Dissertations / Theses on the topic 'Pathogenic fungi – Molecular aspects'

A antracnose é uma das principais doenças que afetam a goiaba no Estado de São Paulo. Tanto Colletotrichum gloeosporioides quanto Colletotrichum acutatum, são relatados como sendo os agentes causais da doença. Os objetivos do trabalho foram caracterizar e identificar 54 isolados de Colletotrichum oriundos de lesões de goiaba, baseados nos aspectos culturais, morfológicos, moleculares e enzimáticos, além da caracterização patogênica de isolados representativos de cada espécie de Colletotrichum identificadas. A caracterização cultural foi avaliada mediante a mensuração do crescimento micelial dos isolados a 25 ºC, além dos aspectos culturais, como a coloração e a topografia das colônias. Na caracterização morfológica foram mensurados comprimento e largura de conídios, bem como avaliados os seus formatos. Oligonucleotídeos específicos foram utilizados na caracterização molecular, visando identificar as espécies dos isolados de Colletotrichum. A caracterização enzimática envolveu a mensuração, in vitro, do halo de degradação dos substratos da amilase, proteinase, celulase, pectinase e lipase. Por fim, alguns isolados representativos e identificados foram utilizados na caracterização patogênica, sendo avaliados os períodos de latência e incubação, a área lesionadas dos frutos e a esporulação. Baseados na coloração das colônias, os isolados foram reunidos em 9 grupos diferentes. Os mesmos puderam se reunidos em dois grupos distintos de acordo com a taxa do crescimento micelial. Os conídios apresentaram os formatos: (i) reto, fusiforme, com ápices afilados, (ii) reto, oblongo, com ápices arredondados, (iii) reto, clavado, afilado em uma extremidade e (iv) reto, com constrição. As dimensões variaram de 11,4 a 16,8 µm de comprimento por 2,6 a 4,9 µm de largura. O uso de oligonucleotídeos específicos permitiu identificar C. acutatum e C. gloeosporioides entre os isolados avaliados. Grande parte dos isolados, 94%, foram identificados como pertencendo à espécie C. gloeosporioides, enquanto que apenas 4% foram identificados como C. acutatum. Em relação à caracterização enzimática, apenas a atividade celulolítica proporcionou diferenças significativas entre C. gloeosporioides e C. acutatum. A patogenicidade dos isolados avaliados mostrou alta variabilidade na severidade da doença nos frutos, contudo não foi possível evidenciar diferenças significativas que distinguissem C. acutatum de C. gloeosporioides. Os períodos de incubação e latência foram menores para os isolados de C. acutatum em relação aos isolados de C. gloeosporioides. C. acutatum produziu quantidade superior de esporos nos frutos inoculados quando comparados a C. gloeosporioides. Observou-se, ainda, correlação positiva entre a área do halo de degradação de pectina, lipídio e amido e a área lesionada dos frutos afetados pelos isolados avaliados.
Anthracnose is one of the major diseases affecting guava in the State of São Paulo. Both Colletotrichum gloeosporioides and Colletotrichum acutatum are reported as the causal agents of the disease. The objectives of this work were to characterize and to identify 54 Colletotrichum isolates from guava, based on cultural, morphological, molecular, enzymatic, and pathogenic aspects. Cultural characterization was achieved by measuring the mycelial growth at 25 ° C, as well as reporting cultural aspects, such as color and topography of the colonies. In the morphological characterization it was measured length and width of conidia, and rated their shapes. CaInt2 and CgInt specific primers were used in the molecular identification of the Colletotrichum isolates. The enzymatic characterization was performed by measuring, in vitro degradation of starch, protein, cellulose, pectin and lipid. Finally some representative and identified isolates were used in the pathogenic characterization, evaluated by latency and incubation periods, diseased area and sporulation. Based on the color of the colonies, the isolates were grouped in 9 different groups. These same isolates showed two distinct growth paterns according to the mycelial growth rates. Conidia showed shapes: (i) straight, fusiform, with acute ends, (ii) straight, oblong, with round ends, (iii) straight, clavate, tapered at one end and (iv) straight, with a constriction in the middle. Conidia size ranged from 11.4 to 16.8 µm in length by 2.6 to 4.9 µm in width. The use of specific primers identified C. acutatum and C. gloeosporioides among the isolates. Most of the isolates (94%) were identified as C. gloeosporioides, while only (6%) were identified as C. acutatum. In the enzymatic characterization, only cellulolytic activity revealed significant differences between C. gloeosporioides and C. acutatum. Pathogenicity of the isolates was highly variable, but could not help to distinguish between C. acutatum and C. gloeosporioides. The incubation and latency periods were shorter for C. acutatum in relation to C. gloeosporioides. C. acutatum produced higher amounts of spores on inoculated fruits compared to C. gloeosporioides. There was also a positive correlation between in vitro degradation of pectin, lipid and starch, and the diseased area for tested isolates.

Maize anthracnose, caused by the fungus Colletotrichum graminicola, is an economically important species contributing to major yield losses. C. graminicola is a hemibiotroph; initially it invades its host while it is alive, and then it switches to destructive necrotrophic growth and the host is killed. Establishment of compatible interactions by biotrophic pathogens is usually associated with suppression of host defenses and cell death, while necrotrophic pathogens typically secrete phytotoxic compounds and induce cell death. To understand the relationship of hemibiotrophy in C. graminicola to biotrophy and necrotrophy, I compared a compatible and an incompatible interaction, utilizing a non-pathogenic mutant strain that is very similar to the wild type in vitro. I developed an assay to visualize in detail living fungal and host cells during pathogenic and nonpathogenic interactions. My results provided evidence that C. graminicola produces diffusible substances during colonization that predispose nearby living host cells for fungal invasion. My observations further suggested that the mutant is nonpathogenic because it fails to produce these substances. To explore the possibility that the C. graminicola mutant is impaired in the production and/or secretion of one or more secondary metabolites (SM), I characterized the range of SM-associated genes in C. graminicola. C. graminicola has a large and diverse repetoire of these genes, indicating significant capacity for the production of SM. I then characterized the global expression of fungal genes during different developmental phases in both compatible and incompatible interactions. I found that SM-associated genes are expressed during early and late stages of maize infection. Secreted proteins and putative effectors were overrepresented among differentially regulated predicted gene products. There were relatively few differences in expression between the mutant and wild type, suggesting that differences between them may relate to post-transcriptional events. The transcriptional analysis indicated that the mutant was defective very early in biotrophy. This study indicates that biotrophy and necrotrophy coexist in this pathosystem in different cells, and that arrays of differentially regulated and locally expressed genes are involved in maintaining this balance. Understanding the nature of induced susceptibility may lead to new therapeutic targets for management of this damaging disease.

In this study, we have demonstrated that inoculation of bean seeds with non-pathogenic binucleate Rhizoctonia (np-BNR) at sowing protected bean seedlings from infection of R. solani. Using quantitative real-time RT-PCR (QRT-PCR), transcript levels of defense genes encoding 1,3-beta-glucanase (GLUC), phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) in one-week old bean seedlings was monitored during np-BNR and R. solani interaction. The results revealed that protection effect of np-BNR correspond to a systemic suppression of these three defense genes' expression from significant higher level elicited by R. solani to the level of non-infected plants. This indicates that bio-protection by np-BNR isolates is not correlated to activation of these three defense genes' expression. Similar suppression was achieved for pre-colonization of bean seedlings with arbuscular mycorrhizal (AM) Glomus introradices on GLUC gene expression, although the AM fungus did not significantly reduce rot symptoms. Possible mechanisms implicated in down-regulation during plant-pathogen and np-BNR or AM interaction are discussed.

