, S1Bt7, S1Bt30 and S1Bt42T) isolated from soil collected from the rhizosphere of a native legume, Amphicarpaea bracteata, were investigated using a polyphasic approach. Colonies were fluorescent, white-yellowish, circular and convex with regular margins on King's B medium. Cells were Gram-reaction-negative, aerobic, non-spore-forming rods. Oxidase- and catalase-positive. The optimal growth temperature of the strains was 37 °C. Phylogenetic analysis of the 16S rRNA gene sequences placed the strains within the genus Pseudomonas. Analysis of the 16S rRNA-rpoD-gyrB concatenated sequences clustered the strains and well separated from Pseudomonas rhodesiae CIP 104664T and Pseudomonas grimontii CFM 97-514T with the type strains of the closest species. Phylogenomic analysis of 92 up-to-date bacterial core gene and matrix-assisted laser desorption/ionization-time-of-flight MS biotyper data confirmed the distinct clustering pattern of these four strains. Digital DNA-DNA hybridization (41.7 %-31.2 %) and average nucleotide identity (91.1 %-87.0 %) values relative to closest validly published Pseudomonas species were below the species delineation thresholds of 70 and 96 %, respectively. Fatty acid composition results validated the taxonomic position of the novel strains in the genus Pseudomonas. Phenotypic characteristics from carbon utilization tests differentiated the novel strains from closely related Pseudomonas species. In silico prediction of secondary metabolite biosynthesis gene clusters in the whole-genome sequences of the four strains revealed the presence of 11 clusters involved in the production of siderophore, redox-cofactor, betalactone, terpene, arylpolyene and nonribosomal peptides. Based on phenotypic and genotypic data, strains S1Bt3, S1Bt7, S1Bt30 and S1Bt42T represent a novel species for which the name Pseudomonas quebecensis sp. nov. is proposed. The type strain is S1Bt42T (=DOAB 746T=LMG 32141T=CECT 30251T). The genomic DNA G+C content is 60.95 mol%.

Forging a symbiosis: transition metal delivery in symbiotic nitrogen fixation.

Symbiotic nitrogen fixation carried out by the interaction between legumes and rhizobia is the main source of nitrogen in natural ecosystems and in sustainable agriculture. For the symbiosis to be viable, nutrient exchange between the partners is essential. Transition metals are among the nutrients delivered to the nitrogen-fixing bacteria within the legume root nodule cells. These elements are used as cofactors for many of the enzymes controlling nodule development and function, including nitrogenase, the only known enzyme able to convert N2 into NH3 . In this review, we discuss the current knowledge on how iron, zinc, copper, and molybdenum reach the nodules, how they are delivered to nodule cells, and how they are transferred to nitrogen-fixing bacteria within.

Widespread Bradyrhizobium distribution of diverse Type III effectors that trigger legume nodulation in the absence of Nod factor.

The establishment of the rhizobium-legume symbiosis is generally based on plant perception of Nod factors (NFs) synthesized by the bacteria. However, some Bradyrhizobium strains can nodulate certain legume species, such as Aeschynomene spp. or Glycine max, independently of NFs, and via two different processes that are distinguished by the necessity or not of a type III secretion system (T3SS). ErnA is the first known type III effector (T3E) triggering nodulation in Aeschynomene indica. In this study, a collection of 196 sequenced Bradyrhizobium strains was tested on A. indica. Only strains belonging to the photosynthetic supergroup can develop a NF-T3SS-independent symbiosis, while the ability to use a T3SS-dependent process is found in multiple supergroups. Of these, 14 strains lacking ernA were tested by mutagenesis to identify new T3Es triggering nodulation. We discovered a novel T3E, Sup3, a putative SUMO-protease without similarity to ErnA. Its mutation in Bradyrhizobium strains NAS96.2 and WSM1744 abolishes nodulation and its introduction in an ernA mutant of strain ORS3257 restores nodulation. Moreover, ectopic expression of sup3 in A. indica roots led to the formation of spontaneous nodules. We also report three other new T3Es, Ubi1, Ubi2 and Ubi3, which each contribute to the nodulation capacity of strain LMTR13. These T3Es have no homology to known proteins but share with ErnA three motifs necessary for ErnA activity. Together, our results highlight an unsuspected distribution and diversity of T3Es within the Bradyrhizobium genus that may contribute to their symbiotic efficiency by participating in triggering legume nodulation.

Harnessing the Potential of Symbiotic Associations of Plants in Phosphate-Deficient Soil for Sustainable Agriculture.

Many plants associate with arbuscular mycorrhizal (AM) fungi for nutrient acquisition, and most legumes also associate with nitrogen-fixing rhizobial bacteria for nitrogen acquisition. The association of plants with AM fungi and rhizobia depends on the perception of lipo-chitooligosaccharides (LCOs) produced by these micro-symbionts. Recent studies reveal that cereals can perceive LCOs better in soil deprived of phosphate (Pi) and nitrogen to activate symbiosis signaling and form efficient AM symbiosis. Nevertheless, the Pi deficiency in the soil hinders the symbiotic association of legumes with rhizobia, ultimately reducing nitrogen fixation. Here, we discuss a mechanistic overview of the factors regulating root nodule symbiosis under Pi-deficient conditions and further emphasize the possible ways to overcome this hurdle. Ignoring the low Pi problem not only can compromise the functionality of the nitrogen cycle by nitrogen fixation through legumes but can also put food security at risk globally. This review aims to bring the scientific community's attention toward the detrimental response of legumes toward Pi-deficient soil for the formation of root nodule symbiosis and hence reduced nitrogen fixation. In this review, we have highlighted the recent studies that have advanced our understanding of these critical areas and discussed some future directions. Furthermore, this review highlights the importance of communicating science with farmers and the agriculture community to fully harness the potential of the symbiotic association of plants in nutrient-deficient soil for sustainable agriculture.