Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-06-13T12:59:08Z No. of bitstreams: 1 resumo.pdf: 16026 bytes, checksum: 08a689ebeadfe931f9bc4172b7695e72 (MD5) / Made available in DSpace on 2017-06-13T12:59:08Z (GMT). No. of bitstreams: 1 resumo.pdf: 16026 bytes, checksum: 08a689ebeadfe931f9bc4172b7695e72 (MD5) Previous issue date: 2003-02-13 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / A variabilidade genética de 20 isolados de Fusarium oxysporum não- patogênicos e patogênicos em feijoeiro foi determinada com base na distribuição do elemento transponível impala e com a técnica de DNA polimórfico amplificado ao acaso (RAPD). A presença de elementos transponíveis impala das subfamílias D e E foi determinada por experimentos de PCR empregando oligonucleotídeos específicos para cada subfamília. Foi observada a presença de representantes das duas subfamílias na maioria dos isolados, sugerindo, portanto, que impala é um antigo componente do genoma de F. oxysporum f. sp. phaseoli. A hibridização do DNA total de cada isolado, clivado com a enzima EcoRI, com um fragmento do elemento impala da subfamíla E, mostrou uma variação nos padrões de bandas dos isolados não-patogênicos, indicando a possível atividade desses elementos. No entanto, no caso dos isolados patogênicos, foram observados padrões de bandas mais homogêneas e alguns isolados apresentaram o mesmo perfil de bandas, indicando que se trata de cópias de impala que possivelmente não são mais capazes de sofrer transposição. Estas cópias inativas são excelentes marcadores genéticos. Um dos isolados patogênicos, Fus4, não apresentou cópias endógenas de impala, o que torna esse isolado um candidato para experimentos de mutagênese insercional usando o vetor pNI160, que apresenta o elemento impala ativo interrompendo o oligonucleotídeos polimorfismo gene niaD. gerou 224 observado foi A análise dos fragmentos compatível dados de polimórficos com o padrão RAPD e 7 de utilizando-se 16 monomórficos. O hibridização. Foram calculadas as distâncias genéticas entre os isolados e estas variaram de 8 a 76%, entre os patogênicos; de 2 a 63%, entre os não-patogênicos, e de 45 a 76%, entre patogênicos e não-patogênicos. Baseados nestas distâncias, quatro grupos foram definidos por análise de agrupamento. Dois grupos foram específicos para patogênicos, um para os não- patogênicos, e um grupo incluiu patogênicos e não-patogênicos. A distância genética observada dentro de um grupo de isolados patogênicos é compatível com os valores de distância genética que definem raças fisiológicas. / The genetic variability of twenty nonpathogenic and pathogenic isolates of Fusarium oxysporum, the causative agent of bean wilt, was analyzed based on the distribution of the transposable element impala and on the random amplification of polymorphic subfamilies DNA D and (RAPD). E of The impala presence was of transposable determined reaction) using specific primers for each subfamily. two subfamilies was observed in most of through elements PCR The presence of belonging (polymerase members of to chain the the isolates, suggesting that it is an old component in the F. oxysporum f. sp. phaseoli genoma. Hybridization of total DNA of each isolate, digested with EcoRI, with impala fragment of subfamily E produced a highly band pattern in the nonpathogenic isolates, indicating the possible activity of these elements. patterns were On the other hand, in the case of the pathogenic isolates, the band more homogeneous and some isolates showed very similar patterns, indicating that these impala copies have lost their capacity to transpose. These inactive copies are suitable as genetic markers. Among the pathogenics isolates, Fus4 did not present endogenous copies of impala and could be used in experiments of insertional viiimutagenesis with the pNI160 plasmid, that harbors the active impala element disrupting the niaD gene. monomorphic The RAPD analysis using 16 primers resulted in 224 polymorphic and 7 fragments. The genetic distances among the isolates based on the presence/absence of the RAPD bands varied between 8 and 76% within the pathogenic isolates, between 2 a 63% within the nonpathogenic isolates and 45 to 76% between the two groups. Based on these distances, four groups were defined by UPGMA analysis. Two groups included within were both the specific pathogenic group of for and pathogenics nonpathogenic pathogenic isolates and isolates.

