Дисертації: "The isolated fungi"

1

Weinstein, Richard Neil. "Ecophysiology of fungi isolated from soil in an Antarctic fellfield ecosystem." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624489.

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Heung, Shing-yan, and 向承恩. "Multilocus sequence typing of Candida albicans strains isolated in Hong Kong." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660005.

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3

April, Trevor Marc. "Hydrocarbon-degrading filamentous fungi isolated from flare pit soils of northern and western Canada." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ40024.pdf.

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4

Ghaderian, Seyed Majid. "The effect of toxic heavy metals upon fungi of the genus Pythium isolated from soil." Thesis, University of Sheffield, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.301558.

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5

Schierbaum, Anna. "Occurrence, distribution and agroactive metabolite production of endophytic fungi isolated from marine and shoreline plants." Thesis, University of Portsmouth, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479128.

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6

Svahn, Stefan. "Analysis of Secondary Metabolites from Aspergillus fumigatus and Penicillium nalgiovense : Antimicrobial Compounds from Filamentous Fungi Isolated from Extreme Environments." Doctoral thesis, Uppsala universitet, Avdelningen för farmakognosi, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-242611.

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This thesis describes the cultivation and extraction of filamentous fungi isolated from extreme environments in the search for new antibiotic compounds. Filamentous fungi are a rich source of medicines including antibiotics, and it is believed that many currently unknown fungal species and bioactive fungal metabolites remain to be discovered. Aspergillus fumigatus and Penicillium nalgiovense strains were isolated from an antibiotic-contaminated riverbed near Hyderabad, India, and soil taken from a penguin’s nest on Paulete Island, Antarctica, respectively. It was anticipated that the extreme conditions within these environments would exert unusual selective pressures on their filamentous fungi, possibly causing the secretion of new bioactive compounds. The cultivation, extraction and analysis of metabolites from the A. fumigatus strain resulted in the isolation of the antimicrobial substance gliotoxin. Subsequent investigations revealed that this strain’s secretion of gliotoxin was increased by as much as 65 % when it was cultivated in the presence of pathogen-associated molecular patterns. These results indicate the existence of a fungal receptor/signaling system for detecting nearby bacteria. The scope for using gliotoxin and the related metabolite bis(methyl)gliotoxin as biomarker metabolites for diagnosing the lethal pulmonary condition invasive aspergillosis was also investigated. Bronchoalveolar lavage fluid from 42 patients with and without possible invasive aspergillosis was extracted and analyzed. The results obtained suggest that gliotoxin and bis(methyl)gliotoxin are not suitable markers for diagnosing invasive aspergillosis. Studies on the P. nalgiovense strain from Antarctica resulted in the isolation of the antifungal agent amphotericin B. The secretion of this compound increased when P. nalgiovense was cultured on a potato-dextrose agar enriched with coconut flakes rather than liquid RPMI 1640 medium. This was the first time amphotericin B was isolated from any organism other than the bacterium Streptomyces nodosus. The results presented in this thesis will be useful in the continuing search for novel bioactive compounds, the diagnosis of fungal infections, and as a source of insight into the interactions between microorganisms. Moreover, they show that even extensively studied fungal genera such as Aspergillus and Penicillium are not completely understood and may produce unexpected or previously unknown bioactive metabolites under appropriate conditions.

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7

Sobreira, Aline Cavalcante Mesquita. "Study chemical and cytotoxic secondary metabolites isolated endophytic fungi from Aroeira-do-SertÃo (Myracrodruon urundeuva Fr. All.):Pseudofusicoccum stromaticum and Lasiodiplodia theobromae." Universidade Federal do CearÃ, 2016. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=16644.

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Empresa Brasileira de Pesquisa AgropecuÃria
Banco do Nordeste do Brasil
Fungos endofÃticos tÃm sido apontados como uma fonte promissora de substÃncias biologicamente ativas, entre as quais compostos com potencial atividade anticÃncer. Neste contexto, o presente trabalho descreve o primeiro estudo de prospecÃÃo quÃmica e citotoxicidade de dois fungos endofÃticos isolados de Aroeira-do-sertÃo (Myracrodruon urundeuva Fr. All.): Pseudofusicoccum stromaticum (MUB58) e Lasiodiplodia theobromae (MUB65). Os extratos de acetato de etila MUB58 e MUB65 foram obtidos atravÃs de partiÃÃo lÃquido-liquido do caldo destas cepas fÃngicas, as quais foram cultivadas por 21 dias em extrato de BD e malte, respectivamente. Os extratos fÃngicos foram inicialmente analisados por cromatografia lÃquida de ultraeficiÃncia acoplada a espectrometria de massas de alta resoluÃÃo (CLUE-EM) e posteriormente submetidos a cromatografia em Sephadex LH-20, seguida de cromatografia lÃquida de alta eficiÃncia com detector de arranjo de diodo. AnÃlise de CLUE-EM permitiu caracterizar a presenÃa de sete metabÃlitos no extrato MUB-58: O-sulfato de colina, xantofusina, 7-hidroxi-1-isocromanona, Ãcido multicÃlico, djalonensona, 3-Ãcido carboxÃlico-8-metoxicarbonila-1-hidroxi-9-oxo-9H-xantona e ciclo-Phe-Leu-Val-Leu-Leu. AlÃm disso, foram isolados quatro metabÃlitos ciclo-L-Phe-D-Leu1-L-Leu2-L-Leu3-L-lle (FC1), 5-hidroxi-metilfurfural (FC2), rotenolona (FC3) e tefrosina (FC4). O composto FC1 Ã inÃdito na literatura, enquanto os compostos FC3 e FC4 estÃo sendo relatados pela primeira vez em fungos endofÃticos. Da mesma forma, a anÃlise CLUE-EM do extrato MUB-65 levou Ã caracterizaÃÃo quÃmica de oito compostos jÃ relatados na espÃcie L. theobromae: 4-hidroximeleina; meleina; (3R, 5R)- 5-hidroxila-de-O-metil-lasiodiplodina; (3R,5R)-hidroxilasiodiplodina; 2,4,6-trimetiloct-2-enoato,1,2,6,8a- tetrahidro-7-hidroxi-1-8a-dimetil-6-oxo-2-naftalenila; lasiodiplodina; lasiojasmonato A e lasiojasmonatos B ou C. Adicionalmente, dois metabÃlitos foram isolados: lasiodiplodina (LT1) e rel-11-12- (7âR*, 4âR*, 2âR*- tetrahidrofuro[1â,2â] piranil)- lasiodiplodina (LT2). O composto LT2 estÃ sendo relatado pela primeira vez na literatura. As estruturas quÃmicas de todas as substÃncias isoladas foram elucidadas por espectrometrias de RessonÃncia MagnÃtica Nuclear de 1H e 13C, infravermelho e CLUE-EM. Os extratos e seus respectivos metabÃlitos isolados foram submetidos ao ensaio de citotoxicidade para avaliaÃÃo dos seus efeitos antiproliferativos frente a uma linhagem celular de cÃncer colorretal (HCT-116). O extrato de MUB58 apresentou significativa atividade (75%) correspondendo a um IC50 de 10,40 Âg.mL-1, enquanto o extrato de MUB65 foi inativo. Os metabÃlitos isolados LT1 (IC50 11,24 Âg.mL-1), FC3 (IC50 5,59 Âg.mL-1) e FC4 (IC50 0,51 Âg.mL-1) apresentaram atividades na faixa de 11,24 a 0,51 Âg.mL-1.
Endophytic fungi have been identified as a promising source of biologically active substances,including compounds with potential anticancer activity. In this context, this paper describes the first study of chemical prospecting and cytotoxicity two endophytic fungi isolated from Aroeira -do-SertÃo(Myracrodruon urundeuva Fr.ll.):Pseudofusicoccum stromaticum (MUB-58) and Lasiodiplodia theobromae (MUB - 65). The ethylacetate extracts MUB-58 and MUB-65 were obta ined by liquid-liquid partition broth these fungal strains which were cultured for 21 days on BD and malt extract , respectively. The fungal extracts were first analyzed by liquid chromatography of ultraefficiency coupled to spectrometry, high resolution mass (CLUE -MS) and subsequently subjected to chromatography on Sephadex LH - 20, followed by liquid chromatography of high efficiency with diode array detector. CLUE - MS analysis allowed to determine the prese nce of metabolites in seven MUB 58 extract: cholin - O - sulfate, xanthofusin, 7-hidroxy -1-isochromanone, multico licacid, djalonensone, 8 - methoxycarbonyl-1-hidroxy-9-oxo-9H-xanthene-3-carboxylix acidand cyclo-Phe-Leu-Val-Leu-Leu. In addition, were isolated four metabolites:cycloL-Phe-D-Leu1-L-Leu2-L-Leu 3-L-Ile (FC1), 5-hidroxy-methilfurfural (FC2), rotenolone (FC3) and tephrosin (FC4).The compound FC1 is unprecedented in the literature, while FC3 and FC4 compounds are being reported for the first time in en dophytic fungi. Likewise, a CLUE - MS analysis the MUB - 65 extract led to characterization of eight chemical compounds already reported in the species L. theobromae:4-hidroxymelein; Mellein;(3R,5R)-5-hidroxyl-de-O-methyl-lasiodiplodin;(3 R,5 R)- hydroxyla siodiplodin; 2,4,6-trimethyloct-2-enoic acid-1,2,6,8a-tetrahydro-7-hydroxy-1-8a-dimethyl-6-oxo-2- naphtalenyl-ester;lasiodiplodin;lasiojasmonate A and lasiojasmonates B ou C. Additionally, two metabolites w ere isolated: lasiodiplodin(LT1) and rel-11-12-(7'R* 4'R*, 2 'R*-tetrahydrofuro [1', 2'] pyranyl)-lasiodiplodin (LT2). The compound LT2 being first reported in the literature. The chemical structures of all isolated compounds were elucidated by spectrometries Nuclear Magnetic Resonance 1 H and 13C, IR and CLUE-MS. The extracts and their isolated metabolites were subjected to the cytotoxicity assay to evaluate their antiproliferative effects against a colorectal cancer cell line (HCT-116). The MUB-58 extract showed significant activity (75%) corresponding to an IC50of 10.40 μg.mL -1, while the MUB65 extract was inactive. The LT1 isolated metabolites (IC5011.24 μg.mL-1), FC3(IC50 5.59 μg.mL-1) and FC4(IC500.51 μg.mL-1) showed activity in the range of 11.24 to 0.51 μg.mL-1.

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Sarkar, Tanushree. "Studies on resistance of trichosanthes dioica and their induction with chemical inducers against fungal pathogen." Thesis, University of North Bengal, 2021. http://ir.nbu.ac.in/handle/123456789/4760.

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9

Hundley, Nicholas James. "Structure Elucidation of Bioactive Compounds Isolated from Endophytes of Alstonia scholaris and Acmena graveolens." Diss., CLICK HERE for online access, 2005. http://contentdm.lib.byu.edu/ETD/image/etd1013.pdf.

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10

Souza, Eduardo Lorensi de. "Produção de mudas e crescimento de Eucalyptus grandis Hill ex maiden inoculado com fungos ectomicorrízicos em área arenizada." Universidade Federal de Santa Maria, 2009. http://repositorio.ufsm.br/handle/1/5492.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico
Eucalyptus. grandis is one of the main species used in forestry, in the Rio Grande do Sul State. The reforestations have been concentrated in low fertility soil areas, especially in phosphorus, such as the Quartzarenic Neosoils, which occur in the south of the state. It represents a problem for the establishment of this culture in field. This forestry species has the capacity of making symbiosis with ectomycorrhizal fungi that increase plant grown and the absorption of water and nutritious. The aim of the work was to evaluate the initial growth of eucalyptus in sandy area and the ectomycorrhizal fungi inoculation isolated, individually or mixed in Neosoil. In the first stage, eucalyptus seedlings were produced in greenhouse, inoculated or no-inoculated with the UFSC-Pt116 isolated, produced in peat or in Neosoil. After 120 days seedlings were transplanted to an area subjected to the process of sanding in São Francisco de Assis and evaluated regarding the survival, height, stem s diameter and tenors of nitrogen, phosphorus and potassium, total phosphorus, inorganic phosphorus, and wood production. In the second stage it was tested, in greenhouse, the effect of ectomycorrhizal fungi inoculation with the UFSC-Pt116, UFSC-Pt188 and UFSC-SA9 isolates, individually or mixed in peat substrate and in Neosoil. Determinations were accomplished regarding stem s diameter and height, dry matter of the aerial portion and the roots, roots volume, mycorrhizal colonization as well as the level of nitrogen, potassium and total, organic and inorganic phosphorus of the aerial portion of plants. In field, the plants produced in Neosoil and inoculated with the isolated UFSC-Pt116 obtained the highest survival, stem s height and diameter, nitrogen level, as well as wood production in relation to the no-inoculated seedlings. In the second study, the plants that received individual inoculation of the isolated UFSC-Pt116 and UFSC-SA9 and that were produced with peat obtained the highest height, stem s diameter, mycorrhizal colonization and dry matter accumulation. The plants that were produced in Neosoil and the individually inoculated with the isolated UFSC-Pt116, UFSC-Pt118 and UFSCSA9 showed highest height, stem s diameter, dry matter of the aerial portion and roots volume. The seedlings produced in Neosoil obtained higher height and diameter than those produced in peat.
No Estado do Rio Grande do Sul o E. grandis é uma das principais espécies utilizadas na silvicultura. Os reflorestamentos têm se concentrado em regiões de solos com baixa fertilidade, especialmente em fósforo, como os Neossolos Quartzarênicos, que ocorrem na metade sul do Estado, tornando-se um problema para o estabelecimento dessa cultura no campo. Esta espécie florestal tem a capacidade de formar simbiose com fungos ectomicorrízicos que auxiliam o crescimento das plantas através do aumento na absorção de nutrientes e água. No presente trabalho teve-se por objetivos avaliar o crescimento inicial do eucalipto em área arenizada e a inoculação dos isolados fúngicos ectomicorrízicos, individualmente ou em mistura em Neossolo. Na primeira etapa do trabalho, foram produzidas mudas de eucalipto em casa de vegetação, que foram inoculadas ou não com o isolado UFSC-Pt116, produzidas em turfa ou em Neossolo. Após 120 dias, essas foram transplantadas para uma área arenizada em São Francisco de Assis e avaliadas quanto à sobrevivência, altura, diâmetro do caule e teores de nitrogênio, fósforo e potássio, fósforo total, fósforo inorgânico, fósforo orgânico e produção de madeira. Na segunda etapa testou-se em casa de vegetação o efeito da inoculação dos isolados de fungos ectomicorrízicos UFSC-Pt116, UFSC-Pt188 e UFSC-SA9, individualmente e em mistura no substrato turfa e no Neossolo. Foram realizadas avaliações quanto à altura e diâmetro do caule, massa seca da parte aérea e das raízes, volume de raízes, percentual de colonização micorrízica e teores de nitrogênio, fósforo e potássio da parte aérea das plantas. No campo, as plantas que foram produzidas no Neossolo e inoculadas com o isolado UFSC-Pt116 obtiveram a maior sobrevivência, altura e diâmetro do caule, teores de nitrogênio, bem como produção de madeira em relação às mudas não inoculadas. No segundo estudo, as plantas que receberam inoculação individual dos isolados UFSC-Pt116 e UFSC-SA9 e foram produzidas com turfa obtiveram a maior altura, diâmetro do caule, colonização micorrízica e acúmulo de massa seca. As plantas que foram produzidas em Neossolo e inoculadas com os isolados UFSC-Pt116, UFSC-Pt188 e UFSC-SA9 individualmente alcançaram a maior altura, diâmetro do caule, massa seca da parte aérea e volume de raízes. As mudas produzidas no Neossolo alcançaram altura e diâmetro maiores que as produzidas na turfa.

