Saprolegnia parasitica is a pathogenic oomycete that cause saprolegniosis. Freshwater fish like salmon and trout species are particularly vulnerable to infection, which is characterized by cotton-like grayish mycelial growth on the surface of the fish. Currently, an effective treatment for this disease is not available. This pathogen has a great impact on freshwater fish species world-wide. An initial step to keep this devastating disease at bay is the ability to detect the responsible pathogen, so that appropriate actions could be taken before it becomes widespread. The development of molecular tools that will accurately and rapidly detect S. parasitica is the main goal of this project. This project is divided into two main sections. The first section describes initial marker design efforts that were focused on the internal transcribed spacer (ITS) regions. Efforts were also made for the collection of field samples and the generation of our own ITS data that includes a number of Saprolegnia spp. Compiled sequence data allowed the identification of unknown samples and the adoption of the clade taxonomic system that other researchers had established for species designations. The accumulated sequence data helped to clarify the taxonomy within the genus Saprolegnia and complemented previous studies. The design of broad specificity PCR primers also allowed a quick initial detection of Saprolegnia spp., which could then be identified to species either by determining ITS nucleotide sequence or by a subsequent step of RFLP marker. Isolates sequence data in the compiled sample collection could be used for validation purposes in further marker development. The second section of the project described the development of higher specificity molecular markers for the detection of S. parasitica. These were based on the study of three different gene loci as potential markers. These included the Pumilio RNA-binding protein (Puf), Glutathionylspermidine synthetase (Gsp) and the thiazole biosynthetic enzyme (Thi4). The nucleotide sequence of each locus was studied to develop suitable PCR primers that were then refined through testing against our isolate collection to improve their specificity for the target species. Saprolegnia parasitica-specific markers were developed for the Puf and Gsp loci and these were further evaluated using our field collected samples. / Graduate

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

Understanding how hyphal organisms grow and develop is essential in order to manipulate mycelial colonies for purposes such as disease prevention and food production. One aspect of hyphal development that is not well understood is hyphal branching. Hyphal organisms branch as a way of creating new hyphal tips required for the search for nutrients, the acquisition of these nutrients and for hyphal fusion events that facilitate communication of signals within a mycelial colony. This investigation focused on the branching process occurring in the fungus N. crassa and in the oomycete A. bisexualis. An induction technique was developed to study branching in N. crassa involving local application of amino acids towards hyphae. This induced a branch to form along the hypha within the field of view. The use of this technique will enable the study of underlying events occurring internally prior to the visible branching stages. The role of Ca²⁺ hyphal branching was investigated in N. crassa suggesting Ca²⁺ is involved in apical dominance of the hyphal tip. This is based on a dose dependent response of increased branch frequency, decreased colony radius and decreased distance between the hyphal tip and the first branch, to the Ca²⁺ channel inhibitor verapamil. The stretch-activated Ca²⁺ channel inhibitors also had an effect on mycelial morphology. Gd³⁺ resulted in an increased branch frequency and a decreased colony radius and La³⁺ resulted in a decreased colony radius. The local application of verapamil towards N. crassa showed an increase in the number of multiple branches forming. Cytoplasmic Ca²⁺ was imaged in hyphae of A. bisexualis and N. crassa showing a tip-high Ca²⁺ gradient in A. bisexualis and Ca²⁺ sequestered into organelles in N. crassa. The role of F-actin in the process of hyphal branching was investigated using Lifeact N. crassa where F-actin could dynamically be seen at the site of both growing and non-growing hyphal branches. The involvement of F-actin at sites of septa development and associated with suspected vesicles was also observed.

