A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control

Soybean seeds are non-sterile and their bacterial population interferes with the enumeration of beneficial bacteria, making it difficult to assess survival under different conditions. Within this context, the principal aims of this work were: (1) to improve a selective media for the enumeration of B. japonicum recovered from inoculated soybean seeds; (2) to establish the most representative mathematical function for B. japonicum mortality on soybean seeds after inoculation; (3) to evaluate if environmental or physiological conditions modify B. japonicum mortality on soybean seeds; and (4) to create a new protocol for quality control of soybean inoculants. We successfully evaluated the combination of pentachloronitrobenzene and vancomycin added to the yeast-mannitol medium to inhibit most fungi and Gram-positive soybean microbiota, thus producing reliable counts of B. japonicum from inoculated soybean seeds. Percentages of recovery and survival factors were obtained and used to construct a two-phase exponential decay non-linear regression function. High temperature and desiccation decreased these parameters, while the optimization of temperature and the use of osmoprotective compounds with inoculants increased them. The use of this protocol minimized heterogeneity between experiments and may be considered more reliable than the simple expression of direct colony count of bacteria recovered from seeds

A simple method to evaluate the number of bradyrhizobia on soybean seeds and its implication on inoculant quality control

Soybean seeds are non-sterile and their bacterial population interferes with the enumeration of beneficial bacteria, making it difficult to assess survival under different conditions. Within this context, the principal aims of this work were: (1) to improve a selective media for the enumeration of B. japonicum recovered from inoculated soybean seeds; (2) to establish the most representative mathematical function for B. japonicum mortality on soybean seeds after inoculation; (3) to evaluate if environmental or physiological conditions modify B. japonicum mortality on soybean seeds; and (4) to create a new protocol for quality control of soybean inoculants. We successfully evaluated the combination of pentachloronitrobenzene and vancomycin added to the yeast-mannitol medium to inhibit most fungi and Gram-positive soybean microbiota, thus producing reliable counts of B. japonicum from inoculated soybean seeds. Percentages of recovery and survival factors were obtained and used to construct a two-phase exponential decay non-linear regression function. High temperature and desiccation decreased these parameters, while the optimization of temperature and the use of osmoprotective compounds with inoculants increased them. The use of this protocol minimized heterogeneity between experiments and may be considered more reliable than the simple expression of direct colony count of bacteria recovered from seeds

Production and function of jasmonates in nodulated roots of soybean plants inoculated with Bradyrhizobium japonicum

Little is known regarding production and function of endogenous jasmonates (JAs) in root nodules of soybean plants inoculated with Bradyrhizobium japonicum. We investigated (1) production of jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) in roots of control and inoculated plants and in isolated nodules; (2) correlations between JAs levels, nodule number, and plant growth during the symbiotic process; and (3) effects of exogenous JA and OPDA on nodule cell number and size. In roots of control plants, JA and OPDA levels reached a maximum at day 18 after inoculation; OPDA level was 1.24 times that of JA. In roots of inoculated plants, OPDA peaked at day 15, whereas JA level did not change appreciably. Shoot dry matter of inoculated plants was higher than that of control at day 21. Chlorophyll a decreased more abruptly in control plants than in inoculated plants, whereas b decreased gradually in both cases. Exogenous JA or OPDA changed number and size of nodule central cells and peripheral cells. Findings from this and previous studies suggest that increased levels of JA and OPDA in control plants are related to senescence induced by nutritional stress. OPDA accumulation in nodulated roots suggests its involvement in "autoregulation of nodulation".Fil: Costanzo, María Emilia. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Andrade, Andrea Mariela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; ArgentinaFil: Tordable, Maria del Carmen. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Cassán, Fabricio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; ArgentinaFil: Abdala, Guillermina Irene. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales; Argentin

Development of sequence characterized amplified region markers for identification of Azospirillum brasilense Az39