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Valero, Rello Ana. "Ochratoxin A in overripe grapes, raisings and special wines in vitro and in vivo studies on fungi isolated from grapes and raisins affected by physical, chemical and biotic agents." Doctoral thesis, Universitat de Lleida, 2007. http://hdl.handle.net/10803/8384.

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Ochratoxin A (OTA) is a fungal secondary metabolite produced by Aspergillus and Penicillium species. It has been detected in a wide range of commodities, including cereals, coffee, grapes, raisins, must and wine. Within grape derivative products, the raisins, red wine and sweet wines have reported to contain the highest OTA levels. Aspergillus section Nigri (A. niger and A. carbonarius) are considered the OTA source in these commodities and they are commonly isolated among other fungi from grapes and raisins.
Starting from this basis the objectives of this thesis were focused into three main aspects: (1) Evaluation of the food products: vine dried fruits and special wines, concerning the mycobiota and OTA occurrence and incidence; (2) Ecophysiological studies of the ochratoxigenic fungi and accompanying mycobiota as affected by environmental conditions; (3) Control and preventive methods such as the evaluation of residual activity of pre-harvest fungicides during grape dehydration and the use of modified atmospheres.
Wine origin and winemaking procedure showed to be determinant for the final OTA content. All special wines analysed from northern European regions were negative for OTA while more than 50% of wines from warmer regions were positive for OTA contamination. The wines with higher OTA levels were fortified musts followed by those made from dried grapes. Acoholic and malo-lactic fermentations, biological 'crianza' (Flor yeast) and the action of Botrytis cinerea in noble rot of grapes may diminish the OTA levels in wine.
In grapes, the presence of Aspergillus section Nigri became predominant at harvest and mainly during sun-drying. Prevalence of Aspergillus section Nigri can be explained by their adaptation to environmental conditions of sun-drying, and by their ability to dominate other fungal species involved when coming into contact with them. Among the Aspergillus section Nigri, A. niger aggregate was dominant, although A. carbonarius increased its inci

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Bastos, Francisco Albuquerque. "OTIMIZAÇÃO DO PROCESSO DE PRODUÇÃO DE TIQUIRA EMPREGANDO ENZIMAS COMERCIAIS E FUNGOS ISOLADOS A PARTIR DOS BEIJUS UTILIZADOS NO MÉTODO TRADICIONAL." Universidade Federal do Maranhão, 2013. http://tedebc.ufma.br:8080/jspui/handle/tede/955.

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Tiquira, a strong content beverage from Maranhão ( Brazil ), obtained from cassava (Manihot esculenta Crantz.) has its production concentrated in several counties of the State, through a handmade process , rather primitive, in which the conversion of starch cassava is taken into fermentable sugars by fungi which is found on the native beijus of manioc where strains are picked at random. Alcoholic fermentation is also made by yeasts found in the environment. Its commercialization is practiced informally, making it difficult to record statistical data of production and the number of producers in the Ministry of Agriculture. In order to optimize this process, commercial enzymes, strains of fungi (Aspergillus niger), were used to in order to convert starch into fermentable wort and pressed yeasts for fermentation. The processes proposed here follow three steps: gelation of starch scarification and subsequent dextrinization the sugars, alcoholic fermentation and distillation of fermented wine. Substituting the mold for commercial enzymes in the proposed circumstances, the manufacturing of tiquira was performed in just 25 hours, whereas when using fungi isolated, the production of tiquira took approximately 136 hours for its processing. As one can see, there was a considerable reduction of time in the two cases presented here in relation to the traditional process, which takes approximately 20 days to achieve the distillate. It was also found that the fermentation and destillation of the wort resulting from the scarification of cassava is equivalent to that of cane sugar, and both processes can be processed with the same equipment by tiquira producers. It was shown that the concentrations of copper and ethyl carbamate were visibly reduced in comparison to the traditional process, enabling a better quality of the beverage, as well as reducing the risks to consumer health. From the experiments it can be concluded that the replacement of the traditional process by commercial enzymes, fungi isolated and pressed yeast are highly and technically recommended primarily to small producers.
A tiquira é uma bebida típica do Maranhão (Brasil), obtida a partir de mandioca (Manihot esculenta, Crantz.), sua produção se concentra em diversos municípios do Estado, através de um processo artesanal, bastante primitivo, no qual a conversão do amido de mandioca em açúcares fermentescíveis é feita por bolores nativos que surgem sobre os beijus de massa de mandioca, onde as cepas são colhidas ao acaso. A fermentação alcoólica também é feita por leveduras selvagens. Tem sua comercialização praticada de modo informal, dificultando o registro de dados estatísticos de produção e número de produtor no Ministério da Agricultura. Com o objetivo de otimizar o referido processo, utilizaram-se, enzimas comerciais, cepas de fungos (Aspergillus níger), para a conversão do amido em mosto fermentescíveis e leveduras prensadas para fermentação. Os processos aqui propostos seguiram três etapas: gelificação do amido com a posterior dextrinização e sacarificação a açúcares, fermentação alcoólica e destilação do vinho fermentado. Substituindo-se os bolores por enzimas comerciais nas circunstâncias propostas, a fabricação da tiquira foi realizada em apenas 25 horas, enquanto que ao utilizar fungos isolados, a produção do aguardente demorou, aproximadamente, 136 horas para o seu processamento. Evidenciou-se assim, uma considerável redução de tempo, nos dois processos aqui apresentados em relação ao processo tradicional, que demora, aproximadamente, 20 dias para a consecução do destilado. Verificou-se também que os processos de fermentação e destilação do mosto oriundos da sacarificação da massa de mandioca são equivalentes ao da cana-de-açúcar, podendo ser processados com os mesmos equipamentos, por pequenos produtores de tiquira. Comprovou-se que as concentrações de cobre e carbamato de etila foram visivelmente reduzidas nos processos propostos em relação ao processo tradicional, possibilitando uma melhor qualidade da bebida, assim como reduzindo os riscos à saúde dos consumidores. A partir dos experimentos realizados conclui-se que a substituição do processo tradicional por enzimas comerciais, fungos isolados e levedura prensada se mostra tecnicamente possível e recomendada, primordialmente, aos pequenos produtores.

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Tonelotto, Mariana. "Produção de celulases, purificação e caracterização bioquímico-cinética da ß-Glicosidase produzida por fungo isolado da região amazônica." Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/7001.

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Financiadora de Estudos e Projetos
The selection of cellulase-producing fungi is one of the possible estrategies for obtaining necessary enzymes to hydrolyze the lignocellulosic material of plant biomass and thereby contribute to the viability of cellulosic ethanol production. The aim of this study was achive a screening of isolated fungi from the Amazon region to assess the production of enzymes related to plant biomass degradation, in order to select a line for production, purification and biochemical, kinetical and and structural biology characterizationof the ß-Galactosidase enzyme. Therefore, this work was undertaken in three stages, first of all it was performed a screening of 40 fungal strains isolated from the Amazon region through the cultivation in solid state fermentation (FES) at 35ºC for 240 hours, using as substrate wheat bran. It was evaluated the production of xylanase, endoglucanase, FPase, pectinase, ß-Glicosidase and total protein, and the fungi that stood out were: P6B2, the best producer of xylanase, P47C3 (Aspergillus niger), the best producer endoglucanase and ß-Glicosidase and P40B3, the best producer of FPase. These three fungi were selected for the second phase of this work for assessment in the production of xylanase, FPase, endoglucanase, ß-Glicosidase and total protein by submerged fermentation (FSm). The fermentation took place for 5 days at 30ºC and 200 rpm with a source of carbon: 1% of wheat bran washed and nutrient medium. The fungi P47C3, which was identified as Aspergillus niger, showed the best production of these enzymes, being selected for the third stage of this project. This last step involved the selection of an enzyme that has not been elucidated its structural biology. Given this fact, we carried out a study of selection of the medium, purification and biochemicalkinetical characterization of ß-Galactosidase. The Aspergillus niger (P47C3) was subjected to submerged for 5 days at 200 rpm at 30ºC. Purification occured in three steps using: ion exchange column SP-Sephadex C-50 and SP TSK-5PW column, and gelfiltration, with the resin Sephacryl S-200. The enzyme ß-Galactosidase showed a molecular weight of 125 kDa, being stable at pH 4,0, with anoptimum temperature of 55ºC. It was evaluated theKmap e Vmáxap of two substrates, PNPG and lactose, being: 2,204 mM-0,285 mM/min and 2,101 mM-0,75mM/min, respectively. The inhibition of hydrolasis of the substrate PNPG by ß-Galactosidase in the presence of galactose inhibitor product showed a Ki value of 5,01 mM. Finally, the ß-Galactosidase was subjected to crystallization conditions, the best conditions occurred in buffer 0,2M Tris- HCl, with the precipitation agent, 12% PEG 4000 at pH 8,6. Therefore, the unpublished protocol for purification of ß-Galactosidase was efficient and it is possible to crystallize this enzyme of isolated fungi from the Amazon region, which showed great potencial for the production of this enzyme and that the future can be used in industrial application and biotechnological innovations.
A seleção de fungos produtores de celulases é uma das possíveis estratégias para a obtenção das enzimas necessárias para hidrolisar o material lignocelulósico da biomassa vegetal e com isso contribuir para a viabilização da produção de etanol celulósico. O objetivo desse trabalho foi realizar um screening dos fungos isolados da região amazônica para a avaliação da produção de enzimas relacionadas à degradação da biomassa vegetal, a fim de selecionar uma linhagem para produção, purificação e caracterização bioquímica, cinética e biologia estrutural da enzima ß-Galactosidase. Dessa forma, esse trabalho foi realizado em três etapas, primeiramente foi realizado um screening de 40 linhagens fúngicas isoladas da região amazônica, através do cultivo em fermentação em estado sólido (FES), a 35°C, por 240 horas, utilizando como substrato o farelo de trigo. Avaliou-se a produção de xilanase, endoglucanase, FPase, pectinase, ßglicosidase e proteínas totais, sendo que os fungos que mais se destacaram foram o: P6B2, melhor produtor de xilanase, P47C3 (Aspergillus niger), melhor produtor de endoglucanase e ß-glicosidase e o P40B3, melhor produtor de FPase. Esses três fungos, foram selecionados para a segunda fase do trabalho para avaliação na produção de xilanase, FPase, endoglucanase, ß-glicosidase e proteínas Totais por fermentação submersa (FSm). A fermentação ocorreu por 5 dias, à 30ºC e 200 rpm tendo como fonte de carbono: 1% de farelo de trigo lavado e meio nutriente. O fungo P47C3, identificado como Aspergillus niger, apresentou melhor produção dessas enzimas, sendo selecionado para a terceira etapa desse projeto. Essa última etapa, envolveu a escolha de uma enzima que não estivesse sua biologia estrutural elucidada. Diante desse fato, realizou-se um estudo de seleção do meio de cultivo, purificação e caracterização bioquimico-cinética da ß-Galactosidase. O fungo Aspergillus niger (P47C3) foi submetido a fermentação submersa, durante 5 dias, à 200 rpm em 30ºC. A purificação ocorreu em três etapas utilizando: colunas de troca iônica SP - Sephadex C-50 e a coluna SP -TSK 5PW; e gel filtração, com a resina Sephacryl S-200. A enzima ß-Galactosidase apresentou uma massa molecular de 125 kDa, sendo estável em pH 4,0, e com temperatura ótima de 55ºC. Avaliou-se a Kmap e Vmáxap de dois substratos, o PNPG e a lactose, sendo: 2,204 mM - 0,285 mM/min e 2,101 mM 0,750 mM/min, respectivamente. A inibição da hidrólise do substrato PNPG pela ß-Galactosidase na presença do produto inibidor galactose apresentou um valor de Ki de 5,01 mM. Por fim, a ß-Galactosidase foi submetida a condições de cristalização, as melhores condições ocorreram em tampão 0,2M Tris-HCl, tendo como agente precipitante, PEG 4000 12% em pH 8,6. Portanto, o protocolo inédito de purificação da ß-Galactosidase foi eficiente, sendo possível cristalizar essa enzima do fungo isolado da região amazônica, o qual apresentou grande potencial para a produção dessa enzima e que futuramente possa ser utilizado em aplicações industriais e inovações biotecnológicas.

