RESUMO
The production of 3-indoleacetic acid (IAA) by plant growth-promoting bacteria (PGPR) stimulates root development and plant growth. In addition, morphological changes such as an increased root ramification and root hair production improves nutrient absorption and biomass accumulation. The objective of this work was to evaluate the effect of IAA-producing strains on rice in an advanced stage of its vegetative cycle. Rice was inoculated with Gluconacetobacter diazotrophicus PAL 5 and its lao- mutant, deficient in auxin production, Azospirillum baldaniorum Sp 245, and Escherichia coli DH10b. Both the mutant and wild-type G. diazotrophicus stimulated root elongation, area, volume, and diameter. However, the lao- mutant strain was the only one capable of increasing the number of roots. In turn, inoculation with A. baldaniorum had no significant effect on plant development. The inoculation with E. coli led to changes in root volume, area, and diameter, and a response that may be related to the stress caused by its presence. We conclude that the inoculation with G. diazotrophicus stimulates the root system's growth independently of their IAA production ability, suggesting that a metabolite other than IAA is responsible for this effect at advanced stages of the rice's vegetative cycle.
Assuntos
Oryza , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Indolacéticos/metabolismo , Oryza/microbiologia , Raízes de Plantas/microbiologiaRESUMO
The enzyme 6-phosphogluconate dehydrogenase catalyzes the conversion of 6-phosphogluconate to ribulose-5-phosphate. It represents an important reaction in the oxidative pentose phosphate pathway, producing a ribose precursor essential for nucleotide and nucleic acid synthesis. We succeeded, for the first time, to determine the three-dimensional structure of this enzyme from an acetic acid bacterium, Gluconacetobacter diazotrophicus (Gd6PGD). Active Gd6PGD, a homodimer (70 kDa), was present in both the soluble and the membrane fractions of the nitrogen-fixing microorganism. The Gd6PGD belongs to the newly described subfamily of short-chain (333 AA) 6PGDs, compared to the long-chain subfamily (480 AA; e.g., Ovis aries, Homo sapiens). The shorter amino acid sequence in Gd6PGD induces the exposition of hydrophobic residues in the C-terminal domain. This distinct structural feature is key for the protein to associate with the membrane. Furthermore, in terms of function, the short-chain 6PGD seems to prefer NAD+ over NADP+ , delivering NADH to the membrane-bound NADH dehydrogenase of the microorganisms required by the terminal oxidases to reduce dioxygen to water for energy conservation. ENZYME: ECnonbreakingspace1.1.1.343. DATABASE: Structural data are available in PDB database under the accession number 6VPB.
Assuntos
Proteínas de Bactérias/metabolismo , Gluconacetobacter/enzimologia , Gluconatos/metabolismo , Fosfogluconato Desidrogenase/metabolismo , Ribulosefosfatos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Gluconacetobacter/genética , Gluconatos/química , Humanos , Modelos Químicos , Modelos Moleculares , Estrutura Molecular , NAD/metabolismo , NADP/metabolismo , Fosfogluconato Desidrogenase/classificação , Fosfogluconato Desidrogenase/genética , Filogenia , Domínios Proteicos , Multimerização Proteica , Ribulosefosfatos/química , Homologia de Sequência de AminoácidosRESUMO
Nowadays, fertilization and pest control are carried out using chemical compounds that contaminate soil and deteriorate human health. Plant growth promoting bacteria endophytes (PGPBEs), are a well-studied group of bacteria that offers benefits to the host plant, such as phytostimulation, biofertilization, and protection against other microorganisms. The study of Gluconacetobacter diazotrophicus-which belongs to PGPBEs-aids the development of alternative strategies of an integrated approach for crop management practices. Ralstonia solanacearum is responsible for bacterial wilt disease. This phytopathogen is of great interest worldwide due to the enormous economic losses it causes. In this study the action of G. diazotrophicus as a growth promoting bacterium in Arabidopsis thaliana seedlings is analyzed, evaluating the antagonistic mechanisms of this beneficial endophytic bacterium during biotic stress produced by R. solanacearum. Effective colonization of G. diazotrophicus was determined through bacterial counting assays, evaluation of anatomical and growth parameters, and pigments quantification. Biocontrol assays were carried out with Ralstonia pseudosolanacearum GMI1000 model strain and R. solanacearum A21 a recently isolated strain. Inoculation of A. thaliana (Col 0) with G. diazotrophicus Pal 5 triggers a set of biochemical and structural changes in roots, stems, and leaves of seedlings. Discrete callose deposits as papillae were observed at specific sites of root hairs, trichomes, and leaf tissue. Upon R. pseudosolanacearum GMI1000 infection, endophyte-treated plants demonstrated being induced for defense through an augmented callose deposition at root hairs and leaves compared with the non-endophyte-treated controls. The endophytic bacterium appears to be able to prime callose response. Roots and stems cross sections showed that integrity of all tissues was preserved in endophyte-treated plants infected with R. solanacearum A21. The mechanisms of resistance elicited by the plant after inoculation with the endophyte would be greater lignification and sclerosis in tissues and reinforcement of the cell wall through the deposition of callose. As a consequence of this priming in plant defense response, viable phytopathogenic bacteria counting were considerably fewer in endophyte-inoculated plants than in not-inoculated controls. Our results indicate that G. diazotrophicus colonizes A. thaliana plants performing a protective role against the phytopathogenic bacterium R. solanacearum promoting the activation of plant defense system.
