Effect of Liquid Biofertilizers on Productivity, Quality and Soil Biota of Fodder Sorghum

The field experiment was conducted during Rabi season of 2022 on sandy clay loam soils at wetland farm of the S.V. Agricultural College, Tirupati, Andhra Pradesh, India, to study the effect of liquid biofertilizers on productivity, quality, and soil biota of fodder sorghum. The treatments were allocated in randomized block design and replicated thrice. The soil was neutral in reaction (6.9 pH), low in available nitrogen (115 kg ha-1), medium in available phosphorus (29 kg ha-1) and low in available potassium (156 kg ha-1) status. The initial soil contains bacteria (13.08 X 107 CFU g-1 soil), fungi (4.04 X 104 CFU g-1 soil) and actinomycetes (1.54 X 103 CFU g-1 soil). The results revealed that the maximum green (32.1 t ha-1) and dry (13.0 t ha-1) fodder yield as well as crude protein (7.3%) were obtained with the application of 75% RDF + Azospirillum + PSB + KSB (Both seed & soil application). Furthermore, this treatment produced a significantly higher soil bacterial (23.2 X 107 CFU g-1 soil), fungal (13.7 X 104 CFU g-1 soil) and actinomycetes (10.3 X 103 CFU g-1 soil) population at harvest.

Influence of Cropping System and Nutrient Management Practices on the Yield, and Economics of Pearl Millet [Pennisetum glaucum]

The Field experiments were conducted to evaluate the effect of cropping systems and nutrient management practices on the yield, and economics of pearl millet in the School of Agricultural Science, Karunya Institute of Technology and Sciences, Coimbatore, India). The experiments were laid out in Factorial Randomized Block Design (FRBD) with two factors (Cropping system and Nutrient management) and three replications for two consecutive seasons in 2022-23. The study comprised three cropping system treatments namely C1 – Sole Pearl millet cropping, C2 - Pearl millet + Black gram intercropping (1:1), C3 – Pearl millet + Cowpea intercropping (1:1) along with four nutrient management treatments viz., N1 - 100% Recommended dose of fertilizer (RDF) @80:40:40 kg of N, P2O5, and K2O/ha., N2– 75% RDF + Azospirillum @2kg/ha and Phosphate solubilizing bacteria (PSB) @2kg/ha, N3 – 75% RDF + Soil application of Azospirillum (2kg/ha) and PSB (2kg/ha) + Foliar spray of 2% urea @ 15 and 35 days after transplanting (DAT), N4 – 75% RDF + Soil application of Azospirillum (2kg/ha) and PSB (2kg/ha) + Foliar spray of Panchagavya3% @ 15 and 35 DAT. The results of the experiment revealed that the intercropping of black gram or cowpea in pearl millet proves to be beneficial and advantageous in terms of, grain yield and returns per rupee invested, rather than going for the sole cropping of pearl millet. On the other hand, reduced application of inorganic recommended dose of fertilizers by 25% with the inclusion of biofertilizers like Azospirillum and phosphate solubilizing bacteria, along with the foliar application of 2% urea or 3% Panchagavya will be an economically viable and environmentally sustainable nutrient management practice for improving the yield of pearl millet.

Response of Bio Fertilizers and Phosphorus on Yield and Economics of Pearl millet (Pennisetum glaucum L.)

A field experiment was conducted during kharif season of 2022, at crop research farm, Department of Agronomy at Sam Higginbottom University of Agriculture, Technology and Sciences, Prayagraj (U.P.).India. The objective is to study the Response of Bio fertilizers and Phosphorus on yield and economics pearl millet. The treatments consisted of three levels of Bio fertilizers (20 g/kg Azotobacter, 20 g/kg Azospirillum, 20 g/kg Azotobacter + Azospirillum) and three levels of phosphorus (35, 40 and 45 kg/ha). The experiment was laid out in randomized block design with the ten treatments combinations replicated thrice. The soil in experimental field was sandy loam in texture, having alkaline reaction (pH 7.7) with very low organic carbon (0.44%), available higher N (171.48 kg/ha), P (27.0 kg/ha) and higher level of K (291.2 kg/ha).The results show significantly higher in yield attributes viz. ear head length (25.25 cm), number of grains/ear head (2010.9),and yield viz. grain yield (3.25 t/ha) and Stover yield (6.27 t/ha). Maximum gross return (113750.00 INR/ha), net return (81010.00 INR/ha) and Benefit - cost ratio (2.47) was found in treatment (T9) with the application of 20 g/ kg Azotobacter + Azospirillum + 45 kg/ha Phosphorous.

