Microbiologia
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11840
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Resultados da Pesquisa
Item Production and regeneration of protoplasts from orchid Mycorrhizal Fungi Epulorhiza repens and Ceratorhiza sp.(Brazilian Archives of Biology and Technology, 2010-01) Coelho, Irene da Silva; Queiroz, Marisa Vieira de; Costa, Maurício Dutra; Kasuya, Maria Catarina Megumi; Araújo, Elza Fernandes deThe aim of this work was to study the standardization of conditions to obtain and regenerate Epulorhiza repens and Ceratorhiza sp. protoplasts. For E. repens, the largest number of protoplasts (8.0 × 106 protoplasts/mL) was obtained in 0.6 M KCl, using 15 mg/mL of Lysing Enzymes, and 2-day-old fungal mycelium. When 0.5 M sucrose was used as osmotic stabilizer, the highest frequency of regeneration was achieved (8.5 %); 80.0 % of protoplasts were nucleated, and 20.0 % anucleated. For Ceratorhiza sp., the largest number of protoplasts (4.0 × 107 protoplasts/mL) was achieved in 0.6 M NaCl, when 15 mg/mL of Lysing Enzymes and 15mg/mL of Glucanex, with 2-day-old fungal mycelium were used. The highest frequency of regeneration was 6.7 % using 0.5 M sucrose as osmotic stabilizer; 88.8 % of protoplasts were nucleated, and 11.2 % anucleated.Item Pectin lyase production by recombinant Penicillium griseoroseum strain 105(Canadian Journal of Microbiology, 2010) Teixeira, Janaina Aparecida; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes de; Cardoso, Patrícia GomesRecombinant Penicillium griseoroseum strain 105 overproduces an extracellular pectin lyase (PL) under the transcriptional control of the strong gpdA promoter of Aspergillus nidulans. Our aim was to evaluate PL production by recombinant P. griseoroseum strain 105 in submerged fermentation system bioreactors BioFloIII and BioFloIV using 2 or 10 L working volumes under different growth conditions and to analyze the production of cellulase, polygalacturonase, pectin methylesterase, and protease. PL overproduction by recombinant P. griseoroseum strain 105 was 112 times higher than that of P. griseoroseum PG63 grown in sugarcane juice. Cellulases and proteases were not detected in the culture filtrate, and evaluation for extracellular proteins in the culture medium by SDS–PAGE showed the presence of a 36 kDa predominant band, similar to the molecular mass estimated from the nucleotide sequence of plg1 gene for PL of P. griseoroseum strain 105. This recombinant strain provides the advantage of PL production, which predominates over other extracellular proteins usually present in most commercial pectinase preparations, using sugarcane juice as a substrate of low cost.Item Development of molecular markers based on retrotransposons for the analysis of genetic variability in Moniliophthora perniciosa(European Journal of Plant Pathology, 2012-11) Santana, Mateus Ferreira; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de; Souza, Jorge Teodoro de; Mizubuti, Eduardo Seiti GomideMoniliophthora perniciosa is a fungus that causes witches’ broom disease (WBD) in the cacao tree (Theobroma cacao). The M. perniciosa genome contains different transposable elements; this prompted an evaluation of the use of its retrotransposons as molecular markers for population studies. The inter-retrotransposon amplified polymorphism (IRAP) and retrotransposon-microsatellite amplified polymorphism (REMAP) techniques were used to study the variability of 70 M. perniciosa isolates from different geographic origins and biotypes. A total of 43 loci was amplified. Cluster analysis of different geographical regions of C biotype revealed two large groups in the state of Bahia, Brazil. Techniques using retrotransposon-based molecular markers showed advantages over previously used molecular techniques for the study of genetic variability in M. perniciosa.Item PacCl, a pH-responsive transcriptional regulator, is essential in the pathogenicity of Colletotrichum lindemuthianum, a causal agent of anthracnose in bean plants(European Journal of Plant Pathology, 2014-08-08) Nogueira, Guilherme Bicalho; Soares, Marcos Antônio; Bazzolli, Denise Mara Soares; Araújo, Elza Fernandes de; Langin, Thierry; Queiroz, Marisa Vieira deIn fungi, the expression of genes encoding proteins related to parasitism is regulated by several factors, including