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1.
Can J Microbiol ; 70(5): 150-162, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38427979

ABSTRACT

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.


Subject(s)
Azospirillum brasilense , Cucumis sativus , Pisum sativum , Rhizobium leguminosarum , Seedlings , Solanum lycopersicum , Solanum lycopersicum/microbiology , Solanum lycopersicum/growth & development , Cucumis sativus/microbiology , Cucumis sativus/growth & development , Seedlings/growth & development , Seedlings/microbiology , Rhizobium leguminosarum/growth & development , Rhizobium leguminosarum/metabolism , Azospirillum brasilense/growth & development , Azospirillum brasilense/metabolism , Pisum sativum/microbiology , Pisum sativum/growth & development , Plant Roots/microbiology , Plant Roots/growth & development , Chemotaxis , Plant Exudates/chemistry , Plant Exudates/metabolism
2.
J Sci Food Agric ; 104(9): 5360-5367, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38324183

ABSTRACT

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.


Subject(s)
Agricultural Inoculants , Azospirillum brasilense , Hot Temperature , Plant Roots , Salt Stress , Zea mays , Zea mays/microbiology , Zea mays/growth & development , Azospirillum brasilense/physiology , Azospirillum brasilense/growth & development , Plant Roots/microbiology , Plant Roots/growth & development , Agricultural Inoculants/physiology , Microbial Viability , Soil Microbiology , Seedlings/microbiology , Seedlings/growth & development
3.
Plant Sci ; 312: 111056, 2021 Nov.
Article in English | MEDLINE | ID: mdl-34620450

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Cadmium/adverse effects , Plant Roots/growth & development , Plant Roots/microbiology , Symbiosis/physiology , Triticum/growth & development , Triticum/microbiology , Seedlings/growth & development , Seedlings/microbiology
4.
Appl Environ Microbiol ; 87(14): e0058221, 2021 06 25.
Article in English | MEDLINE | ID: mdl-33962983

ABSTRACT

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.


Subject(s)
Ammonia/metabolism , Azospirillum brasilense/metabolism , Glutamate-Ammonia Ligase/metabolism , Setaria Plant/microbiology , Azospirillum brasilense/genetics , Azospirillum brasilense/growth & development , Pheophytins/metabolism , Protein Processing, Post-Translational , Setaria Plant/growth & development , Symbiosis
5.
Lett Appl Microbiol ; 72(5): 626-633, 2021 May.
Article in English | MEDLINE | ID: mdl-33354785

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Glycine max/microbiology , Plant Leaves/microbiology , Microscopy, Confocal , Microscopy, Electron, Scanning , Plant Roots/microbiology
6.
Sci Rep ; 10(1): 20408, 2020 11 23.
Article in English | MEDLINE | ID: mdl-33230149

ABSTRACT

Continuous cropping lowers the production and quality of ramie (Boehmeria nivea L. Gaud). This study aimed to reveal the metagenomic and metabolomic changes between the healthy- and obstacle-plant after a long period of continuous cropping. After 10 years of continuous cropping, ramie planted in some portions of the land exhibited weak growth and low yield (Obstacle-group), whereas, ramie planted in the other portion of the land grew healthy (Health-group). We collected rhizosphere soil and root samples from which measurements of soil chemical and plant physiochemical properties were taken. All samples were subjected to non-targeted gas chromatograph-mass spectrometer (GS/MS) metabolome analysis. Further, metagenomics was performed to analyze the functional genes in rhizospheric soil organisms. Based on the findings, ramie in Obstacle-group were characterized by shorter plant height, smaller stem diameter, and lower fiber production than that in Health-group. Besides, the Obstacle-group showed a lower relative abundance of Rhizobiaceae, Lysobacter antibioticus, and Bradyrhizobium japonicum, but a higher relative abundance of Azospirillum lipoferum and A. brasilense compared to the Health-group. Metabolomic analysis results implicated cysteinylglycine (Cys-Gly), uracil, malonate, and glycerol as the key differential metabolites between the Health- and Obstacle-group. Notably, this work revealed that bacteria such as Rhizobia potentially synthesize IAA and are likely to reduce the biotic stress of ramie. L. antibioticus also exerts a positive effect on plants in the fight against biotic stress and is mediated by metabolites including orthophosphate, uracil, and Cys-Gly, which may serve as markers for disease risk. These bacterial effects can play a key role in plant resistance to biotic stress via metabolic and methionine metabolism pathways.


