<p dir="ltr">Romaine lettuce is a vegetable crop that is frequently contaminated with and often implicated in mass outbreaks of human-pathogenic <i>Escherichia coli</i>. To date, research has focused on the specific pathogens responsible for those outbreaks and consumer- and retail-level interventions to eliminate those pathogens or to mitigate their spread. However, in many cases, no singular food source can be identified. Food microbiomes, particularly those associated phylloplanes, are a growing interest area and may be key in identifying the conditions that allow for proliferation of pathogenic organisms like <i>E. coli</i> O157:H7. Further, examination of dominant components of the lettuce microbiome may reveal suitable candidates that can be encouraged or engineered to outcompete or indicate the presence of pathogens. Until recently, most microbiome studies focused on bacteria; less understood are the fungi of these microbiomes. Many fungi have myriad applications in the prevention and mitigation of both human and plant diseases. Thus, to effectively prevent pathogenic <i>E. coli </i>outbreaks, a fundamental understanding about the fungi that cohabit the lettuce microbiome is paramount. The most frequently isolated yeast in the romaine lettuce microbiome is an undescribed yeast, revealing the dearth of information regarding nonpathogenic fungi in food systems. First, the novel yeast <i>Sporobolomyces lactucae</i> is described using a multi-locus phylogeny through genealogical gene concordance, with a discussion on the potential ecological range. Second, the genome of <i>S. lactucae </i>strain HU9244 is assembled and annotated with transcriptomic information to help guide target gene selection for biotechnology, particularly in identifying candidates for reporter genes that may assist in the detection of <i>E. coli </i>O157:H7 via olfactory or visual cues. Third, the distribution of the yeast <i>S. lactucae</i> is determined, finding the organism in various climate types and potentially on other leafy greens as well, indicating its suitability for <i>in situ</i> detection of foodborne pathogens such as <i>E. coli</i> O157:H7 commonly found on romaine lettuce and other leafy greens.</p>

Arbuscular mycorrhizal (AM) fungi are ubiquitous symbionts of terrestrial plant species with associations predominantly characterized as mutualistic. In addition to well-documented enhancement of host growth response, more recent analyses have demonstrated the conferral of host benefits under numerous biotic and abiotic stressors. However, much of the established evidence originates from studies involving limited AM fungal diversity. Accordingly, this study sought to evaluate the potential effects of inoculation on plant host physiological traits within a growth chamber environment, investigate potential correlations between host trait responses, & assess the degree of phylogenetic signal observed in trait responses due to the presence of AM fungi. Overall, inoculation did not result in meaningfully different effects in host trait responses relative to controls. The effects of unique inoculum identity were also not meaningfully different from one another, although some instances of deviation from this trend were observed. Trait correlations were also largely absent after accounting for species relatedness. Further, model selection criteria tended to endorse an effect of unique inoculum identity but was not suggestive of effects due to evolutionary history. The presently described experimental implementation of AM phylogenetic diversity, comprising 36 taxa across 8 families, contributes to a greater contextual understanding of the AM symbiosis and offers an approach suitable for future studies.

<p> Over the last two decades, mycoflorostic studies undertaken in Guyanese forests have uncovered hundreds of new fungal species and genera. One of the recovered fungal families was the Xylariaceae, although most were not identified to species. Members of this family play ecological roles as decomposers, endophytes, and pathogens of vascular plants and grass species.  In addition, this group is increasingly recognized as a significant source of novel metabolic products with potential for applications in medicine, agriculture, and industrial biofuel. Given its  potential, we took a more targeted approach to the documentation of this group. Our sampling  efforts drawing on more than two decades of field collections yielded ca. 90 species in 12 genera, including a putative pathogen, Xylaria karyophthora of the seeds of Chlorocardium spp (Greenheart). Despite the significance of Greenheart to the Guyanese economy, little is known  about the biology and ecology of this fungus. Due to the lack of available resources to study this  fungus, our objectives were two-fold: first, to sequence and annotate the genome of X.  karyophthora to provide a resource for genome-centric explorations, and to use this genome to  infer the biology and ecology of this fungus. We focused on identifying and characterizing  secretomes, viz. carbohydrate-active enzymes (CAZymes) and secondary metabolites  biosynthetic gene clusters (SMBGCs) to infer the nutritional strategy of this fungus. Our results  suggest that X. karyophthora has the capacity to act as both an endophyte and a pathogen. To  make further inferences about the population, we used SSR markers to elucidate the genetic  diversity and population structure of X. karyophthora. X. karyophthora populations have high  genetic diversity, potentially exploiting both outcrossing and inbreeding reproductive strategies, and demonstrate a pattern consistent with human-mediated spread. This work will contribute  information on new species and records of Xylariaceous fungi and their allies from Guyana with  particular emphasis on unraveling the epidemiology, genetic diversity, and population structure  of X. karyophthora </p>

