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

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

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.

Effectiveness of Bacillus subtilis ANT01 and Rhizobium sp. 11B on the control of fusarium wilt in pineapple (Ananas comosus).

Pineapple (Ananas comosus) is commonly infected by Fusarium oxysporum, causal agent of the fusarium wilt disease. Conventionally, growers use synthetic fungicides to control the disease, which lead to environmental pollution, hazardous effects on non-target organisms and risks on human health. The aim of this work was to assess the effectiveness of Bacillus subtilis ANT01 and Rhizobium sp. 11B to control fusarium wilt on pineapple plants. Four treatments derived from a complete factorial design were tested under field conditions. Treatments composed of B. subtilis ANT01 and the combination B. subtilis ANT01-Rhizobium sp. 11B decreased disease severity by 94.4% and 86.1%, respectively. On the other hand, the treatment prepared with Rhizobium sp. 11B alone showed a reduction of 75.0%. Size of leaves and nutritional condition (SPAD units) of the biocontrol agents-treated plants showed no statistical differences. Moreover, B. subtilis ANT01 decreased by 46% the initial soil population of F. oxysporum, while Rhizobium sp. 11B, B. subtilis ANT01 plus Rhizobium sp. 11B and control, showed a population reduction of 12.5%, 24.2% and 23.0%, respectively. These results make evident the potential of B. subtilis ANT01 as biocontrol agent of the pathogen under field conditions.

The radiation of nodulated Chamaecrista species from the rainforest into more diverse habitats has been accompanied by a reduction in growth form and a shift from fixation threads to symbiosomes.

All non-Mimosoid nodulated genera in the legume subfamily Caesalpinioideae confine their rhizobial symbionts within cell wall-bound 'fixation threads' (FTs). The exception is the large genus Chamaecrista in which shrubs and subshrubs house their rhizobial bacteroids more intimately within symbiosomes, whereas large trees have FTs. This study aimed to unravel the evolutionary relationships between Chamaecrista growth habit, habitat, nodule bacteroid type, and rhizobial genotype. The growth habit, bacteroid anatomy, and rhizobial symbionts of 30 nodulated Chamaecrista species native to different biomes in the Brazilian state of Bahia, a major centre of diversity for the genus, was plotted onto an ITS-trnL-F-derived phylogeny of Chamaecrista. The bacteroids from most of the Chamaecrista species examined were enclosed in symbiosomes (SYM-type nodules), but those in arborescent species in the section Apoucouita, at the base of the genus, were enclosed in cell wall material containing homogalacturonan (HG) and cellulose (FT-type nodules). Most symbionts were Bradyrhizobium genotypes grouped according to the growth habits of their hosts, but the tree, C. eitenorum, was nodulated by Paraburkholderia. Chamaecrista has a range of growth habits that allow it to occupy several different biomes and to co-evolve with a wide range of (mainly) bradyrhizobial symbionts. FTs represent a less intimate symbiosis linked with nodulation losses, so the evolution of SYM-type nodules by most Chamaecrista species may have (i) aided the genus-wide retention of nodulation, and (ii) assisted in its rapid speciation and radiation out of the rainforest into more diverse and challenging habitats.

New Inoculation Strategy for Legume Based on Rhizobium-Metabolite Co-encapsulation.

The new strategies that are trying to be developed to protect microorganisms for a successful application have generated various types of granulated, powdered, or liquid formulations. In this work, we have developed a rhizobial encapsulation system for legumes accompanied by metabolites to enhance microorganism-plant communication. This novel way of producing a biofertilizer for legumes was developed based on alginate, a degradable compound that allows environmentally friendly use. This way of generating an inoculant allows it designing by making different molecular combinations for different purposes, being a double inoculant, biological and molecular.

Effect of rhizobium and gibberellin on the production of hydroponic green forage of red clover (Trifolium pratense L.) variety quiñequeli.

The purpose of the present investigation was to determine the effect of rhizobium and gibberellin on the production of hydroponic green forage from red clover (Trifolium pratense L.) variety quiñequeli, four variables were measured: plant height, biomass weight, grass weight and root weight. The treatments were T0: 0%, T1: 10%, T2: 20%, T3: 30% and T4: 40% of Rhizobium before germination and Gibberellin T0: 0g, T1: 2.4g; T2: 3.3g; T3: 4.3 and T4: 5.3g each treatment with 6 repetitions, three applications on days 5, 10, 15 and 20 of growth. Data were analyzed with DCA, ANOVA and DUNCAN's multiple comparisons test; the results obtained were: first measurement with rhizobium without gibberellin there were no statistical differences, second and third measurement with Gibberellin application did not present statistical differences and the fourth measurement presented statistical difference (α=0.05), average height of the plant with a mean of 12.82 cm, T4 was higher, in biomass a statistical difference was obtained with a mean of 3.056 kg, T3 was higher, weight of grass and root did not present statistical differences; concluding that the use of rhizobium and gibberellin could be a usable alternative in the production of hydroponic green fodder, to alleviate the problems of fodder scarcity in dry season, its use being recommended in high Andean livestock.

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.

The outstanding diversity of rhizobia microsymbionts of common bean (Phaseolus vulgaris L.) in Mato Grosso do Sul, central-western Brazil, revealing new Rhizobium species.

Common bean is considered a legume of great socioeconomic importance, capable of establishing symbioses with a wide variety of rhizobial species. However, the legume has also been recognized for its low efficiency in fixing atmospheric nitrogen. Brazil is a hotspot of biodiversity, and in a previous study, we identified 13 strains isolated from common bean (Phaseolus vulgaris) nodules in three biomes of Mato Grosso do Sul state, central-western Brazil, that might represent new phylogenetic groups, deserving further polyphasic characterization. The phylogenetic tree of the 16S rRNA gene split the 13 strains into two large clades, seven in the R. etli and six in the R. tropici clade. The MLSA with four housekeeping genes (glnII, gyrB, recA, and rpoA) confirmed the phylogenetic allocation. Genomic comparisons indicated eight strains in five putative new species and the remaining five as R. phaseoli. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) comparing the putative new species and the closest neighbors ranged from 81.84 to 92.50% and 24.0 to 50.7%, respectively. Other phenotypic, genotypic, and symbiotic features were evaluated. Interestingly, some strains of both R. etli and R. tropici clades lost their nodulation capacity. The data support the description of the new species Rhizobium cerradonense sp. nov. (CNPSo 3464T), Rhizobium atlanticum sp. nov. (CNPSo 3490T), Rhizobium aureum sp. nov. (CNPSo 3968T), Rhizobium pantanalense sp. nov. (CNPSo 4039T), and Rhizobium centroccidentale sp. nov. (CNPSo 4062T).

Characterization of plant growth-promoting bacteria isolated from rhizosphere of lentil (Lens culinaris L.) grown in two different soil orders of eastern India.

Lentil, which is an important grain legume, can be co-inoculated with plant growth-promoting rhizobia and rhizobacteria to boost nitrogen fixation, increase biomass, and a possibility for early nodulation. The goal of the ongoing study was to identify plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of lentil growing soils in eastern India. Sixteen rhizosphere bacteria were isolated from two different soil orders, and their capacity to solubilize phosphate and generate hydrogen cyanide (HCN), siderophore, and indole acetic acid (IAA) was assessed. The three best strains were selected for compatibility study with twenty Rhizobium isolated from lentil root nodules. The isolated rhizobacteria were able to produce ammonia and different mycolytic enzymes. Isolate B3 produced the highest amount of IAA and siderophore; the highest amount of phosphate solubilized by PSB1 strain; and isolates AB1, AB2, B3, PS2, and PSB2 produced considerable amount of HCN gas. Among all the isolates, B3, PSB1, and PS2 performed better based on different plant growth-promoting abilities. These three bacterial isolates showed compatible reaction with most of the Rhizobium strains. Isolates B3, PS2, and PSB1 were identified as Bacillus subtilis (MT729775), Pseudomonas palmensis (MT729782), and Paraburkholderia caribenis (MZ956803), respectively. Lentil shoot weight, root length, nodule number, N uptake, and P uptake were increased in the pot culture experiment when inoculated with these strains. PGPR strain B3 performed best among the three strains in the pot culture experiment. Strain B3 can be used as potential biofertilizer along with compatible Rhizobium species for better production of lentil.

