Fire blight is a destructive plant disease caused by Erwinia amylovora affecting pome fruit trees, and responsible for large yield declines, long phytosanitary confinements, and high economic losses. In Portugal, the first major fire blight outbreaks occurred in 2010 and 2011, and although later considered eradicated, the emergence of other outbreaks in recent years stressed the need to characterize the E. amylovora populations associated with these outbreaks. In this regard, CRISPR genotyping, assessment of three virulence markers, and semi-quantitative virulence bioassays, were carried out to determine the genotype, and assess the virulence of thirty-six E. amylovora isolates associated with outbreaks occurring between 2010 and 2017 and affecting apple and pear orchards located in the country central-west, known as the main producing region of pome fruits in Portugal. The data gathered reveal that 35 E. amylovora isolates belong to one of the widely-distributed CRISPR genotypes (5-24-38 / D-a-α) regardless the host species, year and region. Ea 680 was the single isolate revealing a new CRISPR genotype due to a novel CR2 spacer located closer to the leader sequence and therefore thought to be recently acquired. Regarding pathogenicity, although dot-blot hybridization assays showed the presence of key virulence factors, namely hrpL (T3SS), hrpN (T3E) and amsG from the amylovoran biosynthesis operon in all E. amylovora isolates studied, pathogenicity bioassays on immature pear slices allowed to distinguish four virulence levels, with most of the isolates revealing an intermediate to severe virulence phenotype. Regardless the clonal population structure of the E. amylovora associated to the outbreaks occurring in Portugal between 2010 and 2017, the different virulence phenotypes, suggests that E. amylovora may have been introduced at different instances into the country. This is the first study regarding E. amylovora in Portugal, and it discloses a novel CRISPR genotype for this bacterium.

Fire blight is a major pome fruit trees disease that is caused by the quarantine phytopathogenic Erwinia amylovora, leading to major losses, namely, in pear and apple productions. Nevertheless, no effective sustainable control treatments and measures have yet been disclosed. In that regard, antimicrobial peptides (AMPs) have been proposed as an alternative biomolecule against pathogens but some of those AMPs have yet to be tested against E. amylovora. In this study, the potential of five AMPs (RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) together with BP100, were assessed to control E. amylovora. Antibiograms, minimal inhibitory, and bactericidal concentrations (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), growth and IC50 were determined and membrane permeabilization capacity was evaluated by flow cytometry analysis and colony-forming units (CFUs) plate counting. For the tested AMPs, the higher inhibitory and bactericidal capacity was observed for RW-BP100 and CA-M (5 and 5–8 µM, respectively for both MIC and MBC), whilst for IC50 RW-BP100 presented higher efficiency (2.8 to 3.5 µM). Growth curves for the first concentrations bellow MIC showed that these AMPs delayed E. amylovora growth. Flow cytometry disclosed faster membrane permeabilization for CA-M. These results highlight the potential of RW-BP100 and CA-M AMPs as sustainable control measures against E. amylovora.

Estratégias alternativas de negação sentencial na região sul do Brasil: aná-lise da influência de fatores pragmáti-cos a partir de dados do projeto VARSUL Alternative sentential negation strategies in the South Region of Brazil: an analysis of the influence of pragmatic factors based on data from the VARSUL Project.Marcos Goldnadel Palavras-chaveDupla negação. ciclo de Jespersen; negação sentencial. AbstractBrazilian Portuguese has three forms of sentential negation seman- Ling., Belo Horizonte, v. 21, n 2, p. 35-74, jul./dez. 2013 37 plausibility of a current hypothesis for the emergence of double negative sentences, some data from Florianópolis were analyzed.

Fire blight is a severe bacterial plant disease that affects important chain-of-value fruit trees such as pear and apple trees. This disease is caused by Erwinia amylovora, a quarantine phytopathogenic bacterium, which, although highly distributed worldwide, still lacks efficient control measures. The green revolution paradigm demands sustainable agriculture practices, for which antimicrobial peptides (AMPs) have recently caught much attention. The goal of this work was to disclose the bioactivity of three peptides mixtures (BP100:RW-BP100, BP100:CA-M, and RW-BP100:CA-M), against three strains of E. amylovora representing distinct genotypes and virulence (LMG 2024, Ea 630 and Ea 680). The three AMPs’ mixtures were assayed at eight different equimolar concentrations ranging from 0.25 to 6 μM (1:1). Results showed MIC and MBC values between 2.5 and 4 μM for every AMP mixture and strain. Regarding cell viability, flow cytometry and alamarBlue reduction, showed high reduction (>25%) of viable cells after 30 min of AMP exposure, depending on the peptide mixture and strain assayed. Hypersensitive response in tobacco plants showed that the most efficient AMPs mixtures and concentrations caused low to no reaction of the plant. Altogether, the AMPs mixtures studied are better treatment solutions to control fire blight disease than the same AMPs applied individually.

Background Fire blight is a destructive disease of pome trees, caused by Erwinia amylovora, leading to high losses of chain-of-values fruits. Major outbreaks were registered between 2010 and 2017 in Portugal, and the first molecular epidemiological characterization of those isolates disclosed a clonal population with different levels of virulence and susceptibility to antimicrobial peptides. Methods This work aimed to further disclose the genetic characterization and unveil the phenotypic diversity of this E. amylovora population, resorting to MLSA, growth kinetics, biochemical characterization, and antibiotic susceptibility. Results While MLSA further confirmed the genetic clonality of those isolates, several phenotypic differences were recorded regarding their growth, carbon sources preferences, and chemical susceptibility to several antibiotics, disclosing a heterogeneous population. Principal component analysis regarding the phenotypic traits allows to separate the strains Ea 630 and Ea 680 from the remaining. Discussion Regardless the genetic clonality of these E. amylovora strains isolated from fire blight outbreaks, the phenotypic characterization evidenced a population diversity beyond the genotype clonality inferred by MLSA and CRISPR, suggesting that distinct sources or environmental adaptations of this pathogen may have occurred. Conclusion Attending the characteristic clonality of E. amylovora species, the data gathered here emphasizes the importance of phenotypic assessment of E. amylovora isolates to better understand their epidemiological behavior, namely by improving source tracking, make risk assessment analysis, and determine strain-specific environmental adaptations, that might ultimately lead to prevent new outbreaks.