Characterisation of filamentous fungal diversity of Trichoderma genus isolated from marine sponge by sequencing ITS-rDNA and MALDI-TOF ICMS

[Excerpt] Filamentous fungi are ubiquitous organisms and can easily be isolated from a wide range of environment. Some efforts have been made to investigate microbial communities in marine sponges of eukaryotes using both approaches of conventional taxonomy (morphology) and molecular taxonomy (Passarini et al. 2011). The classification of some filamentous fungi taxa can be difficult to achieve based on these methods. Currently, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Intact Cell Mass Spectrometry (MALDI-TOF ICMS) has been widely used to identify and characterise a large number of taxa of filamentous fungi (Santos et al. 2010). The aim of this study was to investigate the diversity of 33 Trichoderma isolated from the southern Atlantic Ocean marine sponge Dragmacidon reticulata using a polyphasic approach

Identification of Penicillia species isolated from southern Atlantic Ocean marine sponge using a polyphasic approach including MALDI-TOF ICMS

In the last years the research concerning marine microorganisms has increased in number. However, the knowledge about the phylogenetic and diversity of this fungal community is still scarce. The reliable identification of filamentous fungi typically implies the analyses of a variety of morphological, biochemical and molecular traits. Recently, Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Intact Cell Mass Spectrometry (MALDl-TOF ICMS) has been used to generate spectra of protein masses in a range from 2000 to 20000 Da that are a taxa specific fingerprinting. This technique has already shown high potentialities to discriminate very dosely related taxa and, it has been used as a new tool in the polyphasic approach to identify this kind of microorganisms. The aim of the present study was investigate the diversity of 19 penicillia isolated from the southern Atlantic Ocean marine sponge Dragmacidon reticulate using a polyphasic approach. The first approach was the sequence of ITS-rDNA gene region and to compare with sequence data available at NCBl-GenBank using BLAST N· The putative penicillia species were further investigated using the phenotypic techniques: macro- and micromorphologies and MALDl-TOF ICMS. From 19 isolates 9 ribotypes were obtained from the sequencing analysis. One ribotype after phenotypic analysis is a clearly new species and the remain ribotypes have their identification confirmed by micro- and macro-morphological and MALDl-TOF ICMS analyses. In conclusions, the MALDl-TOF ICMS is a sound technique for fungal identification and the new species is now under study to be formally described

Bioprospecção de Composto Citotóxico derivado de Extratos Fúngicos do Gênero Aspergillus

Dissertação de mestrado apresentada ao Programa de Pós-Graduaçãoem Biociências, do Instituto Latino-Americano de Ciências da Vida e da Natureza, da Universidade Federal da Integração Latino-Americana, como requisito parcial à obtenção do título de Mestre em Ciências, área de concentração Biociências.A corrida para descoberta de fontes alternativas e mais sustentáveis para obtenção de antitumorais, como o ativo paclitaxel, é uma necessidade da indústria farmacêutica. Embora muitos esforços estejam sendo feitos nesse sentido, ainda não houve a disponibilização de fontes alternativas que não estejam relacionadas às plantas, as quais correspondem às atuais produtoras do composto paclitaxel. Estudos demonstram que fungos endofíticos do gênero Aspergillus são capazes de sintetizar o composto citotóxico paclitaxel invitro. Considerando que as descobertas recentes sugerem que o paclitaxel é um produto da existência e expressão de genes microbianos e que sua biossíntese não depende da planta hospedeira, podendo assim ser realizada por microrganismos não endofíticos, novos horizontes podem ser explorados. Desse modo, o presente projeto teve como objetivo principal realizar a bioprospecção deste composto citotóxico/antitumoral em fungos filamentosos do gênero Aspergillus, não endofíticos, isolados de efluentes industriais têxteis. Três fungos do gênero Aspergillus spp. (ITF-021; ITF-027; ITF-030) foram estudados. Todos os isolados apresentaram significativa atividade citotóxica na proliferação celular in vitro de células tumorais de pulmão H460. Extratos fúngicos dos isolados foram submetidos à análise por HPLC, bem como por HPLC-MS/MS. Foi possível demonstrar que os isolados fúngicos não sintetizaram o composto paclitaxel, entretanto foram capazes de sintetizar outra substância desconhecida com atividade citotóxica. Os resultados do presente estudo demonstram a necessidade de mais pesquisas para se compreender as vias da biossíntese do paclitaxel em fungos não endofíticos, para que assim seja possível afirmar que princípio ativo é um composto de origem microbiana e não de origem vegetal ou fruto da simbiose existente entre microrganismos e vegetais. Novos estudos podem ser realizados para caracterizar o composto ativo com propriedades citotóxicas contra células tumorais H460, o qual foi sintetizado pelos isolados, abrindo portas para a descoberta de um possível novo fármaco potencialmente aplicável na área médica ou de uma nova via de obtenção de outro composto antitumoral conhecido.Unila e Prati-Donaduzz

