Thesis (doctoral)--Vereinigte Friedrichs-Universität Halle-Wittenberg, 1910. / Reprinted from: Annales mycologici, 8. Jahrg., 1910. Lebenslauf. Includes bibliographical references.

Thesis (doctoral)--Vereinigte Friedrichs-Universität Halle-Wittenberg, 1910. / Reprinted from: Annales mycologici, 8. Jahrg., 1910. Lebenslauf. Includes bibliographical references.

This thesis explores the biosynthesis of two secondary metabolites, fluoroacetate and longianone, and involves the synthesis and feeding of deuterated putative intermediates. The bacterium Streptomyces cattleya produces fluoroacetate and 4- fluorothreonine; the mechanism by which C-F bond formation occurs is unknown. The stereochemistry of the fluorination event was investigated by feeding [2,2,3,3-(^-2)H(_4)]-succinate and (2R)-[l-(^2)H(_2)]- and (2S)-[l-(^2)H(_2)]-glycerols. The chirality of the resultant [2-(^2)H]-fluoroacetate was determined by chiral liquid crystal (^2)H-NMR and the fluorination was demonstrated to proceed with retention of stereochemistry. Longianone is produced by the slow growing fungus Xylaria longiana. This simple bicyclic metabolite is an isomer of the notorious fungal toxin patulin. Putative deuterated intermediates were administered to the fungus and the longianone produced analysed by (^2)H-NMR. It was demonstrated that longianone is biosynthesised from 6-methylsalicylic acid in a pathway closely related to that found in patulin biosynthesis.

This thesis is divided into two parts and covers biosynthetic studies on two secondary metabolites, tropic acid in Part I and piliformic acid, in Part II.(S)-Tropic acid is the acid moiety of the alkaloids hyoscyamine and scopolamine, which are produced by a number of plants of the Solanacae family. An intriguing rearrangement of the L-phenylalanine side chain gives rise to the isopropanoid (S)-tropic acid skeleton. The detailed nature of the rearrangement has however remained elusive despite continued interest over the years. In chapter two the identification of intermediates between L-phenylalanine and (S)-tropic acid is discussed, which has placed (R)-D-phenyllactic acid as an immediate precursor. The stereochemical features of the rearrangement are described in chapter 3 and finally in chapter 4 a mechanism for the rearrangement is proposed. This is based on information obtained from the incorporation of various isotopically labelled precursors to tropic acid into two of the minor alkaloids, 3a-2'-hydroxyacetoxytropane and 3a- phenylacetoxytropane. This work was carried out in collaboration with Dr Richard Robins at the AFRC Institute of Food Research in Norwich. Piliformic acid is elaborated by the slow growing fungus Poronia piliformis. The incorporation of a number of isotopically labelled substrates into piliformic acid has revealed a mixed biosynthetic origin, comprising C(_8) and C(_3) fragments. These have been shown to be of acetogenic and citric acid cycle origins respectively. The C(_8) fragment has been further demonstrated to be a degradation product of a longer chain fatty acid. The mode of coupling of the two fragments has been investigated and suggests the intermediacy of a novel a-carboxyoctanoate. A pathway for the assembly of piliformic acid, involving a 1,3-hydrogen shift, is proposed, consistent with the above findings. These results are the subject of chapter 6.

This thesis deals with the monitoring of fungal organisms involved in the decay of bases of common ash trees (Fraxinus excelsior) affected by the ash dieback (Hymenoscyphus fraxineus) infection. Monitoring was carried out in winter 2018 on 50 trees. Samples were taken from tree bases and sorted into 5 health categories. The total area of samples and the area affected by rot was measured in a laboratory. Fungal organisms were isolated from infected and healthy wood and identified by DNA extraction and sequencing. Finally, the determined species were statistically evaluated by a combination of constrained and unconstrained ordination methods. In total 436 pure cultures of fungal organisms were isolated. 23 species of fungal organisms were determined, of which 21 were in symptomatic wood (with a visible rot) and 5 in asymptomatic (healthy) wood. Xylaria polymorpha was confirmed to be present in most trees, it was found in 27 samples - 16× in asymptomatic wood and 11× in symptomatic wood. Hymenoscyphus fraxineus was isolated once from the 1st health category and once from the 5th health category from symptomatic wood.

