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No. of bitstreams: 1 Dissertação_Completa_Henia_VF-2.pdf: 4622430 bytes, checksum: d09b1f29bbf344aeaacc5347c72ee181 (MD5) / CNPq e Fapesb / “ESTUDO QUÍMICO E AVALIAÇÃO DAS ATIVIDADES ANTIMICROBIANA, ANTICOLINESTERÁSICA E LARVICIDA DO FUNGO Curvularia lunata E LEVANTAMENTO QUIMIOTAXONÔMICO DO COMPLEXO Curvularia- Bipolaris-Cochliobolus” A revisão bibliográfica sobre os metabólitos secundários isolados de espécies fúngicas dos gêneros Curvularia, Bipolaris e Cochliobolus foi realizada com o objetivo de comparar o perfil metabólico deste três gêneros visando contribuir para a classificação taxonômica deste complexo. Como resultados, foram encontrados metabólitos secundários pertencentes às classes dos policetídeos (incluindo antraquinonas, lactonas macrociclicas e compostos fenólicos), terpenoides (sesterpenoies e sesquiterpenoides), esteróides e alcalóides. Os policetídeos como as lactonas macrociclicas curvularinas foram descritas somente para fungos do gênero Curvularia e alguns Cochliobolus, enquanto que os sesterpenoides foram encontrados nos gêneros Bipolaris e Cochliobolus, permitindo sugerir que as lactonas sejam marcadores quimiotaxonômicos para o gênero Curvularia e as espécies de Cochliobolus relacionadas (teleomorficas), e que os sesterpenoides são marcadores do gênero Bipolaris e os teleomorfos do gênero Cochliobolus. A análise dos extratos oriundos do cultivo de Curvularia lunata em diferentes meios de cultura foi realizada para verificação da influencia da fonte de carbono sobre a produção de metabólitos secundários. Os métodos utilizando a cromatografia em camada delgada comparativa, cromatografia líquida de alta eficiência e ressonância magnética nuclear de hidrogênio permitiram estimar a diversidade química conforme a fonte de carbono usada. Os meios de cultura Czapeck, batata e dextrose e extrato de malte apresentaram mais sinais nos cromatogramas e nos espectros de RMN de 1H com relação aos demais, por isto estes meios foram considerados os meio de culturas ideais para o estudo do metabolismo secundário. Além disso, foi possível inferir a presença da 11-α-metoxicurvularina nos extratos do fungo cultivado em batata e dextrose e farinha de arroz. Os extratos obtidos foram submetidos a ensaios biológicos (atividade antimicrobiana, anticolinesterásica e larvicida). Os extratos obtidos do cultivo do fungo em batata e dextrose apresentaram atividade contra a levedura Candida albicans, enquanto os extratos oriundos do meio contendo extrato de levedura XV mostraram atividade antibacteriana contra Micrococcus luteus, Staphylococcus aureus e Bacillus subtilis. Com relação à atividade anticolinesterasica, todos os extratos brutos oriundos do micélio continham um sinal com fator de retenção igual 0.7 que mostrou inibição da atividade da enzima acetilcolinesterase, destacando os extratos fúngicos resultantes do cultivo em meio contendo extrato de malte e meio Czapeck enriquecido com 2% de extrato de levedura por apresentarem maior halo de inibição. Nenhum dos extratos apresentou atividade contra larvas do mosquito Aedes aegypti. Finalmente o fungo Curvularia lunata foi cultivado em larga escala em meio contendo extrato de malte selecionado pela sua atividade anticolinesterasica. A investigação química inicial resultou no isolamento de três derivados perilenoquinônicos: dois já descritos na literatura como dihidroalterperilenol e alterperilenol, mas obtidos pela primeira vez no gênero Curvularia e o perileno de estrutura inédita, identificado como 12-metil- stemfitriol. / “CHEMICAL STUDY AND BIOLOGICAL ASSAYS (ANTIMOCROBIAL, ANTICHOLINESTERASIC AND INSECTICIDAL) FROM THE FUNGUS Curvularia lunata AND QUIMIOTAXONOMIC SEARCH FROM THE FUNGAL COMPLEX Curvularia-Bipolaris-Cochliobolus” A literature revision of secondary metabolites isolated from fungal species of Curvularia, Bipolaris and Cochliobolus genera was performed with the aim of get their metabolic profile in order to contribute to the taxonomic classification on this complex. As result were found secondary metabolites belonging to the polyketides (including anthraquinones, macrocyclic lactones and phenolic), terpenoids (mainly sesterpenoids), steroids and alkaloids. Polyketides like the macrocyclic lactone curvularins were described only for fungi from the genus Curvularia and the related teleomorphic Cochliobolus species, while sesterpenoids such as ophiobolins was described for Bipolaris species as well as their related Cochliobolus, suggesting these lactones and sesterpenoids as quimiotaxonomic marker