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Sample records for rhizobia nodulating lotus

  1. Identification of Lotus rhizobia by direct DNA hybridization of crushed root nodules

    SciTech Connect

    Cooper, J.E.; Bjourson, A.J.; Thompson, J.K.

    1987-07-01

    Hybridization of crushed Lotus pedunculatus root nodules with /sup 32/P-labeled total genomic DNA probes was used to identify Rhizobium loti and Bradyrhizobium sp. (Lotus rhizobia). Probes always hybridized with homologous target DNA and frequency with DNAs of other strains from the same genus. Intergeneric hybridization did not occur. Results were comparable to those from colony hybridization.

  2. The temperature-sensitive brush mutant of the legume Lotus japonicus reveals a link between root development and nodule infection by rhizobia.

    PubMed

    Maekawa-Yoshikawa, Makoto; Müller, Judith; Takeda, Naoya; Maekawa, Takaki; Sato, Shusei; Tabata, Satoshi; Perry, Jillian; Wang, Trevor L; Groth, Martin; Brachmann, Andreas; Parniske, Martin

    2009-04-01

    The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26 degrees C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and reduced cell expansion. Reciprocal grafting of brush with wild-type plants indicated that this genotype only affected the root and that the shoot phenotype was a secondary effect. The root and nodulation phenotype cosegregated as a single Mendelian trait and the BRUSH gene could be mapped to the short arm of chromosome 2. At 18 degrees C, the brush root anatomy was rescued and similar to the wild type, and primary root length, number of infection threads, and nodule formation were partially rescued. Superficially, the brush root phenotype resembled the ethylene-related thick short root syndrome. However, treatment with ethylene inhibitor did not recover the observed phenotypes, although brush primary roots were slightly longer. The defects of brush in root architecture and infection thread development, together with intact nodule architecture and complete absence of symptoms from shoots, suggest that BRUSH affects cellular differentiation in a tissue-dependent way.

  3. The Temperature-Sensitive brush Mutant of the Legume Lotus japonicus Reveals a Link between Root Development and Nodule Infection by Rhizobia[C][W][OA

    PubMed Central

    Maekawa-Yoshikawa, Makoto; Müller, Judith; Takeda, Naoya; Maekawa, Takaki; Sato, Shusei; Tabata, Satoshi; Perry, Jillian; Wang, Trevor L.; Groth, Martin; Brachmann, Andreas; Parniske, Martin

    2009-01-01

    The brush mutant of Lotus japonicus exhibits a temperature-dependent impairment in nodule, root, and shoot development. At 26°C, brush formed fewer nodules, most of which were not colonized by rhizobia bacteria. Primary root growth was retarded and the anatomy of the brush root apical meristem revealed distorted cellular organization and reduced cell expansion. Reciprocal grafting of brush with wild-type plants indicated that this genotype only affected the root and that the shoot phenotype was a secondary effect. The root and nodulation phenotype cosegregated as a single Mendelian trait and the BRUSH gene could be mapped to the short arm of chromosome 2. At 18°C, the brush root anatomy was rescued and similar to the wild type, and primary root length, number of infection threads, and nodule formation were partially rescued. Superficially, the brush root phenotype resembled the ethylene-related thick short root syndrome. However, treatment with ethylene inhibitor did not recover the observed phenotypes, although brush primary roots were slightly longer. The defects of brush in root architecture and infection thread development, together with intact nodule architecture and complete absence of symptoms from shoots, suggest that BRUSH affects cellular differentiation in a tissue-dependent way. PMID:19176723

  4. Phylogenetic relationship of Lotus uliginosus symbionts with bradyrhizobia nodulating genistoid legumes.

    PubMed

    Lorite, María J; Videira e Castro, Isabel; Muñoz, Socorro; Sanjuán, Juan

    2012-02-01

    Lotus species are legumes with potential for pastures in soils with low-fertility and environmental constraints. The aim of this work was to characterize bacteria that establish efficient nitrogen-fixing symbiosis with the forage species Lotus uliginosus. A total of 39 isolates were obtained from nodules of L. uliginosus naturally growing in two different locations of Portugal. Molecular identification of the isolates plus the commercial inoculant strain NZP2039 was performed by REP-PCR, 16S rRNA RFLP, and 16S rRNA, glnII and recA sequence analyses. Limited genetic diversity was found among the L. uliginosus symbionts, which showed a close phylogenetic relationship with the species Bradyrhizobium japonicum. The symbiotic nifH, nodA and nodC gene sequences were closely related with the corresponding genes of various Bradyrhizobium strains isolated from Lupinus and other genistoid legumes and therefore were phylogenetically separated from other Lotus spp. rhizobia. The L. uliginosus bradyrhizobia were able to nodulate and fix nitrogen in association with L. uliginosus, could nodulate Lotus corniculatus with generally poor nitrogen-fixing efficiency, formed nonfixing nodules in Lotus tenuis and Lupinus luteus roots and were unable to nodulate Glycine soja or Glycine max. Thus, L. uliginosus rhizobia seem closely related to B. japonicum biovar genistearum strains.

  5. Genetic diversity of root nodule bacteria nodulating Lotus corniculatus and Anthyllis vulneraria in Sweden.

    PubMed

    Ampomah, Osei Yaw; Huss-Danell, Kerstin

    2011-06-01

    Very little is known about the genetic diversity and phylogeny of rhizobia nodulating Lotus species in northern temperate regions. We have therefore studied the genetic diversity among a total of 61 root nodule bacteria isolated from Lotus corniculatus and Anthyllis vulneraria from different geographic sites and habitats in Sweden by restriction fragment length polymorphism (RFLP) of the internal transcribed spacer between their 16S rRNA and 23S rRNA (IGS) region. A high diversity consisting of 26 IGS types from 54 L. corniculatus isolates and five IGS types from seven A. vulneraria isolates was found. The 16S rRNA sequences and phylogeny of representatives of the different IGS types showed four interesting exceptions from the majority of the isolates belonging to the genus Mesorhizobium: Two isolates were both found to be closely related to Rhodococcus spp., and two other isolates showed close relationship with Geobacillus spp. and Paenibacillus spp., respectively. The nodA sequences and phylogeny showed that all the isolates, including those not belonging to the traditional rhizobia genera, harbored nodA sequences which were typical of Mesorhizobium loti. Generally, the 16S rRNA and nodA phylogenetic trees were not congruent in that isolates with similar 16S rRNA sequences were associated with isolates harboring different nodA sequences. All the isolates were confirmed to nodulate L. corniculatus in an inoculation test. This is the first report of members of these non-rhizobia genera being able to nodulate legumes, and we suggest that they may have acquired their nodulating properties through lateral gene transfer. Copyright © 2011 Elsevier GmbH. All rights reserved.

  6. Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

    PubMed

    Sullivan, J T; Eardly, B D; van Berkum, P; Ronson, C W

    1996-08-01

    Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes.

  7. Four unnamed species of nonsymbiotic rhizobia isolated from the rhizosphere of Lotus corniculatus.

    PubMed Central

    Sullivan, J T; Eardly, B D; van Berkum, P; Ronson, C W

    1996-01-01

    Previously, we found that genetically diverse rhizobia nodulating Lotus corniculatus at a field site devoid of naturalized rhizobia had symbiotic DNA regions identical to those of ICMP3153, the inoculant strain used at the site (J. T. Sullivan, H. N. Patrick, W. L. Lowther, D. B. Scott, and C. W. Ronson, Proc. Natl. Acad. Sci. USA 92:8985-8989, 1995). In this study, we characterized seven nonsymbiotic rhizobial isolates from the rhizosphere of L. corniculatus. These included two from plants at the field site sampled by Sullivan et al. and five from plants at a new field plot adjacent to that site. The isolates did not nodulate Lotus species or hybridize to symbiotic gene probes but did hybridize to genomic DNA probes from Rhizobium loti. Their genetic relationships with symbiotic isolates obtained from the same sites, with inoculant strain ICMP3153, and with R. loti NZP2213T were determined by three methods. Genetic distance estimates based on genomic DNA-DNA hybridization and multilocus enzyme electrophoresis were correlated but were not consistently reflected by 16S rRNA nucleotide sequence divergence. The nonsymbiotic isolates represented four genomic species that were related to R. loti; the diverse symbiotic isolates from the site belonged to one of these species. The inoculant strain ICMP3153 belonged to a fifth genomic species that was more closely related to Rhizobium huakuii. These results support the proposal that nonsymbiotic rhizobia persist in soils in the absence of legumes and acquire symbiotic genes from inoculant strains upon introduction of host legumes. PMID:8702274

  8. Nodulation outer proteins: double-edged swords of symbiotic rhizobia

    USDA-ARS?s Scientific Manuscript database

    Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation is the result of a complex bacterial infection process, which depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial nodulation outer proteins (Nops)...

  9. The integral membrane protein SEN1 is required for symbiotic nitrogen fixation in Lotus japonicus nodules.

    PubMed

    Hakoyama, Tsuneo; Niimi, Kaori; Yamamoto, Takeshi; Isobe, Sawa; Sato, Shusei; Nakamura, Yasukazu; Tabata, Satoshi; Kumagai, Hirotaka; Umehara, Yosuke; Brossuleit, Katja; Petersen, Thomas R; Sandal, Niels; Stougaard, Jens; Udvardi, Michael K; Tamaoki, Masanori; Kawaguchi, Masayoshi; Kouchi, Hiroshi; Suganuma, Norio

    2012-01-01

    Legume plants establish a symbiotic association with bacteria called rhizobia, resulting in the formation of nitrogen-fixing root nodules. A Lotus japonicus symbiotic mutant, sen1, forms nodules that are infected by rhizobia but that do not fix nitrogen. Here, we report molecular identification of the causal gene, SEN1, by map-based cloning. The SEN1 gene encodes an integral membrane protein homologous to Glycine max nodulin-21, and also to CCC1, a vacuolar iron/manganese transporter of Saccharomyces cerevisiae, and VIT1, a vacuolar iron transporter of Arabidopsis thaliana. Expression of the SEN1 gene was detected exclusively in nodule-infected cells and increased during nodule development. Nif gene expression as well as the presence of nitrogenase proteins was detected in rhizobia from sen1 nodules, although the levels of expression were low compared with those from wild-type nodules. Microscopic observations revealed that symbiosome and/or bacteroid differentiation are impaired in the sen1 nodules even at a very early stage of nodule development. Phylogenetic analysis indicated that SEN1 belongs to a protein clade specific to legumes. These results indicate that SEN1 is essential for nitrogen fixation activity and symbiosome/bacteroid differentiation in legume nodules.

  10. Nodulation genes and type III secretion systems in rhizobia

    USDA-ARS?s Scientific Manuscript database

    For establishment of symbiosis, rhizobia and legumes have to communicate. Specific signaling starts with the release of flavonoids by the plant. All rhizobia encode at least one NodD protein, which responds to the presence of specific flavonoids by activation of nodulation genes. In Bradyrhizobium j...

  11. Biogenic amines in rhizobia and legume root nodules.

    PubMed

    Fujihara, Shinsuke

    2009-01-01

    Root-nodule bacteria (rhizobia) are of great importance for nitrogen acquisition through symbiotic nitrogen fixation in a wide variety of leguminous plants. These bacteria differ from most other soil microorganisms by taking dual forms, i.e. a free-living form in soils and a symbiotic form inside of host legumes. Therefore, they should have a versatile strategy for survival, whether inhabiting soils or root nodules formed through rhizobia-legume interactions. Rhizobia generally contain large amounts of the biogenic amine homospermidine, an analog of spermidine which is an essential cellular component in most living systems. The external pH, salinity and a rapid change in osmolarity are thought to be significant environmental factors affecting the persistence of rhizobia. The present review describes the regulation of homospermidine biosynthesis in response to environmental stress and its possible functional role in rhizobia. Legume root nodules, an alternative habitat of rhizobia, usually contain a variety of biogenic amines besides homospermidine and the occurrence of some of these amines is closely associated with rhizobial infections. In the second half of this review, novel biogenic amines found in certain legume root nodules and the mechanism of their synthesis involving cooperation between the rhizobia and host legume cells are also described.

  12. Characterization of strains unlike Mesorhizobium loti that nodulate lotus spp. in saline soils of Granada, Spain.

    PubMed

    Lorite, María J; Muñoz, Socorro; Olivares, José; Soto, María J; Sanjuán, Juan

    2010-06-01

    Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.

  13. Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

    PubMed Central

    Zgadzaj, Rafal; Garrido-Oter, Ruben; Jensen, Dorthe Bodker; Koprivova, Anna; Schulze-Lefert, Paul; Radutoiu, Simona

    2016-01-01

    Lotus japonicus has been used for decades as a model legume to study the establishment of binary symbiotic relationships with nitrogen-fixing rhizobia that trigger root nodule organogenesis for bacterial accommodation. Using community profiling of 16S rRNA gene amplicons, we reveal that in Lotus, distinctive nodule- and root-inhabiting communities are established by parallel, rather than consecutive, selection of bacteria from the rhizosphere and root compartments. Comparative analyses of wild-type (WT) and symbiotic mutants in Nod factor receptor5 (nfr5), Nodule inception (nin) and Lotus histidine kinase1 (lhk1) genes identified a previously unsuspected role of the nodulation pathway in the establishment of different bacterial assemblages in the root and rhizosphere. We found that the loss of nitrogen-fixing symbiosis dramatically alters community structure in the latter two compartments, affecting at least 14 bacterial orders. The differential plant growth phenotypes seen between WT and the symbiotic mutants in nonsupplemented soil were retained under nitrogen-supplemented conditions that blocked the formation of functional nodules in WT, whereas the symbiosis-impaired mutants maintain an altered community structure in the nitrogen-supplemented soil. This finding provides strong evidence that the root-associated community shift in the symbiotic mutants is a direct consequence of the disabled symbiosis pathway rather than an indirect effect resulting from abolished symbiotic nitrogen fixation. Our findings imply a role of the legume host in selecting a broad taxonomic range of root-associated bacteria that, in addition to rhizobia, likely contribute to plant growth and ecological performance. PMID:27864511

  14. Swimming and swarming motility properties of peanut-nodulating rhizobia.

    PubMed

    Vicario, Julio C; Dardanelli, Marta S; Giordano, Walter

    2015-01-01

    Motility allows populations of bacteria to rapidly reach and colonize new microniches or microhabitats. The motility of rhizobia (symbiotic nitrogen-fixing bacteria that nodulate legume roots) is an important factor determining their competitive success. We evaluated the effects of temperature, incubation time, and seed exudates on swimming and swarming motility of five strains of Bradyrhizobium sp. (peanut-nodulating rhizobia). Swimming motility was increased by exudate exposure for all strains except native Pc34. In contrast, swarming motility was increased by exudate exposure for native 15A but unchanged for the other four strains. All five strains displayed the ability to differentiate into swarm cells. Morphological examination by scanning electron microscopy showed that the length of the swarm cells was variable, but generally greater than that of vegetative cells. Our findings suggest the importance of differential motility properties of peanut-nodulating rhizobial strains during agricultural inoculation and early steps of symbiotic interaction with the host.

  15. Grafting analysis indicates that malfunction of TRICOT in the root causes a nodulation-deficient phenotype in Lotus japonicus.

    PubMed

    Suzaki, Takuya; Kawaguchi, Masayoshi

    2013-03-01

    Leguminous plants develop root nodules in symbiosis with soil rhizobia. Nodule formation occurs following rhizobial infection of the host root that induces dedifferentiation of some cortical cells and the initiation of a new developmental program to form nodule primordia. In a recent study, we identified a novel gene, TRICOT (TCO), that acts as a positive regulator of nodulation in Lotus japonicus. In addition to its role in nodulation, tco mutant plants display pleiotropic defects including abnormal shoot apical meristem formation. Here, we investigated the effect of the tco mutation on nodulation using a grafting approach. The results strongly indicate that the nodulation-deficient phenotype of the mutant results from malfunction of the TCO gene in the root.

  16. The trehalose utilization gene thuA ortholog in Mesorhizobium loti does not influence competitiveness for nodulation on Lotus spp.

    PubMed

    Ampomah, Osei Yaw; Jensen, John Beck

    2014-03-01

    Competitiveness for nodulation is a desirable trait in rhizobia strains used as inoculant. In Sinorhizobium meliloti 1021 mutation in either of the trehalose utilization genes thuA or thuB influences its competitiveness for root colonization and nodule occupancy depending on the interacting host. We have therefore investigated whether mutation in the thuA ortholog in Mesorhizobium loti MAFF303099 also leads to a similar competitive phenotype on its hosts. The results show that M. loti thuA mutant Ml7023 was symbiotically effective and was as competitive as the wild type in colonization and nodule occupancy on Lotus corniculatus and Lotus japonicus. The thuA gene in M. loti was not induced during root colonization or in the infection threads unlike in S. meliloti, despite its induction by trehalose and high osmolarity in in vitro assays.

  17. Characterization of Strains unlike Mesorhizobium loti That Nodulate Lotus spp. in Saline Soils of Granada, Spain ▿ †

    PubMed Central

    Lorite, María J.; Muñoz, Socorro; Olivares, José; Soto, María J.; Sanjuán, Juan

    2010-01-01

    Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts. PMID:20435777

  18. Knockdown of LjIPT3 influences nodule development in Lotus japonicus.

    PubMed

    Chen, Yaping; Chen, Wei; Li, Xueliu; Jiang, Huawu; Wu, Pingzhi; Xia, Kuaifei; Yang, Yali; Wu, Guojiang

    2014-01-01

    Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (β-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

  19. Enzymes of the Glyoxylate Cycle in Rhizobia and Nodules of Legumes 1

    PubMed Central

    Johnson, Gordon V.; Evans, Harold J.; Ching, Temay

    1966-01-01

    The relatively high level of fatty acids in soybean nodules and rhizobia from soybean nodules suggested that the glyoxylate cycle might have a role in nodule metabolism. Several species of rhizobia in pure culture were found to have malate synthetase activity when grown on a number of different carbon sources. Significant isocitrate lyase activity was induced when oleate, which presumably may act as an acetyl CoA precursor, was utilized as the principle carbon source. Malate synthetase was active in extracts of rhizobia from nodules of bush bean (Phaseolus vulgaris L.), cowpea (Vigna sinensis L.), lupine (Lupinus angustifolius L.) and soybean (Glycine max L. Merr.). Activity of malate synthetase was, however, barely detectable in rhizobia from alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.) and pea (Pisum sativum L.) nodules. Appreciable isocitrate lyase activity was not detected in rhizobia from nodules nor was it induced by depletion of endogenous substrates by incubation of excised bush bean nodules. Although rhizobia has the potential for the formation of the key enzymes of the glyoxylate cycle, the absence of isocitrate lyase activity in bacteria isolated from nodules indicated that the glyoxylate cycle does not operate in the symbiotic growth of rhizobia and that the observed high content of fatty acids in nodules and nodule bacteria probably is related to a structural role. PMID:16656404

  20. Cessation of photosynthesis in Lotus japonicus leaves leads to reprogramming of nodule metabolism

    PubMed Central

    Flemetakis, Emmanouil

    2013-01-01

    Symbiotic nitrogen fixation (SNF) involves global changes in gene expression and metabolite accumulation in both rhizobia and the host plant. In order to study the metabolic changes mediated by leaf–root interaction, photosynthesis was limited in leaves by exposure of plants to darkness, and subsequently gene expression was profiled by real-time reverse transcription–PCR (RT–PCR) and metabolite levels by gas chromatography–mass spectrometry in the nodules of the model legume Lotus japonicus. Photosynthetic carbon deficiency caused by prolonged darkness affected many metabolic processes in L. japonicus nodules. Most of the metabolic genes analysed were down-regulated during the extended dark period. In addition to that, the levels of most metabolites decreased or remained unaltered, although accumulation of amino acids was observed. Reduced glycolysis and carbon fixation resulted in lower organic acid levels, especially of malate, the primary source of carbon for bacteroid metabolism and SNF. The high amino acid concentrations together with a reduction in total protein concentration indicate possible protein degradation in nodules under these conditions. Interestingly, comparisons between amino acid and protein content in various organs indicated systemic changes in response to prolonged darkness between nodulated and non-nodulated plants, rendering the nodule a source organ for both C and N under these conditions. PMID:23404899

  1. New nodulation mutants responsible for infection thread development in Lotus japonicus.

    PubMed

    Yano, Koji; Tansengco, Myra L; Hio, Taihei; Higashi, Kuniko; Murooka, Yoshikatsu; Imaizumi-Anraku, Haruko; Kawaguchi, Masayoshi; Hayashi, Makoto

    2006-07-01

    Legume plants develop specialized root organs, the nodules, through a symbiotic interaction with rhizobia. The developmental process of nodulation is triggered by the bacterial microsymbiont but regulated systemically by the host legume plants. Using ethylmethane sulfonate mutagenesis as a tool to identify plant genes involved in symbiotic nodule development, we have isolated and analyzed five nodulation mutants, Ljsym74-3, Ljsym79-2, Ljsym79-3, Ljsym80, and Ljsym82, from the model legume Lotus japonicus. These mutants are defective in developing functional nodules and exhibit nitrogen starvation symptoms after inoculation with Mesorhizobium loti. Detailed observation revealed that infection thread development was aborted in these mutants and the nodules formed were devoid of infected cells. Mapping and complementation tests showed that Ljsym74-3, and Ljsym79-2 and Ljsym79-3, were allelic with reported mutants of L. japonicus, alb1 and crinkle, respectively. The Ljsym82 mutant is unique among the mutants because the infection thread was aborted early in its development. Ljsym74-3 and Ljsym80 were characterized as mutants with thick infection threads in short root hairs. Map-based cloning and molecular characterization of these genes will help us understand the genetic mechanism of infection thread development in L. japonicus.

  2. Genetic diversity of rhizobia nodulating native Vicia spp. in Sweden.

    PubMed

    Ampomah, Osei Yaw; Huss-Danell, Kerstin

    2016-05-01

    Despite the recognition that Rhizobium leguminosarum sv. viciae is the most common symbiont of Vicia species worldwide, there is no available information on rhizobia nodulating native Vicia species in Sweden. We have therefore studied the genetic diversity and phylogeny of root nodule bacteria isolated from V. cracca, V. hirsuta, V. sepium, V. tetrasperma and V. sylvatica growing in different locations in Sweden as well as an isolate each from V. cracca in Tromsø, Norway, and V. multicaulis in Siberia, Russia. Out of 25 isolates sampled from the six Vicia species in 12 different locations, there were 14 different genotypes based on the atpD, recA and nodA gene phylogenies. All isolates were classified into Rhizobium leguminosarum sv. viciae group based on the concatenated atpD and recA phylogeny and the nodA phylogeny.

  3. Glutamine synthetase I-deficiency in Mesorhizobium loti differentially affects nodule development and activity in Lotus japonicus.

    PubMed

    Chungopast, Sirinapa; Thapanapongworakul, Pilunthana; Matsuura, Hiroyuki; Van Dao, Tan; Asahi, Toshimasa; Tada, Kuninao; Tajima, Shigeyuki; Nomura, Mika

    2014-03-01

    In this study, we focused on the effect of glutamine synthetase (GSI) activity in Mesorhizobium loti on the symbiosis between the host plant, Lotus japonicus, and the bacteroids. We used a signature-tagged mutant of M. loti (STM30) with a transposon inserted into the GSI (mll0343) gene. The L. japonicus plants inoculated with STM30 had significantly more nodules, and the occurrence of senesced nodules was much higher than in plants inoculated with the wild-type. The acetylene reduction activity (ARA) per nodule inoculated with STM30 was lowered compared to the control. Also, the concentration of chlorophyll, glutamine, and asparagine in leaves of STM30-infected plants was found to be reduced. Taken together, these data demonstrate that a GSI deficiency in M. loti differentially affects legume-rhizobia symbiosis by modifying nodule development and metabolic processes.

  4. Genetic diversity of rhizobia nodulating lentil (Lens culinaris) in Bangladesh.

    PubMed

    Rashid, Md Harun-or; Schäfer, Holger; Gonzalez, Javier; Wink, Michael

    2012-03-01

    In order to determine the bacterial diversity and the identity of rhizobia nodulating lentil in Bangladesh, we performed a phylogenetic analysis of housekeeping genes (16S rRNA, recA, atpD and glnII) and nodulation genes (nodC, nodD and nodA) of 36 bacterial isolates from 25 localities across the country. Maximum likelihood (ML) and Bayesian analyses based on 16S rRNA sequences showed that most of the isolates (30 out of 36) were related to Rhizobium etli and Rhizobium leguminosarum. Only these thirty isolates were able to re-nodulate lentil under laboratory conditions. The protein-coding housekeeping genes of the lentil nodulating isolates showed 89.1-94.8% genetic similarity to the corresponding genes of R. etli and R. leguminosarum. The same analyses showed that they split into three distinct phylogenetic clades. The distinctness of these clades from closely related species was also supported by high resolution ERIC-PCR fingerprinting and phenotypic characteristics such as temperature tolerance, growth on acid-alkaline media (pH 5.5-10.0) and antibiotic sensitivity. Our phylogenetic analyses based on three nodulation genes (nodA, nodC and nodD) and cross-inoculation assays confirmed that the nodulation genes are related to those of R. leguminosarum biovar viciae, but clustered in a distinct group supported by high bootstrap values. Thus, our multi-locus phylogenetic analysis, DNA fingerprinting and phenotypic characterizations suggest that at least three different clades are responsible for lentil nodulation in Bangladesh. These clades differ from the R. etli-R. leguminosarum group and may correspond to novel species in the genus Rhizobium. Copyright © 2011 Elsevier GmbH. All rights reserved.

  5. Nodulation outer proteins: double-edged swords of symbiotic rhizobia.

    PubMed

    Staehelin, Christian; Krishnan, Hari B

    2015-09-15

    Rhizobia are nitrogen-fixing bacteria that establish a nodule symbiosis with legumes. Nodule formation depends on signals and surface determinants produced by both symbiotic partners. Among them, rhizobial Nops (nodulation outer proteins) play a crucial symbiotic role in many strain-host combinations. Nops are defined as proteins secreted via a rhizobial T3SS (type III secretion system). Functional T3SSs have been characterized in many rhizobial strains. Nops have been identified using various genetic, biochemical, proteomic, genomic and experimental approaches. Certain Nops represent extracellular components of the T3SS, which are visible in electron micrographs as bacterial surface appendages called T3 (type III) pili. Other Nops are T3 effector proteins that can be translocated into plant cells. Rhizobial T3 effectors manipulate cellular processes in host cells to suppress plant defence responses against rhizobia and to promote symbiosis-related processes. Accordingly, mutant strains deficient in synthesis or secretion of T3 effectors show reduced symbiotic properties on certain host plants. On the other hand, direct or indirect recognition of T3 effectors by plant cells expressing specific R (resistance) proteins can result in effector triggered defence responses that negatively affect rhizobial infection. Hence Nops are double-edged swords that may promote establishment of symbiosis with one legume (symbiotic factors) and impair symbiotic processes when bacteria are inoculated on another legume species (asymbiotic factors). In the present review, we provide an overview of our current understanding of Nops. We summarize their symbiotic effects, their biochemical properties and their possible modes of action. Finally, we discuss future perspectives in the field of T3 effector research. © 2015 Authors; published by Portland Press Limited.

  6. Phytosulfokine Is Involved in Positive Regulation of Lotus japonicus Nodulation.

    PubMed

    Wang, Chao; Yu, Haixiang; Zhang, Zhongming; Yu, Liangliang; Xu, Xiaoshu; Hong, Zonglie; Luo, Li

    2015-08-01

    Phytosulfokine (PSK) is a tyrosine-sulfated peptide that is widely distributed in plants, participating in cell proliferation, differentiation, and innate immunity. The potential role of PSK in nodulation in legumes has not been reported. In this work, five PSK precursor genes were identified in Lotus japonicas, designated as LjPSK1 to LjPSK5. Three of them (LjPSK1, LjPSK4, and LjPSK5) were found to be expressed in nitrogen-fixing root nodules. LjPSK1 and LjPSK4 were not induced at the early stage of nodulation. Interestingly, while the expression of LjPSK4 was also found in spontaneous nodules without rhizobial colonization, LjPSK1 was not induced in these pseudo nodules. Promoter-β-glucuronidase analysis revealed that LjPSK1 was highly expressed in enlarged symbiotic cells of nodules. Exogenous addition of 1 1M synthetic PSK peptide resulted in increased nodule numbers per plant. Consistently, the number of mature nodules but not the events of rhizobial infection and nodule initiation was increased by overexpressing LjPSK1 in transgenic hairy roots, in which the expression of jasmonate-responsive genes was found to be repressed. These results suggest that PSK is a new peptide signal that regulates nodulation in legumes, probably through cross-talking with other phytohormones.

  7. Polymorphic infection and organogenesis patterns induced by a Rhizobium leguminosarum isolate from Lotus root nodules are determined by the host genotype.

    PubMed

    Gossmann, Jasmin A; Markmann, Katharina; Brachmann, Andreas; Rose, Laura E; Parniske, Martin

    2012-10-01

    To sample the natural variation in genes controlling compatibility in the legume-rhizobium symbiosis, we isolated rhizobia from nodules of endemic Lotus species from 21 sites across Europe. The majority of isolates were identified as Mesorhizobium- or Bradyrhizobium-related and formed nitrogen-fixing root nodules on Lotus corniculatus and L. pendunculatus, respectively, thus confirming previously defined cross-inoculation groups. Rhizobium leguminosarum (Rl) strain Norway, isolated from L. corniculatus nodules, displayed an exceptional phenotypic variation on different Lotus genotypes. On L. burttii, Rl Norway formed infected nodules, whereas tumors and elongated infected swellings were induced on L. glaber and L. japonicus ecotype Nepal, respectively. A symbiosis- and Nod-factor-responsive promoter:uidA fusion was strongly and rapidly induced in L. japonicus Gifu, but infection threads or signs of nodule organogenesis were absent. This complex phenotypic pattern was not mimicked by either of three engineered R. leguminosarum bv viciae strains producing different Nod-factor variants. Intriguingly, Rl Norway formed infection threads on Pisum sativum cv Sparkle, but failed to induce organogenesis. Rl Norway thus uncovered variation in symbiotic capabilities among diploid Lotus species and ecotypes that are obscured by optimally adapted M. loti strains. These contrasting infection and organogenesis phenotypes reveal recent diversification of recognition determinants in Lotus.

  8. Expression of the CLE-RS3 gene suppresses root nodulation in Lotus japonicus.

    PubMed

    Nishida, Hanna; Handa, Yoshihiro; Tanaka, Sachiko; Suzaki, Takuya; Kawaguchi, Masayoshi

    2016-09-01

    Cell-to-cell communication, principally mediated by short- or long-range mobile signals, is involved in many plant developmental processes. In root nodule symbiosis, a mutual relationship between leguminous plants and nitrogen-fixing rhizobia, the mechanism for the autoregulation of nodulation (AON) plays a key role in preventing the production of an excess number of nodules. AON is based on long-distance cell-to-cell communication between roots and shoots. In Lotus japonicus, two CLAVATA3/ESR-related (CLE) peptides, encoded by CLE-ROOT SIGNAL 1 (CLE-RS1) and -RS2, act as putative root-derived signals that transmit signals inhibiting further nodule development through interaction with a shoot-acting receptor-like kinase HYPERNODULATION ABERRANT ROOT FORMATION 1 (HAR1). Here, an in silico search and subsequent expression analyses enabled us to identify two new L. japonicus CLE genes that are potentially involved in nodulation, designated as CLE-RS3 and LjCLE40. Time-course expression patterns showed that CLE-RS1/2/3 and LjCLE40 expression is induced during nodulation with different activation patterns. Furthermore, constitutive expression of CLE-RS3 significantly suppressed nodule formation in a HAR1-dependent manner. TOO MUCH LOVE, a root-acting regulator of AON, is also required for the CLE-RS3 action. These results suggest that CLE-RS3 is a new component of AON in L. japonicus that may act as a potential root-derived signal through interaction with HAR1. Because CLE-RS2, CLE-RS3 and LjCLE40 are located in tandem in the genome and their expression is induced not only by rhizobial infection but also by nitrate, these genes may have duplicated from a common gene.

  9. Intracellular catalytic domain of symbiosis receptor kinase hyperactivates spontaneous nodulation in absence of rhizobia.

    PubMed

    Saha, Sudip; Dutta, Ayan; Bhattacharya, Avisek; DasGupta, Maitrayee

    2014-12-01

    Symbiosis Receptor Kinase (SYMRK), a member of the Nod factor signaling pathway, is indispensible for both nodule organogenesis and intracellular colonization of symbionts in rhizobia-legume symbiosis. Here, we show that the intracellular kinase domain of a SYMRK (SYMRK-kd) but not its inactive or full-length version leads to hyperactivation of the nodule organogenic program in Medicago truncatula TR25 (symrk knockout mutant) in the absence of rhizobia. Spontaneous nodulation in TR25/SYMRK-kd was 6-fold higher than rhizobia-induced nodulation in TR25/SYMRK roots. The merged clusters of spontaneous nodules indicated that TR25 roots in the presence of SYMRK-kd have overcome the control over both nodule numbers and their spatial position. In the presence of rhizobia, SYMRK-kd could rescue the epidermal infection processes in TR25, but colonization of symbionts in the nodule interior was significantly compromised. In summary, ligand-independent deregulated activation of SYMRK hyperactivates nodule organogenesis in the absence of rhizobia, but its ectodomain is required for proper symbiont colonization.

  10. Down-regulated Lotus japonicus GCR1 plants exhibit nodulation signalling pathways alteration.

    PubMed

    Rogato, Alessandra; Valkov, Vladimir Totev; Alves, Ludovico Martins; Apone, Fabio; Colucci, Gabriella; Chiurazzi, Maurizio

    2016-06-01

    G Protein Coupled Receptor (GPCRs) are integral membrane proteins involved in various signalling pathways by perceiving many extracellular signals and transducing them to heterotrimeric G proteins, which further transduce these signals to intracellular downstream effectors. GCR1 is the only reliable plant candidate as a member of the GPCRs superfamily. In the legume/rhizobia symbiotic interaction, G proteins are involved in signalling pathways controlling different steps of the nodulation program. In order to investigate the putative hierarchic role played by GCR1 in these symbiotic pathways we identified and characterized the Lotus japonicus gene encoding the seven transmembrane GCR1 protein. The detailed molecular and topological analyses of LjGCR1 expression patterns that are presented suggest a possible involvement in the early steps of nodule organogenesis. Furthermore, phenotypic analyses of independent transgenic RNAi lines, showing a significant LjGCR1 expression down regulation, suggest an epistatic action in the control of molecular markers of nodulation pathways, although no macroscopic symbiotic phenotypes could be revealed.

  11. Cropping history affects nodulation and symbiotic efficiency of distinct hairy vetch genotypes with resident soil rhizobia

    USDA-ARS?s Scientific Manuscript database

    Presence of compatible rhizobia strains is essential for nodulation and BNF of hairy vetch (Vicia villosa, HV). We evaluated how past HV cultivation affects nodulation and nitrogen fixation across host genotypes. Five groups of HV genotypes were inoculated with soil dilutions from six paired fields,...

  12. Proteome Analysis. Novel Proteins Identified at the Peribacteroid Membrane from Lotus japonicus Root Nodules1

    PubMed Central

    Wienkoop, Stefanie; Saalbach, Gerhard

    2003-01-01

    The peribacteroid membrane (PBM) forms the structural and functional interface between the legume plant and the rhizobia. The model legume Lotus japonicus was chosen to study the proteins present at the PBM by proteome analysis. PBM was purified from root nodules by an aqueous polymer two-phase system. Extracted proteins were subjected to a global trypsin digest. The peptides were separated by nanoscale liquid chromatography and analyzed by tandem mass spectrometry. Searching the nonredundant protein database and the green plant expressed sequence tag database using the tandem mass spectrometry data identified approximately 94 proteins, a number far exceeding the number of proteins reported for the PBM hitherto. In particular, a number of membrane proteins like transporters for sugars and sulfate; endomembrane-associated proteins such as GTP-binding proteins and vesicle receptors; and proteins involved in signaling, for example, receptor kinases, calmodulin, 14-3-3 proteins, and pathogen response-related proteins, including a so-called HIR protein, were detected. Several ATPases and aquaporins were present, indicating a more complex situation than previously thought. In addition, the unexpected presence of a number of proteins known to be located in other compartments was observed. Two characteristic protein complexes obtained from native gel electrophoresis of total PBM proteins were also analyzed. Together, the results identified specific proteins at the PBM involved in important physiological processes and localized proteins known from nodule-specific expressed sequence tag databases to the PBM. PMID:12644660

  13. The genetic and biochemical basis for nodulation of legumes by rhizobia

    SciTech Connect

    Pueppke, S.G.

    1996-05-01

    Soil bacteria of the genera Azorhizobium, Bradyrhizobium, and Rhizobium are collectively termed rhizobia. They share the ability to penetrate legume roots and elicit morphological responses that lead to the appearance of nodules. Bacteria within these symbiotic structures fix atmosphere nitrogen and thus are of immense ecological and agricultural significance. Although modern genetic analysis of rhizobia began less than 20 years ago, dozens of nodulation genes have now been identified, some in multiple species of rhizobia. These genetic advances have led to the discovery of a host surveillance system encoded by nodD and to the identification of Nod factor signals. These derivatives of oligochitin are synthesized by the protein products of nodABC, nodFE, NodPQ, and other nodulation genes: they provoke symbiotic responses on the part of the host and have generated immense interest in recent years. The symbiotic functions of other nodulation genes are nonetheless uncertain, and there remain significant gaps in the knowledge of several large groups of rhizobia with interesting biological properties. This review focuses on the nodulation genes of rhizobia, with particular emphasis on the concept of biological specificity of symbiosis with legume host plants. 419 refs.

  14. Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

    PubMed Central

    Sánchez, Maximo; Ramírez-Bahena, Martha-Helena; Peix, Alvaro; Lorite, María J.; Sanjuán, Juan; Monza, Jorge

    2014-01-01

    Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium. The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium, for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T). PMID:24271211

  15. Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus.

    PubMed

    Sánchez, Maximo; Ramírez-Bahena, Martha-Helena; Peix, Alvaro; Lorite, María J; Sanjuán, Juan; Velázquez, Encarna; Monza, Jorge

    2014-03-01

    Strain S658(T) was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium. The closest related species was, in both cases, Phyllobacterium trifolii PETP02(T) with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658(T) differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA-DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium, for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658(T)( = LMG 27289(T) = CECT 8230(T)).

  16. The REL3-mediated TAS3 ta-siRNA pathway integrates auxin and ethylene signaling to regulate nodulation in Lotus japonicus.

    PubMed

    Li, Xiaolin; Lei, Mingjuan; Yan, Zhongyuan; Wang, Qi; Chen, Aimin; Sun, Jie; Luo, Da; Wang, Yanzhang

    2014-01-01

    The ta-siRNA pathway is required for lateral organ development, including leaf patterning, flower differentiation and lateral root growth. Legumes can develop novel lateral root organs--nodules--resulting from symbiotic interactions with rhizobia. However, ta-siRNA regulation in nodule formation remains unknown. To explore ta-siRNA regulation in nodule formation, we investigated the roles of REL3, a key component of TAS3 ta-siRNA biogenesis, during nodulation in Lotus japonicus. We characterized the symbiotic phenotypes of the TAS3 ta-siRNA defective rel3 mutant, and analyzed the responses of the rel3 mutant to auxin and ethylene in order to gain insight into TAS3 ta-siRNA regulation of nodulation. The rel3 mutant produced fewer pink nitrogen-fixing nodules, with substantially decreased infection frequency and nodule initiation. Moreover, the rel3 mutant was more resistant than wild-type to 1-naphthaleneacetic acid (NAA) and N-1-naphthylphthalamic acid (NPA) in root growth, and exhibited insensitivity to auxins but greater sensitivity to auxin transport inhibitors during nodulation. Furthermore, the rel3 mutant has enhanced root-specific ethylene sensitivity and altered responses to ethylene during nodulation; the low-nodulating phenotype of the rel3 mutant can be restored by ethylene synthesis inhibitor L-α-(2-aminoethoxyvinyl)-glycine (AVG) or action inhibitor Ag(+). The REL3-mediated TAS3 ta-siRNA pathway regulates nodulation by integrating ethylene and auxin signaling.

  17. Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells.

    PubMed

    Abreu, Isidro; Saéz, Ángela; Castro-Rodríguez, Rosario; Escudero, Viviana; Rodríguez-Haas, Benjamín; Senovilla, Marta; Larue, Camille; Grolimund, Daniel; Tejada-Jiménez, Manuel; Imperial, Juan; González-Guerrero, Manuel

    2017-07-21

    Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is a M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone. This article is protected by copyright. All rights reserved.

  18. Growth and Survival of Mesorhizobium loti Inside Acanthamoeba Enhanced Its Ability to Develop More Nodules on Lotus corniculatus.

    PubMed

    Karaś, Magdalena A; Turska-Szewczuk, Anna; Trapska, Dominika; Urbanik-Sypniewska, Teresa

    2015-08-01

    The importance of protozoa as environmental reservoirs of pathogens is well recognized, while their impact on survival and symbiotic properties of rhizobia has not been explored. The possible survival of free-living rhizobia inside amoebae could influence bacterial abundance in the rhizosphere of legume plants and the nodulation competitiveness of microsymbionts. Two well-characterized strains of Mesorhizobium: Mesorhizobium loti NZP2213 and Mesorhizobium huakuii symbiovar loti MAFF303099 were assayed for their growth ability within the Neff strain of Acanthamoeba castellanii. Although the association ability and the initial uptake rate of both strains were similar, recovery of viable M. huakuii MAFF303099 after 4 h postinfection decreased markedly and that of M. loti NZP2213 increased. The latter strain was also able to survive prolonged co-incubation within amoebae and to self-release from the amoeba cell. The temperature 28 °C and PBS were established as optimal for the uptake of Mesorhizobium by amoebae. The internalization of mesorhizobia was mediated by the mannose-dependent receptor. M. loti NZP2213 bacteria released from amoebae developed 1.5 times more nodules on Lotus corniculatus than bacteria cultivated in an amoebae-free medium.

  19. Symbiotic Efficiency of Native Rhizobia Nodulating Common Bean (Phaseolus vulgaris L.) in Soils of Western Kenya

    PubMed Central

    Kawaka, Fanuel; Dida, Mathews M.; Opala, Peter A.; Ombori, Omwoyo; Maingi, John; Osoro, Newton; Muthini, Morris; Amoding, Alice; Mukaminega, Dative; Muoma, John

    2014-01-01

    This study was conducted to determine the abundance and symbiotic efficiency of native rhizobia nodulating common bean in Kisumu and Kakamega, Kenya. Soil sampling was carried out in three farms that had been used for growing common bean for at least two seasons and one fallow land with no known history of growing common bean or inoculation. Abundance of soil rhizobia and symbiotic efficiency (SE) were determined in a greenhouse experiment. Native rhizobia populations ranged from 3.2 × 101 to 3.5 × 104 cells per gram of soil. Pure bacterial cultures isolated from fresh and healthy root nodules exhibited typical characteristics of Rhizobium sp. on yeast extract mannitol agar media supplemented with Congo red. Bean inoculation with the isolates significantly (p < 0.05) increased the shoot dry weight and nitrogen (N) concentration and content. The SE of all the native rhizobia were higher when compared to a reference strain, CIAT 899 (67%), and ranged from 74% to 170%. Four isolates had SE above a second reference strain, Strain 446 (110%). Our results demonstrate the presence of native rhizobia that are potentially superior to the commercial inoculants. These can be exploited to enhance bean inoculation programmes in the region. PMID:27355005

  20. Symbiotic Efficiency of Native Rhizobia Nodulating Common Bean (Phaseolus vulgaris L.) in Soils of Western Kenya.

    PubMed

    Kawaka, Fanuel; Dida, Mathews M; Opala, Peter A; Ombori, Omwoyo; Maingi, John; Osoro, Newton; Muthini, Morris; Amoding, Alice; Mukaminega, Dative; Muoma, John

    2014-01-01

    This study was conducted to determine the abundance and symbiotic efficiency of native rhizobia nodulating common bean in Kisumu and Kakamega, Kenya. Soil sampling was carried out in three farms that had been used for growing common bean for at least two seasons and one fallow land with no known history of growing common bean or inoculation. Abundance of soil rhizobia and symbiotic efficiency (SE) were determined in a greenhouse experiment. Native rhizobia populations ranged from 3.2 × 10(1) to 3.5 × 10(4) cells per gram of soil. Pure bacterial cultures isolated from fresh and healthy root nodules exhibited typical characteristics of Rhizobium sp. on yeast extract mannitol agar media supplemented with Congo red. Bean inoculation with the isolates significantly (p < 0.05) increased the shoot dry weight and nitrogen (N) concentration and content. The SE of all the native rhizobia were higher when compared to a reference strain, CIAT 899 (67%), and ranged from 74% to 170%. Four isolates had SE above a second reference strain, Strain 446 (110%). Our results demonstrate the presence of native rhizobia that are potentially superior to the commercial inoculants. These can be exploited to enhance bean inoculation programmes in the region.

  1. Competition between rhizobia under different environmental conditions affects the nodulation of a legume.

    PubMed

    Ji, Zhao Jun; Yan, Hui; Cui, Qing Guo; Wang, En Tao; Chen, Wen Feng; Chen, Wen Xin

    2017-03-01

    Mutualistic symbiosis and nitrogen fixation of legume rhizobia play a key role in ecological environments. Although many different rhizobial species can form nodules with a specific legume, there is often a dominant microsymbiont, which has the highest nodule occupancy rates, and they are often known as the "most favorable rhizobia". Shifts in the most favorable rhizobia for a legume in different geographical regions or soil types are not well understood. Therefore, in order to explore the shift model, an experiment was designed using successive inoculations of rhizobia on one legume. The plants were grown in either sterile vermiculite or a sandy soil. Results showed that, depending on the environment, a legume could select its preferential rhizobial partner in order to establish symbiosis. For perennial legumes, nodulation is a continuous and sequential process. In this study, when the most favorable rhizobial strain was available to infect the plant first, it was dominant in the nodules, regardless of the existence of other rhizobial strains in the rhizosphere. Other rhizobial strains had an opportunity to establish symbiosis with the plant when the most favorable rhizobial strain was not present in the rhizosphere. Nodule occupancy rates of the most favorable rhizobial strain depended on the competitiveness of other rhizobial strains in the rhizosphere and the environmental adaptability of the favorable rhizobial strain (in this case, to mild vermiculite or hostile sandy soil). To produce high nodulation and efficient nitrogen fixation, the most favorable rhizobial strain should be selected and inoculated into the rhizosphere of legume plants under optimum environmental conditions. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. Expression of a complete soybean leghemoglobin gene in root nodules of transgenic Lotus corniculatus.

    PubMed

    Stougaard, J; Petersen, T E; Marcker, K A

    1987-08-01

    The complete soybean leghemoglobin lbc(3) gene was transferred into the legume Lotus corniculatus using an Agrobacterium rhizogenes vector system. Organ-specific expression of the soybean gene was observed in root nodules formed on regenerated transgenic plants after infection with Rhizobium loti. The primary transcript was processed in the same way as in soybean nodules and the resulting mRNA was translated into Lbc(3) protein. Quantitative determination of the Lbc(3) protein in nodules of transgenic plants indicated that the steady-state level of the soybean protein is comparable to that of endogenous Lotus leghemoglobin.

  3. Mesorhizobium helmanticense sp. nov., isolated from Lotus corniculatus nodules.

    PubMed

    Marcos-García, Marta; Menéndez, Esther; Ramírez-Bahena, Marta Helena; Mateos, Pedro F; Peix, Álvaro; Velazquez, Encarna; Rivas, Raúl

    2017-07-01

    In this study, three strains belonging to the genus Mesorhizobium, CSLC115NT, CSLC19N and CSLC37N, isolated from Lotus corniculatus nodules in Spain, were characterized. Their 16S rRNA gene sequences were closely related to those of Mesorhizobium metallidurans STM 2683T, Mesorhizobium tianshanense A-1BST, Mesorhizobium tarimense CCBAU 83306T, Mesorhizobium gobiense CCBAU 83330T and Mesorhizobium caraganae CCBAU 11299T with similarity values higher than 99.7 %. The analysis of concatenated recA and glnII genes showed that the most closely related type strains were M. metallidurans STM 2683T, M. tianshanense A-1BST and M. tarimense CCBAU 83306T with 96, 95 and 94 % similarity values in the recA gene and 95, 94 and 94 % in the glnII gene, respectively. M. metallidurans LMG 24485T, M. tianshanense USDA 3592T and M. tarimense LMG 24338T showed means of 44, 41 and 42 % DNA-DNA relatedness, respectively, with respect to strain CSLC115NT. The major fatty acids were those from summed feature 8 (C18 : 1ω7c/C18 : 1ω6c), C16 : 0 and C18 : 1ω7c 11-methyl. The results of phenotypic characterization support that the L. corniculatus nodulating strains analysed in this work belong to a novel species of the genus Mesorhizobium for which the name Mesorhizobium helmanticense sp. nov. is proposed, and the type strain is CSLC115NT (= LMG 29734T=CECT 9168T).

  4. Osmoadaptative responses in the rhizobia nodulating Acacia isolated from south-eastern Moroccan Sahara.

    PubMed

    Essendoubi, Mohammed; Brhada, Fatiha; Eljamali, Jamal Eddine; Filali-Maltouf, Abdelkarim; Bonnassie, Sylvie; Georgeault, Sylvie; Blanco, Carlos; Jebbar, Mohamed

    2007-03-01

    Four strains of rhizobia nodulating Acacia were isolated from the Moroccan desert soil by trapping with seedlings of Acacia gummifera and Acacia raddiana, and were studied for their ability to tolerate high salinity and dryness conditions. The strains MDSMC 2, MDSMC 18 and MDSMC 50 were halotolerant (they tolerated up to 1 M NaCl) and they accumulated glutamate and mannosucrose. The synthesis of the latter solute, which is the major endogenous osmolyte, is partially repressed in the presence of glycine betaine. The strain MDSMC 34 was less halotolerant (growth inhibited by a concentration greater than 0.5 M NaCl), and accumulated trehalose (as the main endogenous osmolyte) and glutamate. Rhizobia from the Moroccan desert soil were highly resistant to desiccation and their tolerance to dryness was stimulated by osmotic pretreatment. Thus, the accumulation of mannosucrose or trehalose by desert rhizobia represents both an osmoadaptative response and a part of a desiccation tolerance mechanism.

  5. Mesorhizobium olivaresii sp. nov. isolated from Lotus corniculatus nodules.

    PubMed

    Lorite, María J; Flores-Félix, José David; Peix, Álvaro; Sanjuán, Juan; Velázquez, Encarna

    2016-12-01

    In this study four Mesorhizobium strains isolated from Lotus corniculatus nodules in Granada (Spain) were characterized. Their 16S rRNA gene sequences were closely related to those of M. albiziae LMG 23507(T) and M. chacoense Pr5(T) showing 99.4 and 99.2% similarity values, respectively. The analysis of concatenated rpoB, recA, atpD and glnII genes showed they formed a cluster with internal similarities higher than 97%. The closest species also were M. albiziae LMG 23507(T) and M. chacoense Pr5(T) showing similarity values lower than 92% in rpoB, recA and glnII genes and lower than 96.5% in the atpD gene. These results indicated that the L. corniculatus strains belong to a new species of genus Mesorhizobium which was confirmed by DNA-DNA hybridization and phenotypic characterization. Therefore a new species with the name Mesorhizobium olivaresii sp. nov. is proposed, and the type strain is CPS13(T) (LMG 29295(T)=CECT 9099(T)). Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr).

    PubMed

    Saldaña, Gustavo; Martinez-Alcántara, Virginia; Vinardell, José M; Bellogín, Ramón; Ruíz-Sainz, José E; Balatti, Pedro Alberto

    2003-07-01

    The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD- nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.

  7. Characterization and diversity of rhizobia nodulating selected tree legumes in Ghana.

    PubMed

    Boakye, Emmanuel Yaw; Lawson, Innocent Yao Dotse; Danso, Seth Kofi Akyea; Offei, Samuel Kwame

    The study was conducted to assess the characteristics and diversity of the rhizobia that nodulate some prominent tree legumes in three soils of Ghana. Five introduced and/or indigenous tree legumes were initially assessed for nodulation in three Ghanaian soils. After 12 weeks of growth in nursery pots the 200 rhizobial strains isolated from their nodules were characterized culturally, metabolically and phenotypically. Sixty of these isolates were selected randomly and their genotypic characteristics determined using PCR-RFLP of 16S rRNA and intergenic spacer (ITS) genes. Each tree legume was nodulated by isolates classified as fast or very fast-growers or by isolates classified as slow- or very slow-growers with 54 % of all the 200 isolates belonging to fast- or very fast-growers. Morphologically, eighty five percent of the colonies formed on yeast extract mannitol agar were wet and gummy while 70 % were acid tolerant, i.e. they were able to grow at a pH of 3.5. Combined restriction of the 16S rRNA genes of the 60 rhizobial isolates with five restriction enzymes clearly distinguished seven different clusters at 80 % similarity level. The majority of A. lebbeck isolates were distinct from those of the Acacias and L. leucocephala. The M. thonningii isolates were related to L. leucocephala isolates. Simple PCR of the ITS DNA provided several distinct band sizes indicating great variation among the isolates and restriction of the ITS with three different enzymes did not yield many further differences. Molecular techniques revealed a great diversity among the rhizobia that nodulate tree legumes in the tropics and this may explain why many introduced and/or indigenous trees are able to form nodules with indigenous rhizobia in this region.

  8. Nitrogen-Fixing Nodules Are an Important Source of Reduced Sulfur, Which Triggers Global Changes in Sulfur Metabolism in Lotus japonicus.

    PubMed

    Kalloniati, Chrysanthi; Krompas, Panagiotis; Karalias, Georgios; Udvardi, Michael K; Rennenberg, Heinz; Herschbach, Cornelia; Flemetakis, Emmanouil

    2015-09-01

    We combined transcriptomic and biochemical approaches to study rhizobial and plant sulfur (S) metabolism in nitrogen (N) fixing nodules (Fix(+)) of Lotus japonicus, as well as the link of S-metabolism to symbiotic nitrogen fixation and the effect of nodules on whole-plant S-partitioning and metabolism. Our data reveal that N-fixing nodules are thiol-rich organs. Their high adenosine 5'-phosphosulfate reductase activity and strong (35)S-flux into cysteine and its metabolites, in combination with the transcriptional upregulation of several rhizobial and plant genes involved in S-assimilation, highlight the function of nodules as an important site of S-assimilation. The higher thiol content observed in nonsymbiotic organs of N-fixing plants in comparison to uninoculated plants could not be attributed to local biosynthesis, indicating that nodules are an important source of reduced S for the plant, which triggers whole-plant reprogramming of S-metabolism. Enhanced thiol biosynthesis in nodules and their impact on the whole-plant S-economy are dampened in plants nodulated by Fix(-) mutant rhizobia, which in most respects metabolically resemble uninoculated plants, indicating a strong interdependency between N-fixation and S-assimilation. © 2015 American Society of Plant Biologists. All rights reserved.

  9. Nitrogen-Fixing Nodules Are an Important Source of Reduced Sulfur, Which Triggers Global Changes in Sulfur Metabolism in Lotus japonicus

    PubMed Central

    Kalloniati, Chrysanthi; Krompas, Panagiotis; Udvardi, Michael K.; Flemetakis, Emmanouil

    2015-01-01

    We combined transcriptomic and biochemical approaches to study rhizobial and plant sulfur (S) metabolism in nitrogen (N) fixing nodules (Fix+) of Lotus japonicus, as well as the link of S-metabolism to symbiotic nitrogen fixation and the effect of nodules on whole-plant S-partitioning and metabolism. Our data reveal that N-fixing nodules are thiol-rich organs. Their high adenosine 5′-phosphosulfate reductase activity and strong 35S-flux into cysteine and its metabolites, in combination with the transcriptional upregulation of several rhizobial and plant genes involved in S-assimilation, highlight the function of nodules as an important site of S-assimilation. The higher thiol content observed in nonsymbiotic organs of N-fixing plants in comparison to uninoculated plants could not be attributed to local biosynthesis, indicating that nodules are an important source of reduced S for the plant, which triggers whole-plant reprogramming of S-metabolism. Enhanced thiol biosynthesis in nodules and their impact on the whole-plant S-economy are dampened in plants nodulated by Fix− mutant rhizobia, which in most respects metabolically resemble uninoculated plants, indicating a strong interdependency between N-fixation and S-assimilation. PMID:26296963

  10. Genotypic alteration and competitive nodulation of Mesorhizobium muleiense against exotic chickpea rhizobia in alkaline soils.

    PubMed

    Zhang, Jun Jie; Yu, Tao; Lou, Kai; Mao, Pei Hong; Wang, En Tao; Chen, Wen Feng; Chen, Wen Xin

    2014-10-01

    Mesorhizobium muleiense, Mesorhizobium mediterraneum and Mesorhizobium ciceri are chickpea (Cicer arietinum L.) rhizobia that share a high similarity of the symbiotic genes nodC and nifH, but they have different geographic distributions. M. muleiense has been isolated and found only in alkaline soils of Xinjiang, China, whereas the other two strains have been found in the Mediterranean and India. To investigate the species stability of M. muleiense during natural evolution and its capability of competitive nodulation against the other two exotic species, re-sampling of nodules in the field and competition experiments between the three species were conducted. The results showed that the predominant microsymbiont associated with chickpea grown in Xinjiang was still M. muleiense, but the predominant genotypes of M. muleiense had changed significantly during the four years since a previous survey. The data also showed that M. mediterraneum and M. ciceri were more competitive than the residential strain of M. muleiense CCBAU 83963(T) in sterilized vermiculite or soils from Xinjiang. However, in non-sterilized soils, M. muleiense was the predominant nodule occupier. These results indicated that natural or adapting evolution of M. muleiense was occurring in fields subjected to changing environmental factors. In addition, the biogeography and symbiotic associations of rhizobia with their host legumes were also influenced by biological factors in the soil, such as indigenous rhizobia and other organisms. Copyright © 2014 Elsevier GmbH. All rights reserved.

  11. Rhizobial gibberellin negatively regulates host nodule number

    PubMed Central

    Tatsukami, Yohei; Ueda, Mitsuyoshi

    2016-01-01

    In legume–rhizobia symbiosis, the nodule number is controlled to ensure optimal growth of the host. In Lotus japonicus, the nodule number has been considered to be tightly regulated by host-derived phytohormones and glycopeptides. However, we have discovered a symbiont-derived phytohormonal regulation of nodule number in Mesorhizobium loti. In this study, we found that M. loti synthesized gibberellic acid (GA) under symbiosis. Hosts inoculated with a GA-synthesis-deficient M. loti mutant formed more nodules than those inoculated with the wild-type form at four weeks post inoculation, indicating that GA from already-incorporated rhizobia prevents new nodule formation. Interestingly, the genes for GA synthesis are only found in rhizobial species that inhabit determinate nodules. Our findings suggest that the already-incorporated rhizobia perform GA-associated negative regulation of nodule number to prevent delayed infection by other rhizobia. PMID:27307029

  12. Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations.

    PubMed

    Tian, Chang Fu; Zhou, Yuan Jie; Zhang, Yan Ming; Li, Qin Qin; Zhang, Yun Zeng; Li, Dong Fang; Wang, Shuang; Wang, Jun; Gilbert, Luz B; Li, Ying Rui; Chen, Wen Xin

    2012-05-29

    The rhizobium-legume symbiosis has been widely studied as the model of mutualistic evolution and the essential component of sustainable agriculture. Extensive genetic and recent genomic studies have led to the hypothesis that many distinct strategies, regardless of rhizobial phylogeny, contributed to the varied rhizobium-legume symbiosis. We sequenced 26 genomes of Sinorhizobium and Bradyrhizobium nodulating soybean to test this hypothesis. The Bradyrhizobium core genome is disproportionally enriched in lipid and secondary metabolism, whereas several gene clusters known to be involved in osmoprotection and adaptation to alkaline pH are specific to the Sinorhizobium core genome. These features are consistent with biogeographic patterns of these bacteria. Surprisingly, no genes are specifically shared by these soybean microsymbionts compared with other legume microsymbionts. On the other hand, phyletic patterns of 561 known symbiosis genes of rhizobia reflected the species phylogeny of these soybean microsymbionts and other rhizobia. Similar analyses with 887 known functional genes or the whole pan genome of rhizobia revealed that only the phyletic distribution of functional genes was consistent with the species tree of rhizobia. Further evolutionary genetics revealed that recombination dominated the evolution of core genome. Taken together, our results suggested that faithfully vertical genes were rare compared with those with history of recombination including lateral gene transfer, although rhizobial adaptations to symbiotic interactions and other environmental conditions extensively recruited lineage-specific shell genes under direct or indirect control through the speciation process.

  13. Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal.

    PubMed

    Sylla, Samba Ndao; Samba, Ramatoulaye Thiaba; Neyra, Marc; Ndoye, Ibrahima; Giraud, Eric; Willems, Anne; de Lajudie, Philippe; Dreyfus, Bernard

    2002-12-01

    A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.

  14. Diversity of Rhizobia Nodulating Phaseolus vulgaris L. in Two Kenyan Soils with Contrasting pHs

    PubMed Central

    Anyango, B.; Wilson, K. J.; Beynon, J. L.; Giller, K. E.

    1995-01-01

    Rhizobia were isolated from two Kenyan soils with pHs of 4.5 and 6.8 and characterized on the basis of their host ranges for nodulation and nitrogen fixation, colony morphologies, restriction fragment fingerprints, and hybridization with a nifH probe. The populations of rhizobia nodulating Phaseolus vulgaris in the two soils were similar in numbers and in effectiveness of N(inf2) fixation but were markedly different in composition. The population in the Naivasha soil (pH 6.8) was dominated by isolates specific in host range for nodulation to P. vulgaris; these all had multiple copies, in most cases four, of the structural nitrogenase gene nifH. Only one of the isolates from this soil formed effective nodules on Leucaena leucocephala, and this isolate had only a single copy of nifH. By contrast, the population in the acid Daka-ini soil (pH 4.5) was composed largely of broad-host-range isolates which had single copies of nifH. The isolates from the Daka-ini soil which were specific to P. vulgaris generally had three copies of nifH, although one isolate had only two copies. These rhizobial isolates are indigenous to Kenyan soils and yet have marked similarities to previously described Rhizobium species from other continents. PMID:16535165

  15. Lotus japonicus Clathrin Heavy Chain1 Is Associated with Rho-Like GTPase ROP6 and Involved in Nodule Formation1[OPEN

    PubMed Central

    Zhu, Maosheng; Duan, Liujiang; Yu, Haixiang; Chang, Xiaojun; Li, Li; Kang, Heng; Feng, Yong; Zhu, Hui; Hong, Zonglie

    2015-01-01

    Mechanisms underlying nodulation factor signaling downstream of the nodulation factor receptors (NFRs) have not been fully characterized. In this study, clathrin heavy chain1 (CHC1) was shown to interact with the Rho-Like GTPase ROP6, an interaction partner of NFR5 in Lotus japonicus. The CHC1 gene was found to be expressed constitutively in all plant tissues and induced in Mesorhizobium loti-infected root hairs and nodule primordia. When expressed in leaves of Nicotiana benthamiana, CHC1 and ROP6 were colocalized at the cell circumference and within cytoplasmic punctate structures. In M. loti-infected root hairs, the CHC protein was detected in cytoplasmic punctate structures near the infection pocket along the infection thread membrane and the plasma membrane of the host cells. Transgenic plants expressing the CHC1-Hub domain, a dominant negative effector of clathrin-mediated endocytosis, were found to suppress early nodulation gene expression and impair M. loti infection, resulting in reduced nodulation. Treatment with tyrphostin A23, an inhibitor of clathrin-mediated endocytosis of plasma membrane cargoes, had a similar effect on down-regulation of early nodulation genes. These findings show an important role of clathrin in the leguminous symbiosis with rhizobia. PMID:25717037

  16. Diversity and symbiotic effectiveness of indigenous rhizobia-nodulating Adesmia bicolor in soils of Central Argentina.

    PubMed

    Bianco, Luciana; Angelini, Jorge; Fabra, Adriana; Malpassi, Rosana

    2013-02-01

    Native perennial legume Adesmia bicolor reveals characteristics that are key to securing persistence under grazing. Literature on the diversity and symbiotic effectiveness of indigenous rhizobia-nodulating A. bicolor in central Argentina is limited. The purpose of this study was therefore to determine phenotypic and genotypic variability as well as biological N-fixation effectiveness in rhizobia isolated from A. bicolor nodules. To this end, repetitive genomic regions were analyzed using ERIC primers. In the greenhouse, plants were grown under a (i) N-fertilized treatment, (ii) N-free control treatment, and (iii) rhizobia inoculation treatment. Dry weight and N-content were analyzed. All isolates belonged to Rhizobium genus and showed high symbiotic effectiveness. The N-content/subterranean N-content ratio in aerial and subterranean parts of inoculated plants was higher than that observed in N-fertilized plants during the vegetative stage. Results from this study demonstrate that symbiosis between native rhizobial strains and A. bicolor is very effective.

  17. New aspect of plant–rhizobia interaction: Alkaloid biosynthesis in Crotalaria depends on nodulation

    PubMed Central

    Irmer, Simon; Podzun, Nora; Langel, Dorothee; Heidemann, Franziska; Kaltenegger, Elisabeth; Schemmerling, Brigitte; Geilfus, Christoph-Martin; Zörb, Christian; Ober, Dietrich

    2015-01-01

    Infection of legume hosts by rhizobial bacteria results in the formation of a specialized organ, the nodule, in which atmospheric nitrogen is reduced to ammonia. Nodulation requires the reprogramming of the plant cell, allowing the microsymbiont to enter the plant tissue in a highly controlled manner. We have found that, in Crotalaria (Fabaceae), this reprogramming is associated with the biosynthesis of pyrrolizidine alkaloids (PAs). These compounds are part of the plant’s chemical defense against herbivores and cannot be regarded as being functionally involved in the symbiosis. PAs in Crotalaria are detectable only when the plants form nodules after infection with their rhizobial partner. The identification of a plant-derived sequence encoding homospermidine synthase (HSS), the first pathway-specific enzyme of PA biosynthesis, suggests that the plant and not the microbiont is the producer of PAs. Transcripts of HSS are detectable exclusively in the nodules, the tissue with the highest concentration of PAs, indicating that PA biosynthesis is restricted to the nodules and that the nodules are the source from which the alkaloids are transported to the above ground parts of the plant. The link between nodulation and the biosynthesis of nitrogen-containing alkaloids in Crotalaria highlights a further facet of the effect of symbiosis with rhizobia on the ecologically important trait of the plant’s chemical defense. PMID:25775562

  18. Genetic diversity and community structure of rhizobia nodulating Sesbania cannabina in saline-alkaline soils.

    PubMed

    Li, Yan; Li, Xiangyue; Liu, Yajing; Wang, En Tao; Ren, Chenggang; Liu, Wei; Xu, Hualing; Wu, Hailong; Jiang, Nan; Li, Yunzhao; Zhang, Xiaoli; Xie, Zhihong

    2016-05-01

    Sesbania cannabina is a plant that grows naturally along the seashores in Rudong County, China (RDC) and it has been introduced into the Yellow River Delta (YRD) as a pioneer plant to improve the saline-alkaline soils. In order to investigate the diversity of S. cannabina rhizobia in these soils, a total of 198 rhizobial isolates were characterized and phylogenetic trees were constructed based on data from multilocus sequence analysis (MLSA) of the housekeeping genes recA, atpD and glnII, as well as 16S rRNA. Symbiotic features were also studied by establishing the phylogeny of the symbiotic genes nodA and nifH, and by performing nodulation assays. The isolates had highly conserved symbiotic genes and were classified into nine genospecies belonging to the genera Ensifer, Agrobacterium, Neorhizobium and Rhizobium. A unique community structure was detected in the rhizobia associated with S. cannabina in the saline-alkaline soils that was characterized by five novel genospecies and four defined species. In addition, Ensifer sp. I was the predominant rhizobia in YRD, whereas Ensifer meliloti and Neorhizobium huautlense were the dominant species in RDC. Therefore, the study demonstrated for the first time that this plant strongly selected the symbiotic gene background but not the genomic background of its microsymbionts. In addition, biogeographic patterns existed in the rhizobial populations associated with S. cannabina, which were mainly correlated with pH and salinity, as well as the mineral nutrient contents. This study provided novel information concerning the interaction between soil conditions, host plant and rhizobia, in addition to revealing the diversity of S. cannabina rhizobia in saline-alkaline soils.