pH. This study reports the structural and functional characterization of the pacCl gene, which encodes the transcription factor PacC of C. lindemuthianum. The pacCl gene showed reduced expression in acidic pH, and its transcription was activated by elevated extracellular pH. The importance of this gene was demonstrated by the development of a pacC1 disruption mutant line of C. lindemuthianum. The mutant line was able to penetrate the host tissue through differentiation of primary hyphae. However, it was not able to cause maceration on the infected plant tissue. The results suggest that PacCl is a regulator of gene activation, and its expression is required for fungal growth in alkaline conditions, as well as for the transcription of genes necessary for the passage from the biotrophic to the necrotrophic phase.Item The minimal regulatory region necessary for the expression of the Penicillium griseoroseum plg1 gene(Annals of Microbiology, 2015-06) Bazzolli, Denise Mara Soares; Reis, Klédna Constância Portes; Teixeira, Janaina Aparecida; Ribon, Andréa Oliveira Barros; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes deThe expression of the Penicillium griseoroseum plg1 gene is induced by citric pectin and repressed by glucose. In this work, the minimal region of the plg1 gene promoter essential for expression in pectin and sucrose plus yeast extract was identified by using constructs containing the gfp ORF under control of the plg1 gene promoter. The fragment A (283 bp) is essential for plg1 expression in sucrose plus yeast extract. Fragment B (309 bp plus 184; core promoter) was critical for expression in pectin and abolished the catabolic repression by glucose. Therefore, the fragment of 776 bp (fragment A and B) is essential for the expression of the plg1 gene in natural inducing conditions (pectin as carbon source) and in sucrose plus yeast extract. The fragment B is a promising minimal promoter usable for heterologous expression in filamentous fungi, since genes that contain it could be activated by the presence of peel from citric fruits (which contains citric pectin) and are not affected by glucose in these agricultural by-products.Item Beginning to understand the role of sugar carriers in Colletotrichum lindemuthianum: the function of the gene mfs1(Journal of Microbiology, 2012-10-12) Pereira, Monalessa Fábia; Santos, Carolina Maria de Araújo dos; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de; Bazzolli, Denise Mara SoaresFungi of the Colletotrichum genus are among the most prominent phytopathogens that cause diseases with a considerable economic impact, such as anthracnose. The hemibiotrophic fungus Colletotrichum lindemuthianum (teleomorph Glomerella cingulata f. sp. phaseoli) is the causal agent of the anthracnose of the common bean; and similarly to other phytopathogens, it uses multiple strategies to gain access to different carbon sources from its host. In this study, we examine mfs1, a newly identified C. lindemuthianum hexose transporter. The mfs1 gene is expressed only during the necrotrophic phase of the fungus’ interaction within the plant and allows it to utilize the available sugars during this phase. The deletion of mfs1 gene resulted in differential growth of the fungus in a medium that contained glucose, mannose or fructose as the only carbon source. This study is the first to describe a hexose transporter in the hemibiotrophic pathogen C. lindemuthianum and to demonstrate the central role of this protein in capturing carbon sources during the necrotrophic development of the plant/pathogen interaction.Item Terminal repeat retrotransposons as DNA markers in fungi(Journal of Basic Microbiology, 2013-02-26) Santana, Mateus Ferreira; Batista, Aline Duarte; Ribeiro, Lílian Emídio; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira deIn this study, we demonstrate that ClIRAP primers designed using the transposable element RetroCl1 sequence from Colletotrichum lindemuthianum can be used to generate an efficient IRAP (inter‐retrotransposon amplified polymorphism) molecular marker to study intra‐ and inter‐species diversity in fungi. It has been previously demonstrated that primers generated from this TRIM‐like element can be used in the Colletotrichum species. We now prove that the RetroCl1 sequence can also be used to analyze diversity in