Evaluation of water stress and co-inoculation of Azospirillum brasilense and Rhizobium tropici in beans (Phaseolus vulgaris l) / Avaliação do estratégio de água e co-inoculação de Azospirillum brasilense e Rhizobium tropici in beans (Phaseolus vulgaris l) / Evaluación del estrés hídrico y la coinoculación de Azospirillum brasilense y Rhizobium tropici en frijoles (Phaseolus vulgaris l)

Climate change has caused major changes in abiotic factors, with water stress as the greatest threat to agricultural production. The measures aimed at alleviating the problems caused by this limiting production factor have occurred through the adoption of sustainable strategies, especially microbial biotechnology, which uses the interactions between the microorganism and the plant, ensuring productive quality and inducing plant resistance to stresses biotic and abiotic. The objective of the present work was to evaluate the biological nitrogen fixation and the development of bean seedlings, with co-inoculation of two types of inoculants, which were subjected to water stress by different pot capacities. The experiment was conducted in a greenhouse, at Universidade Paranaense - UNIPAR, from April to June 2019. The experimental design was completely randomized (DIC), with 5 replications, 16 treatments and 80 experimental units. The cultivar used was SCS Riqueza. The parameters evaluated were pot capacity (25%, 50%, 75% and 90%); small, large and total nodules, shoot and root length, dry and fresh weight, total carbon and nitrogen. The evaluation of the morphological parameters of the bean seedlings indicated that the co- inoculation technique promoted beneficial effects for the dry mass parameters of shoot, nodule and root. The analysis of the percentage of carbon and nitrogen in the tissues of the seedlings provided an increase in the concentration of these elements in treatments that involved co-inoculation (Azospirillum brasilensis and Rhizobium tropici) with pot capacities of 25 and 75% (CV), demonstrating that the association of microorganisms is beneficial in the limiting water situation.(AU)

A mudança climática tem causado grandes mudanças nos fatores abióticos, sendo o estresse hídrico a maior ameaça à produção agrícola. As medidas destinadas a aliviar os problemas causados por este fator limitante de produção ocorreram através da adoção de estratégias sustentáveis, especialmente a biotecnologia microbiana, que utiliza as interações entre o microorganismo e a planta, garantindo a qualidade produtiva e induzindo a resistência da planta ao estresse biótico e abiótico. O objetivo do presente trabalho foi avaliar a fixação biológica de nitrogênio e o desenvolvimento de mudas de feijão, com co-inoculação de dois tipos de inoculantes, que foram submetidos ao estresse hídrico por diferentes capacidades de vaso. A experiência foi realizada em uma estufa, na Universidade Paranaense - UNIPAR, de abril a junho de 2019. O projeto experimental foi completamente randomizado (DIC), com 5 réplicas, 16 tratamentos e 80 unidades experimentais. A cultivar utilizada foi a SCS Riqueza. Os parâmetros avaliados foram a capacidade do vaso (25%, 50%, 75% e 90%); nódulos pequenos, grandes e totais, comprimento do rebento e da raiz, peso seco e fresco, carbono total e nitrogênio. A avaliação dos parâmetros morfológicos das mudas de feijão indicou que a técnica de co-inoculação promoveu efeitos benéficos para os parâmetros de massa seca do turião, nódulo e raiz. A análise da porcentagem de carbono e nitrogênio nos tecidos das mudas proporcionou um aumento na concentração destes elementos nos tratamentos que envolveram a co-inoculação (Azospirillum brasilensis e Rhizobium tropici) com capacidades de vaso de 25 e 75% (CV), demonstrando que a associação de microorganismos é benéfica na situação limite da água.(AU)

El cambio climático ha provocado importantes cambios en los factores abióticos, siendo el estrés hídrico la mayor amenaza para la producción agrícola. Las medidas encaminadas a paliar los problemas causados por este factor limitante de la producción se han producido mediante la adopción de estrategias sostenibles, especialmente la biotecnología microbiana, que utiliza las interacciones entre el microorganismo y la planta, asegurando la calidad productiva e induciendo la resistencia de la planta a los estreses bióticos y abióticos. El objetivo del presente trabajo fue evaluar la fijación biológica de nitrógeno y el desarrollo de plántulas de frijol, con la co-inoculación de dos tipos de inoculantes, que fueron sometidos a estrés hídrico por diferentes capacidades de maceta. El experimento se realizó en un invernadero, en la Universidade Paranaense - UNIPAR, de abril a junio de 2019. El diseño experimental fue completamente al azar (DIC), con 5 repeticiones, 16 tratamientos y 80 unidades experimentales. El cultivar utilizado fue SCS Riqueza. Los parámetros evaluados fueron capacidad de maceta (25%, 50%, 75% y 90%); nódulos pequeños, grandes y totales, longitud de brotes y raíces, peso seco y fresco, carbono y nitrógeno total. La evaluación de los parámetros morfológicos de las plántulas de frijol indicó que la técnica de coinoculación promovió efectos beneficiosos para los parámetros de masa seca de brotes, nódulos y raíces. El análisis del porcentaje de carbono y nitrógeno en los tejidos de las plántulas proporcionó un aumento en la concentración de estos elementos en los tratamientos que involucraron la coinoculación (Azospirillum brasilensis y Rhizobium tropici) con capacidades de maceta de 25 y 75% (CV), demostrando que la asociación de microorganismos es beneficiosa en la situación de agua limitante.(AU)