ARAÚJO, Linda Brenna Ribeiro. Potencial genético de variedades tradicionais de feijão-caupi e avaliação para resistência à murcha-de-Fusarium. 2017. 80 f. Dissertação (Mestrado em Agronomia/Fitotecnia) - Universidade Federal do Ceará, Fortaleza, 2017. / Submitted by Deocleciano Xavier (dixavier.ufc@gmail.com) on 2017-08-21T18:35:50Z No. of bitstreams: 1 Dissertação Linda Brenna R Araújo_FINAL.pdf: 1356498 bytes, checksum: b4e907dfc4ac85e53f13d1e09c6d0d55 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-08-29T20:36:01Z (GMT) No. of bitstreams: 1 Dissertação Linda Brenna R Araújo_FINAL.pdf: 1356498 bytes, checksum: b4e907dfc4ac85e53f13d1e09c6d0d55 (MD5) / Made available in DSpace on 2017-08-29T20:36:02Z (GMT). No. of bitstreams: 1 Dissertação Linda Brenna R Araújo_FINAL.pdf: 1356498 bytes, checksum: b4e907dfc4ac85e53f13d1e09c6d0d55 (MD5) Previous issue date: 2017 / Traditional varieties are an important source of germplasm for several crops, and may present alleles that have long been lost, such as those that confer resistance to biotic and abiotic stresses. In the state of Ceará, the cultivation of the cowpea is highlighted because it is mainly carried out by family farmers who make use of these varieties, and their great adaptation to the region of cultivation and to the management adopted by the farmers can be used in the development of cultivars that better meet their demands. Thus, this work was carried out to evaluate the genetic potential of traditional varieties of cowpea collected from family farmers in the state of Ceará. In order to achieve this objective, laboratory analyzes, field and greenhouse tests were carried out. The laboratory analyzes consisted of the evaluation of the genetic diversity of the varieties through ISSR markers. The field experiments consisted of tests for the evaluation of morphological and agronomic descriptors in two environments: Fortaleza and Madalena, in the state of Ceará. The experiment in greenhouse was conducted to evaluate the existence of sources of resistance to Fusarium wilt by the root-dipping inoculation method. The ISSR markers selected for the study were efficient in identifying the genetic variability of the species, presenting high values of polymorphism and polymorphism information content (PIC). Even so, the genetic distance found between the studied varieties ranged from 0,05 to 0,31, values considered low and that suggest a narrow genetic base of the species. Some traditional varieties evaluated showed higher grain yield than commercial cultivars for the evaluated environments (3286.66 tons/ha), indicating a high agronomic potential and a great environmental adaptation. They presented greater genetic variability for the morphological characters evaluated, when compared to the molecular data. Regarding the severity of the disease, the varieties formed three distinct groups. The group with the lowest severity of the disease (25,00 to 32,50 %) consisted of thirty-seven varieties, the second group with intermediate values of severity (33,75 to 37,50 %) consisted of eight varieties and in the third Group were the five varieties that presented the highest values of severity (41,25 to 51,25 %). The varieties with the best results for agronomic traits, resistance to Fusarium wilt and greater genetic distance are promising for use in breeding programs. / As variedades tradicionais constituem importante fonte de germoplasma para diversas culturas, podendo apresentar alelos há muito perdidos, como os que conferem resistência a estresses bióticos e abióticos. No estado do Ceará, o cultivo do feijão-caupi tem destaque por ser realizado principalmente por agricultores familiares que fazem uso dessas variedades, e a grande adaptação delas à região de cultivo e ao manejo adotado pelos agricultores pode ser utilizada no desenvolvimento de cultivares que atendam melhor às suas demandas. Assim, objetivou-se com esse trabalho avaliar o potencial genético e a resistência à murcha-de-Fusarium de variedades tradicionais de feijão-caupi coletadas junto a agricultores familiares no estado do Ceará. Para se alcançar tal objetivo foram realizadas análises em laboratório, ensaios em campo e em casa de vegetação. As análises em laboratório constaram da avaliação da diversidade genética das variedades através de marcadores ISSR. Os experimentos de campo constaram de ensaios para a avaliação de descritores morfológicos e agronômicos em dois ambientes: Fortaleza e Madalena, no estado do Ceará. O ensaio em casa de vegetação foi realizado para avaliar a existência de fontes de resistência à murcha-de-Fusarium através do método de dipping. Os marcadores ISSR selecionados para o estudo foram eficientes na identificação da variabilidade genética dos genótipos, apresentando altos valores de polimorfismo e conteúdo de informação polimórfica (PIC). Ainda assim, a distância genética encontrada entre as variedades estudadas variou de 0,05 a 0,31, valores considerados baixos e que sugerem uma estreita base genética dos genótipos avaliados. Algumas variedades tradicionais avaliadas apresentaram produtividade de grãos superior às cultivares comerciais para os ambientes avaliados (3286,66 ton/ha), o que indica um alto potencial agronômico e uma grande adaptação ambiental. Elas apresentaram maior variabilidade genética para os caracteres morfológicos avaliados, quando comparados aos dados moleculares. Com relação à severidade da doença, as variedades formaram três grupos distintos. O grupo com a menor severidade da doença (25,00 a 32,50 %) foi constituído por trinta e sete variedades, o segundo grupo com valores intermediários de severidade (33,75 a 37,50 %) foi constituído por oito variedades e no terceiro grupo ficaram as cinco variedades que apresentaram os maiores valores de severidade (41,25 a 51,25 %). As variedades com melhores resultados para os caracteres agronômicos, de resistência à murcha-de-Fusarium e maior distância genética são promissoras para uso em programas de melhoramento genético.