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1.
Rev. argent. microbiol ; 52(1): 50-60, mar. 2020. graf
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1155685

ABSTRACT

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.

2.
Article | IMSEAR | ID: sea-189625

ABSTRACT

In March 2016, the EU Commission presented a proposal for new regulations on fertilising material. The regulation includes product rules for a wide range of organic and inorganic products. Microbial biostimulants is one of the categories of products that are included. Biostimulants, in the draft EU regulation, are defined as fertilising materials that affect nutrient processes independently of the product's own nutrient content and with the purpose of improving nutrient utilisation, tolerance for abiotic stress or quality of the crop. Positive list in which species of these bacterial genera are listed: Azotobacter spp, Rhizobium spp., Azospirillum spp and Mycorrhizal fungi are a part of the regulation. Since the import and use of these organisms are the responsibility of both the Norwegian Food Safety Authority and the Norwegian Environment Agency, they asked VKM to submit a joint report on effects on health (humans, plants and animals), biodiversity and dispersal, quality of agricultural land and on soil environment. Conclusions: Health risks: Based upon our literature review, we have found no indication of any specific diseases in plants, animals or humans induced by the discussed microorganisms. A few reported cases of human disease are caused through wound infections or injections in immunocompromised patients. These represent a situation where any microorganism may induce infections and is not specific for the agents discussed in this report. In summary, the risk of any disease caused by the discussed microorganisms is considered negligible. Environmental risks: In soil the biodiversity, competition, adaptation and functional redundancy of microorganisms are extremely high. This means that introduced microorganisms have a very small chance for establishing, and even less so for affecting biodiversity and soil functioning. Introduction of nitrogen fixing species or fungi that can transport P to plants (mycorrhiza) will lead to an increase in the primary production. However, even a large increased activity for these processes will not outcompete naturally occurring symbiotic N-fixation or growth of inherently non-mycorrhizal plant species. Thus, the risks associated with introduced non-pathogenic microorganisms are very low.

3.
Entramado ; 13(2): 250-257, jul.-dic. 2017. tab, graf
Article in Spanish | LILACS-Express | LILACS | ID: biblio-1090152

ABSTRACT

Resumen La gallinaza fresca, debido a su rápida disponibilidad está siendo ampliamente utilizada como fuente exclusiva de nutrientes, especialmente nitrógeno (N), en el cultivo de cebolla (Allium fistulosum), sin embargo, problemas de toxicidad por sales y acumulación de metales pesados en plantas han sido detectados. El objetivo de este trabajo fue estimar el efecto de la gallinaza fresca y compostada sobre las bacterias Azotobacter sp. y Azospirillum sp., así como en el número de esporas de hongos micorrízicos arbusculares (HMA), en el cultivo de cebolla. En el experimento se utilizó un diseño de bloques al azar con cuatro tratamientos y tres réplicas a partir de requerimientos en fertilización del cultivo y el análisis de suelos. Los tratamientos fueron: T1-Gallinaza fresca + Fertilizante foliar líquido quelatado-(14 Ton/ha=400 gr/ sitio gallinaza fresca + complejo biofertilizante a base de N, P y aminoácidos-1 L/ha.), T2-Gallinaza compostada + Fertilizante foliar líquido quelatado-14 Ton/ha=400 gr/sitio gallinaza fresca + (complejo biofertilizante a base de N, P y aminoácidos-1 L/ha.), T3-Fertilizante de síntesis química: fertirrigación con complejo de fertilizante 20-20-20 + elementos menores (10 g/sitio=350 kg/ha) y T4-Fertilizante de síntesis química: Aplicación en el suelo con complejo de fertilizante 20-20-20+elementos menores (20 g/sitio=700 kg/ha.). Los resultados obtenidos indican que la gallinaza cruda tiene un impacto positivo en el rendimiento del cultivo de cebolla y la esporulación de HMA (91 esporas por gamo de solo), un efecto negativo en Azotobacter sp. con mejor desarrollo en la gallinaza compostada (1,0 x107 UFC) y en Azospirillum sp. sin respuesta relevante, sin embargo fue más abundante que Azotobacter sp.en cada uno de los tratamientos.