Improved extraction method to evaluate the degradation of selected PAHs by marine fungi grown in fermentative medium

An improved method to evaluate the degradation of polycyclic aromatic hydrocarbons (PAHs) for filamentous fungi is hereby proposed. The PAH adsorption through fungal mycelia implies the use of an exhaustive extraction, which is usually performed through a combination of different techniques applied separately to the liquid medium and to the mycelia. This work proposes a simplified extraction procedure with reduced analysis time and sample manipulation, thereby guaranteeing its precision and accuracy. The selected PAHs, pyrene and benzo[a]pyrene, were quantified by gas chromatography using a mass selective detector operating in single ion monitoring mode. The method was validated presenting accuracy within the scope of this study and it was used for evaluating the potential of PAHs degradation by thirteen fungi derived from marine environment. This study selected the fungus Aspergillus sclerotiorum CBMAI 849 for being able to degrade 85% of pyrene and 61% of benzo[a]pyrene after 4 and 8 days, respectively, and the fungus Mucor racemosus CBMAI 847 for being able to degrade 44% of benzo[a]pyrene after 8 daysO estudo propõe um método otimizado para avaliar a degradação de hidrocarbonetos policíclicos aromáticos (HPAs) por fungos filamentosos. A adsorção dos HPAs pelo micélio requer o emprego de uma extração exaustiva, normalmente realizada através da combinação de diferentes técnicas aplicadas, separadamente, ao meio líquido e ao micélio. Este trabalho sugere uma simplificação da etapa de extração, reduzindo o tempo de análise e a manipulação da amostra e garantindo a precisão e a exatidão do método. Os HPAs selecionados, pireno e benzo[a]pireno, foram quantificados por cromatografia gasosa usando detector seletivo de massas operando no modo de monitoramento seletivo de íons. O método foi validado apresentando exatidão no âmbito estudado e utilizado para a avaliação do potencial de degradação de HPAs por treze fungos derivados de ambiente marinho. O estudo selecionou o fungo Aspergillus sclerotiorum CBMAI 849 pela capacidade de degradação de 85% de pireno e 61% de benzo[a]pireno após 4 e 8 dias, respectivamente, e o fungo Mucor racemosus CBMAI 847 pela capacidade de degradação de 44% de benzo[a]pireno após 8 dias564570Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Marine-derived Filamentous Fungi And Their Potential Application For Polycyclic Aromatic Hydrocarbon Bioremediation.

Eight marine-derived fungi that were previously selected for their abilities to decolorize RBBR dye were subjected to pyrene and benzo[a]pyrene degradation. The fungus Aspergillus sclerotiorum CBMAI 849 showed the best performance with regard to pyrene (99.7%) and benzo[a]pyrene (76.6%) depletion after 8 and 16 days, respectively. Substantial amounts of benzo[a]pyrene (>50.0%) depletion were also achieved by Mucor racemosus CBMAI 847. Therefore, these two fungal strains were subjected to metabolism evaluation using the HPLC-DAD-MS technique. The results showed that A. sclerotiorum CBMAI 849 and M. racemosus CBMAI 847 were able to metabolize pyrene to the corresponding pyrenylsulfate and were able to metabolize benzo[a]pyrene to benzo[a]pyrenylsulfate, suggesting that the mechanism of hydroxylation is mediated by a cytochrome P-450 monooxygenase, followed by conjugation with sulfate ions. Because these fungi were adapted to the marine environment, the strains that were used in the present study are considered to be attractive targets for the bioremediation of saline environments, such as ocean and marine sediments that are contaminated by PAHs.62364-7