Thesis (MScEng (Process Engineering))--University of Stellenbosch, 2006. / Filamentous fungi have been employed for production of heterologous proteins such as enzymes, antibiotics and vaccines due to their good secretion capacities and effective posttranslational modifications of these proteins. With an improvent in recombinant DNA technologies it has become possible to express many useful proteins in species such as the Aspergilli. However the submerged cultivation of filamentous fungi is complicated by the difficulties in mixing and oxygen and nutrient transfer in the highly viscous culture fluids that result. The purpose of the project was to investigate the potential of simultaneous control of morphology and production of enzymes in the dimorphic fungus, Mucor circinelloides, in order to overcome problems associated with the submerged cultivation of filamentous fungi. Dimorphic M. circinelloides, a zygomycete in the order Mucorales, occurs in a filamentous form or a yeast-like morphology in response to environmental conditions. Recently, advances were made in transformation of Mucor, and it has become possible to transform M. circinelloides to express heterologous proteins. The first example of a strong, regulated promoter from M. circinelloides being used for recombinant protein production was the expression of the glucose oxidase gene (from Aspergillus niger) under the control of the glyceradehyde-3-phosphate dehydrogenase (gpd1) promoter. Glucose oxidase (GOX) is an enzyme used to prevent oxidation of foods to extend shelf-life, to produce low-kilojoule beverages and to measure glucose levels in medical diagnostic applications. The scope of this project was to establish the conditions for yeast and filamentous growth of M. circinelloides in order to allow control of morphology, and to evaluate enzyme production under these conditions. Enzyme production of the GOX producing mutant strain, that was recently constructed, was compared to that of a wild type M.circinelloides strain. M. circinelloides was cultured in two-stage batch fermentations, firstly a yeast stage and then a filamentous stage. The yeast morphology was induced by anaerobic conditions while the filamentous morphology was achieved by exposure to air. The enzyme, biomass and metabolite production of the glucose-oxidase producing mutant strain and the wild type were monitored during the two-stage fermentations. GOX from the mutant and native amylase activity levels from the wild type were compared to each other and to other production systems for these enzymes. The morphology could be maintained in a yeast form under N2 with addition of ergosterol and Tween 80. The GOX activity levels in the culture fluid were comparable to some of the unoptimized GOX production systems in literature, but much lower than the optimized, recombinant GOX production systems that employ certain yeasts, or Aspergilli or Penicillium. The intracellular GOX levels were almost 6-fold higher than the extracellular levels which was unexpected as GOX is usually well-secreted. The morphological control improved the morphology for the initial yeast-stage of the fermentation but did not improve the morphology during the filamentous, enzyme-producing stage and it decreased the biomass yield and enzyme production by 50%. The constraint of Mucor to its yeast-like form did not improve the broth homogeneity or enzyme production and increased the time required for enzyme production. In this study M. circinelloides did not perform that well against other species already used to produce these enzymes. However, M. circinelloides could be used to produce enzymes from zygomycetes that systems such as A. niger do not produce well.

Research regarding the distribution and structure of fungal communities in caves is lacking. The current study examines fungal communities in Kartchner Caverns, a mineralogically diverse cave located in the Whetstone Mountains, Arizona, USA. The first study examines culturable fungal diversity from speleothem surfaces. Twenty-one fungal genera represented by 43 genotypes and 53 distinct morphological taxonomic units (MTU) were recovered from 15 speleothems. Analysis of DGGE profiles indicated a significant effect of sampling site on community structure. The second study examined fungal diversity from speleothem and rock wall surfaces using the 454 FLX Titanium sequencing platform using the rDNA internal transcribed spacer 1 (ITS1) as a genetic marker. Fungal diversity was estimated and compared between speleothem and rock wall surfaces and its variation with distance from the natural entrance of the cave was quantified. Effects of environmental factors and nutrient concentrations in speleothem drip water at different sample sites on fungal diversity were also examined. Sequencing revealed 2219 fungal operational taxonomic units (OTUs) at 95% similarity. Speleothems supported a higher fungal richness and diversity than rock walls, but community membership and the taxonomic distribution of fungal OTUs did not differ significantly. OTU richness and diversity were negatively correlated with distance from the natural cave entrance. Community membership and taxonomic distribution of fungal OTUs differed significantly between the front and back of the cave. There was no observed effect of drip water nutrient concentration on fungal community structure. The third study examined fungal community structure from bat guano over the course of a year. There was no significant difference in fungal OTU richness, diversity, or community membership and taxonomic affiliations among sampling times. There were no significant differences in nutrient concentrations of guano samples among sampling times. Nutrient concentration did have a significant effect on community structure, especially the level of nitrogen and calcium.