Abstract Poultry manure application is highly used as exclusive nutrient sources, especially N, in anion culture (A. fistulosum), However there has been detected toxicity problems by salts and heavy metals in plants. The aim of this study was to estimate the effect of fresh poultry manure and composted on Azotobacter sp., Azospirillum sp. and arbuscular mycorrhizal fungi (AMF). The treatments were performed under completely randomized design with 4 treatments and 3 replications according to fertilization requirements and soil analysis. Treatments were formed as follows: Tl-Poultry manure + Foliar liquid fertilizer quelated-14 ton.ha-1=400 grsite-1 poultry manure + complex biofertilizer of N, P and aminoacids-1 L.ha-1, T2-Composted poultry manure + Foliar liquid fertilizer quelated-14 ton.ha-1=400 grsite-1 poultry manure + complex biofertilizer of N, P and aminoacids-1 L.ha-1, T3-Chemical fertilizer: fertirrigation with complex biofertilizer 20-20-20 + minor elements (l0 grsite-1 =350 kg ha-1) and T4-Chemical fertilizer: Application in the soil with complex biofertilizer 20-20-20 + minor elements (20 kg ha-1=700 kg ha-1.). We observed that anion culture and AMF spores (91 spores by 1g soil) have a significant respond to poultry manure. Azotobacter sp. was negatively affected by poultry manure but positively influenced by composted poultry manure (l.0 xl07 CFU). We no observed a relevant respond of Azospirillum sp., although, it was more abundant than Azotobacter sp. in all treatments.


Resumo O esterco de galinha fresco, pela sua rápida disponibilidade é utilizada como fornecedor de nutrientes, especialmente N, na cultura da cebola (A. fistulosum), No entanto, problemas de toxicidade por sais e acumulação de metais pesados em plantas tem sido detectado. O objetivo deste trabalho foi estimar o efeito do esterco de galinha fresco e decomposto sobre as bactérias Azotobacter sp. e Azospirillum sp. e o número de esporos de fungos micorrízicos arbusculares (FMA) na cultura da cebola. O delineamento experimental foi de blocos ao acaso, com quatro tratamento e três repetições, segundo os requerimentos nutricionais da cultura e a análise de solo. Os tratamentos foram os seguintes: Tl-Cama aviaria fresca + fertilizante foliar líquido quelatado- (14 Ton/ha=400 gr/local-cama aviaria fresca + complexo biofertilizante baseado em N, P y aminoácidos-1 L/ha.), T2-Cama aviaria + fertilizante foliar líquido quelatado-14 Ton/ha=400 gr/local-Cama aviaria + (complexo biofertilizante baseado em N, P y aminoácidos-1 L/ha.), T3-Fertilizante de síntese química: fertirrigação com complexo de fertilizante 20-20-20 + elementos menores (10 g/local=350 kg/ha) y T4-Fertilizante de síntese química: Aplicação no solo com complexo de fertilizante 20-20-20 + elementos menores (20 g/local=700 kg/ha.).Os resultados aqui obtidos mostram efeito positivo do esterco de galinha no rendimento da cultura da cebola e da esporulação dos HMA (91 esporos por gama de solo), efeito negativo em Azotobacter sp., se desenvolvendo melhor no esterco de galinha decomposta (1,0 x107 UFC) e sem resposta relevante em Azospirillum sp., embora tenha sido mais abundante do que Azotobacter sp. em todos os tratamentos.

4.
Rev. argent. microbiol ; 49(4): 356-365, Dec. 2017. tab
Article in Spanish | LILACS | ID: biblio-1041798

ABSTRACT

El cacao (Theobroma cacao L.) es nativo de América del Sur y representa uno de los recursos «bioculturales¼ más significativos de Mesoamérica, ya que es una región donde se domesticó y tuvo relevancia como bebida ritual y como moneda en muchas culturas prehispánicas hasta la llegada de los españoles, quienes difundieron su uso en el mundo y lo convirtieron en una de las mercancías «commodity¼ más consumidas. Mediante este trabajo se propone una alternativa para atender la problemática de las plantaciones a través de la introducción de una diversidad amplia de cultivares de cacao en sistemas agroforestales tradicionales, en sinergia con la inoculación de bacterias edáficas fijadoras de nitrógeno y solubilizadoras de fósforo insoluble. En una parcela agroforestal tradicional se introdujeron plantas de cacao injertadas de cuatro cultivares y se dispusieron 3 tratamientos: aplicación de biofertilizante, aplicación de fertilizante químico y testigo. Se registraron la altura, el diámetro basal, el número de hojas y el número de ramas a los 2 y 12 meses, y se caracterizó la población de microorganismos asociada alrededor del tallo bajo la copa de las plantas. Los resultados de crecimiento muestran un buen potencial para los 4 cultivares estudiados y se observó que la biofertilización generó efectos significativos en algunos de los indicadores de crecimiento de las plantas de cacao. Así, las asociaciones vegetales en un sistema agroforestal podrían ser favorables para potenciar el desarrollo de frutos y resistencia a plagas y enfermedades.


