O estudo das interações planta-microrganismos tem sido de grande interesse ao longo dos últimos anos. Atualmente, as interações que ocorrem entre microrganismos que vivem em estreita relação também vêm merecendo grande atenção, pois forças competitivas e mutualísticas podem induzir a produção de novos metabólitos bioativos. Portanto, estudar interações existentes entre os microrganismos endofíticos que colonizam uma mesma planta parece ser uma estratégia promissora para a obtenção de substâncias quimicamente diferentes, eventualmente bioativas. Através da utilização de culturas mistas de microrganismos, o presente trabalho contribuiu para o conhecimento da relação existente entre os fungos endofíticos SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) e SS84 (Phoma betae), isolados da planta medicinal Smallanthus sonchifolius (yacon), e sua implicação no aumento da diversidade química de produtos naturais microbianos, com o intuito de se identificar metabólitos secundários anticancerígenos. Para isso, os fungos foram cultivados em culturas singles e mistas em meios de cultivo líquidos e semi-sólidos. Foram utilizados diferentes meios de cultura, diferentes maneiras de se estabelecer o cultivo misto e diferentes formas de extração. Os extratos foram analisados química e biologicamente. Após fracionamento, foram isoladas cinco substâncias: afidicolina (I), 3-desóxi-afidicolina (II), estenfiperilenol (III), alterperilenol (IV) e alternariol monometil éter (V), sendo as duas primeiras de origem terpênica e as outras de origem policetídica. As substâncias I e II foram produzidas pelo fungo SS67, sendo que a produção de I aparentemente aumentou nas culturas mistas líquidas com SS13 e SS84, diminuindo consideravelmente na cultura mista com SS77. Devido à afidicolina ser um composto altamente citotóxico, os cultivos do fungo SS67 originaram extratos muito ativos frente aos ensaios de citotoxicidade em células cancerígenas. A substância III, primeiramente, só foi detectada por CLAE-DAD na cultura mista dos fungos SS67 e SS77, e a produção da substância IV foi maior nessa cultura mista que na cultura simples do fungo SS77 (meio fermentativo de extrato de malte). Provavelmente esses compostos foram produzidos por SS77 em resposta à presença de SS67. O extrato obtido durante esse cultivo misto foi o que apresentou maior atividade citotóxica frente à linhagem celular MDAMB-435 (câncer de mama). Posteriormente, a substância III foi também isolada da cultura simples do fungo SS77 cultivado em outras condições (meio fermentativo PDB), juntamente com a substância V. Os experimentos de antagonismo em placa de Petri envolvendo esses dois fungos revelaram, ainda, a presença de vários outros compostos na zona de inibição, que não correspondem às substâncias previamente isoladas de meio líquido, e podem ser responsáveis pelo efeito antagônico observado em meio semi-sólido. Os experimentos de atividade antagônica dos metabólitos produzidos pelos fungos evidenciaram que muitos compostos ativos, provavelmente, são produzidos em quantidades ínfimas, o que impossibilita a detecção por CLAE-DAD. Além disso, verificou-se que a substância I não possui atividade antifúngica significativa contra os fungos SS13, SS50 e SS77 e que a inibição de SS67 por SS77 ocorre devido à produção de compostos difusíveis em meio semi-sólido, e ainda, muito provavelmente, pela produção da substância III e IV em meio líquido, além de outros policetídeos. A produção de metabólitos secundários por fungos endofíticos deve ocorrer em consequência do papel ecológico que desempenham na natureza. Assim, a utilização de culturas mistas desses microrganismos deve induzir a produção de compostos que não seriam produzidos em condições não naturais. / The study of plant-microbe interactions has been of great interest over the past years. Currently, the interactions that occur among organisms that live in close relationship also have been receiving great attention because mutualistic and competitive forces may induce the production of new bioactive metabolites. Therefore, studying the interactions between endophytic microorganisms that colonize the same plant seems to be a promising strategy for obtaining chemically different substances that might also be bioactive. Through the utilization of mixed microbial cultures, this study contributed to knowledge of the relationship among the endophytic fungi SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) and SS84 (Phoma betae), isolated from the medicinal plant Smallanthus sonchifolius (yacon), and its role in the increase of the chemical diversity of microbial natural products, in order to identify anticancer secondary metabolites. For this aim, the fungi were grown in single and mixed cultures, both in liquid and semi-solid media. Different media, different approaches to establish the mixed cultures and different extraction methods were