for Curvularia and Bipolaris genera, respectively. An analysis of extracts derived from cultivation of Curvularia lunata on several media was performed to investigate the effect of carbon source on secondary metabolism of the fungus. As analytical strategy based on TLC (thin layer chromatography), high performance liquid chromatography with diode array detection and nuclear magnetic resonance of hydrogen allowed to get an estimative about chemical diversity under different carbon sources. Malt extract, Czapeck plus 2% of yeast extract and potato and dextrose extracts were the media that exhibited the most signals in chromatograms and 1H NMR spectra, indicating them as the ideal culture media for secondary metabolism studies. Furthermore, we can suggest that the compounds 11-α-methoxycurvularin were found in fungal extracts derived from the cultivation of Curvularia in potato and dextrose and rice flour media. In addition, those extracts were also evaluated for their biological activities (antimicrobial, anticolinesterásico and insecticidal). Extracts obtained from fungal growth in potato and dextrose media exhibited activity against the yeast Candida albicans, while extracts derived from the yeast extract cultivation showed antibacterial activity against M. luteus, S. aureus and B. subtillis. With respect the anticholinesterasic activity, all mycelial extract had a signal (Rf 0.7) that inhibited the enzyme AChE activity, being the fungal extracts from malt extracts and czapeck plus 2% yeast extract that had a marked inhibition halo. None of these XVII extracts had an activity against larval stage of the mosquito Aedes aegypti. Finally, the fungus Curvularia lunata was cultivated in large scale on malt extract medium, selected for its anticholinesterasic activity. Initial chemical investigation had resulted in the isolation of three perylene derivates: known dihydroalterperylenol and alterperylenol but being described for the first time in Curvularia genera and the novel perylene having a metabolic group, which was named as 12-methyl-stemphytriol.

Sorghum grain mold and anthracnose are two major diseases of sorghum (Sorghum bicolor) that constrain sorghum production worldwide. Grain mold is caused by several species of fungi, but the two most common are Curvularia lunata and Fusarium thapsinum. Isolates of these two species were used to inoculate panicles of selected sorghum cultivars in green house and field experimentations. Panicles were sprayed at the time of anthesis with conidial suspensions of the two fungal species individually or in a mixture and with water to serve as a control. Samples were collected 48 hours after inoculation for RNA extraction. In greenhouse studies, four cultivars (Tx2911, Sureno, SC170 and RTx430) were used while thirteen cultivars were grown in the field experiments. Gene expression was measured for the following genes using real time polymerase chain reactions (rt-PCR): PR10, β-glucanase, chitinase, thaumatin, sormatin, phenyalanine ammonia lyase (PAL), obtusifoliol 14α-demethylase (Obtus), antifungal protein (AFP), apoptosis related protein (Apop) and leucine rich repeat (LRR). Seed germination tests in field grown cultivars indicated that germination rates for SC279-14E, SC660 and Sureno were not greatly influenced by grain mold. Covering the panicles with bags served to protect them against grain mold pathogens. The seed mycoflora test showed that Fusarium thapsinum was the most frequently recovered species and there were more species present in non-covered panicles. The response of sorghum cultivars to grain mold infection involves multiple defense genes. Real time PCR used to study the expression of sorghum defense in greenhouse grown plants showed that mRNA encoding PR-10, a small 10 kDa protein, was highly expressed in the glumes and spikelets of resistant cultivars Tx2911 and Sureno and constitutively in leaves. The expression of some other defense genes like beta-glucanase, chitinase and AFP was variable. Sormatin was not expressed. Expression of β-glucanase, chitinase, and PR10 was higher in field than in greenhouse experiments. A second area of research involved tagging of a resistance gene for sorghum anthracnose. Three AFLP markers (Xtxa607, Xtxa3181 and Xtxa4327) and three SSRs (Xtxp3, Xtxp55 and Xtxp72) were identified. These markers were loosely linked to the resistance genes. The markers are located on linkage group B. The results suggest that markers located 20-30 cM on one side or the other of those tested should provide useful tags for the resistance gene.

