Production of potato seed tubers under the effect of Trichoderma sp. and rhizobacteria in greenhouse conditions / Produção de tubérculos de batata-semente sob efeito de Trichoderma sp. e rizobactérias em casa de vegetação

Potato (Solanum tuberosum L.) is one of the main crops in the Andean region and due to environmental aspects, the use of biocontrol agents is considered a safe way to produce potato seed tubers.The objective of the study was to evaluate potato inoculation with Trichoderma sp. as a matrix and rhizobacteria Bacillus simplex and Azotobacter sp. on the growth of potato seedlings from in vitro culture, for the production of seed tubers in a greenhouse. The inoculation of microorganisms was carried out in pots, using five potato genotypes for processing. The inoculation treatments were: control, Trichoderma sp., Trichoderma sp. + Azotobacter sp., Trichoderma sp. + Bacillus simplex, Trichoderma sp. + B. simplex + Azotobacter sp. The potato genotypes were cv. Unica (CIP392797.22), cv. Bicentenaria, the advanced clones CIP 396311.1, CIP 399101.1, and the experimental clone UH-09 from the Universidad Nacional José Faustino Sánchez Carrión. A completely randomized design was used, under a factorial arrangement and comparisons between treatments were made at p<0.05. All inoculant treatments exceeded the control in number and weight of tubers per plant as well as in tuber size. Inoculations of Trichoderma sp. alone or with Azotobacter sp. increased plant height, number of leaves per plant and vegetative uniformity; inoculations with the Trichoderma sp. + B. simplex + Azotobacter sp. consortium, improved the dry weight of the foliage, number of shoots per plant and vegetative vigor. There were significant interactions between potato genotypes and inoculant treatments for plant uniformity, vegetative vigor, and the foliage's dry weight. Coinoculation with Trichoderma sp. and some bacterial strains promote the growth of in vitro potato seedlings, increasing the size and weight of the seed tubers and plant biomass, indicating an interrelation between fungi and bacteria that influence the production of potatoes in a greenhouse.(AU)

A batata (Solanum tuberosum L.) é uma das principais culturas da região andina e devido aos aspectos ambientais, o uso de agentes de biocontrole é considerado uma forma segura de produzir tubérculos de batata-semente. O objetivo do trabalho foi avaliar a inoculação de batata com Trichoderma sp. como matriz e rizobactérias Bacillus simplex e Azotobacter sp. sobre o crescimento de mudas de batata provenientes de cultivo in vitro, para produção de tubérculos-semente em casa de vegetação. A inoculação dos microrganismos foi realizada em vasos, utilizando-se cinco genótipos de batata para processamento. Os tratamentos de inoculação foram: testemunha, Trichoderma sp., Trichoderma sp. + Azotobacter sp., Trichoderma sp. + B.simplex, Trichoderma sp. + B.simplex + Azotobacter sp. Os genótipos de batata foram cv. Única (CIP392797.22), cv. Bicentenaria, os clones avançados CIP396311.1, CIP399101.1, e o clone experimental UH-9 da Universidade Nacional José Faustino Sánchez Carrión. O delineamento foi inteiramente casualizado, em esquema fatorial e as comparações entre os tratamentos foram feitas com p<0,05. Todos os tratamentos com inoculantes excederam o controle em número e peso de tubérculos por planta, bem como em tamanho de tubérculo. Inoculações de Trichoderma sp. sozinho ou com Azotobactersp. aumento da altura das plantas, número de folhas por planta e uniformidade vegetativa; inoculações com o Trichoderma sp. + B.simplex + Azotobacter sp. consorcio, melhorou a massa seca da folhagem, número de brotações por planta e vigor vegetativo. Houve interações significativas entre genótipos de batata e tratamentos inoculantes para uniformidade e vigor vegetativo, e para a massa seca da folhagem. Coinoculação com Trichoderma sp. e algumas cepas bacterianas promovem o crescimento de mudas de batata in vitro, aumentando o tamanho e o peso dos tubérculos-semente e da biomassa vegetal, indicando que existe inter-relação entre fungos e bactérias que influenciam a produção de batata em casa de vegetação.(AU)

Interaction with Soil Bacteria Affects the Growth and Amino Acid Content of Piriformospora indica.

