Molecular identification of Azospirillum spp.: limitations of 16S rRNA and qualities of rpoD as genetic markers

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been sub-jected to intensive research due to their biotechnological potential as crop inoculants. Phylogeneticanalysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsis-tencies and low confidence often arise when working with close species. In this work, it was observedthat these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list ofhousekeeping genes, the correlation between pairwise gene and whole-genome similarities was exam-ined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for thePCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 typestrains of different species. The sequences obtained were used for inferring a phylogenetic tree of thegenus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla iso-lated from many different locations of Argentine. In addition, several strains that might represent novelspecies were detected. The results indicate that the sequencing of rpoD is a suitable alternative methodfor a confident molecular identification in Azospirillum spp.Inst. de Microbiología y Zoología Agrícola IMyZAFil: Maroniche, Guillermo Andrés. Unidad Integrada Balcarce-Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias-Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garcia, Julia Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Salcedo, Florencia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentin

Friends or foes in the rhizosphere: traits of fluorescent Pseudomonas that hinder Azospirillum brasilense growth and root colonization

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.Fil: Maroniche, Guillermo Andrés. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Diaz, Pablo Rafael. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; ArgentinaFil: Borrajo, María Paula. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Valverde, Claudio Fabián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentin

Denitrification-derived nitric oxide modulates biofilm formation in Azospirillum brasilense

Azospirillum brasilense is a rhizobacterium that provides beneficial effects on plants when they colonize roots. The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with surfaces in response to appropriate signals. Nitric oxide (NO) is a signaling molecule implicated in numerous processes in bacteria, including biofilm formation or dispersion, depending on genera and lifestyle. Azospirillum brasilense Sp245 produces NO by denitrification having a role in root growth promotion. We analyzed the role of endogenously produced NO on biofilm formation in A. brasilense Sp245 and in a periplasmic nitrate reductase mutant (napA::Tn5; Faj164) affected in NO production. Cells were statically grown in media with nitrate or ammonium as nitrogen sources and examined for biofilm formation using crystal violet and by confocal laser microscopy. Both strains formed bio- films, but the mutant produced less than half compared with the wild type in nitrate medium showing impaired nitrite production in this condition. NO measurements in biofilm confirmed lower values in the mutant strain. The addition of a NO donor showed that NO influences biofilm formation in a dose-dependent manner and reverses the mutant phenotype, indicating that Nap positively regulates the formation of biofilm in A. brasilense Sp245.Fil: Arruebarrena Di Palma, Andrés. Universidad Nacional de Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pereyra, Cintia Mariana. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Moreno Ramírez, Lizbeth. Benemérita Universidad Autónoma de Puebla; MéxicoFil: Xiqui Vazquez, Maria L.. Benemérita Universidad Autónoma de Puebla; MéxicoFil: Baca, Beatriz E.. Benemérita Universidad Autónoma de Puebla; MéxicoFil: Pereyra, María Alejandra. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lamattina, Lamattina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Creus, Cecilia Mónica. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; Argentina. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

In vitro PGPR properties and osmotic tolerance of different Azospirillum native strains and their effects on growth of maize under drought stress

Osmotic variations in the soil can affect bacterial growth diminishing the number ofinoculated bacteria. In a scenario of water deficit having tolerant bacteria would bebeneficial to achieve a better response of the plant to stress. Thus, selection of moreresistant bacteria could be useful to design new inoculants to be used in arid zones. In thissense, a group of Azospirillum isolates deposited in INTA collection was characterized inorder to select strains tolerant to osmotic stress. The results obtained demonstrated that Az19 strain has similar in vitro PGPR characteristics to Az39, the most used strain inArgentina for inoculants industries, with the advantage of a better tolerance to osmotic andsalt stress. Inoculation of maize plants with this strain resulted in a better response againstwater deficit compared to Az39 strain, encouraging us to further study the behavior of thisstrain in greenhouse and field trials in view of developing new inoculants suitable for areaswith water deficit.Fil: García, Julia E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronomicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Maroniche, Guillermo Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Creus, Cecilia Mónica. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Suarez Rodriguez, Ramón. Universidad Autónoma del Estado de Morelos; MéxicoFil: Ramirez Trujillo, José Augusto. Universidad Autónoma del Estado de Morelos; MéxicoFil: Groppa, María Daniela. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Nitric Oxide as a signal molecule in intra- and extra-cellular bacteria-plant interactions

All plants live in intimate association with many microorganisms. These microorganisms colonize the surfaces, the intercellular spaces within tissues or even the inside of plant cells (Brencic and Winans, 2005). Some of the most complex interactions that terrestrial plants experience occur between the roots and their surrounding environment (Bais et al., 2006). It is precisely in the soil where these interactions are subjected to a plethora of conditions that determine the success or failure of root colonization, leading to effects on plant developmental processes. Soil has been divided into three main zones according proximity to the root: (1) rhizoplane or the root surface, (2) rhizosphere or the soil under root “influence,” and (3) bulk soil (Manthey et al., 1994). Bacteria that inhabit soils can affect plant growth and development. The interactions they establish with roots vary from beneficial, deleterious, or neutral effects on plants (Hirsch et al., 2003). The distribution of microorganisms in the rhizosphere can be classified into four main categories related to root proximity and intimacy: (1) bacteria living in the soil near roots, using metabolites “leaked” from roots as carbon (C) and N sources, (2) bacteria colonizing the rhizoplane, (3) bacteria residing in root tissue, inhabiting spaces between cortical cells, and (4) bacteria living inside cells in specialized root structures or nodules (Gray and Smith, 2005). The latter are generally represented by two groups: the legume-rhizobia and the woody plant- Frankia associations (Hallman et al., 1997; Gray et al., 2005). A more simple and convenient classification based on the preferred colonizing site is proposed by Gray and Smith (2005) dividing plant growth-promoting rhizobacteria (PGPR) into extracellular PGPR (ePGPR), and intracellular PGPR (iPGPR), the latter existing inside root cells generally in specialized nodular structures. classification based on the preferred colonizing site is proposed by Gray and Smith (2005) dividing plant growth-promoting rhizobacteria (PGPR) into extracellular PGPR (ePGPR), and intracellular PGPR (iPGPR), the latter existing inside root cells generally in specialized nodular structures. associations (Hallman et al., 1997; Gray et al., 2005). A more simple and convenient classification based on the preferred colonizing site is proposed by Gray and Smith (2005) dividing plant growth-promoting rhizobacteria (PGPR) into extracellular PGPR (ePGPR), and intracellular PGPR (iPGPR), the latter existing inside root cells generally in specialized nodular structures.Fil: Arruebarrena Di Palma, Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Lamattina, Lorenzo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata; Argentin

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.Fil: Pagnussat, Luciana Anabella. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Maroniche, Guillermo Andrés. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Curatti, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentin

Molecular identification of Azospirillum spp.: Limitations of 16S rRNA and qualities of rpoD as genetic markers

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp.Fil: Maroniche, Guillermo Andrés. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: García, Julia E.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Salcedo, Maria Florencia. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Cs.agrarias. Departamento de Introducción A Las Cs.agrarias. Area Biomolecular; Argentin

Potencial de rizobacterias nativas de Argentina para el control de Meloidogyne javanica

Biocontrol of the nematode Meloidogyne javanica was studied using the Argentinean strains Pseudomonas fluorescens MME3, TAE4, TAR5 and ZME4 and Bacillus sp. B7S, B9T and B19S. Pseudomonas protegens CHA0 was used as a positive control. Egg hatching and juvenile mortality were evaluated in vitro by exposure of nematodes to bacterial suspensions or their cell-free supernatants (CFS). The effect of bacteria on nematode infestation of lettuce was also studied. results showed that most of the tested strains and CFS reduced egg hatching and juvenile survival in vitro. The bacterial suspension of Bacillus sp. B9T produced the lowest hatching of eggs. Juvenile mortality was higher when M. javanica was exposed to Bacillus sp. than to Pseudomonas spp. suspensions. Except for CFS of B9T, all filtrates inhibited hatching at levels similar to or higher than the biocontrol strain P. protegens CHA0. The CFS of CHA0 showed the highest level of juvenile mortality followed by Bacillus sp. strains and P. fluorescens TAE4. None of the inoculated rhizobacteria reverted the negative effect of infestation on the aerial dry weight of lettuce plants. However, inoculation impacted on reproduction of M. javanica by reducing the development of galls and egg masses on roots and diminishing the number of individuals both on roots and in the substrate, as well as the reproduction factor. These results show that most of the analyzed native strains can control the nematode M. javanica. Among them, P. fluorescens TAE4 and Bacillus sp. B9T showed the most promising performances for the biocontrol of this pathogen and have a potential use in the formulation of commercial products.Fil: Borrajo, María Paula. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Mondino, Eduardo Ariel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Maroniche, Guillermo Andrés. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Fernández, Macarena. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata; ArgentinaFil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentin

Endogenous ascorbate restrains apoplastic peroxidase activity during sunflower leaf development

Several apoplastic enzymes have been implicated in the control of elongation growth of plant cells. Among them, peroxidases contribute to both loosening and stiffening of the cell wall. They appear to be regulated by various mechanisms, including the action of extracellular inhibitors. To obtain evidence of the role of the enzyme–inhibitor interaction during leaf development, the intercellular washing fluids from Helianthus annuus leaves of different ages were isolated using standard methods of vacuum infiltration and centrifugation. Peroxidase activities, assessed using tetramethylbenzidine as substrate, increased during leaf development, reaching a maximum value after the leaves were fully expanded. An inhibitor, chemically characterised as ascorbate, co-localised with the enzyme in the apoplast. Moreover, there was a strong negative correlation between the action of peroxidase and the micromolar concentration of ascorbate in the apoplastic fluid. The results show that in growing leaves, the in planta ascorbate concentration is able to restrain peroxidase enzyme activity. Then, at the time of growth cessation, the loss of extracellular ascorbate relieves the inhibition on this enzyme that contributes to wall fixation.Fil: Pinedo, Marcela Lilian. Universidad Nacional de Mar del Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lechner, L.. Universidad Nacional de Mar del Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Creus, Cecilia Mónica. Instituto Nacional de Tecnología Agropecuaria; Argentina. Universidad Nacional de Mar del Plata; ArgentinaFil: Simontacchi, Marcela Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; ArgentinaFil: Aguirrezábal, Luis Adolfo Nazareno. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Fisiología Vegetal. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Fisiología Vegetal; Argentin