Os fungos endofíticos têm sido reconhecidos pela sua grande importância para as plantas hospedeiras, pois podem conferir proteção contra insetos herbívoros e patógenos, promover o crescimento vegetal, além de produzir metabólitos secundários com atividades biológicas diversas, entre outros. A cana-de-açúcar é uma cultura de grande importância social e econômica no Brasil, especialmente para o estado de São Paulo. Ultimamente esta cultura vem recebendo especial atenção devido ao crescente aumento da demanda de matéria prima, principalmente em função do acréscimo no consumo de etanol como biocombustível. Fungos do gênero Penicillium habitam os tecidos e a rizosfera de cana-de-açúcar, onde podem estabelecer associações mutualísticas com a planta e conferir diversos benefícios. Dentro deste contexto, estudos que avaliem a interação de Penicillium spp. com cana-de-açúcar são bastante promissores para geração de conhecimentos que auxiliem na otimização da agricultura. Dessa forma, o presente trabalho teve como objetivos a avaliação do potencial biotecnológico dos endofíticos de raiz e da rizosfera, do gênero Penicillium, pertencentes à comunidade fúngica de cana-de-açúcar, por meio de ensaios de antagonismo, produção de enzimas, solubilização de fosfato inorgânico e produção de ácido indol acético; assim como o estudo da interação de um isolado de P. pinophilum com cana-de-açúcar a partir do desenvolvimento de um sistema de transformação genética mediada pela bactéria Agrobacterium tumefaciens. Tanto a análise da atividade antimicrobiana como a produção de metabólitos apresentaram extensa variação fisiológica entre os isolados avaliados. Um isolado da espécie P. pinophilum (linhagem 44) foi escolhido para ser usado na transformação genética por mostrar-se superior estatisticamente em relação aos demais isolados nos ensaios anteriores. Para aumento da eficiência deste sistema de transformação foram avaliados diferentes parâmetros, dentre eles: tempo de co-cultivo (24 e 48 horas), concentração do indutor acetoseringona (200 M e 400 M) e tipos de membrana (papel filtro e náilon). O sistema de agrotransformação apresentou alta eficiência (482 transformantes por 107 conídios), gerando uma elevada quantidade de transformantes resistentes à higromicina B e expressando GFP. Dentre os parâmetros avaliados, a combinação que deu origem aos melhores resultados de transformação envolveu o co-cultivo por 48 horas sobre membrana de náilon, em meio de cultura contendo 200 M de acetoseringona. A interação fungo-planta foi avaliada a partir da inoculação de P. pinophilum linhagem selvagem e transformantes, em plântulas de cana-de-açúcar, seguida da análise por microscopia óptica de epifluorescência e reisolamento. Os resultados revelaram a natureza não patogênica desse fungo, uma vez que ele foi capaz de colonizar endofiticamente cana-de-açúcar e persistir nas raízes desta planta, sem levar ao desenvolvimento de qualquer sintoma de doença. Além disso, os ensaios de agrotransformação deram origem a uma biblioteca com mil e cem transformantes insercionais, o que constitui uma ferramenta importante para o estudo molecular do metabolismo secundário desse fungo endofítico e poderá contribuir para o entendimento da interação do complexo fungo-cana-de-açúcar, possibilitando no futuro a sua aplicação no melhoramento vegetal e exploração do seu potencial biotecnológico. / Endophytic fungi have been recognized for its great importance for the host plants, they may provide protection against herbivores and pathogens, promote plant growth, and produce secondary metabolites with biological activity, among other benefits. Sugarcane is a socially and economically important crop in Brazil, especially for the state of São Paulo. Lately, this culture has received special attention due to the growing demand for raw materials, mainly due to the increase in consumption of ethanol as a biofuel. Fungi Penicillium inhabit the tissues and rhizosphere of sugarcane, where they can establish mutualistic associations with the plant and provide several benefits. Within this context, studies evaluating the interaction of Penicillium spp. with sugar cane are very promising to generate knowledge in order to assist in the agriculture optimization. Thus, this study aimed to evaluate the biotechnological potential of endophytic Penicillium from root and rhizosphere, belonging to the fungal community of sugarcane, through tests of antagonism, enzyme production, solubilization of inorganic phosphate and indole acetic acid production, as well as studying the interaction of an isolate of P. pinophilum with sugarcane using the development of a system for genetic transformation mediated by Agrobacterium tumefaciens. Both the analysis of antimicrobial activity and the production of metabolites showed extensive physiological variation among isolates. An isolate of the species P. pinophilum (strain 44) was chosen to be used in genetic transformation for being statistically superior than the other strains in previous trials. Different parameters were evaluated to increase the efficiency of this transformation system, among them: co-culture time (24 and 48 hours), concentration of the inducer acetosyringone (200 µM and 400 µM) and types of membrane (filter paper and nylon). Agrotransformation system showed high efficiency, generating a high amount of hygromycin B resistant transformants that expressed GFP. Among the factors evaluated, the combination that showed the best results involved the transformation with a co-cultivation for 48 hours on a nylon membrane, in culture medium containing 200 µM of acetosyringone. The plant-fungus interaction was assessed from the inoculation of wild type and transformants P. pinophilum in seedlings of sugarcane followed by analysis by epifluorescence microscopy and reisolation. Results revealed the non-pathogenic nature of this fungus, since it was capable of endophytically colonize sugarcane and persisted in the roots of this plant, without developing any symptoms of illness. In addition, agrotransformation tests gave rise to a library with a thousand and one hundred insertional transformants, which is an important tool for molecular study of secondary metabolism of endophytic fungus, and may contribute to the comprehension of the complex interaction of fungus-sugarcane, allowing its future application in plant breeding and exploitation of their biotechnological potential.

