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Evidence for the endophytic colonization of Phaseolus vulgaris(common bean) roots by the diazotroph Herbaspirillum seropedicae
Schmidt, M.A.;Souza, E.M.;Baura, V.;Wassem, R.;Yates, M.G.;Pedrosa, F.O.;Monteiro, R.A.;
Brazilian Journal of Medical and Biological Research , 2011, DOI: 10.1590/S0100-879X2011007500004
Abstract: herbaspirillum seropedicae is an endophytic diazotrophic bacterium, which associates with important agricultural plants. in the present study, we have investigated the attachment to and internal colonization of phaseolus vulgaris roots by the h. seropedicae wild-type strain smr1 and by a strain of h. seropedicae expressing a red fluorescent protein (dsred) to track the bacterium in the plant tissues. two-day-old p. vulgaris roots were incubated at 30°c for 15 min with 6 x 108 cfu/ml h. seropedicae smr1 or ram4. three days after inoculation, 4 x 104 cells of endophytic h. seropedicae smr1 were recovered per gram of fresh root, and 9 days after inoculation the number of endophytes increased to 4 x 106 cfu/g. the identity of the recovered bacteria was confirmed by amplification and sequencing of the 16srrna gene. furthermore, confocal microscopy of p. vulgaris roots inoculated with h. seropedicae ram4 showed that the bacterial cells were attached to the root surface 15 min after inoculation; fluorescent bacteria were visible in the internal tissues after 24 h and were found in the central cylinder after 72 h, showing that h. seropedicae ram4 is capable of colonizing the roots of the dicotyledon p. vulgaris. determination of dry weight of common bean inoculated with h. seropedicae smr1 suggested that this bacterium has a negative effect on the growth of p. vulgaris.
Structural interaction between GFP-labeled diazotrophic endophytic bacterium Herbaspirillum seropedicae RAM10 and pineapple plantlets 'Vitória'
Baldotto, Lílian Estrela Borges;Olivares, Fábio Lopes;Bressan-Smith, Ricardo;
Brazilian Journal of Microbiology , 2011, DOI: 10.1590/S1517-83822011000100015
Abstract: the events involved in the structural interaction between the diazotrophic endophytic bacterium herbaspirillum seropedicae, strain ram10, labeled with green fluorescent protein, and pineapple plantlets 'vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. after 6 hours of inoculation, h. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. moreover, within 1 day after inoculation the bacteria were colonizing the shoots. in this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. the penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. after 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of h. seropedicae.
Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis
Salles, Joana Falc?o;Gitahy, Patrícia de Medeiros;Sk?t, Leif;Baldani, José Ivo;
Brazilian Journal of Microbiology , 2000, DOI: 10.1590/S1517-83822000000300001
Abstract: the goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. the endophytic nitrogen-fixing bacteria gluconacetobacter diazotrophicus strain br11281 and herbaspirillum seropedicae strain br11335 were used as models. the cry3a gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid n-free medium. the presence of the cry gene was detected by southern-blot using an internal fragment of 1.0 kb as a probe. the production of d-endotoxin by the recombinant h. seropedicae strain was detected by dot blot while for g. diazotrophicus the western-blot technique was used. in both cases, a specific antibody raised against the b. thuringiensis toxin was applied. the d-endotoxin production showed by the g. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3a gene was fused to a nif promoter. in the case of h. seropedicae the d-endotoxin expression was not affected by the promoter (rhi) used. these results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.
Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from Bacillus thuringiensis  [cached]
Salles Joana Falc?o,Gitahy Patrícia de Medeiros,Sk?t Leif,Baldani José Ivo
Brazilian Journal of Microbiology , 2000,
Abstract: The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a cry gene from Bacillus thuringiensis, envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria Gluconacetobacter diazotrophicus strain BR11281 and Herbaspirillum seropedicae strain BR11335 were used as models. The cry3A gene was transferred by conjugation using a suicide plasmid and the recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the cry gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of delta-endotoxin by the recombinant H. seropedicae strain was detected by dot blot while for G. diazotrophicus the Western-blot technique was used. In both cases, a specific antibody raised against the B. thuringiensis toxin was applied. The delta-endotoxin production showed by the G. diazotrophicus recombinant strain was dependent on the nitrogen fixing conditions since the cry3A gene was fused to a nif promoter. In the case of H. seropedicae the delta-endotoxin expression was not affected by the promoter (rhi) used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests.
Early colonization pattern of maize (Zea mays L. Poales, Poaceae) roots by Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae)
Monteiro, Rose A.;Schmidt, Maria A.;Baura, Valter A. de;Balsanelli, Eduardo;Wassem, Roseli;Yates, Marshall G.;Randi, Marco A.F.;Pedrosa, Fábio O.;Souza, Emanuel M. de;
Genetics and Molecular Biology , 2008, DOI: 10.1590/S1415-47572008005000007
Abstract: the bacterium herbaspirillum seropedicae is an endophytic diazotroph found in several plants, including economically important poaceous species. however, the mechanisms involved in the interaction between h. seropedicae and these plants are not completely characterized. we investigated the attachment of herbaspirillum to maize roots and the invasion of the roots by this bacterium using h. seropedicae strain smr1 transformed with the suicide plasmid putkandsred, which carries a mini-tn5 transposon containing the gene for the discosoma red fluorescent protein (dsred) constitutively expressed together with the kanamycin resistance gene. integration of the mini-tn5 into the bacterial chromosome yielded the mutant h. seropedicae strain ram4 which was capable of expressing dsred and could be observed on and inside fresh maize root samples. confocal microscopy of maize roots inoculated with h. seropedicae three days after germination showed that h. seropedicae cell were attached to the root surface 30 min after inoculation, were visible in the internal tissues after twenty-four hours and in the endodermis, the central cylinder and xylem after three days.
Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies
Silva-Froufe, Lúcia Gracinda da;Boddey, Robert Michael;Reis, Veronica Massena;
Brazilian Journal of Microbiology , 2009, DOI: 10.1590/S1517-83822009000400018
Abstract: the species gluconacetobacterdiazotrophicus, herbaspirillum seropedicae and h. rubrisubalbicans are endophytic n2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. however, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in n free semi-solid media which are only semi-selective for the species/genera [the most probable number (mpn) technique] and each culture must be further subjected to several tests to identify the isolates at the species level. the use of species-specific polyclonal antibodies with the indirect elisa (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. this study was performed to investigate the viability of adapting the indirect elisa technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. the results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-a columns which removed non-specific immuno-globulins. it was possible to quantify the three bacterial species in the brazilian sugarcane variety sp 70-1143 in numbers above 105 cells per g fresh weight in roots, rhizomes and leaves. the numbers of the different bacterial species evaluated using the elisa technique were found to be higher than when the same populations were evaluated using the mpn technique, reaching 1400 times greater for g. diazotrophicus and 225 times greater for herbaspirillum spp. these results constitute the first quantification of herbaspirillum using immunological techniques.
Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses  [PDF]
Fábio O. Pedrosa ,Rose Adele Monteiro,Roseli Wassem,Leonardo M. Cruz,Ricardo A. Ayub,Nelson B. Colauto,Maria Aparecida Fernandez,Maria Helena P. Fungaro,Edmundo C. Grisard,Mariangela Hungria,Humberto M. F. Madeira,Rubens O. Nodari,Clarice A. Osaku,Maria Luiza Petzl-Erler,Hernán Terenzi,Luiz G. E. Vieira,Maria Berenice R. Steffens,Vinicius A. Weiss,Luiz F. P. Pereira,Marina I. M. Almeida,Lysangela R. Alves,Anelis Marin,Luiza Maria Araujo,Eduardo Balsanelli,Valter A. Baura,Leda S. Chubatsu,Helisson Faoro,Augusto Favetti,Geraldo Friedermann,Chirlei Glienke,Susan Karp,Vanessa Kava-Cordeiro,Roberto T. Raittz,Humberto J. O. Ramos,Enilze Maria S. F. Ribeiro,Liu Un Rigo,Saul N. Rocha,Stefan Schwab,Anilda G. Silva,Eliel M. Souza,Michelle Z. Tadra-Sfeir,Rodrigo A. Torres,Audrei N. G. Dabul,Maria Albertina M. Soares,Luciano S. Gasques,Ciela C. T. Gimenes,Juliana S. Valle,Ricardo R. Ciferri,Luiz C. Correa,Norma K. Murace,Jo?o A. Pamphile,Eliana Valéria Patussi,Alberto J. Prioli,Sonia Maria A. Prioli,Carmem Lúcia M. S. C. Rocha,Olívia Márcia N. Arantes,Márcia Cristina Furlaneto,Leandro P. Godoy,Carlos E. C. Oliveira,Daniele Satori,Laurival A. Vilas-Boas,Maria Angélica E. Watanabe,Bibiana Paula Dambros,Miguel P. Guerra,Sandra Marisa Mathioni,Karine Louise Santos,Mario Steindel,Javier Vernal,Fernando G. Barcellos,Rubens J. Campo,Ligia Maria O. Chueire,Marisa Fabiana Nicolás,Lilian Pereira-Ferrari,José L. da Concei??o Silva,Nereida M. R. Gioppo,Vladimir P. Margarido
PLOS Genetics , 2011, DOI: 10.1371/journal.pgen.1002064
Abstract: The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species.
Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides  [PDF]
Eduardo Balsanelli, Thalita Regina Tuleski, Valter Antonio de Baura, Marshall Geoffrey Yates, Leda Satie Chubatsu, Fabio de Oliveira Pedrosa, Emanuel Maltempi de Souza, Rose Adele Monteiro
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0077001
Abstract: Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.
The genome of the endophytic bacterium H. frisingense GSF30T identifies diverse strategies in the Herbaspirillum genus to interact with plants  [PDF]
Daniel Straub,Anton Hartmann,Uwe Ludewig
Frontiers in Microbiology , 2013, DOI: 10.3389/fmicb.2013.00168
Abstract: The diazotrophic, bacterial endophyte Herbaspirillum frisingense GSF30T has been identified in biomass grasses grown in temperate climate, including the highly nitrogen-efficient grass Miscanthus. Its genome was annotated and compared with related Herbaspirillum species from diverse habitats, including H. seropedicae, and further well-characterized endophytes. The analysis revealed that Herbaspirillum frisingense lacks a type III secretion system that is present in some related Herbaspirillum grass endophytes. Together with the lack of components of the type II secretion system, the genomic inventory indicates distinct interaction scenarios of endophytic Herbaspirillum strains with plants. Differences in respiration, carbon, nitrogen and cell wall metabolism among Herbaspirillum isolates partially correlate with their different habitats. Herbaspirillum frisingense is closely related to strains isolated from the rhizosphere of phragmites and from well water, but these lack nitrogen fixation and metabolism genes. Within grass endophytes, the high diversity in their genomic inventory suggests that even individual plant species provide distinct, highly diverse metabolic niches for successful endophyte-plant associations.
Initial performance of maize in response to NPK fertilization combined with Herbaspirillum seropedicae
Baldotto, Marihus Altoé;Baldotto, Lílian Estrela Borges;Santana, Rogério Batista;Marciano, Cláudio Roberto;
Revista Ceres , 2012, DOI: 10.1590/S0034-737X2012000600015
Abstract: the inoculation with plant growth-promoting bacteria can be a technological approach useful for increasing the production of maize. the objective of this study was to evaluate the initial performance of maize in response to application of doses of npk combined with the inoculation of the diazotrophic bacteria herbaspirillum seropedicae in an greenhouse experiment. the experiment consisted of six fertilizer levels: 0, 25, 50, 75, 100 and 200% of the recommended dose of npk applied to maize inoculated and non-inoculated with h. seropedicae. at 30 days after the treatment application, the growth characteristics and nutritional status of the plants were evaluated. plant development was influenced by fertilization, but it was enhanced by combination with the bacteria, which resulted in significant increases in the dry mass of shoots (7%) and leaf area (9%) when compared with non-inoculated plants. the results showed increases in the concentration of n (11%), p (30%) and k (17%) of maize plants in response to bacterial inoculation together with npk compared with plants that were applied fertilize only. the greater consistency and stability response of the host plant to bacterization in the presence of chemical fertilizer indicate a promissory biotechnological approach for improving the initial growth and adaptation of maize to the cultivation environment.
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