Exploration of the effect of soil bacteria on growth and metabolism of beneficial root endophytic fungi is relevant to promote favorable associations between microorganisms of the plant rhizosphere. Hence, the interaction between the plant-growth-promoting fungus Piriformospora indica and different soil bacteria was investigated. The parameters studied were fungal growth and its amino acid composition during the interaction. Fungus and bacteria were confronted in dual cultures in Petri dishes, either through agar or separated by a Perspex wall that only allowed the bacterial volatiles to be effective. Fungal growth was stimulated by Azotobacter chroococcum, whereas Streptomyces anulatus AcH 1003 inhibited it and Streptomyces sp. Nov AcH 505 had no effect. To analyze amino acid concentration data, targeted metabolomics was implemented under supervised analysis according to fungal-bacteria interaction and time. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model clearly discriminated P. indica-A. chroococcum and P. indica-S. anulatus interactions, according to the respective score plot in comparison to the control. The most observable responses were in the glutamine and alanine size groups: While Streptomyces AcH 1003 increased the amount of glutamine, A. chroococcum decreased it. The fungal growth and the increase of alanine content might be associated with the assimilation of nitrogen in the presence of glucose as a carbon source. The N-fixing bacterium A. chroococcum should stimulate fungal amino acid metabolism via glutamine synthetase-glutamate synthase (GS-GOGAT). The data pointed to a stimulated glycolytic activity in the fungus observed by the accumulation of alanine, possibly via alanine aminotransferase. The responses toward the growth-inhibiting Streptomyces AcH 1003 suggest an (oxidative) stress response of the fungus.

Azotobacter chroococcum as a potentially useful bacterial biofertilizer for cotton (Gossypium hirsutum): Effect in reducing N fertilization / Azotobacter chroococcum como biofertilizante bacteriano potencialmente útil para el algodón (Gossypium hirsutum L.): efecto en la reducción de la fertilización nitrogenada

The aim of this research was to evaluate whether the application of two plant growth-promoting (rhizo)bacteria might reduce nitrogen fertilization doses in cotton. We used strains Azotobacter chroococcum AC1 and AC10 for their proven ability to promote seed germination and cotton growth. These microorganisms were characterized by their plant growth-promoting activities. Then, we conducted a glasshouse study to evaluate the plant growth promoting ability of these strains with reduced doses of urea fertilization in cotton. Results revealed that both strains are capable of fixing nitrogen, solubilizing phosphorus, synthesizing indole compounds and producing hydrolytic enzymes. After 12 weeks, the glasshouse experiment showed that cotton growth was positively influenced due to bacterial inoculation with respect to chemical fertilization. Notably, we observed that microbial inoculation further influenced plant biomass (p<0.05) than nitrogen content. Co-inoculation, interestingly, exhibited a greater beneficial effect on plant growth parameters compared to single inoculation. Moreover, similar results without significant statistical differences were observed among bacterial co-inoculation plus 50% urea and 100% fertilization. These findings suggest that coinoculation of A. chroococcum strains allow to reduce nitrogen fertilization doses up to 50% on cotton growth. Our results showed that inoculation with AC1 and AC10 represents a viable alternative to improve cotton growth while decreasing the N fertilizer dose and allows to alleviate the environmental deterioration related to N pollution.

El objetivo de esta investigación fue evaluar si la aplicación de 2 (rizo)bacterias promotoras del crecimiento vegetal podría reducir la dosis de fertilizante nitrogenado en el cultivo de algodón. Se usaron las cepas Azotobacter chroococcum AC1 y AC10 por su habilidad para promover la germinación de semillas y el crecimiento del algodonero. Estos microorganismos fueron caracterizados sobre la base de sus actividades de promoción del crecimiento vegetal. Luego se realizó un estudio de invernadero con plantas de algodón para evaluar la capacidad de promoción del crecimiento vegetal de dichas cepas con dosis reducidas de urea. Los resultados revelaron que ambas cepas son capaces de fijar nitrógeno, solubilizar fósforo, sintetizar compuestos indólicos y producir enzimas hidrolíticas. Después de 12 semanas, el experimento de invernadero permitió observar que el crecimiento del algodón fue influido positivamente por la inoculación bacteriana con respecto a la fertilización química. En particular, se evidenció que la inoculación microbiana impactó más en la biomasa vegetal (p<0,05) que en el contenido de nitrógeno. Curiosamente, la coinoculación exhibió un mayor efecto positivo sobre los parámetros de crecimiento en comparación con la inoculación simple. Además, se observaron resultados similares, sin diferencias estadísticamente significativas, entre la coinoculación bacteriana más del 50% de urea y el 100% de fertilización. Estos hallazgos indican que la coinoculación de las cepas de A. chroococcum AC1 y AC10 permitiría reducir las dosis de fertilización nitrogenada del cultivo de arroz en hasta el 50% y aliviar, de esta manera, el deterioro ambiental relacionado con la contaminación por N.