RESUMO
AIMS: This study aimed to explore the possibility of using the Gluconacin from Gluconacetobacter diazotrophicus strain PAL5 in the biological control of diverse sugarcane phytopathogenic bacteria. METHODS AND RESULTS: An in silico analysis was first employed to determine the phylogenetic relationship between Gram-negative/positive bacteriocin producers and Gluconacin. The analysis showed that this trait is widespread among tested bacterial species and a well-conserved gene within the Acetobacteraceae family. The bacteriocin gene (GDI_0415) present in the genome of strain PAL5 was than cloned in pDEST™17 and expressed in Escherichia coli BL21-AI™. A bioassay showed growth inhibition of Xanthomonas albilineans by the recombinant bacteriocin. Subsequent bioassays indicated different levels of antagonistic activity against the majority of the sugarcane phytopathogenic bacteria (Xanthomonas axonopodis pv. vasculorum, Acidovorax avenae subsp. avenae, Pseudomonas syringae pv. syringae, Xanthomonas vasicola pv. vasculorum). In addition, the bacteriocin was also antagonistic to some beneficial bacterial strains belonging to G. diazotrophicus and endophytic Bacillus species, which also colonize sugarcane plants. CONCLUSIONS: The GDI_0415 gene, responsible for the production of Gluconacin, is well conserved within the Acetobacteraceae family and presented antagonistic activity against phytopathogenic and a few beneficial sugarcane bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of a recombinant protein, named Gluconacin, opens new avenues for the agro-biotechnology application in agriculture, mainly with regard to the sugarcane crop.
RESUMO
For centuries, the Acetobacteraceae is known as a family that harbors many species of organisms of biotechnological importance for industry. Nonetheless, since 1988 representatives of this family have also been described as nitrogen fixing bacteria able to plant growth promotion by a variety of mechanisms. Nitrogen fixation is a biological process that guarantees that the atmospheric N2 is incorporated into organic matter by several bacterial groups. Most representatives of this group, also known as diazotrophic, are generally associated with soil rhizosphere of many plants and also establishing a more specific association living inside roots, leaves, and others plants tissues as endophyte. Their roles as plant growth-promoting microorganisms are generally related to increase in plant biomass, phosphate and other mineral solubilization, and plant pathogen control. Here, we report many of these plant growth-promoting processes related to nitrogen fixing species already described in Acetobacteraceae family, especially Gluconacetobacter diazotrophicus and their importance to agriculture. In addition, a brief review of the state of art of the phylogenetics, main physiological and biochemical characteristics, molecular and functional genomic data of this group of Acetobacteraceae is presented.
Assuntos
Acetobacteraceae/genética , Acetobacteraceae/fisiologia , Agricultura , Gluconacetobacter/fisiologia , Fixação de Nitrogênio , Microbiologia do Solo , Biomassa , Endófitos , Filogenia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , RizosferaRESUMO
Se determinó la capacidad de fijar nitrógeno mediante la actividad reductora de acetileno para 8 cepas de G. diazotrophicus aisladas de diferentes ecosistemas empleando el medio LGI-P. Además, se determinó la producción de auxinas a través del método de Salkowski y se analizó la influencia del aminoácido triptófano y del AIA en la actividad de la nitrogenasa. El triptófano, al igual que otros aminoácidos y las diferentes concentraciones de AIA, inhiben en distinta medida la actividad de la enzima solo parcialmente. Se demostró que las condiciones de nitrofijación no afectan la producción de AIA en esta bacteria. Esta relación entre ambas capacidades fisiológicas beneficiosas para los cultivos agrícolas pudiera tener gran importancia ya que pueden desarrollarse paralelamente, y potenciar la acción beneficiosa hacia la planta, basada en la dinitrofijación y la producción de auxinas estimuladoras del crecimiento vegetal.
