RESUMEN

Bradyrhizobium is known for fixing atmospheric nitrogen in symbiosis with agronomically important crops. This study focused on two groups of strains, each containing eight natural variants of the parental strains, Bradyrhizobium japonicum SEMIA 586 (=CNPSo 17) or Bradyrhizobium diazoefficiens SEMIA 566 (=CNPSo 10). CNPSo 17 and CNPSo 10 were used as commercial inoculants for soybean crops in Brazil at the beginning of the crop expansion in the southern region in the 1960s-1970s. Variants derived from these parental strains were obtained in the late 1980s through a strain selection program aimed at identifying elite strains adapted to a new cropping frontier in the central-western Cerrado region, with a higher capacity of biological nitrogen fixation (BNF) and competitiveness. Here, we aimed to detect genetic variations possibly related to BNF, competitiveness for nodule occupancy, and adaptation to the stressful conditions of the Brazilian Cerrado soils. High-quality genome assemblies were produced for all strains. The core genome phylogeny revealed that strains of each group are closely related, as confirmed by high average nucleotide identity values. However, variants accumulated divergences resulting from horizontal gene transfer, genomic rearrangements, and nucleotide polymorphisms. The B. japonicum group presented a larger pangenome and a higher number of nucleotide polymorphisms than the B. diazoefficiens group, possibly due to its longer adaptation time to the Cerrado soil. Interestingly, five strains of the B. japonicum group carry two plasmids. The genetic variability found in both groups is discussed considering the observed differences in their BNF capacity, competitiveness for nodule occupancy, and environmental adaptation.IMPORTANCEToday, Brazil is a global leader in the study and use of biological nitrogen fixation with soybean crops. As Brazilian soils are naturally void of soybean-compatible bradyrhizobia, strain selection programs were established, starting with foreign isolates. Selection searched for adaptation to the local edaphoclimatic conditions, higher efficiency of nitrogen fixation, and strong competitiveness for nodule occupancy. We analyzed the genomes of two parental strains of Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens and eight variant strains derived from each parental strain. We detected two plasmids in five strains and several genetic differences that might be related to adaptation to the stressful conditions of the soils of the Brazilian Cerrado biome. We also detected genetic variations in specific regions that may impact symbiotic nitrogen fixation. Our analysis contributes to new insights into the evolution of Bradyrhizobium, and some of the identified differences may be applied as genetic markers to assist strain selection programs.

Asunto(s)

Bradyrhizobium , Genoma Bacteriano , Glycine max , Fijación del Nitrógeno , Filogenia , Simbiosis , Bradyrhizobium/genética , Bradyrhizobium/clasificación , Bradyrhizobium/aislamiento & purificación , Fijación del Nitrógeno/genética , Brasil , Glycine max/microbiología , Simbiosis/genética , Variación Genética , Adaptación Fisiológica/genética , Nódulos de las Raíces de las Plantas/microbiología , Microbiología del Suelo , Genómica

RESUMEN

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.

Asunto(s)

Rhizobium , Simbiosis , Fabaceae/microbiología , Guías como Asunto , Fijación del Nitrógeno , Rhizobium/genética , Rhizobium/clasificación , Nódulos de las Raíces de las Plantas/microbiología

RESUMEN

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.

Asunto(s)

Filogenia , Rhizobium , Microbiología del Suelo , Simbiosis , Vicia faba , Vicia faba/microbiología , Rhizobium/genética , Rhizobium/aislamiento & purificación , Rhizobium/clasificación , México , Proteínas Bacterianas/genética , Nódulos de las Raíces de las Plantas/microbiología , Suelo/química , N-Acetilglucosaminiltransferasas/genética , Oxidorreductasas/genética , Rec A Recombinasas/genética , Familia de Multigenes

RESUMEN

In the conducted studies, the moorphological and physiological properties of nodule bacteria of lupine were studied. Lupine plants were grown under the conditions of a microfield experiment on a typical medium loamy urban soil. In the study, a pure culture of Bradyrhizobium lupini was isolated. Then, the morphological properties of nodule bacteria cells and the chemical composition of cell membranes of nodule bacteria were determined. The acid resistance and physiological properties of lupine nodule bacteria were also determined, as well as the ratio of Bradyrhizobium lupini to antibiotics. All studies were carried out according to generally accepted methods. The results of the research showed that during the cultivation of lupine on a typical urban soil, nodule bacteria Bradyrhizobium lupini were isolated, which can be characterized as gram-negative, non-spore-forming rods that do not exhibit amylolytic activity. It was revealed that the rhizobia of nodule bacteria are not acid-resistant. Nodule bacteria turned out to be the least resistant to polymyxin, then to levomycetin, and Bradyrhizobium lupini showed the greatest resistance to tetracycline.