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-26T16:49:22Z No. of bitstreams: 1 TeseRMDA.pdf: 4251540 bytes, checksum: 7f125ff1cf0456ee6c35cc31853e3ee2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T18:22:59Z (GMT) No. of bitstreams: 1 TeseRMDA.pdf: 4251540 bytes, checksum: 7f125ff1cf0456ee6c35cc31853e3ee2 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-11-08T18:23:07Z (GMT) No. of bitstreams: 1 TeseRMDA.pdf: 4251540 bytes, checksum: 7f125ff1cf0456ee6c35cc31853e3ee2 (MD5) / Made available in DSpace on 2016-11-08T18:23:17Z (GMT). No. of bitstreams: 1 TeseRMDA.pdf: 4251540 bytes, checksum: 7f125ff1cf0456ee6c35cc31853e3ee2 (MD5) Previous issue date: 2016-07-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Several aromatic polyketide derivatives from Ginger (Zingiber officinale), like zingerone, 6-gingerol and 6-shogaol were obtained by biocatalytic reactions and semi-synthesis and both their antifungal and cysteine-proteinase inhibitory properties were investigated. The derivatives obtained by semi-synthesis were evaluated in the form of a first screening for inhibition property of growth of phytopathogenic fungi Colletotrichum gloesporioides and Fusararium oxysparum. The biotransformation of four compounds: zingerone purchased from Sigma Aldrich, 6-gingerol isolated from ginger and its derivatives 4-methyl-6-gingerol and 6-shogaol was investigated using whole cells of C. gloesporioides and showed recognition of the fungal pool of enzymes capable of using such compounds as substrates. All experiments were conducted in sterile liquid medium BD (Potato and Dextrose) under agitation (120 rpm) and constant temperature (30° C ), obtaining the methanol extract of mycelial mass (MM), the aqueous extract (Aq) and the ethyl acetate extract (Ac) of culture fluid. The ethyl acetate extract was analyzed by GC-MS and ESI-MS/MS using LC-MS techniques. The fungus C. gloesporioides proved able to promote a reaction of aromatic oxidative coupling of zingerone, forming a dimer of this molecule. This product was isolated, identified by NMR spectroscopy and high resolution mass spectrometry. The biotransformation experiment was also made with isotopically labeled zingerone with deuterium. Analysis of the ethyl acetate extract (Ac) by (-) ESI-MS / MS using LC-IT-MS showed that dimer formation since the first day once the isotopically labeled fragments were detected. The aromatic oxidative coupling reaction also occurs in the biotransformation to phenolic compound 6-gingerol by C. gloesporioides. It is probable that side chain of 6-gingerol was used as carbon source forming an alcohol with molecular mass 168 Da. The dimer of the alcohol appears as biotransformation product. Among the proposed semi-synthesis reactions, the insertion of an oxime ether subunit from zingerone as starting material was obtained. Despite the molecular modeling indicates good complementarity with the catalytic site model cysteinyl preoteases, the product obtained does not show inhibitory activity against the enzymes L and V to 25 uM. The oximine derivative of 8-gingerol, presented halo of inhibition against the proliferation of fungus C.gloesporioides in disk diffusion test on PDA (Potato Dextrose Agar) in a Petri dish. The ethyl acetate extract (Ac) from the tenth day showed a good result of inhibition of cathepsin V (80%). There are studies that investigate the relationship between the antifungal activity of compounds of the property of inhibiting cysteine-preoteases as papain and cathepsin B and L. / A obtenção de novos derivados de policetídeos aromáticos encontrados no gengibre (Zingiber officinale), como a zingerona, o 6-gingerol e o 6-shogaol, empregando-se reações de biocatálise e semi-síntese foram alcançados, bem como a avaliação frente à cisteíno-proteases. Os derivados obtidos por semi-síntese foram avaliados na forma de um primeiro screening quanto à capacidade de inibição do crescimento dos fungos fitopatogênicos como Colletotrichum gloesporioides e Fusararium oxysparum. A biotransformação de quatro compostos: a zingerona, obtida comercialmente, a biotransformação do 6-gingerol, isolado do gengibre e de seus derivados 4-metil-6- gingerol e 6-shogaol por células totais de C. gloesporioides foi investigada tendo-se o reconhecimento do pool enzimático do fungo capaz de utilizar tais compostos como substrato. Todos os experimentos foram conduzidos em meio líquido estéril BD (Batata e Dextrose) sob a agitação de 120 rpm e temperatura de 30°C, obtendo-se o extrato metanólico da massa micelial (MM), o extrato aquoso (Aq), sendo o sobrenadante após obtenção do extrato em acetato de etila (Ac) do fluido da cultura. Os extratos Ac foram analisados por GC-MS e ESI-MS/MS usando técnicas de LC-MS. O fungo C. gloesporioides mostrou ser capaz em promover uma reação de acoplamento oxidativo aromático da zingerona, formando um dímero desta molécula, tal produto foi isolado e a estrutura química proposta com as análises dos espectros de RMN obtidos bem como por espectrometria de massas de alta resolução; o experimento de biotransformação também foi realizado com a zingerona marcada com deutério. A análise do extrato Ac por (-) ESI-MS/MS usando LC-IT-MS mostrou que desde o primeiro dia há formação do dímero o que foi confirmado pela formação de fragmentos isotopicamente marcados. A reação de acoplamento oxidativo aromático também ocorre na biotransformação para o composto fenólico 6-gingerol pelo C. gloesporioides, sendo que, provavelmente há consumo da cadeia lateral pelo fungo como fonte de carbono formando um álcool com a massa molecular de 168 Da, sendo o dímero desse álcool que aparece como produto de biotransformação. Dentre a proposta de reação de semi-síntese, o produto da reação de inserção da subunidade éter de oxima, tendo zingerona como material de partida foi obtido e apesar da modelagem molecular indicar boa complementariedade com o modelo de sítio catalítico de cisteinil-preoteases, o produto obtido não apresentou atividade de inibição contra as enzimas L e V a 25 μM. O derivado oximínico do 8-gingerol, apresentou halo de inibição contra a proliferação do fungo C.gloesporioides em teste de difusão em disco em meio BDA (Batata-Dextrose-Ágar) em placa de Petri, sendo que existem trabalhos que investigam a relação entre a atividade antifúngica de compostos com a propriedade de inibição de cisteínopreoteases como papaína e catepsina B e L. O extrato acetato de etila (Ac) do décimo dia mostrou um bom resultado de inibição da catepsina V (80%).

