Desarrollo de marcadores moleculares del tipo SCAR para la identificación de Azospirillum brasilense Az39 / Development of sequence characterized amplified región markers for identification of Azospirillum brasilense Az39

Resumen Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantespara la formulación de bioinsumos en América del Sur desde hace más de 30 a˜nos. Esta cepapuede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tiposde estrés en las plantas inoculadas, lo que determina un aumento de la productividad de culti-vos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitanconfirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control decalidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología enbase molecular que permita la identificación certera de A. brasilense Az39. Con la secuenciacompleta del genoma y mediante herramientas bioinformáticas, se pudieron reconocer frag-mentos de ADN presentes únicamente en el genoma de Az39. Se dise˜naron cebadores dirigidosa amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicosúnicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimode 105UFC/ml (4,5 ng/l ADN) o de 102UFC/ml (0,88 ng/l ADN) o una concentración mínimade 0,098 ng/l ADN, dependiendo del método de extracción utilizado. Los cebadores fueronevaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojandoresultados positivos, tanto en muestras directas como así también en pruebas confirmatoriasa partir de colonias aisladas de tales productos. La metodología desarrollada en este trabajo,permite la detección certera de A. brasilense Az39 en cultivos puros o mezclas complejas demicroorganismos.

Abstract 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/pl ADN) or 102 CFU/ml (0.88 ng/pl DNA) or in a minimal concentration of 0.098 ng/pl 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.

[Development of sequence characterized amplified region markers for identification of Azospirillum brasilense Az39]. / 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.

Multiplex and quantitative PCR targeting SCAR markers for strain-level detection and quantification of biofertilizers.

Biofertilizers are the eco-friendly bio-input being used to sustain the agriculture by reducing the chemical inputs and improving the soil health. Quality is the major concern of biofertilizer technology which often leads to poor performance in the field and thereby loses the farmers' faith. To authenticate the strain as well as its presumed cell load of a commercial product, sequence characterized amplified region (SCAR) markers were developed for three biofertilizer strains viz., Azospirillum brasilense (Sp7), Bacillus megaterium (Pb1) and Azotobacter chroococcum (Ac1). We evaluated the feasibility of multiplex-PCR and quantitative real-time PCR for SCAR marker-based quality assessment of the product as well as the persistence of the strains during crop growth. We showed that multiplex PCR can concurrently discriminate the strains based on the amplicons' size and detects up to 104 cells per g or per ml of carrier-based or liquid formulation of biofertilizer, respectively. The detection limit of quantitative PCR targeting SCAR markers is 103 cells per g or ml of biofertilizer. Both the PCR methods detected and quantified them in the maize rhizosphere. Hence SCAR marker-based quality assessment would be a sensitive tool to monitor the biofertilizer production as well as its persistence in the inoculated crop rhizosphere.

Screening of plant growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands.

Sorghum [Sorghum bicolor (L.) Moench] is a multipurpose grass cultivated in drylands due to its adaptation to drought. However the characteristics of sorghum-associated bacteria are not known in the Brazilian drylands. The aim of this study was to isolate and evaluate the plant growth promotion potential bacteria from field-grown sorghum under two irrigation and manure application levels in a Brazilian semi-arid reagion. Sorghum was irrigated with 3 or 1 mm day-1 and fertilized or not with liquid goat manure. Bacteria were obtained from surface-disinfected roots applying two nitrogen-free semi-solid media. The bacteria were evaluated for the presence of nifH gene, 16S rRNA sequences, calcium-phosphate solubilization, production of auxins and siderophores and for sorghum growth promotion. We obtained 20 out of 24 positive bacteria for nifH. The isolates were classified as in six different genera. All isolates produced auxins "in vitro", six bacteria produced siderophores and three Enterobacteriaceae solubilized calcium-phosphate. At least ten bacteria resulted in the increased total N content in the sorghum shoots, comparable to fertilization with 50 mg N plant-1 week-1 and to inoculation with Azospirillum brasilense Ab-V5. Enterobacter sp. ESA 57 was the best sorghum plant-growth promoting bacteria isolated in this study.

