Growth-promoting Bacterium Azospirillum Brasilense Research Articles

Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

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

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. 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. 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.

Synergic association of the consortium Arthrospira maxima with the microalga growth-promoting bacterium Azospirillum cultured under the stressful biogas composition.

The present study evaluates the association of the blue-green microalga Arthrospira maxima (Spirulina), which is known for its CO2 fixation, biomass, and high-value metabolite production, with the microalga growth-promoting bacterium Azospirillum brasilense under the stressful composition of biogas. The results demonstrated that A. maxima co-cultured with A. brasilense under the high CO2 (25%) and methane (CH4; 75%) concentrations of biogas recorded a CO2 fixation rate of 0.24 ± 0.03g L-1 days-1, thereby attaining a biomass production of 1.8 ± 0.03g L-1. Similarly, the biochemical composition quality of this microalga enhanced the attainment of higher contents of carbohydrates, proteins, and phycocyanin than cultured alone. However, metabolites other than tryptophan (Trp) and indole-3-acetic acid could have supported this beneficial interaction. Overall, the results demonstrate that this prokaryotic consortium of A. maxima-A. brasilense established a synergic association under biogas, which represents a sustainable strategy to improve the bio-refinery capacity of this microalga and increase the usefulness of A. brasilense in multiple economic sectors.

Microalga-bacteria interaction mitigates adverse effects on microalga produced by ZnO nanoparticles

Zinc oxide nanoparticles (ZnO NPs) used in myriad industries have become an emerging contaminant in various environments. Interactions of microalgae and bacteria, in suspension systems and immobilized, have been proposed for several biotechnology applications, such as wastewater treatment. However, the presence of NPs can affect the performance of these microorganisms during nutrient removal. This study assessed the effect of metal NPs on microalgae˗bacteria interaction, immobilized or in suspension. The effect of two concentrations of ZnO NPs (0.1 and 0.5 ppm) on the growth and photosynthetic pigments of the microalga Chlorella sorokiniana cultured in suspension or immobilized in alginate beads, alone or in the presence of the microalgae growth-promoting bacterium Azospirillum brasilense, was evaluated, and the effect of these NPs on indole-3-acetic acid (IAA) production by A. brasilense was also tested. The presence of ZnO NPs affected the growth of both microorganisms when cultured alone in suspension, and the IAA production by A. brasilense was reduced by 98 %. Nevertheless, when the two microorganisms were co-cultured in suspension, the negative effect of 0.1 ppm of ZnO NPs in C. sorokiniana was mitigated by A. brasilense, indicating a positive interaction between the two microorganisms. Furthermore, adding synthetic IAA enhances microalgae growth and photosynthetic pigment production regardless of NP concentration. On the other hand, immobilization of the cells produces a dose-response effect in the microorganisms, regardless of the partnerʼs presence. The results showed the positive effect of A. brasilense on stress mitigation of C. sorokiniana when 0.1 ppm of ZnO NPs are present and similar to the effect produced by synthetic IAA when the microorganisms are present in a suspension system. On the other hand, the immobilization of cells in alginate beads represents a physical barrier that acts as protection against the stress produced by ZnO NPs.

Using Geospatial Information to Map Yield Gain from the Use of Azospirillum brasilense in Furrow

The application of biological products in agricultural crops has become increasingly prominent. The growth-promoting bacterium Azospirillum brasilense has been used as an alternative to promote greater yield in maize crops. In the context of precision agriculture, interpreting geospatial data has allowed for monitoring the effect of the application of products that increase the yield of corn crops. The objective of this work was to evaluate the potential of Kriging techniques and spectral models through images in estimating the gain in yield of maize crop after applying A. brasilense. Analyses were carried out in two commercial areas treated with A. brasilense. The results revealed that models of yield prediction by Kriging with a high volume of training data estimated the yield gain with a root-mean-square error deviation (RMSE%), mean absolute percentage error (MAPE%), and R2 to be 6.67, 5.42, and 0.88, respectively. For spectral models with a low volume of training data, yield gain was estimated with RMSE%, MAPE%, and R2 to be 9.3, 7.71, and 0.80, respectively. The results demonstrate the potential to map the spatial distribution of productivity gains in corn crops following the application of A. brasilense.

Differences in Exudates Between Strains of Chlorella sorokiniana Affect the Interaction with the Microalga Growth-Promoting Bacteria Azospirillum brasilense : Differences in Exudates Between Strains of Chlorella sorokiniana Affect the Interaction with the Microalga Growth-Promoting Bacteria Azospirillum brasilense.