Sorghum grain mold and anthracnose are two major diseases of sorghum (Sorghum bicolor) that constrain sorghum production worldwide. Grain mold is caused by several species of fungi, but the two most common are Curvularia lunata and Fusarium thapsinum. Isolates of these two species were used to inoculate panicles of selected sorghum cultivars in green house and field experimentations. Panicles were sprayed at the time of anthesis with conidial suspensions of the two fungal species individually or in a mixture and with water to serve as a control. Samples were collected 48 hours after inoculation for RNA extraction. In greenhouse studies, four cultivars (Tx2911, Sureno, SC170 and RTx430) were used while thirteen cultivars were grown in the field experiments. Gene expression was measured for the following genes using real time polymerase chain reactions (rt-PCR): PR10, β-glucanase, chitinase, thaumatin, sormatin, phenyalanine ammonia lyase (PAL), obtusifoliol 14α-demethylase (Obtus), antifungal protein (AFP), apoptosis related protein (Apop) and leucine rich repeat (LRR). Seed germination tests in field grown cultivars indicated that germination rates for SC279-14E, SC660 and Sureno were not greatly influenced by grain mold. Covering the panicles with bags served to protect them against grain mold pathogens. The seed mycoflora test showed that Fusarium thapsinum was the most frequently recovered species and there were more species present in non-covered panicles. The response of sorghum cultivars to grain mold infection involves multiple defense genes. Real time PCR used to study the expression of sorghum defense in greenhouse grown plants showed that mRNA encoding PR-10, a small 10 kDa protein, was highly expressed in the glumes and spikelets of resistant cultivars Tx2911 and Sureno and constitutively in leaves. The expression of some other defense genes like beta-glucanase, chitinase and AFP was variable. Sormatin was not expressed. Expression of β-glucanase, chitinase, and PR10 was higher in field than in greenhouse experiments. A second area of research involved tagging of a resistance gene for sorghum anthracnose. Three AFLP markers (Xtxa607, Xtxa3181 and Xtxa4327) and three SSRs (Xtxp3, Xtxp55 and Xtxp72) were identified. These markers were loosely linked to the resistance genes. The markers are located on linkage group B. The results suggest that markers located 20-30 cM on one side or the other of those tested should provide useful tags for the resistance gene.