Subject(s)
Azospirillum brasilense/metabolism , Azospirillum lipoferum/metabolism , Boehmeria/metabolism , Bradyrhizobium/metabolism , Lysobacter/metabolism , Soil/chemistry , Azospirillum brasilense/growth & development , Azospirillum lipoferum/growth & development , Boehmeria/microbiology , Bradyrhizobium/growth & development , Crops, Agricultural , Dipeptides/metabolism , Gas Chromatography-Mass Spectrometry , Glycerol/metabolism , Humans , Lysobacter/growth & development , Malonates/metabolism , Metabolomics/methods , Metagenomics/methods , Methionine/metabolism , Phosphates/metabolism , Rhizosphere , Soil Microbiology , Stress, Physiological , Uracil/metabolism
7.
Arch Microbiol ; 202(5): 1193-1201, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32078698

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Azospirillum brasilense/radiation effects , Light , Plant Leaves/microbiology , Plants/microbiology
8.
FEMS Microbiol Lett ; 367(4)2020 02 01.
Article in English | MEDLINE | ID: mdl-32105306

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/physiology , Biofilms/growth & development , Extracellular Polymeric Substance Matrix/metabolism , Azospirillum brasilense/growth & development , Bacterial Outer Membrane Proteins/metabolism , DNA, Bacterial/metabolism , Flagellin/metabolism , Kinetics , Microscopy, Confocal , Microscopy, Fluorescence , Polysaccharides, Bacterial/metabolism
9.
Molecules ; 24(24)2019 Dec 17.
Article in English | MEDLINE | ID: mdl-31861075

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/metabolism , Lignin/chemistry , Azospirillum brasilense/growth & development , Biofilms , Nitrogen/metabolism , Nitrogen Fixation , Plant Development , Spectroscopy, Fourier Transform Infrared , Symbiosis
11.
BMC Microbiol ; 19(1): 101, 2019 05 17.
Article in English | MEDLINE | ID: mdl-31101077

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Oxygen/metabolism , Azospirillum brasilense/metabolism , Chemotaxis , Models, Theoretical , Rhizosphere
12.
PLoS One ; 14(4): e0215332, 2019.
Article in English | MEDLINE | ID: mdl-30998695

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Genotype , Germination , Nitrogen/metabolism , Seeds/growth & development , Zea mays/growth & development , Seeds/genetics , Zea mays/genetics
13.
World J Microbiol Biotechnol ; 35(2): 19, 2019 Jan 17.
Article in English | MEDLINE | ID: mdl-30656428

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Biofilms/growth & development , Flagella/genetics , Azospirillum brasilense/genetics , Azospirillum brasilense/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Culture Media/chemistry , Flagella/metabolism , Hydrodynamics , Mutation
14.
Appl Microbiol Biotechnol ; 103(5): 2193-2203, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30685816

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/growth & development , Gelatin/chemistry , Germination/drug effects , Polyvinyl Alcohol/chemistry , Seeds/growth & development , Starch/chemistry , Zea mays/growth & development , Agriculture/methods , Biodegradation, Environmental , Crops, Agricultural/growth & development , Permeability , Seeds/drug effects , Soil Microbiology , Zea mays/microbiology
15.
Arch Microbiol ; 201(3): 325-335, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30617456

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/physiology , Bradyrhizobium/physiology , Droughts , Glycine max/growth & development , Glycine max/microbiology , Stress, Physiological/physiology , Azospirillum brasilense/growth & development , Bradyrhizobium/growth & development , Plant Leaves/microbiology , Plant Root Nodulation/physiology , Plant Roots/microbiology , Water/metabolism
16.
FEBS J ; 286(6): 1214-1229, 2019 03.
Article in English | MEDLINE | ID: mdl-30633437

ABSTRACT

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.


Subject(s)
ADP Ribose Transferases/metabolism , Ammonium Compounds/metabolism , Azospirillum brasilense/metabolism , Bacterial Proteins/metabolism , Cation Transport Proteins/metabolism , N-Glycosyl Hydrolases/metabolism , PII Nitrogen Regulatory Proteins/metabolism , ADP Ribose Transferases/genetics , Azospirillum brasilense/genetics , Azospirillum brasilense/growth & development , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cation Transport Proteins/genetics , Gene Expression Regulation, Bacterial , N-Glycosyl Hydrolases/chemistry , N-Glycosyl Hydrolases/genetics , PII Nitrogen Regulatory Proteins/genetics , Protein Binding , Protein Conformation , Signal Transduction
18.
J Basic Microbiol ; 59(1): 38-53, 2019 Jan.
Article in English | MEDLINE | ID: mdl-30320901

ABSTRACT

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.


