Characterization and efficacy of bacterial strains for biological control of soil-borne diseases caused by Phytophthora cactorum and Meloidogyne javanica on Rosaceous plants

S'avaluaren 58 soques de Pseudomonas fluorescens i Pantoea agglomerans per la seva eficàcia en el biocontrol de la malaltia causada per l'oomicet Phytophthora cactorum en maduixera i pel nematode formador de gal·les Meloidogyne javanica en el portaempelt GF-677. Es desenvolupà un mètode ex vivo d'inoculació de fulla amb l'objectiu de seleccionar soques bacterianes com a agents de control biològic de P. cactorum en maduixera. Tres soques de P. fluorescens es seleccionaren com a soques eficaces en el biocontrol del patogen en fulles i en la reducció de la malaltia en plantes de maduixera. La combinació de soques semblà millorar la consistència del biocontrol en comparació amb les soques aplicades individualment. Tres soques de P. fluorescens es seleccionaren per la seva eficàcia en la reducció de la infecció de M. javanica en portaempelts GF-677. La combinació d'aquestes soques no incrementà l'eficàcia del biocontrol, però semblà reduir la seva variabilitat.

Fire blight (Erwinia amylovora) of rosaceous plants. Pathogen virulence and selection and characterization of biological control agents

El fuego bacteriano, causado por Erwinia amylovora, es una enfermedad muy importante a nivel comercial y económico porque afecta a plantas de la familia de las rosáceas y es especialmente agresiva en manzano (Pyrus malus) y peral (Pyrus communis), así como en plantas ornamentales (Crataegus, Cotoneaster o Pyracantha). Esta enfermedad está distribuida por todo el mundo en zonas climáticas templadas de Amércia del Norte, Nueva Zelanda, Japón, Israel, Turquí y Europa. En España, el fuego bacteriano fue detectado por primera vez en 1995 en el norte del País (Euskadi) y más tarde en nuevos focos aparecidos en otras áreas. La enfermedad puede ser controlada comercialmente mediante la aplicación de pesticidas quimicos (derivados de cobre, antibioticos). Sin embargo, muchos de los productos químicos presentan baja actividad o causan fitotoxicidad, y la estreptomicina, el producto más eficaz, esta prohibido en muchos países, incluyendo España. Por tanto, en ausencia de apropiados agentes químicos, el control biológico se contempla como una buena alternativa. En el presente trabajo, un agente de control biológico, Pseudomonas fluorescens EPS62e, ha sido seleccionada de entre 600 aislados de las especies P. fluorescens y Pantoea agglomerans obtenidos de flores, frutos y hojas de plantas de la familia de las rosáceas durante una prospección llevada a cabo en varias áreas geográficas de España. La cepa ha sido seleccionada por su capacidad de suprimir la infecciones producidas por E. amylovora frutos inmaduros, flores y brotes de peral en condiciones de ambiente controlado, presentando unos niveles de control similares a los obtenidos mediante el control químico usando derivados de cobre o antibióticos. La cepa además ha mostrado la capacidad de colonizar y sobrevivir en flores y heridas producidas en frutos inmaduros en condiciones de ambiento controlado pero también en flores en condiciones de campo. La exclusión de E. amylovora medinate la colonización de la superficie, el consumo de nutrientes, y la interacción entre las células del patógeno y del agente de biocontrol es la principal causa de la inhibición del fuego bacteriano por la cepa EPS62e. Estas características constituyen aspectos interesantes para un desarrollo efectivo de la cepa EPS62e como un agente de biocontrol del fuego bacteriano en condiciones comerciales.

Biosensor-based diagnostics of contaminated groundwater: assessment and remediation strategy

Shallow groundwater beneath a former airfield site in southern England has been heavily contaminated with a wide range of chlorinated solvents. The feasibility of using bacterial biosensors to complement chemical analysis and enable cost-effective, and focussed sampling has been assessed as part of a site evaluation programme. Five different biosensors, three metabolic (Vibrio fischeri, Pseudomonas fluorescens 10568 and Escherichia coli HB101) and two catabolic (Pseudomonas putida TVA8 and E. coli DH5alpha), were employed to identify areas where the availability and toxicity of pollutants is of most immediate environmental concern. The biosensors used showed different sensitivities to each other and to the groundwater samples tested. There was generally a good agreement with chemical analyses. The potential efficacy of remediation strategies was explored by coupling sample manipulation to biosensor tests. Manipulation involved sparging and charcoal treatment procedures to simulate remediative engineering solutions. Sparging was sufficient at most locations. (C) 2004 Elsevier Ltd. All rights reserved.

Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment

The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 106 mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.

Thioquinolobactin, a pseudomonas siderophore with antifungal and anti-pythium activity

Under conditions of iron limitation Pseudomonas fluorescens ATCC 17400 produces two siderophores, pyoverdine, and a second siderophore quinolobactin, which itself results from the hydrolysis of the unstable molecule 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid (thioquinolobactin). Pseudomonas fluorescens ATCC 17400 also displays a strong in vitro antagonism against the Oomycete Pythium, which is repressed by iron, suggesting the involvement of a siderophore(s). While a pyoverdine-negative mutant retains most of its antagonism, a thioquinolobactin-negative mutant only slowed-down Pythium growth, and a double pyoverdine-, thioquinolobactin-negative mutant, which does not produce any siderophore, totally lost its antagonism against Pythium. The siderophore thioquinolobactin could be purified and identified from spent medium and showed anti-Pythium activity, but it was quickly hydrolysed to quinolobactin, which we showed has no antimicrobial activity. Analysis of antagonism-affected transposon mutants revealed that genes involved in haem biosynthesis and sulfur assimilation are important for the production of thioquinolobactin and the expression of antagonism.

Pseudomonas genomes: diverse and adaptable

Members of the genus Pseudomonas inhabit a wide variety of environments, which is reflected in their versatile metabolic capacity and broad potential for adaptation to fluctuating environmental conditions. Here, we examine and compare the genomes of a range of Pseudomonas spp. encompassing plant, insect and human pathogens, and environmental saprophytes. In addition to a large number of allelic differences of common genes that confer regulatory and metabolic flexibility, genome analysis suggests that many other factors contribute to the diversity and adaptability of Pseudomonas spp. Horizontal gene transfer has impacted the capability of pathogenic Pseudomonas spp. in terms of disease severity (Pseudomonas aeruginosa) and specificity (Pseudomonas syringae). Genome rearrangements likely contribute to adaptation, and a considerable complement of unique genes undoubtedly contributes to strain- and species-specific activities by as yet unknown mechanisms. Because of the lack of conserved phenotypic differences, the classification of the genus has long been contentious. DNA hybridization and genome-based analyses show close relationships among members of P. aeruginosa, but that isolates within the Pseudomonas fluorescens and P. syringae species are less closely related and may constitute different species. Collectively, genome sequences of Pseudomonas spp. have provided insights into pathogenesis and the genetic basis for diversity and adaptation.

Evolutionary resurrection of flagellar motility via rewiring of the nitrogen regulation system

A central process in evolution is the recruitment of genes to regulatory networks. We engineered immotile strains of the bacterium Pseudomonas fluorescens that lack flagella due to deletion of the regulatory gene fleQ. Under strong selection for motility, these bacteria consistently regained flagella within 96 hours via a two-step evolutionary pathway. Step 1 mutations increase intracellular levels of phosphorylated NtrC, a distant homologue of FleQ, which begins to commandeer control of the fleQ regulon at the cost of disrupting nitrogen uptake and assimilation. Step 2 is a switch-of-function mutation that redirects NtrC away from nitrogen uptake and towards its novel function as a flagellar regulator. Our results demonstrate that natural selection can rapidly rewire regulatory networks in very few, repeatable mutational steps.

Selective Oxidations of Organoboron Compounds Catalyzed by Baeyer-Villiger Monooxygenases

The applicability of Baeyer-Villiger monooxygenases (BVMOs) in organoboron chemistry has been explored through testing chemo-and enantioselective oxidations of a variety of boron-containing aromatic and vinylic compounds. Several BVMOs, namely: phenylacetone monooxygenase (PAMO), M446G PAMO mutant, 4-hydroxyacetophenone monooxygenase (HAPMO) and cyclohexanone monooxygenase (CHMO) were used in this study. The degree of chemoselectivity depends on the type of BVMO employed, in which the biocatalysts prefer boron-carbon oxidation over Baeyer-Villiger oxidation or epoxidation. Interestingly, it was discovered that PAMO can be used to perform kinetic resolution of boron-containing compounds with good enantioselectivities. These findings extend the known biocatalytic repertoire of BVMOs by showing a new family of compounds that can be oxidized by these enzymes.