Growth-promoting microorganisms in the development of orchid seedlings of Phalaenopsis, Cymbidium, and Dendrobium genera

The demand for sustainable agricultural production systems is increasing, and using growth-promoting microorganisms in plants has stood out because it decreases or even replaces chemical fertilizer use, reducing production costs. This study aimed to evaluate the response of some microorganisms applied to the seedlings of primary orchids cultivated in Brazil (Phalaenopsis sp. 'Taisuco Swan', Cymbidium atropurpureum, and Dendrobium secundum). The experimental design was completely randomized. There were seven treatments (absence of microorganisms ­ control, Trichoderma sp. in sodium alginate, Trichoderma sp. in clay, Trichoderma sp. in sodium alginate and clay, Trichoderma sp. in a liquid medium, Azospirillum brasilense + Bacillus subtilis in a liquid medium, and Bacillus pumilus in a liquid medium), four replications, and three plants per plot. The seedlings were grown in a greenhouse and evaluated 190 days after microorganism inoculation. The evaluation of morpho-physiological characteristics differed according to the particularities of each genus. The Bacillus pumilus and Azospirillum brasilense + Bacillus subtilis rhizobacteria in a liquid medium for Phalaenopsis sp. 'Taisuco Swan' and the Trichoderma sp. fungus in a liquid medium for Cymbidium atropurpureum increased seedling growth and development. Azospirillum brasilense + Bacillus subtilis in a liquid medium for the Dendrobium secundum orchid promoted more root biomass. Using beneficial microorganisms in orchid cultivation is promising, and seedling growth and development depend on their inoculation and the morpho-physiological characteristics of each plant.

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.

Laguncularia racemosa (Combretaceae) y bacterias terrestres asociadas medio siglo después de la contaminación crónica por hidrocarburos / Laguncularia racemosa (Combretaceae) and associated ground bacteria half a century after chronic oil pollution

La evaluación del impacto del petróleo en la interacción suelo-raíz de la planta de mangle blanco, Laguncularia racemosa, es básica para identificar los cambios en la actividad microbiana y en el potencial biotecnológico para la remediación de histosoles contaminados. El objetivo de la investigación fue evaluar la distribución espacial de los hidrocarburos totales del petróleo (HTP) en el suelo orgánico, la densidad poblacional de bacterias promotoras del crecimiento vegetal, así como la respiración microbiana en el rizoplano (RI) en la rizósfera (RZ) y en suelo no rizosférico (SNR) de L. racemosa. Una superficie de 8 000 m2 de un histosol afectado desde 1967 y 1968 se evaluó por derrame crónico de petróleo y lodos de perforación provenientes del pozo petrolero La Venta 248. Se seleccionaron 15 árboles de L. racemosa y se extrajeron muestras del RI, RZ y SNR. Los HTP se extrajeron en equipo soxhlet con diclorometano durante ocho horas y se cuantificaron por gravimetría. La cantidad promedio extraída de HTP permitió la diferenciación de cuatro suelos (S) en el área evaluada, con valor promedio para el S1 de 1 797 mg kg-1 (no contaminado para la normativa mexicana) y tres suelos contaminados: S2 con 3 294, S3: 5 249 y S4: 10 389 mg kg-1. Los resultados evidencian diferencias estadísticas (Duncan, P ≤ 0.05) entre medias de las variables evaluadas. La mayor acumulación de HTP fue 22 962 mg kg-1, se extrajo del SNR en el S4. Las mayores densidades de bacterias fijadoras de N, solubilizadoras de P, Azospirillum y Azotobacter fueron bioestimuladas por la presencia de niveles altos de HTP en el suelo, sin embargo la respiración microbiana fue inhibida. Los resultados sugieren que L. racemosa es sostenible en suelos con petróleo intemperizado, y es un bioestimulador de la actividad microbiana para la atenuación natural.