Asunto(s)

Bradyrhizobium , Lupinus , Rhizobiaceae , Lupinus/microbiología , Nódulos de las Raíces de las Plantas/microbiología , Suelo , Bradyrhizobium/fisiología , Simbiosis/fisiología , Microbiología del Suelo

RESUMEN

Coriariaceae are a small plant family of 14-17 species and subspecies that currently have a global but disjunct distribution. All species can form root nodules in symbiosis with diazotrophic Frankia cluster-2 strains, which form the earliest divergent symbiotic clade within this bacterial genus. Studies on Frankia cluster-2 mostly have focused on strains occurring in the northern hemisphere. Except for one strain from Papua New Guinea, namely Candidatus Frankia meridionalis Cppng1, no complete genome of Frankia associated with Coriaria occurring in the southern hemisphere has been published thus far, yet the majority of the Coriariaceae species occur here. We present field sampling data of novel Frankia cluster-2 strains, representing two novel species, which are associated with Coriaria arborea and Coriaria sarmentosa in New Zealand, and with Coriaria ruscifolia in Patagonia (Argentina), in addition to identifying Ca. F. meridionalis present in New Zealand. The novel Frankia species were found to be closely related to both Ca. F. meridionalis, and a Frankia species occurring in the Philippines, Taiwan, and Japan. Our data suggest that the different Frankia cluster-2 species diverged early after becoming symbiotic circa 100 million years ago.

Asunto(s)

Frankia , Filogenia , Simbiosis , Frankia/genética , Frankia/clasificación , Genoma Bacteriano , Nueva Zelanda , Argentina , Filogeografía , Nódulos de las Raíces de las Plantas/microbiología , Análisis de Secuencia de ADN , ADN Bacteriano/genética

RESUMEN

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

Asunto(s)

Bradyrhizobium , Filogenia , ARN Ribosómico 16S , Microbiología del Suelo , Vigna , Bradyrhizobium/genética , Bradyrhizobium/clasificación , Bradyrhizobium/fisiología , Bradyrhizobium/aislamiento & purificación , Brasil , Vigna/microbiología , ARN Ribosómico 16S/genética , Inoculantes Agrícolas/genética , Inoculantes Agrícolas/fisiología , Inoculantes Agrícolas/clasificación , ADN Bacteriano/genética , Simbiosis , Nódulos de las Raíces de las Plantas/microbiología , Adaptación Fisiológica , Glycine max/microbiología , Estrés Fisiológico

RESUMEN

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis.

Asunto(s)

Ácidos Grasos , Medicago sativa , Fosfatidilcolinas , Nodulación de la Raíz de la Planta , Sinorhizobium meliloti , Simbiosis , Sinorhizobium meliloti/fisiología , Sinorhizobium meliloti/genética , Medicago sativa/microbiología , Medicago sativa/genética , Nodulación de la Raíz de la Planta/genética , Ácidos Grasos/metabolismo , Ácidos Grasos/biosíntesis , Fosfatidilcolinas/metabolismo , Fosfatidilcolinas/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , Mutación , Polisacáridos Bacterianos/metabolismo , Polisacáridos Bacterianos/biosíntesis , Fijación del Nitrógeno

RESUMEN

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.

Asunto(s)

Bradyrhizobium , Burkholderiaceae , Fabaceae , Rhizobium , Rhizobium/genética , ARN Ribosómico 16S/genética , Fabaceae/microbiología , Bosques , Burkholderiaceae/genética , Filogenia , Simbiosis , Nódulos de las Raíces de las Plantas/microbiología , ADN Bacteriano/genética , Análisis de Secuencia de ADN

RESUMEN

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.