Molecular diversity of fungal and bacterial communities in the marine sponge Dragmacidon reticulatum

The present work aimed to investigate the diversity of bacteria and filamentous fungi of southern Atlantic Ocean marine sponge Dragmacidon reticulatum using cultivation-independent approaches. Fungal ITS rDNA and 18S gene analyses (DGGE and direct sequencing approaches) showed the presence of representatives of three order (Polyporales, Malasseziales, and Agaricales) from the phylum Basidiomycota and seven orders belonging to the phylum Ascomycota (Arthoniales, Capnodiales, Dothideales, Eurotiales, Hypocreales, Pleosporales, and Saccharomycetales). On the other hand, bacterial 16S rDNA gene analyses by direct sequencing approach revealed the presence of representatives of seven bacterial phyla (Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes, Lentisphaerae, Chloroflexi, and Planctomycetes). Results from statistical analyses (rarefaction curves) suggested that the sampled clones covered the fungal diversity in the sponge samples studied, while for the bacterial community additional sampling would be necessary for saturation. This is the first report related to the molecular analyses of fungal and bacterial communities by cultivation-independent approaches in the marine sponges D. reticulatum. Additionally, the present work broadening the knowledge of microbial diversity associated to marine sponges and reports innovative data on the presence of some fungal genera in marine samples.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Fungal communities in the garden chamber soils of leaf‐cutting ants

Leaf‐cutting ants modify the properties of the soil adjacent to their nests. Here, we examined whether such an ant‐altered environment impacts the belowground fungal communities. Fungal diversity and community structure of soil from the fungus garden chambers of Atta sexdens rubropilosa and Atta bisphaerica, two widespread leaf‐cutting ants in Brazil, were determined and compared with non‐nest soils. Culture‐dependent methods revealed similar species richness but different community compositions between both types of soils. Penicillium janthinellum and Trichoderma spirale were the prevalent isolates in fungus chamber soils and non‐nest soils, respectively. In contrast to cultivation methods, analyses of clone libraries based on the internal transcribed spacer (ITS) region indicated that richness of operational taxonomic units significantly differed between soils of the fungus chamber and non‐nest soils. FastUnifrac analyses based on ITS sequences further revealed a clear distinction in the community structure between both types of soils. Plectania milleri and an uncultured Clavariaceae fungus were prevalent in fungus chamber soils and non‐nest soils, respectively. FastUnifrac analyses also revealed that fungal community structures of soil from the garden chambers markedly differed among ant species. Our findings suggest that leaf‐cutting ants affect fungal communities in the soil from the fungus chamber in comparison to non‐nest soils541111861196FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

Molecular Analysis Of Microbial Diversity In Corrosion Samples From Energy Transmission Towers.

Microbial diversity in corrosion samples from energy transmission towers was investigated using molecular methods. Ribosomal DNA fragments were used to assemble gene libraries. Sequence analysis indicated 10 bacterial genera within the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. In the two libraries generated from corroded screw-derived samples, the genus Acinetobacter was the most abundant. Acinetobacter and Clostridium spp. dominated, with similar percentages, in the libraries derived from corrosion scrapings. Fungal clones were affiliated with 14 genera belonging to the phyla Ascomycota and Basidiomycota; of these, Capnobotryella and Fellomyces were the most abundant fungi observed. Several of the microorganisms had not previously been associated with biofilms and corrosion, reinforcing the need to use molecular techniques to achieve a more comprehensive assessment of microbial diversity in environmental samples.27435-4