Orientador: Lourdes Campaner dos Santos / Resumo: Este trabalho apresenta o estudo dos efeitos de modificadores epigenéticos e eliciadores químicos na diversificação metabólica de Curvularia lunata e Fusarium fujikuroi, fungos isolados de Paepalanthus chiquitensis (Eriocaulaceae). Os fungos foram cultivados em PDB (caldo batata dextrose), e as seguintes condições foram investigadas: (PDB + SAHA) ácido hidroxâmico suberoilanilida, (PDB + AZA) 5-azacitidina, (PDB + AA) ácido anacárdico, PDB + Cu2+ e PDB + Mg2+. Essas condições foram comparadas com o controle PDB. O estudo químico de C. lunata, resultou no isolamento das novas γ-lactamas espirocíclicas C3 e C4, e outros cinco compostos conhecidos: triticonas E (C1) e F (C2), ácido 5-O-metilcurvulínico (C5), ácido curvulínico (C6) e curvulina (C7). As estruturas foram elucidadas por análises espectroscópicas e pela comparação com dados da literatura. Além disso, um estudo computacional foi realizado para determinar a configuração absoluta do C‒3' de C3 e C4. As triticonas C1 e C2 apresentaram boa atividade antibacteriana para Escherichia coli, com CIM de 62,5 μg/mL. Dos eliciadores químicos utilizados, a suplementação com Mg2+ foi a condição que apresentou as diferenças mais significativas no perfil metabólico de F. fujikuroi. O extrato PDB + Mg2+ preparado do cultivo em escala ampliada foi purificado por métodos cromatográficos e resultou no isolamento e identificação das substâncias: ácido indolacético (F3) e seus derivados F4, F8 e F17, assim como a auxina ácido fenilacético ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Eriocaulaceae, Paepalanthus chiquitensis, Fusarium fujikuroi, Curvularia lunata, fungo endofítico, epigenética / Doutor

O Andropogon L. é uma gramínea forrageira, amplamente distribuída em áreas de Cerrado e com períodos de seca prolongada. Apresenta características de ser denso com grandes touceiras e com inflorescência plumosa, tendo uma elevada capacidade na disseminação de suas sementes. Por sua vez, as sementes são consideradas principais fontes de abrigo e transporte de agentes patogênicos para áreas livres de doenças. No Tocantins, não há registros de trabalhos que relacionem a incidência de fungos em sementes de Andropogon como causadores de doenças em culturas de importância agrícola. Da mesma forma, não há pesquisas sobre o transporte, transmissibilidade e patogenicidade de fungos associados à suas sementes. O trabalho objetivou avaliar a qualidade sanitária de sementes do Andropogon, a transmissão de fungos via semente-plântula e a patogenicidade à plantas de outras espécies de importância agrícola, e também realizar a caracterização morfológica e molecular de Curvularia sp. isolados de sementes de Andropogon. Os experimentos foram conduzidos no Laboratório de Pesquisa em Fitopatologia, Laboratório de Controle Biológico de Doenças e Casa de Vegetação da Universidade Federal do Tocantins. No capítulo 1, utilizou-se o método blotter test para a avaliação da sanidade das sementes com e sem desinfestação. A incidência dos fungos foi avaliada a partir da análise individual das sementes com auxílio de microscópio estereoscópico e ótico. A germinação das sementes foi avaliada após 10 dias de instalação do teste, juntamente com a identificação de fungos associados às sementes não germinadas. Para os fungos detectados na análise sanitária avaliou-se a capacidade de transmissão via semente-plântula. A patogenicidade dos fungos oriundos das sementes de Andropogon foi avaliada por meio da inoculação na própria planta e também foi avaliada a capacidade destes fungos em infectar outras plantas de interesse econômico. No capítulo 2, a identificação morfológica foi realizada a partir de observações macro e micromorfológicas utilizando-se como base as características descritas na literatura quanto ao aspecto da colônia e conídios de Curvularia sp. A caracterização molecular foi realizada a partir da extração do DNA, amplificação e sequenciamento da região do gene Clg2p. A transmissão foi avaliada a partir da semeadura de sementes sem tratamento com fungicidas, onde ao final de 40 dias observou-se sintomas típicos de mancha foliar de Curvularia. A patogenicidade foi avaliada a partir da inoculação de suspensão de conídios de Curvularia sp. nas folhas de plantas sadias, observando ao final de 10 dias, se houve sintomas do patógeno. Foram identificados