used. The extracts were analyzed chemically and biologically. After the fractionation, five compounds were isolated: aphidicolin (I), 3-deoxy-aphidicolin (II), stemphyperylenol (III), alterperylenol (IV) and alternariol monomethyl ether (V). Compounds I and II are terpene derivatives, while III, IV and V are polyketide derivatives. The substances I and II were produced by the fungus SS67, and the production of I apparently increased in liquid mixed cultures with SS13 and SS84, decreasing considerably in mixed culture with SS77. Due to the high cytotoxic activity of aphidicolin (I), the cultures of the fungus SS67 originated extracts highly active in the cytotoxicity assays in cancer cells. Substance III was only detected by HPLC-DAD in the mixed culture between the fungi SS67 and SS77, and the production of compound IV was higher in this mixed culture when compared to the single culture of the fungus SS77 (malt extract medium). Probably these compounds were produced by SS77 in response to the presence of SS67. The extract obtained during this mixed culture showed the highest cytotoxic activity against the cell line MDAMB-435 (breast cancer). In a subsequent fermentation in PDB medium, compound III was also isolated from the single culture of the fungus SS77 grown, along with compound V. Additionally, antagonism experiments in Petri dishes with these two fungi revealed the presence of several other compounds in the inhibition zone, which does not correspond to the substances previously isolated from the liquid medium, and may be responsible for the antagonistic effect observed in semi-solid medium. The experiments of antagonistic activity of metabolites produced by fungi showed that many active compounds are probably produced in very small quantities, making it impossible to detect by HPLC-DAD. Moreover, it was found that aphidicolin (I) did not have significant antifungal activity against the fungi SS13, SS50 and SS77 and that the SS67 inhibition by SS77 is due to the production of diffusible compounds in semi-solid medium, and likely, due to the production of compound III and IV in liquid medium, and other polyketides. The production of secondary metabolites by endophytic fungi probably occurs as a result of the ecological role they play in nature. Thus, the use of mixed cultures of these microorganisms may induce the production of compounds that would not be produced under unnatural conditions.

O estudo das interações planta-microrganismos tem sido de grande interesse ao longo dos últimos anos. Atualmente, as interações que ocorrem entre microrganismos que vivem em estreita relação também vêm merecendo grande atenção, pois forças competitivas e mutualísticas podem induzir a produção de novos metabólitos bioativos. Portanto, estudar interações existentes entre os microrganismos endofíticos que colonizam uma mesma planta parece ser uma estratégia promissora para a obtenção de substâncias quimicamente diferentes, eventualmente bioativas. Através da utilização de culturas mistas de microrganismos, o presente trabalho contribuiu para o conhecimento da relação existente entre os fungos endofíticos SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) e SS84 (Phoma betae), isolados da planta medicinal Smallanthus sonchifolius (yacon), e sua implicação no aumento da diversidade química de produtos naturais microbianos, com o intuito de se identificar metabólitos secundários anticancerígenos. Para isso, os fungos foram cultivados em culturas singles e mistas em meios de cultivo líquidos e semi-sólidos. Foram utilizados diferentes meios de cultura, diferentes maneiras de se estabelecer o cultivo misto e diferentes formas de extração. Os extratos foram analisados química e biologicamente. Após fracionamento, foram isoladas cinco substâncias: afidicolina (I), 3-desóxi-afidicolina (II), estenfiperilenol (III), alterperilenol (IV) e alternariol monometil éter (V), sendo as duas primeiras de origem terpênica e as outras de origem policetídica. As substâncias I e II foram produzidas pelo fungo SS67, sendo que a produção de I aparentemente aumentou nas culturas mistas líquidas com SS13 e SS84, diminuindo consideravelmente na cultura mista com SS77. Devido à afidicolina ser um composto altamente citotóxico, os cultivos do fungo SS67 originaram extratos muito ativos frente aos ensaios de citotoxicidade em células cancerígenas. A substância III, primeiramente, só foi detectada por CLAE-DAD na cultura mista dos fungos SS67 e SS77, e a produção da substância IV foi maior nessa cultura mista que na cultura simples do fungo SS77 (meio fermentativo de extrato de malte). Provavelmente esses compostos foram produzidos por SS77 em resposta à presença de SS67. O extrato obtido durante esse cultivo misto foi o que apresentou maior atividade citotóxica frente à linhagem celular MDAMB-435 (câncer de mama). Posteriormente, a substância III foi também isolada da cultura