Taproot decline (TRD), caused by an undescribed species of Xylaria, is an emerging root disease of soybean in Mississippi. Xylaria sp. isolates were collected from soybean roots and used to characterize TRD distribution as well as optimal growth temperature, pathogenicity, alternative host range, in vitro pathogenicity, and fungicide sensitivity. The 24 selected Xylaria sp. isolates from the 2016 collection had a mean optimal growth temperature of 26.7°C, and were pathogenic to soybean; however, differences in virulence occurred among isolates. Five selected Xylaria sp. isolates produced stroma on six hosts: corn, cotton, peanut, rice, sorghum, and wheat. Xylaria sp. colonized corn, cotton, and soybean seed in vitro; however, only reduced germination in soybean. Three selected Xylaria sp. isolates were exposed to fungicide-amended potato dextrose agar with concentrations up to 100 ppm of commercial products typically used in soybean production systems. Xylaria sp. isolates were insensitive to all active ingredients except thiophanate-methyl.

Os produtos naturais marinhos são apontados com uma das fontes de substâncias bioativas mais importantes para a descoberta de novos fármacos. Neste ambiente, os organismos estão em constante interação ecológica por meio da produção de metabólitos secundários. Fungos endofíticos e cianobactérias representam grupos de micro-organismos que realizam a biossíntese de substâncias com características químicas únicas e atividades biológicas potentes. Entretanto, quando retirados de seu habitat natural, esses seres microbianos geralmente perdem sua capacidade metabólica através de um fenômeno denominado silenciamento gênico, no qual genes biossintéticos deixam de ser transcritos devido a motivos ainda indeterminados. Esse mecanismo genético é intermediado, dentre outros fatores, pelas enzimas DNAmetiltransferase (DNA-MT) e Histona-desacetilase (HDAC). Deste modo, seus inibidores têm sido utilizados com sucesso para promover a eliciação de substâncias que não seriam produzidas em condições laboratoriais. Outra importante abordagem na pesquisa de produtos naturais têm sido a desreplicação baseada na fragmentação (MS/MS) para identificação de substâncias ou análogos. As redes moleculares (molecular networking) constituem uma nova abordagem na qual dados de espectrometria de massas são agrupados de acordo com as semelhanças entre os padrões de fragmentação, formando famílias de moléculas, permitindo a rápida visualização do perfil químico de várias amostras ao mesmo tempo. Deste modo, este trabalho apresenta o isolamento e identificação de metabólitos inéditos a partir de fungos endofíticos e cianobactérias oriundos do ambiente marinho. Para isto, técnicas de eliciação epigenética foram utilizadas em ambos os grupos de organismos, e a desreplicação via redes moleculares foi utilizada em cianobactérias. Fungos endofíticos associados à macroalga vermelha Bostrychia tenella foram alvo de estudos químicos e epigenéticos. As linhagens Xylaria sp. e Nigrospora oryzae foram submetidas ao cultivo em meio sólido arroz, o que resultou no isomento da substância citocalasina D e de um derivado potencialmente inédito da griseofulvina. A linhagem Penicillium decaturense foi cultivada em meio líquido PDB resultando no isolamento da 10,11-deidrocurvularina e possíveis análogos. Experimentos com inibidores epigenéticos (butirato de sódio e procaína) promoveram a modulação do perfil químico desta linhagem, ao estimular a produção de metabólitos não expressos em condições normais de cultivo. Ainda, a linhagem Acremonium sp. produziu várias substâncias quando cultivada em meio de líquido Czapek sob a influência de procaína, sendo uma delas potencialmente inédita e derivada da classe de metabólitos das brevianamidas. Frações orgânicas da cianobactéria Schizothrix sp., coletada no Panamá, foram analisadas em LC-MS/MS e os dados gerados foram utilizados para a criação de redes moleculares. Este estudo resultou na identificação dos metabólitos barbamida, hectoclorina, curacinas A e D, curazole, acetato de malingamida D, dolastatina 10 e carmaficina B. Ainda, análogos das substâncias curazole, dolastatina D e dois análogos inéditos das carmaficinas foram propostos. A cianobactéria Moorea producens JHB, coletada na Jamaica, foi submetida ao cultivo sob influência do ii composto butirato de sódio, e produziu dois metabólitos inéditos, propostos de acordo com os dados de fragmentação, como sendo derivados da jamaicamida e da hectoclorina, num tipo de biossíntese cruzada. Portanto, este trabalho confirma os fungos endofíticos e cianobactérias marinhos como promissores quanto a exploração do metabolismo secundário. / Marine natural products are pointed out as one of the most important sources of bioactive compounds for drug discovery. In this environment, organisms are in constantly interaction ecological through the production of secondary metabolites. Endophytic fungi and cyanobacteria represent groups of microorganisms that perform biosynthesis of substances with unique chemical features and potent biological activities. However, when removed from their natural habitat, these microbial beings