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14

Lau, Ching-lai, and 劉清麗. "Identification of pathogenic fungal isolates by ITS sequencing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/193540.

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In clinical microbiology laboratories, the conventional method for identification of pathogenic fungi is based on fungal culture and observation of fungal phenotypic characters. However, it is time-consuming, subjective and unreliable due to the long incubation period and variations in fungal colony morphology. Thus, there is a need for a rapid, objective and accurate identification of pathogenic fungal isolates. ITS regions are most commonly used targets for molecular identification of fungal pathogens because of the optimal inter- and intra-species variations and large copies in fungal genome. In this study, twenty-two clinical fungal isolates were identified using the phenotypic method and ITS sequencing. The results showed that there were only thirteen isolates identified to species level by phenotypic method, while others were only differentiated in genus level. Due to the poor differentiation based on the conventional phenotypic approach, misidentification of fungal pathogens occasionally occurred. However, ITS sequencing successfully achieved accurate species-level identification of all fungal isolates. The results were demonstrated in phylogenetic trees with high bootstrap support. In conclusion, ITS sequencing is a rapid and reliable for the identification of pathogenic fungal isolates.
published_or_final_version
Microbiology
Master
Master of Medical Sciences

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15

Clipston, Julie. "An investigation into the production by marine-derived fungi of secondary metabolites with pesticidal activities." Thesis, University of Portsmouth, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.343340.

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16

Westwell, Jane Marie. "Effect of spore immobilisation of the pathogenicity of a Cochliobolus isolate." Thesis, University of Westminster, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240167.

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17

McCabe, Bernadette K., of Western Sydney Macarthur University, and Faculty of Business and Technology. "Production of cellulolytic enzymes using immobilised anaerobic fungi." THESIS_FBT_XXX_Mccabe_B.xml, 1998. http://handle.uws.edu.au:8081/1959.7/83.

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An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch.
Doctor of Philosophy (PhD)

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18

Giraldo, López Dixie Alejandra. "Taxonomic study of clinical and environmental isolates of arthroconidial, acremonium-like and ochroconis-like fungi." Doctoral thesis, Universitat Rovira i Virgili, 2015. http://hdl.handle.net/10803/292251.

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En aquets tesis es vol enfocar en l'estudi dels géneres fúngics Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium i Verruconis. Alguns d'ells són ocasionalment aïllats de mostres clíbiques, encara què, l'espectre real de les seves espècies en l'area clínica es poc coneguda, sumant-li la seva difícil identificació i complexa taxonomia. Es van estudiar un total de 248 aïllats, 131 obtingust de mostres clíniques o de sòls i 117 corresponents a soques tipus o de referència de col.leccions internacionals de cultius. La caracterizació fenotípica dels aïllats es va realitzar a traves de l'observació de les seves característiques macroscópiques i microscópiques. La identificació molecular, mitjacant la seqüenciació de les regions ribosomals ITS i LSU. Per obtenir una millor resolució filogenética entre algunes espècies, es van seqüenciar regions parcials d'altres gens (actina, tubulina, factor de elongació, RNA polimerasa i quitina sintasa). L'activitat in vitro d'alguns antifúngics va a ser evaluata front a espècies de Arthrographis, Arthropsis, Ochroconis i Scytalidium. Apart de les espècies comunment conegudes com a patògens oportunistes A. kalrae i V. gallopava, altres espècies no conegudas previament a partir de mostres clíniques vàren ser identificades (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum i Ochroconis cordanae). Incluint les noves espècies descrites aquí: Acremonium dimorphosporum, A. moniliforme, Arthrographis chlamydospora, A.
pseudostrictum i S. subulatum. Adicionalment, quatre géneres nous, Acremoniopsis, Brunneomyces, Cervusimilis i Collarina i 16 nous taxons, Acremoniopsis suttonii, Acremonium asperulatum, A. citrinum, A: parvum, A: pilosum, A. variecolor, Brunneomyces brunnescens, B. hominis, B. europaeus, Cervusimilis alba, Collarina aurantiaca, Ochroconis icarus, Sarocladium gamsii, S. implicatum, S. summerbellii i S. terricola, vàren ser descrits de mostres de diferents origens. En general, les proves de sensibilitat antifúngica mostrar que la terbinafina va ser la droga més activa davant les espècies avaluades, excepte per S. cuboideum, on el posaconazol va a mostrar la millor activitat En esta tesis nos enfocamos en el estudio de los géneros fúngicos Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium y Verruconis. Algunos de ellos son ocasionalmente aislados de muestras clínicas, sin embargo, el espectro real de sus especies en el área clínica es poco conocido, sumado a su difícil identificación y compleja taxonomía. Se estudiaron un total de 248 aislados, 131 obtenidos de muestras clínicas o de suelos y 117 correspondientes a cepas tipo o de referencia de colecciones internacionales de cultivos. La caracterización fenotípica de los aislados se realizó a través de la observación de sus características macroscópicas y microscópicas, y la identificación molecular, mediante la secuencación de las regiones ribosomales ITS y LSU. Para obtener una mejor resolución filogenética entre algunas especies, se secuenciaron regiones parciales de otros genes (actina, tubulina, factor de elongación, RNA polimerasa II y quitina sintasa). La actividad in vitro de varios antifúngicos fue evaluada frente a especies de Arthrographis, Arthropsis, Ochroconis y Scytalidium. Aparte de las especies comunmente reportadas como patógenos oportunistas, Arthrographis kalrae y Verruconis gallopava, otras especies no reportadas previamente a partir de muestras clínicas fueron identificadas (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum y Ochroconis cordanae), incluyendo las nuevas especies descritas aquí:
Ochroconis olivacea, O. ramosa, Sarocladium bifurcatum, S. hominis, S. pseudostrictum y S. subulatum. Adicionalmente, cuatro géneros nuevos, Acremoniopsis, Brunneomyces, Cervusimilis y Collarina, y 16 nuevos taxones, Acremoniopsis suttonii, Acremonium asperulatum, A. citrinum, A. parvum, A. pilosum, A: variecolor, Brunneomyces brunnescens, B. hominis, B. europaeus, Cervusimilis alba, Collarina aurantiaca, Ochroconis icarus, Sarocladium gamsii, S. implicatum, S. sumerbellii y S. terricola, fueron descritos de muestras de diversos orígenes. En general, las pruebas de sensibilidad antifúngica revelaron que la terbinafina fué la droga más activa frente a las especies evaluadas, excepto para S. cuboideum, donde el posaconazol mostró la mejor actividad. In this thesis, we have studied the fungal genera Acremonium, Arthrographis, Arthropsis, Ochroconis, Sarocladium, Scytalidium and Verruconis. Some of them are occasionally recovered from clinical specimens, but the real spectrum of their species in the clinical setting is poorly known. Furthermore, the scarce morphological differentiation of their species make difficult their identification and taxonomy. A total of 248 isolates were studied. 131 obtained from clinical or soil samples and 117 corresponding to type or reference strains from international culture collections. Phenotypical characterization was performed by the observation of their macroscopic and microscopic features in artificial media. Molecular identification was assessed by sequencing of two ribosomal regions (ITS and partial LSU). To obtain deeper phylogenetic resolution of some species, fragments of different loci were also used (actin, tubulin, translation elongation factor, RNA polymerase II and chitin syntase). The in vitro activity of several antifungal drugs was evaluated against Arthrographis, Arthropsis, Ochroconis and Scytalidium species. Apart from the commonly reported opportunistic species Arthrographis kalrae and Verruconis gallopava, other species never associated before to clinical specimens were identified (Arthropsis hispanica, Sarocladium terricola, Scytalidium cuboideum, Ochroconis

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19

Mativandlela, Sannah Patience Nkam. "Evaluation of isolates and identified phenolics from pelargonium sidiodes against tuberculosis, other bacteria and fungi." Pretoria : [s.n.], 2005. http://upetd.up.ac.za/thesis/available/etd-02132006-113925.

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20

Keyser, Chad Alton. "Development of a Laboratory Based System for Selecting Insect Pathogenic Fungi with Greatest Potential for Success in the Field." DigitalCommons@USU, 2010. https://digitalcommons.usu.edu/etd/604.

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Many insects are important agricultural pests, and active control is necessary to keep them at abeyance. The naturally occurring entomopathogenic fungus Metarhizium is a promising tool to control pest insects, and its use avoids the well-known harmful side effects of chemical pesticides. Thousands of unique isolates of Metarhizium exist throughout the world. These isolates vary widely in their ability to cause infection and to tolerate stressful habitats. The research reported here tests the THESIS: A laboratory-based system can be devised that identifies, from among many Metarhizium isolates, those isolates with the greatest potential for successful biological control of pest insects in the field. The study was built on the testing of four hypotheses: (1) Laboratory bioassays using target pest insects will distinguish highly virulent strains of Metarhizium from less virulent strains, (2) Quantity and quality of mass-produced pathogenic fungi will vary among species and strains of Metarhizium, (3) The tolerance to ultraviolet radiation will vary among species and strains of Metarhizium, (4) The effect of temperature on growth rates and survival of both Metarhizium spores and hyphae will vary among isolates and species. These hypotheses test four field-relevant traits using a panel of ten isolates of Metarhizium isolates. Seven sets of laboratory experiments were devised to define the range of responses within the traits covered by the hypotheses. This series of general laboratory tests was developed to assist in identifying fungal isolates with high potential for field use. These tests included evaluation of each isolate's (a) insect pathogenicity, (b) mass–production capabilities, (c) tolerance to high temperatures, (d) tolerance to UV-B radiation, (e) rate of vegetative growth, (f) rate of spore germination, and (g) an evaluation of presence or absence of a post–stress growth inhibition. The application of this protocol to the isolates used in this study indicates that four isolates have high field potential, i.e., DWR 203, DWR 346, DWR 356 and ARSEF 324, and three of these were tested in a field trial. By following the procedures outlined in this thesis, selection of “good” isolates can be accomplished in the laboratory, and a successful isolate can be identified from the abundance of isolates present in nature.

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21

Mativandlela, S. P. N. (Sannah Patience Nkami). "Evaluation of isolates and identified phenolics from Pelargonium Sidoides against Mycobacterium Tuberculosis, other bacteria and fungi." Diss., University of Pretoria, 2005. http://hdl.handle.net/2263/28446.

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Anecdotal evidence of two South African Geranium species (Pelargonium reniforme and Pelargonium sidoides) from the United Kingdom with regard to plants being used against tuberculosis, which lacked scientific evidence’ prompted us to investigate these two plants for their antimicrobial properties. The German herbal remedy (‘Umckaloabo’) is prepared from these two plant species and is currently being sold for bronchitis. Acetone, chloroform and ethanol extracts were investigated against three bacteria (pathogens causing bronchitis), three fungi (fungal species associated with the upper and lower respiratory tract) and Mycobacterium tuberculosis. This is the first report on the extracts’ activity against Moraxella catarrhalis, and three fungi (Asperigilus niger, Rhizopus stolonifer and Fusarium oxysporum). Acetone and ethanol root extracts of P. sidoides and its combination with P. reniforme exhibited activity against bacteria at 5.0 mg/ml concentration. The fungi were significantly inhibited by the acetone and ethanol extracts of P. reniforme and the ethanol extract of P. sidoides at a concentration of 5.0 mg/ml. Antituberculosis activity was observed on acetone, chloroform and ethanol root extract of P. reniforme and chloroform extract of P. sidoides at 5.0 mg/ml concentration. The isolation and purification of compounds were attempted using two different approaches, of which the second approach resulted in isolation of four compounds and two flavonoids. One flavonoid (epigallocatechin) is isolated for the first time from P. sidoides. Laboratory investigations showed no activity of compounds isolated against M. tuberculosis. As Mycobacteria are intracellular pathogens, antimycobacterial activities may be due to either direct or indirect effects. Though the compounds in this study did not show antituberculosis activity, it can be speculated that the anecdotal evidence of TB-patients could be due to their immunostimulant activity.
Dissertation (MSc (Plant Physiology))--University of Pretoria, 2005.
Plant Science
unrestricted

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22

Tondello, Alessandra. "Edophytes Watching: combining molecular and microscopy approches to isolate, identify, tag, and monitor fungi and bacteria inside plants." Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3427059.

