More diverse rhizobial communities can lead to higher symbiotic nitrogen fixation rates, even in nitrogen-rich soils.

Symbiotic nitrogen (N) fixation (SNF) by legumes and their rhizobial partners is one of the most important sources of bioavailable N to terrestrial ecosystems. While most work on the regulation of SNF has focussed on abiotic drivers such as light, water and soil nutrients, the diversity of rhizobia with which individual legume partners may play an important but under-recognized role in regulating N inputs from SNF. By experimentally manipulating the diversity of rhizobia available to legumes, we demonstrate that rhizobial diversity can increase average SNF rates by more than 90%, and that high rhizobial diversity can induce increased SNF even under conditions of high soil N fertilization. However, the effects of rhizobial diversity, the conditions under which diversity effects were the strongest, and the likely mechanisms driving these diversity effects differed between the two legume species we assessed. These results provide evidence that biodiversity-ecosystem function relationships can occur at the scales of an individual plant and that the effects of rhizobial diversity may be as important as long-established abiotic factors, such as N availability, in driving terrestrial N inputs via SNF.

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.

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.

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.

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.

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.

Bacterial exopolysaccharide amendment improves the shelf life and functional efficacy of bioinoculant under salinity stress.

AIM: Bacterial exopolysaccharides (EPS) possess numerous properties beneficial for the growth of microbes and plants under hostile conditions. The study aimed to develop a bioformulation with bacterial EPS to enhance the bioinoculant's shelf life and functional efficacy under salinity stress. METHODS AND RESULTS: High EPS-producing and salt-tolerant bacterial strain (Bacillus haynessi SD2) exhibiting auxin-production, phosphate-solubilization, and biofilm-forming ability, was selected. EPS-based bioformulation of SD2 improved the growth of three legumes under salt stress, from which pigeonpea was selected for further experiments. SD2 improved the growth and lowered the accumulation of stress markers in plants under salt stress. Bioformulations with varying EPS concentrations (1% and 2%) were stored for 6 months at 4°C, 30°C, and 37°C to assess their shelf life and functional efficacy. The shelf life and efficacy of EPS-based bioformulation were sustained even after 6 months of storage at high temperature, enhancing pigeonpea growth under stress in both control and natural conditions. However, the efficacy of non EPS-based bioformulation declined following four months of storage. The bioformulation (with 1% EPS) modulated bacterial abundance in the plant's rhizosphere under stress conditions. CONCLUSION: The study brings forth a new strategy for developing next-generation bioformulations with higher shelf life and efficacy for salinity stress management in pigeonpea.

Biocontrol Methods for the Management of Sclerotinia sclerotiorum in Legumes: A Review.

Sclerotinia sclerotiorum is an economically damaging fungal pathogen that causes Sclerotinia stem rot in legumes, producing enormous yield losses. This pathogen is difficult to control due to its wide host spectrum and ability to produce sclerotia, which are resistant bodies that can remain active for long periods under harsh environmental conditions. Here, the biocontrol methods for the management of S. sclerotiorum in legumes are reviewed. Bacillus strains, which synthesized lipopeptides and volatile organic compounds, showed high efficacies in soybean plants, whereas the highest efficacies for the control of the pathogen in alfalfa and common bean were observed when using Coniothyrium minitans and Streptomyces spp., respectively. The biocontrol efficacies in fields were under 65%, highlighting the lack of strategies to achieve a complete control. Overall, although most studies involved extensive screenings using different biocontrol agent concentrations and application conditions, there is a lack of knowledge regarding the specific antifungal mechanisms, which limits the optimization of the reported methods.

Bacillus licheniformis and Bacillus velezensis from Rhizosphere of Clerodendrum infortunatum L. Promote Plant Growth and Resistance to Sclerotium rolfsii in Vigna unguiculata (L.) Walp.

In the current study, thirty bacterial strains isolated from the rhizosphere of Clerodendrum infortunatum L. were evaluated for the properties related to the plant growth promotion and disease resistance. Here, all the selected strains were screened for its antagonistic effect towards the phytopathogen Sclerotium rolfsii and also for the production of bioactive compounds known to promote the plant growth. Among these isolates, CiRb1 and CiRb16 were observed to have a broad range of plant beneficial features and were identified as Bacillus licheniformis and Bacillus velezensis respectively. Both the isolates were also demonstrated to produce the volatile organic compounds (VOCs) responsible for the growth enhancement in Brassica nigra (L.) and growth inhibition of S. rolfsii. Talc based formulations made out of both B. licheniformis and B. velezensis were further demonstrated to augment the plant growth and protection against S. rolfsii in Vigna unguiculata (L.) Walp. By the GC-MS based analysis, undecane could also be detected in the methanolic extracts prepared from both B. licheniformis and B. velezensis. Here, the selected rhizobacterial isolates were found to promote the plant growth and disease resistance through both direct and VOC mediated mechanisms. The results of the study hence reveal both B. licheniformis and B. velezensis have the potential in field application to promote the growth and control of plant diseases.