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

Characterization of glutamine synthetase from the ammonium-excreting strain HM053 of Azospirillum brasilense / Caracterização da glutamina sintetase da estipe excretora de amônio HM053 de Azospirillum brasilense

Glutamine synthetase (GS), encoded by glnA, catalyzes the conversion of L-glutamate and ammonium to L-glutamine. This ATP hydrolysis driven process is the main nitrogen assimilation pathway in the nitrogen-fixing bacterium Azospirillum brasilense. The A. brasilense strain HM053 has poor GS activity and leaks ammonium into the medium under nitrogen fixing conditions. In this work, the glnA genes of the wild type and HM053 strains were cloned into pET28a, sequenced and overexpressed in E. coli. The GS enzyme was purified by affinity chromatography and characterized. The GS of HM053 strain carries a P347L substitution, which results in low enzyme activity and rendered the enzyme insensitive to adenylylation by the adenilyltransferase GlnE.

A glutamina sintetase (GS), codificada por glnA, catalisa a conversão de L-glutamato e amônio em L-glutamina. Este processo dependente da hidrólise de ATP é a principal via de assimilação de nitrogênio na bactéria fixadora de nitrogênio Azospirillum brasilense. A estirpe HM053 de A. brasilense possui baixa atividade GS e excreta amônio no meio sob condições de fixação de nitrogênio. Neste trabalho, os genes glnA das estirpes do tipo selvagem e HM053 foram clonados em pET28a, sequenciados e superexpressos em E. coli. A enzima GS foi purificada por cromatografia de afinidade e caracterizada. A GS da estirpe HM053 possui uma substituição P347L que resulta em baixa atividade enzimática e torna a enzima insensível à adenililação pela adenililtransferase GlnE.

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.

Valor nutritivo da Coastcross-1 inoculada com Azospirillum brasilense / Nutritive value of Coastcross-1 inoculated with Azospirillum brasilense

Objetivou-se com este trabalho avaliar a taxa de produção de forragem e o valor nutritivo da Coastcross-1, inoculada com Azospirillum brasilense, fertilizada com diferentes doses de N e submetida ao regime de corte. O delineamento experimental foi o de blocos ao acaso, com três repetições, em esquema fatorial. Os fatores foram o uso da inoculação (não inoculada, inoculada somente no plantio e reinoculada no segundo ano), níveis de N (0, 100 e 200kg/ha/ano de N) e os períodos do ano. Avaliaram-se a taxa de produção e a composição botânica da forragem, a composição morfológica, o teor de proteína bruta, a digestibilidade in situ da matéria orgânica e os nutrientes digestíveis totais da Coastcross-1. A inoculação, quando não associada ao adubo nitrogenado, ocasionou aumento na taxa de produção de forragem, na participação da Coastcross-1 e na produção de nutrientes digestíveis totais. Houve redução nos teores de proteína bruta da Coastcross-1, quando a gramínea adubada com 200kg/ha/ano de N foi inoculada. A inoculação tem efeito positivo na Coastcross-1 não submetida à adubação nitrogenada.(AU)

The objective of this study was to evaluate the forage yield rate and nutritive value of Coastcross-1 inoculated with Azospirillum brasilense and fertilized with different levels of nitrogen subjected to cut. The experimental design was randomized blocks with three replications, in a factorial scheme. The factors were the inoculation (without inoculation, inoculated just in the implantation and reinoculated in the second year), levels of nitrogen (0, 100 and 200kg/ha/year of N) and periods of the year. Forage yield rate and botanical composition; morphological composition, crude protein content, in situ digestibility of organic matter and the total digestible nutrients of Coastcross-1 were evaluated. Inoculation increased the forage yield rate, Coastcross-1 contribution and production of total digestible nutrients, when the pasture was not fertilized with N. There was reduction in Coastcross-1 crude protein content when fertilized with 200kg/ha/yr of N. The inoculation has a positive effect on Coastcross-1 not submitted to nitrogen fertilization.(AU)