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This work aims at investigating plant-endophyte interactions under many different standpoints. We took into consideration bacterial as well as fungal microorganisms. Regarding the fungi, endophytic species occurring in orchid plants either as possible mycorrhizal partner as well as other internal colonizers were studied. We analyzed three different species of wild orchids found in the Euganean Hills area, North-Eastern Italy, namely Orchis militaris, Spiranthes spiralis and Orchis purpurea, which are mostly regarded as species in endangered status and for which little is known on the nature and presence of symbionts. An approach involving both molecular methods and microscopy techniques was used. Fungal isolation was performed from surface sterilized roots to obtain pure mycelia. A molecular approach allowed us to amplify the Internal transcribed spacer (ITS) region, starting both from root portions of the orchid plants and from pure cultured mycelia. This genetic region varies relatively little within species but dramatically between species and is easy to amplify because of its high copy number. In addition relatively few primers sets are needed due to the highly conserved SSU and LSU flanking regions. Different amplicons were obtained and analyzed from each species, at first upon their different ARDRA profiles obtained by enzymatic digestions. Representative cases were sequenced and results were examined by BLAST. Fungi of mycorrhizal nature and an additional series of endophytic ones, which are an important component of fungal biodiversity, were found. With different microscopy approaches (Fluorescence, Confocal and Transmission Electron Microscopy) we localized fungi within plant tissues and investigated their features. The hyphal septa found in Orchis militaris and Spiranthes spiralis samples were of basidiomycete type, which was confirmed by the results obtained from DNA extraction, ITS amplification and sequencing. In parallel bacterial endophytes were considered, starting from a previous work in which the coexistence of rhizobia with diverse, endophytic bacterial taxa within nodules of wild legume plants had been demonstrated, using molecular and microscopy-based approaches. In this work in order to co-localize the relevant endophytes inside plant tissues, different fluorescent proteins were used as markers for the different kinds of bacteria. Bacterial strains tagged with GFP were obtained using the pUTgfr2X plasmid, a delivery system for a mini-Tn5 trasposon, expressing kanamycin resistance and the GFP protein. While to obtain bacteria tagged with the rfp gene, a replacement of the gfp with a rfp gene was made starting from plasmid pRL765gfp, obtaining pRL765rfp. Both pRL765rfp and pUT gfp2X vectors were used to incorporate the GFP or RFP cassettes into the chromosome of R. leguminosarum bv. trifolii. In both cases plasmids were introduced by biparental mating. Pseudomonas sp. Hs1::gfp from a wild type Pseudomonas sp. isolated from wild legume nodules was obtained introducing pUTgfp2X by biparental mating. In parallel, to tag an Enterobacter agglomerans also isolated from legumes, pRL765rfp was introduced by electroporation. The four bacterial strains constructed were used to inoculate seedlings of Trifolium repens in nodulation tests. Tagged bacteria were localized on the surface and within plant tissues using Confocal microscopy. Subsequently Pseudomonas sp. Hs1::gfp and Enterobacter agglomerans pRL765rfp were used as co-inoculant strains during nodulation tests performed with seeds of wild legume plants from Sardinia. Their ability to be true endophytes was investigated using jointly standard colony isolation methods and direct PCR amplification of prokaryotic DNA from nodules and other tissues. We found that Pseudomonas sp. Hs1::gfp, upon root inoculation was able to invade one of the wild species of legumes (Tetragonolobus purpureus) and be traslocated to its aerial portions.
Questo lavoro ha come obbiettivo, lo studio con diversi approcci delle interazioni tra piante ed endofiti, sono perciò stati esaminati sia microrganismi batterici che fungini. Per quanto riguarda i microrganismi fungini, sono state studiate le specie endofitiche riscontrabili in piante di orchidea, sia come possibili partner micorrizici che come generici colonizzatori interni. Abbiamo analizzato tre diverse specie di orchidee selvatiche, considerate in pericolo, ritrovate nell’area dei Colli Euganei (Orchis militaris, Spiranthes spiralis e Orchis purpurea) per le quali poco si conosce riguardo alla presenza ed alla natura dei simbionti. Per questo studio è stato scelto un approccio che prevedeva l’utilizzo sia di metodologie molecolari che di tecniche di microscopia; inoltre per ottenere miceli fungini in coltura pura si è provveduto a isolarli dalle radici delle piante sterilizzate in superficie. L’approccio molecolare ci ha permesso di amplificare la regione ITS, partendo sia da miceli in coltura pura che direttamente da porzioni di radici. La regione ITS è una regione facilmente amplificabile con un numero relativamente esiguo di primers, per il suo alto numero di copie e per l’alta conservazione delle regioni che la fiancheggiano. Ulteriormente questa regione ci permette di ottenere interessanti informazioni, in quanto varia relativamente poco all’interno delle specie, ma molto tra specie diverse. Per ogni specie di orchidea analizzata sono stati ottenuti diversi ampliconi, che sono stati differenziati in base ai lori diversi profili ARDRA in seguito a digestione enzimatica. I casi più rappresentativi sono stati sequenziati e i risultati sono stati analizzati su piattaforma BLAST. Mediante analisi di omologie di sequenze sono stati identificati alcuni funghi di natura micorrizica e una serie di altri funghi endofitici, che risultano essere una componente importante della biodiversità fungina all’interno dei tessuti delle piante. Diversi tipi di microscopia: a fluorescenza, confocale ed elettronica a trasmissione sono stati usati per localizzare gli stessi funghi all’interno dei tessuti e analizzarne le caratteristiche. I setti (dolipori) ritrovati nelle ife fungine all’ interno dei campioni di Orchis militaris Spiranthes spiralis erano caratteristici dei basidiomiceti. Queste osservazioni hanno perciò permesso di confermare i risultai ottenuti dalle indagini molecolari. Partendo da un precedente lavoro nel quale è stata dimostrata la coesistenza di rizobi e altri batteri endofitici all’interno di noduli di leguminose selvatiche, per questo progetto si sono considerati anche gli endofiti di tipo batterico. In particolare per localizzare gli endofiti all’interno dei tessuti delle piante si è deciso di utilizzare dei marker fluorescenti diversi per tipi differenti di batteri endofitici e rhizobi. In alcuni ceppi batterici è stato inserito il gene codificante la proteina GFP usando un plasmide pUTgfr2X. Questo sistema trasporta un mini trasposone-Tn5, che esprime oltre alla proteina fluorescente anche la resistenza alla kanamicina. Per ottenere i ceppi marcati con RFP si è dovuto manipolare il plasmide pRL765gfp sostituendo il gene codificante la GFP con quello per la RFP, ricavando così un nuovo plasmide chiamato pRL765rfp. Per quanto riguarda i rizobi sia pRL765rfp che pUTgfp2X sono stati introdotti per coniugazione in R. leguminosarum bv. trifolii, così che i geni codificanti le proteine fluorescenti si integrassero nel cromosoma. Considerando invece le specie endofitiche diverse dai rizobi, un ceppo di Pseudomonas sp. Hs1::gfp è stato ottenuto introducendo per coniugazione in Pseudomonas sp. wt il plasmide pUTgfp2X. Pseudomonas sp. wt era stato precedentemente isolato da noduli di leguminose selvatiche così come Enterobacter agglomerans. Quest’ultimo ceppo (Enterobacter agglomerans) è stato marcato con la RFP introducendo il plasmide pRL765rfp per elettroporazione. I quattro ceppi batterici ottenuti sono stati utilizzati per inoculare piantine di Trifolium repens nei test di nodulazione, utilizzando il microscopio confocale è stato possibile localizzare i batteri sulla superficie delle radici o all’interno delle stesse Successivamente gli stessi endofiti marcati, utilizzati nei test di nodulazione su T. repens (Pseudomonas sp. Hs1::gfp e Enterobacter agglomerans pRL765rfp), sono stati co-inoculati in plantule di leguminose selvatiche della Sardegna per investigare la loro abilità di essere dei veri endofiti. Per questa ultima parte del lavoro si sono utilizzate comuni tecniche di isolamento e amplificazione del DNA dei procarioti tramite PCR. Pseudomonas sp. Hs1::gfp in particolare è in grado di colonizzare una delle specie analizzate (Tetragonolobus purpureus) e di essere traslocato nelle parti aeree.

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Entz, Susan Carol, and University of Lethbridge Faculty of Arts and Science. "Molecular methods and isolates of the entomopathogenic fungus Metarhizium anisolpliae for environmentally sustainable control of grasshoppers in Canada." Thesis, Lethbridge, Alta. : University of Lethbridge, Faculty of Arts and Science, 2005, 2005. http://hdl.handle.net/10133/249.

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Metarhizium anisoplia var. acridum, a gyphomycetous fungus registered worldwide for grasshopper and locust control, is currently under consideration as a worldwide for grasshopper and locust control, is currently under consideration as a potential alternative to chemical insecticides for grasshopper control in Canada. Research in this thesis has contributed data required for the registration of biological control agents in Canada. A diagnostic PCR assay was developed for the specific detection of M. anisopliae var. acridum DNA. The assay was highly sensitive and effective for the detection of fungal DNA in infected grasshoppers. A survey of southern Alberta soils conducted in the spring of 2004 revealed the presence of Metarhizium spp. at low natural incidennce. Two indigenous isolates demonstrated pathogenicity when bioassayed against laboratory-reared and field collected grasshoppers. One of the isolates demonstrated virulence comparable to a commercial isolate. An analysis of historical weather data revealed that summer weather in the Prairie provinces should not preclude the efficacy of M. anisopliae var. acridum under local conditions.
xv, 127 leaves : ill. (some col.) ; 28 cm.

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24

Schreuder, Wouter. "Characterization and pathogenicity of South African isolates of Fusarium oxysporum f. sp. melonis." Thesis, Stellenbosch : Stellenbosch University, 2000. http://hdl.handle.net/10019.1/51651.