different fungi. IRAP profiles were successfully generated for 27 fungi species from 11 different orders, and intra‐species genetic variability was detected in six species. The ClIRAP primers facilitate the use of the IRAP technique for a variety of fungi without prior knowledge of the genome.Item Boto, a class II transposon in Moniliophthora perniciosa, is the first representative of the PIF/ Harbinger superfamily in a phytopathogenic fungus(Microbiology, 2012-10-24) Pereira, Jorge Fernando; Almeida, Ana Paula Morais Martins; Cota, Júnio; Pamphile, João Alencar; Silva, Gilvan Ferreira da; Araújo, Elza Fernandes de; Gramacho, Karina Peres; Brommonschenkel, Sérgio Hermı́nio; Pereira, Gonçalo Amarante Guimarães; Queiroz, Marisa Vieira deBoto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6–12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.Item Endophytic bacteria isolated from common bean (Phaseolus vulgaris) exhibiting high variability showed antimicrobial activity and quorum sensing inhibition(Current Microbiology, 2015-07-23) Lopes, Ralf Bruno Moura; Vanetti, Maria Cristina Dantas; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira de; Costa, Leonardo Emanuel de OliveiraEndophytic bacteria play a key role in the biocontrol of phytopathogenic microorganisms. In this study, genotypic diversity was analyzed via repetitive element PCR (rep-PCR) of endophytic isolates of the phylum Actinobacteria that were previously collected from leaves of cultivars of common bean (Phaseolus vulgaris). Considerable variability was observed, which has not been reported previously for this phylum of endophytic bacteria of the common bean. Furthermore, the ethanol extracts from cultures of various isolates inhibited the growth of pathogenic bacteria in vitro, especially Gram-positive pathogens. Extracts from cultures of Microbacterium testaceum BAC1065 and BAC1093, which were both isolated from the ‘Talismã’ cultivar, strongly inhibited most of the pathogenic bacteria tested. Bean endophytic bacteria were also demonstrated to have the potential to inhibit the quorum sensing of Gram-negative bacteria. This mechanism may regulate the production of virulence factors in pathogens. The ability to inhibit quorum sensing has also not been reported previously for endophytic microorganisms of P. vulgaris. Furthermore, M. testaceum with capacity to inhibit quorum sensing appears to be widespread in common bean. The genomic profiles of M. testaceum were also analyzed via pulsed-field gel electrophoresis, and greater differentiation was observed using this method than rep-PCR; in general, no groups were formed based on the cultivar of origin. This study showed for the first time that endophytic bacteria from common bean plants exhibit high variability and may be useful for the development of strategies for the biological control of diseases in this important legume plant.Item Use of the IRAP marker to study genetic variability in Pseudocercospora fijiensis populations(Current Microbiology, 2013-11-05) Queiroz, Casley Borges de; Santana, Mateus Ferreira; Silva, Gilvan Ferreira da; Mizubuti, Eduardo Seiti Gomide; Araújo, Elza Fernandes de; Queiroz, Marisa Vieira dePseudocercospora fijiensis is the etiological agent of black Sigatoka, which is currently considered as one of the most destructive banana diseases in all locations where it occurs. It is estimated that a large portion of the P. fijiensis genome consists of transposable elements, which allows researchers to use transposon-based molecular markers in the analysis of genetic variability in populations of this pathogen. In this context, the inter-retrotransposon-amplified polymorphism (IRAP) was used to study the genetic variability in P. fijiensis populations from different hosts and different geographical origins in Brazil. A total of 22 loci were amplified and 77.3 % showed a polymorphism. Cluster analysis revealed two major groups in Brazil. The observed genetic diversity (H E) was 0.22, and through molecular analysis of variance, it was determined that the greatest genetic variability occurs within populations. The discriminant analysis of principal components revealed no structuring related to the geographical origin of culture of the host. The IRAP-based marker system is a suitable tool for the study of genetic variability in P. fijiensis.