Resposta de plantas micropropagadas de abacaxizeiro à inoculação de bactérias diazotróficas em casa de vegetação

O objetivo deste trabalho foi selecionar e avaliar bactérias diazotróficas isoladas de abacaxizeiro (Ananas comosus (L.) Merril) no desenvolvimento de cultivares micropropagadas da mesma espécie em casa de vegetação. Plantas da cultivar Perolera foram submetidas à inoculação com Asaia bogorensis (AB219) e cultivadas em tubetes, durante 145 dias, com as misturas: casca de arroz carbonizada, folha de carnaubeira triturada e vermicomposto; casca de arroz carbonizada, pó da casca do coco maduro e vermicomposto; casca de arroz carbonizada, vermiculita e vermicomposto. Plantas da cultivar Primavera receberam inóculos com o AB219 e bactérias relacionadas a Burkholderia cepacia (AB202 e AB213), enquanto plantas das cultivares Pérola e Smooth Cayenne receberam AB219 e AB213, sendo cultivadas, por 140 dias, em tubetes com a mistura de vermicomposto e vermiculita. A colonização dos abacaxizeiros pelas bactérias diazotróficas foi confirmada. As plantas da cultivar Perolera cresceram melhor em casca de arroz carbonizada, vermiculita e vermicomposto e responderam positivamente ao AB219. Já as plantas da cultivar Primavera não apresentaram resposta significativa à inoculação com AB219, AB202 e AB213. Houve incremento de 23,1% a 38,5% na matéria seca de raízes das plantas da cultivar Pérola na presença de AB213 e AB219, respectivamente. A presença de AB213 incrementou em 15,2% a matéria seca da parte aérea das plantas da cultivar Smooth Cayenne. Os resultados revelam a eficiência de bactérias diazotróficas na promoção do crescimento de abacaxizeiros.

Cross talk between 2,4-diacetylphloroglucinol-producing biocontrol pseudomonads on wheat roots.

The performance of Pseudomonas biocontrol agents may be improved by applying mixtures of strains which are complementary in their capacity to suppress plant diseases. Here, we have chosen the combination of Pseudomonas fluorescens CHA0 with another well-characterized biocontrol agent, P. fluorescens Q2-87, as a model to study how these strains affect each other's expression of a biocontrol trait. In both strains, production of the antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a crucial factor contributing to the suppression of root diseases. DAPG acts as a signaling compound inducing the expression of its own biosynthetic genes. Experimental setups were developed to investigate whether, when combining strains CHA0 and Q2-87, DAPG excreted by one strain may influence expression of DAPG-biosynthetic genes in the other strain in vitro and on the roots of wheat. DAPG production was monitored by observing the expression of lacZ fused to the biosynthetic gene phlA of the respective strain. Dual-culture assays in which the two strains were grown in liquid medium physically separated by a membrane revealed that Q2-87 but not its DAPG-negative mutant Q2-87::Tn5-1 strongly induced phlA expression in a DeltaphlA mutant of strain CHA0. In the same way, phlA expression in a Q2-87 background was induced by DAPG produced by CHA0. When coinoculated onto the roots of wheat seedlings grown under gnotobiotic conditions, strains Q2-87 and CHA0, but not their respective DAPG-negative mutants, were able to enhance phlA expression in each other. In summary, we have established that two nonrelated pseudomonads may stimulate each other in the expression of an antimicrobial compound important for biocontrol. This interpopulation communication occurs in the rhizosphere, i.e., at the site of pathogen inhibition, and is mediated by the antimicrobial compound itself acting as a signal exchanged between the two pseudomonads.

Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.

Caracterização da comunidade bacteriana endofítica de citros por isolamento, PCR específico e DGGE

O objetivo deste trabalho foi caracterizar a comunidade bacteriana endofítica de plantas assintomáticas (escapes) e afetadas pela clorose variegada dos citros (CVC) por meio de isolamento em meio de cultura, técnica de gradiente desnaturante em gel de eletroforese (DGGE) e detecção de Methylobacterium mesophilicum e Xyllela fastidiosa por meio de PCR específico, para estudar esta comunidade e sua relação com a ocorrência da CVC. A análise da comunidade bacteriana via DGGE permitiu a detecção de X. fastidiosa, bem como Klebsiella sp. e Acinetobacter sp. como endófitos de citros. Foram observados também Curtobacterium sp., Pseudomonas sp., Enterobacter sp. e Bacillus spp. Utilizando primers específicos, Methylobacterium mesophilicum e X. fastidiosa também foram observadas, reforçando hipóteses de que estas bactérias podem estar interagindo no interior da planta hospedeira.

A quorum sensing regulated small volatile molecule reduces acute virulence and promotes chronic infection phenotypes.