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Thesis (PhD(Agric))--University of Stellenbosch, 2000.
ENGLISH ABSTRACT: The purpose of this study was to characterize the race and vegetative compatibility of Fusarium oxysporum f. sp. melonis (FOM) isolates collected in the major melon producing areas, to report on their geographical distribution, and their possible relatedness to isolates from other countries. Seventy two FOM isolates obtained from 30 fields in 17 melon producing regions were race-typed using the differential cultivars Topmark (susceptible to all races), Doublon (Fomi), CM 17187 (Fom2) and Perlita (Fom3) and grouped by means of vegetative compatibility. All isolates belonged to vegetative compatibility group 0134, indicating a high degree of genetic homogeneity among the South African FOM population. Fifty four isolates were identified as race 0, eight as race 1, and 10 as race 2. Race 0 occurred in 15 of the regions whereas race 1 was sporadically recovered. Race 2, on the other hand, was obtained only from four fields located in one geographical region. Perlita plants (carrying the gene Fom3) inoculated with local isolates ofrace 0 and race 2 and reference isolates of race 0 became stunted, their leaves turned yellow, and became thickened and brittle. These results suggested that Fom3 in Perlita confers a tolerant reaction compared to the resistant reaction of gene FornI in Doublon. The disease reaction of cultivar Perlita to FOM was therefore reinvestigated. Twenty isolates, including the four FOM races (0, 1, 2, and 1,2) obtained from different countries, were used. The differential cultivars were included to verify virulence of the isolates. Perlita plants inoculated with three isolates of race 2 remained asymptomatic. The remaining race 2 and 0 isolates, induced severe stunting of Perlita plants, but mean percentage stunting values did not differ significantly (P = 0.05) and ranged between 25.1 and 50.0. Leaves of stunted plants were chlorotic, thickened and brittle. Disease reaction of Perlita was verified at a lower inoculum concentration with two race 2 (pipette method) and two race 0 isolates (root dip method). Results proved that Fom3 does not confer similar resistance towards race 0 and some race 2 isolates as FornI in Doublon. Cultivars possessing Fom3, should therefore be considered tolerant to FOM races 0 and 2. The ability of a nit mutant isolate, generated from FOM race 0 which belongs to VCG 0134, to change its virulence during infection of melon plants, was investigated under quarantine. Seedlings of melon cultivars Imperial 45 and Early Sweet (no resistance genes), Amber (Fom2) and Fiata (FomI, Fom2) were consecutively grown in two cement troughs in a gauzehouse. Each planting was terminated when plants had advanced Fusarium wilt or after the fruit were harvested. In the first planting, Imperial 45 seedlings were transplanted and artificially inoculated with the nil mutant isolate. In the consecutive plantings, seeds were sown in the infested soil to enable natural infection. For each crop, representative plants showing Fusarium wilt were selected for isolation. All F. oxysporum isolates recovered were single-spored and their nit mutant and VCG status verified. Virulence of the labelled isolates was determined using differential cultivars. In trough A, all plants of the susceptible cultivars Imperial 45 and Early Sweet crops showed Fusarium wilt. The labelled isolates recovered from the selected plants were all designated race O. In the first crop (planting No.5) of the resistant cultivar Amber, 6.7% of the plants developed Fusarium wilt. In the second Amber crop the disease incidence increased to 56.6%, and to 81.8% in the final crop. Contrary to the susceptible cultivars, only race 2 isolates were obtained from the symptomatic Amber plants. Similar data were found with the susceptible cultivar Imperial 45 and the resistant cultivar Amber in trough B. Planting of Fiata caused a dramatic reduction in Fusarium wilt incidence in trough B. However, 1.2% of plants were affected by Fusarium wilt in the first Fiata crop (planting No.6), whereas 4% of the plants were symptomatic in the final planting. From these symptomatic Fiata plants only race 1,2 isolates were obtained. These findings, and the fact that the symptomatic plants represented a substantial proportion of the first Amber (approximately 7-15%) and Fiata (approximately 2%) crops, provedthat changes in the race structure of this fungal pathogen occurred rapidly when confronted with a resistant cultivar. The potential of RAPD analysis to differentiate between the isolates displaying virulence changes was evaluated. Four F. oxysporum f. sp. niveum isolates were included as an outgroup. A histopathological study was conducted to verify whether these isolates retain their ability to behave as true vascular pathogens. The three primers used clearly distinguished the 12 FOM isolates from the four F. oxysporum f. sp. niveum isolates. However, the primers showed a highly conserved and characteristic banding pattern for the FOM isolates which represented three physiological races (race 0, race 2, race 1,2), indicating that RAPD analysis cannot detect race-specific groupings in FOM. Disease reactions on the three differential cultivars confirmed the virulence of FOM isolates. The histopathological data furthermore proved that the two FOM races (race 2, race 1,2), which derived from the race 0 parent isolate, retained their ability to behave as true vascular pathogens.
AFRIKAANSE OPSOMMING: DIE KARAKTERISERING EN PATOGENESITEIT VAN SUID-AFRIKAANSE ISOLATE VAN FUSARIUMOXYSPORUMF. SP.MELONIS Die doel van die studie was om Fusarium oxysporum f. sp. melonis (FOM) isolate wat in die hoof spanspekproduserende gebiede versamel is, volgens ras en vegetatiewe verenigbaarheid te karakteriseer, en hul geografiese verspreiding en verwantskap met isolate van ander lande aan te dui. Twee en sewentig FOM isolate afkomstig vanaf 30 landerye wat 17 spanspekproduserende areas verteenwoordig, is gebruik. Die differensiële kultivars Topmark (vatbaar vir alle rasse), Doublon (Forni), CM 17187 (Fom2) en Perlita (Fond) is gebruik om die rasbepalings te doen asook om die vegetatiewe verenigbare groepe (VVG) te bepaal. Al die isolate is as VVG 0134 geklassifiseer, wat 'n hoë mate van genetiese homogenesiteit binne die Suid-Afrikaanse populasie aandui. Vier en vyftig isolate is as ras 0, agt as ras 1 en 10 as ras 2 geklassifiseer. Ras 0 is vanaf 15 gebiede afkomstig, terwyl ras 1 sporadies voorgekom het. Ras 2 is vanuit vier landerye binne dieselfde geografiese gebied verkry. Plante van die kultivar Perlita wat met plaaslike isolate van ras 0 en 2, asook verwysings-isolate van ras 0 geïnokuleer is, het verdwerg voorgekom. Die blare van die plante het vergeel, verdik en bros voorgekom. Hierdie siekte reaksie het aangedui dat Fond in Perlita toleransie bewerkstellig in teenstelling met die weerstandbiedende reaksie van geen Fomi in Doublon. Die siekte reaksie van Perlita teenoor FOM is dus verder ondesoek. Hiervoor is 20 isolate wat al vier FOM rasse insluit (0, 1, 2, en 1,2), en van verskillende wêrelddele afkomstig is, gebruik. Die virulensie van die isolate is met die differensiële kultivars bevestig. Drie van die ras 2 isolate het geen siektesimptome op Perlita veroorsaak nie. Die ander ras 2 isolate, en al die ras 0 isolate, het egter die Perlita plante aansienlik verdwerg en die blare vergeel en verdik. Laasgenoemde groep isolate het 'n gemiddelde verdwergingsindeks van tussen 25.1% en 50.0% veroorsaak. Die siekte reaksie by Perlita is verder bevestig deur plante teen 'n laer inokulumdigtheid van twee ras 2 (pipet metode), en twee ras 0 (wortel-doop metode) isolate, te inokuleer. Uit die resultate was dit duidelik dat die weerstand wat Fom3 teenoor ras 0 en sommige ras 2 isolate verskaf, van FornI verskil. Kultivars wat oor die weerstandsgeen Fom3 beskik moet dus as tolerant beskou word. 'n Ondersoek is geloods na die vermoë van 'n nil mutant isolaat, genereer vanaf die wilde ras 0 isolaat van FOM (VVG 0134), om onder kwarantyn sy virulensie gedurende infeksie van spanspekplante te verander. Saailinge van die spanspekkultivars Early Sweet (geen weerstandsgene), Amber (Fom2) en Fiata (FornI, Fom2) is opeenvolgens in twee sement trêe in 'n gaashuis verbou. Die afsonderlike aanplantings is beëindig sodra gevorderde Fusarium-verwelksimptome verkry is, of nadat vrugte ge-oes is. Vir die eerste aanplanting is oorgeplante Imperial 45 saailinge kunsmatig met die nil mutant isolaat geïnokuleer. Tydens die opeenvolgende aanplantings is saad direk in die besmette grond gesaai ten einde natuurlike infeksie te verkry. Met elke aanplanting is isolasies gedoen vanaf verteenwoordigende plante wat Fusarium-verwelksimptome getoon het. Alle F. oxysporum isolate wat verkry is, is ge-enkelspoor en hul nit mutant status en VVG is bevestig. Virulensie van die gemerkte isolate is bepaal deur inokulasie van die differensiële kultivars. Alle plante van die vatbare Imperial 45 en Early Sweet kultivars wat in trog A geplant is, het Fusarium-verwelksimptome getoon. Die gemerkte isolate wat vanaf die verteenwoordigende plante verkry is, is almal as ras 0 geklassifiseer. Tydens die eerste aanplanting van die weerstandbiedende kultivar, Amber (aanplanting No.5), het 6.7% van die plante Fusarium-verwe1ksimptome ontwikkel. Tydens die tweede en derde aanplanting van Amber het die frekwensie van siektevoorkoms verhoog na 56.6% en 81.8 %, onderskeidelik. In teenstelling met die vatbare kultivars, is slegs ras 2 vanuit die Amber plante met siektesimptome verkry. Soortgelyke resultate is met Imperial 45 en Amber in trog B verkry. Aanplanting van kultivar Fiata het egter 'n dramatiese verlaging in die voorkoms van Fusarium-verwelk bewerkstellig. Tydens die eerste Fiata aanplanting (aanplanting No.6) het 1.2% plante Fusarium-verwelksimptome ontwikkel, en 4% tydens die laaste aanplanting. Vanaf hierdie plante is slegs ras 1,2 isolate verkry. Hierdie bevindings, en die feit dat 'n aansienlike hoeveelheid van die Amber (ongeveer 7-15%) en Fiata plante (ongeveer 2%) siektesimptome getoon het, bewys dat FOM vinnig van virulensie verander wanneer die patogeen 'n weerstanbiedende kultivar infekteer. Die vermoë van RAPD analise om tussen isolate wat in virulensie verander het, te onderskei, is ondersoek. Vier isolate van F. oxysporum f. sp. niveum is as 'n buite-groep ingesluit. Om te bevestig dat die isolate wat van ras verander het wel egte vaskulêre patogene is, is 'n histopatologiese ondersoek gedoen. Die drie inleiers wat gebruik is, het die 12 FOM isolate duidelik van die vier F. oxysporum f. sp. niveum isolate onderskei. Die 12 FOM isolate wat drie fiosologiese rasse (ras 0, ras 2, ras 1,2) verteenwoordig het, is egter saam gegroepeer, wat aandui dat hierdie metode nie tussen rasse van FOM kan onderskei nie. Inokulasiestudies met die differensiële kultivars het die virulensie van die isolate bevestig. Die histopatologiese ondersoek het verder bewys dat beide FOM rasse (ras 2, ras 1,2) wat vanaf die wilde tipe ras ° isolaat ontstaan het, hul vermoë behou het om as egte vaskulêre patogene op te tree.

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Júnior, Giovani Marcio Coura. "Análise integrada das variáveis virulência e produção de conídios na seleção de fungos entomopatogênicos para o desenvolvimento de biopesticidas." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/11/11146/tde-16082017-135321/.

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Os fungos entomopatogênicos do gênero Metarhizium e Beauveria compreendem um importante grupo de patógenos de artrópodes-praga. A seleção de isolados de fungos promissores é a primeira e uma das mais importantes etapas no desenvolvimento de um biopesticida, pois alguns isolados podem apresentar alta virulência e não necessariamente boa produção em substrato e vice-versa, sendo interessante a combinação desses dois parâmetros para a viabilidade comercial de um produto. A dificuldade de criação ou manutenção de algumas espécies de pragas em laboratório é um limitante para a condução de testes de virulência, tornando-se interessante a utilização de espécies modelo, de fácil criação, nas etapas preliminares de seleção. Nesse sentido, este estudo objetivou selecionar isolados com alta produção de conídios e virulência, comparando a eficiência de controle de M. anisopliae e B. bassiana às pragas alvo Mahanarva fimbriolata e Bemisia tabaci Biótipo B, respectivamente, com a mortalidade em Tenebrio molitor. Inicialmente, foram selecionados 50 isolados a partir de 100 isolados de cada gênero, baseado em avaliações visuais do crescimento e esporulação em meio de cultivo em placas de Petri. Estes isolados selecionados foram cultivados em arroz parboilizado para quantificação do rendimento produtivo de conídios. Os 25 isolados mais produtivos de cada espécie de fungo foram utilizados nos bioensaios com T. molitor. Posteriormente, os cinco isolados que causaram maior e menor mortalidade de cada gênero, foram utilizados nos bioensaios com as respectivas pragas-alvo. A variação no rendimento de conídios de Beauveria spp., foi de 0,3 a 7,7 x 109 conídios.grama de arroz-1 e de Metarhizium spp. foi de 0,1 a 2,5 x 109 conídios.grama de arroz-1. A mortalidade confirmada de larvas de T. molitor por Beauveria spp., variou de 5,5 a 96,4% e por M. anisopliae variou de 29,1 a 89,1%. Alguns isolados causaram mortalidade elevada tanto no inseto modelo quanto na praga-alvo, porém, não foi verificada uma relação entre a virulência para as duas espécies. Da mesma forma, não foi observada associação entre os parâmetros produção de conídios e virulência. O isolado ESALQ 4958 de B. bassiana se destacou nos dois bioensaios apresentando mortalidades elevadas de ninfas de B. tabaci Biótipo B. Nos bioensaios utilizando ninfas de M. fimbriolata, ESALQ 1641 foi o isolado que apresentou os maiores percentuais de mortalidade nos dois bioensaios. Analisando conjuntamente as variáveis produção de conídios e virulência a T. molitor e a espécie alvo, os isolados ESALQ 540 (B. bassiana) e ESALQ 1116 (M. anisopliae) se destacaram por apresentarem valores elevados para todas as variáveis de interesse. Os resultados reforçam a necessidade de uma análise conjunta destas variáveis com um peso diferenciado para cada variável na seleção de isolados para utilização em produtos microbianos para o controle de pragas.
The genus Metarhizium spp. and Beauveria spp. are important entomopathogenic fungi used to control arthropod pests. The selection of promising fungal isolates is the first and one of the most important steps on the development of a biopesticide, since some isolates may present high virulence and not necessarily good production in substrate and vice-versa, being the combination of these two parameters important for the commercial viability. Difficulties of rearing or maintaining some species of pests in laboratory are limitations for the conduction of virulence tests, justifying the use of easy to breed model species on the preliminary steps of selection. Therefore, this study aimed to select isolates with high conidia production and virulence, comparing the control efficiency of M. anisopliae and B. bassiana to the target pests, Mahanarva fimbriolata and Bemisia tabaci biotype B, respectively, with mortality in Tenebrio molitor. At first, 50 isolates were selected from 100 isolates of each genus, based on growth and sporulation in culture medium on Petri dishes. These isolates were grown in parboiled rice to quantify the yield of conidia. The 25 most productive isolates of each fungus species were used in the bioassays with T. molitor. After, the five isolates that caused higher and lower mortality of each genus were used in the bioassays with the respective target pests. Beauveria spp. conidia yield ranged from 0.3 to 7.7 x 109 conidia.grams of rice-1 and Metarhizium spp. from 0.1 to 2.5 x 109 conidia.gram of rice-1. The confirmed mortality of T. molitor larvae by Beauveria spp. varied from 5.5 to 96.4% and M. anisopliae varied from 29.1 to 89.1%. Some isolates caused high mortality in both, model insect and the target pest; however, no relationship between the virulence of both species was observed. Similarly, there was no association between the parameters conidia production and virulence. The B. bassiana isolate ESALQ 4958 in both bioassays presented high mortalities of B. tabaci Biotype B. In bioassays using M. fimbriolata nymphs, ESALQ 1641 was the isolate that presented the highest mortalities in both bioassays. Analyzing the variables, conidia production and virulence to T. molitor and the target species, the isolates ESALQ 540 (B. bassiana) and ESALQ 1116 (M. anisopliae) showed high values for all variables of interest. The results reinforce the necessity of a joint analysis of these variables with different weight for each one in the selection of isolates, aiming to use them in microbial products for pest control.

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Wilson, Richard. "Endophytic fungi from four tree species in New Brunswick and a comparison of two methods of identification of Leptostroma isolates of Pinus resinosa, morphology and molecular probing." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0003/NQ29475.pdf.

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27

Oliveira, Geovana Gomez de. "Trichoderma spp. no crescimento vegetal e no biocontrole de sclerotinia slcerotiorum e de patógenos em sementes de cártamo(Carthamus tinctorius)." Universidade Federal de Santa Maria, 2007. http://repositorio.ufsm.br/handle/1/5145.