  19. Diversity of rhizobia nodulating wild shrubs of Sicily and some neighbouring islands.

    PubMed

    Cardinale, Massimiliano; Lanza, Angela; Bonnì, Maria Laura; Marsala, Salvatore; Puglia, Anna Maria; Quatrini, Paola

    2008-10-01

    Legume shrubs have great potential for rehabilitation of semi-arid degraded soils in Mediterranean ecosystems as they establish mutualistic symbiosis with N-fixing rhizobia. Eighty-eight symbiotic rhizobia were isolated from seven wild legume shrubs native of Sicily (Southern Italy) and grouped in operational taxonomic units (OTU) by analysis of the ribosomal internal transcribed spacer (ITS) polymorphism. Partial sequencing of 16S rRNA gene of representative isolates of each OTU revealed that most Genisteae symbionts are related to Bradyrhizobium canariense, B. japonicum and B. elkanii. Teline monspessulana was the only Genistea nodulated by Mesorhizobium strains, and Anagyris foetida (Thermopsideae) was promiscuosly nodulated by Rhizobium, Mesorhizobium, Agrobacterium and Bradyrhizobium strains. Analysis of the nodulation gene nodA assigned most Mediterranean Genisteae bradyrhizobia to clade II but also to clades IV, I and III, which included, so far, sequences of (sub)tropical and Australian isolates. The high diversity and low host specificity observed in most wild legumes isolates suggest that preferential associations may establish in the field depending on differences in the benefits conferred to the host and on competition ability. Once identified, these beneficial symbiosis can be exploited for rehabilitation of arid, low productive and human-impacted soils of the Mediterranean countries.

  20. Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia.

    PubMed

    Delgadillo, Julián; Lafuente, Alejandro; Doukkali, Bouchra; Redondo-Gómez, Susana; Mateos-Naranjo, Enrique; Caviedes, Miguel A; Pajuelo, Eloísa; Rodríguez-Llorente, Ignacio D

    2015-01-01

    The rhizobia-legume interaction has been proposed as an interesting and appropriate tool for rhizostabilization of soils contaminated with heavy metals. One of the main requirements to use this symbiosis is the availability of tolerant and symbiotically effective rhizobia. The aim of this work was to improve the symbiotic properties of the arsenic-resistant wild-type strain Ensifer medicae MA11 in Cu-contaminated substrates. The copAB genes from a Cu-resistant Pseudomonas fluorescens strain were expressed in E. medicae MA11 under the control of the nifH promoter. The resulting strain E. medicae MA11-copAB was able to alleviate the toxic effect of Cu in Medicago truncatula. At 300 µM Cu, root and shoot dry matter production, nitrogen content, number of nodules and photosynthetic rate were significantly reduced in plants inoculated with the wild-type strain. However, these parameters were not altered in plants inoculated with the genetically modified strain. Moreover, nodules elicited by this strain were able to accumulate twofold the Cu measured in nodules formed by the wild-type strain. In addition, the engineered E. medicae strain increased Cu accumulation in roots and decreased the content in shoots. Thus, E. medicae MA11-copAB increased the capacity of M. truncatula to rhizostabilize Cu, decreasing the translocation factor and avoiding metal entry into the food chain. The plasmid containing the nifH promoter-copAB construct could be a useful biotool for Cu rhizostabilization using legumes, since it can be transferred to different rhizobia microsymbionts of authoctonous legumes growing in Cu-contaminated soils.

  1. Shoot HAR1 mediates nitrate inhibition of nodulation in Lotus japonicus.

    PubMed

    Okamoto, Satoru; Kawaguchi, Masayoshi

    2015-01-01

    Nitrate is a major environmental factor in the inhibition of nodulation. In a model legume Lotus japonicus, a CLV1-like receptor kinase, HAR1, mediates nitrate inhibition and autoregulation of nodulation. Autoregulation of nodulation involves root-to-shoot-to-root long-distance communication, and HAR1 functions in shoots. However, it remains elusive where HAR1 functions in the nitrate inhibition of nodulation. We performed grafting experiments with the har1 mutant under various nitrate conditions, and found that shoot HAR1 is critical for the inhibition of nodulation at 10 mM nitrate. Combined with our recent finding that the nitrate-induced CLE-RS2 glycopeptide binds directly to the HAR1 receptor, this result suggests that CLE-RS2/HAR1 long-distance signaling plays an important role in the both nitrate inhibition and the autoregulation of nodulation.

  2. NODULE INCEPTION Directly Targets NF-Y Subunit Genes to Regulate Essential Processes of Root Nodule Development in Lotus japonicus

    PubMed Central

    Soyano, Takashi; Kouchi, Hiroshi; Hirota, Atsuko; Hayashi, Makoto

    2013-01-01

    The interactions of legumes with symbiotic nitrogen-fixing bacteria cause the formation of specialized lateral root organs called root nodules. It has been postulated that this root nodule symbiosis system has recruited factors that act in early signaling pathways (common SYM genes) partly from the ancestral mycorrhizal symbiosis. However, the origins of factors needed for root nodule organogenesis are largely unknown. NODULE INCEPTION (NIN) is a nodulation-specific gene that encodes a putative transcription factor and acts downstream of the common SYM genes. Here, we identified two Nuclear Factor-Y (NF-Y) subunit genes, LjNF-YA1 and LjNF-YB1, as transcriptional targets of NIN in Lotus japonicus. These genes are expressed in root nodule primordia and their translational products interact in plant cells, indicating that they form an NF-Y complex in root nodule primordia. The knockdown of LjNF-YA1 inhibited root nodule organogenesis, as did the loss of function of NIN. Furthermore, we found that NIN overexpression induced root nodule primordium-like structures that originated from cortical cells in the absence of bacterial symbionts. Thus, NIN is a crucial factor responsible for initiating nodulation-specific symbiotic processes. In addition, ectopic expression of either NIN or the NF-Y subunit genes caused abnormal cell division during lateral root development. This indicated that the Lotus NF-Y subunits can function to stimulate cell division. Thus, transcriptional regulation by NIN, including the activation of the NF-Y subunit genes, induces cortical cell division, which is an initial step in root nodule organogenesis. Unlike the legume-specific NIN protein, NF-Y is a major CCAAT box binding protein complex that is widespread among eukaryotes. We propose that the evolution of root nodules in legume plants was associated with changes in the function of NIN. NIN has acquired functions that allow it to divert pathways involved in the regulation of cell division to

  3. Phenotypic and genotypic characterization of rhizobia from diverse geographical origin that nodulate Pachyrhizus species.

    PubMed

    Rodríguez-Navarro, Dulce N; Camacho, María; Leidi, Eduardo O; Rivas, Raúl; Velázquez, Encarna

    2004-11-01

    Legumes from the genus Pachyrhizus, commonly known as yam bean, are cultivated in several countries from the American continent and constitute an alternative source for sustainable starch, oil and protein production. The endosymbionts of these legumes have been poorly studied although it is known that this legume is nodulated by fast and slow growing rhizobia. In this study we have analyzed a collection of strains isolated in several countries using different phenotypic and molecular methods. The results obtained by SDS-PAGE analysis, LPS profiling and TP-RAPD fingerprinting showed the high diversity of the strains analyzed, although all of them presented slow growth in yeast mannitol agar (YMA) medium. These results were confirmed using 16S-23S internal transcribed spacer (ITS) region and complete sequencing of the 16S rRNA gene, showing that most strains analyzed belong to different species of genus Bradyrhizobium. Three strains were closely related to B. elkanii and the rest of the strains were related to the phylogenetic group constituted by B. japonicum, B. liaoningense, B. yuanmingense and B. betae. These results support that the study of rhizobia nodulating unexplored legumes in different geographical locations will allow the discovery of new species able to establish legume symbioses.

  4. Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[W][OPEN

    PubMed Central

    Held, Mark; Hou, Hongwei; Miri, Mandana; Huynh, Christian; Ross, Loretta; Hossain, Md Shakhawat; Sato, Shusei; Tabata, Satoshi; Perry, Jillian; Wang, Trevor L.; Szczyglowski, Krzysztof

    2014-01-01

    Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1. PMID:24585837

  5. Application of Multilocus Sequence Typing To Study the Genetic Structure of Megaplasmids in Medicago-Nodulating Rhizobia

    USDA-ARS?s Scientific Manuscript database

    A multilocus sequence typing (MLST) analysis was used to examine the relatedness and distribution of genotypic variants of the two large extrachromosomal replicons in Medicago-nodulating rhizobia (Sinorhizobium meliloti and S. medicae). One goal was to develop a strategy for the characterization of...

  6. Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling

    PubMed Central

    Suzuki, Akihiro; Suriyagoda, Lalith; Shigeyama, Tamaki; Tominaga, Akiyoshi; Sasaki, Masayo; Hiratsuka, Yoshimi; Yoshinaga, Aya; Arima, Susumu; Agarie, Sakae; Sakai, Tatsuya; Inada, Sayaka; Jikumaru, Yusuke; Kamiya, Yuji; Uchiumi, Toshiki; Abe, Mikiko; Hashiguchi, Masatsugu; Akashi, Ryo; Sato, Shusei; Kaneko, Takakazu; Tabata, Satoshi; Hirsch, Ann M.

    2011-01-01

    Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling. PMID:21930895

  7. Isolation and Characterization of Alfalfa-Nodulating Rhizobia Present in Acidic Soils of Central Argentina and Uruguay

    PubMed Central

    del Papa, María F.; Balagué, Laura J.; Sowinski, Susana Castro; Wegener, Caren; Segundo, Eduardo; Abarca, Francisco Martínez; Toro, Nicolás; Niehaus, Karsten; Pühler, Alfred; Aguilar, O. Mario; Martínez-Drets, Gloria; Lagares, Antonio

    1999-01-01

    We describe the isolation and characterization of alfalfa-nodulating rhizobia from acid soils of different locations in Central Argentina and Uruguay. A collection of 465 isolates was assembled, and the rhizobia were characterized for acid tolerance. Growth tests revealed the existence of 15 acid-tolerant (AT) isolates which were able to grow at pH 5.0 and formed nodules in alfalfa with a low rate of nitrogen fixation. Analysis of those isolates, including partial sequencing of the genes encoding 16S rRNA and genomic PCR-fingerprinting with MBOREP1 and BOXC1 primers, demonstrated that the new isolates share a genetic background closely related to that of the previously reported Rhizobium sp. Or191 recovered from an acid soil in Oregon (B. D. Eardly, J. P. Young, and R. K. Selander, Appl. Environ. Microbiol. 58:1809–1815, 1992). Growth curves, melanin production, temperature tolerance, and megaplasmid profiles of the AT isolates were all coincident with these characteristics in strain Or191. In addition to the ability of all of these strains to nodulate alfalfa (Medicago sativa) inefficiently, the AT isolates also nodulated the common bean and Leucaena leucocephala, showing an extended host range for nodulation of legumes. In alfalfa, the time course of nodule formation by the AT isolate LPU 83 showed a continued nodulation restricted to the emerging secondary roots, which was probably related to the low rate of nitrogen fixation by the largely ineffective nodules. Results demonstrate the complexity of the rhizobial populations present in the acidic soils represented by a main group of N2-fixing rhizobia and a second group of ineffective and less-predominant isolates related to the AT strain Or191. PMID:10103231

  8. Lotus corniculatus nodulation specificity is changed by the presence of a soybean lectin gene

    PubMed

    van Rhijn P; Goldberg; Hirsch

    1998-08-01

    Plant lectins have been implicated as playing an important role in mediating recognition and specificity in the Rhizobium-legume nitrogen-fixing symbiosis. To test this hypothesis, we introduced the soybean lectin gene Le1 either behind its own promoter or behind the cauliflower mosaic virus 35S promoter into Lotus corniculatus, which is nodulated by R. loti. We found that nodulelike outgrowths developed on transgenic L. corniculatus plant roots in response to Bradyrhizobium japonicum, which nodulates soybean and not Lotus spp. Soybean lectin was properly targeted to L. corniculatus root hairs, and although infection threads formed, they aborted in epidermal or hypodermal cells. Mutation of the lectin sugar binding site abolished infection thread formation and nodulation. Incubation of bradyrhizobia in the nodulation (nod) gene-inducing flavonoid genistein increased the number of nodulelike outgrowths on transgenic L. corniculatus roots. Studies of bacterial mutants, however, suggest that a component of the exopolysaccharide surface of B. japonicum, rather than Nod factor, is required for extension of host range to the transgenic L. corniculatus plants.

  9. Nodule Ultrastructure and Initial Growth of Anadenanthera peregrina (L.) Speg. var. falcata (Benth.) Altschul Plants Infected with Rhizobia

    PubMed Central

    GROSS, E.; CORDEIRO, L.; CAETANO, F. H.

    2002-01-01

    The anatomy and ultrastructure of root nodules of Anadenanthera peregrina var. falcata (Leguminosae‐Mimosoideae) were analysed, as was plant growth. To ensure that nodules developed, seedlings were inoculated with a mixture of six strains of rhizobia. Nodules were produced that differed in appearance—and probably also effectiveness—but their structure was similar and they showed characteristics typical of indeterminate nodules, such as persistent meristematic tissue and a gradient of cells at different stages of development. Many starch grains were present in inner cortex cells and interstitial cells of infected tissue. Infected cells were densely packed with bacteroids, which contained many poly‐β‐hydroxybutyrate granules. The high incidence of these granules, together with high levels of starch accumulation in interstitial cells, suggested low N2‐fixation efficiency of the rhizobia isolates used for inoculation. In the symbiosomes of early‐senescent infected cells, reticulum‐like structures, small vesicles and a fibrillar material were observed; these may be related to bacteroid degradation. In the cytoplasm of late‐senescent infected cells, many vesicles and membrane‐like structures were observed, probably associated with membrane degradation of bacteroids and peribacteroids. The total biomass of plants inoculated with rhizobia was low and their xylopodia and shoots had low levels of N compared with non‐inoculated plants fertilized with ammonium nitrate. However, inoculated plants did not show N‐deficiency symptoms and grew better than non‐inoculated plants without N fertilization. These growth results, together with ultrastructural observations of nodules, suggest that nitrogen fixation of rhizobia isolates associated with Anadenanthera peregrina var. falcata roots is poor. PMID:12197515

  10. Lentil (Lens culinaris Medik.) nodulates with genotypically and phenotypically diverse rhizobia in Ethiopian soils.

    PubMed

    Tena, Wondwosen; Wolde-Meskel, Endalkachew; Degefu, Tulu; Walley, Fran

    2017-01-01

    Forty-eight lentil-nodulating rhizobia were isolated from soil samples collected from diverse agro-ecological locations in Ethiopia, and characterized based on 76 phenotypic traits. Furthermore, 26 representative strains were selected and characterized using multilocus sequence analyses (MLSA) of core (16S rRNA, recA, atpD, glnII and gyrB) and symbiotic (nodA and nifH) genes. Numerical analysis of phenotypic characteristics showed that the 48 test strains fell into three major distinct clusters. The phylogenetic trees based on 16S rRNA genes showed that they belong to the Rhizobium genus. Our phylogenetic reconstruction based on combined gene trees (recA, atpD and glnII) supported three distinct sub-lineages (Clades I-III). While genospecies I and II could be classified with Rhizobium etli and Rhizobium leguminosarum, respectively, genospecies III, might be an unnamed genospecies within the genus Rhizobium. Phylogenetic reconstruction based on the symbiosis-related genes supported a single cluster, indicating differences in the evolutionary histories between chromosomal and symbiotic genes. Overall, these results confirmed the presence of a great diversity of lentil-nodulating Rhizobium species in Ethiopia, inviting further exploration. Moreover, the differences in symbiotic effectiveness of the test strains indicated the potential for selecting and using them as inoculants to improve the productivity of lentil in the country. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Phylogenetic multilocus sequence analysis of native rhizobia nodulating faba bean (Vicia faba L.) in Egypt.

    PubMed

    Youseif, Sameh H; Abd El-Megeed, Fayrouz H; Ageez, Amr; Cocking, Edward C; Saleh, Saleh A

    2014-12-01

    The taxonomic diversity of forty-two Rhizobium strains, isolated from nodules of faba bean grown in Egypt, was studied using 16S rRNA sequencing, multilocus sequence analyses (MLSA) of three chromosomal housekeeping loci and one nodulation gene (nodA). Based on the 16S rRNA gene sequences, most of the strains were related to Rhizobium leguminosarum, Rhizobium etli, and Rhizobium radiobacter (syn. Agrobacterium tumefaciens). A maximum likelihood (ML) tree built from the concatenated sequences of housekeeping proteins encoded by glnA, gyrB and recA, revealed the existence of three distinct genospecies (I, II and III) affiliated to the defined species within the genus Rhizobium/Agrobacterium. Seventeen strains in genospecies I could be classified as R. leguminosarum sv. viciae. Whereas, a single strain of genospecies II was linked to R. etli. Interestingly, twenty-four strains of genospecies III were identified as A. tumefaciens. Strains of R. etli and A. tumefaciens have been shown to harbor the nodA gene and formed effective symbioses with faba bean plants in Leonard jar assemblies. In the nodA tree, strains belonging to the putative genospecies were closely related to each other and were clustered tightly to R. leguminosarum sv. viciae, supporting the hypothesis that symbiotic and core genome of the species have different evolutionary histories and indicative of horizontal gene transfer among these rhizobia.

  12. Leguminous plants: inventors of root nodules to accommodate symbiotic bacteria.

    PubMed

    Suzaki, Takuya; Yoro, Emiko; Kawaguchi, Masayoshi

    2015-01-01

    Legumes and a few other plant species can establish a symbiotic relationship with nitrogen-fixing rhizobia, which enables them to survive in a nitrogen-deficient environment. During the course of nodulation, infection with rhizobia induces the dedifferentiation of host cells to form primordia of a symbiotic organ, the nodule, which prepares plants to accommodate rhizobia in host cells. While these nodulation processes are known to be genetically controlled by both plants and rhizobia, recent advances in studies on two model legumes, Lotus japonicus and Medicago truncatula, have provided great insight into the underlying plant-side molecular mechanism. In this chapter, we review such knowledge, with particular emphasis on two key processes of nodulation, nodule development and rhizobial invasion. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. A novel ABA insensitive mutant of Lotus japonicus with a wilty phenotype displays unaltered nodulation regulation.

    PubMed

    Biswas, Bandana; Chan, Pick Kuen; Gresshoff, Peter M

    2009-05-01

    An ABA insensitive mutant, Beyma, was isolated in Lotus japonicus MG-20 from an EMS mutagenesis population using root growth inhibition to applied ABA as the screening criterion. (The name 'Beyma' was taken from the Australian Aboriginal language, Wagiman, beyma, meaning 'drying up'.) The stable mutant that segregates as a dominant Mendelian mutation is insensitive to ABA induced inhibition of germination, vegetative growth, stomatal opening, as well as nodulation. Tissue ABA levels were normal, suggesting a sensitivity rather than biosynthesis mutation. It is slow-growing (50-70% of wild-type MG-20) and has a near-constitutive wilty phenotype associated with its inability to regulate stomatal opening. Whilst showing a wide range of ABA insensitive phenotypes, Beyma did not show alteration of nodule number control, as, in the absence of added ABA, the number and patterning (but not size) of nodules formed in the mutant were similar to that of MG-20. Split root experiments on MG-20 showed that application of ABA on one side of the root inhibited nodulation locally but not systemically. We propose that ABA is not involved directly in systemic autoregulation of nodulation (AON).

  14. Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus.

    PubMed

    Kapranov, P; Routt, S M; Bankaitis, V A; de Bruijn, F J; Szczyglowski, K

    2001-06-01

    Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes. Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner. Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L. japonicus late nodulin Nlj16. Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules. The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene. This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene. We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules.

  15. Site-specific distribution and competitive ability of indigenous bean-nodulating rhizobia isolated from organic fields in Minnesota.

    PubMed

    Wongphatcharachai, Manoosak; Wang, Ping; Staley, Christopher; Chun, Chan Lan; Ferguson, John A; Moncada, Kristine M; Sheaffer, Craig C; Sadowsky, Michael J

    2015-11-20

    Organic dry bean production systems have received increasing interest in many regions of the US, including Minnesota. Thus, improving biological N2 fixation would be highly beneficial for organic crop production. To date, only limited work has been done to select efficient N2-fixing rhizobia for organic dry bean production. In this study, soil samples from 25 organic fields in Minnesota, with a previous cropping history of dry beans, soybeans or both, were collected during May to July 2012. Genetic diversity of indigenous dry bean-rhizobia (511 isolates) was determined by using horizontal, fluorophore-enhanced, repetitive, extragenic, and palindromic-PCR (HFERP) DNA fingerprinting and isolates were classified as belonging to 58 different genotypes. The more abundant rhizobia isolated from bean nodules comprised 35.6% of the population. None of the isolates were identical to commonly-used commercial strains used in the U.S., including Rhizobium tropici CIAT899. Seventeen predominant genotypes were shown to represent two main species, Rhizobium leguminosarum bv. phaseoli (67.1%) and Rhizobium etli (30.2%). One of the indigenous strains, orgK9, displayed efficient N2-fixation and competitive ability relative to the commercial strains tested. The lack of large numbers of indigenous dry bean-rhizobia at most study sites will be useful to avoid competition problems between inoculant strains and indigenous rhizobia. This will allow inoculation with highly effective N2-fixing rhizobia, thus resulting in improved crop productivity. Our results highlight the existence of site-specific rhizobial genotypes in different organic fields and identify strains that may prove useful as novel inoculants for organic dry bean production systems. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. micro RNA 172 (miR172) signals epidermal infection and is expressed in cells primed for bacterial invasion in Lotus japonicus roots and nodules.

    PubMed

    Holt, Dennis B; Gupta, Vikas; Meyer, Dörte; Abel, Nikolaj B; Andersen, Stig U; Stougaard, Jens; Markmann, Katharina

    2015-10-01

    Legumes interact with rhizobial bacteria to form nitrogen-fixing root nodules. Host signalling following mutual recognition ensures a specific response, but is only partially understood. Focusing on the stage of epidermal infection with Mesorhizobium loti, we analysed endogenous small RNAs (sRNAs) of the model legume Lotus japonicus to investigate their involvement in host response regulation. We used Illumina sequencing to annotate the L. japonicus sRNA-ome and isolate infection-responsive sRNAs, followed by candidate-based functional characterization. Sequences from four libraries revealed 219 novel L. japonicus micro RNAs (miRNAs) from 114 newly assigned families, and 76 infection-responsive sRNAs. Unlike infection-associated coding genes such as NODULE INCEPTION (NIN), a micro RNA 172 (miR172) isoform showed strong accumulation in dependency of both Nodulation (Nod) factor and compatible rhizobia. The genetics of miR172 induction support the existence of distinct epidermal and cortical signalling events. MIR172a promoter activity followed a previously unseen pattern preceding infection thread progression in epidermal and cortical cells. Nodule-associated miR172a expression was infection-independent, representing the second of two genetically separable activity waves. The combined data provide a valuable resource for further study, and identify miR172 as an sRNA marking successful epidermal infection. We show that miR172 acts upstream of several APETALA2-type (AP2) transcription factors, and suggest that it has a role in fine-tuning AP2 levels during bacterial symbiosis.

  17. Monitoring the colonization and infection of legume nodules by Micromonospora in co-inoculation experiments with rhizobia.

    PubMed

    Benito, Patricia; Alonso-Vega, Pablo; Aguado, Carolina; Luján, Rafael; Anzai, Yojiro; Hirsch, Ann M; Trujillo, Martha E

    2017-09-08

    The discovery that the actinobacterium Micromonospora inhabits nitrogen-fixing nodules raised questions as to its potential ecological role. The capacity of two Micromonospora strains to infect legumes other than their original host, Lupinus angustifolius, was investigated using Medicago and Trifolium as test plants. Compatible rhizobial strains were used for coinoculation of the plants because Micromonospora itself does not induce nodulation. Over 50% of nodules from each legume housed Micromonospora, and using 16S rRNA gene sequence identification, we verified that the reisolated strains corresponded to the microorganisms inoculated. Entry of the bacteria and colonization of the plant hosts were monitored using a GFP-tagged Lupac 08 mutant together with rhizobia, and by using immunogold labeling. Strain Lupac 08 was localized in plant tissues, confirming its capacity to enter and colonize all hosts. Based on studying three different plants, our results support a non-specific relationship between Micromonospora and legumes. Micromonospora Lupac 08, originally isolated from Lupinus re-enters root tissue, but only when coinoculated with the corresponding rhizobia. The ability of Micromonospora to infect and colonize different legume species and function as a potential plant-growth promoting bacterium is relevant because this microbe enhances the symbiosis without interfering with the host and its nodulating and nitrogen-fixing microbes.

  18. Effect of Co-Inoculation with Mycorrhiza and Rhizobia on the Nodule Trehalose Content of Different Bean Genotypes

    PubMed Central

    Ballesteros-Almanza, L; Altamirano-Hernandez, J; Peña-Cabriales, J.J; Santoyo, G; Sanchez-Yañez, J.M; Valencia-Cantero, E; Macias-Rodriguez, L; Lopez-Bucio, J; Cardenas-Navarro, R; Farias-Rodriguez, R

    2010-01-01

    Studies on Rhizobium-legume symbiosis show that trehalose content in nodules under drought stress correlates positively with an increase in plant tolerance to this stress. Fewer reports describe trehalose accumulation in mycorrhiza where, in contrast with rhizobia, there is no flux of carbohydrates from the microsymbiont to the plant. However, the trehalose dynamics in the Mycorrhiza-Rhizobium-Legume tripartite symbiosis is unknown. The present study explores the role of this tripartite symbiosis in the trehalose content of nodules grown under contrasting moisture conditions. Three wild genotypes (P. filiformis, P. acutifolis and P. vulgaris) and two commercial genotypes of Phaseolus vulgaris (Pinto villa and Flor de Mayo) were used. Co-inoculation treatments were conducted with Glomus intraradices and a mixture of seven native rhizobial strains, and trehalose content was determined by GC/MS. The results showed a negative effect of mycorrhizal inoculation on nodule development, as mycorrhized plants showed fewer nodules and lower nodule dry weight compared to plants inoculated only with Rhizobium. Mycorrhizal colonization was also higher in plants inoculated only with Glomus as compared to plants co-inoculated with both microsymbionts. In regard to trehalose, co-inoculation negatively affects its accumulation in the nodules of each genotype tested. However, the correlation analysis showed a significantly positive correlation between mycorrhizal colonization and nodule trehalose content. PMID:21253462

  19. Identification of fast-growing rhizobia nodulating tropical legumes from Puerto Rico as Rhizobium gallicum and Rhizobium tropici.

    PubMed

    Zurdo-Piñeiro, José Luis; Velázquez, Encarna; Lorite, María José; Brelles-Mariño, Graciela; Schröder, Eduardo C; Bedmar, Eulogio J; Mateos, Pedro F; Martínez-Molina, Eustoquio

    2004-08-01

    Fifteen isolates from several nodulated tropical legumes from Puerto Rico (USA) were characterised by their phenotypic, molecular and symbiotic features. The identification of isolates was based on a polyphasic approach, including phenotypic characteristics, 16S rRNA sequencing, Low molecular weight (LMW) RNA profiles, Two Primers-RAPD patterns, and restriction patterns from 16S rDNA molecules. Despite of the variety of hosts included in this study the 15 isolates were separated into only two groups that corresponded to Rhizobium gallicum and Rhizobium tropici. This work shows that R. gallicum and R. tropici nodulate legume plants, such as Sesbania, Caliandra, Poitea, Piptadenia, Neptunia and Mimosa species, that were not previously considered as hosts for these rhizobia. Moreover, some of these host plants can be nodulated by both species. The results confirm the great promiscuity of R. tropici and also support the hypothesis that the species R. gallicum may be native from America or cosmopolitan and worldwide spread.

  20. Nodulation of cowpeas and survival of cowpeas Rhizobia in acid, aluminum-rich soils. [Vigna unguiculata; Rhizobium

    SciTech Connect

    Hartel, P.G.; Whelan, A.M.; Alexander, M.

    1983-01-01

    A study was undertaken to determine whether the reduced nodulation of cowpeas (Vigna unguiculata (L.) Walp) grown in certain acid, Alrich soils resulted from the poor survival of the potentially infective rhizobia. Two strains of Rhizobium capable of nodulating cowpeas were used. The lowest pH for growth in defined liquid medium was 4.2 for one strain and 3.9 for the other. Only the latter was Al tolerant and could grow in a defined liquid medium containing 50 ..mu..M KAl(SO/sub 4/)/sub 2/. The survival of the bacteria and their ability to nodulate cowpeas in three soils were measured after the soils were amended with Ca or Al salts to give pH values ranging from 5.7 to 4.1 and extractable-Al concentrations from < 0.1 to 3.7 cmol(p/sup +/)/kg of soil. Only small differences in survival in 7 or 8 weeks were noted between the two strains. Plants inoculated with the Al-sensitive strain bore significantly fewer nodules in the more acid, Al-rich soils than in the same soils with higher pH values and less extractable Al. No significant reduction in nodule number was evident for plants inoculated with the Al-tolerant strain and grown in the more acid, Al-rich soils compared to cowpeas grown in the same soils with higher pH values and less extractable Al. It is suggested that the Al content of soil is not a major factor in the survival of cowpea rhizobia but that it does have a significant effect on nodulation. 24 references, 3 figures, 2 tables.

  1. Medicago truncatula natural resistance-associated macrophage Protein1 is required for iron uptake by rhizobia-infected nodule cells.

    PubMed

    Tejada-Jiménez, Manuel; Castro-Rodríguez, Rosario; Kryvoruchko, Igor; Lucas, M Mercedes; Udvardi, Michael; Imperial, Juan; González-Guerrero, Manuel

    2015-05-01

    Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II.

  2. Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus-Mesorhizobium loti symbiosis.

    PubMed

    Fukudome, Mitsutaka; Calvo-Begueria, Laura; Kado, Tomohiro; Osuki, Ken-Ichi; Rubio, Maria Carmen; Murakami, Ei-Ichi; Nagata, Maki; Kucho, Ken-Ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

    2016-09-01

    Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia-legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5'-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  3. Overexpression of alfalfa cytosolic glutamine synthetase in nodules and flowers of transgenic Lotus japonicus plants.

    PubMed

    Suárez, Ramón; Márquez, Judith; Shishkova, Svetlana; Hernández, Georgina

    2003-03-01

    Legumes can obtain nitrogen from symbiotic nitrogen fixation in root nodules. The glutamine synthetase/glutamate synthase cycle is responsible for the initial nitrogen assimilation. This work reports the analysis of transgenic Lotus japonicus plants with the chimeric gene containing the alfalfa cytosolic glutamine synthetase (GS1) (EC 6.3.1.2) gene controlled by the Sesbania rostrata leghemoglobin gene promoter (Srglb3p). Surprisingly, all of the transgenic primary transformants analysed were sterile. Two transformants designated GS39 and GS44 were further analysed. GS in nodules of GS39 and GS44 plants was upregulated, at the level of transcript and protein. The transgenic plants had 2-fold higher nodule GS activity and similar root GS activity compared to control plants. The GS39 and GS44 sterile plants showed morphological alterations in pollen grains and in ovules. An increase in GS transcript abundance and enzyme activity was measured during early and late stages of flower development of GS plants. Flowers of GS plants showed higher glutamine content, resulting in an increased glutamine/glutamate ratio. The GS transcript and protein were detected in ovules. These data indicate that overexpression of GS1 in reproductive organs critically affects their development and might be a reason for sterility of L. japonicus plants.

  4. Does Lowering Glutamine Synthetase Activity in Nodules Modify Nitrogen Metabolism and Growth of Lotus japonicus?1

    PubMed Central

    Harrison, Judith; Pou de Crescenzo, Marie-Anne; Sené, Olivier; Hirel, Bertrand

    2003-01-01

    A cDNA encoding cytosolic glutamine synthetase (GS) from Lotus japonicus was fused in the antisense orientation relative to the nodule-specific LBC3 promoter of soybean (Glycine max) and introduced into L. japonicus via transformation with Agrobacterium tumefaciens. Among the 12 independent transformed lines into which the construct was introduced, some of them showed diminished levels of GS1 mRNA and lower levels of GS activity. Three of these lines were selected and their T1 progeny was further analyzed both for plant biomass production and carbon and nitrogen (N) metabolites content under symbiotic N-fixing conditions. Analysis of these plants revealed an increase in fresh weight in nodules, roots and shoots. The reduction in GS activity was found to correlate with an increase in amino acid content of the nodules, which was primarily due to an increase in asparagine content. Thus, this study supports the hypothesis that when GS becomes limiting, other enzymes (e.g. asparagine synthetase) that have the capacity to assimilate ammonium may be important in controlling the flux of reduced N in temperate legumes such as L. japonicus. Whether these alternative metabolic pathways are important in the control of plant biomass production still remains to be fully elucidated. PMID:12970491

  5. Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants.

    PubMed

    Granada, Camille E; Strochein, Marcos; Vargas, Luciano K; Bruxel, Manuela; de Sá, Enilson Luiz Saccol; Passaglia, Luciane M P

    2014-06-01

    This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species.

  6. Genetic diversity and symbiotic compatibility among rhizobial strains and Desmodium incanum and Lotus spp. plants

    PubMed Central

    Granada, Camille E.; Strochein, Marcos; Vargas, Luciano K.; Bruxel, Manuela; de Sá, Enilson Luiz Saccol; Passaglia, Luciane M.P.

    2014-01-01

    This work aimed to evaluate the symbiotic compatibility and nodulation efficiency of rhizobia isolated from Desmodium incanum, Lotus corniculatus, L. subbiflorus, L. uliginosus and L. glaber plants by cross-inoculation. Twelve reference strains and 21 native isolates of rhizobia were genetically analyzed by the BOX-PCR technique, which showed a high genetic diversity among the rhizobia studied. The isolates were also characterized based on their production of indolic compounds and siderophores, as well as on their tolerance to salinity. Fifteen of the 33 rhizobia analyzed were able to produce indolic compounds, whereas 13 produced siderophores. All the tested rhizobia were sensitive to high salinity, although some were able to grow in solutions of up to 2% NaCl. Most of the native rhizobia isolated from L. uliginosus were able to induce nodulation in all plant species studied. In a greenhouse experiment using both D. incanum and L. corniculatus plants, the rhizobia isolate UFRGS Lu2 promoted the greatest plant growth. The results demonstrate that there are native rhizobia in the soils of southern Brazil that have low host specificity and are able to induce nodulation and form active nodules in several plant species. PMID:25071405

  7. PII Overexpression in Lotus japonicus Affects Nodule Activity in Permissive Low-Nitrogen Conditions and Increases Nodule Numbers in High Nitrogen Treated Plants.

    PubMed

    D'Apuzzo, Enrica; Valkov, Vladimir Totev; Parlati, Aurora; Omrane, Selim; Barbulova, Ani; Sainz, Maria Martha; Lentini, Marco; Esposito, Sergio; Rogato, Alessandra; Chiurazzi, Maurizio

    2015-04-01

    We report here the first characterization of a GLNB1 gene coding for the PII protein in leguminous plants. The main purpose of this work was the investigation of the possible roles played by this multifunctional protein in nodulation pathways. The Lotus japonicus LjGLB1 gene shows a significant transcriptional regulation during the light-dark cycle and different nitrogen availability, conditions that strongly affect nodule formation, development, and functioning. We also report analysis of the spatial profile of expression of LjGLB1 in root and nodule tissues and of the protein's subcellular localization. Transgenic L. japonicus lines overexpressing the PII protein were obtained and tested for the analysis of the symbiotic responses in different conditions. The uncoupling of PII from its native regulation affects nitrogenase activity and nodule polyamine content. Furthermore, our results suggest the involvement of PII in the signaling of the nitrogen nutritional status affecting the legumes' predisposition for nodule formation.

  8. Comparative symbiotic performance of native rhizobia of the Flooding Pampa and strains currently used for inoculating Lotus tenuis in this region.

    PubMed

    Sannazzaro, Analía Inés; Bergottini, Verónica Mariel; Paz, Rosalía Cristina; Castagno, Luis Nazareno; Menéndez, Ana Bernardina; Ruiz, Oscar Adolfo; Pieckenstain, Fernando Luis; Estrella, María Julia

    2011-02-01

    The Flooding Pampa (FP) is the most important area for cattle breeding in Argentina. In this region, persistence and yield of typical forage legumes are strongly limited by soil salinity and alkalinity, which affect around 30% of the total area. Instead, naturalized Lotus tenuis is the main forage legume in this region. Rhizobial strains currently used for inoculating L. tenuis in the FP are exotic or native from non-saline soils of this region, their taxonomic identity being unknown. Assuming that rhizobia native from the most restrictive environments are well adapted to adverse conditions, the use of such isolates could improve the productivity of L. tenuis in the FP. Hence, the goal of this study was to evaluate the symbiotic efficiency of selected L. tenuis rhizobia native from the FP, as compared with strains currently used for field inoculation of this legume. Under non-stressing conditions, the symbiotic performance of native strains of FP exceeded those ones currently used for L. tenuis. Moreover, the symbiotic performance of the native strain ML103 was considerably high under salt stress, compared with strains currently used as inoculants. Analysis of 16S rRNA gene sequencing revealed that unclassified rhizobia currently used for field inoculation of L. tenuis and native strains grouped with the genus Mesorhizobium. As a whole, results obtained demonstrate that soils of the FP are a source of efficient and diverse rhizobia that could be used as a sustainable agronomic tool to formulate inoculants that improve forage yield of L. tenuis in this region.

  9. Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the Southern African crotalarioid clade Lotononis s.l.

    PubMed

    Ardley, Julie K; Reeve, Wayne G; O'Hara, Graham W; Yates, Ron J; Dilworth, Michael J; Howieson, John G

    2013-07-01

    The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of diverse chromosomal and symbiotic lineages. The

  10. Nodule morphology, symbiotic specificity and association with unusual rhizobia are distinguishing features of the genus Listia within the southern African crotalarioid clade Lotononis s.l.

    PubMed Central

    Ardley, Julie K.; Reeve, Wayne G.; O'Hara, Graham W.; Yates, Ron J.; Dilworth, Michael J.; Howieson, John G.

    2013-01-01

    Background and Aims The legume clade Lotononis sensu lato (s.l.; tribe Crotalarieae) comprises three genera: Listia, Leobordea and Lotononis sensu stricto (s.s.). Listia species are symbiotically specific and form lupinoid nodules with rhizobial species of Methylobacterium and Microvirga. This work investigated whether these symbiotic traits were confined to Listia by determining the ability of rhizobial strains isolated from species of Lotononis s.l. to nodulate Listia, Leobordea and Lotononis s.s. hosts and by examining the morphology and structure of the resulting nodules. Methods Rhizobia were characterized by sequencing their 16S rRNA and nodA genes. Nodulation and N2 fixation on eight taxonomically diverse Lotononis s.l. species were determined in glasshouse trials. Nodules of all hosts, and the process of infection and nodule initiation in Listia angolensis and Listia bainesii, were examined by light microscopy. Key Results Rhizobia associated with Lotononis s.l. were phylogenetically diverse. Leobordea and Lotononis s.s. isolates were most closely related to Bradyrhizobium spp., Ensifer meliloti, Mesorhizobium tianshanense and Methylobacterium nodulans. Listia angolensis formed effective nodules only with species of Microvirga. Listia bainesii nodulated only with pigmented Methylobacterium. Five lineages of nodA were found. Listia angolensis and L. bainesii formed lupinoid nodules, whereas nodules of Leobordea and Lotononis s.s. species were indeterminate. All effective nodules contained uniformly infected central tissue. Listia angolensis and L. bainesii nodule initials occurred on the border of the hypocotyl and along the tap root, and nodule primordia developed in the outer cortical layer. Neither root hair curling nor infection threads were seen. Conclusions Two specificity groups occur within Lotononis s.l.: Listia species are symbiotically specific, while species of Leobordea and Lotononis s.s. are generally promiscuous and interact with rhizobia of

  11. Abundance and diversity of soybean-nodulating rhizobia in black soil are impacted by land use and crop management.

    PubMed

    Yan, Jun; Han, Xiao Zeng; Ji, Zhao Jun; Li, Yan; Wang, En Tao; Xie, Zhi Hong; Chen, Wen Feng

    2014-09-01

    To investigate the effects of land use and crop management on soybean rhizobial communities, 280 nodule isolates were trapped from 7 fields with different land use and culture histories. Besides the known Bradyrhizobium japonicum, three novel genospecies were isolated from these fields. Grassland (GL) maintained a higher diversity of soybean bradyrhizobia than the other cultivation systems. Two genospecies (Bradyrhizobium spp. I and III) were distributed widely in all treatments, while Bradyrhizobium sp. II was found only in GL treatment. Cultivation with soybeans increased the rhizobial abundance and diversity, except for the soybean monoculture (S-S) treatment. In monoculture systems, soybeans favored Bradyrhizobium sp. I, while maize and wheat favored Bradyrhizobium sp. III. Fertilization decreased the rhizobial diversity indexes but did not change the species composition. The organic carbon (OC) and available phosphorus (AP) contents and pH were the main soil parameters positively correlated with the distribution of Bradyrhizobium spp. I and II and Bradyrhizobium japonicum and negatively correlated with Bradyrhizobium sp. III. These results revealed that different land uses and crop management could not only alter the diversity and abundance of soybean rhizobia, but also change interactions between rhizobia and legume or nonlegume plants, which offered novel information about the biogeography of rhizobia.

  12. Sesbania herbacea-Rhizobium huautlense Nodulation in Flooded Soils and Comparative Characterization of S. herbacea-Nodulating Rhizobia in Different Environments.

    PubMed

    Wang, E T; Martínez-Romero, E

    2000-07-01

    The nodulation of S. herbacea was compared under flooded and non-flooded conditions in two different soils. One soil was from a flooded field in Sierra de Huautla, the native habitat of this legume, while the other soil was from a well-drained field in Cuernavaca, where rhizobia were found to nodulate the introduced S. herbacea plants. Nodulation of the plants was completely eliminated by flooding in the Cuernavaca soil, whereas nodules were obtained in the same soil under non-flooded conditions. In contrast, nodules were formed in Huautla soil under both flooded and non-flooded conditions. Most isolates, except isolate HS2, from Huautla soil and water were identified as R. huautlense by colony morphology, growth rate, PCR-RFLP of 16S rRNA genes, MLEE, cellular plasmid contents, and RFLP of nifH and nodDAB genes. Isolate HS2 was identified as Mesorhizobium sp. Isolates from Cuernavaca soil were different from R. huautlense in many aspects and were classified into five rDNA types within the genera Mesorhizobium, Rhizobium, and Sinorhizobium by PCR-RFLP of 16S rRNA genes. R. huautlense is a water Rhizobium species. Growth by denitrification under oxygen limitation or with ethanol was observed for R. huautlense bacteria but not for the isolates from Cuernavaca. In an interstrain nodulation competitive assay under both flooded and non-flooded conditions, R. huautlense strain S02 completely inhibited the nodulation of Mesorhizobium sp. Sn2, an isolate from Cuernavaca. From these results, we conclude that R. huautlense has the unique ability to nodulate S. herbacea not only in flooded soils, but in non-flooded soils as well.

  13. Comparison of rhizobia that nodulate Medicago laciniata and Medicago truncatula present in a single Tunisian arid soil.

    PubMed

    Badri, Y; Zribi, K; Badri, M; Huguet, T; van Berkum, P; Aouani, M E

    2007-02-01

    The rhizobia present in a single arid region Tunisian soil that nodulate Medicago laciniata and Medicago truncatula were compared. All isolates, 40 from each host, were Sinorhizobium meliloti based on 16S rRNA polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) patterns and subsequent confirmation by sequence analysis of the 16S rRNA genes in four representatives from each host species. There was no apparent relationship between Medicago host species of isolation and the nodulating rhizobial genome as determined by repetitive extragenic palandromic PCR. The isolates of M. laciniata were distinguished from those of M. truncatula present in the same soil by variation in PCR-RFLP of nifDK, indicating that this dissimilarity is originally genetic and not geographic. While forming effective symbioses with their own respective isolates, both M. laciniata and M. truncatula formed ineffective true nodules, nodule-like structures, or no nodules at all in cross-inoculation tests, as confirmed by the histological observations.

  14. CYTOKININ OXIDASE/DEHYDROGENASE3 Maintains Cytokinin Homeostasis during Root and Nodule Development in Lotus japonicus1[OPEN

    PubMed Central

    Heckmann, Anne B.; Kelly, Simon

    2016-01-01

    Cytokinins are required for symbiotic nodule development in legumes, and cytokinin signaling responses occur locally in nodule primordia and in developing nodules. Here, we show that the Lotus japonicus Ckx3 cytokinin oxidase/dehydrogenase gene is induced by Nod factor during the early phase of nodule initiation. At the cellular level, pCkx3::YFP reporter-gene studies revealed that the Ckx3 promoter is active during the first cortical cell divisions of the nodule primordium and in growing nodules. Cytokinin measurements in ckx3 mutants confirmed that CKX3 activity negatively regulates root cytokinin levels. Particularly, tZ and DHZ type cytokinins in both inoculated and uninoculated roots were elevated in ckx3 mutants, suggesting that these are targets for degradation by the CKX3 cytokinin oxidase/dehydrogenase. The effect of CKX3 on the positive and negative roles of cytokinin in nodule development, infection and regulation was further clarified using ckx3 insertion mutants. Phenotypic analysis indicated that ckx3 mutants have reduced nodulation, infection thread formation and root growth. We also identify a role for cytokinin in regulating nodulation and nitrogen fixation in response to nitrate as ckx3 phenotypes are exaggerated at increased nitrate levels. Together, these findings show that cytokinin accumulation is tightly regulated during nodulation in order to balance the requirement for cell divisions with negative regulatory effects of cytokinin on infection events and root development. PMID:26644503

  15. [Characterization of rhizobia causing nodules on leguminous trees native to Uruguay using the rep-PCR technique].

    PubMed

    Rodríguez, A; Frioni, L

    2003-01-01

    Methods for identifying and following microorganisms in the environment such as soils, water and plant association have been highly developed in recent years. In this study, we used rep-PCR for the characterization of Rhizobium bacteria isolated from legume trees native of Uruguay which permitted to follow them in plant inoculation assays. Among the primers used, ERIC and BOX1AR, the latter allowed the differentiation of isolates from different legumes and the finding of a high level of homology among rhizobia that nodulate the same legume species. Besides, it demonstrated that the profiles of the isolates recovered from nodules of Acacia caven, were identical to the ones used as inoculants; this confirmed the efficiency of these methods to follow these diazotrophs in the environment.

  16. Two MicroRNAs Linked to Nodule Infection and Nitrogen-Fixing Ability in the Legume Lotus japonicus1[W

    PubMed Central

    De Luis, Ana; Markmann, Katharina; Cognat, Valérie; Holt, Dennis B.; Charpentier, Myriam; Parniske, Martin; Stougaard, Jens; Voinnet, Olivier

    2012-01-01

    Legumes overcome nitrogen shortage by developing root nodules in which symbiotic bacteria fix atmospheric nitrogen in exchange for host-derived carbohydrates and mineral nutrients. Nodule development involves the distinct processes of nodule organogenesis, bacterial infection, and the onset of nitrogen fixation. These entail profound, dynamic gene expression changes, notably contributed to by microRNAs (miRNAs). Here, we used deep-sequencing, candidate-based expression studies and a selection of Lotus japonicus mutants uncoupling different symbiosis stages to identify miRNAs involved in symbiotic nitrogen fixation. Induction of a noncanonical miR171 isoform, which targets the key nodulation transcription factor Nodulation Signaling Pathway2, correlates with bacterial infection in nodules. A second candidate, miR397, is systemically induced in the presence of active, nitrogen-fixing nodules but not in that of noninfected or inactive nodule organs. It is involved in nitrogen fixation-related copper homeostasis and targets a member of the laccase copper protein family. These findings thus identify two miRNAs specifically responding to symbiotic infection and nodule function in legumes. PMID:23071252

  17. Interkingdom Responses to Bacterial Quorum Sensing Signals Regulate Frequency and Rate of Nodulation in Legume-Rhizobia Symbiosis.

    PubMed

    Palmer, Andrew G; Mukherjee, Arijit; Stacy, Danielle M; Lazar, Stephen; Ané, Jean-Michel; Blackwell, Helen E

    2016-11-17

    Density-dependent phenotypic switching in bacteria, the phenomenon of quorum sensing (QS), is instrumental in many pathogenic and mutualistic behaviors. In many Gram-negative bacteria, QS is regulated by N-acylated-l-homoserine lactones (AHLs). Synthetic analogues of these AHLs hold significant promise for regulating QS at the host-symbiont interface. Regulation depends on refined temporal and spatial models of quorums under native conditions. Critical to this is an understanding of how the presence of these signals may affect a prospective host. We screened a library of AHL analogues for their ability to regulate the legume-rhizobia mutualistic symbiosis (nodulation) between Medicago truncatula and Sinorhizobium meliloti. Using an established QS-reporter line of S. meliloti and nodulation assays with wild-type bacteria, we identified compounds capable of increasing either the rate of nodule formation or total nodule number. Most importantly, we identified compounds with activity exclusive to either host or pathogen, underscoring the potential to generate QS modulators selective to bacteria with limited effects on a prospective host. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgars have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain

    USDA-ARS?s Scientific Manuscript database

    Common bean and Medicago rhizobia isolated from five locations on the island of Lanzarote, the Canary Islands, by partial analysis of 10 chromosomal genes were shown to exhibit close similarity to Sinorhizobium meliloti. Several bean isolates from Lanzarote, mainland Spain and Tunisia nodulated Leu...

  19. A phytase gene is overexpressed in root nodules cortex of Phaseolus vulgaris-rhizobia symbiosis under phosphorus deficiency.

    PubMed

    Lazali, Mohamed; Zaman-Allah, Mainassara; Amenc, Laurie; Ounane, Ghania; Abadie, Josiane; Drevon, Jean-Jacques

    2013-08-01

    Phosphorus is an essential nutrient for rhizobial symbioses to convert N2 into NH4 usable for N nutrition in legumes and N cycle in ecosystems. This N2 fixation process occurs in nodules with a high energy cost. Phytate is the major storage form of P and accounts for more than 50 % of the total P in seeds of cereals and legumes. The phytases, a group of enzymes widely distributed in plant and microorganisms, are able to hydrolyze a variety of inositol phosphates. Recently, phytase activity was discovered in nodules. However, the gene expression localization and its role in N2-fixing nodules are still unknown. In this work, two recombinant inbred lines (RILs) of common bean (Phaseolus vulgaris L.), selected as contrasting for N2 fixation under P deficiency, namely RILs 115 (P-efficient) and 147 (P-inefficient) were inoculated with Rhizobium tropici CIAT 899, and grown under hydroaeroponic conditions with sufficient versus deficient P supply. With in situ RT-PCR methodology, we found that phytase transcripts were particularly abundant in the nodule cortex and infected zone of both RILs. Under P deficiency, phytase transcripts were significantly more abundant for RIL115 than for RIL147, and more in the outer cortex than in the infected zone. Additionally, the high expression of phytase among nodule tissues for the P-deficient RIL115 was associated with an increase in phytase (33 %) and phosphatase (49 %) activities and efficiency in use of the rhizobial symbiosis (34 %). It is argued that phytase activity in nodules would contribute to the adaptation of the rhizobia-legume symbiosis to low-P environments.

  20. De novo alanine synthesis by bacteroids of Mesorhizobium loti is not required for nitrogen transfer in the determinate nodules of Lotus corniculatus.

    PubMed

    Kumar, Shalini; Bourdès, Alexandre; Poole, Philip

    2005-08-01

    Deletion of both alanine dehydrogenase genes (aldA) in Mesorhizobium loti resulted in the loss of AldA enzyme activity from cultured bacteria and bacteroids but had no effect on the symbiotic performance of Lotus corniculatus plants. Thus, neither indeterminate pea nodules nor determinate L. corniculatus nodules export alanine as the sole nitrogen secretion product.

  1. Lotus hosts delimit the mutualism-parasitism continuum of Bradyrhizobium.

    PubMed

    Regus, J U; Gano, K A; Hollowell, A C; Sofish, V; Sachs, J L

    2015-02-01

    Symbioses are modelled as evolutionarily and ecologically variable with fitness outcomes for hosts shifting on a continuum from mutualism to parasitism. In a classic example, rhizobia fix atmospheric nitrogen for legume hosts in exchange for photosynthetic carbon. Rhizobial infection often enhances legume growth, but hosts also incur interaction costs because of root tissues and or metabolites needed to support symbionts in planta. Rhizobia exhibit genetic variation in symbiotic effectiveness, and ecological changes in light or mineral nitrogen availability can also alter the benefits of rhizobial infection for hosts. The net effects of symbiosis thus can range from mutualistic to parasitic in a context-dependent manner. We tested the extent of the mutualism-parasitism continuum in the legume-rhizobium symbiosis and the degree to which host investment can shape its limits. We infected Lotus strigosus with sympatric Bradyrhizobium genotypes that vary in symbiotic effectiveness. Inoculations occurred under different mineral nitrogen and light regimes spanning ecologically relevant ranges. Net growth benefits of Bradyrhizobium infection varied for Lotus and were reduced or eliminated dependent on Bradyrhizobium genotype, mineral nitrogen and light availability. But we did not detect parasitism. Lotus proportionally reduced investment in Bradyrhizobium as net benefit from infection decreased. Lotus control occurred primarily after infection, via fine-scale modulation of nodule growth, as opposed to control over initial nodulation. Our results show how divestment of symbiosis by Lotus can prevent shifts to parasitism.

  2. Effects of Endogenous Salicylic Acid on Nodulation in the Model Legumes Lotus japonicus and Medicago truncatula1[W

    PubMed Central

    Stacey, Gary; McAlvin, Crystal Bickley; Kim, Sung-Yong; Olivares, José; Soto, María José

    2006-01-01

    The exogenous addition of salicylic acid (SA) was previously shown to inhibit indeterminate but not determinate-type nodulation. We sought to extend these results by modulating endogenous levels of SA through the transgenic expression of salicylate hydroxylase (NahG) in both stably transformed Lotus japonicus and composite Medicago truncatula plants. NahG expression in L. japonicus resulted in a marked reduction of SA levels. This reduction correlated with an increase in the number of infections and mean nodule number when compared to controls. However, a complicating factor was that NahG-expressing plants had greater root growth. Spot inoculations of NahG-expressing L. japonicus plants confirmed increased nodulation in these plants. Consistent with the reported inhibitory effects of exogenous SA on indeterminate-type nodulation, NahG expression in M. truncatula plants led to enhanced nodulation and infection. These data point to an important role for SA-mediated plant defense pathways in controlling nodule formation on both determinate and indeterminate nodule-forming hosts. PMID:16798946

  3. Phylogenetic multilocus sequence analysis of indigenous slow-growing rhizobia nodulating cowpea (Vigna unguiculata L.) in Greece.

    PubMed

    Tampakaki, Anastasia P; Fotiadis, Christos T; Ntatsi, Georgia; Savvas, Dimitrios

    2017-04-01

    Cowpea (Vigna unguiculata) is a promiscuous grain legume, capable of establishing efficient symbiosis with diverse symbiotic bacteria, mainly slow-growing rhizobial species belonging to the genus Bradyrhizobium. Although much research has been done on cowpea-nodulating bacteria in various countries around the world, little is known about the genetic and symbiotic diversity of indigenous cowpea rhizobia in European soils. In the present study, the genetic and symbiotic diversity of indigenous rhizobia isolated from field-grown cowpea nodules in three geographically different Greek regions were studied. Forty-five authenticated strains were subjected to a polyphasic approach. ERIC-PCR based fingerprinting analysis grouped the isolates into seven groups and representative strains of each group were further analyzed. The analysis of the rrs gene showed that the strains belong to different species of the genus Bradyrhizobium. The analysis of the 16S-23S IGS region showed that the strains from each geographic region were characterized by distinct IGS types which may represent novel phylogenetic lineages, closely related to the type species of Bradyrhizobium pachyrhizi, Bradyrhizobium ferriligni and Bradyrhizobium liaoningense. MLSA analysis of three housekeeping genes (recA, glnII, and gyrB) showed the close relatedness of our strains with B. pachyrhizi PAC48(T) and B. liaoningense USDA 3622(T) and confirmed that the B. liaoningense-related isolate VUEP21 may constitute a novel species within Bradyrhizobium. Moreover, symbiotic gene phylogenies, based on nodC and nifH genes, showed that the B. pachyrhizi-related isolates belonged to symbiovar vignae, whereas the B. liaoningense-related isolates may represent a novel symbiovar. Copyright © 2017 Elsevier GmbH. All rights reserved.

  4. Construction of a Lotus japonicus late nodulin expressed sequence tag library and identification of novel nodule-specific genes.

    PubMed Central

    Szczyglowski, K; Hamburger, D; Kapranov, P; de Bruijn, F J

    1997-01-01

    A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 110 differentially displayed polymerase chain reaction products were cloned and analyzed. Of 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodule-specific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation. PMID:9276951

  5. Growth and nitrogen fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon metabolism.

    PubMed

    López, Miguel; Herrera-Cervera, Jose A; Iribarne, Carmen; Tejera, Noel A; Lluch, Carmen

    2008-04-18

    Lotus japonicus and Medicago truncatula model legumes, which form determined and indeterminate nodules, respectively, provide a convenient system to study plant-Rhizobium interaction and to establish differences between the two types of nodules under salt stress conditions. We examined the effects of 25 and 50mM NaCl doses on growth and nitrogen fixation parameters, as well as carbohydrate content and carbon metabolism of M. truncatula and L. japonicus nodules. The leghemoglobin (Lb) content and nitrogen fixation rate (NFR) were approximately 10.0 and 2.0 times higher, respectively, in nodules of L. japonicus when compared with M. truncatula. Plant growth parameters and nitrogenase activity decreased with NaCl treatments in both legumes. Sucrose was the predominant sugar quantified in nodules of both legumes, showing a decrease in concentration in response to salt stress. The content of trehalose was low (less than 2.5% of total soluble sugars (TSS)) to act as an osmolyte in nodules, despite its concentration being increased under saline conditions. Nodule enzyme activities of trehalose-6-phosphate synthase (TPS) and trehalase (TRE) decreased with salinity. L. japonicus nodule carbon metabolism proved to be less sensitive to salinity than in M. truncatula, as enzymatic activities responsible for the carbon supply to the bacteroids to fuel nitrogen fixation, such as sucrose synthase (SS), alkaline invertase (AI), malate dehydrogenase (MDH) and phosphoenolpyruvate carboxylase (PEPC), were less affected by salt than the corresponding activities in barrel medics. However, nitrogenase activity was only inhibited by salinity in L. japonicus nodules.

  6. Phenotypic and genotypic characterizations of rhizobia isolated from root nodules of peanut (Arachis hypogaea L.) grown in Moroccan soils.

    PubMed

    El-Akhal, M Rabie; Rincon, Ana; Mourabit, Nourdin El; Pueyo, Jose J; Barrijal, Said

    2009-10-01

    The phenotypic and genotypic characterization of sixty-two rhizobial isolates obtained from nodules of Arachis hypogaea in north-western Morocco was performed. Their physiological and biochemical properties revealed a great deal of diversity among them. Isolates were classified into two major groups based on the numerical analysis of their phenotypic and genotypic characteristics. Isolates in the first group were alkali- and salt-sensitive, slow or extra-slow growers; they did not use disaccharides as carbon source and varied in the use of amino acids. ARDRA analysis of the 16S rDNA region grouped them together with reference strains belonging to the genus Bradyrhizobium. In the second group, isolates were fast growers, acid-sensitive, and alkali- and salt-tolerant; they used both mono and disaccharides as carbon sources, and methionine was the only amino acid they could metabolize as a nitrogen source. ARDRA analysis grouped them with fast-growing reference strains. Both groups exhibited a range of variability in tolerance to heavy metals. The Intergenic Spacer (IGS)-PCR fingerprinting analysis confirmed a high genotypic diversity at the strain level. This characterization provides a basis for the selection of peanut-nodulating rhizobia which may have applications in formulating appropriate inocula for improving peanut crop yield on Moroccan soils, including saline and acidic marginal areas.

  7. Ribotyping of rhizobia nodulating Acacia mangium and Paraserianthes falcataria from different geographical areas in Indonesia using PCR-RFLP-SSCP (PRS) and sequencing.

    PubMed

    Clapp, J P; Mansur, I; Dodd, J C; Jeffries, P

    2001-04-01

    Acacia mangium and Paraserianthes falcataria are leguminous tree species widely grown for timber in Indonesia and other tropical countries, yet little is known about the identity of their rhizobial symbionts. Polymerase chain reaction-restriction fragment length polymorphism-single-strand conformational polymorphism (PRS) analysis of the 16S rRNA gene was used along with sequencing to assess the diversity of 57 rhizobia isolated from nodules of A. mangium and P. falctaria in Indonesia. In total, 26 rhizobia isolated from A. mangium were analysed by PRS and sequencing. The PRS patterns indicated that 12 (46%) clustered with Bradyrhizobium elkanii, 13 (50%) with B. lianoningense/japonicum and one (4%) with Mesorhizobium loti. Thirty-one isolates were analysed from P. falcataria: five (16%) clustered with B. elkanii and 26 (84%) with B. lianoningense/japonicum. These results were confirmed by phylogenetic analysis of sequences. Intraspecific diversity of the 16S rRNA genes from rhizobia nodulating A. mangium and P. falcataria revealed by PRS was low, only one genotype was found within the isolates that clustered with B. elkanii and two within the B. liaoningense/japonicum group. These Bradyrhizobium species are apparently ubiquitous throughout the Indonesian archipelago and it is clear why the two tree species are able to successfully establish outside their native range without the need for inoculation with indigenous rhizobia.

  8. Characterization of Rhizobia from Ineffective Alfalfa Nodules: Ability to Nodulate Bean Plants [Phaseolus vulgaris (L.) Savi.] †

    PubMed Central

    Eardly, Bertrand D.; Hannaway, David B.; Bottomley, Peter J.

    1985-01-01

    This study was initiated to characterize Rhizobium isolates obtained from root nodules of ineffectively nodulated, field-grown alfalfa (Medicago sativa L.) plants. The purpose was to determine if these isolates possessed characteristics which would explain either their ineffectiveness in N2 fixation or their apparent ability to tolerate the moderately acid soil conditions from which they originated. Isolates were characterized by analysis of growth rate, 39°C tolerance, acid production on conventional media, and symbiotic performance. All isolates were ineffective in N2 fixation on alfalfa, and they contained one or more anomalous characteristics. These included either slow growth rate, lack of 39°C tolerance, or lack of acid production on conventional media. Infectiveness tests on a broad range of legumes revealed that the isolates formed root nodules on M. sativa, Medicago lupulina L., and Phaseolus vulgaris (L.) Savi. (common bean). These results provide evidence that, in some situations, ineffective nodulation of M. sativa in the field may be due to the presence of promiscuous, native Rhizobium species. PMID:16346942

  9. A MAP Kinase Kinase Interacts with SymRK and Regulates Nodule Organogenesis in Lotus japonicus[C][W

    PubMed Central

    Chen, Tao; Zhu, Hui; Ke, Danxia; Cai, Kai; Wang, Chao; Gou, Honglan; Hong, Zonglie; Zhang, Zhongming

    2012-01-01

    The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis. PMID:22353370

  10. Genetic Diversity and Symbiotic Efficiency of Nodulating Rhizobia Isolated from Root Nodules of Faba Bean in One Field

    PubMed Central

    Lan, Qin; Wang, Ke; Liu, Ming; Peng, Dan; Zhang, Xiaoping; Chen, Qiang; Zhao, Ke; Zeng, Xiangzhong; Xu, Kai Wei

    2016-01-01

    Thirty-one nodulating rhizobium strains were collected from root nodules of spring and winter type faba bean cultivars grown in micro ecoarea, i.e. the same field in Chengdu plain, China. The symbiotic efficiency and phylogeny of these strains were studied. Effectively nitrogen fixing strains were isolated from both winter type and spring type cultivars. Based on phylogenetic analysis of 16S rRNA gene and concatenated sequence of atpD, glnII and recA genes, the isolates were assigned as Rhizobium anhuiense and a potential new Rhizobium species. The isolates were diverse on symbiosis related gene level, carrying five, four and three variants of nifH, nodC and nodD, respectively. Strains carrying similar gene combinations were trapped by both winter and spring cultivars, disagreeing with the specificity of symbiotic genotypes to reported earlier faba bean ecotypes. PMID:27936180

  11. Nodulation gene mutants of Mesorhizobium loti R7A-nodZ and nolL mutants have host-specific phenotypes on Lotus spp.

    PubMed

    Rodpothong, Patsarin; Sullivan, John T; Songsrirote, Kriangsak; Sumpton, David; Cheung, Kenneth W J-T; Thomas-Oates, Jane; Radutoiu, Simona; Stougaard, Jens; Ronson, Clive W

    2009-12-01

    Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a DeltanodSDeltanolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7ADeltanodZ and R7ADeltanolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene induction. In planta complementation experiments further suggested that the acetylfucose was important for balanced signaling in response to Nod factor by the L. japonicus NFR1/NFR5 receptors. Overall the results reveal differences in the sensitivity of plant perception with respect to signaling leading to root hair deformation and nodule primordium development versus infection thread formation and rhizobial entry.

