Soybean-nodulating strains with low intrinsic competitiveness for nodulation, good symbiotic performance, and stress-tolerance isolated from soybean-cropped soils in Argentina

Soybean is the most important oilseed in the world, cropped in 120-130 million hectares each year. The three most important soybean producers are Argentina, Brazil, and United States, where soybean crops are routinely inoculated with symbiotic N2-fixing Bradyrhizobium spp. This extended inoculation gave rise to soybean-nodulating allochthonous populations (SNAPs) that compete against new inoculant for nodulation, thus impairing yield responses. Competitiveness depends on intrinsic factors contributed by genotype, extrinsic ones determined by growth and environmental conditions, and strain persistence in the soil. To assess these factors in Argentinean SNAPs, we studied 58 isolates from five sites of the main soybean cropping area. BOX-A1R DNA fingerprint distributed these isolates in 10 clades that paralleled the pHs of their original soils. By contrast, reference Bradyrhizobium spp. strains, including those used as soybean-inoculants, were confined to a single clade. More detailed characterization of a subset of 11 SNAP-isolates revealed that five were Bradyrhizobium japonicum, two Bradyrhizobium elkanii, two Rhizobium radiobacter (formerly Agrobacterium tumefaciens), one Bradyrhizobium diazoefficiens, and one Paenibacillus glycanilyticus-which did not nodulate when inoculated alone, and therefore was excluded from further characterization. The remaining subset of 10 SNAP-isolates was used for deeper characterization. All SNAP-isolates were aluminum- and heat-tolerant, and most of them were glyphosate-tolerant. Meanwhile, inoculant strains tested were sensitive to aluminum and glyphosate. In addition, all SNAP-isolates were motile to different degrees. Only three SNAP-isolates were deficient for N2-fixation, and none was intrinsically more competitive than the inoculant strain. These results are in contrast to the general belief that rhizobia from soil populations evolved as intrinsically more competitive for nodulation and less N2-fixing effective than inoculants strains. Shoot:root ratios, both as dry biomass and as total N, were highly correlated with leaf ureide contents, and therefore may be easy indicators of N2-fixing performance, suggesting that highly effective N2-fixing and well-adapted strains may be readily selected from SNAPs. In addition, intrinsic competitiveness of the inoculants strains seems already optimized against SNAP strains, and therefore our efforts to improve nodules occupation by inoculated strains should focus on the optimization of extrinsic competitiveness factors, such as inoculant formulation and inoculation technology.Fil: Iturralde, Esteban Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Covelli, Julieta Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Alvarez, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Pérez Giménez, Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Arrese Igor, Cesar. Universidad Pública de Navarra; EspañaFil: Lodeiro, Aníbal R.. Universidad Nacional de La Plata. Facultad de Ciencias Exactas; Argentin

Soybean lectin enhances biofilm formation by Bradyrhizobium japonicum in the absence of plants

Soybean lectin (SBL) purified fromsoybean seeds by affinity chromatography strongly bound to Bradyrhizobium japonicum USDA 110 cell surface. This lectin enhanced biofilm formation by B. japonicum in a concentration-dependent manner. Presence of galactose during biofilm formation had different effects in the presence or absence of SBL. Biofilms were completely inhibited in the presence of both SBL and galactose, while in the absence of SBL, galactose was less inhibitory. SBL was very stable, since its agglutinating activity of B. japonicum cells as well as of human group A+ erythrocytes was resistant to preincubation for one week at 60°C. Hence, we propose that plant remnants might constitute a source of this lectin, which might remain active in soil and thus favor B. japonicum biofilm formation in the interval between soybean crop seasons.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

An inoculant Bradyrhizobium strain with increased motility improves yield of soybean crops