Landscape management has altered the population dynamics of many species residing in old growth ecosystems. As a result, fragmentations in residual old oak habitats in Sweden have led to the decline of endangered and long-lived polypores including the bright orange Hapalopilus croceus. Since little is known about this species, the aim was to investigate its growth, mating, and population biology using molecular and ecological methods. Specifically, what its growth patterns are, what its mating type is, and whether there is genetic variation among sampled populations in Sweden and the Baltic area. Fruiting body samples were collected from 34 localities in Sweden and 6 localities in neighboring Baltic States. Heterokaryotic mycelia were then cultivated from these samples and the genomes sequenced. 42 single-spore isolates from SLU's culture bank were also revived and cultured. These homokaryons were then crossed to determine mating type. Mating type was examined using morphological observations and calculated via mating matrix. To examine growth rate and establishment, H. croceus mycelia were grown in comparison to Fomitopsis pinicola mycelia. To explore the population biology, the potential for gene flow was analyzed among Swedish and Baltic isolates. As a result, H. croceus was found to be slow growing, exhibit a tetra polar mating type, and with limited genetic variation in the Baltic Sea area. Its slow growth could provide a possible insight into colonization strategy, whereas its multiallelic mating system could possibly increase outcrossing in the future. Minimal genetic variation could result from previous connectivity between oaks, where the fungus may have outbred readily and colonized more hosts, maintaining a more uniform genetic structure. However, recent habitat alteration has not been analyzed for this species, nor has community interaction among other fungi and fruiting dynamics. Thus, further examination of these factors would help expand this study and possibly provide conservation and management guidelines for future projects.

The fungal microbiome, or “mycobiome” of plants is diverse and important to host health, but the fluxes of fungi among plant hosts and with the surrounding environment are poorly understood. In chapter two, we employed sterile culture techniques and spatial sampling to examine leaves as possible vectors for transfer of their endophytic fungi from the canopy to substrate on the forest floor, as predicted by the Foraging Ascomycete hypothesis. Some foliar endophytic fungal species are also present as wood-decomposing fungi on the forest floor, that transfer of mycelium across these two life history stages can occur, that endophytic life history stages are buffered from environmental conditions in comparison to wood-decomposing fungi, and that spatial linkages between the two life history stages can be observed. In another study, described in chapter 3, wood and leaf wood endophytes were sampled across a 25 ha plot, to explore landscape patterns of mycobiomes, and to explore the concept of a core microbiome in aerial plant tissues. We found that core microbiomes may be observed in a real ecological setting, but that the concept of core must be carefully defined and that some level of buffering from disturbance may be necessary to allow core microbiomes to assemble. In chapter four, we return to examine some of the assumptions and implications of the Foraging Ascomycete hypothesis, with an agent-based model. We model the conditions under which dispersal through falling leaves may represent a fitness-enhancing dispersal strategy for fungi, and that deforestation as is currently underway throughout the world may have impacts on fungi that rely upon a canopy- inhabiting life stage for dispersal. In chapter five, some challenges associated with environmental sampling of microbes using illumina© MiSeq sequences are critically examined. We find that biases introduced by random sampling at various stages of IVenvironmental DNA extraction and illumina© MiSeq sequencing are not well corrected by currently accepted bioinformatic algorithms. In addition, information loss from differential extraction, PCR amplification, and sequencing success, requires that users of MiSeq read libraries to interpret read abundances carefully. This dissertation includes previously published, co-authored material.