  12. Nodulation of Sesbania Species by Rhizobium (Agrobacterium) Strain IRBG74 and Other Rhizobia

    USDA-ARS?s Scientific Manuscript database

    Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridisation with R. ...

  13. Introduction of a novel pathway for IAA biosynthesis to rhizobia alters vetch root nodule development.

    PubMed

    Camerini, Serena; Senatore, Beatrice; Lonardo, Enza; Imperlini, Esther; Bianco, Carmen; Moschetti, Giancarlo; Rotino, Giuseppe L; Campion, Bruno; Defez, Roberto

    2008-07-01

    We introduced into Rhizobium leguminosarum bv. viciae LPR1105 a new pathway for the biosynthesis of the auxin, indole-3-acetic acid (IAA), under the control of a stationary phase-activated promoter active both in free-living bacteria and bacteroids. The newly introduced genes are the iaaM gene from Pseudomonas savastanoi and the tms2 gene from Agrobacterium tumefaciens. Free-living bacteria harbouring the promoter-iaaMtms2 construct release into the growth medium 14-fold more IAA than the wild-type parental strain. This IAA overproducing R. l. viciae, the RD20 strain, elicits the development of vetch root nodules containing up to 60-fold more IAA than nodules infected by the wild-type strain LPR1105. Vetch root nodules derived from RD20 are fewer in number per plant, heavier in terms of dry weight and show an enlarged and more active meristem. A significant increase in acetylene reduction activity was measured in nodules elicited in vetch by RD20.

  14. Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain.

    PubMed

    Iglesias, Olga; Rivas, Raúl; García-Fraile, Paula; Abril, Adriana; Mateos, Pedro F; Martinez-Molina, Eustoquio; Velázquez, Encarna

    2007-12-01

    Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.

  15. Ectopic expression of miR156 represses nodulation and causes morphological and developmental changes in Lotus japonicus.

    PubMed

    Wang, Ying; Wang, Zhishuo; Amyot, Lisa; Tian, Lining; Xu, Ziqin; Gruber, Margaret Y; Hannoufa, Abdelali

    2015-04-01

    The effects of microRNA156 overexpression on general plant architecture, branching, flowering time and nodulation were investigated in the model legume, Lotus japonicus. We cloned an miR156 homolog, LjmiR156a, from L. japonicus, and investigated its SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes and its biological function at enhancing vegetative biomass yield, extending flowering time, and its impact on nodulation. Thirteen potential targets for LjmiR156 were identified in vitro and their expression profiles were determined in aerial and underground parts of mature plants, including genes coding for eight SPLs, one WD-40, one RNA-directed DNA polymerase, two transport proteins, and one histidine-phosphotransfer protein. Two SPL and one WD-40 cleavage targets for LjmiR156-TC70253, AU089191, and TC57859-were identified. Transgenic plants with ectopic expression of LjmiR156a showed enhanced branching, dramatically delayed flowering, underdeveloped roots, and reduced nodulation. We also examined the transcript levels of key genes involved in nodule organogenesis and infection thread formation to determine the role of miR156 in regulating symbiosis. Overexpression of LjmiR156a led to repression of several nodulation genes during the early stages of root development such as three ENOD genes, SymPK, POLLUX, CYCLOPS, Cerberus, and Nsp1, and the stimulation of NFR1. Our results show that miR156 regulates vegetative biomass yield, flowering time and nodulation by silencing downstream target SPLs and other genes, suggesting that the miR156 regulatory network could be modified in forage legumes (such as alfalfa and trefoils) and in leafy vegetables (like lettuce and spinach) to positively impact economically valuable crop species.

  16. NENA, a Lotus japonicus homolog of Sec13, is required for rhizodermal infection by arbuscular mycorrhiza fungi and rhizobia but dispensable for cortical endosymbiotic development.

    PubMed

    Groth, Martin; Takeda, Naoya; Perry, Jillian; Uchida, Hisaki; Dräxl, Stephan; Brachmann, Andreas; Sato, Shusei; Tabata, Satoshi; Kawaguchi, Masayoshi; Wang, Trevor L; Parniske, Martin

    2010-07-01

    Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant's common symbiosis signaling network. We performed a microscopy screen for Lotus japonicus mutants defective in AM development and isolated a mutant, nena, that aborted fungal infection in the rhizodermis. NENA encodes a WD40 repeat protein related to the nucleoporins Sec13 and Seh1. Localization of NENA to the nuclear rim and yeast two-hybrid experiments indicated a role for NENA in a conserved subcomplex of the nuclear pore scaffold. Although nena mutants were able to form pink nodules in symbiosis with Mesorhizobium loti, root hair infection was not observed. Moreover, Nod factor induction of the symbiotic genes NIN, SbtM4, and SbtS, as well as perinuclear calcium spiking, were impaired. Detailed phenotypic analyses of nena mutants revealed a rhizobial infection mode that overcame the lack of rhizodermal responsiveness and carried the hallmarks of crack entry, including a requirement for ethylene. CCaMK-dependent processes were only abolished in the rhizodermis but not in the cortex of nena mutants. These data support the concept of tissue-specific components for the activation of CCaMK.

  17. NENA, a Lotus japonicus Homolog of Sec13, Is Required for Rhizodermal Infection by Arbuscular Mycorrhiza Fungi and Rhizobia but Dispensable for Cortical Endosymbiotic Development[C][W

    PubMed Central

    Groth, Martin; Takeda, Naoya; Perry, Jillian; Uchida, Hisaki; Dräxl, Stephan; Brachmann, Andreas; Sato, Shusei; Tabata, Satoshi; Kawaguchi, Masayoshi; Wang, Trevor L.; Parniske, Martin

    2010-01-01

    Legumes form symbioses with arbuscular mycorrhiza (AM) fungi and nitrogen fixing root nodule bacteria. Intracellular root infection by either endosymbiont is controlled by the activation of the calcium and calmodulin-dependent kinase (CCaMK), a central regulatory component of the plant’s common symbiosis signaling network. We performed a microscopy screen for Lotus japonicus mutants defective in AM development and isolated a mutant, nena, that aborted fungal infection in the rhizodermis. NENA encodes a WD40 repeat protein related to the nucleoporins Sec13 and Seh1. Localization of NENA to the nuclear rim and yeast two-hybrid experiments indicated a role for NENA in a conserved subcomplex of the nuclear pore scaffold. Although nena mutants were able to form pink nodules in symbiosis with Mesorhizobium loti, root hair infection was not observed. Moreover, Nod factor induction of the symbiotic genes NIN, SbtM4, and SbtS, as well as perinuclear calcium spiking, were impaired. Detailed phenotypic analyses of nena mutants revealed a rhizobial infection mode that overcame the lack of rhizodermal responsiveness and carried the hallmarks of crack entry, including a requirement for ethylene. CCaMK-dependent processes were only abolished in the rhizodermis but not in the cortex of nena mutants. These data support the concept of tissue-specific components for the activation of CCaMK. PMID:20675572

  18. Small-Subunit rRNA Genotyping of Rhizobia Nodulating Australian Acacia spp.

    PubMed Central

    Lafay, Bénédicte; Burdon, Jeremy J.

    2001-01-01

    The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998). Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to Rhizobium tropici. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989–3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU r

  19. Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean.

    PubMed

    Cardoso, Aline Assis; Andraus, Michel de Paula; Borba, Tereza Cristina de Oliveira; Martin-Didonet, Claudia Cristina Garcia; Ferreira, Enderson Petrônio de Brito

    This study aimed to evaluate the tolerance to salinity and temperature, the genetic diversity and the symbiotic efficiency of rhizobia isolates obtained from wild genotypes of common bean cultivated in soil samples from the States of Goiás, Minas Gerais and Paraná. The isolates were subjected to different NaCl concentrations (0%, 1%, 2%, 4% and 6%) at different temperatures (28°C, 33°C, 38°C, 43°C and 48°C). Genotypic characterization was performed based on BOX-PCR, REP-PCR markers and 16S rRNA sequencing. An evaluation of symbiotic efficiency was carried out under greenhouse conditions in autoclaved Leonard jars. Among 98 isolates about 45% of them and Rhizobium freirei PRF81 showed a high tolerance to temperature, while 24 isolates and Rhizobium tropici CIAT899 were able to use all of the carbon sources studied. Clustering analysis based on the ability to use carbon sources and on the tolerance to salinity and temperature grouped 49 isolates, R. tropici CIAT899 and R. tropici H12 with a similarity level of 76%. Based on genotypic characterization, 65% of the isolates showed an approximately 66% similarity with R. tropici CIAT899 and R. tropici H12. About 20% of the isolates showed symbiotic efficiency similar to or better than the best Rhizobium reference strain (R. tropici CIAT899). Phylogenetic analysis of the 16S rRNA revealed that two efficient isolates (ALSG5A1 and JPrG6A8) belong to the group of strains used as commercial inoculant for common bean in Brazil and must be assayed in field experiments.

  20. Medicago truncatula Natural Resistance-Associated Macrophage Protein1 Is Required for Iron Uptake by Rhizobia-Infected Nodule Cells1[OPEN

    PubMed Central

    Tejada-Jiménez, Manuel; Castro-Rodríguez, Rosario; Kryvoruchko, Igor; Lucas, M. Mercedes; Udvardi, Michael; Imperial, Juan; González-Guerrero, Manuel

    2015-01-01

    Iron is critical for symbiotic nitrogen fixation (SNF) as a key component of multiple ferroproteins involved in this biological process. In the model legume Medicago truncatula, iron is delivered by the vasculature to the infection/maturation zone (zone II) of the nodule, where it is released to the apoplast. From there, plasma membrane iron transporters move it into rhizobia-containing cells, where iron is used as the cofactor of multiple plant and rhizobial proteins (e.g. plant leghemoglobin and bacterial nitrogenase). MtNramp1 (Medtr3g088460) is the M. truncatula Natural Resistance-Associated Macrophage Protein family member, with the highest expression levels in roots and nodules. Immunolocalization studies indicate that MtNramp1 is mainly targeted to the plasma membrane. A loss-of-function nramp1 mutant exhibited reduced growth compared with the wild type under symbiotic conditions, but not when fertilized with mineral nitrogen. Nitrogenase activity was low in the mutant, whereas exogenous iron and expression of wild-type MtNramp1 in mutant nodules increased nitrogen fixation to normal levels. These data are consistent with a model in which MtNramp1 is the main transporter responsible for apoplastic iron uptake by rhizobia-infected cells in zone II. PMID:25818701

  1. Iron-induced nitric oxide leads to an increase in the expression of ferritin during the senescence of Lotus japonicus nodules.

    PubMed

    Chungopast, Sirinapa; Duangkhet, Mallika; Tajima, Shigeyuki; Ma, Jian Feng; Nomura, Mika

    2017-01-01

    Iron is an essential nutrient for legume-rhizobium symbiosis and accumulates abundantly in the nodules. However, the concentration of free iron in the cells is strictly controlled to avoid toxicity. It is known that ferritin accumulates in the cells as an iron storage protein. During nodule senescence, the expression of the ferritin gene, Ljfer1, was induced in Lotus japonicus. We investigated a signal transduction pathway leading to the increase of Ljfer1 in the nodule. The Ljfer1 promoter of L. japonicus contains a conserved Iron-Dependent Regulatory Sequence (IDRS). The expression of Ljfer1 was induced by the application of iron or sodium nitroprusside, which is a nitric oxide (NO) donor. The application of iron to the nodule increased the level of NO. These data strongly suggest that iron-induced NO leads to increased expression of Ljfer1 during the senescence of L. japonicus nodules. Copyright © 2016 Elsevier GmbH. All rights reserved.

  2. CAROTENOID CLEAVAGE DIOXYGENASE 7 modulates plant growth, reproduction, senescence, and determinate nodulation in the model legume Lotus japonicus

    PubMed Central

    Liu, Junwei; Novero, Mara; Charnikhova, Tatsiana; Ferrandino, Alessandra; Schubert, Andrea; Ruyter-Spira, Carolien; Bonfante, Paola; Lovisolo, Claudio; Bouwmeester, Harro J.; Cardinale, Francesca

    2013-01-01

    Strigolactones (SLs) are newly identified hormones that regulate multiple aspects of plant development, infection by parasitic weeds, and mutualistic symbiosis in the roots. In this study, the role of SLs was studied for the first time in the model plant Lotus japonicus using transgenic lines silenced for CAROTENOID CLEAVAGE DIOXYGENASE 7 (LjCCD7), the orthologue of Arabidopsis More Axillary Growth 3. Transgenic LjCCD7-silenced plants displayed reduced height due to shorter internodes, and more branched shoots and roots than the controls, and an increase in total plant biomass, while their root:shoot ratio remained unchanged. Moreover, these lines had longer primary roots, delayed senescence, and reduced flower/pod numbers from the third round of flower and pod setting onwards. Only a mild reduction in determinate nodule numbers and hardly any impact on the colonization by arbuscular mycorrhizal fungi were observed. The results show that the impairment of CCD7 activity in L. japonicus leads to a phenotype linked to SL functions, but with specific features possibly due to the peculiar developmental pattern of this plant species. It is believed that the data also link determinate nodulation, plant reproduction, and senescence to CCD7 function for the first time. PMID:23567864

  3. Rhizobia from Lanzarote, the Canary Islands, That Nodulate Phaseolus vulgaris Have Characteristics in Common with Sinorhizobium meliloti Isolates from Mainland Spain▿

    PubMed Central

    Zurdo-Piñeiro, José Luis; García-Fraile, Paula; Rivas, Raúl; Peix, Alvaro; León-Barrios, Milagros; Willems, Anne; Mateos, Pedro Francisco; Martínez-Molina, Eustoquio; Velázquez, Encarna; van Berkum, Peter

    2009-01-01

    The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote. PMID:19218416

  4. Phenotypic, genomic and phylogenetic characteristics of rhizobia isolated from root nodules of Robinia pseudoacacia (black locust) growing in Poland and Japan.

    PubMed

    Mierzwa, Bozena; Wdowiak-Wróbel, Sylwia; Małek, Wanda

    2009-09-01

    Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.

  5. Genetic diversity of indigenous soybean-nodulating rhizobia in response to locally-based long term fertilization in a Mollisol of Northeast China.

    PubMed

    Yan, Jun; Chen, WenFeng; Han, XiaoZeng; Wang, EnTao; Zou, WenXiu; Zhang, ZhiMing

    2017-01-01

    The influences of five different fertilizer treatments on diversity of rhizobia in soybean nodule were investigated in a long-term experiment with with four replicates: (1) control (without fertilization), (2) balanced NPK fertilizer (NPK), and (3-5) unbalanced chemical fertilizers without one of the major elements (NP, PK, and NK) in Mollisol in Northeast China. The highest soybean yield was observed in the NPK treatment. Total of 200 isolates were isolated and grouped into four Bradyrhizobium genospecies corresponding to B. japonicum, B. diazoefficiens, B. ottawaense and Bradyrhizobium sp. I, based upon the multilocus sequence analysis of 6 housekeeping genes. The Bradyrhizobium sp. I was extensively distributed throughout the study site and was recorded as the dominant soybean rhizobia (82.5-87.5%). Except the NK treatment, the other fertilizer treatments had no effect on rhizobial species composition. Compared with the CK treatment, all the fertilizer treatments decreased species richness, diversity and evenness. The soil organic carbon contents, available N content and pH were the key soil factors to rhizobial community structure. Results suggest that long-term fertilization can decrease rhizobial species diversity, while balanced fertilization with NPK is the most suitable fertilization regime if taking both soybean yields and rhizobial diversity into account.

  6. Phytohormone regulation of legume-rhizobia interactions.

    PubMed

    Ferguson, Brett J; Mathesius, Ulrike

    2014-07-01

    The symbiosis between legumes and nitrogen fixing bacteria called rhizobia leads to the formation of root nodules. Nodules are highly organized root organs that form in response to Nod factors produced by rhizobia, and they provide rhizobia with a specialized niche to optimize nutrient exchange and nitrogen fixation. Nodule development and invasion by rhizobia is locally controlled by feedback between rhizobia and the plant host. In addition, the total number of nodules on a root system is controlled by a systemic mechanism termed 'autoregulation of nodulation'. Both the local and the systemic control of nodulation are regulated by phytohormones. There are two mechanisms by which phytohormone signalling is altered during nodulation: through direct synthesis by rhizobia and through indirect manipulation of the phytohormone balance in the plant, triggered by bacterial Nod factors. Recent genetic and physiological evidence points to a crucial role of Nod factor-induced changes in the host phytohormone balance as a prerequisite for successful nodule formation. Phytohormones synthesized by rhizobia enhance symbiosis effectiveness but do not appear to be necessary for nodule formation. This review provides an overview of recent advances in our understanding of the roles and interactions of phytohormones and signalling peptides in the regulation of nodule infection, initiation, positioning, development, and autoregulation. Future challenges remain to unify hormone-related findings across different legumes and to test whether hormone perception, response, or transport differences among different legumes could explain the variety of nodules types and the predisposition for nodule formation in this plant family. In addition, the molecular studies carried out under controlled conditions will need to be extended into the field to test whether and how phytohormone contributions by host and rhizobial partners affect the long term fitness of the host and the survival and

  7. Diversity of rhizobia from nodules of the leguminous tree Gliricidia sepium, a natural host of Rhizobium tropici.

    PubMed

    Acosta-Durán, Carlos; Martínez-Romero, Esperanza

    2002-08-01

    The Rhizobium species that nodulate the legume tree Gliricidia sepium were analyzed by phenotypic characteristics (including nodule formation in different hosts), PCR-RFLP patterns and sequences of 16S rRNA genes, multilocus enzyme electrophoresis, and plasmid patterns. Strains of Rhizobium tropici type A and B, Sinorhizobium spp., and Rhizobium etli bv. phaseoli were encountered in G. sepium nodules and their presence depended on the site sampled.

  8. Diversity and biogeography of rhizobia isolated from root nodules of Glycine max grown in Hebei Province, China.

    PubMed

    Li, Qin Qin; Wang, En Tao; Zhang, Yun Zeng; Zhang, Yan Ming; Tian, Chang Fu; Sui, Xin Hua; Chen, Wen Feng; Chen, Wen Xin

    2011-05-01

    A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S-23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline-saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.

  9. The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules.

    PubMed

    Carsolio, C; Campos, F; Sánchez, F; Rocha-Sosa, M

    1994-12-01

    In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.

  10. Genetic diversity of nodulating and non-nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China.

    PubMed

    Wu, Li Juan; Wang, Hai Qing; Wang, En Tao; Chen, Wen Xin; Tian, Chang Fu

    2011-06-01

    A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.

  11. Lotus japonicus SUNERGOS1 encodes a predicted subunit A of a DNA topoisomerase VI that is required for nodule differentiation and accommodation of rhizobial infection

    PubMed Central

    Yoon, Hwi Joong; Hossain, Md Shakhawat; Held, Mark; Hou, Hongwei; Kehl, Marilyn; Tromas, Alexandre; Sato, Shusei; Tabata, Satoshi; Andersen, Stig Uggerhøj; Stougaard, Jens; Ross, Loretta; Szczyglowski, Krzysztof

    2014-01-01

    A symbiotic mutant of Lotus japonicus, called sunergos1-1 (suner1-1), originated from a har1-1 suppressor screen. suner1-1 supports epidermal infection by Mesorhizobium loti and initiates cell divisions for organogenesis of nodule primordia. However, these processes appear to be temporarily stalled early during symbiotic interaction, leading to a low nodule number phenotype. This defect is ephemeral and near wild-type nodule numbers are reached by suner1-1 at a later point after infection. Using an approach that combined map-based cloning and next-generation sequencing we have identified the causative mutation and show that the suner1-1 phenotype is determined by a weak recessive allele, with the corresponding wild-type SUNER1 locus encoding a predicted subunit A of a DNA topoisomerase VI. Our data suggest that at least one function of SUNER1 during symbiosis is to participate in endoreduplication, which is an essential step during normal differentiation of functional, nitrogen-fixing nodules. PMID:24661810

  12. Core and symbiotic genes reveal nine Mesorhizobium genospecies and three symbiotic lineages among the rhizobia nodulating Cicer canariense in its natural habitat (La Palma, Canary Islands).

    PubMed

    Armas-Capote, Natalia; Pérez-Yépez, Juan; Martínez-Hidalgo, Pilar; Garzón-Machado, Víctor; Del Arco-Aguilar, Marcelino; Velázquez, Encarna; León-Barrios, Milagros

    2014-03-01

    Cicer canariense is a threatened perennial wild chickpea endemic to the Canary Islands. In this study, rhizobia that nodulate this species in its natural habitats on La Palma (Canary Islands) were characterised. The genetic diversity and phylogeny were estimated by RAPD profiles, 16S-RFLP analysis and sequencing of the rrs, recA, glnII and nodC genes. 16S-RFLP grouped the isolates within the Mesorhizobium genus and distinguished nine different ribotypes. Four branches included minority ribotypes (3-5 isolates), whereas another five contained the predominant ribotypes that clustered with reference strains of M. tianshanense/M. gobiense/M. metallidurans, M. caraganae, M. opportunistum, M. ciceri and M. tamadayense. The sequences confirmed the RFLP groupings but resolved additional internal divergence within the M. caraganae group and outlined several potential novel species. The RAPD profiles showed a high diversity at the infraspecific level, except in the M. ciceri group. The nodC phylogeny resolved three symbiotic lineages. A small group of isolates had sequences identical to those of symbiovar ciceri and were only detected in M. ciceri isolates. Another group of sequences represented a novel symbiotic lineage that was associated with two particular chromosomal backgrounds. However, nodC sequences closely related to symbiovar loti predominated in most isolates, and they were detected in several chromosomal backgrounds corresponding to up to nine Mesorhizobium lineages. The results indicated that C. canariense is a promiscuous legume that can be nodulated by several rhizobial species and symbiotypes, which means it will be important to determine the combination of core and symbiotic genes that produce the most effective symbiosis. Copyright © 2013 Elsevier GmbH. All rights reserved.

  13. Genetic diversity, community structure and distribution of rhizobia in the root nodules of Caragana spp. from arid and semi-arid alkaline deserts, in the north of China.

    PubMed

    Li, Mao; Li, Yán; Chen, Wen Feng; Sui, Xin Hua; Li, Ying; Li, Yàn; Wang, En Tao; Chen, Wen Xin

    2012-06-01

    The genetic diversity of 88 Caragana nodule rhizobial isolates, collected from arid and semi-arid alkaline sandy soils in the north of China, was assessed by PCR-RFLP of the 16S rRNA gene and the 16S-23S IGS, as well as the phylogenies of housekeeping genes (atpD, glnII and recA) and symbiotic genes (nodC and nifH). Of the 88 strains, 69 were placed in the genus Mesorhizobium, 16 in Rhizobium and 3 in Bradyrhizobium. Mesorhizobium amorphae, Mesorhizobium septentrionale, Mesorhizobium temperatum and Rhizobium yanglingense were the four predominant microsymbionts associated with Caragana spp. in the surveyed regions, and M. septentrionale was widely distributed among the sampling sites. Phylogenies of nodC and nifH genes showed that two kinds of symbiotic genes existed, corresponding to Mesorhizobium and Rhizobium, respectively. Available phosphorous (P) and potassium (K) contents were the main soil factors correlated with the distribution of these rhizobia in the sampling regions. Positive correlations between the available higher P content/lower K content and the dominance of Mesorhizobium species (M. temperatum, M. amorphae and M. septentrionale), and between the lower P content/higher K content and the dominance of R. yanglingense were found.

  14. Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the Lotus japonicus–Mesorhizobium loti symbiosis

    PubMed Central

    Fukudome, Mitsutaka; Calvo-Begueria, Laura; Kado, Tomohiro; Osuki, Ken-ichi; Rubio, Maria Carmen; Murakami, Ei-ichi; Nagata, Maki; Kucho, Ken-ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

    2016-01-01

    Leghemoglobins transport and deliver O2 to the symbiosomes inside legume nodules and are essential for nitrogen fixation. However, the roles of other hemoglobins (Hbs) in the rhizobia–legume symbiosis are unclear. Several Lotus japonicus mutants affecting LjGlb1-1, a non-symbiotic class 1 Hb, have been used to study the function of this protein in symbiosis. Two TILLING alleles with single amino acid substitutions (A102V and E127K) and a LORE1 null allele with a retrotransposon insertion in the 5′-untranslated region (96642) were selected for phenotyping nodulation. Plants of all three mutant lines showed a decrease in long infection threads and nodules, and an increase in incipient infection threads. About 4h after inoculation, the roots of mutant plants exhibited a greater transient accumulation of nitric oxide (NO) than did the wild-type roots; nevertheless, in vitro NO dioxygenase activities of the wild-type, A102V, and E127K proteins were similar, suggesting that the mutated proteins are not fully functional in vivo. The expression of LjGlb1-1, but not of the other class 1 Hb of L. japonicus (LjGlb1-2), was affected during infection of wild-type roots, further supporting a specific role for LjGlb1-1. In conclusion, the LjGlb1-1 mutants reveal that this protein is required during rhizobial infection and regulates NO levels. PMID:27443280

  15. Identification of several soybean cytosolic glutamine synthetase transcripts highly or specifically expressed in nodules: expression studies using one of the corresponding genes in transgenic Lotus corniculatus.

    PubMed

    Marsolier, M C; Debrosses, G; Hirel, B

    1995-01-01

    A DNA fragment containing sequences hybridizing to the 5' region of GS15, a gene encoding soybean cytosolic glutamine synthetase, was isolated from a soybean genomic library. Mapping and partial sequence analysis of the genomic clone revealed that it encodes a cytosolic GS gene, GS21, which is different from GS15. In parallel, a number of cDNA clones encoding cytosolic GS were isolated using the coding region of pGS20 as a probe (pGS20 is a cDNA clone which corresponds to a transcript of the GS15 gene). Two new full-length cDNAs designated pGS34 and pGS38 were isolated and sequenced. In the 5' non-coding region a strong homology was found between the two clones and the GS21 gene. However, none of these sequences were identical, which suggests that there are at least three members in this group of genes. In order to determine their relative levels of transcription, specific sequences from pGS34, pGS38 and GS21 were used in an RNAse protection assay. This experiment clearly showed that GS21 and the gene encoding pGS38 are specifically expressed in young or mature nodules, whereas the gene encoding pGS34 is highly transcribed in nodules and constitutively expressed at a lower level in other soybean organs. In order to further analyse the molecular mechanisms controlling GS21 transcription, different fragments of the promoter region were fused to the Escherichia coli reporter gene encoding beta-glucuronidase (GUS) and the constructs were introduced into Lotus corniculatus via Agrobacterium rhizogenes-mediated transformation. Analysis of GUS activity showed that the GS21 promoter-GUS constructs were expressed in the vasculature of all vegetative organs. This result is discussed in relation to species-specific metabolic and developmental characteristics of soybean and Lotus.

  16. Knockdown of LjALD1, AGD2-like defense response protein 1, influences plant growth and nodulation in Lotus japonicus.

    PubMed

    Chen, Wei; Li, Xueliu; Tian, Lu; Wu, Pingzhi; Li, Meiru; Jiang, Huawu; Chen, Yaping; Wu, Guojiang

    2014-11-01

    The discovery of the enzyme L,L-diaminopimelate aminotransferase (LL-DAP-AT, EC 2.6.1.83) uncovered a unique step in the L-lysine biosynthesis pathway in plants. In Arabidopsis thaliana, LL-DAP-AT has been shown to play a key role in plant-pathogen interactions by regulation of the salicylic acid (SA) signaling pathway. Here, a full-length cDNA of LL-DAP-AT named as LjALD1 from Lotus japonicus (Regel) Larsen was isolated. The deduced amino acid sequence shares 67% identity with the Arabidopsis aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (AtALD1) and is predicted to contain the same key elements: a conserved aminotransferase domain and a pyridoxal-5'-phosphate cofactor binding site. Quantitative real-time PCR analysis showed that LjALD1 was expressed in all L. japonicus tissues tested, being strongest in nodules. Expression was induced in roots that had been infected with the symbiotic rhizobium Mesorhizobium loti or treated with SA agonist benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid. LjALD1 Knockdown exhibited a lower SA content, an increased number of infection threads and nodules, and a slight reduction in nodule size. In addition, compared with wild-type, root growth was increased and shoot growth was suppressed in LjALD1 RNAi plant lines. These results indicate that LjALD1 may play important roles in plant development and nodulation via SA signaling in L. japonicus. © 2014 Institute of Botany, Chinese Academy of Sciences.

  17. LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

    PubMed

    Duan, Guilan; Hakoyama, Tsuneo; Kamiya, Takehiro; Miwa, Hiroki; Lombardo, Fabien; Sato, Shusei; Tabata, Satoshi; Chen, Zheng; Watanabe, Toshihiro; Shinano, Takuro; Fujiwara, Toru

    2017-03-09

    Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp(292) to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nM. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not Mo delivery to the nodules. This article is protected by copyright. All rights reserved.

  18. How legumes recognize rhizobia.

    PubMed

    Via, Virginia Dalla; Zanetti, María Eugenia; Blanco, Flavio

    2016-01-01

    Legume plants have developed the capacity to establish symbiotic interactions with soil bacteria (known as rhizobia) that can convert N2 to molecular forms that are incorporated into the plant metabolism. The first step of this relationship is the recognition of bacteria by the plant, which allows to distinguish potentially harmful species from symbiotic partners. The main molecular determinant of this symbiotic interaction is the Nod Factor, a diffusible lipochitooligosaccharide molecule produced by rhizobia and perceived by LysM receptor kinases; however, other important molecules involved in the specific recognition have emerged over the years. Secreted exopolysaccharides and the lipopolysaccharides present in the bacterial cell wall have been proposed to act as signaling molecules, triggering the expression of specific genes related to the symbiotic process. In this review we will briefly discuss how transcriptomic analysis are helping to understand how multiple signaling pathways, triggered by the perception of different molecules produced by rhizobia, control the genetic programs of root nodule organogenesis and bacterial infection. This knowledge can help to understand how legumes have evolved to recognize and establish complex ecological relationships with particular species and strains of rhizobia, adjusting gene expression in response to identity determinants of bacteria.

  19. Analysis of Mesorhizobium loti glycogen operon: effect of phosphoglucomutase (pgm) and glycogen synthase (g/gA) null mutants on nodulation of Lotus tenuis.

    PubMed

    Lepek, Viviana C; D'Antuono, Alejandra L; Tomatis, Pablo E; Ugalde, Juan E; Giambiagi, Susana; Ugalde, Rodolfo A

    2002-04-01

    The phosphoglucomutase (pgm) gene codes for a key enzyme required for the formation of UDP-glucose and ADP-glucose, the sugar donors for the biosynthesis of glucose containing polysaccharides. A Mesorhizobium loti pgm null mutant obtained in this study contains an altered form of lipopolysaccharide (LPS), lacks exopolysaccharide (EPS), beta cyclic glucan, and glycogen and is unable to nodulate Lotus tenuis. The nonnodulating phenotype of the pgm mutant was not due to the absence of glycogen, since a glycogen synthase (glgA) null mutant effectively nodulates this legume. In M. loti, pgm is part of the glycogen metabolism gene cluster formed by GlgP (glycogen phosphorylase), glgB (glycogen branching), glgC (ADP-glucose pyrophosphorylase), glgA, pgm, and glgX (glycogen debranching). The genes are transcribed as a single transcript from glgP to at least pgm under the control of a strong promoter (promoter I) upstream of glgP. An alternative promoter (promoter II), mapping in a 154-bp DNA fragment spanning 85 bp upstream of the glgA start codon and the first 69 bp of the glgA coding region, controls the expression of glgA and pgm, independently of the rest of the upstream genes. Primer extension experiments showed that transcription starts 19 bp upstream of the glgA start codon.

  20. Protocols for growing plant symbioses; rhizobia.

    PubMed

    Gourion, Benjamin; Bourcy, Marie; Cosson, Viviane; Ratet, Pascal

    2013-01-01

    Legume plants are used as a protein source for human and animal nutrition. The high protein content of legume plants is achieved via the establishment of a root symbiosis with rhizobia that allows the reduction of atmospheric nitrogen. In recent years, M. truncatula has been used as a legume model in view of its small, diploid genome, self-fertility, and short life cycle, as well as availability of various genomic and genetic tools. The choice and use of this model legume plant in parallel with the other model legume Lotus japonicus for molecular studies has triggered extensive studies that have now identified the molecular actors corresponding to the first steps of the plant-bacterial interaction. The use of this plant as model in an increasing number of laboratories has resulted in the development of numerous protocols to study the establishment of the symbiosis. The media and growth conditions used in our laboratory to nodulate wild-type or transgenic plants as well as wild-type plants with transgenic hairy root system are described below.

  1. SymRK defines a common genetic basis for plant root endosymbioses with arbuscular mycorrhiza fungi, rhizobia, and Frankiabacteria

    PubMed Central

    Gherbi, Hassen; Markmann, Katharina; Svistoonoff, Sergio; Estevan, Joan; Autran, Daphné; Giczey, Gabor; Auguy, Florence; Péret, Benjamin; Laplaze, Laurent; Franche, Claudine; Parniske, Martin; Bogusz, Didier

    2008-01-01

    Root endosymbioses vitally contribute to plant nutrition and fitness worldwide. Nitrogen-fixing root nodulation, confined to four plant orders, encompasses two distinct types of associations, the interaction of legumes (Fabales) with rhizobia bacteria and actinorhizal symbioses, where the bacterial symbionts are actinomycetes of the genus Frankia. Although several genetic components of the host–symbiont interaction have been identified in legumes, the genetic basis of actinorhiza formation is unknown. Here, we show that the receptor-like kinase gene SymRK, which is required for nodulation in legumes, is also necessary for actinorhiza formation in the tree Casuarina glauca. This indicates that both types of nodulation symbiosis share genetic components. Like several other legume genes involved in the interaction with rhizobia, SymRK is also required for the interaction with arbuscular mycorrhiza (AM) fungi. We show that SymRK is involved in AM formation in C. glauca as well and can restore both nodulation and AM symbioses in a Lotus japonicus symrk mutant. Taken together, our results demonstrate that SymRK functions as a vital component of the genetic basis for both plant–fungal and plant–bacterial endosymbioses and is conserved between legumes and actinorhiza-forming Fagales. PMID:18316735

  2. Functional analysis of chimeric lysin motif domain receptors mediating Nod factor-induced defense signaling in Arabidopsis thaliana and chitin-induced nodulation signaling in Lotus japonicus.

    PubMed

    Wang, Wei; Xie, Zhi-Ping; Staehelin, Christian

    2014-04-01

    The expression of chimeric receptors in plants is a way to activate specific signaling pathways by corresponding signal molecules. Defense signaling induced by chitin from pathogens and nodulation signaling of legumes induced by rhizobial Nod factors (NFs) depend on receptors with extracellular lysin motif (LysM) domains. Here, we constructed chimeras by replacing the ectodomain of chitin elicitor receptor kinase 1 (AtCERK1) of Arabidopsis thaliana with ectodomains of NF receptors of Lotus japonicus (LjNFR1 and LjNFR5). The hybrid constructs, named LjNFR1-AtCERK1 and LjNFR5-AtCERK1, were expressed in cerk1-2, an A. thaliana CERK1 mutant lacking chitin-induced defense signaling. When treated with NFs from Rhizobium sp. NGR234, cerk1-2 expressing both chimeras accumulated reactive oxygen species, expressed chitin-responsive defense genes and showed increased resistance to Fusarium oxysporum. In contrast, expression of a single chimera showed no effects. Likewise, the ectodomains of LjNFR1 and LjNFR5 were replaced by those of OsCERK1 (Oryza sativa chitin elicitor receptor kinase 1) and OsCEBiP (O. sativa chitin elicitor-binding protein), respectively. The chimeras, named OsCERK1-LjNFR1 and OsCEBiP-LjNFR5, were expressed in L. japonicus NF receptor mutants (nfr1-1; nfr5-2) carrying a GUS (β-glucuronidase) gene under the control of the NIN (nodule inception) promoter. Upon chitin treatment, GUS activation reflecting nodulation signaling was observed in the roots of NF receptor mutants expressing both chimeras, whereas a single construct was not sufficient for activation. Hence, replacement of ectodomains in LysM domain receptors provides a way to specifically trigger NF-induced defense signaling in non-legumes and chitin-induced nodulation signaling in legumes. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  3. Common symbiosis genes CERBERUS and NSP1 provide additional insight into the establishment of arbuscular mycorrhizal and root nodule symbioses in Lotus japonicus.

    PubMed

    Nagae, Miwa; Takeda, Naoya; Kawaguchi, Masayoshi

    2014-01-01

    Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) share several common symbiotic components, and many of the common symbiosis mutants block the entry of symbionts into the roots. We recently reported that CERBERUS (an E3 ubiquitin ligase) and NSP1 (a GRAS family transcription factor), required for RNS, also modulate AMS development in Lotus japonicus. The novel common symbiosis mutants, cerberus and nsp1, have low colonization of arbuscular mycorrhiza (AM) fungi, caused by a defect in internal hyphal elongation and by a decreased fungal entry into the roots, respectively. Here, we showed that CERBERUS was induced at the sites of symbiotic fungal or bacterial infection. NSP1 has been implicated in a strigolactone biosynthesis gene DWARF27 expression. Nevertheless, in nsp1, DWARF27 was induced by inoculation with AM fungi, implying the existence of a NSP1-independent regulatory mechanism of strigolactone biosynthesis during AMS establishment. These results support functional analysis of CERBERUS and NSP1, and also contribute to elucidation of common mechanisms in AMS and RNS.

  4. Common symbiosis genes CERBERUS and NSP1 provide additional insight into the establishment of arbuscular mycorrhizal and root nodule symbioses in Lotus japonicus

    PubMed Central

    Nagae, Miwa; Takeda, Naoya; Kawaguchi, Masayoshi

    2014-01-01

    Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) share several common symbiotic components, and many of the common symbiosis mutants block the entry of symbionts into the roots. We recently reported that CERBERUS (an E3 ubiquitin ligase) and NSP1 (a GRAS family transcription factor), required for RNS, also modulate AMS development in Lotus japonicus. The novel common symbiosis mutants, cerberus and nsp1, have low colonization of arbuscular mycorrhiza (AM) fungi, caused by a defect in internal hyphal elongation and by a decreased fungal entry into the roots, respectively. Here, we showed that CERBERUS was induced at the sites of symbiotic fungal or bacterial infection. NSP1 has been implicated in a strigolactone biosynthesis gene DWARF27 expression. Nevertheless, in nsp1, DWARF27 was induced by inoculation with AM fungi, implying the existence of a NSP1-independent regulatory mechanism of strigolactone biosynthesis during AMS establishment. These results support functional analysis of CERBERUS and NSP1, and also contribute to elucidation of common mechanisms in AMS and RNS. PMID:24705023

  5. Shoot-derived cytokinins systemically regulate root nodulation.

    PubMed

    Sasaki, Takema; Suzaki, Takuya; Soyano, Takashi; Kojima, Mikiko; Sakakibara, Hitoshi; Kawaguchi, Masayoshi

    2014-09-19

    Legumes establish symbiotic associations with nitrogen-fixing bacteria (rhizobia) in root nodules to obtain nitrogen. Legumes control nodule number through long-distance communication between roots and shoots, maintaining the proper symbiotic balance. Rhizobial infection triggers the production of mobile CLE-RS1/2 peptides in Lotus japonicus roots; the perception of the signal by receptor kinase HAR1 in shoots presumably induces the production of an unidentified shoot-derived inhibitor (SDI) that translocates to roots and blocks further nodule development. Here we show that, CLE-RS1/2-HAR1 signalling activates the production of shoot-derived cytokinins, which have an SDI-like capacity to systemically suppress nodulation. In addition, we show that LjIPT3 is involved in nodulation-related cytokinin production in shoots. The expression of LjIPT3 is activated in an HAR1-dependent manner. We further demonstrate shoot-to-root long-distance transport of cytokinin in L. japonicus seedlings. These findings add essential components to our understanding of how legumes control nodulation to balance nutritional requirements and energy status.

  6. Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum

    PubMed Central

    Rincón-Rosales, Reiner; Lloret, Lourdes; Ponce, Edith; Martínez-Romero, Esperanza

    2009-01-01

    Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum. A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA–DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima (Rhizobium, Sinorhizobium and Mesorhizobium) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella. PMID:19120461

  7. Plasmid transfer systems in the rhizobia.

    PubMed

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  8. Explaining coexistence of nitrogen fixing and non-fixing rhizobia in legume-rhizobia mutualism using mathematical modeling.

    PubMed

    Moyano, G; Marco, D; Knopoff, D; Torres, G; Turner, C

    2017-10-01

    In the mutualism established between legumes and soil bacteria known as rhizobia, bacteria from soil infect plants roots and reproduce inside root nodules where they fix atmospheric N2 for plant nutrition, receiving carbohydrates in exchange. Host-plant sanctions against non N2 fixing, cheating bacterial symbionts have been proposed to act in the legume-Rhizobium symbiosis, to preserve the mutualistic relationship. Sanctions include decreased rhizobial survival in nodules occupied by cheating rhizobia. Previously, a simple population model experimentally based showed that the coexistence of fixing and cheating rhizobia strains commonly found in field conditions is possible, and that the inclusion of sanctions leads to the extinction of cheating strains in soil. Here, we extend the previous model to include other factors that could complicate the sanction scenario, like horizontal transmission of symbiotic plasmids, turning non-nodulating strains into nodulating rhizobia, and competition between fixing and cheating strains for nodulation. In agreement with previous results, we show that plant populations persist even in the presence of cheating rhizobia without incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N2 from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions leads to the unrealistic extinction of cheater strains in soil. Our results agree with increasing experimental evidence and theoretical work showing that mutualisms can persist in presence of cheating partners. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Verification and rapid identification of soybean rhizobia in Indian soils.

    PubMed

    Annapurna, K; Balakrishnan, N; Vital, L

    2007-04-01

    Sixty root nodule isolates of soybean rhizobia indigenous to eight field sites in India were characterized using PCR-RFLP for repeated sequence RSalpha a 1195-bp DNA fragment, indole acetic acid production, and nitrogenase activity. Site-dependent variations were observed in terms of IAA production and nitrogenase activities. RSalpha was conserved in slow-growing soybean rhizobia across locations and sites and was absent in other Rhizobiaceae members and other bacterial genera. The results suggest that RSalpha can be a useful molecular marker for slow-growing soybean rhizobia. The study also showed the low presence of soybean nodulating fast growers in Indian soils.

  10. Lotus japonicus ARPC1 is required for rhizobial infection.

    PubMed

    Hossain, Md Shakhawat; Liao, Jinqiu; James, Euan K; Sato, Shusei; Tabata, Satoshi; Jurkiewicz, Anna; Madsen, Lene H; Stougaard, Jens; Ross, Loretta; Szczyglowski, Krzysztof

    2012-10-01

    Remodeling of the plant cell cytoskeleton precedes symbiotic entry of nitrogen-fixing bacteria within the host plant roots. Here we identify a Lotus japonicus gene encoding a predicted ACTIN-RELATED PROTEIN COMPONENT1 (ARPC1) as essential for rhizobial infection but not for arbuscular mycorrhiza symbiosis. In other organisms ARPC1 constitutes a subunit of the ARP2/3 complex, the major nucleator of Y-branched actin filaments. The L. japonicus arpc1 mutant showed a distorted trichome phenotype and was defective in epidermal infection thread formation, producing mostly empty nodules. A few partially colonized nodules that did form in arpc1 contained abnormal infections. Together with previously described L. japonicus Nck-associated protein1 and 121F-specific p53 inducible RNA mutants, which are also impaired in the accommodation of rhizobia, our data indicate that ARPC1 and, by inference a suppressor of cAMP receptor/WASP-family verpolin homologous protein-ARP2/3 pathway, must have been coopted during evolution of nitrogen-fixing symbiosis to specifically mediate bacterial entry.

  11. Plasmid-linked nif and “nod” genes in fast-growing rhizobia that nodulate Glycine max, Psophocarpus tetragonolobus, and Vigna unguiculata

    PubMed Central

    Broughton, William J.; Heycke, Nina; Z.A., Heiner Meyer; Pankhurst, Clive E.

    1984-01-01

    Forty-nine fast-growing Rhizobium strains from the nodules of 26 different tropical legume genera were screened to find isolates that would (i) nodulate, e.g., winged beans, so producing large nodules for RNA and protein isolation; (ii) also nodulate various small-seeded legumes, thus allowing screening of large numbers of mutants; and (iii) harbor plasmids containing nif structural genes as well as other functions involved in nodulation. On the basis of six different criteria, this rhizobial group appeared intermediate between classical fast- and slow-growing organisms, yet all contained plasmids. Plasmid numbers varied from one to five. Hybridizations between DNA prepared from nifDH and the putatative “nod” region of R. meliloti and these plasmids bound to nitrocellulose filters suggested that nif-nod genes are linked on a single sym plasmid. A broad-host-range strain containing a single sym plasmid was chosen for further study. Its plasmid, pMPIK3030a, was isolated on cesium chloride gradients and cloned in the cosmid pJB8, and the overlapping fragments were mapped by homology with the nif and nod regions of R. meliloti. As the wild-type plasmid pMPIK3030a was not self-transmissible, confirmation that the nod genes detected by homology were responsible for nodulation was obtained by introducing the mobilization functions of RP4 (together with Tn5) and selecting transconjugants resistant to kanamycin and neomycin. Transconjugants (obtained at a frequency of about 10-6 per recipient) in Agrobacterium tumefaciens cured of the Ti plasmid produced ineffective nodules on Vigna unguiculata, those in nonnodulating (Nod-) R. meliloti were partially effective, while those in Nod-R. leguminosarum were often fully effective. Images PMID:16593465

  12. Medicago truncatula NIN Is Essential for Rhizobial-Independent Nodule Organogenesis Induced by Autoactive Calcium/Calmodulin-Dependent Protein Kinase1

    PubMed Central

    Marsh, John F.; Rakocevic, Alexandra; Mitra, Raka M.; Brocard, Lysiane; Sun, Jongho; Eschstruth, Alexis; Long, Sharon R.; Schultze, Michael; Ratet, Pascal; Oldroyd, Giles E.D.

    2007-01-01

    The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11∷GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules. PMID:17369436

  13. The Thiamine Biosynthesis Gene THI1 Promotes Nodule Growth and Seed Maturation.

    PubMed

    Nagae, Miwa; Parniske, Martin; Kawaguchi, Masayoshi; Takeda, Naoya

    2016-11-01

    Thiamine (vitamin B1) is essential for living organisms. Unlike animals, plants can synthesize thiamine. In Lotus japonicus, the expression of two thiamine biosynthesis genes, THI1 and THIC, was enhanced by inoculation with rhizobia but not by inoculation with arbuscular mycorrhizal fungi. THIC and THI2 (a THI1 paralog) were expressed in uninoculated leaves. THI2-knockdown plants and the transposon insertion mutant thiC had chlorotic leaves. This typical phenotype of thiamine deficiency was rescued by an exogenous supply of thiamine. In wild-type plants, THI1 was expressed mainly in roots and nodules, and the thi1 mutant had green leaves even in the absence of exogenous thiamine. THI1 was highly expressed in actively dividing cells of nodule primordia. The thi1 mutant had small nodules, and this phenotype was rescued by exogenous thiamine and by THI1 complementation. Exogenous thiamine increased nodule diameter, but the level of arbuscular mycorrhizal colonization was unaffected in the thi1 mutant or by exogenous thiamine. Expression of symbiotic marker genes was induced normally, implying that mainly nodule growth was delayed in the thi1 mutant. Furthermore, this mutant formed many immature seeds with reduced seed weight. These results indicate that thiamine biosynthesis mediated by THI1 enhances nodule enlargement and is required for seed development in L. japonicus. © 2016 American Society of Plant Biologists. All Rights Reserved.

  14. Movement of rhizobia inside tobacco and lifestyle alternation from endophytes to free-living rhizobia on leaves.

    PubMed

    Ji, Kui-Xian; Chi, Feng; Yang, Ming-Feng; Shen, Shi-Hua; Jing, Yu-Xiang; Dazzo, Frank B; Cheng, Hai-Ping

    2010-02-01

    Rhizobia are well-known for their ability to infect and nodulate legume roots, forming a nitrogen-fixing symbiosis of agricultural importance. In addition, recent studies have shown that rhizobia can colonize roots and aerial plant tissues of rice as a model plant of the Graminaceae family. Here we show that rhizobia can invade tobacco, a model plant belonging to the Solanaceae family. Inoculation of seedling roots with five GFP-tagged rhizobial species followed by microscopy and viable plating analyses indicated their colonization of the surface and interior of the whole vegetative plant. Blockage of ascending epiphytic migration by coating the hypocotyls with Vaseline showed that the endophytic rhizobia can exit the leaf interior through stomata and colonize the external phyllosphere habitat. These studies indicate rhizobia can colonize both below and above-ground tissues of tobacco using a dynamic invasion process that involves both epiphytic and endophytic lifestyles.

  15. Characterization of the papilionoid-Burkholderia interaction in the Fynbos biome: The diversity and distribution of beta-rhizobia nodulating Podalyria calyptrata (Fabaceae, Podalyrieae).

    PubMed

    Lemaire, Benny; Van Cauwenberghe, Jannick; Verstraete, Brecht; Chimphango, Samson; Stirton, Charles; Honnay, Olivier; Smets, Erik; Sprent, Janet; James, Euan K; Muasya, A Muthama

    2016-02-01

    The South African Fynbos soils are renowned for nitrogen-fixing Burkholderia associated with diverse papilionoid legumes of the tribes Crotalarieae, Hypocalypteae, Indigofereae, Phaseoleae and Podalyrieae. However, despite numerous rhizobial studies in the region, the symbiotic diversity of Burkholderia has not been investigated in relation to a specific host legume and its geographical provenance. This study analyzed the diversity of nodulating strains of Burkholderia from the legume species Podalyria calyptrata. Diverse lineages were detected that proved to be closely related to Burkholderia taxa, originating from hosts in other legume tribes. By analyzing the genetic variation of chromosomal (recA) and nodulation (nodA) sequence data in relation to the sampling sites we assessed the geographical distribution patterns of the P. calyptrata symbionts. Although we found a degree of genetically differentiated rhizobial populations, a correlation between genetic (recA and nodA) and geographic distances among populations was not observed, suggesting high rates of dispersal and rhizobial colonization within Fynbos soils.

  16. Competitiveness of a Bradyrhizobium sp. strain in soils containing indigenous rhizobia.

    PubMed

    Bogino, Pablo; Banchio, Erika; Bonfiglio, Carlos; Giordano, Walter

    2008-01-01

    The success of rhizobial inoculation on plant roots is often limited by several factors, including environmental conditions, the number of infective cells applied, the presence of competing indigenous (native) rhizobia, and the inoculation method. Many approaches have been taken to solve the problem of inoculant competition by naturalized populations of compatible rhizobia present in soil, but so far without a satisfactory solution. We used antibiotic resistance and molecular profiles as tools to find a reliable and accurate method for competitiveness assay between introduced Bradyrhizobium sp. strains and indigenous rhizobia strains that nodulate peanut in Argentina. The positional advantage of rhizobia soil population for nodulation was assessed using a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed an increase in nodule number per plant and nodule occupancy for strains established in vermiculite. In field experiments, only 9% of total nodules were formed by bacteria inoculated by direct coating of seed, whereas 78% of nodules were formed by bacteria inoculated in the furrow at seeding. In each case, the other nodules were formed by indigenous strains or by both strains (inoculated and indigenous). These findings indicate a positional advantage of native rhizobia or in-furrow inoculated rhizobia for nodulation in peanut.

  17. Specificity in Legume-Rhizobia Symbioses.

    PubMed

    Andrews, Mitchell; Andrews, Morag E

    2017-03-26

    Most species in the Leguminosae (legume family) can fix atmospheric nitrogen (N₂) via symbiotic bacteria (rhizobia) in root nodules. Here, the literature on legume-rhizobia symbioses in field soils was reviewed and genotypically characterised rhizobia related to the taxonomy of the legumes from which they were isolated. The Leguminosae was divided into three sub-families, the Caesalpinioideae, Mimosoideae and Papilionoideae. Bradyrhizobium spp. were the exclusive rhizobial symbionts of species in the Caesalpinioideae, but data are limited. Generally, a range of rhizobia genera nodulated legume species across the two Mimosoideae tribes Ingeae and Mimoseae, but Mimosa spp. show specificity towards Burkholderia in central and southern Brazil, Rhizobium/Ensifer in central Mexico and Cupriavidus in southern Uruguay. These specific symbioses are likely to be at least in part related to the relative occurrence of the potential symbionts in soils of the different regions. Generally, Papilionoideae species were promiscuous in relation to rhizobial symbionts, but specificity for rhizobial genus appears to hold at the tribe level for the Fabeae (Rhizobium), the genus level for Cytisus (Bradyrhizobium), Lupinus (Bradyrhizobium) and the New Zealand native Sophora spp. (Mesorhizobium) and species level for Cicer arietinum (Mesorhizobium), Listia bainesii (Methylobacterium) and Listia angolensis (Microvirga). Specificity for rhizobial species/symbiovar appears to hold for Galega officinalis (Neorhizobium galegeae sv. officinalis), Galega orientalis (Neorhizobium galegeae sv. orientalis), Hedysarum coronarium (Rhizobium sullae), Medicago laciniata (Ensifer meliloti sv. medicaginis), Medicago rigiduloides (Ensifer meliloti sv. rigiduloides) and Trifolium ambiguum (Rhizobium leguminosarum sv. trifolii). Lateral gene transfer of specific symbiosis genes within rhizobial genera is an important mechanism allowing legumes to form symbioses with rhizobia adapted to particular soils

  18. Specificity in Legume-Rhizobia Symbioses

    PubMed Central

    Andrews, Mitchell; Andrews, Morag E.

    2017-01-01

    Most species in the Leguminosae (legume family) can fix atmospheric nitrogen (N2) via symbiotic bacteria (rhizobia) in root nodules. Here, the literature on legume-rhizobia symbioses in field soils was reviewed and genotypically characterised rhizobia related to the taxonomy of the legumes from which they were isolated. The Leguminosae was divided into three sub-families, the Caesalpinioideae, Mimosoideae and Papilionoideae. Bradyrhizobium spp. were the exclusive rhizobial symbionts of species in the Caesalpinioideae, but data are limited. Generally, a range of rhizobia genera nodulated legume species across the two Mimosoideae tribes Ingeae and Mimoseae, but Mimosa spp. show specificity towards Burkholderia in central and southern Brazil, Rhizobium/Ensifer in central Mexico and Cupriavidus in southern Uruguay. These specific symbioses are likely to be at least in part related to the relative occurrence of the potential symbionts in soils of the different regions. Generally, Papilionoideae species were promiscuous in relation to rhizobial symbionts, but specificity for rhizobial genus appears to hold at the tribe level for the Fabeae (Rhizobium), the genus level for Cytisus (Bradyrhizobium), Lupinus (Bradyrhizobium) and the New Zealand native Sophora spp. (Mesorhizobium) and species level for Cicer arietinum (Mesorhizobium), Listia bainesii (Methylobacterium) and Listia angolensis (Microvirga). Specificity for rhizobial species/symbiovar appears to hold for Galega officinalis (Neorhizobium galegeae sv. officinalis), Galega orientalis (Neorhizobium galegeae sv. orientalis), Hedysarum coronarium (Rhizobium sullae), Medicago laciniata (Ensifer meliloti sv. medicaginis), Medicago rigiduloides (Ensifer meliloti sv. rigiduloides) and Trifolium ambiguum (Rhizobium leguminosarum sv. trifolii). Lateral gene transfer of specific symbiosis genes within rhizobial genera is an important mechanism allowing legumes to form symbioses with rhizobia adapted to particular soils

  19. Rhizobial strain involvement in plant growth, nodule protein composition and antioxidant enzyme activities of chickpea-rhizobia symbioses: modulation by salt stress.

    PubMed

    Mhadhbi, Haythem; Jebara, Moez; Limam, Férid; Aouani, Mohamed Elarbi

    2004-09-01

    Mesorhizobium ciceri, Mesorhizobium mediterraneum and Sinorhizobium medicae strains showed different symbiotic performances when inoculated to chickpea (Cicer arietinum L., cv. chetoui) at unstressed conditions and under salt stress. The analysis of nodular proteic composition and antioxidant enzyme activities revealed a polymorphism of patterns on SDS and native PAGE suggesting a potential dependence on the bacterial partner. Salt effect was analysed on plant growth, nitrogen fixation and antioxidant enzymes. M. ciceri, the most efficient strain, seemed to allow a best tolerance to chickpea plants under salt stress. This constraint did not affect the nodular superoxide dismutase (SOD, E.C. 1.15.1.1) activity of the symbiosis implicating the latter strain. This symbiosis showed the least decrease for the nodule protein level and the catalase (CAT, E.C. 1.11.1.6) activity, and the highest increase of peroxidase (POX, E.C. 1.11.1.7) activity that seemed to be related with the tolerance to salt.

  20. The abundance and diversity of legume-nodulating rhizobia in 28-year-old plantations of tropical, subtropical, and exotic tree species: a case study from the Forest Reserve of Bandia, Senegal.

    PubMed

    Sene, Godar; Thiao, Mansour; Samba-Mbaye, Ramatoulaye; Khasa, Damase; Kane, Aboubacry; Mbaye, Mame Samba; Beaulieu, Marie-Ève; Manga, Anicet; Sylla, Samba Ndao

    2013-01-01

    Several fast-growing and multipurpose tree species have been widely used in West Africa to both reverse the tendency of land degradation and restore soil productivity. Although beneficial effects have been reported on soil stabilization, there still remains a lack of information about their impact on soil microorganisms. Our investigation has been carried out in exotic and native tree plantations of 28 years and aimed to survey and compare the abundance and genetic diversity of natural legume-nodulating rhizobia (LNR). The study of LNR is supported by the phylogenetic analysis which clustered the isolates into three genera: Bradyrhizobium, Mesorhizobium, and Sinorhizobium. The results showed close positive correlations between the sizes of LNR populations estimated both in the dry and rainy seasons and the presence of legume tree hosts. There were significant increases in Rhizobium spp. population densities in response to planting with Acacia spp., and high genetic diversities and richness of genotypes were fittest in these tree plantations. This suggests that enrichment of soil Rhizobium spp. populations is host specific. The results indicated also that species of genera Mesorhizobium and Sinorhizobium were lacking in plantations of non-host species. By contrast, there was a widespread distribution of Bradyrhizobium spp. strains across the tree plantations, with no evident specialization in regard to plantation type. Finally, the study provides information about the LNR communities associated with a range of old tree plantations and some aspects of their relationships to soil factors, which may facilitate the management of man-made forest systems that target ecosystem rehabilitation and preservation of soil biota.

  1. Diversity in the rhizobia associated with Phaseolus vulgaris L. in Ecuador, and comparisons with Mexican bean rhizobia.

    PubMed

    Bernal, G; Graham, P H

    2001-06-01

    Common beans (Phaseolus vulgaris L.) have centers of origin in both Mesoamerica and Andean South America, and have been domesticated in each region for perhaps 5000 years. A third major gene pool may exist in Ecuador and Northern Peru. The diversity of the rhizobia associated with beans has also been studied, but to date with an emphasis on the Mesoamerican center of origin. In this study we compared bean rhizobia from Mexico and Andean South America using both phenotypic and phylogenetic approaches. When differences between the rhizobia of these two regions were shown, we then examined the influence of bean cultivar on the most probable number (MPN) count and biodiversity of rhizobia recovered from different soils. Three clusters of bean rhizobia were distinguished using phenotypic analysis and principal-component analysis of Box AIR-PCR banding patterns. They corresponded principally to isolates from Mexico, and the northern and southern Andean regions, with isolates from southern Ecuador exhibiting significant genetic diversity. Rhizobia from Dalea spp., which are infective and effective on beans, may have contributed to the apparent diversity of rhizobia recovered from the Mesoamerican region, while the rhizobia of wild Phaseolus aborigineus from Argentina showed only limited similarity to the other bean rhizobia tested. Use of P. vulgaris cultivars from the Mesoamerican and Andean Phaseolus gene pools as trap hosts did not significantly affect MPN counts of bean rhizobia from the soils of each region, but did influence the diversity of the rhizobia recovered. Such differences in compatibility of host and Rhizobium could be a factor in the poor reputation for nodulation and N2 fixation in this crop.

  2. The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation.

    PubMed

    Mongiardini, Elías J; Ausmees, Nora; Pérez-Giménez, Julieta; Julia Althabegoiti, María; Ignacio Quelas, Juan; López-García, Silvina L; Lodeiro, Aníbal R

    2008-08-01

    The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.

  3. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes.

    PubMed

    Nelson, Matthew S; Sadowsky, Michael J

    2015-01-01

    The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity.

  4. Secretion systems and signal exchange between nitrogen-fixing rhizobia and legumes

    PubMed Central

    Nelson, Matthew S.; Sadowsky, Michael J.

    2015-01-01

    The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses. Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation. Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity. PMID:26191069

  5. Lotus japonicus symRK-14 uncouples the cortical and epidermal symbiotic program.

    PubMed

    Kosuta, Sonja; Held, Mark; Hossain, Md Shakhawat; Morieri, Giulia; Macgillivary, Amanda; Johansen, Christopher; Antolín-Llovera, Meritxell; Parniske, Martin; Oldroyd, Giles E D; Downie, Allan J; Karas, Bogumil; Szczyglowski, Krzysztof

    2011-09-01

    SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

  6. Transport and metabolism in legume-rhizobia symbioses.

    PubMed

    Udvardi, Michael; Poole, Philip S

    2013-01-01

    Symbiotic nitrogen fixation by rhizobia in legume root nodules injects approximately 40 million tonnes of nitrogen into agricultural systems each year. In exchange for reduced nitrogen from the bacteria, the plant provides rhizobia with reduced carbon and all the essential nutrients required for bacterial metabolism. Symbiotic nitrogen fixation requires exquisite integration of plant and bacterial metabolism. Central to this integration are transporters of both the plant and the rhizobia, which transfer elements and compounds across various plant membranes and the two bacterial membranes. Here we review current knowledge of legume and rhizobial transport and metabolism as they relate to symbiotic nitrogen fixation. Although all legume-rhizobia symbioses have many metabolic features in common, there are also interesting differences between them, which show that evolution has solved metabolic problems in different ways to achieve effective symbiosis in different systems.

  7. Molecular diversity of native rhizobia trapped by five field pea genotypes in Indian soils.

    PubMed

    Wadhwa, K; Dudeja, S S; Yadav, R K

    2011-02-01

    Five pea cultivars; HFP 4, HVP 3-5, HFP 9426, Jayanti and Hariyal, being grown in CCS Haryana Agricultural University farm were used to isolate native rhizobia. Selected 54 rhizobia, from all cultivars, were authenticated as rhizobia by plant infectivity test. Along with nodulation, symbiotic effectiveness in terms of symbiotic ratios showed wide range of effectiveness of pea rhizobia from 1.11 to 5.0. DNA of all the 54 rhizobia was extracted and amplified by PCR, using ERIC and 16S rDNA primers. Dendrogram based on ERIC profiles of these 54 rhizobia showed the formation of 13 subclusters at 80% level of similarity. Dendrogram based on RFLP of 16S rDNA by three restriction endonucleases; Msp I, Csp 6I and Rsa I; also formed 13 subclusters at 80% level of similarity. However, positioning of subclusters was different from that of ERIC based dendrogram. Majority of the isolates i.e. 64.8% by ERIC profiles and 44.4% by RFLP of 16S rDNA formed one cluster. Isolates from same nodule were not 100% similar. Considering each cluster representing a rhizobial genotype, both techniques used to assess molecular diversity indicated the presence of 13 genotypes of field pea rhizobia in CCS Haryana Agricultural University farm soil. Two pea rhizobial genotypes were able to nodulate all the five pea cultivars. Furthermore, high strain richness index (0.43-0.5) of field pea rhizobia was observed by both the techniques.

  8. Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase.

    PubMed

    Okamoto, Satoru; Shinohara, Hidefumi; Mori, Tomoko; Matsubayashi, Yoshikatsu; Kawaguchi, Masayoshi

    2013-01-01

    Leguminous plants establish a symbiosis with rhizobia to enable nitrogen fixation in root nodules under the control of the presumed root-to-shoot-to-root negative feedback called autoregulation of nodulation. In Lotus japonicus, autoregulation is mediated by CLE-RS genes that are specifically expressed in the root, and the receptor kinase HAR1 that functions in the shoot. However, the mature functional structures of CLE-RS gene products and the molecular nature of CLE-RS/HAR1 signalling governed by these spatially distant components remain elusive. Here we show that CLE-RS2 is a post-translationally arabinosylated glycopeptide derived from the CLE domain. Chemically synthesized CLE-RS glycopeptides cause significant suppression of nodulation and directly bind to HAR1 in an arabinose-chain and sequence-dependent manner. In addition, CLE-RS2 glycopeptide specifically produced in the root is found in xylem sap collected from the shoot. We propose that CLE-RS glycopeptides are the long sought mobile signals responsible for the initial step of autoregulation of nodulation.

  9. Comprehensive Comparative Genomic and Transcriptomic Analyses of the Legume Genes Controlling the Nodulation Process

    PubMed Central

    Qiao, Zhenzhen; Pingault, Lise; Nourbakhsh-Rey, Mehrnoush; Libault, Marc

    2016-01-01

    Nitrogen is one of the most essential plant nutrients and one of the major factors limiting crop productivity. Having the goal to perform a more sustainable agriculture, there is a need to maximize biological nitrogen fixation, a feature of legumes. To enhance our understanding of the molecular mechanisms controlling the interaction between legumes and rhizobia, the symbiotic partner fixing and assimilating the atmospheric nitrogen for the plant, researchers took advantage of genetic and genomic resources developed across different legume models (e.g., Medicago truncatula, Lotus japonicus, Glycine max, and Phaseolus vulgaris) to identify key regulatory protein coding genes of the nodulation process. In this study, we are presenting the results of a comprehensive comparative genomic analysis to highlight orthologous and paralogous relationships between the legume genes controlling nodulation. Mining large transcriptomic datasets, we also identified several orthologous and paralogous genes characterized by the induction of their expression during nodulation across legume plant species. This comprehensive study prompts new insights into the evolution of the nodulation process in legume plant and will benefit the scientific community interested in the transfer of functional genomic information between species. PMID:26858743

  10. Comprehensive Comparative Genomic and Transcriptomic Analyses of the Legume Genes Controlling the Nodulation Process.

