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.

Investigation into the potential mechanism of Bacillus amyloliquefaciens in the fermentation of broad bean paste by metabolomics and transcriptomics.

Pixian broad bean paste is a renowned fermented seasoning. The fermentation of broad bean is the most important process of Pixian broad bean paste. To enhance the flavor of tank-fermented broad bean paste, salt-tolerant Bacillus amyloliquefaciens strain was inoculated, resulting in an increase in total amount of volatile compounds, potentially leading to different flavor characteristics. To investigate the fermentation mechanism, monoculture simulated fermentation systems were designed. Metabolomics and transcriptomics were used to explore Bacillus amyloliquefaciens' transcriptional response to salt stress and potential aroma production mechanisms. The results highlighted different metabolite profiles under salt stress, and the crucial roles of energy metabolism, amino acid metabolism, reaction system, transportation system in Bacillus amyloliquefaciens' hypersaline stress response. This study provides a scientific basis for the industrial application of Bacillus amyloliquefaciens and new insights into addressing the challenges of poor flavor quality in tank fermentation products.

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.

Rhizobium determinants of rhizosphere persistence and root colonization.

Bacterial persistence in the rhizosphere and colonization of root niches are critical for the establishment of many beneficial plant-bacteria interactions including those between Rhizobium leguminosarum and its host legumes. Despite this, most studies on R. leguminosarum have focused on its symbiotic lifestyle as an endosymbiont in root nodules. Here, we use random barcode transposon sequencing to assay gene contributions of R. leguminosarum during competitive growth in the rhizosphere and colonization of various plant species. This facilitated the identification of 189 genes commonly required for growth in diverse plant rhizospheres, mutation of 111 of which also affected subsequent root colonization (rhizosphere progressive), and a further 119 genes necessary for colonization. Common determinants reveal a need to synthesize essential compounds (amino acids, ribonucleotides, and cofactors), adapt metabolic function, respond to external stimuli, and withstand various stresses (such as changes in osmolarity). Additionally, chemotaxis and flagella-mediated motility are prerequisites for root colonization. Many genes showed plant-specific dependencies highlighting significant adaptation to different plant species. This work provides a greater understanding of factors promoting rhizosphere fitness and root colonization in plant-beneficial bacteria, facilitating their exploitation for agricultural benefit.

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.

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.

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.

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.

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.

Multiphasic investigations imply transfer of orange-/red-pigmented strains of the bean pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens to a new species as C. aurantiacum sp. nov., elevation of the poinsettia pathogen C. flaccumfaciens pv. poinsettiae to the species level as C. Poinsettiae sp. nov., and synonymy of C. albidum with C. citreum.

Curtobacterium flaccumfaciens (Microbacteriaceae), a plant-pathogenic coryneform species includes five pathovars with valid names and a number of proposed - but unvalidated - new members. In this study, phenotypic features and DNA similarity indexes were investigated among all C. flaccumfaciens members. Results showed that the C. flaccumfaciens pv. poinsettiae strains causing bacterial canker of Euphorbia pulcherrima in the USA as well as the orange-/red-pigmented strains of C. flaccumfaciens pv. flaccumfaciens pathogenic on dry beans in Iran are too distinct from each other and from the type strain of the species to be considered members of C. flaccumfaciens. Hence, the latter two groups were elevated at the species level as C. poinsettiae sp. nov. (ATCC 9682T = CFBP 2403T = ICMP 2566T = LMG 3715T = NCPPB 854T as type strain), and C. aurantiacum sp. nov. (50RT = CFBP 8819T = ICMP 22071T as type strain). Within the emended species C. flaccumfaciens comb. nov., yellow-pigmented strains causing bacterial wilt of dry beans and those causing bacterial canker of Euphorbia pulcherrima in Europe were retained as C. flaccumfaciens pv. flaccumfaciens and C. flaccumfaciens pv. poinsettiae, respectively; while taxonomic position of the sugar beet pathogen C. flaccumfaciens pv. beticola ATCC BAA144PT was confirmed. The newly described onion pathogen C. allii was also reclassified as C. flaccumfaciens pv. allii with the pathotype strain LMG 32517PT. Furthermore, C. flaccumfaciens pv. basellae causing bacterial leaf spot of malabar spinach (Basella rubra) was transferred to C. citreum pv. basellae with ATCC BAA143PT as pathotype.

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.

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.

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.

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.

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 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.

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.