Cocoa plant (Theobroma cacao L.) is native from South America and it represents one of the most significant "bio-cultural" resources of Mesoamerica, since it is a region where it was domesticated and had a relevance as ritual drink and as currency in many pre-hispanic cultures until the arrival of the Spaniards who spread its use worldwide, and became it one of the most consumed commodity goods. Through this research, an alternative is proposed to address the problem of cultivars through the introduction of a wide variety of cocoa plants in traditional agroforestry systems, in synergy with the inoculation of nitrogen-fixing and insoluble phosphor solubilizing edaphic bacterial consortia. Four cultivars of improved grafted cocoa plants were introduced in a traditional agroforestry plot and three fertilization treatments were applied: application of biofertilizer, application of chemical fertilizer and control. Measurements of height, stem diameter, number of leaves and branches were recorded at 2 and 12 months after planting and rhizosphere microbial populations were characterized. Growth results showed good potential for all studied cultivars and it was observed that biofertilization foresees significant effects in some of the growth indicators of cocoa plant. Thereby, plant associations in an agroforestry system could be favorable to promote fruit development and resistance to pests and diseases.


Subject(s)
Cacao , Agricultural Inoculants , South America , Cacao/microbiology , Cacao/growth & development , Forestry , Environment , Mexico
5.
An. acad. bras. ciênc ; 89(2): 1027-1040, Apr.-June 2017. tab, graf
Article in English | LILACS | ID: biblio-886675

ABSTRACT

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.


Subject(s)
Arachis/growth & development , Arachis/microbiology , Pseudomonas fluorescens/physiology , Azospirillum brasilense/physiology , Fertilizers , Rhizobium/physiology , Seeds/growth & development , Seeds/microbiology , Soil Microbiology , Azotobacter/physiology , Bacillus megaterium/physiology , Bacillus subtilis/physiology , Plant Roots/growth & development , Plant Roots/microbiology , Plant Leaves , Seedlings/growth & development , Seedlings/microbiology
6.
Rev. biol. trop ; 65(1): 21-30, Jan.-Mar. 2017. tab, ilus
Article in Spanish | LILACS | ID: biblio-897522

ABSTRACT

ResumenLa industria petrolera ha generado derrames crónicos de petróleo y su acumulación en Gleysoles en zonas anegadas en el estado de Tabasco, en el sureste de México. El anegamiento es un factor que limita el uso de tecnologías de remediación por el alto costo y los bajos niveles de degradación del petróleo, sin embargo, Leersia hexandra Sw. es un pasto que crece en estas zonas contaminadas con petróleo intemperizado. El objetivo del estudio fue evaluar la densidad de bacterias, producción de biomasa vegetal y fitorremediación de L.hexandra en suelo contaminado con petróleos fresco e intemperizado, bajo condiciones experimentales de anegamiento. Se realizaron dos experimentos (E1 y E2) en un túnel de plástico. El E1 se basó en ocho dosis: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 y 180 000 mg kg-1 base seca (b.s.) de hidrocarburos totales de petróleo fresco (HTPF), y en el E2 se evaluaron cinco dosis: 14 173, 28 400, 50 598, 75 492 y 112 142 mg kg-1 b. s. de hidrocarburos totales de petróleo intemperizado (HTPI), con ocho repeticiones en cada experimento, además se utilizó un testigo con 2 607 mg kg-1 b. s. de HTP de origen biogénico. Las variables evaluadas a los tres y seis meses fueron 1) densidad microbiana de las bacterias fijadoras de nitrógeno de vida libre totales (BFN), del grupo Azospirillum (AZP) y Azotobacter (AZT), por cuenta viable en placa seriada; 2) producción de materia seca total (MS), se cuantificó por el peso seco por gravimetría, y 3) el porcentaje de descontaminación de los hidrocarburos (DSC) por extracción en equipo soxhlet. En suelos con HTPF, la población de BFN, AZP y AZT se estimuló hasta cinco veces más que el tratamiento testigo a los tres y seis meses; sin embargo, concentraciones de 150 000 y 180 000 mg kg-1 b. s. inhibieron entre un 70 y 89 % la densidad bacteriana. A su vez, en suelos con PI, la inhibición se registró hasta en un 90 %, a excepción del tratamiento con 14 173 mg kg-1 b. s., el cual estimuló las BNF y AZT en 2 y 0.10 veces más que testigo, respectivamente. La producción de MS fue continua en los experimentos hasta los seis meses, con valores de 63 y 89 g en PF y PI, respectivamente; sin diferencias significativas con el testigo (p ≤ 0.05). El DSC alcanzó valores del 66 % al 87 % en HTPF como HTPI a los seis meses, respectivamente. Estos resultados demuestran la habilidad del L. hexandra para desarrollar una rizósfera con alta densidad de BFN, producir biomasa vegetal y fitorremediar Gleysoles con petróleo fresco e intemperizado en ambientes tropicales inundados.