Azospirillum brasilense Az39 es utilizada por empresas productoras de inoculantes para la formulación de bioinsumos en América del Sur desde hace más de 30 años. Esta cepa puede promover el crecimiento, desarrollo, así como la capacidad de tolerar diferentes tipos de estrés en las plantas inoculadas, lo que determina un aumento de la productividad de cultivos de interés agronómico. En la actualidad, no existen protocolos en Argentina que permitan confirmar la identidad de Az39 en productos comerciales a nivel de laboratorios de control de calidad de inoculantes. Por ello, el objetivo de este trabajo fue desarrollar una metodología en base molecular que permita la identificación certera de A. brasilense Az39. Con la secuencia completa del genoma y mediante herramientas bioinformáticas, se pudieron reconocer fragmentos de ADN presentes únicamente en el genoma de Az39. Se diseñaron cebadores dirigidos a amplificar por PCR dichas secuencias. Como resultado se observaron los productos específicos únicamente en la presencia de la cepa de interés. La reacción pudo detectar un título mínimo de 105 UFC/ml (4,5 ng/μl ADN) o de 102 UFC/ml (0,88 ng/μl ADN) o una concentración mínima de 0,098 ng/μl ADN, dependiendo del método de extracción utilizado. Los cebadores fueron evaluados en el análisis de productos comerciales obtenidos del mercado nacional, arrojando resultados positivos, tanto en muestras directas como así también en pruebas confirmatorias a 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 de microorganismos.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.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Coniglio, Anahí. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: López, Gastón Alberto. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: Gualpa, José L. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: Molina, Romina M. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: Rosas, Susana Beatriz. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola (IMYZA); ArgentinaFil: Mora, Verónica. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; ArgentinaFil: Cassán, Fabricio. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción Planta-Microorganismo; Argentin

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.Fil: Coniglio, Nayla Anahí. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Lopez, Gaston Alberto. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Gualpa, José Luis. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Molina, Romina Micaela. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Rosas, Susana Beatriz. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Mora, Maria Veronica. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; ArgentinaFil: Cassán, Fabricio. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Investigaciones Agrobiotecnologicas. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Investigaciones Agrobiotecnologicas.; Argentin

The benefits of foliar inoculation with Azospirillum brasilense in soybean are explained by an auxin signaling model

Azospirillum sp. is one of the most studied genera of plant growth-promoting rhizobacteria (PGPR). The ability of Azospirillum sp. to promote plant growth has been associated with its ability to produce several phytohormones, such as auxins, gibberellins and cytokinins, but mainly indole-3-acetic acid (IAA). It has been propoosed that the production of IAA explains the positive effects of co-inoculation with Azospirillum sp. on the rhizobia-legume symbiosis. In this study, we constructed an IAA-deficient mutant of A. brasilense Az39 (ipdC−) by using a restriction-free cloning method. We inoculated soybean seeds with 1·106 cfu·seed−1 of Bradyrhizobium japonicum E109 and co-inoculating leaves at the V3 stage with 1·108 cfu.plant−1 of A. brasilense Az39 wt or ipdC− or inoculated leaves with 20 μg.plant−1 synthetic IAA. The results confirmed soybean growth promotion as there was increased total plant and root length, aerial and root dry weight, number of nodules on the primary root, and an increase in the symbiosis established with B. japonicum E109. Nodule weight also increased after foliar co-inoculation with the IAA- producer A. brasilense Az39. The exogenous application of IAA decreased aerial and root length, as well as the number of nodules on primary roots in comparison with the Az39 wt strain. These results allow us to propose a biological model of response to foliar co-inoculation of soybean with IAA-producing rhizobacteria. This model clearly shows that both the presence of microorganism as part of the colonization process and the production of IAA in situ are co-responsible, via plant signaling molecules, for the positive effects on plant growth and symbiosis establishment.Inst. de Microbiología y Zoología Agrícola IMyZAFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola. Laboratorio de Bacterias Promotoras del Crecimiento Vegetal; ArgentinaFil: Gualpa, Jose Luis. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; ArgentinaFil: López, Gastón A. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; ArgentinaFil: Molina, Romina M. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; ArgentinaFil: Carletti, Susana. Universidad Nacional de Luján. Departamento de Ciencias Básicas; ArgentinaFil: Cassán, Fabricio D. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; Argentin

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.Instituto de Microbiología y Zoología AgrícolaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Zawoznik, Myriam. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento Química Biológica; ArgentinaFil: Lopez De Sabando, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Agencia De Extensión Rural Tandil; ArgentinaFil: Perez, Gonzalo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Pergamino. Agencia De Extensión Rural Bolivar; ArgentinaFil: Gualpa, José Luis. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; ArgentinaFil: Carletti, Susana M. Universidad Nacional de Luján. Departamento de Ciencias Básicas; ArgentinaFil: Cassán, Fabricio D. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Ciencias Naturales. Laboratorio de Fisiología Vegetal y de la Interacción planta-microorganismo; Argentin