Rhizophagus intraradices and Azospirillum brasilense improve growth of herbaceous plants and soil biological activity in revegetation of a recovering coal-mining area.

We assessed, in a field experiment, the effects of arbuscular mycorrhizal fungi (Rhizophagus intraradices) and plant growth-promoting bacteria (Azospirillum brasilense) on the soil biological activity and the growth of key pioneer species used in the revegetation of coal-mining areas undergoing recovery. We applied four inoculation treatments to the pioneer plant species (Lablab purpureus, Paspalum notatum, Crotalaria juncea, Neonotonia wightii, Stylosanthes guianensis, Andropogon gayanus and Trifolium repens) used in the recovery process: NI (Control - Non-inoculated), AZO (A. brasilense), AMF (R. intraradices), and co-inoculation of AZO and AMF. On the 75th and 180th days, we measured plant dry mass, mycorrhizal colonization, N and P concentration, and accumulation in plant tissue. We collected soil to quantify glomalin content and soil enzyme activity. After 180 days, we did a phytosociological characterization of the remaining spontaneous plants.The both microorganisms, singly or co-inoculated, promoted increases in different fractions of soil glomalin, acid phosphatase activity, and fluorescein diacetate activity at 75 and 180 days. The inoculation was linked to higher plant biomass production (62-89%) and increased plant P and N accumulation by 34-75% and 70-85% at 180 days, compared with the non-inoculated treatment. Among the pioneer species sown Crotalaria juncea produced the highest biomass at the 75th and 180th days (67% and 76% of all biomass), followed by Lablab purpureus (3% and 0.5%), while the other species failed to establish. At 180 days, we observed twenty spontaneous plant species growing in the area, primarily from the Poaceae family (74%). That suggests that the pioneer species present in the area do not hinder the ecological succession process. Inoculation of R. intraradices and A. brasilense, isolated or combined, increases soil biological activity, growth, and nutrient accumulation in key pioneer plant species, indicating the potential of that technique for the recovery of lands degraded by coal mining.

Plant seedlings of peas, tomatoes, and cucumbers exude compounds that are needed for growth and chemoattraction of Rhizobium leguminosarum bv. viciae 3841 and Azospirillum brasilense Sp7.

This study characterizes seedling exudates of peas, tomatoes, and cucumbers at the level of chemical composition and functionality. A plant experiment confirmed that Rhizobium leguminosarum bv. viciae 3841 enhanced growth of pea shoots, while Azospirillum brasilense Sp7 supported growth of pea, tomato, and cucumber roots. Chemical analysis of exudates after 1 day of seedling incubation in water yielded differences between the exudates of the three plants. Most remarkably, cucumber seedling exudate did not contain detectable sugars. All exudates contained amino acids, nucleobases/nucleosides, and organic acids, among other compounds. Cucumber seedling exudate contained reduced glutathione. Migration on semi solid agar plates containing individual exudate compounds as putative chemoattractants revealed that R. leguminosarum bv. viciae was more selective than A. brasilense, which migrated towards any of the compounds tested. Migration on semi solid agar plates containing 1:1 dilutions of seedling exudate was observed for each of the combinations of bacteria and exudates tested. Likewise, R. leguminosarum bv. viciae and A. brasilense grew on each of the three seedling exudates, though at varying growth rates. We conclude that the seedling exudates of peas, tomatoes, and cucumbers contain everything that is needed for their symbiotic bacteria to migrate and grow on.

Thermal and salt stress effects on the survival of plant growth-promoting bacteria Azospirillum brasilense in inoculants for maize cultivation.

BACKGROUND: The plant growth-promoting bacteria (PGPB) Azospirillum brasilense is widely used as an inoculant for important grass crops, providing numerous benefits to the plants. However, one limitation to develop viable commercial inoculants is the control of PGPB survival, requiring strategies that guarantee their survival during handling and field application. The application of sublethal stress appears to be a promising strategy to increase bacterial cells tolerance to adverse environmental conditions since previous stress induces the activation of physiological protection in bacterial cell. In this work, we evaluated the effects of thermal and salt stresses on the survival of inoculant containing A. brasilense Ab-V5 and Ab-V6 strains and we monitored A. brasilense viability in inoculated maize roots after stress treatment of inoculant. RESULTS: Thermal stress application (> 35 °C) in isolated cultures for both strains, as well as salt stress [sodium chloride (NaCl) concentrations > 0.3 mol L-1], resulted in growth rate decline. The A. brasilense enumeration in maize roots obtained by propidium monoazide quantitative polymerase chain reaction (PMA-qPCR), for inoculated maize seedlings grown in vitro for 7 days, showed that there is an increased number of viable cells after the salt stress treatment, indicating that A. brasilense Ab-V5 and Ab-V6 strains are able to adapt to salt stress (0.3 mol L-1 NaCl) growth conditions. CONCLUSION: Azospirillum brasilense Ab-V5 and Ab-V6 strains had potential for osmoadaptation and salt stress, resulting in increased cell survival after inoculation in maize plants. © 2024 Society of Chemical Industry.