Initial growth in maize in compliance of Azospirillum brasilense inoculation and nitrogen rates / Crescimento inicial do milho em função da inoculação de Azospirillum brasilense e doses de nitrogênio

Investigate the effects of the interaction between nitrogen fertilization and inoculation with diazotrophic bacteria becomes important, especially for maize, highly exacting in N. Thus, the aim of this study was to evaluate the initial growth of maize in compliance A. brasilense inoculation and nitrogen rates. The experiment was conducted in a greenhouse, in Mato Grosso do Sul State University, Aquidauana University Unit, between the months of march and April of 2014. It was used completely randomized design in a 2x4 factorial arrangement with four replications. The treatments consisted of the absence and presence of inoculation with A. brasilense and four levels of nitrogen fertilization (0, 25, 50, 100 kg ha-1 N). At 40 days after sowing the following variables were evaluated: leaf area, plant height, stem diameter, dry mass of shoot and of root. There was interaction between doses of nitrogen and inoculation with A. brasilense for most of the evaluated variables. Inoculation with A. brasilense provides improvements in the establishment and initial development of maize. In the presence of inoculation with A. brasilense, the development of maize increases linearly the administration of the nitrogen doses.

Investigar os efeitos da interação entre adubação nitrogenada e inoculação com bactérias diazotróficas tornou-se importante, sobretudo para a cultura do milho, altamente exigente em N. Assim, o objetivo deste trabalho foi verificar o crescimento inicial do milho em função de inoculação A. brasilense e doses de nitrogênio. O experimento foi desenvolvido em estufa agrícola, na Universidade Estadual de Mato Grosso do Sul, Unidade Universitária de Aquidauana, entre os meses de março a abril de 2014. Utilizou-se delineamento experimental inteiramente casualizado em esquema fatorial 2x4, com quatro repetições. Os tratamentos constaram da combinação da ausência e presença de inoculação com A. brasilense e quatro níveis de adubação nitrogenada (0; 25; 50; 100 kg ha-1 de N). Aos 40 dias após a semeadura foram avaliadas as seguintes variáveis: área foliar, altura de plantas, diâmetro do colmo, massa seca da parte aérea e massa seca das raízes. Houve interação entre doses de nitrogênio e inoculação com A. brasilense para a maioria das variáveis avaliadas. A inoculação com A. brasilense proporciona melhorias no estabelecimento e no desenvolvimento inicial do milho. Na presença de inoculação com A. brasilense, o desenvolvimento de milho aumenta linearmente a função das doses de nitrogênio.

Tiller density and tillering on Brachiaria brizantha cv. Marandu pastures inoculated with Azospirillum brasilense / Densidade populacional e perfilhamento em pastagens de Brachiaria brizantha cv. Marandu inoculada com Azospirillum brasilense

The objective of this study is to verify the population density and the dynamics of tillering in the Marandu palisade grass sward subjected to nitrogen (N) fertilization strategies, characterized by the N supply via urea or bacterial inoculant (Azospirillum brasilense). The treatments comprised of four nitrogen fertilization strategies: (A) Without fertilization, (B) 80 kg N/ha, (C) inoculant (A. brasilense), and (D) 80 kg N/ha + inoculant, distributed in a randomized complete block design, with three replications. The nitrogen supply strategies were evaluated during six periods: October, November, and December (2012) as well as January, March, and April (2013). The nitrogen dose or inoculant had no effect on the tiller appearance rate (TAR), tiller mortality rate (TMR), tiller survival rate (TSR), or tiller population density (TPD). However, these variables were influenced by the season. The TAR and TSR were higher at the beginning of the experimental period (October) and lower towards the end of the period (March-April), whereas, TMR and TPD exhibited the opposite behavior, with lower values in October and higher from January onward. Neither the nitrogen nor the inoculant influenced the population dynamics of the tillers in Marandu palisade grass.(AU)

