Chromosomal polymorphism of the Ceratocystis fimbriata species complex in Brazil.

Ceratocystis fimbriata is an important pathogen that causes wilt in several plant species. Despite the importance of this pathogen, knowledge about its karyotypic polymorphism and genomic architecture is limited. The main objective of this study was to investigate the karyotype of isolates of the C. fimbriata species complex from different host plants and geographical origins in Brazil. First, the identity of the isolates was confirmed conducting multilocus sequence analysis (MLSA) phylogeny using ß-tubulin (TUBB), translation elongation factor 1α (TEF-1α) and mating-type (MAT1 and MAT2) gene sequences. To investigate the chromosomal polymorphism, two conditions of pulsed-field gel electrophoresis (PFGE) were used and the karyotypes of the isolates obtained. The retrotransposon-microsatellite amplified polymorphism (REMAP) molecular marker was utilized to assess the genetic variability among isolates. In the MLSA utilizing the concatenated gene sequences, Ceratocystis cacaofunesta and C. fimbriata formed separate clades, but considerable variation among C. fimbriata isolates was observed. Polymorphism in chromosome number and size was found, indicating the existence of genomic differences among isolates and occurrence of chromosomal rearrangements in the species complex. The number of chromosomes varied from seven to nine and the estimated minimum chromosome sizes were estimated to be between 2.7 and 6.0 Mbp. Small polymorphic chromosomes ranging from 1.2 to 1.8 Mbp were observed in all isolates, raising the hypothesis that they could be supernumerary chromosomes. REMAP analysis revealed a high genetic variability and that isolates from the same host tend to group together in a same cluster. Our results bring new insights into the chromosomal diversity and genome organization of the C. fimbriata complex.

New Lager Brewery Strains Obtained by Crossing Techniques Using Cachaça (Brazilian Spirit) Yeasts.

The development of hybrids has been an effective approach to generate novel yeast strains with optimal technological profile for use in beer production. This study describes the generation of a new yeast strain for lager beer production by direct mating between two Saccharomyces cerevisiae strains isolated from cachaça distilleries: one that was strongly flocculent, and the other with higher production of acetate esters. The first step in this procedure was to analyze the sporulation ability and reproductive cycle of strains belonging to a specific collection of yeasts isolated from cachaça fermentation vats. Most strains showed high rates of sporulation, spore viability, and homothallic behavior. In order to obtain new yeast strains with desirable properties useful for lager beer production, we compare haploid-to-haploid and diploid-to-diploid mating procedures. Moreover, an assessment of parental phenotype traits showed that the segregant diploid C2-1d generated from a diploid-to-diploid mating experiment showed good fermentation performance at low temperature, high flocculation capacity, and desirable production of acetate esters that was significantly better than that of one type lager strain. Therefore, strain C2-1d might be an important candidate for the production of lager beer, with distinct fruit traces and originating using a non-genetically modified organism (GMO) approach.IMPORTANCE Recent work has suggested the utilization of hybridization techniques for the generation of novel non-genetically modified brewing yeast strains with combined properties not commonly found in a unique yeast strain. We have observed remarkable traits, especially low temperature tolerance, maltotriose utilization, flocculation ability, and production of volatile aroma compounds, among a collection of Saccharomyces cerevisiae strains isolated from cachaça distilleries, which allow their utilization in the production of beer. The significance of our research is in the use of breeding/hybridization techniques to generate yeast strains that would be appropriate for producing new lager beers by exploring the capacity of cachaça yeast strains to flocculate and to ferment maltose at low temperature, with the concomitant production of flavoring compounds.

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.

Isolation and regeneration of Penicillium brevicompactum protoplasts - DOI: 10.4025/actascibiolsci.v26i4.1529 / Isolamento e regeneração de protoplastos de Penicillium brevicompactum - DOI: 10.4025/actascibiolsci.v26i4.1529

The isolation and regeneration of fungal protoplasts is a key step for the establishment of transformation systems, electrophoretic karyotype analysis and fusion between strains, all techniques of broad application on improvement programs of filamentous fungi. To establish conditions for the isolation and regeneration of ,em>Penicillium brevicompactum protoplasts, that is an excellent pectinase producer, different lytic enzymes and osmotic stabilizers were tested. P. brevicompactum protoplasts were obtained at a larger scale when their mycelium was digested with 15mg.mL-1 of Glucanex (Novo Nordisk) and 0.8 mol.L1 NaCl as osmotic stabilizer. The best osmotic stabilizer for the regeneration of P. brevicompactum protoplasts was 0.8 mol.L1 KCl, with a regeneration frequency of 36.58%. This protocol can be applied in genetic analysis of this Penicillium species which, to date has been poorly characterized

O isolamento e regeneração de protoplastos de fungos é um passo fundamental para o estabelecimento de sistemas de transformação, análise do cariótipo molecular e fusão entre linhagens, que são técnicas de ampla aplicação em programas de melhoramento para fungos filamentosos. Neste trabalho foram testados diferentes preparações enzimáticas e estabilizadores osmóticos para estabelecer condições otimizadas de isolamento e regeneração de protoplastos de Penicillium brevicompactum, que é um excelente produtor de pectinases. Protoplastos de P. brevicompactum foram obtidos em maior quantidade quando o micélio foi digerido com 15mg.mL-1 de Glucanex (Novo Nordisk) em NaCl 0,8 mol.L-1 como estabilizador osmótico. O melhor estabilizador osmótico para a regeneração dos protoplastos foi KCl 0.8 mol.L1 apresentando uma freqüência de regeneração de 36,58%. Esse protocolo pode ser utilizado em análises genéticas para essa espécie de Penicillium cujos estudos têm sido pouco reportados