The microalga Chlorella sorokiniana and the microalgae growth-promoting bacteria (MGPB) Azospirillum brasilense have a mutualistic interaction that can begin within the first hours of co-incubation; however, the metabolites participating in this initial interaction are not yet identified. Nuclear magnetic resonance (NMR) was used in the present study to characterize the metabolites exuded by two strains of C. sorokiniana (UTEX 2714 and UTEX 2805) and A. brasilense Cd when grown together in an oligotrophic medium. Lactate and myo-inositol were identified as carbon metabolites exuded by the two strains of C. sorokiniana; however, only the UTEX 2714 strain exuded glycerol as the main carbon compound. In turn, A. brasilense exuded uracil when grown on the exudates of either microalga, and both microalga strains were able to utilize uracil as a nitrogen source. Interestingly, although the total carbohydrate content was higher in exudates from C. sorokiniana UTEX 2805 than from C. sorokiniana UTEX 2714, the growth of A. brasilense was greater in the exudates from the UTEX 2714 strain. These results highlight the fact that in the exuded carbon compounds differ between strains of the same species of microalgae and suggest that the type, rather than the quantity, of carbon source is more important for sustaining the growth of the partner bacteria.

Inoculation of corn seeds with Azospirillum brasilense in different temperatures

Growth-promoting bacteria Azospirillum brasilense, used as an inoculant in corn culture, can be severely affected by the exposure temperature, with a lethal effect above 35°C, when cultivated alone under laboratory conditions. Such effects may limit the associative interaction between plant-bacteria, with reduced inoculation efficiency, resulting in a lower growth rate of the plant and an increase in oxidative stress. Thus, the objective of the research was to evaluate the efficiency of the inoculation process with A. brasilense in seeds and in the initial growth of seedlings of two corn cultivars submitted to different temperatures. Were utilized corn hybrids seed Syn 488 and Syn 505. The experimental design was completely randomized in a 2 x 4 factorial scheme (with and without inoculation of A. brasilense x 4 sowing temperatures: 20, 25, 30, and 35ºC), with four replications. The inoculation efficiency in corn seedlings submitted to different temperatures was evaluated through the following tests: germination, first count, seedling length and dry weight. In addition, responses at the biochemical level of the interaction (temperatures x inoculation) for the content of photosynthetic pigments and hydrogen peroxide (H2O2), antioxidant enzymes and lipid peroxidation were evaluated. The inoculation with A. brasilense changed the morphological and biochemical responses of corn seedlings, and the inoculation process was efficient for all temperatures tested. The best results were under temperatures 25, 30, and 35°C. Plant and bacteria when exposed to temperature conditions (30°C inoculated) showed a significant increase in plant biomass and activity of antioxidant enzymes.

Natural occurrence of Azospirillum brasilense in petunia with capacity to improve plant growth and flowering.

To evaluate the natural occurrence of the plant growth-promoting bacterium Azospirillum brasilense and petunia plants, local strains were isolated and characterized by biochemical and molecular methods. Three strains were assessed in greenhouse conditions using Petunia × hybrida Ultra™. Treatments: Plants without bacterial inoculation or chemical fertilization; fertilized with NPK and KNO3 ; and independently inoculated with the strains 2A1, 2A2, and 2E1 by submerging their roots in a bacterial suspension (~106 CFU·ml-1 ). Root length, dry weight of roots and shoots, leaf area, leaf greenness, and nutrient content were evaluated. The number of days from transplanting to the opening of the first flower and the number of flowers per plant were also determined. As a result, five isolates were characterized as A. brasilense, showing the capacity to produce indoles and siderophores, to solubilize phosphate, nitrogenase activity, and nifH-PCR amplification. In general, all the parameters of the plant assay were improved in plants inoculated with A. brasilense, with variations among the strains, as well as the onset of flowering and the number of flowers per plant, compared with uninoculated or fertilized plants. This is the first report on the natural occurrence of A. brasilense in petunia with the capacity to improve plant growth and flowering.

Expression and function of the cdgD gene, encoding a CHASE\u2013PAS-DGC-EAL domain protein, in Azospirillum brasilense

The plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

Inoculation With Growth-Promoting Bacteria Azospirillum brasilense and Its Effects on Productivity and Nutritional Accumulation of Wheat Cultivars

