Biodiversidade e bioprospecção de actinomicetos da planta Vochysia divergens (Cambará)

Resum

Gênero Microbispora : reclassificação filogenética, bioprospecção e identificação de metabólitos secundários

Orientadora : Profª Drª Chirlei GlienkeCo-orientadora : Drª Josiane Gomes Figueiredo e Dr. Jurgen RohrTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 27/08/2015Inclui referênciasÁrea de concentraçãoResumo: A ioprospecção de microrganismos hoje é a fonte mais promissora para obtenção de novos compostos bioativos. Actinomicetos do solo pertencentes ao gênero Streptomyces, produzem uma vasta gama de compostos amplamente utilizados pela industría farmacêutica e agronômica. Porém, devido ao grande número de pesquisas envolvendo estes microrganismos a probablidade da descoberta de novos compostos a partir de linhagens isoladas do solo torna-se cada vez menos provável, levando a exploração de actinomicetos de outras fontes naturais, como por exemplo, isolados de plantas medicinais. Neste contexto, o presente trabalho objetivou o isolamento, bioprospecção, e a identificação de actinomicetos endofíticos da planta medicinal Vochysia divergens. O trabalho foi dividido em 3 capítulos voltados a bioprospecção e avaliação da atividade biológica dos isolados endofíticos - principalmente pertencentes ao gênero Microbispora - seguido de identificação dos compostos produzidos pela linhagem mais promissora e posterior estudo filogenético dos isolados pertencentes ao gênero Microbispora. O primeiro capítulo, intitulado "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens", explorou o isolamento e biodiversidade de actinomicetos isolados da planta Vochysia divergens, bem como a capacidade de seis isolados em produzir compostos com atividade antibacteriana, antioxidante e antitumoral. O segundo capítulo, "Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?- Carbolines and Indoles with Biological Activity" objetivou o isolamento e identificação de compostos produzidos pela linhagem de Microbispora sp. LGMB259. No trabalho foram identificados sete compostos, quatro ?-carbolinas e três indóis, sendo que o composto 1-vinil- ?-carbolina-3-ácido carboxílico foi responsável pela atividade biológica desta linhagem, o qual apresentou atividade antibacteriana, antifúngica e citotóxica. O último capítulo, intitulado "Multilocus Sequence Analysis of the Genus Microbispora" baseou-se em análise multigênica utilizando os genes 16S rRNA, 23S rRNA, gyrB e rpoB para o estudo filogenético dentro do gênero Microbispora. As análises realizadas sugerem que as espécies M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica e M. thermorosea são espécies distintas da espécie M. rosea; diferentemente do proposto na literatura por Miyadoh et al. (1990). M. aerata e M. thermodiastica compartilharam elevada similaridade genética e provavelmente pertencem a mesma espécie, assim como M. indica e M. rosea. As linhagens endofíticas pertencentes aos clusters Microbispora sp. 1, Microbispora sp. 2 e Microbispora sp. 3 são diferentes das espécies de Microbispora previamente descritas, sendo que mais estudos são necessários para a descrição das mesmas como novas espécies. Foi proposto também a análise concatenada dos genes gyrB-rpoB como uma alternativa à técnica de hibridização de DNA para a identificação de espécies dentro do gênero Microbispora, e o valor de 98,0% como o valor de homologia mínimo para classificar os isolados de Microbispora como pertencentes a mesma espécie. Palavras-chave: Vochysia divergens - Gênero Microbispora - metabólitos secundários - ?- carbolinas - reclassificação filogenéticaAbstract: The Bioprospecting of Microorganisms is the most promising source for new bioactive compounds. Actinomycetes - mainly that belonging to the genus Streptomyces - isolated from soil produced a wide range of bioactive compounds widely used by the pharmaceutical and agronomic industries. However, due to the large number of research involving these microorganisms the probablidade of discovery novel compounds isolated from soil strains is increasingly less, leading to exploration of actinomycetes isolated from others natural sources such as, endophytic isolated by medicial plants. In this context, our objectives were the isolation, bioprospecting, and the identification of endophytic actinomycetes from medicinal plant Vochysia divergens. The work was divided into three chapters and includes bioprospecting and avaliation of biological activity of endophytic isolates - mainly belonging to the genus Microbispora - followed by identification of compounds produced by the most promising strain and subsequent phylogenetic study of isolates belonging to Microbispora genus. The first chapter, entitled "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens" is about the isolation and biodiversity of actinomycetes from the medicinal plant Vochysia divergens, and the ability of six isolates to produce compounds with antibacterial, antioxidant and antitumor activities. The second chapter, "Microbispora sp. LGMB259 Endophytic actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?-Carbolines and Indoles with Biological Activity" aimed to isolate and identify compounds produced by the strain Microbispora sp. LGMB259. We identified seven compounds produced by this strain - four ?-carbolines and three indoles- wherein the compound 1-vinyl-?-carboline-3-carboxylic acid was responsible for the biological activities of this strain, which showed antibacterial, antifungal and cytotoxic activities. The last chapter, entitled "Multilocus Sequence Analysis of the Genus Microbispora" was based on multigene analysis using the 16S rRNA, 23S rRNA, gyrB and rpoB for phylogenetic studies in the Microbispora genus. The analyzes suggest that the species M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica and M. thermorosea are distinct from M. rosea; different by proposed for Miyadoh et al. (1990). However M. aerata and M. thermodiastatica probably are the same species, as well M. indica and M. rosea. Endophytic isolates belongs to the clusters Microbispora sp. 1, Microbispora sp. 2 and Microbispora sp. 3 are different from the Microbispora species previous described and futures studies are required to describe these species. It is also proposed the concatenated analyses of gyrB-rpoB genes as a useful alternative to DNA-DNA hybridization for the identification and phylogenetic analysis in the Microbispora genus, and values less than 98,0% to characterizate and determine relationship at species level. Key-words: Vochysia divergens - Microbispora Genus - secondary metabolites - ?- carbolines - phylogenetic reclassificatio