A significant number of environmental microorganisms can cause serious, even fatal, acute and chronic infections in humans. The severity and outcome of each type of infection depends on the expression of specific bacterial phenotypes controlled by complex regulatory networks that sense and respond to the host environment. Although bacterial signals that contribute to a successful acute infection have been identified in a number of pathogens, the signals that mediate the onset and establishment of chronic infections have yet to be discovered. We identified a volatile, low molecular weight molecule, 2-amino acetophenone (2-AA), produced by the opportunistic human pathogen Pseudomonas aeruginosa that reduces bacterial virulence in vivo in flies and in an acute mouse infection model. 2-AA modulates the activity of the virulence regulator MvfR (multiple virulence factor regulator) via a negative feedback loop and it promotes the emergence of P. aeruginosa phenotypes that likely promote chronic lung infections, including accumulation of lasR mutants, long-term survival at stationary phase, and persistence in a Drosophila infection model. We report for the first time the existence of a quorum sensing (QS) regulated volatile molecule that induces bistability phenotype by stochastically silencing acute virulence functions in P. aeruginosa. We propose that 2-AA mediates changes in a subpopulation of cells that facilitate the exploitation of dynamic host environments and promote gene expression changes that favor chronic infections.

Induction of systemic resistance in tomato by the autochthonous phylloplane resident Bacillus cereus

The objective of this work was to verify if the induced resistance mechanism is responsible for the capacity of a phylloplane resident bacteria (Bacillus cereus), isolated from healthy tomato plants, to control several diseases of this crop. A strain of Pseudomonas syringae pv. tomato was used as the challenging pathogen. The absence of direct antibiosis of the antagonist against the pathogen, the significant increase in peroxidases activity in tomato plants exposed to the antagonist and then inoculated with the challenging pathogen, as well as the character of the protection, are evidences wich suggest that biocontrol efficiency presented by the antagonist in previous works might be due to induced systemic resistance (ISR).

Regulation of antibiotic production in root-colonizing Peudomonas spp. and relevance for biological control of plant disease.

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

Quorum-sensing systems in staphylococci as therapeutic targets.

The staphylococci are an ever-present threat in our world, capable of causing a wide range of infections, and are a persistent presence in the clinical environment. As the number of antimicrobial compounds effective against staphylococci decreases, because of the acquisition and spread of antibiotic resistance, there is a growing need for novel therapeutic molecules. Intra and inter-species communication (quorum sensing) is a biologically significant phenomenon that has been associated with virulence, intracellular survival, and biofilm formation. Quorum sensing molecules of staphylococci and other species (e.g. Pseudomonas aeruginosa) can inhibit virulence factor production and/or growth of staphylococci, leading to the possibility that interference with staphylococcal quorum-sensing systems could be a way of controlling the diverse infections caused by the staphylococci. In this article, we discuss the potential of quorum-sensing systems of staphylococci as therapeutic targets.

Plant- and microbe-derived compounds affect the expression of genes encoding antifungal compounds in a pseudomonad with biocontrol activity.

We have investigated the impacts of 63 different low-molecular-weight compounds, most of them plant derived, on the in vitro expression of two antifungal biosynthetic genes by the plant-protecting rhizobacterium Pseudomonas fluorescens CHA0. The majority of the compounds tested affected the expression of one or both antifungal genes. This suggests that biocontrol activity in plant-beneficial pseudomonads is modulated by plant-bacterium signaling.

Low immunoglobulin levels in patients with cystic fibrosis: reduced inflammation or sign of common variable immunodeficiency syndrome?

Introduction: In children with cystic fibrosis (CF), low immunoglobulin (IgG) levels have been reported to be associated with significantly less severe lung disease. However, decreased IgG can be a sign for common variable immunodeficiency (CVID) and affect clinical outcome. The aim of this study was to analyze clinical and serological data of patients having low IgG levels in routine blood tests at annual assessment, particularly their antibody response to polysaccharide antigens. Method: Retrospective chart review of demographic data of CF patients followed at the pediatric CF clinic throughout 2009. Clinical parameters (genotype, pancreas sufficiency, FEV1), presence of Pseudomonas aeruginosa (PA) and number of exacerbations per year were correlated with immunoglobulin and vaccination antibodies levels (antibodies to pneumococcal serotypes 14, 19, 23, 1, 5 and 7F measured by enzyme-linked immune-sorbent assay). Results: 4 out of 60 patients (6.7%) had lower IgG-levels for age. Ages ranged from 1 year 8 months to 11 years, 2 boys, 2 girls. Three patients were delF508 homozygotes, one heterozygote composite delF508/G542X. All were pancreatic insufficient. FEV1 ranged from 74 to 108%. One patient never had colonization by PA, 2 had intermittent PA colonization and one was chronically infected. After conjugated vaccination all patients had protective antibodies against serotypes 14, 19, 23F. For serotypes not included in the vaccine, only one patient had protective titers for 1 out of 3 serotypes. None of the patients had received unconjugated pneumococcal vaccine. There was no significant clinical difference in FEV1, PA colonization or number of exacerbations according to IgG and vaccination antibody levels. Conclusion: Cystic Fibrosis patients with low immunoglobulin levels have normal antibody response to protein antigens. However, despite recurrent infections, there seems to be delayed or deficient antibody response to polysaccharide antigens. Prospective studies are needed to evaluate the development of polysaccharide antibody responses in CF-patients to monitor for CVID. With early detection of CF by newborn screening program, long term follow up could be started early in childhood.

