Os fungos fitopatogênicos pertencentes ao gênero Fusarium são conhecidos causadores de doenças de plantas em diversos hospedeiros. Dentre os quais se destaca o tomateiro, atacado pelas três raças conhecidas de Fusarium oxysporum f. sp. lycopersici (Fol), causadoras da murcha vascular. Para o controle dessa doença, o emprego de microrganismos, como os isolados antagonistas de Trichoderma sp. pode ser uma alternativa ao emprego de agroquímicos. No presente trabalho foi avaliada a patogenicidade de dez isolados de Fol em cultivares diferenciadoras de tomateiro, sendo que apenas quatro se mostraram patogênicos a cultivar suscetível, e nenhum deles foi diagnosticado como pertencente à raça 3. Os resultados obtidos nos testes de confronto direto com os isolados de Trichoderma sp. apresentaram alta variabilidade em relação à capacidade micoparasítica, com os melhores resultados de sobreposição para os isolados T3, T8 e T17. Nestes casos foi observada evidência de sobreposição da colônia do hospedeiro para pelo menos seis dos nove isolados antagonistas. Em relação à capacidade inibitória destes isolados, os resultados de maior inibição de crescimento da colônia do fitopatógeno foram obtidos para os isolados T2 e T3. A linhagem 34970 de Fol foi a mais resistente às ações antagonistas dos isolados de Trichoderma sp., e o isolado TO 11 sofreu as maiores médias de inibição. Em teste de produção de metabólitos voláteis somente os isolados Fusarium 23, Fusarium 27 e 34970 de Fol, foram inibidos após 120 horas de teste. No controle biológico da fusariose do tomateiro, provocada pelo isolado TO 245 de Fol, as plântulas tratadas com os isolados T6 e T17 de Trichoderma sp. apresentaram menor incidência de doença, com notas iguais a 1,25 e 0,75 respectivamente em comparação a 1,87 do grupo controle, de acordo com a escala de severidade de doença de Vakalounakis et al. (2004). Os tratamentos com os isolados T6 e T17 em substrato contaminado com o isolado 1205/2 de Fol apresentaram valor de 0,50 e 0,38, de acordo com a escala, em comparação com 0,50 do tratamento controle. As médias de alturas, comprimento e peso seco das raízes não apresentaram diferença estatística para nenhum dos tratamentos, porém o peso seco da parte aérea foi significativamente maior para as plantas tratadas com o isolado T6 em substrato infestado pelo isolado 1205/2 de Fol. Todos os isolados patogênicos pareados com as linhagens padrão dos grupos de compatibilidade vegetativa apresentaram sinais de heterocariose com a linhagem padrão 34970 correspondente ao GCV 0030, indicando uma possível similaridade genética entre os isolados utilizados. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Phytopathogenic fungi belonging to Fusarium genera are known as the agents responsible of many diseases in plants. Among these, tomato plants are attacked by the 3 races of Fusarium oxysporum f. sp. lycopersici (Fol), which causes vascular wilt. To control this disease, many techniques have been employed, mostly the use of resistant cultivars and chemical fungicides. Alternatively, the use of antagonistic microorganisms for biological control and decrease of inoculum of plant pathogens by introducing mass production of Trichoderma sp. has been studied for many years. In this study we evaluated the pathogenicity of 10 isolates of Fol on differential cultivars of tomato, where only four of ten isolates proved pathogenic to susceptible cultivar to this disease and none of which was diagnosed as belonging to race 3. The results obtained in tests of direct confrontation with Trichoderma sp. isolates showed high variability in relation to mycoparasitism, with the best results for agressiveness the isolates T3, T8 and T17. In these cases it was observed evidence of growth of the host colony for at least six of nine isolates tested. Regarding the inhibitory effect of these isolates, the results of greater inhibition of colony growth of plant pathogenic fungus were obtained for the isolated T3 and T2. The strain 34970 of Fol was the least inhibited by isolates of Trichoderma sp., and isolated TO 11 showed the highest average of inhibition. With regards to the production of volatile metabolites, only the isolates Fusarium 23, Fusarium 27 and 34970 of Fol were inhibited after 120 hours of testing. In the biological control of Fusarium wilt of tomato caused by isolate TO 245, seedlings treated with isolates T6 and T17 of Trichoderma sp. showed a lower incidence of disease, with 1.25 and 0.75 respectively compared to 1.87 of the control group in accordance with scale of severity of disease by Vakalounakis et al. (2004). The treatments with the isolates T6 and T17 in substrate inoculated with isolate 1205/2 showed values of 0.5 and 0.375 according to the scale, compared with 0.5 in the control treatment without the presence of Trichoderma sp. The average height, length and dry weight of roots did not differ significantly for any treatment. The dry weight of shoot was significantly higher for plants treated with isolate T6 in substrate infested by the isolated 1205/2. All the pathogenic isolates of Fol were paired with the standard strains of vegetative compatibility groups and showed signs of heterokaryosis with the standard strain 34970 corresponding to VCG 0030, indicating a possible genetic similarity among the isolates used.

