Interacciones tempranas entre <i>Bradyrhizobium japonicum</i> y soja: efectos de la escasez de N y la distribución de los rizobios sobre la raiz

Dado que una simbiosis exitosa requiere bajos niveles de N biodisponible en el suelo y que ciertas evidencias sugieren que la escasez de nutrientes podría favorecer la capacidad infectiva de los rizobios sobre las raíces de las leguminosas (por ejemplo Kijne et al., 1988; Brelles Mariño et al., 1996; Lodeiro et al, 2000), puede plantearse la alternativa de preparar inoculantes en medios de alta relación C/N. Los medios de cultivo empleados comúnmente para la obtención de caldos de rizobios para inoculantes son diversos, pero en todos los casos están bien balanceados y se procura obtener con ellos altas concentraciones bacterianas, con lo cual generalmente se trata además de medios ricos. Esta condición podría dar por resultado inoculantes poco infectivos no sólo por la abundancia de nutrientes, sino porque los sistemas de percepción del quorum en estos cultivos densos podrían determinar la liberación de bradioxetina y la posterior inhibición de la expresión de los genes nod (Loh et al, 2001; 2002). El método más común de aplicación de los inoculantes en soja consiste en ponerlos en contacto con las semillas antes de sembrar y permitir su adhesión a las mismas. Este método es muy conveniente desde el punto de vista práctico debido a su simplicidad y a que no requiere de operaciones adicionales ni equipos especiales. Sin embargo, la inoculación sobre las semillas puede constituir otro problema para la eficiencia final obtenida con cualquier inoculante por su mala distribución en el suelo debida a los problemas de movilidad que ya hemos analizado. Por lo tanto, debería compararse esta metodología de aplicación del inoculante con otras que lo distribuyan mejor en el sustrato en el cual crecen las raíces. En vista de estos problemas hemos enfocado nuestro trabajo de Tesis al estudio de la eficiencia de los rizobios para nodular, asociada a aspectos del metabolismo bacteriano bajo diversos grados de limitación de N y a la distribución de los rizobios respecto de la zona infectable de la raíz. En particular, es de nuestro interés investigar si la limitación en N favorece o no el desarrollo de las etapas tempranas de preinfección e infección y encarar el análisis de la competitividad de rizobios cultivados bajo nuestras condiciones de limitación o suficiencia de N frente a poblaciones como las naturalizadas en los suelos.Tesis digitalizada en SEDICI gracias a la Biblioteca Central de la Facultad de Ciencias Exactas (UNLP).Facultad de Ciencias Exacta

Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens

Bradyrhizobium diazoefficiens, a nitrogen- fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZor nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. AB. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ54, was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.Instituto de Biotecnologia y Biologia Molecula

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin

Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Improved soybean root association of N-starved Bradyrhizobium japonicum

In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day-1 in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH4)2SO4] limited to only 20 μM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH4)2SO4. The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.Facultad de Ciencias Exacta

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin

Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Stimulation of adhesiveness, infectivity, and competitiveness for nodulation of Bradyrhizobium japonicum by its pretreatment with soybean seed lectin

Soybean seed lectin stimulates adsorption of Bradyrhizobium japonicum to its host roots. Pretreatment of the rhizobia with soybean seed lectin for at least 6-12 h previous to their interaction with the plants was required to detect the stimulatory effect. This activity could be observed with as few as 1000 soybean seed lectin molecules per bacterium, and required specific carbohydrate binding. Infectivity and competitiveness for nodulation were also stimulated by preincubation of the rhizobia either with soybean seed meal extract or soybean seed lectin, the extract being more effective in enhancing competitiveness.Instituto de Biotecnologia y Biologia MolecularFacultad de Ciencias Exacta

Improved soybean root association of N-starved Bradyrhizobium japonicum

In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day-1 in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH4)2SO4] limited to only 20 μM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH4)2SO4. The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.Facultad de Ciencias Exacta

