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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.
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.
Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy
Silva, Lúcia Gracinda da;Miguens, Flávio Costa;Olivares, Fabio Lopes;
Brazilian Journal of Microbiology , 2003, DOI: 10.1590/S1517-83822003000500023
Abstract: the interaction between sugar cane plantlets and h. seropedicae was investigated using high pressure freezing followed by freeze substitution. microscopical observation showed consistent differences between this approaches when compared with the conventional preparation, specially related to appearance of the bacteria cell and the endophytic attachment to the host cell wall.
The type III secretion system is necessary for the development of a pathogenic and endophytic interaction between Herbaspirillum rubrisubalbicans and Poaceae
Maria A Schmidt, Eduardo Balsanelli, Helisson Faoro, Leonardo M Cruz, Roseli Wassem, Valter A de Baura, Vinicius Weiss, Marshall G Yates, Humberto M F Madeira, Lilian Pereira-Ferrari, Maria H P Fungaro, Francine M de Paula, Luiz F P Pereira, Luiz G E Vieira, Fábio L Olivares, Fábio O Pedrosa, Emanuel M de Souza, Rose A Monteiro
BMC Microbiology , 2012, DOI: 10.1186/1471-2180-12-98
Abstract: A 21?kb DNA region of the H. rubrisubalbicans genome contains a cluster of 26 hrp/hrc genes encoding for the type three secretion system (T3SS) proteins. To investigate the contribution of T3SS to the plant-bacterial interaction process we generated mutant strains of H. rubrisubalbicans M1 carrying a Tn5 insertion in both the hrcN and hrpE genes. H. rubrisulbalbicans hrpE and hrcN mutant strains of the T3SS system failed to cause the mottled stripe disease in the sugarcane susceptible variety B-4362. These mutant strains also did not produce lesions on Vigna unguiculata leaves. Oryza sativa and Zea mays colonization experiments showed that mutations in hrpE and hrcN genes reduced the capacity of H. rubrisulbalbicans to colonize these plants, suggesting that hrpE and hrcN genes are involved in the endophytic colonization.Our results indicate that the T3SS of H. rubrisubalbicans is necessary for the development of the mottled stripe disease and endophytic colonization of rice.
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.
Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans
Nogueira, Eduardo de Matos;Vinagre, Fabiano;Masuda, Hana Paula;Vargas, Claudia;Pádua, Vania Lúcia Muniz de;Silva, Felipe Rodrigues da;Santos, Renato V. dos;Baldani, José Ivo;Ferreira, Paulo Cavalcanti Gomes;Hemerly, Adriana Silva;
Genetics and Molecular Biology , 2001, DOI: 10.1590/S1415-47572001000100027
Abstract: several brazilian sugarcane varieties have the ability to grow with little addition of inorganic nitrogen fertilizers, showing high contributions of biological nitrogen fixation (bnf). a particular type of nitrogen-fixing association has been described in this crop, where endophytic diazotrophs such as gluconacetobacter diazotrophicus and herbaspirillum spp. colonize plant tissues without causing disease symptoms. in order to gain insight into the role played by the sugarcane in the interaction between this plant and endophytic diazotrophs, we investigated gene expression profiles of sugarcane plants colonized by g. diazotrophicus and h. rubrisubalbicans by searching the sugarcane expressed sequence tag sucest database (http://sucest.lad.ic.unicamp.br/en/). we produced an inventory of sugarcane genes, candidates for exclusive or preferential expression during the nitrogen-fixing association. this data suggests that the host plant might be actively involved in the establishment of the interaction with g. diazotrophicus and h. rubrisubalbicans.
Expression of sugarcane genes induced by inoculation with Gluconacetobacter diazotrophicus and Herbaspirillum rubrisubalbicans  [cached]
Nogueira Eduardo de Matos,Vinagre Fabiano,Masuda Hana Paula,Vargas Claudia
Genetics and Molecular Biology , 2001,
Abstract: Several Brazilian sugarcane varieties have the ability to grow with little addition of inorganic nitrogen fertilizers, showing high contributions of Biological Nitrogen Fixation (BNF). A particular type of nitrogen-fixing association has been described in this crop, where endophytic diazotrophs such as Gluconacetobacter diazotrophicus and Herbaspirillum spp. colonize plant tissues without causing disease symptoms. In order to gain insight into the role played by the sugarcane in the interaction between this plant and endophytic diazotrophs, we investigated gene expression profiles of sugarcane plants colonized by G. diazotrophicus and H. rubrisubalbicans by searching the sugarcane expressed sequence tag SUCEST Database (http://sucest.lad.ic.unicamp.br/en/). We produced an inventory of sugarcane genes, candidates for exclusive or preferential expression during the nitrogen-fixing association. This data suggests that the host plant might be actively involved in the establishment of the interaction with G. diazotrophicus and H. rubrisubalbicans.
Diversidade de bactérias diazotróficas endofíticas dos gêneros Herbaspirillum e Burkholderia na cultura do arroz inundado
Rodrigues, Luciana da Silva;Baldani, Vera Lucia Divan;Reis, Veronica Massena;Baldani, José Ivo;
Pesquisa Agropecuária Brasileira , 2006, DOI: 10.1590/S0100-204X2006000200012
Abstract: the objective of this work was to evaluate the diversity of endophytic diazotrophic bacteria of the genera herbaspirillum and burkholderia, in two rice varieties, considered of high (ir 42) and low (iac 4440) contribution on bnf. two experiments were conducted in greenhouse conditions, in order to study the association of endophytic diazotrophic bacteria with wetland rice varieties, which were planted in two types of soil: one from rio de janeiro state and another from goiás state, brazil. bacterial population (in different parts) and physiological stages of the plants were evaluated, followed by the both genera strains isolation using culture media. the isolated bacteria were characterized based on morphological and physiological aspects. high bacterial counts were detected (106 cells g-1 fresh weight), especially in root samples of both rice varieties. the majority of the isolates were used for characterization, and the data obtained from the similarity matrix showed the presence of isolates belonging to herbaspirillum seropedicae, while other isolates were clustered into the burkholderia genus.
Comparative molecular analysis of Herbaspirillum strains by RAPD, RFLP, and 16S rDNA sequencing
Soares-Ramos, Juliana R.L.;Ramos, Humberto J.O.;Cruz, Leonardo M.;Chubatsu, Leda S.;Pedrosa, Fábio O.;Rigo, Liu U.;Souza, Emanuel M.;
Genetics and Molecular Biology , 2003, DOI: 10.1590/S1415-47572003000400019
Abstract: herbaspirillum spp. are endophytic diazotrophic bacteria associated with important agricultural crops. in this work, we analyzed six strains of h. seropedicae (z78, m2, za69, za95, z152, and z67) and one strain of h. rubrisubalbicans (m4) by restriction fragment length polymorphism (rflp) using hindiii or drai restriction endonucleases, random amplified polymorphic dna (rapd), and partial sequencing of 16s rdna. the results of these analyses ascribed the strains studied to three distinct groups: group i, consisting of m2 and m4; group ii, of za69; and group iii, of za95, z78, z67, and z152. rapd fingerprinting showed a higher variability than the other methods, and each strain had a unique electrophoretic pattern with five of the six primers used. interestingly, h. seropedicae m2 was found by all analyses to be genetically very close to h. rubrisubalbicans m4. our results show that rapd can distinguish between all herbaspirillum strains tested.
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