Iron acquisition with the natural siderophore enantiomers pyochelin and enantio-pyochelin in Pseudomonas species.

The bacterial siderophore pyochelin is composed of salicylate and two cysteine-derived heterocycles, the second of which is modified by reduction and N-methylation during biosynthesis. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation, whereas the second cysteine remains in its L-configuration. Stereochemistry is opposite in the Pseudomonas fluorescens siderophore enantio-pyochelin, in which the first ring originates from L-cysteine and the second ring from D-cysteine. Both siderophores promote growth of the producer organism during iron limitation and induce the expression of their biosynthesis genes by activating the transcriptional AraC-type regulator PchR. However, neither siderophore is functional as an iron carrier or as a transcriptional inducer in the other species, demonstrating that both processes are highly stereospecific. Stereospecificity of pyochelin/enantio-pyochelin-mediated iron uptake is ensured at two levels: (i) by the outer membrane siderophore receptors and (ii) by the cytosolic PchR regulators.

Biocontrole da queima-das-bainhas e do nematoide-das-galhas e promoção de crescimento de plantas de arroz por rizobactérias

O objetivo deste trabalho foi avaliar a compatibilidade entre rizobactérias biocontroladoras pré-selecionadas e o efeito de suas combinações sobre a queima-das-bainhas (Rhizoctonia solani), a meloidoginose (Meloidogyne graminicola) e a promoção de crescimento de plantas de arroz. A compatibilidade foi determinada pela antibiose. O efeito das combinações de isolados foi avaliado por microbiolização de sementes de arroz, cultivar El Paso L144, com suspensões das rizobactérias DFs185 (Pseudomonas synxantha), DFs223 (P. fluorescens), DFs306 (ainda não identificada), DFs416 e DFs418 (Bacillus sp.). Essas rizobactérias foram usadas isoladamente ou combinadas em arranjos de dois, três e quatro isolados. O isolado DFs223 não foi combinado com nenhum outro por ser incompatível com os demais isolados. Cinco combinações de rizobactérias (DFs185/418, DFs306/416, DFs306/418, DFs416/418, DFs185/306/418) e o isolado DFs306 destacaram-se por reduzir a reprodução de M. graminicola e promover o crescimento das plantas. A combinação DFs185/306 apresentou os melhores resultados quanto ao controle de R. solani e à promoção de crescimento. A combinação DFs306/416 proporcionou os melhores resultados para o controle das duas doenças.

Signal transduction in plant-beneficial rhizobacteria with biocontrol properties.

Biological control of root pathogens--mostly fungi--can be achieved by the introduction of selected bacterial inoculants acting as 'biopesticides'. Successful inoculants have been identified among Gram-negative and Gram-positive bacteria, often belonging to Pseudomonas spp. and Bacillus spp., respectively. Biocontrol activity of a model rhizobacterium, P. fluorescens CHAO, depends to a considerable extent on the synthesis of extracellular antimicrobial secondary metabolites and exoenzymes, thought to antagonize the pathogenicity of a variety of phytopathogenic fungi. The regulation of exoproduct formation in P. fluorescens (as well as in other bacteria) depends essentially on the GacS/GacA two-component system, which activates a largely unknown signal transduction pathway. However, recent evidence indicates that GacS/GacA control has a major impact on target gene expression at a post-transcriptional level, involving an mRNA target sequence (typically near the ribosome binding site), two RNA binding proteins (designated RsmA and RsmE), and a regulatory RNA (RsmZ) capable of binding RsmA. The expression and activity of the regulatory system is stimulated by at least one low-molecular-weight signal. The timing and specificity of this switch from primary to secondary metabolism are essential for effective biocontrol.

Phylogenetic position of Fit insect toxin producing pseudomonads

Fit produced by Pseudomonas fluorescens CHA0 is a novel insect toxin in root colonizing pseudomonads, of which a homologue is described in Photorhabdus species.However, occurrence and abundance of insect pathogenicity in plant-associated pseudomonads is still unclear. An extensive screening outside the P. fluorescens complex identified strains of Pseudomonas chlororaphis as further Fit toxin producing candidates. Sequences of five different P. chlororaphis strains generated in this study were used to reconstruct the evolutionary history of the Fit toxin gene and to analyse its mode of evolution. We found that P. chlororaphis is closely associated with a small subgroup of 2,4-diacetylphloroglucinol and pyoluteorin- producing pseudomonads, both when analyzing four housekeeping genes and the nucleotide sequences for the Fit toxin gene. Additionally, we identified purifying selection to be the predominant mode of Fit toxin evolution.