Phoma medicaginis is a necrotrophic fungal pathogen, commonly found infecting Medicago truncatula and M. sativa in temperate regions of Australia. To identify, characterize and differentiate eight P. medicaginis isolates from Western Australia, morphological phenotypes and five gene regions (actin, â- tubulin, calmodulin, internal transcribed spacer, translation elongation factor 1-á) were examined. Sequence comparisons showed that specimens isolated from M. truncatula in Western Australia formed a group that was consistently different from, but closely allied to, a P. medicaginis var. medicaginis type specimen. Characterization of three P. medicaginis genotypes showed that all exhibited a narrow host range, causing disease only in M. sativa and M. truncatula among eight commonly cultivated legume species sampled. Infection of 85 M. truncatula accessions showed a continuous distribution in disease phenotypes, with the majority of accessions susceptible. Differences in disease phenotypes suggest that M. truncatula harbours specific and diverse sources of resistance to individual P. medicaginis genotypes. To characterize the genetic basis of resistance to P. medicaginis two F2 populations derived from crosses between the resistant accession SA27063 and the susceptible accessions SA3054 and A17 were phenotyped for disease symptoms. Highly significant recessive QTLs for resistance to P. medicaginis OMT5 were identified in each mapping population. In SA27063 x A17 a QTL named resistance to the necrotroph Phoma medicaginis one (rnpm1) was identified on the short arm of LG4. In SA27063 x SA3054 a QTL (rnpm2) was identified on the long arm of LG8. Further fine mapping of the areas surrounding the QTLs is underway to identify the genes underlying rnpm1 and rnpm2. Examination of the recombination frequencies between genetic markers on the long arms of chromosomes 4 and 8 in the SA27063 x A17 cross revealed an apparent genetic linkage between these chromosomes. Subsequent analysis of other crosses showed this unexpected linkage relationship is characteristic for genetic maps derived from A17. Furthermore F1 individuals derived from crosses involving A17 showed 50% pollen viability or less. This semisterility and the unexpected linkage relationships provide good evidence for a reciprocal translocation in A17 between chromosomes four and eight. The implications of the distinctive chromosomal rearrangement in A17 on genetic mapping, genome sequencing and comparative mapping are discussed. The Mt16kOLI1plus microarray was used to identify transcriptional changes in M. truncatula expressed in defence against P. medicaginis. Three-hundred-and-thirty-four differentially expressed transcripts showed a change of two-fold or more in either the resistant or susceptible interaction, and most of the Phoma-regulated genes could be assigned to functional categories which have been reported to be involved in plant defence responses. RT-qPCR and HPLCUV confirmed involvement of the octadecanoid and phenylpropanoid pathways in response to P. medicaginis infection. Faster induction of lipoxygenase genes and constitutively higher levels of certain phenolic metabolites were observed in resistant plants.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
A emergência e reemergência de doenças infecciosas é impulsionada por vários fatores e a busca de patógenos em amostras animais podem oferecer oportunidades para estudos eco-epidemiológicos e também dados sobre a evolução dos patógenos. O objetivo deste estudo foi avaliar a ocorrência de fungos patogênicos importantes em saúde pública, em exemplares de animais silvestres mortos por atropelamento no estado de Santa Catarina e identificar e mapear áreas de risco para a infecção humana. Grande parte destes fungos apresenta em comum dimorfismo, distribuição geográfica restrita e produção de conídios infectantes que são aspirados pelo hospedeiro por meio das vias respiratórias. Cães e tatus são apontados como transmissores de Paracoccidioides brasiliensis, os morcegos ao Histoplasma spp., assim como as fezes de pombos ao Cryptococcus spp.. No presente trabalho foram analisadas 1063 amostras de pulmão, fígado, baço, pele e coração de 297 animais silvestres, para detecção de Paracoccidioides brasiliensis, Histoplasma capsulatum e Cryptococcus spp. pela técnica de Reação em Cadeia de Polimerase (PCR). Utilizou-se primers universais para detecção de fungos em geral e obteve-se positividade em 102 amostras de 59 animais. Para a análise de P. brasiliensis, utilizou-se os primers específicos, obtendo oito amostras positivas em cinco animais (quatro Oxymycterus spp. e um Euryoryzomys russatus). Não houve a detecção molecular para Histoplasma spp.. Foi possível a identificação de três amostras para Cryptococcus spp.. O sequenciamento foi realizado, porém em 89 amostras de 49 animais foi possível somente a identificação em Fungal sp. (GenBank KT923226.1), duas amostras para Cryptococcus neoformans (GenBank KY107218.1) obtidas de Oxymycterus spp. e Akodon spp. e três amostras de Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1) obtidas de Gracilinanus spp., Oxymycterus spp. e Philander spp. Importante salientar que houve coinfecção de P. brasiliensis e Cryptococcus neoformans em amostra de um Oxymycterus spp. Esta pesquisa mostra a importância dos animais silvestres na transmissão de doenças e auxilia no mapeamento dos locais de ocorrência de determinados patógenos e doenças em uma região ainda não avaliada.
The emergence and reemergence of infectious diseases is propelled by many diverse factors and the search for pathogens in animal samples may offer opportunity for eco-epidemiologic studies as well as data on the evolution of pathogens. The objective of this study was to evaluate in samples of road-killed wild animals the occurrence of pathogenic fungi of importance for public health. A great part of these fungi presented, in common, dimorphism, restricted geographic distribution and production of conidia infecting, which are aspirated by the host by means of their respiratory tract. Dogs and armadillos are normally related to the transmission of Paracoccidioides brasiliensis, bats to Histoplasma spp., as well as pigeons feces to Cryptococcus spp.. In this study we analyzed 1063 samples of organs of 297 wild animals for the detection of Paracoccidioides brasiliensis, Histoplasma capsulatum and Cryptococcus spp. by the technique of Polymerase Chain Reaction (PCR). Universal primers were employed for the detection of fungi in general and positivity was obtained in 102 samples from 59 animals. For the P. brasiliensis analysis was used specific primers, resulting in eight positive samples from five animals (four Oxymycterus spp. and one Euryoryzomys russatus). There was no molecular detection to Histoplasma spp.. Was possible the identification of three samples to Cryptococcus spp.. The sequencing was performed, however in 89 samples from 49 animals was possible to identify Fungal sp. (GenBank KT923226.1), two samples for Cryptococcus neoformans (GenBank KY107218.1) obtained from Oxymycterus spp. and Akodon spp. and three samples from Gracilinanus spp., Oxymycterus spp. and Philander spp. were positive for Aspergillus penicillioides (GenBank KP131612.1 e KP997215.1). Is important emphasize the coinfection with P. brasiliensis and Cryptococcus neoformans in a sample from Oxymycterus spp.. This research shows the importance of the wild animals in transmissions of diseases and assists in the mapping of pathogen and disease sites in a region that has not yet been evaluated.
FAPESP: 2015/17519-4

Colletotrichum species are considered one of the most economically important plant pathogens. They cause many losses in tropical, subtropical and temperate regions affecting a wide range of plant species. In tropical and subtropical regions C. gloeosporioides and C. acutatum are associated with significant losses on pre and post-harvest anthracnoses. There are still many features to understand about Colletotrichum biology and its systematics. The accurate identification of species involved with each anthracnose is of high relevance to establish management strategies to control the disease. Although the great advances on Colletotrichum systematics, species complex such as C. gloeosporioides and C. acutatum are used in a broad sense in Brazil. These complexes were recently investigated and showed to be highly genetic and geographic variable. In this study multigene analysis were carried out based on ITS, GAPDH, CHS-1, TUB2 and CAL or HIS3 partial sequences for strains of C. gloesporioides and C. acutatum complexes collected from fruit crops in Brazil. Strains from different countries and exepitypes and others sequences available on GenBank from the species accepted on both complexes were added on dataset. Six strains from C. gloeosporiodes complex and five for C. acutatum were selected based on multigene phylogeny to investigate the pathogenicity through inoculations on detached fruit. The multigene phylogenies showed the occurrence of species in Brazil related to those complexes with a high genetic variability among them. The phylogeny of Brazilian strains belonging to the C. gloeosporioides complex showed that C. siamense represents the most genetically and host-specific variable clade. In contrast, C. asianum clade grouped only strains isolated from mango. The strains from this clade used on pathogenic test were not able to infect avocado and one of the strains caused symptoms only on mango. All strains from Brazil grouped in one subclade within the C. fructicola clade and seem to represent a genetically distinct group. C. theobromicola is first reported causing anthracnose on acerola fruit. Three new species (C. polyphialidicum, C. paranaense and C. pruni) belonging to the C. acutatum complex were recognized and their morphologic descriptions were provided. The pathogenic test for the strains in the C. acutatum complex showed their cross infection ability, but in some cases the larger lesions were produced on the original host. Most brazilian strains from C. acutatum complex grouped in one subclade within the C. nymphaeae clade and seem to be genetically distinct.
Fungos do gênero Colletotrichum são considerados um dos mais importantes economicamente na Fitopatologia. Espécies desse gênero são encontradas amplamente disseminadas e estão associadas a diversas espécies de plantas hospedeiras. Em regiões tropicais e subtropicais, espécies dos complexos C. gloeosporioides e C. acutatum são a principal causa das antracnoses em pré e póscolheita de frutos e consequentemente causam significantivas perdas. Ainda há muitos aspectos a serem compreendidos sobre o gênero Colletotrichum, como a biologia e a sistemática. A acurada identificação das espécies associadas a antracnoses é de suma importância para o estabelecimento de estratégias de controle. No entanto, apesar dos grandes avanços na sistemática desse gênero, complexos de espécies como aquelas citadas acima são tratados de modo genérico no Brasil. Estes complexos de espécies foram recentemente estudados e considerados geneticamente e geograficamente variáveis. Neste sentido, o presente trabalho teve como objetivo caracterizar isolados de Colletotrichum spp. associados a diferentes frutos e regiões do Brasil por meio de análise filogenética. Para análise multilocus, foram utilizadas sequências parciais dos genes ITS, GAPDH, CHS-1, TUB2 and CAL ou HIS3. Sequências de espécies-tipos disponíveis no GenBank e de isolados de diferentes países foram adicionadas ao conjunto de dados. Com base nos resultados obtidos por meio de filogenia multilocus, seis isolados do complexo C. gloeosporiodes e cinco do complexo C. acutatum foram escolhidos para testes de patogenicidade cruzada. A espécie C. siamense, pertencente ao complexo C. gloeosporioides, foi a mais variável geneticamente e quanto ao hospedeiro de origem. Diferentemente, apenas isolados obtidos de manga se agruparam no clado C. asianum. Isolados agrupados neste clado não infectaram abacate e um dos isolados (CPC 20969) causou sintomas apenas em manga. No clado C. fructicola, isolados coletados no Brasil se agruparam em um subclado e parecem representar um grupo geneticamente distinto. A espécie C. theobromicola é relatada pela primeira vez em acerola. Foram identificadas três novas espécies, C. polyphialidicum, C. paranaense e C. pruni, pertencentes ao complexo C. acutatum. Isolados brasileiros agrupados no clado C. nymphaeae parecem representar um grupo geneticamente distinto, todos se agruparam em um subclado. Isolados do complexo C. acutatum utilizados no teste de patogenicidade provocaram sintomas nos hospedeiros testados, porém, em algumas inoculações, as lesões foram maiores no hospedeiro de origem.