Abstract:The oil industry has generated chronic oil spills and their accumulation in wetlands of the state of Tabasco, in Southeastern Mexico. Waterlogging is a factor that limits the use of remediation technologies because of its high cost and low levels of oil degradation. However, Leersia hexandra is a grass that grows in these contaminated areas with weathered oil. The aim of the study was to evaluate the bacteria density, plant biomass production and phytoremediation of L. hexandra in contaminated soil. For this, two experiments in plastic tunnel were performed with fresh (E1) and weathered petroleum (E2) under waterlogging experimental conditions. The E1 was based on eight doses: 6 000, 10 000, 30 000, 60 000, 90 000, 120 000, 150 000 and 180 000 mg.kg-1 dry basis (d. b.) of total petroleum hydrocarbons fresh (TPH-F), and the E2, that evaluated five doses: 14 173, 28 400, 50 598, 75 492 and 112 142 mg. kg-1 d. b. of total petroleum hydrocarbons weathered (TPH-W); a control treatment with 2 607 mg.kg-1 d. b. was used. Each experiment, with eight replicates per treatment, evaluated after three and six months: a) microbial density of total free-living nitrogen-fixing bacteria (NFB) of Azospirillum (AZP) and Azotobacter group (AZT), for viable count in serial plate; b) dry matter production (DMP), quantified gravimetrically as dry weight of L. hexandra; and c) the decontamination percentage of hydrocarbons (PDH) by Soxhlet extraction. In soil with TPH-F, the NFB, AZP y AZT populations were stimulated five times more than the control both at the three and six months; however, concentrations of 150 000 and 180 000 mg.kg-1 d. b. inhibited the bacterial density between 70 and 89 %. Likewise, in soil with TPH-W, the FNB, AZP and AZT inhibitions were 90 %, with the exception of the 14 173 mg.kg-1 d. b. treatment, which stimulated the NFB and AZT in 2 and 0.10 times more than the control, respectively. The DMP was continued at the six months in the experiments, with values of 63 and 89 g in fresh and weathered petroleum, respectively; had no significant differences with the control (p≤0.05). The PDH reached values of 66 to 87 % both TPH-F and TPH-W at six months, respectively. These results demonstrated the ability the L. hexandra rhizosphere to stimulate the high NFB density, vegetal biomass production and phytoremediation of contaminated soils (with fresh and weathered petroleum), in a tropical waterlogging environment. Rev. Biol. Trop. 65 (1): 21-30. Epub 2017 March 01.


Subject(s)
Soil Pollutants/chemistry , Biodegradation, Environmental , Petroleum Pollution/prevention & control , Poaceae/microbiology , Poaceae/chemistry , Reference Values , Soil/chemistry , Soil Microbiology , Azotobacter/growth & development , Time Factors , Petroleum/analysis , Colony Count, Microbial , Reproducibility of Results , Azospirillum/growth & development , Biomass , Hydrocarbons/analysis , Hydrocarbons/chemistry
7.
Braz. j. med. biol. res ; 50(1): e5492, 2017. graf
Article in English | LILACS | ID: biblio-839240

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/metabolism , Flow Cytometry/methods , Herbaspirillum/metabolism , Hydroxybutyrates/metabolism , Plant Roots/microbiology , Polyesters/metabolism , Microscopy, Fluorescence
8.
Rev. colomb. biotecnol ; 18(2): 40-47, jul.-dic. 2016. ilus, tab
Article in Spanish | LILACS-Express | LILACS | ID: biblio-959838

ABSTRACT

Esta investigación tuvo como objetivo cuantificar bacterias diazótrofas y comparar fisicoquímicamente suelos rizosféricos de tres cacaotales (Theobroma cacao L.) del Departamento Norte de Santander, Colombia; para lo cual se caracterizaron, diferenciándose en área cultivada, manejo agronómico y edad del cultivo. A partir de diluciones seriadas de las muestras y empleando la técnica de Número Más Probable (NMP), en medios de cultivo semiselectivos (NFb, JMV, LGI, JNFb) semisólidos, se cuantificaron las diazótrofas, evaluando como positivo la formación de una película subsuperficial en el medio contenido en viales sellados; muestras pares se enviaron al laboratorio Bioambiental (UNET) para los análisis fisicoquímicos. Como resultado, las muestras evaluadas mostraron deficiencias en el porcentaje de materia orgánica y elementos como Potasio, Fósforo y Magnesio. Se reportaron estadísticamente diferencias altamente significativas en NMP. La mayor cuantificación de diazótrofas se reportó en la finca Florilandia, que se caracterizó por tener riego por goteo. La mayor cuantificación de diazotrófas se registró en los medios NFb y JMV, demostrándose una mayor presencia de los presuntos géneros Azospirillum sp. y Burkholderia sp. los cuales son fácilmente aislados de suelos rizosféricos, a diferencia de los géneros Herbaspirillum sp. y Gluconacetobacter sp. que por su carácter endófito suelen ser menos predominantes en este tipo de muestras. Se concluye además, que las características fisicoquímicas del suelo, la humedad y las relaciones climáticas al momento de la toma de las muestras, condicionan la cantidad de exudados de las raíces y por tanto son factores que condicionaron la presencia de diazótrofas en las muestras.


