Submitted in part fulfilment of the requirements for the Degree of Master of Technology: Biotechnology, Durban Institute of Technology, 2004. / Various methods have been developed for the analysis of Fusarium and its toxins. Advances in molecular biology can lead to efficient characterization of this group of fungi. This study was undertaken to examine random amplified polymorphic DNA, volatile compound production and hydrolytic enzyme production by 19 Fusarial isolates. These techniques were employed to assess their abilities in differentiating Fusarium species and F. verticillioides strains and extending the analysis to discriminate toxin producing capabilities amongst these fungi

Fusarium circinatum is the causal agent of the disease known as pitch canker of pine. The fungus causes resinous cankers on stems and branches of mature trees, dieback of female flowers and cones, as well as root rot and pre- and post emergence damping off of seedlings. Little is known regarding the epidemiology and biology of F. circinatum in South African pine seedling nurseries, where the fungus has been causing major economic losses since its introduction into the country in the early 1990s. The objectives of this study were, therefore, to study the infection biology and epidemiology of F. circinatum on pine seedlings, the organism’s saprophytic biology. I also considered approaches to rapidly diagnose plants infected by F. circinatum and its relatedness to other species. Much research has been done on the pitch canker disease and the causal agent F. circinatum. Chapter 1 of this thesis aimed to summarize the available knowledge on the pitch canker fungus and its biology, ecology and epidemiology. Trials to screen for resistance of Pinus spp. to the pitch canker fungus have been conducted by many research groups and also by industries that rely on Pinus spp. for pulp and wood production. In Chapter 2, parameters for such trials, including optimal wounding methods, spore concentrations, plant physiological considerations and time elapsed between wounding and inoculation, were investigated. Temperature and ambient humidity are considered important factors in plant disease epidemiology. The effect of these factors on pitch canker epidemics has not yet been studied. In Chapter 3, a survey of disease incidence in pine nurseries from different geographic areas in South Africa with different climates is presented. This was done by correlating disease incidence data from the nurseries with temperature and humidity measurements. The saprophytic biology of a plant pathogen is of great importance in its epidemiology. The extent of any plant pathogen’s saprophytic survival determines the initial inoculum levels at the onset of an epidemic. In Chapter 4, I investigated the saprophytic growth and survival of F. circinatum in various substrates, temperatures and moisture regimes. Fusarium circinatum is morphologically similar to fungi referred to as Fusarium subglutinans sensu lato. Distinguishing F. circinatum from other species in this group has in the past required pathogenicity tests and sexual crosses, which are labour intensive and time consuming. In Chapter 5, a molecular diagnostic technique, based on real-time PCR, with which identification of Fusarium spp. commonly occurring in South African nurseries is possible, was developed. Fusarium circinatum and other fungi referred to as F. subglutinans sensu lato are members of the Gibberella fujikuroi species complex. Molecular taxonomic studies have shown that F. subflutinans sensu lato is a polyphyletic taxon. The objective of the study presented in Chapter 6 was to resolve the taxon phylogenetically with the use of multiple loci. The studies in the individual chapters of this thesis present individual aspects of the biology, ecology, epidemiology and molecular ecology of Fusarium circinatum. Each chapter represents an independent entity and consequently repetition between chapters has been unavoidable. / Dissertation (MSc)--University of Pretoria, 2007. / Microbiology and Plant Pathology / Unrestricted