Made available in DSpace on 2014-06-12T15:03:57Z (GMT). No. of bitstreams: 2 arquivo4377_1.pdf: 7022466 bytes, checksum: ddcd0ae8a9c48ea3d7bc2290ffa19a0c (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2000 / Quinze isolados de Fusarium oxysporum f.sp. passiflorae provenientes do nordeste do Brasil foram analisados quanto a sua variabilidade genética utilizando a técnica de RAPD (Random Amplified Polymorphic DNA) e em termos de características morfofisiológicas. Os isolados foram obtidos da Micoteca-URM do Departamento de Micologia/UFPE, Recife-PE. Na primeira etapa deste trabalho, foram observadas as características morfológicas (coloração e aspecto da colônia) e fisiológicas (crescimento e taxa de crescimento micelial dos isolados), utilizando-se dois meios de cultura, o BDA e o CZA. As características micromorfológicas também foram observadas, após microcultivos, utilizando o meio BDA. Os resultados obtidos, indicaram que houve maior variação no crescimento e também na expressão da coloração micelial dos isolados colonizados em meio BDA. As amostras apresentaram uma variação significativa em relação as taxas de crescimento entre os meios CZA e BDA. Para a investigação do polimorfismo de DNA dos isolados utilizando-se a técnica de RAPD, foram testados 57 primers dos quais quatro foram selecionados considerando-se a geração de bandas mais fortes, mais definidas e em maior número. Os primers selecionados geraram um total de 448 bandas, permitindo a construção de uma matriz de similaridade. Observou-se elevado polimorfismo genético, pois pelo menos uma banda não estava presente em todas as amostras não havendo presença de bandas monomórficas. O coeficiente médio de similaridade obtido para todas as amostras foi de 0.62 (62%). O dendrograma gerado a partir dos dados da matriz revelou uma alta variabilidade genética entre os isolados, pertencentes a mesma formae specialis. Aparentemente, não existe correlação entre a taxa de crescimento micelial e agrupamento dos isolados no dendrograma

Doctora en Bioquímica / Las giberelinas (GAs) son fitohormonas diterpénicas sintetizadas como metabolitos secundarios por algunos hongos, entre ellos Fusarium fujikuroi, patógeno del arroz que produce grandes cantidades de ácido giberélico (GA3). Esta especie forma parte del complejo taxonómico Gibberella fujikuroi que incluye otras 10 especies relacionadas filogenéticamente denominadas poblaciones de apareamiento (A-K). Aunque todas las especies del complejo G. fujikuroi contienen genes de la biosíntesis de GAs la producción de estos diterpenos se ha descrito casi exclusivamente en F. fujikuroi, con excepción de dos cepas de Fusarium proliferatum y una cepa de Fusarium konzum que sintetiza muy bajas cantidades de GAs. Con el objeto de investigar si existen otras especies productoras de GAs en el complejo G. fujikuroi, se caracterizaron varias cepas de Fusarium sacchari (aislado de la caña de azúcar), de Fusarium konzum (aislado de pastos de praderas) y de Fusarium subglutinans (aislado del maíz). F. sacchari es una especie cercanamente relacionada con F. fujikuroi, agrupada en el clado Asiático del complejo en tanto que F. konzum y F. subglutinans son especies filogenéticamente alejadas de F. fujikuroi agrupadas en el clado Americano. Las cepas de F. sacchari investigadas presentaron diferencias con respecto a su capacidad de sintetizar GAs. Cinco aislados (B-12756; B-1732, B-7610, B- 1721 and B-1797) resultaron activos sintetizando principalmente GA3 (2,76-28,4 mg/mL) mientras que otros dos (B-3828 and B-1725) resultaron inactivos. Las etapas catalizadas por la geranilgeranil difosfato sintasa (GGS2) y/o la ent-kaureno sintasa (CPS/KS) son limitantes en las cepas productoras ya que los niveles de GA3 aumentaron 2,9 veces por complementación con los genes ggs2 y cps/ks de F. fujikuroi. Con respecto a F. konzum, de los seis aislados que se investigaron tres (I-10653; I-11616; I-11893) sintetizaron GAs, principalmente GA1, un producto final diferente al de las cepas activas de F. sacchari. Además, los niveles de GAs sintetizados por F. konzum fueron muy bajos (menos de 0,1 mg/mL). Tres cepas de F. konzum resultaron inactivas y no presentaron actividad de ninguna oxidasa de GAs lo que sugiere que los genes respectivos no se expresan. Las dos cepas de F. subglutinans ensayadas no sintetizan GAs ni presentan actividad de las oxidasas. Estos resultados indican que la capacidad de sintetizar GAs está presente en otras especies del complejo G. fujikuroi además de F. fujikuroi, pero puede diferir significativamente entre cepas. Finalmente se investigó la biosíntesis de GAs en un conjunto de 19 cepas híbridas (CxD) provenientes de un cruce entre F. fujikuroi y F. proliferatum, dos especies muy cercanas filogenéticamente agrupadas en el clado Asiático del complejo G. fujikuroi. Las dos cepas parentales (F. fujikuroi C-1995 y F. proliferatum D-4854) contienen los genes de la biosíntesis de GAs pero difieren en su capacidad biosintética: C-1995 sintetiza GAs, principalmente GA3, mientras que D-4854 no produce GAs. Las cepas híbridas no presentaron un patrón de segregación Mendeliano 1:1 como se esperaba sino que se encontró en la progenie un patrón de tres fenotipos: 8 híbridos producen GA3 en cantidades similares a la cepa parental C-1995 en tanto que 6 cepas resultaron inactivas para la biosíntesis de GAs. Además de los fenotipos parentales una parte de la progenie (5 cepas) produjo pequeñas cantidades de GAs, principalmente GA1 y presenta bajos niveles de expresión de los genes. Estos resultados evidencian que el cruce interespecies puede generar nuevos patrones de metabolitos secundarios, en este caso de GAs. El conjunto de resultados obtenidos sugiere que la capacidad de sintetizar GAs en niveles significativos estaría restringida a las especies de Fusarium agrupadas en el clado Asiático del complejo G. fujikuroi / Gibberellins (GAs) are diterpene phytohormones synthesized as secondary metabolites by some fungi like Fusarium fujikuroi, a plant pathogen that produces high levels of gibberellic acid (GA3). This Fusarium species belongs to Gibberella fujikuroi, a taxonomic species complex that includes 10 other phylogenetically related species denominated mating populations (A-K). Even when all the species within the G. fujikuroi complex contain GA biosynthesis genes, the production of these diterpenes has been almost exclusively found in F. fujikuroi, except for two Fusarium proliferatum strains and one Fusarium konzum isolate that synthesizes very low GA levels. In order to find out if other species within the G. fujikuroi complex synthesize GAs, several strains of three Fusarium species were characterized: Fusarium sacchari (isolated from sugar cane), F. konzum (isolated from prairie grasses) and Fusarium subglutinans (isolated from maize). F. sacchari is closely related to F. fujikuroi and grouped in the Asian clade of the complex while F. konzum and F. subglutinans are phylogenetically distant species grouped in the American clade. Analyzed F. sacchari strains differed in their