M.Sc. (Botany) / The genus Phomopsis is an important fungal genus due to its widespread pathogenicity on a wide variety of hosts and its complex anamorphic morphology. Three species of the genus Phomopsis viz. Phomopsis citri, Phomopsis leptostromiformis and Phomopsis zeicola, were studied in artificial culture and on host plants in vitro. The culture of Phomopsis citri failed to form conidiomata in culture and this species was studied intensively. As the species in this genus are difficult to distinguish and identify, the species named above were compared in an attempt to define taxonomically usable distinguishing characters. The morphology and ontogeny of the colonies, conidiomata and conidia, the karyology of these species, pathogenicity to cultivars of Lupinus spp. and Zea mays, were studied and the pectic enzymes were analyzed. Optical-, scanning electron- and transmission electron microscopic techniques were used in addition to the enzyme analysis. The conidiomata were typically stromatic and often irregularly multiloculate and could arise from more than one type of primordium. The most common primordia were ringshaped structures but hyphal aggregations were also found. Conidiogenesis was phialidic and the a-conidia of both species examined were shown to be uninucleate. The {3-conidia of Phomopsis leptrostromiformis were similarly uninucleate but failed to germinate. Their function is thus still unknown. Phomopsis zeicola failed to form {3-conidia. Transmission electron microscopy showed differences in conidial morphology between these species which, together with pectic enzyme analysis, could be good characters to separate species in this genus. The implications of heterokaryosis are discussed in assessing the potential value of these parameters and further investigations will have to be done.

No description available.

" August 2002." / Bibliography: leaves 218-235. / viii, 235 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The pathogenicity of Phomopsis taxon 1 is examined in relation to symptom expression and bud loss on grapevines. Phomopsis taxon 1-specific DNA probe, pT1P180, and taxon 2-specific probe, pT1P25, were used to detect Phomopsis taxon 1 and Phomopsis taxon 2 in infected buds, canes and shoots in glasshouse and field experiments. Experiments confirm the isolates of taxon 1 examined did not cause leaf or shoot symptoms associated with Phomopsis cane and leaf spot, and that taxon 2 is more virulent than taxon 1. Suggests that taxon 1 (Diaporthe) is an endophyte which does not cause harm to the grapevine and that chemical control is not warranted for control of taxon 1 on grapevine. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

" August 2002." / Bibliography: leaves 218-235. / viii, 235 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The pathogenicity of Phomopsis taxon 1 is examined in relation to symptom expression and bud loss on grapevines. Phomopsis taxon 1-specific DNA probe, pT1P180, and taxon 2-specific probe, pT1P25, were used to detect Phomopsis taxon 1 and Phomopsis taxon 2 in infected buds, canes and shoots in glasshouse and field experiments. Experiments confirm the isolates of taxon 1 examined did not cause leaf or shoot symptoms associated with Phomopsis cane and leaf spot, and that taxon 2 is more virulent than taxon 1. Suggests that taxon 1 (Diaporthe) is an endophyte which does not cause harm to the grapevine and that chemical control is not warranted for control of taxon 1 on grapevine. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2002

Natural products have, historically, played an important role in drug discovery. Nevertheless, drug resistance, pathogen evolution, and global climate change threaten human health and nearly all current anti-infective treatments on the market today. It is undeniable that new drug discovery efforts are needed with increasing urgency. Bolstered by a rich history of discovering treatments in the world around us, natural products chemists continue to look to the environment with increasing understanding and emerging technologies that allow efficient, effective isolation of new chemical entities. This thesis will describe one such endeavor. Focusing on fungal natural products, herein is described the isolation and structure elucidation of new, bio-active natural products. Further, the development and implementation of a large fungal screening program will be discussed, the results of which stand to advance microbial drug discovery in the Baker lab for years to come.

M.S.