[EN] Citrus spp. (Rutaceae) are the most widely produced fruits and they are grown in over a hundred countries. During postharvest, major losses are caused by fungal diseases, that for decades have been controlled by treatments with synthetic fungicides. However, their use is continuously more restricted by distributors and supermarket chains, which lower the Maximum Residue Level (MRL) to one third of the authorized or even ban the use of certain fungicides. This has loss of efficacy of treatments and development of resistant strains of pathogens. Alternatives are necessary to substitute or combine the use of synthetic fungicides. The objective of the present work was to evaluate alternative treatments against green and blue molds, caused by Penicillium digitatum (Pers.: Fr.) Sacc. and Penicillium italicum Wehmer, respectively. Food additives and GRAS substances were tested in in vivo preliminary tests against green and blue molds. Potassium sorbate (PS) (Chapter 2) and sodium benzoate (SB) (Chapter 1) were the most effective. 60-s dips in 3% (w/v) SB above 50°C reduced by 90% green and blue mold incidence on 'Valencia' oranges after 7 days at 20°C. This treatment was also effective on 'Lanelate' oranges, 'Fino' lemons and 'Ortanique' mandarins, but not on 'Clemenules' mandarins. Heated solutions combining SB with low doses of imazalil (IMZ) were synergistic. In semi-commercial trials with 'Marisol' Clementine mandarins, the combination PS+IMZ at 20°C allowed a significant reduction of the IMZ doses still controlling green mold. A new colorimetric method that employed extraction of PS from macerated fruit, followed by reaction with 2-thiobarbituric acid (Chapter 3) showed that in oranges treated with PS and stored at 15°C, residues declined initially rapidly and later more slowly, until residues stopped declining after 6 d. A brief double-dip rinse in tap water, immediately after immersion of lemons in a 2% (w/v) PS, removed more than 90% of PS residue. Exposure at 33ºC with 15 kPa CO2 for 24 h or 30 kPa O2 for 48 h effectively controlled both green and blue molds after 7 days of incubation at 20ºC, but control of both diseases lowered after 15 days (Chapter 4). Combining PS dip treatments with gas exposure showed a synergistic effect, that maintained the efficacy of combined treatments during 15 days. In cold storage, after 14, 28, and 42 days, green mold incidence on 'Clemenules' and 'Ortanique' mandarins treated with PS dips and 30kPa O2 for 48h was reduced by 100, 96 and 68%, and 100, 97 and 79%, respectively. Again, a synergistic effect between treatments was observed (Chapter 5). Postharvest green mold and blue molds were effectively controlled by fumigation of lemons and oranges for 6 h at 22ºC with two applied dosages of 3000 ¿L L-1 of ammonia that was injected initially and again 2 h later (Chapter 6). Ammonia fumigation controlled an IMZ-resistant isolate of P. digitatum. When fruit were first immersed in 10 or 30 mg L-1 IMZ (about 10% of typical commercial rates) before ammonia fumigation, a single fumigation with 1500 ¿L L-1 of ammonia was adequate to control both diseases and the increase in effectiveness was additive or synergistic. All these treatments, at different degrees, could replace synthetic fungicides or augment IMZ performance in citrus postharvest decay management. The development of green and blue molds on the most commercially important citrus species and cultivars was assessed for four consecutive harvest seasons and associated with fruit quality parameters (Chapter 7). Fruit susceptibility of the cultivars to green mold in increasing order was Nova, Sanguinelli, Ortanique, Lanelate, Navelate, Fortune, Clemenules, Valencia, W. Navel, Oronules, Clemenpons, Marisol, Salustiana, Hernandina, and Nadorcott. Peel characteristics were the most influencing quality parameters on the susceptibility of citrus fruit to green and blue molds. / [ES] Los cítricos (Citrus spp. fam. Rutaceae) son la fruta más consumida a nivel mundial y se cultivan en más de un centenar de países. Durante el manejo en poscosecha, la mayor parte de las pérdidas se deben a enfermedades fúngicas, que durante décadas se han controlado mediante tratamientos con fungicidas de síntesis. Sin embargo, su uso está continuamente más restringido por parte de los distribuidores y cadenas de supermercados, que reducen el Límite Máximo de Residuos (LMR) a un tercio del establecido o incluso prohíben el uso de ciertos fungicidas. Esto ha provocado la pérdida de eficacia de los tratamientos y el desarrollo de cepas de patógenos resistentes a los fungicidas. Se necesitan pues alternativas para sustituir o combinar con el uso de fungicidas de síntesis. El objetivo de este trabajo fue evaluar tratamientos alternativos contra las podredumbres verde y azul, causadas por Penicillium digitatum (Pers.: Fr.) Sacc. y Penicillium italicum Wehmer, respectivamente. Aditivos alimentarios y sustancias GRAS se probaron en test in vivo preliminaries contra los mohos verde y azul. El benzoato sódico (BS; Capítulo 1) y el sorbato potásico (SP; Capítulo 2) fueron los más eficaces. Baños de 60 s en 3% (p/v) BS sobre 50°C redujeron en un 90% la incidencia de las podredumbres verde y azul en naranjas 'Valencia Late' tras 7 días a 20°C. Estos tratamientos fueron también efectivos en naranjas 'Lanelate', limones 'Fino' y mandarinas híbridas 'Ortanique', pero no en mandarinas 'Clemenules'. La combinación de baños calientes de BS con dosis bajas de imazalil (IMZ) fueron sinérgicas. Un nuevo método colorimétrico que empleó la extracción del SP de la fruta macerada, seguido de una reacción con ácido 2-tiobabitúrico (Capítulo 3) mostró que en naranjas tratadas con SP almacenadas a 15°C, los residuos descendieron rápidamente al inicio y lentamente más tarde, hasta que dejaron de descender tras 6 días. Un doble aclarado rápido con agua corriente inmediatamente después de la inmersión de limones en SP al 2% (p/v), eliminó más del 90% del residuo de SP. Los choques gaseosos a 33ºC y 15kPa CO2 durante 24 h o 30 kPa O2 durante 48 h controlaron de manera eficaz las podredumbres verde y azul tras 7 días de incubación a 20ºC, pero el control de las dos podredumbres descendió tras 15 días (Capítulo 4). En almacenamiento en frío, tras 14, 28 y 42 días, la incidencia de moho verde en mandarinas 'Clemenules' y 'Ortanique' tratadas con baños con SP y 30kPa O2 durante 48h se redujo en un 100, 96 y 68%, y 100, 97 y 79%, respectivamente. De nuevo, una fuerte sinergia se observó entre los tratamientos (Capítulo 5). Las podredumbres verde y azul se controlaron