Thesis (MSc)--University of Stellenbosch, 2006.
ENGLISH ABSTRACT: Fish infections caused by pathogenic Flavobacterium spp. are a major problem in the aquaculture industry worldwide, often leading to large economic losses. Thirty-two Flavobacterium spp. isolates, obtained from various diseased fish species and biofilm growth, were characterised genetically using 16S rRNA gene sequencing, 16S rRNA gene PCR restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD) PCR, repetitive extragenic palindromic (REP) element PCR, plasmid profiling, whole cell protein (WCP) and outer membrane protein (OMP) analyses. The biofilm-forming capability of five genetically heterogeneous Flavobacterium spp. study isolates was investigated using a modified microtiter-plate adherence assay, as well as flow cell studies. Experimental infection studies with Mozambique tilapia (Oreochromis mossambicus) were carried out in order to determine the virulence of the Flavobacterium spp. study isolates. 16S rRNA gene sequence analysis showed the Flavobacterium spp. study isolates were closely related, and 97% sequence similarity was shared with published F. johnsoniae sequences. A high degree of genetic heterogeneity was displayed by the Flavobacterium spp. study isolates following RAPD-PCR, REP-PCR and OMP analysis, however, based on the results obtained by plasmid profiling and WCP analysis, the isolates appeared genetically very homogeneous. The biofilm phenotype was displayed by all five Flavobacterium spp. isolates tested and varied from weakly to strongly adherent. No specific correlation was observed between the RAPD, REP and/or OMP profiles and degree of adherence displayed by Flavobacterium spp. isolates. However, a specific WCP profile (profile B), exhibited by 48% of the Flavobacterium spp. isolates, was linked to strong adherence. Experimental infection studies showed that Flavobacterium spp. isolates displayed variable levels of virulence, which could not be linked to biofilm formation, nor specific genotypes. This is the first reported isolation and characterisation of Flavobacterium spp. isolated from diseased fish in Southern Africa, and there appears to be significant diversity amongst the isolates which is not geographically linked nor host related.
AFRIKAANSE OPSOMMING: Visinfeksies veroorsaak deur Flavobacterium spp. is problematies in die akwakultuur industrie wêreldwyd en lei tot groot ekonomiese verliese. Twee en dertig Flavobacterium spp. isolate, geïsoleer vanaf verskye geïnfekteerde visspesies en biofilm groei, was geneties gekarakteriseer met behulp van 16S rRNS geenvolgorde, 16S rRNS geen PKR restriksie fragment lengte polimorfisme (RFLP), toevallig geamplifiseerde polimorfiese DNS (TGPD) PKR, herhaalde ekstrageniese palindromiese (HEP) element PKR, plasmied profilering, heelsel protein (HSP) en buite membraan protein (BMP) analise. Die vermoë van vyf geneties heterogene Flavobacterium spp. isolate om biofilms te vorm was ondersoek met behulp van ‘n gemodifiseerde mikrotiterplaat vashegtings toets asook vloei-sel studies. Eksperimentele infeksie studies was uitgevoer op bloukurpers (Oreochromis mossambicus) om die virulensie van die Flavobacterium spp. studie isolate te toets. 16S rRNS geenvolgorde analise het getoon dat die Flavobacterium spp. studie isolate naby verwant was, en het 97% ooreenstemming getoon met gepubliseerde F. johnsoniae volgordes. TGPD-PKR, HEP-PKR en BMP analise het ‘n hoë graad van heterogeniteit tussen die Flavobacterium spp. studie isolate aangetoon, egter, op grond van plasmied profilering en HSP analise, was die studie isolate geneties baie homogeen. Die biofilm fenotipe was getoon deur al die getoetsde Flavobacterium spp. isolate en het gevarieer van swak tot sterk vashegting. Geen spesifieke korrelasie was waargeneem tussen die TGPD, HEP en/of BMP profiele en graad van vashegting vertoon deur Flavobacterium spp. isolate nie, maar ‘n spesifieke HSP profiel (profiel B), getoon deur 48% van die Flavobacterium spp. isolate, was verbind met sterk vashegting. Eksperimentele infeksie studies het getoon dat Flavobacterium spp. isolate varierende grade van virulensie vertoon het en wat met biofilm formasie of spesifieke genotipes geassosieer kon word nie. Hierdie is die eerste gedokumenteerde isolasie en karakterisering van Flavobacterium spp. geïsoleer van geïnfekteerde vis in Suider Afrika, en daar is beduidende diversiteit tussen die isolate wat nie geografies of gasheer geassosieerd is nie.