Azotobacter chroococcum as a potentially useful bacterial biofertilizer for cotton (Gossypium hirsutum): Effect in reducing N fertilization.

The aim of this research was to evaluate whether the application of two plant growth-promoting (rhizo)bacteria might reduce nitrogen fertilization doses in cotton. We used strains Azotobacter chroococcum AC1 and AC10 for their proven ability to promote seed germination and cotton growth. These microorganisms were characterized by their plant growth-promoting activities. Then, we conducted a glasshouse study to evaluate the plant growth promoting ability of these strains with reduced doses of urea fertilization in cotton. Results revealed that both strains are capable of fixing nitrogen, solubilizing phosphorus, synthesizing indole compounds and producing hydrolytic enzymes. After 12 weeks, the glasshouse experiment showed that cotton growth was positively influenced due to bacterial inoculation with respect to chemical fertilization. Notably, we observed that microbial inoculation further influenced plant biomass (p<0.05) than nitrogen content. Co-inoculation, interestingly, exhibited a greater beneficial effect on plant growth parameters compared to single inoculation. Moreover, similar results without significant statistical differences were observed among bacterial co-inoculation plus 50% urea and 100% fertilization. These findings suggest that co-inoculation of A. chroococcum strains allow to reduce nitrogen fertilization doses up to 50% on cotton growth. Our results showed that inoculation with AC1 and AC10 represents a viable alternative to improve cotton growth while decreasing the N fertilizer dose and allows to alleviate the environmental deterioration related to N pollution.

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

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

A New Player in the Biorefineries Field: Phasin PhaP Enhances Tolerance to Solvents and Boosts Ethanol and 1,3-Propanediol Synthesis in Escherichia coli.

The microbial production of biofuels and other added-value chemicals is often limited by the intrinsic toxicity of these compounds. The phasin PhaP from the soil bacterium Azotobacter sp. strain FA8 is a polyhydroxyalkanoate granule-associated protein that protects recombinant Escherichia coli against several kinds of stress. PhaP enhances growth and poly(3-hydroxybutyrate) synthesis in polymer-producing recombinant strains and reduces the formation of inclusion bodies during overproduction of heterologous proteins. In this work, the heterologous expression of this phasin in E. coli was used as a strategy to increase tolerance to several biotechnologically relevant chemicals. PhaP was observed to enhance bacterial fitness in the presence of biofuels, such as ethanol and butanol, and other chemicals, such as 1,3-propanediol. The effect of PhaP was also studied in a groELS mutant strain, in which both GroELS and PhaP were observed to exert a beneficial effect that varied depending on the chemical tested. Lastly, the potential of PhaP and GroEL to enhance the accumulation of ethanol or 1,3-propanediol was analyzed in recombinant E. coli Strains that overexpressed either groEL or phaP had increased growth, reflected in a higher final biomass and product titer than the control strain. Taken together, these results add a novel application to the already multifaceted phasin protein group, suggesting that expression of these proteins or other chaperones can be used to improve the production of biofuels and other chemicals.IMPORTANCE This work has both basic and applied aspects. Our results demonstrate that a phasin with chaperone-like properties can increase bacterial tolerance to several biochemicals, providing further evidence of the diverse properties of these proteins. Additionally, both the PhaP phasin and the well-known chaperone GroEL were used to increase the biosynthesis of the biotechnologically relevant compounds ethanol and 1,3-propanediol in recombinant E. coli These findings open the road for the use of these proteins for the manipulation of bacterial strains to optimize the synthesis of diverse bioproducts from renewable carbon sources.