Bioluminescent sensor for naphthalene in air: Cell immobilization and evaluation with a dynamic standard atmosphere generator

A dynamic atmosphere generator with a naphthalene emission source has been constructed and used for the development and evaluation of a bioluminescence sensor based on the bacteria Pseudomonas fluorescens HK44 immobilized in 2% agar gel (101 cell mL(-1)) placed in sampling tubes. A steady naphthalene emission rate (around 7.3 nmol min(-1) at 27 degrees C and 7.4 mLmin(-1) of purified air) was obtained by covering the diffusion unit containing solid naphthalene with a PTFE filter membrane. The time elapsed from gelation of the agar matrix to analyte exposure (""maturation time"") was found relevant for the bioluminescence assays, being most favorable between 1.5 and 3 h. The maximum light emission, observed after 80 min, is dependent on the analyte concentration and the exposure time (evaluated between 5 and 20 min), but not on the flow rate of naphthalene in the sampling tube, over the range of 1.8-7.4 nmol min(-1). A good linear response was obtained between 50 and 260 nmol L-1 with a limit of detection estimated in 20 nmol L-1 far below the recommended threshold limit value for naphthalene in air. (c) 2008 Elsevier B.V. All rights reserved.

Vitpunktsförskjutningens påverkan på ICC-profiler vid användande av optiska vitmedel

The connection between fluorescence and color management still is a relatively unexplored area. Thefollowing composition analyses the theory that when printing on fluorescent ink jet paper using ICCprofilesa more yellow print will appear. In theory the ICC-profile compensate for blue light that fluorescefrom the paper with the complementary color, yellow. The human eye apprehands this fluorescentlight as white.When categorizing a number of ink jet qualities some tests to indicate change of color in the final printwere made. Five qualities were chosen for further analyses, each with different white point. TheTC3.5CMYK target was prined on an ink jet printer and measured with different filters for the measurementequipment, GretagMacbeth Spectrolino. U- and UV-filters were used during the testings.From the measurement data ICC-profiles were made to make it possible to watch how the profiles compensatefor the blue change of color that appeared in the data. Although a distinct change of color intoblue were discernible in the measured data there is no evident proof that this cause a final print thatis more yellow. Other conclusions and results are to be read at the end of this composition.

Avaliação, identificação e atividade enzimática de bactérias psicotróficas presentes no leite cru refrigerado