Orientador: Virgínia Bodelão Richini-Pereira
Resumo: A emergência e reemergência de doenças infecciosas é impulsionada por vários fatores e a busca de patógenos em amostras animais podem oferecer oportunidades para estudos eco-epidemiológicos e também dados sobre a evolução dos patógenos. O objetivo deste estudo foi avaliar a ocorrência de fungos patogênicos importantes em saúde pública, em exemplares de animais silvestres mortos por atropelamento no estado de Santa Catarina e identificar e mapear áreas de risco para a infecção humana. Grande parte destes fungos apresenta em comum dimorfismo, distribuição geográfica restrita e produção de conídios infectantes que são aspirados pelo hospedeiro por meio das vias respiratórias. Cães e tatus são apontados como transmissores de Paracoccidioides brasiliensis, os morcegos ao Histoplasma spp., assim como as fezes de pombos ao Cryptococcus spp.. No presente trabalho foram analisadas 1063 amostras de pulmão, fígado, baço, pele e coração de 297 animais silvestres, para detecção de Paracoccidioides brasiliensis, Histoplasma capsulatum e Cryptococcus spp. pela técnica de Reação em Cadeia de Polimerase (PCR). Utilizou-se primers universais para detecção de fungos em geral e obteve-se positividade em 102 amostras de 59 animais. Para a análise de P. brasiliensis, utilizou-se os primers específicos, obtendo oito amostras positivas em cinco animais (quatro Oxymycterus spp. e um Euryoryzomys russatus). Não houve a detecção molecular para Histoplasma spp.. Foi possível a identificação de três amos... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: Yeasts, like most organisms, have to survive in highly variable and hostile environments. Survival therefore requires adaptation to the changing external conditions. On the molecular level, specific adaptation to specific environmental conditions requires the yeast to be able: (i) to sense all relevant environmental parameters; (ii) to relay the perceived signals to the interior of the cell via signal transduction networks; and (iii) to implement a specific molecular response by modifying enzyme activities and by regulating transcription of the appropriate genes. The availability of nutrients is one of the major trophic factors for all unicellular organisms, including yeast. Saccharomyces cerevisiae senses the nutritional composition of the media and implements a specific developmental choice in response to the level of essential nutrients. In conditions in which ample nutrients are available, S. cerevisiae will divide mitotically and populate the growth environment. If the nutrients are exhausted, diploid S. cerevisiae cells can undergo meiosis, which produces four ascospores encased in an ascus. These ascospores are robust and provide the yeast with a means to survive adverse environmental conditions. The ascospores can lie dormant for extended periods of time until the onset of favourable growth conditions, upon which the spores will germinate, mate and give rise to a new yeast population. However, S. cerevisiae has a third developmental option, referred to as pseudohyphal and invasive growth. In growth conditions in which nutrients are limited, but not exhausted, the yeast can undergo a morphological switch, altering its budding pattern and forming chains of elongated cells that can penetrate the growth substrate to forage for nutrients. The focus of this study was on elements of the signal transduction networks regulating invasive growth in S. cerevisiae. Some components of the signal transduction pathways are well characterised, while several transcription factors that are regulated via these pathways remain poorly studied. In this study, the RMEt gene was identified for its ability to enhance starch degradation and invasive growth when present on a multiple copy plasmid. Rme1 p had previously been identified as a repressor of meiosis and, for this reason, the literature review focuses on the regulation of the meiotic process. In particular, the review focuses on the factors governing entry into meiosis in response to nutrient starvation and ploidy. Also, the transcriptional regulation of the master initiator of meiosis, IMEt, and the action of Ime1 p are included in the review. The experimental part of the study entailed a genetic analysis of the role of Rme1 p in invasive growth and starch metabolism. Epistasis analysis was conducted of Rme1 p and elements of the MAP Kinase module, as well as of the transcription factors, Mss11p, Msn1p/Mss10p, Tec1p, Phd1p and F108p. Rme1p is known to bind to the promoter of CLN2, a G1-cyclin, and enhances its expression. Therefore, the cell cyclins CLN1 and CLN2 were included in the study. The study revealed that Rme1 p functions independently or downstream of the MAP Kinase cascade and does not require Cln1 p or Cln2p to induce invasive growth. FL011/MUC1 encodes a cell wall protein that is required for invasive growth. Like the above-mentioned factors, Rme1 p requires FL011 to induce invasive growth. We identified an Rme1 p binding site in the promoter of FL011. Overexpression of Rme1p was able to induce FL01t expression, despite deletions of mss11, msn1, ttos, tee1 and phd1. In the inverse experiment, these factors were able to induce FL011 expression in an rme1 deleted strain. This would indicate that Rme1 p does not function in a hierarchical signalling system with these factors, but could function in a more general role to modify transcription.
AFRIKAANSE OPSOMMING: Die natuur is hoogs veranderlik en alle organismes, insluitende gis, moet by die omgewing kan aanpas om te kan oorleef. Baie eksterne faktore beïnvloed die ontwikkeling van die gissel. Vir die gis om by spesifieke omgewingstoestande aan te pas, moet die gis op 'n molekulêre vlak: (i) al die omgewingsparameters waarneem; (ii) die waargenome omgewingsparameters as seine na die selkern deur middel van seintransduksieweë gelei; en (iii) transkripsie van gene aktiveer of onderdruk en ensiemaktiwiteit reguleer om sodoende die gepaste molekulêre respons te implementeer. Die beskikbaarheid van voedingstowwe in die omgewing is een van die belangrikste omgewingseine wat eensellige organismes moet kan waarneem. Saccharomyces cerevisiae kan spesifieke ontwikkelingsopsies, na gelang van die voedingstowwe wat beskikbaar is, uitoefen. In groeiomstandighede waar daar 'n oorvloed van voedingstowwe is, verdeel S. cerevisiae d.m.v. mitose en vesprei dit deur die omgewing. Sodra die voedingstowwe uitgeput is, word mitose onderdruk. Diploïede S. cerevisiae inisieer meiose, wat aanleiding tot die vorming van vier spore gee. Hierdie spore bevat slegs die helfte van die ouer se chromosome en kan gevolglik met 'n ander spoor paar om weer 'n diploïede gissel te vorm. Die spore is bestand teen strawwe omgewingstoestande en kan vir lang tye oorleef. Wanneer die spoor aan gunstige groeitoestande blootgestel word, ontkiem dit om aan 'n nuwe giskolonie oorsprong te gee. S. cerevisiae het egter 'n derde ontwikkelingsopsie, naamlik pseudohife-differensiëring. Wanneer die beskikbaarheid van voedingstowwe in die omgewing afneem, maar nog nie uitgeput is nie, ondergaan die gis 'n morfologiese verandering. Hierdie verandering word gekenmerk deur selverlenging, nl. botselle wat slegs aan die een punt van die gissel vorm en dogterselle wat aan die moerderselle geheg bly. Dit lei tot die vorming van kettings van selle wat van die giskolonie af weggroei. Voorts kan die selkettings ook die groeisubstraat binnedring. Dit staan as penetrasie-groei bekend en laat die gis toe om na nuwe voedingsbronne te soek. Hierdie studie het op die elemente van seintransduksieweë, wat by penetrasiegroei betrokke is, gefokus. Sekere komponente van die seintransduksieweë is reeds goed gekarakteriseer, terwyl ander komponente nog grootliks onbekend is. In hierdie studie, word 'n rol vir RME1 in die verbetering van styselafbraak en penetrasiegroei geïdentifiseer. Aangesien Rme1 p voorheen as 'n onderdrukker van meiose geïdentifiseer is, is 'n litetaruurstudie oor die inisiasie van meiose saamgestel. Die faktore wat meiose induseer, naamlik 'n gebrek aan voedingstowwe en die sel se ploïedie, word bespreek. Die regulering van die meester inisieerder van meiosie, IME1, asook die proteïene waarmee Ime1p reageer, is ook in die studie ingesluit. Die eksperimentele deel van die studie behels die genetiese analise van Rme1 p tydens penetrasiegroei en styselhidroliese. 'n Epistase-analise tussen Rme1 p en elemente van die MAP-Kinasemodule, asook van die transkripsie faktore Mss11 p, Msn1p/Mss10p, Tec1p, Phd1p en F108p, is onderneem. Rme1p is bekend om aan die promotor van CLN2 te bind en transkripsie te induseer. Daarom is die selsikliene CLN1 en CLN2 in die studie ingesluit. Die studie dui daarop dat Rme1 ponafhanklik van die MAP-Kinasemodule funksioneer en nie Cln1 p en Cln2p benodig om penetrasiegroei te induseer nie. FL011/MUC1 kodeer vir 'n selwandproteïen wat noodsaaklik vir pentrasiegroei is. Soos in die geval van die bogenoemde faktore, benodig Rme1 p FL011 om penetrasiegroei te kan induseer. Ten spyte van mss11-, msn1-, ttos-, tec1- en phd1- delesies, kan ooruitdrukking van Rme1p die transkripsie van FL011 induseer. In die omgekeerde eksperiment kon die bogenoemde faktore FL011-transkripsie ten spyte van 'n rme1 delesie induseer. Die resultate dui daarop dat Rme1 p nie in 'n hiërargiese pad funksioneer nie, maar dat dit waarskynlik 'n meer algemene rol deur transkripsiemodifisering vervul.