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

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.

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

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.

Agronomical performance of potato (Solanum tuberosum L.) cv. 'Unica' under inoculation with native rhizobacteria and application of acetyl salicylic acid / Desempenho agronômico da batata (Solanum tuberosum L.) cv. 'Unica' sob inoculação com rhizobacterias nativas e aplicação de ácido acetil salicílico

Peru is center of origin of potato and its wild relatives, so it is convenient to develop new technologies of agronomical management with less impact on the ecosystem, as in the use of pesticides and chemical fertilizers in potato is increasing in developing countries. The objective of the work was to evaluate the agronomic performance of potato cv. 'Unica' under inoculation with PGPR rhizobacteria isolated from the rhizosphere of Andean potatoes and on the effect of the application of acetylsalicylic acid in field conditions of the Peruvian coast. Inoculation of four bacterial isolates were evaluated: Bacillus simplex B13, Bacillus amyloliquefaciens Bac 15Mb, Azotobacter sp. Azo 16M2 and Pantoea sp. DZ22, as well as a dose of 0.4 mM acetyl salicylic acid as promoter treatments for plant growth versus a control with no inoculation. A randomized complete block design was used in a sandy loam class soil, slightly alkaline, with low organic content and free of salts. There was a relative humidity between 54 and 71%, while the average air temperatures ranged between 13 and 21 ºC in San Vicente de Cañete, Lima (coordinates UTM 18L3540788550372). Inoculated plants with bacterial strains at sowing time and control were evaluated at flowering in plots of 40 plants with three replications for several physiological and productive parameters. Means were separated statistically through Duncan's multiple range test and processed with Infostat software. Inoculation with strain Azotobacter sp. Azo 16M2, showed significant differences (p<0.05) with respect to control without inoculation, increasing tuber yield and commercial tuber weight per hectare and with less damage by the leafminer fly (Liriomyza huidobrensis) vis-à-vis the control. Also application of acetyl salicylic acid showed higher percentage of inflorescences (p<0.05) compared to the control, while statistical differences were not found between bacterial strains...

Peru é o centro de origem da batata e seus parentes silvestres, por isso é conveniente desenvolver novas tecnologias de gestão agronômica com menor impacto no ecossistema, como o uso de pesticidas e fertilizantes químicos em batata está aumentando nos países em desenvolvimento. O objetivo do trabalho foi avaliar o desempenho agronômico da cv. 'Unica' sob inoculação com rizobactérias PGPR isoladas da rizosfera de batata andina e pelo efeito da aplicação de ácido acetilsalicílico nas condições de campo da costa peruana. Avaliou-se a inoculação de quatro isolados bacterianos: Bacillus simplex B13, B. amyloliquefaciens Bac 15Mb, Azotobacter sp. Azo 16M2 e Pantoea sp. DZ22 bem como uma dose de 0,4 mM de ácido acetilsalicílico como promotores de tratamentos para o crescimento das plantas versus um controle sem inoculação. Utilizou-se um delineamento em blocos casualizados, em um solo franco-arenoso,  ligeiramente alcalino, de baixo conteúdo orgânico e livre de sais. O clima durante o ensaio apresentou uma umidade relativa entre 54 e 71%, temperaturas médias entre 13 e 21 ºC em San Vicente de Cañete, Lima (coordenadas UTM  18L3540788550372). As plantas foram inoculadas com estirpes bacterianas no momento da semeadura e na floração e os tratamentos foram avaliados em parcelas de 40 plantas com três repetições para diversos parâmetros fisiológicos e produtivos. Os dados foram analisados estatisticamente através do teste de Duncan e processados com o software Infostat. A inoculação com a estirpe Azo 16M2, apresentou diferenças significativas (p<0,05) em relação ao controle sem inoculação, aumentando a produção de tubérculos e o peso comercial de tubérculos por hectare e com menor dano pela mosca minadora (Liriomyza huidobrensis) em relação ao controle. Também a aplicação de ácido acetilsalicílico apresentou maior porcentagem de inflorescências (p<0,05) em relação ao controle, enquanto que diferenças estatísticas não foram encontradas entre...

