RESUMO

The search for new drug-producing microorganisms is one of the most promising situations in current world scientific scenarios. The use of molecular biology as well as the cloning of protein and compound genes is already well established as the gold standard method of increasing productivity. Aiming at this increase in productivity, this work aims at the cloning, purification and in silico analysis of l-asparaginase from Fusarium proliferatum in Komagataella phaffii (Pichia pastoris) protein expression systems. The l-asparaginase gene (NCBI OQ439985) has been cloned into Pichia pastoris strains. Enzyme production was analyzed via the quantification of aspartic B-hydroxamate, followed by purification on a DEAE FF ion exchange column. The in silico analysis was proposed based on the combined use of various technological tools. The enzymatic activity found intracellularly was 2.84 IU/g. A purification factor of 1.18 was observed. The in silico analysis revealed the position of five important amino acid residues for enzymatic activity, and likewise, it was possible to predict a monomeric structure with a C-score of 1.59. The production of the enzyme l-asparaginase from F. proliferatum in P. pastoris was demonstrated in this work, being of great importance for the analysis of new methodologies in search of the production of important drugs in therapy.

RESUMO

Plant-associated fungi are considered a vast source for biotechnological processes whose potential has been poorly explored. The interactions and diversity of sugarcane, one of the most important crops in Brazil, have been rarely studied, mainly concerning fungal communities and their interactions with transgenic plants. Taking this into consideration, the purpose of this study was, based on culture dependent strategy, to determine the structure and diversity of the fungal community (root endophytes and rhizosphere) associated with two varieties of sugarcane, a non-genetically modified (SP80-1842) variety and its genetically modified counterpart (IMI-1, expressing imazapyr herbicide resistance). For this, the sugarcane varieties were evaluated in three sampling times (3, 10 and 17 months after planting) under two crop management (weeding and herbicide treatments). In addition, a strain of Trichoderma virens, an endophyte isolated from sugarcane with great potential as a biological control, growth promotion and enzyme production agent, was selected for the fungal-plant interaction assays. The results of the isolation, characterization and evaluation of fungal community changes showed that the sugarcane fungal community is composed of at least 35 different genera, mostly in the phylum Ascomycota. Many genera are observed at very low frequencies among a few most abundant genera, some of which were isolated from specific plant sites (e.g., the roots or the rhizosphere). An assessment of the possible effects upon the fungal community showed that the plant growth stage was the only factor that significantly affected the community's structure. Moreover, if transgenic effects are present, they may be minor compared to other natural sources of variation. The results of interaction studies using the Green fluorescent protein (GFP)-expressing T. virens strain T.v.223 revealed that this fungus did not promote any phenotypic changes in the host plant and was found mostly in the roots where it formed a dense mycelial cover and was able to penetrate the intercellular spaces of the root epidermis upper layers. The ability of T. virens to colonize plant roots suggests a potential for protecting plant health, inhibiting pathogens or inducing systemic resistance.

Assuntos

Endófitos/fisiologia , Saccharum/microbiologia , Trichoderma/fisiologia , Biodiversidade , Produção Agrícola , DNA Fúngico/genética , Resistência a Herbicidas/genética , Filogenia , Raízes de Plantas/microbiologia , Raízes de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/microbiologia , Plantas Geneticamente Modificadas/fisiologia , Rizosfera , Saccharum/genética , Saccharum/fisiologia , Análise de Sequência de DNA , Simbiose/genética , Simbiose/fisiologia , Trichoderma/genética

RESUMO

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 µM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

Assuntos

Agrobacterium tumefaciens/genética , Ascomicetos/genética , Citrus/microbiologia , Técnicas de Transferência de Genes , Mutagênese Insercional/métodos , Doenças das Plantas/microbiologia , Transformação Genética , Agrobacterium tumefaciens/fisiologia , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Micélio/genética , Micélio/crescimento & desenvolvimento , Micélio/metabolismo