O meloeiro (Cucumis melo L.) é uma cultura de grande importância econômica para o comércio de exportação brasileiro e é cultivada principalmente na região Nordeste. A produção da cultura pode ser limitada por uma doença das partes aéreas, denominada oídio, sendo no Brasil, causada pelo fungo Podosphaera xanthii. Este patógeno apresenta diversas raças definidas com base na reação de um conjunto de cultivares diferenciadoras de meloeiro. Dentre estes genótipos, o acesso PI 414723 é resistente à maior parte das raças e a linhagem Védrantais é suscetível. O presente trabalho teve como objetivos: (i) estudar a herança da resistência às raças 1, 3 e 5 de P. xanthii em indivíduos da geração F2 do cruzamento PI 414723 x Védrantais e (ii) mapear os genes de resistência a estas raças com base em marcadores de polimorfismo de comprimento de fragmentos amplificados (AFLP), de repetições de sequências simples (SSR) e análogos de genes de resistência (RGA) também nesta população. A herança da resistência foi analisada em 87 indivíduos F2 cultivados em condições de casa-de-vegetação. As três raças foram inoculadas em seis regiões eqüidistantes da nervura central em quatro folhas de cada planta. Plantas foram classificadas como resistentes ou suscetíveis com base em avaliações visuais do desenvolvimento do fungo nas folhas. As plantas foram classificadas como suscetíveis quando houve reprodução abundante de conídios e resistentes quando a reprodução foi inexistente ou escassa. Frequências de indivíduos resistentes e suscetíveis indicaram que a resistência às três raças é controlada por um gene dominante de efeito maior. Um mapa genético foi construído compreendendo 1.469 cM, consistindo de 207 marcadores (139 AFLP, 47 SSR, 18 RGA e três fenotípicos) e com uma distância média de 7,4 cM entre marcadores distribuídos em 12 grupos de ligação. Análises de co-segregação com marcadores indicaram que os genes de resistência estão localizados no grupo de ligação II. Em adição a isto, as análises indicaram ligação completa entre os genes de resistência às raças 1 e 5, sendo este gene denominado Pm-x1.5. Já o gene de resistência à raça 3 (Pm-x3) foi localizado a 5,1 cM dos demais. Um marcador AFLP (H35M75_156) foi localizado entre os dois genes a 1,3 cM de Pm-x1.5 e 3,8 cM de Pm-x3. Este é o primeiro relato da localização genética de genes de resistência às raças 3 e 5 em PI 414723 e também o primeiro relato do mapeamento de marcadores RGA gerados pela técnica TRAP em meloeiro.
Melon (Cucumis melo L.) is a crop of great economic importance for the export trade in Brazil and is cultivated mainly in the Northeast. Crop yield can be affected by a disease of the aerial parts, called powdery mildew that in Brazil is caused by the fungus Podosphaera xanthii. This pathogen has several races characterized based on the reaction of a set of differential melon cultivars. Among these genotypes, the plant introgression PI 414723 is resistant to most races and the breeding line Védrantais is susceptible. This study aimed to: (i) study the inheritance of resistance to races 1, 3 and 5 of P. xanthii in the F2 generation from the cross PI 414723 x Védrantais, and (ii) map resistance genes to these races in this same population based on amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR) and resistance gene analog (RGA) markers. The inheritance of resistance was analyzed on 87 F2 individuals grown under greenhouse conditions. The three races were inoculated simultaneously on four leaves of each plant. Plants were classified as resistant or susceptible based on visual assessments of fungal growth on the leaves. Plants were considered susceptible when there was abundant production of conidia and resistant when the production was scarce or non-existent. The frequencies of resistant and susceptible individuals indicated that resistance to all three races is controlled by a dominant major gene. A genetic map was constructed comprising 1469 cM, consisting of 207 markers (139 AFLP, 47 SSR, 18 RGA, and three phenotypic) with an average distance of 7.4 cM between markers distributed in 12 linkage groups. Co-segregation analysis with markers indicated that the resistance genes are located on linkage group II. Moreover, the analysis indicated complete linkage between resistance to races 1 and 5, and this gene was denominated Pm-x1.5. The gene for resistance to race 3 (Pm-x3) was located at 5.1 cM from Pmx1.5. An AFLP marker (H35M75_156) was located between the two genes at 1.3 cM from Pmx1.5 and 3.8 cM from Pm-x3. These is the first report on the location of resistance genes to races 3 and 5 in PI 414723, and also the first report of RGA markers mapping using the TRAP technique in melon.

Les Penicilliums sont des champignons filamenteux appartenant au genre Ascomycota. Ces champignons ont été utilisés par l’homme pour la production de nourriture depuis des siècles. Plus récemment, ils ont aussi été utilisés dans l’industrie biotechnologique pour la production de composés chimiques d’intérêts pharmaceutiques. Certaines espèces de Penicillium sont par ailleurs des moisissures contaminants certains aliments, d’autres sont des pathogènes de plantes, y compris de certains fruits. Leur génomique est globalement peut connue. Dans cette étude, nous avons analysé les génomes de deux espèces nouvellement séquencées, Penicillium roqueforti et Penicillium camemberti. Nous reportons ici le développement d’une nouvelle méthodologie pour l’amélioration et la validation d’assemblage de génomes en utilisant une technologie permettant l’observation de molécules d’ADN unique, le Peignage Moléculaire. En utilisant cette méthode, nous avons amélioré l’assemblage de Penicillium roqueforti. Ce manuscrit décrit aussi de multiples occurrences d’un transfert horizontal d’un ilot génomique de plus de cinq cent kilobases entre plusieurs Penicillium. Ce cas de transfert horizontal indique une fréquence d’échange latéral de matériel génétique plus forte qu’attendue. Enfin nous présentons un inventaire préliminaire du potentiel génomique pour la production de métabolites secondaires dans ces importants Penicillium alimentaires
Penicillium are filamentous fungi belonging to the Ascomycota genus. Penicillium species have been used by Man for centuries in food making processes. More recently they have also been used in the biotechnology industry for the production of compounds of pharmaceutical interest. Some Penicillium species are food spoilage agents, pathogens of plants including fruits. Aspects of their genomics are largely unknown. In this study, we analysed the genomes of two newly sequenced species, Penicillium roqueforti and Penicillium camemberti. Here we report the development of a new methodology for improving and validating genome assembly using an original single DNA molecule technology, Molecular Combing. Using this methodology we were able to produce a high quality genome assembly of Penicillium roqueforti. This work also reports the multiple and recurrent horizontal transfer of a large genomic island of over half a megabase between several Penicillium species. This horizontal transfer indicates a higher frequency of lateral genetic exchange between cheesemaking fungi than previously expected. Finally, we present an early assessment of the genomic potential for secondary metabolite production in these important food associated penicilliums

No presente trabalho, foi utilizada uma coleção de 14 isolados de Sporisorium scitamineum coletados em diferentes regiões canavieiras, para estudar a diversidade genética por RFLP da região telomérica de maneira comparativa a marcadores AFLP. Os teliósporos, esporos de resistência do fungo, foram coletados a partir do sintoma mais característico da planta infectada que é formação do chicote. Os teliósporos são diplóides e quando germinam dão origem ao probasídio, onde, por meiose formam-se os esporídios haplóides. Estes quando compatíveis sexualmente voltam a se fundir formando um micélio dicariótico infectivo. A fase do ciclo de vida escolhida para as análises foram os derivados haplóides de cada linhagem dicariótica obtida a partir dos teliósporos. Na técnica de AFLP foram encontrados 40 loci polimórficos (3%) entre 1311 analisados obtidos a partir de 2 enzimas de corte raro, 1 de corte frequente e 19 combinações de primers. A técnica de RFLP da região telomérica foi comparativamente mais eficiente, no qual foram utilizadas três enzimas de restrição que geraram 102 loci, sendo 36 polimórficos (34,3%). O agrupamento com base nos coeficientes de similaridade e os resultados de atribuição pelo programa Structure revelaram dois grupos genotípicos homogêneos, tanto quando os marcadores foram analisados separadamente como na análise conjunta. Não houve agrupamento por localidade, mas ficou claro que o fluxo desses isolados é baixo. Os derivados haplóides de tipos de reação sexual opostos de cada linhagem dicariótica (teliósporo) permaneceram dentro do mesmo grupo, com exceção de uma única linhagem. Desta forma, de maneira geral na natureza, a fusão entre os esporídios deve acontecer entre aqueles que estão mais próximos. Uma análise molecular de diversidade genética em isolados obtidos em diferentes regiões do mundo por outros autores revelou uma alta homogeneidade entre eles, de forma que somente em localidades da Ásia foi possível revelar, com os marcadores utilizados, alguma diversidade genética. Nossos resultados indicam que a escolha do marcador foi fundamental para revelar diversidade entre os isolados de S. scitamineum, sendo que o RFLP-tel revelou um fingerprinting de DNA quase que específico para cada isolado, sendo assim mais apropriado do que os descritos anteriormente. A quantidade de loci AFLP necessária para revelar polimorfismo foi mais alta do que para RFLP-tel. Uma segunda contribuição deste trabalho foi a detecção de heterozigotos quando consideramos a análise conjunta de derivados haplóides por teliósporo. Um alto grau de homozigosidade foi detectado quando as análises foram realizadas considerando o comportamento dicariótico como diplóide, no entanto, loci heterozigotos puderam ser encontrados. Esta é a primeira vez que um estudo desta natureza foi realizado com isolados de Sporisorium scitamineum do Brasil.
The genetic diversity of Sporisorium scitamineum, the sugarcane smut agent, was characterized in a 14 isolates collection. The isolates were obtained from various sugarcane growing areas and RFLP of the telomeric region was used as molecular marker, compared with AFLP. Teliospores were collected from the whip of infected plants. Teliospores are diploids and germinate producing a probasidium where meiosis takes place originating four haploid cells. These cells if sexually compatible can fuse and a dikaryotic mycelium is formed. The haploid phase, derived from each dikaryotic line obtained from teliósporos, was used to perform the analysis. Our results showed that 40 polymorphic loci (3%) were described among 1,311 analyzed. These were obtained with two rare-cutter restriction enzymes, one frequent-cutter restriction enzyme and 19 primers combinations. The RFLP markers were more efficient when compared to AFLP to reveal polymorphisms. Three restriction enzymes produced 102 loci, from which 36 were polymorphic (34.3%). Clustering using similarity coefficients and results obtained by Structure software revealed two genotypic groups. The analyses were performed with individual markers and combining RFLP and AFLP markers. Locations were not responsible for clustering, and low flux of isolates was evident. The opposite sexual types haploid derivatives originated from the same teliospore were clustered together, with only one exception. This implies that sporideos that are located close together are more likely to fuse. Our data suggest that choosing RFLP as markers were the key for unrevealing diversity among isolates of S. scitamineum. RFLP-tel revealed almost unique DNA fingerprintings to various isolates. A second contribution of this work was that heterozygous were detected when considering a combined analyses of haploids derivatives from the same teliospore. This is the first time a study of this nature was organized with Brazilian isolates of S. scitamineum.

