Diversidade e bioprospecção de fungos endofíticos de Vochysia divergens e Stryphnodendron adstringens do Pantanal e do Cerrado Sul Matogrossense

Orientadora: Profª Dra. Chirlei GlienkeCoorientadora: Dra. Daiani Cristina SaviDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 28/02/2018Inclui referênciasResumo: Os fungos endofíticos são uma das principais fontes de metabólitos secundários com vasta atividade biológica, particularmente aqueles associados a plantas medicinais. Os biomas Pantanal e Cerrado no Brasil têm alta riqueza associada, principalmente em relação à flora, e muitas das plantas encontradas nesses biomas são usadas pela comunidade local devido a suas propriedades medicinais. Portanto, catalogar a comunidade endofítica de plantas encontradas nessas regiões e explorar o potencial biotecnológico desses microrganismos é de grande importância. Os objetivos do presente trabalho foram analisar a comunidade de fungos endofíticos de duas plantas medicinais encontradas no Pantanal e Cerrado e avaliar seu potencial antimicrobiano. Um total de 1116 fungos endofíticos foram isolados das plantas Vochysia divergens, presente na região do Pantanal, e Stryphnodendron adstringens encontrada no Cerrado, em duas coletas, a primeira realizada em Janeiro e a segunda para o estudo de biodiversidade realizado em Junho de 2016. Os isolados endofíticos foram agrupados em 124 grupos com base em seus morfotipos e cada representante foi identificado por análise da região ITS (Internal Transcribed Spacer). As análises de diversidade foram realizadas com 1079 isolados obtidos na segunda coleta e foram utilizados: riqueza e abundância absoluta, índices de diversidade de Shannon (H '), índice de Simpson (D-1) e índice de uniformidade de Pielou (J). O screening do potencial antimicrobiano foi realizado através de cultura pareada contra fitopatógenos e overlap contra patógenos humanos. Para isolados que apresentaram potencial no screening foi realizada a produção do extrato em dois meios de cultivos, Extrato de Malte e Czapeck, e os metabólitos secundários extraídos foram avaliados novamente contra microrganismos fitopatogênicos e bactérias e levedura de importância clínica. O índice de riqueza foi alto para ambas as plantas, no entanto, a comunidade endofítica não difere entre as plantas, Diaporthe foi o gênero dominante em ambas as plantas. Os testes antimicrobianos revelaram o alto potencial dos isolados Diaporthe sp. LGMF1548 e LGMF1583 e Neofusicoccum brasiliensis LGMF1535, com atividade contra Staphylococcus aureus resistente à meticilina (MRSA), Klebssiella pneumoniae (KPC) e Candida albicans. Contra fitopatógenos o isolado Diaporthe sp. LGMF1631 inibiu 90% do crescimento micelial de Phyllosticta citricarpa e 70% de Colletotrichum abscissum. Esses resultados demonstram a elevada diversidade de endofíticos, filogenia e a atividade antimicrobiana de fungos endofíticos associados às plantas V. divergens e S. adstringens. Nossos dados também sugerem que essas plantas servem como hospedeiras de novas espécies e gêneros, os quais produzem compostos de interesse.Abstract: The endophytic fungi are a main source of interest in metabolites, particularly those associated with medicinal plants. The Pantanal region and Brazilian savannah (Cerrado) in Brazil has high richness associated and many plants used for a local community because of their medicinal properties. Therefore exploring the bioprospecting potential of endophytic microorganisms in this region is important. The goals of the present work were to analyze the community endophytic fungi in medicinal plants of Pantanal and Cerrado, and their antimicrobial potential. A total of 1116 endophytic fungi were isolated from two different medicinal plants, Vochysia divergens in the region of Pantanal and Stryphnodendron adstringens, in the Cerrado and in two collects, the first realized in January and the second for biodiversity study realized in June of 2016. These isolates were grouped in 124 clades based in their morphotypes. The ITS (Internal Transcribed Spacer) sequence was used for species identification. The analyses were realized with one isolated each group. The diversity analyses was performed with 1079 isolates obtained in the second collect, and were used: absolute richness and abundance, indices of diversity of Shannon (H') and Simpson's index (D-1), and Pielou's evenness index (J). Screening for antimicrobial potential was determined by dual culture against phytopathogens and overlap against human pathogens. The production of crude extract were performed in Malt Extract and Czapeck media, derived from endophytic fungal selected in the screening, was tested against pathogenic microorganisms for inhibition of micelial growth for phytopathogens and in inhibition zone for clinical pathogens. In phylogenetic analyses were obtained 28 genera, with possible new genera. The richness index was high for both plants and the most part of the isolates belonging to Diaporthe genus, however, the endophytic community not differ between the plants. The antimicrobial tests showed the isolates Diaporthe sp. LGMF1548 and LGMF1583 and Neofusicoccum brasiliensis LGMF1535 with the best results against methicilineresistent Staphylococcus aureus, Klebssiella pneumoniae (KPC) and Candida albicans, for tests against phytopathogens the isolate Diaporthe sp. LGMF1631 inhibited 90% the mycelial growth of the Phyllosticta citricarpa and 70% of the Colletotrichum abscissum. These results demonstrate the biodiversity, phylogeny, and antimicrobial activity of endophytic fungi associated with the plants V. divergens and S. adstringens, and reveals the potential for discovery news taxa and compounds of interesting from the endophytes in these plant