The objective of this research was to quantify diazotrophic bacteria and compare physicochemically rhizospheric soils of three cocoa plantations (Theobroma cacao L.) in Norte de Santander Department, Colombia; for which they were characterized, differing in cultivated area, agronomic management and crop age. From serial dilutions of the samples and using the technique of Most Probable Number (MPN), In semisolid culture media (NFb, JMV, LGI, JNFb), the diazotrophs were quantified, evaluating as positive the formation of a subsurface film in the medium contained in sealed vials; equal samples were sent to the Bioambiental laboratory (UNET) for physicochemical analyzes. As a result, the evaluated samples showed deficiencies in the percentage of organic matter and elements such as Potassium, Phosphorus and Magnesium. Statistically highly significant differences in MPN were reported. The highest quantification of diazotrophs was reported in the Florilandia farm, which was characterized by drip irrigation. The highest quantification of diazotrophs was recorded in the media NFb and JMV, demonstrating a greater presence of the presumed genera Azospirillum sp. and Burkholderia sp. which are easily isolated from rhizospheric soils, unlike the genera Herbaspirillum sp. and Gluconacetobacter sp. which by their endophytic character tend to be less predominant in this type of samples. It is also concluded that the physicochemical characteristics of the soil, humidity and climatic relationships at the moment of sampling, condition the amount of root exudates and therefore are factors that conditioned the presence of diazotrophs in the samples.

9.
Braz. j. microbiol ; 47(3): 542-550, July-Sept. 2016. tab, graf
Article in English | LILACS | ID: lil-788956

ABSTRACT

ABSTRACT The main objective of the present study was to isolate phytohormone-producing, phosphate-solubilizing strains of Azospirillum from wheat to be used as inoculants for plant growth promotion. Five Azospirillum strains were isolated from the rhizosphere of field-grown wheat (Triticum aestivum L.), and it was confirmed by BOX-polymerase chain reaction (PCR) that the isolates were different and not re-isolates of the same strain. Sequence analysis of the PCR-amplified 16S rRNA gene indicated that four isolates showed maximum similarity to Azospirillum brasilense and one isolate showed maximum similarity to Azospirillum zeae. This is the first report indicating the presence of an A. zeae like isolate in the wheat rhizosphere in Pakistan. The bacterial isolates were characterized for their plant growth-promoting traits, phosphate solubilization, and indole-3-acetic acid (IAA) production. None of the isolates showed phosphate solubilization activity in the commonly used Pikovskaya medium. However, all strains (except AzoK4) exhibited ability to solubilize tricalcium phosphate (TCP) in modified Pikovskaya medium in which sucrose was replaced by Na-malate, as well as in TCP-supplemented Luria-Bertani (LB) medium. Organic acids, such as acetic, citric, lactic, malic, and succinic acids, were detected in culture supernatants of the tested Azospirillum strains. All strains exhibited ability to produce IAA in the growth medium, except Azospirillum sp. AzoK1. Among the strains tested, the maximum IAA production (30.49 ± 1.04 mg L-1) and phosphate solubilization (105.50 ± 4.93 mg L-1) were shown by a pure culture of Azospirillum sp. AzoK2. In pot experiments, single-strain inocula of Azospirillum sp. AzoK1 and AzoK2 improved wheat plant growth.


Subject(s)
Plant Growth Regulators/biosynthesis , Triticum/microbiology , Azospirillum/classification , Azospirillum/physiology , Rhizosphere , Pakistan , Phylogeny , Sequence Analysis, DNA , Phosphorus Acids/metabolism , Genes, Bacterial , Nitrogen/metabolism
10.
Article in English | IMSEAR | ID: sea-177044

ABSTRACT

The effect of the co-inoculation of arbuscular mycorrhizal fungi (AMF) and Azospirillum on micro-propagated banana seedlings development during their adaptation phase was determined. At the time of transplanting, banana seedlings were inoculated with an indigenous mycorrhizal inoculum containing 10 spores/g at four doses: 0, 50, 100 and 200 g. Seventy days after fungal inoculation, 20 ml of Azospirillum in four concentrations (0, 106, 107 and 108 CFU/ml) were applied. Finally, after 98 days from the start of the experiment a second dose (40 ml) of Azospirillum in the concentrations mentioned above was inoculated. Plants were harvested 5 months after transplanting and the growth and nutritional parameters were evaluated. The analysis of the data showed that banana plants co-inoculated with 200 g of AMF and 1.5E8 CFU/ml of Azospirillum presented greater development, an increase of 7 times in height, 4 times in perimeter, 16 times in leaf area, 12 times in aerial biomass, and 8 times in root biomass relative to control plants. The results achieved were due to synergism between fungus-bacteria when inoculated at higher doses, with lower doses stimulating growth is minimal. The co-inoculation in high doses demonstrates adequate support and cooperative effect between HMA and Azospirillum crops. In addition, co-inoculation promotes optimal nutritional status because microorganisms allowed plants achieve greater absorption of phosphorus and nitrogen relative to those treated with single inoculation and the control.