Turnera subulata L. is a plant belonging to the Turneraceae family and is popularly known in Brazil as chanana. The genus Turnera has about 120 species distributed in the Americas and Africa, being the most representative of the family Turneraceae. Some species of Turnera are highly used in popular medicine for different types of inflammatory diseases. The association between living beings is a vital condition for species unable to achieve alone, as a means of survival for obtaining nutrients and defenses against predatory species. Among the microorganisms, the fungi are those who are most often associated with plants. Endophytic are microorganisms that inhabit the bodies of plants that, at some period of their life cycle, colonize internal tissues of the plant, without causing any apparent damage to its host. From the fragments of leaves from Turnera subulata L. could be isolated a filamentous fungus that was identified as the fungus Phomopsis sp. through traditional microscopy and molecular techniques. In a statistical evaluation it was noted that the endophytic Phomopsis sp. presents a higher growth in BDA and BSA media, as well as in the presence of light. The steroid ergosterol could be isolated from the hexane extract of this fungus and identified by the NMR techniques. In tests of antagonism of the endophytic Phomopsis sp. against phytopathogens, it was observed halum of inhibition against Rhizoctonia sp., Colletotrichum sp. and Pestalotiopsis sp. Concerning the antioxidant activity it was observed that the chloroform extract was more effective than the hexane one. On the other hand all the extracts from the mycelium of Phomopsis sp. and its ethylacetate extract from the cultured filtrate showed no antimicrobial activity against strains of Escherichia coli, Staphylococcus aureus, Candida albicans, Candida tropicalis and Candida glabrata. Therefore it was concluded that Phomopsis sp. may act as endophytic of T. subulata. it has shown to grow best in BDA and BSA medium, as well as under continuous lightining. It produces and accumulates ergosterol in his micelium. It promotes inhibition zone of growth when tested against phytopathogens. His hexanic and chloroform extrat has shown very litte antioxidant activity / Turnera subulata L. é uma planta pertencente à família Turneraceae e popularmente conhecida no Brasil como chanana. O gênero Turnera apresenta cerca de 120 espécies espalhadas nas Américas e África, sendo o mais representativo da família Turneraceae. Algumas espécies de Turnera são altamente utilizadas na medicina popular para diferentes tipos de doenças inflamatórias. A associação entre os seres vivos é uma condição vital para espécies incapazes de conseguirem, sozinhas, meios de sobrevivência como obtenção de nutrientes e defesas contra espécies predadoras. Entre os microrganismos, os fungos são os que se encontram mais frequentemente associados às plantas. Microrganismos endofíticos são os organismos que habitam órgãos de plantas sem causar dano aparente a seu hospedeiro. A partir dos fragmentos de folhas de Turnera subulata L. isolou-se um fungo filamentoso que foi identificado através de características morfológicas (morfoculturais) e moleculares como sendo Phomopsis sp. O isolado obtido apresentou um crescimento superior nos meios BDA (Batata-Dextrose-Ágar) e BSA (Batata-Sacarose-Ágar), com relações à cinco meios testados, sendo a presença de luz constante um fator de estímulo. Foi identificada por RMN a presença de ergosterol no extrato hexânico das céluas do Phomopsis sp. cultivado em meio líquido BD (Batata-dextrose). Em testes de antagonismo do endofítico Phomopsis sp. contra patógenos de plantas, observou-se halo de inibição deste frente a Rhizoctonia sp., Colletotrichum sp. e Pestalotiopsis sp. Nos resultados da avaliação do percentual de atividade antioxidante dos extratos do micélio em hexano e clorofórmio pôde-se observar que extrato em hexano teve menor atividade que o extrato em clorofórmio. Os extratos hexânico, clorofórmico, acetato etílico e metanólico do micélio de Phomopsis sp. e o extrato acetato etílico do filtrado de cultura desse fungo isento de células não apresentaram atividade antimicrobiana contra as cepas de Escherichia coli, Staphylococcus aureus, Candida albicans, Candida tropicalis e Candida glabrata. Com este trabalho, portanto, podemos concluir que o Phomopsis sp. pode atuar como endófito de T. subulata, apresenta um melhor crescimento em meios BDA e BSA, bem como em presença de iluminação contínua, apresenta ergosterol em sua composição, é capaz de promover halo de inibição frente à alguns patógenos de plantas e que seus extratos celulares hexânico e clorofórmico apresentam uma pequena atividade antioxidante

