Aníbal R. Lodeiro

Comparison of drought tolerance in nitrogen-fixing and inorganic nitrogen-grown common beans.

In this work, we evaluated how the use of alternative N sources affects drought-stress tolerance in common beans. To this end, plants were cultivated employing either N(2) fixation or two levels of inorganic nitrogen: 1 mM NH(4)NO(3) (limiting) or 10 mM NH(4)NO(3) (sufficient). Drought was imposed by withholding watering at 30 days after planting (DAP) - coinciding with flowering. At 20 DAP, growth and N content were significantly higher in NH(4)NO(3)-sufficient plants than in N(2)-fixing and NH(4)NO(3)-limited beans. At later times, only N(2)-fixing and NH(4)NO(3)-sufficient plants continued assimilating N and growing, with the NH(4)NO(3)-sufficient plants being consistently bigger. After 10 days of stress (40 DAP), desiccation was evident, but only NH(4)NO(3)-sufficient plants suffered drought-induced senescence. After 20 days of stress (50 DAP), N content increased in NH(4)NO(3)-sufficient but not in N(2)-fixing beans, despite the latters lesser state of wilt. Pod dry weight dropped 43% in NH(4)NO(3)-sufficient beans with respect to well-watered plants, while remaining constant in N(2)-fixing beans. Under drought conditions, the number of pods limited pod yield regardless of the nitrogen source used; nevertheless, the translocation of soluble matter to pods continued in both NH(4)NO(3)-sufficient and N(2)-fixing beans. We conclude that common beans grown under conditions of N(2) fixation were more drought tolerant than those provided with sufficient levels of NH(4)NO(3). The most stress-sensitive traits in these plants were the incorporation of N into their shoots and the number of pods remaining on them.

The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia to plant roots but not in nodulation

The effect of the rhizobium adhesion protein RapA1 on Rhizobium leguminosarum bv. trifolii adsorption to Trifolium pratense (red clover) roots was investigated. We altered RapA1 production by cloning its encoding gene under the plac promoter into the stable vector pHC60. After introducing this plasmid in R. leguminosarum bv. trifolii, three to four times more RapA1 was produced, and two to five times higher adsorption to red clover roots was obtained, as compared with results for the empty vector. Enhanced adsorption was also observed on soybean and alfalfa roots, not related to R. leguminosarum cross inoculation groups. Although the presence of 1 mM Ca2+ during rhizobial growth enhanced adsorption, it was unrelated to RapA1 level. Similar effects were obtained when the same plasmid was introduced in Rhizobium etli for its adsorption to bean roots. Although root colonization by the RapA1-overproducing strain was also higher, nodulation was not enhanced. In addition, in vitro biofilm formation was similar to the wild-type both on polar and on hydrophobic surfaces. These results suggest that RapA1 receptors are present in root but not on inert surfaces, and that the function of this protein is related to rhizosphere colonization.

Effects of N-starvation and C-source on Bradyrhizobium japonicum exopolysaccharide production and composition, and bacterial infectivity to soybean roots

The exopolysaccharide (EPS) is an extracellular molecule that in Bradyrhizobium japonicum affects bacterial efficiency to nodulate soybean. Culture conditions such as N availability, type of C-source, or culture age can modify the amount and composition of EPS. To better understand the relationship among these conditions for EPS production, we analyzed their influence on EPS in B. japonicum USDA 110 and its derived mutant ΔP22. This mutant has a deletion including the 3′ region of exoP, exoT, and the 5′ region of exoB, and produces a shorter EPS devoid of galactose. The studies were carried out in minimal media with the N-source at starving or sufficient levels, and mannitol or malate as the only C-source. Under N-starvation there was a net EPS accumulation, the levels being similar in the wild type and the mutant with malate as the C-source. By contrast, the amount of EPS diminished in N-sufficient conditions, being poyhydroxybutyrate accumulated with culture age. Hexoses composition was the same in both N-situations, either with mannitol or malate as the only C-source, in contrast to previous observations made with different strains. This result suggests that the change in EPS composition in response to the environment is not general in B. japonicum. The wild type EPS composition was 1 glucose:0.5 galactose:0.5 galacturonic acid:0.17 mannose. In ΔP22 the EPS had no galactose but had galacturonic acid, thus indicating that it was not produced from oxidation of UDP-galactose. Infectivity was lower in ΔP22 than in USDA 110. When the mutant infectivity was compared between N-starved or N-sufficient cultures, the N-starved were not less infective, despite the fact that the amounts of altered EPS produced by this mutant under N-starvation were higher than in N-sufficiency. Since this altered EPS does not bind soybean lectin, the interaction of EPS with this protein was not involved in increasing ΔP22 infectivity under N-starvation.

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).

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 [(NH(4))(2)SO(4)] limited to only 20 microM. 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 (NH(4))(2)SO(4). 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.

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.

Swarming motility in Bradyrhizobium japonicum.

Flagellar-driven bacterial motility is an important trait for colonization of natural environments. Bradyrhizobium japonicum is a soil species that possesses two different flagellar systems: one subpolar and the other lateral, each with a filament formed by a different set of flagellins. While synthesis of subpolar flagellins is constitutive, translation of lateral flagellins was detected in rhizobia grown with l-arabinose, but not with d-mannitol as sole carbon source, independently of whether bacteria were in liquid or semisolid medium. We characterized swarming of B. japonicum in semisolid medium and found that this motility was faster with l-arabinose than with d-mannitol. By using mutants with deletions in each flagellin set, we evaluated the contribution of each flagellum system to swarming in semisolid culture media, and in soil. Mutants devoid of either of the flagella were affected in swarming in culture media, with this impairment being stronger for mutants without lateral flagella. In sterile soil at 100% or 80% field capacity, flagellar-driven motility of mutants able to swim but impaired in swarming was similar to wild type, indicating that swimming was the predominant movement here.

Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

Bradyrhizobium japonicum USDA 110 has five polyhydroxyalkanoate (PHA) synthases (PhaC) annotated in its genome: bll4360 (phaC1), bll6073 (phaC2), blr3732 (phaC3), blr2885 (phaC4), and bll4548 (phaC5). All these proteins possess the catalytic triad and conserved amino acid residues of polyester synthases and are distributed into four different PhaC classes. We obtained mutants in each of these paralogs and analyzed phaC gene expression and PHA production in liquid cultures. Despite the genetic redundancy, only phaC1 and phaC2 were expressed at significant rates, while PHA accumulation in stationary-phase cultures was impaired only in the ΔphaC1 mutant. Meanwhile, the ΔphaC2 mutant produced more PHA than the wild type under this condition, and surprisingly, the phaC3 transcript increased in the ΔphaC2 background. A double mutant, the ΔphaC2 ΔphaC3 mutant, consistently accumulated less PHA than the ΔphaC2 mutant. PHA accumulation in nodule bacteroids followed a pattern similar to that seen in liquid cultures, being prevented in the ΔphaC1 mutant and increased in the ΔphaC2 mutant in relation to the level in the wild type. Therefore, we used these mutants, together with a ΔphaC1 ΔphaC2 double mutant, to study the B. japonicum PHA requirements for survival, competition for nodulation, and plant growth promotion. All mutants, as well as the wild type, survived for 60 days in a carbon-free medium, regardless of their initial PHA contents. When competing for nodulation against the wild type in a 1:1 proportion, the ΔphaC1 and ΔphaC1 ΔphaC2 mutants occupied only 13 to 15% of the nodules, while the ΔphaC2 mutant occupied 81%, suggesting that the PHA polymer is required for successful competitiveness. However, the bacteroid content of PHA did not affect the shoot dry weight accumulation.

Swimming performance of Bradyrhizobium diazoefficiens is an emergent property of its two flagellar systems

Many bacterial species use flagella for self-propulsion in aqueous media. In the soil, which is a complex and structured environment, water is found in microscopic channels where viscosity and water potential depend on the composition of the soil solution and the degree of soil water saturation. Therefore, the motility of soil bacteria might have special requirements. An important soil bacterial genus is Bradyrhizobium, with species that possess one flagellar system and others with two different flagellar systems. Among the latter is B. diazoefficiens, which may express its subpolar and lateral flagella simultaneously in liquid medium, although its swimming behaviour was not described yet. These two flagellar systems were observed here as functionally integrated in a swimming performance that emerged as an epistatic interaction between those appendages. In addition, each flagellum seemed engaged in a particular task that might be required for swimming oriented toward chemoattractants near the soil inner surfaces at viscosities that may occur after the loss of soil gravitational water. Because the possession of two flagellar systems is not general in Bradyrhizobium or in related genera that coexist in the same environment, there may be an adaptive tradeoff between energetic costs and ecological benefits among these different species.

Competition for Nodulation

Nitrogen (N) is the nutrient that most often becomes limiting for plant growth. Soybean may obtain this nutrient from the air, thanks to its ability to perform a symbiosis with bacteria of the genera Bradyrhizobium (B. japonicum, B. elkanii, and B. liaoningense), Sinorhizobium (S. fredii and S. xinjiangense) and Mesorhizobium (M. tianshanense). These bacterial species are collectively known as soybean-nodulating rhizobia, but only B. japonicum, B. elkanii, and S. fredii were used as commercial inoculants for soybean crops, with B. japonicum being the most widely employed. In this symbiosis the rhizobial partner reduces the atmospheric N2 to NH3 in a reaction catalyzed by the nitrogenase enzymatic complex, while the plant partner supplies the C sources that provide the energy required for the N2 reduction reaction. Since atmospheric N2 is an unlimited source of N, the process of N2 fixation is of great potential for sustainable agriculture, and in the special case of legumes, the symbiosis is so efficient that in hydroponic culture the plant may satisfy all its N needs without resorting to any other N source. In addition, this symbiosis is a biological process that does not require fossil energy consumption, and does not leak any contaminant byproduct to the biosphere. Therefore, the inoculation of legume crops with selected rhizobial strains of high N2 fixation performance is an extended practice in agriculture since decades ago. In parallel, the industry of inoculants is very active, commercializing a variety of formulations with different strains and combinations with other plant-promoting rhizobacterial species such as Azospirillum brasilense or Pseudomonas fluorescens. For the farmers, inoculating a legume crop with active rhizobia is a simple procedure, and its economic cost is much lower than applying chemical fertilizers. All these advantages are, however, obscured by the fact that in field crops the symbiotic N2 fixation seldom provides the expected results, and the plants may consume the N from the soil. Several factors account for this low performance of N2 fixation in field crops. In energetic terms, N2 fixation is more costly for the plant than soil N uptake and therefore soil N is preferred when this source is not limiting, or when N2 fixation is inefficient (Salon et al., 2009). This may be appreciated if one takes into account that the symbiosis only occurs in a specialized organ known as root nodule. It is there where the rhizobia differentiate into the state able to reduce N2 −the bacteroid− and where the O2 concentration is lowered at levels compatible with nitrogenase activity (Patriarca et al., 2004). Therefore, rhizobia must infect the roots and trigger the development of nodules, which finally will be occupied by the rhizobia. During the earliest steps of nodule development and root infection (Ferguson et al., 2010), the plant-rhizobia relationship is more similar to a pathogenesis than to a mutualistic symbiosis: rhizobia invade plant tissues, consume plant energy resources,