    PubMed

    Qiao, Zhenzhen; Pingault, Lise; Nourbakhsh-Rey, Mehrnoush; Libault, Marc

    2016-01-01

    Nitrogen is one of the most essential plant nutrients and one of the major factors limiting crop productivity. Having the goal to perform a more sustainable agriculture, there is a need to maximize biological nitrogen fixation, a feature of legumes. To enhance our understanding of the molecular mechanisms controlling the interaction between legumes and rhizobia, the symbiotic partner fixing and assimilating the atmospheric nitrogen for the plant, researchers took advantage of genetic and genomic resources developed across different legume models (e.g., Medicago truncatula, Lotus japonicus, Glycine max, and Phaseolus vulgaris) to identify key regulatory protein coding genes of the nodulation process. In this study, we are presenting the results of a comprehensive comparative genomic analysis to highlight orthologous and paralogous relationships between the legume genes controlling nodulation. Mining large transcriptomic datasets, we also identified several orthologous and paralogous genes characterized by the induction of their expression during nodulation across legume plant species. This comprehensive study prompts new insights into the evolution of the nodulation process in legume plant and will benefit the scientific community interested in the transfer of functional genomic information between species.

  11. The glutathione peroxidase gene family of Lotus japonicus: characterization of genomic clones, expression analyses and immunolocalization in legumes.

    PubMed

    Ramos, Javier; Matamoros, Manuel A; Naya, Loreto; James, Euan K; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2009-01-01

    Despite the multiple roles played by antioxidants in rhizobia-legume symbioses, little is known about glutathione peroxidases (GPXs) in legumes. Here the characterization of six GPX genes of Lotus japonicus is reported. Expression of GPX genes was analysed by quantitative reverse transcription-polymerase chain reaction in L. japonicus and Lotus corniculatus plants exposed to various treatments known to generate reactive oxygen and/or nitrogen species. LjGPX1 and LjGPX3 were the most abundantly expressed genes in leaves, roots and nodules. Compared with roots, LjGPX1 and LjGPX6 were highly expressed in leaves and LjGPX3 and LjGPX6 in nodules. In roots, salinity decreased GPX4 expression, aluminium decreased expression of the six genes, and cadmium caused up-regulation of GPX3, GPX4 and GPX5 after 1 h and down-regulation of GPX1, GPX2, GPX4 and GPX6 after 3-24 h. Exposure of roots to sodium nitroprusside (a nitric oxide donor) for 1 h increased the mRNA levels of GPX4 and GPX6 by 3.3- and 30-fold, respectively. Thereafter, the GPX6 mRNA level remained consistently higher than that of the control. Immunogold labelling revealed the presence of GPX proteins in root and nodule amyloplasts and in leaf chloroplasts of L. japonicus and other legumes. Labelling was associated with starch grains. These results underscore the differential regulation of GPX expression in response to cadmium, aluminium and nitric oxide, and strongly support a role for GPX6 and possibly other GPX genes in stress and/or metabolic signalling.

  12. [Diversity and resistance of rhizobia isolated from Caragana intermedia in Maowusu sandland].

    PubMed

    Gao, Lifeng; Deng, Xin; Wang, Hongxin; Hu, Zhiang

    2004-01-01

    Fifteen rhizobia strains were isolated from wild shrubby legume Caragana intermedia in Maowusu sandland. A dendrogram was constructed based on esterase profiles, showing a rich diversity of these rhizobia. Many biochemical characteristics were detected, including acid or alkali production, catalase activity, utilization of sole carbon sources, and resistance to salt, acid-alkali and temperature variation. The results indicated that all the rhizobia strains isolated from Caragana intermedia could excrete H+ on YMA agar and produce catalase. 73.3% strains could tolerate NaCl stress at 3% concentration, and 80% strains could grow at 50 degrees C. Except the difference in lactose and starch utilization, rhizobia strains had no bias on the rest carbon sources. However, the difference in resistance to stress existed among strains, which might be related to the adaptation of rhizobia to diverse landscapes in Maowusu sandland. It was revealed that rhizobia nodulating Caragana intermedia could be used as a new germplasm to fix nitrogen under severe environment.

  13. Increased accumulation of trehalose in rhizobia cultured under 1% oxygen

    SciTech Connect

    Hoelzle, I.; Streeter, J.G. )

    1990-10-01

    The growth of rhizobia under 1% O{sub 2} induced the accumulation of {alpha},{alpha}-trehalose, and the effect of low O{sub 2} was independent of medium composition and Rhizobium species. Trehalose concentration in cells declined rapidly when microaerobic cultures were supplied with 21% O{sub 2}. Trehalose formation in nodules may be induced by the microaerobic environment.

  14. The Ribosomal RNA is a Useful Marker to Visualize Rhizobia Interacting with Legume Plants

    ERIC Educational Resources Information Center

    Rinaudi, Luciana; Isola, Maria C.; Giordano, Walter

    2004-01-01

    Symbiosis between rhizobia and leguminous plants leads to the formation of nitrogen-fixing root nodules. In the present article, we recommend the use of the ribosomal RNA (rRNA) isolated from legume nodules in an experimental class with the purpose of introducing students to the structure of eukaryotic and prokaryotic ribosomes and of…

  15. The Ribosomal RNA is a Useful Marker to Visualize Rhizobia Interacting with Legume Plants

    ERIC Educational Resources Information Center

    Rinaudi, Luciana; Isola, Maria C.; Giordano, Walter

    2004-01-01

    Symbiosis between rhizobia and leguminous plants leads to the formation of nitrogen-fixing root nodules. In the present article, we recommend the use of the ribosomal RNA (rRNA) isolated from legume nodules in an experimental class with the purpose of introducing students to the structure of eukaryotic and prokaryotic ribosomes and of…

  16. Plant genes involved in harbouring symbiotic rhizobia or pathogenic nematodes.

    PubMed

    Damiani, Isabelle; Baldacci-Cresp, Fabien; Hopkins, Julie; Andrio, Emilie; Balzergue, Sandrine; Lecomte, Philippe; Puppo, Alain; Abad, Pierre; Favery, Bruno; Hérouart, Didier

    2012-04-01

    The establishment and development of plant-microorganism interactions involve impressive transcriptomic reprogramming of target plant genes. The symbiont (Sinorhizobium meliloti) and the root knot-nematode pathogen (Meloidogyne incognita) induce the formation of new root organs, the nodule and the gall, respectively. Using laser-assisted microdissection, we specifically monitored, at the cell level, Medicago gene expression in nodule zone II cells, which are preparing to receive rhizobia, and in gall giant and surrounding cells, which play an essential role in nematode feeding and constitute the typical root swollen structure, respectively. We revealed an important reprogramming of hormone pathways and C1 metabolism in both interactions, which may play key roles in nodule and gall neoformation, rhizobia endocytosis and nematode feeding. Common functions targeted by rhizobia and nematodes were mainly down-regulated, whereas the specificity of the interaction appeared to involve up-regulated genes. Our transcriptomic results provide powerful datasets to unravel the mechanisms involved in the accommodation of rhizobia and root-knot nematodes. Moreover, they raise the question of host specificity and the evolution of plant infection mechanisms by a symbiont and a pathogen.

  17. Poly-3-hydroxybutyrate (PHB) supports survival and reproduction in starving rhizobia.

    PubMed

    Ratcliff, William C; Kadam, Supriya V; Denison, Robert Ford

    2008-09-01

    The carbon that rhizobia in root nodules receive from their host powers both N(2) fixation, which mainly benefits the host, and rhizobium reproduction. Rhizobia also store energy in the lipid poly-3-hydroxybutyrate (PHB), which may enhance rhizobium survival when they are carbon limited, either in nodules or in the soil between hosts. There can be a conflict of interest between rhizobia and legumes over the rate of PHB accumulation, due to a metabolic tradeoff between N(2) fixation and PHB accumulation. To quantify the benefits of PHB to carbon-limited rhizobia, populations of genetically uniform rhizobia with high vs. low PHB (confirmed by flow cytometry) were generated by fractionating Sinorhizobium meliloti via density gradient centrifugation, and also by harvesting cells at early vs. late stationary phase. These rhizobia were starved for 165 days. PHB use during starvation was highly predictive of both initial reproduction and long-term population maintenance. Cultured S. meliloti accumulated enough PHB to triple their initial population size when starved, and to persist for c. 150 days before the population fell below its initial value. During the first 21 days of nodule growth, undifferentiated S. meliloti within alfalfa nodules accumulated enough PHB to support significant increases in reproduction and survival during starvation.

  18. CERBERUS and NSP1 of Lotus japonicus are common symbiosis genes that modulate arbuscular mycorrhiza development.

    PubMed

    Takeda, Naoya; Tsuzuki, Syusaku; Suzaki, Takuya; Parniske, Martin; Kawaguchi, Masayoshi

    2013-10-01

    Arbuscular mycorrhizal symbiosis (AMS) and root nodule symbiosis (RNS) are mutualistic plant-microbe interactions that confer nutritional benefits to both partners. Leguminous plants possess a common genetic system for intracellular symbiosis with AM fungi and with rhizobia. Here we show that CERBERUS and NSP1, which respectively encode an E3 ubiquitin ligase and a GRAS transcriptional regulator and which have previously only been implicated in RNS, are involved in AM fungal infection in Lotus japonicus. Hyphal elongation along the longitudinal axis of the root was reduced in the cerberus mutant, giving rise to a lower colonization level. Knockout of NSP1 decreased the frequency of plants colonized by AM fungi or rhizobia. CERBERUS and NSP1 showed different patterns of expression in response to infection with symbiotic microbes. A low constitutive level of CERBERUS expression was observed in the root and an increased level of NSP1 expression was detected in arbuscule-containing cells. Induction of AM marker gene was triggered in both cerberus and nsp1 mutants by infection with symbiotic microbes; however, the mutants showed a weaker induction of marker gene expression than the wild type, mirroring their lower level of colonization. The common symbiosis genes are believed to act in an early signaling pathway for recognition of symbionts and for triggering early symbiotic responses. Our quantitative analysis of symbiotic phenotypes revealed developmental defects of the novel common symbiosis mutants in both symbioses, which demonstrates that common symbiosis mechanisms also contribute to a range of functions at later or different stages of symbiont infection.

  19. Agrobacterium strains isolated from root nodules of common bean specifically reduce nodulation by Rhizobium gallicum.

    PubMed

    Mrabet, Moncef; Mnasri, Bacem; Romdhane, Samir Ben; Laguerre, Gisèle; Aouani, Mohamed Elarbi; Mhamdi, Ridha

    2006-05-01

    In a previous work, we showed that non-nodulating agrobacteria strains were able to colonize root nodules of common bean. Both rhizobia and agrobacteria co-existed in the infected nodules. No impact on symbiosis was found in laboratory conditions when using sterile gravel as a support for growth. In this study, soil samples originating from different geographic and agronomic regions in Tunisia were inoculated with a mixture of agrobacteria strains isolated previously from root nodules of common bean. A significant effect on nodulation and vegetal growth of common bean was observed. Characterization of nodulating rhizobia and comparison with non-inoculated controls showed a biased genetic structure. It seemed that Rhizobium gallicum was highly inhibited, whereas nodulation by Sinorhizobium medicae was favored. Co-inoculation of non-sterile soils with R. gallicum and agrobacteria confirmed these findings. In vitro antibiosis assays indicated that agrobacteria exercised a significant antagonism against R. gallicum.

  20. Rhizobial Diversity and Nodulation Characteristics of the Extremely Promiscuous Legume Sophora flavescens.

    PubMed

    Jiao, Yin Shan; Liu, Yuan Hui; Yan, Hui; Wang, En Tao; Tian, Chang Fu; Chen, Wen Xin; Guo, Bao Lin; Chen, Wen Feng

    2015-12-01

    In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class β-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-β-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

  1. Sanctions and mutualism stability: why do rhizobia fix nitrogen?

    PubMed Central

    West, Stuart A; Kiers, E Toby; Simms, Ellen L; Denison, R Ford

    2002-01-01

    Why do rhizobia expend resources on fixing N(2) for the benefit of their host plant, when they could use those resources for their own reproduction? We present a series of theoretical models which counter the hypotheses that N(2) fixation is favoured because it (i) increases the exudation of useful resources to related rhizobia in the nearby soil, or (ii) increases plant growth and therefore the resources available for rhizobia growth. Instead, we suggest that appreciable levels of N(2) fixation are only favoured when plants preferentially supply more resources to (or are less likely to senesce) nodules that are fixing more N(2) (termed plant sanctions). The implications for different agricultural practices and mutualism stability in general are discussed. PMID:11934359

  2. Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with LMW RNA group II Sinorhizobium meliloti of Medicago, Melilotus and Trigonella from soils of mainland Spain

    USDA-ARS?s Scientific Manuscript database

    Several isolates from nodules of Phaseolus vulgaris grown in soil of Lanzarote, an island of the Canaries, had electrophoretic LMW RNA patterns identical with a less common pattern within S. meliloti (assigned as group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northe...

  3. Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation and biotechnology.

    PubMed

    Zahran, H H

    2001-10-04

    Wild legumes (herb or tree) are widely distributed in arid regions and actively contribute to soil fertility in these environments. The N2-fixing activity and tolerance to drastic conditions may be higher in wild legumes than in crop legumes. The wild legumes in arid zones harbor diverse and promiscuous rhizobia in their root-nodules. Specificity existed only in few rhizobia from wild legumes, however, the majority of them are with wide host range. Based on phenotypic characteristics and molecular techniques (protein profiles, polysaccharides, plasmids, DNA-DNA hybridization, 16SrRNA, etc.), the root-nodule bacteria that was isolated from wild legumes had been classified into four genera (Rhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium). The rhizobia of wild legumes in arid zones, exhibit higher tolerance to the prevailing adverse conditions, e.g. salt stress, elevated temperatures and desiccation. These rhizobia may be used to inoculate wild, as well as, crop legumes, cultivated in reclaimed desert lands. Recent reports indicated that the wild-legume rhizobia formed successful symbioses with some grain legumes. Moreover, intercropping of some N2-fixing tree legumes (e.g. Lablab, Leucaena, Sesbania, etc.) to pasture grasses improved biomass yield and herb quality. In recent years, the rhizobia of wild legumes turn the attention of biotechnologists. These bacteria may have specific traits that can be transferred to other rhizobia through genetic engineering tools or used to produce industrially important compounds. Therefore, these bacteria are very important from both economic and environmental points of view.

  4. Quorum Sensing in Nitrogen-Fixing Rhizobia

    PubMed Central

    González, Juan E.; Marketon, Melanie M.

    2003-01-01

    Members of the rhizobia are distinguished for their ability to establish a nitrogen-fixing symbiosis with leguminous plants. While many details of this relationship remain a mystery, much effort has gone into elucidating the mechanisms governing bacterium-host recognition and the events leading to symbiosis. Several signal molecules, including plant-produced flavonoids and bacterially produced nodulation factors and exopolysaccharides, are known to function in the molecular conversation between the host and the symbiont. Work by several laboratories has shown that an additional mode of regulation, quorum sensing, intercedes in the signal exchange process and perhaps plays a major role in preparing and coordinating the nitrogen-fixing rhizobia during the establishment of the symbiosis. Rhizobium leguminosarum, for example, carries a multitiered quorum-sensing system that represents one of the most complex regulatory networks identified for this form of gene regulation. This review focuses on the recent stream of information regarding quorum sensing in the nitrogen-fixing rhizobia. Seminal work on the quorum-sensing systems of R. leguminosarum bv. viciae, R. etli, Rhizobium sp. strain NGR234, Sinorhizobium meliloti, and Bradyrhizobium japonicum is presented and discussed. The latest work shows that quorum sensing can be linked to various symbiotic phenomena including nodulation efficiency, symbiosome development, exopolysaccharide production, and nitrogen fixation, all of which are important for the establishment of a successful symbiosis. Many questions remain to be answered, but the knowledge obtained so far provides a firm foundation for future studies on the role of quorum-sensing mediated gene regulation in host-bacterium interactions. PMID:14665677

  5. Ensifer meliloti bv. lancerottense establishes nitrogen-fixing symbiosis with Lotus endemic to the Canary Islands and shows distinctive symbiotic genotypes and host range.

    PubMed

    León-Barrios, Milagros; Lorite, María José; Donate-Correa, Javier; Sanjuán, Juan

    2009-09-01

    Eleven strains were isolated from root nodules of Lotus endemic to the Canary Islands and they belonged to the genus Ensifer, a genus never previously described as a symbiont of Lotus. According to their 16S rRNA and atpD gene sequences, two isolates represented minority genotypes that could belong to previously undescribed Ensifer species, but most of the isolates were classified within the species Ensifer meliloti. These isolates nodulated Lotus lancerottensis, Lotus corniculatus and Lotus japonicus, whereas Lotus tenuis and Lotus uliginosus were more restrictive hosts. However, effective nitrogen fixation only occurred with the endemic L. lancerottensis. The E. meliloti strains did not nodulate Medicago sativa, Medicago laciniata Glycine max or Glycine soja, but induced non-fixing nodules on Phaseolus vulgaris roots. nodC and nifH symbiotic gene phylogenies showed that the E. meliloti symbionts of Lotus markedly diverged from strains of Mesorhizobium loti, the usual symbionts of Lotus, as well as from the three biovars (bv. meliloti, bv. medicaginis, and bv. mediterranense) so far described within E. meliloti. Indeed, the nodC and nifH genes from the E. meliloti isolates from Lotus represented unique symbiotic genotypes. According to their symbiotic gene sequences and host range, the Lotus symbionts would represent a new biovar of E. meliloti for which bv. lancerottense is proposed.

  6. A Novel Interaction between CCaMK and a Protein Containing the Scythe_N Ubiquitin-Like Domain in Lotus japonicus1[C][W][OA

    PubMed Central

    Kang, Heng; Zhu, Hui; Chu, Xiaojie; Yang, Zhenzhen; Yuan, Songli; Yu, Dunqiang; Wang, Chao; Hong, Zonglie; Zhang, Zhongming

    2011-01-01

    In the Rhizobium-legume symbiosis, calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator for both rhizobial infection and nodule organogenesis. Deregulation of CCaMK by either a point mutation in the autophosphorylation site or the deletion of the carboxyl-terminal regulatory domain results in spontaneous nodule formation without rhizobia. However, the underlying biochemical mechanisms are poorly understood. Here, using the kinase domain of CCaMK as a bait in yeast two-hybrid screening, we identify a novel protein, CIP73 (for CCaMK-interacting protein of approximately 73 kD), that interacts with CCaMK. CIP73 contains a Scythe_N ubiquitin-like domain and belongs to the large ubiquitin superfamily. Deletion and mutagenesis analysis demonstrate that CIP73 could only interact with CCaMK when the calmodulin-binding domain and three EF-hand motifs are removed from the kinase domain. The amino-terminal 80 amino acid residues (80–160) of CCaMK are required for interacting with CIP73 in yeast cells. On the other hand, protein pull-down assay and bimolecular fluorescence complementation assay in Nicotiana benthamiana show that the full-length CCaMK could interact with CIP73 in vitro and in planta. Importantly, CCaMK phosphorylates the amino terminus of CIP73 in a Ca2+/calmodulin-dependent manner in vitro. CIP73 transcripts are preferentially expressed in roots, and very low expression is detected in leaves, stems, and nodules. The expression in roots is significantly decreased after inoculation of Mesorhizobium loti. RNA interference knockdown of CIP73 expression by hairy root transformation in Lotus japonicus led to decreased nodule formation, suggesting that CIP73 performed an essential role in nodulation. PMID:21209278

  7. Deletion of the SACPD-C locus alters the symbiotic relationship between Bradyrhizobium japonicum USDA110 and soybean, resulting in elicitation of plant defense response and nodulation defects

    USDA-ARS?s Scientific Manuscript database

    Legumes form symbiotic association with soil-dwelling bacteria collectively called rhizobia. This association results in the formation of nodules, unique plant-derived organs, within which the rhizobia are housed. Rhizobia encoded-nitrogenase facilitates the conversation of atmospheric nitrogen int...

  8. Phenotypic and molecular assessment of chickpea rhizobia from different chickpea cultivars of India.

    PubMed

    Sharma, Anu; Bandamaravuri, Kishore Babu; Sharma, Anjana; Arora, Dillip K

    2017-10-01

    In the present study, heterogeneity in natural chickpea rhizobia populations associated with 18 different chickpea (Cicer arientinum) cultivars of India was investigated. Physiological diversity of 20 chickpea rhizobia was characterized based on phenotypic parameters such as Bromothymol blue (BTB) test, pH, temperature and salinity tolerance. Based on response to BTB test and pH tolerance, all chickpea rhizobia were further divided into slow growers/alkali producers (14 isolates) and fast growers/acid producers (6 isolates). The temperature (upto 40 °C) and salinity (NaCl) tolerance (upto 6%) tests provided a wide description of physiological diversity among the rhizobial isolates. The intrinsic antibiotic resistance of each isolate against 14 different antibiotics distinguished all chickpea rhizobia into five clades at the level of 80% similarity coefficient. Further, based on UPGMA phylogeny of carbon utilization profile, all isolates were dispersed into six clusters at the level of 85% similarity coefficient, which indicated a remarkable variability among the rhizobia. The evaluation of nodule-forming efficiency of all isolates revealed that the isolate ACR15 was more competent for nodule formation than all other isolates. The representative strain from each carbon metabolic cluster was further subjected for molecular identification through 16S rRNA gene characterization. Neighbour-joining method-based phylogeny of 16S rRNA gene sequence revealed a high degree of species diversity among the isolates. Further, the prominent nodule-forming isolate such as ACR15 was identified as Mesorhizobium ciceri, while other isolates showed similarity with other species of Mesorhizobium genus. The present study contributed to the knowledge that besides M. ciceri and M. mediterraneum, chickpea can also be nodulated by many other native chickpea rhizobia which indicates the impact of exploration of promising native populations. These findings may support the further

  9. Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan.

    PubMed

    Mierzwa, Bozena; Łotocka, Barbara; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Gnat, Sebastian; Małek, Wanda

    2010-05-01

    The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99-100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97-98% for nodC, and 97-100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.

  10. Tripartite symbiosis of Sophora tomentosa, rhizobia and arbuscular mycorhizal fungi.

    PubMed

    Toma, Maíra Akemi; Soares de Carvalho, Teotonio; Azarias Guimarães, Amanda; Martins da Costa, Elaine; Savana da Silva, Jacqueline; de Souza Moreira, Fatima Maria

    2017-06-29

    Sophora tomentosa is a pantropical legume species with potential for recovery of areas degraded by salinization, and for stabilization of sand dunes. However, few studies on this species have been carried out, and none regarding its symbiotic relationship with beneficial soil microorganisms. Therefore, this study aimed to evaluate the diversity of nitrogen-fixing bacteria isolated from nodules of Sophora tomentosa, and to analyze the occurrence of colonization of arbuscular mycorrhizal fungi on the roots of this legume in seafront soil. Thus, seeds, root nodules, and soil from the rhizosphere of Sophora tomentosa were collected. From the soil samples, trap cultures with this species were established to extract spores and to evaluate arbuscular mycorhizal fungi colonization in legume roots, as well as to capture rhizobia. Rhizobia strains were isolated from nodules collected in the field or from the trap cultures. Representative isolates of the groups obtained in the similarity dendrogram, based on phenotypic characteristics, had their 16S rRNA genes sequenced. The legume species showed nodules with indeterminate growth, and reddish color, distributed throughout the root. Fifty-one strains of these nodules were isolated, of which 21 were classified in the genus Bacillus, Brevibacillus, Paenibacillus, Rhizobium and especially Sinorhizobium. Strains closely related to Sinorhizobium adhaerens were the predominant bacteria in nodules. The other genera found, with the exception of Rhizobium, are probably endophytic bacteria in the nodules. Arbuscular mycorrhizal fungi was observed colonizing the roots, but arbuscular mycorhizal fungi spores were not found in the trap cultures. Therefore Sophora tomentosa is associated with both arbuscular mycorhizal fungi and nodulating nitrogen-fixing bacteria. Copyright © 2017 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  11. Direct and interactive effects of light and nutrients on the legume-rhizobia mutualism

    NASA Astrophysics Data System (ADS)

    Lau, Jennifer A.; Bowling, Evan James; Gentry, Lowell E.; Glasser, Paul A.; Monarch, Elizabeth A.; Olesen, Whitney M.; Waxmonsky, Jillian; Young, Ryan Thomas

    2012-02-01

    The relative abundance of traded resources can alter the outcomes of symbioses, potentially shifting mutually-beneficial interactions into interactions that are detrimental to one or both partners. We manipulated the legume-rhizobia symbiosis and light and nutrient levels to investigate how the net growth benefits to both partners shift across environments differing in the availability of both traded resources. In low nutrient, high light environments, rhizobia increased mean plant biomass by 62%. In contrast, when plants were light-limited, rhizobia did not increase above-ground biomass and reduced mean below-ground biomass by 46%. Similarly, rhizobia only increased plant biomass under low nutrient conditions. Resource availability also affected nodule traits correlated with rhizobia fitness, with light-limited plants producing fewer and smaller nodules. Our results suggest that the growth benefits to both partners in the legume-rhizobia symbiosis depend on the availability of both traded resources. We also detected evidence, however, that plants may reduce investment in symbionts as the net benefits of association decrease, potentially limiting how far this interaction shifts toward parasitism in low-light, high nutrient environments.

  12. Synergism and context dependency of interactions between arbuscular mycorrhizal fungi and rhizobia with a prairie legume.

    PubMed

    Larimer, Anna L; Clay, Keith; Bever, James D

    2014-04-01

    Biotic interactions play primary roles in major theories of the distribution and abundance of species, yet the nature of these biotic interactions can depend upon the larger ecological community. Leguminous plants, for example, commonly associate with both arbuscular mycorrhizal fungi (AMF) and rhizobia bacteria, and the pairwise interactions may depend upon the presence or identity of the third partner. To determine if the dynamics of plant-AMF and plant-rhizobia interactions are affected by the alternate symbiont, we manipulated the presence and identity of each symbiont, as well as levels of the nutrients supplied by each symbiont (nitrogen and phosphorus), on the growth of prairie legume Amorpha canescens. We found strong synergistic effects of AMF and rhizobia inoculation on plant biomass production that were independent of nutrient levels. AMF and rhizobia responses were each influenced by the other, but not in the same direction. AMF infection increased root nodule number and mass, but rhizobia inoculation decreased AMF hyphal colonization of roots. The relative benefits of each combination of symbionts depended upon phosphorus level. The effect of nitrogen was also contingent on the biotic environment where nitrogen addition decreased nodulation, but this decrease was reduced with coinfection by AMF. Our results demonstrate a strong contingency on the co-occurrence of AMF and rhizobia for the long-term fitness of A. canescens, and suggest that the belowground community is critical for the success of this species in tallgrass prairies.

  13. Plasmid profiles of mercuric chloride tolerant rhizobia from horse gram (Macrotyloma uniflorum).

    PubMed

    Edulamudi, Prabhavati; Johnson, Antony A M; Divi, Venkata Ramana Sai Gopal; Konada, Veera Mallaiah

    2012-03-01

    Thirty two rhizobia were isolated from the fresh healthy root nodules of horse gram. They were found to be highly salt tolerant. They were identified as rhizobia by cultural, biochemical and 16S rRNA sequence. The sequences of the four selected isolates were deposited in the NCBI GenBank. The obtained accession numbers were GQ483457, GQ483458, GQ483459 and GQ483460. All the rhizobia were able to grow at 10 ppm mercuric chloride concentration. Four isolates HGR-11, 16, 30 and 31 were used to study the effect of different concentrations of mercuric chloride on the growth of rhizobia. These isolates were able to grow at 30 ppm concentration also. In these isolates, HGR-11 and HGR-30 showed maximum growth at 20 ppm than at control. These isolates contained one mega plasmid (-22 kb) at 20 ppm mercuric chloride concentration.

  14. Nitric oxide detoxification in the rhizobia-legume symbiosis.

    PubMed

    Sánchez, Cristina; Cabrera, Juan J; Gates, Andrew J; Bedmar, Eulogio J; Richardson, David J; Delgado, María J

    2011-01-01

    NO (nitric oxide) is a signal molecule involved in diverse physiological processes in cells which can become very toxic under certain conditions determined by its rate of production and diffusion. Several studies have clearly shown the production of NO in early stages of rhizobia-legume symbiosis and in mature nodules. In functioning nodules, it has been demonstrated that NO, which has been reported as a potent inhibitor of nitrogenase activity, can bind Lb (leghaemoglobin) to form LbNOs (nitrosyl-leghaemoglobin complexes). These observations have led to the question of how nodules overcome the toxicity of NO. On the bacterial side, one candidate for NO detoxification in nodules is the respiratory Nor (NO reductase) that catalyses the reduction of NO to nitrous oxide. In addition, rhizobial fHbs (flavohaemoglobins) and single-domain Hbs which dioxygenate NO to form nitrate are candidates to detoxify NO under free-living and symbiotic conditions. On the plant side, sHbs (symbiotic Hbs) (Lb) and nsHbs (non-symbiotic Hbs) have been proposed to play important roles as modulators of NO levels in the rhizobia-legume symbiosis. In the present review, current knowledge of NO detoxification by legume-associated endosymbiotic bacteria is summarized.

  15. Fate map of Medicago truncatula root nodules.

    PubMed

    Xiao, Ting Ting; Schilderink, Stefan; Moling, Sjef; Deinum, Eva E; Kondorosi, Eva; Franssen, Henk; Kulikova, Olga; Niebel, Andreas; Bisseling, Ton

    2014-09-01

    Legume root nodules are induced by N-fixing rhizobium bacteria that are hosted in an intracellular manner. These nodules are formed by reprogramming differentiated root cells. The model legume Medicago truncatula forms indeterminate nodules with a meristem at their apex. This organ grows by the activity of the meristem that adds cells to the different nodule tissues. In Medicago sativa it has been shown that the nodule meristem is derived from the root middle cortex. During nodule initiation, inner cortical cells and pericycle cells are also mitotically activated. However, whether and how these cells contribute to the mature nodule has not been studied. Here, we produce a nodule fate map that precisely describes the origin of the different nodule tissues based on sequential longitudinal sections and on the use of marker genes that allow the distinction of cells originating from different root tissues. We show that nodule meristem originates from the third cortical layer, while several cell layers of the base of the nodule are directly formed from cells of the inner cortical layers, root endodermis and pericycle. The latter two differentiate into the uninfected tissues that are located at the base of the mature nodule, whereas the cells derived from the inner cortical cell layers form about eight cell layers of infected cells. This nodule fate map has then been used to re-analyse several mutant nodule phenotypes. This showed, among other things, that intracellular release of rhizobia in primordium cells and meristem daughter cells are regulated in a different manner.

  16. Lupine leghemoglobin I: expression in transgenic Lotus and tobacco tissues.

    PubMed

    Strózycki, P M; Karłowski, W M; Dessaux, Y; Petit, A; Legocki, A B

    2000-03-01

    The proximal parts of the promoters of the genes for symbiotic-type hemoglobins are generally conserved, but the promoter of the lbI gene of lupine (LulbI) shows some unusual structural features. It lacks typical organ-specific elements characteristic of all the leghemoglobin gene promoters described thus far. We have analysed its functional activity in transgenic Lotus corniculatus. A fusion construct between the lbI promoter and the GUS reporter gene was expressed mainly in the central zone of the root nodule, but the product was also detected in the non-nodule root zone and in roots in tissue culture. In roots of transgenic tobacco, the activity of the promoter was only 24% lower than in Lotus nodules. LulbI promoter activity was also detected in tobacco leaves. Lupine hemoglobin I has a higher sequence identity to symbiotic-type hemoglobins and thus it groups within the "Class II" hemoglobins.

  17. Estimating Divergence Times and Substitution Rates in Rhizobia

    PubMed Central

    Chriki-Adeeb, Rim; Chriki, Ali

    2016-01-01

    Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth–death, birth–death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes. PMID:27168719

  18. Estimating Divergence Times and Substitution Rates in Rhizobia.

    PubMed

    Chriki-Adeeb, Rim; Chriki, Ali

    2016-01-01

    Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth-death, birth-death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes.

  19. R gene-controlled host specificity in the legume-rhizobia symbiosis

    USDA-ARS?s Scientific Manuscript database

    Leguminous plants can enter into root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. An intriguing but still poorly understood property of the symbiosis is its host specificity, which is controlled at multiple levels involving both rhizobial and host genes. Here we report the...

  20. Mechanistic action of gibberellins in legume nodulation.

    PubMed

    Hayashi, Satomi; Gresshoff, Peter M; Ferguson, Brett J

    2014-10-01

    Legume plants are capable of entering into a symbiotic relationship with rhizobia bacteria. This results in the formation of novel organs on their roots, called nodules, in which the bacteria capture atmospheric nitrogen and provide it as ammonium to the host plant. Complex molecular and physiological changes are involved in the formation and establishment of such nodules. Several phytohormones are known to play key roles in this process. Gibberellins (gibberellic acids; GAs), a class of phytohormones known to be involved in a wide range of biological processes (i.e., cell elongation, germination) are reported to be involved in the formation and maturation of legume nodules, highlighted by recent transcriptional analyses of early soybean symbiotic steps. Here, we summarize what is currently known about GAs in legume nodulation and propose a model of GA action during nodule development. Results from a wide range of studies, including GA application, mutant phenotyping, and gene expression studies, indicate that GAs are required at different stages, with an optimum, tightly regulated level being key to achieve successful nodulation. Gibberellic acids appear to be required at two distinct stages of nodulation: (i) early stages of rhizobia infection and nodule primordium establishment; and (ii) later stages of nodule maturation. © 2014 Institute of Botany, Chinese Academy of Sciences.

  1. Blue light does not inhibit nodulation in Sesbania rostrata.

    PubMed

    Shimomura, Aya; Arima, Susumu; Hayashi, Makoto; Maymon, Maskit; Hirsch, Ann M; Suzuki, Akihiro

    2017-01-02

    Earlier, we reported that root nodulation was inhibited by blue light irradiation of Lotus japonicus. Because some legumes do not establish nodules exclusively on underground roots, we investigated whether nodule formation in Sesbania rostrata, which forms both root and "stem" nodules following inoculation with Azorhizobium caulinodans, is inhibited by blue light as are L. japonicus nodules. We found that neither S. rostrata nodulation nor nitrogen fixation was inhibited by blue light exposure. Moreover, although A. caulinodans proliferation was not affected by blue light irradiation, bacterial survival was decreased. Therefore, blue light appears to impose different responses depending on the legume-rhizobial symbiosis.

  2. Diversity and symbiotic effectiveness of beta-rhizobia isolated from sub-tropical legumes of a Brazilian Araucaria Forest.

    PubMed

    Lammel, Daniel R; Cruz, Leonardo M; Carrer, Helaine; Cardoso, Elke J B N

    2013-12-01

    While the occurrence of Betaproteobacteria occupying the nodules of tropical legumes has been shown, little is known about subtropical areas. Araucaria Forest is a subtropical endangered ecosystem, and a better understanding of the legume-rhizobial symbionts may allow their use in land reclamation. The 16S rRNA gene of bacteria isolated from nine leguminous species was sequenced and their nodulation tested in Mimosa scabrella and Phaseolus vulgaris. 196 isolates were identified as eight genotypes: Pantoea, Pseudomonas, Bradyrhizobium sp1-2, Rhizobium, and Burkholderia sp1-3. The majority of the isolates from native plants (87 %) were taxonomically related to β-rhizobia, namely Burkholderia, however the legumes Galactia crassifolia and Collea speciosa were nodulated by both α and β-rhizobia, and Acacia dealbata, an exotic plant, only by α-rhizobia. The nifH genes of some isolates were sequenced and N-fixing potential shown by the acetylene reduction test. Most of the isolates nodulated the test plants, some were effective in M. scabrella, but all presented low efficiency in the exotic promiscuous legume P. vulgaris. Pantoea and Pseudomonas did not nodulate and probably are endophytic bacteria. The presented data shows diversity of α, β and γ-Proteobacteria in nodules of subtropical legumes, and suggests host specificity with β-rhizobia. Potential isolates were found for M. scabrella, indicating that a high N-fixing strain may be further inoculated in plants for use in reforestation.

  3. An experimental and modelling exploration of the host-sanction hypothesis in legume-rhizobia mutualism.

    PubMed

    Marco, Diana E; Carbajal, Juan P; Cannas, Sergio; Pérez-Arnedo, Rebeca; Hidalgo-Perea, Angeles; Olivares, José; Ruiz-Sainz, José E; Sanjuán, Juan

    2009-08-07

    Despite the importance of mutualism as a key ecological process, its persistence in nature is difficult to explain since the existence of exploitative, "cheating" partners that could erode the interaction is common. By analogy with the proposed policing strategy stabilizing intraspecific cooperation, host sanctions against non-N(2) fixing, cheating symbionts have been proposed as a force stabilizing mutualism in legume-Rhizobium symbiosis. Following this proposal, penalizations would include decreased nodular rhizobial viability and/or early nodule senescence in nodules occupied by cheating rhizobia. In this work, we analyse the stability of Rhizobium-legume symbiosis when non-fixing, cheating strains are present, using an experimental and modelling approach. We used split-root experiments with soybean plants inoculated with two rhizobial strains, a cooperative, normal N(2) fixing strain and an isogenic non-fixing, "perfect" cheating mutant derivative that lacks nitrogenase activity but has the same nodulation abilities inoculated to split-root plants. We found no experimental evidence of functioning plant host sanctions to cheater rhizobia based on nodular rhizobia viability and nodule senescence and maturity molecular markers. Based on these experiments, we developed a population dynamic model with and without the inclusion of plant host sanctions. We show that plant populations persist in spite of the presence of cheating rhizobia without the need of incorporating any sanction against the cheater populations in the model, under the realistic assumption that plants can at least get some amount of fixed N(2) from the effectively mutualistic rhizobia occupying some nodules. Inclusion of plant sanctions leads to the unrealistic effect of ultimate extinction of cheater strains in soil. Our simulation results are in agreement with increasing experimental evidence and theoretical work showing that mutualisms can persist in presence of cheating partners.

  4. The analysis of core and symbiotic genes of rhizobia nodulating Vicia from different continents reveals their common phylogenetic origin and suggests the distribution of Rhizobium leguminosarum strains together with Vicia seeds.

    PubMed

    Alvarez-Martínez, Estela R; Valverde, Angel; Ramírez-Bahena, Martha Helena; García-Fraile, Paula; Tejedor, Carmen; Mateos, Pedro F; Santillana, Nery; Zúñiga, Doris; Peix, Alvaro; Velázquez, Encarna

    2009-08-01

    In this work, we analysed the core and symbiotic genes of rhizobial strains isolated from Vicia sativa in three soils from the Northwest of Spain, and compared them with other Vicia endosymbionts isolated in other geographical locations. The analysis of rrs, recA and atpD genes and 16S-23S rRNA intergenic spacer showed that the Spanish strains nodulating V. sativa are phylogenetically close to those isolated from V. sativa and V. faba in different European, American and Asian countries forming a group related to Rhizobium leguminosarum. The analysis of the nodC gene of strains nodulating V. sativa and V. faba in different continents showed they belong to a phylogenetically compact group indicating that these legumes are restrictive hosts. The results of the nodC gene analysis allow the delineation of the biovar viciae showing a common phylogenetic origin of V. sativa and V. faba endosymbionts in several continents. Since these two legume species are indigenous from Europe, our results suggest a world distribution of strains from R. leguminosarum together with the V. sativa and V. faba seeds and a close coevolution among chromosome, symbiotic genes and legume host in this Rhizobium-Vicia symbiosis.

  5. Molecular diversity and phylogeny of rhizobia associated with Lablab purpureus (Linn.) grown in Southern China.

    PubMed

    Chang, Yue Li; Wang, En Tao; Sui, Xin Hua; Zhang, Xiao Xia; Chen, Wen Xin

    2011-06-01

    As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes.

  6. Phylogenetic relationships and diversity of β-rhizobia associated with Mimosa species grown in Sishuangbanna, China.

    PubMed

    Liu, Xiao Yun; Wu, Wei; Wang, En Tao; Zhang, Bin; Macdermott, Jomo; Chen, Wen Xin

    2011-02-01

    In order to investigate the genetic diversity of rhizobia associated with various exotic and invasive species in tropical mainland China, 116 bacterial isolates were obtained from Mimosa root nodules collected from Sishuangbanna and Yuanjiang districts of Yunnan province. Isolated rhizobia were characterized by RFLP analysis of 16S rRNA genes, SDS-PAGE of whole-cell proteins and BOX-PCR. Most of the isolated strains were identified as β-rhizobia belonging to diverse populations of Burkholderia and Cupriavidus, and the phylogenetic relationships of their 16S rRNA gene sequences showed that they were closely related to one of four β-rhizobia species: Burkholderia phymatum, B. mimosarum, B. caribensis or Cupriavidus taiwanensis. Additionally, among the 116 isolates, 53 different whole-cell SDS-PAGE profiles and 30 distinct BOX-PCR genotypic patterns were detected, which demonstrated the genetic and phenotypic diversity found within these Burkholderia and Cupriavidus strains. To the best of our knowledge, this is the first report that β-rhizobia are extant and possibly widespread on the Chinese mainland and nodulate easily with Mimosa plants. We also find it especially interesting that this appears to be the first report from mainland China of Cupriavidus symbionts of Mimosa. These records enrich our knowledge and understanding of the geographical distribution and diversity of these bacteria.

  7. Characterization of indigenous rhizobia from caatinga

    PubMed Central

    Pires e Teixeira, Fernanda Cíntia; Borges, Wardsson Lustrino; Xavier, Gustavo Ribeiro; Rumjanek, Norma Gouvêa

    2010-01-01

    The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results. PMID:24031482

  8. Characterization of indigenous rhizobia from caatinga.

    PubMed

    Pires E Teixeira, Fernanda Cíntia; Borges, Wardsson Lustrino; Xavier, Gustavo Ribeiro; Rumjanek, Norma Gouvêa

    2010-01-01

    The aim of this study was to characterize rhizobial isolates from Cratylia mollis Mart. ex Benth, Calliandra depauperata Benth. and Mimosa tenuiflora (Willd.) Poir. by means of rhizobial colonies morphology and restriction analysis of the 16S ribosomal gene (16S rDNA-ARDRA). Nodules were collected in the field and from plants cultivated in a greenhouse experiment using Caatinga soil samples. Sixty seven isolates were described by morphological analysis. Forty seven representative isolates were used for ARDRA analysis using seven restriction enzymes. We observed high diversity of both slow and fast-growing rhizobia that formed three morpho-physiological clusters. A few fast-growing isolates formed a group of strains of the Bradyrhizobium type; however, most of them diverged from the B. japonicum and B. elkanii species. Cratylia mollis nodule isolates were the most diverse, while all Mimosa tenuiflora isolates displayed fast growth with no pH change and were clustered into groups bearing 100% similarity, according to ARDRA results.

  9. [Genetic diversity of rhizobia isolated from common legumes in the Karst area. Northwest Guangxi].

    PubMed

    Liu, Lu; He, Xun-yang; Xie, Qiang; Wang, Ke-lin

    2015-12-01

    Legumes, with a strong resistance to the adverse environmental conditions, are pioneer plants in degraded habitats, and play an important role in ecosystem restoration. In this study, the nodulation characteristics of 24 legumes were surveyed in the Karst area of Northwest Guangxi. A total of 39 nodule samples were collected from 15 legumes, the DNA was extracted and the 16S rDNA and nifH gene were amplified. A phylogenetic tree was then constructed to analyze the genetic diversity of rhizobia. The results showed that 15 legumes were nodulated, of which 14 belonged to the Papilionoideae, one to the Mimosaceae, and none to the Caesalpinoideae. No nodules were found on some legumes that were reported as nodulated, which might result from soil water stress in Karst. BLAST result and phylogenetic analyse indicated that most of the legumes were associated with rhizobia that belonged to the genus Bradyrhizobium, with the exception of two samples from Callerya nitida that were associated with the genus Mesorhizobium. In the phylogenetic tree, the sequences obtained from the same plot or the sequences from the same host species clustered together in most cases. This finding suggested that host selection and the ecological environment are the major factors that influence the genotype of rhizobia.

  10. Two distinct EIN2 genes cooperatively regulate ethylene signaling in Lotus japonicus.

    PubMed

    Miyata, Kana; Kawaguchi, Masayoshi; Nakagawa, Tomomi

    2013-09-01

    Leguminous plants establish a mutualistic symbiosis with bacteria, collectively referred to as rhizobia. Host plants positively and negatively regulate the symbiotic processes to keep the symbiosis at an appropriate level. Although the plant hormone ethylene is known as a negative regulator of symbiotic processes, the molecular mechanisms of ethylene signaling remain unresolved, especially in the model plant Lotus japonicus. Here, we identified two genes, LjEIN2-1 and LjEIN2-2, from L. japonicus. These genes share moderate similarity in their amino acid sequences, are located on different chromosomes and are composed of different numbers of exons. Suppression of either LjEIN2-1 or LjEIN2-2 expression significantly promoted the root growth of transformed plants on plates containing 1-amino-cyclopropane-carboxylic acid (ACC), the biosynthetic precursor of ethylene. Simultaneous suppression of both LjEIN2-1 and LjEIN2-2 markedly increased the ethylene insensitivity of transgenic roots and resulted in an increased nodulation phenotype. These results indicate that LjEIN2-1 and LjEIN2-2 concertedly regulate ethylene signaling in L. japonicus. We also observed that Nod factor (NF) induced the expression of the ethylene-responsive gene LjACO2, and simultaneous treatment with NF and ACC markedly increases its transcript level compared with either NF or ACC alone. Because LjACO2 encodes ACC oxidase, which is a key enzyme in ethylene biosynthesis, this result suggests the existence of an NF-triggered negative feedback mechanism through ethylene signaling.

  11. The effect of acidity on the distribution and symbiotic efficiency of rhizobia in Lithuanian soils

    NASA Astrophysics Data System (ADS)

    Lapinskas, E. B.

    2007-04-01

    The distribution and symbiotic efficiency of nodule bacteria Rhizobium leguminosarum_bv. trifolii F., Sinorhizobium meliloti D., Rhizobium galegae L., and Rhizobium leguminosarum bv. viciae F. in Lithuanian soils as dependent on the soil acidity were studied in the long-term field, pot, and laboratory experiments. The critical and optimal pH values controlling the distribution of rhizobia and the symbiotic nitrogen fixation were determined for every bacterial species. The relationship was found between the soil pH and the nitrogen-fixing capacity of rhizobia. A positive effect of liming of acid soils in combination with inoculation of legumes on the efficiency of symbiotic nitrogen fixation was demonstrated.

  12. Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

    PubMed

    Gnat, Sebastian; Małek, Wanda; Oleńska, Ewa; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Łotocka, Barbara; Wójcik, Magdalena

    2015-01-01

    The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99-100% in the case of nodAC and nifH genes, and 98-99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar "glycyphyllae", based on nodA and nodC genes sequences.

  13. Phylogeny of Symbiotic Genes and the Symbiotic Properties of Rhizobia Specific to Astragalus glycyphyllos L.

    PubMed Central

    Gnat, Sebastian; Małek, Wanda; Oleńska, Ewa; Wdowiak-Wróbel, Sylwia; Kalita, Michał; Łotocka, Barbara; Wójcik, Magdalena

    2015-01-01

    The phylogeny of symbiotic genes of Astragalus glycyphyllos L. (liquorice milkvetch) nodule isolates was studied by comparative sequence analysis of nodA, nodC, nodH and nifH loci. In all these genes phylograms, liquorice milkvetch rhizobia (closely related to bacteria of three species, i.e. Mesorhizobium amorphae, Mesorhizobium septentrionale and Mesorhizobium ciceri) formed one clearly separate cluster suggesting the horizontal transfer of symbiotic genes from a single ancestor to the bacteria being studied. The high sequence similarity of the symbiotic genes of A. glycyphyllos rhizobia (99–100% in the case of nodAC and nifH genes, and 98–99% in the case of nodH one) points to the relatively recent (in evolutionary scale) lateral transfer of these genes. In the nodACH and nifH phylograms, A. glycyphyllos nodule isolates were grouped together with the genus Mesorhizobium species in one monophyletic clade, close to M. ciceri, Mesorhizobium opportunistum and Mesorhizobium australicum symbiovar biserrulae bacteria, which correlates with the close relationship of these rhizobia host plants. Plant tests revealed the narrow host range of A. glycyphyllos rhizobia. They formed effective symbiotic interactions with their native host (A. glycyphyllos) and Amorpha fruticosa but not with 11 other fabacean species. The nodules induced on A. glycyphyllos roots were indeterminate with apical, persistent meristem, an age gradient of nodule tissues and cortical vascular bundles. To reflect the symbiosis-adaptive phenotype of rhizobia, specific for A. glycyphyllos, we propose for these bacteria the new symbiovar “glycyphyllae”, based on nodA and nodC genes sequences. PMID:26496493

  14. Failure to fix nitrogen by non-reproductive symbiotic rhizobia triggers host sanctions that reduce fitness of their reproductive clonemates.

    PubMed

    Oono, Ryoko; Anderson, Carolyn G; Denison, R Ford

    2011-09-07

    The legume-rhizobia symbiosis is a classical mutualism where fixed carbon and nitrogen are exchanged between the species. Nonetheless, the plant carbon that fuels nitrogen (N(2)) fixation could be diverted to rhizobial reproduction by 'cheaters'--rhizobial strains that fix less N(2) but potentially gain the benefit of fixation by other rhizobia. Host sanctions can decrease the relative fitness of less-beneficial reproductive bacteroids and prevent cheaters from breaking down the mutualism. However, in certain legume species, only undifferentiated rhizobia reproduce, while only terminally differentiated rhizobial bacteroids fix nitrogen. Sanctions were, therefore, tested in two legume species that host non-reproductive bacteroids. We demonstrate that even legume species that host non-reproductive bacteroids, specifically pea and alfalfa, can severely sanction undifferentiated rhizobia when bacteroids within the same nodule fail to fix N(2). Hence, host sanctions by a diverse set of legumes play a role in maintaining N(2) fixation.

  15. Phylogenetic Diversity of Ammopiptanthus Rhizobia and Distribution of Rhizobia Associated with Ammopiptanthus mongolicus in Diverse Regions of Northwest China.

    PubMed

    Zhao, Liang; Wang, Xinye; Huo, Haibo; Yuan, Guiji; Sun, Yali; Zhang, Dehui; Cao, Ying; Xu, Lin; Wei, Gehong

    2016-07-01

    Aiming to investigate the diversity and distribution of rhizobia associated with Ammopiptanthus, an endangered evergreen legume widely distributed in deserts, we characterized a total of 219 nodule isolates from nine sampling sites in Northwest China with different soil characteristics based upon restriction fragment length polymorphism (RFLP) analysis of 16S ribosomal RNA (rRNA) and symbiotic genes (nodC and nifH). Ten isolates representing different 16S rRNA-RFLP types were selected for further sequence analyses of 16S rRNA and four housekeeping genes. As results, nine genospecies belonging to the genera Ensifer, Neorhizobium, Agrobacterium, Pararhizobium, and Rhizobium could be defined among the isolates. The nodC and nifH phylogenies of 14 isolates representing different symbiotic-RFLP types revealed five lineages linked to Ensifer fredii, Ensifer meliloti, Rhizobium leguminosarum, Mesorhizobium amorphae, and Rhizobium gallicum, which demonstrated the various origins and lateral transfers of symbiotic genes between different genera and species. The rhizobial diversities of Ammopiptanthus mongolicus varied among regions, and the community compositions of rhizobia associated with A. mongolicus were significantly different in wild and cultured fields. Constrained correspondence analysis showed that the distribution of A. mongolicus rhizobia could be explained by available potassium content and that the assembly of symbiotic types was mainly affected by available phosphorus content and carbon-nitrogen ratio.

  16. Symbiotic potential and survival of native rhizobia kept on different carriers

    PubMed Central

    Ruíz-Valdiviezo, Víctor Manuel; Canseco, Lucía María Cristina Ventura; Suárez, Luis Antonio Castillo; Gutiérrez-Miceli, Federico Antonio; Dendooven, Luc; Rincón-Rosales, Reiner

    2015-01-01

    Native rhizobia are ideal for use as commercial legume inoculants. The characteristics of the carrier used to store the inoculants are important for the survival and symbiotic potential of the rhizobia. The objective of this study was to investigate the effects of peat (PEAT), perlite sugarcane bagasse (PSB), carboxymethyl cellulose plus starch (CMCS), and yeast extract mannitol supplemented with mannitol (YEMM) on the survival, nodulation potential and N2 fixation capacity of the native strains Sinorhizobium mexicanum ITTG R7T and Rhizobium calliandrae LBP2-1T and of the reference strain Rhizobium etli CFN42T. A factorial design (4 × 3) with four repetitions was used to determine the symbiotic potential of the rhizobial strains. The survival of the strains was higher for PEAT (46% for strain LBP2-1T, 167% for strain CFN42T and 219% for strain ITTG R7T) than for the other carriers after 240 days, except for CFN42T kept on CMCS (225%). All the strains kept on the different carriers effectively nodulated common bean, with the lowest number of nodules found (5 nodules) when CFN42T was kept on CMCS and with the highest number of nodules found (28 nodules) when ITTG R7T was kept on PSB. The nitrogenase activity was the highest for ITTG R7T kept on PEAT (4911 μmol C2H4 per fresh weight nodule h−1); however, no activity was found when the strains were kept on YEMM. Thus, the survival and symbiotic potential of the rhizobia depended on the carrier used to store them. PMID:26413054

  17. Symbiotic potential and survival of native rhizobia kept on different carriers.

    PubMed

    Ruíz-Valdiviezo, Víctor Manuel; Canseco, Lucía María Cristina Ventura; Suárez, Luis Antonio Castillo; Gutiérrez-Miceli, Federico Antonio; Dendooven, Luc; Rincón-Rosales, Reiner

    2015-01-01

    Native rhizobia are ideal for use as commercial legume inoculants. The characteristics of the carrier used to store the inoculants are important for the survival and symbiotic potential of the rhizobia. The objective of this study was to investigate the effects of peat (PEAT), perlite sugarcane bagasse (PSB), carboxymethyl cellulose plus starch (CMCS), and yeast extract mannitol supplemented with mannitol (YEMM) on the survival, nodulation potential and N2 fixation capacity of the native strains Sinorhizobium mexicanum ITTG R7(T) and Rhizobium calliandrae LBP2-1(T) and of the reference strain Rhizobium etli CFN42(T). A factorial design (4 × 3) with four repetitions was used to determine the symbiotic potential of the rhizobial strains. The survival of the strains was higher for PEAT (46% for strain LBP2-1(T), 167% for strain CFN42(T) and 219% for strain ITTG R7(T)) than for the other carriers after 240 days, except for CFN42(T) kept on CMCS (225%). All the strains kept on the different carriers effectively nodulated common bean, with the lowest number of nodules found (5 nodules) when CFN42(T) was kept on CMCS and with the highest number of nodules found (28 nodules) when ITTG R7(T) was kept on PSB. The nitrogenase activity was the highest for ITTG R7(T) kept on PEAT (4911 μmol C2H4 per fresh weight nodule h(-1)); however, no activity was found when the strains were kept on YEMM. Thus, the survival and symbiotic potential of the rhizobia depended on the carrier used to store them.

  18. Competition among rhizobium species for nodulation of Leucaena leucocephala in two tropical soils

    SciTech Connect

    Moawad, H.; Bohlool, B.B.

    1984-07-01

    The successful nodulation of legumes by a Rhizobium strain is determined by the competitve ability of that strain against the mixture of other native and inoculant rhizobia. Competition among six Leucaena rhizobial strains in single and multistrain inoculants were studied. Field inoculation trials were conducted in an oxisol and a mollisol soil, both of which contained indigenous Leucaena-nodulating rhizobia. Strain-specific fluorescent antibodies were used for the identification of the strains in Leucaena nodules. Mixtures of three recommended inoculum strains for Leucaena species (TAL82, TAL582, and TAL1145) were used in peat-based inocula either alone or with one of the three other strains isolated from the sites, B213, B214, and B215. Each of these latter three strains was also used as single-strain inocula to study their competition with the native rhizobia in the two soil systems. In the oxisol soil, strains B213 and B215, when used as single-strain inocula, outcompeted the native rhizobia and formed 92 and 62% of the nodules, respectively. Strain B214 was the least competitive in oxisol soil, where it formed 30% of the nodules, and the best in mollisol soil, where it formed 70% of the nodules. The most successful competitor for nodulation in multistrain inocula was strain TAL1145, which outcompeted native and other inoculum Leucaena rhizobia is both soils. None of the strains in single or multistrain inoculants was capable of completely overcoming the resident rhizobia, which formed 4 to 70% of the total nodules in oxisol soil and 12 to 72% in mollisol soil. No strong relationship was detected between the size of the rhizosphere population of a strain and its successful occupation of nodules. 24 references.

  19. NODULES WITH ACTIVATED DEFENSE 1 is required for maintenance of rhizobial endosymbiosis in Medicago truncatula.

    PubMed

    Wang, Chao; Yu, Haixiang; Luo, Li; Duan, Liujian; Cai, Liuyang; He, Xinxing; Wen, Jiangqi; Mysore, Kirankumar S; Li, Guoliang; Xiao, Aifang; Duanmu, Deqiang; Cao, Yangrong; Hong, Zonglie; Zhang, Zhongming

    2016-10-01

    The symbiotic interaction between legume plants and rhizobia results in the formation of root nodules, in which symbiotic plant cells host and harbor thousands of nitrogen-fixing rhizobia. Here, a Medicago truncatula nodules with activated defense 1 (nad1) mutant was identified using reverse genetics methods. The mutant phenotype was characterized using cell and molecular biology approaches. An RNA-sequencing technique was used to analyze the transcriptomic reprogramming of nad1 mutant nodules. In the nad1 mutant plants, rhizobial infection and propagation in infection threads are normal, whereas rhizobia and their symbiotic plant cells become necrotic immediately after rhizobia are released from infection threads into symbiotic cells of nodules. Defense-associated responses were detected in nad1 nodules. NAD1 is specifically present in root nodule symbiosis plants with the exception of Morus notabilis, and the transcript is highly induced in nodules. NAD1 encodes a small uncharacterized protein with two predicted transmembrane helices and is localized at the endoplasmic reticulum. Our data demonstrate a positive role for NAD1 in the maintenance of rhizobial endosymbiosis during nodulation.

  20. Rhizobia survival in seeds coated with polyvinyl alcohol (PVA) electrospun nanofibres.

    PubMed

    Damasceno, Raquel; Roggia, Isabel; Pereira, Claudio; de Sá, Enilson

    2013-11-01

    The electrospinning technique of rhizobia immobilization in nanofibres is an innovative and promising alternative for reducing the harmful effects of environmental stress on bacteria strains in a possible inoculant nanotechnology product for use in agriculture. The use of polyvinyl alcohol (PVA) shows up as an effective polymer in cell encapsulation because of its physical characteristics, such as viscosity and power of scattering. The aim of these studies has been to evaluate the survival of rhizobia incorporated in PVA nanofibres, which were applied to soybean seed and then subjected to different storage times and exposure to fungicide. The maintenance of the symbiotic characteristics of the incorporated bacterial strains was also evaluated, noting the formation of nodules in the soybean seedlings. No significant differences in the cell survival at 0 h and after 24 h of storage were observed. After 48 h, a significant difference in the bacterial cell concentration of the seeds affixed with PVA nanofibres was observed. Exposure to the fungicide decreased the viability of the bacteria strains even when coated with the nanofibres. A larger number of nodules formed in soybean seedlings from seeds inoculated with rhizobia incorporated in PVA nanofibres than from seeds inoculated with rhizobia without PVA. Thus, the electrospinning technique is a great alternative to the usual protector inoculants because of its unprecedented capacity to control the release of bacteria.

  1. Plant dependence on rhizobia for nitrogen influences induced plant defenses and herbivore performance.

    PubMed

    Dean, Jennifer M; Mescher, Mark C; De Moraes, Consuelo M

    2014-01-21

    Symbiotic rhizobia induce many changes in legumes that could affect aboveground interactions with herbivores. We explored how changing the intensity of Bradyrhizobium japonicum, as modulated by soil nitrogen (N) levels, influenced the interaction between soybean (Glycine max) and herbivores of different feeding guilds. When we employed a range of fertilizer applications to manipulate soil N, plants primarily dependent on rhizobia for N exhibited increased root nodulation and higher levels of foliar ureides than plants given N fertilizer; yet all treatments maintained similar total N levels. Soybean podworm (Helicoverpa zea) larvae grew best on plants with the highest levels of rhizobia but, somewhat surprisingly, preferred to feed on high-N-fertilized plants when given a choice. Induction of the defense signaling compound jasmonic acid (JA) by H. zea feeding damage was highest in plants primarily dependent on rhizobia. Differences in rhizobial dependency on soybean did not appear to affect interactions with the phloem-feeding soybean aphid (Aphis glycines). Overall, our results suggest that rhizobia association can affect plant nutritional quality and the induction of defense signaling pathways and that these effects may influence herbivore feeding preferences and performance-though such effects may vary considerably for different classes of herbivores.

  2. Genetic Diversity and Symbiotic Phenotype of Hairy Vetch Rhizobia in Japan.

    PubMed

    Yuan, Kun; Miwa, Hiroki; Iizuka, Maki; Yokoyama, Tadashi; Fujii, Yoshiharu; Okazaki, Shin

    2016-06-25

    Hairy vetch (Vicia villosa Roth) is a leguminous crop widely used as green manure and a cover crop in Japan. It exhibits strong weed-suppressing activity, high resistance to insect pests, and the ability to fix nitrogen through symbiotic interactions with soil bacteria known as rhizobia. Few studies have investigated the rhizobia that form nodules on hairy vetch in Japan, and the biological resources available for selecting high nitrogen-fixing rhizobia are limited. In the present study, we isolated 110 hairy vetch rhizobia from 13 different areas in Japan. Based on their 16S rRNA gene sequences, 73% of the isolates were identified as Rhizobium leguminosarum. A comparative analysis of nodC and 16S rRNA gene phylogenies revealed that several isolates possessed congruent nodC sequences despite having divergent 16S rRNA gene sequences, suggesting that the horizontal transfer of nod genes occurred during the evolution of rhizobia. Inoculation tests showed that isolates closely related to R. leguminosarum had better plant growth-promoting effects than other strains, thereby providing a promising agricultural resource for inoculating crops.

  3. The Plant Growth Promoting Substance, Lumichrome, Mimics Starch, and Ethylene-Associated Symbiotic Responses in Lotus and Tomato Roots

    PubMed Central

    Gouws, Liezel M.; Botes, Eileen; Wiese, Anna J.; Trenkamp, Sandra; Torres-Jerez, Ivone; Tang, Yuhong; Hills, Paul N.; Usadel, Björn; Lloyd, James R.; Fernie, Alisdair R.; Kossmann, Jens; van der Merwe, Margaretha J.

    2012-01-01

    Symbiosis involves responses that maintain the plant host and symbiotic partner’s genetic program; yet these cues are far from elucidated. Here we describe the effects of lumichrome, a flavin identified from Rhizobium spp., applied to lotus (Lotus japonicus) and tomato (Solanum lycopersicum). Combined transcriptional and metabolite analyses suggest that both species shared common pathways that were altered in response to this application under replete, sterile conditions. These included genes involved in symbiosis, as well as transcriptional and metabolic responses related to enhanced starch accumulation and altered ethylene metabolism. Lumichrome priming also resulted in altered colonization with either Mesorhizobium loti (for lotus) or Glomus intraradices/G. mossea (for tomato). It enhanced nodule number but not nodule formation in lotus; while leading to enhanced hyphae initiation and delayed arbuscule maturation in tomato. PMID:22701462

  4. A mechanistic molecular test of the plant-sanction hypothesis in legume-rhizobia mutualism

    NASA Astrophysics Data System (ADS)

    Marco, Diana E.; Pérez-Arnedo, Rebeca; Hidalgo-Perea, Ángeles; Olivares, José; Ruiz-Sainz, José E.; Sanjuán, Juan

    2009-09-01

    The origin and persistence of mutualism is difficult to explain because of the widespread occurrence of exploitative, 'cheating' partners. As a policing strategy stabilising intraspecific cooperation, host sanctions against non-N 2 fixing, cheating symbionts have been proposed to stabilise mutualism in legume-rhizobium symbiosis. Mechanism of penalisations would include decreased nodular rhizobial viability and/or early nodule senescence. We tested these potential mechanisms of penalisations in split-root experiments using two soybean varieties and two rhizobial strains, a cooperative, normal N 2-fixing strain and an isogenic non-fixing derivative. We found no differences in the number of viable rhizobia recovered from nodules and no differential expression of a nodular senescence molecular marker. Thus, our results do not support the hypothesis of plant sanctions acting against cheating rhizobia in our experimental conditions.

  5. New Betaproteobacterial Rhizobium Strains Able To Efficiently Nodulate Parapiptadenia rigida (Benth.) Brenan

    PubMed Central

    Taulé, Cecilia; Zabaleta, María; Mareque, Cintia; Platero, Raúl; Sanjurjo, Lucía; Sicardi, Margarita; Frioni, Lillian; Battistoni, Federico

    2012-01-01

    Among the leguminous trees native to Uruguay, Parapiptadenia rigida (Angico), a Mimosoideae legume, is one of the most promising species for agroforestry. Like many other legumes, it is able to establish symbiotic associations with rhizobia and belongs to the group known as nitrogen-fixing trees, which are major components of agroforestry systems. Information about rhizobial symbionts for this genus is scarce, and thus, the aim of this work was to identify and characterize rhizobia associated with P. rigida. A collection of Angico-nodulating isolates was obtained, and 47 isolates were selected for genetic studies. According to enterobacterial repetitive intergenic consensus PCR patterns and restriction fragment length polymorphism analysis of their nifH and 16S rRNA genes, the isolates could be grouped into seven genotypes, including the genera Burkholderia, Cupriavidus, and Rhizobium, among which the Burkholderia genotypes were the predominant group. Phylogenetic studies of nifH, nodA, and nodC sequences from the Burkholderia and the Cupriavidus isolates indicated a close relationship of these genes with those from betaproteobacterial rhizobia (beta-rhizobia) rather than from alphaproteobacterial rhizobia (alpha-rhizobia). In addition, nodulation assays with representative isolates showed that while the Cupriavidus isolates were able to effectively nodulate Mimosa pudica, the Burkholderia isolates produced white and ineffective nodules on this host. PMID:22226956

  6. Conditional requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis.

    PubMed

    Kelly, Simon J; Muszyński, Artur; Kawaharada, Yasuyuki; Hubber, Andree M; Sullivan, John T; Sandal, Niels; Carlson, Russell W; Stougaard, Jens; Ronson, Clive W

    2013-03-01

    Rhizobial surface polysaccharides are required for nodule formation on the roots of at least some legumes but the mechanisms by which they act are yet to be determined. As a first step to investigate the function of exopolysaccharide (EPS) in the formation of determinate nodules, we isolated Mesorhizobium loti mutants affected in various steps of EPS biosynthesis and characterized their symbiotic phenotypes on two Lotus spp. The wild-type M. loti R7A produced both high molecular weight EPS and lower molecular weight (LMW) polysaccharide fractions whereas most mutant strains produced only LMW fractions. Mutants affected in predicted early biosynthetic steps (e.g., exoB) formed nitrogen-fixing nodules on Lotus corniculatus and L. japonicus 'Gifu', whereas mutants affected in mid or late biosynthetic steps (e.g., exoU) induced uninfected nodule primordia and, occasionally, a few infected nodules following a lengthy delay. These mutants were disrupted at the stage of infection thread (IT) development. Symbiotically defective EPS and Nod factor mutants functionally complemented each other in co-inoculation experiments. The majority of full-length IT observed harbored only the EPS mutant strain and did not show bacterial release, whereas the nitrogen-fixing nodules contained both mutants. Examination of the symbiotic proficiency of the exoU mutant on various L. japonicus ecotypes revealed that both host and environmental factors were linked to the requirement for EPS. These results reveal a complex function for M. loti EPS in determinate nodule formation and suggest that EPS plays a signaling role at the stages of both IT initiation and bacterial release.