Azospirillum brasilense, a Beneficial Soil Bacterium: Isolation and Cultivation.

Bacteria of the genus Azospirillum comprise 15 species to date, with A. brasilense the best studied species in the genus. Azospirillum are soil bacteria able to promote the growth of plants from 113 species spanning 35 botanical families. These non-pathogenic and beneficial bacteria are ubiquitous in soils and inhabit the roots of diverse plants. These bacteria are microaerophilic, able to fix nitrogen under free-living conditions, motile, and able to navigate in gradients of various chemicals, including oxygen. These physiological traits are used to isolate these soil bacteria from soil and plant root samples, providing isolates that can be used for studying microbial physiology and plant growth promotion. © 2017 by John Wiley & Sons, Inc.

Labeled Azospirillum brasilense wild type and excretion-ammonium strains in association with barley roots.

Soil bacteria colonization in plants is a complex process, which involves interaction between many bacterial characters and plant responses. In this work, we labeled Azospirillum brasilense FP2 (wild type) and HM053 (excretion-ammonium) strains by insertion of the reporter gene gusA-kanamycin into the dinitrogenase reductase coding gene, nifH, and evaluated bacteria colonization in barley (Hordeum vulgare). In addition, we determined inoculation effect based on growth promotion parameters. We report an uncommon endophytic behavior of A. brasilense Sp7 derivative inside the root hair cells of barley and highlight the promising use of A. brasilense HM053 as plant growth-promoting bacterium.

[INFLUENCE OF AZOSPIRILLUM BRASILENSE 10/1 ON ASSOCIATIVE NITROGEN FIXATION AND INTRAVARIETAL POLYMORPHISM OF SPRING TRITICALE].

It is shown, that the perspective Ukrainian sorts of spring triticale characterizes by considerable polymorphism by associative N2-fixing ability in root zone of plants. Application of active strain Azospirillum brasilense 10/1 promotes the decline of variability of this sign within the limits of sort, increase potential nitrogen activity is on the average in 3,2-4,7 times and also distributing normalizations in the selections of the inoculated plants.

[Immunochemical Detection of Azospirilla in Soil with Genus-Specific Antibodies].

Immunoelectrophoresis and immunodiffusion analysis with antibodies to whole intact cells of the type strain of nitrogen-fixing soil bacteria Azospirillum brasilense Sp7 revealed at least three conservative surface immunogenic proteins of azospirilla. Cross-reactions with these proteins made it possible to use the above antibodies for detection of azospirilla as a genus-specific probe conjugated with horseradish peroxidase as an enzymatic label. Direct immune-enzyme analysis of soil suspensions (typical chernozem, Saratov oblast) confirmed applicability of the conjugates based on genus-specific antibodies to the surface proteins of azospirilla for direct detection of this bacterial genus in environmental samples. These results provide a basis for broad application of this method for analysis of Azospirillum occurrence in soil.

[Biofilm Formation by the Nonflagellated flhB1 Mutant of Azospirillum brasilense Sp245].

Azospirillum brasilense Sp245 with mixed flagellation are able to form biofilms on various surfaces. A nonflagellated mutant of this strain with inactivated chromosomal copy of the flhB gene (flhB1) was shown to exhibit specific traits at the later stages of biofilm formation on a hydrophilic (glass) surface. Mature biofilms of the flhB1::Omegon-Km mutant Sp245.1063 were considerably thinner than those of the parent strain Sp245. The biofilms of the mutant were more susceptible to the forces of hydrodynamic shear. A. brasilense Sp245 cells in biofilms were not found to possess lateral flagella. Cells with polar flagella were, however, revealed by atomic force microscopy of mature native biofilms of strain Sp245. Preservation of a polar flagellum (probably nonmotile) on the cells of A. brasilense Sp245 may enhance the biofilm stability.