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.Instituto de Biotecnologia y Biologia Molecula

Analysis of the role of the two flagella of Bradyrhizobium japonicum in competition for nodulation of soybean

Bradyrhizobium japonicum has two types of flagella. One has thin filaments consisting of the 33-kDa flagellins FliCI and FliCII (FliCI-II) and the other has thick filaments consisting of the 65-kDa flagellins FliC1, FliC2, FliC3, and FliC4 (FliC1-4). To investigate the roles of each flagellum in competition for nodulation, we obtained mutants deleted in fliCI-II and/or fliC1-4 in the genomic backgrounds of two derivatives from the reference strain USDA 110: the streptomycin-resistant derivative LP 3004 and its more motile derivative LP 3008. All mutations diminished swimming motility. When each mutant was co-inoculated with the parental strain on soybean plants cultivated in vermiculite either at field capacity or flooded, their competitiveness differed according to the flagellin altered. ΔfliCI-II mutants were more competitive, occupying 64-80% of the nodules, while ΔfliC1-4 mutants occupied 45-49% of the nodules. Occupation by the nonmotile double mutant decreased from 55% to 11% as the water content of the vermiculite increased from 85% to 95% field capacity to flooding. These results indicate that the influence of motility on competitiveness depended on the water status of the rooting substrate.Instituto de Biotecnologia y Biologia Molecula

Retention and release of nutrients and dissolved organic carbon in a nutrient-rich stream: A mass balance approach

The relevance of fluvial systems to process nutrients and carbon is widely accepted, but their role as sinks and sources of nutrients and dissolved organic carbon (DOC) is still under discussion especially in non-forested and highly productive streams. In this study, we used a mass balance approach at a reach scale in a Pampean stream to elucidate the major sources of water, nutrients and DOC as well as to determine net in-stream retention efficiencies of nutrients and DOC under different hydrological conditions. We measured conductivity, conservative ions (chloride and calcium), soluble reactive phosphorus (SRP), nitrate (NO3), nitrite (NO2), ammonium (NH4) and DOC at the end-point of a reach of Las Flores stream (site A), at two upstream tributaries (B1 and B2), and at each potential hydrological contributors to stream flow (groundwater, overland and subsurface flows, and rainfall). In addition, we monitored one storm event where we collected samples during the rising and the recession limb of the hydrograph. Stream flow originated from groundwater (≈50%), upstream tributaries (B1 and B2) at baseflow, whereas overland flow contributed >20% during high flows. During baseflow, groundwater provided NO3 to stream water, while B2, which received a point input of a dairy industry, was the main source of SRP and NH4. Conversely, SRP and NH4 were provided by B1, overland flow and subsurface flow during high flows. Overland flow also contributed DOC during high flow periods. Mass balance estimates revealed that the reach acts as a source of DOC, SRP and NO3 (21.4, 37.4 and 53.5% mean net in-stream release, respectively) and a sink of NH4 (−36.8% mean net in-stream retention). Relevant in-stream processes may be nutrient uptake (as in the case of SRP and NH4) and biotic production (DOC), as well as decomposition (SRP) and nitrification (NH4) in this Pampean stream. Our results stress the relevance of nutrient and DOC generation processes within the channel in non-forested and highly productive streams.Fil: Feijoó, Claudia Silvina. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Messetta, María Laura. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Hegoburu, Cecilia. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Gómez Vázquez, Alicia. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Guerra López, José Rodolfo. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Mas Pla, Josep. Universidad de Girona. Institut Catala de Recerca de L' Aigua; España. Universidad de Girona; EspañaFil: Rigacci, Laura Natalia. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: García, Victoria Julieta. Universidad Nacional de Luján. Instituto de Ecología y Desarrollo Sustentable. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ecología y Desarrollo Sustentable; ArgentinaFil: Butturini, Andrea. Universidad de Barcelona; Españ