  7. Biodiversity and biogeography of rhizobia associated with common bean (Phaseolus vulgaris L.) in Shaanxi Province.

    PubMed

    Wang, Li; Cao, Ying; Wang, En Tao; Qiao, Ya Juan; Jiao, Shuo; Liu, Zhen Shan; Zhao, Liang; Wei, Ge Hong

    2016-05-01

    The biodiversity and biogeography of rhizobia associated with bean in Shaanxi Province were investigated. A total of 194 bacterial isolates from bean nodules collected from 13 sampling sites were characterized based on phylogenetic analyses of the 16S rRNA gene, the housekeeping genes recA, glnII and atpD, and the symbiotic genes nodC and nifH. Fifteen genospecies belonging to the genera Rhizobium, Agrobacterium, Ensifer, Bradyrhizobium and Ochrobactrum were defined among the isolates, with Rhizobium sp. II, Agrobacterium sp. II, E. fredii and R. phaseoli being the dominant groups. Four symbiotic gene lineages corresponding to Rhizobium sp. I, Rhizobium sp. II, R. phaseoli and B. liaoningense were detected in the nodC and nifH sequence analyses, indicating different origins for the symbiotic genes and their co-evolution with the chromosome of the bacteria. Moreover, the Ensifer isolates harbored symbiotic genes closely related to bean-nodulating Pararhizobium giardinii, indicating possible lateral gene transfer from Rhizobium to Ensifer. Correlation of rhizobial community composition with moisture, temperature, intercropping, soil features and nutrients were detected. All the results demonstrated a great diversity of bean rhizobia in Shaanxi that might be due to the adaptable evolution of the bean-nodulating rhizobia subjected to the diverse ecological conditions in the area. Copyright © 2016 Elsevier GmbH. All rights reserved.

  8. Multiple steps control immunity during the intracellular accommodation of rhizobia

    PubMed Central

    Berrabah, Fathi; Ratet, Pascal; Gourion, Benjamin

    2015-01-01

    Medicago truncatula belongs to the legume family and forms symbiotic associations with nitrogen fixing bacteria, the rhizobia. During these interactions, the plants develop root nodules in which bacteria invade the plant cells and fix nitrogen for the benefit of the plant. Despite massive infection, legume nodules do not develop visible defence reactions, suggesting a special immune status of these organs. Some factors influencing rhizobium maintenance within the plant cells have been previously identified, such as the M. truncatula NCR peptides whose toxic effects are reduced by the bacterial protein BacA. In addition, DNF2, SymCRK, and RSD are M. truncatula genes required to avoid rhizobial death within the symbiotic cells. DNF2 and SymCRK are essential to prevent defence-like reactions in nodules after bacteria internalization into the symbiotic cells. Herein, we used a combination of genetics, histology and molecular biology approaches to investigate the relationship between the factors preventing bacterial death in the nodule cells. We show that the RSD gene is also required to repress plant defences in nodules. Upon inoculation with the bacA mutant, defence responses are observed only in the dnf2 mutant and not in the symCRK and rsd mutants. In addition, our data suggest that lack of nitrogen fixation by the bacterial partner triggers bacterial death in nodule cells after bacteroid differentiation. Together our data indicate that, after internalization, at least four independent mechanisms prevent bacterial death in the plant cell. These mechanisms involve successively: DNF2, BacA, SymCRK/RSD and bacterial ability to fix nitrogen. PMID:25682610

  9. Boston Scientific Lotus valve.

    PubMed

    Meredith, Ian T; Hood, Kristin L; Haratani, Nicole; Allocco, Dominic J; Dawkins, Keith D

    2012-09-01

    As a result of recent randomised controlled trials and registry observations, transcatheter aortic valve replacement (TAVR) enjoys growing appeal for the treatment of patients at high or extreme risk from surgical aortic valve replacement. However, the current technologies and techniques have important limitations, including risk of stroke, vascular complications and paravalvular aortic regurgitation, which may in turn influence survival. While careful patient selection and screening may improve outcomes, new valve designs and iterations are required. The Lotus aortic valve replacement system is a new fully repositionable device designed to facilitate more precise delivery and minimise paravalvular regurgitation. The safety and efficacy of the Lotus valve are being studied systematically in the REPRISE clinical trial programme.

  10. Binding of isolated plant lectin by rhizobia during episodes of reduced gravity obtained by parabolic flight

    NASA Technical Reports Server (NTRS)

    Henry, R. L.; Green, P. D.; Wong, P. P.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1990-01-01

    Development of a legume root nodule is a complex process culminating in a plant/bacterial symbiosis possessing the capacity for biological dinitrogen fixation. Formation of root nodules is initiated by the binding and stabilization of rhizobia to plant root hairs, mediated in part by a receptor/ligand recognition system composed of lectins on the plant root surface and lectin-binding sites on the rhizobial cell surface. The dinitrogen fixation activity of these root nodules may be an important feature of enclosed, space-based life support systems, and may provide an ecological method to recycle nitrogen for amino acid production. However, the effects on nodule development of varied gravitational fields, or of root nutrient delivery hardware, remain unknown. We have investigated the effects of microgravity on root nodule formation, with preliminary experiments focused upon the receptor/ligand component. Microgravity, obtained during parabolic flight aboard NASA 930, has no apparent effect on the binding of purified lectin to rhizobia, a result that will facilitate forthcoming experiments using intact root tissues.

  11. Prospecting metal-tolerant rhizobia for phytoremediation of mining soils from Morocco using Anthyllis vulneraria L.

    PubMed

    El Aafi, N; Saidi, N; Maltouf, A Filali; Perez-Palacios, P; Dary, M; Brhada, F; Pajuelo, E

    2015-03-01

    The aim of this work was using the legume plant Anthyllis vulneraria L. (ecotype metallicolous) as a trap plant, in order to isolate metal-tolerant rhizobial strains from metal-contaminated soils from Morocco, with pollution indexes spanning three orders of magnitude. As bioindicator, soil bacterial density was inversely correlated to the pollution index. Forty-three bulk soil bacteria and sixty two bacteria from nodules were isolated. The resistance of bacteria from nodules to heavy metals was four to ten times higher than that of bulk soil bacteria, reaching high maximum tolerable concentrations for Cd (2 mM), Cu (2 mM), Pb (7 mM), and Zn (3 mM). Besides, some strains show multiple metal-tolerant abilities and great metal biosorption onto the bacterial surface. Amplification and restriction analysis of ribosomal 16S rDNA (ARDRA) and 16S ribosomal DNA (rDNA) sequencing were used to assess biodiversity and phylogenetic position among bacteria present in nodules. Our results suggest that a great diversity of non-rhizobial bacteria (alpha- and gamma-proteobacteria) colonize nodules of Anthyllis plants in contaminated soils. Taking together, our results evidence that, in polluted soils, rhizobia can be displaced by non-rhizobial (and hence, non-fixing) strains from nodules. Thus, the selection of metal-resistant rhizobia is a key step for using A. vulneraria symbioses for in situ phytoremediation.

  12. Binding of isolated plant lectin by rhizobia during episodes of reduced gravity obtained by parabolic flight

    NASA Technical Reports Server (NTRS)

    Henry, R. L.; Green, P. D.; Wong, P. P.; Guikema, J. A.; Spooner, B. S. (Principal Investigator)

    1990-01-01

    Development of a legume root nodule is a complex process culminating in a plant/bacterial symbiosis possessing the capacity for biological dinitrogen fixation. Formation of root nodules is initiated by the binding and stabilization of rhizobia to plant root hairs, mediated in part by a receptor/ligand recognition system composed of lectins on the plant root surface and lectin-binding sites on the rhizobial cell surface. The dinitrogen fixation activity of these root nodules may be an important feature of enclosed, space-based life support systems, and may provide an ecological method to recycle nitrogen for amino acid production. However, the effects on nodule development of varied gravitational fields, or of root nutrient delivery hardware, remain unknown. We have investigated the effects of microgravity on root nodule formation, with preliminary experiments focused upon the receptor/ligand component. Microgravity, obtained during parabolic flight aboard NASA 930, has no apparent effect on the binding of purified lectin to rhizobia, a result that will facilitate forthcoming experiments using intact root tissues.

  13. Nodule characterization: subsolid nodules.

    PubMed

    Raad, Roy A; Suh, James; Harari, Saul; Naidich, David P; Shiau, Maria; Ko, Jane P

    2014-01-01

    In this review, we focus on the radiologic, clinical, and pathologic aspects primarily of solitary subsolid pulmonary nodules. Particular emphasis will be placed on the pathologic classification and correlative computed tomography (CT) features of adenocarcinoma of the lung. The capabilities of fluorodeoxyglucose positron emission tomography-CT and histologic sampling techniques, including CT-guided biopsy, endoscopic-guided biopsy, and surgical resection, are discussed. Finally, recently proposed management guidelines by the Fleischner Society and the American College of Chest Physicians are reviewed.

  14. Biotin biosynthesis, transport and utilization in rhizobia.

    PubMed

    Guillén-Navarro, Karina; Encarnación, Sergio; Dunn, Michael F

    2005-05-15

    Biotin, a B-group vitamin, performs an essential metabolic function in all organisms. Rhizobia are alpha-proteobacteria with the remarkable ability to form a nitrogen-fixing symbiosis in combination with a compatible legume host, a process in which the importance of biotin biosynthesis and/or transport has been demonstrated for some rhizobia-legume combinations. Rhizobia have also been used to delimit the biosynthesis, metabolic effects and, more recently, transport of biotin. Molecular genetic analysis shows that an orthodox biotin biosynthesis pathway occurs in some rhizobia while others appear to synthesize the vitamin using alternative pathways. In addition to its well established function as a prosthetic group for biotin-dependent carboxylases, we are beginning to delineate a role for biotin as a metabolic regulator in rhizobia.

  15. Response to temperature stress in rhizobia.

    PubMed

    Alexandre, Ana; Oliveira, Solange

    2013-08-01

    It is well established that soil is a challenging environment for bacteria, where conditions may change rapidly and bacteria have to acclimate and adapt in order to survive. Rhizobia are an important group of soil bacteria due to their ability to establish atmospheric nitrogen-fixing symbioses with many legume species. Some of these legumes are used to feed either humans or cattle and therefore the use of rhizobia can reduce the need for synthetic N-fertilizers. Several environmental factors shape the composition and the activity of rhizobia populations in the rhizosphere. Soil pH and temperature are often considered to be the major abiotic factors in determining the bacterial community diversity. The present review focuses on the current knowledge on the molecular bases of temperature stress response in rhizobia. The effects of temperature stress in the legume-rhizobia symbioses are also addressed.

  16. Regulatory patterns of a large family of defensin-like genes expressed in nodules of Medicago truncatula

    USDA-ARS?s Scientific Manuscript database

    Root nodules are the symbiotic organ of legumes that house nitrogen-fixing bacteria. Many genes are specifically induced in nodules during the interactions between the host plant and symbiotic rhizobia. Information regarding the regulation of expression for most of these genes is lacking. One of the...

  17. Life histories of symbiotic rhizobia and mycorrhizal fungi.

    PubMed

    Denison, R Ford; Kiers, E Toby

    2011-09-27

    Research on life history strategies of microbial symbionts is key to understanding the evolution of cooperation with hosts, but also their survival between hosts. Rhizobia are soil bacteria known for fixing nitrogen inside legume root nodules. Arbuscular mycorrhizal (AM) fungi are ubiquitous root symbionts that provide plants with nutrients and other benefits. Both kinds of symbionts employ strategies to reproduce during symbiosis using host resources; to repopulate the soil; to survive in the soil between hosts; and to find and infect new hosts. Here we focus on the fitness of the microbial symbionts and how interactions at each of these stages has shaped microbial life-history strategies. During symbiosis, microbial fitness could be increased by diverting more resources to individual reproduction, but that may trigger fitness-reducing host sanctions. To survive in the soil, symbionts employ sophisticated strategies, such as persister formation for rhizobia and reversal of spore germination by mycorrhizae. Interactions among symbionts, from rhizobial quorum sensing to fusion of genetically distinct fungal hyphae, increase adaptive plasticity. The evolutionary implications of these interactions and of microbial strategies to repopulate and survive in the soil are largely unexplored.

  18. Ecological Indicators of Native Rhizobia in Tropical Soils †

    PubMed Central

    Woomer, Paul; Singleton, Paul W.; Bohlool, B. Ben

    1988-01-01

    The relationship between environment and abundance of rhizobia was described by determining the populations of root nodule bacteria at 14 diverse sites on the island of Maui. Mean annual rainfall at the sites ranged from 320 to 1,875 mm, elevation from 37 to 1,650 m, and soil pH from 4.6 to 7.9. Four different soil orders were represented in this study: inceptisols, mollisols, ultisols, and an oxisol. The rhizobial populations were determined by plant infection counts of five legumes (Trifolium repens, Medicago sativa, Vicia sativa, Leucaena leucocephala, and Macroptilium atropurpureum). Populations varied from 1.1 to 4.8 log10 cells per g of soil. The most frequently occurring rhizobia were Bradyrhizobium spp., which were present at 13 of 14 sites with a maximum of 4.8 log10 cells per g of soil. Rhizobium trifolii and R. leguminosarum occurred only at higher elevations. The presence of a particular Rhizobium or Bradyrhizobium sp. was correlated with the occurrence of its appropriate host legume. Total rhizobial populations were significantly correlated with mean annual rainfall, legume cover and shoot biomass, soil temperature, soil pH, and phosphorus retention. Regression models are presented which describe the relationship of legume hosts, soil climate, and soil fertility on native rhizobial populations. PMID:16347624

  19. Co-inoculation Effect of Rhizobia and Plant Growth Promoting Rhizobacteria on Common Bean Growth in a Low Phosphorus Soil

    PubMed Central

    Korir, Hezekiah; Mungai, Nancy W.; Thuita, Moses; Hamba, Yosef; Masso, Cargele

    2017-01-01

    Nitrogen (N) fixation through legume-Rhizobium symbiosis is important for enhancing agricultural productivity and is therefore of great economic interest. Growing evidence indicates that other soil beneficial bacteria can positively affect symbiotic performance of rhizobia. Nodule endophytic plant growth promoting rhizobacteria (PGPR) were isolated from common bean nodules from Nakuru County in Kenya and characterized 16S rDNA partial gene sequencing. The effect of co-inoculation of rhizobium and PGPR, on nodulation and growth of common bean (Phaseolus vulgaris L.) was also investigated using a low phosphorous soil under greenhouse conditions. Gram-positive nodule endophytic PGPR belonging to the genus Bacillus were successfully isolated and characterized. Two PGPR strains (Paenibacillus polymyxa and Bacillus megaterium), two rhizobia strains (IITA-PAU 987 and IITA-PAU 983) and one reference rhizobia strain (CIAT 899) were used in the co-inoculation study. Two common bean varieties were inoculated with Rhizobium strains singly or in a combination with PGPR to evaluate the effect on nodulation and growth parameters. Co-inoculation of IITA-PAU 987 + B. megaterium recorded the highest nodule weight (405.2 mg) compared to IITA-PAU 987 alone (324.8 mg), while CIAT 899 + B. megaterium (401.2 mg) compared to CIAT 899 alone (337.2 mg). CIAT 899 + B. megaterium recorded a significantly higher shoot dry weight (7.23 g) compared to CIAT 899 alone (5.80 g). However, there was no significant difference between CIAT 899 + P. polymyxa and CIAT 899 alone. Combination of IITA-PAU 987 and B. megaterium led to significantly higher shoot dry weight (6.84 g) compared to IITA-PAU 987 alone (5.32 g) but no significant difference was observed when co-inoculated with P. polymyxa. IITA-PAU 983 in combination with P. polymyxa led to significantly higher shoot dry weight (7.15 g) compared to IITA-PAU 983 alone (5.14 g). Plants inoculated with IITA-PAU 987 and B. megaterium received 24.0 % of

  20. Co-inoculation Effect of Rhizobia and Plant Growth Promoting Rhizobacteria on Common Bean Growth in a Low Phosphorus Soil.

    PubMed

    Korir, Hezekiah; Mungai, Nancy W; Thuita, Moses; Hamba, Yosef; Masso, Cargele

    2017-01-01

    Nitrogen (N) fixation through legume-Rhizobium symbiosis is important for enhancing agricultural productivity and is therefore of great economic interest. Growing evidence indicates that other soil beneficial bacteria can positively affect symbiotic performance of rhizobia. Nodule endophytic plant growth promoting rhizobacteria (PGPR) were isolated from common bean nodules from Nakuru County in Kenya and characterized 16S rDNA partial gene sequencing. The effect of co-inoculation of rhizobium and PGPR, on nodulation and growth of common bean (Phaseolus vulgaris L.) was also investigated using a low phosphorous soil under greenhouse conditions. Gram-positive nodule endophytic PGPR belonging to the genus Bacillus were successfully isolated and characterized. Two PGPR strains (Paenibacillus polymyxa and Bacillus megaterium), two rhizobia strains (IITA-PAU 987 and IITA-PAU 983) and one reference rhizobia strain (CIAT 899) were used in the co-inoculation study. Two common bean varieties were inoculated with Rhizobium strains singly or in a combination with PGPR to evaluate the effect on nodulation and growth parameters. Co-inoculation of IITA-PAU 987 + B. megaterium recorded the highest nodule weight (405.2 mg) compared to IITA-PAU 987 alone (324.8 mg), while CIAT 899 + B. megaterium (401.2 mg) compared to CIAT 899 alone (337.2 mg). CIAT 899 + B. megaterium recorded a significantly higher shoot dry weight (7.23 g) compared to CIAT 899 alone (5.80 g). However, there was no significant difference between CIAT 899 + P. polymyxa and CIAT 899 alone. Combination of IITA-PAU 987 and B. megaterium led to significantly higher shoot dry weight (6.84 g) compared to IITA-PAU 987 alone (5.32 g) but no significant difference was observed when co-inoculated with P. polymyxa. IITA-PAU 983 in combination with P. polymyxa led to significantly higher shoot dry weight (7.15 g) compared to IITA-PAU 983 alone (5.14 g). Plants inoculated with IITA-PAU 987 and B. megaterium received 24.0 % of

  1. Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants.

    PubMed

    Kubo, Mie; Ueda, Hiroaki; Park, Pyoyun; Kawaguchi, Masayoshi; Sugimoto, Yukihiro

    2009-03-01

    Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.

  2. Genetic Diversity and Symbiotic Efficiency of Indigenous Common Bean Rhizobia in Croatia

    PubMed Central

    Pohajda, Ines; Babić, Katarina Huić; Rajnović, Ivana; Kajić, Sanja

    2016-01-01

    Summary Nodule bacteria (rhizobia) in symbiotic associations with legumes enable considerable entries of biologically fixed nitrogen into soil. Efforts are therefore made to intensify the natural process of symbiotic nitrogen fixation by legume inoculation. Studies of field populations of rhizobia open up the possibility to preserve and probably exploit some indigenous strains with hidden symbiotic or ecological potentials. The main aim of the present study is to determine genetic diversity of common bean rhizobia isolated from different field sites in central Croatia and to evaluate their symbiotic efficiency and compatibility with host plants. The isolation procedure revealed that most soil samples contained no indigenous common bean rhizobia. The results indicate that the cropping history had a significant impact on the presence of indigenous strains. Although all isolates were found to belong to species Rhizobium leguminosarum, significant genetic diversity at the strain level was determined. Application of both random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC- -PCR) methods resulted in similar grouping of strains. Symbiotic efficiency of indigenous rhizobia as well as their compatibility with two commonly grown bean varieties were tested in field experiments. Application of indigenous rhizobial strains as inoculants resulted in significantly different values of nodulation, seed yield as well as plant nitrogen and seed protein contents. The most abundant nodulation and the highest plant nitrogen and protein contents were determined in plants inoculated with R. leguminosarum strains S17/2 and S21/6. Although, in general, the inoculation had a positive impact on seed yield, differences depending on the applied strain were not determined. The overall results show the high degree of symbiotic efficiency of the specific indigenous strain S21/6. These results indicate different

  3. Genetic Diversity and Symbiotic Efficiency of Indigenous Common Bean Rhizobia in Croatia.

    PubMed

    Pohajda, Ines; Babić, Katarina Huić; Rajnović, Ivana; Kajić, Sanja; Sikora, Sanja

    2016-12-01

    Nodule bacteria (rhizobia) in symbiotic associations with legumes enable considerable entries of biologically fixed nitrogen into soil. Efforts are therefore made to intensify the natural process of symbiotic nitrogen fixation by legume inoculation. Studies of field populations of rhizobia open up the possibility to preserve and probably exploit some indigenous strains with hidden symbiotic or ecological potentials. The main aim of the present study is to determine genetic diversity of common bean rhizobia isolated from different field sites in central Croatia and to evaluate their symbiotic efficiency and compatibility with host plants. The isolation procedure revealed that most soil samples contained no indigenous common bean rhizobia. The results indicate that the cropping history had a significant impact on the presence of indigenous strains. Although all isolates were found to belong to species Rhizobium leguminosarum, significant genetic diversity at the strain level was determined. Application of both random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC- -PCR) methods resulted in similar grouping of strains. Symbiotic efficiency of indigenous rhizobia as well as their compatibility with two commonly grown bean varieties were tested in field experiments. Application of indigenous rhizobial strains as inoculants resulted in significantly different values of nodulation, seed yield as well as plant nitrogen and seed protein contents. The most abundant nodulation and the highest plant nitrogen and protein contents were determined in plants inoculated with R. leguminosarum strains S17/2 and S21/6. Although, in general, the inoculation had a positive impact on seed yield, differences depending on the applied strain were not determined. The overall results show the high degree of symbiotic efficiency of the specific indigenous strain S21/6. These results indicate different symbiotic

  4. Denitrification ability of rhizobial strains isolated from Lotus sp.

    PubMed

    Monza, Jorge; Irisarri, Pilar; Díaz, Pedro; Delgado, Ma Jesús; Mesa, Socorro; Bedmar, Eulogio J

    2006-01-01

    Ten rhizobial strains isolated from Lotus sp. have been characterized by their ability to denitrify. Out of the 10 strains, the five slow-growing isolates grew well under oxygen-limiting conditions with nitrate as a sole nitrogen source, and accumulated nitrous oxide in the growth medium when acetylene was used to inhibit nitrous oxide reductase activity. All five strains contained DNA homologous to the Bradyrhizobium japonicum nirK, norBDQ and nosZ genes. In contrast, fast-growing lotus rhizobia were incapable of growing under nitrate-respiring conditions, and did not accumulate nitrous oxide in the growth medium. DNA from each of the five fast-growing strains showed a hybridization band with the B. japonicum nirK gene but not with norBDQ and nosZ genes. Partial 16S rDNA gene sequencing revealed that fast-growing strains could be identified as Mesorhizobium loti species and the slow-growers as Bradyrhizobium sp.

  5. Potential of Native Rhizobia in Enhancing Nitrogen Fixation and Yields of Climbing Beans (Phaseolus vulgaris L.) in Contrasting Environments of Eastern Kenya.

    PubMed

    Koskey, Gilbert; Mburu, Simon W; Njeru, Ezekiel M; Kimiti, Jacinta M; Ombori, Omwoyo; Maingi, John M

    2017-01-01

    Climbing bean (Phaseolus vulgaris L.) production in Kenya is greatly undermined by low soil fertility, especially in agriculturally prolific areas. The use of effective native rhizobia inoculants to promote nitrogen fixation could be beneficial in climbing bean production. In this study, we carried out greenhouse and field experiments to evaluate symbiotic efficiency, compare the effect of native rhizobia and commercial inoculant on nodulation, growth and yield parameters of mid-altitude climbing bean (MAC 13 and MAC 64) varieties. The greenhouse experiment included nine native rhizobia isolates, a consortium of native isolates, commercial inoculant Biofix, a mixture of native isolates + Biofix, nitrogen treated control and a non-inoculated control. In the field experiments, the treatments included the best effective native rhizobia isolate ELM3, a consortium of native isolates, a commercial inoculant Biofix, a mixture of native isolates + Biofix, and a non-inoculated control. Remarkably, four native rhizobia isolates ELM3, ELM4, ELM5, and ELM8 showed higher symbiotic efficiencies compared to the Biofix. Interestingly, there was no significant difference in symbiotic efficiency between the two climbing bean varieties. Field results demonstrated a significant improvement in nodule dry weight and seed yields of MAC 13 and MAC 64 climbing bean varieties upon rhizobia inoculation when compared to the non-inoculated controls. Inoculation with ELM3 isolate resulted to the highest seed yield of 4,397.75 kg ha(-1), indicating 89% increase over non-inoculated control (2,334.81 kg ha(-1)) and 30% increase over Biofix (3,698.79 kg ha(-1)). Farm site significantly influenced nodule dry weight and seed yields. This study, therefore, revealed the potential of native rhizobia isolates to enhance delivery of agroecosystem services including nitrogen fixation and bean production. Further characterization and mapping of the native isolates will be imperative in development of

  6. Potential of Native Rhizobia in Enhancing Nitrogen Fixation and Yields of Climbing Beans (Phaseolus vulgaris L.) in Contrasting Environments of Eastern Kenya

    PubMed Central

    Koskey, Gilbert; Mburu, Simon W.; Njeru, Ezekiel M.; Kimiti, Jacinta M.; Ombori, Omwoyo; Maingi, John M.

    2017-01-01

    Climbing bean (Phaseolus vulgaris L.) production in Kenya is greatly undermined by low soil fertility, especially in agriculturally prolific areas. The use of effective native rhizobia inoculants to promote nitrogen fixation could be beneficial in climbing bean production. In this study, we carried out greenhouse and field experiments to evaluate symbiotic efficiency, compare the effect of native rhizobia and commercial inoculant on nodulation, growth and yield parameters of mid-altitude climbing bean (MAC 13 and MAC 64) varieties. The greenhouse experiment included nine native rhizobia isolates, a consortium of native isolates, commercial inoculant Biofix, a mixture of native isolates + Biofix, nitrogen treated control and a non-inoculated control. In the field experiments, the treatments included the best effective native rhizobia isolate ELM3, a consortium of native isolates, a commercial inoculant Biofix, a mixture of native isolates + Biofix, and a non-inoculated control. Remarkably, four native rhizobia isolates ELM3, ELM4, ELM5, and ELM8 showed higher symbiotic efficiencies compared to the Biofix. Interestingly, there was no significant difference in symbiotic efficiency between the two climbing bean varieties. Field results demonstrated a significant improvement in nodule dry weight and seed yields of MAC 13 and MAC 64 climbing bean varieties upon rhizobia inoculation when compared to the non-inoculated controls. Inoculation with ELM3 isolate resulted to the highest seed yield of 4,397.75 kg ha−1, indicating 89% increase over non-inoculated control (2,334.81 kg ha−1) and 30% increase over Biofix (3,698.79 kg ha−1). Farm site significantly influenced nodule dry weight and seed yields. This study, therefore, revealed the potential of native rhizobia isolates to enhance delivery of agroecosystem services including nitrogen fixation and bean production. Further characterization and mapping of the native isolates will be imperative in development of

  7. Legume growth-promoting rhizobia: an overview on the Mesorhizobium genus.

    PubMed

    Laranjo, Marta; Alexandre, Ana; Oliveira, Solange

    2014-01-20

    The need for sustainable agricultural practices is revitalizing the interest in biological nitrogen fixation and rhizobia-legumes symbioses, particularly those involving economically important legume crops in terms of food and forage. The genus Mesorhizobium includes species with high geographical dispersion and able to nodulate a wide variety of legumes, including important crop species, like chickpea or biserrula. Some cases of legume-mesorhizobia inoculant introduction represent exceptional opportunities to study the rhizobia genomes evolution and the evolutionary relationships among species. Complete genome sequences revealed that mesorhizobia typically harbour chromosomal symbiosis islands. The phylogenies of symbiosis genes, such as nodC, are not congruent with the phylogenies based on core genes, reflecting rhizobial host range, rather than species affiliation. This agrees with studies showing that Mesorhizobium species are able to exchange symbiosis genes through lateral transfer of chromosomal symbiosis islands, thus acquiring the ability to nodulate new hosts. Phylogenetic analyses of the Mesorhizobium genus based on core and accessory genes reveal complex evolutionary relationships and a high genomic plasticity, rendering the Mesorhizobium genus as a good model to investigate rhizobia genome evolution and adaptation to different host plants. Further investigation of symbiosis genes as well as stress response genes will certainly contribute to understand mesorhizobia-legume symbiosis and to develop more effective mesorhizobia inoculants.

  8. Environmental Signals and Regulatory Pathways That Influence Exopolysaccharide Production in Rhizobia

    PubMed Central

    Janczarek, Monika

    2011-01-01

    Rhizobia are Gram-negative bacteria that can exist either as free-living bacteria or as nitrogen-fixing symbionts inside root nodules of leguminous plants. The composition of the rhizobial outer surface, containing a variety of polysaccharides, plays a significant role in the adaptation of these bacteria in both habitats. Among rhizobial polymers, exopolysaccharide (EPS) is indispensable for the invasion of a great majority of host plants which form indeterminate-type nodules. Various functions are ascribed to this heteropolymer, including protection against environmental stress and host defense, attachment to abiotic and biotic surfaces, and in signaling. The synthesis of EPS in rhizobia is a multi-step process regulated by several proteins at both transcriptional and post-transcriptional levels. Also, some environmental factors (carbon source, nitrogen and phosphate starvation, flavonoids) and stress conditions (osmolarity, ionic strength) affect EPS production. This paper discusses the recent data concerning the function of the genes required for EPS synthesis and the regulation of this process by several environmental signals. Up till now, the synthesis of rhizobial EPS has been best studied in two species, Sinorhizobium meliloti and Rhizobium leguminosarum. The latest data indicate that EPS synthesis in rhizobia undergoes very complex hierarchical regulation, in which proteins engaged in quorum sensing and the regulation of motility genes also participate. This finding enables a better understanding of the complex processes occurring in the rhizosphere which are crucial for successful colonization and infection of host plant roots. PMID:22174640

  9. Thyroid nodule

    MedlinePlus

    ... has grown Another possible treatment is an ethanol (alcohol) injection into the nodule to shrink ... Division of Metabolism, Endocrinology & Nutrition, University of Washington School of Medicine, ...

  10. Manganese nodules

    USGS Publications Warehouse

    Hein, James R.; Harff, Jan; Petersen, Sven; Thiede, Jorn

    2016-01-01

    The existence of manganese (Mn) nodules (Fig. 1) has been known since the late 1800s when they were collected during the Challenger expedition of 1873–1876. However, it was not until after WWII that nodules were further studied in detail for their ability to adsorb metals from seawater. Many of the early studies did not distinguish Mn nodules from Mn crusts. Economic interest in Mn nodules began in the late 1950s and early 1960s when John Mero finished his Ph.D. thesis on this subject, which was published...

  11. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant.

    PubMed

    Silva, F V; Simões-Araújo, J L; Silva Júnior, J P; Xavier, G R; Rumjanek, N G

    2012-04-01

    The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium.

  12. Autophosphorylation is essential for the in vivo function of the Lotus japonicus Nod factor receptor 1 and receptor-mediated signalling in cooperation with Nod factor receptor 5.

    PubMed

    Madsen, Esben B; Antolín-Llovera, Meritxell; Grossmann, Christina; Ye, Juanying; Vieweg, Syndi; Broghammer, Angelique; Krusell, Lene; Radutoiu, Simona; Jensen, Ole N; Stougaard, Jens; Parniske, Martin

    2011-02-01

    Soil-living rhizobia secrete lipochitin oligosaccharides known as Nod factors, which in Lotus japonicus are perceived by at least two Nod-factor receptors, NFR1 and NFR5. Despite progress in identifying molecular components critical for initial legume host recognition of the microsymbiont and cloning of downstream components, little is known about the activation and signalling mechanisms of the Nod-factor receptors themselves. Here we show that both receptor proteins localize to the plasma membrane, and present evidence for heterocomplex formation initiating downstream signalling. Expression of NFR1 and NFR5 in Nicotiana benthamiana and Allium ampeloprasum (leek) cells caused a rapid cell-death response. The signalling leading to cell death was abrogated using a kinase-inactive variant of NFR1. In these surviving cells, a clear interaction between NFR1 and NFR5 was detected in vivo through bimolecular fluorescence complementation (BiFC). To analyse the inter- and intramolecular phosphorylation events of the kinase complex, the cytoplasmic part of NFR1 was assayed for in vitro kinase activity, and autophosphorylation on 24 amino acid residues, including three tyrosine residues, was found by mass spectrometry. Substitution of the phosphorylated amino acids of NFR1 identified a single phosphorylation site to be essential for NFR1 Nod-factor signalling in vivo and kinase activity in vitro. In contrast to NFR1, no in vitro kinase activity of the cytoplasmic domain of NFR5 was detected. This is further supported by the fact that a mutagenized NFR5 construct, substituting an amino acid essential for ATP binding, restored nodulation of nfr5 mutant roots. © 2010 The Authors. The Plant Journal © 2010 Blackwell Publishing Ltd.

  13. Evolutionary Dynamics of Nitrogen Fixation in the Legume–Rhizobia Symbiosis

    PubMed Central

    Fujita, Hironori; Aoki, Seishiro; Kawaguchi, Masayoshi

    2014-01-01

    The stabilization of host–symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost–benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume–rhizobia symbioses, which is extendable to other single-host, multiple

  14. Evolutionary dynamics of nitrogen fixation in the legume-rhizobia symbiosis.

    PubMed

    Fujita, Hironori; Aoki, Seishiro; Kawaguchi, Masayoshi

    2014-01-01

    The stabilization of host-symbiont mutualism against the emergence of parasitic individuals is pivotal to the evolution of cooperation. One of the most famous symbioses occurs between legumes and their colonizing rhizobia, in which rhizobia extract nutrients (or benefits) from legume plants while supplying them with nitrogen resources produced by nitrogen fixation (or costs). Natural environments, however, are widely populated by ineffective rhizobia that extract benefits without paying costs and thus proliferate more efficiently than nitrogen-fixing cooperators. How and why this mutualism becomes stabilized and evolutionarily persists has been extensively discussed. To better understand the evolutionary dynamics of this symbiosis system, we construct a simple model based on the continuous snowdrift game with multiple interacting players. We investigate the model using adaptive dynamics and numerical simulations. We find that symbiotic evolution depends on the cost-benefit balance, and that cheaters widely emerge when the cost and benefit are similar in strength. In this scenario, the persistence of the symbiotic system is compatible with the presence of cheaters. This result suggests that the symbiotic relationship is robust to the emergence of cheaters, and may explain the prevalence of cheating rhizobia in nature. In addition, various stabilizing mechanisms, such as partner fidelity feedback, partner choice, and host sanction, can reinforce the symbiotic relationship by affecting the fitness of symbionts in various ways. This result suggests that the symbiotic relationship is cooperatively stabilized by various mechanisms. In addition, mixed nodule populations are thought to encourage cheater emergence, but our model predicts that, in certain situations, cheaters can disappear from such populations. These findings provide a theoretical basis of the evolutionary dynamics of legume-rhizobia symbioses, which is extendable to other single-host, multiple

  15. nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

    PubMed Central

    Stacey, G; Luka, S; Sanjuan, J; Banfalvi, Z; Nieuwkoop, A J; Chun, J Y; Forsberg, L S; Carlson, R

    1994-01-01

    The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal. Images PMID:8300517

  16. NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes.

    PubMed

    Couzigou, Jean-Malo; Zhukov, Vladimir; Mondy, Samuel; Abu el Heba, Ghada; Cosson, Viviane; Ellis, T H Noel; Ambrose, Mike; Wen, Jiangqi; Tadege, Million; Tikhonovich, Igor; Mysore, Kirankumar S; Putterill, Joanna; Hofer, Julie; Borisov, Alexei Y; Ratet, Pascal

    2012-11-01

    During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ.

  17. Manganese nodules

    USGS Publications Warehouse

    Hein, James R.; Harff, Jan; Petersen, Sven; Thiede, Jorn

    2016-01-01

    The existence of manganese (Mn) nodules (Figure 1) has been known since the late 1800s when they were collected during the Challenger expedition of 1873–1876. However, it was not until after WWII that nodules were further studied in detail for their ability to adsorb metals from seawater. Many of the early studies did not distinguish Mn nodules from Mn crusts. Economic interest in Mn nodules began in the late 1950s and early 1960s when John Mero finished his Ph.D. thesis on this subject, which was published in the journal Economic Geology (Mero, 1962) and later as a book (Mero, 1965). By the mid-1970s, large consortia had formed to search for and mine Mn nodules that occur between the Clarion and Clipperton fracture zones (CCZ) in the NE Pacific (Figure 2). This is still the area considered of greatest economic potential in the global ocean because of high nickel (Ni), copper (Cu), and Mn contents and the dense distribution of nodules in the area. While the mining of nodules was fully expected to begin in the late 1970s or early 1980s, this never occurred due to a downturn in the price of metals on the global market. Since then, many research cruises have been undertaken to study the CCZ nodules, and now 15 contracts for exploration sites have been given or are pending by the International Seabed Authority (ISA). Many books and science journal articles have been published summarizing the early work (e.g., Baturin, 1988; Halbach et al., 1988), and research has continued to the present day (e.g., ISA, 1999; ISA, 2010). Although the initial attraction for nodules was their high Ni, Cu, and Mn contents, subsequent work has shown that nodules host large quantities of other critical metals needed for high-tech, green-tech, and energy applications (Hein et al., 2013; Hein and Koschinsky, 2014).

  18. Hormonal Control of Lateral Root and Nodule Development in Legumes

    PubMed Central

    Bensmihen, Sandra

    2015-01-01

    Many plants can establish symbioses with nitrogen-fixing bacteria, some of which lead to nodulation, including legumes. Indeed, in the rhizobium/legume symbiosis, new root organs, called nodules, are formed by the plant in order to host the rhizobia in protective conditions, optimized for nitrogen fixation. In this way, these plants can benefit from the reduction of atmospheric dinitrogen into ammonia by the hosted bacteria, and in exchange the plant provides the rhizobia with a carbon source. Since this symbiosis is costly for the plant it is highly regulated. Both legume nodule and lateral root organogenesis involve divisions of the root inner tissues, and both developmental programs are tightly controlled by plant hormones. In fact, most of the major plant hormones, such as auxin, cytokinins, abscisic acid, and strigolactones, control both lateral root formation and nodule organogenesis, but often in an opposite manner. This suggests that the sensitivity of legume plants to some phytohormones could be linked to the antagonism that exists between the processes of nodulation and lateral root formation. Here, we will review the implication of some major phytohormones in lateral root formation in legumes, compare them with their roles in nodulation, and discuss specificities and divergences from non-legume eudicot plants such as Arabidopsis thaliana. PMID:27135340

  19. Characteristics of bacteroids in indeterminate nodules of the leguminous tree Leucaena glauca.

    PubMed

    Ishihara, Hironobu; Koriyama, Hiroki; Osawa, Atsushi; Zehirov, Grigor; Yamaura, Masatoshi; Kucho, Ken-ichi; Abe, Mikiko; Higashi, Shiro; Kondorosi, Eva; Mergaert, Peter; Uchiumi, Toshiki

    2011-01-01

    Rhizobia establish symbiosis with legumes. Bacteroids in indeterminate nodules of Inverted Repeat Lacking Clade (IRLC) legumes undergo terminal differentiation caused by Nodule-specific Cysteine-Rich peptides (NCRs). Microscopic observations of bacteroids and the detection of NCRs in indeterminate nodules of the non-IRLC legume Leucaena glauca were performed. A portion of the bacteroids showed moderate cell elongation, loss of membrane integrity, and multiple nucleoids. The symbiosome contained multiple bacteroids and NCR-like peptides were not detectable. These results indicate that bacteroid differentiation in L. glauca is different from that in IRLC legumes although both hosts form indeterminate nodules.

  20. Differential effectiveness of novel and old legume-rhizobia mutualisms: implications for invasion by exotic legumes.

    PubMed

    Rodríguez-Echeverría, Susana; Fajardo, Susana; Ruiz-Díez, Beatriz; Fernández-Pascual, Mercedes

    2012-09-01

    The degree of specialization in the legume-rhizobium mutualism and the variation in the response to different potential symbionts are crucial factors for understanding the process of invasion by exotic legumes and the consequences for the native resident plants and bacteria. The enhanced novel mutualism hypothesis predicts that exotic invasive legumes would take advantage of native rhizobia present in the invaded soils. However, recent studies have shown that exotic legumes might become invasive by using exotic introduced microsymbionts, and that they could be a source of exotic bacteria for native legumes. To unravel the role of novel and old symbioses in the progress of invasion, nodulation and symbiotic effectiveness were analyzed for exotic invasive plants and native co-occurring legumes in a Mediterranean coastal dune ecosystem. Although most of the studied species nodulated with bacteria from distant origins these novel mutualisms were less effective in terms of nodulation, nitrogenase activity and plant growth than the interactions of plants and bacteria from the same origin. The relative effect of exotic bradyrhizobia was strongly positive for exotic invasive legumes and detrimental for native shrubs. We conclude that (1) the studied invasive legumes do not rely on novel mutualisms but rather need the co-introduction of compatible symbionts, and (2) since exotic rhizobia colonize native legumes in invaded areas, the lack of effectiveness of these novel symbiosis demonstrated here suggests that invasion can disrupt native belowground mutualisms and reduce native legumes fitness.

  1. Genetic diversity and distribution of rhizobia associated with the medicinal legumes Astragalus spp. and Hedysarum polybotrys in agricultural soils.

    PubMed

    Yan, Hui; Ji, Zhao Jun; Jiao, Yin Shan; Wang, En Tao; Chen, Wen Feng; Guo, Bao Lin; Chen, Wen Xin

    2016-03-01

    With the increasing cultivation of medicinal legumes in agricultural fields, the rhizobia associated with these plants are facing new stresses, mainly from fertilization and irrigation. In this study, investigations on the nodulation of three cultivated medicinal legumes, Astragalus mongholicus, Astragalus membranaceus and Hedysarum polybotrys were performed. Bacterial isolates from root nodules of these legumes were subjected to genetic diversity and multilocus sequence analyses. In addition, the distribution of nodule bacteria related to soil factors and host plants was studied. A total 367 bacterial isolates were obtained and 13 genospecies were identified. The predominant microsymbionts were identified as Mesorhizobium septentrionale, Mesorhizobium temperatum, Mesorhizobium tianshanense, Mesorhizobium ciceri and Mesorhizobium muleiense. M. septentrionale was found in most root nodules especially from legumes grown in the barren soils (with low available nitrogen and low organic carbon contents), while M. temperatum was predominant in nodules where the plants were grown in the nitrogen-rich fields. A. mongholicus tended to be associated with M. septentrionale, M. temperatum and M. ciceri in different soils, while A. membranaceus and H. polybotrys tended to be associated with M. tianshanense and M. septentrionale, respectively. This study showed that soil fertility may be the main determinant for the distribution of rhizobia associated with these cultured legume plants.

  2. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1.

    PubMed

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T; Hocart, Charles H; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-08-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation.

  3. Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

    PubMed Central

    Ng, Jason Liang Pin; Hassan, Samira; Truong, Thy T.; Hocart, Charles H.; Laffont, Carole; Frugier, Florian; Mathesius, Ulrike

    2015-01-01

    Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation. PMID:26253705

  4. [Milker's nodules].

    PubMed

    Hansen, S K; Mertz, H; Krogdahl, A S; Veien, N K

    1997-01-20

    Milker's nodule is a parapox virus infection seen mostly on the hands of dairy farmers. We saw 15 cases over a period of two years in the County of North Jutland. Clinically, milker's nodule goes through a papular, a nodular and a crusted stage. Most patients were seen when the infection was in the nodular stage, an often painful condition requiring treatment. Three patients developed an erythema multiformelike secondary eruption. Lesions from nine patients were removed for histological examination. The histology of all lesions was consistent with milker's nodule. In three of seven patients parapox virus was demonstrated by electron microscopy. Treatment was commonly curettage followed by cauterization.

  5. A comparative study of different factors involved in mass cultivation of rhizobia, using shakers and fermentors.

    PubMed

    Gulati, S L

    1978-01-01

    Growth was proportionally linear to increasing load of inoculum although an inoculum load of 6 to 8% was optimum to obtain uniform number of viable cells, beyond which the number of viable cells did not increase. From the point of view of contamination with other microorganisms and the nodulating ability of cultures, fermentor cultures were better than shake cultures. When growth of Rhizobium was studied in relation to unit of mannitol consumed, it was observed that fermentors are more economical for culturing rhizobia than shakers.

  6. Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

    PubMed

    Fterich, A; Mahdhi, M; Lafuente, A; Pajuelo, E; Caviedes, M A; Rodriguez-Llorente, I D; Mars, M

    2012-06-01

    A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

  7. Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation.

    PubMed

    Montiel, Jesús; Arthikala, Manoj-Kumar; Cárdenas, Luis; Quinto, Carmen

    2016-05-18

    Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant's response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis.

  8. Legume NADPH Oxidases Have Crucial Roles at Different Stages of Nodulation

    PubMed Central

    Montiel, Jesús; Arthikala, Manoj-Kumar; Cárdenas, Luis; Quinto, Carmen

    2016-01-01

    Plant NADPH oxidases, formerly known as respiratory burst oxidase homologues (RBOHs), are plasma membrane enzymes dedicated to reactive oxygen species (ROS) production. These oxidases are implicated in a wide variety of processes, ranging from tissue and organ growth and development to signaling pathways in response to abiotic and biotic stimuli. Research on the roles of RBOHs in the plant’s response to biotic stresses has mainly focused on plant-pathogen interactions; nonetheless, recent findings have shown that these oxidases are also involved in the legume-rhizobia symbiosis. The legume-rhizobia symbiosis leads to the formation of the root nodule, where rhizobia reduce atmospheric nitrogen to ammonia. A complex signaling and developmental pathway in the legume root hair and root facilitate rhizobial entrance and nodule organogenesis, respectively. Interestingly, several reports demonstrate that RBOH-mediated ROS production displays versatile roles at different stages of nodulation. The evidence collected to date indicates that ROS act as signaling molecules that regulate rhizobial invasion and also function in nodule senescence. This review summarizes discoveries that support the key and versatile roles of various RBOH members in the legume-rhizobia symbiosis. PMID:27213330

  9. The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth.

    PubMed

    Hobecker, Karen Vanesa; Reynoso, Mauricio Alberto; Bustos-Sanmamed, Pilar; Wen, Jiangqi; Mysore, Kirankumar S; Crespi, Martín; Blanco, Flavio Antonio; Zanetti, María Eugenia

    2017-08-01

    Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2 Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development. © 2017 American Society of Plant Biologists. All Rights Reserved.

  10. Reassimilation of ammonium in Lotus japonicus.

    PubMed

    Betti, Marco; García-Calderón, Margarita; Pérez-Delgado, Carmen M; Credali, Alfredo; Pal'ove-Balang, Peter; Estivill, Guillermo; Repčák, Miroslav; Vega, José M; Galván, Francisco; Márquez, Antonio J

    2014-10-01

    This review summarizes the most recent results obtained in the analysis of two important metabolic pathways involved in the release of internal sources of ammonium in the model legume Lotus japonicus: photorespiratory metabolism and asparagine breakdown mediated by aparaginase (NSE). The use of photorespiratory mutants deficient in plastidic glutamine synthetase (GS2) enabled us to investigate the transcriptomics and metabolomic changes associated with photorespiratory ammonium accumulation in this plant. The results obtained indicate the existence of a coordinate regulation of genes involved in photorespiratory metabolism. Other types of evidence illustrate the multiple interconnections existing among the photorespiratory pathway and other processes such as intermediate metabolism, nodule function, and secondary metabolism in this plant, all of which are substantially affected in GS2-deficient mutants because of the impairment of the photorespiratory cycle. Finally, the importance of asparagine metabolism in L. japonicus is highlighted because of the fact that asparagine constitutes the vast majority of the reduced nitrogen translocated between different organs of this plant. The different types of NSE enzymes and genes which are present in L. japonicus are described. There is a particular focus on the most abundant K(+)-dependent LjNSE1 isoform and how TILLING mutants were used to demonstrate by reverse genetics the importance of this particular isoform in plant growth and seed production. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  11. Allelopatic effects of cyanobacteria extracts containing microcystins on Medicago sativa-Rhizobia symbiosis.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Lahrouni, Majida; El Ghazali, Issam; Saqrane, Sanaa; Vasconcelos, Vitor; Oudra, Brahim

    2011-03-01

    The eutrophication of water leads to massive blooms of cyanobacteria potentially producers of highly toxic substances: cyanotoxins, especially microcystins (MC). The contamination of water used for irrigation by these toxins, can cause several adverse effects on plants and microorganisms. In this work, we report the phytotoxic effects of microcystins on the development of symbiosis between the leguminous plant Medicago sativa (Alfalfa) and rhizobia strains. The exposure of rhizobial strains to three different concentrations 0.01, 0.05 and 0.1 μg MC ml(-1) led to decrease on the bacteria growth. The strains of rhizobia Rh L1, Rh L2, Rh L3 and Rh L4 reduced their growth to, respectively, 20.85%, 20.80%, 33.19% and 25.65%. The chronic exposure of alfalfa seeds and seedlings to different MC concentrations affects the whole stages of plant development. The germination process has also been disrupted with an inhibition, which reaches 68.34% for a 22.24 μg MC ml(-1). Further, seedlings growth and photosynthetic process were also disrupted. The toxins reduced significantly the roots length and nodule formation and leads to an oxidative stress. Thus, the MCs contained in lake water and used for irrigation affect the development of symbiosis between M. sativa and Rhizobia.

  12. Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria.

    PubMed

    Tampakaki, Anastasia P

    2014-01-01

    Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80's and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts.

  13. Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria

    PubMed Central

    Tampakaki, Anastasia P.

    2014-01-01

    Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80's and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts. PMID:24723933

  14. Genetic Characterization of Soybean Rhizobia Isolated from Different Ecological Zones in North-Eastern Afghanistan.

    PubMed

    Habibi, Safiullah; Ayubi, Abdul Ghani; Ohkama-Ohtsu, Naoko; Sekimoto, Hitoshi; Yokoyama, Tadashi

    2017-03-31

    Seventy rhizobial isolates were obtained from the root nodules of two soybean (Glycine max) cultivars: Japanese cultivar Enrei and USA cultivar Stine3300, which were inoculated with different soil samples from Afghanistan. In order to study the genetic properties of the isolates, the DNA sequences of the 16S rRNA gene and symbiotic genes (nodD1 and nifD) were elucidated. Furthermore, the isolates were inoculated into the roots of two soybean cultivars, and root nodule numbers and nitrogen fixation abilities were subsequently evaluated in order to assess symbiotic performance. Based on 16S rRNA gene sequences, the Afghanistan isolates obtained from soybean root nodules were classified into two genera, Bradyrhizobium and Ensifer. Bradyrhizobium isolates accounted for 54.3% (38) of the isolates, and these isolates had a close relationship with Bradyrhizobium liaoningense and B. yuanmingense. Five out of the 38 Bradyrhizobium isolates showed a novel lineage for B. liaoningense and B. yuanmingense. Thirty-two out of the 70 isolates were identified as Ensifer fredii. An Ensifer isolate had identical nodD1 and nifD sequences to those in B. yuanmingense. This result indicated that the horizontal gene transfer of symbiotic genes occurred from Bradyrhizobium to Ensifer in Afghanistan soil. The symbiotic performance of the 14 tested isolates from the root nodules of the two soybean cultivars indicated that Bradyrhizobium isolates exhibited stronger acetylene reduction activities than Ensifer isolates. This is the first study to genetically characterize soybean-nodulating rhizobia in Afghanistan soil.

  15. Lung Nodules: Overview

    MedlinePlus

    ... Research & Science Education & Training Home Conditions Lung Nodules Lung Nodules Make an Appointment Find a Doctor Ask ... Kern, MD (June 01, 2016) What is a lung nodule? A lung nodule is also called a ...

  16. Lung Nodules: Overview

    MedlinePlus

    ... Research & Science Education & Training Home Conditions Lung Nodules Lung Nodules Make an Appointment Find a Doctor Ask ... Kern, MD (June 01, 2016) What is a lung nodule? A lung nodule is also called a ...

  17. Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes.

    PubMed

    Thies, J E; Singleton, P W; Bohlool, B B

    1991-01-01

    significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N(2)-fixing crops indicates a potential for improving inoculation technology, the N(2) fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia.

  18. Influence of the Size of Indigenous Rhizobial Populations on Establishment and Symbiotic Performance of Introduced Rhizobia on Field-Grown Legumes †

    PubMed Central

    Thies, Janice E.; Singleton, Paul W.; Bohlool, B. Ben

    1991-01-01

    significantly increased 85% of the time. Yield was significantly increased in only 6% of the observations when numbers of indigenous rhizobia were greater than 10 cells g of soil-1. A significant response to N application, significant increases in nodule parameters, and greater than 50% nodule occupancy by inoculant rhizobia did not necessarily coincide with significant inoculation responses. No less than a doubling of nodule mass and 66% nodule occupancy by inoculant rhizobia were required to significantly increase the yield of inoculated crops over that of uninoculated crops. However, lack of an inoculation response was common even when inoculum strains occupied the majority of nodules. In these trials, the symbiotic yield of crops was, on average, only 88% of the maximum yield potential, as defined by the fertilizer N treatment. The difference between the yield of N-fertilized crops and that of N2-fixing crops indicates a potential for improving inoculation technology, the N2 fixation capacity of rhizobial strains, and the efficiency of symbiosis. In this study, we show that the probability of enhancing yield with existing inoculation technology decreases dramatically with increasing numbers of indigenous rhizobia. PMID:16348393

  19. Genotypic and phenotypic diversity of rhizobia isolated from Lathyrus japonicus indigenous to Japan.

    PubMed

    Aoki, Seishiro; Kondo, Tetsuya; Prévost, Danielle; Nakata, Sayuri; Kajita, Tadashi; Ito, Motomi

    2010-11-01

    Sixty-one rhizobial strains from Lathyrus japonicus nodules growing on the seashore in Japan were characterized and compared to two strains from Canada. The PCR-based method was used to identify test strains with novel taxonomic markers that were designed to discriminate between all known Lathyrus rhizobia. Three genomic groups (I, II, and III) were finally identified using RAPD, RFLP, and phylogenetic analyses. Strains in genomic group I (related to Rhizobium leguminosarum) were divided into two subgroups (Ia and Ib) and subgroup Ia was related to biovar viciae. Strains in subgroup Ib, which were all isolated from Japanese sea pea, belonged to a distinct group from other rhizobial groups in the recA phylogeny and PCR-based grouping, and were more tolerant to salt than the isolate from an inland legume. Test strains in genomic groups II and III belonged to a single clade with the reference strains of R. pisi, R. etli, and R. phaseoli in the 16S rRNA phylogeny. The PCR-based method and phylogenetic analysis of recA revealed that genomic group II was related to R. pisi. The analyses also showed that genomic group III harbored a mixed chromosomal sequence of different genomic groups, suggesting a recent horizontal gene transfer between diverse rhizobia. Although two Canadian strains belonged to subgroup Ia, molecular and physiological analyses showed the divergence between Canadian and Japanese strains. Phylogenetic analysis of nod genes divided the rhizobial strains into several groups that reflected the host range of rhizobia. Symbiosis between dispersing legumes and rhizobia at seashore is discussed.

  20. nip, a symbiotic Medicago truncatula mutant that forms root nodules with aberrant infection threads and plant defense-like response.

    PubMed

    Veereshlingam, Harita; Haynes, Janine G; Penmetsa, R Varma; Cook, Douglas R; Sherrier, D Janine; Dickstein, Rebecca

    2004-11-01

    To investigate the legume-Rhizobium symbiosis, we isolated and studied a novel symbiotic mutant of the model legume Medicago truncatula, designated nip (numerous infections and polyphenolics). When grown on nitrogen-free media in the presence of the compatible bacterium Sinorhizobium meliloti, the nip mutant showed nitrogen deficiency symptoms. The mutant failed to form pink nitrogen-fixing nodules that occur in the wild-type symbiosis, but instead developed small bump-like nodules on its roots that were blocked at an early stage of development. Examination of the nip nodules by light microscopy after staining with X-Gal for S. meliloti expressing a constitutive GUS gene, by confocal microscopy following staining with SYTO-13, and by electron microscopy revealed that nip initiated symbiotic interactions and formed nodule primordia and infection threads. The infection threads in nip proliferated abnormally and very rarely deposited rhizobia into plant host cells; rhizobia failed to differentiate further in these cases. nip nodules contained autofluorescent cells and accumulated a brown pigment. Histochemical staining of nip nodules revealed this pigment to be polyphenolic accumulation. RNA blot analyses demonstrated that nip nodules expressed only a subset of genes associated with nodule organogenesis, as well as elevated expression of a host defense-associated phenylalanine ammonia lyase gene. nip plants were observed to have abnormal lateral roots. nip plant root growth and nodulation responded normally to ethylene inhibitors and precursors. Allelism tests showed that nip complements 14 other M. truncatula nodulation mutants but not latd, a mutant with a more severe nodulation phenotype as well as primary and lateral root defects. Thus, the nip mutant defines a new locus, NIP, required for appropriate infection thread development during invasion of the nascent nodule by rhizobia, normal lateral root elongation, and normal regulation of host defense-like responses

  1. Effects of co-inoculation with arbuscular mycorrhizal fungi and rhizobia on soybean growth as related to root architecture and availability of N and P.

    PubMed

    Wang, Xiurong; Pan, Qiang; Chen, Fengxian; Yan, Xiaolong; Liao, Hong

    2011-04-01

    Soybean plants can form tripartite symbiotic associations with rhizobia and arbuscular mycorrhizal (AM) fungi, but little is known about effects of co-inoculation with rhizobia and AM fungi on plant growth, or their relationships to root architecture as well as nitrogen (N) and phosphorus (P) availability. In the present study, two soybean genotypes contrasting in root architecture were grown in a field experiment to evaluate relationships among soybean root architecture, AMF colonization, and nodulation under natural conditions. Additionally, a soil pot experiment in greenhouse was conducted to investigate the effects of co-inoculation with rhizobia and AM fungi on soybean growth, and uptake of N and P. Our results indicated that there was a complementary relationship between root architecture and AMF colonization in the field. The deep root soybean genotype had greater AMF colonization at low P, but better nodulation with high P supply than the shallow root genotype. A synergistic relationship dependent on N and P status exists between rhizobia and AM fungi on soybean growth. Co-inoculation with rhizobia and AM fungi significantly increased soybean growth under low P and/or low N conditions as indicated by increased shoot dry weight, along with plant N and P content. There were no significant effects of inoculation under adequate N and P conditions. Furthermore, the effects of co-inoculation were related to root architecture. The deep root genotype, HN112, benefited more from co-inoculation than the shallow root genotype, HN89. Our results elucidate new insights into the relationship between rhizobia, AM fungi, and plant growth under limitation of multiple nutrients, and thereby provides a theoretical basis for application of co-inoculation in field-grown soybean.

  2. Molybdate in Rhizobial Seed-Coat Formulations Improves the Production and Nodulation of Alfalfa.

    PubMed

    Zhou, Jiqiong; Deng, Bo; Zhang, Yingjun; Cobb, Adam B; Zhang, Zhao

    2017-01-01

    Rhizobia-legume symbiosis is the most well researched biological nitrogen fixation system. Coating legume seeds with rhizobia is now a recognized practical measure for improving the production of legume corp. However, the efficacy of some commercial rhizobia inoculants cannot be guaranteed in China due to the low rate of live rhizobia in these products. A greenhouse experiment was conducted to assess the effects of different rhizobial inoculant formulations on alfalfa productivity and nitrogen fixation. Two rhizobia strains, (ACCC17631 and ACCC17676), that are effective partners with alfalfa variety Zhongmu No. 1 were assessed with different concentrations of ammonium molybdate in seed-coat formulations with two different coating adhesives. Our study showed that the growth, nodulation, and nitrogen fixation ability of the plants inoculated with the ACCC17631 rhizobial strain were greatest when the ammonium molybdate application was0.2% of the formulation. An ammonium molybdate concentration of 0.1% was most beneficial to the growth of the plants inoculated with the ACCC17676 rhizobial strain. The sodium carboxymethyl cellulose and sodium alginate, used as coating adhesives, did not have a significant effect on alfalfa biomass and nitrogen fixation. However, the addition of skimmed milk to the adhesive improved nitrogenase activity. These results demonstrate that a new rhizobial seed-coat formulation benefitted alfalfa nodulation and yield.

  3. Molybdate in Rhizobial Seed-Coat Formulations Improves the Production and Nodulation of Alfalfa

    PubMed Central

    Zhou, Jiqiong; Deng, Bo; Zhang, Yingjun; Cobb, Adam B.; Zhang, Zhao

    2017-01-01

    Rhizobia-legume symbiosis is the most well researched biological nitrogen fixation system. Coating legume seeds with rhizobia is now a recognized practical measure for improving the production of legume corp. However, the efficacy of some commercial rhizobia inoculants cannot be guaranteed in China due to the low rate of live rhizobia in these products. A greenhouse experiment was conducted to assess the effects of different rhizobial inoculant formulations on alfalfa productivity and nitrogen fixation. Two rhizobia strains, (ACCC17631 and ACCC17676), that are effective partners with alfalfa variety Zhongmu No. 1 were assessed with different concentrations of ammonium molybdate in seed-coat formulations with two different coating adhesives. Our study showed that the growth, nodulation, and nitrogen fixation ability of the plants inoculated with the ACCC17631 rhizobial strain were greatest when the ammonium molybdate application was0.2% of the formulation. An ammonium molybdate concentration of 0.1% was most beneficial to the growth of the plants inoculated with the ACCC17676 rhizobial strain. The sodium carboxymethyl cellulose and sodium alginate, used as coating adhesives, did not have a significant effect on alfalfa biomass and nitrogen fixation. However, the addition of skimmed milk to the adhesive improved nitrogenase activity. These results demonstrate that a new rhizobial seed-coat formulation benefitted alfalfa nodulation and yield. PMID:28099471

  4. [Symbiotic efficiency and genetic diversity of the rhizobia isolated from Leucaena leucocephala in Liangshan Prefecture].

    PubMed

    Xu, Kaiwei; Zhang, Xiaoping; Chen, Yuanxue; Gu, Feiyue; Zhou, Dehai; Tang, Chengyi

    2014-05-04

    We analyzed the symbiotic efficiency and genetic diversity of rhizobia isolated from Leucaena leucocephala in Liangshan Prefecture of SichuanProvince. We studied genetic diversity of these isolates with 16S rRNA RFLP, BOX-PCR and AFLP fingerprinting, and constructed phylogenetic tree based on the concatenated sequences of the four housekeeping genes 16S rRNA, recA, atpD and glnII. The nodulation ability and the symbiotic efficiency of the isolates were tested by plant inoculation assay on their original host plant. Genetic diversity and phylogenetic tree indicate that 26 isolates were assigned as Sinorhizobium, 3 Bradyrhizobium, 3 Rhizobium and 1 Mesorhizobium. SCAU203 might represent a new Rhizobium group, SCAU211 might represent a new Bradyrhizobium group, the other three representative strains were located in three phylogenic branches and closely related to S. americanum, M. plurifarium and R. huautlense, respectively. In the nodulation and symbiotic efficiency assay, only 2 of the 20 isolates promoted the growth of L. leucocephala, but 3 isolates had a growth slowing effect on the host, while the other isolates (84%) were ineffective on symbiotic nitrogen fixation. The majority of rhizobia isolated from L. leucocephala in Liangshan Prefecture were ineffective on symbiotic nitrogen fixation.

  5. Physiological and antioxidant responses of Medicago sativa-rhizobia symbiosis to cyanobacterial toxins (Microcystins) exposure.

    PubMed

    El Khalloufi, Fatima; Oufdou, Khalid; Lahrouni, Majida; Faghire, Mustapha; Peix, Alvaro; Ramírez-Bahena, Martha Helena; Vasconcelos, Vitor; Oudra, Brahim

    2013-12-15

    Toxic cyanobacteria in freshwaters can induce potent harmful effects on growth and development of plants irrigated with contaminated water. In this study, the effect of cyanobacteria extract containing Microcystins (MC) on Medicago sativa-rhizobia symbiosis was investigated in order to explore plants response through biomass production, photosynthetic pigment and antioxidant enzymes analysis: Peroxidase (POD), Polyphenoloxidase (PPO) and Catalase (CAT). Alfalfa plants were inoculated with two endosymbiotic rhizobial strains: RhOL1 (MC less sensitive strain) and RhOL3 (MC more sensitive strain), to evaluate the rhizobial contribution on the plant response cultured under cyanobacterial toxins stress. The two rhizobia strains were identified as Ensifer meliloti by sequence analysis of their rrs and atpD genes. The chronic exposure to MC extract showed shoot, root and nodules dry weight decrease, in both symbiosis cultures. The rate of decline in plants inoculated with RhOL3 was higher than that in symbiosis with RhOL1 mainly at 20 μg L(-1) of MC. Cyanotoxins also reduced photosynthetic pigment content and generated an oxidative stress observed at cellular level. POD, PPO and CAT activities were significantly increased in leaves, roots and nodules of alfalfa plants exposed to MC. These enzyme activities were higher in plants inoculated with RhOL3 especially when alfalfa plants were exposed to 20 μg L(-1) of MC. The present paper reports new scientific finding related to the behavior of rhizobia-M. sativa associations to MC (Microcystins) for later recommendation concerning the possible use of these symbiosis face to crops exposure to MC contaminated water irrigation.

  6. Insights into post-transcriptional regulation during legume-rhizobia symbiosis.

    PubMed

    Reynoso, Mauricio Alberto; Blanco, Flavio Antonio; Zanetti, María Eugenia

    2013-02-01

    During the past ten years, changes in the transcriptome have been assessed at different stages of the legume-rhizobia association by the use of DNA microarrays and, more recently, by RNA sequencing technologies. These studies allowed the identification of hundred or thousand of genes whose steady-state mRNA levels increase or decrease upon bacterial infection or in nodules as compared with uninfected roots. However, transcriptome based-approaches do not distinguish between mRNAs that are being actively translated, stored as messenger ribonucleoproteins (mRNPs) or targeted for degradation. Despite that the increase in steady-state levels of an mRNA does not necessarily correlate with an increase in abundance or activity of the encoded protein, this information has been commonly used to select genes that are candidates to play a role during nodule organogenesis or bacterial infection. Such criterion does not take into account the post-transcriptional mechanisms that contribute to the regulation of gene expression. One of such mechanisms, which has significant impact on gene expression, is the selective recruitment of mRNAs to the translational machinery.  Here, we review the post-transcriptional mechanisms that contribute to the regulation of gene expression in the context of the ecological and agronomical important symbiotic interaction established between roots of legumes and the nitrogen fixing bacteria collectively known as rhizobia. In addition, we discuss how the development of new technologies that allow the assessment of these regulatory layers would help to understand the genetic network governing legume rhizobia symbiosis.

  7. Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China.

    PubMed

    Zhao, Liang; Fan, Miaochun; Zhang, Dehui; Yang, Ruiping; Zhang, Feilong; Xu, Lin; Wei, Xiuli; Shen, Yaoyao; Wei, Gehong

    2014-09-01

    A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China. Copyright © 2014 Elsevier GmbH. All rights reserved.

  8. Nitrate Effects on Nodule Oxygen Permeability and Leghemoglobin (Nodule Oximetry and Computer Modeling).

    PubMed Central

    Denison, R. F.; Harter, B. L.

    1995-01-01

    Two current hypotheses to explain nitrate inhibition of nodule function both involve decreased O2 supply for respiration in support of N2 fixation. This decrease could result from either (a) decreased O2 permeability (PO) of the nodule cortex, or (b) conversion of leghemoglobin (Lb) to an inactive, nitrosyl form. These hypotheses were tested using alfalfa (Medicago sativa L. cv Weevlchek) and birdsfoot trefoil (Lotus corniculatus L. cv Fergus) plants grown in growth pouches under controlled conditions. Nodulated roots were exposed to 10 mM KNO3 or KCI. Fractional oxygenation of Lb under air (FOLair), relative concentration of functional Lb, apparent PO, and O2-saturated central zone respiration rate were all monitored by nodule oximetry. Apparent PO and FOLair in nitrate-treated nodules decreased to <50% of values for KCI controls within 24 h, but there was no decrease in functional Lb concentration during the first 72 h. In nitrate-treated alfalfa, but not in birdsfoot trefoil, FOLair, apparent PO, and O2-saturated central zone respiration rate decreased during each light period and recovered somewhat during the subsequent dark period. This species difference could be explained by greater reliance on photoreduction of nitrate in alfalfa than in birdsfoot trefoil. Computer simulations extended the experimental results, showing that previously reported decreases in apparent PO of Glycine max nodules with nitrate exposure cannot be explained by hypothetical decreases in the concentration or O2 affinity of Lb. PMID:12228439

  9. Diversity and Evolution of Hydrogenase Systems in Rhizobia

    PubMed Central

    Baginsky, Cecilia; Brito, Belén; Imperial, Juan; Palacios, José-Manuel; Ruiz-Argüeso, Tomás

    2002-01-01

    Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (hup) systems in Bradyrhizobium japonicum and Rhizobium leguminosarum bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of hup gene clusters from Bradyrhizobium sp. (Lupinus), Bradyrhizobium sp. (Vigna), and Rhizobium tropici and Azorhizobium caulinodans strains with the aim of defining the extent of diversity in hup gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using hupS, hupE, hupUV, hypB, and hoxA probes showed a diversity of intraspecific hup profiles within Bradyrhizobium sp. (Lupinus) and Bradyrhizobium sp. (Vigna) strains and homogeneous intraspecific patterns within R. tropici and A. caulinodans strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of hupS and hupL clustered R. leguminosarum and R. tropici hup sequences together with those from B. japonicum and Bradyrhizobium sp. (Lupinus) strains, suggesting a common origin. In contrast, Bradyrhizobium sp. (Vigna) hup sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source. PMID:12324339

  10. Determination of the hydrogenase status of individual legume nodules by a methylene blue reduction assay.

    PubMed

    Lambert, G R; Hanus, F J; Russell, S A; Evans, H J

    1985-08-01

    We adapted a method for the rapid screening of colonies of free-living Rhizobium japonicum for hydrogenase activity to determine the hydrogenase status of individual soybean nodules. Crude bacteroid suspensions from nodules containing strains known to be hydrogen uptake positive (Hup) caused a localized decolorization of filter paper disks, whereas suspensions from nodules arising from inoculation with hydrogen uptake-negative (Hup) mutants or strains did not decolorize the disks. The reliability of the method was demonstrated by its successful application to 29 slow-growing rhizobia. The Hup phenotype on methylene blue filters agreed with that determined amperometrically with either methylene blue or oxygen as the electron acceptor.

  11. Characterization of rhizobia isolated from Albizia spp. in comparison with microsymbionts of Acacia spp. and Leucaena leucocephala grown in China.

    PubMed

    Wang, Feng Qin; Wang, En Tao; Zhang, Yong Fa; Chen, Wen Xin

    2006-09-01

    This is the first systematic study of rhizobia associated with Albizia trees. The analyses of PCR-RFLP and sequencing of 16S rRNA genes, SDS-PAGE of whole-cell proteins and clustering of phenotypic characters grouped the 31 rhizobial strains isolated from Albizia into eight putative species within the genera Bradyrhizobium, Mesorhizobium and Rhizobium. Among these eight rhizobial species, five were unique to Albizia and the remaining three were shared with Acacia and Leucaena, two legume trees coexisting with Albizia in China. These results indicated that Albizia species nodulate with a wide range of rhizobial species and had preference of microsymbionts different from Acacia and Leucaena. The definition of four novel groups, Mesorhizobium sp., Rhizobium sp. I, Rhizobium sp. II and "R. giardinii", indicates that further studies with enlarged rhizobial population are necessary to better understand the diversity and to clarify the taxonomic relationships of Albizia-associated rhizobia.

  12. The Symbiotic Performance of Chickpea Rhizobia Can Be Improved by Additional Copies of the clpB Chaperone Gene.

    PubMed

    Paço, Ana; Brígido, Clarisse; Alexandre, Ana; Mateos, Pedro F; Oliveira, Solange

    2016-01-01

    The ClpB chaperone is known to be involved in bacterial stress response. Moreover, recent studies suggest that this protein has also a role in the chickpea-rhizobia symbiosis. In order to improve both stress tolerance and symbiotic performance of a chickpea microsymbiont, the Mesorhizobium mediterraneum UPM-Ca36T strain was genetically transformed with pPHU231 containing an extra-copy of the clpB gene. To investigate if the clpB-transformed strain displays an improved stress tolerance, bacterial growth was evaluated under heat and acid stress conditions. In addition, the effect of the extra-copies of the clpB gene in the symbiotic performance was evaluated using plant growth assays (hydroponic and pot trials). The clpB-transformed strain is more tolerant to heat shock than the strain transformed with pPHU231, supporting the involvement of ClpB in rhizobia heat shock tolerance. Both plant growth assays showed that ClpB has an important role in chickpea-rhizobia symbiosis. The nodulation kinetics analysis showed a higher rate of nodule appearance with the clpB-transformed strain. This strain also induced a greater number of nodules and, more notably, its symbiotic effectiveness increased ~60% at pH5 and 83% at pH7, compared to the wild-type strain. Furthermore, a higher frequency of root hair curling was also observed in plants inoculated with the clpB-transformed strain, compared to the wild-type strain. The superior root hair curling induction, nodulation ability and symbiotic effectiveness of the clpB-transformed strain may be explained by an increased expression of symbiosis genes. Indeed, higher transcript levels of the nodulation genes nodA and nodC (~3 folds) were detected in the clpB-transformed strain. The improvement of rhizobia by addition of extra-copies of the clpB gene may be a promising strategy to obtain strains with enhanced stress tolerance and symbiotic effectiveness, thus contributing to their success as crop inoculants, particularly under

  13. A Phaseolus vulgaris NADPH oxidase gene is required for root infection by Rhizobia.

    PubMed

    Montiel, Jesús; Nava, Noreide; Cárdenas, Luis; Sánchez-López, Rosana; Arthikala, Manoj-Kumar; Santana, Olivia; Sánchez, Federico; Quinto, Carmen

    2012-10-01

    Plant NADPH oxidases [respiratory burst oxidase homologs (RBOHs)] have emerged as key players in the regulation of plant-pathogen interactions. Nonetheless, their role in mutualistic associations, such as the rhizobia-legume symbiosis, is poorly understood. In this work, nine members of the Phaseolus vulgaris Rboh gene family were identified. The transcript of one of these, PvRbohB, accumulated abundantly in shoots, roots and nodules. PvRbohB promoter activity was detected in meristematic regions of P. vulgaris roots, as well as during infection thread (IT) progression and nodule development. RNA interference (RNAi)-mediated PvRbohB down-regulation in transgenic roots reduced reactive oxygen species (ROS) production and lateral root density, and greatly impaired nodulation. Microscopy analysis revealed that progression of the ITs was impeded at the base of root hairs in PvRbohB-RNAi roots. Furthermore, the few nodules that formed in PvRbohB-down-regulated roots displayed abnormally wide ITs and reduced nitrogen fixation. These findings indicate that this common bean NADPH oxidase is crucial for successful rhizobial colonization and probably maintains proper IT growth and shape.

  14. The role of the plant cytoskeleton in the interaction between legumes and rhizobia.

    PubMed

    Timmers, A C J

    2008-08-01

    The study of the symbiotic interaction between rhizobia and legumes represents a major theme in plant biology. This interaction results in the formation of nodules, root organs in which the bacteria reduce atmospheric nitrogen into ammonia, which can subsequently be utilized by the plant. The execution of the different developmental stages observed during nodule ontogenesis involves many cellular processes with significant roles for the plant cytoskeleton. A challenging question in cell biology is how the cytoskeleton organizes itself into the dynamic arrays required for cell differentiation and functioning. Nodulation is, particularly, well qualified as an experimental system for cytoskeleton research because an early essential step of the plant/microbe interaction takes place in surface-exposed root hairs, well suited for cell biological in vivo experimentation. Moreover, the changes in the organization of the cytoskeleton can be elicited by a well-defined molecule, the Nod factor, or by bacterial inoculation, thus providing the researcher with the possibility of controlling the cytoskeletal changes in target cells. In addition, the well-known cytology of the symbiotic interaction facilitates the correlation between the changes in the organization of the plant cytoskeleton with both histological and cellular changes. In this review, the current knowledge on the role of the plant cytoskeleton during nodulation is summarized, with emphasis on the interaction between Medicago truncatula and Sinorhizobium meliloti.

  15. Ascending migration of endophytic rhizobia, from roots to leaves, inside rice plants and assessment of benefits to rice growth physiology.

    PubMed

    Chi, Feng; Shen, Shi-Hua; Cheng, Hai-Ping; Jing, Yu-Xiang; Yanni, Youssef G; Dazzo, Frank B

    2005-11-01

    Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 x 10(10) rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the world's most important cereal crops.

  16. NODULE ROOT and COCHLEATA Maintain Nodule Development and Are Legume Orthologs of Arabidopsis BLADE-ON-PETIOLE Genes[W][OA

    PubMed Central

    Couzigou, Jean-Malo; Zhukov, Vladimir; Mondy, Samuel; Abu el Heba, Ghada; Cosson, Viviane; Ellis, T.H. Noel; Ambrose, Mike; Wen, Jiangqi; Tadege, Million; Tikhonovich, Igor; Mysore, Kirankumar S.; Putterill, Joanna; Hofer, Julie; Borisov, Alexei Y.; Ratet, Pascal

    2012-01-01

    During their symbiotic interaction with rhizobia, legume plants develop symbiosis-specific organs on their roots, called nodules, that house nitrogen-fixing bacteria. The molecular mechanisms governing the identity and maintenance of these organs are unknown. Using Medicago truncatula nodule root (noot) mutants and pea (Pisum sativum) cochleata (coch) mutants, which are characterized by the abnormal development of roots from the nodule, we identified the NOOT and COCH genes as being necessary for the robust maintenance of nodule identity throughout the nodule developmental program. NOOT and COCH are Arabidopsis thaliana BLADE-ON-PETIOLE orthologs, and we have shown that their functions in leaf and flower development are conserved in M. truncatula and pea. The identification of these two genes defines a clade in the BTB/POZ-ankyrin domain proteins that shares conserved functions in eudicot organ development and suggests that NOOT and COCH were recruited to repress root identity in the legume symbiotic organ. PMID:23136374

  17. The Nodulation of Alfalfa by the Acid-Tolerant Rhizobium sp. Strain LPU83 Does Not Require Sulfated Forms of Lipochitooligosaccharide Nodulation Signals▿

    PubMed Central

    Torres Tejerizo, Gonzalo; Del Papa, María Florencia; Soria-Diaz, M. Eugenia; Draghi, Walter; Lozano, Mauricio; Giusti, María de los Ángeles; Manyani, Hamid; Megías, Manuel; Gil Serrano, Antonio; Pühler, Alfred; Niehaus, Karsten; Lagares, Antonio; Pistorio, Mariano

    2011-01-01

    The induction of root nodules by the majority of rhizobia has a strict requirement for the secretion of symbiosis-specific lipochitooligosaccharides (nodulation factors [NFs]). The nature of the chemical substitution on the NFs depends on the particular rhizobium and contributes to the host specificity imparted by the NFs. We present here a description of the genetic organization of the nod gene cluster and the characterization of the chemical structure of the NFs associated with the broad-host-range Rhizobium sp. strain LPU83, a bacterium capable of nodulating at least alfalfa, bean, and Leucena leucocephala. The nod gene cluster was located on the plasmid pLPU83b. The organization of the cluster showed synteny with those of the alfalfa-nodulating rhizobia, Sinorhizobium meliloti and Sinorhizobium medicae. Interestingly, the strongest sequence similarity observed was between the partial nod sequences of Rhizobium mongolense USDA 1844 and the corresponding LPU83 nod genes sequences. The phylogenetic analysis of the intergenic region nodEG positions strain LPU83 and the type strain R. mongolense 1844 in the same branch, which indicates that Rhizobium sp. strain LPU83 might represent an early alfalfa-nodulating genotype. The NF chemical structures obtained for the wild-type strain consist of a trimeric, tetrameric, and pentameric chitin backbone that shares some substitutions with both alfalfa- and bean-nodulating rhizobia. Remarkably, while in strain LPU83 most of the NFs were sulfated in their reducing terminal residue, none of the NFs isolated from the nodH mutant LPU83-H were sulfated. The evidence obtained supports the notion that the sulfate decoration of NFs in LPU83 is not necessary for alfalfa nodulation. PMID:20971905

  18. A Legume Genetic Framework Controls Infection of Nodules by Symbiotic and Endophytic Bacteria

    PubMed Central

    Zgadzaj, Rafal; James, Euan K.; Kelly, Simon; Kawaharada, Yasuyuki; de Jonge, Nadieh; Jensen, Dorthe B.; Madsen, Lene H.; Radutoiu, Simona

    2015-01-01

    Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule. PMID:26042417

  19. A legume genetic framework controls infection of nodules by symbiotic and endophytic bacteria.

    PubMed

    Zgadzaj, Rafal; James, Euan K; Kelly, Simon; Kawaharada, Yasuyuki; de Jonge, Nadieh; Jensen, Dorthe B; Madsen, Lene H; Radutoiu, Simona

    2015-06-01

    Legumes have an intrinsic capacity to accommodate both symbiotic and endophytic bacteria within root nodules. For the symbionts, a complex genetic mechanism that allows mutual recognition and plant infection has emerged from genetic studies under axenic conditions. In contrast, little is known about the mechanisms controlling the endophytic infection. Here we investigate the contribution of both the host and the symbiotic microbe to endophyte infection and development of mixed colonised nodules in Lotus japonicus. We found that infection threads initiated by Mesorhizobium loti, the natural symbiont of Lotus, can selectively guide endophytic bacteria towards nodule primordia, where competent strains multiply and colonise the nodule together with the nitrogen-fixing symbiotic partner. Further co-inoculation studies with the competent coloniser, Rhizobium mesosinicum strain KAW12, show that endophytic nodule infection depends on functional and efficient M. loti-driven Nod factor signalling. KAW12 exopolysaccharide (EPS) enabled endophyte nodule infection whilst compatible M. loti EPS restricted it. Analysis of plant mutants that control different stages of the symbiotic infection showed that both symbiont and endophyte accommodation within nodules is under host genetic control. This demonstrates that when legume plants are exposed to complex communities they selectively regulate access and accommodation of bacteria occupying this specialized environmental niche, the root nodule.

  20. The MicroRNA390/TAS3 Pathway Mediates Symbiotic Nodulation and Lateral Root Growth1[OPEN

    PubMed Central

    Bustos-Sanmamed, Pilar; Mysore, Kirankumar S.

    2017-01-01

    Legume roots form two types of postembryonic organs, lateral roots and symbiotic nodules. Nodule formation is the result of the interaction of legumes with rhizobia and requires the mitotic activation and differentiation of root cells as well as an independent, but coordinated, program that allows infection by rhizobia. MicroRNA390 (miR390) is an evolutionarily conserved microRNA that targets the Trans-Acting Short Interference RNA3 (TAS3) transcript. Cleavage of TAS3 by ARGONAUTE7 results in the production of trans-acting small interference RNAs, which target mRNAs encoding AUXIN RESPONSE FACTOR2 (ARF2), ARF3, and ARF4. Here, we show that activation of the miR390/TAS3 regulatory module by overexpression of miR390 in Medicago truncatula promotes lateral root growth but prevents nodule organogenesis, rhizobial infection, and the induction of two key nodulation genes, Nodulation Signaling Pathway1 (NSP1) and NSP2. Accordingly, inactivation of the miR390/TAS3 module, either by expression of a miR390 target mimicry construct or mutations in ARGONAUTE7, enhances nodulation and rhizobial infection, alters the spatial distribution of the nodules, and increases the percentage of nodules with multiple meristems. Our results revealed a key role of the miR390/TAS3 pathway in legumes as a modulator of lateral root organs, playing opposite roles in lateral root and nodule development. PMID:28663332

  1. Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry 1

    PubMed Central

    Tjepkema, John D.; Cartica, Robert J.

    1982-01-01

    Parasponia is the first non-legume genus proven to form nitrogen-fixing root nodules induced by rhizobia. Infiltration with India ink demonstrated that intercellular air spaces are lacking in the inner layers of the nodule cortex. Oxygen must diffuse through these layers to reach the cells containing the rhizobia, and it was calculated that most of the gradient in O2 partial pressure between the atmosphere and rhizobia occurs at the inner cortex. This was confirmed by O2 microelectrode measurements which showed that the O2 partial pressure was much lower in the zone of infected cells than in the cortex. Measurements of nitrogenase activity and O2 uptake as a function of temperature and partial pressure of O2 were consistent with diffusion limitation of O2 uptake by the inner cortex. In spite of the presumed absence of leghemoglobin in nodules of Parasponia rigida Merr. and Perry, energy usage for nitrogen fixation was similar to that in legume nodules. The results demonstrate that O2 regulation in legume and Parasponia nodules is very similar and differs from O2 regulation in actionorhizal nodules. Images PMID:16662284

  2. A nonRD receptor-like kinase prevents nodule early senescence and defense-like reactions during symbiosis.

    PubMed

    Berrabah, Fathi; Bourcy, Marie; Eschstruth, Alexis; Cayrel, Anne; Guefrachi, Ibtissem; Mergaert, Peter; Wen, Jiangqi; Jean, Viviane; Mysore, Kirankumar S; Gourion, Benjamin; Ratet, Pascal

    2014-09-01

    Rhizobia and legumes establish symbiotic interactions leading to the production of root nodules, in which bacteria fix atmospheric nitrogen for the plant's benefit. This symbiosis is efficient because of the high rhizobia population within nodules. Here, we investigated how legumes accommodate such bacterial colonization. We used a reverse genetic approach to identify a Medicago truncatula gene, SymCRK, which encodes a cysteine-rich receptor-like kinase that is required for rhizobia maintenance within the plant cells, and performed detailed phenotypic analyses of the corresponding mutant. The Medicago truncatula symCRK mutant developed nonfunctional and necrotic nodules. A nonarginine asparate (nonRD) motif, typical of receptors involved in innate immunity, is present in the SymCRK kinase domain. Similar to the dnf2 mutant, bacteroid differentiation defect, defense-like reactions and early senescence were observed in the symCRK nodules. However, the dnf2 and symCRK nodules differ by their degree of colonization, which is higher in symCRK. Furthermore, in contrast to dnf2, symCRK is not a conditional mutant. These results suggest that in M. truncatula at least two genes are involved in the symbiotic control of immunity. Furthermore, phenotype differences between the two mutants suggest that two distinct molecular mechanisms control suppression of plant immunity during nodulation.

  3. GS52 Ecto-Apyrase Plays a Critical Role during Soybean Nodulation1[W][OA

    PubMed Central

    Govindarajulu, Manjula; Kim, Sung-Yong; Libault, Marc; Berg, R. Howard; Tanaka, Kiwamu; Stacey, Gary; Taylor, Christopher G.

    2009-01-01

    Apyrases are non-energy-coupled nucleotide phosphohydrolases that hydrolyze nucleoside triphosphates and nucleoside diphosphates to nucleoside monophosphates and orthophosphates. GS52, a soybean (Glycine soja) ecto-apyrase, was previously shown to be induced very early in response to inoculation with the symbiotic bacterium Bradyrhizobium japonicum. Overexpression of the GS52 ecto-apyrase in Lotus japonicus increased the level of rhizobial infection and enhanced nodulation. These data suggest a critical role for the GS52 ecto-apyrase during nodulation. To further investigate the role of GS52 during nodulation, we used RNA interference to silence GS52 expression in soybean (Glycine max) roots using Agrobacterium rhizogenes-mediated root transformation. Transcript levels of GS52 were significantly reduced in GS52 silenced roots and these roots exhibited reduced numbers of mature nodules. Development of the nodule primordium and subsequent nodule maturation was significantly suppressed in GS52 silenced roots. Transmission electron micrographs of GS52 silenced root nodules showed that early senescence and infected cortical cells were devoid of symbiosome-containing bacteroids. Application of exogenous adenosine diphosphate to silenced GS52 roots restored nodule development. Restored nodules contained bacteroids, thus indicating that extracellular adenosine diphosphate is important during nodulation. These results clearly suggest that GS52 ecto-apyrase catalytic activity is critical for the early B. japonicum infection process, initiation of nodule primordium development, and subsequent nodule organogenesis in soybean. PMID:19036836

  4. Selection and characterization of coal mine autochthonous rhizobia for the inoculation of herbaceous legumes.

    PubMed

    Hernández, Anabel González; de Moura, Ginaini Doin; Binati, Renato Leal; Nascimento, Francisco Xavier Inês; Londoño, Diana Morales; Mamede, Ana Carolina Peixoto; da Silva, Emanuela Pille; de Armas, Rafael Dutra; Giachini, Admir José; Rossi, Márcio José; Soares, Cláudio Roberto Fonsêca Sousa

    2017-04-05

    Coal open pit mining in the South of Santa Catarina state (Brazil) was inappropriately developed, affecting approximately 6.700 ha. Re-vegetation is an alternative for the recovery of these areas. Furthermore, the use of herbaceous legumes inoculated with nitrogen fixing bacteria is motivated due to the difficulty implementing a vegetation cover in these areas, mainly due to low nutrient availability. Therefore, the aim of this work was to evaluate, among 16 autochthonous rhizobia isolated from the coal mining areas, those with the greatest potential to increase growth of the herbaceous legumes Vicia sativa and Calopogonium mucunoides. Tests were conducted in greenhouse containing 17 inoculation treatments (16 autochthonous rhizobia + Brazilian recommended strain for each plant species), plus two treatments without inoculation (with and without mineral nitrogen). After 60 days, nodulation, growth, N uptake, and symbiotic efficiency were evaluated. Isolates characterization was assessed by the production of indole acetic acid, ACC deaminase, siderophores, and inorganic phosphate solubilization. The classification of the isolates was performed by 16 S rDNA gene sequencing. Only isolates UFSC-M4 and UFSC-M8 were able to nodulate C. mucunoides. Among rhizobia capable of nodulating V. sativa, only UFSC-M8 was considered efficient. It was found the presence of more than one growth-promoting attributes in the same organism, and isolate UFSC-M8 presented all of them. Isolates were classified as belonging to Rhizobium, Burkholderia and Curtobacterium. The results suggest the inoculation of Vicia sativa with strain UFSC-M8, classified as Rhizobium sp., as a promising alternative for the revegetation of coal mining degraded areas.

  5. Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity, Adaptations, and Host Specificity

    PubMed Central

    Grönemeyer, Jann L.; Kulkarni, Ajinkya; Berkelmann, Dirk; Hurek, Thomas

    2014-01-01

    The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizobia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX repetitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sampling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqingense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates' performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations. PMID:25239908

  6. Rhizobia Indigenous to the Okavango Region in Sub-Saharan Africa: Diversity, Adaptations, and Host Specificity.

    PubMed

    Grönemeyer, Jann L; Kulkarni, Ajinkya; Berkelmann, Dirk; Hurek, Thomas; Reinhold-Hurek, Barbara

    2014-12-01

    The rhizobial community indigenous to the Okavango region has not yet been characterized. The isolation of indigenous rhizobia can provide a basis for the formulation of a rhizobial inoculant. Moreover, their identification and characterization contribute to the general understanding of species distribution and ecology. Isolates were obtained from nodules of local varieties of the pulses cowpea, Bambara groundnut, peanut, hyacinth bean, and common bean. Ninety-one of them were identified by BOX repetitive element PCR (BOX-PCR) and sequence analyses of the 16S-23S rRNA internally transcribed spacer (ITS) and the recA, glnII, rpoB, and nifH genes. A striking geographical distribution was observed. Bradyrhizobium pachyrhizi dominated at sampling sites in Angola which were characterized by acid soils and a semihumid climate. Isolates from the semiarid sampling sites in Namibia were more diverse, with most of them being related to Bradyrhizobium yuanmingense and Bradyrhizobium daqingense. Host plant specificity was observed only for hyacinth bean, which was nodulated by rhizobia presumably representing yet-undescribed species. Furthermore, the isolates were characterized with respect to their adaptation to high temperatures, drought, and local host plants. The adaptation experiments revealed that the Namibian isolates shared an exceptionally high temperature tolerance, but none of the isolates showed considerable adaptation to drought. Moreover, the isolates' performance on different local hosts showed variable results, with most Namibian isolates inducing better nodulation on peanut and hyacinth bean than the Angolan strains. The local predominance of distinct genotypes implies that indigenous strains may exhibit a better performance in inoculant formulations.

  7. Mutualistic rhizobia reduce plant diversity and alter community composition.

    PubMed

    Keller, Kane R

    2014-12-01

    Mutualistic interactions can be just as important to community dynamics as antagonistic species interactions like competition and predation. Because of their large effects on both abiotic and biotic environmental variables, resource mutualisms, in particular, have the potential to influence plant communities. Moreover, the effects of resource mutualists such as nitrogen-fixing rhizobia on diversity and community composition may be more pronounced in nutrient-limited environments. I experimentally manipulated the presence of rhizobia across a nitrogen gradient in early assembling mesocosm communities with identical starting species composition to test how the classic mutualism between nitrogen-fixing rhizobia and their legume host influence diversity and community composition. After harvest, I assessed changes in α-diversity, community composition, β-diversity, and ecosystem properties such as inorganic nitrogen availability and productivity as a result of rhizobia and nitrogen availability. The presence of rhizobia decreased plant community diversity, increased community convergence (reduced β-diversity), altered plant community composition, and increased total community productivity. These community-level effects resulted from rhizobia increasing the competitive dominance of their legume host Chamaecrista fasciculata. Moreover, different non-leguminous species responded both negatively and positively to the presence of rhizobia, indicating that rhizobia are driving both inhibitory and potentially facilitative effects in communities. These findings expand our understanding of plant communities by incorporating the effects of positive symbiotic interactions on plant diversity and composition. In particular, rhizobia that specialize on dominant plants may serve as keystone mutualists in terrestrial plant communities, reducing diversity by more than 40%.

  8. Ectopic expression of miR160 results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of symbiotic nodule development in soybean.

    PubMed

    Turner, Marie; Nizampatnam, Narasimha Rao; Baron, Mathieu; Coppin, Stéphanie; Damodaran, Suresh; Adhikari, Sajag; Arunachalam, Shivaram Poigai; Yu, Oliver; Subramanian, Senthil

    2013-08-01

    Symbiotic root nodules in leguminous plants result from interaction between the plant and nitrogen-fixing rhizobia bacteria. There are two major types of legume nodules, determinate and indeterminate. Determinate nodules do not have a persistent meristem, while indeterminate nodules have a persistent meristem. Auxin is thought to play a role in the development of both these types of nodules. However, inhibition of rootward auxin transport at the site of nodule initiation is crucial for the development of indeterminate nodules but not determinate nodules. Using the synthetic auxin-responsive DR5 promoter in soybean (Glycine max), we show that there is relatively low auxin activity during determinate nodule initiation and that it is restricted to the nodule periphery subsequently during development. To examine if and what role auxin plays in determinate nodule development, we generated soybean composite plants with altered sensitivity to auxin. We overexpressed microRNA393 to silence the auxin receptor gene family, and these roots were hyposensitive to auxin. These roots nodulated normally, suggesting that only minimal/reduced auxin signaling is required for determinate nodule development. We overexpressed microRNA160 to silence a set of repressor auxin response factor transcription factors, and these roots were hypersensitive to auxin. These roots were not impaired in epidermal responses to rhizobia but had significantly reduced nodule primordium formation, suggesting that auxin hypersensitivity inhibits nodule development. These roots were also hyposensitive to cytokinin and had attenuated expression of key nodulation-associated transcription factors known to be regulated by cytokinin. We propose a regulatory feedback loop involving auxin and cytokinin during nodulation.

  9. The identification of novel loci required for appropriate nodule development in Medicago truncatula.

    PubMed

    Domonkos, Agota; Horvath, Beatrix; Marsh, John F; Halasz, Gabor; Ayaydin, Ferhan; Oldroyd, Giles E D; Kalo, Peter

    2013-10-11

    The formation of functional symbiotic nodules is the result of a coordinated developmental program between legumes and rhizobial bacteria. Genetic analyses in legumes have been used to dissect the signaling processes required for establishing the legume-rhizobial endosymbiotic association. Compared to the early events of the symbiotic interaction, less attention has been paid to plant loci required for rhizobial colonization and the functioning of the nodule. Here we describe the identification and characterization of a number of new genetic loci in Medicago truncatula that are required for the development of effective nitrogen fixing nodules. Approximately 38,000 EMS and fast neutron mutagenized Medicago truncatula seedlings were screened for defects in symbiotic nitrogen fixation. Mutant plants impaired in nodule development and efficient nitrogen fixation were selected for further genetic and phenotypic analysis. Nine mutants completely lacking in nodule formation (Nod-) represented six complementation groups of which two novel loci have been identified. Eight mutants with ineffective nodules (Fix-) represented seven complementation groups, out of which five were new monogenic loci. The Fix- M. truncatula mutants showed symptoms of nitrogen deficiency and developed small white nodules. Microscopic analysis of Fix- nodules revealed that the mutants have defects in the release of rhizobia from infection threads, differentiation of rhizobia and maintenance of persistence of bacteria in nodule cells. Additionally, we monitored the transcriptional activity of symbiosis specific genes to define what transcriptional stage of the symbiotic process is blocked in each of the Fix- mutants. Based on the phenotypic and gene expression analysis a functional hierarchy of the FIX genes is proposed. The new symbiotic loci of M. truncatula isolated in this study provide the foundation for further characterization of the mechanisms underpinning nodulation, in particular the later

  10. Ethylene, a Hormone at the Center-Stage of Nodulation

    PubMed Central

    Guinel, Frédérique C.

    2015-01-01

    Nodulation is the result of a beneficial interaction between legumes and rhizobia. It is a sophisticated process leading to nutrient exchange between the two types of symbionts. In this association, within a nodule, the rhizobia, using energy provided as photosynthates, fix atmospheric nitrogen and convert it to ammonium which is available to the plant. Nodulation is recognized as an essential process in nitrogen cycling and legume crops are known to enrich agricultural soils in nitrogenous compounds. Furthermore, as they are rich in nitrogen, legumes are considered important as staple foods for humans and fodder for animals. To tightly control this association and keep it mutualistic, the plant uses several means, including hormones. The hormone ethylene has been known as a negative regulator of nodulation for almost four decades. Since then, much progress has been made in the understanding of both the ethylene signaling pathway and the nodulation process. Here I have taken a large view, using recently obtained knowledge, to describe in some detail the major stages of the process. I have not only reviewed the steps most commonly covered (the common signaling transduction pathway, and the epidermal and cortical programs), but I have also looked into steps less understood (the pre-infection step with the plant defense response, the bacterial release and the formation of the symbiosome, and nodule functioning and senescence). After a succinct review of the ethylene signaling pathway, I have used the knowledge obtained from nodulation- and ethylene-related mutants to paint a more complete picture of the role played by the hormone in nodule organogenesis, functioning, and senescence. It transpires that ethylene is at the center of this effective symbiosis. It has not only been involved in most of the steps leading to a mature nodule, but it has also been implicated in host immunity and nodule senescence. It is likely responsible for the activation of other hormonal

  11. Ethylene, a Hormone at the Center-Stage of Nodulation.

    PubMed

    Guinel, Frédérique C

    2015-01-01

    Nodulation is the result of a beneficial interaction between legumes and rhizobia. It is a sophisticated process leading to nutrient exchange between the two types of symbionts. In this association, within a nodule, the rhizobia, using energy provided as photosynthates, fix atmospheric nitrogen and convert it to ammonium which is available to the plant. Nodulation is recognized as an essential process in nitrogen cycling and legume crops are known to enrich agricultural soils in nitrogenous compounds. Furthermore, as they are rich in nitrogen, legumes are considered important as staple foods for humans and fodder for animals. To tightly control this association and keep it mutualistic, the plant uses several means, including hormones. The hormone ethylene has been known as a negative regulator of nodulation for almost four decades. Since then, much progress has been made in the understanding of both the ethylene signaling pathway and the nodulation process. Here I have taken a large view, using recently obtained knowledge, to describe in some detail the major stages of the process. I have not only reviewed the steps most commonly covered (the common signaling transduction pathway, and the epidermal and cortical programs), but I have also looked into steps less understood (the pre-infection step with the plant defense response, the bacterial release and the formation of the symbiosome, and nodule functioning and senescence). After a succinct review of the ethylene signaling pathway, I have used the knowledge obtained from nodulation- and ethylene-related mutants to paint a more complete picture of the role played by the hormone in nodule organogenesis, functioning, and senescence. It transpires that ethylene is at the center of this effective symbiosis. It has not only been involved in most of the steps leading to a mature nodule, but it has also been implicated in host immunity and nodule senescence. It is likely responsible for the activation of other hormonal

  12. Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens.

    PubMed

    Pankhurst, C E; Broughton, W J; Wieneke, U

    1983-08-01

    Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively. Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R. meliloti, indicating that nif genes were not present on these plasmids. The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R. leguminosarum, R. meliloti and Agrobacterium tumefaciens. All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a. Transfer of pRlo2037a to R. loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L. pedunculatus. Determinants for flavolan resistance, believed to be necessary for effective nodulation of L. pedunculatus, were not carried on pRlo2037a. These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R. loti strain NZP2037.

  13. Generation of 7137 non-redundant expressed sequence tags from a legume, Lotus japonicus.

    PubMed

    Asamizu, E; Nakamura, Y; Sato, S; Tabata, S

    2000-04-28

    For comprehensive analysis of genes expressed in a model legume, Lotus japonicus, a total of 22,983 5' end expressed sequence tags (ESTs) were accumulated from normalized and size-selected cDNA libraries constructed from young (2 weeks old) plants. The EST sequences were clustered into 7137 non-redundant groups. Similarity search against public non-redundant protein database indicated that 3302 groups showed similarity to genes of known function, 1143 groups to hypothetical genes, and 2692 were novel sequences. Homologues of 5 nodule-specific genes which have been reported in other legume species were contained in the collected ESTs, suggesting that the EST source generated in this study will become a useful tool for identification of genes related to legume-specific biological processes. The sequence data of individual ESTs are available at the web site: http://www.kazusa.or.jp/en/plant/lotus/EST/.

  14. Importance of rhizobia in Agriculture: potential of the commercial inoculants and native strains for improving legume yields in different land-use systems

    NASA Astrophysics Data System (ADS)

    Lesueur, D.; Atieno, M.; Mathu, S.; Herrmann, L.

    2012-04-01

    Legumes play an important role in the traditional diets of many regions throughout the world because they provide a multitude of benefits to both the soil and other crops grown in combination with them or following them in several cropping systems. The ability of legumes to fix atmospheric nitrogen in association with rhizobia gives them the capacity to grow in very degraded soils. But do we have to systematically inoculate legumes? For example our results suggested that the systematic inoculation of both cowpea and green gram in Kenya with commercial inoculants to improve yields is not really justified, native strains performing better than inoculated strains. But when native rhizobia nodulating legumes are not naturally present, application of rhizobial inoculants is very commonly used. Our results showed that the utilization of effective good-quality rhizobial inoculants by farmers have a real potential to improve legume yields in unfertile soils requesting high applications of mineral fertilizers. For example an effective soybean commercial inoculants was tested in different locations in Kenya (in about 150 farms in 3 mandate areas presenting different soil characteristics and environmental conditions). Application of the rhizobial inoculant significantly increased the soybean yields in all mandate areas (about 75% of the farms). Nodule occupancy analysis showed that a high number of nodules occupied by the inoculated strain did not obviously lead to an increase of soybean production. Soil factors (pH, P, C, N…) seemed to affect the inoculant efficiency whether the strain is occupying the nodules or not. Our statistic analysis showed that soil pH significantly affected nodulation and yield, though the effect was variable depending on the region. We concluded that the competitiveness of rhizobial strains might not be the main factor explaining the effect (or lack of) of legumes inoculation in the field. Another study was aiming to assess if several factors

  15. Study of the basis for the competitiveness of Rhizobium japonicum in the nodulation of soybean

    SciTech Connect

    Bauer, W.

    1990-06-01

    The overall goal of our studies was to identify cellular and molecular characteristics of rhizobia that are important to root colonization and symbiotic infection in competition with indigenous soil microbes. Rhizobia were found to respond to specific host flavonoids as chemoattractants as well as nodulation gene inducers. Motility and chemotaxis contributed significantly to the efficiency of symbiotic interactions, but not to colonization of root tips, where bacterial dispersal depended primarily on passive movement by root cell elongation. Co-inoculation of Rhizobium nod gene mutants with each other and with the wild-type showed that the mutants could help the wild-type, but not each other, to initiate nodules. Mutants with defective host specificity genes were the only ones capable of helping the wild-type in an additive or synergistic manner. Alfalfa was shown to have rapid, systemic feedback regulation suppressing nodule formation in younger parts of the root system in response to initiation of the first nodules. Root mucilage and exudate components were found to affect attachment of rhizobia to root surfaces. Methods for optimal surface sterilization of legume seeds were examined. 5 refs.

  16. Increasing Nitrogen Fixation and Seed Development in Soybean Requires Complex Adjustments of Nodule Nitrogen Metabolism and Partitioning Processes.

    PubMed

    Carter, Amanda M; Tegeder, Mechthild

    2016-08-08

    Legumes are able to access atmospheric di-nitrogen (N2) through a symbiotic relationship with rhizobia that reside within root nodules. In soybean, following N2 fixation by the bacteroids, ammonia is finally reduced in uninfected cells to allantoin and allantoic acid [1]. These ureides present the primary long-distance transport forms of nitrogen (N), and are exported from nodules via the xylem for shoot N supply. Transport of allantoin and allantoic acid out of nodules requires the function of ureide permeases (UPS1) located in cells adjacent to the vasculature [2, 3]. We expressed a common bean UPS1 transporter in cortex and endodermis cells of soybean nodules and found that delivery of N from nodules to shoot, as well as seed set, was significantly increased. In addition, the number of transgenic nodules was increased and symbiotic N2 fixation per nodule was elevated, indicating that transporter function in nodule N export is a limiting step in bacterial N acquisition. Further, the transgenic nodules showed considerable increases in nodule N assimilation, ureide synthesis, and metabolite levels. This suggests complex adjustments of nodule N metabolism and partitioning processes in support of symbiotic N2 fixation. We propose that the transgenic UPS1 plants display metabolic and allocation plasticity to overcome N2 fixation and seed yield limitations. Overall, it is demonstrated that transporter function in N export from nodules is a key step for enhancing atmospheric N2 fixation and nodule function and for improving shoot N nutrition and seed development in legumes.

  17. Nitrate inhibits soybean nodulation by regulating expression of CLE genes.

    PubMed

    Lim, Chae Woo; Lee, Young Woo; Lee, Sung Chul; Hwang, Cheol Ho

    2014-12-01

    Nitrogen compounds such as nitrate act as a potential inhibitor for legume nodulation. In this study, we isolated a new CLE gene, GmNIC2, from nitrate-treated roots, which shares high sequence homology with nitrate-induced CLE gene GmNIC1. Similar to GmNIC1, the expression level of GmNIC2 was not significantly altered in roots by rhizobial inoculation and was much higher in young nodules than in roots. In addition, overexpression of GmNIC2 led to similar nodulation inhibition of transgenic hairy roots to that of GmNIC1, which occurred in GmNARK-dependent manner and at the local level. By analyzing GmNARK loss-of-function mutant, SS2-2, it was found that expression levels of GmNIC1 and GmNIC2 in the SS2-2 roots were lower than in the wild type (WT) roots in response to nitrate. In contrast to GmNIC1 and GmNIC2, expressions of GmRIC1 and GmRIC2 genes that are related to the autoregulation of nodulation (AON) were strongly suppressed both of the soybeans during all periods of nitrate treatment and even were not induced by additional inoculation with rhizobia. Taken together, the results of this study suggest that GmNIC2, as an active homologous gene located in chromosome 13, acts locally to suppress nodulation, like GmNIC1, and nitrate inhibition of nodulation is led by fine-tuned regulation of both nitrate-induced CLEs and rhizobia-induced CLEs. Copyright © 2014. Published by Elsevier Ireland Ltd.

  18. Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization.

    PubMed

    Regus, John U; Gano, Kelsey A; Hollowell, Amanda C; Sachs, Joel L

    2014-04-22

    Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic.

  19. Efficiency of partner choice and sanctions in Lotus is not altered by nitrogen fertilization

    PubMed Central

    Regus, John U.; Gano, Kelsey A.; Hollowell, Amanda C.; Sachs, Joel L.

    2014-01-01

    Eukaryotic hosts must exhibit control mechanisms to select against ineffective bacterial symbionts. Hosts can minimize infection by less-effective symbionts (partner choice) and can divest of uncooperative bacteria after infection (sanctions). Yet, such host-control traits are predicted to be context dependent, especially if they are costly for hosts to express or maintain. Legumes form symbiosis with rhizobia that vary in symbiotic effectiveness (nitrogen fixation) and can enforce partner choice as well as sanctions. In nature, legumes acquire fixed nitrogen from both rhizobia and soils, and nitrogen deposition is rapidly enriching soils globally. If soil nitrogen is abundant, we predict host control to be downregulated, potentially allowing invasion of ineffective symbionts. We experimentally manipulated soil nitrogen to examine context dependence in host control. We co-inoculated Lotus strigosus from nitrogen depauperate soils with pairs of Bradyrhizobium strains that vary in symbiotic effectiveness and fertilized plants with either zero nitrogen or growth maximizing nitrogen. We found efficient partner choice and sanctions regardless of nitrogen fertilization, symbiotic partner combination or growth season. Strikingly, host control was efficient even when L. strigosus gained no significant benefit from rhizobial infection, suggesting that these traits are resilient to short-term changes in extrinsic nitrogen, whether natural or anthropogenic. PMID:24573843

  20. Ethylene Inhibitors Restore Nodulation to sym 5 Mutants of Pisum sativum L. cv Sparkle 12

    PubMed Central

    Fearn, Jeffrey C.; LaRue, Thomas A.

    1991-01-01

    The sym 5 mutants of pea, Pisum sativum L. cv Sparkle, do not differ in growth habit from their normal parent and nodulate poorly at a root temperature of 20°C. If inhibitors of ethylene formation or action (Co2+, aminoethoxyvinylglycine, or Ag+) are added to the substrate, nodulation of the sym 5 mutants is increased. Similar treatments of four other mutant sym lines do not restore nodulation. When Ag+ is added to the substrate from 4 days before to 4 days after inoculation with rhizobia, nodulation of sym 5 mutants is increased. The roots of the mutant need only be exposed to Ag+ for 4 hours to significantly increase nodule numbers. The content of free 1-aminocyclopropane-1-carboxylic acid and the production of ethylene in the lateral roots of sym 5 mutants do not differ from Sparkle. PMID:16668158

  1. Basis for the competitiveness of Rhizobium japonicum in nodulation of soybean. Final progress report

    SciTech Connect

    Evans, W.R.; Bauer, W.D.

    1986-07-30

    This study sought to identify molecular, genetic and environmental factors most crucial to the ability of an inoculated strain of rhizobia to nodulate soybean roots in the face of competition from indigenous microorganisms. Co-inoculation of a more efficient strain with a less-efficient strain resulted in the more efficient strain occupying a higher percentage of nodules. When culture conditions became less stringent the less efficient strain became more competitive. The number of infections formed was related in a direct manner to nodulation efficiency. The strain which was more nodulation efficient also was more infection efficient. That the number of infections per se, as determined in a single inoculum, can not be the only factor in determining the competency was indicated by experiments in which one strain formed more total infection yet nodule occupancy was equally distributed. 5 figs., 9 tabs.

  2. Detection of S-nitrosothiol and nitrosylated proteins in Arachis hypogaea functional nodule: response of the nitrogen fixing symbiont.

    PubMed

    Maiti, Debasis; Sarkar, Tuhin Subhra; Ghosh, Sanjay

    2012-01-01

    To detect the presence of NO, ROS and RNS in nodules of crack entry legumes, we used Arachis hypogaea functional nodule. The response of two cognate partner rhizobia was compared towards NO and GSNO using S. meliloti and Bradyrhizobium sp NC921001. ROS, NO, nitrosothiol and bacteroids were detected by fluorescence microscopy. Redox enzymes and thiol pools were detected biochemically. Nitrosothiols were found to be present but ROS and NO were absent in A. hypogaea nodule. A number of S-nitrosylated proteins were also detected. The total thiol pool and most of the redox enzymes were low in nodule cytosolic extract but these were found to be high in the partner microorganisms indicating partner rhizobia could protect the nodule environment against the nitrosothiols. Both S. meliloti and Bradyrhizobium sp NC921001 were found to contain GSNO reductase. Interestingly, there was a marked difference in growth pattern between S. meliloti and Bradyrhizobium sp in presence of sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Bradyrhizobium sp was found to be much more tolerant to NO donor compounds than the S. meliloti. In contrast, S. meliloti showed resistance to GSNO but was sensitive to SNP. Together our data indicate that nodule environment of crack entry legumes is different than the nodules of infection mode entry in terms of NO, ROS and RNS. Based on our biochemical characterization, we propose that exchange of redox molecules and reactive chemical species is possible between the bacteroid and nodule compartment.

  3. The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules

    PubMed Central

    Barraza, Aarón; Contreras-Cubas, Cecilia; Estrada-Navarrete, Georgina; Reyes, José L.; Juárez-Verdayes, Marco A.; Avonce, Nelson; Quinto, Carmen; Díaz-Camino, Claudia; Sanchez, Federico

    2016-01-01

    Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant. PMID:27847509

  4. The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules.

    PubMed

    Barraza, Aarón; Contreras-Cubas, Cecilia; Estrada-Navarrete, Georgina; Reyes, José L; Juárez-Verdayes, Marco A; Avonce, Nelson; Quinto, Carmen; Díaz-Camino, Claudia; Sanchez, Federico

    2016-01-01

    Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

  5. Why so strong for the lotus leaf?

    NASA Astrophysics Data System (ADS)

    Guo, Zhiguang; Liu, Weimin; Su, Bao-Lian

    2008-11-01

    The authors discussed the potential reasons why the lotus leaf is so strong by means of scanning electron microscopy (SEM). The results showed that the good mechanical properties of lotus leaf should be attributed to its architecture, such as paralleled microtubes structure, umbrellalike structure, and hierarchically layered hexagon structure. The important observation from this work is that the surface of the rear face of the lotus leaf seems to be constituted by the layers of hexagons whose hierarchical pilling up of size decreases as we go deeper from surface. This is a typical fractal-like phenomenon.

  6. Lotus Base: An integrated information portal for the model legume Lotus japonicus

    PubMed Central

    Mun, Terry; Bachmann, Asger; Gupta, Vikas; Stougaard, Jens; Andersen, Stig U.

    2016-01-01

    Lotus japonicus is a well-characterized model legume widely used in the study of plant-microbe interactions. However, datasets from various Lotus studies are poorly integrated and lack interoperability. We recognize the need for a comprehensive repository that allows comprehensive and dynamic exploration of Lotus genomic and transcriptomic data. Equally important are user-friendly in-browser tools designed for data visualization and interpretation. Here, we present Lotus Base, which opens to the research community a large, established LORE1 insertion mutant population containing an excess of 120,000 lines, and serves the end-user tightly integrated data from Lotus, such as the reference genome, annotated proteins, and expression profiling data. We report the integration of expression data from the L. japonicus gene expression atlas project, and the development of tools to cluster and export such data, allowing users to construct, visualize, and annotate co-expression gene networks. Lotus Base takes advantage of modern advances in browser technology to deliver powerful data interpretation for biologists. Its modular construction and publicly available application programming interface enable developers to tap into the wealth of integrated Lotus data. Lotus Base is freely accessible at: https://lotus.au.dk. PMID:28008948

  7. Nitrate inhibition of legume nodule growth and activity. II. Short term studies with high nitrate supply

    SciTech Connect

    Streeter, J.G.

    1985-02-01

    Soybean plants (Glycine max (L.) Merr) were grown in sand culture with 2 millimolar nitrate for 37 days and then supplied with 15 millimolar nitrate for 7 days. Control plants received 2 millimolar nitrate and 13 millimolar chloride and, after the 7-day treatment period, all plants were supplied with nil nitrate. The temporary treatment with high nitrate inhibited nitrogenase (acetylene reduction) activity by 80% whether or not Rhizobium japonicum bacteroids had nitrate reductase (NR) activity. The pattern of nitrite accumulation in nodules formed by NR/sup +/ rhizobia was inversely related to the decrease and recovery of nitrogenase activity. However, nitrite concentration in nodules formed by NR/sup -/ rhizobia appeared to be too low to explain the inhibition of nitrogenase. Nodules on plants treated with 15 millimolar nitrate contained higher concentrations of amino N and, especially, ureide N than control nodules and, after withdrawal of nitrate, reduced N content of treated and control nodules returned to similar levels. The accumulation of N/sub 2/ fixation products in nodules in response to high nitrate treatment was observed with three R. japonicum strains, two NR/sup +/ and one NR/sup -/.

  8. Transcript profiling of common bean nodules subjected to oxidative stress.

    PubMed

    Ramírez, Mario; Guillén, Gabriel; Fuentes, Sara I; Iñiguez, Luis P; Aparicio-Fabre, Rosaura; Zamorano-Sánchez, David; Encarnación-Guevara, Sergio; Panzeri, Dario; Castiglioni, Bianca; Cremonesi, Paola; Strozzi, Francesco; Stella, Alessandra; Girard, Lourdes; Sparvoli, Francesca; Hernández, Georgina

    2013-11-01

    Several environmental stresses generate high amounts of reactive oxygen species (ROS) in plant cells, resulting in oxidative stress. Symbiotic nitrogen fixation (SNF) in the legume-rhizobia symbiosis is sensitive to damage from oxidative stress. Active nodules of the common bean (Phaseolus vulgaris) exposed to the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride hydrate), which stimulates ROS accumulation, exhibited reduced nitrogenase activity and ureide content. We analyzed the global gene response of nodules subjected to oxidative stress using the Bean Custom Array 90K, which includes probes from 30,000 expressed sequence tags (ESTs). A total of 4280 ESTs were differentially expressed in stressed bean nodules; of these, 2218 were repressed. Based on Gene Ontology analysis, these genes were grouped into 42 different biological process categories. Analysis with the PathExpress bioinformatic tool, adapted for bean, identified five significantly repressed metabolic pathways related to carbon/nitrogen metabolism, which is crucial for nodule function. Quantitative reverse transcription (qRT)-PCR analysis of transcription factor (TF) gene expression showed that 67 TF genes were differentially expressed in nodules exposed to oxidative stress. Putative cis-elements recognized by highly responsive TF were detected in promoter regions of oxidative stress regulated genes. The expression of oxidative stress responsive genes and of genes important for SNF in bacteroids analyzed in stressed nodules revealed that these conditions elicited a transcriptional response. © 2013 Scandinavian Plant Physiology Society.

  9. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9

    PubMed Central

    Wang, Longxiang; Wang, Longlong; Tan, Qian; Fan, Qiuling; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming; Duanmu, Deqiang

    2016-01-01

    The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus. PMID:27630657

  10. Efficient Inactivation of Symbiotic Nitrogen Fixation Related Genes in Lotus japonicus Using CRISPR-Cas9.

    PubMed

    Wang, Longxiang; Wang, Longlong; Tan, Qian; Fan, Qiuling; Zhu, Hui; Hong, Zonglie; Zhang, Zhongming; Duanmu, Deqiang

    2016-01-01

    The targeted genome editing technique, CRISPR/Cas9 system, has been widely used to modify genes of interest in a predictable and precise manner. In this study, we describe the CRISPR/Cas9-mediated efficient editing of representative SNF (symbiotic nitrogen fixation) related genes in the model legume Lotus japonicus via Agrobacterium-mediated stable or hairy root transformation. We first predicted nine endogenous U6 genes in Lotus and then demonstrated the efficacy of the LjU6-1 gene promoter in driving expression of single guide RNAs (sgRNAs) by using a split yellow fluorescence protein (YFP) reporter system to restore the fluorescence in Arabidopsis protoplasts. Next, we chose a customized sgRNA targeting SYMRK (symbiosis receptor-like kinase) loci and achieved ~35% mutagenic efficiency in 20 T0 transgenic plants, two of them containing biallelic homozygous mutations with a 2-bp deletion near the PAM region. We further designed two sgRNAs targeting three homologous leghemoglobin loci (LjLb1, LjLb2, LjLb3) for testing the possibility of generating multi-gene knockouts. 20 out of 70 hairy root transgenic plants exhibited white nodules, with at least two LjLbs disrupted in each plant. Compared with the constitutively active CaMV 35S promoter, the nodule-specific LjLb2 promoter was also effective in gene editing in nodules by hairy root transformation. Triple mutant knockout of LjLbs was also obtained by stable transformation using two sgRNAs. Collectively, these studies demonstrate that the CRISPR/Cas9 system should greatly facilitate functional analyses of SNF related genes in Lotus japonicus.

  11. Genetic Characterization of Soybean Rhizobia Isolated from Different Ecological Zones in North-Eastern Afghanistan

    PubMed Central

    Habibi, Safiullah; Ayubi, Abdul ghani; Ohkama-Ohtsu, Naoko; Sekimoto, Hitoshi; Yokoyama, Tadashi

    2017-01-01

    Seventy rhizobial isolates were obtained from the root nodules of two soybean (Glycine max) cultivars: Japanese cultivar Enrei and USA cultivar Stine3300, which were inoculated with different soil samples from Afghanistan. In order to study the genetic properties of the isolates, the DNA sequences of the 16S rRNA gene and symbiotic genes (nodD1 and nifD) were elucidated. Furthermore, the isolates were inoculated into the roots of two soybean cultivars, and root nodule numbers and nitrogen fixation abilities were subsequently evaluated in order to assess symbiotic performance. Based on 16S rRNA gene sequences, the Afghanistan isolates obtained from soybean root nodules were classified into two genera, Bradyrhizobium and Ensifer. Bradyrhizobium isolates accounted for 54.3% (38) of the isolates, and these isolates had a close relationship with Bradyrhizobium liaoningense and B. yuanmingense. Five out of the 38 Bradyrhizobium isolates showed a novel lineage for B. liaoningense and B. yuanmingense. Thirty-two out of the 70 isolates were identified as Ensifer fredii. An Ensifer isolate had identical nodD1 and nifD sequences to those in B. yuanmingense. This result indicated that the horizontal gene transfer of symbiotic genes occurred from Bradyrhizobium to Ensifer in Afghanistan soil. The symbiotic performance of the 14 tested isolates from the root nodules of the two soybean cultivars indicated that Bradyrhizobium isolates exhibited stronger acetylene reduction activities than Ensifer isolates. This is the first study to genetically characterize soybean-nodulating rhizobia in Afghanistan soil. PMID:28321006

  12. Drought Stress Responses in Soybean Roots and Nodules

    PubMed Central

    Kunert, Karl J.; Vorster, Barend J.; Fenta, Berhanu A.; Kibido, Tsholofelo; Dionisio, Giuseppe; Foyer, Christine H.

    2016-01-01

    Drought is considered to be a major threat to soybean production worldwide and yet our current understanding of the effects of drought on soybean productively is largely based on studies on above-ground traits. Although the roots and root nodules are important sensors of drought, the responses of these crucial organs and their drought tolerance features remain poorly characterized. The symbiotic interaction between soybean and rhizobia facilitates atmospheric nitrogen fixation, a process that provides essential nitrogen to support plant growth and development. Symbiotic nitrogen fixation is important for sustainable agriculture, as it sustains plant growth on nitrogen-poor soils and limits fertilizer use for crop nitrogen nutrition. Recent developments have been made in our understanding of the drought impact on soybean root architecture and nodule traits, as well as underpinning transcriptome, proteome and also emerging metabolome information, with a view to improve the selection of more drought-tolerant soybean cultivars and rhizobia in the future. We conclude that the direct screening of root and nodule traits in the field as well as identification of genes, proteins and also metabolites involved in such traits will be essential in order to gain a better understanding of the regulation of root architecture, bacteroid development and lifespan in relation to drought tolerance in soybean. PMID:27462339

  13. Biodiversity and Biogeography of Rhizobia Associated with Soybean Plants Grown in the North China Plain ▿ †

    PubMed Central

    Zhang, Yan Ming; Li, Ying; Chen, Wen Feng; Wang, En Tao; Tian, Chang Fu; Li, Qin Qin; Zhang, Yun Zeng; Sui, Xin Hua; Chen, Wen Xin

    2011-01-01

    As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain. PMID:21784912

  14. Nod factors stimulate seed germination and promote growth and nodulation of pea and vetch under competitive conditions.

    PubMed

    Kidaj, Dominika; Wielbo, Jerzy; Skorupska, Anna

    2012-03-20

    Nod factors are lipochitooligosaccharide (LCO) produced by soil bacteria commonly known as rhizobia acting as signals for the legume plants to initiate symbiosis. Nod factors trigger early symbiotic responses in plant roots and initiate the development of specialized plant organs called nodules, where biological nitrogen fixation takes place. Here, the effect of specific LCO originating from flavonoid induced Rhizobium leguminosarum bv. viciae GR09 culture was studied on germination, plant growth and nodulation of pea and vetch. A crude preparation of GR09 LCO significantly enhanced symbiotic performance of pea and vetch grown under laboratory conditions and in the soil. Moreover, the effect of GR09 LCOs seed treatments on the genetic diversity of rhizobia recovered from vetch and pea nodules was presented.

  15. Use of potato extract broth for culturing root-nodule bacteria.

    PubMed

    Martyniuk, Stefan; Oroń, Jadwiga

    2011-01-01

    Liquid media containing potato extract and 1% of glucose or sucrose were used to culture root-nodule bacteria (rhizobia) in shaken Erlenmeyer flasks. For comparison, these bacteria were also cultured in yeast extract-mannitol broth (YEMB) as a standard medium. Proliferation of rhizobia was monitored by measuring optical densities (OD550) of the cultures and by plate counting of the viable cells (c.f.u) of the bacteria. In general, multiplication of the rhizobia in potato extract-glucose broth (PEGB) and potato extract-sucrose broth (PESB) was markedly faster, as indicated by higher values of OD550, than in YEMB. The numbers of R. leguminosarum by. vicae GGL and S. meliloti 330 in PEGB and PEGB were high and ranged from 1.2 x 10(10) to 4.9 x 10(10) mL(-1) after 48 h of incubation at 28 degrees C. B. japonicum B3S culture in PEGB contained 6.4 x 10(9) c.f.u. ml(-1) after 72 h of incubation. PEGB and YEMB cultures of the rhizobia were similar with respect to their beneficial effects on nodulation of the host-plants of these bacteria.

  16. Transition Metal Transport in Plants and Associated Endosymbionts: Arbuscular Mycorrhizal Fungi and Rhizobia

    PubMed Central

    González-Guerrero, Manuel; Escudero, Viviana; Saéz, Ángela; Tejada-Jiménez, Manuel

    2016-01-01

    Transition metals such as iron, copper, zinc, or molybdenum are essential nutrients for plants. These elements are involved in almost every biological process, including photosynthesis, tolerance to biotic and abiotic stress, or symbiotic nitrogen fixation. However, plants often grow in soils with limiting metallic oligonutrient bioavailability. Consequently, to ensure the proper metal levels, plants have developed a complex metal uptake and distribution system, that not only involves the plant itself, but also its associated microorganisms. These microorganisms can simply increase metal solubility in soils and making them more accessible to the host plant, as well as induce the plant metal deficiency response, or directly deliver transition elements to cortical cells. Other, instead of providing metals, can act as metal sinks, such as endosymbiotic rhizobia in legume nodules that requires relatively large amounts to carry out nitrogen fixation. In this review, we propose to do an overview of metal transport mechanisms in the plant–microbe system, emphasizing the role of arbuscular mycorrhizal fungi and endosymbiotic rhizobia. PMID:27524990

  17. Transition Metal Transport in Plants and Associated Endosymbionts: Arbuscular Mycorrhizal Fungi and Rhizobia.

    PubMed

    González-Guerrero, Manuel; Escudero, Viviana; Saéz, Ángela; Tejada-Jiménez, Manuel

    2016-01-01

    Transition metals such as iron, copper, zinc, or molybdenum are essential nutrients for plants. These elements are involved in almost every biological process, including photosynthesis, tolerance to biotic and abiotic stress, or symbiotic nitrogen fixation. However, plants often grow in soils with limiting metallic oligonutrient bioavailability. Consequently, to ensure the proper metal levels, plants have developed a complex metal uptake and distribution system, that not only involves the plant itself, but also its associated microorganisms. These microorganisms can simply increase metal solubility in soils and making them more accessible to the host plant, as well as induce the plant metal deficiency response, or directly deliver transition elements to cortical cells. Other, instead of providing metals, can act as metal sinks, such as endosymbiotic rhizobia in legume nodules that requires relatively large amounts to carry out nitrogen fixation. In this review, we propose to do an overview of metal transport mechanisms in the plant-microbe system, emphasizing the role of arbuscular mycorrhizal fungi and endosymbiotic rhizobia.

  18. Genetic diversity of Rhizobia isolates from Amazon soils using cowpea (Vigna unguiculata) as trap plant

    PubMed Central

    Silva, F.V.; Simões-Araújo, J.L.; Silva Júnior, J.P.; Xavier, G.R.; Rumjanek, N.G.

    2012-01-01

    The aim of this work was to characterize rhizobia isolated from the root nodules of cowpea (Vigna unguiculata) plants cultivated in Amazon soils samples by means of ARDRA (Amplified rDNA Restriction Analysis) and sequencing analysis, to know their phylogenetic relationships. The 16S rRNA gene of rhizobia was amplified by PCR (polymerase chain reaction) using universal primers Y1 and Y3. The amplification products were analyzed by the restriction enzymes HinfI, MspI and DdeI and also sequenced with Y1, Y3 and six intermediate primers. The clustering analysis based on ARDRA profiles separated the Amazon isolates in three subgroups, which formed a group apart from the reference isolates of Bradyrhizobium japonicum and Bradyrhizobium elkanii. The clustering analysis of 16S rRNA gene sequences showed that the fast-growing isolates had similarity with Enterobacter, Rhizobium, Klebsiella and Bradyrhizobium and all the slow-growing clustered close to Bradyrhizobium. PMID:24031880

  19. Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

    PubMed Central

    Elliott, Geoffrey N.; Chen, Wen-Ming; Bontemps, Cyril; Chou, Jui-Hsing; Young, J. Peter W.; Sprent, Janet I.; James, Euan K.

    2007-01-01

    nodulate papilioinoid legumes. Conclusions Papilionoid legumes from widely different tribes can be nodulated by β-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules. PMID:17881339

  20. Endoreduplication-mediated initiation of symbiotic organ development in Lotus japonicus.

    PubMed

    Suzaki, Takuya; Ito, Momoyo; Yoro, Emiko; Sato, Shusei; Hirakawa, Hideki; Takeda, Naoya; Kawaguchi, Masayoshi

    2014-06-01

    Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells. © 2014. Published by The Company of Biologists Ltd.

  1. The role of PHB metabolism in the symbiosis of rhizobia with legumes.

    PubMed

    Trainer, Maria A; Charles, Trevor C

    2006-07-01

    The carbon storage polymer poly-beta-hydroxybutyrate (PHB) is a potential biodegradable alternative to plastics, which plays a key role in the cellular metabolism of many bacterial species. Most species of rhizobia synthesize PHB but not all species accumulate it during symbiosis with legumes; the reason for this remains unclear, although it was recently shown that a metabolic mutant of a nonaccumulating species retains the capacity to store PHB in symbiosis. Although the precise roles of PHB metabolism in these bacteria during infection, nodulation, and nitrogen fixation are not determined, the elucidation of these roles will influence our understanding of the metabolic nature of the symbiotic relationship. This review explores the progress that was made in determining the biochemistry and genetics of PHB metabolism. This includes the elucidation of the PHB cycle, variations in PHB metabolism among rhizobial species, and the implications of these variations, while proposing a model for the role of PHB metabolism and storage in symbiosis.