Quantification of Azospirillum brasilense FP2 Bacteria in Wheat Roots by Strain-Specific Quantitative PCR.

Azospirillum is a rhizobacterial genus containing plant growth-promoting species associated with different crops worldwide. Azospirillum brasilense strains exhibit a growth-promoting effect by means of phytohormone production and possibly by N2 fixation. However, one of the most important factors for achieving an increase in crop yield by plant growth-promoting rhizobacteria is the survival of the inoculant in the rhizosphere, which is not always achieved. The objective of this study was to develop quantitative PCR protocols for the strain-specific quantification of A. brasilense FP2. A novel approach was applied to identify strain-specific DNA sequences based on a comparison of the genomic sequences within the same species. The draft genome sequences of A. brasilense FP2 and Sp245 were aligned, and FP2-specific regions were filtered and checked for other possible matches in public databases. Strain-specific regions were then selected to design and evaluate strain-specific primer pairs. The primer pairs AzoR2.1, AzoR2.2, AzoR5.1, AzoR5.2, and AzoR5.3 were specific for the A. brasilense FP2 strain. These primer pairs were used to monitor quantitatively the population of A. brasilense in wheat roots under sterile and nonsterile growth conditions. In addition, coinoculations with other plant growth-promoting bacteria in wheat were performed under nonsterile conditions. The results showed that A. brasilense FP2 inoculated into wheat roots is highly competitive and achieves high cell numbers (∼10(7) CFU/g [fresh weight] of root) in the rhizosphere even under nonsterile conditions and when coinoculated with other rhizobacteria, maintaining the population at rather stable levels for at least up to 13 days after inoculation. The strategy used here can be applied to other organisms whose genome sequences are available.

Search for endophytic diazotrophs in barley seeds.

Eight endophytic isolates assigned to Pseudomonas, Azospirillum, and Bacillus genera according to pheno-genotypic features were retrieved from barley seeds under selective pressure for nitrogen-fixers. Genetic relationships among related isolates were investigated through RAPD. Six isolates displayed nitrogen-fixing ability, while all could biosynthesize indolacetic acid in vitro and showed no antibiosis effects against Azospirillum brasilense Az39, a recognized PGPR.

Physiological and biochemical characterization of Azospirillum brasilense strains commonly used as plant growth-promoting rhizobacteria.

Azospirillum is a plant growth-promoting rhizobacteria (PGPR) genus vastly studied and utilized as agriculture inoculants. Isolation of new strains under different environmental conditions allows the access to the genetic diversity and improves the success of inoculation procedures. Historically, the isolation of this genus has been performed by the use of some traditional culture media. In this work we characterized the physiology and biochemistry of five different A. brasilense strains, commonly used as cereal inoculants. The aim of this work is to contribute to pose into revision some concepts concerning the most used protocols to isolate and characterize this bacterium. We characterized their growth in different traditional and non-traditional culture media, evaluated some PGPR mechanisms and characterized their profiles of fatty acid methyl esters and carbon-source utilization. This work shows, for the first time, differences in both profiles, and ACC deaminase activity of A. brasilense strains. Also, we show unexpected results obtained in some of the evaluated culture media. Results obtained here and an exhaustive knowledge revision revealed that it is not appropriate to conclude about bacterial species without analyzing several strains. Also, it is necessary to continue developing studies and laboratory techniques to improve the isolation and characterization protocols.

Search for endophytic diazotrophs in barley seeds

Eight endophytic isolates assigned to Pseudomonas, Azospirillum, and Bacillus genera according to pheno-genotypic features were retrieved from barley seeds under selective pressure for nitrogen-fixers. Genetic relationships among related isolates were investigated through RAPD. Six isolates displayed nitrogen-fixing ability, while all could biosynthesize indolacetic acid in vitro and showed no antibiosis effects against Azospirillum brasilense Az39, a recognized PGPR.