O Brasil tem uma posição de destaque mundial na produção de frutas e alguns grãos, os quais são comercializados in natura ou na forma de produtos processados. O processamento têm várias vantagens, porém gera grandes quantidades de resíduos, os quais têm sido aproveitados para a alimentação animal e geração de energia. Entretanto, muito tem sido relatado sobre o potencial bioativo desses materiais, dentre eles a ação fungitóxica de alguns de seus compostos sobre fungos fitopatogênicos. Assim, o objetivo desse trabalho foi avaliar o potencial de extratos de resíduos agroindustriais de abacate, uva, café, manga, abacaxi, maracujá e caju na inibição do crescimento micelial in vitro dos fungos Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani e Phomopsis sp., bem como a composição química do resíduo mais promissor. Os resíduos foram liofilizados, moídos e submetidos a extração com etanol 80% (etanol:água; 8:2, v/v) em banho de ultrassom (extrato denominado bruto ou EB). O EB também foi tratado com a resina Amberlite XAD-2, visando à eliminação de açúcares e interferentes, dando assim origem ao extrato semi-purificado (EP). Os extratos que apresentaram alto rendimento foram avaliados quanto à atividade antifúngica in vitro e teor de compostos fenólicos totais. A amostra que apresentou os melhores índices de inibição foi selecionada para dar continuidade ao estudo, sendo a mesma fracionada em coluna de gel Sephadex LH-20. Os extratos e as frações ativas, detectadas pelo ensaio de bioautografia, foram analisadas quanto a composição química pelas técnicas de HPLC e GC/MS. Na análise de fenólicos totais os maiores teores encontrados foram para a casca de abacate, enquanto que para o ensaio de inibição de crescimento micelial in vitro o melhor resultado foi para a semente de abacate, tanto para o EB quanto para o EP. Desta maneira, a semente de abacate foi selecionada para as etapas posteriores. Para o fracionamento em gel Sephadex LH-20 foi eleito o EP para F. pallidoroseum enquanto que para os demais fungos foi eleito o EB. No fracionamento do EP obtiveram-se 13 frações, sendo que as frações 3 e 4 foram ativas, enquanto que no do EB obtiveram-se nove, sendo as frações 3, 4 e 5 ativas. Pela técnica de HPLC foram detectados em comum nas frações 3, 4 e 5 do EB dois compostos majoritários, e nas frações 3 e 4 do EP, sete compostos, os quais não puderam ser identificados pelos padrões comerciais disponíveis. Já pela técnica de GC/MS foi possível a identificação de quatro compostos em comum nas frações ativas do EB e de 11 nas frações do EP. Dentre os compostos presentes nas frações ativas, foram identificados ácidos graxos, os quais têm sido reconhecidos por apresentarem ação antifúngica. Assim, pode-se concluir que os resíduos agroindustriais estudados são fontes de compostos com atividade antifúngica, podendo assim ser uma alternativa para o controle de fungos fitopatogênicos à cultura da soja / Brazil holds an important position in the world production of fruit and some grains, which are commercialized in natura or in processed products. The processing has several advantages, but generates large amounts of by-products which have been used for animal feed and power generation. However, bioactive potential of these materials have been reported, including the fungitoxic effect. The goal of this study was to evaluate the potential of agro-industrial by-products extracts of avocado, grape, coffee, mango, pineapple, passion fruit and cashew to inhibit in vitro mycelial growth of fungi Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani and Phomopsis sp. as well as the chemical composition of the most promising by-products. The by-products were freeze-dried, milled and the extraction was done with ethanol 80% (ethanol:water, 8:2, v/v) in an ultrasound bath (called crude extract or CE). The CE was also treated with Amberlite XAD-2 to eliminate sugars and interfering compounds so that the semi-purified extract (PE) could be obtained. Extracts with high yield were evaluated to in vitro antifungal activity and phenolic compounds content. Samples with the best inhibition rates were selected to continue this study, and it was fractioned on gel Sephadex LH-20. The extracts and the active fractions, detected by bioautography, were analyzed by HPLC and GC/MS. The highest phenolic content was found in the avocado peel, meanwhile for the mycelial growth inhibition in vitro, the best result was found in the avocado seeds for both extracts (CE and PE). Thus, avocado seed was selected for subsequent steps. For fractionation on Sephadex LH-20 gel, PE was chosen for F. pallidoroseum and CE for the other fungi. For PE, 13 fractions were obtained in which fractions 3 and 4 were active. For CE, 9 fractions were obtained and the actives were 3, 4 and 5. In CE, HPLC technique detected two major compounds in common in fractions 3, 4 and 5. In PE, seven major compounds were detected in fractions 3 and 4, which could not be identified by commercial available standards. By the GC/MS technique was possible to identify four compounds in common in the active CE fractions and 11 in PE. Among the compounds presented in the active fractions, fatty acids were identified. It has been reported that antifungal action has been found in these compounds. Therefore, the studied agroindustrial by-products are sources of compounds with antifungal activity and they can be used as an alternative to control phytopathogenic fungi on soybean