Azospirillum brasilense Az39 restricts cadmium entrance into wheat plants and mitigates cadmium stress.

In this work, we tested if inoculation with the plant growth-promoting rhizobacteria Azospirillum brasilense strain Az39 alleviates Cd2+ stress in wheat seedlings grown under controlled conditions. Growth, total N, N-related metabolites/enzymes, and oxidative stress parameters were measured. Additionally, the usefulness of a real-time PCR protocol to screen the preferred colonization site of the introduced microorganism was evaluated. Inoculated plants demonstrated mitigation of cadmium-induced adverse effects on plant growth and less reactive oxygen species accumulation in their roots by the end of the experiment, 28 days after sowing. Cd addition resulted in lower NO3- content in the leaves and higher NO3- content in the roots, and a significant rise in NH4+ concentration in both organs in uninoculated plants; in inoculated plants, NH4+ content in the roots did not vary. A. brasilense Az39 enhanced NO levels in wheat root tips, and more adventitious roots and root hairs were observed in inoculated plants. Despite having a more developed root system, inoculated plants showed lower Cd levels in their roots compared to non-inoculated plants. Inoculation with this PGPR favored ion homeostasis in the roots of metal-exposed plants, decreasing Cd/Fe ratio. We corroborated A. brasilense Az39 preference for wheat exorhizosphere using a real-time PCR-based method targeting the nifA gene.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

Localization and survival of Azospirillum brasilense Az39 in soybean leaves.

In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial micro-organisms to crops under field conditions. Colonization efficiency by such micro-organisms largely depends on their ability to survive when applied on the leaves. In this work, we evaluated the survival and localization of Azospirillum brasilense Az39 (Az39) in excised soybean leaves. Scanning electron microscopy and confocal laser scanning microscopy of a red fluorescent-transformed variant of Az39 were used to determine bacterial localization, while the most probable number and plate count methods were applied for bacterial quantification. Microscopic observations indicated a decrease in the number of Az39 cells on the leaf surface at 24 h after treatment, whereas midribs and cell-cell junctions of the inner leaf epidermis became highly populated zones. The presence of Az39 inside xylem vessels was corroborated at 6 h after bacterization. Az39 population did not significantly decrease throughout 24 h. We could visualize Az39 cells on the surface and in internal tissues of soybean leaves and recover them through culture methodologies. These results evidence the survival capacity of Az39 on and inside leaves and suggest a previously unnoticed endophytic potential for this well-known plant growth-promoting rhizobacteria strain.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense.

Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

Evaluation of growth and motility in non-photosynthetic Azospirillum brasilense exposed to red, blue, and white light.

Azospirillum brasilense is a non-photosynthetic rhizobacterium that promotes the growth of plants. In this work, we evaluated the effects of different light qualities on the growth, viability, and motility in combination to other culture conditions such as temperature or composition of the culture medium. Exponential cultures of A. brasilense Az39 were inoculated by drop-plate method on nutritionally rich (LB) or chemically defined (MMAB) media in the presence or absence of Congo Red indicator (CR) and exposed continuously to white light (WL), blue light (BL), and red light (RL), or maintained in dark conditions (control). The exposure to BL or WL inhibited growth, mostly in LB medium at 36 °C. By contrast, the exposure to RL showed a similar behavior to the control. Swimming motility was inhibited by exposure to WL and BL, while exposure to RL caused only a slight reduction. The effects of WL and BL on plant growth-promoting rhizobacteria should be considered in the future as deleterious factors that could be manipulated to improve the functionality of foliar inoculants, as well as the bacterial effects on the leaf after inoculation.