CRK12: A Key Player in Regulating the Phaseolus vulgaris-Rhizobium tropici Symbiotic Interaction.

Cysteine-rich receptor-like kinases (CRKs) are a type of receptor-like kinases (RLKs) that are important for pathogen resistance, extracellular reactive oxygen species (ROS) signaling, and programmed cell death in plants. In a previous study, we identified 46 CRK family members in the Phaseolus vulgaris genome and found that CRK12 was highly upregulated under root nodule symbiotic conditions. To better understand the role of CRK12 in the Phaseolus-Rhizobia symbiotic interaction, we functionally characterized this gene by overexpressing (CRK12-OE) and silencing (CRK12-RNAi) it in a P. vulgaris hairy root system. We found that the constitutive expression of CRK12 led to an increase in root hair length and the expression of root hair regulatory genes, while silencing the gene had the opposite effect. During symbiosis, CRK12-RNAi resulted in a significant reduction in nodule numbers, while CRK12-OE roots showed a dramatic increase in rhizobial infection threads and the number of nodules. Nodule cross sections revealed that silenced nodules had very few infected cells, while CRK12-OE nodules had enlarged infected cells, whose numbers had increased compared to controls. As expected, CRK12-RNAi negatively affected nitrogen fixation, while CRK12-OE nodules fixed 1.5 times more nitrogen than controls. Expression levels of genes involved in symbiosis and ROS signaling, as well as nitrogen export genes, supported the nodule phenotypes. Moreover, nodule senescence was prolonged in CRK12-overexpressing roots. Subcellular localization assays showed that the PvCRK12 protein localized to the plasma membrane, and the spatiotemporal expression patterns of the CRK12-promoter::GUS-GFP analysis revealed a symbiosis-specific expression of CRK12 during the early stages of rhizobial infection and in the development of nodules. Our findings suggest that CRK12, a membrane RLK, is a novel regulator of Phaseolus vulgaris-Rhizobium tropici symbiosis.

Vinasse as a substrate for inoculant culture and soil fertigation: Advancing the circular and green economy.

Vinasse is a by-product with a key role in circular economy. In this work, we analyze sugarcane vinasse as culture medium for obtaining single and mixed inoculants. Trichoderma harzianum MT2 was cultured in single and sequential co-culture with Pseudomonas capeferrum WCS358 or Rhizobium sp. N21.2. Fungal biomass in single culture was more than three folds higher in vinasse than in a standard medium, and was higher in co-culture with Rhizobium sp. N21.2 than with P. capeferrum WCS358. Bacterial growths in vinasse, in particular P. capeferrum WCS358, were improved in co-culture with T. harzianum MT2. Residual vinasses, obtained after microbial growth, presented almost neutral pH and lower conductivities and toxicity than raw vinasse. Fertigation with residual vinasses modifies characteristics of soil evidenced in the total N, cation exchange capacity, urease and acid phosphatase, and microbial metabolic diversity, in comparison to raw vinasse. In general, soil fertigation with residual vinasse from co-culture with P. capeferrum WCS358 is more similar to irrigation with water. Treatment evaluation indicates that vinasse is suitable for the production of mixed inoculants containing T. harzianum. The co-culture with P. capeferrum WCS358 improves the characteristics of the residual vinasse allowing a fertigation with less detrimental effect in soil in comparison to Rhizobium sp. N21.2. Obtaining valuable biomass of single or mixed inoculants in vinasse with lower ecological impact is relevant for the circular and green economy.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

Rhizobacteria control damping-off and promote growth of lima bean with and without co-inoculation with Rhizobium tropici CIAT899.