Asunto(s)

Bradyrhizobium , Pilotos , Rhizobium , Vigna , Humanos , Vigna/genética , Vigna/microbiología , Simbiosis/genética , Rhizobium/genética , ARN Ribosómico 16S/genética , Nódulos de las Raíces de las Plantas/microbiología , Bradyrhizobium/genética , Fijación del Nitrógeno , Filogenia

RESUMEN

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.

Asunto(s)

Acacia , Bradyrhizobium , Rhizobium , Rhizobium/genética , Plantones , Fijación del Nitrógeno , Simbiosis/genética , Filogenia , Nódulos de las Raíces de las Plantas/microbiología , Bradyrhizobium/genética

RESUMEN

This study aimed to evaluate the resilience of phytophysiognomies under influence of iron mining by assessing the occurrence, diversity, and symbiotic efficiency of native communities of nitrogen-fixing bacteria that nodulate leguminous plants (rhizobia) in soils of an area revegetated with grass after iron mining activities and in the phytophysiognomies in adjacent areas (Canga, Atlantic Forest, Cerrado, and Eucalyptus-planted forest). Experiments for capturing rhizobia through two species of promiscuous plants, siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata), were conducted in a greenhouse. The rhizobial strains isolated were characterized phenotypically, genetically (16S rRNA sequencing and BOX-PCR fingerprinting), and regard symbiotic efficiency of biological nitrogen fixation (BNF) compared to mineral nitrogen and reference strains. Cowpea captured a higher density of rhizobia than siratro did. However, most of the strains captured by siratro had greater symbiotic efficiency. The revegetated area proved to be the community most efficient in N2 fixation and was also the most diverse, whereas Canga was the least diverse. For the two trap species, the predominant genus captured in the revegetated area and in the phytophysiognomies was Bradyrhizobium. The greater symbiotic efficiency and the high genetic diversity of the rhizobial community in the revegetated area indicate the effectiveness of the soil rehabilitation process. The revegetated area and the phytophysiognomies proved to harbor strains with high biotechnological potential. Results indicate that the high functional redundancy of this group of bacteria contributes to the resilience of these phytophysiognomies and the revegetated area.

Asunto(s)

Bradyrhizobium , Fabaceae , Rhizobium , Vigna , Hierro , ARN Ribosómico 16S/genética , Fabaceae/genética , Simbiosis , Minería , Filogenia , Nódulos de las Raíces de las Plantas/microbiología

RESUMEN

Biological nitrogen fixation (BNF) is a key process for the N input in agriculture, with outstanding economic and environmental benefits from the replacement of chemical fertilizers. However, not all symbioses are equally effective in fixing N2, and a major example relies on the high contribution associated with the soybean (Glycine max), contrasting with the low rates reported with the common bean (Phaseolus vulgaris) crop worldwide. Understanding these differences represents a major challenge that can help to design strategies to increase the contribution of BNF, and next-generation sequencing (NGS) analyses of the nodule and root microbiomes may bring new insights to explain differential symbiotic performances. In this study, three treatments evaluated in non-sterile soil conditions were investigated in both legumes: (i) non-inoculated control; (ii) inoculated with host-compatible rhizobia; and (iii) co-inoculated with host-compatible rhizobia and Azospirillum brasilense. In the more efficient and specific symbiosis with soybean, Bradyrhizobium presented a high abundance in nodules, with further increases with inoculation. Contrarily, the abundance of the main Rhizobium symbiont was lower in common bean nodules and did not increase with inoculation, which may explain the often-reported lack of response of this legume to inoculation with elite strains. Co-inoculation with Azospirillum decreased the abundance of the host-compatible rhizobia in nodules, probably because of competitiveness among the species at the rhizosphere, but increased in root microbiomes. The results showed that several other bacteria compose the nodule microbiomes of both legumes, including nitrogen-fixing, growth-promoters, and biocontrol agents, whose contribution to plant growth deserves further investigation. Several genera of bacteria were detected in root microbiomes, and this microbial community might contribute to plant growth through a variety of microbial processes. However, massive inoculation with elite strains should be better investigated, as it may affect the root microbiome, verified by both relative abundance and diversity indices, that might impact the contribution of microbial processes to plant growth.