Nitrogen fertilization and leaf spraying with Azospirillum brasilense in wheat: effects on mineral nutrition and yield / Adubação nitrogenada e pulverização foliar de Azospirillum brasilense em trigo: efeitos na nutrição mineral e produtividade

The use of Azospirillum brasilense has the potential to improve plant nitrogen (N) use efficiency, while a better understanding of alternative management practices with inoculation is necessary. The aim was to examine the effects of the leaf application of A. brasilense in association with nitrogen fertilization on the wheat crop. The experiment was conducted in Lidianópolis, Paraná, Brazil, in a completely randomized block design with four replications. The treatments included four doses of A. brasilense for leaf application (0, 200, 400, and 600 ml ha-1) and four doses of N (0, 40, 80, and 120 kg N ha-1). The nutritional content, yield components, quality, and yield of the wheat crop were evaluated. There was no interaction among the factors, nor did the treatments have an isolated effect on spike length, the number of spikelets per spike, spikes per m2, thousand grain weight, and test weight. However, doses of A. brasilense increased calcium and magnesium absorption at 283 and 380 ml ha-1, respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha-1, respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha-1, respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf.

O uso de Azospirillum brasilense apresenta potencial em melhorar a eficiência de uso do nitrogênio (N), sendo necessário melhor compreensão de formas alternativas de inoculação, visto que o tratamento químico de sementes pode comprometer a eficiência das bactérias. O objetivo foi avaliar os efeitos do A. brasilense aplicado via foliar associado a adubação nitrogenada na cultura do trigo. O experimento foi implantado em Lidianópolis, no estado do Paraná, Brasil, em blocos completos com tratamentos ao acaso e quatro repetições, sendo os tratamentos: quatro doses de A. brasilense via foliar (0, 200, 400 e 600 ml ha-1) e quatro doses de N (0, 40, 80 e 120 kg N ha-1). Foram avaliados os teores nutricionais, componentes de rendimento, qualidade e produtividade. Não houve interação entre os fatores, tampouco efeito isolado dos tratamentos para comprimento da espiga, número de espiguetas por espiga, espigas por m², massa de mil grãos e peso de hectolitro. Todavia as doses de A. brasilense, aumentaram a absorção de Ca e Mg até a dose de 283 e 380 ml ha-1, respectivamente. De modo similar, o N aplicado aumentou os teores foliares de cálcio, magnésio, além de cobre até a dose de 61, 47 e 49 kg ha-1 de N, respectivamente. O N também incrementou o número de grãos por espiga e a produtividade até a dose 56 e 54 kg N ha-1, respectivamente, porém a eficiência de uso

Nitrogen fertilization and leaf spraying with Azospirillum brasilense in wheat: effects on mineral nutrition and yield / Adubação nitrogenada e pulverização foliar de Azospirillum brasilense em trigo: efeitos na nutrição mineral e produtividade

The use of Azospirillum brasilense has the potential to improve plant nitrogen (N) use efficiency, while a better understanding of alternative management practices with inoculation is necessary. The aim was to examine the effects of the leaf application of A. brasilense in association with nitrogen fertilization on the wheat crop. The experiment was conducted in Lidianópolis, Paraná, Brazil, in a completely randomized block design with four replications. The treatments included four doses of A. brasilense for leaf application (0, 200, 400, and 600 ml ha-1) and four doses of N (0, 40, 80, and 120 kg N ha-1). The nutritional content, yield components, quality, and yield of the wheat crop were evaluated. There was no interaction among the factors, nor did the treatments have an isolated effect on spike length, the number of spikelets per spike, spikes per m2, thousand grain weight, and test weight. However, doses of A. brasilense increased calcium and magnesium absorption at 283 and 380 ml ha-1, respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha-1, respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha-1, respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf.(AU)