eficazmente mediante la fumigación de limones y naranjas durante 6 h a 22ºC con dos dosis de 3000 ¿L L-1 amoniaco, inyectadas al inicio del tratamiento y 2 h después (Capítulo 6). El tratamiento con amoniaco controló una cepa de P. digitatum resistente a IMZ. Cuando la fruta se bañó en 10 o 30 mg L-1 IMZ (un 10% de la dosis comercial) antes de la fumigación con amoniaco, una sola aplicación con 1500 ¿L L-1 amoniaco fue suficiente para controlar las dos podredumbres, y el incremento en la eficacia fue aditivo o sinérgico. Todos estos tratamientos, a diferentes niveles, podrían sustituir a los fungicidas de síntesis o aumentar la eficacia de los tratamientos con IMZ en el manejo poscosecha de los cítricos. El desarrollo de las podredumbres verde y azul en las especies y variedades de cítricos más importantes comercialmente se evaluó durante cuatro campañas consecutivas y se relacionó con los parámetros de calidad de la fruta (Capítulo 7). La susceptibilidad de los cultivares en orden creciente fue Nova, Sanguinelli, Ortanique, Lanelate, Navelate, Fortune, Clemenules, Valencia, W. Navel, Oronules, Clemenpons, Marisol, Salustiana, Hernandina, y Nadorcott. Las características de la corteza fueron los parámetros de calidad / [CAT] Els cítrics (Citrus spp. fam. Rutaceae) son la fruita més consumida a nivell mundial i es cultiven en un centenar de països. Durant la poscollita, la major part de les pèrdues es deuen a malalties fúngiques, que durant dècades s'han controlat mitjançant tractaments amb fungicides de síntesi. No obstant, l'ús d'aquestes pràctiques està contínuament més restringit per part dels distribuïdors y cadenes de supermercats, que redueixen el Límit Màxim de Residus (LMR) a un terç del permès per la legislació o inclús prohibeixen l'ús d'alguns fungicides. Açò ha provocat la minora de l'eficàcia dels tractaments poscollita i el desenvolupament de soques dels patògens resistents als fungicides. Es necessari per lo tant trobar i implementar alternatives per a substituir o combinar l'ús de fungicides de síntesi. L'objectiu d'aquest treball ha sigut avaluar tractaments alternatius contra les podridures verda i blava, causades per Penicillium digitatum (Pers.: Fr.) Sacc. i Penicillium italicum Wehmer, respectivament. Additius alimentaris i substàncies GRAS es provaren en test in vivo preliminars contra les podridures verda i blava. El benzoat sòdic (BS; Capítol 1) i el sorbat potàsic (SP; Capítol 2) foren els més eficaços. Solucions calentes de BS combinades amb baixes dosis d'imazalil (IMZ) foren sinèrgiques. Esta combinació va reduir l'incidència de les podridures verda i blava en quasi el 100% en taronges 'Valencia Late' després de 8 setmanes a 5°C i 7 dies a 20°C. En assajos semicomercials mandarines 'Marisol', la combinació SP+IMZ a 20°C va permetre una baixada significativa de la dosis de IMZ sense perdre eficàcia contra la podridura verda. Un nou mètode colorimètric que inclou l'extracció del SP de la fruita macerada, seguit d'una reacció amb àcid 2-tiobabitúric (Capítol 3) mostrà que en taronges tractades amb SP conservades a 15°C, els residus descendiren ràpidament al inici i lentament mes tard, fins que deixaren de descendir després de 6 dies. Un doble rentat ràpid amb aigua corrent immediatament després de d'immersió de llimones en SP al 2% (p/v), eliminà més del 90% del residu de SP. Els xocs gasosos a 33ºC i 15kPa CO2 durant 24 h o 30 kPa O2 durant 48 h controlaren de manera eficaç les podridures verda i blava després de 7 dies d'incubació a 20ºC, però el control de les dos podridures descendí després de 15 dies (Capítol 4). La combinació de banys amb SP i xocs gasosos mostrà un efecte sinèrgic, que mantingué l'eficàcia dels tractaments combinats durant 15 dies. En conservacions en fred, després de 14, 28 i 42 dies, d'incidència de podridura verda en mandarines 'Clemenules' i 'Ortanique' tractades en banys amb SP i 30kPa O2 durant 48h es reduí en un 100, 96 i 68%, i 100, 97 i 79%, respectivament. De nou, una forta sinergia s'observà entre els tractaments (Capítol 5). Les podridures verda i blava es controlaren eficaçment mitjançant la fumigació de llimones i taronges durant 6 h a 22ºC amb dos dosis de 3000 ¿L L-1 amoníac, injectades al inici del tractament i 2 h després (Capítol 6). El tractament amb amoníac controlà una soca de P. digitatum amb un alt nivell de resistència a IMZ. Tots estos tractaments, a diferents nivells, podrien substituir als fungicides de síntesis o augmentar l'eficàcia dels tractaments amb IMZ en el maneig poscollita dels cítrics. El desenvolupament de les podridures verda i blava en les especies y varietats de cítrics més importants comercialment s'avaluà durant quatre campanyes consecutives i es relacionà amb els paràmetres de qualitat de la fruita (Capítol 7). La susceptibilitat dels distints cultivars en ordre creixent fon Nova, Sanguinelli, Ortanique, Lanelate, Navelate, Fortune, Clemenules, Valencia, W. Navel, Oronules, Clemenpons, Marisol, Salustiana, Hernandina, i Nadorcott. Les característiques de la pell foren els paràmetres de qualitat més influents en la susceptibilitat dels cítrics a les pod / Montesinos Herrero, CI. (2016). Evaluation of alternatives to synthetic fungicides to control citrus postharvest diseases [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/63662 / TESIS

To assess the effects of gamma radiation at low doses, spores of Penicillium expansum NRRL 971 and Penicillium patulum NRRL 989 were exposed to doses of 0, 50, 100, 150, and 200 krads. The amount of patulin for each culture was quantitatively determined and growth at each dose was evaluated. The results showed a 31% increase in the amount of patulin synthesis after a dose of 150 krads for Penicillium expansum At 200 krads, both fungi showed low levels of patulin production and inhibited growth. Eight color mutants of P. expansum and eight color mutants of P. patulum were produced by exposing spores of each fungus to doses of 150 and 200 krads of gamma radiation. An evaluation of patulin production and growth for each mutant showed that color mutation could result in mutants with an enhanced ability to grow or produce increased levels of patulin. When grown at a subatmospheric pressure of 320 mm Hg, P. expansum produced 30% more patulin per flask, when compared to control cultures. At 160 mm Hg, P. expansum showed a 75% decrease over the control cultures. P. patulum was greatly affected by the temperature of 15℃ and showed decreases both in growth and patulin production. Effective control of growth and patulin synthesis was found for these fungi at 160 mm Hg.