Poster y resumenSoybean production is very important in Argentina, where more than 20 million hectares are sowed annually with this crop. Since soybean plants possess a very high N-demand, it is crucial that this agricultural activity is developed in a sustainable way because otherwise, this crop could deplete Nnutrition from the soils, leading to erosion, compaction, and flooding. Soybean roots are nodulated by Bradyrhizobium spp., which may fix atmospheric N2 in symbiosis with the plant, thus contributing to keep the N-status of the soil. For this reason and due to their low cost, Bradyrhizobium spp. are widely used in inoculants for soybean crops. However, the efficiency of inoculants is low due to the competition exerted by bradyrhizobia resident in the soil. Among factors that affect the competition for nodulation is the self-propelled motility of the rhizobia. Previously, we developed an artificial selection method to obtain bradyrhizobial strains with higher motility. B. japonicum E109 is the strain recommend by INTA for inoculants production. Therefore, we used E109 to increase its motility, and hereby we obtained the derived B. japonicum E109 m+ strain. This strain possesses 50% more motility than its parental strain in semisolid agar medium, and has the same growth kinetics as the wild type, ruling out the possibility that the increased spreading of E109 m+ in semisolid agar be due to faster growth. B. diazoefficiens have two flagella systems, one subpolar and another lateral, characterized by flagellins of different molecular weights. The subpolar flagellum has constitutive expression and the lateral is inducible with L-arabinose as carbon source, but not with D-mannitol as carbon source. However, E109 m+ expressed both flagella with D-mannitol, as observed with SDS-PAGE of purified flagellins, in agreement with previous results obtained with B. diazoefficiens USDA 110. B. japonicum E109 m+ nodulated soybean and after that, bacteria recovered from nodules maintained the higher motility phenotype. Field trials were performed to estimate yield when the soybean plots were inoculated with E109 m+ or the E109 parental strain. Experiments were carried out in San Antonio de Areco, Province of Buenos Aires, in a soil with a competitive resident Bradyrhizobium spp. population, employing a randomized complete block design that included uninoculated controls. Grain yields were compared by ANOVA, which indicated that inoculation with E109 m+ led to significantly higher yield than inoculation with E109 wild type. Our results suggested that inoculation of soybean with improved motility strains could increase soybean yield by enhancing competition for nodulation in a sustainable way.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Colla, Delfina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Brignoli, Damián.Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Iturralde, Esteban Tomás.Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Covelli, Julieta Mariana. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo; ArgentinaFil: Althabegoiti, María Julia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Perticari, Alejandro. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Lodeiro, Aníbal Roberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biotecnología y Biología Molecular; Argentina. Universidad Nacional de La Plata. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Lodeiro, Aníbal Roberto. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Laboratorio de Genética; Argentin

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

Soybean lectin enhances biofilm formation by Bradyrhizobium japonicum in the absence of plants

Soybean lectin (SBL) purified fromsoybean seeds by affinity chromatography strongly bound to Bradyrhizobium japonicum USDA 110 cell surface. This lectin enhanced biofilm formation by B. japonicum in a concentration-dependent manner. Presence of galactose during biofilm formation had different effects in the presence or absence of SBL. Biofilms were completely inhibited in the presence of both SBL and galactose, while in the absence of SBL, galactose was less inhibitory. SBL was very stable, since its agglutinating activity of B. japonicum cells as well as of human group A+ erythrocytes was resistant to preincubation for one week at 60°C. Hence, we propose that plant remnants might constitute a source of this lectin, which might remain active in soil and thus favor B. japonicum biofilm formation in the interval between soybean crop seasons.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Soybean Seed Lectin Prevents the Accumulation of S-Adenosyl Methionine Synthetase and the S1 30S Ribosomal Protein in <i>Bradyrhizobium japonicum</i> Under C and N Starvation

Soybean lectin (SBL) participates in the recognition between Bradyrhizobium japonicum and soybean although its role remains unknown. To search for changes in the proteome in response to SBL, B. japonicum USDA 110 was incubated for 12 h in a C- and N-free medium with or without SBL (10 μg ml ⁻¹), and the soluble protein profiles were compared. Two polypeptides, S-adenosyl-methionine synthetase (MetK) and the 30S ribosomal protein S1 (RpsA), were found only in the fractions from rhizobia incubated without SBL. Transcript levels of metK and rpsA were not correlated with polypeptide levels, indicating that there was regulation at translation. In support of this proposal, the 5′ translation initiation-region of rpsA mRNA contained folding elements as those involved in regulation of its translation in other species. Disappearance of MetK and RpsA from the soluble protein fractions of SBL-treated rhizobia suggests that SBL might have attenuated the nutritional stress response of B. japonicum.Instituto de Biotecnología y Biología 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

Lack of Galactose or Galacturonic Acid in <i>Bradyrhizobium japonicum</i> USDA 110 Exopolysaccharide Leads to Different Symbiotic Responses in Soybean