O Brasil tem uma posição de destaque mundial na produção de frutas e alguns grãos, os quais são comercializados in natura ou na forma de produtos processados. O processamento têm várias vantagens, porém gera grandes quantidades de resíduos, os quais têm sido aproveitados para a alimentação animal e geração de energia. Entretanto, muito tem sido relatado sobre o potencial bioativo desses materiais, dentre eles a ação fungitóxica de alguns de seus compostos sobre fungos fitopatogênicos. Assim, o objetivo desse trabalho foi avaliar o potencial de extratos de resíduos agroindustriais de abacate, uva, café, manga, abacaxi, maracujá e caju na inibição do crescimento micelial in vitro dos fungos Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani e Phomopsis sp., bem como a composição química do resíduo mais promissor. Os resíduos foram liofilizados, moídos e submetidos a extração com etanol 80% (etanol:água; 8:2, v/v) em banho de ultrassom (extrato denominado bruto ou EB). O EB também foi tratado com a resina Amberlite XAD-2, visando à eliminação de açúcares e interferentes, dando assim origem ao extrato semi-purificado (EP). Os extratos que apresentaram alto rendimento foram avaliados quanto à atividade antifúngica in vitro e teor de compostos fenólicos totais. A amostra que apresentou os melhores índices de inibição foi selecionada para dar continuidade ao estudo, sendo a mesma fracionada em coluna de gel Sephadex LH-20. Os extratos e as frações ativas, detectadas pelo ensaio de bioautografia, foram analisadas quanto a composição química pelas técnicas de HPLC e GC/MS. Na análise de fenólicos totais os maiores teores encontrados foram para a casca de abacate, enquanto que para o ensaio de inibição de crescimento micelial in vitro o melhor resultado foi para a semente de abacate, tanto para o EB quanto para o EP. Desta maneira, a semente de abacate foi selecionada para as etapas posteriores. Para o fracionamento em gel Sephadex LH-20 foi eleito o EP para F. pallidoroseum enquanto que para os demais fungos foi eleito o EB. No fracionamento do EP obtiveram-se 13 frações, sendo que as frações 3 e 4 foram ativas, enquanto que no do EB obtiveram-se nove, sendo as frações 3, 4 e 5 ativas. Pela técnica de HPLC foram detectados em comum nas frações 3, 4 e 5 do EB dois compostos majoritários, e nas frações 3 e 4 do EP, sete compostos, os quais não puderam ser identificados pelos padrões comerciais disponíveis. Já pela técnica de GC/MS foi possível a identificação de quatro compostos em comum nas frações ativas do EB e de 11 nas frações do EP. Dentre os compostos presentes nas frações ativas, foram identificados ácidos graxos, os quais têm sido reconhecidos por apresentarem ação antifúngica. Assim, pode-se concluir que os resíduos agroindustriais estudados são fontes de compostos com atividade antifúngica, podendo assim ser uma alternativa para o controle de fungos fitopatogênicos à cultura da soja / Brazil holds an important position in the world production of fruit and some grains, which are commercialized in natura or in processed products. The processing has several advantages, but generates large amounts of by-products which have been used for animal feed and power generation. However, bioactive potential of these materials have been reported, including the fungitoxic effect. The goal of this study was to evaluate the potential of agro-industrial by-products extracts of avocado, grape, coffee, mango, pineapple, passion fruit and cashew to inhibit in vitro mycelial growth of fungi Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani and Phomopsis sp. as well as the chemical composition of the most promising by-products. The by-products were freeze-dried, milled and the extraction was done with ethanol 80% (ethanol:water, 8:2, v/v) in an ultrasound bath (called crude extract or CE). The CE was also treated with Amberlite XAD-2 to eliminate sugars and interfering compounds so that the semi-purified extract (PE) could be obtained. Extracts with high yield were evaluated to in vitro antifungal activity and phenolic compounds content. Samples with the best inhibition rates were selected to continue this study, and it was fractioned on gel Sephadex LH-20. The extracts and the active fractions, detected by bioautography, were analyzed by HPLC and GC/MS. The highest phenolic content was found in the avocado peel, meanwhile for the mycelial growth inhibition in vitro, the best result was found in the avocado seeds for both extracts (CE and PE). Thus, avocado seed was selected for subsequent steps. For fractionation on Sephadex LH-20 gel, PE was chosen for F. pallidoroseum and CE for the other fungi. For PE, 13 fractions were obtained in which fractions 3 and 4 were active. For CE, 9 fractions were obtained and the actives were 3, 4 and 5. In CE, HPLC technique detected two major compounds in common in fractions 3, 4 and 5. In PE, seven major compounds were detected in fractions 3 and 4, which could not be identified by commercial available standards. By the GC/MS technique was possible to identify four compounds in common in the active CE fractions and 11 in PE. Among the compounds presented in the active fractions, fatty acids were identified. It has been reported that antifungal action has been found in these compounds. Therefore, the studied agroindustrial by-products are sources of compounds with antifungal activity and they can be used as an alternative to control phytopathogenic fungi on soybean