The evaluation of the impact of oil on the soil-root interaction of the white mangrove plant, Laguncularia racemosa is essential to identify changes in microbial activity and biotechnological potential for remediation contaminated histosols. The objective was to evaluate the spatial distribution of total petroleum hydrocarbons (THP) in organic soil, the population density of plant growth promoting bacteria, also in microbial respiration in the rhizoplane (RI), in the rhizosphere (RZ) and in non-rhizospheric soil (NRS) of L. racemosa. An area of 8 000 m2 of an affected histosol was evaluated, during 1967 and 1968, by chronic oil spill and drilling mud from the La Venta 248 oil well. Fifteen trees of this species were selected to obtain samples of the RI, RZ and NRS. The TPH were extracted in soxhlet with dichloromethane for eight hours and quantified by gravimetry. The average amount extracted from TPH allowed the differentiation of four soils (S) from the evaluated area, the average values were for S1: 1 797 mg kg-1 (not contaminated for Mexican regulations) and three contaminated soils, the values are S2: 3 294, S3: 5 249, and S4: 10 389 mg kg-1. The results show statistical differences (Duncan, P ≤ 0.05) between means of the evaluated variables. The greatest accumulation of TPH was 22 962 mg kg-1, it was extracted from the NRS in S4. The highest densities of N-fixing bacteria, P solubilizers, Azospirillum and Azotobacter were biostimulated by the presence of high levels of THP in the soil, however microbial respiration was inhibited. The results suggest that L. racemosa is sustainable in soils with weathering oil, and is a biostimulator of microbial activity for natural attenuation.

Microorganisms in Biostimulants

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.

Efecto de inoculantes bacterianos edáficos mixtos en el desarrollo temprano de cultivares mejorados de cacao (Theobroma cacao L.) en un sistema agroforestal tradicional del norte de Oaxaca, México / Effect of mixed edaphic bacterial inoculants in the early development of improved cocoa cultivars (Theobroma cacao L.) in a traditional agroforestry system of Oaxaca, Mexico

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.

Efecto de la gallinaza sobre Azotobacter sp., Azospirillum sp. y hongos micorrízicos arbusculares en un cultivo de cebolla (Allium fistulosum) / Effect of poultry manure on Azotobacter sp., Azospirillum sp. and arbuscular mycorrhizal fungi in anion culture (A. fistulosum) / Efeito do esterco de galinha fresco sobre Azotobacter sp., Azospirillum sp. e fungos micorrízicos arbusculares na cultura da cebola (A. fistulosum)

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.

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.

Uso de Leersia hexandra (Poaceae) en la fitorremediación de suelos contaminados con petróleo fresco e intemperizado / Use of Leersia hexandra (Poaceae) for soil phytoremediation in soils contaminated with fresh and weathered oil

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.

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.

Cuantificación de bacterias diazótrofas aisladas de suelos cacaoteros (Theobroma cacao L.), por la técnica de Número Más Probable (NMP) / Quantification of diazotrophs bacteria isolated from cocoa soils (Theobroma cacao L.), by the technique of Most Probable Number (MPN)

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.

Isolation and identification by 16S rRNA sequence analysis of plant growth-promoting azospirilla from the rhizosphere of wheat

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.

Effect of Arbuscular mycorrhizal fungi (AMF) and Azospirillum on growth and nutrition of banana plantlets during acclimatization phase.

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.

Development and formulation of azospirillum lipoferum and pseudomonas fluorescens as effective biological agents for enhanced agro-productivity / Desenvolvimento e formulação de azospirillum lipoferum e pseudomonas fluorescens enquanto agentes biológicos eficazes para uma agro-produtividade melhorada

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.

Effect of Azospirillum spp. and Azotobacter spp. on the growth and yield of strawberry (Fragaria vesca) in hydroponic system under different nitrogen levels.

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.

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.

Effects of saline tolerant Azospirillum species on the growth parameters of mangrove seedlings.

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.