[Obtaining the phage mini-antibodies and their use for detection of microbial cells by using an electro-acoustic sensor].

The phage mini-antibodies to bacterial cells of strain Azospirillum brasilense Sp245 were obtained and the possibility of using them for detection of microbial cells by means of a lateral field excited piezoelectric resonator was studied. It has been found that the frequency dependencies of the real and imaginary parts of the electrical impedance of the resonator loaded by the cell suspension A. brasilense Sp245 with the mini-antibodies, significantly differ from those of the resonator with the control cell suspension without mini-antibodies. The concentration limit of possible determination of the microbial cells in their interaction with the mini-antibodies is equal to 10(3) cells/ml. It has been ascertained that detection of A. brasilense Sp245 cells using the mini-antibodies is possible even in the presence of other cultures, for example, E. coli BL-Ril and A. brasilense Sp7 cells. Therefore, it has been shown for the first time that detection of microbial cells by an electro-acoustic sensor is feasible.

[Isolation and purification of Mn-peroxidase from Azospirillum brasilense Sp245].

Homogenous Mn-peroxidase of a 26-fold purity grade was isolated from a culture of Azospirillum brasilense Sp245 cultivated on a medium containing 0.1 mM pyrocatechol. The molecular weight of the enzyme is 43 kD as revealed by electrophoresis in SDS-PAAG. It was shown that the use of pyrocatechol and 2,2'-azino-bis(3-ethylbenzotiazoline-6-sulfonate) at concentrations of 0.1 and I mM as inductors increased the Mn-peroxidase activity by a factor of 3.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

Azospirillum formosense sp. nov., a diazotroph from agricultural soil.

A gram-negative, spiral or rod-shaped, non-spore-forming diazotrophic bacterium, designated CC-Nfb-7(T), was isolated from agricultural soil in Yunlin County, Taiwan. 16S rRNA gene sequence analysis showed that strain CC-Nfb-7(T) was most closely related to Azospirillum brasilense DSM 1690(T) (97.4 % 16S rRNA gene sequence similarity), Azospirillum rugosum IMMIB AFH-6(T) (96.8 %) and Azospirillum oryzae JCM 21588(T) (96.6 %); <96.5 % 16S rRNA gene sequence similarity was found with all other members of the genus Azospirillum. DNA-DNA relatedness between strain CC-Nfb-7(T) and A. brasilense DSM 1690(T), A. rugosum DSM 19657(T) and A. oryzae JCM 21588(T) was 38.9, 30.1 and 31.8 %, respectively. The respiratory quinone was ubiquinone Q-10. The major fatty acids were summed feature 8 (consisting of C(18 : 1)ω7c and/or C(18 : 1)ω6c), summed feature 3 (consisting of C(16 : 1)ω7c and/or C(16 : 1)ω6c), summed feature 2 (consisting of C(14 : 0) 3-OH and/or iso-C(16 : 1) I), C(16 : 0), C(18 : 0) 2-OH and C(16 : 0) 3-OH. The polar lipids consisted mainly of phosphatidylglycerol, phosphatidylcholine and one unidentified phospholipid. Furthermore, moderate amounts of phosphatidylethanolamine, phosphatidyldimethylethanolamine and one unidentified aminophospholipid were also detected. Strain CC-Nfb-7(T) could be distinguished from members of phylogenetically related species by differences in phenotypic properties. On the basis of morphological, chemotaxonomic and phylogenetic data, strain CC-Nfb-7(T) represents a novel species within the genus Azospirillum, for which we propose the name Azospirillum formosense sp. nov. The type strain is CC-Nfb-7(T) ( = BCRC 80273(T) = JCM 17639(T) = DSM 24137(T)).

Structural and functional peculiarities of the lipopolysaccharide of Azospirillum brasilense SR55, isolated from the roots of Triticum durum.