ability to synthesize GAs. Five isolates (B-12756; B-1732, B- 7610, B-1721 and B-1797) were active and synthesized mainly GA3 (2,76-28,4 mg/mL) while two others (B-3828 and B-1725) were inactive. The steps catalyzed by geranylgeranyl diphosphate synthase (GGS2) and/or ent-kaurene synthase (CPS/KS) were limiting in F. sacchari active strains since it was found that GA3 levels increased by 2.9 fold upon complementation with ggs2 and cps/ks genes from F. fujikuroi. For F. konzum, six isolates were analyzed of which three (I-10653; I-11616; I- 11893) synthesized GAs, mainly GA1, a different final product than that synthesized by F. sacchari active strains. GA levels formed by F. konzum isolates were very low (less than 0,1 mg/mL). Three F. konzum strains were inactive in GA biosynthesis and contained no GA oxidase activities suggesting that the respective genes were not expressed. For F. subglutinans the two isolates assayed did not synthesize GAs and lacked activity of the GA oxidases. These results evidence that GA biosynthesis is present in other species within the G. fujikuroi complex besides F. fujikuroi but may differ significantly between isolates. Finally, GA biosynthesis was investigated in a group of 19 hybrid strains (CxD) from an interspecies cross between F. fujikuroi y F. proliferatum, two closely related species that belong to the American clade in the G. fujikuroi complex. Both parental strains (F. fujikuroi C-1995 and F. proliferatum D-4854) contain the GA biosynthetic genes but differed in their ability to produce GAs: C-1995 synthesizes GAs, mainly GA3, while D-4854 did not produce GAs. The hybrid strains did not show a Mendelian 1:1 segregation pattern as expected but we found in the progeny a three phenotype pattern: 8 CxD hybrids produced GA3 at similar levels than the parental strain C-1995 while 6 strains were inactive for GA biosynthesis. Besides the parental phenotypes 5 progeny strains produced low GA levels, mainly GA1 and showed low expression of the respective genes. These findings indicate that interspecies crosses may generate new profiles of secondary metabolites, like the GAs. Altogether obtained results suggest that GA biosynthesis at significant levels would be restricted to the Fusarium species of the Asian clade in the G. fujikuroi complex / FONDECYT; FONDAP; MECESUP

Thesis (PhD)--University of Pretoria, 2006. / Microbiology and Plant Pathology / Unrestricted

Fusarium moniliforme Sheldon a common fungal contaminant of maize throughout the world has been associated with diseases in both man and animals. The structure of the fumonisins, a family of structurally related mycotoxins isolated from cultures associated with the high incidence of human oesophageal cancer in the Transkei region in South Africa and with equine leucoencephalomalacia, a neurological disorder in horses and donkeys, has been established. These mycotoxins in the case of fumonisin B3 and B4 consist of the diester formed by the C(14) and C(15) hydroxy groups of 2-amino-12,16-dimethyl-3,10,14,15-tetrahydroxyicosane and the C(10) deoxy analogue, respectively, with the Si carboxy group of propane-1,2,3-tricarboxylic acid. The aim of the synthetic study outlined in this dissertation is the development and implementation of methodology for the synthesis of the C(1)-C(8) unit of the C20 backbone of fumonisin B3 and B4 with the appropriate stereochemistry. Retrosynthetic analysis of the C20 backbone identifies (6S,7S)-7-amino-1,6-octanediol as the target structure. The use of t-butyldimethylsilyl