The genus Phytophthora contains a large number of species that are known plant pathogens of a variety of important crops. Phytophthora sojae, a hemibiotroph, causes approximately 1-2 billion dollars (US) of lost soybean world-wide each year. P. infestans, the causative agent of the Irish potato famine, is responsible for over 5 billion dollars (US) worth of lost potato each year. These destructive plant pathogens facilitate pathogenesis through the use of small secreted proteins known as effector proteins. A large subset of effector proteins is able to translocate into host cells and target plant defense pathways. P. sojae Avr1b is able to suppress cell death triggered by BAX and hydrogen peroxide. The W-domain of Avr1b is responsible for this functionality, and is recognized by the Rps1b gene product to induce effector triggered immunity. These oomycete effector proteins translocate into host cells via a highly conserved N-terminal motif known as RXLR-dEER without the use of any pathogen encoded machinery. In fungi an RXLR-like motif exists, [R,K,H] X [L,F,Y,M,~I] X, that is able to facilitate translocation without pathogen encoded machinery. Both functional RXLR and RXLR-like motifs are able to bind phosphatidylinositol-3-phosphate (PtdIns- 3-P) to mediate entry into host cells. The use of novel inhibitory mechanisms has shown effector entry can be blocked either by sequestering PtdIns-3-P on the outer leaflet of plant and animal cells or by competitive inhibition of the binding pocket of the RXLR or RXLR-like motifs. / Ph. D.

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. The genome of Hyaloperonospora arabidopsidis (Hpa), the causative agent of downy mildew of Arabidopsis, contains at least 134 candidate RXLR effector genes. These genes contain an RXLR motif required for effector entry into host cells. Only a small subset of these candidate effectors is conserved in related oomycetes. Here, we describe a comparative functional characterization of the Hpa RXLR effector HaRxL96 and a homologous gene, PsAvh163, from the soybean pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress or activate immune responses in soybean, Nicotiana, and Arabidopsis. Several SA-responsive defense genes are suppressed in Arabidopsis Col:HaRxL96 and Col:PsAvh163 during an incompatible interaction with Hpa Emoy2. Both effectors are localized to the nucleus and cytoplasm of plant cells. Nuclear localization of both effectors is required for proper virulence functions, including suppression of basal resistance and RPP4-mediated immunity to virulent and avirulent Hpa, respectively. In addition, both effectors interact with plant U-box (PUB) proteins that are conserved between diverse plant species. The targeted PUB proteins are negative regulators of plant immunity in Arabidopsis. These experiments demonstrate that evolutionarily-conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's primary host. / Ph. D.

No description available.

The genus Phytophthora comprises numerous plant pathogens in both natural and managed ecosystems. For Phytophthora spp. that infect roots, dispersal occurs in soil water through a combination of advection and swimming of specialized motile propagules (zoospores). Specific biological and physico-chemical processes, however, remain poorly understood, due to difficulties in studying phenomena in opaque media and lack of a theoretical framework for analyzing transport of motile microorganisms. The goal of this research was to elucidate the impacts of advection and swimming on zoospore movement in a saturated, ideal soil. The work was accomplished in two stages, (i) conceptualization of 3-dimensional topography and flow field heterogeneity at the subpore-scale, and (ii) observation of behavior of zoospore suspensions infiltrated into saturated media. Chapter 2 introduces a 3-dimensional particle tracking method and presents two studies investigating particle transport in simplified 'ideal pores'. The first study describes 'avoidance' by latex microspheres of a volume surrounding orthogonal grain contacts and the second describes 'capture', translation, and retention of microspheres under conditions unfavorable to deposition. Chapter 3 expands on the first study and demonstrates, with the aid of computational fluid dynamics, that low flow zones associated with orthogonal grain contacts are minimally connected to the main flow. Thus, probability of entry into these regions for large, non-Brownian particles by advection alone is low. In zoospore infiltration experiments, zoospore plumes 'converged' rather than dispersing as expected. To assess the possibility of zoospore auto-aggregation driving this 'convergence', Chapter 4 delves into the 'pattern swimming' observed in free-swimming zoospore suspensions, concluding that the concentrating is an example of bioconvection. Chapter 5 introduces a conceptual model to explain the anomalous zoospore plume behavior. Random walk simulations replicated plume convergence but were less successful at modeling anisotropic dispersion. At low infiltration rates (<100 μm s⁻¹), simulations predict that zoospores will remain at or near the soil surface, resulting in greater opportunity to find host tissues or to be transported with surface water. Further investigation is necessary to develop a robust theoretical framework with appropriate conceptualization of the subpore hydrodynamic environment for predicting transport of zoospores and other motile microorganisms in porous media. / Graduation date: 2011