O uso de Azospirillum brasilense apresenta potencial em melhorar a eficiência de uso do nitrogênio (N), sendo necessário melhor compreensão de formas alternativas de inoculação, visto que o tratamento químico de sementes pode comprometer a eficiência das bactérias. O objetivo foi avaliar os efeitos do A. brasilense aplicado via foliar associado a adubação nitrogenada na cultura do trigo. O experimento foi implantado em Lidianópolis, no estado do Paraná, Brasil, em blocos completos com tratamentos ao acaso e quatro repetições, sendo os tratamentos: quatro doses de A. brasilense via foliar (0, 200, 400 e 600 ml ha-1) e quatro doses de N (0, 40, 80 e 120 kg N ha-1). Foram avaliados os teores nutricionais, componentes de rendimento, qualidade e produtividade. Não houve interação entre os fatores, tampouco efeito isolado dos tratamentos para comprimento da espiga, número de espiguetas por espiga, espigas por m², massa de mil grãos e peso de hectolitro. Todavia as doses de A. brasilense, aumentaram a absorção de Ca e Mg até a dose de 283 e 380 ml ha-1, respectivamente. De modo similar, o N aplicado aumentou os teores foliares de cálcio, magnésio, além de cobre até a dose de 61, 47 e 49 kg ha-1 de N, respectivamente. O N também incrementou o número de grãos por espiga e a produtividade até a dose 56 e 54 kg N ha-1, respectivamente, porém a eficiência de uso(AU)

Valorization of Lignin as an Immobilizing Agent for Bioinoculant Production using Azospirillum brasilense as a Model Bacteria.

Plant growth-promoting bacteria (PGPB) have been largely considered as beneficial in harsh and limiting environments given their effects on alleviating plant stress. For practical applications, most of the PGPB are prepared in immobilization matrices to improve the stability and benefits of bacteria. Despite the long list of immobilizing agents/carriers tested to date, a long list of desired requirements is yet to be achieved. Here, lignin stands as a scarcely tested immobilizer for bioinoculants with great potential for this purpose. The aim of this work was to demonstrate the feasibility of lignin as a carrier of the nitrogen-fixing Azospirillum brasilense. These bacteria were cultured in liquid media with recovered organosolv lignin added for bacterial immobilization. Then, lignin was recovered and the immobilized biomass was quantified gravimetrically by DNA extraction and serial dilution plating. Fluorescent microscopy as well as Congo red agar plating showed the immobilization of the bacterial cells in the lignin matrix and crystal violet dyeing showed the biofilms formation in lignin particles. A high number of cells were counted per gram of dried lignin. Lignin can be readily used as low-cost, health-safe bioinoculant carrier to be used in soil and agricultural applications.

Modeling aerotaxis band formation in Azospirillum brasilense.

BACKGROUND: Bacterial chemotaxis, the ability of motile bacteria to navigate gradients of chemicals, plays key roles in the establishment of various plant-microbe associations, including those that benefit plant growth and crop productivity. The motile soil bacterium Azospirillum brasilense colonizes the rhizosphere and promotes the growth of diverse plants across a range of environments. Aerotaxis, or the ability to navigate oxygen gradients, is a widespread behavior in bacteria. It is one of the strongest behavioral responses in A. brasilense and it is essential for successful colonization of the root surface. Oxygen is one of the limiting nutrients in the rhizosphere where density and activity of organisms are greatest. The aerotaxis response of A. brasilense is also characterized by high precision with motile cells able to detect narrow regions in a gradient where the oxygen concentration is low enough to support their microaerobic lifestyle and metabolism. RESULTS: Here, we present a mathematical model for aerotaxis band formation that captures most critical features of aerotaxis in A. brasilense. Remarkably, this model recapitulates experimental observations of the formation of a stable aerotactic band within 2 minutes of exposure to the air gradient that were not captured in previous modeling efforts. Using experimentally determined parameters, the mathematical model reproduced an aerotactic band at a distance from the meniscus and with a width that matched the experimental observation. CONCLUSIONS: Including experimentally determined parameter values allowed us to validate a mathematical model for aerotactic band formation in spatial gradients that recapitulates the spatiotemporal stability of the band and its position in the gradient as well as its overall width. This validated model also allowed us to capture the range of oxygen concentrations the bacteria prefer during aerotaxis, and to estimate the effect of parameter values (e.g. oxygen consumption rate), both of which are difficult to obtain in experiments.

Azospirillum brasilense promotes increases in growth and nitrogen use efficiency of maize genotypes.