Wood ants use resin to protect themselves against pathogens

Social life is generally associated with an increased exposure to pathogens and parasites, due to factors such as high population density, frequent physical contact and the use of perennial nest sites. However, sociality also permits the evolution of new collective behavioural defences. Wood ants, Formica paralugubris, commonly bring back pieces of solidified coniferous resin to their nest. Many birds and a few mammals also incorporate green plant material into their nests. Collecting plant material rich in volatile compounds might be an efficient way to fight bacteria and fungi. However, no study has demonstrated that this behaviour has a positive effect on survival. Here, we provide the first experimental evidence that animals using plant compounds with antibacterial and antifungal properties survive better when exposed to detrimental micro-organisms. The presence of resin strongly improves the survival of F. paralugubris adults and larvae exposed to the bacteria Pseudomonas fluorescens, and the survival of larvae exposed to the entomopathogenic fungus Metarhizium anisopliae. These results show that wood ants capitalize on the chemical defences which have evolved in plants to collectively protect themselves against pathogens.

Ação antagônica de rizobactérias contra Phytophthora parasitica e p. citrophthora e seu efeito no desenvolvimento de plântulas de citros

O antagonismo de Pseudomonas putida biovar A (C1-1B), P. putida biovar B (Santa Bárbara), P. fluorescens (C2-8C e RA2), Bacillus subtilis (OG e RC2) e Flavobacterium sp. (CIS/NA) contra Phytophthora parasitica e P. citrophthora , agentes da podridão radicular dos citros, foi avaliado através da inibição do crescimento micelial (cultura pareada) e redução na percentagem de infecção da doença em mudas de citros (tratamento de sementes com rizobactérias). Na seleção preliminar, 33 isolados bacterianos foram testados. Sementes de citros pré-germinadas foram tratadas por imersão nas suspensões das bactérias (10(9) ufc/ml), e plantadas em tubetes contendo solo natural infestado com o fitopatógeno (50 ml de suspensão/ kg de solo). A avaliação da percentagem de infecção foi efetuada após 15 dias. In vitro, os isolados bacterianos RC2, OG, CIS/NA e C1-1B foram os mais ativos inibidores do crescimento micelial de Phytophthora. Em condições de casa de vegetação, todos os isolados proporcionaram redução na percentagem de infecção da doença em todos os ensaios realizados. Promoção de crescimento de plantas foi verificada pela inoculação de plântulas com as linhagens OG, RC2, CiS/Na e C1-1B.

Evolutionary patchwork of an insecticidal toxin shared between plant-associated pseudomonads and the insect pathogens Photorhabdus and Xenorhabdus.

BACKGROUND: Root-colonizing fluorescent pseudomonads are known for their excellent abilities to protect plants against soil-borne fungal pathogens. Some of these bacteria produce an insecticidal toxin (Fit) suggesting that they may exploit insect hosts as a secondary niche. However, the ecological relevance of insect toxicity and the mechanisms driving the evolution of toxin production remain puzzling. RESULTS: Screening a large collection of plant-associated pseudomonads for insecticidal activity and presence of the Fit toxin revealed that Fit is highly indicative of insecticidal activity and predicts that Pseudomonas protegens and P. chlororaphis are exclusive Fit producers. A comparative evolutionary analysis of Fit toxin-producing Pseudomonas including the insect-pathogenic bacteria Photorhabdus and Xenorhadus, which produce the Fit related Mcf toxin, showed that fit genes are part of a dynamic genomic region with substantial presence/absence polymorphism and local variation in GC base composition. The patchy distribution and phylogenetic incongruence of fit genes indicate that the Fit cluster evolved via horizontal transfer, followed by functional integration of vertically transmitted genes, generating a unique Pseudomonas-specific insect toxin cluster. CONCLUSIONS: Our findings suggest that multiple independent evolutionary events led to formation of at least three versions of the Mcf/Fit toxin highlighting the dynamic nature of insect toxin evolution.