Recentemente, l’ocratossina A (OTA) è stata segnalata in formaggio grattugiato confezionato: si ritiene sia stata prodotta dallo sviluppo di alcuni funghi durante il periodo di maturazione. Gli studi presentati in questa tesi hanno avuto lo scopo di migliorare la conoscenza dei funghi, Aspergilli e Penicilli, associati al formaggio di tipo grana. Si è dunque provveduto alla loro identificazione tramite approccio multifasico, eseguendo anche prove ecologiche per definire i fabbisogni delle specie presumibilmente dominanti. Obiettivo finale sarà quello di predire il rischio di contaminazione da micotossine alle condizioni ambientali di stagionatura del prodotto. Aspergillus puulaauensis e alcune specie del genere Penicillium (P.crustosum and P. solitum) sono state rilevate con maggior frequenza, mentre non sono state rilevate nei campioni di crosta le specie P. nordicum e P. verrucosum. In ogni caso, le condizioni ambientali dei locali di maturazione (15-22°C; 72-88% UR) sono risultate favorevoli allo sviluppo di queste ultime, dando adito a preoccupazione circa una possibile contaminazione da OTA; tuttavia, il ruolo di altre specie micotossigene, ad esempio P. crustosum, non va sottovalutato. Le azioni di contrasto verso questi funghi micotossigeni dovrebbero, innanzitutto, ridurre l’inoculo presente nell’aria: l’ozono sembra efficace in tal senso; inoltre, l’utilizzo della luce blu potrebbe ridurre notevolmente la crescita fungina sulla superficie dei formaggi e delle scaffalature. Le informazioni fornite in questa tesi saranno utili ai produttori e agli altri operatori della filiera di questo prodotto, aiutandoli ad attuare una miglior gestione del rischio e a garantire un prodotto sicuro e di elevata qualità ai consumatori. / Ochratoxin A (OTA) was detected a few years ago in Italian packed grated cheese, and supposed to be produced by fungal growth during cheese ripening. The works managed and presented in this thesis aimed to improve knowledge about mycotoxigenic Aspergilli and Penicillia associated to Italian grana cheese. A multiphasic approach was applied for their identification and ecological trials were organised to define fungal needs of the expected dominant species. The objective was to predict the risk of mycotoxin contamination during the ripening condition of grana cheese. Aspergillus puulaauensis and some Penicillium spp (P.crustosum and P. solitum) were the prevalent species associated to grana cheese, while neither P. nordicum, nor P. verrucosum were detected in cheese rind. However, the environmental conditions of the ripening rooms (15-22°C; 72-88% RH) are favourable to P. nordicum and P. verrucosum, reinforcing concern about possible OTA contamination, but other mycotoxigenic species such as P. crustosum must not be neglected. Actions against those mycotoxigenic fungi should mainly reduce total inoculum in the environmental air: ozone is reported to be effective in this sense; moreover, blue light may significantly reduce fungal growth on cheese and shelves surfaces. Information provided therein will be useful for producers and stakeholders to perform a better risk management and guarantee a safe, high-quality product to consumers.

Les Penicilliums sont des champignons filamenteux appartenant au genre Ascomycota. Ces champignons ont été utilisés par l’homme pour la production de nourriture depuis des siècles. Plus récemment, ils ont aussi été utilisés dans l’industrie biotechnologique pour la production de composés chimiques d’intérêts pharmaceutiques. Certaines espèces de Penicillium sont par ailleurs des moisissures contaminants certains aliments, d’autres sont des pathogènes de plantes, y compris de certains fruits. Leur génomique est globalement peut connue. Dans cette étude, nous avons analysé les génomes de deux espèces nouvellement séquencées, Penicillium roqueforti et Penicillium camemberti. Nous reportons ici le développement d’une nouvelle méthodologie pour l’amélioration et la validation d’assemblage de génomes en utilisant une technologie permettant l’observation de molécules d’ADN unique, le Peignage Moléculaire. En utilisant cette méthode, nous avons amélioré l’assemblage de Penicillium roqueforti. Ce manuscrit décrit aussi de multiples occurrences d’un transfert horizontal d’un ilot génomique de plus de cinq cent kilobases entre plusieurs Penicillium. Ce cas de transfert horizontal indique une fréquence d’échange latéral de matériel génétique plus forte qu’attendue. Enfin nous présentons un inventaire préliminaire du potentiel génomique pour la production de métabolites secondaires dans ces importants Penicillium alimentaires. / Penicillium are filamentous fungi belonging to the Ascomycota genus. Penicillium species have been used by Man for centuries in food making processes. More recently they have also been used in the biotechnology industry for the production of compounds of pharmaceutical interest. Some Penicillium species are food spoilage agents, pathogens of plants including fruits. Aspects of their genomics are largely unknown. In this study, we analysed the genomes of two newly sequenced species, Penicillium roqueforti and Penicillium camemberti. Here we report the development of a new methodology for improving and validating genome assembly using an original single DNA molecule technology, Molecular Combing. Using this methodology we were able to produce a high quality genome assembly of Penicillium roqueforti. This work also reports the multiple and recurrent horizontal transfer of a large genomic island of over half a megabase between several Penicillium species. This horizontal transfer indicates a higher frequency of lateral genetic exchange between cheesemaking fungi than previously expected. Finally, we present an early assessment of the genomic potential for secondary metabolite production in these important food associated penicilliums.