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The cultivation of flowers is, in Brazil, very recent and little known species have a high ornamental potential, Cartamus is such a species and there are cultivars for the production of oil and as well as for ornamental pourposes. Being a recent crop in Brazil, little is known about its associated diseases, specially those that affect the seeds and the soil borne, such as white mold that causes great losses to several crops. Six experiments were conducted with the objectives of testing the reaction of the crop to isolates of Sclerotinia sclerotiorum, the biocontrol of S. sclerotiorum and of pathogens associated to the seeds, and the effect of the biocontrollers on cartamus plant growth. Isolates of the pathogen from chrisantemum, lettuce, soybean, and carrots, as well as isolates of Trichoderma sp. (ETSR 20 e TC 1.15) and comercial products of Trichoderma spp. (Agrotrich and Trichodel®) were used. In the reaction test, the cartamus crop was more severely attacked by the isolate obtained from lettuce, which when incorporated to commercial substrate did not promote the development of the disease in plant, but reduced plant growth. Trichodel® was the product that promoted the highest growth of plants when incorporated to the substrate, even in the absence of the pathogen. The product more efficient in controlling seed pathogens was Agrotrich. In the growth of seedlings, the isolates ETSR20 and TC1.15 were the best, when applied to the seeds and the latter promoted the best emergency of seeds in commercial substrate. Therefore, the Trichoderma based products can be used in the control of seed pathogens and growth promotion of cartamus plants as seed or substrate treatment. There are differences in disease severity on cartamus among isolates of S. sclerotiorum from different crops and its presence reduces the crop s growth, even in the absence of visible symptoms
A floricultura, no Brasil, é relativamente recente, existindo espécies pouco conhecidas com alto potencial ornamental. O cártamo é uma delas e possui cultivares tanto para a produção de óleo quanto para ornamentação. Por ser uma cultura recente no Brasil, pouco se conhece sobre as doenças associadas a ela, especialmente as que afetam as sementes e as veiculadas pelo solo, como o mofo branco, que causa grandes prejuízos a diversas culturas. Seis experimentos foram conduzidos com o objetivo de testar a reação da cultura a isolados de Sclerotinia sclerotiorum, o biocontrole de patógenos associados às sementes e de Sclerotinia sclerotiorum e o efeito dos biocontroladores no crescimento de plantas de cártamo. Foram utilizados isolados de Sclerotinia sclerotiorum das culturas do crisântemo, alface, soja e cenoura, e isolados de Trichoderma spp. (ETSR 20 e TC 1.15) e formulados comerciais à base de Trichoderma spp. (Agrotrich e Trichodel®). No teste de reação, a cultura do cártamo foi mais severamente atacada pelo isolado proveniente da alface, o qual, quando incorporado ao substrato comercial, não promoveu o desenvolvimento da doença nas plantas, porém prejudicou seu crescimento. Trichodel® foi o produto que maior crescimento proporcionou às plantas quando incorporado ao substrato, mesmo sem a presença do patógeno. O produto mais eficiente no controle dos patógenos das sementes foi o formulado comercial Agrotrich. No crescimento de plântulas, os isolados ETSR 20 e TC 1.15 se sobressaíram quando aplicados às sementes e este obteve melhor resultado na emergência de plântulas em substrato. Assim, os produtos a base de Trichoderma são viáveis para controle de patógenos de sementes e crescimento de plantas tanto no tratamento de sementes como do substrato. Existem diferenças na severidade da doença em cártamo entre isolados de Sclerotinia sclerotiorum oriundos de diferentes culturas e sua presença reduz o crescimento da cultura, mesmo na ausência de sintomas visíveis

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28

Farias, Emanuela Ximenes. "Study of secondary metabolites of cytotoxic potential isolates of the fungus Paecilomyces lilacinus recovered from marine sediments of costa cearense." Universidade Federal do CearÃ, 2014. http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=11874.

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Este trabalho descreve o estudo de bioprospecÃÃo de metabÃlitos secundÃrios citotÃxicos do PecÃm-CE. Inicialmente, foi realizado o estudo cinÃtico da produÃÃo de metabÃlitos secundÃrios por P. lilacinus cultivado no meio de batata-dextrose (BD) durante 7, 14 21 e 28 dias. AtravÃs da anÃlise cromatogrÃfica dos extratos, bem como dos resultados de atividade citotÃxica dos mesmos, frente Ã linhagem de cÃlula tumoral HCT -116 (cÃncer de cÃlon), foi possÃvel selecionar o extrato oriundo do crescimento do fungo por 14 dias (inibiÃÃo de crescimento:85,7 %) como o mais citotÃxico. O fracionamento bioguiado do extrato orgÃnico do fungo cultivado por 14 dias em grande escala levou ao isolamento dos esterÃides wortimanina e 11-deacetoxiwortimanina, ambos inÃditos no gÃnero Paecilomyces . Os metabÃlitos secundÃrios foram isolados at ravÃs de mÃtodos cromatogrÃficos usuais e CLAE (Cromatografia lÃquida de alta eficiÃncia). A caracterizaÃÃo estrutural foi realizada atravÃs do uso de mÃtodos espectromÃtricos, especialmente espectrometria de massas, ressonÃncia magnÃtica nuclear (1H e 13C) e infravermelho, alÃm de comparaÃÃo de dados da literatura. TambÃm foi realizado um estudo de desreplicaÃÃo do extrato, o qual sugeriu a presenÃa dos compostos ciclosinefungina, estatana A, virescenosideo -D, (+)-esclerotiorina, 2'-Amino-2'-deoxiguanosina , fumaramidimicina e deoxinivalenol, todos inÃditos no gÃnero Paecilomyces produzidos por Paecilomyces lilacinus (cepa BRF053) recuperado de sedimentos da praia.
The bioprospection of cytotoxic secondary metabolites produced by Paecilomyces lilacinus(fungal strain BRF053) recovered from sediments collected at PecÃm beach, CearÃ state, was investigated. First, the production of compounds by the fungal strain was performed by culturing the microorganism in potato-dextrose broth (PDB) for 7,14,21 and 28 days. Both HPLC analysis and cytotoxic activity(tumor cell line HCT-116) of the extracts from each period of culture revealed 1 4 days as the optimum time for producing cytotoxic compounds (growth inhibition 85.7 %). Bioguided fractionation of the organic extract from the fungus cultured in large scale under the same conditions (14 days) allowed the isolation of the steroids wortimann in and 11-deacetox ywortiman n in, both new compounds in Paecilomyces. These compounds were isolated by chromatography column and HPLC, and their structures were elucidated by spectrometric methods (HRMS, 1H, 13C NMR, and IR) besides comparison with literature data. Dereplication of the extract by LC -MS suggested the presence of cyclosinefungin, stanna A, virescenoside-D, (+)-esclerotiorin, 2'-Amino-2'-deoxiguanosine, fumaramidimicin e deoxy nivalenol, all of the new compounds in Paecilomyces.

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29

Woltjen, Christine D. "Responding to industry needs from the field to the greenhouse: Dieback and cankers of Gleditsia triacanthos var. inermis and characterization of an Ohio isolate of Melon necrotic spot virus and its vector, Olpidium bornovanus, collected from Cucumis sativ." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1276549930.

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30

Chaves, Ana Filipa Amaral. "Biofilm Interactions Between Filamentous Fungi and Bacteria Isolated from Drinking Water." Master's thesis, 2014. https://repositorio-aberto.up.pt/handle/10216/90008.

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31

Chiu, Yu-Chiao, and 邱毓喬. "The identification and xylanase genes characterization of two isolated rumen fungi." Thesis, 2016. http://ndltd.ncl.edu.tw/handle/13862572276774388932.

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32

Chaves, Ana Filipa Amaral. "Biofilm Interactions Between Filamentous Fungi and Bacteria Isolated from Drinking Water." Dissertação, 2014. https://repositorio-aberto.up.pt/handle/10216/90008.

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33

Chen, Yo-Chia, and 陳又嘉. "Studies of Obligate anaerobic fungi isolated from ruminants and their cellulolytic ability." Thesis, 1997. http://ndltd.ncl.edu.tw/handle/17049197515203357459.

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碩士
國立臺灣大學
農業化學系
85
Obligate anaerobic fungi are found in gastrointestinal trace of the ruminants. It had been reported that five genera and sixteen species were isolated from many ruminants before 1996. The obligate anaerobic fungi have been known to possess diverse plant polysaccharide hydrolase activities. The Neocallimastix cellulases have been shown to form a large multienzyme complex, which exhibits very high activity against crystalline cellulose. In this study, I isolated seven isolates of obligate anaerobic fungi from the feces of llama, water baffulo and Taiwan native animals Formosan Sika deer and Taiwan yellow bos. The seven isolates were cultured on modified basal medium with filter paper as the carbon source. According to their morphological characters, D-21, X-26, X-34, Y-15, Y-24 and L-87 were classified to genera Neocallimastix, and X-55 isolate was similar to genera Caecomyces. In my study, I have also compared cellulolytic ability with the crude extracts of the six Neocallimastix isolates and the commercial cellulases of Trichoderma viride (B.M.) and Aspergillus niger (Sigma). The activity of the complete cellulase complex was measured by using Whatman No. 1 filter paper. This assay is recommended by the Commission on Biotechnology (IUPAC) for measurement of activity of total cellulase or true cellulase. The cellulolytic activity of N. frontalis X-26 was 0.02 IU/mg which of T. viride and A. niger were 0.035 and 0.015 IU/mg. The endoglucanase activity of N. frontalis D-21 was 0.460 IU/mg which of T. viride and A. niger were 0.545 and 0.148 IU/ mg protein. This result suggests the anaerobic fungi exhibits very high activity against cellulose. I report here the cellulolytic activites of six isolates and provide that obligate anaerobic fungi can also be found in gastrointestinal trace of the Taiwan native ruminants - Formosan Sika deer and Taiwan yellow bos.

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34

Troeh, Zahra Ifnou. "Diversity and efficacy of arbuscular mycorrhizal (AM) fungi isolated from soils of soybean fields /." 2006.

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35

Li, Han-Jen, and 李漢楨. "Study of chitins isolated from fungi combined with platelet lysate on chronic wound healing." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/48868441626375130595.

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碩士
臺北醫學大學
生醫材料暨工程研究所
97
In the present study, SACCHACHITIN and RHIZOCHITIN were used to evaluate the effectiveness in accelerating chronic wound healing, by employing a cell line in vitro model and a chemical-induced diabetic rat in vivo model. For In vitro, Hs68 fibroblast cells were cultured in high-glucose medium (110 mM glucose) for 24 hours and then SACCHACHITIN and RHIZOCHITIN (100, 250, and 500 μg/ml) were supplemented as suspension to the medium. After 7 days, we observed both SACCHACHITIN and RHIZOCHITIN resumed cell viability and cell mobility of Hs68 cell in high-glucose condition. In in vivo model, full-thickness skin wounds (4 cm2) were created on the dorsum of wistar diabetic rats. The cutaneous wounds were treated with membrane of SACCHACHITIN or RHIZOCHITIN, and the area of wounds was analysed at 3, 7, 14, 21 days. The data in vivo indicated both SACCHACHITIN and RHIZOCHITIN induced wound healing, significantly. Furthermore, new wound dressings were designed to combine with GF18, a mixture of human platelet growth factor, with SACCHACHITIN or RHIZOCHITIN, and the effects of the novel wound dressings on diabetic wound healing were evaluated. The data in vivo indicated the addition of GF18 optimized the recovery effect of SACCHACHITIN and RHIZOCHITIN. Transforming growth factor-beta1(TGF-β1) was assayed by ELISA in the samples of wound area. As a result, SACCHACHITIN, RHIZOCHITIN, GF18 and new wound dressings all significantly increased the production of TGF-β1 in different stages of the healing process.

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36

Esteves, Maria Prata e. Castro de Sena. "Anticancer activity of marine-derived fungi extracts and isolated compound in human cancer cell lines." Master's thesis, 2014. https://repositorio-aberto.up.pt/handle/10216/77834.

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37

Esteves, Maria Prata e. Castro de Sena. "Anticancer activity of marine-derived fungi extracts and isolated compound in human cancer cell lines." Dissertação, 2014. https://repositorio-aberto.up.pt/handle/10216/77834.

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38

Nunes, Cátia Fernandes. "Selection of glyphosate-degrading microorganisms isolated from vineyard soils." Master's thesis, 2020. http://hdl.handle.net/10198/24073.

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Glyphosate is the most widely used herbicide worldwide. It is a non-selective, systemic, post-emergence herbicide that controls more weed species than any other. Its extensively use have been led to the contamination of both terrestrial and aquatic ecosystems, becoming a huge environmental concern worldwide. Bioremediation using glyphosate-degrading microorganisms could be a promising approach to overcome the risks associated to the accumulation of this herbicide in the environment. Hence, this work aims to isolate bacteria and fungi from vineyard soil's exposed to glyphosate and identify potential strains for bioremediation of glyphosate-polluted soils. Accordingly, microorganisms were isolated from soils collected in three different slope positions (top, middle and bottom), and were further screened for their ability to grow in medium containing glyphosate as the sole phosphorus source. Overall, 477 glyphosate-tolerant morphotypes (325 bacteria and 152 fungi) were isolated from vineyard soil’s. Slope position showed to influenced significantly the diversity of bacteria but also some soil physicochemical properties. Contents on Extractable phosphorus (E.P.) in soils affected significantly the composition of the whole microbial community, in particular of bacteria. Among the strains screened, 55% of the bacteria and 33 % of the fungi showed the capacity to utilize glyphosate efficiently. The sequencing of the 16S (for bacteria) and ITS (for fungi) region of rRNA, identified Erwinia billingiae (strain B105), Pseudomonas fragi (strains B328 and B295), Pseudomonas sp. 1 (strain B93) and Trichoderma sp. 2 (strain F54), as the best potential candidates for bioremediation of glyphosate-polluted soils. Their bioremediation effectiveness must be evaluated in future studies.
O glifosato é o herbicida mais usado em todo o mundo. É um herbicida não seletivo, sistêmico e pós-emergência que controla mais espécies de ervas daninhas do que qualquer outro herbicida. O seu uso extensivo tem levado à contaminação de ecossistemas terrestres e aquáticos, tornando-se uma grande preocupação ambiental em todo o mundo. A biorremediação com microrganismos degradadores do glifosato pode ser uma abordagem promissora para superar os riscos associados à acumulação deste herbicida no meio ambiente. Assim, este trabalho tem como objetivo isolar bactérias e fungos de solos de vinhas expostas ao glifosato e identificar potenciais estirpes para a biorremediação de solos poluídos com glifosato. Deste modo, microrganismos foram isolados de amostras de solos de três posições de declive diferentes (topo, meio e fundo), e foram ainda avaliados quanto à sua capacidade de crescer em meio com glifosato como única fonte de fósforo. No geral, 477 morfotipos tolerantes ao glifosato (325 bactérias e 152 fungos) foram isolados do solo das vinhas. A diferença de declives mostrou influenciar significativamente a diversidade de bactérias, mas também algumas propriedades físico-químicas do solo. Os conteúdos de fósforo extraível (E.P.) nos solos afetaram significativamente a composição de toda a comunidade microbiana, em particular de bactérias. Entre as estirpes testadas, 55% das bactérias e 33% dos fungos mostraram capacidade de utilizar o glifosato de forma eficiente. O sequenciamento da região 16S (para bactérias) e ITS (para fungos) do rRNA, identificou Erwinia billingiae (estirpe B105), Pseudomonas fragi (estirpes B328 e B295), Pseudomonas sp. 1 (estirpe B93) e Trichoderma sp. 2 (estirpe F54), como os melhores candidatos potenciais para biorremediação de solos poluídos com glifosato. A sua eficácia de biorremediação deve ser avaliada em estudos futuros.