11.
Biosci. j. (Online) ; 32(3): 670-683, may/june 2016. graf, ilus, tab
Article in English | LILACS | ID: biblio-965508

ABSTRACT

Biofertilizer is a group of beneficial microorganisms used for improving the productivity of soil by fixing atmospheric nitrogen or by solubilizing soil phosphorus. They also stimulate plant growth through synthesis of growth promoting substances. In this present study, Azospirillum lipoferum is grown in Nitrogen free Bromothymol blue (Nfb) medium and Pseudomonas fluorescens in King's B medium. Bioprocess condition was optimized for both of the culture and found that Pseudomonas fluorescens has shown highest growth at 300C in pH 8 after 72 hours of incubation where as Azospirillum lipoferum showed highest cell concentration at 310C in pH 7, with incubation period of 72 hours. The optimized culture is mixed with different formulations of powder and liquid carrier such as Saw dust, Rice husk, Date seed powder, Matka khad, Jiwamrit and Beejamrit respectively. Shelf life study for 0, 30, 60, 90 and 120 days by cell counting and spread plate method showed that shelf life of the biofertilizer produced from Powder and liquid carriers had high amount of viable microbial population up to 120 days storage. Among biofertilizer based bio inoculants, Saw dust showed maximum population of 77x109cfu/ml for Azospirillum lipoferum and 72 x 109 CFU/ml for Pseudomonas strain on 120th day and the liquid carrier Matka khad showed 85x109 cfu/ml for Azospirillum lipoferum and 78 x 109 CFU/ml for Pseudomonas fluorescens.


Biofertilizante é um grupo de microorganismos benéficos utilizados para melhorar a produtividade do solo através da fixação de azoto atmosférico ou por solubilização de fósforo no solo. Eles também estimulam o crescimento vegetal através de síntese de substâncias promotoras do crescimento. No presente estudo, Azospirillum lipoferum é cultivado em um meio de azul de bromotimol sem nitrogênio (Nfb) e Pseudomonas fluorescens num meio de King's B. A condição de bioprocesso foi optimizada para ambas as culturas e descobriram que Pseudomonas fluorescens mostraram maior crescimento a 300ºC em pH 8 após 72 horas de incubação, enquanto que Azospirillum lipoferum mostraram maior concentração de células a 310ºC em pH 7, com um período de incubação de 72 horas. A cultura optimizada é misturada com diferentes formulações de pó e veículo líquido tal como serragem, casca de arroz, pó de semente de tâmaras, Matka khad, Jiwamrit e Beejamrit respectivamente. O estudo do prazo de validade para 0, 30, 60, 90 e 120 dias por contagem celular e método de espalhamento em placa mostrou que o prazo de validade do biofertilizante produzido a partir do pó e veículos líquidos teve grande quantidade de população microbiana viável até 120 dias de armazenamento. Entre inoculantes biológicos de base biofertilizantes, a serragem mostrou população máxima de 77x109 CFU/ml para Azospirillum lipoferum e 72 x 109 CFU/ml para a estirpe Pseudomonas no 120º dia e um veículo líquido Matka khad mostrou 85x109 CFU/ml para Azospirillum lipoferum e 78x109 CFU/ml para Pseudomonas fluorescens.


Subject(s)
Soil , Pseudomonas fluorescens , Azospirillum lipoferum , Fertilizers
12.
Article in English | IMSEAR | ID: sea-176884

ABSTRACT

It is well established fact that bacterial species promotes plant growth. This growth enhancing activity was believed to be through different mechanisms such as synthesis of phytohormones, nitrogen-fixing, and biological control. For this reason, in this present investigation we have isolated and identified Azotobacter spp. and Azospirillum spp. through macromorphologically and micromorphologically in order to assess its effect on growth and yield of strawberry (Fragaria vesca) an Albion variety in hydroponic system. The inoculation and co-inoculation of bacterial culture was performed in combination with three nitrogen levels (50, 100 and 150 ppm), growth parameters such as plant height, root length, fresh and dry weight of root and aerial parts, leaf area, chlorophyll content, nutrient content, solid soluble, caliber, yield/plant. It is observed that in T8 (co-inoculation in100 ppm N) group showed significantly increase in plant height (18.57cm), chlorophyll content (48.57 Soil Plant Analysis Development-SPAD), fresh root weight (25.82g) and dry root weight (5.93g), while in treatment group T5 (Azotobacter spp. 100ppm of Nitrogen) and T6 (Azotobacter spp. 150ppm of Nitrogen) showed significant increase in root length, leaf area, dry and fresh weights of aerial parts. The N content of leaf for all treatments was in the ranges of 2.42 - 2.83 % that is suitable for cultivation. Similarly, the treatment group T5 and T6 showed increase in yield per plant and soluble solids content. So, Azotobacter and Nitrogen treatment has growth related benefits in strawberries under hydroponic system.