O ambiente marinho apresenta uma rica biodiversidade e, por ser ainda pouco explorado configura-se como uma fonte potencial de novos organismos da flora e fauna marinhas, o que possibilita a descoberta de estruturas distintas e biologicamente ativas. Microrganismos marinhos como cianobactérias e fungos possuem um metabolismo secundário rico, o qual produz substâncias bioativas. Em virtude do exposto, um dos objetivos deste trabalho foi avaliar química e biologicamente as linhagens de cianobactérias marinhas Cyanobium sp. CENA178, Cyanobium sp. CENA181 e Oxynema sp. CENA135 provenientes de manguezais da Ilha do Cardoso, do Estado de São Paulo. Foram identificados os aminoácidos alanina, treonina e valina presentes na fração ACN:H2O (40:60) da linhagem Oxynema sp. CENA135. Por meio das análises realizadas em CG-EM, a fração n-hexano/acetato de etila (9:1) da linhagem Cyanobium sp. CENA178 apresentou os componentes: 2,4-bis (1,1-dimetiletil) fenol, (Z)-7-hexadeceno 6,10,14-trimetil-2-pentadecanona e eicosano; a fração n-hexano da linhagem Cyanobium sp. CENA181 apresentou como componente majoritário o neoftadieno, o qual também foi encontrado na fração acetato de etila da linhagem Oxynema sp. CENA135, juntamente com os componentes majoritários: heptadecano, [R-[R*,R*-(E)]]- 3,7,11,15-tetrametil-2-hexadeceno e octadecino. Com relação à triagem biológica, as linhagens Cyanobium sp. CENA178 e Cyanobium sp. CENA181, apresentaram atividade anticolinesterásica moderada e fraca, respectivamente. Concomitantemente foi realizada uma co-cultura entre a linhagem de cianobactéria Cyanobium sp. CENA181 e o fungo endofítico marinho Penicillium decaturense, onde foi isolada do extrato bruto do fungo P. decaturense a substância 10,11-deidrocurvularina e como produto da co-cultura foi obtida a substância curvularina. Deste modo, foi demonstrado que houve interação química entre os microrganismos devido a produção de diferentes metabólitos secundários. Adicionalmente, foram investigadas nove linhagens de cianobactérias do gênero Symploca quanto à análise química e biológica, provenientes do Panamá, Samoa Americana e uma da coleção de cultura de Pasteur (PCC8002). Por meio da análise de redes moleculares (molecular networking) foram identificadas três substâncias conhecidas: apratoxina A; palmiramida A e curacina D. Das frações A2143 E e F foi isolada uma substância inédita denominada de caracolamida A. Da fração G foi isolada uma substância conhecida pertencente à classe dos polimetoxi-alquenos isotáticos denominada de 4,6,8,10,12,14,16,18,20,22-decametóxi-heptacos-1-eno. A substância inédita caracolamida A foi comparada com o padrão feniletilamina e ambas demonstraram ter atividade neuromoduladora em concentrações subnanomolares, possuindo efeitos similares na oscilação e frequência dos canais de Ca2+. Os resultados apresentados neste trabalho acrescentam dados químicos e biológicos às espécies estudadas e enriquecem a área de Produtos Naturais Marinhos. / The marine environment contains a rich biodiversity and, since it is relatively underexplored, is a great source for finding new organisms including marine flora and fauna. This enables the discovery of chemicals with distinct and structures and biological activity. Marine microorganisms, such as cyanobacteria and fungi, have rich secondary metabolism, which yield biologically active molecules. Accordingly, one aim of this work was to evaluate the chemical profiles and biological activities of the compounds isolated from marine cyanobacteria strains Cyanobium sp. CENA178, Cyanobium sp. CENA181 and Oxynema sp. CENA135 from Ilha do Cardoso mangrove, State of São Paulo. The results indicate that it would help to adopt different laboratory cultivation methods for growing cyanobacteria strains to mimic the natural habitat and increase the opportunity to obtain new secondary metabolites. Aminoacids alanine, threonine and valine were identified of the fraction ACN:H2O (40:60) of the strain Oxynema sp. CENA135. Through the analyzes performed in CG-MS, the hexane/ethyl acetate (9:1) fraction of the strain Cyanobium sp. CENA178 showed the components: 2,4-bis (1,1-dimethylethyl) phenol, (Z)-7-hexadecene, 6,10,14-trimethyl-2-pentadecanone and eicosane. From the hexane fraction of Cyanobium sp. CENA181, neophytadiene was observed as the major component, and this was also found in the ethyl acetate fraction of Oxynema sp. CENA135 strain along with heptadecane, 2-hexadecene, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]] and 1-octadecyne. Cyanobium sp. CENA178 and Cyanobium sp. CENA181 showed moderate and weak anticholinesterase activity, respectively. Simultaneously, a co-culture was performed using Cyanobium sp. CENA181 and the marine endophytic fungus, Penicillium decaturense. The compound 10,11-dehydrocurvularin was isolated from the crude extract of P. decaturense, and curvularin from only the co-culture. Therefore, it was clear that the microorganisms exhibited an interaction leading to the different production of secondary metabolites. Nine species of cyanobacteria of the genus Symploca from Panama, American Samoa and one from Pasteur culture collection (PCC8002) were investigated to yield new natural products. Through molecular networking analysis three known compounds were identified: apratoxin A; palmiramide A and curacin D. Caracolamide A is a new compound isolated from fractions A2143 E and F. A known compound isolated from the same organism is called 4,6,8,10,12,14,16,18,20,22-decamethoxy-heptacos-1-ene, and it is an isotactic polymethoxy-1-alkene. The new compound caracolamide A demonstrated neuromodulatory activity at subnanomolar concentrations, and displayed similar effects as a phenylethylamine standard on the oscillation amplitude and frequency in Ca2+ channels. The results presented in this work provide chemical and biological information about the species studied, and enrich marine natural products research