Agricultural practices that allow a productive increase in a sustainable manner are becoming increasingly necessary to feed an ever-growing global population. The inoculation with Azospirillum brasilense has the potential to reduce the use of synthetic mineral fertilizers with efficient capacity to promote plant growth and increase nutrition. Therefore, this research was developed to investigate the potential use of A. brasilense to increase the accumulation of macro- and micronutrients and its influence on grain yield, plant height, and spike population in two wheat cultivars (CD1104 and CD150), under irrigated conditions in the Brazilian Cerrado. The study was carried out in a Rhodic Hapludox under a no-tillage system. The experiment was designed in randomized blocks with six replications, arranged in a 2 × 2 factorial scheme: two cultivars (CD150 and CD1104) and two levels of inoculation (control and with A. brasilense). The inoculation with A. brasilense provided greater accumulation of micronutrients in the aerial part of the wheat. In the cultivar CD1104, B and Cu had an accumulation 27.7 and 57.4% higher compared with those of the control without bacteria. In the cultivar CD150, Azospirillum increased the accumulation of B, Fe, and Mn by 43.8, 49.9, and 22%, respectively, and reduced Cu by 21.9%, compared with those of the control. The cultivar CD150 has greater efficiency to accumulate N (+35.5 kg N ha−1) as compared with the cultivar CD1104. Interactions between inoculation and cultivars resulted in greater accumulation of S and K in the shoot of the wheat cultivar CD150, as well greater accumulation of Cu in CD1104. In growth assessments, inoculation or cultivars did not statistically influence wheat grain yield and spike population. Howevere, for plant height, the CD1104 genotype has 13.1% bigger plant height on average than that of the CD150 genotype. Inoculation can contribute more sustainably to wheat nutrition.

The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense.

The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status.IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors.

Biofertilization with Macrocystis pyrifera algae extracts combined with PGPR-enhanced growth in Lactuca sativa seedlings

To improve crop yields the application of fertilizers to provide nutrients to soils is recommended. An alternative option to substitute nutritional supplements of chemical origin is the use of biofertilizers. This study evaluated the effectiveness of a biofertilizer of natural origin, Macrocystis pyrifera algal extracts and its combination with the plant growth–promoting bacterium Azospirillum brasilense, on the germination and establishment of lettuce seedlings (Lactuca sativa) under two different water irrigation treatments. Results demonstrated that germination energy and germination power improved upon inoculation of lettuce seeds with the seaweed extract. After 7 days of culture, seedlings showed increased root growth which would help to ensure their establishment. The application of bacteria and algae individually or in combination exerted a positive effect on lettuce plant growth compared with controls (without applications). In addition, treatments with the brown seaweed extract favoured seedlings adaptation to an unfavourable environment (water deficit) by increasing their root mass and protecting them from the negative effects imposed by stress. These results suggest that the use of M. pyrifera extract to improve plant growth and to provide stress protection may be considered an interesting tool for agricultural purposes. Nevertheless, when A. brasilense (whose plant growth–promoting properties are largely known) is combined with the brown seaweed extract, a new biofertilizing formula is obtained and proposed for future seed inoculation technology, based on the promising effects observed in this work.

Compatibility of Azospirillum brasilense with Pesticides Used for Treatment of Maize Seeds

Seed treatment with chemical pesticides is commonly used as an initial plant protection procedure against pests and diseases. However, the use of such chemicals may impair the survival and performance of beneficial microorganisms introduced via inoculants, such as the plant growth-promoting bacterium Azospirillum brasilense. We assessed the compatibility between the most common pesticide used in Brazil for the treatment of maize seeds, composed of two fungicides, and one insecticide, with the commercial strains Ab-V5 and Ab-V6 of A. brasilense, and evaluated the impacts on initial plant development. The toxicity of the pesticide to A. brasilense was confirmed, with an increase in cell mortality after only 24 hours of exposure in vitro. Seed germination and seedling growth were not affected neither by the A. brasilense nor by the pesticide. However, under greenhouse conditions, the pesticide affected root volume and dry weight and root-hair incidence, but the toxicity was alleviated by the inoculation with A. brasilense for the root volume and root-hair incidence parameters. In maize seeds inoculated with A. brasilense, the pesticide negatively affected the number of branches, root-hair incidence, and root-hair length. Therefore, new inoculant formulations with cell protectors and the development of compatible pesticides should be searched to guarantee the benefits of inoculation with plant growth-promoting bacteria.

Auxin-dependent alleviation of oxidative stress and growth promotion of Scenedesmus obliquus C1S by Azospirillum brasilense

There is currently an increasing interest in the use of microalgae for wastewater treatment and the use of its biomass as a feedstock for biofuels. Both of these applications are often performed more efficiently by microalgal-bacteria consortia. However, the mechanisms that account for the stability and robustness of this kind of interactions are poorly understood. In this study, we confirmed the growth promotion activity of the plant growth-promoting bacterium Azospirillum brasilense Sp245 on the microalgae Scenedesmus obliquus C1S. We show that this activity is critically dependent on bacterial indole-3 acetic acid (IAA) production, which results in a decrease in algal reactive oxygen species levels, higher cell densities and ameliorates algal cells bleaching after nitrogen deprivation. We also show a close inter-species interaction between both partners and an active expression of the bacterial Indole-3-pyruvate decarboxylase (ipdC) gene involved in production of IAA when co-cultivated.This study extends the current knowledge of the mechanisms underlying bacteria-microalgae consortia to improve their technological applications and to better understand ecological relationships in the environment.

Morphological Changes in Sugarcane Crop Induced by the Plant Growth-Promoting Bacterium Azospirillum brasilense

N fertilizers make the total cost of production of biofuels, sugar and bioelectricity expensive in the sugar-energy industry. Microbes could replace N fertilizers in the nutritional management of sugarcane crop by capturing N2 from the atmosphere. We accordingly conducted this scientific study to investigate the response of sugarcane crop to Azospirillum brasilense. We tested the strain Az-V5 at the doses of 0, 50, 100, 200 and 400 mL ha−1, by spraying it into the planting furrow, 7, 14 and 21 days after crop planting into 20-l pots filled with sieved Rhodic Hapludox, with a loamy texture. In order to examine biologically modulated morphological changes in the host plant material, namely sugarcane variety ‘RB86-7515,’ we harvested and then assessed 90-day-old plants for plant height, stalk diameter, number of tillers, number of leaves, leaf area, tiller length, dry matter of shoots and root dry matter. Sugarcane plants treated with the bioagent 14 and 21 days after crop planting had lower mean values of plant height, number of leaves, leaf area, dry matter of shoots and root dry matter. Therefore, applying A. brasilense into the planting furrow, instead of spraying it on sugarcane leaves, could protect it for a longer period from biotic and abiotic factors influencing cell viability, root colonization and ultimately symbiosis. The conclusion is, therefore, application of A. brasilense into planting furrow is the best environmentally friendly and low-cost strategy to promote growth and development in sugarcane plants at the beginning of their life cycle.

Phasin PhaP1 is involved in polyhydroxybutyrate granules morphology and in controlling early biopolymer accumulation in Azospirillum brasilense Sp7

Phasins are amphiphilic proteins involved in the regulation of the number and size of polyhydroxybutyrate (PHB) granules. The plant growth promoting bacterium Azospirillum brasilense Sp7 accumulates high quantities of bioplastic PHB as carbon and energy source. By analyzing the genome, we identified six genes that code for proteins with a Phasin_2 domain. To understand the role of A. brasilense Sp7 PhaP1 (PhaP1Abs) on PHB synthesis, the phaP1 gene (AMK58_RS17065) was deleted. The morphology of the PHB granules was analyzed by transmission electron microscopy (TEM) and the PHB produced was quantified under three different C:N ratios in cultures subjected to null or low-oxygen transfer. The results showed that PhaP1Abs is involved in PHB granules morphology and in controlling early biopolymer accumulation. Using RT-PCR it was found that phasin genes, except phaP4, are transcribed in accordance with the C:N ratio used for the growth of A. brasilense. phaP1, phaP2 and phaP3 genes were able to respond to the growth conditions tested. This study reports the first analysis of a phasin protein in A. brasilense Sp7.

Early Changes in Nutritional Conditions Affect Formation of Synthetic Mutualism Between Chlorella sorokiniana and the Bacterium Azospirillum brasilense.

The effect of three different nutritional conditions during the initial 12h of interaction between the microalgae Chlorella sorokiniana UTEX 2714 and the plant growth-promoting bacterium Azospirillum brasilense Cd on formation of synthetic mutualism was assessed by changes in population growth, production of signal molecules tryptophan and indole-3-acetic acid, starch accumulation, and patterns of cell aggregation. When the interaction was supported by a nutrient-rich medium, production of both signal molecules was detected, but not when this interaction began with nitrogen-free (N-free) or carbon-free (C-free) media. Overall, populations of bacteria and microalgae were larger when co-immobilized. However, the highest starch production was measured in C. sorokiniana immobilized alone and growing continuously in a C-free mineral medium. In this interaction, the initial nutritional condition influenced the time at which the highest accumulation of starch occurred in Chlorella, where the N-free medium induced faster starch production and the richer medium delayed its accumulation. Formation of aggregates made of microalgae and bacteria occurred in all nutritional conditions, with maximum at 83h in mineral medium, and coincided with declining starch content. This study demonstrates that synthetic mutualism between C. sorokiniana and A. brasilense can be modulated by the initial nutritional condition, mainly by the presence or absence of nitrogen and carbon in the medium in which they are interacting.

Dry micro-polymeric inoculant of Azospirillum brasilense is useful for producing mesquite transplants for reforestation of degraded arid zones

Massive clear-cutting of wild stands of mesquite trees in the Mexican part of the Sonoran Desert result from high demand for this wood by the charcoal industry. Consequently, there is a need to develop techniques for reforestation of this tree in the desert and maintain its natural diversity at the same time. An outdoor nursery procedure to produce mesquite transplants from diversely originated seeds for reforestation of arid zones was developed. This procedure involved: 1) inoculation of the seedlings in the nursery with the plant growth-promoting bacteria (PGPB) Azospirillum brasilense immobilized in dry microbeads of alginate, and 2) developing a reliable way to monitor plant development and aerial volume in the nursery for the entire growth period of seven months before transplantation. Dry microbeads containing the PGPB and maintained at room temperature were tested for survival of bacteria for up to seven months. These dry microbeads maintained sufficient population levels of A. brasilense to inoculate the plant for the entire period. Inoculation with the PGPB enhanced all growth parameters of the plants, including biomass, aerial volume, root system, and chlorophyll pigments, but not the auxiliary photosynthetic pigments. The PGPB was specifically identified colonizing the roots of the transplants by fluorescent in situ hybridization for the entire growth period. Measuring a few simple parameters allowed development of a workable model for plant growth. This model was confirmed by data obtained from sacrificed plants whose parameters were measured directly. This study shows that outdoor nursery cultivation of inoculated mesquite transplants is feasible.

Role of ethylene and related gene expression in the interaction between strawberry plants and the plant growth-promoting bacterium Azospirillum brasilense.

Induced systemic resistance (ISR) is one of the indirect mechanisms of growth promotion exerted by plant growth-promoting bacteria, and can be mediated by ethylene (ET). We assessed ET production and the expression of related genes in the Azospirillum-strawberry plant interaction. Ethylene production was evaluated by gas chromatography in plants inoculated or not with A.brasilense REC3. Also, plants were treated with AgNO3 , an inhibitor of ET biosynthesis; with 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ET biosynthesis; and with indole acetic acid (IAA). Plant dry biomass and the growth index were determined to assess the growth-promoting effect of A.brasilense REC3 in strawberry plants. Quantitative real time PCR (qRT-PCR) was performed to analyse relative expression of the genes Faetr1, Faers1 and Faein4, which encode ET receptors; Factr1 and Faein2, involved in the ET signalling pathway; Faacs1 encoding ACC synthase; Faaco1 encoding ACC oxidase; and Faaux1 and Faami1 for IAA synthesis enzymes. Results showed that ET acts as a rapid and transient signal in the first 12h post-treatment. A.brasilense REC3-inoculated plants had a significantly higher growth index compared to control plants. Modulation of the genes Faetr1, Faers1, Faein4, Factr1, Faein2 and Faaco1 indicated activation of ET synthesis and signalling pathways. The up-regulation of Faaux1 and Faami1 involved in IAA synthesis suggested that inoculation with A.brasilense REC3 induces production of this auxin, modulating ET signalling. Ethylene production and up-regulation of genes associated with ET signalling in strawberry plants inoculated with A.brasilense REC3 support the priming activation characteristic of ISR. This type of resistance and the activation of systemic acquired resistance previously observed in this interaction indicate that both are present in strawberry plants, could act synergistically and increase protection against pathogens.

The plant growth promoting bacterium Azospirillum brasilense is vertically transmitted in Phaseolus vulgaris (common bean)

Azospirillum brasilense is a plant growth-promoting bacterium that is utilized as a bio-fertilizer worldwide. Inoculation of crops with A. brasilense can lead to a substantial boost in crop yield, although this enhancement is variable. Sometimes crop production is enhanced as much as 70% and other times enhancement does not occur at all. To solve this problem, a greater understanding of A. brasilense – plant interactions is needed. While the root/rhizosphere ecology of A. brasilense has been studied extensively there are no significant studies that address whether A. brasilense populates above ground plant tissues. In this study, we use a combination of qPCR, transcriptomics, confocal microscopy, and culturing techniques to map the in-planta distribution of a GFP-expressing A. brasilense in Phaseolus vulgaris (common bush bean). We show that A. brasilense constitute part of the plant bacterial microflora well above the root system. During P. vulgaris development A. brasilense was shown to actively move to developing bean seeds where it forms a significant intercellular population. GFP-expressing A. brasilense was shown to vertically transmit to successive plant generations demonstrating that A. brasilense in the seed constitute an effective inoculum. The ability of A. brasilense to not only colonize roots but to also actively migrate to developing seeds indicates the existence of a more complex symbiotic relationship than previously understood.