generally lose their metabolic capacity through a phenomenon called gene silencing, in which biosynthetic genes are no longer transcribed due to reasons still undetermined. This genetic mechanism is brokered, among other factors, by the enzyme DNA methyltransferase (DNA-MT) and histone deacetylase (HDAC). Thus, their inhibitors have been used successfully to promote the elicitation of substances that would not be produced under laboratory conditions. Another important approach in the natural products research field have been dereplication based on the fragmentation (MS/MS) for the identification of substances or analogues. The molecular networking is a new approach in which data from mass spectrometry are grouped according to the similarities between the patterns of fragmentation, forming families of molecules, allowing rapid visualization of the chemical profile of several samples simultaneously. Thus, this work presents the isolation and identification of novel metabolites from endophytic fungi and cyanobacteria originating from the marine environment. For this propose, epigenetic elicitation techniques were used in both groups of organisms and the molecular networks via dereplication was used in cyanobacteria. Endophytic fungi associated with red seaweed Bostrychia tenella were subjected to chemical and epigenetic studies. Xylaria sp. and Nigrospora oryzae strains were cultured in solid medium rice, resulting in isolation of substance of cytochalasin D and a potentially novel derivative of griseofulvin. Penicillium decaturense strain was grown in PDB liquid medium resulting in the isolation of 10,11- deidrocurvularina and possible analogues. Experiments with epigenetic inhibitors (sodium butyrate and procaine) promoted the modulation of the chemical profile of this strain, to stimulate the production of metabolites not expressed under normal culture conditions. Moreover, Acremonium sp. produced various substances when grown in liquid medium under the influence of Czapek procaine, one of novel and potentially derived from the class of metabolites brevianamides. Organic fractions of the cyanobacteria Schizothrix sp., collected in Panama, were analized by LC-MS/MS and the data generated were used to create molecular networks. This study resulted in the identification of metabolites barbamide, hectochlorin, curacins A and D, curazole malyngamide D acetate, dolastatin 10 and carmaphycin B. Also, analogs of curazole, dolastatin 10 and carmaphycins A and B have been proposed. Cyanobacteria Moorea producens JHB, collected in Jamaica, was grown under the influence of sodium butyrate, and produced two new proposed metabolites in accordance with the fragmentation data as being derived from jamaicamide and hectochlorin, in a sort of crossed biosynthesis. Therefore, this work corroborates marine endophytic fungi and cyanobacteria as promising for exploration of secondary metabolism.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2017-06-06T19:00:54Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Fungos endolinquênios associados á Cladonia curta Ahti & Marcelli no pampa gaúcho- Brasil.pdf: 2358078 bytes, checksum: ad400f5271deb54c851824e68ecbedd3 (MD5) / Made available in DSpace on 2017-06-06T19:00:54Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Fungos endolinquênios associados á Cladonia curta Ahti & Marcelli no pampa gaúcho- Brasil.pdf: 2358078 bytes, checksum: ad400f5271deb54c851824e68ecbedd3 (MD5) Previous issue date: 2017-05-03 / Os liquens são considerados uma associação simbiótica composta por um micobionte, um ou mais fotobiontes e inclusive um terceiro simbionte Basidiomycota, representam um dos estilos de vida de maior sucesso para fungos e uma das formas mais bem sucedidas da simbiose na natureza. Liquens exploram todos os ambientes naturais e se adaptam a condições extremas como as causadas pelas pertubações humanas, tornando-se importantes bioindicadores da qualidade ambiental. Cladonia curta & Marcelli é uma espécie de líquen escamoso da família Cladoniaceae distribuídos em três locais ao sudeste do Brasil, em florestas e no Cerrado e coletado no bioma Pampa durante o presente estudo, tornando-se um registro de nova ocorrência. Por outro lado, existem estudos que demonstram a presença de fungos que ocorrem dentro dos talos aparentemente saudáveis dos liquens, conhecidos como endoliquênicos, associados principalmente ao fotobionte. Durante os últimos 30 anos, fungos endoliquênicos têm sido estudados com base em métodos dependentes do cultivo in vitro e o uso de ferramentas moleculares empregadas principalmente na sua detecção e identificação. Assim o objetivo do presente trabalho foi conhecer a diversidade de fungos endoliquênicos abrigados no talo de Cladonia curta e suas relações filogenéticas com outros fungos endófitos. As análises filogenéticas baseadas na amplificação das regiões ITS do RDNA e do gene da ß-Tubulina infere as linhagens de fungos endoliquênicos no gênero Xylaria. Do mesmo modo, as características morfológicas das colônias e dos estromas anamórficos obtidos em cultura confirmam esta classificação. Nossos resultados baseados em marcadores moleculares fornecem