No presente estudo, duas espécies de fungos isoladas do ambiente marinho tiveram seus extratos brutos ativos contra Staphylococcus aureus e Candida albicans para uma espécie de P. citrinum e, atividade citotóxica e contra Mycobacterium tuberculosis para uma espécie de P. oxalicum. Estas foram estudadas com a finalidade de otimizar suas condições de crescimento para produção de metabólitos secundários. Foram realizadas análises multivariadas utilizando planejamento fatorial fracionário. Foram obtidas duas condições ótimas de crescimento para as duas linhagens de Penicillium. O crescimento em maior volume em condições de cultura otimizadas para P. oxalicum permitiu observar a presença de três metabólitos secundários no extrato do meio de cultura. Dois puderam ser isolados e identificados: a meleagrina 52 e a oxalina 26. A metodologia utilizada para se obter uma maior quantidade de metabólitos secundários proporcionou, ainda, o incremento em cerca de 150% na área do pico da oxalina. A partir do crescimento em maior volume em condições de cultura otimizadas para P. citrinum foi possível observar presença de pelo menos onze diferentes metabólitos na análise dos extratos obtidos do meio de cultura. Foi possível identificar e isolar quatro compostos já conhecidos: a (8E)-1-(2,3-diidropirrol-1-il)-2-metildec-8-eno-1,3-diona 56; a 1-(2,3-diidropirrol-1-il)-2-metildecano-1,3-diona 58; a 2-((E)-hept-5-enil)-6,7,8,8a-tetraidro-3-metilpirrolo[2,1-b][1,3]oxazin-4-ona 59 e a citrinina 31. Também foram isolados dois novos alcalóides indólicos contendo um grupo nitro na molécula que foram nomeadas de citrinalinas A 60 e B 62. Foi realizado, também, um estudo com P. citrinum visando a maior produção das citrinalinas, que possibilitou o incremento na produção da citrinalina B. / In this study, two species of fungi isolated from the marine environment had their active extracts against Staphylococcus aureus and Candida albicans to a strain of P. citrinum, and activity cytotoxic and against Mycobacterium tuberculosis to a strain of P. oxalicum. This studied aims the optimization their growth conditions for the production of secondary metabolites. Multivariate analysis using a fractional factorial design were used to establish optimal growth conditions for both Penicillium strains. Two optimal growth conditions were obtained for both Penicillium strains. A largest growth volume of P. oxalicum using optimized conditions enabled the detection of three secondary metabolites in the culture media crude extract. Two of such compounds were isolated and identified: meleagrin 52 and oxaline 26. The methodology used to increase the production of secondary metabolites by P. citrinum enabled an increase of 150% in the peak area of oxaline. A largest growth volume of P. citrinum led to the detection of eleven different metabolites in the culture media. Four of these compounds were isolated and identified as the known (8E)-1-(2,3-dihydropyrrol-1-yl)-2-methyldec-8-ene-1,3-dione 56; the 1-(2,3-dihydropyrrol-1-yl)-2-methyldecane-1,3-dione 58; the 2-((E)-hept-5-enyl)-6,7,8,8a-tetrahydro-3-methylpyrrolo[2,1-b][1,3]oxazin-4-one 59 and citrinin 31. Two new indole alkaloids containing a nitro group were also isolated and identified, named citrinalins A 60 and B 62. A further study with P. citrinum aiming an enhanced production of citrinalins allowed a significant increase in the production of citrinalin B.

Made available in DSpace on 2015-03-26T13:39:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 5369129 bytes, checksum: 9b335fdfaa7871edcc12363d6834d034 (MD5) Previous issue date: 2010-02-24 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The green mold, caused by Penicillium digitatum, is a major disease in the postharvest of citrus fruit. In this work the effects of alternatives to agrochemicals in the control of green mold in 'Ponkan' tangerines and 'Valencia' oranges were tested. Physiologically mature fruits were superficially wounded in the equatorial region with a tube of 9 mm in diameter filled with needles and inoculated with a conidial suspension of P. digitatum at a concentration of 1x106 conidia/mL. After air drying, they were sprayed to full wetting with distilled water (control), distilled water + 8 mL/L Tween 20, imazalil (200 mL EC/100 Magnate ® 500 L of water), thiabendazole (103 mL Tecto ® SC/100 L of water), methyl thiophanate (70 g Cercobin® 700 WP/100 L of water), chitosan 10 mL/L of water or essential oils of garlic 10 mL/L of water, clove 5 mL/L water, neem 10 mL/L of water, long pepper 5 mL/L of water and citric biomass 10 mL/L of water. To the essential oils and chitosan were added 8 mL/L Tween 20. Then the fruits were packed in groups of three in polystyrene trays and kept at 21 ± 1 º C and 85-90% RH for 8 ('Ponkan' tangerine) or 7 days ('Valencia' orange). The fruits were analyzed for incidence and severity of illness, incubation period and latent period of the fungus, loss of fresh weight and CO2 production. In the case of 'Ponkan' tangerine, citric biomass was able to reduce the incidence of 76.3% compared to the control, in the third day, and also reduce the severity in 44%, in the fourth day. For the incubation period, except for fruits treated with garlic oil, there is formation of distinct groups of averages between the control and those treated with alternative products, indicating capacity to delay the onset of symptoms. Citric biomass stood out, showing similar results to the fungicide methyl thiophanate, delaying the incubation period in a day in relation to the water. The same was observed for the latent period, in which the fruits treated with alternative products, except garlic oil, stood in a group other than the control. Although the fruits of all treatments overcome the control, imazalil was able to better maintain the turgidity of the fruit, showing the lower values of loss of fresh weight on the eighth day of evaluation (5.2%). The mass loss and CO2 production correlated positively with the incidence and severity, in other words, the largest mass losses and higher respiratory rates result from high values of incidence and severity. In the case of 'Valencia' orange, citric biomass and chitosan reduced the incidence and severity during the entire experimental period. On the seventh day after inoculation, the incidence was reduced by 73.7% and 67.4% by citric biomass and chitosan, respectively, in comparison to control water, while the severity was reduced by 59.6% and 62%, respectively. On the other hand, neem oil, long pepper oil and garlic oil were ineffective in controlling the pathogen. Imazalil and methyl thiophanate totally controlled the disease during the evaluation period. The loss of fresh weight was increasing over time, however, fruits of all treatments had acceptable values for the marketing, located in the range 3 until 5%. As for CO2 production, fungicides maintained the respiratory rate low, followed in ascending order by chitosan and subsequently by other products. Thus, the citric biomass has good potential for control of green mold on 'Ponkan', while citric biomass and chitosan are capable of controlling the disease in 'Valencia' orange. / O bolor verde, causado pelo fungo Penicillium digitatum, é uma das principais doenças na pós-colheita dos frutos cítricos. Neste trabalho foram testados os efeitos de produtos alternativos aos agroquímicos no controle do bolor verde em tangerinas Poncã e em laranjas Valência . Frutos fisiologicamente maduros foram feridos superficialmente na região equatorial com um tubo de 9 mm de diâmetro preenchido por agulhas e inoculados com uma suspensão