Conditional sanctioning in a legume-Rhizobium mutualism.

Legumes are high in protein and form a valuable part of human diets due to their interaction with symbiotic nitrogen-fixing bacteria known as rhizobia. Plants house rhizobia in specialized root nodules and provide the rhizobia with carbon in return for nitrogen. However, plants usually house multiple rhizobial strains that vary in their fixation ability, so the plant faces an investment dilemma. Plants are known to sanction strains that do not fix nitrogen, but nonfixers are rare in field settings, while intermediate fixers are common. Here, we modeled how plants should respond to an intermediate fixer that was otherwise isogenic and tested model predictions using pea plants. Intermediate fixers were only tolerated when a better strain was not available. In agreement with model predictions, nodules containing the intermediate-fixing strain were large and healthy when the only alternative was a nonfixer, but nodules of the intermediate-fixing strain were small and white when the plant was coinoculated with a more effective strain. The reduction in nodule size was preceded by a lower carbon supply to the nodule even before differences in nodule size could be observed. Sanctioned nodules had reduced rates of nitrogen fixation, and in later developmental stages, sanctioned nodules contained fewer viable bacteria than nonsanctioned nodules. This indicates that legumes can make conditional decisions, most likely by comparing a local nodule-dependent cue of nitrogen output with a global cue, giving them remarkable control over their symbiotic partners.

Legume-microbiome interactions unlock mineral nutrients in regrowing tropical forests.

Legume trees form an abundant and functionally important component of tropical forests worldwide with N2-fixing symbioses linked to enhanced growth and recruitment in early secondary succession. However, it remains unclear how N2-fixers meet the high demands for inorganic nutrients imposed by rapid biomass accumulation on nutrient-poor tropical soils. Here, we show that N2-fixing trees in secondary Neotropical forests triggered twofold higher in situ weathering of fresh primary silicates compared to non-N2-fixing trees and induced locally enhanced nutrient cycling by the soil microbiome community. Shotgun metagenomic data from weathered minerals support the role of enhanced nitrogen and carbon cycling in increasing acidity and weathering. Metagenomic and marker gene analyses further revealed increased microbial potential beneath N2-fixers for anaerobic iron reduction, a process regulating the pool of phosphorus bound to iron-bearing soil minerals. We find that the Fe(III)-reducing gene pool in soil is dominated by acidophilic Acidobacteria, including a highly abundant genus of previously undescribed bacteria, Candidatus Acidoferrum, genus novus. The resulting dependence of the Fe-cycling gene pool to pH determines the high iron-reducing potential encoded in the metagenome of the more acidic soils of N2-fixers and their nonfixing neighbors. We infer that by promoting the activities of a specialized local microbiome through changes in soil pH and C:N ratios, N2-fixing trees can influence the wider biogeochemical functioning of tropical forest ecosystems in a manner that enhances their ability to assimilate and store atmospheric carbon.

Outbreak of Listeria monocytogenes in hospital linked to a fava bean product, Finland, 2015 to 2019.

Listeria monocytogenes (Lm) is a bacterium widely distributed in the environment. Listeriosis is a severe disease associated with high hospitalisation and mortality rates. In April 2019, listeriosis was diagnosed in two hospital patients in Finland. We conducted a descriptive study to identify the source of the infection and defined a case as a person with a laboratory-confirmed Lm serogroup IIa sequence type (ST) 37. Six cases with Lm ST 37 were notified to the Finnish Infectious Diseases Registry between 2015 and 2019. Patient interviews and hospital menus were used to target traceback investigation of the implicated foods. In 2021 and 2022, similar Lm ST 37 was detected from samples of a ready-to-eat plant-based food product including fava beans. Inspections by the manufacturer and the local food control authority indicated that the food products were contaminated with Lm after pasteurisation. Our investigation highlights the importance that companies producing plant-based food are subject to similar controls as those producing food of animal origin. Hospital menus can be a useful source of information that is not dependent on patient recall.

Perennial Baki™ Bean Safety for Human Consumption: Evidence from an Analysis of Heavy Metals, Folate, Canavanine, Mycotoxins, Microorganisms and Pesticides.