Fish skin is a metabolically active tissue that responds quickly to stressors and is the first line of defence against physical damage. Club cells, characteristic components of Ostariophysian fish skin, release their contents into the surrounding water upon rupture (e.g. during predation). These chemical cues act as public information of predation risk. Despite the assumption that club cells evolved under the selective force of predation, studies demonstrated that predation has no effect on club cell investment. Rather, club cell production is stimulated by skin penetrating pathogens and parasites. The experiments in this thesis investigate the responses of fish skin to manipulated pathogen risk. In the first experiment, fathead minnows (Pimephales promelas) were exposed to varying infective risk from two pathogen species that differ in pathogenicity, Saprolegnia ferax and S. parasitica. Although there was no difference in club cell density between fish exposed to the two Saprolegnia species, fish exposed to high concentrations of the pathogens had smaller club cells than those exposed to low concentrations. These results are the first to demonstrate a pathogen effect on the size of club cells. The second experiment investigated whether the physical presence of the pathogen was necessary for an alteration in epidermal parameters or whether Saprolegnia parastitica conditioned water was the only stimulus necessary to evoke a change. Results indicated a lack of treatment effect on club cell density, club cell size or epidermal thickness. The third experiment investigated the timing of club cell changes following a pathogen challenge. Although fish exposed to the Saprolegnia ferax treatment had higher club cell density than fish exposed to the control, there was no difference in club cell density between fish sacrificed on day 3, 6, 9 or 12. A portion of the test population for the third experiment was infected with black spot disease. When analyzed separately, trematode infected fish had smaller club cells than those that were uninfected. In light of inconsistent epidermal responses to pathogen challenges, and comparison with other studies, assessment of environmental stressors and population differences that may affect experimental outcomes and potentially interact with infectious agents is advised.

Fish skin is a metabolically active tissue that responds quickly to stressors and is the first line of defence against physical damage. Club cells, characteristic components of Ostariophysian fish skin, release their contents into the surrounding water upon rupture (e.g. during predation). These chemical cues act as public information of predation risk. Despite the assumption that club cells evolved under the selective force of predation, studies demonstrated that predation has no effect on club cell investment. Rather, club cell production is stimulated by skin penetrating pathogens and parasites. The experiments in this thesis investigate the responses of fish skin to manipulated pathogen risk. In the first experiment, fathead minnows (Pimephales promelas) were exposed to varying infective risk from two pathogen species that differ in pathogenicity, Saprolegnia ferax and S. parasitica. Although there was no difference in club cell density between fish exposed to the two Saprolegnia species, fish exposed to high concentrations of the pathogens had smaller club cells than those exposed to low concentrations. These results are the first to demonstrate a pathogen effect on the size of club cells. The second experiment investigated whether the physical presence of the pathogen was necessary for an alteration in epidermal parameters or whether Saprolegnia parastitica conditioned water was the only stimulus necessary to evoke a change. Results indicated a lack of treatment effect on club cell density, club cell size or epidermal thickness. The third experiment investigated the timing of club cell changes following a pathogen challenge. Although fish exposed to the Saprolegnia ferax treatment had higher club cell density than fish exposed to the control, there was no difference in club cell density between fish sacrificed on day 3, 6, 9 or 12. A portion of the test population for the third experiment was infected with black spot disease. When analyzed separately, trematode infected fish had smaller club cells than those that were uninfected. In light of inconsistent epidermal responses to pathogen challenges, and comparison with other studies, assessment of environmental stressors and population differences that may affect experimental outcomes and potentially interact with infectious agents is advised.