The capacity to fix nitrogen of 8 strains of G. diazotrophicus from different ecosystems was determined by acetylene reduction assay using LGIP medium. Moreover, auxins production was determined by Salkowskiâs method and the influence of triptophan and indoleacetic acid (IAA) in the nitrogenase activity were analyzed. The triptophan as other aminoacids and different concentrations of IAA, inhibit at different levels the nitrogen fixation only partially. There were demonstrated that the nitrogen fixation conditions do not affect the auxins production of this bacteria. This relationship between both crop beneficial physiological capacities should be a great importance since they may be parallely developed, and enhance the beneficial action to the plant, based on dinitrogen fixation and stimulating plant growth auxins production.
Assuntos
Nitrogênio/farmacologia , Nitrogênio/química , NitrogênioRESUMO
A new role for the plant growth-promoting nitrogen-fixing endophytic bacteria Gluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions. Living G.diazotrophicus possess and/or produce elicitor molecules which activate the sugarcane defense response resulting in the plant resistance to X. albilineans, in this particular case controlling the pathogen transmission to emerging agamic shoots. A total of 47 differentially expressed transcript derived fragments (TDFs) were identified by cDNA-AFLP. Transcripts showed significant homologies to genes of the ethylene signaling pathway (26%), proteins regulates by auxins (9%), beta-1,3 Glucanase proteins (6%) and ubiquitin genes (4%), all major signaling mechanisms. Results point toward a form of induction of systemic resistance in sugarcane-G. diazotrophicus interactions which protect the plant against X. albilineans attack.
RESUMO
The effects of soil moisture on the survival of three diazotrophic bacteria species (Azospirillum amazonense, Gluconacetobacter diazotrophicus and Azospirillum brasilense) were tested. Soil moisture had little influence on the survival of A. brasilense, which is considered a free-living species. On the other hand, increased soil moisture extended the survival of the endophytes A. amazonense and G. diazotrophicus. These results indicate that nitrogen-fixing endophytic species are more affected by soil moisture than associative nitrogen-fixing species.
Neste trabalho foi avaliado o efeito da umidade do solo na sobrevivência de três espécies de bactérias diazotróficas (Azospirillum amazonense, Gluconacetobacter diazotrophicus e Azospirillum brasilense). O teor de umidade apresentou pouca influência na sobrevivência de A. brasilense, considerada uma espécie cosmopolita, enquanto A. amazonense e G. diazotrophicus, consideradas endofíticas, aumentaram o período de culturabilidade na presença de umidade no solo. Os resultados demonstram que o teor de umidade do solo possui maior influência nas espécies endofíticas, em comparação às espécies associativas.
RESUMO
The inoculation technique with endophytic diazotrophic bacteria in sugarcane has been shown as an alternative practice to plant growth promotion. The aim of this work was to evaluate the biological nitrogen fixation (BNF) contribution by different strains of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus in sugarcane plant inoculated from seeds. The seeds were planted in pots filled with non-sterile soil, inoculated with the bacterial strains and grown 10 months outside of the greenhouse. The BNF contribution of the inoculated bacteria varied depending on the plant species used as a control. The highest BNF contribution as well as the highest populations of reisolated bacteria was observed with inoculation of H. seropedicae strains. The roots appeared to be the preferential tissues for the establishment of the inoculated species.
A técnica de inoculação com bactérias diazotróficas endofíticas na cana-de-açúcar apresenta-se como uma prática alternativa para promover o crescimento vegetal menos dependente da adubação nitrogenada. Este trabalho teve como objetivo avaliar a contribuição da fixação biológica de nitrogênio (FBN) por diferentes estirpes de Herbaspirillum seropedicae e Gluconacetobacter diazotrophicus inoculadas em plantas de cana-de-açúcar originadas de semente. As sementes foram plantadas em vasos com solo não estéril, inoculadas com as diferentes bactérias e mantidas por 10 meses ao ar livre. As maiores contribuições da FBN ocorreram com a inoculação de estirpes Herbaspirillum seropedicae, e dependeram da espécie vegetal utilizada como testemunha. As raízes apresentaram-se como o órgão vegetal preferencial para o estabelecimento das espécies inoculadas.