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39

Fernandes, Susana Maria da Fonseca. "The role of abiotic and biotic factors on biofilm formation by filamentous fungi isolated from drinking water." Master's thesis, 2018. https://hdl.handle.net/10216/113499.

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40

Fernandes, Susana Maria da Fonseca. "The role of abiotic and biotic factors on biofilm formation by filamentous fungi isolated from drinking water." Dissertação, 2018. https://hdl.handle.net/10216/113499.

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41

Fernandes, Susana Maria da Fonseca. "The role of abiotic and biotic factors on biofilm formation by filamentous fungi isolated from drinking water." Dissertação, 2002. https://repositorio-aberto.up.pt/handle/10216/113499.

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42

Eyjolfsdottir, Gudridur Gyda. "Fungi isolated from stained wood associated with bark beetle galleries in timber trees in New Zealand, Norway and Western Canada." 1990. http://hdl.handle.net/1993/7205.

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Fungi isolated from stained wood, mostly coniferous and recently attacked by bark beetles, in New Zealand, Norway, Western Canada, and the U. S.A. were grown in culture under prescribed conditions to determine their specific characteristics and taxonomic relationships. The study resulted in the preparation of detailed descriptions of 36 taxa which represent species of the genera Acremonium, Aphanocladium, Beauveria, Chalara, Dipodascus, Engyodontium, Gliocladium, Graphium, Hyalodendron, Hyalopesotum, Hyalorhinocladiella, Leptodontidium, Mariannaea, Monocillium, Phaeoisaria, Phialographium, Phialophora, Pitomyces, Rhinocladiella, Verticillium, Volutella, and taxonomic genus 1. Of these, nine are proposed as new, and are to be found in Acremonium, Beauveria, Gliocladium, Graphium, Hyalopesotum (synanamorph Hyalorhinocladiella), Monocillium, Phialographium (synanamorph Phialophora), and one for which the new genus will be erected. In addition, to accommodate one of these species, the genus Erostella was re-established, and its type, E. minima, and the only other previously described species, E. fraxinopennsylvanica, were included for comparison with E. novae-zelandiae sp. nov. prop. Wood-staining fungi, especially members of the Ophistomatales and their anamorphs, many of which cause blueing of the sapwood of economically important timber trees, have been the subject of numerous studies. However, other fungi which occur in association with the wood-staining organisms in and around bark beetle galleries have largely been ignored, especially if they are non-staining. This investigation sought at least partially to redress this neglect. This study is a taxonomic investigation of various fungi from the bark beetle galleries. Its aim was to identify the more poorly known entities and thus add information as to the nature of the bark beetle-host tree-microorganism ecosystem.

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43

Cantrell, Isabella Cardona. "Effects of Preinoculation with VAM fungi isolated from different sites on plant tolerance to salinity in soils amended with sodium chloride." Thesis, 2000. http://hdl.handle.net/1957/33109.

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The hypothesis that inoculation of transplants with vesicular-arbuscular mycorrhizal (VAM) fungi before planting into saline soils would alleviate salt effects on growth and productivity was tested on lettuce (Lactuca sativa L.) and onion (Allium cepa L.). A secondary hypothesis was that the fungi isolated from a saline soil would be more effective than those from a nonsaline soil. VAM inocula from a high-and a low-salt soil were trap-cultured, their propagules quantified, adjusted, and added to a pasteurized growth medium in which seeds germinated and seedlings grew for a few weeks. These seedlings, once colonized by VAM fungi, were transplanted into saline soil. Seedlings were exposed to high concentrations of NaCl at the time of transplant; in this respect, our technique aimed to simulate conditions of high salinity prevalent in soils affected by NaCl. Preinoculated lettuce and onion transplants grown for 10 weeks had increased shoot biomass compared with nonVAM plants at all salinity (NaCl) levels tested. Leaves of VAM lettuce at the highest salt level were significantly greener than those of the nonVAM lettuce. NonVAM onions were stunted due to available P deficiency in the soil, but inoculation with VAM fungi alleviated P deficiency and salinity effects except at the highest salinity level; nevertheless, VAM onions were significantly larger at all salinity levels. Increasing the level of available P by weekly applications to nonVAM plants partially alleviated the salinity effects on onion growth. VAM fungi from the saline soil site were not more effective in ameliorating the reduction on plant growth caused by salt than those from the nonsaline site. Colonization of roots and length of soil hyphae produced by the test fungi decreased with increasing salt. Results indicate that preinoculation of transplants with VAM fungi can effectively alleviate deleterious effects of saline soils on crop productivity.
Graduation date: 2000

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44

Falconi, Cesar E. "Epiphytic yeasts isolated from apple leaves to control of gray and blue mold fruit rots of apple." Thesis, 1996. http://hdl.handle.net/1957/34028.

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Eight phylloplane yeasts were isolated from backyard apple trees in Corvallis, OR. Yeast isolates were classified to genus or species level. All isolates were tested in vitro for antagonistic activity against the postharvest pathogens Botrytis cinerea and Penicillium expansum. Of these isolates, Aureobasidium pullulans, Sporobolomyces roseus Rhodotorula sp., consistently reduced mycelial growth of B. cinerea and P. expansum in nutrient yeast dextrose agar (pH 4.5 or 7.0) incubated for 8 or 30 days at 24 or 1 C, respectively. These three yeasts also were evaluated for their ability to suppress spore germination of B. cinerea and P. expansum in a gradient of apple juice concentrations and to suppress development of gray and blue mold lesions in inoculated fruits of Golden Delicious apple. Germination of B. cinerea and P. expansum was reduced significantly (P��0.05) when incubated with the yeast isolates in 100 or 50% apple juice, but not in 0, 1 or 10% apple juice. S. roseus and A. pullulans reduced significantly (P��0.05) the size of gray mold lesions in wounded fruit stored at 5 C and 24 C by 63 to 72 and 81 to 90%, respectively, when compared to the nontreated control. Size of blue mold lesions in fruit stored at 5 and 24 C also were reduced significantly (P��0.05) by 66 to 38 and 74 to 63%, respectively, when pre-treated with S. roseus and A. pullulans. In general, fruit rot suppression by some yeasts isolated in this study was similar in magnitude to suppression obtained by Cryptococcus laurentii isolate 87-108, a yeast with commercial potential to suppress postharvest rots of pome fruits. Pretreatment of apple wounds with washed cells of A. pullulans, S. roseus, Rhodotorula sp., resulted in disease suppression, but treatment of wounds with cell-free culture supernatant of these isolates did not affect lesion development. Population size of A. pullulans, S. roseus, and C. laurentii increased in apple wounds incubated at 5 or 24 C for up to 25 days, indicating that they colonized the wound site. Data collected in this study support the hypothesis that yeast isolates antagonize fruit pathogens by competing for nutrients in wounds on fruit surfaces. The isolates of A. pullulans and S. roseus show promise for commercial development.
Graduation date: 1997

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45

Kwinda, Grace Thiambi. "Detection and molecular identification of Mucorales isolated from spoilt agricultural commodities collected in fresh produce markets in Gauteng province, South Africa." Diss., 2014. http://hdl.handle.net/10500/19632.

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Fruit and vegetables are often spoilt during storage, handling and transportation due to microorganisms. The common spoilage causes are fungi within the order Mucorales, the largest order of the class Zygomycetes. Such spoilage can result in reduced food supplies, poor quality and severe losses to producers and traders. The study was to investigate the type of Mucorales prevalent in various commodities and in a particular market than others. Fifty infected papaya, peaches and strawberries were collected at five occasions from large, medium and small markets. Isolation was done aseptically in a biosafety cabinet. Mucorales were identified morphologically, through culture based tests and molecular techniques. Mucorales isolated are Rhizopus stolonifer, Mucor circinelloides and Mucor racemosus. Mucorales were isolated at a higher rate in samples collected from the small market than other two markets. Spoilage in all three markets is assumed to be influenced by lack of modified temperatures in the storage room.
Life and Consumer Sciences
M. Sc. (Life Sciences)

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46

Valayil, Jinu Mathew. "Structure Elucidation and Biological Evaluation of a Novel Steroidal Saponin, Cholestanol Glucoside Isolated from Saraca Asoca Enodophytic Fuungus, Lasiodiplodia Theobromae." Thesis, 2015. http://etd.iisc.ac.in/handle/2005/3549.

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Although the molecular mechanisms underlying the onset and progression of cancer has been unraveled to a great extend, cancer continues to remain a leading cause of death around the world. Clinical efficacy of the existing anticancer drugs are largely compromised by the inherent and acquired resistance of cancer cell types and the severe side effects evoked by chemotherapeutic agents. Hence, the search for novel anticancer drugs with minimum side effects remains an active area of cancer research. Although molecular targeted drugs are preferred over the conventional cytotoxic chemotherapy, the screening of natural compounds with cytotoxic potentialities continues as they can serve as lead structures for the development of tumor selective anticancer drugs. Plants and microorganisms have been the prominent sources of therapeutic agents. Microorganisms being readily renewable, inexhaustible sources of diverse bioactive secondary metabolites are preferred over plants as sources of bioactive compounds. Endophytes are microorganisms that reside within the living tissue of host plant and they enhance the survival value of the host plant by mediating various stress tolerance mechanisms. Endophytic fungi have gained attention as potential sources of bioactive secondary metabolites following the discovery of a taxol producing endophytic fungus Taxomyces adrenae, from Taxus brevifolia. Moreover, endophytes occupy a unique biological niche in which they maintain a balanced interaction with the host organism and other co-inhabiting microorganisms. All these factors contribute to the chemical diversity of the metabolites they produce. Plants restricted to extreme or unique habitats or those with ethnobotanical value are likely to lodge endophytes that possess a unique hoard of secondarymetabolites. Saraca asoca is a traditionalmedicinal plant with its occurrence restricted to countries such as India, Sri Lanka, Burma and Malaysia. The purpose of the present study is to explore the endophytic fungal population associated with S. asoca in search of novel anticancer lead structures. S. asoca was found to house a diverse endophytic fungal population belonging to 37 different species. Identification of the fungal isolates was based on ITS (internal transcribed spacer region) sequence analysis as well as colony and spore characteristics. The organic extracts of all fungal species were assessed for their in vitro cytotoxicities in three human cancer cell lines, HeLa, HepG2 and PC3 byMTT assay. 18 species exhibited remarkable cytotoxic activities, among which Pestalotiopsis sp. 6 exhibited themost significant cytotoxicity. The strain with second highest activity was Lasiodiplodia theobromae. In order to identify the active principle present in the organic extracts of Pestalotiopsis sp. 6 and L. theobromae, the organic extracts were chromatographed on TLC plates and individual compounds were recovered by scraping off from the TLC plates and extracting with methanol. The cytotoxicity assay of the TLC purified compounds suggested the cytotoxic activity of Pestalotiopsis sp.6 to be a synergetic effect of two or more compounds whereas the cytotoxicity of L. theobromae organic extract was largely due to a single compound. Hence the active principle present in L. theobromae organic extract was purified by bioassay - guided column chromatography. Repeated chromatography of the crude extract using three silica gel columns resulted in the isolation of anticancer compound. Based on the analysis of ESI-MS, IR, NMR and UV spectral data, the isolated compound was identified as a novel steroidal saponin, cholestan-3-O-¯-Dglucopyranoside (cholestanol glucoside - CG). The in vitro cytotoxic effects of CG towards seven human cancer cell lines, HeLa, HepG2, PC3, U251,MCF 7, OVCAR3 and A549 were examined. Among the cell lines screened, HeLa cells weremost vulnerable to CG treatment, with an IC50 value of 3.2 ¹M. Hence themode of cell death induction in HeLa cells by CG was further investigated. Analysis of cell cycle progression by propidium iodide (PI) staining revealed that CG arrests the cells in S phase of cell cycle prior to the induction of cell death. The morphological and biochemical features of apoptosis were investigated by nuclear staining, DNA fragmentation assay and Annexin V-FITC/ PI dual staining. All these results suggested that CG effectively induced apoptosis in HeLa cells in a concentration dependent manner. It was also found that CG treatment induced remarkable ROS generation and mitochondrial membrane potential loss. The pretreatment of cells with an antioxidant, N-acetyl cysteine (NAC), blocked CG induced ROS generation, mitochondrialmembrane depolarization and apoptotic cell death. Hence it could be concluded that CG kills the cancer cells by augmenting their basal oxidative stress and hence is less likely to be toxic to normal cells. Moreover, a high Bax to Bcl-2 ratio, high levels of Apaf-1 and p53, activation of procaspase-3 and procaspase-9 and cleavage of PARP were observed in CG treated HeLa cells. Taken together, our results suggested that CG induced apoptosis in HeLa cells via ROS mediated mitochondria dependent pathway. Biosynthesis of secondarymetabolites by filamentous fungi is influenced by the availability of nutrient factors. Therefore, it is essential to optimize the culturemedium components to ensure a maximum and consistent yield of desired metabolite by the fungal isolate. We designed a chemically defined production medium for CG production by L. theobromae. Carbon source, nitrogen source and microelements in the production medium were further optimized in stationary flask cultures to improve the mycelial growth and yield of CG by L. theobromae. The conventional one-factor at a time (OFAT)method was employed for the optimization of carbon and nitrogen sourceswhose contribution effects towards the final yield are large. Response surface methodology (RSM) was employed for the optimization of microelements. Optimization of culturemedium enhanced the yield of CG from 10mg L¡1 to 50mg L¡1. Various secondarymetabolites are produced by organisms in response to different stress conditions. This knowledge has been exploited in plant cell culture systems to increase the yield of particular secondary metabolites by artificial implementation of stress conditions. We investigated the effect of oxidative, osmotic and heat shock stresses on the production of CG by L. theobromae. Heat shock and osmotic stresses in liquid cultures were found to enhance the yield of CG by 1.2-fold, relative to the controls. Oxidative stress by both menadione and H2O2 enhanced the yield by 1.8-fold compared to the controls. Thus oxidative stress proved to be an efficient enhancer of CG production by L. theobromae. These findings ensure a large scale, cost-effective production of CG.