Thesis (DTech (Chemical Engineering))--Cape Peninsula University of Technology, 2017. / The high rate of industrialisation in most developing countries has brought about challenges of wastewater management especially in the mineral processing industry. Cyanide has been used in base metal extraction processes due to its lixiviant properties thus, its presence in wastewater generated is inevitable. Furthermore, partial and/or the use of unsuitable treatment methods for such wastewater is a potential hazard to both human and the environment. There are several reports on biotechnological treatments of cyanide containing wastewater but few mineral processing industries have adopted this approach. Hence, the thermodynamic study of biodegradation of cyanide containing wastewater was undertaken. The primary aim of this study was to explore the application of bioenergetic models and biological stoichiometry to determine the functionality and thermodynamic requirements for cyanide degrading isolate (Fusarium oxysporum EKT01/02), grown exclusively on Beta vulgaris, for a system designed for the bioremediation of cyanidation wastewater. Chapter 2 reviews some of the applicable thermodynamic parameters such as enthalpy, entropy, heat of combustion, heat capacity, Gibbs energy, including stoichiometry models in relation to their applicability for microbial proliferation in cyanidation wastewater. The chapter places emphasis on the application of agro-industrial waste as a suitable replacement for refined carbon sources for microbial proliferation in bioremediation systems because such systems are environmentally benign. The choice of using agro-industrial waste is due to organic waste properties, i.e. agro-industrial waste is rich in nutrients and is generated in large quantities. Chapter 3 presents the materials and various standardised methods used to address the research gaps identified in chapter 2. For an organism to degrade free cyanide in wastewater, it must be able to survive and perform its primary function in the presence of such a toxicant. Chapter 4 exemplifies both molecular and biochemical characteristics of Fusarium oxysporum EKT01/02 isolated from the rhizosphere of Zea mays contaminated with a cyanide based pesticide. The molecular analyses confirmed the fungal isolate to be Fusarium oxysporum EKT01/02 and the nucleotide sequence of the isolates were deposited with National Centre for Biotechnology Information (NCBI) with accession numbers KU985430 and KU985431. The biochemical analyses revealed a wide substrate utilisation mechanism of the isolate dominated by aminopeptidase including nitrate assimilation capabilities. A preliminary investigation showed free cyanide degradation efficiency of 77.6% (100 mg CN-/L) after 5 days by the isolate. The excess production of extracellular polymeric substance (EPS) was attributed to the isolates’ strive to protect itself from cyanide toxicity.

Submitted by Jean Medeiros (jeanletras@uepb.edu.br) on 2017-07-26T14:00:45Z No. of bitstreams: 1 PDF - Nadja Gláucia de Melo Souza.pdf: 13497516 bytes, checksum: 9022b8a01973d688cc5a2d32ec30a609 (MD5) / Approved for entry into archive by Secta BC (secta.csu.bc@uepb.edu.br) on 2017-08-29T15:30:12Z (GMT) No. of bitstreams: 1 PDF - Nadja Gláucia de Melo Souza.pdf: 13497516 bytes, checksum: 9022b8a01973d688cc5a2d32ec30a609 (MD5) / Made available in DSpace on 2017-08-29T15:30:13Z (GMT). No. of bitstreams: 1 PDF - Nadja Gláucia de Melo Souza.pdf: 13497516 bytes, checksum: 9022b8a01973d688cc5a2d32ec30a609 (MD5) Previous issue date: 2017-03-30 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Maize seeds are susceptible to Fusarium spp., which cause rotting, damping off and corn kernel contamination by mycotoxins. Plants produce antimicrobial compounds that can be extracted and used as an alternative method in the management of diseases. The objective of this work was to obtain an alternative product, through the use of vegetal extracts associated with powders (rock powder, kaolin and ash) for the control of Fusarium spp. associated with maize seeds. For this, were evaluated the fungitoxicity of extracts of fruits of chilli pepper, eucalyptus leaves, black pepper fruits and neem leaves to Fusarium spp.; and the effect of the extracts on the germination of maize seeds. The tested plant extracts are fungitoxic to Fusarium spp. causing total inhibition of the growth of the pathogens from the concentration of 5%. The treatment of the seeds with the extracts (20%) did not cause decrease in germination, and the extract of chilli pepper promoted the germination of the seeds. The black pepper extract (10%) had fungitoxic effect to F. verticillioides (F159 and F311) in maize cv. Sertanejo. The extracts tested (10% and 20%) did not inhibit F. oxysporum (F01) in maize cv. Sertanejo. Black pepper extract + rock powder, solvent + rock powder and solvent + kaolin accelerated the germination of maize cv. Sertanejo. All formulations tested decreased the incidence of F. verticillioides F311 in maize var. Sertanejo. Extract of black pepper + rock powder and black pepper extract had a positive effect on the control of damping off caused by Fusarium spp., and ind in the seedling development. It is concluded that the alternative product formulated with black pepper extract + rock powder and the black pepper extract have potentiality to be used in the alternative control of Fusarium spp. in maize seeds. / Sementes de milho são suscetíveis a Fusarium spp., que causam o apodrecimento, tombamento das plântulas e a contaminação dos grãos por micotoxinas. Plantas produzem compostos antimicrobianos que podem ser extraídos e utilizados como método alternativo no manejo de doenças. O objetivo do trabalho foi obter um produto alternativo, por meio da utilização de extratos vegetais associados a pós (pó de rocha, caulim e cinza) para o controle de Fusarium spp. associados às sementes de milho. Para tanto, foi avaliada a fungitoxicidade de extratos de frutos de pimenta malagueta, folhas de eucalipto, frutos de pimenta do reino e folhas de neem a Fusarium spp.; e o efeito dos extratos na germinação de sementes de milho. Os extratos vegetais testados são fungitóxicos a Fusarium spp., causando inibição total do crescimento dos patógenos a partir da concentração de 5%. O tratamento das sementes com os extratos (20%) não causou diminuição da germinação, sendo que o extrato de pimenta malagueta promoveu a germinação das sementes. O extrato de pimenta do reino (10%) teve efeito fungitóxico a F. verticillioides (F159 e F311) em sementes de milho cv. Sertanejo. Os extratos testados (10% e 20%) não inibiram F. oxysporum (F01) nas sementes de milho cv. Sertanejo. Extrato de pimenta do reino + pó de rocha, solvente + pó de rocha e solvente + caulim aceleraram a germinação das sementes de milho cv. Sertanejo. Todas as formulações testadas diminuíram a incidência de F. verticillioides F311 nas sementes de milho var. Sertanejo. Extrato de pimenta do reino + pó de rocha e extrato de pimenta do reino tiveram efeito positivo no controle do tombamento causado por Fusarium spp.; e na promoção de desenvolvimento das plântulas. Conclui-se que o produto alternativo formulado com extrato de pimenta do reino + pó de rocha e o extrato de pimenta do reino têm potencialidade para serem utilizados no controle alternativo de Fusarium spp. em sementes de milho.