de conídios de P. digitatum na concentração de 1x106 conídios/mL. Após a secagem ao ar, foram pulverizados até o molhamento completo com água destilada (testemunha), água destilada + 8 mL/L de tween 20, imazalil (200 mL de Magnate® 500 EC/100 L de água), thiabendazole (103 mL de Tecto® SC/100 L de água), tiofanato metílico (70 g de Cercobin® 700 WP/100 L de água), quitosana 10 mL/L de água ou óleos essenciais de alho 10 mL/L de água, cravo-da-índia 5 mL/L de água, nim 10 mL/L de água, pimenta longa 5 mL/L de água e biomassa cítrica 10 mL/L de água. Os óleos essenciais e a quitosana foram acrescidos de 8 mL/L de tween 20. Em seguida, os frutos foram acondicionados em grupos de três em bandejas de poliestireno expandido e mantidos em câmara a 21±1 ºC e 85-90% UR por 8 (tangerina Poncã ) ou 7 dias (laranja Valência ). Os frutos foram analisados quanto à incidência e severidade da doença, período de incubação e período latente do fungo, perda de massa da matéria fresca e produção de CO2. No caso da tangerina Poncã , a biomassa cítrica foi capaz de reduzir a incidência em 76,3%, em comparação à testemunha, no terceiro dia, e reduzir também a severidade em 44%, no quarto dia. Para o período de incubação, com exceção dos frutos tratados com óleo de alho, observa-se formação de grupos de médias distintos entre a testemunha e aqueles tratados com produtos alternativos, indicando capacidade de atrasar o surgimento de sintomas da doença. A biomassa cítrica destacou-se, apresentando resultado semelhante ao fungicida tiofanato metílico, atrasando o período de incubação em um dia em relação à água. O mesmo foi observado para o período latente, em que os frutos tratados com produtos alternativos, exceto óleo de alho, situaram-se em um grupo distinto da testemunha. Embora os frutos de todos os tratamentos superassem a testemunha, o imazalil foi capaz de melhor preservar a turgidez dos frutos, apresentando os menores valores de perda de massa da matéria fresca no oitavo dia de avaliação (5,2%). A perda de massa e a produção de CO2 se correlacionaram positivamente com a incidência e severidade, ou seja, as maiores perdas de massa e maiores taxas respiratórias decorrem de altos valores de incidência e severidade. No caso da laranja Valência , biomassa cítrica e quitosana reduziram a incidência e severidade durante todo o período experimental. No sétimo dia após a inoculação, a incidência foi reduzida em 73,7% e 67,4% pela biomassa cítrica e quitosana, respectivamente, em comparação com a testemunha água, enquanto a severidade foi reduzida em 59,6% e 62%, respectivamente. Por outro lado, óleo de nim, óleo de pimenta longa e óleo de alho foram ineficazes em controlar o patógeno. Imazalil e tiofanato metílico controlaram totalmente a doença durante o período de avaliação. A perda de massa da matéria fresca foi crescente ao longo do tempo, porém, os frutos de todos os tratamentos apresentaram valores aceitáveis para a comercialização, situados na faixa de 3 a 5%. Quanto à produção de CO2, os fungicidas mantiveram a taxa respiratória dos frutos baixa, seguidos em ordem crescente pela quitosana e, posteriormente, pelos demais produtos. Dessa maneira, conclui-se que a biomassa cítrica tem bom potencial para controle do bolor verde em tangerina Poncã , enquanto biomassa cítrica e quitosana são capazes de controlar a doença em laranja Valência .

No presente estudo, duas espécies de fungos isoladas do ambiente marinho tiveram seus extratos brutos ativos contra Staphylococcus aureus e Candida albicans para uma espécie de P. citrinum e, atividade citotóxica e contra Mycobacterium tuberculosis para uma espécie de P. oxalicum. Estas foram estudadas com a finalidade de otimizar suas condições de crescimento para produção de metabólitos secundários. Foram realizadas análises multivariadas utilizando planejamento fatorial fracionário. Foram obtidas duas condições ótimas de crescimento para as duas linhagens de Penicillium. O crescimento em maior volume em condições de cultura otimizadas para P. oxalicum permitiu observar a presença de três metabólitos secundários no extrato do meio de cultura. Dois puderam ser isolados e identificados: a meleagrina 52 e a oxalina 26. A metodologia utilizada para se obter uma maior quantidade de metabólitos secundários proporcionou, ainda, o incremento em cerca de 150% na área do pico da oxalina. A partir do crescimento em maior volume em condições de cultura otimizadas para P. citrinum foi possível observar presença de pelo menos onze diferentes metabólitos na análise dos extratos obtidos do meio de cultura. Foi possível identificar e isolar quatro compostos já conhecidos: a (8E)-1-(2,3-diidropirrol-1-il)-2-metildec-8-eno-1,3-diona 56; a 1-(2,3-diidropirrol-1-il)-2-metildecano-1,3-diona 58; a 2-((E)-hept-5-enil)-6,7,8,8a-tetraidro-3-metilpirrolo[2,1-b][1,3]oxazin-4-ona 59 e a citrinina 31. Também foram isolados dois novos alcalóides indólicos contendo um grupo nitro na molécula que foram nomeadas de citrinalinas A 60 e B 62. Foi realizado, também, um estudo com P. citrinum visando a maior produção das citrinalinas, que possibilitou o incremento na produção da citrinalina B. / In this study, two species of fungi isolated from the marine environment had their active extracts against Staphylococcus aureus and Candida albicans to a strain of P. citrinum, and activity cytotoxic and against Mycobacterium tuberculosis to a strain of P. oxalicum. This studied aims the optimization their growth conditions for the production of secondary metabolites. Multivariate analysis using a fractional factorial design were used to establish optimal growth conditions for both Penicillium strains. Two optimal growth conditions were obtained for both Penicillium strains. A largest growth volume of P. oxalicum using optimized conditions enabled the detection of three secondary metabolites in the culture media crude extract. Two of such compounds were isolated and identified: meleagrin 52 and oxaline 26. The methodology used to increase the production of secondary metabolites by P. citrinum enabled an increase of 150% in the peak area of oxaline. A largest growth volume of P. citrinum led to the detection of eleven different metabolites in the culture media. Four of these compounds were isolated and identified as the known (8E)-1-(2,3-dihydropyrrol-1-yl)-2-methyldec-8-ene-1,3-dione 56; the 1-(2,3-dihydropyrrol-1-yl)-2-methyldecane-1,3-dione 58; the 2-((E)-hept-5-enyl)-6,7,8,8a-tetrahydro-3-methylpyrrolo[2,1-b][1,3]oxazin-4-one 59 and citrinin 31. Two new indole alkaloids containing a nitro group were also isolated and identified, named citrinalins A 60 and B 62. A further study with P. citrinum aiming an enhanced production of citrinalins allowed a significant increase in the production of citrinalin B.