Exopolysaccharide (EPS) and lipopolysaccharide (LPS) from Bradyrhizobium japonicum are important for infection and nodulation of soybean (Glycine max), although their roles are not completely understood. To better understand this, we constructed mutants in B. japonicum USDA 110 impaired in galactose or galacturonic acid incorporation into the EPS without affecting the LPS. The derivative LP 3010 had a deletion of lspL-ugdH and produced EPS without galacturonic acid whereas LP 3013, with an insertion in exoB, produced EPS without galactose. In addition, the strain LP 3017, with both mutations, had EPS devoid of both galactosides. The missing galactosides were not replaced by other sugars. The defects in EPS had different consequences. LP 3010 formed biofilms and nodulated but was defective in competitiveness for nodulation; and, inside nodules, the peribacteroid membranes tended to fuse, leading to the merging of symbiosomes. Meanwhile, LP 3013 and LP 3017 were unable to form biofilms and produced empty pseudonodules but exoB suppressor mutants were obtained when LP 3013 plant inoculation was supplemented with wild-type EPS. Similar phenotypes were observed with all these mutants in G. soja. Therefore, the lack of each galactoside in the EPS has a different functional effect on the B. japonicum-soybean symbiosis.Facultad de Ciencias ExactasInstituto de Biotecnologia y Biologia Molecula

Biofertilización con Bradyrhizobium japonicum para la agricultura sustentable: aspectos ecofisiológicos del problema de la competición para la nodulación

El cultivo de soja es de gran importancia en nuestro país, sin embargo requiere un aporte importante de N para obtener buenos rendimientos, el cual, si se quiere promover la agricultura sustentable a largo plazo, no debería salir exclusivamente de la fertilidad del suelo, ni aún siendo suplementado por fertilizantes químicos. Una manera de abastecer N de forma sustentable y económica para lograr altos rindes podría ser la utilización del potencial de los rizobios fijadores de N2. Así, la biofertilización ha sido la técnica utilizada desde los comienzos de este cultivo, como una forma de mantener los niveles de N en el suelo. Sin embargo, en suelos con historial de cultivo de soja, a lo largo de las sucesivas biofertilizaciones se va perdiendo la respuesta del rendimiento. Este hecho se observa dado que las biofertilizaciones facilitan el desarrollo de poblaciones de bacterias noduladoras de soja en el suelo, la cuales pueden adaptarse a las condiciones edáficas, formando las denominadas poblaciones alóctonas. Estas poblaciones, en algunos casos, se han señalado como poblaciones de (bradi)rizobios muy competitivas para la nodulación pero deficientes en la FBN, por lo cual impedirían el ingreso a los nódulos de los bradirrizobios inoculados durante la biofertilización. Esto, a su vez, explicaría por qué no se obtienen respuestas en el rendimiento luego de la biofertilización. A este fenómeno se lo ha denominado “el problema de la competición para nodulación”, el cual, por su extensión geográfica y ubicuidad en distintos agroecosistemas, constituye uno de los puntos clave de la mala performance general de la biofertilización. Por lo tanto, es necesario conocerlo y encarar mejoras para evitarlo. Como todo proceso biológico, la competición para la nodulación depende de diversos factores, entre ellos podemos mencionar las características propias de la población bacteriana del suelo y otros inherentes a la cepa inoculada, como así también factores ambientales y la posición de los (bradi)rizobios en el suelo. Con respecto al último punto, la movilidad de los (bradi)rizobios suele ser un punto en discusión y no está claro hasta qué punto este factor influye en la competitividad para nodular. B. japonicum, la especie utilizada generalmente en los inoculantes para soja, posee flagelación mixta, por lo cual es de esperar que los dos sistemas de flagelos tengan algún rol dentro del ciclo de vida de este microorganismo en el ambiente edáfico. En nuestro laboratorio ya se ha caracterizado la natación de esta cepa, pero dadas las condiciones del medio edáfico es posible que este bradirrizobio utilice allí un tipo de desplazamiento caracterizado por ser en superficie, el swarming. A partir de estos interrogantes se plantean los objetivos de esta Tesis: .Profundizar nuestra comprensión de las variables que condicionan y determinan el fenómeno de la competición para nodulación. .Evaluar las poblaciones alóctonas de (bradi)rizobios noduladores de soja presentes en suelos argentinos en base a su genotipo, su capacidad simbiótica, competitiva y de adaptación a su medio de origen. .Observar el swarming en B. japonicum, caracterizarlo en laboratorio y averiguar si este tipo de movilidad es el utilizado en el medio edáfico.Facultad de Ciencias Exacta