Promoção de crescimento e controle de tombamento de plântulas de pepino por rizobactérias

O objetivo deste trabalho foi avaliar o efeito de rizobactérias, no crescimento de plântulas de pepino e no controle de tombamento, causado por Pythium aphanidermatum. Foram realizados em laboratório ensaios de: degradação de 1-aminociclopropano-1-carboxilato (ACC); colonização das raízes de plântulas de pepino; e pareamento de culturas. A identificação dos melhores isolados foi feita pela determinação das seqüências do gene 16S rDNA. Trinta e sete isolados, dos 165 testados, aumentaram a massa de matéria seca das plantas de pepino em até 63%. Desses, somente um isolado (N13 - Pseudomonas fluorescens) reduziu o tombamento de plântulas em 25%; 21 isolados inibiram o crescimento micelial de P. aphanidermatum, colonizaram o sistema radicular das plantas de pepino e cresceram em presença de ACC como única fonte de nitrogênio. Dos dez isolados que apresentaram resultados satisfatórios, cinco foram identificados como pertencentes aos gêneros Bacillus, quatro Pseudomonas e um Stenotrophomonas. Dos 165 isolados de rizobactérias testados, sete possuem potencial para promover o crescimento de plantas de pepino e um para controlar o tombamento causado por P. aphanidermatum.

Utilização de bactérias ácido-lácticas isoladas do trato intestinal de tilápia-do-nilo como probiótico

O objetivo deste trabalho foi isolar bactérias ácido-lácticas do intestino de tilápias-do-nilo, e avaliar seu potencial probiótico. Foram isoladas cepas de bactérias ácido-lácticas, e foi avaliada a inibição aos patógenos in vitro. As cepas com os melhores resultados foram identificadas e utilizadas no experimento de colonização do trato intestinal de tilápias-do-nilo, via suplementação na dieta, em delineamento inteiramente ao acaso, com três tratamentos e quatro repetições. Foram avaliados: o total de bactérias, as bactérias ácido-lácticas, Vibrio ssp. e Pseudomonas ssp. A cepa com melhor resultado foi utilizada na infecção experimental, em delineamento inteiramente ao acaso, em esquema fatorial 2x3: dieta suplementada com a cepa e dieta-controle; e os peixes não submetidos à injeção, peixes submetidos à injeção de solução salina e à injeção de Enterococcus durans, com três repetições. Foram avaliados os parâmetros hematológicos. As duas cepas identificadas foram: Lactobacillus plantarum e Lactobacillus brevis, que colonizaram o trato intestinal de tilápias, contudo L. plantarum teve menor número total de bactérias e de Pseudomonas ssp. Foi observado maior número total de eritrócitos, trombócitos, leucócitos, linfócitos, neutrófilos e monócitos, em peixes alimentados com L. plantarum e submetidos à injeção de E. durans. O L. plantarum tem efeito probiótico e melhora o sistema imune das tilápias.

Manipulation of salicylate content in Arabidopsis thaliana by the expression of an engineered bacterial salicylate synthase.

Salicylic acid (SA) plays a central role as a signalling molecule involved in plant defense against microbial attack. Genetic manipulation of SA biosynthesis may therefore help to generate plants that are more disease-resistant. By fusing the two bacterial genes pchA and pchB from Pseudomonas aeruginosa, which encode isochorismate synthase and isochorismate pyruvate-lyase, respectively, we have engineered a novel hybrid enzyme with salicylate synthase (SAS) activity. The pchB-A fusion was expressed in Arabidopsis thaliana under the control of the constitutive cauliflower mosaic virus (CaMV) 35S promoter, with targeting of the gene product either to the cytosol (c-SAS plants) or to the chloroplast (p-SAS plants). In p-SAS plants, the amount of free and conjugated SA was increased more than 20-fold above wild type (WT) level, indicating that SAS is functional in Arabidopsis. P-SAS plants showed a strongly dwarfed phenotype and produced very few seeds. Dwarfism could be caused by the high SA levels per se or, perhaps more likely, by a depletion of the chorismate or isochorismate pools of the chloroplast. Targeting of SAS to the cytosol caused a slight increase in free SA and a significant threefold increase in conjugated SA, probably reflecting limited chorismate availability in this compartment. Although this modest increase in total SA content did not strongly induce the resistance marker PR-1, it resulted nevertheless in enhanced disease resistance towards a virulent isolate of Peronospora parasitica. Increased resistance of c-SAS lines was paralleled with reduced seed production. Taken together, these results illustrate that SAS is a potent tool for the manipulation of SA levels in plants.