13.
Braz. j. microbiol ; 45(2): 621-625, Apr.-June 2014. ilus, tab
Article in English | LILACS | ID: lil-723126

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/isolation & purification , Bacillus/isolation & purification , Endophytes/isolation & purification , Hordeum/microbiology , Nitrogen Fixation , Pseudomonas/isolation & purification , Seeds/microbiology , Antibiosis , Azospirillum brasilense/classification , Azospirillum brasilense/genetics , Azospirillum brasilense/metabolism , Bacillus/classification , Bacillus/genetics , Bacillus/metabolism , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Endophytes/classification , Endophytes/genetics , Endophytes/metabolism , Indoleacetic Acids/metabolism , Molecular Typing , Pseudomonas/classification , Pseudomonas/genetics , Pseudomonas/metabolism , Random Amplified Polymorphic DNA Technique , /genetics , Sequence Analysis, DNA
14.
Braz. j. med. biol. res ; 45(12): 1135-1140, Dec. 2012. ilus, tab
Article in English | LILACS | ID: lil-659653

ABSTRACT

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.


Subject(s)
Adenosine Triphosphate/metabolism , Azospirillum brasilense/enzymology , Bacterial Proteins/metabolism , Ketoglutaric Acids/metabolism , Transcription Factors/metabolism , beta-Galactosidase/metabolism , Azospirillum brasilense/metabolism , Genetic Vectors , Plasmids
15.
J Environ Biol ; 2012 Sept; 33(5): 933-939
Article in English | IMSEAR | ID: sea-148452

ABSTRACT

Five species of Azospirillum isolated from Manakkudi mangrove ecosystem were subjected for their efficiency to find out their growth parameters potential for the successful establishment of mangrove seedlings. Of the isolated five Azospirillum species, Azospirillum lipoferum (60%) was found to be the dominant one. But the level of maximum indole acetic acid (IAA) production (19.8mg.ml-1) and nitrogen fixation (5.9 C2H2hr-1) was identified with A. brasilense. Further, A. brasilense showed significant (p<0.05) level of increased growth parameters [maximum root length (29.55%), average root length (7.39%), total Chl (55.36%), carrotenoids (28.57%), Chl b (37.50%), carbohydrates (90.91%) and total amino acids (78.95%)] in Avicennia officinals when compared with control group. Further, A. brasilense also showed significant (p<0.05) level of increased growth parameters [average number of primary roots (40%), average biomass (44.44%), average shoot biomass (55.56%), total Chl (20%), Chl b (77.78%) and carotenoid (1.54%)] in C. decandra seedlings when compared with control group. Similarly, the average number of primary roots (23.08%), average root biomass (15.52%), average shoot biomass (15.30%), carbohydrate (20%) and total amino acids (44.44%) were found significant (p<0.05) in A. irakense inoculated R. apiculata seedlings. In conclusion, Azospirillum brasilense was found better for the growth of Avicennia officinalis and Ceriops decandra seedlings, but Azospirillum irakense was found better for Rhizophora apiculata seedlings.

16.
Article in English | IMSEAR | ID: sea-162848

ABSTRACT

Aim: The present study reflects the effect of Arbuscular Mycorrhizae (AM) fungi and other bioinoculants on the growth of cotton seedlings. Study Design: Screening of efficient biofertilizers from undisturbed forest soils to improve the crop yield of cotton in barren lands of Mahabubnagar District. Place and Duration of Study: Department of Microbiology, Palamuru University, Mahabubnagar, Andhra Pradesh, India, between May 2011 and February 2012. Methodology: Mahyco hybrid variety, the most widely cultivated variety in Mahabubnagar District, Andhra Pradesh, India was selected among the sixteen varieties of cotton seeds for this study. Soil samples were analyzed for physicochemical characteristics. Seven different isolates of AM fungi were maintained as pure cultures in laboratory, which were isolated from different Agroforestry tree rhizosphere soils. Among these pure cultures, R1- R2 has shown maximum colonization with Mahyco variety and this isolate was identified as Glomus mosseae. Mahyco hybrid variety was also tested with three different bioinoculants (Rhizobium sp., Azospirillum sp., Bacillus sp.) along with the combination of AM pure culture of R1-R2. These three potential bioinoculants were identified based on 16S rRNA gene sequence. Preliminarily Mahyco hybrid variety was investigated with individual pure cultures of AM fungi and with other bioinoculants. R1-R2 was taken in single, dual, triple and multi combinations with other three bioinoculants. Results: In single combination M+R1-R2 showed the best growth by M+Rhizobium, followed by M+Bacillus and in dual combination M+R1-R2+Rhizobium and in triple combination M+R1-R2+Rhizobium+Azospirillum and in multicombination i.e. M+R1- R2+Rhizobium+Azospirillum+Bacillus showed the best growth among all the combinations. Conclusion: The multicombination mediates increased the cotton growth characteristics. The effect of multicombination was not significantly different in treatment affected by various varieties. Inoculation of multicombination along with AM pure culture resulted significant increase in shoot and root length of cotton plant. So multicombination was proved to be superior.