Global food production relies on annual grain crops. The reliability and productivity of these crops are threatened by adaptations to climate change and unsustainable rates of soil loss associated with their cultivation. Perennial grain crops, which do not require planting every year, have been proposed as a transformative solution to these challenges. Perennial grain crops typically rely on wild species as direct domesticates or as sources of perenniality in hybridization with annual grains. Onobrychis spp. (sainfoins) are a genus of perennial legumes domesticated as ancient forages. Baki™ bean is the tradename for pulses derived from sainfoins, with ongoing domestication underway to extend demonstrated benefits to sustainable agriculture. This study contributes to a growing body of evidence characterizing the nutritional quality of Baki™ bean. Through two studies, we investigated the safety of Baki™ bean for human consumption. We quantified heavy metals, folate, and canavanine for samples from commercial seed producers, and we quantified levels of mycotoxins, microorganisms, and pesticides in samples from a single year and seed producer, representing different varieties and production locations. The investigated analytes were not detectable or occurred at levels that do not pose a significant safety risk. Overall, this study supports the safety of Baki™ bean for human consumption as a novel pulse crop.

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.

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.

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.

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%.

Euphorbia helioscopia a Putative Plant Reservoir of Pathogenic Curtobacterium flaccumfaciens.

The endophyte EHF3 strain was isolated in Algeria from an Euphorbia helioscopia plant growing in a fallow field. The strain was characterized by biochemical and physiological tests and assayed for the production of secondary metabolites involved in biocontrol, for plant growth promotion ability and for pathogenicity. The strain was identified by BIOLOG test as Curtobacterium flaccumfaciens. Biochemical and physiological characterization revealed that the strain was able to secrete protease, caseinase and amylase enzymes, to grow up to 37 °C and at pH values 5 to 9. C. flaccumfaciens EHF3 strain was incapable of solubilizing phosphorus and to produce IAA, HCN siderophores and phenazine compounds. The strain showed a moderate swimming and swarming motility and produced biofilm. EHF3 strain was positive at the hypersensitivity test on tobacco plants and induced symptoms on three varieties of bean resembling to those of the bacterial wilt disease induced by C. flaccumfaciens pv. flaccumfaciens. The preliminary data reported in this study, regarding the detection of a pathogenic C. flaccumfaciens strain, as endophyte of a E. helioscopia plant, highlight the role of non-host plants as reservoir of this bacterial pathogen.

Rhizobium as Biotechnological Tools for Green Solutions: An Environment-Friendly Approach for Sustainable Crop Production in the Modern Era of Climate Change.

Modern and industrialized agriculture enhanced farm output during the last few decades, but it became possible at the cost of agricultural sustainability. Industrialized agriculture focussed only on the increase in crop productivity and the technologies involved were supply-driven, where enough synthetic chemicals were applied and natural resources were overexploited with the erosion of genetic diversity and biodiversity. Nitrogen is an essential nutrient required for plant growth and development. Even though nitrogen is available in large quantities in the atmosphere, it cannot be utilized by plants directly with the only exception of legumes which have the unique ability to fix atmospheric nitrogen and the process is known as biological nitrogen fixation (BNF). Rhizobium, a group of gram-negative soil bacteria, helps in the formation of root nodules in legumes and takes part in the BNF. The BNF has great significance in agriculture as it acts as a fertility restorer in soil. Continuous cereal-cereal cropping system, which is predominant in a major part of the world, often results in a decline in soil fertility, while legumes add nitrogen and improve the availability of other nutrients too. In the present context of the declining trend of the yield of some important crops and cropping systems, it is the need of the hour for enriching soil health to achieve agricultural sustainability, where Rhizobium can play a magnificent role. Though the role of Rhizobium in biological nitrogen fixation is well documented, their behaviour and performance in different agricultural environments need to be studied further for a better understanding. In the article, an attempt has been made to give an insight into the behaviour, performance and mode of action of different Rhizobium species and strains under versatile conditions.

Biosorption and Symbiotic Potential of Horse Gram Rhizobia in Soils Contaminated with Cobalt.

The current study aims evaluation of biosorption and symbiotic potential of horse gram plants associated with rhizobia inspite of Cobalt (Co) metal stress, and these rhizobia strains play a pivotal role in the phytoremediation of Co heavy metal-contaminated soils. Horse gram rhizobial isolates HGR-4, HGR-6, HGR-13 and HGR-25 were able to tolerate 1000 µg g-1 Co supplemented in culture media and also 100 µg g-1 in Co supplemented soil. The plants nodulated with the isolates from the study have shown higher nodulation, nitrogen and leghaemoglobin content in the potted experiment on par with the control plants. Atomic absorption spectroscopic analysis of Co content in horse gram plants inoculated with these four isolates showed maximum biosorption of Co among the bacterial root nodules. Application of these strains can be potentially aid the phytoextraction of Co from contaminated soils on association with horse gram plants.