Subject(s)
Acyl-Butyrolactones/metabolism , Azospirillum brasilense/physiology , Bradyrhizobium/physiology , Glycine max/microbiology , Microbial Interactions/physiology , Quorum Sensing/physiology , Symbiosis/physiology , Azospirillum brasilense/genetics , Azospirillum brasilense/growth & development , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biofilms/growth & development , Bradyrhizobium/genetics , Bradyrhizobium/growth & development , Brazil , Coculture Techniques , Gene Expression Regulation, Bacterial , Quorum Sensing/genetics , Repressor Proteins/genetics , Symbiosis/genetics , Trans-Activators/genetics , Transcription Factors/genetics
19.
FEMS Microbiol Ecol ; 94(12)2018 12 01.
Article in English | MEDLINE | ID: mdl-30299474

ABSTRACT

Bacteria of the Azospirillum and Pseudomonas genera are ubiquitous members of the rhizosphere, where they stimulate plant growth. Given the outstanding capacity of pseudomonads to antagonize other microorganisms, we analyzed the interaction between these two bacterial groups to identify determinants of their compatibility. We could establish that, when in direct contact, certain Pseudomonas strains produce lethality on Azospirillum brasilense cells using an antibacterial type 6 secretion system. When analyzing the effect of Pseudomonas spp. diffusible metabolites on A. brasilense growth on King's B medium, we detected strong inhibitory effects, mostly mediated by siderophores. On Congo Red medium, both inhibitory and stimulatory effects were induced by unidentified compounds. Under this condition, Pseudomonas protegens CHA0 produced a Gac/Rsm-regulated antibiotic which specifically inhibited A. brasilense Sp7 but not Sp245. This effect was not associated with the production of 2,4-diacetylphloroglucinol. The three identified antagonism determinants were also active in vivo, producing a reduction of viable cells of A. brasilense in the roots of wheat seedlings when co-inoculated with pseudomonads. These results are relevant to the understanding of social dynamics in the rhizosphere and might aid in the selection of strains for mixed inoculants.


Subject(s)
Antibiosis/physiology , Azospirillum brasilense/growth & development , Plant Roots/microbiology , Pseudomonas/metabolism , Azospirillum brasilense/metabolism , Rhizosphere , Seedlings/microbiology , Siderophores/metabolism , Triticum/microbiology , Type VI Secretion Systems/physiology
20.
Microb Ecol ; 76(2): 430-442, 2018 Aug.
Article in English | MEDLINE | ID: mdl-29327073

ABSTRACT

Mutualism interactions of microalgae with other microorganisms are widely used in several biotechnological processes since symbiotic interaction improves biotechnological capabilities of the microorganisms involved. The interaction of the bacterium Azospirillum brasilense was assessed with three microalgae genus, Scenedesmus, Chlorella, and Chlamydomonas, during CO2 fixation under high CO2 concentrations. The results in this study have demonstrated that A. brasilense maintained a mutualistic interaction with the three microalgae assessed, supported by the metabolic exchange of indole-3-acetic acid (IAA) and tryptophan (Trp), respectively. Besides, CO2 fixation increased, as well as growth and cell compound accumulation, mainly carbohydrates, in each microalgae evaluated, interacting with the bacterium. Overall, these results propose the mutualism interaction of A. brasilense with microalgae for improving biotechnological processes based on microalgae as CO2 capture and their bio-refinery capacity.


Subject(s)
Azospirillum brasilense/metabolism , Carbon Cycle , Carbon Dioxide/metabolism , Chlamydomonas reinhardtii/metabolism , Chlorella vulgaris/metabolism , Microalgae/metabolism , Scenedesmus/metabolism , Azospirillum brasilense/growth & development , Biomass , Cell Count , Cells, Immobilized , Chlamydomonas reinhardtii/growth & development , Chlorella vulgaris/growth & development , Culture Media , Indoleacetic Acids/metabolism , Microalgae/growth & development , Microbial Interactions/physiology , Plant Growth Regulators/metabolism , Symbiosis , Tryptophan/metabolism
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