17.
J Environ Biol ; 2012 May; 33(3): 597-602
Article in English | IMSEAR | ID: sea-146743

ABSTRACT

Seasonal variations of population dynamics of free living nitrogen fixing bacteria, Azospirillum in relation to chemical parameters in Manakkudi mangrove eco system was assessed in root and rhizosphere soil samples of mangroves and mangrove associated plants. In rhizosphere soil and root samples, the counts of Azospirillum were recorded maximum in Acrostichum aureum as 8.63±0.92 x104 and 115.48±17.36 x 104 CFU g-1, respectively. The counts of Azospirillum in non-rhizosphere soil varied from 0.01±0.001x 104 to 5.77±0.92x 104 CFU g-1 and found maximum in February and minimum in March and September. Azospirillum counts in water samples were found maximum (2.24 x 104 CFU l-1) in February. During seasonal variations maximum counts of Azospirillum were recorded during southwest monsoon season in Avicennia officinalis (1.40 x 104 CFU g-1 ) followed by Rhizophora mucronata (1.07 x 104 CFU g-1). The average maximum population density of Azospirillum counts was found during non monsoon season (9.73 x 104 CFU g-1 ) and the average maximum population density of Azospirillum counts was found with the mangrove associated root samples (13.73 x 104 CFU g-1 ). Of the selected isolates Azospirillum lipoferum (60%) was found to be predominant followed by Azospirillum brasilense (25%), Azospirillum irakense (5%), Azospirillum halopraeferens (5%) and Azospirillum amazonense (5%). Of the isolated species, A. halopraeferens exhibited better growth at 35 g l-1 NaCl. The level of Fe, Cu, Zn and Mn were varied from 0.91 to 15.93 ppm. The level of Mn (12.13 ppm) was found maximum during non-monsoon of rhizosphere soil sample. Highest rainfall (192.80 mm) and atmospheric temperature (25.10 oC) were recorded during south west monsoon and non monsoon seasons. The increased population density was greatly influenced by the pH (r=+0.686). The present finding provides enough information on the nitrogen flow through biological process in Manakkudi mangrove ecosystem which can be useful for the effective implementation of mangrove management plan.

18.
Braz. j. med. biol. res ; 45(2): 113-117, Feb. 2012. ilus, tab
Article in English | LILACS | ID: lil-614577

ABSTRACT

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.


Subject(s)
Azospirillum brasilense/metabolism , Bacterial Proteins/metabolism , Nitrogen Fixation/genetics , Transcription Factors/metabolism , Azospirillum brasilense/chemistry , Azospirillum brasilense/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Carrier Proteins/genetics , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/isolation & purification
19.
Rev. colomb. biotecnol ; 12(2): 103-115, dic. 2010. tab, graf
Article in Spanish | LILACS | ID: lil-590777

ABSTRACT

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...


Subject(s)
Carica/growth & development , Carica/embryology , Carica/physiology , Carica/genetics , Carica/microbiology , Carica/chemistry , Fertilizers/analysis , Fertilizers/adverse effects , Fertilizers/microbiology , Azospirillum brasilense/isolation & purification , Azospirillum brasilense/growth & development , Azospirillum brasilense/physiology , Azospirillum brasilense/genetics , Azospirillum brasilense/immunology , Azospirillum brasilense/chemistry
20.
Article in English | IMSEAR | ID: sea-161206

ABSTRACT

Altogether 30 Azospirillum strains isolated from paddy rhizosphere soil were identified as nitrogen fixers. A preliminary screening of all the species isolated from soils were made for nitrogen fixing activity.Among 30 strains three species of Azospirillum showed promising activity. These strains were classified as a unique grass by RAPD fingerprinting restriction enzyme analysis and 16S rRNA gene sequencing. Phylogenetic analysis of the 16S rRNA gene indicated that these diazotrophic strains belonged to the genus Azospirillum and were closely related to A. brasilense (with 84% similarity), A.amazonense (78% similarity) and A. halopraferens (93% similarity).

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