Objetivou-se com este estudo verificar a densidade populacional e a dinâmica do perfilhamento em dosséis de capim Marandu submetidos a estratégias de adubação nitrogenada, caracterizadas pelo fornecimento de N via ureia ou inoculante (Azospirillum brasilense). Os tratamentos foram quatro estratégias de adubação nitrogenada: sem fertilização, 80kg N/ha, inoculante (A. brasilense) e 80kg N/ha + inoculante, distribuídos em um delineamento de blocos completos ao acaso, com três repetições por tratamento. As estratégias de aporte nitrogenado foram avaliadas em seis épocas: outubro, novembro e dezembro de 2012; e janeiro, março e abril de 2013. Não foi verificada influência da dose de nitrogênio ou do inoculante sobre a taxa de aparecimento (TAP), a taxa de mortalidade (TMP), a taxa de sobrevivência (TSP) e a densidade populacional de perfilhos (DPP). No entanto, essas variáveis foram influenciadas pela época do ano. A TAP e a TSP apresentaram maiores valores no início do período experimental (outubro) e menor valor ao final do período (março a abril). A TMP e a DPP expressaram respostas opostas, com menores valores em outubro e maiores a partir de janeiro. Não houve influência do nitrogênio e do inoculante sobre a dinâmica populacional de perfilhos em capim Marandu.(AU)

Compatibility of Azospirillum brasilense and Pseudomonas fluorescens in growth promotion of groundnut ( Arachis hypogea L)

ABSTRACT We attempted to study the compatibility among plant beneficial bacteria in the culture level by growing them near in the nutrient agar plates. Among all the bacteria tested, Rhizobium was found to inhibit the growth of other bacteria. From the compatible group of PGPR, we have selected one biofertilizer (Azospirillum brasilense strain TNAU) and one biocontrol agent (Pseudomonas fluorescens strain PF1) for further studies in the pot culture. We have also developed a bioformulation which is talc powder based, for individual bacteria and mixed culture. This formulation was used as seed treatment, soil application, seedling root dip and foliar spray in groundnut crop in vitro germination conditions. A. brasilense was found to enhance the tap root growth and P. fluorescens, the lateral root growth. The other growth parameters like shoot growth, number of leaves were enhanced by the combination of both of the bacteria than their individual formulations. Among the method of application tested in our study, soil application was found to be the best in yielding better results of plant growth promotion.

A simple and efficient method for poly-3-hydroxybutyrate quantification in diazotrophic bacteria within 5 minutes using flow cytometry

The conventional method for quantification of polyhydroxyalkanoates based on whole-cell methanolysis and gas chromatography (GC) is laborious and time-consuming. In this work, a method based on flow cytometry of Nile red stained bacterial cells was established to quantify poly-3-hydroxybutyrate (PHB) production by the diazotrophic and plant-associated bacteria, Herbaspirillum seropedicae and Azospirillum brasilense. The method consists of three steps: i) cell permeabilization, ii) Nile red staining, and iii) analysis by flow cytometry. The method was optimized step-by-step and can be carried out in less than 5 min. The final results indicated a high correlation coefficient (R2=0.99) compared to a standard method based on methanolysis and GC. This method was successfully applied to the quantification of PHB in epiphytic bacteria isolated from rice roots.

Microorganisms associated to tomato seedlings growing in saline culture act as osmoprotectant

Less than 0.5% of total water in the world is available for human consumption and agriculture. The major part of the world's water is saline and salinity in soils interferes in germination of seeds and the posterior development of the plant. In order to increase the osmotolerance of tomato, seedlings were associated with Azospirillum brasilense Cd, Azospirillum brasilense Cd transformed bacteria with a plasmid harboring a trehalose biosynthesis gene-fusion or Chlorella vulgaris. Two plant culture media: Hydroponic and Murashige and Skoog were tested. In the first set of studies seedlings were associated to single free cells meanwhile in a second set single and combined free cells were studied. A positive interaction between transformed Azospirillum and Chlorella vulagris and tomato plants was observed. Seedlings showed a salt concentration tolerance, as sodium chloride, up to 200 mM. According to our results, the association of plants with A. brasilense Cd-BIF and C. vulgaris is a viable approach to increase their salt tolerance and biomass, as consequence the possible use of sea water to irrigate horticultural plants.

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.

Effect of ATP and 2-oxoglutarate on the in vitro interaction between the NifA GAF domain and the GlnB protein of Azospirillum brasilense

Azospirillum brasilense is a diazotroph that associates with important agricultural crops and thus has potential to be a nitrogen biofertilizer. The A. brasilense transcription regulator NifA, which seems to be constitutively expressed, activates the transcription of nitrogen fixation genes. It has been suggested that the nitrogen status-signaling protein GlnB regulates NifA activity by direct interaction with the NifA N-terminal GAF domain, preventing the inhibitory effect of this domain under conditions of nitrogen fixation. In the present study, we show that an N-terminal truncated form of NifA no longer required GlnB for activity and lost regulation by ammonium. On the other hand, in trans co-expression of the N-terminal GAF domain inhibited the N-truncated protein in response to fixed nitrogen levels. We also used pull-down assays to show in vitro interaction between the purified N-terminal GAF domain of NifA and the GlnB protein. The results showed that A. brasilense GlnB interacts directly with the NifA N-terminal domain and this interaction is dependent on the presence of ATP and 2-oxoglutarate.

Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

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...

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state / Caracterização molecular de bactérias fixadoras de nitrogênio isoladas de plantas brasileiras no estado de São Paulo

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.

Different responses of the GlnB and GlnZ proteins upon in vitro uridylylation by the Azospirillum brasilense GlnD protein

Azospirillum brasilense is a diazotroph found in association with important agricultural crops. In this organism, the regulation of nitrogen fixation by ammonium ions involves several proteins including the uridylyltransferase/uridylyl-removing enzyme, GlnD, which reversibly uridylylates the two PII proteins, GlnB and GlnZ, in response to the concentration of ammonium ions. In the present study, the uridylylation/deuridylylation cycle of A. brasilense GlnB and GlnZ proteins by GlnD was reconstituted in vitro using the purified proteins. The uridylylation assay was analyzed using non-denaturing polyacrylamide gel electrophoresis and fluorescent protein detection. Our results show that the purified A. brasilense GlnB and GlnZ proteins were uridylylated by the purified A. brasilense GlnD protein in a process dependent on ATP and 2-oxoglutarate. The dependence on ATP for uridylylation was similar for both proteins. On the other hand, at micromolar concentration of 2-oxoglutarate (up to 100 µM), GlnB uridylylation was almost twice that of GlnZ, an effect that was not observed at higher concentrations of 2-oxoglutarate (up to 10 mM). Glutamine inhibited uridylylation and stimulated deuridylylation of both GlnB and GlnZ. However, glutamine seemed to inhibit GlnZ uridylylation more efficiently. Our results suggest that the differences in the uridylylation pattern of GlnB and GlnZ might be important for fine-tuning of the signaling pathway of cellular nitrogen status in A. brasilense.

Efecto de la biofertilización en vivero del cacao (Theobroma cacao L) con Azospirillum brasilense tarrand, Krieg et Dõbereiner y Glomus intraradices Schenk et Smith / Effect of cocoa (theobroma cacao L) biofertilitation in nursely whit Azospirillum brasilense Tarrand, Krieg et D”bereiner and Glomus intraradices Schenk et Smith

Theobroma cacao L. es una especie originaria de América y ha estado ligada al desarrollo de diversas culturas indígenas en las regiones tropicales húmedas. Su semilla se utiliza para la elaboración de alimentos, bebidas y golosinas y su demanda se ha incrementado cuando se cultiva sin agroquímicos. La nutrición de la planta mediante biofertilizantes microbianos es una alternativa para incrementar la oferta de cacao orgánico. En este trabajo se identificó el aporte de dos microsimbiontes en el desarrollo vegetal y nutrimental del cacao en dos condiciones de suelo del Soconusco, Chiapas, México, uno de ellos tratado con bromuro de metilo y otro sin tratar. Las semillas de cacao se inocularon con Azospirillum brasilense y Glomus intraradices, solos o combinados al momento de la siembra. Se registraron variables morfológicas y fisiológicas del rendimiento y el contenido de N2, P y Ca²+ en el tejido vegetal cada 30 días durante seis meses. Los resultados indicaron una respuesta diferencial entre condiciones de suelo y microsimbiontes en la asignación de materia seca. Los órganos de la planta más modificados fueron la raíz y la lámina foliar. Las plantas inoculadas mostraron mayor concentración de N2 en suelo no tratado. G. intraradices fue más efectivo en promover la incorporación de P en suelo no tratado y de Ca²+ en ambas condiciones del suelo.

Mode of utilization of amino acids as growth substrates by Azospirillum brasilense.

The study was undertaken to analyze the rate of uptake and utilization of various amino acids by Azospirillum brasilense Sp81 (RG) in a basal mineral salts solution under non-nitrogen fixing condition. These amino acids including other nitrogenous compounds were tested for both N- and C-sources. The kinetic constants (Km and Vmax) of uptake of some amino acids (e.g. lysine, arginine, proline, glutamine and glutamic acid) were exploited using a Hanes-Woolf plot, and discussed in the context of nitrogen starvation or both carbon and nitrogen starvation. To summarize all the kinetic data for these amino acids strongly suggested that the mode of these amino acids utilization in this bacterium followed the same general pattern, although the quantitative differences were there. A single amino acid was able to satisfy the nitrogen needs of this bacterium in basal mineral salts solution, and this possibility could be considered for the cost-effective growth medium for this bacterium in the biotechnological industry.