Une méthode permettant de caractériser l’ensemble du métabolome secondaire de moisissures a été appliquée à la caractérisation des métabolomes de Penicillium nordicum, Penicillium verrucosum et Fusarium graminearum. Le substrat représentant l’unique source de carbone et d’azote des moisissures, chacun des champignons ont été mis en culture sur trois types de grains de blé: (i) grains naturels, (ii) grains marqués à 97% de 13C, et (iii) grains marqués à 53% 13C et 97% de 15N. Les extraits ont été analysés par HRMS. Les métabolites secondaires ont été spécifiquement détectés et leurs formules brutes ont été caractérisées. La caractérisation de nouveaux métabolites secondaires a ensuite été assistées par la génération de réseaux moléculaires de similarités MS/MS. L’étude de P. verrucosum et P. nordicum a permis de détecter 98 et 92 métabolites secondaires respectivement. Parmi eux, 80% étaient inconnus. La génération de réseaux moléculaires a permis de mettre en évidence un groupe de 25 composés se fragmentant de manière similaire. Seize de ces composés ont été identifiés comme étant des dérivés de fungisporines, des métabolites suspectés d’intervenir dans la croissance aérienne des champignons. Des analyses structurales ont permis de caractériser de nouveaux composés potentiellement impliqués dans l’infestation des denrées alimentaires. Le marquage du métabolome de F. graminearum par des isotopes stables a permis de mettre en évidence la production de 37 métabolites secondaires dont 29 inconnus lorsque le champignon se développe in vitro. Des analyses par MSn ont permis d’élucider les structures des fusaristatines C et D. / Characterization of fungal secondary metabolomes became a great challenge in the last decades due to both the emergence of fungal threats, and the industrial interest of many natural products; In view of this, we recently developed an analytical strategy for fungal secondary metabolome characterization (Cano P. et al. Anal. Chem. (2013) 85:8412) based on untargeted MS metabolomics applied to labeled samples. This strategy has been here validated by application to the analysis of the complex secondary metabolomes of Penicillium verrucosum and Penicillium nordicum. HRMS acquisitions performed on specific isotopically labelled samples, MS/MS experiments and in-silico emerging tools such as molecular networks, allowed to characterize 181 metabolites, including 80% of new compounds, and the structural determination of seven potential new mycotoxins. Penicillium verrucosum (NRRL 5571) and Penicillium nordicum (NRRL 6062) were grown on wheat grains (Triticum aestivum) presenting different isotopic enrichments: (i) naturally enriched grains, (ii) 97% 13C, and (iii) 53% 13C / 97% 15N. Extracts of each culture were analyzed by HPLC coupled to a LTQ-Orbitrap mass spectrometer equipped with electrospray ionization, operating in the positive or the negative mode. Metabolites were then specifically detected according to the specific isotopic pattern of their respective isotopic enrichments. Known secondary metabolites were annotated using the Antibase database, then identified by comparison with standard compounds when available. Unknown secondary metabolites were annotated using molecular networks of MS/MS similarities (Watrous J. et al.; PNAS (2012) 109 E1743). Wheat grains representing the only source of carbon and nitrogen for fungal growth, the produced fungal secondary metabolites were either unlabeled (naturally enriched cultures), singly labeled (13C cultures) or doubly labeled (13C/15N cultures). This feature allowed discrimination of fungal metabolites against non-fungal compounds which remained unlabeled in the three substrates. Fungal origin was further confirmed by analysis of a control 12C wheat extract (without fungus). Furthermore, the comparison of m/z ratios of a same metabolite detected in the three different cultures, led to the unambiguous determination of the number of carbon and nitrogen atoms and therefore to the unambiguous characterization of its chemical formula. This approach previously developed and validated on a well characterized fungus, has been here successfully applied to the characterization of the complex and unknown secondary metabolomes of P. verrucosum and P. nordicum. Analyses of the two studied fungal strains allowed the detection of 181 secondary metabolites. Interestingly, only 20% of them are suspected to match known metabolites according to databases, meaning that 80% of this metabolome is unknown. To enhance unknown identification efficiency, a molecular network of MS/MS similarities has been generated from our data. A group of 24 metabolites with highly similar MS/MS spectra was highlighted on P. nordicum and P. verrucosum. Fifteen of them were identified as cyclic tetrapeptides from the fungisporin family. Tandem mass spectrometry experiments were performed to characterize the structure of these secondary metabolites. To the best of our knowledge, this is the first time these molecules are pointed out on these Penicillium species. More interestingly, seven of the other metabolites display some similarities with fungisporins, but have never been detected on fungal metabolomes. Furthermore, although the two studied strains are genetically close, these new metabolites seem to be strain specific.