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

Quality control of Bradyrhizobium inoculant strains: detection of nosZ and correlation of symbiotic efficiency with soybean leaf chlorophyll levels

Greenhouse gas emissions, such as N2O from excessive N-fertilizer use, are of concern. Symbiotic N2-fixation by pulses such as soybean might mitigate this issue, for which inoculants carrying locally adapted Bradyrhizobium strains are recommended. In the frame of this goal, enhancing the quality control of these inoculants is required on two key aspects: determining the presence of nosZ, for the strains being able to reduce N2O, and assessing N2-fixation potential. Previously it was demonstrated that, in soybean leaves, N-contents are well correlated with chlorophyll contents. However, no such correlations were made with either N obtained from N2-fixation or with nodules mass, which is an indicator of nodular activity. Here we aimed to leverage the correlation between N and chlorophyll levels to develop a simple and non-destructive laboratory method to be applied in quality control of inoculants, able to assess the N2-fixing capacity of rhizobial strains. To establish such correlations, we cultivated soybeans in vermiculite with N-free nutrient solution, and inoculated them with various Bradyrhizobium field isolates that displayed a range of symbiotic N2-fixing capacities. Subsequently, we measured chlorophyll with a portable chlorophyllometer, and correlated these measures with symbiotic parameters. Moreover, we tested for the presence of nosZ by PCR. We observed significant correlations between chlorophyll and shoot nitrogen obtained from N2-fixation and, in addition, we corroborated that chlorophyll contents were significantly correlated also with nodules mass. Two B. diazoefficiens strains stood out and possessed nosZ. In contrast, B. elkanii and B. japonicum isolates displayed lower chlorophyll and symbiotic performance, and lacked nosZ. Our findings highlight the potential of measuring chlorophyll contents and testing for the presence of nosZ as two straightforward techniques that may enhance laboratory tests for quality control, enabling selection of superior and safe locally isolated strains for soybean inoculants without increased production costs

Soybean Lectin Enhances Biofilm Formation by Bradyrhizobium japonicum in the Absence of Plants

Soybean lectin (SBL) purified from soybean 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

A Rhizobium leguminosarum CHDL- (Cadherin-Like-) Lectin Participates in Assembly and Remodeling of the Biofilm Matrix

In natural environments most bacteria live in multicellular structures called biofilms. These cell aggregates are enclosed in a self-produced polymeric extracellular matrix, which protects the cells, provides mechanical stability and mediates cellular cohesion and adhesion to surfaces. Although important advances were made in the identification of the genetic and extracellular factors required for biofilm formation, the mechanisms leading to biofilm matrix assembly, and the roles of extracellular proteins in these processes are still poorly understood. The symbiont Rhizobium leguminosarum requires the synthesis of the acidic exopolysaccharide and the PrsDE secretion system to develop a mature biofilm. PrsDE is responsible for the secretion of the Rap family of proteins that share one or two Ra/CHDL (cadherin-like-) domains. RapA2 is a calcium-dependent lectin with a cadherin-like β sheet structure that specifically recognizes the exopolysaccharide, either as a capsular polysaccharide (CPS) or in its released form [extracellular polysaccharide (EPS)]. In this study, using gain and loss of function approaches combined with phenotypic and microscopic studies we demonstrated that RapA lectins are involved in biofilm matrix development and cellular cohesion. While the absence of any RapA protein increased the compactness of bacterial aggregates, high levels of RapA1 expanded distances between cells and favored the production of a dense matrix network. Whereas endogenous RapA(s) are predominantly located at one bacterial pole, we found that under overproduction conditions, RapA1 surrounded the cell in a way that was reminiscent of the capsule. Accordingly, polysaccharide analyses showed that the RapA lectins promote CPS formation at the expense of lower EPS production. Besides, polysaccharide analysis suggests that RapA modulates the EPS size profile. Collectively, these results show that the interaction of RapA lectins with the polysaccharide is involved in rhizobial biofilm matrix assembly and remodeling.Fil: Vozza, Nicolas Federico. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Abdian, Patricia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Russo, Daniela Marta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mongiardini, Elias Javier. 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: Lodeiro, Anibal. 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: Molin, Soren. Technical University of Denmark; DinamarcaFil: Zorreguieta, Ángeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Induction by Bradyrhizobium diazoefficiens of Different Pathways for Growth in D-mannitol or L-arabinose Leading to Pronounced Differences in CO2 Fixation, O2 Consumption, and Lateral-Flagellum Production