Extended storage of refrigerated milk can lead to reduced quality of raw and processed milk, which is a consequence of the growth and metabolic activities of psychrotrophic bacteria, able to grow under 7oC or lower temperatures. Although most of these microorganisms are destroyed by heat treatment, some have the potential to produce termoresistant proteolytic and lipolytic enzymes that can survive even UHT processing and reduce the processed products quality. Recently, the IN 51 determineds that milk should be refrigerated and stored at the farm what increased the importance of this group of microorganisms. In this work, psychrotrophic bacteria were isolated from 20 communitarian bulk tanks and 23 individual bulk tanks from dairy farms located at Zona da Mata region of Minas Gerais State and from southeastern Rio de Janeiro. Selected milk dilutions were plated on standard agar and after incubation for 10 days at 7oC, five colonies were isolated, firstly using nutrient agar and after using McConkey agar for 24 hours at 21oC. The isolates were identified by morphology, Gram stain method, catalase production, fermentative/oxidative metabolism and by API 20E, API 20NE, API Staph, API Coryne or API 50 CH (BioMerieux). In order to ensure reproductibility, API was repeated for 50% of the isolates. Species identification was considered when APILAB indexes reached 75% or higher. 309 strains were isolated, 250 Gram negative and 59 Gram positive. 250 Gram negative isolates were identified as: Acinetobacter spp. (39), Aeromonas spp. (07), A. Hydrophila (16), A. sobria (1), A. caviae (1), Alcaligenes feacalis (1), Burkholderia cepacia (12), Chryseomonas luteola (3), Enterobacter sp. (1), Ewingella americana(6), Hafnia alvei (7), Klebsiella sp. (1), Klebsiella oxytoca (10), Yersinia spp. (2), Methylobacterium mesophilicum (1), Moraxella spp. (4), Pantoea spp. (16), Pasteurella sp. (1), Pseudomonas spp. (10), P. fluorescens (94), P. putida (3), Serratia spp. (3), Sphigomonas paucomobilis (1). Five isolates kept unidentified. Pseudomonas was the predominant bacteria found (43%) and P. fluorescens the predominant species (37.6%), in accordance with previous reports. Qualitative analysis of proteolytic and lipolytic activity was based on halo formation using caseinate agar and tributirina agar during 72 hours at 21oC and during 10 days at 4°C, 10oC and 7°C. Among 250 Gram negative bacteria found, 104 were identified as Pseudomonas spp. and 60,57% of this group showed proteolytic and lipolytic acitivities over all four studied temperatures. 20% of Acinetobacter, Aeromonas, Alcaligenes, Burkholderia, Chryseomonas, Methylobacterium, Moraxella presented only lipolytic activity. Some isolates presented enzymatic activity in one or more studied temperatures. Among Gram positive bacteria, 30.51% were proteolytic and lipolytic at 10oC, 8.47% were proteolytic at 7oC, 10oC, and 21oC, 8.47% were proteolytic at all studied temperatures (4oC, 7oC, 10oC and 21oC) and 3.38% were proteolytic only at 21oC. At 4oC, only one isolate showed proteolytic activity and six isolates were lipolytic. In relation to Gram negative microorganisms, 4% were proteolytic and lipolytic at 7oC, 10oC and 21oC, 10% were proteolytic at 10oC and 4.4% were lipolytic at 4oC, 7oC, 10oC and 21oC, while 6.4% of all isolates were proteolytic and lipolytic at 10oC and 21oC as well as lipolytic at 4oC and 7oC. These findings are in accordance with previous researches that pointed out Pseudomonas as the predominant psycrotrophic flora in stored refrigerated raw milk

Pulmonary, microbiological and hematological changes in Crotalus durissus terrificus (Serpentes, Viperidae) parasitized by nematodes of the genus Rhabdias (Nematoda, Rhabdiasidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Avaliação de populações de possíveis rizobactérias em solos sob espécies florestais

Embora estudos recentes relatem a utilização de RCPC (Rizobactérias Promotoras de Crescimento de Plantas) no Brasil, raríssimos trabalhos avaliam a presença natural dessas espécies bacterianas no solo. O objetivo deste trabalho foi avaliar a ocorrência de RPCP em duas amostras de solo sob diferentes tipos de manejo, através da construção e do seqüenciamento de bibliotecas de DNA metagenômico. Utilizaram-se oligonucleotídeos específicos para amplificação da região hipervariável do espaço intergênico dos genes ribossomais 16S-23S de DNA extraído de diferentes solos, sob Eucalyptus sp. e sob mata. Os fragmentos obtidos foram inseridos em vetor e clonados. As bibliotecas geraram 495 clones, que foram seqüenciados e identificados através de comparações realizadas pelo software Blast. O solo sob Eucalyptus sp. apresentou maior número de RPCP do que sob mata. Os filos Actinobacteria e Proteobacteria eram maiores no solo sob Eucalyptus sp., estando o filo Firmicutes ausente no solo sob mata. Somente oito espécies diferentes de RPCP foram detectadas: Bacillus subtilis, Bacillus megaterium, Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium sp., Frankia sp., Pseudomonas fluorescens e Pseudomonas gladioli. O trabalho forneceu valiosos dados sobre a presença de RPCP em solos com espécies florestais e sua possível utilização em reflorestamentos, assim como para o melhor conhecimento desses microrganismos nos solos do Brasil.

Implicações do parasitismo por nematódeos do gênero Rhabdias(Nematoda, Rhabdiasidae) em Crotalus durissus terrificus (Serpentes, Viperidae): alterações pulmonares, microbiológicas e hematológicas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

PHYSICO-CHEMICAL, SPECTROSCOPICAL AND THERMAL CHARACTERIZATION OF BIODIESEL OBTAINED BY ENZYMATIC ROUTE AS A TOOL TO SELECT THE MOST EFFICIENT IMMOBILIZED LIPASE

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45 degrees C and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and H-1 NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.