My dissertation research examined the effect of the cultivation of insect-resistant Bacillus thuringiensis (Bt) maize on the soil environment with a goal of understanding how to obtain a balance between technological advancement and maintenance of a healthy soil ecosystem. Although Bt plants may help to reduce pesticide use, conferring benefits to farm workers and the environment, there are still unresolved questions about how the cultivation of Bt plants affects soil organisms. For this dissertation project, I used 14 different genotypes of Bt maize and non-Bt maize (Zea mays) to investigate the effects of transgenic Bt plants on the colonization ability, abundance, and diversity of symbiotic arbuscular mycorrhizal fungi (AMF) in the soil ecosystem over time. My greenhouse studies demonstrated that Bt maize plants exhibited reduced AMF colonization across multiple Bt genotypes and that effects were most pronounced when fertilizer levels were limited and spore density was high. In addition, I found that although differences in AMF colonization between Bt and non-Bt maize were difficult to detect in the field, spore density was reduced in Bt field plots after just one growing season. When I tested the effect of plot history on AMF and plant growth, I found that Bt and non-Bt maize plants had higher leaf chlorophyll content when grown in plots previously cultivated with the same maize line as the previous year, indicative of a positive feedback effect. I also examined potential mechanisms contributing to the reduced AMF colonization observed in Bt maize in greenhouse studies and determined that follow-up experiments should continue to investigate differences in root apoplastic invertase activity and root permeability in Bt and non-Bt maize. Future investigations would also benefit from examining potential differences in root exudate profiles and volatile organic compounds between Bt and non-Bt cultivars. Taken together, my dissertation results suggest that, while difficult to detect in the field, reductions in AMF colonization in Bt maize roots may be ecologically significant as they could lead to a decrease in the abundance of AMF propagules in the soil over time, potentially impacting soil structure and function in areas where Bt crop cultivation is high.

Thesis by publication.
"August 2006"
Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences & Dept. of Chemistry & Biomolecular Sciences), 2007.
Bibliography: leaves 157-183.
1. Introduction -- 1.1. Proteomics and two-dimensional electrophoresis -- 1.2. A proteomic approach to study the filamentous fungus Trichoderma -- 1.3. Aims of the thesis -- 2. Materials and methods -- 3. Results and discussion -- 3.1. Method development for the display and identification of fungal proteins by 2DE and mass spectrometry -- 3.2. Discovery of novel determinants in the biological control of phytopathogens by Trichoderma atroviride -- 3.3. Summary and concluding remarks.
Trichoderma harzianum and T. atroviride are filamentous fungi commonly found in soil. Both display biocontrol capabilities against a range of phytopathogenic fungi including Rhizoctonia solani and Botrytis cinerea which are known pests of hundreds of commercially important crops including tomatoes, potatoes, beans, cucumber, strawberries, cotton and grapes. These Trichoderma species secrete a combination of enzymes degrading cell walls and antibiotics to overgrow and kill fungal phytopathogens. They are seen as an environmentally friendly alternative to chemical fungicides currengly used on crops.
A proteomic approach was taken to separate and identify proteins from a strain of T. harzianum with well established biocontrol properties. Several methods were developed in this thesis to display the whole proteome content and several subcellular proteome fractions from T. harzianum. Proteins were separated by two-dimensional electrophoresis and identified by mass spectrometric methods. The resulting proteomic maps represent the first extensive array of cellular and sub-cellular proteomes for T. harzianum.
Cellular protein patterns of T. atroviride (T. harzianum P1) grown on media containing either glucose or R. solani cell walls were compared by differential gel electrophoresis to identify a suite of new proteins involved in the biological control response. Twenty four T. atroviride protein spots up-regulated in the presence of the R. solani cell walls were identified by mass spectrometry and N-terminal sequencing. Proteins identified from this study included previously implicated enzymes degrading cell walls and three novel proteases, vacuolar serine protease, vacuolar protease A and trypsin-like protease. The genes encoding two of these proteases, vacuolar protease A and vacuolar serine protease have been cloned by degenerate primer PCR and genomic walking PCR and sequenced. The gene sequences and protein sequences derived from these genes have been partially characterised.
Mode of access: World Wide Web.
194 leaves ill

Indiana University-Purdue University Indianapolis (IUPUI)
Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.

碩士
國防醫學院
病理及寄生蟲學研究所
90
Superficial pathogenic fungi are a group of fungi responsible for the infection of skin or superficial mucosa, and dermatophytes and some Candida spp. constitute the majority of the pathogen. In order to survey the current prevalence of superficial pathogenic fungi in northern Taiwan and Kinmen, and set up a supplement method for identification, this study was performed by using molecular diagnostic method. Between August 2001 and April 2002, 153 specimens were collected from 118 patients of dermatomycoses in northern Taiwan and Kinmen, and 81 specimens were collected from 61 patients of vaginitis in northern Taiwan between December 2001 and April 2002. Totally, 154 isolates obtained from the specimens of dermatomycoses and 35 strains from the specimens of vaginitis were identified. In the specimens of ill persons suffering from dermatomycoses, dermatophytes ( 40.3% ) were the most fungi identified, followed by moulds ( 35.7% ). In the specimens of ill persons suffering from vaginitis, yeasts ( 91.4% ) were the most organisms identified. The most common dermatophytes isolated were T. rubrum and Trichosporon spp. in nondermatophytes. The most common fungi identified in vaginitis were C. albicans ( 82.9% ), followed by C. glabrata ( 5.7% ). The overall positive rate of dermatophytes found in dermatomycoses was 58%. Mixed fungal florae have been demonstrates in some clinical specimens. For patients suffering from dermatomycoses, pure cultures from the different lesions of the same case could be obtained from only 15.2% ( 5/33 ) of cases. The kinds of species identified in Kinmen were more than those in northern Taiwan, but the overall positive rate of dermatophytes in northern Taiwan was higher than that in Kinmen.

Under storage conditions of ambient temperature and relative humidity in South Africa, seed-associated mycoflora proliferates. Fusarium moniliforme is ubiquitous in newly-harvested maize, persisting for variable periods in storage, while Aspergillus flavus may represent the final group of species in the succession of aspergilli after grain storage under high temperature and/or high humidity. Many strains of these fungi produce toxigenic secondary metabolites (mycotoxins) under local storage conditions. Since pathogenic fungi may be present within the tissues of stored seeds, these contaminants will not be eradicated by external fungicide treatment, therefore a possible alternative is biological control. The aim of the present investigation was to ascertain whether certain strains and/or species of Trichoderma have potential as biocontrol agents against the seed-associated pathogenic fungi, Aspergillus flavus and Fusarium moniliforme. A study of the fungal growth in dual cultures revealed that from nine isolates of Trichoderma spp. (T harzianum and T viride), four had a noticeable inhibitory effect on the growth of the pathogenic fungi. Scanning electron microscopical investigation of fungal interaction demonstrated no obvious hyphal penetration by - Trichoderma spp. In addition, significant alteration of Fusarium hyphae, with pronounced collapse and loss of turgor, and production of aberrant conidial heads and microheads by A. flavus were observed. Evidence derived from some biochemical studies revealed that antibiosis (by production of extracellular enzymes, volatile compounds and possible antibiotics) is probably the mechanism involved in the antagonistic effect of the four aggressive Trichoderma spp. The in vitro studies demonstrated that the use of Trichoderma spp. as biocontrol agents against A. flavus and F. moniliforme appears promising.
Thesis (M.Sc.)-University of Natal, 1995.