Rhizoctonia solani compromises the production of lima bean, an alternative and low-input food source in many tropical regions. Inoculation of bacterial strains has been used, but research on their biocontrol and growth promotion potential on lima bean is scarce. The objective of this study was to evaluate the effects of inoculation with rhizobacterial strains of the genera Bacillus, Brevibacillus, Paenibacillus, Burkholderia, Pseudomonas, and Rhizobium in combination or not with N2-fixing Rhizobium tropici on the control of damping-off disease and growth promotion in lima bean plants. Greenhouse experiments were conducted to evaluate the inoculation with bacterial strains with biocontrol potential in combination or not with R. tropici in substrate infected with R. solani CML 1846. Growth promotion of these strains was also assessed. Strains of Brevibacillus (UFLA 02-286), Pseudomonas (UFLA 02-281 and UFLA 04-885), Rhizobium (UFLA 04-195), and Burkholderia (UFLA 04-227) co-inoculated with the strain CIAT 899 (Rhizobium tropici) were the most effective in controlling R. solani, reducing the disease incidence in 47-60% on lima bean. The promising strains used in the biocontrol assays were also responsive in promoting growth of lima bean under disease and sterile conditions. A positive synergistic effect of co-inoculation of different genera contributed to plant growth, and these outcomes are important first steps to improve lima bean production.

Rhizobia induce SYMRK endocytosis in Phaseolus vulgaris root hair cells.

MAIN CONCLUSION: PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis, which rely on the phosphorylation status of T589, the endocytic YXXØ motif and the kinase activity of the receptor. Legume-rhizobia nodulation is a complex developmental process. It initiates when the rhizobia-produced Nod factors are perceived by specific LysM receptors present in the root hair apical membrane. Consequently, SYMRK (Symbiosis Receptor-like Kinase) becomes active in the root hair and triggers an extensive signaling network essential for the infection process and nodule organogenesis. Despite its relevant functions, the underlying cellular mechanisms involved in SYMRK signaling activity remain poorly characterized. In this study, we demonstrated that PvSYMRK-EGFP undergoes constitutive and rhizobia-induced endocytosis. We found that in uninoculated roots, PvSYMRK-EGFP is mainly associated with the plasma membrane, although intracellular puncta labelled with PvSymRK-EGFP were also observed in root hair and nonhair-epidermal cells. Inoculation with Rhizobium etli producing Nod factors induces in the root hair a redistribution of PvSYMRK-EGFP from the plasma membrane to intracellular puncta. In accordance, deletion of the endocytic motif YXXØ (YKTL) and treatment with the endocytosis inhibitors ikarugamycin (IKA) and tyrphostin A23 (TyrA23), as well as brefeldin A (BFA), drastically reduced the density of intracellular PvSYMRK-EGFP puncta. A similar effect was observed in the phosphorylation-deficient (T589A) and kinase-dead (K618E) mutants of PvSYMRK-EGFP, implying these structural features are positive regulators of PvSYMRK-EGFP endocytosis. Our findings lead us to postulate that rhizobia-induced endocytosis of SYMRK modulates the duration and amplitude of the SYMRK-dependent signaling pathway.

AUR1 and its pals: orchestration of intracellular rhizobia infection in legume for nitrogen fixation.

KEY MESSAGE: We highlight the newly emerged regulatory role of a mitotic kinase AUR1, its activator, and its microtubule-associated proteins (MAPs) in infection thread formation for root nodule symbiosis.

Not just passengers, but co-pilots! Non-rhizobial nodule-associated bacteria promote cowpea growth and symbiosis with (brady)rhizobia.