Le phoma (Phoma macdonaldii / Leptosphaeria linquistii) et le phomopsis (Phomopsis / Diaporthe helianthi) sont deux champignons pathogènes majeurs du tournesol. Dans un contexte d’objectif de réduction de la lutte chimique, une meilleure compréhension des interactions peuplement-agents pathogènes-environnement-conduite de culture est nécessaire. Des essais menés au champ et en serre à Auzeville en 2010 et 2011 ont permis de décomposer les effets de la conduite de culture du tournesol sur l’apparition et le développement des deux maladies. Les variables clés de l’architecture des plantes en peuplement et du microclimat, modifiées par la conduite de culture (choix variétal, densité de semis, fertilisation azoté, irrigation) ont été décrites et mises en relation avec les composantes de la maladie. Ainsi, pour le phomopsis, le rôle déterminant du microclimat résultant du niveau de développement de la couverture foliaire lors des phases initiales d’infection a été confirmé. La taille des feuilles et le diamètre des tiges sont des caractères déterminants de la progression des dégâts sur feuille et tige. Les conséquences des attaques de phomopsis sur feuille en termes de mécanismes de nuisibilité ont été analysées finement et modélisées. Pour le phoma, le rôle du statut nutritionnel azoté dans la progression des attaques sur tige et collet a été confirmé. La fonction régulatrice du phoma vis-à-vis des attaques de phomopsis sur feuille a été quantifiée. Sur le plan de la modélisation, plusieurs approches ont été menées : (i) Evaluation de la qualité prédictive d’Asphodel, modèle épidémiologique prévoyant les émissions de ascospores de Phomopsis ; application à différentes conduites de culture et extension au phoma pour la partie inoculum primaire ; (ii) développement d’un modèle prédictif de l’incidence du phoma prenant en compte les effets climatiques, microclimatiques et agronomiques ; (iii) proposition d’une modélisation conceptuelle des interactions plante-agent(s) pathogène(s)-environnement-conduite de culture. Ce travail pourra, à terme, contribuer à la conception de stratégies de gestion des principales maladies du tournesol, à l’échelle de la parcelle ou du territoire. / Phoma (Phoma macdonaldii / Leptosphaeria linquistii) and phomopsis (Phomopsis / Diaporthe helianthi) are two major sunflower fungal diseases. In a context of reduction of pesticides, a better understanding of the interactions between crop canopy, pathogens, environment and cropping practices is needed. Field and greenhouse experiments set up at Auzeville in 2010 and 2011 allowed to dissect the impact of sunflower crop management on disease appearance and development. Key variables of plant architecture and microclimate, modified by cropping practices (variety choice, sowing density, nitrogen fertilization, irrigation) were described and related to disease components. Thereby, for phomopsis, the key role of microclimate resulting from the level of development of leaf area during the initial stages of infection was confirmed. Leaf length and stem diameter are key characters for necrosis progression on leaf and stem. The impact of phomopsis leaf infection in term of damage mechanism was analysed and modelled. For phoma, the effect of nitrogen nutrition status on necrosis progression on stem and stem base was confirmed. The regulatory function of phoma towards phomopsis leaf infection was quantified. In terms of modelling, several approaches were developed: (i) evaluation of the predictive quality of Asphodel, an epidemiological model predicting Phomopsis ascospore release; application to different cropping practices and extension to phoma for primary inoculum; (ii) development of a predictive model for phoma incidence taking into account the climatic, microclimatic and agronomic effects; (iii) proposal of a conceptual model of the interactions between crop canopy, pathogens, environment and cropping practices. This study could contribute to the design of integrated management strategies for the main sunflower diseases, at field or at territory scale.