simples do fungo SS77 cultivado em outras condições (meio fermentativo PDB), juntamente com a substância V. Os experimentos de antagonismo em placa de Petri envolvendo esses dois fungos revelaram, ainda, a presença de vários outros compostos na zona de inibição, que não correspondem às substâncias previamente isoladas de meio líquido, e podem ser responsáveis pelo efeito antagônico observado em meio semi-sólido. Os experimentos de atividade antagônica dos metabólitos produzidos pelos fungos evidenciaram que muitos compostos ativos, provavelmente, são produzidos em quantidades ínfimas, o que impossibilita a detecção por CLAE-DAD. Além disso, verificou-se que a substância I não possui atividade antifúngica significativa contra os fungos SS13, SS50 e SS77 e que a inibição de SS67 por SS77 ocorre devido à produção de compostos difusíveis em meio semi-sólido, e ainda, muito provavelmente, pela produção da substância III e IV em meio líquido, além de outros policetídeos. A produção de metabólitos secundários por fungos endofíticos deve ocorrer em consequência do papel ecológico que desempenham na natureza. Assim, a utilização de culturas mistas desses microrganismos deve induzir a produção de compostos que não seriam produzidos em condições não naturais. / The study of plant-microbe interactions has been of great interest over the past years. Currently, the interactions that occur among organisms that live in close relationship also have been receiving great attention because mutualistic and competitive forces may induce the production of new bioactive metabolites. Therefore, studying the interactions between endophytic microorganisms that colonize the same plant seems to be a promising strategy for obtaining chemically different substances that might also be bioactive. Through the utilization of mixed microbial cultures, this study contributed to knowledge of the relationship among the endophytic fungi SS13 (Papulaspora immersa), SS50 (Fusarium oxysporum), SS67 (Nigrospora sphaerica), SS77 (Alternaria tenuissima) and SS84 (Phoma betae), isolated from the medicinal plant Smallanthus sonchifolius (yacon), and its role in the increase of the chemical diversity of microbial natural products, in order to identify anticancer secondary metabolites. For this aim, the fungi were grown in single and mixed cultures, both in liquid and semi-solid media. Different media, different approaches to establish the mixed cultures and different extraction methods were used. The extracts were analyzed chemically and biologically. After the fractionation, five compounds were isolated: aphidicolin (I), 3-deoxy-aphidicolin (II), stemphyperylenol (III), alterperylenol (IV) and alternariol monomethyl ether (V). Compounds I and II are terpene derivatives, while III, IV and V are polyketide derivatives. The substances I and II were produced by the fungus SS67, and the production of I apparently increased in liquid mixed cultures with SS13 and SS84, decreasing considerably in mixed culture with SS77. Due to the high cytotoxic activity of aphidicolin (I), the cultures of the fungus SS67 originated extracts highly active in the cytotoxicity assays in cancer cells. Substance III was only detected by HPLC-DAD in the mixed culture between the fungi SS67 and SS77, and the production of compound IV was higher in this mixed culture when compared to the single culture of the fungus SS77 (malt extract medium). Probably these compounds were produced by SS77 in response to the presence of SS67. The extract obtained during this mixed culture showed the highest cytotoxic activity against the cell line MDAMB-435 (breast cancer). In a subsequent fermentation in PDB medium, compound III was also isolated from the single culture of the fungus SS77 grown, along with compound V. Additionally, antagonism experiments in Petri dishes with these two fungi revealed the presence of several other compounds in the inhibition zone, which does not correspond to the substances previously isolated from the liquid medium, and may be responsible for the antagonistic effect observed in semi-solid medium. The experiments of antagonistic activity of metabolites produced by fungi showed that many active compounds are probably produced in very small quantities, making it impossible to detect by HPLC-DAD. Moreover, it was found that aphidicolin (I) did not have significant antifungal activity against the fungi SS13, SS50 and SS77 and that the SS67 inhibition by SS77 is due to the production of diffusible compounds in semi-solid medium, and likely, due to the production of compound III and IV in liquid medium, and other polyketides. The production of secondary metabolites by endophytic fungi probably occurs as a result of the ecological role they play in nature. Thus, the use of mixed cultures of these microorganisms may induce the production of compounds that would not be produced under unnatural conditions.