Orientador: Plínio Trabasso / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T11:49:19Z (GMT). No. of bitstreams: 1 Souza_Marcelade_M.pdf: 1154397 bytes, checksum: c435b972ac36d284080e07e6b6b20a6e (MD5) Previous issue date: 2014 / Resumo: Este trabalho teve por objetivo comparar o desempenho das técnicas de microarranjo de DNA (DNA microarray), amplificação circular isotérmica (Loop mediated isothermal amplification (LAMP)), PCR em tempo real e sequenciamento de DNA para a identificação das espécies de Fusarium, agentes de infecção da corrente sanguínea, isolados de pacientes com doenças onco-hematológicas do Hospital de Clínicas da Universidade Estadual de Campinas (HC-Unicamp) e do Hospital de Clínicas da Faculdade de Medicina da Universidade de São Paulo (FMUSP) em um estudo de corte retrospectivo. Foram selecionados 21 amostras de Fusarium isoladas de sangue, sendo 17 amostras da micoteca do Laboratório de Investigação em Fungos (LIF) da Faculdade de Ciências Médicas da Unicamp (FCM-Unicamp) e três amostras da micoteca do Laboratório de Microbiologia da FMUSP. As metodologias de DNA microarray, LAMP e PCR em tempo real, identificaram 17 das 21 amostras como complexo Fusarium solani e quatro como complexo Fusarium não - solani, apresentando um alto desempenho, uma concordância de 100% entre elas e uma sensibilidade de 100% para as metodologias DNA microarray, LAMP e PCR em tempo real e uma especificidade de 100% para a metodologia de LAMP. Esses dados foram validados pela metodologia de sequenciamento de DNA, a qual identificou 17 amostras como complexo Fusarium solani e quatro como Fusarium complexo não ¿ solani, sendo três Fusarium napiforme e um Fusarium oxysporum, concordando com os resultados encontrados nas três metodologias aplicadas neste trabalho. A técnica de LAMP demonstrou ser mais acessível do que as técnicas de DNA microarray e PCR em tempo real por ser mais rápida, indicando ser uma metodologia promissora para ser utilizada na rotina de diagnóstico de fungos filamentosos, auxiliando em uma terapia apropriada de uma forma rápida e específica, proporcionando assim uma melhora na assistência prestada ao paciente / Abstract: This study aimed to compare the performance of the techniques of DNA microarray, loop mediated isothermal amplification (LAMP), real-time PCR and DNA sequencing for identification of species of Fusarium as agents of bloodstream infection, isolated from patients with hematologic malignancies cared for at the university hospital of State University of Campinas and the university hospital of University of Sao Paulo, in a retrospective cross study. In the study, 21 Fusarium isolates from blood were selected, being 17 samples of mycology collection of the Laboratory for Research on Fungi (LIF), Faculty of Medical Sciences, Campinas (FCM-Unicamp) and three samples of mycology collection of the Laboratory of Microbiology of FMUSP. The techniques of DNA microarray, LAMP and Real-Time PCR identified 17 of the 21 samples as Fusarium solani, and 4 isolates as Fusarium non-solani complex, showing a high performance, a 100% agreement among them and a sensitivity of 100% for the DNA microarray, LAMP and real-time PCR methods and a 100% specificity for the LAMP method. These data were validated by the DNA sequencing technique, which identified 17 isolates as Fusarium solani complex and not four as Fusarium non-solani complex, being three Fusarium napiforme and one Fusarium oxysporum, agreeing with the findings in the three methodologies applied in this work. The LAMP technique proved to be more accessible than the techniques of DNA microarray and real-time PCR to be faster, indicating a promising methodology to be used in routine diagnosis of filamentous fungi, aiding in proper therapy rapid and specific manner, thereby providing an improvement to the care of patients / Mestrado / Clinica Medica / Mestra em Clínica Médica

Distribution of Fusarium wilt of muskmelon and watermelon in southwestern Ontario was studied. Particular attention was paid to morphological and physiological variations of the isolates obtained. Morphological variations were based on comparison in culture with a selected standard. Physiological variations were detected by pathogenicity experiments, and a study of assimilation of various carbon and nitrogen compounds. Some further aspects of the biology of the organisms were investigated. An experiment was carried out, employing several muskmelons and watermelon varieties, to compare their resistance under field conditions. / Thesis / Master of Science (MS)

No description available.

No description available.

Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.

Fusarium verticillioides is one of the most important fungal pathogens of maize and has also received increasing attention due to its ability to produce various secondary metabolites, including fumonisin B1 (FB1) and bikaverin. However, little is known about the regulatory mechanisms associated with F. verticillioides secondary metabolism. In this study, I utilized functional genomics, forward and reverse genetics, proteomics, and high efficiency homologous recombination, to better understand the complex secondary metabolism regulations in F. verticillioides. First, using the reverse genetics approach, I characterized a putative protein phosphatase gene, CPP1 as a negative regulator of FB1 biosynthesis. CPP1 gene deletion also affected multiple phenotypes such as radial growth, conidia germination rates, macroconidia formation, and hyphal swelling. Through gene complementation, I also demonstrated that the F. verticillioides CPP1 and Neurospora crassa wild-type ppe-1 gene are functionally conserved. Second, I used proteomics and quantitative real-time (qRT)-PCR, to advance our understanding of genes associated with fumonisin production. I analyzed the proteomic changes associated with the mutation in FCC1, a key positive regulator of fumonisins biosynthesis. I isolated proteins that were significantly up-regulated in either the wild-type or the fcc1 mutant, and transcriptional profiles of the genes corresponding to the selected proteins were analyzed via qRT-PCR. These genes showing expression patterns concomitant with fumonisin biosynthesis can be identified as primary targets for functional analysis. Next, I utilized REMI (Restriction Enzyme Mediated Integration) to isolate GAC1 gene, which encodes a GTPase activating protein, that serve as a negative regulator of bikaverin biosynthesis in F. verticillioides. AREA and PKS4 are downstream genes that are regulated positively and negatively by GAC1, respectively. Lastly, I generated a highly efficient homologous recombination strain of F. verticillioides. In eukaryotes, KU70 and KU80 play important roles in nonhomologous end-joining process, which leads to a high percentage of ectopic integration events during fungal transformation. By generating a KU70 gene deletion mutant (SF41), I have established a resource that will contribute to functional genomic research in F. verticillioides.