Molecular basis and regulation of insect pathogenicity in plant-beneficial pseudomonads

Les bactéries du genre Pseudomonas ont la capacité étonnante de s'adapter à différents habitats et d'y survivre, ce qui leur a permis de conquérir un large éventail de niches écologiques et d'interagir avec différents organismes hôte. Les espèces du groupe Pseudomonas fluorescens peuvent être facilement isolées de la rhizosphère et sont communément connues comme des Pseudomonas bénéfiques pour les plantes. Elles sont capables d'induire la résistance systémique des plantes, d'induire leur croissance et de contrer des phytopathogènes du sol. Un sous-groupe de ces Pseudomonas a de plus développé la capacité d'infecter et de tuer certaines espèces d'insectes. Approfondir les connaissances sur l'interaction de ces bactéries avec les insectes pourraient conduire au développement de nouveaux biopesticides pour la protection des cultures. Le but de cette thèse est donc de mieux comprendre la base moléculaire, l'évolution et la régulation de la pathogénicité des Pseudomonas plante-bénéfiques envers les insectes. Plus spécifiquement, ce travail a été orienté sur l'étude de la production de la toxine insecticide appelée Fit et sur l'indentification d'autres facteurs de virulence participant à la toxicité de la bactérie envers les insectes. Dans la première partie de ce travail, la régulation de la production de la toxine Fit a été évaluée par microscopie à épifluorescence en utilisant des souches rapportrices de Pseudomonas protegens CHA0 qui expriment la toxine insecticide fusionnée à une protéine fluorescente rouge, au site natif du gène de la toxine. Celle-ci a été détectée uniquement dans l'hémolymphe des insectes et pas sur les racines des plantes, ni dans les milieux de laboratoire standards, indiquant une production dépendante de l'hôte. L'activation de la production de la toxine est contrôlée par trois protéines régulatrices dont l'histidine kinase FitF, essentielle pour un contrôle précis de l'expression et possédant un domaine "senseur" similaire à celui de la kinase DctB qui régule l'absorption de carbone chez les Protéobactéries. Il est donc probable que, durant l'évolution de FitF, un réarrangement de ce domaine "senseur" largement répandu ait contribué à une production hôte-spécifique de la toxine. Les résultats de cette étude suggèrent aussi que l'expression de la toxine Fit est plutôt réprimée en présence de composés dérivés des plantes qu'induite par la perception d'un signal d'insecte spécifique. Dans la deuxième partie de ce travail, des souches mutantes ciblant des facteurs de virulence importants identifiés dans des pathogènes connus ont été générées, dans le but d'identifier ceux avec une virulence envers les insectes atténuée. Les résultats ont suggéré que l'antigène O du lipopolysaccharide (LPS) et le système régulateur à deux composantes PhoP/PhoQ contribuent significativement à la virulence de P. protegens CHA0. La base génétique de la biosynthèse de l'antigène O dans les Pseudomonas plante-bénéfiques et avec une activité insecticide a été élucidée et a révélé des différences considérables entre les lignées suite à des pertes de gènes ou des acquisitions de gènes par transfert horizontal durant l'évolution de certaines souches. Les chaînes latérales du LPS ont été montrées comme vitales pour une infection des insectes réussie par la souche CHA0, après ingestion ou injection. Les Pseudomonas plante-bénéfiques, avec une activité insecticide sont naturellement résistants à la polymyxine B, un peptide antimicrobien modèle. La protection contre ce composé antimicrobien particulier dépend de la présence de l'antigène O et de la modification du lipide A, une partie du LPS, avec du 4-aminoarabinose. Comme les peptides antimicrobiens cationiques jouent un rôle important dans le système immunitaire des insectes, l'antigène O pourrait être important chez les Pseudomonas insecticides pour surmonter les mécanismes de défense de l'hôte. Le système PhoP/PhoQ, connu pour contrôler les modifications du lipide A chez plusieurs bactéries pathogènes, a été identifié chez Pseudomonas chlororaphis PCL1391 et P. protegens CHA0. Pour l'instant, il n'y a pas d'évidence que des modifications du lipide A contribuent à la pathogénicité de cette bactérie envers les insectes. Cependant, le senseur-kinase PhoQ est requis pour une virulence optimale de la souche CHA0, ce qui suggère qu'il régule aussi l'expression des facteurs de virulence de cette bactérie. Les découvertes de cette thèse démontrent que certains Pseudomonas associés aux plantes sont de véritables pathogènes d'insectes et donnent quelques indices sur l'évolution de ces microbes pour survivre dans l'insecte-hôte et éventuellement le tuer. Les résultats suggèrent également qu'une recherche plus approfondie est nécessaire pour comprendre comment ces bactéries sont capables de contourner ou surmonter la réponse immunitaire de l'hôte et de briser les barrières physiques pour envahir l'insecte lors d'une infection orale. Pour cela, les futures études ne devraient pas uniquement se concentrer sur le côté bactérien de l'interaction hôte-microbe, mais