\u3cem\u3ePhaeophleospora vochysiae\u3c/em\u3e Savi & Glienke sp. nov. Isolated from \u3cem\u3eVochysia divergens\u3c/em\u3e Found in the Pantanal, Brazil, Produces Bioactive Secondary Metabolites

Microorganisms associated with plants are highly diverse and can produce a large number of secondary metabolites, with antimicrobial, anti-parasitic and cytotoxic activities. We are particularly interested in exploring endophytes from medicinal plants found in the Pantanal, a unique and widely unexplored wetland in Brazil. In a bio-prospecting study, strains LGMF1213 and LGMF1215 were isolated as endophytes from Vochysia divergens, and by morphological and molecular phylogenetic analyses were characterized as Phaeophleospora vochysiae sp. nov. The chemical assessment of this species reveals three major compounds with high biological activity, cercoscosporin (1), isocercosporin (2) and the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone (3). Besides the isolation of P. vochysiae as endophyte, the production of cercosporin compounds suggest that under specific conditions this species causes leaf spots, and may turn into a pathogen, since leaf spots are commonly caused by species of Cercospora that produce related compounds. In addition, the new compound 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-methylcyclohex-2-enone showed considerable antimicrobial activity and low cytotoxicity, which needs further exploration

Analysis of the genetic diversity of Candida isolates obtained from diabetic patients and kidney transplant recipients

Yeasts of the genu

Antibacterial Activity of Endophytic Actinomycetes Isolated from the Medicinal Plant \u3cem\u3eVochysia divergens\u3c/em\u3e (Pantanal, Brazil)

Endophytic actinomycetes from medicinal plants produce a wide diversity of secondary metabolites (SM). However, to date, the knowledge about endophytes from Brazil remains scarce. Thus, we analyzed the antimicrobial potential of 10 actinomycetes isolated from the medicinal plant Vochysia divergens located in the Pantanal sul-mato-grossense, an unexplored wetland in Brazil. Strains were classified as belonging to the Aeromicrobium, Actinomadura, Microbacterium, Microbispora, Micrococcus, Sphaerisporangium, Streptomyces, and Williamsia genera, through morphological and 16S rRNA phylogenetic analyzes. A susceptibility analysis demonstrated that the strains were largely resistant to the antibiotics oxacillin and nalidixic acid. Additionally, different culture media (SG and R5A), and temperatures (28 and 36°C) were evaluated to select the best culture conditions to produce the active SM. All conditions were analyzed for active metabolites, and the best antibacterial activity was observed from metabolites produced with SG medium at 36°C. The LGMB491 (close related to Aeromicrobium ponti) extract showed the highest activity against methicillin-resistant Staphylococcus aureus (MRSA), with a MIC of 0.04 mg/mL, and it was selected for SM identification. Strain LGMB491 produced 1-acetyl-β-carboline (1), indole-3-carbaldehyde (2), 3-(hydroxyacetyl)-indole (4), brevianamide F (5), and cyclo-(L-Pro-L-Phe) (6) as major compounds with antibacterial activity. In this study, we add to the knowledge about the endophytic community from the medicinal plant V. divergens and report the isolation of rare actinomycetes that produce highly active metabolites

Endophytes of Brazilian Medicinal Plants with Activity against Phytopathogens

Plant diseases caused by phytopathogens are responsible for significant crop losses worldwide. Resistance induction and biological control have been exploited in agriculture due to their enormous potential. In this study, we investigated the antimicrobial potential of endophytic fungi of leaves and petioles of medicinal plants Vochysia divergens and Stryphnodendron adstringens located in two regions of high diversity in Brazil, Pantanal, and Cerrado, respectively. We recovered 1,304 fungal isolates and based on the characteristics of the culture, were assigned to 159 phenotypes. One isolate was selected as representative of each phenotype and studied for antimicrobial activity against phytopathogens. Isolates with better biological activities were identified based on DNA sequences and phylogenetic analyzes. Among the 159 representative isolates, extracts from 12 endophytes that inhibited the mycelial growth (IG) of Colletotrichum abscissum (≥40%) were selected to expand the antimicrobial analysis. The minimum inhibitory concentrations (MIC) of the extracts were determined against citrus pathogens, C. abscissum, Phyllosticta citricarpa and Xanthomonas citri subsp. citri and the maize pathogen Fusarium graminearum. The highest activity against C. abscissum were from extracts of Pseudofusicoccum stromaticum CMRP4328 (IG: 83% and MIC: 40 μg/mL) and Diaporthe vochysiae CMRP4322 (IG: 75% and MIC: 1 μg/mL), both extracts also inhibited the development of post-bloom fruit drop symptoms in citrus flowers. The extracts were promising in inhibiting the mycelial growth of P. citricarpa and reducing the production of pycnidia in citrus leaves. Among the isolates that showed activity, the genus Diaporthe was the most common, including the new species D. cerradensis described in this study. In addition, high performance liquid chromatography, UV detection, and mass spectrometry and thin layer chromatography analyzes of extracts produced by endophytes that showed high activity, indicated D. vochysiae CMRP4322 and P. stromaticum CMRP4328 as promising strains that produce new bioactive natural products. We report here the capacity of endophytic fungi of medicinal plants to produce secondary metabolites with biological activities against phytopathogenic fungi and bacteria. The description of the new species D. cerradensis, reinforces the ability of medicinal plants found in Brazil to host a diverse group of fungi with biotechnological potential

Gênero Microbispora : reclassificação filogenética, bioprospecção e identificação de metabólitos secundários

Orientadora : Profª Drª Chirlei GlienkeCo-orientadora : Drª Josiane Gomes Figueiredo e Dr. Jurgen RohrTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciencias Biológicas (Microbiologia, Parasitologia e Patologia Básica). Defesa: Curitiba, 27/08/2015Inclui referênciasÁrea de concentraçãoResumo: A ioprospecção de microrganismos hoje é a fonte mais promissora para obtenção de novos compostos bioativos. Actinomicetos do solo pertencentes ao gênero Streptomyces, produzem uma vasta gama de compostos amplamente utilizados pela industría farmacêutica e agronômica. Porém, devido ao grande número de pesquisas envolvendo estes microrganismos a probablidade da descoberta de novos compostos a partir de linhagens isoladas do solo torna-se cada vez menos provável, levando a exploração de actinomicetos de outras fontes naturais, como por exemplo, isolados de plantas medicinais. Neste contexto, o presente trabalho objetivou o isolamento, bioprospecção, e a identificação de actinomicetos endofíticos da planta medicinal Vochysia divergens. O trabalho foi dividido em 3 capítulos voltados a bioprospecção e avaliação da atividade biológica dos isolados endofíticos - principalmente pertencentes ao gênero Microbispora - seguido de identificação dos compostos produzidos pela linhagem mais promissora e posterior estudo filogenético dos isolados pertencentes ao gênero Microbispora. O primeiro capítulo, intitulado "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens", explorou o isolamento e biodiversidade de actinomicetos isolados da planta Vochysia divergens, bem como a capacidade de seis isolados em produzir compostos com atividade antibacteriana, antioxidante e antitumoral. O segundo capítulo, "Microbispora sp. LGMB259 Endophytic Actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?- Carbolines and Indoles with Biological Activity" objetivou o isolamento e identificação de compostos produzidos pela linhagem de Microbispora sp. LGMB259. No trabalho foram identificados sete compostos, quatro ?-carbolinas e três indóis, sendo que o composto 1-vinil- ?-carbolina-3-ácido carboxílico foi responsável pela atividade biológica desta linhagem, o qual apresentou atividade antibacteriana, antifúngica e citotóxica. O último capítulo, intitulado "Multilocus Sequence Analysis of the Genus Microbispora" baseou-se em análise multigênica utilizando os genes 16S rRNA, 23S rRNA, gyrB e rpoB para o estudo filogenético dentro do gênero Microbispora. As análises realizadas sugerem que as espécies M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica e M. thermorosea são espécies distintas da espécie M. rosea; diferentemente do proposto na literatura por Miyadoh et al. (1990). M. aerata e M. thermodiastica compartilharam elevada similaridade genética e provavelmente pertencem a mesma espécie, assim como M. indica e M. rosea. As linhagens endofíticas pertencentes aos clusters Microbispora sp. 1, Microbispora sp. 2 e Microbispora sp. 3 são diferentes das espécies de Microbispora previamente descritas, sendo que mais estudos são necessários para a descrição das mesmas como novas espécies. Foi proposto também a análise concatenada dos genes gyrB-rpoB como uma alternativa à técnica de hibridização de DNA para a identificação de espécies dentro do gênero Microbispora, e o valor de 98,0% como o valor de homologia mínimo para classificar os isolados de Microbispora como pertencentes a mesma espécie. Palavras-chave: Vochysia divergens - Gênero Microbispora - metabólitos secundários - ?- carbolinas - reclassificação filogenéticaAbstract: The Bioprospecting of Microorganisms is the most promising source for new bioactive compounds. Actinomycetes - mainly that belonging to the genus Streptomyces - isolated from soil produced a wide range of bioactive compounds widely used by the pharmaceutical and agronomic industries. However, due to the large number of research involving these microorganisms the probablidade of discovery novel compounds isolated from soil strains is increasingly less, leading to exploration of actinomycetes isolated from others natural sources such as, endophytic isolated by medicial plants. In this context, our objectives were the isolation, bioprospecting, and the identification of endophytic actinomycetes from medicinal plant Vochysia divergens. The work was divided into three chapters and includes bioprospecting and avaliation of biological activity of endophytic isolates - mainly belonging to the genus Microbispora - followed by identification of compounds produced by the most promising strain and subsequent phylogenetic study of isolates belonging to Microbispora genus. The first chapter, entitled "Antitumor, antioxidant and antibacterial activities of secondary metabolites extracted by endophytic actinomycetes isolated from Vochysia divergens" is about the isolation and biodiversity of actinomycetes from the medicinal plant Vochysia divergens, and the ability of six isolates to produce compounds with antibacterial, antioxidant and antitumor activities. The second chapter, "Microbispora sp. LGMB259 Endophytic actinomycete Isolated from Vochysia divergens (Pantanal, Brazil) Producing ?-Carbolines and Indoles with Biological Activity" aimed to isolate and identify compounds produced by the strain Microbispora sp. LGMB259. We identified seven compounds produced by this strain - four ?-carbolines and three indoles- wherein the compound 1-vinyl-?-carboline-3-carboxylic acid was responsible for the biological activities of this strain, which showed antibacterial, antifungal and cytotoxic activities. The last chapter, entitled "Multilocus Sequence Analysis of the Genus Microbispora" was based on multigene analysis using the 16S rRNA, 23S rRNA, gyrB and rpoB for phylogenetic studies in the Microbispora genus. The analyzes suggest that the species M. amethystogenes, M. chromogenes, M. karnatakensis, M. parva, M. aerata, M. thermodiastatica and M. thermorosea are distinct from M. rosea; different by proposed for Miyadoh et al. (1990). However M. aerata and M. thermodiastatica probably are the same species, as well M. indica and M. rosea. Endophytic isolates belongs to the clusters Microbispora sp. 1, Microbispora sp. 2 and Microbispora sp. 3 are different from the Microbispora species previous described and futures studies are required to describe these species. It is also proposed the concatenated analyses of gyrB-rpoB genes as a useful alternative to DNA-DNA hybridization for the identification and phylogenetic analysis in the Microbispora genus, and values less than 98,0% to characterizate and determine relationship at species level. Key-words: Vochysia divergens - Microbispora Genus - secondary metabolites - ?- carbolines - phylogenetic reclassificatio

Bioprospecting of elite plant growth-promoting bacteria for the maize crop

The use of plant growth-promoting bacteria (PGPB), which aims to replace chemical fertilizers and biological control, is a goal for achieving agriculture sustainability. In this scenario, our goal was to identify and evaluate the potential of bacteria isolated from maize roots to promote plant growth and be used as inoculants. We evaluated 173 bacterial strains isolated from the maize (Zea mays L.) rhizosphere for the properties of their PGPB in vitro. Twelve strains were positive for siderophores, indole acetic acid (IAA) production, biological nitrogen fixation (BNF), and phosphate solubilization. Sequence analysis of 16S rRNA identified these strains as belonging to the genera Cellulosimicrobium, Stenotrophomonas, Enterobacter, and Bacillus. The elite strains were evaluated under greenhouse conditions upon the inoculation of two maize hybrids, ATL100 and KWX628. The ability of the isolates to promote plant growth was dependent on the maize genotype; Enterobacter sp. LGMB208 showed the best ability to promote growth of hybrid ATL100, while Enterobacter sp. strains LGMB125, LGMB225, and LGMB274 and Cellulosimicrobium sp. strain LGMB239 showed the best ability to promote growth of hybrid KWX628. The results highlight the potential of bacterial genera little explored as maize PGPB but indicate the need to investigate their interactions with different plant genotypes