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

Diversidade e bioprospecção de fungos endofíticos de Vochysia divergens e Stryphnodendron adstringens do Pantanal e do Cerrado Sul Matogrossense

Orientadora: Profª Dra. Chirlei GlienkeCoorientadora: Dra. Daiani Cristina SaviDissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Microbiologia, Parasitologia e Patologia. Defesa : Curitiba, 28/02/2018Inclui referênciasResumo: Os fungos endofíticos são uma das principais fontes de metabólitos secundários com vasta atividade biológica, particularmente aqueles associados a plantas medicinais. Os biomas Pantanal e Cerrado no Brasil têm alta riqueza associada, principalmente em relação à flora, e muitas das plantas encontradas nesses biomas são usadas pela comunidade local devido a suas propriedades medicinais. Portanto, catalogar a comunidade endofítica de plantas encontradas nessas regiões e explorar o potencial biotecnológico desses microrganismos é de grande importância. Os objetivos do presente trabalho foram analisar a comunidade de fungos endofíticos de duas plantas medicinais encontradas no Pantanal e Cerrado e avaliar seu potencial antimicrobiano. Um total de 1116 fungos endofíticos foram isolados das plantas Vochysia divergens, presente na região do Pantanal, e Stryphnodendron adstringens encontrada no Cerrado, em duas coletas, a primeira realizada em Janeiro e a segunda para o estudo de biodiversidade realizado em Junho de 2016. Os isolados endofíticos foram agrupados em 124 grupos com base em seus morfotipos e cada representante foi identificado por análise da região ITS (Internal Transcribed Spacer). As análises de diversidade foram realizadas com 1079 isolados obtidos na segunda coleta e foram utilizados: riqueza e abundância absoluta, índices de diversidade de Shannon (H '), índice de Simpson (D-1) e índice de uniformidade de Pielou (J). O screening do potencial antimicrobiano foi realizado através de cultura pareada contra fitopatógenos e overlap contra patógenos humanos. Para isolados que apresentaram potencial no screening foi realizada a produção do extrato em dois meios de cultivos, Extrato de Malte e Czapeck, e os metabólitos secundários extraídos foram avaliados novamente contra microrganismos fitopatogênicos e bactérias e levedura de importância clínica. O índice de riqueza foi alto para ambas as plantas, no entanto, a comunidade endofítica não difere entre as plantas, Diaporthe foi o gênero dominante em ambas as plantas. Os testes antimicrobianos revelaram o alto potencial dos isolados Diaporthe sp. LGMF1548 e LGMF1583 e Neofusicoccum brasiliensis LGMF1535, com atividade contra Staphylococcus aureus resistente à meticilina (MRSA), Klebssiella pneumoniae (KPC) e Candida albicans. Contra fitopatógenos o isolado Diaporthe sp. LGMF1631 inibiu 90% do crescimento micelial de Phyllosticta citricarpa e 70% de Colletotrichum abscissum. Esses resultados demonstram a elevada diversidade de endofíticos, filogenia e a atividade antimicrobiana de fungos endofíticos associados às plantas V. divergens e S. adstringens. Nossos dados também sugerem que essas plantas servem como hospedeiras de novas espécies e gêneros, os quais produzem compostos de interesse.Abstract: The endophytic fungi are a main source of interest in metabolites, particularly those associated with medicinal plants. The Pantanal region and Brazilian savannah (Cerrado) in Brazil has high richness associated and many plants used for a local community because of their medicinal properties. Therefore exploring the bioprospecting potential of endophytic microorganisms in this region is important. The goals of the present work were to analyze the community endophytic fungi in medicinal plants of Pantanal and Cerrado, and their antimicrobial potential. A total of 1116 endophytic fungi were isolated from two different medicinal plants, Vochysia divergens in the region of Pantanal and Stryphnodendron adstringens, in the Cerrado and in two collects, the first realized in January and the second for biodiversity study realized in June of 2016. These isolates were grouped in 124 clades based in their morphotypes. The ITS (Internal Transcribed Spacer) sequence was used for species identification. The analyses were realized with one isolated each group. The diversity analyses was performed with 1079 isolates obtained in the second collect, and were used: absolute richness and abundance, indices of diversity of Shannon (H') and Simpson's index (D-1), and Pielou's evenness index (J). Screening for antimicrobial potential was determined by dual culture against phytopathogens and overlap against human pathogens. The production of crude extract were performed in Malt Extract and Czapeck media, derived from endophytic fungal selected in the screening, was tested against pathogenic microorganisms for inhibition of micelial growth for phytopathogens and in inhibition zone for clinical pathogens. In phylogenetic analyses were obtained 28 genera, with possible new genera. The richness index was high for both plants and the most part of the isolates belonging to Diaporthe genus, however, the endophytic community not differ between the plants. The antimicrobial tests showed the isolates Diaporthe sp. LGMF1548 and LGMF1583 and Neofusicoccum brasiliensis LGMF1535 with the best results against methicilineresistent Staphylococcus aureus, Klebssiella pneumoniae (KPC) and Candida albicans, for tests against phytopathogens the isolate Diaporthe sp. LGMF1631 inhibited 90% the mycelial growth of the Phyllosticta citricarpa and 70% of the Colletotrichum abscissum. These results demonstrate the biodiversity, phylogeny, and antimicrobial activity of endophytic fungi associated with the plants V. divergens and S. adstringens, and reveals the potential for discovery news taxa and compounds of interesting from the endophytes in these plant