evidências de que fungos endoliquênios isolados de Cladonia curta estão intimamente relacionados com fungos endófitos e saprofíticos; no entanto, os isolados se mostram mais estreitamente relacionados com fungos endófitos, sugerindo que a sua associação ao talo não é puramente acidental e respaldando a relação filogenética entre três estilos de vida, como previamanete relatado por outros autores. Estudos são necessários para demosntrar que isolados Xylaria spp. e Xylaria berteri sejam conspecíficos. Além disso, nõs sugerimos o uso do meio de cultura MS como alternativa nos esforços para conhecer a grande diversidade de fungos endófitos que tecidos vivos de liquens. Finalmente, a diversidade e a prevalência dos fungos endoliquênicos continua sendo estudada sendo este o primeiro relato de isolamento e identificação de fungos endoliquênicos no Brasil. / Lichens are considered a symbiotic association between a mycobionte, one or more photobionts and even a third Basidiomycota symbiont, represent one of the most successful lifestyles for fungi and one of the most successful forms of symbiosis in nature. Lichens explore all natural environments and adapt to extreme conditions such as those caused by human disturbances, becoming important bioindicators of environmental quality. Short Cladonia & Marcelli is a species of scaly lichen of the family Cladoniaceae distributed in three places to the southeast of Brazil, in forests and in the Cerrado and collected in the Pampa biome during the present study, becoming a record of new occurrence. On the other hand, there are studies that demonstrate the presence of fungi that occur within apparently healthy lichens of lichens, known as endolynetics, associated mainly with photobiont. During the last 30 years, endoliquenic fungi have been studied based on in vitro culture-dependent methods and the use of molecular tools mainly used for their detection and identification. Thus the objective of the present work was to know the diversity of endoliquenic fungi harbored in Cladonia curta thallus and their phylogenetic relationships with other endophytic fungi. Phylogenetic analyzes based on the amplification of the ITS regions of the rDNA and the β-Tubulin gene infer the endolichenic fungi lineages in the genus Xylaria. Likewise, the morphological characteristics of the colonies and the anamorphic stromatas obtained in culture confirm this classification. Our results based on molecular markers provide evidence that isolated endolivhenic fungi of C. curta are closely related to endophytic and saprophytic fungi; However, isolates are more closely related to endophyte fungi, suggesting that their association to the stem is not purely accidental and supporting the phylogenetic relationship between three lifestyles, as previously reported by other authors. Additional studies are needed to demonstrate that isolated Xylaria spp. And Xylaria berteri are conspecific. In addition, we suggest the use of the MS culture medium as an alternative in efforts to know the great diversity of endophytic fungi that live lichen tissues. Finally, the diversity and prevalence of endolichenic fungi continues to be studied, with this the first report of isolation and identification of endolichenicnic fungi in Brazil.

Os produtos naturais marinhos são apontados com uma das fontes de substâncias bioativas mais importantes para a descoberta de novos fármacos. Neste ambiente, os organismos estão em constante interação ecológica por meio da produção de metabólitos secundários. Fungos endofíticos e cianobactérias representam grupos de micro-organismos que realizam a biossíntese de substâncias com características químicas únicas e atividades biológicas potentes. Entretanto, quando retirados de seu habitat natural, esses seres microbianos geralmente perdem sua capacidade metabólica através de um fenômeno denominado silenciamento gênico, no qual genes biossintéticos deixam de ser transcritos devido a motivos ainda indeterminados. Esse mecanismo genético é intermediado, dentre outros fatores, pelas enzimas DNAmetiltransferase (DNA-MT) e Histona-desacetilase (HDAC). Deste modo, seus inibidores têm sido utilizados com sucesso para promover a eliciação de substâncias que não seriam produzidas em condições laboratoriais. Outra importante abordagem na pesquisa de produtos naturais têm sido a desreplicação baseada na fragmentação (MS/MS) para identificação de substâncias ou análogos. As redes moleculares (molecular networking) constituem uma nova abordagem na qual dados de espectrometria de massas são agrupados de acordo com as semelhanças entre os padrões de fragmentação, formando famílias de moléculas, permitindo a rápida visualização do perfil químico de várias amostras ao mesmo tempo. Deste modo, este trabalho apresenta o isolamento e identificação de metabólitos inéditos a partir de fungos endofíticos e cianobactérias oriundos do ambiente marinho. Para isto, técnicas de eliciação epigenética foram utilizadas em ambos os grupos de organismos, e a desreplicação via redes moleculares foi utilizada em cianobactérias. Fungos endofíticos associados à macroalga vermelha Bostrychia tenella foram alvo de estudos