Asunto(s)

Microbiota , Phaseolus , Rhizobium , Fertilizantes , Nitrógeno , Fijación del Nitrógeno , Phaseolus/microbiología , Raíces de Plantas/microbiología , Rhizobium/fisiología , Nódulos de las Raíces de las Plantas/microbiología , Suelo , Glycine max/microbiología , Simbiosis

RESUMEN

The introduction of a forage legume into a tropical pasture should decrease the need for N fertilizer, provided biological N2 fixation (BNF) contributes enough to compensate for exported N. Macrotyloma axillare (perennial horsegram) is a suitable legume for composing mixed pastures, and our hypothesis is that the isolation of indigenous rhizobia from roots and rhizosphere is the way of achieving an efficient inoculant to maximize BNF to the legume. Nodules and rhizosphere soil taken from M. axillare grown in a mixed pasture with palisade grass were sampled and used in a trap host assay using Leonard jars containing a mixture of vermiculite and sand. A total of ten bacteria were initially isolated using this technique. The isolates were then used in two experiments to evaluate the inoculation responses on the perennial horsegram in greenhouse conditions to which nodulation, plant growth, and shoot N accumulation were measured. Phylogenetic analyses based on 16S rRNA and recA placed all strains within genus Bradyrhizobium, some of them not previously described. The best strain provided more than 120 nodules and more than 65 mg of nodules per plant. Strain BR14182 was considered the most promising given the high dry matter and N accumulation in plant shoots. This study provides the first analysis of Bradyrhizobium diversity nodulating M. axillare in Brazil and provided evidence of the role of inoculation in incrementing the plant-rhizobium symbiosis in a forage legume.

Asunto(s)

Bradyrhizobium , Fabaceae , Rhizobium , Nódulos de las Raíces de las Plantas/microbiología , ARN Ribosómico 16S/genética , Filogenia , Fijación del Nitrógeno , Fabaceae/microbiología , Simbiosis/genética , ADN Bacteriano/genética

RESUMEN

Bradyrhizobium is a heterogeneous bacterial genus capable of establishing symbiotic associations with a broad range of legume hosts, including species of economic and environmental importance. This study was focused on the taxonomic and symbiovar definition of four strains - CNPSo 4026T, WSM 1704T, WSM 1738T and WSM 4400T - previously isolated from nodules of legumes in Western Australia and South Africa. The 16S rRNA gene phylogenetic tree allocated the strains to the Bradyrhizobium elkanii supergroup. The multilocus sequence analysis (MLSA) with partial sequences of six housekeeping genes - atpD, dnaK, glnII, gyrB, recA and rpoB - did not cluster the strains under study as conspecific to any described Bradyrhizobium species. Average nucleotide identity and digital DNA-DNA hybridization values were calculated for the four strains of this study and the closest species according to the MLSA phylogeny with the highest values being 95.46 and 62.20 %, respectively; therefore, both being lower than the species delineation cut-off values. The nodC and nifH phylogenies included strains WSM 1738T and WSM 4400T in the symbiovars retamae and vignae respectively, and also allowed the definition of three new symbiovars, sv. cenepequi, sv. glycinis, and sv. cajani. Analysis of morphophysiological characterization reinforced the identification of four novel proposed Bradyrhizobium species that are accordingly named as follows: Bradyrhizobium cenepequi sp. nov. (CNPSo 4026T=WSM 4798T=LMG 31653T), isolated from Vigna unguiculata; Bradyrhizobium semiaridum sp. nov. (WSM 1704T=CNPSo 4028T=LMG 31654T), isolated from Tephrosia gardneri; Bradyrhizobium hereditatis sp. nov. (WSM 1738T=CNPSo 4025T=LMG 31652T), isolated from Indigofera sp.; and Bradyrhizobium australafricanum sp. nov. (WSM 4400T=CNPSo 4015T=LMG 31648T) isolated from Glycine sp.