aussi étudier l'infection du point de vue de l'hôte. Les connaissances gagnées sur la pathogénicité envers les insectes des Pseudomonas plante-bénéfiques donnent un espoir pour une future application en agriculture, pour protéger les plantes, non seulement contre les maladies, mais aussi contre les insectes ravageurs. -- Pseudomonas bacteria have the astonishing ability to survive within and adapt to different habitats, which has allowed them to conquer a wide range of ecological niches and to interact with different host organisms. Species of the Pseudomonas fluorescens group can readily be isolated from plant roots and are commonly known as plant-beneficial pseudomonads. They are capable of promoting plant growth, inducing systemic resistance in the plant host and antagonizing soil-borne phytopathogens. A defined subgroup of these pseudomonads evolved in addition the ability to infect and kill certain insect species. Profound knowledge about the interaction of these particular bacteria with insects could lead to the development of novel biopesticides for crop protection. This thesis thus aimed at a better understanding of the molecular basis, evolution and regulation of insect pathogenicity in plant-beneficial pseudomonads. More specifically, it was outlined to investigate the production of an insecticidal toxin termed Fit and to identify additional factors contributing to the entomopathogenicity of the bacteria. In the first part of this work, the regulation of Fit toxin production was probed by epifluorescence microscopy using reporter strains of Pseudomonas protegens CHAO that express a fusion between the insecticidal toxin and a red fluorescent protein in place of the native toxin gene. The bacterium was found to express its insecticidal toxin only in insect hemolymph but not on plant roots or in common laboratory media. The host-dependent activation of Fit toxin production is controlled by three local regulatory proteins. The histidine kinase of this regulatory system, FitF, is essential for the tight control of toxin expression and shares a sensing domain with DctB, a sensor kinase regulating carbon uptake in Proteobacteria. It is therefore likely that shuffling of a ubiquitous sensor domain during the evolution of FitF contributed to host- specific production of the Fit toxin. Findings of this study additionally suggest that host-specific expression of the Fit toxin is mainly achieved by repression in the presence of plant-derived compounds rather than by induction upon perceiving an insect-specific signal molecule. In the second part of this thesis, mutant strains were generated that lack factors previously shown to be important for virulence in prominent pathogens. A screening for attenuation in insect virulence suggested that lipopolysaccharide (LPS) O-antigen and the PhoP-PhoQ two-component regulatory system significantly contribute to virulence of P. protegens CHAO. The genetic basis of O-antigen biosynthesis in plant-beneficial pseudomonads displaying insect pathogenicity was elucidated and revealed extensive differences between lineages due to reduction and horizontal acquisition of gene clusters during the evolution of several strains. Specific 0 side chains of LPS were found to be vital for strain CHAO to successfully infect insects by ingestion or upon injection. Insecticidal pseudomonads with plant-beneficial properties were observed to be naturally resistant to polymyxin B, a model antimicrobial peptide. Protection against this particular antimicrobial compound was dependent on the presence of O-antigen and modification of the lipid A portion of LPS with 4-aminoarabinose. Since cationic antimicrobial peptides play a major role in the immune system of insects, O-antigenic polysaccharides could be important for insecticidal pseudomonads to overcome host defense mechanisms. The PhoP-PhoQ system, which is well-known to control lipid A modifications in several pathogenic bacteria, was identified in Pseudomonas chlororaphis PCL1391 and P. protegens CHAO. No evidence was found so far that lipid A modifications contribute to insect pathogenicity in this bacterium. However, the sensor kinase PhoQ was required for full virulence of strain CHAO suggesting that it additionally regulates the expression of virulence factors in this bacterium. The findings of this thesis demonstrate that certain plant-associated pseudomonads are true insect pathogens and give some insights into how these microbes evolved to survive within and eventually kill the insect host. Results however also point out that more in-depth research is needed to know how exactly these fascinating bacteria manage to bypass or overcome host immune responses and to breach physical barriers to invade insects upon oral infection. To achieve this, future studies should not only focus on the bacterial side of the microbe-host interactions but also investigate the infection from a host-oriented view. The knowledge gained about the entomopathogenicity of plant-beneficial pseudomonads gives hope for their future application in agriculture to protect plants not only against plant diseases but also against insect pests.