Bradyrhizobium diazoefficiens, a soybean N2-fixing symbiont, constitutes the basic input in one of the most prominent inoculant industries worldwide. This bacterium may be cultured with D-mannitol or L-arabinose as carbon-plus-energy source (C-source) with similar specific growth rates, but with higher biomass production with D-mannitol. To better understand the bacterium’s carbon metabolism, we analyzed, by liquid chromatography and tandem mass spectrometry (MS), the whole set of proteins obtained from cells grown on each C-source. Among 3,334 proteins identified, 266 were overproduced in D-mannitol and 237 in L-arabinose, but among these, only 22% from D-mannitol cultures and 35% from L-arabinose cultures were annotated with well defined functions. In the D-mannitol-differential pool we found 19 enzymes of the pentose-phosphate and Calvin–Benson–Bassham pathways and accordingly observed increased extracellular-polysaccharide production by D-mannitol grown bacteria in a CO2-enriched atmosphere. Moreover, poly-3-hydroxybutyrate biosynthesis was increased, suggesting a surplus of reducing power. In contrast, the L-arabinose-differential pool contained 11 enzymes of the L-2-keto-3-deoxyarabonate pathway, 4 enzymes for the synthesis of nicotinamide-adenine dinucleotide from aspartate, with those cultures having a threefold higher O2-consumption rate than the D-mannitol cultures. The stoichiometric balances deduced from the modeled pathways, however, resulted in similar O2 consumptions and ATP productions per C-mole of substrate. These results suggested higher maintenance-energy demands in L-arabinose, which energy may be used partly for flagella-driven motility. Since B. diazoefficiens produces the lateral-flagella system in only L-arabinose, we calculated the O2-consumption rates of a lafR::Km mutant devoid of lateral flagella cultured in L-arabinose or D-mannitol. Contrary to that of the wild-type, the O2-consumption rate of this mutant was similar on both C-sources, and accordingly outcompeted the wild-type in coculture, suggesting that the lateral flagella behaved as parasitic structures under these conditions. Proteomic data are available via ProteomeXchange with identifier PXD008263

Phylogenomic Analyses of <i>Bradyrhizobium</i> Reveal Uneven Distribution of the Lateral and Subpolar Flagellar Systems, Which Extends to <i>Rhizobiales</i>

Dual flagellar systems have been described in several bacterial genera, but the extent of their prevalence has not been fully explored. Bradyrhizobium diazoefficiens USDA 110T possesses two flagellar systems, the subpolar and the lateral flagella. The lateral flagellum of Bradyrhizobium displays no obvious role, since its performance is explained by cooperation with the subpolar flagellum. In contrast, the lateral flagellum is the only type of flagella present in the related Rhizobiaceae family. In this work, we have analyzed the phylogeny of the Bradyrhizobium genus by means of Genome-to-Genome Blast Distance Phylogeny (GBDP) and Average Nucleotide Identity (ANI) comparisons of 128 genomes and divided it into 13 phylogenomic groups. While all the Bradyrhizobium genomes encode the subpolar flagellum, none of them encodes only the lateral flagellum. The simultaneous presence of both flagella is exclusive of the B. japonicum phylogenomic group. Additionally, 292 Rhizobiales order genomes were analyzed and both flagellar systems are present together in only nine genera. Phylogenetic analysis of 150 representative Rhizobiales genomes revealed an uneven distribution of these flagellar systems. While genomes within and close to the Rhizobiaceae family only possess the lateral flagellum, the subpolar flagellum is exclusive of more early-diverging families, where certain genera also present both flagella

Pleiotropic Effects of PhaR Regulator in <i>Bradyrhizobium diazoefficiens</i> Microaerobic Metabolism

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia