Azospirillum Brasilense Az39 Research Articles

Formulation technology: Macrocystis pyrifera extract is a suitable support/medium for Azospirillum brasilense

Sustainable agricultural strategies include the use of inoculants that enhance plant growth (and therefore yield) through the action of living microorganisms. The extract of Macrocystis pyrifera, the largest and fastest growing macroalgal species, could be a suitable alternative as a support medium for the design of such inoculants, since it may ensure the long-term viability of the microorganisms. Moreover, certain physiological and biochemical aspects of this extract may prove beneficial when applied to plants. This study characterized and analyzed Macrocystis pyrifera in terms of its nutritional and hormonal composition and its metabolomic profile. Formulations made with different extract concentrations were evaluated for their impact on the growth, viability and cell survival of inoculant strain Azospirillum brasilense Az39. The results show that the most promising algae extracts (AE) are those formulated at 3 and 30 %. They contain a variety of mineral nutrients, such as magnesium, calcium, phosphorus, ferrum, zinc and boron. They also have a high concentration of organic matter and a high carbon/nitrogen ratio. These nutrients, as well as the production of phytohormones and compounds derived from alginic acid, promoted bacterial growth and viability in bacterium-algae formulations over a period of twenty-eight days. Several hormones were detected in the pure AE, in Az39 grown in NFb, and in the combinations of both (AE + Az39). Although these hormones (gibberellins, auxins and cytokinins) were found in all the samples, pure AE had the highest gibberellin content. Cytokinin production was additive in the formulations. Moreover, recovery of A. brasilense cells was significant in in vitro and in vivo assays on Zea mays seeds. These findings provide evidence in favor of using algae extracts as a renewable bioresource for the design of eco-friendly and effective inoculants.

Genome-based reclassification of Azospirillum brasilense Az39 as the type strain of Azospirillum argentinense sp. nov.

Strain Az39T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20-38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5-0.9 to 1.8-2.2 µm. They contain C16 : 0, C18 : 0 and C18 : 1 ω7c/ω6c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum, showing a close relationship with Azospirillum baldaniorum Sp245T, Azospirillum brasilense Sp7T and Azospirillum formosense CC-Nfb-7T. Housekeeping gene analysis revealed that Az39T, together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245T, A. brasilense Sp7T and A. formosense CC-Nfb-7T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Az39T and the aforementioned type strains revealed values below 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39T, Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5 %. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39T, along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39T (=LBPCV39T=BR 148428T=CCCT 22.01T).

Effect of Azospirillum brasilense on the in vitro germination of Eustoma grandiflorum (Raf.) Schinn.(Gentianaceae)

Lisianthus (Eustoma grandiflorum (Raf) Shinn.) is an excellent ornamental cut variety because it has flowers with good vase life. Seed propagation is difficult due to the long production time (it takes up to 100 days for transplanting) and the small size of its seeds. In vitro germination may produce healthy and homogeneous seedlings and may be used as a model system to study the effect of plant growth promoting rhizobacteria. Azospirillum brasilense is widely studied for its ability to colonize and promote various plant species growth, especially by increasing root development and plant biomass. This work aimed to evaluate physiological, morphological and anatomical aspects involved in vitro germination of E. grandiflorum inoculated with Azospirillum brasilense Az39. Seeds of E. grandiflorum (F1 double big. Arena III Pink) were disinfected with 10% NaClO and culture in MS medium. Inoculation of A. brasilense Az39 was performed with 106 and 107 cfu on seed surface at sowing. Non-inoculated seeds were used as controls. Biofertilization generated earliness and higher germination percentages. Furthermore, bacterization with 106 cfu significantly increased number of leaves, leaf area, fresh and dry weight of seedlings, length and number of roots, diameter of the vascular bundle, leaves thickness and root thickness, among other parameters. These results suggested that inoculation of lisianthus with A. brasilense Az39 could improve seedlings and lead to a shorter time production.

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.

Improvement of Maize Yield by Foliar Application of Azospirillum brasilense Az39

Azospirillum brasilense is a plant growth promoting bacteria (PGPB) widely used as an inoculant in diverse species, including maize (Zea mays L.) to improve their growth, development and productivity. Its ability to survive to the presence of herbicides has been demonstrated. Currently, the effectiveness of foliar application of biological formulations takes advantage of the intrinsic capacity of bacteria, regardless of the edaphic conditions of the soil. The objective of this work was to evaluate the effects of the foliar inoculation of A. brasilense Az39 in maize crop with different combinations of herbicides to increase production and to improve sustainable management practices. The field study was performed during three successive seasons. In the growing seasons between 2015 and 2018, different combinations of glyphosate and atrazine were evaluated with and without the foliar inoculation of Az39. A higher yield and total chlorophyll content were observed, together with a lower damage of membranes in the treatment with foliar inoculation of Az39, one application of atrazine and two applications of glyphosate. The yield varied between seasons but in all of them, the foliar inoculations with Az39 benefited the crop. Therefore, application of A. brasilense Az39 improves the performance of the maize crop under suitable combinations of herbicides based upon cost and environmental risk. These results, obtained with PGPB native strains, demonstrate its complementary benefits for the development of sustainable crop production practices.

Rhizobacteria improve the germination and modify the phenolic compound profile of pepper (Capsicum annum L.)

The percentage of germination and the vigor of seeds are major determinants for an efficient seedling production to successfully establishment a crop. Some Capsicum annuum germinate within a few days, but others take until 21 days. Pepper seeds have significant amounts of free and bound polyphenols that are modified during the germination process. Phenolic compounds are used by seed to scavenge free radicals, they can also work as signal compounds or they can be related to plant's defense participating in the induced systemic resistance (ISR). However, the effect of plant growth-promoting rhizobacteria (PGPR) on pepper seed germination and the changes on phenolic profile has not been examined. In this study, we evaluated the transformation of phenolic compounds during seed germination produced by the inoculation with native PGPR on pepper seeds germination. Five bacterial strains: Cellulosimicrobium 60I1, Ochrobactrum 53F, Enterobacter 64S1, Pseudomonas 42P4 and Azospirillum brasilense Az39 were investigated. Our results showed that seed inoculation enhanced significantly seed germination, seedling growth, root length, and diameter of pepper seedling. In addition, changes in the profile of free and bound phenolic compounds were observed in seeds inoculated with PGPR. The free phenolic compounds were higher in the seedling inoculated with 60I1, 42P4 or the consortium with respect to control. The phenolic acid increased in seed inoculated suggests an elicitation of phenylpropanoid pathway related to ISR. The free flavonoids, naringenin, and rutin were present in the control treatment, while they were not detectable in the inoculated treatments. These could be used as a carbon source by PGPR during the germination or converted into flavonols that could protect the plants under abiotic stress. The tyrosol was present in 60I1 and in 60I1+42P4 treatment. The most effective strains were Cellulosimicrobium 60I1 and Pseudomonas 42P4 that increased their potential when it was used together.

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 24h 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 6h after bacterization. Az39 population did not significantly decrease throughout 24h. 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.

Day and blue light modify growth, cell physiology and indole-3-acetic acid production of Azospirillum brasilense Az39 under planktonic growth conditions.

In this work, we evaluated the effects of light on growth, cell physiology and stress response of Azospirillum brasilense Az39, a non-photosynthetic rhizobacteria, under planktonic growth conditions. Exponential cultures of Az39 were exposed to blue (BL), red (RL) and daylight (DL) or maintained in darkness for 24, 48 and 72h. The biomass production and indole 3-acetic acid (IAA) biosynthesis increased by exposition to DL. Conversely, BL decreased IAA concentration through a direct effect on the molecule. The DL increased superoxide dismutase activity, hydrogen peroxide and thiobarbituric acid reactive substances levels, but the last one was also increased by BL. Both DL and BL increased cell aggregation but only BL increased biofilm formation. We demonstrated that both BL and DL are stress effectors for A. brasilense Az39 under planktonic growth conditions. The DL increased biomass production, IAA biosynthesis and bacterial response to stress, whereas BL induced cell aggregation and biofilms formation, but decreased the IAA concentration by photooxidation. Blue light and DL changes growth capacity, cell physiology and plant growth promotion ability of A. brasilense Az39 and these changes could be considered to improve the production and functionality of biofertilizers.

Immobilization of Bradyrhizobium and Azospirillum in alginate matrix for long time of storage maintains cell viability and interaction with peanut.

Immobilizarion of PGPR for agricultural applications aims to provide temporary physical protection from stressful environmental conditions and thegradual release of cells for successful root colonization, release the cells gradually. In this work, we immobilized Bradyrhizobium sp. SEMIA6144 or Azospirillum brasilense Az39 cells in 2% alginate beads prepared by ionic gelation process, and then stored up to 12 months at 4 °C. Alginate matrix showed interaction with the immobilized bacteria (FTIR), allowed a constant release of cells, and improved their viability and capability to interact with Arachis hypogaea. Cell number into beads reached 107 CFU.bead-1; however, viability decreased from 4 months of storage for Az39, while it was maintained up to 12 months for SEMIA6144, showing a low metabolic activity measured by the MTT assay. Adhesion of SEMIA6144 and Az39 from new beads to peanut root was 11.5% and 16%, respectively, higher than non-immobilized bacteria. Peanut inoculation with 12 months storage SEMIA6144 beads significantly increased root length and biomass at 30 days of growth, and under restrictive water condition (RWC), nodulation and total plant N content increased compared with liquid inoculation. Our results demonstrate that immobilization of SEMIA6144 and Az39 in alginate matrix is a potential alternative to enhance peanut growth even under RWC. KEY POINTS: • Alginate encapsulation enhances viability of SEMIA6144 or Az39 under storage at 4 °C for 1 year. • Alginate beads 2% ensure the gradual release of the microorganisms. • Cells from beads stored for long periods present chemotaxis and adhesion to peanut root. • Peanut inoculation with 1-year-old SEMIA6144 beads improves nodulation and growth in RWC.

Pseudomonas fluorescens and Azospirillum brasilense Increase Yield and Fruit Quality of Tomato Under Field Conditions

Crop inoculation with plant growth-promoting rhizobacteria (PGPR) is a sustainable alternative to diminish the excessive use of chemical fertilizers in agriculture. However, there is little information about PGPR inoculation effects under field conditions and even less on industrial tomato production. We aimed to study the effects of a sole inoculation at seedling stage with Pseudomonas fluorescens Rt6M10, Azospirillum brasilense Az39, and their combination on growth and yield of two industrial tomato varieties UCO 14 (UCO) and Harris Moran 3861 (HM). We compared these PGPR inoculation treatments with chemically fertilized and non-fertilized (control) seedlings under field conditions. We found that inoculation with Rt6M10, Az39, and their combination increased seedling root dry weight by 62%, 41%, and 23%, respectively and shoot dry weight by 29%, 23%, and 2%, respectively compared with non-inoculated control, improving tolerance to transplant stress. Inoculation with Rt6M10, Az39, and their combination increased stem diameter by 15%, 16%, and 13%, respectively, while Rt6M10 and the combination treatments increased leaf chlorophyll and carotenoid levels compared with non-inoculated plants. Az39 increased fruit number (35%) and fruit weight (38%) per plant in HM, whereas in UCO variety, the increase was the highest (48% and 49%, respectively). Seedling inoculation increase fruit firmness and equatorial and polar fruit diameter by 24%, 10%, and 12%, respectively in HM and by 21%, 14%, and 14%, respectively in UCO. Overall, bio-inoculation with Rt6M10 and/or Az39 was beneficial for tomato seedlings at transplanting and supported fruit yield and quality (total soluble solid content, pH, and titratable acidity) equally or better than chemically fertilized seedlings.

Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39

Azospirillum brasilense Az39 has been used since more than 30 years by several companies in South America for biofertilizers production. This strain may promote plants growth and development, as well as the ability of inoculated plants to tolerate environmental stresses, which determines an increase in the productivity under field conditions. At present, there are no protocols in Argentina to confirm the identity of Az39 in commercial products; however, such biofertilizers are formulated almost exclusively with this strain. Therefore, the objective of this paper was to develop a molecular methodology that allows the accurate identification of A. brasilense Az39. Using the complete genome sequence and several bioinformatics tools, fragments of DNA present only in the Az39 genome were recognized. A set of PCR primers to amplify these sequences were designed, and the specific products were observed only in the strain of our interest. The sensitivity of the methodology was evaluated, where the strain could be detected up to a titer of 105 CFU/ml (4.5 ng/μl ADN) or 102 CFU/ml (0.88 ng/μl DNA) or in a minimal concentration of 0.098 ng/μl DNA, depending on the DNA extraction methodology used. Primers were tested against direct samples of commercial inoculants and cultures, in both cases there were specifics products, both in direct samples and in confirmatory tests from isolated colonies from those products. The procedure presented in this paper allows the accurate identification of A. brasilense Az39 in pure cultures, mixtures of microorganisms, and commercial biofertilizers.

Azospirillum brasilense Az39, a model rhizobacterium with AHL quorum-quenching capacity.

The aim of this research was to analyse the quorum-sensing (QS) and quorum-quenching (QQ) mechanisms based on N-acyl-l-homoserine lactones (AHLs) in Azospirillum brasilense Az39, a strain with remarkable capacity to benefit a wide range of crops under agronomic conditions. We performed an in silico and invitro analysis of the quorum mechanisms in A. brasilense Az39. The results obtained invitro using the reporter strains Chromobacterium violaceum and Agrobacterium tumefaciens and liquid chromatography coupled with mass-mass spectrometry analysis showed that although Az39 does not produce AHL molecules, it is capable of degrading them by at least two hypothetical enzymes identified by bioinformatics approach, associated with the bacterial cell. In Az39 cultures supplemented with 500nmoll-1 of the C3 unsubstituted AHLs (C4, C6, C8, C10, C12, C14), AHL levels were lower than in noninoculated LB media controls. Similar results were observed upon the addition of AHLs with hydroxy (OH-) and keto (oxo-) substitutions in C3. These results not only demonstrate the ability of Az39 to degrade AHLs. They also show the wide spectrum of molecules that can be degraded by this bacterium. Although A. brasilense Az39 is a silent bacterium unable to produce AHL signals, it is able to interrupt the communications between other bacteria and/or plants by a QQ activity. This is the first report confirming by unequivocal methodology the ability of A. brasilense, one of the most agriculturally used benefic bacteria around the world, to degrade AHLs by a QQ mechanism.

Performance of Bradyrhizobium and Bradyrhizobium–Azospirillum in Alleviating the Effects of Water-Restrictive Conditions During the Early Stages of Arachis hypogaea Growth

Fil: Cesari, Adriana Belen. Universidad Nacional de Rio Cuarto. Facultad de Ciencias Exactas, Fisicoquimicas y Naturales. Departamento de Biologia Molecular. Seccion Quimica Biologica; Argentina. Consejo Nacional de Investigaciones Cientificas y Tecnicas; Argentina

Impact of double inoculation with Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39 on soybean plants grown under arsenic stress

Inoculation practice with plant growth-promoting bacteria (PGPB) has been proposed as a good biotechnological tool to enhance plant performance and alleviate heavy metal/metalloid stress. Soybean is often cultivated in soil with high arsenic (As) content or irrigated with As-contaminated groundwater, which causes deleterious effects on its growth and yield, even when it was inoculated with rhizobium. Thus, the effect of double inoculation with known PGPB strains, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39 was evaluated in plants grown in pots under controlled conditions and treated with As. First, the viability of these co-cultivated bacteria was assayed using a flow cytometry analysis using SYTO9 and propidium iodide (PI) dyes. This was performed in vitro to evaluate the bacterial population dynamic under 25 μM AsV and AsIII treatment. A synergistic effect was observed when bacteria were co-cultured, since mortality diminished, compared to each growing alone. Indole acetic acid (IAA) produced by A. brasilense Az39 would be one of the main components involved in B. japonicum E109 mortality reduction, mainly under AsIII treatment. Regarding in vivo assays, under As stress, plant growth improvement, nodule number and N content increase were observed in double inoculated plants. Furthermore, double inoculation strategy reduced As translocation to aerial parts thus improving As phytostabilization potential of soybean plants. These results suggest that double inoculation with B. japonicum E109 and A. brasilense Az39 could be a safe and advantageous practice to improve growth and yield of soybean exposed to As, accompanied by an important metalloid phytostabilization.

Regulation of IAA Biosynthesis in Azospirillum brasilense Under Environmental Stress Conditions.

Indole-3-acetic acid (IAA) is one of the most important molecules produced by Azospirillum sp., given that it affects plant growth and development. Azospirillum brasilense strains Sp245 and Az39 (pFAJ64) were pre-incubated in MMAB medium plus 100mg/mL L-tryptophan and treated with or exposed to the following (a) abiotic and (b) biotic stress effectors: (a) 100mM NaCl or Na2SO4, 4.0% (w/v) PEG6000, 0.5mM H2O2, 0.1mM abscisic acid, 0.1mM 1-aminocyclopropane 1-carboxylic acid, 45°C or daylight, and (b) 4.0% (v/v) filtered supernatant of Pseudomonas savastanoi (Ps) or Fusarium oxysporum (Fo), 0.1mM salicylic acid (SA), 0.1mM methyl jasmonic acid (MeJA), and 0.01% (w/v) chitosan (CH). After 30 and 120min of incubation, biomass production, cell viability, IAA concentration (µg/mL), and ipdC gene expression were measured. Our results show that IAA production increases with daylight or in the presence of PEG6000, ABA, SA, CH, and Fo. On the contrary, exposure to 45°C or treatment with H2O2, NaCl, Na2SO4, ACC, MeJA, and Ps decrease IAA biosynthesis. In this report, growth and IAA biosynthesis in A. brasilense under biotic and abiotic stress conditions are discussed from the point of view of their role in bacterial lifestyle and their potential application as bioproducts.

Improvement of soybean grain nutritional quality under foliar inoculation with Azospirillum brasilense strain Az39

Legumes depend on biological nitrogen fixation through symbiosis with rhizobia to meet their nitrogen requirements. Certain plant growth promoting rizobacteria such as Azospirillum may cooperate in the establishment and maintenance of effective legume-rhizobia symbiosis. The aim of this work was to assess if foliar inoculation of soybean with Azospirillum brasilense Az39 has advantages over inoculation with this bacterium at sowing in combination with Bradyrhizobium japonicum E109 inoculation. To test this, glasshouse and field experiments were carried out. Higher shoot biomass was observed at V6 and R2 stages under foliar inoculation of A. brasilense Az39 as compared to coinoculation at sowing. Additionally, increased root dry biomass and higher nodule number and nodule fresh weight per plant were found at V6. Leghemoglobin levels in nodules were significantly greater in foliar-inoculated plants at both stages (V6 and R2) as compared with seed-inoculated plants. In line with these positive effects of foliar inoculation with A. brasilense Az39 on soybean growth and nodulation, grains harvested from foliar-inoculated plants had higher nitrogen and protein contents than those harvested from plants coinoculated at sowing. This was corroborated for two soybean varieties cultivated at two different locations. Our findings may open new insights into soybean agricultural technology.

Carotenoid production and phenotypic variation in Azospirillum brasilense

We assessed the occurrence of phenotypic variation in Azospirillum brasilense strains Sp7, Cd, Sp245, Az39 and phv2 during growth in rich media, screening for variants altered in colony pigmentation or extracellular polysaccharide (EPS) production. Previous studies showed that EPS-overproducing variants of Sp7 appear frequently following starvation or growth in minimal medium. In contrast, no such variants were detected during growth in rich media in the tested strains except for few variants of phv2. Regarding alteration in colony pigmentation (from pink to white in strain Cd and from white to pink in the others), strain Sp7 showed a relatively high frequency of variation (0.009–0.026%). Strain Cd showed a lower frequency of alteration in pigmentation (0–0.008%), and this type of variation was not detected in the other strains. In A. brasilense, carotenoid synthesis is controlled by two RpoE sigma factors and their cognate ChrR anti-sigma factors, the latter acting as negative regulators of carotenoid synthesis. Here, all tested (n = 28) pink variants of Sp7 carried mutations in one of the anti-sigma factor genes, chrR1. Our findings indicate that, in A. brasilense, phenotypic variation is strain- and environment-dependent and support the central role of ChrR1 in regulation of carotenoid production.

Biofertilization with Azospirillum brasilense improves in vitro culture of Handroanthus ochraceus, a forestry, ornamental and medicinal plant

Biofertilization with plant growth-promoting rhizobacteria is a potential alternative to plant productivity. Here, in vitro propagation of Handroanthus ochraceus (yellow lapacho), a forest crop with high economic and environmental value, was developed using the Azospirillum brasilense strains Cd and Az39 during rhizogenesis. Epicotiles of in vitro plantlets were multiplied in Woody Plant Medium (WPM). For rooting, elongated shoots were transferred to auxin-free Murashige-Skoog medium with Gamborg's vitamins and WPM, both at half salt concentration (½MSG and ½WPM), and inoculated with Cd or Az39 at the base of each shoot. Anatomical studies were performed using leaves cleared and stained with safranin for optical microscopy and leaves and roots metalized with gold-palladium for scanning electron microscopy (SEM). In ½WPM auxin-free medium, A. brasilense Cd inoculation produced 55% of rooting, increased root fresh and dry weight (45% and 77%, respectively), and led to lower stomata size and density with similar proportion of open and closed stomata. Both strains selectively increased the size or density of glandular trichomes in ½MSG. Moreover, bacteria were detected on the root surface by SEM. In conclusion, the difference in H. ochraceus response to A. brasilense inoculation depends on the strain and the plant culture media. Cd strain enhanced rooting in auxin-free ½WPM and produced plantlets with features similar to those expected in ex vitro plants. This work presents an innovative in vitro approach using beneficial plant-microorganism interaction as an ecologically compatible strategy in plant biotechnology.

Effect of arsenic on tolerance mechanisms of two plant growth-promoting bacteria used as biological inoculants

Bacterial ability to colonize the rhizosphere of plants in arsenic (As) contaminated soils is highly important for symbiotic and free-living plant growth-promoting rhizobacteria (PGPR) used as inoculants, since they can contribute to enhance plant As tolerance and limit metalloid uptake by plants. The aim of this work was to study the effect of As on growth, exopolysaccharide (EPS) production, biofilm formation and motility of two strains used as soybean inoculants, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39. The metabolism of arsenate (As(V)) and arsenite (As(III)) and their removal and/or possible accumulation were also evaluated. The behavior of both bacteria under As treatment was compared and discussed in relation to their potential for colonizing plant rhizosphere with high content of the metalloid. B. japonicum E109 growth was reduced with As(III) concentration from 10μM while A. brasilense Az39 showed a reduction of growth with As(III) from 500μM. EPS and biofilm production increased significantly under 25μM As(III) for both strains. Moreover, this was more notorious for Azospirillum under 500μM As(III), where motility was seriously affected. Both bacterial strains showed a similar ability to reduce As(V). However, Azospirillum was able to oxidize more As(III) (around 53%) than Bradyrhizobium (17%). In addition, both strains accumulated As in cell biomass. The behavior of Azospirillum under As treatments suggests that this strain would be able to colonize efficiently As contaminated soils. In this way, inoculation with A. brasilense Az39 would positively contribute to promoting growth of different plant species under As treatment.

Complete Genome Sequence of the Model Rhizosphere Strain Azospirillum brasilense Az39, Successfully Applied in Agriculture.

We present the complete genome sequence of Azospirillum brasilense Az39, isolated from wheat roots in the central region of Argentina and used as inoculant in extensive and intensive agriculture during the last four decades. The genome consists of 7.39 Mb, distributed in six replicons: one chromosome, three chromids, and two plasmids.