Los mecanismos que contribuyen a evitar la herbivoría inducen una respuesta selectiva negativa en los herbívoros, mientras que los que contribuyen a su tolerancia inducen una respuesta selectiva positiva en los mismos. De ahí que las plantas que los poseen se clasifiquen como de baja o de alta palatabilidad, respectivamente. Los mecanismos de evitación compiten por recursos para el crecimiento, lo cual limitaría la productividad de las especies de baja palatabilidad. Por otra parte los mecanismos de evitación disminuyen la calidad de la broza (mantillo y raíces muertas), lo que limitaría la velocidad de descomposición y mineralización de las especies de baja palatabilidad. El objetivo de la presente tesis fue comparar la productividad y la velocidad de descomposición y mineralización de dos especies nativas del Caldenal, una de alta palatabilidad (Poa ligularis) y la otra de baja palatabilidad (Stipa tenuissima). Complementariamente se midió la densidad de tejido, la longevidad, la eficiencia de reabsorción de nitrógeno (N) y la eficiencia de uso de N, a nivel foliar y radical. La metodología abarcó mediciones periódicas de la biomasa aérea y subterránea por el método de la cosecha; incubaciones de hojas, raíces y suelo en condiciones in situ y/o en condiciones de laboratorio; y determinaciones del contenido de N y/o fósforo en hojas, raíces y suelo. P. ligularis fue más productiva, tanto en la parte aérea como en la parte subterránea. S. tenuissima presentó tejidos más densos a nivel foliar y radical, y más longevos y con mayor eficiencia de reabsorción y de uso de nitrógeno a nivel radical. Las hojas de P. ligularis se descompusieron y liberaron nutrientes más rápidamente que las hojas de S. tenuissima, pero las raíces de ambas especies se descompusieron y liberaron nutrientes a un ritmo similar. El concentración de N inorgánico fue similar o mayor en el suelo subyacente a P. ligularis. La tasa de mineralización in situ fue similar o mayor en el suelo subyacente a S. tenuissima, pero la tasa de mineralización potencial de nitrógeno fue superior en el suelo subyacente a P. ligularis. El N retenido en la biomasa microbiana del suelo fue similar o mayor en S. tenuissima. Si bien P. ligularis produjo broza de más calidad y en mayor cantidad que S. tenuissima, dichas diferencias no se tradujeron en forma consistente en una mayor velocidad de descomposición y de liberación de nutrientes, ni en una mayor tasa de mineralización y de concentración de nitrógeno en el suelo in situ en la primera que en la última especie. La estrechez de las diferencias en la calidad de la broza, sumado a episodios de escasez de agua recurrentes que limitarían la expresión de tales diferencias, explicaría las inconsistencias en las diferencias entre la gramínea de alta y la de baja palatabilidad en la dinámica del N mineral en el suelo subyacente a las a las mismas. / Mechanisms that help to avoid herbivory induce a negative selective response in herbivores, whereas those who contribute to herbivory tolerance induce a positive selective response in them. Plants having these mechanisms are classified as palatable or unpalatable species, respectively. Avoidance mechanisms compete for growth resources, which would limit the productivity of unpalatable species. On the other hand, the mechanisms of avoidance diminish litter quality, which would limit decomposition and mineralization speed of unpalatable species. The objective of the present thesis was to compare productivity, and decomposition and mineralization speed of two species native to the Caldenal, a palatable (Poa ligularis) and an unpalatable (Stipa tenuissima) grass species. In addition, tissue density, longevity, nitrogen (N) resorption efficiency and N use efficiency at leaf and root level were measured. The methodology included periodic measurements of above - and belowground biomass by the harvest method; in situ and/or laboratory incubations of leaves, roots and soil samples; as well as determinations of the content of N and / or phosphorus in leaves, roots and soil samples. P. ligularis was more productive, both aboveground and belowground. S. tenuissima presented more dense leaf and root tissues. The later species presented also longer tissue longevity and higher N resorption and N use efficiencies at root level. The leaves of P. ligularis decomposed and liberated nutrients faster than the leaves of S. tenuissima, but the roots of both species decomposed and liberated nutrients to a similar rate. Inorganic N content was similar or higher in the soil under P. ligularis. In situ net N mineralization was similar or higher in the soil under S. tenuissima, but potential net N mineralization was always higher in the soil under P. ligularis. N retained in the soil microbial biomass was higher in the soil under S. tenuissima. Poa ligularis produced more litter and litter of higher quality than S. tenuissima. However, these differences did not consistently translated either in faster decomposition and nutrient release rate or higher in situ net N mineralization and soil N content. Limited variation between species in the chemical composition of litter in combination to frequent moisture limitation episodes, which tend to reduce the influence of species on nutrient dynamics, may explain the inconsistencies in the differences between the palatable and the unpalatable grass in mineral N dynamic in the soil under them.