A grande diversidade de organismos que hoje encontramos em nosso planeta se deve à adaptação às diferentes condições ambientais de cada nicho ecológico existente e à resposta adaptativa originária das mudanças dessas condições. Pode-se considerar que a etapa inicial do processo de adaptação seja a reprogramação da expressão gênica dum organismo como resposta imediata a uma nova condição ambiental. De fato parte do genoma de todos os organismos é dedicada à codificação de proteínas relacionadas ao controle dos efeitos nocivos criados por diferentes tipos de estresse como choque térmico, ou osmótico, estresse oxidativo, ou aqueles resultantes de altas concentrações de íons de metais pesados. De forma semelhante, a ausência, ou exaustão, de fontes de macronutrientes, como carbono, nitrogênio, fósforo ou enxofre, exige uma reorganização do padrão de expressão gênica para adequação às novas condições nutricionais, também sendo considerada um estresse ambiental. Visto que a maioria dos estudos de análise da expressão gênica em resposta a estresses ambientais realizados em fungos se refere às leveduras unicelulares Saccharomyces cerevisiae e Schizossacharomyces pombe, nos propusemos a estudar tal resposta no fungo filamentoso multicelular Trichoderma reesei. Dessa forma analisamos por meio da técnica de \"microarrays\" de cDNAs a expressão gênica de aproximadamente 2.000 transcritos desse organismo em resposta a choque térmico, à alta concentração de íons de cádmio II e à ausência de fonte de carbono, ou nitrogênio, por período de 2 horas. Em geral, as respostas aos estresses se compuseram da regulação negativa da transcrição de genes envolvidos em processos com alta demanda de energia como a síntese protéica, evidenciada pela repressão da expressão de genes de proteínas ribossomais e do anabolismo. Em contrapartida, genes codificando proteínas relacionadas à defesa celular, como chaperonas, tiveram sua expressão induzida. As respostas ao choque térmico e ao tratamento com cádmio II se mostraram bastantes semelhantes, enquanto a ausência de fonte de nitrogênio também induziu a expressão de genes relacionados à degradação de proteínas e nucleotídeos. Genes relacionados à utilização de reservas lipídicas foram induzidos tanto na ausência de fonte de carbono quanto de nitrogênio. Foram identificados reguladores transcricionais e componentes de vias de sinalização celular com expressão diferenciada frente a esses diferentes estresses ambientais. A maior parte dos genes cuja expressão se alterou em função dos diversos estresses ambientais estudados ainda não tem função celular conhecida, sendo essa observação, portanto, uma contribuição importante para sua anotação funcional. Uma vez que o fungo filamentoso Trichoderma reesei vem se tornando um organismo de valor biotecnológico por sua característica de alto poder de síntese e secreção de proteínas, esperamos que os dados apresentados forneçam um maior entendimento dos processos celulares desse organismo e possam subsidiar futuros projetos visando uma melhor adaptação do mesmo a ambientes industriais. / The diversity of organisms found today in our planet is due to their adaptation to different environmental conditions present in each ecological niche, and to the adaptative response originated from changes in those conditions. The first step in the adaptation process is considered to be the reprogramming of gene expression as an immediate response to a new environmental condition. A fraction of the genome from all living organisms is dedicated to encoding proteins related to the control of deleterious effects created by different types of stresses like heat or osmotic shock, oxidative stress, or by the presence of high concentrations of heavy metal ions. Similarly, the absence or exhaustion of macronutrients as carbon, nitrogen, phosphorous or sulphur sources demand new patterns of gene expression in order to the organisms survive in a limited nutritional condition, which is also considered an environmental stress. Once the gene expression analyses in fungi as a response to environmental stresses have been widely studied in the yeasts Saccharomyces cerevisiae and Schizossacharomyces pombe, we proposed to study such response in the multicellular filamentous fungus Trichoderma reesei. To this purpose, we have utilized the cDNA microarray technique to analyze the gene expression of approximately 2,000 T. reesei transcripts in response to heat shock, to high concentration of cadmium II ions and to a 2-hour absence of carbon or nitrogen source. As a general response to the four studied stresses, we observed on one hand a negative transcriptional regulation of genes involved in processes that demand great amounts of energy, i.e. a negative regulation of protein synthesis, indicated by strong repression of ribosomal protein genes transcription, as well as a negative regulation of anabolism. On the other hand, genes that encode proteins associated with cellular defense, like chaperones, had their expression induced. The responses to heat shock and to cadmium poisoning were quite similar while nitrogen source absence also induced the expression of genes related to protein and nucleotide degradation. Genes implicated in the consumption of lipid reserves were induced in the absence of both carbon and nitrogen sources. We identified some transcription regulators as well as components of signal transduction pathways that have differential patterns of gene expression caused by these different environmental stresses. Most of the genes that had their expression altered in response to the studied environmental stresses has no known function yet. Their expression patterns towards such stresses are therefore an important contribution to their functional annotation. Since the filamentous fungus Trichoderma reesei has become a microorganism of biotechnological value for its high capacity of synthesis and secretion of proteins, we expect that the data presented on this work can provide a better understanding of its cellular processes and may support future projects for a better adaptation of this organism to industrial conditions.