AIMS: To isolate and characterize non-rhizobial nodule-associated bacteria (NAB) from cowpea root-nodules regarding their performance of plant-growth-promoting mechanisms and their ability to enhance cowpea growth and symbiosis when co-inoculated with bradyrhizobia. METHODS AND RESULTS: Sixteen NAB were isolated, identified, and in vitro evaluated for plant growth promotion traits. The ability to promote cowpea growth was analyzed when co-inoculated with Bradyrhizobium pachyrhizi BR 3262 in sterile and non-sterile substrates. The 16S rRNA gene sequences analysis revealed that NAB belonged to the genera Chryseobacterium (4), Bacillus (3), Microbacterium (3), Agrobacterium (1), Escherichia (1), Delftia (1), Pelomonas (1), Sphingomonas (1), and Staphylococcus (1). All strains produced different amounts of auxin siderophores and formed biofilms. Twelve out of the 16 strains carried the nifH, a gene associated with nitrogen fixation. Co-inoculation of NAB (ESA 424 and ESA 29) with Bradyrhizobium pachyrhizi BR 3262 significantly promoted cowpea growth, especially after simultaneous inoculation with the three strains. CONCLUSIONS: NAB are efficient cowpea growth promoters and can improve the efficiency of the symbiosis between cowpea and the N2-fixing microsymbiont B. pachyrhizi BR 3262, mainly under a specific triple microbial association.

Discovering the genetic modules controlling root nodule symbiosis under abiotic stresses: salinity as a case study.

Legumes form a symbiotic association with rhizobia and fix atmospheric nitrogen in specialized root organs known as nodules. It is well known that salt stress inhibits root nodule symbiosis by decreasing rhizobial growth, rhizobial infection, nodule number, and nitrogenase activity in diverse legumes. Despite this knowledge, the genetic and molecular mechanisms governing salt stress's inhibition of nodulation and nitrogen fixation are still elusive. In this Viewpoint, we summarize the most recent knowledge of the genetic mechanisms that shape this symbiosis according to the salt levels in the soil. We emphasize the relevance of modulating the activity of the transcription factor Nodule Inception to properly shape the symbiosis with rhizobia accordingly. We also highlight the knowledge gaps that are critical for gaining a deeper understanding of the molecular mechanisms underlying the adaptation of the root nodule symbiosis to salt-stress conditions. We consider that filling these gaps can help to improve legume nodulation and harness its ecological benefits even under salt-stress conditions.

Diversity of rhizobia, symbiotic effectiveness, and potential of inoculation in Acacia mearnsii seedling production.

Black wattle (Acacia mearnsii) is a forest species of significant economic importance in southern Brazil; as a legume, it forms symbiotic associations with rhizobia, fixing atmospheric nitrogen. Nonetheless, little is known about native rhizobia in soils where the species is cultivated. Therefore, this study aimed to evaluate the diversity and symbiotic efficiency of rhizobia nodulating A. mearnsii in commercial planting areas and validate the efficiency of a potential strain in promoting seedling development. To this end, nodules were collected from four A. mearnsii commercial plantations located in Rio Grande do Sul State, southern Brazil. A total of 80 rhizobia isolates were obtained from black wattle nodules, and thirteen clusters were obtained by rep-PCR. Higher genetic diversity was found within the rhizobial populations from the Duas Figueiras (H' = 2.224) and Seival (H' = 2.112) plantations. Twelve isolates were evaluated belonging to the genus Bradyrhizobium, especially to the species Bradyrhizobium guangdongense. The principal component analysis indicated an association between rhizobia diversity and the content of clay, Ca, Mg, and K. Isolates and reference strains (SEMIA 6163 and 6164) induced nodulation and fixed N via symbiosis with black wattle plants after 60 days of germination. The isolates DF2.4, DF2.3, DF3.3, SEMIA 6164, SEMIA 6163, CA4.3, OV3.4, and OV1.4 showed shoot nitrogen accumulation values similar to the N + control treatment. In the second experiment (under nursery conditions), inoculation with the reference strain SEMIA 6164 generally improved the growth of A. mearnsii seedlings, reinforcing its efficiency even under production conditions.