RESUMO
The inoculation technique with endophytic diazotrophic bacteria in sugarcane has been shown as an alternative practice to plant growth promotion. The aim of this work was to evaluate the biological nitrogen fixation (BNF) contribution by different strains of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus in sugarcane plant inoculated from seeds. The seeds were planted in pots filled with non-sterile soil, inoculated with the bacterial strains and grown 10 months outside of the greenhouse. The BNF contribution of the inoculated bacteria varied depending on the plant species used as a control. The highest BNF contribution as well as the highest populations of reisolated bacteria was observed with inoculation of H. seropedicae strains. The roots appeared to be the preferential tissues for the establishment of the inoculated species.
A técnica de inoculação com bactérias diazotróficas endofíticas na cana-de-açúcar apresenta-se como uma prática alternativa para promover o crescimento vegetal menos dependente da adubação nitrogenada. Este trabalho teve como objetivo avaliar a contribuição da fixação biológica de nitrogênio (FBN) por diferentes estirpes de Herbaspirillum seropedicae e Gluconacetobacter diazotrophicus inoculadas em plantas de cana-de-açúcar originadas de semente. As sementes foram plantadas em vasos com solo não estéril, inoculadas com as diferentes bactérias e mantidas por 10 meses ao ar livre. As maiores contribuições da FBN ocorreram com a inoculação de estirpes Herbaspirillum seropedicae, e dependeram da espécie vegetal utilizada como testemunha. As raízes apresentaram-se como o órgão vegetal preferencial para o estabelecimento das espécies inoculadas.
RESUMO
The inoculation technique with endophytic diazotrophic bacteria in sugarcane has been shown as an alternative practice to plant growth promotion. The aim of this work was to evaluate the biological nitrogen fixation (BNF) contribution by different strains of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus in sugarcane plant inoculated from seeds. The seeds were planted in pots filled with non-sterile soil, inoculated with the bacterial strains and grown 10 months outside of the greenhouse. The BNF contribution of the inoculated bacteria varied depending on the plant species used as a control. The highest BNF contribution as well as the highest populations of reisolated bacteria was observed with inoculation of H. seropedicae strains. The roots appeared to be the preferential tissues for the establishment of the inoculated species.
A técnica de inoculação com bactérias diazotróficas endofíticas na cana-de-açúcar apresenta-se como uma prática alternativa para promover o crescimento vegetal menos dependente da adubação nitrogenada. Este trabalho teve como objetivo avaliar a contribuição da fixação biológica de nitrogênio (FBN) por diferentes estirpes de Herbaspirillum seropedicae e Gluconacetobacter diazotrophicus inoculadas em plantas de cana-de-açúcar originadas de semente. As sementes foram plantadas em vasos com solo não estéril, inoculadas com as diferentes bactérias e mantidas por 10 meses ao ar livre. As maiores contribuições da FBN ocorreram com a inoculação de estirpes Herbaspirillum seropedicae, e dependeram da espécie vegetal utilizada como testemunha. As raízes apresentaram-se como o órgão vegetal preferencial para o estabelecimento das espécies inoculadas.
RESUMO
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.
Este estudo teve como objetivo avaliar o potencial de uso de bactérias diazotróficas endofíticas como vetores para a expressão de genes cry de Bacillus thuringiensis. As bactérias diazotróficas endofíticas Gluconacetobacter diazotrophicus estirpe BR11281 e Herbaspirillum seropedicae estirpe BR11335 foram usadas como modelo. O gene cry3A foi transferido através de um plasmídeo suicída por conjugação e os recombinantes foram selecionados pela sua capacidade de fixar nitrogênio em meio semi-sólido sem N. A presença do gene cry3A no genoma dos transconjugantes foi detectada através da técnica de "Southern blot" utilizando como sonda um fragmento de 1,0 kb, interno ao gene cry3A. A produção de delta-endotoxina pelos transconjugantes foi detectada por "Dot blot" em de H. seropedicae e por "Western blot" em G. diazotrophicus, usando-se o anticorpo específico para a toxina de B. thuringiensis. A avaliação da produção da delta-endotoxina mostrou que a expressão do gene cry3A em G. diazotrophicus é dependente do processo de fixação de nitrogênio por estar fusionado a um promotor nif nesta bactéria. No caso de H. seropedicae a expressão não foi influenciada pelo promotor de rizosfera (rhi) usado. Os resultados obtidos sugerem que estas bactérias diazotróficas endofíticas podem ser usadas como vetores para expressar genes com atividade entomopatogênica, visando o controle de pragas de cana-de-açúcar ou outras plantas de interesse econômico.