Agronomical performance of potato (Solanum tuberosum L.) cv. 'Unica' under inoculation with native rhizobacteria and application of acetyl salicylic acid / Desempenho agronômico da batata (Solanum tuberosum L.) cv. 'Unica' sob inoculação com rhizobacterias nativas e aplicação de ácido acetil salicílico

Peru is center of origin of potato and its wild relatives, so it is convenient to develop new technologies of agronomical management with less impact on the ecosystem, as in the use of pesticides and chemical fertilizers in potato is increasing in developing countries. The objective of the work was to evaluate the agronomic performance of potato cv. 'Unica' under inoculation with PGPR rhizobacteria isolated from the rhizosphere of Andean potatoes and on the effect of the application of acetylsalicylic acid in field conditions of the Peruvian coast. Inoculation of four bacterial isolates were evaluated: Bacillus simplex B13, Bacillus amyloliquefaciens Bac 15Mb, Azotobacter sp. Azo 16M2 and Pantoea sp. DZ22, as well as a dose of 0.4 mM acetyl salicylic acid as promoter treatments for plant growth versus a control with no inoculation. A randomized complete block design was used in a sandy loam class soil, slightly alkaline, with low organic content and free of salts. There was a relative humidity between 54 and 71%, while the average air temperatures ranged between 13 and 21 ºC in San Vicente de Cañete, Lima (coordinates UTM 18L3540788550372). Inoculated plants with bacterial strains at sowing time and control were evaluated at flowering in plots of 40 plants with three replications for several physiological and productive parameters. Means were separated statistically through Duncan's multiple range test and processed with Infostat software. Inoculation with strain Azotobacter sp. Azo 16M2, showed significant differences (p<0.05) with respect to control without inoculation, increasing tuber yield and commercial tuber weight per hectare and with less damage by the leafminer fly (Liriomyza huidobrensis) vis-à-vis the control. Also application of acetyl salicylic acid showed higher percentage of inflorescences (p<0.05) compared to the control, while statistical differences were not found between bacterial strains...(AU)

Peru é o centro de origem da batata e seus parentes silvestres, por isso é conveniente desenvolver novas tecnologias de gestão agronômica com menor impacto no ecossistema, como o uso de pesticidas e fertilizantes químicos em batata está aumentando nos países em desenvolvimento. O objetivo do trabalho foi avaliar o desempenho agronômico da cv. 'Unica' sob inoculação com rizobactérias PGPR isoladas da rizosfera de batata andina e pelo efeito da aplicação de ácido acetilsalicílico nas condições de campo da costa peruana. Avaliou-se a inoculação de quatro isolados bacterianos: Bacillus simplex B13, B. amyloliquefaciens Bac 15Mb, Azotobacter sp. Azo 16M2 e Pantoea sp. DZ22 bem como uma dose de 0,4 mM de ácido acetilsalicílico como promotores de tratamentos para o crescimento das plantas versus um controle sem inoculação. Utilizou-se um delineamento em blocos casualizados, em um solo franco-arenoso,  ligeiramente alcalino, de baixo conteúdo orgânico e livre de sais. O clima durante o ensaio apresentou uma umidade relativa entre 54 e 71%, temperaturas médias entre 13 e 21 ºC em San Vicente de Cañete, Lima (coordenadas UTM  18L3540788550372). As plantas foram inoculadas com estirpes bacterianas no momento da semeadura e na floração e os tratamentos foram avaliados em parcelas de 40 plantas com três repetições para diversos parâmetros fisiológicos e produtivos. Os dados foram analisados estatisticamente através do teste de Duncan e processados com o software Infostat. A inoculação com a estirpe Azo 16M2, apresentou diferenças significativas (p<0,05) em relação ao controle sem inoculação, aumentando a produção de tubérculos e o peso comercial de tubérculos por hectare e com menor dano pela mosca minadora (Liriomyza huidobrensis) em relação ao controle. Também a aplicação de ácido acetilsalicílico apresentou maior porcentagem de inflorescências (p<0,05) em relação ao controle, enquanto que diferenças estatísticas não foram encontradas entre...(AU)

A phasin with extra talents: a polyhydroxyalkanoate granule-associated protein has chaperone activity.

Phasins are proteins associated to intracellular polyhydroxyalkanoate granules that affect polymer accumulation and the number and size of the granules. Previous work demonstrated that a phasin from Azotobacter sp FA-8 (PhaPAz ) had an unexpected growth-promoting and stress-protecting effect in Escherichia coli, suggesting it could have chaperone-like activities. In this work, in vitro and in vivo experiments were performed in order to investigate this possibility. PhaPAz was shown to prevent in vitro thermal aggregation of the model protein citrate synthase and to facilitate the refolding process of this enzyme after chemical denaturation. Microscopy techniques were used to analyse the subcellular localization of PhaPAz in E. coli strains and to study the role of PhaPAz in in vivo protein folding and aggregation. PhaPAz was shown to colocalize with inclusion bodies of PD, a protein that aggregates when overexpressed. A reduction in the number of inclusion bodies of PD was observed when it was coexpressed with PhaPAz or with the known chaperone GroELS. These results demonstrate that PhaPAz has chaperone-like functions both in vitro and in vivo in E. coli recombinants, and suggests that phasins could have a general protective role in natural polyhydroxyalkanoate producers.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants.

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of ß-galactosidase activity constitutively. Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of ß-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to ß-galactosidase activity. The method was found to be sensitive enough to determine 1.7 × 10(6) CFU mL(-1) in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

A phasin with many faces: structural insights on PhaP from Azotobacter sp. FA8.

Phasins are a group of proteins associated to granules of polyhydroxyalkanoates (PHAs). Apart from their structural role as part of the PHA granule cover, different structural and regulatory functions have been found associated to many of them, and several biotechnological applications have been developed using phasin protein fusions. Despite their remarkable functional diversity, the structure of these proteins has not been analyzed except in very few studies. PhaP from Azotobacter sp. FA8 (PhaPAz) is a representative of the prevailing type in the multifunctional phasin protein family. Previous work performed in our laboratory using this protein have demonstrated that it has some very peculiar characteristics, such as its stress protecting effects in recombinant Escherichia coli, both in the presence and absence of PHA. The aim of the present work was to perform a structural characterization of this protein, to shed light on its properties. Its aminoacid composition revealed that it lacks clear hydrophobic domains, a characteristic that appears to be common to most phasins, despite their lipid granule binding capacity. The secondary structure of this protein, consisting of α-helices and disordered regions, has a remarkable capacity to change according to its environment. Several experimental data support that it is a tetramer, probably due to interactions between coiled-coil regions. These structural features have also been detected in other phasins, and may be related to their functional diversity.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

The use of lacZ marker in enumeration of Azotobacter chroococcum in carrier based inoculants

A transconjugant of Azotobacter chroococcum Mac 27 tagged with lac Z(A. chroococcum Mac27 L) was found to possess high levels of β-galactosidase activity constitutively.Further, the lac Z marker was found to be stably integrated into the chromosome of the A. chroococcum Mac 27 and did not have any adverse effect on growth, nitrogen fixation and excretion of ammonia. A quick method to determine the viable cell number in broth culture and carrier based inoculants has been developed on the basis of β-galactosidase assay. It was found that there was a direct relationship between the number of cell as determined by standard plate count and intensity of colour that developed upon degradation of ONPG due to β-galactosidase activity .The method was found to be sensitive enough to determine 1.7 x 10(6) CFU mL-1 in broth culture as well as carrier based Azotobacter inoculants. Further, it was observed that when A. chroococcum Mac27 L was inoculated on Brassica campestris, it could be detected in the presence of other bacteria capable of growing on Burks agar medium containing X-gal on the basis of lac Z genetic marker.

Genotypic characterization of Azotobacteria isolated from Argentinean soils and plant-growth-promoting traits of selected strains with prospects for biofertilizer production.

