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      History on the biological nitrogen fixation research in graminaceous plants: special emphasis on the Brazilian experience

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          Abstract

          This review covers the history on Biological Nitrogen Fixation (BNF) in Graminaceous plants grown in Brazil, and describes research progress made over the last 40 years, most of whichwas coordinated by Johanna Döbereiner. One notable accomplishment during this period was the discovery of several nitrogen-fixing bacteria such as the rhizospheric (Beijerinckia fluminensis and Azotobacter paspali), associative (Azospirillum lipoferum, A. brasilense, A. amazonense) and the endophytic (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis and B. tropica). The role of these diazotrophs in association with grasses, mainly with cereal plants, has been studied and a lot of progress has been achieved in the ecological, physiological, biochemical, and genetic aspects. The mechanisms of colonization and infection of the plant tissues are better understood, and the BNF contribution to the soil/plant system has been determined. Inoculation studies with diazotrophs showed that endophytic bacteria have a much higher BNF contribution potential than associative diazotrophs. In addition, it was found that the plant genotype influences the plant/bacteria association. Recent data suggest that more studies should be conducted on the endophytic association to strengthen the BNF potential. The ongoing genome sequencing programs: RIOGENE (Gluconacetobacter diazotrophicus) and GENOPAR (Herbaspirillum seropedicae) reflect the commitment to the BNF study in Brazil and should allow the country to continue in the forefront of research related to the BNF process in Graminaceous plants.

          Translated abstract

          A presente revisão aborda a história da Fixação Biológica de Nitrogênio (FBN) em Gramíneas no Brasil, procurando mostrar a evolução da pesquisa na área iniciada a mais de 40 anos sob a liderança da pesquisadora Johanna Döbereiner. Um aspecto marcante deste período foi a descoberta de diversas bactérias fixadoras de nitrogênio atmosférico tais com as rizosféricas (Beijerinckia fluminensis e Azotobacter paspali), associativas (Azospirillum lipoferum, A. brasilense, A. amazonense) e as endofíticas (Herbaspirillum seropedicae, H. rubrisubalbicans, Gluconacetobacter diazotrophicus, Burkholderia brasilensis e B. tropica). O papel destas bactérias diazotróficas em associação com as gramíneas, especialmente os cereais, tem sido estudado e muito se avançou sobre os aspectos ecológicos, fisiológicos, bioquímicos e genéticos. Os mecanismos de colonização e infecção dos tecidos das plantas foram melhor entendidos e a contribuição da FBN para o sistema solo-planta foi determinado. Estudos de inoculação de cereais com bactérias diazotróficas, têm mostrado que as endofíticas têm um maior potencial de contribuição da FBN e que o genótipo da planta influencia na associação da planta/bactéria. Os avanços alcançados apontam para uma maior exploração e entendimento desta associação endofítica. Os programas de sequenciamento do genoma: RIOGENE (Gluconacetobacter diazotrophicus) e GENOPAR (Herbaspirillum seropedicae) mostram a importância da FBN no Brasil e devem permitir que o país continue na fronteira do conhecimento em relação ao processo de FBN em gramíneas.

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          Endophytic colonization and in planta nitrogen fixation by a Herbaspirillum sp. isolated from wild rice species.