e quantificados nas sementes de Andropogon L. fungos dos gêneros Alternaria sp., Bipolaris sp., Curvularia sp., Fusarium sp., Phoma sp., Aspergillus sp., Cladosporium sp., Penicillium sp. e Rhizopus sp. A realização da desinfestação das sementes reduziu os fungos presentes nas sementes. O fungo Curvularia sp. foi transmitido das sementes para as plantas de Andropogon. As sementes de Andropogon L. transportaram e disseminaram fungos que uma vez inoculados causaram infecção na própria planta e em outras culturas de importância econômica, tais como: arroz, feijão-caupi, melancia, melão, milho, sorgo e aos capins marandu, mombaça, piatã e quicuia. Baseado em marcadores morfológicos e moleculares, o fungo identificado com elevada incidência associado às sementes de Andropogon coletadas em diferentes regiões produtoras agrícolas, trata-se de Curvularia lunata. C. lunata é transmitido para as plantas de Andropogon via semente, sendo patogênico a esta espécie de gramínea forrageira, causando manchas necróticas foliares. / Andropogon L. is a forage grass, widely distributed in Cerrado areas and with prolonged drought periods. It presents dense characteristics with large clumps and with plumose inflorescence, with high capacity in the seeds dissemination. In turn, seeds are considered the main sources of shelter and transport of pathogens to disease-free areas. In Tocantins, there are no studies records relate the fungi incidence in Andropogon grass seeds as diseases cause in agricultural importance crops. Likewise, there is no research about the transport, transmissibility and pathogenicity of fungi associated with their seeds. The work aim was evaluate the sanitary quality of Andropogon grass seeds, the fungi transmission by seed-seedlings and the fungis pathogenicity to other species plants of agricultural importance, and also perform the Curvularia sp. morphological and molecular characterization, isolated from Andropogon grass seeds. The experiments were conducted at the Phytopathology Research Laboratory, Biologic Control of Disease Laboratory and green house of Tocantins Federal University. In chapter 1, the blotter test method was used to evaluate seed health with and without disinfestation. The fungi incidence was evaluated from seeds individual analysis using stereoscopic and optical microscope. The seeds germination was evaluated after 10 days after test installation, together with the fungi identification associated with non-germinated seeds. For the detected fungus in the sanitary analysis was evaluated the transmission capacity of seed-seedling. The fungi pathogenicity from the Andropogon seeds grass was evaluated by inoculation in the plant itself and was also evaluated the ability of these fungi to infect other plants of economic interest. In chapter 2, the morphological identification was performed from macro and micromorphological observations using as basis the characteristics described in literature regarding the aspect of the colony and conidia of Curvularia sp. Molecular characterization was performed from DNA extraction, amplification and sequencing gene Clg2p region. Transmission was evaluated from seed sowing whitout treatment with fungicides, where at the end of 40 days typical leaf spot symptoms of Curvularia. The pathogenicity was evaluated from the inoculation of conidia suspension on leaves of healthy plants, observing at the end 10 days, if there were symptoms of the pathogen. Were identified and quantified in the seeds of Andropogon L. fungi of the genera Alternaria sp., Bipolaris sp., Curvularia sp., Fusarium sp., Phoma sp., Aspergillus sp., Cladosporium sp., Penicillium sp. and Rhizopus sp. The seed disinfestation reduced the fungi present in the seeds. The fungus Curvularia sp. it was transmitted seed to Andropogon plant. Andropogon L. seeds carried and spread fungi the once inoculated caused infection in the plant itself and other economically important crops, such as rice, cowpea, watermelon, melon, corn, sorghum and grasses marandu, mombaça, piatã and quicuia. Based on morphological and molecular markers, the fungus identified with high incidence associated with Andropogon seeds collected in different agricultural producing regions, is Curvularia lunata. C. lunata is transmitted to plants of Andropogon by seed, being pathogenic to this species of forage grass, causing foliar necrotic spots.