Made available in DSpace on 2014-06-12T15:50:48Z (GMT). No. of bitstreams: 1 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Lectinas são proteínas que se ligam a carboidratos e glicoconjugados. Rizomas de Microgramma vaccinifolia têm ampla utilização na medicina popular no tratamento de hemoptises e hematúria. Os objetivos deste trabalho foram isolar, caracterizar, avaliar as atividades tóxica, antibacteriana e antifúngica da lectina de rizoma de M. vaccinifolia (MvRL) e identificar as raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici por biologia molecular. As proteínas do extrato de rizoma (ER) foram fracionadas com sulfato de amônio fornecendo a fração 0-60% (F0-60%). Atividade hemaglutinante (AH) e concentração de proteína foram determinadas em ER e F0-60%. MvRL foi isolada por cromatografia da F0-60% em Sephadex G-25. A AH de MvRL foi avaliada em presença de carboidratos, glicoproteínas, preparações contendo carboidrato das raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici, em diferentes temperaturas, valores de pH e na presença de íons. Eletroforese em gel de poliacrilamida de MvRL foi realizada em condições nativas (PAGE) e desnaturadas (SDS-PAGE). O efeito de MvRL sobre Artemia salina, bactérias e fungos foi também avaliado. O DNA das raças 1, 2 e 3 de Fusarium oxysporum f. sp. lycopersici foi isolado utilizando marcadores moleculares ISSR e RAPD. MvRL aglutinou eritrócitos humanos e sua AH foi inibida por manose, soro fetal bovino e preparações de F. oxysporum f.sp. lycopersici. A AH de MvRL foi termoestável, ativa em pH 5,0 e dependente de íons. PAGE revelou MvRL como uma proteína ácida e SDS-PAGE revelou banda polipeptídica glicosilada de massa molecular 17 kDa. Cromatografia de gel filtração definiu a massa molecular nativa de MvRL como 100 kDa indicando a lectina como um agregado molecular. MvRL foi tóxica sobre A. salina (CL50 de 154,16 μg/mL), não exibiu atividade antibacteriana e apresentou atividade antifúngica. MvRL foi mais ativa em inibir o crescimento da raça 3 de F. oxysporum f.sp. lycopersici. Os marcadores moleculares foram adequados para avaliar a variabilidade genética das raças. Em conclusão MvRL é uma lectina com atividade antifúngica e o efeito sobre F. oxysporum f.sp. lycopersici foi diferente para as três raças

Submitted by Gabriela Lopes (gmachadolopesufpel@gmail.com) on 2018-06-28T15:02:41Z No. of bitstreams: 1 TESE_Patricia Migliorini.pdf: 2682211 bytes, checksum: 8bdf76d1693cb07dba37b177863fdbef (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-07-03T18:36:36Z (GMT) No. of bitstreams: 1 TESE_Patricia Migliorini.pdf: 2682211 bytes, checksum: 8bdf76d1693cb07dba37b177863fdbef (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2018-07-03T18:38:23Z (GMT) No. of bitstreams: 1 TESE_Patricia Migliorini.pdf: 2682211 bytes, checksum: 8bdf76d1693cb07dba37b177863fdbef (MD5) / Made available in DSpace on 2018-07-03T18:38:30Z (GMT). No. of bitstreams: 1 TESE_Patricia Migliorini.pdf: 2682211 bytes, checksum: 8bdf76d1693cb07dba37b177863fdbef (MD5) Previous issue date: 2018-03-19 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / O presente trabalho teve por objetivo: a) avaliar o efeito de diferentes doses e fontes de Si por meio do recobrimento de sementes sobre a qualidade física, fisiológica e sanitária de sementes assim como, determinar a concentração de silício e cálcio absorvidos em plântulas de feijão; b) avaliar o desempenho de plântulas inoculadas com Fusarium oxysporum f. sp. phaseoli e recobertas com diferentes fontes de Si, durante a emergência e na expressão dos mecanismos bioquímicos, envolvidos nas respostas pós infecção em diferentes momentos e; c) avaliar o efeito do recobrimento de sementes com Si em resposta à infecção em uma cultivar suscetível e resistente ao Colletotrichum lindemuthianum, sobre o potencial fisiológico e sanitário de sementes e plântulas, durante o desenvolvimento inicial da cultura do feijão. Testou-se diferentes doses (0; 45; 90; 135 e 180g Si 100 kg-1 de sementes) e fontes de Si [Agrosilício®, silicato de cálcio e casca de arroz carbonizada (CAC)] via recobrimento de sementes, inoculados ou não pelo método de restrição hídrica. O recobrimento das sementes composto por Agrosilício®, CAC e silicato de cálcio não altera a qualidade física das sementes e proporciona plântulas vigorosas, sem comprometer a emergência final. Sementes recobertas com doses de silicato de cálcio e CAC elevam a concentração de Si nas raízes, mas reduzem a concentração de cálcio (Ca), enquanto que o Agrosilício® aumenta a concentração de Si em folhas e do Ca nas raízes. Diferentes fontes e doses de Si não controlam patógenos associados às sementes de feijão. A associação do F. oxysporum f. sp. phaseoli nas sementes prejudica a emergência e crescimento das plântulas. O Si tem pouco efeito nas respostas em plântulas não inoculadas, mas eleva a concentração de lignina. Na presença do F. oxysporum f. sp. phaseoli, o Si desencadeia melhor desempenho inicial das plântulas, favorece e potencializa os mecanismos de defesa. A fonte de Si fornecido pelo Agrosilício® proporciona uma antecipação rápida inicial da peroxidase o que pode ter amenizado e atrasado os danos do patógeno nas plântulas. A CAC como fonte de Si, embora tenha contribuído para a potencialização das enzimas de defesa nos períodos mais avançados da infecção, não se mostrou eficiente no controle da incidência da doença. O recobrimento das sementes com Si diminui a incidência e a severidade de C. lindemuthianum e proporciona maior crescimento das plântulas de feijão. O efeito de respostas potencializadas pelo Si tem maior expressividade na cultivar de maior suscetibilidade. / The a im of this study was: a) to evaluate the effect of different doses and sources of Si through the coating of seeds on the physical quality, physiological and sanitary conditions of seeds as, to determine the concentration of silicon and calcium absorbed in