Asunto(s)

Bradyrhizobium , Fabaceae , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Fabaceae/microbiología , Ácidos Grasos/química , Genes Bacterianos , Filogenia , ARN Ribosómico 16S/genética , Nódulos de las Raíces de las Plantas/microbiología , Análisis de Secuencia de ADN , Sudáfrica , Verduras , Australia Occidental

RESUMEN

The development of a symbiotic nitrogen-fixing nodule in legumes involves infection and organogenesis. Infection begins when rhizobia enter a root hair through an inward structure, the infection thread (IT), which guides the bacteria towards the cortical tissue. Concurrently, organogenesis takes place by inducing cortical cell division (CCD) at the infection site. Genetic analysis showed that both events are well-coordinated; however, the dynamics connecting them remain to be elucidated. To visualize the crossroads between IT and CCD, we benefited from the fact that, in Phaseolus vulgaris nodulation, where the first division occurs in subepidermal cortical cells located underneath the infection site, we traced a Rhizobium etli strain expressing DsRed, the plant cytokinesis marker YFP-PvKNOLLE, a nuclear stain and cell wall auto-fluorescence. We found that the IT exits the root hair to penetrate an underlying subepidermal cortical (S-E) cell when it is concluding cytokinesis.

Asunto(s)

Phaseolus , Rhizobium , División Celular , Phaseolus/microbiología , Proteínas de Plantas/genética , Nodulación de la Raíz de la Planta , Raíces de Plantas/genética , Rhizobium/genética , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis/genética

RESUMEN

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

Asunto(s)

Metalotioneína/metabolismo , Phaseolus , Proteínas de Plantas/metabolismo , Rhizobium/crecimiento & desarrollo , Nódulos de las Raíces de las Plantas , Simbiosis , Phaseolus/metabolismo , Phaseolus/microbiología , Nódulos de las Raíces de las Plantas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología

RESUMEN

The neotropical genus Calliandra is of great importance to ecology and agroforestry, but little is known about its nodulation or its rhizobia. The nodulation of several species from two restricted diversity centres with native/endemic species (Eastern Brazil and North-Central America) and species widespread in South America, as well as their nodule structure and the molecular characterization of their rhizobial symbionts based on phylogeny of the 16S rRNA, recA and nodC gene, is reported herein. Species representative of different regions were grown in Brazilian soil, their nodulation observed, and their symbionts characterized. Calliandra nodules have anatomy that is typical of mimosoid nodules regardless of the microsymbiont type. The rhizobial symbionts differed according to the geographical origin of the species, i.e. Alphaproteobacteria (Rhizobium) were the exclusive symbionts from North-Central America, Betaproteobacteria (Paraburkholderia) from Eastern Brazil, and a mixture of both nodulated the widespread species. The symbiont preferences of Calliandra species are the result of the host co-evolving with the "local" symbiotic bacteria that thrive in the different edaphoclimatic conditions, e.g. the acidic soils of NE Brazil are rich in acid-tolerant Paraburkholderia, whereas those of North-Central America are typically neutral-alkaline and harbour Rhizobium. It is hypothesized that the flexibility of widespread species in symbiont choice has assisted in their wider dispersal across the neotropics.

Asunto(s)

Fabaceae , Interacciones Microbiota-Huesped , Rhizobium , Nódulos de las Raíces de las Plantas , Microbiología del Suelo , Brasil , Burkholderiaceae , ADN Bacteriano/genética , Fabaceae/microbiología , Filogenia , ARN Ribosómico 16S/genética , Rhizobium/genética , Nódulos de las Raíces de las Plantas/microbiología , Análisis de Secuencia de ADN , Simbiosis

RESUMEN

Phaseolus vulgaris is a grain cultivated in vast areas of different countries. It is an excellent alternative to the other legumes in the Venezuelan diet and is of great agronomic interest due to its resistance to soil acidity, drought, and high temperatures. Phaseolus establishes symbiosis primarily with Rhizobium and Ensifer species in most countries, and this rhizobia-legume interaction has been studied in Asia, Africa, and the Americas. However, there is currently no evidence to show that rhizobia nodulate the endemic cultivars of P. vulgaris in Venezuela. Therefore, we herein investigated the phylogenetic diversity of plant growth-promoting and N2-fixing nodulating bacteria isolated from the root nodules of P. vulgaris cultivars in a different agroecosystem in Venezuela. In comparisons with other countries, higher diversity was found in isolates from P. vulgaris nodules, ranging from α- and ß-proteobacteria. Some isolates belonging to several new phylogenetic lineages within Bradyrhizobium, Ensifer, and Mesorhizobium species were also specifically isolated at some topographical regions. Additionally, some isolates exhibited tolerance to high temperature, acidity, alkaline pH, salinity stress, and high Al levels; some of these characteristics may be related to the origin of the isolates. Some isolates showed high tolerance to Al toxicity as well as strong plant growth-promoting and antifungal activities, thereby providing a promising agricultural resource for inoculating crops.