The development of cultivars with an improved nitrogen use efficiency (NUE) together with the application of plant growth-promoting bacteria is considered one of the main strategies for reduction of fertilizers use. In this sense, this study: i) evaluated the effect of Azospirillum brasilense on the initial development of maize genotypes; ii) investigated the influence of A. brasilense inoculation on NUE under nitrogen deficit; and iii) sought for more NUE genotypes with higher responsiveness to A. brasilense inoculation. Twenty-seven maize genotypes were evaluated in three independent experiments. The first evaluated the initial development of maize genotypes with and without A. brasilense (strain Ab-V5) inoculation of seeds on germination paper in a growth chamber. The second and third experiments were carried out in a greenhouse using Leonard pots and pots with substrate, respectively, and the genotypes were evaluated at high nitrogen, low nitrogen and low nitrogen plus A. brasilense Ab-V5 inoculation. The inoculation of seeds with A. brasilense Ab-V5 intensified plant growth, improved biochemical traits and raised NUE under nitrogen deficit. The inoculation of seeds with A. brasilense can be considered an economically viable and environmentally sustainable strategy for maize cultivation.

Polar flagellum of the alphaproteobacterium Azospirillum brasilense Sp245 plays a role in biofilm biomass accumulation and in biofilm maintenance under stationary and dynamic conditions.

Bacteria Azospirillum brasilense may swim and swarm owing to the rotation of a constitutive polar flagellum (Fla) and inducible lateral flagella (Laf). They also construct sessile biofilms on various interfaces. As compared to the wild-type strain Sp245, the previously characterized Fla- Laf- flhB1 mutant Sp245.1063 accumulated less biomass in mature biofilms, which also were susceptible to the forces of hydrodynamic shear. In this study, we compared biofilms formed by strain Sp245 and its previously constructed derivatives on the interfaces between a minimal (malate-salt medium, or MSM) or rich (LB) liquid growth medium and a hydrophilic (glass) or hydrophobic (polystyrene) solid surface under static or dynamic conditions. In all experimental settings, the alterations in Sp245.1063's mature biofilm traits were partially (in MSM) or completely (in LB) rescued in the complemented mutant Sp245.1063 (pRK415-150177), which received the pRK415-borne coding sequence for the putative FlhB1 protein of the flagellar type III secretion system. Although Laf were not found in the biofilms of azospirilla, Fla was present on the biofilm cells of the complemented mutant Sp245.1063 (pRK415-150177) and other studied strains, which had normal flagellation on liquid and solid nutritional media. Accordingly, mature biofilms of these strains contained more biomass and were significantly more resistant to shaking at 140 rpm, as compared to the biofilms of the flagella-free mutant bacteria. These data proved that the polar flagellum of A. brasilense Sp245 plays a significant positive role in biofilm biomass increase and in biofilm stabilization.

The ammonium transporter AmtB and the PII signal transduction protein GlnZ are required to inhibit DraG in Azospirillum brasilense.

The ammonium-dependent posttranslational regulation of nitrogenase activity in Azospirillum brasilense requires dinitrogenase reductase ADP-ribosyl transferase (DraT) and dinitrogenase reductase ADP-glycohydrolase (DraG). These enzymes are reciprocally regulated by interaction with the PII proteins, GlnB and GlnZ. In this study, purified ADP-ribosylated Fe-protein was used as substrate to study the mechanism involved in the regulation of A. brasilense DraG in vitro. The data show that DraG is partially inhibited by GlnZ and that DraG inhibition is further enhanced by the simultaneous presence of GlnZ and AmtB. These results are the first to demonstrate experimentally that DraG inactivation requires the formation of a ternary DraG-GlnZ-AmtB complex in vitro. Previous structural data have revealed that when the DraG-GlnZ complex associates with AmtB, the flexible T-loops of the trimeric GlnZ bind to AmtB and become rigid; these molecular events stabilize the DraG-GlnZ complex, resulting in DraG inactivation. To determine whether restraining the flexibility of the GlnZ T-loops is a limiting factor in DraG inhibition, we used a GlnZ variant that carries a partial deletion of the T-loop (GlnZΔ42-54). However, although the GlnZΔ42-54 variant was more effective in inhibiting DraG in vitro, it bound to DraG with a slightly lower affinity than does wild-type GlnZ and was not competent to completely inhibit DraG activity either in vitro or in vivo. We, therefore, conclude that the formation of a ternary complex between DraG-GlnZ-AmtB is necessary for the inactivation of DraG.

Development of biodegradable coatings for maize seeds and their application for Azospirillum brasilense immobilization.