M.Sc.
Polygalacturonase-inhibiting proteins (PGIPs) are cell wall-associated plant proteins that inhibit endopolygalacturonases from phytopathogenic fungi. It has been proposed that pgip encoding genes could be utilised for engineering increased resistance in transgenic crops against important fungal pathogens such as Botrytis cinerea. During this study a pgip gene from Malus domestica cv Granny Smith apple fruit was cloned by the degenerate and inverse polymerase chain reaction (PCR) techniques. An alignment of the pear and bean PGIP sequences was used to design degenerate PCR primers in highly conserved regions. Degenerate PCR allowed the amplification of a 351bp internal fragment of the pgip gene, termed ipgip. The DNA sequence of ipgip was used to design inverse PCR primers. A Southern blot of apple genomic DNA probed with the ipgip fragment was used to identify restriction enzyme sites for inverse PCR. Inverse PCR enabled cloning of the remainder of the gene, from which a composite pgip gene sequence was constructed. The composite apple pgip gene comprised an open reading frame of 990bp that is predicted to encode a 330 amino acid polypeptide. The polypeptide contains a putative 24 amino acid N-terminal leader sequence that may function as a signal peptide for secretion. The deduced apple PGIP contains nine cysteine residues and seven potential N-linked glycosylation sites. Ten loosely conserved leucine-rich repeat motifs characteristic of PG1Ps were identified in the apple PGIP sequence. The apple PGIP showed 97% and 55% amino acid identity to the pear and bean PGIPs, respectively. The full-length apple pgip gene was re-isolated from genomic DNA by PCR using primers designed to the 5' and 3' ends of the composite pgip gene. The apple pgip gene was cloned into a plant transformation vector and transformed into tobacco by Agrobacterium-mediated transformation. Phenotypically normal transgenic tobacco plants were produced. Stable transgene insertion into the transgenic tobacco genomes was verified by PCR and Southern blot analyses. Sequence analysis of the pgip construct used for transformation revealed two potential mutations in the deduced amino acid sequence. The substitutions of Asp residues with Asn and Tyr at positions 43 and 196, respectively, could interfere with the secondary structure of the expressed transgene protein. To test whether the apple PGIP was effective against Botrytis cinerea, protein extracts were prepared from apple fruit and transgenic tobacco and tested for inhibitory activity against B. cinerea polygalacturonases. Biochemical assays showed that a heat-denaturable PGIP extract prepared from apple fruit inhibited the polygalacturonases produced by a virulent isolate of Botrytis cinerea grown on pectin and apple cell walls. Protein extracts prepared from transgenic tobacco did not show any inhibitory activity towards Botrytis polygalacturonases. This suggests the absence of active PGIP in the extracts possibly due to inefficient transcription of the transgene or due to the introduced mutations.

Tese de doutoramento em Biologia (Biologia Molecular), apresentada à Universidade de Lisboa através da Faculdade de Ciências, 2007
Grapevine (Vitis) species are the most economically important fruit crop worldwide but they are prone to several diseases, being fungi pathogens the major problem of its cultivation around the world. Vitis vinifera response against pathogens is a complex process that differs within cultivars. The mechanisms that enable the plant to reduce the disease incidence are not fully understood. A better knowledge of the complexity of the resistant capability of cultivars, such as Regent, is determinant for an overall understanding of the resistance mechanism and for the definition of improvement strategies for highly susceptible crop species. Transcript and metabolic profiling of in-field growing Regent (resistant) and Trincadeira (sensitive) cultivars were achieved through cDNA microarray and 1HNMR. Plants were collected prior to flowering, this way transcript and metabolic differences between both cultivars revealed their behaviour for the same environmental conditions. The results from the present work suggest that Regent has an intrinsic resistance capability. Transcripts coding for a tonoplast intrinsic protein, J2P and several transcripts associated with defence were found up regulated in Regent, suggesting higher resistance to stress. The regulation of transcripts coding for phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase and WD-repeat protein-like also suggests a constitutive accumulation of phenolic compounds in Regent. The regulation of transcripts coding for TIP suggest increased resistance to drought stress. Regent cultivar presents higher abundances of myo-inositol, glutamine, glutamate, alanine, and caffeic acid derivative which could be associated with its inherent resistance to fungi presented. Myo-inositol and alanine could act as stress signalling molecules, enabling a faster response in the case of pathogen attack. High glutamine abundance could suggest increased activity of PAL and phenylpropanoid metabolism. Caffeic acid derivatives may be related to the resistance response after pathogen attack, since constitutive phenolic compounds are known to confer resistance, either directly or indirectly, through activation of post-infection responses.
A cultura da videira é uma actividade de importância económica mundial, com grande relevância em Portugal. Esta cultura é afectada por diversos patógenos, nomeadamente fungos. A videira apresenta mecanismos de defesa complexos que diferem entre cultivares. Estes mecanismos que conferem à planta capacidade de reduzir a incidência das infecções por patógenos são pouco conhecidos. Um maior conhecimento das capacidades de defesa apresentadas por algumas cultivares resistentes como a Regent, é determinante, para o melhoramento de cultivares sensíveis, sobretudo se forem comercialmente importantes. A aplicação de técnicas de biologia molecular e análise química, como os microarrays de cDNA e o 1H NMR à caracterização de transcritos e metabolitos em larga escala, permitiu o desenvolvimento de disciplinas científicas como a transcritómica e a metabolómica que fornecem grande quantidade de dados. Neste trabalho, foram aplicadas técnicas de caracterização do transcritoma e metaboloma o que permitiu a diferenciação entre duas castas de videira, Regent e Trincadeira, que têm sido descritas como variedades resistente e sensível, respectivamente, a patógenos. Foram analisadas folhas de ambas as castas de videira, colhidas no campo antes da formação de inflorescências. As diferenças encontradas na expressão de transcritos e na abundância de metabolitos refletem a resposta das duas castas às mesmas condições ambientais no campo. Para a comparação da expressão génica foi construída uma biblioteca de cDNA utilizando folhas de cada uma das castas Trincadeira e Regent. Para tal, foi desenvolvido um novo método para a síntese da biblioteca de cDNA, baseado na técnica de PCR. Este método inovador conjuga o melhor de duas das tecnologias líder na área, nomeadamente a recombinação e a troca de template pela transcriptase reversa. Este protocolo permitiu a eliminação dos passos de digestão e sub-clonagem, que são normalmente necessários, e a obtenção rápida de clones de cDNA para a aplicação em microarrays. Foram comparados os transcritomas foliares de ambas as castas, e os transcritos que apresentaram uma variação estatísticamente significativa foram analisados. Após sequenciação foram identificados transcritos relacionados com a fotossíntese, produção de energia, stress e defesa, regulação da transcrição e tradução, regulação da expressão génica nos cloroplastos, transporte celular e modulação proteica. Os metabolitos identificados a partir da análise do metaboloma, e cuja abundância permite a diferenciação das duas castas, são o mio-inositol, a glutamina, o glutamato, a alanina, derivados do ácido cafeico, a glucose, o ácido succínico e um composto fenólico de identidade desconhecida. Os resultados deste trabalho sugerem que a casta Regent é intrinsecamente mais resistente do que a casta Trincadeira. Esta característica poderá estar relacionada com a activação de genes relacionados com processos de defesa e que codificam para proteínas como a phenylalanine ammonia lyase, S-adenosyl methionine synthase, subtilisin -like protease, WD-repeat protein like, tonoplast intrinsic protein e J2P. A maior abundância de metabolitos como o mio-inositol, glutamina, glutamato, alanina e derivados do ácido cafeico, metabolitos que são normalmente produzidos após o ataque por fungos patogénicos, vem também confirmar a maior resistência verificada na casta Regent. A sobre-expressão de transcritos codificantes de uma subtilisin like protease pode ser relevante quando a casta Regent é infectada por um patógeno, visto que estas proteínas participam na regulação de processos biológicos como o reconhecimento de agentes patogénicos, com subsequente activação de respostas de defesa. A sobre-expressão de transcritos codificantes de uma S-adenosyl-L methionine synthase pode estar relacionada com uma maior produção na casta Regent de S-adenosyl methionine, sendo que esta molécula pode ser percursora ou participar na biossíntese de classes de fenilpropanóides e poliaminas, compostos que participam em mecanismos de defesa. Por outro lado, a sobreexpressão de transcritos que codificam uma phenylalanine ammonia lyase sugere uma maior acumulação de compostos fenólicos. A sobre-expressão de transcritos que codificam uma WD-repeat protein –like pode estar também associada a uma maior acumulação de compostos fenólicos nesta casta. Uma maior acumulação de derivados do ácido cafeico pode estar relacionada com a regulação da phenylalanine ammonia lyase e, provavelmente, com a regulação da S-adenosyl-L methionine synthase. A acumulação de derivados do acído cafeico na Regent sugere uma maior resistência desta casta uma vez que a ocorrência constitutiva de compostos fenólicos está relacionada com a formação de barreiras químicas ao estabelecimento e propagação da infecção e pode proteger a planta em caso de ataque de patógenos. A acumulação de compostos fenólicos é reconhecida por conferir uma maior resistência directamente ou indirectamente pela activação de respostas de defesa. Por outro lado, a acumulação de transcritos que codificam uma tonoplast intrinsic protein pode estar relacionada com uma maior capacidade de resposta em caso de stress hídrico, levando a uma maior resistência. O mio-inositol pode actuar como uma molécula de sinalização, permitindo uma rápida resposta de defesa em caso de ataque de um patógeno. Por um lado, a glutamina pode estar directamente relacionada com uma maior actividade da phenylalanine ammonia lyase, uma vez que ajuda a célula a reciclar os iões amónio da fenilalanina. Por outro lado, o glutamato é um precursor de duas moléculas de resposta a stress, nomeadamente a prolina e a glutationa. A alanina, cuja acumulação tem sido detectada em resposta a diversos stresses, pode funcionar como uma molécula sinalizadora induzindo uma rápida resposta de defesa. A casta Trincadeira é uma casta muito sensível a infecções por patógenos. Esta casta apresentou uma sobre-expressão de transcritos relacionados com a fotossíntese, de transcritos associados com o mecanismo antioxidante e de transcritos codificantes para a NADH dehydrogenase subunit A, sugerindo que, para as mesmas condições ambientais, esta cultivar apresenta um menor controlo da regulação fotossintética relativamente à cultivar Regent. Desta forma, necessita da acção do sistema anti-oxidante para controlar os radicais livres de oxigénio que se formam nas reacções fotossintéticas prevenindo assim os danos foto-oxidativos. Esta casta apresenta também uma maior acumulação de glucose e ácido succínico e uma sobre-expressão de transcritos codificantes para uma glyoxysomal NAD-malate dehydrogenase e para um putativo factor de transcrição relacionado com processos de desenvolvimento em plantas tais como o desenvolvimento dos frutos e a senescência. Este factor de transcrição apresenta elevada homologia com o gene VvMSA, que controla a expressão de um transportador de hexoses o qual possui elevada afinidade para a glucose. A elevada acumulação de glucose nesta casta sugere, por um lado, um elevado transporte de glucose para as folhas. Por outro lado a sobre-expressão de um transcrito codificante para uma glyoxysomal NAD-malate dehydrogenase e a elevada acumulação de ácido succínico e glucose sugerem uma maior conversão de lípidos em carbohidratos. Os resultados deste trabalho permitem formular a hipótese de que as plantas da casta Regent são intrinsecamente mais resistentes ao stress. A sua resistência a patógenos, nomeadamente fungos, poderá estar relacionada com a presença constitutiva de compostos fenólicos e com uma rápida indução da resposta de defesa quando em contacto com um patógeno.
Fundação para a Ciência e a Tecnologia (FCT, SFRH / BD / 12403 / 2003)