          Nitrogen-fixing bacteria were isolated from the stems of wild and cultivated rice on a modified Rennie medium. Based on 16S ribosomal DNA (rDNA) sequences, the diazotrophic isolates were phylogenetically close to four genera: Herbaspirillum, Ideonella, Enterobacter, and Azospirillum. Phenotypic properties and signature sequences of 16S rDNA indicated that three isolates (B65, B501, and B512) belong to the Herbaspirillum genus. To examine whether Herbaspirillum sp. strain B501 isolated from wild rice, Oryza officinalis, endophytically colonizes rice plants, the gfp gene encoding green fluorescent protein (GFP) was introduced into the bacteria. Observations by fluorescence stereomicroscopy showed that the GFP-tagged bacteria colonized shoots and seeds of aseptically grown seedlings of the original wild rice after inoculation of the seeds. Conversely, for cultivated rice Oryza sativa, no GFP fluorescence was observed for shoots and only weak signals were observed for seeds. Observations by fluorescence and electron microscopy revealed that Herbaspirillum sp. strain B501 colonized mainly intercellular spaces in the leaves of wild rice. Colony counts of surface-sterilized rice seedlings inoculated with the GFP-tagged bacteria indicated significantly more bacterial populations inside the original wild rice than in cultivated rice varieties. Moreover, after bacterial inoculation, in planta nitrogen fixation in young seedlings of wild rice, O. officinalis, was detected by the acetylene reduction and (15)N(2) gas incorporation assays. Therefore, we conclude that Herbaspirillum sp. strain B501 is a diazotrophic endophyte compatible with wild rice, particularly O. officinalis.
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            Azospirillum– plant relationships: environmental and physiological advances (1990–1996)

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              Burkholderia, a genus rich in plant-associated nitrogen fixers with wide environmental and geographic distribution.

              The genus Burkholderia comprises 19 species, including Burkholderia vietnamiensis which is the only known N(2)-fixing species of this bacterial genus. The first isolates of B. vietnamiensis were recovered from the rhizosphere of rice plants grown in a phytotron, but its existence in natural environments and its geographic distribution were not reported. In the present study, most N(2)-fixing isolates recovered from the environment of field-grown maize and coffee plants cultivated in widely separated regions of Mexico were phenotypically identified as B. cepacia using the API 20NE system. Nevertheless, a number of these isolates recovered from inside of maize roots, as well as from the rhizosphere and rhizoplane of maize and coffee plants, showed similar or identical features to those of B. vietnamiensis TVV75(T). These features include nitrogenase activity with 10 different carbon sources, identical or very similar nifHDK hybridization patterns, very similar protein electrophoregrams, identical amplified 16S rDNA restriction (ARDRA) profiles, and levels of DNA-DNA reassociation higher than 70% with total DNA from strain TVV75(T). Although the ability to fix N(2) is not reported to be a common feature among the known species of the genus Burkholderia, the results obtained show that many diazotrophic Burkholderia isolates analyzed showed phenotypic and genotypic features different from those of the known N(2)-fixing species B. vietnamiensis as well as from those of B. kururiensis, a bacterium identified in the present study as a diazotrophic species. DNA-DNA reassociation assays confirmed the existence of N(2)-fixing Burkholderia species different from B. vietnamiensis. In addition, this study shows the wide geographic distribution and substantial capability of N(2)-fixing Burkholderia spp. for colonizing diverse host plants in distantly separated environments.
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                Author and article information

                Journal
                aabc
                Anais da Academia Brasileira de Ciências
                An. Acad. Bras. Ciênc.
                Academia Brasileira de Ciências (Rio de Janeiro, RJ, Brazil )
                0001-3765
                1678-2690
                September 2005
                : 77
                : 3
                : 549-579
                Affiliations
                [01] Seropédica Rio de Janeiro orgnameEmbrapa Agrobiologia Brasil
                Article
                S0001-37652005000300014 S0001-3765(05)07700314
                10.1590/S0001-37652005000300014
                16127558
                a1b851cb-288d-4070-997c-743e10b5c743

                This work is licensed under a Creative Commons Attribution 4.0 International License.

                History
                : 06 April 2005
                : 27 December 2004
                Page count
                Figures: 0, Tables: 0, Equations: 0, References: 218, Pages: 31
                Product

                SciELO Brazil

                Categories
                Agrarian Sciences

                bacterial endophytes,diazotrophs,semi-solid N-free medium,inoculation,cereals,grass plants,bactéria endofítica,diazotrófica,meio semi-sólido,inoculação,cereais,planta forrageira

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