The objective of this study was to develop biodegradable coatings for agriculture crop seeds based on starch, gelatin, and polyvinyl alcohol (PVA). Developed materials were characterized according to their microstructures, barrier properties, influence on germination of maize seeds, and ability to sustain Azospirillum brasilense Ab-V5 viability in coated maize seeds. The coatings were obtained employing different proportions of starch, gelatin, and PVA, ranging from 0 to 3.0 g/100 g of each material, respectively. Samples formulated with the pure polymers showed the highest values of water absorption capacity, solubility, and water vapor permeability, and the ternary mixtures showed the lowest values. Single polymer formulations and the binary starch-gelatin mixture (CS50GL50) favored maize seeds germination compared to the uncoated maize seeds. In addition, seed coating obtained from CS50GL50 formulation resulted in A. brasilense Ab-V5 viability in coated seeds up to 15 days after bacterial immobilization, being considered a promising low-cost, biodegradable, and renewable source material to be used in agriculture.

Can co-inoculation of Bradyrhizobium and Azospirillum alleviate adverse effects of drought stress on soybean (Glycine max L. Merrill.)?

Harnessing the beneficial potential of plant growth-promoting rhizobacteria may be an alternative strategy to improve plant tolerance to drought stress. The effect of inoculation with Bradyrhizobium japonicum and Azospirillum brasilense either alone or in combination on the plant growth and drought tolerance of soybean [Glycine max (L.) Merrill.] was investigated in this study in greenhouse conditions. Treatments were arranged in a randomized block design in a 3 × 4 factorial: three irrigation regimes [100% of pot capacity-PC (well-watered control), 50% of PC (moderate stress) and 25% of PC (severe stress)] and four inoculation treatments [control (non-inoculated), inoculation with B. japonicum, inoculation with A. brasilense, and co-inoculation with B. japonicum and A. brasilense]. Leaf relative water content, cell membrane stability, root nodulation, plant growth, and morphophysiological indexes were recorded. The inoculation of soybean plants with B. japonicum and A.brasilense either alone or in combination improved leaf membrane stability under drought stress conditions when compared to non-inoculated plants; however, this lower damage to cell membranes was not sufficient to maintain the leaf water content of the plant under drought stress. Plants co-inoculated with B. japonicum and A.brasilense improved the root nodulation under severe drought conditions. Inoculation of B. japonicum and A. brasilense either alone or in combination reduced the pod abortion rate under moderate drought stress, but had no effect under severe drought stress. In summary, the co-inoculation of A. brasilense and B. japonicum alleviate adverse effects limited by drought stress to the growth of soybeans.Author: Please check and confirm that the authors [Elijanara Raissa Silva, Carlos Eduardo Silva Oliveira, Alan Mario Zuffo, Eduardo Pradi Vendruscolo] and their initials have been correctly identified and amend if necessary.The authors were correctly identified.

Quorum sensing communication: Bradyrhizobium-Azospirillum interaction via N-acyl-homoserine lactones in the promotion of soybean symbiosis.

Quorum-sensing (QS) mechanisms are important in intra- and inter-specific communication among bacteria. We investigated QS mechanisms in Bradyrhizobium japonicum strain CPAC 15 and Azospirillum brasilense strains Ab-V5 and Ab-V6, used in commercial co-inoculants for the soybean crop in Brazil. A transconjugant of CPAC 15-QS with partial inactivation of N-acyl-homoserine lactones (AHLs) was obtained and several parameters were evaluated; in vitro, CPAC 15 and the transconjugant differed in growth, but not in biofilm formation, and no differences were observed in the symbiotic performance in vivo. The genome of CPAC 15 carries functional luxI and luxR genes and low amounts of three AHL molecules were detected: 3-OH-C12-AHL, 3-OH-C14-AHL, and 3-oxo-C14-AHL. Multiple copies of luxR-like genes, but not of luxI are present in the genomes of Ab-V5 and Ab-V6, and differences in gene expression were observed when the strains were co-cultured with B. japonicum; we may infer that the luxR-genes of A. brasilense may perceive the AHL molecules of B. japonicum. Soybean symbiotic performance was improved especially by co-inoculation with Ab-V6, which, contrarily to Ab-V5, did not respond to the AHLs of CPAC 15. We concluded that A. brasilense Ab-V5, but not Ab-V6, responded to the QS signals of CPAC 15, and that the synergistic interaction may be credited, at least partially, to the QS interaction. In addition, we confirmed inter- and intra-species QS communication between B. japonicum and A. brasilense and, for Azospirillum, at the strain level, impacting several steps of the symbiosis, from cell growth to plant nodulation and growth.