The genetic diversity among 31 putative Azotobacter isolates obtained from agricultural and non-agricultural soils was assessed using rep-PCR genomic fingerprinting and identified to species level by ARDRA and partial 16S rRNA gene sequence analysis. High diversity was found among the isolates, identified as A. chroococcum, A. salinestris, and A. armeniacus. Selected isolates were characterized on the basis of phytohormone biosynthesis, nitrogenase activity, siderophore production, and phosphate solubilization. Indole-3 acetic-acid (IAA), gibberellin (GA3) and zeatin (Z) biosynthesis, nitrogenase activity, and siderophore production were found in all evaluated strains, with variation among them, but no phosphate solubilization was detected. Phytohormones excreted to the culture medium ranged in the following concentrations: 2.2-18.2 µ g IAA mL(-1), 0.3-0.7 µ g GA3 mL(-1), and 0.5-1.2 µ g Z mL(-1). Seed inoculations with further selected Azotobacter strains and treatments with their cell-free cultures increased the number of seminal roots and root hairs in wheat seedlings. This latter effect was mimicked by treatments with IAA-pure solutions, but it was not related to bacterial root colonization. Our survey constitutes a first approach to the knowledge of Azotobacter species inhabiting Argentinean soils in three contrasting geographical regions. Moreover, this phenotypic characterization constitutes an important contribution to the selection of Azotobacter strains for biofertilizer formulations.

Evaluación de la estabilidad de Trichoderma sp. y Azotobacter sp. conservados por diferentes métodos / Evaluation of the stability of Trichoderma sp. and Azotobacter sp. preserved by different methods

En el Laboratorio de Microbiología Aplicada de la Universidad Francisco de Paula Santander (Cúcuta, Colombia) constantemente ingresan al Banco de Cepas, cultivos microbianos de interés biotecnológico, especialmente del sector agrícola, los cuales son utilizados en las actividades de docencia e investigación. Por esta razón, existe el interés de mantener viables a través del tiempo estos cultivos microbianos, para lo cual se realizó en esta investigación, la evaluación de la estabilidad de los aislados de Trichoderma sp. y Azotobacter sp., utilizando las técnicas de conservación en viales con solución salina estéril (0,85% NaCl) en refrigeración (4°C) y temperatura ambiente (30°C), suelo estéril en refrigeración (4°C), comparados con la metodología de repiques sucesivos como tratamiento control. Los resultados no mostraron diferencias significativas según el Test de Duncan (P≤0,05) en la tasa de supervivencia microbiana entre los métodos de conservación. Sin embargo, se observó en las técnicas de viales con solución salina estéril y suelo estéril mantenidos en refrigeración, mayor estabilidad en la concentración celular durante los cuatros meses de evaluación, sin registrar contaminación en los cultivos. Así mismo, se registró un óptimo crecimiento macroscópico de los cultivos microbianos y sus características microscópicas se mantuvieron estables en estos dos métodos de conservación. Por esta razón, se seleccionaron como técnicas de conservación de estos microorganismos, teniendo en cuenta además, ventajas como la facilidad de la técnica, disponibilidad de equipos, materiales y personal con los que se cuenta en el laboratorio.

In the Laboratory of Applied Microbiology of the University Francisco de Paula Santander (Cúcuta, Colombia), strains of microbial cultures of biotechnological interest are constantly entering, particularly from the agricultural sector, which are used for teaching and research activities. There is an interest in keeping them viable over time, which was the main reason of this research: To evaluate the stability of the isolates of Trichoderma sp. and Azotobacter sp., using different conservation techniques: vials with saline solution (0,85% NaCl) in two temperature conditions: refrigeration (4°C) and room temperature (30°C), sterile soil under refrigeration (4°C), and successive peals methodology as the control treatment. Results showed no significant differences according to Duncan test (P ≤ 0.05) in the microbial survival ratio between preservation methods. However, a greater stability in cell concentration was observed with the techniques of vials with sterile saline solution and sterile soil -both under refrigeration-, during the four-month evaluation, with no register of contamination on cultures. Also, there was an optimum growth of microbial cultures macroscopically and the microscopic characteristics were stable in these two methods of preservation for both strains. For this reason, we suggest these conservation techniques for the two microorganisms, considering further advantages such as ease of technology, availability of equipment, materials and personnel available in the laboratory.

Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation

Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 ºC with an inoculum of 1 x 10(6) spores and yielded 1500 active units (UµmL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 ºC and yielded 40 UµmL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 ºC, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase.

Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae.

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.