Over the years, secondary metabolites have proven to be significant in the discovery of novel antibiotics and/or lead compounds. Various secondary metabolites have been reported to be produced by fungus of the genus, Alternaria. Here we report the isolation of secondary metabolites by the bioassay-guided fractionation of the ethyl acetate extract of fermentation cultures of the fungus, Alternaria tenuissima. This fungal strain was isolated from the soil underlying the lichen, Peltigera didactyla; and the lichen was collected from Wapusk National Park, a location in Northern Manitoba. The compounds isolated from the fungus include deoxyphomalone, dimethyl 4-methyl-2,6-pyridinedicarboxylate, stemphyperylenol and N-Methyl pyrrolidinone. Their structures were determined by comprehensive analysis of their spectroscopic data including FT-IR, mass spectrometry, 1D and 2D NMR; and their bioactivities were tested against E. coli cells. Some of the compounds demonstrated some bioactivity. The taxonomic identity of the fungus was confirmed by ITS sequencing of its ribosomal DNA.

Over the years, secondary metabolites have proven to be significant in the discovery of novel antibiotics and/or lead compounds. Various secondary metabolites have been reported to be produced by fungus of the genus, Alternaria. Here we report the isolation of secondary metabolites by the bioassay-guided fractionation of the ethyl acetate extract of fermentation cultures of the fungus, Alternaria tenuissima. This fungal strain was isolated from the soil underlying the lichen, Peltigera didactyla; and the lichen was collected from Wapusk National Park, a location in Northern Manitoba. The compounds isolated from the fungus include deoxyphomalone, dimethyl 4-methyl-2,6-pyridinedicarboxylate, stemphyperylenol and N-Methyl pyrrolidinone. Their structures were determined by comprehensive analysis of their spectroscopic data including FT-IR, mass spectrometry, 1D and 2D NMR; and their bioactivities were tested against E. coli cells. Some of the compounds demonstrated some bioactivity. The taxonomic identity of the fungus was confirmed by ITS sequencing of its ribosomal DNA.

<p>  </p> <p>Plant-parasitic nematodes are amongst the most important pathogens impacting crops. Potatoes and soybean are vital crops for rural livelihoods and essential for food security, but their cyst nematode parasites remain a significant constraint globally. <em>Globodera</em> and <em>Heterodera </em>species are amongst the most damaging and internationally recognized quarantine pests of these crops, causing up to 80% yield loss. Their second stage juveniles (J2s) penetrate the host plant root tips and establish a residence close to the vascular bundle from where they extract nutrients as they complete their life cycle. Restrictions of control chemicals have led to an urgent need for alternative control strategies for cyst nematodes. Biological control is a promising alternative control measure, and fungi possess many characteristics that could make them great biological control agents of cyst nematodes. Most nematode populations are thought to be regulated by their natural enemy community. It is unclear which fungi are best adapted as natural enemies of these parasites and how they might do this. This project aimed to (a) characterize and compare the mycobiome diversity of cyst nematode species; (b) isolate and characterize fungi associated with potato and soybean cyst nematodes (c) screen isolated fungi for their efficacy as biocontrol agents against cyst nematodes; and (d) describe any isolated fungi identified as new and having potential for cyst nematode antagonism. Cyst populations were collected from different regions and screened for fungi using culture-based methods. For our next-generation sequence data analysis, we found differences in fungal community assemblages between center of origin of the potato cyst nematode (Peru; South America) and the regions where these nematodes were introduced such as Europe and North America.  There was no significant difference in fungal community assemblages of cysts collected between the years 2019 and 2020. We characterized fungi associated with the cyst nematodes and found that the most frequently isolated genera were <em>Fusarium, Penicillium, Cylindrocarpon, Phoma, Aspergillus </em>and<em> Verticillium</em>. Filtrates from <em>Trichoderma</em> sp. 2, <em>Alternaria alternata, </em>and <em>Fusarium acaciae-mearnsii </em>were toxic on SCN eggs while <em>Purpureucillium lilacinum, Fusarium proliferatum</em> and <em>Aureobasidium</em> <em>ellingtonae </em>sp. nov. were toxic on PCN eggs and juveniles. A new species of <em>Aureobasidium</em> isolated from the potato cyst nematode (<em>Globodera ellingtonae</em>) for the first time, and having biocontrol potentials against this nematode, was also identified, and described. </p>