and t-butyldiphenylsilyl, methoxymethyl and benzyl protecting groups in the proposed synthetic route, the stereochemical control in the creation of the stereogenic centres using Sharpless asymmetric epoxidation-kinetic resolution, and the introduction of the amino group, were initially investigated using pentane-1,5-diol as starting material. The findings were then applied in the synthesis of (2 S,3S)-3-benzyloxy-2-(t-butyloxycarbonyl)amino-8-[(t-butyldimethylsilyl)oxy]-octane, the synthetic intermediate corresponding to the target structure, from hexane-1,6-diol using t-butyldimethylsilyl and benzyl protecting groups. The structures of 3-epi-fumonisin B3 and -fumonisin B4 were deduced from 1H and 13C NMR data and confirmed by the data for a model compound, (5R,6S)-6-amino-1,5-heptanediol, prepared from pentane-1,5-diol using the methodology established earlier in the dissertation. / Dissertation (MSc (Chemistry))--University of Pretoria, 2007. / Chemistry / unrestricted

Research investigates the chemical basis for Fusarium head blight (FHB) resistance and initiating development of a screening test for resistant wheat genotypes. The focus is on minimizing cost of screening and gaining chemical approach against FHB. Wheat bran/agar plates (8% bran, w/v) prepared from hard red spring wheat with different susceptibility to FHB were inoculated with F. graminearum. Fusarium plaque diameters and ergosterol levels after 4 days of growth were significantly lower (p< 0.05) on plates prepared from genotypes with low FHB susceptibility than from high FHB susceptible genotypes. F. graminearum growth was lower, when methanol-soluble compounds (MSC) extracted from a low FHB susceptibility genotype, Glenn, were added to high susceptibility genotype, Samson. Wheat bran/agar plates enriched with linoleic acid significantly (p<0.05) reduced the growth rate of F. graminearum in both Glenn and Samson genotypes. Oxygenated fatty acids, including monohydroxy- and dihydroxy- fatty acids were identified in the MSC.

The highly toxic fungal metabolite, neosolaniol monoacetate, was isolated and purified from cultures of Fusarium sambucinum. Since little is known about its toxic properties, the biological effects of this trichothecene were compared to those caused by diacetoxy-scirpenol in male Wistar rats. The lesions caused by the two toxins were very similar. Chronic exposure to either toxin led to a significant decrease (P<0.05) in red blood cell counts and a significant increase (P<0.05) in platelet size. The major pathological lesions observed were atrophy of the actively dividing cells of the bone marrow, thymus, spleen and lymph nodes. The reported species difference in T-2 toxin toxicity was investigated by determining the deacylation rate of T-2 toxin to HT-2 toxin, one of the first steps in the detoxification of this trichothecene. The high deacylation rate catalysed by rat microsomes correlated with the low sensitivity of this species to T-2 toxin, whereas the low deacylation rates with cat and monkey microsomes agreed with their high sensitivity. In contrast to this, the apparently high toxicity of T-2 toxin to humans does not correlate with the high deacylation rate observed in human hepatic microsomes. Involvement of the UDP-glucuronyltransferases in the detoxification of T-2 toxin was studied with rat and pig hepatic microsomes. T-2 toxin and two of its metabolites, HT-2 toxin and T-2 tetraol, did not appear to act as substrates for these enzymes under the in vitro conditions used.