Eficàcia d'un biopreparat de microorganismes nadius de muntanya sobre el nematode Meloidogyne javanica en cultiu de tomàquet

Es va determinar l’efecte nematicida d’un biopreparat fet a partir de material de mantell de bosc autòcton, tècnica coneguda com a Activació de Microorganismes de Muntanya, sobre una població de Meloidogyne javanica, en cultiu de tomàquet cv. Durinta, en condicions de semicamp i sota hivernacle. Es van realitzar 5 aplicacions de biopreparat actiu (MMactivat) i el mateix biopreparat esterilitzat en autoclau (MMautoclavat) en un volum d’1L de sorra de riera autoclavada a raó de 8,4 mL per planta. La primera aplicació es va fer just desprès de trasplantar el conreu i una setmana abans d’inocular 1 J2·cm-3 de sòl, i els quatre tractaments restants amb cadència setmanal. Paral·lelament, es va determinar les unitats formadores de colònia de fongs i bacteris del biopreparat actiu i esterilitzat, així com la presència de coliformes fecals. Al final de l’assaig, desprès que el nematode completés una generació, es va avaluar el pes sec aeri i el pes fresc de les arrels, el nombre d’ous per planta, i la freqüència d’aïllament de fongs endòfits d’arrel i tija de tomaquera. El pH del biopreparat era de 3,7. Les mostres contenien nivells superiors a 107 UFC ml-1 tant de fongs com de bactèries. No es va detectar la presència de coliformes totals obtenint un valor < 3 NMP/mL amb p≤0,05. No es van aïllar colònies de bacteris termodúrics ni de Pseudomonas aureginosa ni P. fluorescens que poguessin actuar com a organismes de control biològic. El biopreparat no va mostrar cap efecte sobre el desenvolupament aeri i radicular de la planta. La producció d’ous en les plantes tractades amb el biopreparat actiu i esterilitzat va ser un 63% i un 67% menor que en el testimoni sense tractar. Es van detectar fongs endòfits en arrel i tija de tomaquera en la majoria de les mostres sense atendre a cap tractament, inclòs el control sense tractar.

Embryonic gene expression of Coregonus palaea (whitefish) under pathogen stress as analyzed by high-throughput RNA-sequencing.

Most fishes produce free-living embryos that are exposed to environmental stressors immediately following fertilization, including pathogenic microorganisms. Initial immune protection of embryos involves the chorion, as a protective barrier, and maternally-allocated antimicrobial compounds. At later developmental stages, host-genetic effects influence susceptibility and tolerance, suggesting a direct interaction between embryo genes and pathogens. So far, only a few host genes could be identified that correlate with embryonic survival under pathogen stress in salmonids. Here, we utilized high-throughput RNA-sequencing in order to describe the transcriptional response of a non-model fish, the Alpine whitefish Coregonus palaea, to infection, both in terms of host genes that are likely manipulated by the pathogen, and those involved in an early putative immune response. Embryos were produced in vitro, raised individually, and exposed at the late-eyed stage to a virulent strain of the opportunistic fish pathogen Pseudomonas fluorescens. The pseudomonad increased embryonic mortality and affected gene expression substantially. For example, essential, upregulated metabolic pathways in embryos under pathogen stress included ion binding pathways, aminoacyl-tRNA-biosynthesis, and the production of arginine and proline, most probably mediated by the pathogen for its proliferation. Most prominently downregulated transcripts comprised the biosynthesis of unsaturated fatty acids, the citrate cycle, and various isoforms of b-cell transcription factors. These factors have been shown to play a significant role in host blood cell differentiation and renewal. With regard to specific immune functions, differentially expressed transcripts mapped to the complement cascade, MHC class I and II, TNF-alpha, and T-cell differentiation proteins. The results of this study reveal insights into how P. fluorescens impairs the development of whitefish embryos and set a foundation for future studies investigating host pathogen interactions in fish embryos.