Asunto(s)

Bacterias/genética , Bacterias/aislamiento & purificación , Variación Genética , Phaseolus/microbiología , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis , Bacterias/clasificación , Fenómenos Fisiológicos Bacterianos , ADN Bacteriano/genética , Fijación del Nitrógeno , Phaseolus/crecimiento & desarrollo , Filogenia , Nodulación de la Raíz de la Planta , Microbiología del Suelo , Venezuela

RESUMEN

In this work, we investigated Bradyrhizobium strains isolated from soils collected from the rhizosphere of native and exotic legumes species inhabiting two ecoclimatic zones - asubtropical-lowland pasture (Pampa Biome) and a volcanic plateau covered by Araucaria Moist Forests (Atlantic Forest Biome). The rhizobial strains were isolated from the nodules of seven native and one exotic legume species used as rhizobium traps. Single-gene (recA, glnII, dnaK) and combined-gene MLSA analyses (dnaK-glnII-gyrB-recA-rpoB) revealed that nearly 85% of the isolates clustered in B. elkanii supergroup, while the remaining (except for two isolates) in B. japonicum supergroup, albeit, in most cases, separately from the type strains of Bradyrhizobium species. As a symbiotic gene marker, a portion of nifD gene was sequenced for 194 strains. In the nifD-tree, an American branch III.3D (104 isolates), was the most numerous among the isolates. A significant portion of the isolates clustered in American groups; subclade III.4 (40 strains), Clade VII (3 strains), and a new Clade XX (4 strains). Most of the remaining strains belonged to a pantropical III.3C branch (39 isolates). On the other hand, identification of isolates belonging, respectively, to Clade I and Clade II may result of spreading of the Australian (Clade I) and European (Clade II) bradyrhizobia following the introduction of their legume hosts. Our study indicated that the American groups predominated in the symbiotic Bradyrhizobium communities in southern Brazil. However, there is a significant component of exotic lineages, resulting from the dispersal of pantropical Fabaceae taxa and the introduction of exotic legumes.

Asunto(s)

Bradyrhizobium , Fabaceae , Bosques , Pradera , Filogenia , Bradyrhizobium/clasificación , Bradyrhizobium/aislamiento & purificación , Brasil , ADN Bacteriano/genética , Fabaceae/microbiología , Genes Bacterianos , ARN Ribosómico 16S/genética , Rizosfera , Nódulos de las Raíces de las Plantas/microbiología , Análisis de Secuencia de ADN , Simbiosis

RESUMEN

The aim of this work was to characterize and identify some bacteria isolated from the root nodules of Retama monosperma grown in Sidi Boubker lead and zinc mine tailings. Very few root nodules were obtained on the root nodules of R. monosperma grown in these soils. The three bacteria isolated from the root nodules were tolerant in vitro to different concentrations of heavy metals, including lead and zinc. The rep-PCR experiments showed that the three isolates have different molecular fingerprints and were considered as three different strains. The analysis of their 16S rRNA gene sequences proved their affiliation to the genus Bradyrhizobium. The analysis and phylogeny of the housekeeping genes atpD, glnII, gyrB, recA, and rpoB confirmed that the closest species was B. valentinum with similarity percentages of 95.61 to 95.82%. The three isolates recovered from the root nodules were slow-growing rhizobia capable to renodulate their original host plant in the presence of Pb-acetate. They were able to nodulate R. sphaerocarpa and Lupinus luteus also but not Glycine max or Phaseolus vulgaris. The phylogeny of the nodA and nodC nodulation genes as well as the nifH gene of the three strains showed that they belong to the symbiovar retamae of the genus Bradyrhizobium. The three strains isolated could be considered for use as inoculum for Retama plants before use in phytoremediation experiments.

Asunto(s)

Bradyrhizobium/metabolismo , Fabaceae/microbiología , Plomo/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Zinc/metabolismo , Bradyrhizobium/clasificación , Bradyrhizobium/genética , Bradyrhizobium/aislamiento & purificación , Minería , Marruecos , Filogenia , Nodulación de la Raíz de la Planta , Glycine max/microbiología