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47

Valayil, Jinu Mathew. "Structure Elucidation and Biological Evaluation of a Novel Steroidal Saponin, Cholestanol Glucoside Isolated from Saraca Asoca Enodophytic Fuungus, Lasiodiplodia Theobromae." Thesis, 2015. http://etd.iisc.ernet.in/2005/3549.

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Although the molecular mechanisms underlying the onset and progression of cancer has been unraveled to a great extend, cancer continues to remain a leading cause of death around the world. Clinical efficacy of the existing anticancer drugs are largely compromised by the inherent and acquired resistance of cancer cell types and the severe side effects evoked by chemotherapeutic agents. Hence, the search for novel anticancer drugs with minimum side effects remains an active area of cancer research. Although molecular targeted drugs are preferred over the conventional cytotoxic chemotherapy, the screening of natural compounds with cytotoxic potentialities continues as they can serve as lead structures for the development of tumor selective anticancer drugs. Plants and microorganisms have been the prominent sources of therapeutic agents. Microorganisms being readily renewable, inexhaustible sources of diverse bioactive secondary metabolites are preferred over plants as sources of bioactive compounds. Endophytes are microorganisms that reside within the living tissue of host plant and they enhance the survival value of the host plant by mediating various stress tolerance mechanisms. Endophytic fungi have gained attention as potential sources of bioactive secondary metabolites following the discovery of a taxol producing endophytic fungus Taxomyces adrenae, from Taxus brevifolia. Moreover, endophytes occupy a unique biological niche in which they maintain a balanced interaction with the host organism and other co-inhabiting microorganisms. All these factors contribute to the chemical diversity of the metabolites they produce. Plants restricted to extreme or unique habitats or those with ethnobotanical value are likely to lodge endophytes that possess a unique hoard of secondarymetabolites. Saraca asoca is a traditionalmedicinal plant with its occurrence restricted to countries such as India, Sri Lanka, Burma and Malaysia. The purpose of the present study is to explore the endophytic fungal population associated with S. asoca in search of novel anticancer lead structures. S. asoca was found to house a diverse endophytic fungal population belonging to 37 different species. Identification of the fungal isolates was based on ITS (internal transcribed spacer region) sequence analysis as well as colony and spore characteristics. The organic extracts of all fungal species were assessed for their in vitro cytotoxicities in three human cancer cell lines, HeLa, HepG2 and PC3 byMTT assay. 18 species exhibited remarkable cytotoxic activities, among which Pestalotiopsis sp. 6 exhibited themost significant cytotoxicity. The strain with second highest activity was Lasiodiplodia theobromae. In order to identify the active principle present in the organic extracts of Pestalotiopsis sp. 6 and L. theobromae, the organic extracts were chromatographed on TLC plates and individual compounds were recovered by scraping off from the TLC plates and extracting with methanol. The cytotoxicity assay of the TLC purified compounds suggested the cytotoxic activity of Pestalotiopsis sp.6 to be a synergetic effect of two or more compounds whereas the cytotoxicity of L. theobromae organic extract was largely due to a single compound. Hence the active principle present in L. theobromae organic extract was purified by bioassay - guided column chromatography. Repeated chromatography of the crude extract using three silica gel columns resulted in the isolation of anticancer compound. Based on the analysis of ESI-MS, IR, NMR and UV spectral data, the isolated compound was identified as a novel steroidal saponin, cholestan-3-O-¯-Dglucopyranoside (cholestanol glucoside - CG). The in vitro cytotoxic effects of CG towards seven human cancer cell lines, HeLa, HepG2, PC3, U251,MCF 7, OVCAR3 and A549 were examined. Among the cell lines screened, HeLa cells weremost vulnerable to CG treatment, with an IC50 value of 3.2 ¹M. Hence themode of cell death induction in HeLa cells by CG was further investigated. Analysis of cell cycle progression by propidium iodide (PI) staining revealed that CG arrests the cells in S phase of cell cycle prior to the induction of cell death. The morphological and biochemical features of apoptosis were investigated by nuclear staining, DNA fragmentation assay and Annexin V-FITC/ PI dual staining. All these results suggested that CG effectively induced apoptosis in HeLa cells in a concentration dependent manner. It was also found that CG treatment induced remarkable ROS generation and mitochondrial membrane potential loss. The pretreatment of cells with an antioxidant, N-acetyl cysteine (NAC), blocked CG induced ROS generation, mitochondrialmembrane depolarization and apoptotic cell death. Hence it could be concluded that CG kills the cancer cells by augmenting their basal oxidative stress and hence is less likely to be toxic to normal cells. Moreover, a high Bax to Bcl-2 ratio, high levels of Apaf-1 and p53, activation of procaspase-3 and procaspase-9 and cleavage of PARP were observed in CG treated HeLa cells. Taken together, our results suggested that CG induced apoptosis in HeLa cells via ROS mediated mitochondria dependent pathway. Biosynthesis of secondarymetabolites by filamentous fungi is influenced by the availability of nutrient factors. Therefore, it is essential to optimize the culturemedium components to ensure a maximum and consistent yield of desired metabolite by the fungal isolate. We designed a chemically defined production medium for CG production by L. theobromae. Carbon source, nitrogen source and microelements in the production medium were further optimized in stationary flask cultures to improve the mycelial growth and yield of CG by L. theobromae. The conventional one-factor at a time (OFAT)method was employed for the optimization of carbon and nitrogen sourceswhose contribution effects towards the final yield are large. Response surface methodology (RSM) was employed for the optimization of microelements. Optimization of culturemedium enhanced the yield of CG from 10mg L¡1 to 50mg L¡1. Various secondarymetabolites are produced by organisms in response to different stress conditions. This knowledge has been exploited in plant cell culture systems to increase the yield of particular secondary metabolites by artificial implementation of stress conditions. We investigated the effect of oxidative, osmotic and heat shock stresses on the production of CG by L. theobromae. Heat shock and osmotic stresses in liquid cultures were found to enhance the yield of CG by 1.2-fold, relative to the controls. Oxidative stress by both menadione and H2O2 enhanced the yield by 1.8-fold compared to the controls. Thus oxidative stress proved to be an efficient enhancer of CG production by L. theobromae. These findings ensure a large scale, cost-effective production of CG.

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48

Kashyap, Srishti. "Isolation, Structure Elucidation and Functional Characterization of a Novel Cytotoxic Secondary Metabolite Phomafuranone, 2-Hydroxy-2, 4-dimethyl-5-[-1-propen-1-yl]-3(2H)-furanone, from Phoma tropica an Endophytic Fungus Isolated from Mappia foetida." Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4135.

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Cancer has become the leading disease-related cause of death in the human population. For example, in the United States, cancer is the second leading cause of death behind cardiovascular disease, and it is projected that cancer will become the leading cause of death in the coming years. The medical treatment of cancer still has many unmet needs. The main curative therapies for cancer, surgery and radiation, are generally only successful if the cancer is found at an early localized stage. Once cancer has progressed to metastatic stage, these therapies are less successful. Hence, chemotherapeutic drugs are used for the treatment of these advanced tumors, particularly in the case of the common epithelial tumors such as lung, colorectal, breast, prostate, and pancreatic cancers. New chemotherapeutic drugs are necessary since most of the cancers acquire resistance towards existing anticancer drugs. Nature has always been an attractive source of new chemotherapeutic agents, as a tremendous chemical diversity is found in millions of species of plants, animals, and microorganisms.Microorganisms (bacteria, fungi, actinomycetes) serve as readily renewable and inexhaustible source of novel bioactive metabolites.Endophyte, a microorganism that reside in theinternal tissues of living plantswithout causing any immediate overt negative effects, are potential sources of novel natural products for exploitation in medicine. Mappia foetidais distributed in western part of peninsular coastal India from Konkanghatsto northern parts of the Kanara, Nilgiris, Anamalis, and Pullneys hills of India. It is a rich source of alkaloids such as camptothecin, 9-methoxy camptothecin, mappicin,sitosterol and lupeol and other natural products having anticancer and antimicrobial properties. Since, the secondary metabolite production in fungal endophytes is greatly influenced by theircompetitive ecological niche and interaction with host metabolism, Mappia foetida was chosen for endophytic fungal isolation. Sirsi, (North Karnataka, India) because of its rich biodiversity was chosen as a location for collection of the plant samples. The organicsolvent extracts obtained from mycelia and culture filtrates of all the endophytic fungal isolates were screened for their cytotoxic activity against HeLa (human cervical carcinoma) cellline. Organic extracts of 8 fungal isolates exhibited significant cytotoxic activity, among which Phoma tropica(S1/3) culture filtrate extract exhibited most significant cytotoxicactivity againstHeLa cell line with IC50 of 25µg/ml. Dichloromethane extract of the Phoma tropica culture filtrate was subjected to bioassay–guided column chromatographic fractionationwhich resulted in the isolation of purified cytotoxic secondary metabolite. Based on the analysis of variousspectroscopic techniques such as NMR, FTIR, LC-MS/MS, HRMS, CHNOS elemental analysis and X-ray diffraction studies, the purified metabolite was identified as 2-Hydroxy-2,4-dimethyl-5-[-1-propen-1-yl]-3(2H)furanone(Phomafuranone).Phomafuranone exhibited significant cytotoxic activity against various cancer cell lines (HeLa, Jurkat, COLO 205, HT-29, HCT-15, HCT 116, A549, A-431 and OVCAR-3) Further studies were undertaken to elucidate the mechanism of cytotoxicityof the purified metabolite on human cancer cell lines. Phomafuranone contains a conjugated unsaturated α, β, γ, δ carbonyl pharmacophore moiety. Polyunsaturated carbonyl compounds are referred to as “Michael acceptors” and they behave as soft electrophiles. Michael acceptors react with strong biological nucleophiles such as thiols.The reactivity of electrophilic Phomafuranone with thiol containing biomolecules like glutathione, cysteine and N-acetyl cysteine was investigated by spectrophotometric methods. Cell cycle progressionanalyses oftreated HCT 116 (colorectal carcinoma) cell line by flow cytometry revealed that Phomafuranone arrested significant proportion of cells in G2/M phase. HCT 116 cells were arrested specifically in early mitotic phase of cell cycle as indicated by time dependent increase in phospo-histone3 (Ser10) levels and nuclear import of Cyclin B1.On treatment cancer cells with Phomafuranone, time dependent depletion of intracellular reduced glutathione levels was observed. Depletion of reduced glutathione inturn led to elevated intracellular ROS levels.Pre-treatment of HCT 116 cell lines with thiol containing antioxidants like N- acetylcysteine (NAC) and reduced glutathione (GSH) completely abrogated itscytotoxic effect, suggesting Phomafuranone inducedthiol–mediated cytotoxicity. The elevated ROS levels led to mitochondrial membrane depolarization as indicated by cytochrome c release in cytosol from mitochondria in a time dependent manner. Cytochrome c release was followed caspase 9 mediated apoptotic cell death. Thus, our results suggest that Phomafuranone induced redox imbalancemediated apoptosis in HCT 116 cell line by intrinsic pathway.

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49

Ianson, David C. "Variation in plant response to inoculation with different isolates of vesicular arbuscular mycorrhizal fungi." Thesis, 1990. http://hdl.handle.net/1957/37325.

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50

McCabe, Bernadette K. "Production of cellulolytic enzymes using immobilised anaerobic fungi." Thesis, 1998. http://handle.uws.edu.au:8081/1959.7/83.

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An investigation was made into the isolation and screening of highly cellulolytic anaerobic fungi and their production of cellulolytic enzymes using immobilised rhizomycelia. A total of 46 anaerobic fungi were isolated on cellulosic substrates from ruminant and non-ruminant herbivores. Primary screening of these isolates was performed using dye release from cellulose-azure which qualitatively detected cellulolytic activity. Twelve isolates were chosen on the basis of their maximum solubilisation rates of the labelled cellulose and then subjected to secondary screening which involved the quantification of enzyme activity. The enzyme mixtures were characterised by carboxymethylcellulase, xylanase, B-glucosidase, B-xylosidase and cellobiase assays, measured by the production of either reducing sugars, p-nitrophenol or glucose. All strains produced a number of enzymes that allowed them to hydrolyse straw and highest enzyme activity was measured in static cultures grown on 0.5% straw. A monocentric isolate, Piromyces strain KSX1 from a red kangaroo, and a cattle polycentric isolate, Orpinomyces strain 478P1, were selected for study of cellulolytic enzyme production on the basis of high fibre digestion capability and amenability toward encapsulation. The immobilised polycentric strain proved to be operationally superior to strain KSX1 as strain 478P1 did not produce any viable growth in the culture liquor. Studies into single batch cultures of free cells of strains KSX1 and 478P1 revealed that the maximum specific rate of B-glucosidase production occurred concomitantly with maximum specific growth rate suggesting that the immobilised fungus must grow for continuous enzyme production to occur. Although the physiology of cellulase synthesis in strains KSX1 and 478P1 was found to be growth-associated, immobilisation of the fungus offered the advantage of the repeat-batch use of cells with the accumulation of extracellular enzymes after each batch. Thus, operational gains were the key issues in assessing the potential application of immobilised anaerobic fungi in the production of cellulolytic enzymes. The repeat-batch system was operationally more efficient than the free cell batch cultures because immobilisation removed the need of reculturing the cells for every single batch.

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