Controle biológico da queima-das-bainhas em arroz pela microbiolização de sementes com bactérias antagonistas

Objetivou-se avaliar o efeito de oito isolados bacterianos pré-selecionados de Pseudomonas synxatha, P. fluorescens, Bacillus subtilis, Bacillus sp. e Stenotrophomonas malthophilia no controle da queima-das-bainhas do arroz, causada por Rhizoctonia solani. Sementes da cultivar El Passo L144 foram imersas em suspensão (A540=0,5) de cada um dos isolados e agitadas por 30 min a 10ºC. Sementes imersas somente em solução salina e em salina mais fungicida (Carboxin + Thiran) foram utilizadas como testemunhas. Foram semeadas 10 sementes por vaso, em quatro repetições, dispostas em delineamento completamente casualizado. Foram realizados três ensaios, sendo que no primeiro foi possível selecionar três isolados como promissores, com reduções na severidade da doença atingindo 50, 33,3 e 16,7 %, respectivamente. Estes isolados foram utilizados nos ensaios posteriores, instalados em casa de vegetação e conduzidos até o ponto de colheita, onde foi possível observar o efeito biocontrolador propiciado, principalmente, pelo isolado de P. fluorescens DFs223, com reduções significativas na severidade da doença chegando a 88 e 91,7% no segundo e terceiro ensaios respectivamente. Em ambos os ensaios, houve incremento tanto do número de panículas quanto do número de perfilhos e da massa seca de raízes em até 42,8, 81,2 e 113% respectivamente, nas plantas tratadas com o isolado de P. fluorescens DFs223.

Chemical sanitizers to control biofilms formed by two Pseudomonas species on stainless steel surface

The biofilm formation of Pseudomonas aeruginosa and Pseudomonas fluorescens on AISI 304 stainless steel in the presence of reconstituted skim milk under different temperatures was conducted, and the potential of three chemical sanitizers in removing the mono-species biofilms formed was compared. Pseudomonas aeruginosa cultivated in skim milk at 28 °C presented better growth rate (10.4 log CFU.mL-1) when compared with 3.7 and 4.2 log CFU.mL-1 for P. aeruginosa and P. fluorescens cultivated at 7 °C, respectively. Pseudomonas aeruginosa formed biofilm when cultivated at 28 °C. However, only the adhesion of P. aeruginosa and P. fluorescens was observed when incubated at 7 °C. The sodium dichloroisocyanurate was the most efficient sanitizer in the reduction of the adhered P. aeruginosa cells at 7 and 28 °C and those on the biofilm, respectively. The hydrogen peroxide was more effective in the reduction of adhered cells of P. fluorescens at 7 °C.

Influência da microbiolização de sementes de feijão sobre a transmissão de Colletrichum Lindemuthianum (Saac e Magn)

Isolados de Pseudomonas veronii (DFs513), Bacillus spp. (DFs093 e DFs348), Bacillus cereus (DFs769), Rodhococcus fascians (DFs843 e DFs912) e Pseudomonas fluorescens (DFs831 e DFs842), selecionados para o controle de Xanthomonas axonopdis pv. phaseoli, bem como a combinação de alguns destes isolados bacterianos, foram avaliados quanto a possível influência sobre a transmissão de Colletotrichum lindemuthianum para plântulas de feijão, a partir de sementes naturalmente infectadas e/ou infestadas. Sementes de dois lotes foram microbiolizadas com suspensões dos biocontroladores, sendo que no primeiro ensaio, em rolo de papel, realizou-se a semeadura com oito repetições de 25 sementes, incubadas a 20 ± 2°C. Os percentuais de germinação das sementes e incidência do patógeno foram avaliados. Em um segundo ensaio avaliou-se a transmissão do patógeno para a planta em ensaio conduzido em substrato esterilizado, incubadas por 10 dias. Realizaram-se contagens diárias das plântulas emergidas, incidência do patógeno, massa seca das folhas e raízes. Para a antibiose in vitro, um isolado de C. lindemuthianum foi confrontado com os isolados biocontroladores. Todos os tratamentos proporcionam aumentos de massa foliar e radicular, tanto considerando o número total quanto por planta. Os isolados, DFs831, DFs842, DFs843 e DFs912 produzem antibióticos em testes in vitro. Todos os tratamentos possibilitaram a redução da transmissão de C. lindemuthianum para plantas em pelo menos um dos ensaios. Porém, o isolado DFs912 (Rhodococcus fascians) destaca-se, por apresentar em todos os experimentos reduções da transmissão que variam de 40 a 67%.

Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease

Quantifying diffusion in biofilms : from model hydrogels to living biofilms

Les biofilms sont des communautés de microorganismes incorporés dans une matrice exo-polymérique complexe. Ils sont reconnus pour jouer un rôle important comme barrière de diffusion dans les systèmes environnementaux et la santé humaine, donnant lieu à une résistance accrue aux antibiotiques et aux désinfectants. Comme le transfert de masse dans un biofilm est principalement dû à la diffusion moléculaire, il est primordial de comprendre les principaux paramètres influençant les flux de diffusion. Dans ce travail, nous avons étudié un biofilm de Pseudomonas fluorescens et deux hydrogels modèles (agarose et alginate) pour lesquels l’autodiffusion (mouvement Brownien) et les coefficients de diffusion mutuels ont été quantifiés. La spectroscopie par corrélation de fluorescence a été utilisée pour mesurer les coefficients d'autodiffusion dans une volume confocal de ca. 1 m3 dans les gels ou les biofilms, tandis que les mesures de diffusion mutuelle ont été faites par cellule de diffusion. En outre, la voltamétrie sur microélectrode a été utilisée pour évaluer le potentiel de Donnan des gels afin de déterminer son impact sur la diffusion. Pour l'hydrogel d'agarose, les observations combinées d'une diminution du coefficient d’autodiffusion et de l’augmentation de la diffusion mutuelle pour une force ionique décroissante ont été attribuées au potentiel de Donnan du gel. Des mesures de l'effet Donnan (différence de -30 mV entre des forces ioniques de 10-4 et 10-1 M) et l'accumulation correspondante d’ions dans l'hydrogel (augmentation d’un facteur de 13 par rapport à la solution) ont indiqué que les interactions électrostatiques peuvent fortement influencer le flux de diffusion de cations, même dans un hydrogel faiblement chargé tel que l'agarose. Curieusement, pour un gel plus chargé comme l'alginate de calcium, la variation de la force ionique et du pH n'a donné lieu qu'à de légères variations de la diffusion de sondes chargées dans l'hydrogel. Ces résultats suggèrent qu’en influençant la diffusion du soluté, l'effet direct des cations sur la structure du gel (compression et/ou gonflement induits) était beaucoup plus efficace que l'effet Donnan. De même, pour un biofilm bactérien, les coefficients d'autodiffusion étaient pratiquement constants sur toute une gamme de force ionique (10-4-10-1 M), aussi bien pour des petits solutés chargés négativement ou positivement (le rapport du coefficient d’autodiffusion dans biofilm sur celui dans la solution, Db/Dw ≈ 85 %) que pour des nanoparticules (Db/Dw≈ 50 %), suggérant que l'effet d'obstruction des biofilms l’emporte sur l'effet de charge. Les résultats de cette étude ont montré que parmi les divers facteurs majeurs qui affectent la diffusion dans un biofilm environnemental oligotrophe (exclusion stérique, interactions électrostatiques et hydrophobes), les effets d'obstruction semblent être les plus importants lorsque l'on tente de comprendre la diffusion du soluté. Alors que les effets de charge ne semblaient pas être importants pour l'autodiffusion de substrats chargés dans l'hydrogel d'alginate ou dans le biofilm bactérien, ils ont joué un rôle clé dans la compréhension de la diffusion à travers l’agarose. L’ensemble de ces résultats devraient être très utiles pour l'évaluation de la biodisponibilité des contaminants traces et des nanoparticules dans l'environnement.