O objetivo do presente trabalho foi estudar a ação de diversos antifúngicos isolados e associados a anfotericina B contra amostras sequenciais ou não recuperadas de pacientes com cromoblastomicose e submetidas ou não a tratamento. A partir de 18 pacientes foram isoladas 39 cepas, submetidas a testes de diluição em ágar contendo anfotericina B, cetoconazol, itraconazol, terbinafina, 5-fluorocitosina, fluconazol ou griseofulvina. Com base nas CIMs e nas concentrações séricas desses antifúngicos verificou-se que as amostras foram, de modo geral, sensíveis ao cetoconazol, a terbinafina e ao itraconazol. Somente 13 amostras foram sensíveis a anfotericina, 7 a 5-FC, 2 ao fluconazol e nenhuma à griseofulvina.Com relação as CFMs, 2 cepas foram sensíveis a AnB, 11 ao itraconazol, 20 ao cetoconazol e 3 a 5-FC. Seis não responderam a terbinafina e 38 ao fluconazol. As combinações de antifúngicos resultaram em interações principalmente aditivas ou indiferentes. O estudo de isolados seqüenciais evidenciou um ponto de corte para sensibilidade desses agentes ao itraconazol, correspondente a ClMs &#8804;0,06&#181;g/ml desse azol. / The aim of the present work was to study the action of several antifungals isolated and combined with amphotericin B against sequential or non-sequential samples in patients infected with chromoblastomycosis and treated or not treated. Thirty nine strains from 18 patients were isolated and submitted to agar dilution testing containing amphotericin B, cetoconazole, itraconazole, terbinafine, 5-flucytosin, fluconazole or griseofulvin. Based on the CIMs and on the serum levels of these antifungals, the isolates were susceptible to ketoconazole, terbinafine and itraconazole. Only 13 of the isolates were susceptible to amphotericin; seven to 5-FC, two to fluconazole and none to a griseofulvin. Regarding the CFMs two strains were susceptible to AnB, 11 for itraconazole, 20 for ketoconazole and three for 5-FC. Six didn\'t respond to terbinafine and 38 for fluconazole. The combination of antifungals resulted in mainly additive or indifferent interaction. The study of the sequential isolated sample showed a breaking point for the susceptibility of these agents to itraconazole, corresponding CIMs &#8804;0,06&#181;g/ml of this azole.

Criptococose é uma doença causada pelo fungo C. neoformans que têm grande importância atualmente, devido ao aumento da população imunocomprometida,. Além disso, existem poucas opções terapêuticas contra micoses profundas. Neste trabalho foi avaliado se a via de biossíntese do triptofano seria um bom alvo para o desenvolvimento de novos antifúngicos. Com o uso da tecnologia de RNA de interferência, concluiu-se que esta via de síntese é essencial para a sobrevivência desta levedura, sendo, portanto, um ótimo alvo. Ainda neste estudo, demonstrou-se que a letalidade decorre da baixa captação de triptofano pelas permeases de aminoácidos, as quais sofrem repressão catabólica pela fonte de nitrogênio e efeito negativo da temperatura. Foram testados dois inibidores específicos que atuam sobre a antranilato sintase e a triptofano sintase, duas enzimas cruciais para a conversão do corismato em triptofano. Ambos compostos causaram inibição do crescimento de C. neoformans e C. gattii. / Cryptococcosis is a disease caused by C. neoformans, currently of great importance due to the increase in immunocompromised population. Furthermore, there are few therapeutic options for treating this disease. This study evaluated the tryptophan biosynthetic pathway as a possible target for the antifungal development. By using RNA interference technology we concluded that this metabolic pathway is essential for the survival of this yeast, and, therefore, it is a good target. In the same study, it was demonstrated that lethality results from the low uptake of the tryptophan amino acid by permeases, which undergo nitrogen catabolite repression and negative effect of temperature. Two specific inhibitors acting on the anthranilate synthase and tryptophan synthase, two key enzymes for the conversion of chorismate into tryptophan were tested. Both compounds caused growth inhibition of C. neoformans and C. gattii.