Azospirillum brasilense SR55, isolated from the rhizosphere of Triticum durum, was classified as serogroup II on the basis of serological tests. Such serogroup affiliation is uncharacteristic of wheat-associated Azospirillum species. The lipid A of A. brasilense SR55 lipopolysaccharide contained 3-hydroxytetradecanoic, 3-hydroxyhexadecanoic, hexadecanoic and octadecenoic fatty acids. The structure of the lipopolysaccharide's O polysaccharide was established, with the branched octasaccharide repeating unit being represented by l-rhamnose, l-3-O-Me-rhamnose, d-galactose and d-glucuronic acid. The SR55 lipopolysaccharide induced deformations of wheat root hairs. The lipopolysaccharide was not involved in bacterial cell aggregation, but its use to pretreat wheat roots was conducive to cell adsorption. This study shows that Azospirillum bacteria can utilise their own lipopolysaccharide as a carbon source, which may give them an advantage in competitive natural environments.

Efecto de la biofertilización y los biorreguladores en la germinación y el crecimiento de carica papaya L / Effect of biofertilization and bioregulators on germination and growth of carica papaya L

Con el objetivo de incrementar y acelerar el proceso de germinación de las semillas y obtener una alta producción y homogeneidad de plántulas de Carica papaya variedad Maradol en vivero, se evaluó el efecto de tres biofertilizantes aplicados solos o en combinación (Azotobacter chroococcum, Azospirillum brasilense y Glomus intraradices), y un biorregulador del crecimiento vegetal, el ácido giberélico (AG3), en la germinación y el crecimiento vegetal. Se realizó un experimento bajo un diseño completamente al azar con ocho tratamientos y tres repeticiones. A las semillas se les aplicó un pretratamiento germinativo con alternancia de temperatura para superar la dormancia. Los tratamientos simples con A. chroococcum y A. brasilense, incrementaron el porcentaje de germinación a 90,28 y 88,89% respectivamente. Además, con la aplicación de los biofertilizantes y el AG3, la velocidad de germinación se incrementó y el tiempo medio de germinación se redujo. La doble aplicación en semillas y foliar de los biofertilizantes y el AG3 en plántulas mejoró el crecimiento vegetal. La población de A. chroococcum fue mayor cuando se inoculó en combinación con G. intraradices. La prevalencia de colonización de las plántulas inoculadas con G. intraradices varió de 18,53 a 26,67%, con el mayor valor registrado para el tratamiento combinado con A. brasilense. Finalmente, aplicando esta metodología se logró acelerar la germinación, obteniéndose una mayor homogeneidad en la emergencia de las plántulas, disminuyendo así el tiempo de permanencia en el vivero.

In order to increase and accelerate the process of seed germination and obtain a high yield and homogeneity of papaya seedlings cv. Maradol in nurseries, we evaluated the effect of three biofertilizers applied single or in combination (Azotobacter chroococcum, Azospirillum brasilense and Glomus intraradices) and a plant growth bioregulator, the gibberellic acid 3 (AG3), on the germination and subsequent growth of papaya seedlings. An experimental design completely random with eight treatments and three replications were used. The application of a pre-germinal treatment with alternating temperature had to be applied to seeds to overcome dormancy. Single biofertilization with A. chroococcum and A. brasilense, promoted the germination percentage 90.28 y 88.89% respectively. Germination rate could be enhanced and the mean germination time was reduced with the application of biofertilizer and AG3. Both applications on seeds and leaves of biofertilizers and AG3, had a positive effect on plant growth. The population of A. chroococcum was higher in the combined inoculation with G. intraradices. The prevalence of colonization of plants inoculated with G. intraradices ranged from 18.53 to 26.67%, with the greatest values recorded for the treatment involving combined inoculation with A. brasilense. Finally, with the application of this methodology the seed germination rate was improved, as well as the uniformity of seedlings emergence...