Fusarium oxysporum Schlecht. is a cosmopolitan species complex that consists of both pathogenic and non-pathogenic members. The pathogenic members are subdivided into formae speciales, based on virulence to specific host species. More than 150 formae speciales have been described, of which F. oxysporum f.sp. cubense (E.F.Smith) Snyder and Hansen (Foc), causal agent of Fusarium wilt of banana, is regarded as one of the economically most important and destructive. According to phenotypic and genotypic markers, Foc has been classified into three races and 24 vegetative compatibility groups, and can be divided into a number of clonal lineages that roughly correspond with VCG groupings. In this thesis, we investigated the evolutionary relationships among VCGs using multi-gene sequencing and MAT genotyping. A PCR-RFLP fingerprint discriminating the Foc lineages and a PCR primer that identified Foc ‘subtropical’ race 4 isolates, was developed. Nine microsatellite markers (SSRs) were applied to a global population of Foc in order to investigate diversity not always detectable using sequencing data. Phylogenetic analysis of isolates representing Foc, various other formae speciales of F. oxysporum and non-pathogenic F. oxysporum of the genes encoding the translation elongation factor-1á (TEF), the mitochondrial small subunit (MtSSU), ribosomal RNA (rRNA), the repeated region encoded on the mitochondrion (MtR) and the intergenic spacer (IGS) gene regions separated these isolates into four clades, two of which included Foc. Within these two clades, Foc separated into six lineages that broadly corresponded to VCGs, while the non-pathogenic isolates of F. oxysporum grouped together in only one of the two clades, with an unknown Foc VCG isolate. The mating type of all isolates was determined and crosses were attempted between isolates harbouring MAT-1 and MAT-2 genes, without success. Cultural, morphological and pathogenic variation among isolates of Foc was unable to identify lineages as species. The separation of Foc isolates into two clades suggested that the banana pathogen evolved during two unrelated events. Factors such as horizontal gene transfer, however, might also have played a part in the pathogen’s evolution, as was evident from the divergent placement of some VCGs and lineages within the phylogenetic trees constructed. The inclusion of other formae speciales of F. oxysporum and non-pathogenic F. oxysporum isolates illustrated the great diversity that exists within the F. oxysporum complex. The inclusion of the Foc isolate of an unknown VCG suggests that the genetic diversity of Foc might be far greater than what is known and what was revealed in this study. The opposite mating types found in the respective lineages indicate a sexual origin for the Fusarium wilt fungus that could account for its polyphyletic nature. Within South Africa, Foc ‘subtropical’ race 4 is regarded the most important constrain to banana production. Conventional control practices for Fusarium wilt of banana are ineffective, and disease management relies heavily on the use of clean planting material and the early detection and isolation of the pathogen, in order to restrict spread to unaffected areas. Identification of Foc typically involves vegetative compatibility assays and pathogenicity testing using a set of differential host cultivars. The development of a PCR-based method for the rapid and accurate identification of Foc ‘subtropical’ race 4 will, therefore, be of great importance. The lack of morphological variation between lineages of Foc, and between pathogenic and non-pathogenic members, as well as the unreliability in race identification in Foc, makes the use of molecular tools a viable alternative. Following DNA isolation, PCR and sequencing of the MtR, the DNA sequence data revealed an 8-bp insertion that was subsequently targeted for the design of a Foc ‘subtropical’ race 4-specific primer. Isolates were positively identified as Foc ‘subtropical’ race 4 with the amplification of an 800-pb fragment. The development of the Foc ‘subtropical’ race 4 primer will aid in rapid and accurate detection of the Fusarium wilt pathogen of banana. The population structure defined according to SSR data of a global population of 239 Foc isolates resembled the structure defined according to multi-gene phylogeny, with some exceptions. Measures of gene and genotypic diversity unequivocally supported the opinion that Asia is the centre of origin of Foc. The presence of unique genotypes in all geographically-defined Foc populations could potentially indicate their evolution outside the centre of origin, although this is highly unlikely. The absence of certain genotypes from the Asian population was either due to insufficient and selective sampling, or it demonstrated the effects of clonal selection in combination with adaptation to the forces of geographic isolation and environmental changes over time. The worldwide collection of Foc mostly consisted of six over represented genotypes, thereby providing support for a clonal genetic structure. It was, however, not possible to reject the hypothesis of a recombining population for the populations representing isolates of Lineage V. The implication of recombination within some Foc lineages may be due to unobserved sexual reproduction in nature or the historical association with a sexual ancestor. When one considers diversity within and among genotypes, a specific genotype was mostly associated with only one or two Foc VCGs, therefore indicating that vegetative compatibility determination, in combination with phylogenetic analyses, is a powerful tool for characterizing isolates causing Fusarium wilt of banana. Results from this study, in combination with the multi-gene phylogeny, clearly indicated the presence of unrelated lineages that most probably represent cryptic species. Copyright 2008, University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. Please cite as follows: Fourie, G 2008, Evolutionary biology of Fusarium oxysporum f.s.p. cubense, MSc dissertation, University of Pretoria, Pretoria, viewed yymmdd < http://upetd.up.ac.za/thesis/available/etd-11192008-094622/> E1216/gm / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

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