  2. Determining the Site of Action of Strigolactones during Nodulation.

    PubMed

    McAdam, Erin L; Hugill, Cassandra; Fort, Sebastien; Samain, Eric; Cottaz, Sylvain; Davies, Noel W; Reid, James B; Foo, Eloise

    2017-09-01

    Strigolactones (SLs) influence the ability of legumes to associate with nitrogen-fixing bacteria. In this study, we determine the precise stage at which SLs influence nodulation. We show that SLs promote infection thread formation, as a null SL-deficient pea (Pisum sativum) mutant forms significantly fewer infection threads than wild-type plants, and this reduction can be overcome by the application of the synthetic SL GR24. We found no evidence that SLs influence physical events in the plant before or after infection thread formation, since SL-deficient plants displayed a similar ability to induce root hair curling in response to rhizobia or Nod lipochitooligosaccharides (LCOs) and SL-deficient nodules appear to fix nitrogen at a similar rate to those of wild-type plants. In contrast, an SL receptor mutant displayed no decrease in infection thread formation or nodule number, suggesting that SL deficiency may influence the bacterial partner. We found that this influence of SL deficiency was not due to altered flavonoid exudation or the ability of root exudates to stimulate bacterial growth. The influence of SL deficiency on infection thread formation was accompanied by reduced expression of some early nodulation genes. Importantly, SL synthesis is down-regulated by mutations in genes of the Nod LCO signaling pathway, and this requires the downstream transcription factor NSP2 but not NIN This, together with the fact that the expression of certain SL biosynthesis genes can be elevated in response to rhizobia/Nod LCOs, suggests that Nod LCOs may induce SL biosynthesis. SLs appear to influence nodulation independently of ethylene action, as SL-deficient and ethylene-insensitive double mutant plants display essentially additive phenotypes, and we found no evidence that SLs influence ethylene synthesis or vice versa. © 2017 American Society of Plant Biologists. All Rights Reserved.

  3. (Study of the basis for the competitiveness of Rhizobium japonicum in the nodulation of soybean): Progress report, (May 1986-May 1987)

    SciTech Connect

    Bauer, W.D.

    1987-01-01

    The overall goal of our current studies is to identify cellular and molecular factors that are of central importance to the colonization and infection of host roots by inoculated rhizobia in competition with indigenous soil microbes. This report covers research compelted between May 1986 and May 1987. Progress was made during the past year in areas of the role of motility and chemotaxis in nodule initiation by rhizobia, the further characterization of bacterial adhesion to host root surfaces, the effects of root exudate on bacterial attachment, the effects of bacteria and bacterial culture filtrates on bacterial attachment, the movement of rhizobia on the surface of the growing host root, and the effects of adding various nod mutants of Rhizobium meliloti on nodule formation by co-inoculated wildtype cells. 1 fig., 5 tabs.

  4. Assess suitability of hydroaeroponic culture to establish tripartite symbiosis between different AMF species, beans, and rhizobia

    PubMed Central

    Tajini, Fatma; Suriyakup, Porntip; Vailhe, Hélène; Jansa, Jan; Drevon, Jean-Jacques

    2009-01-01

    Background Like other species of the Phaseoleae tribe, common bean (Phaseolus vulgaris L.) has the potential to establish symbiosis with rhizobia and to fix the atmospheric dinitrogen (N2) for its N nutrition. Common bean has also the potential to establish symbiosis with arbuscular mycorrhizal fungi (AMF) that improves the uptake of low mobile nutrients such as phosphorus, from the soil. Both rhizobial and mycorrhizal symbioses can act synergistically in benefits on plant. Results The tripartite symbiosis of common bean with rhizobia and arbuscular mycorrhizal fungi (AMF) was assessed in hydroaeroponic culture with common bean (Phaseolus vulgaris L.), by comparing the effects of three fungi spp. on growth, nodulation and mycorrhization of the roots under sufficient versus deficient P supplies, after transfer from initial sand culture. Although Glomus intraradices Schenck & Smith colonized intensely the roots of common bean in both sand and hydroaeroponic cultures, Gigaspora rosea Nicolson & Schenck only established well under sand culture conditions, and no root-colonization was found with Acaulospora mellea Spain & Schenck under either culture conditions. Interestingly, mycorrhization by Glomus was also obtained by contact with mycorrhized Stylosanthes guianensis (Aubl.) sw in sand culture under deficient P before transfer into hydroaeroponic culture. The effect of bean genotype on both rhizobial and mycorrhizal symbioses with Glomus was subsequently assessed with the common bean recombinant inbreed line 7, 28, 83, 115 and 147, and the cultivar Flamingo. Significant differences among colonization and nodulation of the roots and growth among genotypes were found. Conclusion The hydroaeroponic culture is a valuable tool for further scrutinizing the physiological interactions and nutrient partitioning within the tripartite symbiosis. PMID:19534785

  5. Novel, highly expressed late nodulin gene (LjNOD16) from Lotus japonicus

    SciTech Connect

    Kapranov, P.; Bruijn, F.J. de; Szczyglowski, K.

    1997-04-01

    We have isolated a Lotus japonicus cDNA corresponding to a highly abundant, late nodule-specific RNA species that encodes a polypeptide with a predicted molecular mass of 15.6 kD. The protein and its corresponding gene were designated NIj16 and LjNOD16, respectively. LjNOD16 was found to be expressed only in the infected cells of L. japonicus nodules. Related DNA sequences could be identified in the genomes of both Glycine max and Medicago sativa. In the latter, a homologous mRNA species was detected in the nodules. Unlike LiNOD16, its alfalfa homologs appear to represent low-abundance mRNA species. However, the proteins corresponding to the LjNOD16 and its alfalfa homolog could be detected at similar levels in nodules but not in roots of both legume species. The predicted amino acid sequence analysis of nodulin NIj16 revealed the presence of a long {alpha}-helical region and a positively charged C terminus. The former domain has a very high propensity to form a coiled-coil type structure, indicating that nodulin NIj16 may interact with an as-yet-unidentified protein target(s) in the nodule-infected cells. Homology searches revealed no significant similarities to any known sequences in the databases, with the exception of two related, anonymous Arabidopsis expressed sequence tags.

  6. The origin of Lotus corniculatus.

    PubMed

    Ross, M D; Jones, W T

    1985-12-01

    Earlier students of the origin of Lotus corniculatus suggested that this tetraploid species arose as an autotetraploid of the closely related diploid species L. tenuis or L. alpinus. More recent studies suggested that L. alpinus and L. japonicus could be ancestral forms. The present study of tannin content, phenolic content, cyanide production, morphology, cytogenetics, Rhizobium specificity and self-incompatibility in the corniculatus group virtually excludes the possibility that L. corniculatus could have arisen through autopolyploidy of L. tenuis or L. alpinus, and suggests that L. corniculatus arose through hybridization of L. alpinus and/or L. tenuis (probably as female parent) with L. uliginosus (probably as male parent), followed by chromosome doubling in the hybrid.

  7. A purple acid phosphatase plays a role in nodule formation and nitrogen fixation in Astragalus sinicus.

    PubMed

    Wang, Jianyun; Si, Zaiyong; Li, Fang; Xiong, Xiaobo; Lei, Lei; Xie, Fuli; Chen, Dasong; Li, Yixing; Li, Youguo

    2015-08-01

    The AsPPD1 gene from Astragalus sinicus encodes a purple acid phosphatase. To address the functions of AsPPD1 in legume-rhizobium symbiosis, its expression patterns, enzyme activity, subcellular localization, and phenotypes associated with its over-expression and RNA interference (RNAi) were investigated. The expression of AsPPD1 was up-regulated in roots and nodules after inoculation with rhizobia. Phosphate starvation reduced the levels of AsPPD1 transcripts in roots while increased those levels in nodules. We confirmed the acid phosphatase and phosphodiesterase activities of recombinant AsPPD1 purified from Pichia pastoris, and demonstrated its ability to hydrolyze ADP and ATP in vitro. Subcellular localization showed that AsPPD1 located on the plasma membranes in hairy roots and on the symbiosomes membranes in root nodules. Over-expression of AsPPD1 in hairy roots inhibited nodulation, while its silencing resulted in nodules early senescence and significantly decreased nitrogenase activity. Furthermore, HPLC measurement showed that AsPPD1 overexpression affects the ADP levels in the infected roots and nodules, AsPPD1 silencing affects the ratio of ATP/ADP and the energy charge in nodules, and quantitative observation demonstrated the changes of AsPPD1 transcripts level affected nodule primordia formation. Taken together, it is speculated that AsPPD1 contributes to symbiotic ADP levels and energy charge control, and this is required for effective nodule organogenesis and nitrogen fixation.

  8. A study on the prevalence of bacteria that occupy nodules within single peanut plants.

    PubMed

    Angelini, Jorge; Ibáñez, Fernando; Taurian, Tania; Tonelli, María Laura; Valetti, Lucio; Fabra, Adriana

    2011-06-01

    In this study, bacteria hosted in root nodules of single plants of legume Arachis hypogaea L. (peanut) cv Tegua Runner growing at field were isolated. The collection of nodule isolates included both fast and slow growing strains. Their genetic diversity was assessed in order to identify the more frequently rhizobial strain associated to nodules from single plants. Molecular fingerprinting of 213 nodular isolates indicated heterogeneity, absence of a dominant genotype and, therefore, of a unique strains highly competitive. Efficient nitrogen-fixing isolates were identified as Bradyrhizobium sp. by phylogenetic analysis of the sequences of their 16S rRNA genes. The genetic diversity of 68 peanut nodulating isolates from all the collected plants was also analyzed. Considering their ERIC-PCR profiles, they were grouped in eighteen different OTUs for 60% similarity cut-off. Results obtained in this study indicate that the genetic diversity of rhizobia occupying nodules from single plant is very high, without the presence of a dominant strain. Therefore, the identification of useful peanut rhizobia for agricultural purposes requires strongly the selection, among the diverse population, of a very competitive genotype in combination with a high-symbiotic performance.

  9. Root nodule bacteria from Clitoria ternatea L. are putative invasive nonrhizobial endophytes.

    PubMed

    Aeron, Abhinav; Chauhan, Puneet Singh; Dubey, Ramesh Chand; Maheshwari, Dinesh Kumar; Bajpai, Vivek K

    2015-02-01

    In this study, bacteria (8 species and 5 genera) belonging to the classes Betaproteobacteria, Gammaproteobacteria, and Sphingobacteria were isolated from root nodules of the multipurpose legume Clitoria ternatea L. and identified on the basis of partial 16S rRNA sequencing. The root nodule bacteria were subjected to phenotypic clustering and diversity studies using biochemical kits, including Hi-Media Carbokit™, Enterobacteriaceae™ identification kit, ERIC-PCR, and 16S ARDRA. All the strains showed growth on Ashby's N-free media over 7 generations, indicative of presumptive nitrogen fixation and further confirmed by amplification of the nifH gene. None of the strains showed the capability to renodulate the host plant, neither alone nor in combination with standard rhizobial strains, which was further confirmed by the absence of nodC bands in PCR assay. The results clearly indicate the common existence of nonrhizobial microflora inside the root nodules of legumes, which were thought to be colonized only by rhizobia and were responsible for N2 fixation in leguminous crops. However, with the recent discovery of nodule endophytes from a variety of legumes, as also observed here, it can be assumed that symbiotic rhizobia are not all alone and that these invasive endophytes belonging to various bacterial genera are more than just opportunistic colonizers of specialized nodule niche.

  10. microRNA160 dictates stage-specific auxin and cytokinin sensitivities and directs soybean nodule development.

    PubMed

    Nizampatnam, Narasimha Rao; Schreier, Spencer John; Damodaran, Suresh; Adhikari, Sajag; Subramanian, Senthil

    2015-10-01

    Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia. The phytohormone cytokinin promotes nodule formation, and recent findings suggest that the phytohormone auxin inhibits nodule formation. Here we show that microRNA160 (miR160) is a key signaling element that determines the auxin/cytokinin balance during nodule development in soybean (Glycine max). miR160 appears to promote auxin activity by suppressing the levels of the ARF10/16/17 family of repressor ARF transcription factors. Using quantitative PCR assays and a fluorescence miRNA sensor, we show that miR160 levels are relatively low early during nodule formation and high in mature nodules. We had previously shown that ectopic expression of miR160 in soybean roots led to a severe reduction in nodule formation, coupled with enhanced sensitivity to auxin and reduced sensitivity to cytokinin. Here we show that exogenous cytokinin restores nodule formation in miR160 over-expressing roots. Therefore, low miR160 levels early during nodule development favor cytokinin activity required for nodule formation. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. Exogenous auxin partially restored proper nodule formation and maturation in STTM160 roots, suggesting that high miR160 activity later during nodule development favors auxin activity and promotes nodule maturation. Therefore, miR160 dictates developmental stage-specific sensitivities to auxin and cytokinin to direct proper nodule formation and maturation in soybean.

  11. Phylogeny of nodulation genes and symbiotic properties of Genista tinctoria bradyrhizobia.

    PubMed

    Kalita, Michał; Stepkowski, Tomasz; Łotocka, Barbara; Małek, Wanda

    2006-08-01

    Pairwise comparisons of Genista tinctoria (dyer's weed) rhizobium nodA, nodC, and nodZ gene sequences to those available in databanks revealed their highest sequence identities to nodulation loci of Bradyrhizobium sp. (Lupinus) strains and rhizobia from other genistoid legumes. On phylogenetic trees, genistoid microsymbionts were grouped together in monophyletic clusters, which suggested that their nodulation genes evolved from a common ancestor. G. tinctoria nodulators formed symbioses not only with the native host, but also with other plants of Genisteae tribe such as: Lupinus luteus, Sarothamnus scoparius, and Chamaecytisus ratisbonensis, and they were classified as the genistoid cross-inoculation group. The dyer's weed root nodules were designated as indeterminate with apical meristem consisting of infected and uninfected cells.

  12. Soybean nodule-enhanced CLE peptides in roots act as signals in GmNARK-mediated nodulation suppression.

    PubMed

    Lim, Chae Woo; Lee, Young Woo; Hwang, Cheol Ho

    2011-09-01

    The number of nodules formed in the roots of leguminous plants is systemically controlled by autoregulation of nodulation (AON). This study characterized two of the CLAVATA3/endosperm-surrounding region (CLE) genes involved in AON signal transduction. The GmRIC1 and GmRIC2 genes initiated expression solely in the roots at approximately 3 days after inoculation (DAI) with Nod factor-producing rhizobia, corresponding to the time point of AON, and the expression was up-regulated by cytokinins. Levels of GmRIC1 and GmRIC2 gene expression were much higher in the supernodulation mutant, SS2-2, than in wild-type (WT) soybeans during nodule development, even after initiation of nitrogen fixation. At 3 DAI, GmRIC2 was induced in the cells of the pericycle and the outer cortex, which undergo cell division to form nodule primordia and spreads from the central region to the whole nodule as it develops. Overexpression of GmRIC1 and GmRIC2 strongly suppressed the nodulation of WT roots as well as transgenic hairy roots in a GmNARK-dependent manner. This systemic suppression of nodulation was caused by the secretion of two CLE proteins into the extracellular space. Double grafting between WT and SS2-2 soybeans showed that signal Q is larger in SS2-2 than in WT roots during nodulation. The results of this study suggest that GmRIC1 and GmRIC2 are good candidates for root-derived signal Q in AON signal transduction.

  13. Lotus Notes Databases: The Foundation of a Virtual Library.

    ERIC Educational Resources Information Center

    Liberman, Kristen; Rich, Jane L.

    1993-01-01

    Describes the transformation of the Lotus Development Corporation's corporate library from a paper-based information service to a virtual library using Lotus Notes as the underlying development platform. Lotus Notes' tracking, broadcast, reference, and discussion applications are presented; and applications being developed by other corporations…

  14. Cell-specific expression of the promoters of two nonlegume hemoglobin genes in a transgenic legume, Lotus corniculatus.

    PubMed

    Andersson, C R; Llewellyn, D J; Peacock, W J; Dennis, E S

    1997-01-01

    The promoters of the hemoglobin genes from the nitrogen-fixing tree Parasponia andersonii and the related nonnitrogen-fixing Trema tomentosa both confer beta-glucuronidase reporter gene expression to the central zone of the nodules of a transgenic legume, Lotus corniculatus. beta-Glucuronidase expression was high in the uninfected interstitial cells and parenchyma of the surrounding boundary layer and was low in the Rhizobium-infected cells. This contrasts with the expression of both the P. andersonii hemoglobin protein in P. andersonii nodules and the endogenous Lotus leghemoglobins that are expressed in the infected cells at very high levels. The expression pattern of the P. andersonii and T. tomentosa hemoglobin promoters in L. corniculatus resembles that of a nonsymbiotic hemoglobin gene from Casuarina glauca, which was introduced into this legume, and suggests that only the nonsymbiotic functions of the P. andersonii promoter are being recognized. Deletion of the distal segments of both the P. andersonii and T. tomentosa promoters identified regions important for the control of their tissue-specific and temporal activity in Lotus. Potential regulatory elements, which enhance nodule expression and suppress nonnodule expression, were also identified and localized to a distal promoter segment. A proximal AAGAG motif is present in the P. andersonii, T. tomentosa, and nonsymbiotic Casuarina hemoglobin genes. Mutation of this motif in the P. andersonii promoter resulted in a significant reduction in both the nodule and root expression levels in L. corniculatus. Some of the regulatory motifs characterized are similar to, but different from, the nodulin motifs of the leghemoglobins.

  15. South african papilionoid legumes are nodulated by diverse burkholderia with unique nodulation and nitrogen-fixation Loci.

    PubMed

    Beukes, Chrizelle W; Venter, Stephanus N; Law, Ian J; Phalane, Francina L; Steenkamp, Emma T

    2013-01-01

    The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.

  16. South African Papilionoid Legumes Are Nodulated by Diverse Burkholderia with Unique Nodulation and Nitrogen-Fixation Loci

    PubMed Central

    Beukes, Chrizelle W.; Venter, Stephanus N.; Law, Ian J.; Phalane, Francina L.; Steenkamp, Emma T.

    2013-01-01

    The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region. PMID:23874611

  17. Relationship between photosynthetic capacity, nitrogen assimilation and nodule metabolism in alfalfa (Medicago sativa) grown with sewage sludge.

    PubMed

    Antolín, M Carmen; Fiasconaro, M Laura; Sánchez-Díaz, Manuel

    2010-10-15

    Sewage sludge has been used as N fertilizer because it contains some of inorganic N, principally as nitrate and ammonium ions. However, sewage sludge addition to legumes could result in impaired nodule metabolism due to the presence of inorganic N from sludge. A greenhouse experiment was conducted to examine the effects of sewage sludge on growth, photosynthesis, nitrogen assimilation and nodule metabolism in alfalfa (Medicago sativa L. cv. Aragón). Plants were grown in pots with a mixture of perlite and vermiculite (2:1, v/v). The experiment included three treatments: (1) plants inoculated with rhizobia and amended with sewage sludge at rate of 10% (w/w) (RS); (2) plants inoculated with rhizobia without any amendment (R); and (3) non-inoculated plants fed with ammonium nitrate (N). N(2)-fixing plants had lower growth and sucrose phosphate synthase activity but higher photosynthesis than nitrate-fed plants because they compensated the carbon cost of the rhizobia. However, sewage sludge-treated plants evidenced a loss of carbon sink strength due to N(2) fixation by means of decreased photosynthetic capacity, leaf chlorophylls and N concentration in comparison to untreated plants. Sewage sludge did no affect nodulation but decreased nodule enzyme activities involved in carbon and N metabolisms that may lead to accumulation of toxic N-compounds.

  18. Pyrroline-5-Carboxylate Reductase in Soybean Nodules 1

    PubMed Central

    Chilson, Oscar P.; Kelly-Chilson, Anne E.; Schneider, Julie D.

    1992-01-01

    Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD+-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP+ and proline. The K1 for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable. PMID:16668837

  19. Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain R7A

    PubMed Central

    2014-01-01

    Mesorhizobium loti strain R7A was isolated in 1993 in Lammermoor, Otago, New Zealand from a Lotus corniculatus root nodule and is a reisolate of the inoculant strain ICMP3153 (NZP2238) used at the site. R7A is an aerobic, Gram-negative, non-spore-forming rod. The symbiotic genes in the strain are carried on a 502-kb integrative and conjugative element known as the symbiosis island or ICEMlSymR7A. M. loti is the microsymbiont of the model legume Lotus japonicus and strain R7A has been used extensively in studies of the plant-microbe interaction. This report reveals that the genome of M. loti strain R7A does not harbor any plasmids and contains a single scaffold of size 6,529,530 bp which encodes 6,323 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25780499

  20. Genome sequence of the Lotus spp. microsymbiont Mesorhizobium loti strain R7A.

    PubMed

    Kelly, Simon; Sullivan, John; Ronson, Clive; Tian, Rui; Bräu, Lambert; Munk, Christine; Goodwin, Lynne; Han, Cliff; Woyke, Tanja; Reddy, Tatiparthi; Huntemann, Marcel; Pati, Amrita; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos; Reeve, Wayne

    2014-01-01

    Mesorhizobium loti strain R7A was isolated in 1993 in Lammermoor, Otago, New Zealand from a Lotus corniculatus root nodule and is a reisolate of the inoculant strain ICMP3153 (NZP2238) used at the site. R7A is an aerobic, Gram-negative, non-spore-forming rod. The symbiotic genes in the strain are carried on a 502-kb integrative and conjugative element known as the symbiosis island or ICEMlSym(R7A). M. loti is the microsymbiont of the model legume Lotus japonicus and strain R7A has been used extensively in studies of the plant-microbe interaction. This report reveals that the genome of M. loti strain R7A does not harbor any plasmids and contains a single scaffold of size 6,529,530 bp which encodes 6,323 protein-coding genes and 75 RNA-only encoding genes. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  1. Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses.

    PubMed

    Appunu, Chinnaswamy; Ganesan, Govindan; Kalita, Michał; Kaushik, Raghavan; Saranya, Balamurugan; Prabavathy, Vaiyapuri Ramalingam; Sudha, Nair

    2011-04-01

    Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I-V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.

  2. A Sinorhizobium meliloti-specific N-acyl homoserine lactone quorum-sensing signal increases nodule numbers in Medicago truncatula independent of autoregulation.

    PubMed

    Veliz-Vallejos, Debora F; van Noorden, Giel E; Yuan, Mengqi; Mathesius, Ulrike

    2014-01-01

    N-acyl homoserine lactones (AHLs) act as quorum sensing signals that regulate cell-density dependent behaviors in many gram-negative bacteria, in particular those important for plant-microbe interactions. AHLs can also be recognized by plants, and this may influence their interactions with bacteria. Here we tested whether the exposure to AHLs affects the nodule-forming symbiosis between legume hosts and rhizobia. We treated roots of the model legume, Medicago truncatula, with a range of AHLs either from its specific symbiont, Sinorhizobium meliloti, or from the potential pathogens, Pseudomonas aeruginosa and Agrobacterium vitis. We found increased numbers of nodules formed on root systems treated with the S. meliloti-specific AHL, 3-oxo-C14-homoserine lactone, at a concentration of 1 μM, while the other AHLs did not result in significant changes to nodule numbers. We did not find any evidence for altered nodule invasion by the rhizobia. Quantification of flavonoids that could act as nod gene inducers in S. meliloti did not show any correlation with increased nodule numbers. The effects of AHLs were specific for an increase in nodule numbers, but not lateral root numbers or root length. Increased nodule numbers following 3-oxo-C14-homoserine lactone treatment were under control of autoregulation of nodulation and were still observed in the autoregulation mutant, sunn4 (super numeric nodules4). However, increases in nodule numbers by 3-oxo-C14-homoserine lactone were not found in the ethylene-insensitive sickle mutant. A comparison between M. truncatula with M. sativa (alfalfa) and Trifolium repens (white clover) showed that the observed effects of AHLs on nodule numbers were specific to M. truncatula, despite M. sativa nodulating with the same symbiont. We conclude that plant perception of the S. meliloti-specific 3-oxo-C14-homoserine lactone influences nodule numbers in M. truncatula via an ethylene-dependent, but autoregulation-independent mechanism.

  3. Identification and structure of the Rhizobium galegae common nodulation genes: evidence for horizontal gene transfer.

    PubMed

    Suominen, L; Roos, C; Lortet, G; Paulin, L; Lindström, K

    2001-06-01

    Rhizobia are soil bacteria able to fix atmospheric nitrogen in symbiosis with leguminous plants. In response to a signal cascade coded by genes of both symbiotic partners, a specific plant organ, the nodule, is formed. Rhizobial nodulation (nod) genes trigger nodule formation through the synthesis of Nod factors, a family of chitolipooligosaccharides that are specifically recognized by the host plant at the first stages of the nodulation process. Here, we present the organization and sequence of the common nod genes from Rhizobium galegae, a symbiotic member of the RHIZOBIACEAE: This species has an intriguing phylogenetic position, being symbiotic among pathogenic agrobacteria, which induce tumors instead of nodules in plant shoots or roots. This apparent incongruence raises special interest in the origin of the symbiotic apparatus of R. galegae. Our analysis of DNA sequence data indicated that the organization of the common nod gene region of R. galegae was similar to that of Sinorhizobium meliloti and Rhizobium leguminosarum, with nodIJ downstream of nodABC and the regulatory nodD gene closely linked to the common nod operon. Moreover, phylogenetic analyses of the nod gene sequences showed a close relationship especially between the common nodA sequences of R. galegae, S. meliloti, and R. leguminosarum biovars viciae and trifolii. This relationship in structure and sequence contrasts with the phylogeny based on 16S rRNA, which groups R. galegae close to agrobacteria and separate from most other rhizobia. The topology of the nodA tree was similar to that of the corresponding host plant tree. Taken together, these observations indicate that lateral nod gene transfer occurred from fast-growing rhizobia toward agrobacteria, after which the symbiotic apparatus evolved under host plant constraint.

  4. Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules.

    PubMed

    Cam, Yvan; Pierre, Olivier; Boncompagni, Eric; Hérouart, Didier; Meilhoc, Eliane; Bruand, Claude

    2012-10-01

    Nitric oxide (NO) is a signalling and defence molecule involved in diverse plant developmental processes, as well as in the plant response to pathogens. NO has also been detected at different steps of the symbiosis between legumes and rhizobia. NO is required for an optimal establishment of the Medicago truncatula-Sinorhizobium meliloti symbiotic interaction, but little is known about the role of NO in mature nodules. Here, we investigate the role of NO in the late steps of symbiosis. Genetic and pharmacological approaches were conducted to modulate the NO level inside root nodules, and their effects on nitrogen fixation and root nodule senescence were monitored. An increase in endogenous NO levels led to a decrease in nitrogen fixation and early nodule senescence, characterized by cytological modifications of the nodule structure and the early expression of a specific senescence marker. By contrast, a decrease in NO levels led to a delay in nodule senescence. Together, our results strongly suggest that NO is a signal in developmental as well as stress-induced nodule senescence. In addition, this work demonstrates the pivotal role of the bacterial NO detoxification response in the prevention of early nodule senescence, and hence the maintenance of efficient symbiosis.

  5. Polyphenol oxidase affects normal nodule development in red clover (Trifolium pratense L.)

    PubMed Central

    Webb, K. Judith; Cookson, Alan; Allison, Gordon; Sullivan, Michael L.; Winters, Ana L.

    2014-01-01

    Polyphenol oxidase (PPO) may have multiple functions in tissues depending on its cellular or tissue localization. Here we use PPO RNAi transformants of red clover (Trifolium pratense) to determine the role PPO plays in normal development of plants, and especially in N2-fixing nodules. In red clover, PPO was not essential for either growth or nodule production, or for nodule function in plants grown under optimal, N-free conditions. However, absence of PPO resulted in a more reduced environment in all tissues, as measured by redox potential, and caused subtle developmental changes in nodules. Leaves and, to a lesser extent nodules, lacking PPO tended to accumulate phenolic compounds. A comparison of nodules of two representative contrasting clones by microscopy revealed that nodules lacking PPO were morphologically and anatomically subtly altered, and that phenolics accumulated in different cells and tissues. Developing nodules lacking PPO were longer, and there were more cell layers within the squashed cell layer (SCL), but the walls of these cells were less thickened and the cells were less squashed. Within the N2-fixing zone, bacteroids appeared more granular and were less tightly packed together, and were similar to developmentally compromised bacteroids elicited by catalase mutant rhizobia reported elsewhere. PMID:25566275

  6. Genome Wide Identification and Expression Profiling of Ethylene Receptor Genes during Soybean Nodulation.

    PubMed

    Wang, Youning; Yuan, Jinhong; Yang, Wei; Zhu, Lin; Su, Chao; Wang, Xiaodi; Wu, Haiyan; Sun, Zhengxi; Li, Xia

    2017-01-01

    It has long been known that the gaseous plant hormone ethylene plays a key role in nodulation in legumes. The perception of ethylene by a family of five membrane-localized receptors is necessary to trigger the ethylene signaling pathway, which regulates various biological responses in Arabidopsis. However, a systematic analysis of the ethylene receptors in leguminous plants and their roles in nodule development is lacking. In this study, we performed a characterization of ethylene receptor genes based on the latest Glycine max genome sequence and a public microarray database. Eleven ethylene receptor family genes were identified in soybean through homology searches, and they were divided into two subgroups. Exon-intron analysis showed that the gene structures are highly conserved within each group. Further analysis of their expression patterns showed that these ethylene receptor genes are differentially expressed in various soybean tissues and organs, including functional nodules. Notably, the ethylene receptor genes showed different responses to rhizobial infection and Nod factors, suggesting a possible role for ethylene receptors and ethylene signaling in rhizobia-host cell interactions and nodulation in soybean. Together, these data indicate the functional divergence of ethylene receptor genes in soybean, and that some of these receptors mediate nodulation, including rhizobial infection, nodule development, and nodule functionality. These findings provide a foundation for further elucidation of the molecular mechanism by which the ethylene signaling pathway regulates nodulation in soybean, as well as other legumes.

  7. Rhizobium pongamiae sp. nov. from root nodules of Pongamia pinnata.

    PubMed

    Kesari, Vigya; Ramesh, Aadi Moolam; Rangan, Latha

    2013-01-01

    Pongamia pinnata has an added advantage of N2-fixing ability and tolerance to stress conditions as compared with other biodiesel crops. It harbours "rhizobia" as an endophytic bacterial community on its root nodules. A gram-negative, nonmotile, fast-growing, rod-shaped, bacterial strain VKLR-01(T) was isolated from root nodules of Pongamia that grew optimal at 28°C, pH 7.0 in presence of 2% NaCl. Isolate VKLR-01 exhibits higher tolerance to the prevailing adverse conditions, for example, salt stress, elevated temperatures and alkalinity. Strain VKLR-01(T) has the major cellular fatty acid as C(18:1) ω7c (65.92%). Strain VKLR-01(T) was found to be a nitrogen fixer using the acetylene reduction assay and PCR detection of a nifH gene. On the basis of phenotypic, phylogenetic distinctiveness and molecular data (16S rRNA, recA, and atpD gene sequences, G + C content, DNA-DNA hybridization etc.), strain VKLR-01(T) = (MTCC 10513(T) = MSCL 1015(T)) is considered to represent a novel species of the genus Rhizobium for which the name Rhizobium pongamiae sp. nov. is proposed. Rhizobium pongamiae may possess specific traits that can be transferred to other rhizobia through biotechnological tools and can be directly used as inoculants for reclamation of wasteland; hence, they are very important from both economic and environmental prospects.

  8. Petal Development in Lotus japonicus.

    PubMed

    Weng, Lin; Tian, Zhaoxia; Feng, Xianzhong; Li, Xin; Xu, Shilei; Hu, Xiaohe; Luo, Da; Yang, Jun

    2011-10-01

    Previous studies have demonstrated that petal shape and size in legume flowers are determined by two separate mechanisms, dorsoventral (DV) and organ internal (IN) asymmetric mechanisms, respectively. However, little is known about the molecular mechanisms controlling petal development in legumes. To address this question, we investigated petal development along the floral DV axis in Lotus japonicus with respect to cell and developmental biology by comparing wild-type legumes to mutants. Based on morphological markers, the entire course of petal development, from initiation to maturity, was grouped to define 3 phases or 13 stages. In terms of epidermal micromorphology from adaxial surface, mature petals were divided into several distinct domains, and characteristic epidermal cells of each petal differentiated at stage 9, while epidermal cells of all domains were observed until stage 12. TCP and MIXTA-like genes were found to be differentially expressed in various domains of petals at stages 9 and 12. Our results suggest that DV and IN mechanisms interplay at different stages of petal development, and their interaction at the cellular and molecular level guides the elaboration of domains within petals to achieve their ideal shape, and further suggest that TCP genes determine petal identity along the DV axis by regulating MIXTA-like gene expression.

  9. Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

    PubMed Central

    Meade, James; Higgins, Patrick; O'Gara, Fergal

    1985-01-01

    The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 104 rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 107 CFU ml−1 (2 × 106 bacteria seed−1) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (107 CFU ml−1) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 109 CFU ml−1 (9.6 × 107 bacteria seed−1). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive. Images PMID:16346769

  10. Stimulation of nodulation in Medicago truncatula by low concentrations of ammonium: quantitative reverse transcription PCR analysis of selected genes.

    PubMed

    Fei, Houman; Vessey, J Kevin

    2009-03-01

    Although mineral nitrogen generally has negative effects on nodulation in legume-rhizobia symbioses, low concentrations of ammonium stimulate nodulation in some legumes. In this study, the effects of ammonium and nitrate on growth, nodulation and expression of 2 nitrogen transport and 12 putative nodulation-related genes of the model symbiosis of Medicago truncatula - Sinorhizobium meliloti are investigated. After 3 weeks of hydroponic growth, whole-plant nodulation was enhanced in all the ammonium treatments and up to three-fold in the 0.5 mM treatment compared with the zero-nitrogen control. Specific nodulation (nodules g(-1) root dry weight) was greatly stimulated in the 0.1 and 0.5 mM NH4+ treatments, to a lower extent in the 0.1 mM NO3- treatment, and inhibited in all other treatments. Expression of the 14 selected genes was observed at 0, 6, 12 and 24 h after exposure to rhizobia and nitrogen. Expression of nitrogen transporter genes increased significantly, but responses of the three genes putatively associated with symbiosis signaling/nodule initiation were mixed. There were infrequent responses of genes coding for an ABA-activated protein kinase or a gibberellin-regulated protein, but an ethylene-responsive element-binding factor showed increased expression in various treatments and sampling times. Three auxin-responsive genes and three cytokinin-responsive genes showed varied responses to ammonium and nitrate. This study indicates that low concentrations of ammonium stimulate nodulation in M. truncatula, but the data were inconclusive in verifying the hypothesis that a relatively high ratio of cytokinin to auxin in roots may be an underlying mechanism in this stimulation of nodulation.

  11. The Lotus effect: superhydrophobicity and metastability.

    PubMed

    Marmur, Abraham

    2004-04-27

    To learn how to mimic the Lotus effect, superhydrophobicity of a model system that resembles the Lotus leaf is theoretically discussed. Superhydrophobicity is defined by two criteria: a very high water contact angle and a very low roll-off angle. Since it is very difficult to calculate the latter for rough surfaces, it is proposed here to use the criterion of a very low wet (solid-liquid) contact area as a simple, approximate substitute for the roll-off angle criterion. It is concluded that nature employs metastable states in the heterogeneous wetting regime as the key to superhydrophobicity on Lotus leaves. This strategy results in two advantages: (a) it avoids the need for high steepness protrusions that may be sensitive to breakage and (b) it lowers the sensitivity of the superhydrophobic states to the protrusion distance.

  12. Isolation and Characterization of Nodule-Associated Exiguobacterium sp. from the Root Nodules of Fenugreek (Trigonella foenum-graecum) and Their Possible Role in Plant Growth Promotion

    PubMed Central

    Rajendran, Geetha; Patel, Maheshwari H.; Joshi, Sanket J.

    2012-01-01

    One of the ways to increase the competitive survivability of rhizobial biofertilizers and thus achieve better plant growth under such conditions is by modifying the rhizospheric environment or community by addition of nonrhizobial nodule-associated bacteria (NAB) that cause better nodulation and plant growth when coinoculated with rhizobia. A study was performed to investigate the most commonly associated nodule-associated bacteria and the rhizospheric microorganisms associated with the Fenugreek (Trigonella foenum-graecum) plant. Isolation of nonrhizobial isolates from root nodules of Fenugreek was carried out along with the rhizospheric isolates. About 64.7% isolates obtained from Fenugreek nodules were gram-negative coccobacilli, 29.41% were gram-positive bacilli, and all rhizospheric isolates except one were gram-positive bacilli. All the isolates were characterized for their plant growth promoting (PGP) activities. Two of the NAB isolates M2N2c and B1N2b (Exiguobacterium sp.) showed maximum positive PGP features. Those NAB isolates when coinoculated with rhizobial strain—S. meliloti, showed plant growth promotion with respect to increase in plant's root and shoot length, chlorophyll content, nodulation efficiency, and nodule dry weight. PMID:22518149

  13. MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges

    PubMed Central

    Godiard, Laurence; Lepage, Agnès; Moreau, Sandra; Laporte, Damien; Verdenaud, Marion; Timmers, Ton; Gamas, Pascal

    2011-01-01

    This study aimed at defining the role of a basic helix–loop–helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria. We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics. We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor. Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots. PMID:21679315

  14. Molecular Characterization of LjABCG1, an ATP-Binding Cassette Protein in Lotus japonicus

    PubMed Central

    Sugiyama, Akifumi; Fukuda, Shoju; Takanashi, Kojiro; Yoshioka, Miki; Yoshioka, Hirofumi; Narusaka, Yoshihiro; Narusaka, Mari; Kojima, Mikiko; Sakakibara, Hitoshi; Shitan, Nobukazu; Sato, Shusei; Tabata, Satoshi; Kawaguchi, Masayoshi; Yazaki, Kazufumi

    2015-01-01

    LjABCG1, a full-size ABCG subfamily of ATP-binding cassette proteins of a model legume, Lotus japonicus, was reported as a gene highly expressed during the early stages of nodulation, but have not been characterized in detail. In this study we showed that the induction of LjABCG1 expression was remarkable by methyl jasmonate treatment, and reporter gene experiments indicated that LjABCG1 was strongly expressed in the nodule parenchyma and cell layers adjacent to the root vascular tissue toward the nodule. LjABCG1 was suggested to be localized at the plasma membrane based on the fractionation of microsomal membranes as well as separation via aqueous two-phase partitioning. The physiological functions of LjABCG1 in symbiosis and pathogenesis were analyzed in homologous and heterologous systems. LjABCG1 knock-down L. japonicus plants did not show clear phenotypic differences in nodule formation, and not in defense against Pseudomonas syringae, either. In contrast, when LjABCG1 was expressed in the Arabidopsis pdr8-1 mutant, the penetration frequency of Phytophthora infestans, a potato late blight pathogen, was significantly reduced in LjABCG1/pdr8-1 than in pdr8-1 plants. This finding indicated that LjABCG1, at least partially, complemented the phenotype of pdr8 in Arabidopsis, suggesting the multiple roles of this protein in plant-microbe interactions. PMID:26418593

  15. Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus.

    PubMed

    Takeda, Naoya; Sato, Shusei; Asamizu, Erika; Tabata, Satoshi; Parniske, Martin

    2009-06-01

    In the arbuscular mycorrhiza (AM) symbiosis, plant roots accommodate Glomeromycota fungi within an intracellular compartment, the arbuscule. At this symbiotic interface, fungal hyphae are surrounded by a plant membrane, which creates an apoplastic compartment, the periarbuscular space (PAS) between fungal and plant cell. Despite the importance of the PAS for symbiotic signal and metabolite exchange, only few of its components have been identified. Here we show that two apoplastic plant proteases of the subtilase family are required for AM development. SbtM1 is the founder member of a family of arbuscular mycorrhiza-induced subtilase genes that occur in at least two clusters in the genome of the legume Lotus japonicus. A detailed expression analysis by RT-PCR revealed that SbtM1, SbtM3, SbtM4 and the more distantly related SbtS are all rapidly induced during development of arbuscular mycorrhiza, but only SbtS and SbtM4 are also up-regulated during root nodule symbiosis. Promoter-reporter fusions indicated specific activation in cells that are adjacent to intra-radical fungal hyphae or in cells that harbour them. Venus fluorescent protein was observed in the apoplast and the PAS when expressed from a fusion construct with the SbtM1 signal peptide or the full-length subtilase. Suppression of SbtM1 or SbtM3 by RNAi caused a decrease in intra-radical hyphae and arbuscule colonization, but had no effect on nodule formation. Our data indicate a role for these subtilases during the fungal infection process in particular arbuscule development.

  16. Production of 3-indolylacetic acid in root nodules and culture by a Rhizobium species isolated from root nodules of the leguminous pulse Phaseolus mungo.

    PubMed

    Ghosh, S; Sengupta, C; Maiti, T K; Basu, P S

    2008-01-01

    The root nodules of Phaseolus mungo (a herbaceous leguminous pulse) contained a high amount of 3-indolylacetic acid (IAA). A tryptophan pool present in the nodule might play the role of precursor for IAA production. From the root nodule a Rhizobium sp. was isolated. The symbiont produced a large amount of IAA (142 microg/mL) from L-tryptophan-supplemented basal medium. The production of IAA by the symbiont was much increased over the control when a L-tryptophan (2 mg/mL) supplemented C-free mineral medium was enriched with mannitol (1 %), L-asparagine (0.3 %) and thiamine hydrochloride (1 microg/mL). The possible role of the rhizobial production of IAA on the rhizobia-legume symbiosis is discussed.

  17. The presence of nodules on legume root systems can alter phenotypic plasticity in response to internal nitrogen independent of nitrogen fixation.

    PubMed

    Goh, Chooi-Hua; Nicotra, Adrienne B; Mathesius, Ulrike

    2016-04-01

    All higher plants show developmental plasticity in response to the availability of nitrogen (N) in the soil. In legumes, N starvation causes the formation of root nodules, where symbiotic rhizobacteria fix atmospheric N2 for the host in exchange for fixed carbon (C) from the shoot. Here, we tested whether plastic responses to internal [N] of legumes are altered by their symbionts. Glasshouse experiments compared root phenotypes of three legumes, Medicago truncatula, Medicago sativa and Trifolium subterraneum, inoculated with their compatible symbiont partners and grown under four nitrate levels. In addition, six strains of rhizobia, differing in their ability to fix N2 in M. truncatula, were compared to test if plastic responses to internal [N] were dependent on the rhizobia or N2 -fixing capability of the nodules. We found that the presence of rhizobia affected phenotypic plasticity of the legumes to internal [N], particularly in root length and root mass ratio (RMR), in a plant species-dependent way. While root length responses of M. truncatula to internal [N] were dependent on the ability of rhizobial symbionts to fix N2 , RMR response to internal [N] was dependent only on initiation of nodules, irrespective of N2 -fixing ability of the rhizobia strains.

  18. Alkalinity of Lanzarote soils is a factor shaping rhizobial populations with Sinorhizobium meliloti being the predominant microsymbiont of Lotus lancerottensis.

    PubMed

    León-Barrios, Milagros; Pérez-Yépez, Juan; Dorta, Paola; Garrido, Ana; Jiménez, Concepción

    2017-02-02

    Lotus lancerottensis is an endemic species that grows widely throughout Lanzarote Island (Canary Is.). Characterization of 48 strains isolated from root nodules of plants growing in soils from eleven locations on the island showed that 38 isolates (79.1%) belonged to the species Sinorhizobium meliloti, whereas only six belonged to Mesorhizobium sp., the more common microsymbionts for the Lotus. Other genotypes containing only one isolate were classified as Pararhizobium sp., Sinorhizobium sp., Phyllobacterium sp. and Bradyrhizobium-like. Strains of S. meliloti were distributed along the island and, in most of the localities they were exclusive or major microsymbionts of L. lancerottensis. Phylogeny of the nodulation nodC gene placed the S. meliloti strains within symbiovar lancerottense and the mesorhizobial strains with the symbiovar loti. Although strains from both symbiovars produced effective N2-fixing nodules, S. meliloti symbiovar lancerottense was clearly the predominant microsymbiont of L. lancerottensis. This fact correlated with the better adaptation of strains of this species to the alkaline soils of Lanzarote, as in vitro characterization showed that while the mesorhizobial strains were inhibited by alkaline pH, S. meliloti strains grew well at pH 9.

  19. Choice of hydrogen uptake (Hup) status in legume-rhizobia symbioses

    PubMed Central

    Annan, Henry; Golding, Amber-Leigh; Zhao, Yinping; Dong, Zhongmin

    2012-01-01

    The H2 is an obligate by-product of N-fixation. Recycling of H2 through uptake hydrogenase (Hup) inside the root nodules of leguminous plants is often considered an advantage for plants. However, many of the rhizobium-legume symbioses found in nature, especially those used in agriculture are shown to be Hup−, with the plants releasing H2 produced by nitrogenase activity from root nodules into the surrounding rhizosphere. Recent studies have suggested that, H2 induces plant-growth-promoting rhizobacteria, which may explain the widespread of Hup− symbioses in spite of the low energy efficiency of such associations. Wild legumes grown in Nova Scotia, Canada, were surveyed to determine if any plant-growth characteristics could give an indication of Hup choice in leguminous plants. Out of the plants sampled, two legumes, Securigera varia and Vicia cracca, showed Hup+ associations. Securigera varia exhibited robust root structure as compared with the other plants surveyed. Data from the literature and the results from this study suggested that plants with established root systems are more likely to form the energy-efficient Hup+ symbiotic relationships with rhizobia. Conversely, Hup− associations could be beneficial to leguminous plants due to H2-oxidizing plant-growth-promoting rhizobacteria that allow plants to compete successfully, early in the growing season. However, some nodules from V. cracca tested Hup+, while others were Hup−. This was similar to that observed in Glycine max and Pisum sativum, giving reason to believe that Hup choice might be affected by various internal and environmental factors. PMID:23139886

  20. Negotiation of mutualism: rhizobia and legumes

    PubMed Central

    Akçay, Erol; Roughgarden, Joan

    2006-01-01

    The evolution and persistence of biological cooperation have been an important puzzle in evolutionary theory. Here, we suggest a new approach based on bargaining theory to tackle the question. We present a mechanistic model for negotiation of benefits between a nitrogen-fixing nodule and a legume plant. To that end, we first derive growth rates for the nodule and plant from metabolic models of each as a function of material fluxes between them. We use these growth rates as pay-off functions in the negotiation process, which is analogous to collective bargaining between a firm and a workers' union. Our model predicts that negotiations lead to the Nash bargaining solution, maximizing the product of players' pay-offs. This work introduces elements of cooperative game theory into the field of mutualistic interactions. In the discussion of the paper, we argue for the benefits of such an approach in studying the question of biological cooperation. PMID:17015340

  1. Dissection of Symbiosis and Organ Development by Integrated Transcriptome Analysis of Lotus japonicus Mutant and Wild-Type Plants

    PubMed Central

    Høgslund, Niels; Radutoiu, Simona; Krusell, Lene; Voroshilova, Vera; Hannah, Matthew A.; Goffard, Nicolas; Sanchez, Diego H.; Lippold, Felix; Ott, Thomas; Sato, Shusei; Tabata, Satoshi; Liboriussen, Poul; Lohmann, Gitte V.; Schauser, Leif; Weiller, Georg F.; Udvardi, Michael K.; Stougaard, Jens

    2009-01-01

    Genetic analyses of plant symbiotic mutants has led to the identification of key genes involved in Rhizobium-legume communication as well as in development and function of nitrogen fixing root nodules. However, the impact of these genes in coordinating the transcriptional programs of nodule development has only been studied in limited and isolated studies. Here, we present an integrated genome-wide analysis of transcriptome landscapes in Lotus japonicus wild-type and symbiotic mutant plants. Encompassing five different organs, five stages of the sequentially developed determinate Lotus root nodules, and eight mutants impaired at different stages of the symbiotic interaction, our data set integrates an unprecedented combination of organ- or tissue-specific profiles with mutant transcript profiles. In total, 38 different conditions sampled under the same well-defined growth regimes were included. This comprehensive analysis unravelled new and unexpected patterns of transcriptional regulation during symbiosis and organ development. Contrary to expectations, none of the previously characterized nodulins were among the 37 genes specifically expressed in nodules. Another surprise was the extensive transcriptional response in whole root compared to the susceptible root zone where the cellular response is most pronounced. A large number of transcripts predicted to encode transcriptional regulators, receptors and proteins involved in signal transduction, as well as many genes with unknown function, were found to be regulated during nodule organogenesis and rhizobial infection. Combining wild type and mutant profiles of these transcripts demonstrates the activation of a complex genetic program that delineates symbiotic nitrogen fixation. The complete data set was organized into an indexed expression directory that is accessible from a resource database, and here we present selected examples of biological questions that can be addressed with this comprehensive and powerful

  2. A nodule-specific lipid transfer protein AsE246 participates in transport of plant-synthesized lipids to symbiosome membrane and is essential for nodule organogenesis in Chinese milk vetch.

    PubMed

    Lei, Lei; Chen, Ling; Shi, Xiaofeng; Li, Yixing; Wang, Jianyun; Chen, Dasong; Xie, Fuli; Li, Youguo

    2014-02-01

    Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.

  3. A Nodule-Specific Lipid Transfer Protein AsE246 Participates in Transport of Plant-Synthesized Lipids to Symbiosome Membrane and Is Essential for Nodule Organogenesis in Chinese Milk Vetch1[C][W][OPEN

    PubMed Central

    Lei, Lei; Chen, Ling; Shi, Xiaofeng; Li, Yixing; Wang, Jianyun; Chen, Dasong; Xie, Fuli; Li, Youguo

    2014-01-01

    Rhizobia in legume root nodules fix nitrogen in symbiosomes, organelle-like structures in which a membrane from the host plant surrounds the symbiotic bacteria. However, the components that transport plant-synthesized lipids to the symbiosome membrane remain unknown. This study identified and functionally characterized the Chinese milk vetch (Astragalus sinicus) lipid transfer protein AsE246, which is specifically expressed in nodules. It was found that AsE246 can bind lipids in vitro. More importantly, AsE246 can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol in vivo. Immunofluorescence and immunoelectron microscopy showed that AsE246 and digalactosyldiacylglycerol localize in the symbiosome membrane and are present in infection threads. Overexpression of AsE246 resulted in increased nodule numbers; knockdown of AsE246 resulted in reduced nodule numbers, decreased lipids contents in nodules, diminished nitrogen fixation activity, and abnormal development of symbiosomes. AsE246 knockdown also resulted in fewer infection threads, nodule primordia, and nodules, while AsE246 overexpression resulted in more infection threads and nodule primordia, suggesting that AsE246 affects nodule organogenesis associated with infection thread formation. Taken together, these results indicate that AsE246 contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis. PMID:24367021

  4. Rhizobia species: A Boon for "Plant Genetic Engineering".

    PubMed

    Patel, Urmi; Sinha, Sarika

    2011-10-01

    Since past three decades new discoveries in plant genetic engineering have shown remarkable potentials for crop improvement. Agrobacterium Ti plasmid based DNA transfer is no longer the only efficient way of introducing agronomically important genes into plants. Recent studies have explored a novel plant genetic engineering tool, Rhizobia sp., as an alternative to Agrobacterium, thereby expanding the choice of bacterial species in agricultural plant biotechnology. Rhizobia sp. serve as an open license source with no major restrictions in plant biotechnology and help broaden the spectrum for plant biotechnologists with respect to the use of gene transfer vehicles in plants. New efficient transgenic plants can be produced by transferring genes of interest using binary vector carrying Rhizobia sp. Studies focusing on the interactions of Rhizobia sp. with their hosts, for stable and transient transformation and expression of genes, could help in the development of an adequate gene transfer vehicle. Along with being biologically beneficial, it may also bring a new means for fast economic development of transgenic plants, thus giving rise to a new era in plant biotechnology, viz. "Rhizobia mediated transformation technology."

  5. Organic acid mediated repression of sugar utilization in rhizobia.

    PubMed

    Iyer, Bhagya; Rajput, Mahendrapal Singh; Jog, Rahul; Joshi, Ekta; Bharwad, Krishna; Rajkumar, Shalini

    2016-11-01

    Rhizobia are a class of symbiotic diazotrophic bacteria which utilize C4 acids in preference to sugars and the sugar utilization is repressed as long as C4 acids are present. This can be manifested as a diauxie when rhizobia are grown in the presence of a sugar and a C4 acid together. Succinate, a C4 acid is known to repress utilization of sugars, sugar alcohols, hydrocarbons, etc by a mechanism termed as Succinate Mediated Catabolite Repression (SMCR). Mechanism of catabolite repression determines the hierarchy of carbon source utilization in bacteria. Though the mechanism of catabolite repression has been well studied in model organisms like E. coli, B. subtilis and Pseudomonas sp., mechanism of SMCR in rhizobia has not been well elucidated. C4 acid uptake is important for effective symbioses while mutation in the sugar transport and utilization genes does not affect symbioses. Deletion of hpr and sma0113 resulted in the partial relief of SMCR of utilization of galactosides like lactose, raffinose and maltose in the presence of succinate. However, no such regulators governing SMCR of glucoside utilization have been identified till date. Though rhizobia can utilize multitude of sugars, high affinity transporters for many sugars are yet to be identified. Identifying high affinity sugar transporters and studying the mechanism of catabolite repression in rhizobia is important to understand the level of regulation of SMCR and the key regulators involved in SMCR.

  6. Rhizobia: a potential biocontrol agent for soilborne fungal pathogens.

    PubMed

    Das, Krishnashis; Prasanna, Radha; Saxena, Anil Kumar

    2017-03-12

    Rhizobia are a group of organisms that are well known for their ability to colonize root surfaces and form symbiotic associations with legume plants. They not only play a major role in biological nitrogen fixation but also improve plant growth and reduce disease incidence in various crops. Rhizobia are known to control the growth of many soilborne plant pathogenic fungi belonging to different genera like Fusarium, Rhizoctonia, Sclerotium, and Macrophomina. Antagonistic activity of rhizobia is mainly attributed to production of antibiotics, hydrocyanic acid (HCN), mycolytic enzymes, and siderophore under iron limiting conditions. Rhizobia are also reported to induce systemic resistance and enhance expression of plant defense-related genes, which effectively immunize the plants against pathogens. Seed bacterization with appropriate rhizobial strain leads to elicitation and accumulation of phenolic compounds, isoflavonoid phytoalexins, and activation of enzymes like L-phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), peroxidase (POX), polyphenol oxidase (PPO), and others involved in phenylpropanoid and isoflavonoid pathways. Development of Rhizobium inoculants with dual attributes of nitrogen fixation and antagonism against phytopathogens can contribute to increased plant growth and productivity. This compilation aims to bring together the available information on the biocontrol facet of rhizobia and identify research gaps and effective strategies for future research in this area.

  7. Plant growth promoting rhizobia: challenges and opportunities.

    PubMed

    Gopalakrishnan, Subramaniam; Sathya, Arumugam; Vijayabharathi, Rajendran; Varshney, Rajeev Kumar; Gowda, C L Laxmipathi; Krishnamurthy, Lakshmanan

    2015-08-01

    Modern agriculture faces challenges, such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Sustainability and environmental safety of agricultural production relies on eco-friendly approaches like biofertilizers, biopesticides and crop residue return. The multiplicity of beneficial effects of microbial inoculants, particularly plant growth promoters (PGP), emphasizes the need for further strengthening the research and their use in modern agriculture. PGP inhabit the rhizosphere for nutrients from plant root exudates. By reaction, they help in (1) increased plant growth through soil nutrient enrichment by nitrogen fixation, phosphate solubilization, siderophore production and phytohormones production (2) increased plant protection by influencing cellulase, protease, lipase and β-1,3 glucanase productions and enhance plant defense by triggering induced systemic resistance through lipopolysaccharides, flagella, homoserine lactones, acetoin and butanediol against pests and pathogens. In addition, the PGP microbes contain useful variation for tolerating abiotic stresses like extremes of temperature, pH, salinity and drought; heavy metal and pesticide pollution. Seeking such tolerant PGP microbes is expected to offer enhanced plant growth and yield even under a combination of stresses. This review summarizes the PGP related research and its benefits, and highlights the benefits of PGP rhizobia belonging to the family Rhizobiaceae, Phyllobacteriaceae and Bradyrhizobiaceae.

  8. Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress.

    PubMed

    Jebara, Salwa; Jebara, Moez; Limam, Férid; Aouani, Mohamed Elarbi

    2005-08-01

    To analyse nodular antioxidant enzyme expression in response to salt stress, Phaseolus vulgaris genotype BAT477 was inoculated with reference strain CIAT899, and treated with 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity were analysed. Results showed that: (1) all parameters, particularly in nodules, were affected by salt treatments, and (2) confirmed preferential growth allocation to roots. The ARA was significantly decreased by salt treatments. Protein dosage confirmed that nodules were more affected by salt treatment than were roots. We analysed superoxide dismutase, catalase, ascorbate peroxidase and peroxidase in nodules, roots and a free rhizobial strain. Our results indicated that SOD and CAT nodular isozymes had bacterial and root origins. The SOD expressed the same CuZn, Fe and Mn SOD isoforms in nodules and roots, whereas in free rhizobia we found only one Fe and Mn SOD. APX and POX nodule and root profiles had only root origins, as no rhizobial band was detected. Under salt stress, plant growth, nitrogen fixation and activities of antioxidant defense enzymes in nodules were affected. Thus, these enzymes appear to preserve symbiosis from stress turned out that NaCl salinity lead to a differential regulation of distinct SOD and POX isoenzyme. So their levels in nodules appeared to be consistent with a symbiotic nitrogen fixing efficiency hypothesis, and they seem to function as the molecular mechanisms underlying the nodule response to salinity.

  9. Cyanobacterial extracts containing microcystins affect the growth, nodulation process and nitrogen uptake of faba bean (Vicia faba L., Fabaceae).

    PubMed

    Lahrouni, Majida; Oufdou, Khalid; Faghire, Mustapha; Peix, Alvaro; El Khalloufi, Fatima; Vasconcelos, Vitor; Oudra, Brahim

    2012-04-01

    The use of irrigation water containing cyanobacterial toxins may generate a negative impact in both yield and quality of agricultural crops causing significant economic losses. We evaluated the effects of microcystins (MC) on the growth, nodulation process and nitrogen uptake of a Faba bean cultivar (Vicia faba L., Fabaceae), particularly the effect of MC on rhizobia-V. faba symbiosis. Three rhizobial strains (RhOF4, RhOF6 and RhOF21), isolated from nodules of local V. faba were tested. The exposure of rhizobia to MC showed that the toxins had a negative effect on the rhizobial growth especially at the highest concentrations of 50 and 100 μg/l. The germination of faba bean seeds was also affected by cyanotoxins. We registered germination rates of 75 and 68.75% at the toxin levels of 50 and 100 μg/l as compared to the control (100%). The obtained results also showed there was a negative effect of MC on plants shoot, root (dry weight) and total number of nodules per plant. Cyanotoxins exposure induced a significant effect on nitrogen assimilation by faba bean seedlings inoculated with selected rhizobial strains RhOF6 and RhOF21, while the effect was not significant on beans seedling inoculated with RhOF4. This behavior of tolerant rhizobia-legumes symbioses may constitute a very important pathway to increase soil fertility and quality and can represent a friendly biotechnological way to remediate cyanotoxins contamination in agriculture.

  10. Cultural conditions required for the induction of an adaptive acid-tolerance response (ATR) in Sinorhizobium meliloti and the question as to whether or not the ATR helps rhizobia improve their symbiosis with alfalfa at low pH.

    PubMed

    Draghi, Walter O; Del Papa, María Florencia; Pistorio, Mariano; Lozano, Mauricio; de Los Angeles Giusti, María; Torres Tejerizo, Gonzalo A; Jofré, Edgardo; Boiardi, José Luis; Lagares, Antonio

    2010-01-01

    Sinorhizobium meliloti associates with Medicago and Melilotus species to develop nitrogen-fixing symbioses. The agricultural relevance of these associations, the worldwide distribution of acid soils, and the remarkable acid sensitivity of the microsymbiont have all stimulated research on the responses of the symbionts to acid environments. We show here that an adaptive acid-tolerance response (ATR) can be induced in S. meliloti, as shown previously for Sinorhizobium medicae, when the bacteria are grown in batch cultures at the slightly acid pH of 6.1. In marked contrast, no increased tolerance to hydrogen ions is obtained if rhizobia are grown in a chemostat under continuous cultivation at the same pH. The adaptive ATR appears as a complex process triggered by an increased hydrogen-ion concentration, but operative only if other--as yet unknown--concomitant factors that depend on the culture conditions are present (although not provided under continuous cultivation). Although the stability of the ATR and its influence on acid tolerance has been characterized in rhizobia, no data have been available on the effect of the adapted state on symbiosis. Coinoculation experiments showed that acid-adapted indicator rhizobia (ATR+) were present in >90% of the nodules when nodulation was performed at pH 5.6, representing a >30% increase in occupancy compared with a control test. We show that the ATR represents a clear advantage in competing for nodulation at low pH. It is not yet clear whether such an effect results from an improved performance in the acid environment during preinfection, an enhanced ability to initiate infections, or both conditions. The practical use of ATR+ rhizobia will depend on validation experiments with soil microcosms and on field testing, as well as on the possibility of preserving the physiology of ATR+ bacteria in inoculant formulations.

  11. Selective recruitment of mRNAs and miRNAs to polyribosomes in response to rhizobia infection in Medicago truncatula.

    PubMed

    Reynoso, Mauricio Alberto; Blanco, Flavio Antonio; Bailey-Serres, Julia; Crespi, Martín; Zanetti, María Eugenia

    2013-01-01

    Translation of mRNAs is a key regulatory step that contributes to the coordination and modulation of eukaryotic gene expression during development or adaptation to the environment. mRNA stability or translatability can be regulated by the action of small regulatory RNAs (sRNAs), which control diverse biological processes. Under low nitrogen conditions, leguminous plants associate with soil bacteria and develop a new organ specialized in nitrogen fixation: the nodule. To gain insight into the translational regulation of mRNAs during nodule formation, the association of mRNAs and sRNAs to polysomes was characterized in roots of the model legume Medicago truncatula during the symbiotic interaction with Sinorhizobium meliloti. Quantitative comparison of steady-state and polysomal mRNAs for 15 genes involved in nodulation identified a group of transcripts with slight or no change in total cellular abundance that were significantly upregulated at the level of association with polysomes in response to rhizobia. This group included mRNAs encoding receptors like kinases required either for nodule organogenesis, bacterial infection or both, and transcripts encoding GRAS and NF-Y transcription factors (TFs). Quantitative analysis of sRNAs in total and polysomal RNA samples revealed that mature microRNAs (miRNAs) were associated with the translational machinery, notably, miR169 and miR172, which target the NF-YA/HAP2 and AP2 TFs, respectively. Upon inoculation, levels of miR169 pronouncedly decreased in polysomal complexes, concomitant with the increased accumulation of the NF-YA/HAP2 protein. These results indicate that both mRNAs and miRNAs are subject to differential recruitment to polysomes, and expose the importance of selective mRNA translation during root nodule symbiosis.

  12. The purple acid phosphatase GmPAP21 enhances internal phosphorus utilization and possibly plays a role in symbiosis with rhizobia in soybean.

    PubMed

    Li, Chengchen; Li, Caifeng; Zhang, Haiyan; Liao, Hong; Wang, Xiurong

    2017-02-01

    Induction of secreted and intracellular purple acid phosphatases (PAPs; EC 3.1.3.2) is widely recognized as an adaptation of plants to phosphorus (P) deficiency. The secretion of PAPs plays important roles in P acquisition. However, little is known about the functions of intracellular PAP in plants and nodules. In this study, we identified a novel PAP gene GmPAP21 in soybean. Expression of GmPAP21 was induced by P limitation in nodules, roots and old leaves, and increased in roots with increasing duration of P starvation. Furthermore, the induction of GmPAP21 in nodules and roots was more intensive than in leaves in both P-efficient genotype HN89 and P-inefficient genotype HN112 in response to P starvation, and the relative expression in the leaves and nodules of HN89 was significantly greater than that of HN112 after P deficiency treatment. Further functional analyses showed that over-expressing GmPAP21 significantly enhanced both acid phosphatase activity and growth performance of hairy roots under P starvation condition, indicating that GmPAP21 plays an important role in P utilization. Moreover, GUS expression driven by GmPAP21 promoter was shown in the nodules besides roots. Overexpression of GmPAP21 in transgenic soybean significantly inhibited nodule growth, and thereby affected plant growth after inoculation with rhizobia. This suggests that GmPAP21 is also possibly involved in regulating P metabolism in nodules. Taken together, our results suggest that GmPAP21 is a novel plant PAP that functions in the adaptation of soybean to P starvation, possibly through its involvement in P recycling in plants and P metabolism in nodules. © 2016 Scandinavian Plant Physiology Society.

  13. OxyR-regulated catalase activity is critical for oxidative stress resistance, nodulation and nitrogen fixation in Azorhizobium caulinodans.

    PubMed

    Zhao, Yue; Nickels, Logan M; Wang, Hui; Ling, Jun; Zhong, Zengtao; Zhu, Jun

    2016-07-01

    The legume-rhizobial interaction results in the formation of symbiotic nodules in which rhizobia fix nitrogen. During the process of symbiosis, reactive oxygen species (ROS) are generated. Thus, the response of rhizobia to ROS is important for successful nodulation and nitrogen fixation. In this study, we investigated how Azorhizobium caulinodans, a rhizobium that forms both root and stem nodules on its host plant, regulates ROS resistance. We found that in-frame deletions of a gene encoding the putative catalase-peroxidase katG or a gene encoding a LysR-family regulatory protein, oxyR, exhibited increased sensitivity to H2O2 We then showed that OxyR positively regulated katG expression in an H2O2-independent fashion. Furthermore, we found that deletion of katG or oxyR led to significant reduction in the number of stem nodules and decrease of nitrogen fixation capacities in symbiosis. Our results revealed that KatG and OxyR are not only critical for antioxidant defense in vitro, but also important for nodule formation and nitrogen fixation during interaction with plant hosts. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. Novel players in the AP2-miR172 regulatory network for common bean nodulation.

    PubMed

    Íñiguez, Luis P; Nova-Franco, Bárbara; Hernández, Georgina

    2015-01-01

    The intricate regulatory network for floral organogenesis in plants that includes AP2/ERF, SPL and AGL transcription factors, miR172 and miR156 along with other components is well documented, though its complexity and size keep increasing. The miR172/AP2 node was recently proposed as essential regulator in the legume-rhizobia nitrogen-fixing symbiosis. Research from our group contributed to demonstrate the control of common bean (Phaseolus vulgaris) nodulation by miR172c/AP2-1, however no other components of such regulatory network have been reported. Here we propose AGLs as new protagonists in the regulation of common bean nodulation and discuss the relevance of future deeper analysis of the complex AP2 regulatory network for nodule organogenesis in legumes.

  15. Bioproduction of ascorbic acid in root nodule and root of the legume pulse Phaseolus mungo.

    PubMed

    Ghosh, S; Maiti, T K; Basu, P S

    2008-05-01

    The root nodules of Phaseolus mungo (L.), a herbaceous leguminous pulse, contain high amounts of ascorbic acid (AsA). A glucose pool present in the nodule might serve as precursor for AsA production. From root nodule, a Rhizobium sp. was isolated. The symbiont produced a large amount of AsA (290.5 microg/ml) from glucose-supplemented basal medium. The production of AsA by the symbiont was much greater than that of the control when the glucose (0.5%)-supplemented mineral medium was enriched with thiamine hydrochloride (20 microg/100 ml), biotin (20 microg/100 ml), and L-asparagine (0.2%). The possible role of the rhizobial production of AsA on rhizobia-legume symbiosis is discussed.

  16. Symbiotic diversity, specificity and distribution of rhizobia in native legumes of the Core Cape Subregion (South Africa).

    PubMed

    Lemaire, Benny; Dlodlo, Oscar; Chimphango, Samson; Stirton, Charles; Schrire, Brian; Boatwright, James S; Honnay, Olivier; Smets, Erik; Sprent, Janet; James, Euan K; Muasya, Abraham M

    2015-02-01

    Rhizobial diversity and host preferences were assessed in 65 native Fynbos legumes of the papilionoid legume tribes Astragaleae, Crotalarieae, Genisteae, Indigofereae, Millettieae, Phaseoleae, Podalyrieae, Psoraleeae and Sesbanieae. Sequence analyses of chromosomal 16S rRNA, recA, atpD and symbiosis-related nodA, nifH genes in parallel with immunogold labelling assays identified the symbionts as alpha- (Azorhizobium, Bradyrhizobium, Ensifer, Mesorhizobium and Rhizobium) and beta-rhizobial (Burkholderia) lineages with the majority placed in the genera Mesorhizobium and Burkholderia showing a wide range of host interactions. Despite a degree of symbiotic promiscuity in the tribes Crotalarieae and Indigofereae nodulating with both alpha- and beta-rhizobia, Mesorhizobium symbionts appeared to exhibit a general host preference for the tribe Psoraleeae, whereas Burkholderia prevailed in the Podalyrieae. Although host genotype was the main factor determining rhizobial diversity, ecological factors such as soil acidity and site elevation were positively correlated with genetic variation within Mesorhizobium and Burkholderia, respectively, indicating an interplay of host and environmental factors on the distribution of Fynbos rhizobia.

  17. klavier (klv), a novel hypernodulation mutant of Lotus japonicus affected in vascular tissue organization and floral induction.

    PubMed

    Oka-Kira, Erika; Tateno, Kumiko; Miura, Kin-ichiro; Haga, Tatsuya; Hayashi, Masaki; Harada, Kyuya; Sato, Shusei; Tabata, Satoshi; Shikazono, Naoya; Tanaka, Atsushi; Watanabe, Yuichiro; Fukuhara, Izumi; Nagata, Toshiyuki; Kawaguchi, Masayoshi

    2005-11-01

    A novel hypernodulation mutant line was isolated from Lotus japonicus Miyakojima MG-20 by irradiation with a helium ion beam. This mutant, named klavier (klv), had roots that were densely covered with small nodules. The nodulation zone of klv was significantly wider than that of the wild type. Grafting experiments showed that klv is impaired in the long-distance shoot-to-root autoregulatory mechanism. Thus the shoot genotype was found to be responsible for the negative regulation of nodule development by KLV. Nodulation of klv showed a higher tolerance to nitrogen (KNO3) than the wild type, which is a common feature of hypernodulating mutants. In addition to an increased number of nodules, the klv mutant showed convex leaf veins on the adaxial leaf surface, markedly delayed flowering and dwarf phenotypes. Microscopic examination of the leaf veins revealed that they were discontinuous. Other phenotypes such as fasciated stems, increased number of flowers and bifurcated pistils were also frequently observed in the klv mutant. Among these phenotypes, hypernodulation, aberrant leaf vein formation and significantly delayed flowering were all linked in a monogenic and recessive manner, indicating that these phenotypes are caused by either a single mutation, or tightly linked mutations. KLV was mapped within 0.29 cM on the long arm of chromosome 1.

  18. Nodulation competitiveness of Ensifer meliloti alfalfa nodule isolates and their potential for application as inoculants.

    PubMed

    Marek-Kozaczuk, Monika; Wielbo, Jerzy; Pawlik, Anna; Skorupska, Anna

    2014-01-01

    Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown in arable soil was used as the basis for selection of potential inoculants. Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA, recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed different symbiotic performance and diversity in the number of plasmids and molecular weight. Isolates that were the most efficient in symbiotic nitrogen fixation were tagged with a constitutively expressed gusA gene carried by a stable plasmid vector pJBA21Tc and used in competition experiments in soil under greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively competed with indigenous soil rhizobia, were selected. The metabolic profiles of these selected strains showed differences in the use of carbon and energy sources. In addition, the LU09 strain exhibited bacteriocin production and LU12 mineral phosphate solubilization, which are valuable traits for soil survival. These strains may be considered as potential biofertilizers for alfalfa cultivation.

  19. Characterization of the Bradyrhizobium japonicum galE gene: its impact on lipopolysaccharide profile and nodulation of soybean.

    PubMed

    Chang, Woo-Suk; Park, Kyoung-Min; Koh, Sung-Cheol; So, Jae-Seong

    2008-03-01

    The galE gene from Bradyrhizobium japonicum 61A101C, a soybean endosymbiont, was cloned and characterized. Its deduced amino-acid sequence showed a high similarity with that of other rhizobia. Functional identification of the galE gene was achieved by complementation of a galE mutant strain, PL2, with a series of pKM subclones. Disruption of the B. japonicum galE gene affects the lipopolysaccharide profile compared with that of the wild type, suggesting that galE is responsible for alteration of lipopolysaccharide structure. Examination of nodule formation by the wild-type and galE mutant revealed that the former displayed normal nodule development on soybean roots, whereas the latter showed no nodule formation at all time points examined except for 20 days after inoculation when <10% of soybean formed pseudo-nodules.

  20. Biometamaterials: Black Ultrathin Gold Film Fabricated on Lotus Leaf

    PubMed Central

    Ebihara, Yuusuke; Ota, Ryoichi; Noriki, Takahiro; Shimojo, Masayuki; Kajikawa, Kotaro

    2015-01-01

    We report on a black metamaterial of gold fabricated on a lotus leaf that was used as a template. In spite of the extremely thin gold coating (10-nm thick) on the lotus leaf, the surface shows reflectivity below 0.01 over the entire visible spectral range. Finite-difference time-domain (FDTD) calculations suggest that the low reflectivity stems from the secondary structures on the lotus leaf, where randomly oriented nanorods are distributed. PMID:26530514

  1. Peroxiredoxins and NADPH-dependent thioredoxin systems in the model legume Lotus japonicus.

    PubMed

    Tovar-Méndez, Alejandro; Matamoros, Manuel A; Bustos-Sanmamed, Pilar; Dietz, Karl-Josef; Cejudo, Francisco Javier; Rouhier, Nicolas; Sato, Shusei; Tabata, Satoshi; Becana, Manuel

    2011-07-01

    Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

  2. Rapid Identification of Rhizobia by Restriction Fragment Length Polymorphism Analysis of PCR-Amplified 16S rRNA Genes

    PubMed Central

    Laguerre, Gisèle; Allard, Marie-Reine; Revoy, Françoise; Amarger, Noelle

    1994-01-01

    Forty-eight strains representing the eight recognized Rhizobium species, two new Phaseolus bean Rhizobium genomic species, Bradyrhizobium spp., Agrobacterium spp., and unclassified rhizobia from various host plants were examined by restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes amplified by polymerase chain reaction (PCR). Twenty-one composite genotypes were obtained from the combined data of the RFLP analysis with nine endonucleases. Species assignments were in full agreement with the established taxonomic classification. Estimation from these data of genetic relationships between and within genera and species correlated well with previously published data based on DNA-rRNA hybridizations and sequence analysis of 16S rRNA genes. This PCR-RFLP method provides a rapid tool for the identification of root nodule isolates and the detection of new taxa. Images PMID:16349165

  3. Variation of proanthocyanidins in Lotus species.

    PubMed

    Sivakumaran, Subathira; Rumball, William; Lane, Geoff A; Fraser, Karl; Foo, Lai Y; Yu, Min; Meagher, Lucy P

    2006-08-01

    The proanthocyanidin (PA) chemistry of 12 Lotus species of previously unknown PA content was examined in comparison with agricultural cultivars of L. pedunculatus, L. corniculatus, and L. tenuis and a "creeping" selection of L. corniculatus. Herbage harvested in winter 2000 and again in spring had extractable PA concentrations, estimations of which varied between 0.2 and 10.9% of dry matter. The four novel Lotus spp. with the highest concentrations were selected for further evaluation together with the agricultural accessions. PA concentrations in herbage were estimated for individual plants harvested in spring 2001 and bulk samples harvested in summer 2002-2003. PA oligomer and polymer fractions were separated by Sephadex LH-20 chromatography from aqueous acetone PA extracts of herbage. The chemical characteristics of the fractions were examined by acid catalyzed degradation with benzyl mercaptan, (13)C nuclear magnetic resonance spectroscopy, electrospray ionization (ESI), and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). A wide variation was found in the chemical composition, mean degree of polymerization (mDP), and polydispersity of PAs from Lotus spp. Fractions from L. americanus, L. corniculatus "creeping selection," and L. pedunculatus consisted predominantly of prodelphinidin (PD) units, whereas PA from L. angustissimus and L. corniculatus consisted predominantly of procyanidin (PC) units. An approximately equal composition in terms of PC and PD units was found in L. parviflorus and L. suaveolens. In L. angustissimus, epicatechin is dominant in both extender and terminal units. In all Lotus PA fractions, the 2,3-cis isomers (epicatechin or epigallocatechin) predominated. Only trace amounts of PA were extracted from L. tenuis. The mDP of the PA fractions ranged from 8 to 97, with high mDP found only for L. pedunculatus and L. americanus. In the ESI-MS and MALDI-TOF-MS of the L. angustissimus PA fraction, ions

  4. Diverse genotypes of Bradyrhizobium nodulate herbaceous Chamaecrista (Moench) (Fabaceae, Caesalpinioideae) species in Brazil.

    PubMed

    Santos, José Miguel Ferreira Dos; Casaes Alves, Patricia Alves; Silva, Verônica Cordeiro; Kruschewsky Rhem, Mariana Ferreira; James, Euan K; Gross, Eduardo

    2017-03-01

    The genus Chamaecrista comprises more than 330 species which are mainly distributed across tropical America, especially in Brazil (256 spp.), the main center of radiation. In this study, nodulation of herbaceous Chamaecrista species that are commonly found growing in different vegetation types in the north eastern Brazilian state of Bahia was assessed together with the diversity of rhizobia isolated from their root nodules. Genetic characterization of the isolates was performed using molecular markers to examine the phylogeny of their "core" (16S rRNA, ITS, recA, glnII, dnaK and gyrB) and symbiosis-related (nifH, nodC) genomes. Nodule morphology, anatomy and ultrastructure were also examined, as was the capacity of the isolates to form nodules on Chamaecrista desvauxii and siratro (Macroptilium atropurpureum). Analysis of 16S rRNA gene sequences demonstrated that the isolates belonged to seven clusters within the genus Bradyrhizobium, and more detailed analyses using sequences of the ITS region and concatenated housekeeping genes grouped the Chamaecrista rhizobia by vegetation type and plant species. These analyses also suggested some potentially novel Bradyrhizobium species, which was corroborated by analyses of their nifH and nodC sequences, as these formed separated branches from all Bradyrhizobium type strains. All the 47 strains tested produced effective nodules on C. desvauxii but none on siratro. Chamaecrista nodules are herein described for the first time in detail: they are indeterminate and structurally similar to others described in the Caesalpinioideae, with infection threads in the invasion and nitrogen fixation zones, and with both infected and uninfected (interstitial) cells in the nitrogen fixation zone.

  5. Quantitative trait locus analysis of symbiotic nitrogen fixation activity in the model legume Lotus japonicus.

    PubMed

    Tominaga, Akiyoshi; Gondo, Takahiro; Akashi, Ryo; Zheng, Shao-Hui; Arima, Susumu; Suzuki, Akihiro

    2012-05-01

    Many legumes form nitrogen-fixing root nodules. An elevation of nitrogen fixation in such legumes would have significant implications for plant growth and biomass production in agriculture. To identify the genetic basis for the regulation of nitrogen fixation, quantitative trait locus (QTL) analysis was conducted with recombinant inbred lines derived from the cross Miyakojima MG-20 × Gifu B-129 in the model legume Lotus japonicus. This population was inoculated with Mesorhizobium loti MAFF303099 and grown for 14 days in pods containing vermiculite. Phenotypic data were collected for acetylene reduction activity (ARA) per plant (ARA/P), ARA per nodule weight (ARA/NW), ARA per nodule number (ARA/NN), NN per plant, NW per plant, stem length (SL), SL without inoculation (SLbac-), shoot dry weight without inoculation (SWbac-), root length without inoculation (RLbac-), and root dry weight (RWbac-), and finally 34 QTLs were identified. ARA/P, ARA/NN, NW, and SL showed strong correlations and QTL co-localization, suggesting that several plant characteristics important for symbiotic nitrogen fixation are controlled by the same locus. QTLs for ARA/P, ARA/NN, NW, and SL, co-localized around marker TM0832 on chromosome 4, were also co-localized with previously reported QTLs for seed mass. This is the first report of QTL analysis for symbiotic nitrogen fixation activity traits.

  6. Solitary Pulmonary Nodule

    DTIC Science & Technology

    2010-05-01

    Benign nodules generally fall into two categories making up 90% of all benign lesions. These are infectious granulomas incorporating 80% of benign...nodules and hamartomas comprising 10% of the remainder. Infectious granulomas are generally caused by fungi endemic to the environment or

  7. Genetic diversity of resident soil rhizobia isolated from nodules of distinct hairy vetch genotypes

    USDA-ARS?s Scientific Manuscript database

    Hairy vetch (Vicia villosa Roth) is widely grown as a legume cover crop throughout the U.S.A., with biological nitrogen fixation (BNF) through symbiosis with Rhizobium leguminosarum biovar viciae (Rlv) being one of the most sought after benefits of its cultivation. This study determined if HV culti...

  8. [Pulmonary nodules and arachnophobia].

    PubMed

    Colinet, B; Dargent, J-L; Fremault, A

    2014-01-01

    Pulmonary nodules are a common reason for consultation and their investigation must always exclude a possible neoplastic cause. This means that, in addition to a thorough history, investigations may be necessary which are sometimes invasive and therefore potentially a cause of iatrogenic harm. The toxic aetiologies for pulmonary nodules are rare. We report a case of a patient with pulmonary nodules occurring predominantly in the right lung, about 1cm in diameter, non-cavitating without calcification, and sometimes surrounded by a peripheral halo. The nodules were a chance finding during preoperative evaluation. After a comprehensive review, a reaction to an inhaled irritant was the preferred hypothesis, specifically overuse of a compound insecticide containing, in addition to the propellant gas and solvent type hydrocarbon - a mixture of piperonyl butoxide, of esbiothrine and permethrin. Removal of this led to the complete disappearance of nodules. Pathological examination identified bronchiolitis obliterans with organising pneumonia accompanied by non-necrotizing granulomas and lipid vacuoles.

  9. Air bubble bursting effect of lotus leaf.

    PubMed

    Wang, Jingming; Zheng, Yongmei; Nie, Fu-Qiang; Zhai, Jin; Jiang, Lei

    2009-12-15

    In this paper, a phenomenon of air bubbles quickly bursting within several milliseconds on a "self-cleaning" lotus leaf was described. This observation prompted the synthesis of artificial surfaces similar to that of the lotus leaf. The artificial leaf surfaces, prepared by photolithography and wet etching, showed a similar air bubble bursting effect. Smooth and rough silicon surfaces with an ordered nanostructure or patterned microstructure were utilized to study the contribution of the micro/nano hierarchical structures to this phenomenon of air bubble bursting. Air bubbles were found to burst on some superhydrophobic surfaces with microstructure (within 220 ms). However, air bubbles burst much more rapidly (within 13 ms) on similar surfaces with micro/nanostructure. The height, width, and spacing of hierarchical structures could also affect air bubble bursting, and the effect of the height was more obvious. When the height of hierarchical structures was around the height found in natural lotus papillae, the width and spacing were significant for air bubble bursting. An original model was proposed to further evaluate the reason why the micro/nano hierarchical rough structures had an excellent air bubble bursting effect, and the validity of the model was theoretically demonstrated.

  10. Genome-wide analysis of ATP-binding cassette (ABC) proteins in a model legume plant, Lotus japonicus: comparison with Arabidopsis ABC protein family.

    PubMed

    Sugiyama, Akifumi; Shitan, Nobukazu; Sato, Shusei; Nakamura, Yasukazu; Tabata, Satoshi; Yazaki, Kazufumi

    2006-10-31

    ATP-binding cassette (ABC) proteins constitute a large family in plants with more than 120 members each in Arabidopsis and rice, and have various functions including the transport of auxin and alkaloid, as well as the regulation of stomata movement. In this report, we carried out genome-wide analysis of ABC protein genes in a model legume plant, Lotus japonicus. For analysis of the Lotus genome sequence, we devised a new method 'domain-based clustering analysis', where domain structures like the nucleotide-binding domain (NBD) and transmembrane domain (TMD), instead of full-length amino acid sequences, are used to compare phylogenetically each other. This method enabled us to characterize fragments of ABC proteins, which frequently appear in a draft sequence of the Lotus genome. We identified 91 putative ABC proteins in L. japonicus, i.e. 43 'full-size', 40 'half-size' and 18 'soluble' putative ABC proteins. The characteristic feature of the composition is that Lotus has extraordinarily many paralogs similar to AtMRP14 and AtPDR12, which are at least six and five members, respectively. Expression analysis of the latter genes performed with real-time quantitative reverse transcription-PCR revealed their putative involvement in the nodulation process.

  11. Improved characterization of nod factors and genetically based variation in LysM Receptor domains identify amino acids expendable for nod factor recognition in Lotus spp.

    PubMed

    Bek, Anita S; Sauer, Jørgen; Thygesen, Mikkel B; Duus, Jens Ø; Petersen, Bent O; Thirup, Søren; James, Euan; Jensen, Knud J; Stougaard, Jens; Radutoiu, Simona

    2010-01-01

    Formation of functional nodules is a complex process depending on host-microsymbiont compatibility in all developmental stages. This report uses the contrasting symbiotic phenotypes of Lotus japonicus and L. pedunculatus, inoculated with Mesorhizobium loti or the Bradyrhizobium sp. (Lotus), to investigate the role of Nod factor structure and Nod factor receptors (NFR) for rhizobial recognition, infection thread progression, and bacterial persistence within nodule cells. A key contribution was the use of 800 MHz nuclear magnetic resonance spectroscopy and ultrahigh-performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry for Nod factor analysis. The Nod factor decorations at the nonreducing end differ between Bradyrhizobium sp. (Lotus) and M. loti, and the NFR1/NFR5 extracellular regions of L. pedunculatus and L. japonicus were found to vary in amino acid composition. Genetic transformation experiments using chimeric and wild-type receptors showed that both receptor variants recognize the structurally different Nod factors but the later symbiotic phenotype remained unchanged. These results highlight the importance of additional checkpoints during nitrogen-fixing symbiosis and define several amino acids in the LysM domains as expendable for perception of the two differentially carbamoylated Nod factors.

  12. Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A new promising Plant Probiotic Bacteria.

    PubMed

    Martínez-Hidalgo, Pilar; Galindo-Villardón, Purificación; Trujillo, Martha E; Igual, José M; Martínez-Molina, Eustoquio

    2014-09-17

    Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed.

  13. Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum.

    PubMed

    Zhang, Yu Jing; Zheng, Wen Tao; Everall, Isobel; Young, J Peter W; Zhang, Xiao Xia; Tian, Chang Fu; Sui, Xin Hua; Wang, En Tao; Chen, Wen Xin

    2015-09-01

    Four rhizobia-like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized species of the genus Rhizobium by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequences of the housekeeping genes atpD, recA and glnII for strain CCBAU 23252(T) showed 86.9 to 95% similarity to those of known species of the genus Rhizobium. All four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from members of closely related species. Based on all the evidence, we propose a novel species, Rhizobium anhuiense sp. nov., and designate CCBAU 23252(T) ( = CGMCC 1.12621(T) = LMG 27729(T)) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a DNA G+C content of 61.1 mol% (Tm).

  14. Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system

    PubMed Central

    Okazaki, Shin; Kaneko, Takakazu; Sato, Shusei; Saeki, Kazuhiko

    2013-01-01

    Root–nodule symbiosis between leguminous plants and nitrogen-fixing bacteria (rhizobia) involves molecular communication between the two partners. Key components for the establishment of symbiosis are rhizobium-derived lipochitooligosaccharides (Nod factors; NFs) and their leguminous receptors (NFRs) that initiate nodule development and bacterial entry. Here we demonstrate that the soybean microsymbiont Bradyrhizobium elkanii uses the type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria, to promote symbiosis. Intriguingly, wild-type B. elkanii, but not the T3SS-deficient mutant, was able to form nitrogen-fixing nodules on soybean nfr mutant En1282. Furthermore, even the NF-deficient B. elkanii mutant induced nodules unless T3SS genes were mutated. Transcriptional analysis revealed that expression of the soybean nodulation-specific genes ENOD40 and NIN was increased in the roots of En1282 inoculated with B. elkanii but not with its T3SS mutant, suggesting that T3SS activates host nodulation signaling by bypassing NF recognition. Root-hair curling and infection threads were not observed in the roots of En1282 inoculated with B. elkanii, indicating that T3SS is involved in crack entry or intercellular infection. These findings suggest that B. elkanii has adopted a pathogenic system for activating host symbiosis signaling to promote its infection. PMID:24082124

  15. Micromonospora from nitrogen fixing nodules of alfalfa (Medicago sativa L.). A new promising Plant Probiotic Bacteria.

    PubMed Central

    Martínez-Hidalgo, Pilar; Galindo-Villardón, Purificación; Igual, José M.; Martínez-Molina, Eustoquio

    2014-01-01

    Biotic interactions can improve agricultural productivity without costly and environmentally challenging inputs. Micromonospora strains have recently been reported as natural endophytes of legume nodules but their significance for plant development and productivity has not yet been established. The aim of this study was to determine the diversity and function of Micromonospora isolated from Medicago sativa root nodules. Micromonospora-like strains from field alfalfa nodules were characterized by BOX-PCR fingerprinting and 16S rRNA gene sequencing. The ecological role of the interaction of the 15 selected representative Micromonospora strains was tested in M. sativa. Nodulation, plant growth and nutrition parameters were analyzed. Alfalfa nodules naturally contain abundant and highly diverse populations of Micromonospora, both at the intra- and at interspecific level. Selected Micromonospora isolates significantly increase the nodulation of alfalfa by Ensifer meliloti 1021 and also the efficiency of the plant for nitrogen nutrition. Moreover, they promote aerial growth, the shoot-to-root ratio, and raise the level of essential nutrients. Our results indicate that Micromonospora acts as a Rhizobia Helper Bacteria (RHB) agent and has probiotic effects, promoting plant growth and increasing nutrition efficiency. Its ecological role, biotechnological potential and advantages as a plant probiotic bacterium (PPB) are also discussed. PMID:25227415

  16. Fixating on metals: new insights into the role of metals in nodulation and symbiotic nitrogen fixation

    PubMed Central

    González-Guerrero, Manuel; Matthiadis, Anna; Sáez, Áez;ngela; Long, Terri A.

    2014-01-01

    Symbiotic nitrogen fixation is one of the most promising and immediate alternatives to the overuse of polluting nitrogen fertilizers for improving plant nutrition. At the core of this process are a number of metalloproteins that catalyze and provide energy for the conversion of atmospheric nitrogen to ammonia, eliminate free radicals produced by this process, and create the microaerobic conditions required by these reactions. In legumes, metal cofactors are provided to endosymbiotic rhizobia within root nodule cortical cells. However, low metal bioavailability is prevalent in most soils types, resulting in widespread plant metal deficiency and decreased nitrogen fixation capabilities. As a result, renewed efforts have been undertaken to identify the mechanisms governing metal delivery from soil to the rhizobia, and to determine how metals are used in the nodule and how they are recycled once the nodule is no longer functional. This effort is being aided by improved legume molecular biology tools (genome projects, mutant collections, and transformation methods), in addition to state-of-the-art metal visualization systems. PMID:24592271

  17. Wild peanut Arachis duranensis are nodulated by diverse and novel Bradyrhizobium species in acid soils.

    PubMed

    Chen, Jing Yu; Gu, Jun; Wang, En Tao; Ma, Xing Xian; Kang, Shi Tong; Huang, Ling Zi; Cao, Xue Ping; Li, Liang Bing; Wu, Yan Ling

    2014-10-01

    Aiming at learning the microsymbionts of Arachis duranensis, a diploid ancestor of cultivated peanut, genetic and symbiotic characterization of 32 isolates from root nodules of this plant grown in its new habitat Guangzhou was performed. Based upon the phylogeny of 16S rRNA, atpD and recA genes, diverse bacteria belonging to Bradyrhizobium yuanmingense, Bradyrhizobium elkanii, Bradyrhizobium iriomotense and four new lineages of Bradyrhizobium (19 isolates), Rhizobium/Agrobacterium (9 isolates), Herbaspirillum (2 isolates) and Burkholderia (2 isolates) were defined. In the nodulation test on peanut, only the bradyrhizobial strains were able to induce effective nodules. Phylogeny of nodC divided the Bradyrhizobium isolates into four lineages corresponding to the grouping results in phylogenetic analysis of housekeeping genes, suggesting that this symbiosis gene was mainly maintained by vertical gene transfer. These results demonstrate that A. duranensis is a promiscuous host preferred the Bradyrhizobium species with different symbiotic gene background as microsymbionts, and that it might have selected some native rhizobia, especially the novel lineages Bradyrhizobium sp. I and sp. II, in its new habitat Guangzhou. These findings formed a basis for further study on adaptation and evolution of symbiosis between the introduced legumes and the indigenous rhizobia. Copyright © 2014 Elsevier GmbH. All rights reserved.

  18. Genetic Characterization of Soybean Rhizobia in Paraguay†

    PubMed Central

    Chen, Lu Shi; Figueredo, Antonio; Pedrosa, Fábio O.; Hungria, Mariangela

    2000-01-01

    The soybean is an exotic plant introduced in Paraguay in this century; commercial cropping expanded after the 1970s. Inoculation is practiced in just 15 to 20% of the cropping areas, but root nodulation occurs in most sites where soybeans grow. Little is known about rhizobial diversity in South America, and no study has been performed in Paraguay until this time. Therefore, in this study, the molecular characterization of 78 rhizobial isolates from soybean root nodules, collected under field conditions in 16 sites located in the two main producing states, Alto Paraná and Itapúa, was undertaken. A high level of genetic diversity was detected by an ERIC-REP-PCR analysis, with the majority of the isolates representing unique strains. Most of the 58 isolates characterized by slow growth and alkaline reactions in a medium containing mannitol as a carbon source were clustered with strains representative of the Bradyrhizobium japonicum and Bradyrhizobium elkanii species, and the 16S ribosomal DNA (rDNA) sequences of 5 of those isolates confirmed the species identities. However, slow growers were highly polymorphic in relation to the reference strains, including five carried in commercial inoculants in neighboring countries, thus indicating that the Paraguayan isolates might represent native bradyrhizobia. Twenty isolates highly polymorphic in the ERIC-REP-PCR profiles were characterized by fast growth and acid reactions in vitro, and two of them showed high 16S rDNA identities with Rhizobium genomic species Q. However, two other fast growers showed high 16S rDNA identity with Agrobacterium spp., and both of these strains established efficient symbioses with soybean plants. PMID:11055970

  19. Inoculation of Woody Legumes with Selected Arbuscular Mycorrhizal Fungi and Rhizobia To Recover Desertified Mediterranean Ecosystems

    PubMed Central

    Herrera, M. A.; Salamanca, C. P.; Barea, J. M.

    1993-01-01

    Revegetation strategies, either for reclamation or for rehabilitation, are being used to recover desertified ecosystems. Woody legumes are recognized as species that are useful for revegetation of water-deficient, low-nutrient environments because of their ability to form symbiotic associations with rhizobial bacteria and mycorrhizal fungi, which improve nutrient acquisition and help plants to become established and cope with stress situations. A range of woody legumes used in revegetation programs, particularly in Mediterranean regions, were assayed. These legumes included both exotic and native species and were used in a test of a desertified semiarid ecosystem in southeast Spain. Screening for the appropriate plant species-microsymbiont combinations was performed previously, and a simple procedure to produce plantlets with optimized mycorrhizal and nodulated status was developed. The results of a 4-year trial showed that (i) only the native shrub legumes were able to become established under the local environmental conditions (hence, a reclamation strategy is recommended) and (ii) biotechnological manipulation of the seedlings to be used for revegetation (by inoculation with selected rhizobia and mycorrhizal fungi) improved outplanting performance, plant survival, and biomass development. PMID:16348838

  20. Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps.

    PubMed

    Fröhlich, Jürgen; Koustiane, Christine; Kämpfer, Peter; Rosselló-Mora, Ramón; Valens, Maria; Berchtold, Manfred; Kuhnigk, Thomas; Hertel, Horst; Maheshwari, Dinesh K; König, Helmut

    2007-01-01

    Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC 33499). The DNA-DNA hybridization revealed a similarity of 66% with E. adhaerens (NCIMB12342(T)). In contrast to the type strain the isolate M3A possesses the capacity to nodulate plant roots. This is the first report on the detailed identification of a rhizobia-related strain from the intestinal tract of animals. Strain M3A has been deposited with two culture collections (DSM10169; ATCC BAA-396).

  1. Phytoremediation of heavy and transition metals aided by legume-rhizobia symbiosis.

    PubMed

    Hao, X; Taghavi, S; Xie, P; Orbach, M J; Alwathnani, H A; Rensing, C; Wei, G

    2014-01-01

    Legumes are important for nitrogen cycling in the environment and agriculture due to the ability of nitrogen fixation by rhizobia. In this review, we introduce an important and potential role of legume-rhizobia symbiosis in aiding phytoremediation of some metal contaminated soils as various legumes have been found to be the dominant plant species in metal contaminated areas. Resistant rhizobia used for phytoremediation could act on metals directly by chelation, precipitation, transformation, biosorption and accumulation. Moreover, the plant growth promoting (PGP) traits of rhizobia including nitrogen fixation, phosphorus solubilization, phytohormone synthesis, siderophore release, and production of ACC deaminase and the volatile compounds of acetoin and 2, 3-butanediol may facilitate legume growth while lessening metal toxicity. The benefits of using legumes inoculated with naturally resistant rhizobia or recombinant rhizobia with enhanced resistance, as well as co-inoculation with other plant growth promoting bacteria (PGPB) are discussed. However, the legume-rhizobia symbiosis appears to be sensitive to metals, and the effect of metal toxicity on the interaction between legumes and rhizobia is not clear. Therefore, to obtain the maximum benefits from legumes assisted by rhizobia for phytoremediation of metals, it is critical to have a good understanding of interactions between PGP traits, the symbiotic plant-rhizobia relationship and metals.

  2. Lotus alianus, a new species from Cabo Verde and nomeenclatural notes on Lotus section Pedrosia (Fabaceae)

    USDA-ARS?s Scientific Manuscript database

    Lotus alianus J.H. Kirkbr., sp. nov., is described and illustrated. It is a rare endemic species from the Republic of Cape Verde, and is found in dry habitats on just two islands, Ilhas de Santo Antao and Sao Vicente. In addition, two species names are synonymized with L. creticus L., and a lectotyp...

  3. Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes.

    PubMed

    Granqvist, Emma; Sun, Jongho; Op den Camp, Rik; Pujic, Petar; Hill, Lionel; Normand, Philippe; Morris, Richard J; Downie, J Allan; Geurts, Rene; Oldroyd, Giles E D

    2015-08-01

    Plants that form root-nodule symbioses are within a monophyletic 'nitrogen-fixing' clade and associated signalling processes are shared with the arbuscular mycorrhizal symbiosis. Central to symbiotic signalling are nuclear-associated oscillations in calcium ions (Ca(2+) ), occurring in the root hairs of several legume species in response to the rhizobial Nod factor signal. In this study we expanded the species analysed for activation of Ca(2+) oscillations, including nonleguminous species within the nitrogen-fixing clade. We showed that Ca(2+) oscillations are a common feature of legumes in their association with rhizobia, while Cercis, a non-nodulating legume, does not show Ca(2+) oscillations in response to Nod factors from Sinorhizobium fredii NGR234. Parasponia andersonii, a nonlegume that can associate with rhizobia, showed Nod factor-induced calcium oscillations to S. fredii NGR234 Nod factors, but its non-nodulating sister species, Trema tomentosa, did not. Also within the nitrogen-fixing clade are actinorhizal species that associate with Frankia bacteria and we showed that Alnus glutinosa induces Ca(2+) oscillations in root hairs in response to exudates from Frankia alni, but not to S. fredii NGR234 Nod factors. We conclude that the ability to mount Ca(2+) oscillations in response to symbiotic bacteria is a common feature of nodulating species within the nitrogen-fixing clade.

  4. Calcium-dependent regulation of genes for plant nodulation in Rhizobium leguminosarum detected by iTRAQ quantitative proteomic analysis.

    PubMed

    Arrigoni, Giorgio; Tolin, Serena; Moscatiello, Roberto; Masi, Antonio; Navazio, Lorella; Squartini, Andrea

    2013-11-01

    Rhizobia, the nitrogen-fixing bacterial symbionts of legumes, represent an agricultural application of primary relevance and a model of plant-microbe molecular dialogues. We recently described rhizobium proteome alterations induced by plant flavonoids using iTRAQ. Herein, we further extend that experimentation, proving that the transient elevation in cytosolic calcium is a key signaling event necessary for the expression of the nodulation (nod) genes. Ca(2+) involvement in nodulation is a novel issue that we recently flagged with genetic and physiological approaches and that hereby we demonstrate also by proteomics. Exploiting the multiple combinations of 4-plex iTRAQ, we analyzed Rhizobium leguminosarum cultures grown with or without the nod gene-inducing plant flavonoid naringenin and in the presence or absence of the extracellular Ca(2+) chelator EGTA. We quantified over a thousand proteins, 189 of which significantly altered upon naringenin and/or EGTA stimulation. The expression of NodA, highly induced by naringenin, is strongly reduced when calcium availability is limited by EGTA. This confirms, from a proteomic perspective, that a Ca(2+) influx is a necessary early step in flavonoid-mediated legume nodulation by rhizobia. We also observed other proteins affected by the different treatments, whose identities and roles in nodulation and rhizobium physiology are likewise discussed.

  5. Compatibility of rhizobial genotypes within natural populations of Rhizobium leguminosarum biovar viciae for nodulation of host legumes.

    PubMed

    Laguerre, Gisèle; Louvrier, Philippe; Allard, Marie-Reine; Amarger, Noëlle

    2003-04-01

    Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy.

  6. Compatibility of Rhizobial Genotypes within Natural Populations of Rhizobium leguminosarum Biovar viciae for Nodulation of Host Legumes

    PubMed Central

    Laguerre, Gisèle; Louvrier, Philippe; Allard, Marie-Reine; Amarger, Noëlle

    2003-01-01

    Populations of Rhizobium leguminosarum biovar viciae were sampled from two bulk soils, rhizosphere, and nodules of host legumes, fava bean (Vicia faba) and pea (Pisum sativum) grown in the same soils. Additional populations nodulating peas, fava beans, and vetches (Vicia sativa) grown in other soils and fava bean-nodulating strains from various geographic sites were also analyzed. The rhizobia were characterized by repetitive extragenomic palindromic-PCR fingerprinting and/or PCR-restriction fragment length polymorphism (RFLP) of 16S-23S ribosomal DNA intergenic spacers as markers of the genomic background and PCR-RFLP of a nodulation gene region, nodD, as a marker of the symbiotic component of the genome. Pairwise comparisons showed differences among the genetic structures of the bulk soil, rhizosphere, and nodule populations and in the degree of host specificity within the Vicieae cross-inoculation group. With fava bean, the symbiotic genotype appeared to be the preponderant determinant of the success in nodule occupancy of rhizobial genotypes independently of the associated genomic background, the plant genotype, and the soil sampled. The interaction between one particular rhizobial symbiotic genotype and fava bean seems to be highly specific for nodulation and linked to the efficiency of nitrogen fixation. By contrast with bulk soil and fava bean-nodulating populations, the analysis of pea-nodulating populations showed preferential associations between genomic backgrounds and symbiotic genotypes. Both components of the rhizobial genome may influence competitiveness for nodulation of pea, and rhizosphere colonization may be a decisive step in competition for nodule occupancy. PMID:12676710

  7. Phylogeny based on 16S rDNA and nifH sequences of Ralstonia taiwanensis strains isolated from nitrogen-fixing nodules of Mimosa pudica, in India.

    PubMed

    Verma, Subhash Chandra; Chowdhury, Soumitra Paul; Tripathi, Anil Kumar

    2004-05-01

    Bacterial symbionts present in the indeterminate-type nitrogen (N)-fixing nodules of Mimosa pudica grown in North and South India showed maximum similarity to Ralstonia taiwanensis on the basis of carbon-source utilization patterns and 16S rDNA sequence. Isolates from the nodules of M. pudica from North India and South India showed identical ARDRA (Amplified Ribosomal DNA Restriction Analysis) patterns with Sau3AI and RsaI, but AluI revealed dimorphy between the North Indian and South Indian isolates. Alignment of 16S rDNA sequences revealed similarity of North Indian isolates with an R. taiwanensis strain isolated from M. pudica in Taiwan, whereas South Indian isolates showed closer relatedness with the isolates from Mimosa diplotricha. Alignment of nifH sequences from both North Indian and South Indian isolates with that of the related isolates revealed their closer affinity to alpha-rhizobia, suggesting that nif genes in the beta-rhizobia might have been acquired from alpha-rhizobia via lateral transfer during co-occupancy of nodules by alpha-rhizobia and progenitors of R. taiwanensis, members of the beta-subclass of Proteobacteria. Immunological cross-reaction of the bacteroid preparation of M. pudica nodules showed strong a positive signal with anti-dinitrogenase reductase antibody, whereas a weak positive cross-reaction was observed with free-living R. taiwanensis grown microaerobically in minimal medium with and without NH4Cl. In spite of the expression of dinitrogenase reductase under free-living conditions, acetylene reduction was not observed under N-free conditions even after prolonged incubation.

  8. Rapid In Situ Evolution of Nodulating Strains for Biserrula pelecinus L. through Lateral Transfer of a Symbiosis Island from the Original Mesorhizobial Inoculant▿

    PubMed Central

    Nandasena, Kemanthi G.; O'Hara, Graham W.; Tiwari, Ravi P.; Howieson, John G.

    2006-01-01

    Diverse rhizobia able to nodulate Biserrula pelecinus evolved following in situ transfer of nodA and nifH from an inoculant to soil bacteria. Transfer of these chromosomal genes and the presence of an identical integrase gene adjacent to a Phe tRNA gene in both the inoculant and recipients indicate that there was lateral transfer of a symbiosis island. PMID:16936054

  9. Ureide assay for measuring nitrogen fixation by nodulated soybean calibrated by sup 15 N methods. [Glycine max

    SciTech Connect

    Herridge, D.F. ); Peoples, M.B. )

    1990-06-01

    We report experiments to quantify the relationships between the relative abundance of ureide-N in root-bleeding sap, vacuum-extracted sap, and hot water extracts of stems and petioles of nodulated soybean (Glycine max (L.) Merrill cv Bragg) and the proportion of plant N derived from nitrogen fixation. Additional experiments examined the effects of plant genotype and strain of rhizobia on these relationships. In each of the five experiments reported, plants of cv Bragg (experiment 1), cv Lincoln (experiments 3, 4, 5), or six cultivars/genotypes (experiment 2) were grown in a sand:vermiculite mixture in large pots in a naturally lit, temperature-controlled glasshouse during summer. Pots were inoculated at sowing with effective Bradyrhizobium japonicum CB 1809 (USDA 136) or with one of 21 different strains of rhizobia. The proportions of plant N derived from nitrogen fixation were determined using {sup 15}N dilution. Results show that assessment of nitrogen fixation by soybean using the ureide technique should now be possible with the standard curves presented, irrespective of genotype or strain of rhizobia occupying the nodules.

  10. 78 FR 39062 - Group Lotus plc; Modification of a Temporary Exemption From an Advanced Air Bag Requirement of...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-06-28

    ... National Highway Traffic Safety Administration Group Lotus plc; Modification of a Temporary Exemption From... Protection. SUMMARY: This notice modifies the temporary exemption granted to Group Lotus plc (Lotus) on March... in the Federal Register a notice granting Group Lotus Plc (Lotus) a temporary exemption from...

  11. Genetic diversity and phylogeny of rhizobia isolated from Caragana microphylla growing in desert soil in Ningxia, China.

    PubMed

    Dai, J; Liu, X; Wang, Y

    2012-08-16

    Rhizobia are soil bacteria with the capacity to induce nitrogen-fixing nodules on the roots or stems of legume plants. A total of 40 bacterial isolates from the root nodules of Caragana microphylla growing in desert soil in Ningxia, China, were analyzed for genetic diversity and phylogenetic position. These isolates were classified into 7 types of 16S ribosomal DNA (rDNA) using polymerase chain reaction-restriction fragment length polymorphism analysis. They were grouped into 4 clades, Rhizobium-Agrobacterium, Sinorhizobium, Phyllobacterium, and Bradyrhizobium, when the phylogenies of 16S rDNA, recA, and atpD genes were applied. Phylogenetic analysis showed that the tree generated from the 16S rDNA sequencing agreed with that produced from the recA and atpD genes. By analyzing phylogenetic relationship using the 3 loci, the isolates in the branches of Phyllobacterium and Sinorhizobium could be identified as P. brassicacearum and S. meliloti. The isolates in the branch of Rhizobium-Agrobacterium were the most abundant microsymbiont of C. microphylla and were designated R. leguminosarum, R. galegae, R. alamii, and A. tumefaciens. Two isolates with low sequence similarity to the known species of Bradyrhizobium might be novel species in this genus.

  12. Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis.

    PubMed

    Laloum, Tom; Baudin, Maël; Frances, Lisa; Lepage, Agnes; Billault-Penneteau, Benjamin; Cerri, Marion R; Ariel, Federico; Jardinaud, Marie-Françoise; Gamas, Pascal; de Carvalho-Niebel, Fernanda; Niebel, Andreas

    2014-09-01

    During endosymbiotic interactions between legume plants and nitrogen-fixing rhizobia, successful root infection by bacteria and nodule organogenesis requires the perception and transduction of bacterial lipo-chitooligosaccharidic signal called Nod factor (NF). NF perception in legume roots leads to the activation of an early signaling pathway and of a set of symbiotic genes which is controlled by specific early transcription factors (TFs) including CYCLOPS/IPD3, NSP1, NSP2, ERN1 and NIN. In this study, we bring convincing evidence that the Medicago truncatula CCAAT-box-binding NF-YA1 TF, previously associated with later stages of rhizobial infection and nodule meristem formation is, together with its closest homolog NF-YA2, also an essential positive regulator of the NF-signaling pathway. Here we show that NF-YA1 and NF-YA2 are both expressed in epidermal cells responding to NFs and their knock-down by reverse genetic approaches severely affects the NF-induced expression of symbiotic genes and rhizobial infection. Further over-expression, transactivation and ChIP-PCR approaches indicate that NF-YA1 and NF-YA2 function, at least in part, via the direct activation of ERN1. We thus propose a model in which NF-YA1 and NF-YA2 appear as early symbiotic regulators acting downstream of DMI3 and NIN and possibly within the same regulatory complexes as NSP1/2 to directly activate the expression of ERN1.

  13. Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

    PubMed

    Abdel-Lateif, Khalid; Vaissayre, Virginie; Gherbi, Hassen; Verries, Clotilde; Meudec, Emmanuelle; Perrine-Walker, Francine; Cheynier, Véronique; Svistoonoff, Sergio; Franche, Claudine; Bogusz, Didier; Hocher, Valérie

    2013-09-01

    Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation. No claim to original French goverment works New Phytologist © 2013 New Phytologist Trust.

  14. Rhizobial Factors Required for Stem Nodule Maturation and Maintenance in Sesbania rostrata-Azorhizobium caulinodans ORS571 Symbiosis▿ †

    PubMed Central

    Suzuki, Shino; Aono, Toshihiro; Lee, Kyung-Bum; Suzuki, Tadahiro; Liu, Chi-Te; Miwa, Hiroki; Wakao, Seiji; Iki, Taichiro; Oyaizu, Hiroshi

    2007-01-01

    The molecular and physiological mechanisms behind the maturation and maintenance of N2-fixing nodules during development of symbiosis between rhizobia and legumes still remain unclear, although the early events of symbiosis are relatively well understood. Azorhizobium caulinodans ORS571 is a microsymbiont of the tropical legume Sesbania rostrata, forming N2-fixing nodules not only on the roots but also on the stems. In this study, 10,080 transposon-inserted mutants of A. caulinodans ORS571 were individually inoculated onto the stems of S. rostrata, and those mutants that induced ineffective stem nodules, as displayed by halted development at various stages, were selected. From repeated observations on stem nodulation, 108 Tn5 mutants were selected and categorized into seven nodulation types based on size and N2 fixation activity. Tn5 insertions of some mutants were found in the well-known nodulation, nitrogen fixation, and symbiosis-related genes, such as nod, nif, and fix, respectively, lipopolysaccharide synthesis-related genes, C4 metabolism-related genes, and so on. However, other genes have not been reported to have roles in legume-rhizobium symbiosis. The list of newly identified symbiosis-related genes will present clues to aid in understanding the maturation and maintenance mechanisms of nodules. PMID:17720818

  15. Nodule-Specific Polypeptides from Effective Alfalfa Root Nodules and from Ineffective Nodules Lacking Nitrogenase 1

    PubMed Central

    Lang-Unnasch, Naomi; Ausubel, Frederick M.

    1985-01-01

    In addition to leghemoglobin, at least nine nodule-specific polypeptides from the alfalfa (Medicago sativa L.)-Rhizobium meliloti symbiosis were identified by immune assay. Some of these polypeptides may be subunits of larger proteins but none appeared to be subunits of the same multimeric protein. All nine of the nodule-specific polypeptides were localized to within the plant cytosol; they were not found in extracts of bacteroids or in the peribacteroid space. At least one of these nodule-specific polypeptides was found to be antigenically related to nodule-specific polypeptides in pea and/or soybean. Ineffective nodules elicited by R. meliloti strains containing mutations in four different genes required for nitrogenase synthesis contained reduced concentrations of leghemoglobin and of several of the nodule-specific polypeptides. Other nodule-specific polypeptides were unaltered or actually enriched in the ineffective nodules. Many of the differences between the ineffective and effective nodules were apparent in nodules harvested shortly after the nodules became visible. These differences were greatly amplified in older nodules. When the four ineffective nodule types were compared to one another, there were clear quantitative differences in the concentrations of several of the nodule-specific polypeptides. These differences suggest that lack of a functional nitrogenase does not have a single direct effect on nodule development. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:16664146

  16. Inducing salt tolerance in mung bean through coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase.

    PubMed

    Ahmad, Maqshoof; Zahir, Zahir A; Asghar, H Naeem; Asghar, M

    2011-07-01

    Twenty-five strains of plant-growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and 10 strains of rhizobia were isolated from rhizosphere soil samples and nodules of mung bean. They were screened in separate trials under salt-stressed axenic conditions. The three most effective strains of PGPR (Mk1, Pseudomonas syringae ; Mk20, Pseudomonas fluorescens ; and Mk25, Pseudomonas fluorescens biotype G) and Rhizobium phaseoli strains M1, M6, and M9 were evaluated in coinoculation for their growth-promoting activity at three salinity levels (original, 4 dS·m(-1), and 6 dS·m(-1)) under axenic conditions. The results showed that salinity stress significantly reduced plant growth but inoculation with PGPR containing ACC deaminase and rhizobia enhanced plant growth, thus reducing the inhibitory effect of salinity. However, their combined application was more effective under saline conditions, and the combination Mk20 × M6 was the most efficient for improving seedling growth and nodulation. The effect of high ethylene concentrations on plant growth and the performance of these strains for reducing the negative impact of saline stress was also evaluated by conducting a classical triple-response bioassay. The intensity of the classical triple response decreased owing to inoculation with these strains, with the root and shoot lengths of inoculated mung bean seedlings increasing and stem diameter decreasing, which is a typical response to the dilution in a classical triple response bioassay. Thus, coinoculation with PGPR containing ACC deaminase and Rhizobium spp. could be a useful approach for inducing salt tolerance and thus improving growth and nodulation in mung bean under salt-affected conditions.

  17. Superhydrophobicity in perfection: the outstanding properties of the lotus leaf

    PubMed Central

    Ditsche-Kuru, Petra; Neinhuis, Christoph; Barthlott, Wilhelm

    2011-01-01

    Summary Lotus leaves have become an icon for superhydrophobicity and self-cleaning surfaces, and have led to the concept of the ‘Lotus effect’. Although many other plants have superhydrophobic surfaces with almost similar contact angles, the lotus shows better stability and perfection of its water repellency. Here, we compare the relevant properties such as the micro- and nano-structure, the chemical composition of the waxes and the mechanical properties of lotus with its competitors. It soon becomes obvious that the upper epidermis of the lotus leaf has developed some unrivaled optimizations. The extraordinary shape and the density of the papillae are the basis for the extremely reduced contact area between surface and water drops. The exceptional dense layer of very small epicuticular wax tubules is a result of their unique chemical composition. The mechanical robustness of the papillae and the wax tubules reduce damage and are the basis for the perfection and durability of the water repellency. A reason for the optimization, particularly of the upper side of the lotus leaf, can be deduced from the fact that the stomata are located in the upper epidermis. Here, the impact of rain and contamination is higher than on the lower epidermis. The lotus plant has successfully developed an excellent protection for this delicate epistomatic surface of its leaves. PMID:21977427

  18. Superhydrophobicity in perfection: the outstanding properties of the lotus leaf.

    PubMed

    Ensikat, Hans J; Ditsche-Kuru, Petra; Neinhuis, Christoph; Barthlott, Wilhelm

    2011-01-01

    Lotus leaves have become an icon for superhydrophobicity and self-cleaning surfaces, and have led to the concept of the 'Lotus effect'. Although many other plants have superhydrophobic surfaces with almost similar contact angles, the lotus shows better stability and perfection of its water repellency. Here, we compare the relevant properties such as the micro- and nano-structure, the chemical composition of the waxes and the mechanical properties of lotus with its competitors. It soon becomes obvious that the upper epidermis of the lotus leaf has developed some unrivaled optimizations. The extraordinary shape and the density of the papillae are the basis for the extremely reduced contact area between surface and water drops. The exceptional dense layer of very small epicuticular wax tubules is a result of their unique chemical composition. The mechanical robustness of the papillae and the wax tubules reduce damage and are the basis for the perfection and durability of the water repellency. A reason for the optimization, particularly of the upper side of the lotus leaf, can be deduced from the fact that the stomata are located in the upper epidermis. Here, the impact of rain and contamination is higher than on the lower epidermis. The lotus plant has successfully developed an excellent protection for this delicate epistomatic surface of its leaves.

  19. Trifolitoxin Production Increases Nodulation Competitiveness of Rhizobium etli CE3 under Agricultural Conditions

    PubMed Central

    Robleto, Eduardo A.; Kmiecik, Kenneth; Oplinger, Edward S.; Nienhuis, James; Triplett, Eric W.

    1998-01-01

    A major barrier to the use of nitrogen-fixing inoculum strains for the enhancement of legume productivity is the inability of commercially available strains to compete with indigenous rhizobia for nodule formation. Despite extensive research on nodulation competitiveness, there are no examples of field efficacy studies of strains that have been genetically improved for nodulation competitiveness. We have shown previously that production of the peptide antibiotic trifolitoxin (TFX) by Rhizobium etli results in significantly increased nodule occupancy values in nonsterile soil in growth chamber experiments (E. A. Robleto, A. J. Scupham, and E. W. Triplett, Mol. Plant-Microbe Interact. 10:228–233, 1997). To determine whether TFX production by Rhizobium etli increases nodulation competitiveness in field-grown plants, seeds of Phaseolus vulgaris were inoculated with mixtures of Rhizobium etli strains at different ratios. The three nearly isogenic inoculum strains used included TFX-producing and non-TFX-producing strains, as well as a TFX-sensitive reference strain. Data was obtained over 2 years for nodule occupancy and over 3 years for assessment of the effect of the TFX production phenotype on grain yield. In comparable mixtures in which the test strain accounted for between 5 and 50% of the inoculum, the TFX-producing strain exhibited at least 20% greater nodule occupancy than the non-TFX-producing strain in both years. The TFX production phenotype had no effect on grain yield over 3 years; the average yields reached 2,400 kg/ha. These results show that addition of the TFX production phenotype significantly increases nodule occupancy under field conditions without adverse effects on grain yield. As we used common inoculation methods in this work, there are no practical barriers to the commercial adoption of the TFX system for agriculture. PMID:9647840

  20. Phylogenetic relationships of rhizobia based on citrate synthase gene sequences.

    PubMed

    Hernández-Lucas, Ismael; Rogel-Hernández, Marco Antonio; Segovia, Lorenzo; Rojas-Jiménez, Keilor; Martínez-Romero, Esperanza

    2004-11-01

    Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined. Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences. However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa. We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

  1. Nod factor supply under water stress conditions modulates cytokinin biosynthesis and enhances nodule formation and N nutrition in soybean.

    PubMed

    Prudent, Marion; Salon, Christophe; Smith, Donald L; Emery, R J Neil

    2016-09-01

    Nod factors (NF) are molecules produced by rhizobia which are involved in the N2-fixing symbiosis with legume plants, enabling the formation of specific organs called nodules. Under drought conditions, nitrogen acquisition by N2-fixation is depressed, resulting in low legume productivity. In this study, we evaluated the effects of NF supply on nitrogen acquisition and on cytokinin biosynthesis of soybean plants grown under drought. NF supply to water stressed soybeans increased the CK content of all organs. The profile of CK metabolites also shifted from t-Z to cis-Z and an accumulation of nucleotide and glucoside conjugates. The changes in CK coincided with enhanced nodule formation with sustained nodule specific activity, which ultimately increased the total nitrogen fixed by the plant.

  2. Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula

    PubMed Central

    van Noorden, Giel E.; Verbeek, Rob; Dinh, Quy Dung; Jin, Jian; Green, Alexandra; Ng, Jason Liang Pin; Mathesius, Ulrike

    2016-01-01

    The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes. PMID:27384556

  3. Molecular Signals Controlling the Inhibition of Nodulation by Nitrate in Medicago truncatula.

    PubMed

    van Noorden, Giel E; Verbeek, Rob; Dinh, Quy Dung; Jin, Jian; Green, Alexandra; Ng, Jason Liang Pin; Mathesius, Ulrike

    2016-07-02

    The presence of nitrogen inhibits legume nodule formation, but the mechanism of this inhibition is poorly understood. We found that 2.5 mM nitrate and above significantly inhibited nodule initiation but not root hair curling in Medicago trunatula. We analyzed protein abundance in M. truncatula roots after treatment with either 0 or 2.5 mM nitrate in the presence or absence of its symbiont Sinorhizobium meliloti after 1, 2 and 5 days following inoculation. Two-dimensional gel electrophoresis combined with mass spectrometry was used to identify 106 differentially accumulated proteins responding to nitrate addition, inoculation or time point. While flavonoid-related proteins were less abundant in the presence of nitrate, addition of Nod gene-inducing flavonoids to the Sinorhizobium culture did not rescue nodulation. Accumulation of auxin in response to rhizobia, which is also controlled by flavonoids, still occurred in the presence of nitrate, but did not localize to a nodule initiation site. Several of the changes included defense- and redox-related proteins, and visualization of reactive oxygen species indicated that their induction in root hairs following Sinorhizobium inoculation was inhibited by nitrate. In summary, the presence of nitrate appears to inhibit nodulation via multiple pathways, including changes to flavonoid metabolism, defense responses and redox changes.

  4. Multiple nodulation genes are up-regulated during establishment of reniform nematode feeding sites in soybean.

    PubMed

    Redding, Nathan Wayne; Agudelo, Paula; Wells, Christina E

    2017-09-15

    The semi-endoparastic reniform nematode (Rotylenchulus reniformis) infects over 300 plant species. Females penetrate host roots and induce formation of complex, multinucleate feeding sites called syncytia. While anatomical changes associated with reniform nematode infection are well documented, little is known about their molecular basis. We grew soybean (Glycine max) in a split-root growth system, inoculated half of each root system with R. reniformis, and quantified gene expression in infected and control root tissue at four dates after inoculation. Over 6,000 genes were differentially expressed between inoculated and control roots on at least one date (FDR = 0.01, |log2FC| ≥ 1), and 507 gene sets were significantly enriched or depleted in inoculated roots (FDR = 0.05). Numerous genes up-regulated during syncytium formation had previously been associated with rhizobia nodulation. These included the nodule-initiating transcription factors CYCLOPS, NSP1, NSP2, and NIN, as well as multiple nodulins associated with the plant-derived peribacteroid membrane. Nodulation-related NIP aquaporins and SWEET sugar transporters were induced, as were plant CLAVATA3/ESR-related (CLE) signaling proteins and cell cycle regulators such as CCS52A and E2F. Nodulins and nodule-associated genes may have ancestral functions in normal root development and mycorrhization that have been co-opted by both parasitic nematodes and rhizobial bacteria to promote feeding site and nodule formation.

  5. Development of molecular tools to monitor conjugative transfer in rhizobia.

    PubMed

    Tejerizo, Gonzalo Torres; Bañuelos, Luis Alfredo; Cervantes, Laura; Gaytán, Paul; Pistorio, Mariano; Romero, David; Brom, Susana

    2015-10-01

    Evolution of bacterial populations has been extensively driven by horizontal transfer events. Conjugative plasmid transfer is considered the principal contributor to gene exchange among bacteria. Several conjugative and mobilizable plasmids have been identified in rhizobia, and two major molecular mechanisms that regulate their transfer have been described, under laboratory conditions. The knowledge of rhizobial plasmid transfer regulation in natural environments is very poor. In this work we developed molecular tools to easily monitor the conjugative plasmid transfer in rhizobia by flow cytometry (FC) or microscopy. 24 cassettes were constructed by combining a variety of promotors, fluorescent proteins and antibiotic resistance genes, and used to tag plasmids and chromosome of donor strains. We were able to detect plasmid transfer after conversion of non-fluorescent recipients into fluorescent transconjugants. Flow cytometry (FC) was optimized to count donor, recipient and transconjugant strains to determine conjugative transfer frequencies. Results were similar, when determined either by FC or by viable counts. Our constructions also allowed the visualization of transconjugants in crosses performed on bean roots. The tools presented here may also be used for other purposes, such as analysis of transcriptional fusions or single-cell tagging. Application of the system will allow the survey of how different environmental conditions or other regulators modulate plasmid transfer in rhizobia. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Growth of Fast- and Slow-Growing Rhizobia on Ethanol

    PubMed Central

    Sadowsky, Michael J.; Bohlool, B. Ben

    1986-01-01

    Free-living soybean rhizobia and Bradyrhizobium spp. (lupine) have the ability to catabolize ethanol. Of the 30 strains of rhizobia examined, only the fast- and slow-growing soybean rhizobia and the slow-growing Bradyrhizobium sp. (lupine) were capable of using ethanol as a sole source of carbon and energy for growth. Two strains from each of the other Rhizobium species examined (R. meliloti, R. loti, and R. leguminosarum biovars phaseoli, trifolii, and viceae) failed to grow on ethanol. One Rhizobium fredii (fast-growing) strain, USDA 191, and one (slow-growing) Bradyrhizobium japonicum strain, USDA 110, grew in ethanol up to concentrations of 3.0 and 1.0%, respectively. While three of the R. fredii strains examined (USDA 192, USDA 194, and USDA 205) utilized 0.2% acetate, only USDA 192 utilized 0.1% n-propanol. None of the three strains utilized 0.1% methanol, formate, or n-butanol as the sole carbon source. PMID:16347190

  7. Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development.

    PubMed

    Groth, Martin; Kosuta, Sonja; Gutjahr, Caroline; Haage, Kristina; Hardel, Simone Liesel; Schaub, Miriam; Brachmann, Andreas; Sato, Shusei; Tabata, Satoshi; Findlay, Kim; Wang, Trevor L; Parniske, Martin

    2013-07-01

    Arbuscular mycorrhiza (AM) fungi form nutrient-acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM-defective mutants via a microscopic screen of an ethyl methanesulfonate-mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild-type-like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

  8. Genome sequence of the Lotus corniculatus microsymbiont Mesorhizobium loti strain R88B.

    PubMed

    Reeve, Wayne; Sullivan, John; Ronson, Clive; Tian, Rui; Bräu, Lambert; Davenport, Karen; Goodwin, Lynne; Chain, Patrick; Woyke, Tanja; Lobos, Elizabeth; Huntemann, Marcel; Pati, Amrita; Mavromatis, Konstantinos; Markowitz, Victor; Ivanova, Natalia; Kyrpides, Nikos

    2014-01-01

    Mesorhizobium loti strain R88B was isolated in 1993 in the Rocklands range in Otago, New Zealand from a Lotus corniculatus root nodule. R88B is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain R88B contains a single scaffold of size 7,195,110 bp which encodes 6,950 protein-coding genes and 66 RNA-only encoding genes. This genome does not harbor any plasmids but contains the integrative and conjugative element ICEMlSym(R7A), also known as the R7A symbiosis island, acquired by horizontal gene transfer in the field environment from M. loti strain R7A. It also contains a mobilizable genetic element ICEMladh(R88B), that encodes a likely adhesin gene which has integrated downstream of ICEMlSym(R7A), and three acquired loci that together allow the utilization of the siderophore ferrichrome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project.

  9. Genome sequence of the Lotus corniculatus microsymbiont Mesorhizobium loti strain R88B

    PubMed Central

    2014-01-01

    Mesorhizobium loti strain R88B was isolated in 1993 in the Rocklands range in Otago, New Zealand from a Lotus corniculatus root nodule. R88B is an aerobic, Gram-negative, non-spore-forming rod. This report reveals the genome of M. loti strain R88B contains a single scaffold of size 7,195,110 bp which encodes 6,950 protein-coding genes and 66 RNA-only encoding genes. This genome does not harbor any plasmids but contains the integrative and conjugative element ICEMlSymR7A, also known as the R7A symbiosis island, acquired by horizontal gene transfer in the field environment from M. loti strain R7A. It also contains a mobilizable genetic element ICEMladhR88B, that encodes a likely adhesin gene which has integrated downstream of ICEMlSymR7A, and three acquired loci that together allow the utilization of the siderophore ferrichrome. This rhizobial genome is one of 100 sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project. PMID:25780496

  10. Transient Nod factor-dependent gene expression in the nodulation-competent zone of soybean (Glycine max [L.] Merr.) roots.

    PubMed

    Hayashi, Satomi; Reid, Dugald E; Lorenc, Michał T; Stiller, Jiri; Edwards, David; Gresshoff, Peter M; Ferguson, Brett J

    2012-10-01

    All lateral organ development in plants, such as nodulation in legumes, requires the temporal and spatial regulation of genes and gene networks. A total mRNA profiling approach using RNA-seq to target the specific soybean (Glycine max) root tissues responding to compatible rhizobia [i.e. the Zone Of Nodulation (ZON)] revealed a large number of novel, often transient, mRNA changes occurring during the early stages of nodulation. Focusing on the ZON enabled us to discard the majority of root tissues and their developmentally diverse gene transcripts, thereby highlighting the lowly and transiently expressed nodulation-specific genes. It also enabled us to concentrate on a precise moment in early nodule development at each sampling time. We focused on discovering genes regulated specifically by the Bradyrhizobium-produced Nod factor signal, by inoculating roots with either a competent wild-type or incompetent mutant (nodC(-) ) strain of Bradyrhizobium japonicum. Collectively, 2915 genes were identified as being differentially expressed, including many known soybean nodulation genes. A number of unknown nodulation gene candidates and soybean orthologues of nodulation genes previously reported in other legume species were also identified. The differential expression of several candidates was confirmed and further characterized via inoculation time-course studies and qRT-PCR. The expression of many genes, including an endo-1,4-β-glucanase, a cytochrome P450 and a TIR-LRR-NBS receptor kinase, was transient, peaking quickly during the initiation of nodule ontogeny. Additional genes were found to be down-regulated. Significantly, a set of differentially regulated genes acting in the gibberellic acid (GA) biosynthesis pathway was discovered, suggesting a novel role of GAs in nodulation. © 2012 The Authors. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  11. A conserved α-proteobacterial small RNA contributes to osmoadaptation and symbiotic efficiency of rhizobia on legume roots.

    PubMed

    Robledo, Marta; Peregrina, Alexandra; Millán, Vicenta; García-Tomsig, Natalia I; Torres-Quesada, Omar; Mateos, Pedro F; Becker, Anke; Jiménez-Zurdo, José I

    2017-07-01

    Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we provide primary insights into the function and activity mechanism of the Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA). Northern blot probing and transcription tracking with fluorescent promoter-reporter fusions unveiled high nfeR1 expression in response to salt stress and throughout the symbiotic interaction. The strength and differential regulation of nfeR1 transcription are conferred by a motif, which is conserved in nfeR1 promoter regions in α-proteobacteria. NfeR1 loss-of-function compromised osmoadaptation of free-living bacteria, whilst causing misregulation of salt-responsive genes related to stress adaptation, osmolytes catabolism and membrane trafficking. Nodulation tests revealed that lack of NfeR1 affected competitiveness, infectivity, nodule development and symbiotic efficiency of S. meliloti on alfalfa roots. Comparative computer predictions and a genetic reporter assay evidenced a redundant role of three identical NfeR1 unpaired anti Shine-Dalgarno motifs for targeting and downregulation of translation of multiple mRNAs from transporter genes. Our data provide genetic evidence of the hyperosmotic conditions of the endosymbiotic compartments. NfeR1-mediated gene regulation in response to this cue could contribute to coordinate nutrient uptake with the metabolic reprogramming concomitant to symbiotic transitions. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. Rhizobial position as a main determinant in the problem of competition for nodulation in soybean.

    PubMed

    López-García, Silvina L; Vázquez, Tirso E E; Favelukes, Gabriel; Lodeiro, Aníbal R

    2002-04-01

    Selected Bradyrhizobium japonicum strains inoculated on soybean seeds often fail to occupy a significant proportion of nodules when a competitor rhizobial population is established in the soil. This competition problem could result from a genetic/ physiological advantage of the adapted soil population over the introduced inoculant or from a positional advantage, as the soil population already occupies the soil profile where the roots will penetrate, whereas the inoculant remains concentrated around the seeds. Here, we have assessed the contribution of these factors with a laboratory model in which a rhizobial population is established in sterile vermiculite. We observed that the wild-type strain B. japonicum LP 3004 was able to grow in pots with N-free plant nutrient solution-watered vermiculite for six or seven generations with a duplication rate of at least 0.7 day(-1). In addition, the rhizobial population persisted for 3 months with 10(6)-10(7) colony-forming units ml(-1) of the vermiculite-retained solution. N-starved, young rhizobial cultures are more efficient in performing severa