RESUMO

BACKGROUND: Sugarcane is one of the most important crops in Brazil, mainly because of its use in biofuel production. Recent studies have sought to determine the role of sugarcane endophytic microbial diversity in microorganism-plant interactions, and their biotechnological potential. Epicoccum nigrum is an important sugarcane endophytic fungus that has been associated with the biological control of phytopathogens, and the production of secondary metabolites. In spite of several studies carried out to define the better conditions to use E. nigrum in different crops, little is known about the establishment of an endophytic interaction, and its potential effects on plant physiology. METHODOLOGY/PRINCIPAL FINDINGS: We report an approach based on inoculation followed by re-isolation, molecular monitoring, microscopic analysis, plant growth responses to fungal colonization, and antimicrobial activity tests to study the basic aspects of the E. nigrum endophytic interaction with sugarcane, and the effects of colonization on plant physiology. The results indicate that E. nigrum was capable of increasing the root system biomass and producing compounds that inhibit the in vitro growth of sugarcane pathogens Fusarium verticillioides, Colletotrichum falcatum, Ceratocystis paradoxa, and Xanthomomas albilineans. In addition, E. nigrum preferentially colonizes the sugarcane surface and, occasionally, the endophytic environment. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates that E. nigrum has great potential for sugarcane crop application because it is capable of increasing the root system biomass and controlling pathogens. The study of the basic aspects of the interaction of E. nigrum with sugarcane demonstrated the facultative endophytism of E. nigrum and its preference for the phylloplane environment, which should be considered in future studies of biocontrol using this species. In addition, this work contributes to the knowledge of the interaction of this ubiquitous endophyte with the host plant, and also to a better use of microbial endophytes in agriculture.

Assuntos

Antifúngicos/metabolismo , Ascomicetos/metabolismo , Endófitos/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Saccharum/crescimento & desenvolvimento , Saccharum/microbiologia , Acetatos , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Ascomicetos/crescimento & desenvolvimento , Ascomicetos/isolamento & purificação , Biomassa , Contagem de Colônia Microbiana , Endófitos/efeitos dos fármacos , Endófitos/crescimento & desenvolvimento , Endófitos/isolamento & purificação , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Epiderme Vegetal/efeitos dos fármacos , Epiderme Vegetal/microbiologia , Epiderme Vegetal/ultraestrutura , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Folhas de Planta/ultraestrutura , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/microbiologia , Saccharum/efeitos dos fármacos , Esporos Fúngicos/ultraestrutura

RESUMO

BACKGROUND: Epicoccum nigrum Link (syn. E. purpurascens Ehrenb. ex Schlecht) is a saprophytic ascomycete distributed worldwide which colonizes a myriad of substrates. This fungus has been known as a biological control agent for plant pathogens and produces a variety of secondary metabolites with important biological activities as well as biotechnological application. E. nigrum produces darkly pigmented muriform conidia on short conidiophores on sporodochia and is a genotypically and phenotypically highly variable species. Since different isolates identified as E. nigrum have been evaluated as biological control agents and used for biocompound production, it is highly desirable that this species name refers to only one lineage. However, according to morphological and genetic variation, E. nigrum present two genotypes that may comprise more than one species. METHODOLOGY/PRINCIPAL FINDINGS: We report the application of combined molecular (ITS and ß-tubulin gene sequence analysis, PCR-RFLP and AFLP techniques), morphometric, physiological, genetic compatibility and recombination analysis to study the taxonomic relationships within an endophytic population that has been identified as E. nigrum. This combined analysis established two genotypes showing morphological, physiological and genetic divergence as well as genetic incompatibility characterized by colony inhibition, strongly indicating that these genotypes correspond to different species. Genotype 1 corresponds to E. nigrum while genotype 2 represents a new species, referred to in this study as Epicoccum sp. CONCLUSIONS/SIGNIFICANCE: This research contributes to the knowledge of the Epicoccum genus and asserts that the classification of E. nigrum as a single variable species should be reassessed. In fact, based on the polyphasic approach we suggest the occurrence of cryptic species within E. nigrum and also that many of the sequences deposited as E. nigrum in GenBank and culture collection of microbial strains should be reclassified, including the reference strain CBS 161.73 sequenced in this work. In addition, this study provides valuable tools for differentiation of Epicoccum species.

Assuntos

Ascomicetos/classificação , Ascomicetos/genética , Variação Genética , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Ascomicetos/enzimologia , Ascomicetos/crescimento & desenvolvimento , Sequência de Bases , Teorema de Bayes , Contagem de Colônia Microbiana , Impressões Digitais de DNA , DNA Intergênico/genética , Proteínas Fúngicas/metabolismo , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Micélio/metabolismo , Filogenia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Recombinação Genética/genética , Especificidade da Espécie , Esporos Fúngicos/citologia , Tubulina (Proteína)/genética

RESUMO

Recently many transposable elements have been identified and characterized in filamentous fungi, especially in species of agricultural, biotechnological and medical interest. Similar to the elements found in other eukaryotes, fungal transposons can be classified as class I elements (retrotransposons) that use RNA and reverse transcriptase and class II elements (DNA transposons) that use DNA. The changes (transposition and recombination) caused by transposons can supply wide-ranging genetic variation, especially for species that do not have a sexual phase. The application of transposable elements to gene isolation and population analysis is an important tool for molecular biology and studies of fungal evolution

Assuntos

Animais , DNA Fúngico , Fungos/genética , Genoma Fúngico/genética , Elementos de DNA Transponíveis