Willow (Salix spp.), a major source of biomass and renewable fiber production, is one of the best choices for short-rotation intensive culture (SRIC) in Canada. Since fungal communities play important roles in the plant’s health status, it is vital to understand their interactions with willows and their roles in the sustainability of SRIC. In this study, fungal diversity of the above-ground organs (stem/leaf) of healthy and diseased willow plants in western Canadian Prairies were assessed using cultural and PCR-denaturing gradient gel electrophoresis (DGGE) techniques. Comparison of the mycoprofiles within established plantations vs. newly introduced cuttings revealed differences in the fungal communities. Ascomycota were mainly isolated, followed by Basidiomicota and Zygomycota. Willow genotypes seem have an influence on the abundance of fungal pathogens and disease severity; among them Charlie (Salix alba x gladfelteri) and SV1 (S. eriocephala) cultivars demonstrated superior performances. Photosynthesis measurements and biomass compositions confirmed these findings. Potentially pathogenic fungi (Dothioraceae, Diaporthaceae, Glomeraceae, and Pleosporaceae) dominated in diseased or symptomatic willows, whereas potentially beneficial fungi (Coniochaetaceae, Hypoceraceae, Nectriaceae, Trichocomaceae, and Agaricaceae) prevailed in healthy plants. In-vivo and greenhouse assays showed that inoculation with potentially pathogenic fungi induced leaf necrosis, anthracnose and open cankers. However, suppression of the latter was still possible using fungal antagonists. Hence, assessment of stem/bark and leaf fungal communities with respect to willow genotypes, cuttings origin, and SRIC location, is useful for the design of an effective management strategy to increase the productivity of the SRIC-biomass systems.

In eukaryotes, the defined loci on each chromosome, the centromeres, accomplish the critical task of correct cell division. In some organisms, centromeres are composed of a euchromatic central core region embedded in a stretch of heterochromatin and the inheritance and maintenance of centromeres are controlled by dynamic epigenetic phenomena. Although the size of centromeres differs between organisms, its organization, and the placement of euchromatic and heterochromatic regions is conserved from the fission yeast, Schizosaccharomyces pombe, to humans, Homo sapiens. However, relatively little is known about centromeres in the filamentous fungi from the Ascomycota, representing the largest group of fungi and fungal pathogens. Further, studies from humans, flies, yeast and plants have shown that the inheritance of centromeres is not strictly guided by centromeric DNA content, which is highly AT-rich, repetitive and constantly evolving. Therefore, it is difficult to align ans assemble the sequenced contigs of centromeric regions of higher eukaryotes, including most filamentous fungi. A genetic technique, tetrad (or octad) analysis has helped to map the centromeres of the filamentous fungus Neurospora crassa early on. The research presented in this dissertation used N. crassa as a model to focus on characterizing different features of centromeres with an emphasis on the centromere-specific histone H3 (CenH3) protein. Data included here represent the first study on centromere-specific proteins in Neurospora, and demonstrate that the central core of the centromeres are heterochromatic, showing enrichment of silent histone marks, which is in contrast to the centromere arrangement in fission yeast. The CenH3 protein, whose deposition on the genome licenses formation or maintenance of centromeres, shows highly divergent N-terminal regions and a conserved histone fold domain (HFD) in all eukaryotes. This bipartite nature of CenH3 is also observed in the Ascomycota, which provides an opportunity for functional complementation assays by replacing Neurospora CenH3 (NcCenH3) with CenH3 genes from other species within the Ascomycota. The results from this experimental approach provide good measures for (1) determining the specific regions of CenH3 required for the assembly of centromeres during meiotic and mitotic cell divisions and (2) analyzing the resistance to changes in the organization of centromeres in N. crassa. The genetic analysis showed that the divergent N-terminal region is essential for the proper assembly of centromeres, and that the conserved carboxy-terminus of CenH3 is important for the process of meiosis but not mitotic cell division. ChIP-seq analyses suggest that the observed loss of Podospora anserina CenH3 (PaCenH3- GFP) from certain N. crassa centromeres does not result in obvious phenotypic defects, e.g. diminished growth or evidence for aneuploidy. Further, the low enrichment of PaCenH3-GFP at certain centromeres is possibly predetermined during meiosis, which results in irreversible and progressive decreases in enrichment. It remains to be determined if this process is random as far as selection of centromeres is concerned. Together the results presented here suggest that during meiosis more stringent structural requirements for centromere assembly apply and that these are dependent on CenH3, and that depletion of CenH3 from centromeres does not critically affect mitosis in the asynchronously dividing nuclei of Neurospora hyphae.
Graduation date: 2013

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach.
Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008