químicos e epigenéticos. As linhagens Xylaria sp. e Nigrospora oryzae foram submetidas ao cultivo em meio sólido arroz, o que resultou no isomento da substância citocalasina D e de um derivado potencialmente inédito da griseofulvina. A linhagem Penicillium decaturense foi cultivada em meio líquido PDB resultando no isolamento da 10,11-deidrocurvularina e possíveis análogos. Experimentos com inibidores epigenéticos (butirato de sódio e procaína) promoveram a modulação do perfil químico desta linhagem, ao estimular a produção de metabólitos não expressos em condições normais de cultivo. Ainda, a linhagem Acremonium sp. produziu várias substâncias quando cultivada em meio de líquido Czapek sob a influência de procaína, sendo uma delas potencialmente inédita e derivada da classe de metabólitos das brevianamidas. Frações orgânicas da cianobactéria Schizothrix sp., coletada no Panamá, foram analisadas em LC-MS/MS e os dados gerados foram utilizados para a criação de redes moleculares. Este estudo resultou na identificação dos metabólitos barbamida, hectoclorina, curacinas A e D, curazole, acetato de malingamida D, dolastatina 10 e carmaficina B. Ainda, análogos das substâncias curazole, dolastatina D e dois análogos inéditos das carmaficinas foram propostos. A cianobactéria Moorea producens JHB, coletada na Jamaica, foi submetida ao cultivo sob influência do ii composto butirato de sódio, e produziu dois metabólitos inéditos, propostos de acordo com os dados de fragmentação, como sendo derivados da jamaicamida e da hectoclorina, num tipo de biossíntese cruzada. Portanto, este trabalho confirma os fungos endofíticos e cianobactérias marinhos como promissores quanto a exploração do metabolismo secundário. / Marine natural products are pointed out as one of the most important sources of bioactive compounds for drug discovery. In this environment, organisms are in constantly interaction ecological through the production of secondary metabolites. Endophytic fungi and cyanobacteria represent groups of microorganisms that perform biosynthesis of substances with unique chemical features and potent biological activities. However, when removed from their natural habitat, these microbial beings generally lose their metabolic capacity through a phenomenon called gene silencing, in which biosynthetic genes are no longer transcribed due to reasons still undetermined. This genetic mechanism is brokered, among other factors, by the enzyme DNA methyltransferase (DNA-MT) and histone deacetylase (HDAC). Thus, their inhibitors have been used successfully to promote the elicitation of substances that would not be produced under laboratory conditions. Another important approach in the natural products research field have been dereplication based on the fragmentation (MS/MS) for the identification of substances or analogues. The molecular networking is a new approach in which data from mass spectrometry are grouped according to the similarities between the patterns of fragmentation, forming families of molecules, allowing rapid visualization of the chemical profile of several samples simultaneously. Thus, this work presents the isolation and identification of novel metabolites from endophytic fungi and cyanobacteria originating from the marine environment. For this propose, epigenetic elicitation techniques were used in both groups of organisms and the molecular networks via dereplication was used in cyanobacteria. Endophytic fungi associated with red seaweed Bostrychia tenella were subjected to chemical and epigenetic studies. Xylaria sp. and Nigrospora oryzae strains were cultured in solid medium rice, resulting in isolation of substance of cytochalasin D and a potentially novel derivative of griseofulvin. Penicillium decaturense strain was grown in PDB liquid medium resulting in the isolation of 10,11- deidrocurvularina and possible analogues. Experiments with epigenetic inhibitors (sodium butyrate and procaine) promoted the modulation of the chemical profile of this strain, to stimulate the production of metabolites not expressed under normal culture conditions. Moreover, Acremonium sp. produced various substances when grown in liquid medium under the influence of Czapek procaine, one of novel and potentially derived from the class of metabolites brevianamides. Organic fractions of the cyanobacteria Schizothrix sp., collected in Panama, were analized by LC-MS/MS and the data generated were used to create molecular networks. This study resulted in the identification of metabolites barbamide, hectochlorin, curacins A and D, curazole malyngamide D acetate, dolastatin 10 and carmaphycin B. Also, analogs of curazole, dolastatin 10 and carmaphycins A and B have been proposed. Cyanobacteria Moorea producens JHB, collected in Jamaica, was grown under the influence of sodium butyrate, and produced two new proposed metabolites in accordance with the fragmentation data as being derived from jamaicamide and hectochlorin, in a sort of crossed biosynthesis. Therefore, this work corroborates marine endophytic fungi and cyanobacteria as promising for exploration of secondary metabolism.

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