bean seedlings; b) to evaluate the performance of seedlings inoculated with Fusarium oxysporum f. sp. phaseoli and covered with different sources of Si, during emergence and in the expression of biochemical mechanisms, involved in post-infection responses at different times and; c) to evaluate the effect of seed coating with Si in response to infection in a susceptible and resistant Colletotrichum lindemuthianum, on the physiological and sanitary potential of seeds and seedlings, during the initial development of the bean culture. Different doses were tested (0; 45; 90; 135 and 180g Si 100 kg-1 of seeds) and sources of Si [Agrosilício®, calcium silicate and rice husk ash (AHC)] by seed coating, whether or not inoculated by the water restriction method. The covering of seeds composed of Agrosilício®, AHC and calcium silicate, does not change the physical quality of the seed, provides vigorous seedlings, without compromising the end emergency. Seeds coated with doses of calcium silicate and AHC, increase the concentration of Si in roots, but reduce the concentration of calcium (Ca), while the Agrosilício® increases the concentration of Si in leaves and Ca in the roots. Different sources and doses of Si do not control pathogens associated with bean seeds. The association of F. oxysporum f. sp. phaseoli in seeds impairs seedling emergence and growth. Si has little effect on responses in uninoculated seedlings, but raises lignin concentration. In the presence of F. oxysporum f. sp. phaseoli, Si triggered better initial seedling performance, favored and enhanced the defense mechanisms. The Si source supplied by Agrosilício® provides early rapid anticipation of peroxidase, which may have ameliorated and delayed the damage of the pathogen in the seedlings. The AHC as a source of Si, although it has contributed to the potentiation of defense enzymes in the later stages of infection was not efficient in controlling the incidence of the disease. The seed coating with Si decreases the incidence and severity of C. lindemuthianum and provides greater growth of the bean seedlings. The effect of responses potentiated by Si has greater expressiveness in the cultivar of greater susceptibility.

In the interactions between fungal endophytes and their hosts, the host may benefit through protection against pathogens and pests, growth promotion and tolerance to abiotic stresses. Non-pathogenic Fusarium oxysporum endophytes of banana have been shown to reduce the damage caused by the Cosmopolitus sordidus and the burrowing nematode Radopholus similis. The mode of protection against the burrowing nematode involves induced resistance, but the molecular basis of this resistance yet to be demonstrated. It has further been reported that protection of the host by multiple endophytes can lead to better control of target pests, probably because of the multiple modes of action involved. This phenomenon, however, has not been fully demonstrated for F. oxysporum endophytes of banana. This study aimed to investigate the molecular and biochemical basis of endophyte protection of East African Highland bananas (EAHB) against C. sordidus and R. similis. Expression of banana defence-related genes following endophyte inoculation and R. similis challenge varied greatly between the nematode-susceptible cv Nabusa and the nematode-tolerant cv Kayinja. In cv Nabusa, only the peroxidase (POX) and lectin genes were responsive to endophyte colonization of roots, or R. similis challenge. POX and lectin activities were significantly down-regulated 2 and 33 days after endophyte inoculation (dai), respectively. In cv Kayinja, endophyte colonization resulted in transient up-regulation of POX and a down-regulation of endochitinase (PR-3), lectin, pectin acetylesterase (PAE), phenylalanine ammonia-lyase (PAL) and PIR7A (peroxidase). Similar to systemic acquired resistance, PR-1 and catalase activities were up-regulated in the cv Kayinja 33 dai. Genes involved in signal transduction, cell wall strengthening, jasmonic acid pathway and defence molecule transport were differentially expressed in endophyte-inoculated plants. The expression profiles of four defence-related genes following endophyte inoculation and R. similis challenge were studied using quantitative real-time PCR. ABC transporter, Β-1,3-glucan synthase, coronatine insensitive 1 (COI1) and lipoxygenase (LOX) were up-regulated following endophyte inoculation. Β-1,3-glucan synthase and COI1 were highly up-regulated following R. similis challenge of endophyte-inoculated plants of the susceptible cv Nabusa, while COI1 and LOX were highly up-regulated following nematode challenge of endophyte-inoculated plants of the tolerant cv Kayinja. However ABC transporter gene activity was not up-regulated following nematode challenge of plants of both cultivars. UP-regulation of phenylpropanoid pathway enzymes PAL, POX and PPO has been observed in roots following colonization by both pathogenic and non-pathogenic fungi. In the current study, endophyte inoculation resulted in down-regulation of PAL activity in both a susceptible (cv Nabusa) and tolerant (cv Yangambi) banana. In cv Nabusa, endophyte inoculation primed PAL activity for up-regulation 30 days post nematode challenge (dpnc). However, in cv Yangambi PAL activity was up-regulated 7 dpnc irrespective of endophyte inoculation. Endophyte inoculation transiently up-regulated POX in cv Nabusa, but activity reduced to the levels in the controls 30 dai. Similar to PAL, R. similis challenge of endophyte-inoculated plants of Nabusa caused significant up-regulation of POX 7 dpnc. Nematode challenge of control plants of cv Yangambi resulted in a non-significant up-regulation of POX compared with non-challenged controls, but a significant up-regulation compared to all endophyte-inoculated plants. PPO activity was transiently up-regulated in cv Nabusa and down-regulated in cv Yangambi 7 dai. For all treatments, PPO activity was significantly reduced between 7 dai and 120 dai (60 dpnc). Fusarium oxysporum endophyte isolates Emb2.4o and V5w2 were successfully marked with benomyl- and chlorate resistance and transformed with fluorescent protein genes, while Eny1.31i, Eny7.11o and V4w5 were marked with benomyl resistance only. Most mutants and fluorescent protein transformants maintained resistance to the selective chemical on PDA and after plant colonization. Benomyl- and chlorate-resistant mutants were successfully used to determine actual plant colonization percentages by inoculated endophytes. Similarly, GFP transformants were successfully used to ascertain the pattern of endophytic root colonization in vivo. In plants dually inoculated with isolates Emb2.4o BR 8 and V5w2 CHR 9, both isolates were recovered from roots and rhizomes 4 weeks after inoculation, but isolate V5w2 CHR 9 proved a better colonizer of the two tissue types. Root colonization by isolate V5w2 CHR 9 was boosted when inoculated dually with Emb2.4o BR 8, while that by Emb2.4o BR 8 was reduced in the presence of V5w2 CHR 9. Where growth advantages were observed for dually inoculated plants, it occurred where plants were challenged with R. similis. In the absence of pests, control plants showed better growth than endophyte-inoculated plants. On the other hand, weevil challenge of control plants resulted in significant reductions in plant height, number of live roots and root fresh weight. Dual endophyte inoculation resulted in a significant reduction in R. similis populations in nematode only challenged plants, compared with plants inoculated with Emb2.4o BR 8 singly and control plants challenged with the nematode. In one replicate banana weevil damage to the outer and inner pseudostem base, and the inner rhizome were significantly reduced for dually-inoculated plants. Copyright / Thesis (PhD)--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Fusarium oxysporum f.sp. cubense Schlecht (Foc), causal agent of Fusarium wilt of banana (Panama disease), is considered to be one of the most serious threats to banana production in the world. There is no effective control measure for Fusarium wilt, except for the replacement of susceptible with resistant banana varieties. However, resistant varieties are not always acceptable to producers and local consumer markets. A greater awareness of the detrimental effect of chemicals on the environment has stimulated research on biological control of plant pathogens. The use of indigenous microorganims, such as non-pathogenic F. oxysporum and the bacterium Pseudomonas fluorescens, therefore, offers not only an environmentally safe but also an economical approach to combat Fusarium wilt of banana as part of an integrated disease management strategy. Non-pathogenic F. oxysporum and P. fluorescens isolates have previously been isolated from the root rhizosphere in disease suppressive soils. These isolates have the ability to reduce the incidence of Fusarium wilt in greenhouse pathogenicity trials. In this study we had hoped to expand on existing knowledge on the biological control of Fusarium wilt of banana with non-pathogenic endophytic F. oxysporum and P. fluorescens. Isolates that significantly suppress disease development in greenhouse trials were tested under field conditions. Physiological and histological studies were also performed to understand the modes of action of putative biological control agents. For the histological investigations, non-pathogenic F. oxysporum isolates were modified with green and red fluorescent proteins. Chapter 1 depicts a general overview of the biological control of Fusarium wilt diseases of agricultural crops. This chapter addresses the biology and pathogenesis of F. oxysporum, before strategies to control Fusarium wilt are discussed. The application of biological control organisms was analysed in terms of potentially useful organisms, where they can be isolated, and their possible modes of action. Finally, factors that influence biological control of Fusarium wilt diseases are discussed. A good source of prospective biocontrol agents is suppressive soils. In Chapter 2, non-pathogenic F. oxysporum isolates were collected from healthy banana roots in disease suppressive soil. Random Fragment Length Polymorphisms of the intergenic spacer region were then applied to group the non-pathogenic F. oxysporum isolates into genotypes, from which candidates were selected for biological control studies. The selected endophytes were then inoculated onto banana roots to determine their ability to act as biocontrol agents against Foc. The isolates that protected banana best against Fusarium wilt in the greenhouse, together with P. fluorescens WCS 417, were tested in the field to determine whether these isolates could effectively reduce disease incidence in an uncontrolled environment. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce systemic resistance in Cavendish banana plants against Foc was investigated in Chapter 3 with the use of a split-root technique. The putative biocontrol agents were inoculated, separately and in combination, on one half of the roots in a split-root experiment, while the other half was challenged by a pathogenic isolate of Foc. Five different phenolic acids were assayed which included total soluble phenolic acids, non-conjugated (free acids) phenolic acids, ester-bound phenolic acids, glycosidebound phenolic acids and cell wall-bound phenolic acids. The knowledge gained will contribute to the understanding of how the biocontrol agents may induce defense responses in banana roots against Foc. Non-pathogenic isolates of F. oxysporum were transformed with the green fluorescent protein (GFP) and DsRed-Express genes in Chapter 4. These isolates were used to visualise their interactions with a GFP-transformed Foc isolate on the banana root in a non-destructive manner by means of confocal laser scanning microscopy (CLSM) in Chapter 5. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce structural changes was also investigated with a split-root system using the CLSM. Antibioses as a mode of action of the two potential biocontrol agents was tested in vitro. Understanding the modes of action of non-pathogenic F. oxysporum and P. fluorescens WCS 417 are important when considering strategies for the implementation of these isolates in an integrated disease management strategy against Fusarium wilt of banana. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Fusarium wilt of banana has a long and devastating history in many of the world’s banana producing countries. The most pronounced damage caused by Fusarium oxysporum f.sp. cubense (Foc), the Fusarium wilt pathogen, occurred during the 20th century in Central America, where tens of thousands of virgin forests were lost to further banana production. No control strategy is effective against Fusarium wilt other than replacement of susceptible by resistant varieties. It is, therefore, important to develop or identify resistant replacements that would not only be able to resist the pathogen, but also be acceptable to consumers. Resistance in wild banana varieties has been identified, and hybrids have been developed by breeding programmes with good resistance to Fusarium wilt. These varieties, unfortunately, appear not to be acceptable replacements for Cavendish bananas, the sweet desert banana variety that serves as the primary export banana and constitutes almost 40% of all bananas planted in the world today. A field selection, GCTCV-218, now proved to be the Cavendish plant with the most resistance to Foc ‘tropical’ race 4 (VCG 0121) has saved the Cavendish-based banana industry in Taiwan from devastation. In this thesis, GCTCV-218 has been evaluated against Foc ‘subtropical’ race 4 (VCG 0120), the primary variant of the pathogen in subtropical banana-producing countries such as South Africa, Australia and the Canary Islands. Defence-associated genes that are differentially expressed and that were up-regulated early in the defence response against the pathogen were isolated and identified. Greenhouse and field trials conducted at the research facilities of the Forestry and Agricultural Biotechnology Institute, University of Pretoria and in Kiepersol, South Africa, respectively, showed that GCTCV-218 had a significantly higher level of disease tolerance against Foc ‘subtropical’ race 4 (VCG 0120) when compared to the commercially grown Williams cultivar. Phenolic assays revealed that total phenolics and cell-wall bound phenolics were expressed at higher levels in GCTCV-218 after pathogen attack and seemed to play an important role in the tolerance of GCTCV-218. It was, therefore, proposed that GCTCV-218 could be considered a replacement for other Cavendish banana varieties planted in South Africa. The genetic basis of defence mechanisms in banana to Foc is unknown. In this investigation, Suppression Subtractive Hybridisation (SSH) was used to construct a cDNA library, containing banana genes that were up-regulated early (3&6 hours after infection), in the GCTCV-218/Foc interaction. The efficiency of the procedure was confirmed by PCR amplification of a known defence gene (endochitinase) present in the subtracted tester material, as well as analysing the reduction of a known housekeeping gene, actin, in the subtracted material compared to unsubtracted material. Southern blot data further provided confidence in the subtraction process. A cDNA library containing 736 gene fragments was constructed and then subjected to a screening procedure to remove false positives that escaped the subtraction process. The screening of a banana cDNA library for defence-related genes involved the development of a high-throughput cDNA microarray technique. This novel technique removed all false positives, such as housekeeping genes that escaped the subtraction as well as clones representing rDNAs. Seventy-nine genes differentially expressed in GCTCV-218 and not in Williams were selected, sequenced and subjected to BLASTX, BLASTN and DBest searches. Of these, several gene fragments showed homology to defence-associated genes, and 20 unique genes fragments were identified. These include two different peroxidases, response regulator 6, catalase 2, metallothionein, pectin acetylesterase (PAE), two different unknown proteins, salt stress, trypsin inhibitor, unspecific monooxygenase cytochrome P450, Bowman Birk proteinase inhibitor, root control, xylanase inhibitor, inhibitor CII, hypothetical protein, putative senescence-associated protein, pathogenesis-related protein 1 (PR1) and ribosomal protein S3a. The significance of the defence reaction to Fusarium wilt diseases in agricultural crops depends on the tempo of plant response. When a host plant is able to respond early to pathogen invasion the pathogen is successfully contained, preventing further spread throughout the plant. The expression of genes with antimicrobial activity, such as endochitinase, suggests an induced biochemical defence response against Foc. The expression of PAE and PR1 results in the deposition of lignin and callose production for cell wall strengthening. Four defence associated genes (catalase 2, pectin acetyl esterase (PAE), PR-1 and endochitinase) were selected for expression profile analysis using Real-time reverse transcriptase PCR, with TaqMan® and Light Cycler technology. All four genes were shown to be differentially expressed in GCTCV-218 at 3 and 6 hrs after infection, confirming SSH results. PR-1 and PAE were induced very early (3 hrs after infection) in the GCTCV-218, while PR3 and catalase 2 followed with a significant induction at 6 hrs after infection. This study concludes that GCTCV-218 is able to respond rapidly in response to Foc infection by activating both a biochemical and structural defence mechanism. / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted