Dissertations / Theses on the topic 'Zoospores'

Phytophthora infestans is a highly destructive plant pathogen and the causal agent of the potato blight disease that devastated Ireland’s potato crops in the 19th century.Today, this disease is still a serious problem, with global crop losses and spending oncontrol measures estimated to exceed £3 billion annually. A key to the success of P. infestans is the dispersal of free-swimming zoospore cells from infected plant tissue into aqueous environments. These cells are specialised infection agents that have evolved an array of tactic responses in order to locate and infect new hosts. An interesting and poorly understood aspect of zoospore behaviour is the phenomenon of auto-aggregation. That is, large numbers of zoospores observed in vitro are seen to form complex, large-scale patterns in the absence of external signals or stimuli. Current competing hypotheses suggest that patterns are formed by one of two distinct, concentrative phenomena: chemotaxis and bioconvection. In this thesis we investigate the mechanics and implications of zoospore auto-aggregation behaviour using an interdisciplinary approach that combines continuum mathematical modelling with laboratory experimental work. We investigate the modelling of chemotactic and bioconvective processes and compare results with our experimental observations. Finally, we present a novel bioconvection-chemotaxis model and thus provide strong evidence to support the hypothesis that auto-aggregation in P. infestans zoospores results from a necessary combination of these processes.

Pythium aphanidermatum (Edson) Fitz., Pythium catenulatum Matthews and Pythium dissotocum Drechs. were compared for responses to amino acids and sugars in tests involving taxis, zoospore encystment and cyst germination in vitro. Comparisons were made between the fungi and for each fungus at these three stages of development, which are parts of the normal sequence leading to infection of host roots or colonisation of non-living substrata from zoospores. Other substances tested for induction of encystment or germination in vitro were cellulose film, crab shell, uronic acids or uronate-containing compounds, partially characterised polysaccharides from plants, root mucilages and cations. Motility attributes of the fungi were compared and, for P. aphanidermatum only, were characterised in the presence of several ions and compounds that interfere with calcium-mediated events (EGTA, dibucaine, trifluoperazine, lanthanum, verapamil, amiloride, A3187 and TMB-8). Amino acids were tested for competitive effects in chemotaxis assays in vitro. Calcium and EGTA were also tested for effects on adhesion of encysted spores to glass slides. Accumulation and encystment of P. aphanidermatum on detached wheat roots was studied on microscope slides, with or without prior treatment of roots with calcium alginate gel, methylene blue, alcian blue, India, ink ruthenium red and lectins that bind residues of fucose, D-glucose, D-mannose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. Many experiments involved video-recording zoospore responses and subsequent analysis of videotapes. Evidence is presented for an effect of calcium and other divalent cations on zoospore motility patterns, cyst adhesion and induction of germination. Several amino acids and sugars elicited zoospore taxis, encystment, or cyst germination but sometimes differently for different fungi or at these different stages of development. Competition experiments enabled some of these responses to be related to proposed receptor functions, and in some cases amino acids could overcome inhibition of germination caused by the presence of calcium-modulators.

This thesis is concerned with an analysis of swimming of oomycete zoospores, particularly in relation to the hypothesis that electrotaxis mediated host root colonisation. A correlation was found between the electrotactic behaviour of zoospores of Phytophthora palmivora and Pythium aphanidermatum and their localisation relative to anodic or cathodic regions of the majority of non-host roots. Cathodotropic P. aphanidermatum zoospores were found to be attracted to the cathode generated at the wound site on monocotyledonous and dicotyledonous plant roots studied. Zoospores of P. aphanidermatum were also found to become gradually less attracted to these wound sites as they dissipated over time. Anodotropic P. palmivora zoospores were found to be repelled by the cathodic wound sites on roots of all plants investigated, with the exception of Petunia hybrida. In addition to this zoospores of P. aphanidermatum were found not exhibit chemotaxis or encystment in gradients of the wound-specific metabolite acetosyringone. This suggests that electrotaxis and chemotaxis both operate in directing zoospore accumulation around roots. This reduction of the conductivity of the bathing medium by the addition of different concentrations of sodium chloride salts did not appear to affect the accumulation of both zoospore species around roots of rye grass. The results of this study supports the hypothesis that zoospores use electrotaxis as one means to locate new plant hosts in the rhizosphere. However, chemotaxis may still augment the regulation of zoospore colonisation and encystment.

Motile zoospores of P. dissotocum and P. catenulatum were added to 20 and 60 mesh silica sand and a sandy loam soil to investigate the duration of motility. Both Pythium species remained motile for up to 24 hours in all soil textures tested. However, the duration and percentage of the motile population varied depending upon the soil type and species tested. The duration of survival of motile and encysted zoospores of P. dissotocum and P. catenulatum in air dried and saturated sterile silica sand was also tested. P. dissotocum, under air dried conditions, did not survive while P. catenulatum was capable of surviving 4 and 16 days as motile and encysted zoospores, respectively. Both fungi could be recovered at high percentages after 5 weeks under saturated conditions. The survival structure is believed to be in the form of a zoospore cyst.

La propagation des maladies des plantes causées par Phytophthora repose notamment sur la dispersion dans le sol de zoospores unicellulaires, polarisées et biflagellées. Le mouvement des zoospores vers un hôte s'appuie sur divers mécanismes tels que la chimiotaxie, l'électrotaxie, la géotaxie négative et la rhéotaxie. Les signaux provenant des particules du sol et de la plante déterminent ce mouvement dirigé lors des premières étapes de la colonisation racinaire. Cependant, les mécanismes de perception du signal par les zoospores, qui conduisent au mouvement dirigé vers l'hôte, restent mal compris. Des questions cruciales subsistent concernant la nature et la spécificité de ces mécanismes par rapport à ceux utilisés par d'autres microorganismes du sol, ainsi que l'influence des caractéristiques dynamiques et morphologiques des zoospores sur leur mouvement guidé.Dans ce contexte, la première partie de ce travail a exploré les capacités de détection des zoospores de Phytophthora parasitica en analysant le répertoire protéique de leur membrane plasmique à l'aide d'une approche protéomique et de la LC-MS/MS. Les protéines d'échantillons membranaires ont été identifiées sur la base du protéome de référence de Phytophthora parasitica, permettant l'identification du répertoire membranaire et une comparaison des profils des fractions issues de corps cellulaires et flagellaires. Trois protéines membranaires clés associées aux mécanismes de détection et de réponse au mouvement des zoospores ont été plus particulièrement étudiées, dont certaines étaient spécifiquement localisées à la membrane flagellaire - une protéine de détection des stérols, une nucléotide cyclase et une Na+/K+ ATPase - supportant l'hypothèse d'un rôle critique des flagelles dans les mécanismes de perception du signal. Pour initier une analyse fonctionnelle de la détection des zoospores, des essais d'immunolocalisation, pharmacologiques et électrophysiologiques ont été initiés.La deuxième partie de cette thèse était basée sur une approche d'imagerie automatisée, pour développer une méthode de quantification de diverses caractéristiques de micronageurs du sol, y compris les zoospores, évoluant en une suspension microbienne et en réponse à un facteur sol/hôte (gradient de potassium). Dans ces conditions, les réponses de mouvement des zoospores ont été caractérisées, et comparées à celles d'autres espèces, montrant qu'il est possible de distinguer les morphologies, les trajectoires, les vitesses, et l'impact du facteur du sol dans une communauté synthétique simple composée de P. parasitica, Vorticella microstoma et Enterobacter aerogenes. Par ailleurs, une analyse biomécanique a été réalisée détaillant des métriques de mouvement des zoospores, telles que la vitesse, la géométrie des trajectoires et les fréquences de battement flagellaire sous le même stimulus, révélant des dynamiques de mouvement spécifiques aux zoospores par rapport à d'autres micronageurs. Ainsi, nous avons constaté que l'augmentation des concentrations de potassium perturbe le schéma de nage des zoospores, généralement caractérisé par des trajectoires longues et rectilignes alternant avec des culbutes périodiques. Les zoospores présentaient alors des trajectoires de plus en plus courtes et circulaires, avec une réduction de la vitesse et une altération du battement des flagelles.Ces résultats combinent des principes biochimiques et biomécaniques pour mieux comprendre les processus fondamentaux qui guident les espèces microbiennes vers des stimuli externes. Ce travail clarifie l'étendue des mécanismes de détection et des réponses de mouvement des zoospores de Phytophthora, améliorant notre compréhension de la phase de pré-colonisation et de la façon dont ces pathogènes se dirigent vers leurs plantes hôtes. Cette approche intégrée fournit des perspectives de compréhension des premiers stades de l'infection, par ailleurs utiles pour développer de nouvelles stratégies de gestion des maladies
The epidemic spread of plant diseases caused by Phytophthora is primarily based on the dispersal of unicellular, biflagellated zoospores in the soil. Zoospore guidance towards host plants relies on diverse mechanisms such as chemotaxis, electrotaxis, negative geotaxis and rheotaxis. Signals from soil particles and host plants critically influence these motion processes, guiding zoospores during the initial stages of root colonization. However, the mechanisms underlying zoospores perception, resulting in the directed motion toward hosts remain unclear. Critical questions include the nature and the specificity of these sensing mechanisms compared to those used by other soil microorganisms, and the extent to which the dynamic and morphological characteristics of zoospores contribute to their guided motion.In this context, the first part of this thesis focused on investigating the sensing capabilities of Phytophthora parasitica zoospores by analyzing their plasma membrane protein repertoire through a proteomic approach. Peptides were detected from membrane samples using LC-MS/MS, and related proteins were identified by mapping against the Phytophthora parasitica reference proteome, allowing for detailed characterization and comparison of the membrane profiles of the zoospore cell body and flagella fractions. Given the naturally polarized structure of zoospores, which exhibit two morphologically distinct flagella responsible for oriented motion, we hypothesized a critical role of flagella in sensing mechanisms. Our analysis identified three prominent membrane proteins associated with sensing and motion response mechanisms in zoospores, some of which were specifically localized to the flagella membrane: a sterol-sensing protein, a nucleotide cyclase and a Na+/K+ ATPase. To start a functional analysis in zoospore sensing, immunolocalization, pharmacological and electrophysiological assays were initiated.The second part of this thesis employed an automated high-content imaging approach to establish a novel method for quantifying diverse characteristics of soil microswimmers, including zoospores, in response to a soil/host factor (potassium gradient). The initial observations focused on the motion responses of zoospores among other species, demonstrating the feasibility to simultaneously distinguish morphologies, trajectories, velocities, and the impacts of the soil factor on a simple synthetic microbial community composed of P. parasitica, Vorticella microstoma, and Enterobacter aerogenes. Following these observations, a detailed biomechanical analysis was conducted to quantify motion metrics, such as velocity, trajectory geometry, and flagellar beating patterns under the same stimulus, revealing specific motion dynamics unique to zoospores compared to other microswimmers. As a result, we found that increasing potassium concentrations disrupt the normal swimming pattern of zoospores, typically characterized by long, straight runs and periodic tumbles. Instead, the zoospores exhibited progressively shorter and more circular trajectories, with reduced velocity and altered flagella beatingThese findings integrate biochemical and biomechanical principles to advance the understanding of the fundamental biological process of microbial guidance toward external cues. Elucidating the sensing mechanisms and motion responses of Phytophthora zoospores enhances our understanding of the pre-colonization phase of plant infection, highlighting how these pathogens move toward hosts. This integrated approach offers valuable insights into early infection stages, potentially guiding new plant disease management strategies

Root cap cells of two cotton species elicited a specific chemotactic response in zoospores of Pythium dissotocum. When roots of cotton seedlings were placed into a suspension of P. dissotocum zoospores, there was immediate attraction, accumulation and encystment exclusively in the root cap cell region. Furthermore, root cap cells remained attractive when isolated from the root: attraction, accumulation, and encystment on individual root cap cells occurred within seconds after contact. Zoospores penetrated and killed isolated root cap cells within 15-30 minutes, and seedlings died within 24 hours. In contrast, zoospores of P. catenulatum, which exhibited a chemotactic response to roots of Bentgrass, were not attracted to and did not infect seedlings or isolated root cap cells of cotton. Preliminary studies indicate that both Pythium species are capable of infecting cotton seedlings in sand culture, though it is not known if either are pathogens on cotton grown in the field.

A citricultura brasileira apresenta números bastante significativos e expressivos, refletindo a grande importância social e econômica que essa atividade agrícola e industrial tem para a economia do país. O Brasil se destaca como o maior produtor de laranja do mundo. As plantas cítricas podem ser produzidas e multiplicadas de várias formas e a enxertia é a forma de propagação vegetativa mais utilizada comercialmente no Brasil. Dentre os porta-enxertos mais empregados, sobressaem-se o citrumeleiro Swingle e a tangerineira Sunki. Apesar das vantagens presentes nesses dois genótipos, a produção deles está sendo limitada por doenças causadas por fungos e estraminipilas. Em citros, as diversas espécies de Phytophthora são responsáveis pelas doenças gomose e podridões, principalmente de radicelas. P. nicotianae é a espécie que mais afeta as plantas cítricas no Brasil. Não obstante a grande importância de P. nicotianae para os porta-enxertos, alguns mecanismos de resistência ou susceptibilidade ainda requerem mais investigações cientificas. O presente trabalho visa a elucidação de alguns mecanismos de resistência dos citros a P. nicotianae, por meio de estudos fisiológicos e bioquímicos dos porta-enxertos, além da ação de meios de cultura e da qualidade da luz no estudo da fisiologia desse estraminipila. Para isso realizou-se a detecção de alguns equivalentes de compostos fenólicos através da técnica HPLC e o comportamento do genótipo resistente frente à possível inibição da síntese de compostos fenólicos através do tratamento das raízes do porta-enxerto Swingle com Pro-Ca; avaliou-se a atração de zoósporos de P. nicotianae por exsudatos radiculares provenientes dos dois porta-enxertos com o uso de uma armadilha adaptada; verificou-se o comportamento do zoósporo, a motilidade e a zoosporogênese frente à ação de vários compostos fenólicos e testou-se a ação de diferentes meios de culturas e da qualidade da luz no crescimento micelial e na esporulação de P. nicotianae. Os resultados revelaram que a tangerineira Sunki possui maiores quantidades de equivalentes em apigenina que o citrumeleiro Swingle nos dois períodos de avaliação, para o HPLC. O porta-enxerto resistente apresentou mais equivalentes de ácido clorogênico que o genótipo susceptível. Quanto ao Pro-Ca, os equivalentes de compostos fenólicos em raízes de plântulas do citrumeleiro Swingle não diferiram daquelas plantas tratadas com água. O Pro-Ca não inibiu e não desativou a síntese de compostos fenólicos. O uso do anticorpo contra a elicitina \"α-plurivorina\" teve resultados intermediários no consumo de água por plântulas de tangerineira Sunki e a atividade zoosporicida dos compostos fenólicos apresentou resultados variáveis no comportamento da motilidade de zoósporos. Os compostos fenólicos escopoletina e tricetinpentametoxi conseguiram paralisar a motilidade e proporcionaram as menores porcentagens de zoosporogênese. O meio de cultura AA permitiu a maior TCMD, seguido de CA e V8-CaCO3-Ágar. Os maiores valores de esporulação foram encontrados nesses dois últimos meios, respectivamente. Todas as faixas de luzes (e a ausência de luz) estudadas no presente trabalho podem ser avaliadas para o crescimento micelial, porém o tratamento claro foi aquele que mais induziu a esporulação de zoósporos de P. nicotianae.
The Brazilian citrus industry has very significant and expressive numbers, reflecting the great social and economic importance of this industrial and agricultural activity has for the economy. Brazil stands out as the world\'s largest orange producer. Citrus plants can be produced and multiplied in various ways and the bud is the form of vegetative propagation more commercially used in Brazil. Among the most commonly used rootstocks, stand up the Swingle citrumelo and Sunki mandarin. Despite the advantages these two genotypes present, their production is being limited by diseases caused by fungi and estraminipilas. In citrus, all species of Phytophthora are responsible for diseases gummosis and fibrous root rot, especially. P. nicotianae is the kind that affects citrus plants in Brazil. Regardless of the great importance of P. nicotianae for rootstocks, some mechanisms of resistance or susceptibility still require further scientific investigation. This study aims to elucidate some mechanisms of resistance of citrus to P. nicotianae, through physiological and biochemical studies of rootstocks, beyond of action culture media and quality of light in the physiological study of this estraminipila. To this was carried out to detect certain equivalent of phenolic compounds by HPLC and genotype resistant behavior against the possible inhibition of the synthesis of phenolic compounds by treatment of the roots of Swingle rootstock with Pro-Ca; evaluated the zoospores attraction P. nicotianae by exudates from the two rootstocks using an adapted trap; it was the zoospore behavior, motility and zoosporogenesis to the action of various phenolic compounds and tested the action of different culture media and quality of light on mycelial growth and sporulation of P. nicotianae. The results for HPLC revealed that Sunki mandarin has larger amounts of apigenin equivalents Swingle citrumelo in that the two periods. The sturdy rootstock had more chlorogenic acid equivalents of the susceptible genotype. As for the Pro-Ca equivalent of phenolic compounds in Swingle seedling roots not differ from those plants treated with water. The Pro-Ca not inhibited and not disabled the synthesis of phenolic compounds. The use of the antibody against elicitin \"α-plurivorin\" had intermediate results in water consumption by Sunki mandarin seedlings and zoosporicide activity of phenolic compounds showed variable results in the behavior of zoospore motility. The scopoletin and tricetinpentametoxi phenolic compounds managed to paralyze the motility and provided the smallest percentages of zoosporogenesis. The culture medium AA allowed greater DMGR, followed by CA and V8-CaCO3-Agar. The highest sporulation values were found in the last two media, respectively. All lights tracks (and the absence of light) studied in this work can be evaluated for mycelial growth, but the treatment light was that more induced sporulation of zoospores of P. nicotianae.

L'objectif de ce projet visait à déterminer si le silicium (Si), reconnu pour son effet prophylactique contre les agents biotrophes, pouvait protéger le soja contre Phytophthora sojae, un oomycète hemi-biotrophe. Le défi initial consistait à développer une méthode d'inoculation qui reproduisait le processus d'infection naturelle tout en permettant aux plants d'absorber le Si. Dans un premier temps, l'inoculation de zoospores dans une solution hydroponique a permis de générer des infections reproductibles conséquentes avec les réponses phénotypiques attendues. Dans un deuxième temps, l'ajout de Si a permis de réduire significativement l'incidence de la maladie tout en augmentant le rendement des plants. Cet effet était surtout manifeste sur les cultivars possédant un niveau de résistance contre P. sojae ou sur ceux absorbant plus de Si. Nous proposons ainsi une nouvelle méthode d'inoculation de P. sojae représentative de l'infection naturelle et démontrons que le Si peut servir pour la lutte contre cet oomycète.

It has been suggested that ectomycorrhizae protect roots from invasion by soilborne plant pathogens. The present study tested this hypothesis with in vitro studies using axenic conditions. When Eucalyptus sieberi . roots were in association with Pisolithus tinctorius, a degree of protection was afforded, whereby lesion lengths caused by Phytophthora cinnamomi in primary roots are reduced. Exudates from P. tinctorius resulted in a significant increase in the number of lateral roots. Lateral root apices were observed as a popul,ar site for P. cinnamomi infection in control roots. By 48 h, lesion development in ectomycorrhizae was 1.96 - 4.58 and 0.26 - 4.75 mm.d-1 (P. tinctorius isolates H445 and M56, respectively), whereas the control lesion extenstion rate was 11.2 mm.d-1. In non-mycorrhizal plants, P. cinnamomi caused a brown lesion, in the stele of the root, extending from the root tip within 12 h. At 5 mm beyond the visible lesion front, the amount and position of hyphae observed varied from root to root. Staining of embedded sections with methylene blue and azure II in borax detected lignin in the xylem and in large amounts in the metaxylem, of all inoculated roots. Lignin was only observed in the xylem of the non-inoculated control roots. Sudan black B detected suberin only within sections of visible lesions. Whole mounts stained with Trypan blue showed most hyphae were arranged randomly along the root surface, with many orientated along cell wall junctions. Hyphae were concentrated at the apex of newly formed lateral roots, and were detected growing intercellulary throughout the cortex and the phloem of primary roots. The influence of the P. tinctorius hyphae on• zoospore germination and mycelial growth, on solid media, varied between the two P. cinnamomi isolates used in this study. The interaction between P. cinnamomi isolate MP112 and P. tinctorius isolate M56 resulted in the greatest inhibition of the pathogen's mycelium. In this reaction, P. cinnamomi hyphae tended to aggregate, but did not stop growing. Zoospore germination of P. cinnamomi isolate MP94048 were suppressed at 0.5 h only, while germination of zoospores of isolate MP112 were significantly reduced by both P. tinctorius isolates for the entire trial. Staining of embedded sections revealed the ectomycorrhizae (P. tinctorius isolate M56) produced more elongated epidermal cells with approximately 30% more primary root tips covered by hyphae, than isolate H445. However, both isolates were observed to have a loose mantle. Isolate M56 produced some defence reactions, detected by aniline blue fluorescence in the cortex, and both isolates resulted in greater lignification in the metaxylem in comparison to the control roots.

Depuis la vulgarisation de certains concepts comme la globalisation ou la mondialisation, le secteur agroalimentaire a connu une expansion fulgurante et un engouement incessant pour la commercialisation d’aliments entre les peuples à travers le monde. Ce phénomène, contribuant significativement à l’accroissement économique des marchés, n’est toutefois pas sans risque. Pendant ce temps, les dangers de sources microbiologiques, notamment les pathogènes, sont véhiculés par des matrices alimentaires et voyagent d’un pays à l’autre, ce qui augmente le risque de contamination pour les consommateurs. Conséquemment, on assiste à une augmentation des cas d’allergies alimentaires, d’intoxications ou de toxi-infections alimentaires dont les agents étiologiques peuvent venir des quatre coins du monde. À cet effet, les organismes réglementaires comme l’Agence canadienne d’inspection des aliments (ACIA), Santé Canada, la Food and Drug Administration (USFDA) américaine ou d’autres autorités internationales compétentes comme l’Organisation des Nations unies pour l’alimentation et l’agriculture(FAO) et l’Organisation mondiale de la santé (OMS) multiplient leurs efforts afin de mettre en place des normes et politiques réglementaires pour aider l’industrie agroalimentaire à renforcer les contrôles depuis la fabrication jusqu’à la commercialisation des aliments. Les dangers microbiologiques venant de pathogènes comme Bacillus cereus demeurent un risque de santé publique majeur qu’il faut maîtriser afin d’assurer la protection des consommateurs. Bien que de nombreuses techniques de contrôle (e.g., additifs alimentaires, haute pression hydrostatique, rayonnements ionisants, procédés thermiques, etc.) ont été développées et utilisées pour assurer la salubrité et l’innocuité des aliments, dans certains cas cela n’a pas permis de produire des aliments totalement exempts de bactéries responsables de la dégradation/altération des aliments et de pathogènes causant des intoxications alimentaires comme c’est le cas avec B. cereus. En effet, cette bactérie pathogène est ubiquitaire, aérobie et anaérobie facultative. Elle est capable de produire dans une grande variété d’aliments et d’ingrédients comme les épices des spores très résistante sain si que différents types de toxines pouvant causer la diarrhée, la nausée, le vomissement et même la mort. Dans cette optique, et vue la grande difficulté à maitriser la contamination des aliments causée par ce pathogène, l’objectif général de cette recherche a été d’utiliser la technologie d’électro-activation, une branche appliquée de l’électrochimie qui s’intéresse notamment à la réactivité des solutions aqueuses, comme méthode alternative et potentiellement efficace pour lutter contre B. cereus afin de produire des aliments plus sécuritaires avec une grande valeur nutritionnelle et organoleptique. Pour y parvenir, des solutions aqueuses de sels d’acides organiques de lactate de calcium, d’ascorbate de calcium et de leur mélange équimolaire ont été électro-activées dans un réacteur soumis à un courant électrique continu avec des intensités de l’ordre de 250, 500 et 750 mA pendant un maximum de temps de 30 minutes afin de produire les acides organiques conjugués respectifs; de l’acide lactique et de l’acide ascorbique. Dans la première partie de ce travail de recherche, les caractéristiques physicochimiques (e.g., pH, acidité titrable, pKa) des solutions électro-activées (SÉA) ont été étudiées et leurs profils moléculaires comparés à ceux d’acides standards respectifs en utilisant différentes techniques (e.g., FTIR, HPLC, DSC, DPPH), ce qui a permis de confirmer la production d’acides organiques conjugués respectifs des sels utilisés. Ces SÉA avaient un pH très bas, une acidité titrable élevée, notamment pour l’ascorbate de calcium et le mélange. En plus, une activité antioxydante élevée a été observée pour la solution électro-activée d’ascorbate de calcium et du mélange. Dans la deuxième partie de l’étude, les SÉA traitées à 250, 500 et 750 m Apendant 10, 20 et 30 min ont été retenues pour être mises en contact avec des cellules végétatives de Bacillus cereus ATCC 14579 en conditions modèles (contact direct) afin d’évaluer leurs effets antimicrobiens sur ce pathogène. Les cellules ont été testées encontact direct avec les SÉA pendant 5, 30 et 60 secondes. Le même traitement a été également réalisé par contact direct avec des acides organiques standards (lactique,ascorbique) pendant 5, 30, 60, et 120 secondes afin de faire des comparaisons. Les SÉA et les acides organiques standards correspondants avaient les mêmes valeurs d’acidité titrable. Par la suite, les cellules ont été observées au microscope (coloration au bleu de méthylène et fluorescence) afin d’évaluer les effets inhibiteurs/destructeurs de ces solutions. Également, les SÉA ont été diluées avec de l’eau distillée pour obtenir des solutions possédant 10 à 90% de l’acidité titrable (force) initiale pour être ensuite testées contre les cellules de B. cereus. Les résultats ont démontré que toutes les SÉA avaient une grande efficacité contre les cellules végétatives de B. cereus. Également, même à des taux de dilution représentant en moyenne 20% de la force initiale des SÉA, l’effet antimicrobien vétait très élevé pour les différentes solutions. L’observation de B. cereus au microscope a permis de confirmer les effets létaux des SÉA. Dans ce volet avec des cellules végétatives de B. cereus, l’efficacité des SÉA a été estimée à une réduction de 4–7 log UFC/mL. En plus, il a été démontré que le pouvoir antibactérien des SÉA était nettement plus élevé que celui des acides lactiques et ascorbiques standards (conventionnels). Dans la troisième partie de cette étude, des solutions électro-activées de lactate de calcium, d’ascorbate de calcium et de leur mélange équimolaire à 750 mA pendant 30 minutes ont été retenues et utilisées contre des spores de Bacillus cereus ATCC 14579 en conditions modèles et dans du saumon Atlantique frais. Les spores traitées ont été analysées à l’aide de microscopes électroniques à balayage et à transmission pour évaluer les effets sporicides des SÉA. Les résultats obtenus ont clairement montré un grand pouvoir sporicide des SÉA utilisées sur les spores de B. cereus avec une réduction de 7 à 9 log en utilisant une population initiale de spores de 109 UFC/mL, dépendamment des conditions évaluées; à savoir : en contact direct (2–30 min), dans du saumon utilisé comme matrice alimentaire(2–7 min), ainsi qu’en combinaison avec de la chaleur modérée de 60, 70, 80 et 90 °C pendant 0.5–2 min. Également, il a été observé que la capacité sporicide des SÉA augmentait avec la température et le temps de contact. La microscopie électronique à balayage et à transmission a permis de constater que les SÉA pouvaient provoquer la destruction totale des cellules de B. cereus, et notamment la perforation de la membrane(cortex et manteau), ainsi que le reflux de différentes composantes de la structure des spores de B. cereus. Tenant compte des résultats obtenus dans cette étude, nous pouvons conclure que les solutions électro-activées à base de lactate de calcium, ascorbate decalcium et leur mélange, notamment celles électro-activées à 750 mA–30 min, pourraient être d’une grande contribution afin de renforcer la capacité de l’industrie alimentaire à lutter contre B. cereus ATCC 14579 et de produire des aliments plus sécuritaires pour le consommateur.
Since the popularization of concepts like globalization, the agri-food sector has experienced a huge expansion and a ceaseless craze for the marketing of food between the peoples worldwide. This phenomenon, contributing significantly to the economic growth ofthe markets, is not without risk, however. Meanwhile, microbiological hazards, including pathogens, are carried through food matrices and travel from one country to another,increasing the risk of contamination for consumers. Consequently, we are also witnessing an increase in cases of food allergies, foodborne illnesses and outbreaks, with etiological agents coming from all over the world. Thus, regulatory organisms such as Canadian Food Inspection Agency (CFIA), Health Canada, United States Food and Drug Administration(USFDA) or competent international authorities like Food and Agriculture Organization ofthe United Nations (FAO) and World Health Organization (WHO) are stepping up efforts to put in place regulatory standards and policies in order to help the food industry to strengthen controls from the processing to the marketing of foods. Microbiological hazards from pathogens like Bacillus cereus remain a major public health risk that must be controlled in order to ensure consumers protection. Although many techniques of control(e.g., food additives, high hydrostatic pressure, ionizing radiation, thermal processes, etc.) have been developed and used to ensure the safety and security of foods, in some instance this has not allowed to produce food products that are completely free of bacteria responsible for degradation/spoilage of food and pathogens causing food poisoning as is the case with B. cereus. Indeed, this pathogenic bacterium is ubiquitous, aerobic and facultative anaerobic. It is able to produce, in a wide variety of foods and ingredients such as spices,highly resistant spores as well as different types of toxins that can cause diarrhea, nausea,vomiting, and even death. In this context, and given the great difficulty in controlling the contamination of food caused by this pathogen, the general objective of this research was touse the electro-activation technology, an applied branch of electrochemistry which is particularly interested in the reactivity of aqueous solutions, as an alternative and potentially effective method to fight against B. cereus in order to produce safer foods with high nutritional and organoleptic values. To achieve this, aqueous solutions of organic acid salts of calcium lactate, calcium ascorbate and their equimolar mixture were electroactivated in a reactor subjected to a direct electric current with intensities of 250, 500 and 750 mA for a maximum time of 30 minutes in a bid to produce the respective conjugated organic acids, lactic acid and ascorbic acid. In the first part of this research work, the physicochemical characteristics (e.g.,pH, titratable acidity, pKa) of the electro-activated solutions (EAS) were studied and their molecular profiles compared to those of respective standard acids using different techniques (e.g., FTIR, HPLC, DSC, DPPH), which helped to confirm the production of conjugated organic acids from the respective salts used. These EAS had a very low pH, ahigh titratable acidity, particularly for the calcium ascorbate and the mixture. In addition, a high antioxidant activity was observed for the electro-activated calcium ascorbate solution and the mixture In the second part of the study, the EAS treated at 250, 500 and 750 mA for 10,20 and 30 min were selected to be brought into contact with vegetative cells of Bacilluscereus ATCC 14579 under model conditions (direct contact) in order to evaluate their antimicrobial effects on this pathogen. The cells were tested in direct contact with the EASfor 5, 30 and 60 seconds. The same treatment was also carried out by direct contact with standard organic acids (lactic, ascorbic) for 5, 30, 60, and 120 seconds in order to make comparisons. The EAS and the corresponding standard organic acids had the same titratable acidity values. There after, the cells were observed under microscope (Methyleneblue and fluorescence) to evaluate the inhibitory / destructive effects of these solutions.Also, the EAS were diluted with distilled water to obtain solutions with 10 to 90% of the initial titratable acidity (strength) to be tested against B. cereus cells. The results demonstrated that all the EAS made were highly effective against the vegetative cells of B.cereus. Also, even at dilution rates averaging 20% of the EAS initial strength, the antimicrobial effect was very high for the different solutions. In addition, the microscopic observation of B. cereus has confirmed the lethal effects of EAS. In this part with the vegetative B. cereus cells, the efficacy of the EAS was estimated to a reduction of 4–7 logCFU/mL. In addition, the antibacterial power of the EAS has been shown to be significantly higher than that of the standard (conventional) lactic and ascorbic acids.In the third part of the study, electro-activated solutions of calcium lactate,calcium ascorbate and their equimolar mixture at 750 mA for 30 min were selected and used against the spores of Bacillus cereus ATCC 14579 under model conditions and in fresh Atlantic salmon. The treated spores were analyzed using scanning and transmission electron microscopes to evaluate the sporicidal effects of EAS. The results obtained clearly showed a great sporicidal power of the EAS used on B. cereus spores with a reduction of 7to 9 log using an initial spore population of 109 CFU/mL, depending on the conditions assessed; namely: in direct contact (2–30 min), in salmon used as a food matrix (2–7 min),as well as in combination with moderate heat of 60, 70, 80 and 90 ℃ for 0.5–2 min. Also, it was observed that the sporicidal capacity of the EAS increased with temperature and contact time. Scanning and transmission electron microscopy showed that the EAS could cause the total destruction of B. cereus cells, including perforation of the membranes(cortex and coat), as well as the reflux of different components of the structure of B. cereus spores. Taking into account the results obtained in this study, we can conclude that the electro-activated solutions made with calcium lactate, calcium ascorbate and their mixture,especially those electro-activated at 750 mA–30 min, could be of a great contribution to reinforce the capacity of the food industry to control B. cereus ATCC 14579 and produces afer foods for the consumer.

Zoospore production of Pythium dissotocum Drechs. in the nutrient solution of hydroponically-grown lettuce, in the greenhouse, was shown to be cyclic. The number of zoospores detected in the nutrient solution was lowest around noontime, (11:00-14:00 hr) and highest around 20:00 hr. Growth chamber studies were conducted to determine the effect of different light periods on zoospore production. Under continuous light or continuous darkness, the population of zoospores in the nutrient solution decreased. But under 12 hours light, and 12 hours darkness or two periods of light each for 3 hours, zoospore populations decreased during the light period but increased during the dark period.

Plant roots generate weak electrical fields in the rhizosphere due to the movement of ions, particularly protons, into and out of their tissues. It has been postulated that zoosporic fungi pathogenic to plants may utilise these electrical fields in targeting suitable host plants to infect. An experimental system is described which permits the application of low voltage electrical fields to zoospore suspensions. The resulting electrotactic response is shown to occur at field strengths of a magnitude similar to those generated by plant roots. In addition, pH taxis and electrophoresis/electroosmosis are shown not to occur in the system used. Electrotaxis is influenced by electrical field strength, ambient pH, and zoospore population density. Zoospores of Phytophthora palmivora swim consistently to the anode of an applied electrical field. Zoospore populations of a relatively high density form multi-cell clumps, or auto-aggregates. This is a result of active taxis and is shown to be density-dependent and influenced by pH. In addition, auto-aggregation is genus-specific and requires calcium ions to occur. It is postulated that auto-aggregation may enhance zoospore population survival. In addition, observations suggest that aggregation on roots, as well as being Ca2+-dependent, acts to enhance inoculum potential, i.e. to increase the number of zoospores reaching a plant root. The electrical activity of various plant roots is assessed. The primary host of Ph. palmivora, Theobroma cacao (cocoa), is shown consistently to display an outward electrical current (proton flow) at its zone of elongation, thus rendering this region anodic (positively charged). The same is true of some non-host plants e.g. Lolium perenne (perennial rye grass), Hordeum vulgare (barley) and Eucalyptus pilularis (eucalyptus).

The work in this thesis was designed to study the role of Ca²⁺ in zoospore biology, and extends previous findings by comparing effects of Ca²⁺ and Ca²⁺ -modulating treatments in zoospores and zoospore cysts of Pythium spp., Phytophthora spp. and Aphanomyces euteiches. Zoospore motility was studied by videomicroscopy to compare the behaviour of Phytophthora infestans, Ph. parasiticia and Ph. plamivora aphanidermatum, Py. dissotocum, Py. catenulatum and A. euteiches. Zoospores of all fungi swam in an extended helix, but the Phytophthora spp. and A. euteiches made frequent changes of direction when swimming in water or buffer, whereas the Pythium spp. did not do so. Chelation of external Ca²⁺ with EGTA or BAPTA buffer caused zoospores of all fungi to swim in a straight path, reduced random turning, and prevented the spores from flanging direction when they collided with obstacles. Phytophthora zoospores accumulated at the surface of suspensions, but this was abolished in the presence of EGTA, whereas Pythium zoospores always accumulated at the base of suspensions. Isotropic backgrounds of some amino acids (e.g. L-glutamic acid) induced an irregular motility pattern; this was overridden by EGTA but restored by subsequent addition of Ca²⁺. These findings are discussed in relation to host-location behaviour by zoospores. A role of Ca²⁺ in germination of cysts, induced by agitation of zoospores of Py. aphanidermatum and Ph. parasitica, was demonstrated by reduced or suppressed germination in the presence of Ca²⁺ -uptake inhibitors (La³⁺, verapamil), Ca²⁺ chelators (BAPTA), calmodulin-antagonists (trifluoperazine, calmidazolium) or compounds that affect intracellular Ca²⁺ stores (caffeine, TMB-8). The critical concentrations and times of application of these treatments were determined, and some divalent cations and organic nutrients (sugars, amino acids) were shown partly to reverse the suppression of germination. A link was demonstrated between Ca²⁺ fluxes and zoospore development into cysts.

The plasmodiophorid organism, Spongospora subterranea f. sp. nasturtii, is the causal agent of crook root disease of watercress (Rorippa nasturtium-aquaticumy. The only current control measure is zinc, used such that levels do not exceed 0.07S~g mrl in effluent water. Laboratory results indicated that zinc acts by actively lysing zoospores of S. subterranea, but does not cause 100% mortality at field concentrations. At 8ug ml", around 20 % of zoospores were seen to survive compared to controls. Of other cations investigated as potential control agents, none were as effective as zinc at equivalent levels. Only cobalt was capable of significant zoospore lysis. Attraction of zoospores to watercress roots and total root extracts appeared to be non-specific when compared to attraction to tobacco (Nicotiana rustica), tomato (Lycopersicon esculentum), and mustard (Brassica napus). Results implied that a general plant attractant was present, and that this did not mask a more specific attractant. A molecular, PeR-based diagnostic test was developed for S. subterranea f. sp. nasturtii. The internal transcribed spacer (ITS) and I8S ribosomal DNA were used to design specific peR primers, capable of detecting DNA directly from zoospores. As little as Sng genomic DNA or 100 zoospores were required as template. Efforts to develop a sampling technique for zoospores in watercress beds were successful based on washing root material followed by peR. DNA analysis also identified a Chytridiomycete-like organism as being consistently associated with crooked roots. However, preliminary studies showed that it did not have a direct role in crook root disease. The I8S ribosomal DNA sequence was' used to infer phylogeny of S. subterranea f. sp. nasturtii, and plasmodiophorids generally, by performing parsimonious and distance-based analyses. Within the plasmodiophorid grouping, S. suhterranea f. sp. nasturtii appeared to be closely related to S. suhterranea f. sp. suhterranea and Plasmodiophora brassicae, based on 270 bases at the 3' end of the gene, whilst Polymyxa species aligned on a separate branch. Based on a complete I8S ribosomal DNA analysis, plasmodiophorids seemed to form a discrete taxonomic grouping, not closely linked to either protists or fungi.

Zoosporic fungi (chytrids) are common within soils. Chytrids contribute to the soil carbon cycle by converting complex carbohydrates to more accessible forms and are also important in biogeochemical cycling of nitrogen and sulphur. However, the roles of chytrids in many soil processes, and their responses to environmental stress, are not well understood. In order to explore the response of chytrids to temperature stress, here we initially examine the effect of increased temperature on chytrid isolates from different geographic and climatic regions. Isolates of Gaertneriomyces semiglobifer from different regions grow at similar rates and have similar patterns of zoospore production at different temperatures. This data allows prediction of the distribution, growth and abundance of the fungus and potential changes due to the effects of climate change. We then examine the effect of the toxic metals Cu (ll), Pb (ll) and Zn (ll) on growth, zoospore production and attachment of chytrids to common organic substrates. The four isolates, representing four orders within the phylum Chytridiomycota, showed greatest sensitivity in growth, attachment and zoospore production in response to Cu (ll) and least sensitivity to Pb (ll). Interestingly, some metals also caused increases in growth, zoospore production and attachment below the toxic threshold concentrations. In particular, Rhizophlyctis rosea increased the number and length of rhizoids when incubated with Pb (ll). Chytrids are known to be widespread and common throughout soils world-wide. Our work allows us to predict that the levels of Cu (ll), Pb (ll) and Zn (ll), found here to be toxic, will be detrimental to soil chytrid populations and reduce colonisation of organic substrates. Toxic effects of metals on the lifecycle of chytrids are expected to reduce the rate of mineralisation of soil organic matter, thereby reducing nutrient availability for the soil microbial loop, to the detriment of ecological function in soils.

In laboratory experiments, Ca²⁺, other divalent cations and various Ca²⁺-modulating treatments were tested for effects on germination of zoospore cysts and of sporangia of three isolates of Phytophthora infestans and sometimes for an isolate of P. palmivora. The aim was to determine whether manipulation of Ca²⁺ or Ca²⁺-mediated events has the potential for control of potato late blight caused by P. infestans. This work was based on previous evidence for a central role of Ca²⁺ in the infection sequence from zoospores of phytopathogenic Phytophthora, Pythium and Aphanomyces spp. Zoospore cyst germination of both P. infestans and P. palmivora was suppressed by early post-encystment addition of the chelators EGTA or BAPTA, indicating a requirement for external Ca²⁺ or other polyvalent cations, by calcium channel blockers (La³⁺, Gd³⁺, verapamil) or by amiloride, indicating a requirement for flux of Ca²⁺ or other ions across the cyst membrane, by calmodulin antagonists (calmidazolium, dibucaine, trifluoperazine) and by intracellular calcium antagonists (caffeine, TMB-8), indicating a role for both calmodulin and for Ca²⁺ release from intracellular stores. Supplements of Ca²⁺ or other divalent cations (Ba²⁺, Mg²⁺) also suppressed cyst germination, but sometimes partly or completely overcame the suppression by other treatments if applied early as post-treatments. Germination of sporangia of P. infestans by hyphal outgrowth (direct germination, at c. 20°C) or zoosporogenesis (indirect germination, at c. 12°C) was suppressed by the same treatments as applied to zoospore cysts. These treatments sometimes caused rapid sporangial death, assessed microscopically by irreversible changes in cytoplasmic organisation. Their suppressive or toxic effects were generally more pronounced when sporangia were incubated to induce indirect rather than direct germination. The suppression caused by Ca²⁺-modulating treatments could be rescued only partly by simultaneous or early post-application of divalent cations. Even potentially mild chemical treatments (0.1% pectin or 5 mM orthophosphate) caused rapid (20-30 min) death of sporangia (especially when incubated for indirect germination). Simultaneous or early post-treatments with divalent cations could only partly reverse the suppression.

With the aim of finding simple and effective disease-control measures, laboratory experiments described in this thesis tested the effects of various cations (Ca²⁺, Mg²⁺, K⁺) and potentially fungitoxic compounds (saponins, gramicidin S, ethanol) on different stages of the life cycle of two pathogenic zoospore-forming fungi, Pythium aphanidermatum and Phytophthora parasitica. Also, nutrient irrigation solutions from various experimental treatments in a large tomato-cropping glasshouse trial at Horticultural Research International (HRI, Yorkshire) were tested for effects on the two fungi in laboratory conditions. Cations, fungitoxic compounds and experimental irrigation solutions were tested for effects on the following aspects of fungal behaviour: mycelial growth, production of sporangia, release of zoospores from sporangia, zoospore motility, zoospore encystment and cyst germination. When tested on individual stages of the life cycle, high concentrations of Ca²⁺ and Mg²⁺ in nutrient broth reduced mycelial growth by Py. aphanidermatum but not Ph. parasitica. Sporangial production by Ph. parasitica in mineral salts solution was unaffected by amendments of Ca²⁺, Mg²⁺ or K⁺, but these amendments suppress the ability of sporangia to subsequently liberate zoospores into water; the exception was 5 mM Ca²⁺ which was markedly enhanced subsequent zoospore release. Increasing concentrations of Ca²⁺, Mg²⁺ and K⁺ in the solution that bathed pre-formed sporangia of either fungus reduced the number of zoospores that were released. These three cations also suppressed the proportion of zoospores that remained motile, and increased the proportion of vortex-encysted zoospores that would germinate. When tested cumulatively on all stages of the life cycle K⁺ was more effective than Ca²⁺ in suppressing the infection related behaviour of both fungi. These experiments suggest that increasing the ratio of K⁺ to Ca²⁺ in bathing solutions should suppress the ability of both fungi to spread and cause disease. The trial undertaken by HRI failed to demonstrate natural suppression in a semi-commercial irrigation system, which was the principal trial objective. Consequently the analyses presented in this thesis were unable to determine at which points of the infection sequence the agents of natural suppression might act.

A proteína de choque térmico Hsp90 é uma chaperone molecular encontrada no citosol. O cDNA incompleto desta proteína foi isolado de uma biblioteca construída a partir de mRNA de células de esporulação de B. emersonii submetidas a choque térmico. Um clone genômico contendo a seqüência completa do gene hsp90 também foi isolado, seqüenciado e caracterizado. A região codificadora do gene hsp90 é interrompida por um único íntron de 184 nucleotídeos. A seqüência de aminoácidos deduzida indicou uma proteína de 71 O resíduos, com massa molecular calculada de 80.792 Da e um pl médio de 4,85. Experimentos de extensão de oligonucleotídeo e RACE-PCR demonstraram um sítio único de início de transcrição localizado a -65 e -70 nucleotídeos do ATG da metionina iniciadora, respectivamente. Motivos similares ao consenso do elemento de choque térmico eucariótico (HSE) e do elemento responsivo a estresse (STRE) foram encontrados na região promotora do gene a -395 e -98 nucleotídeos do ATG, respectivamente. Experimentos de \"Northern blot\" revelaram que o mRNA para a Hsp90 apresenta níveis máximos aos 90 minutos da fase de esporulação do fungo. Análise por \"western blot\" mostrou que a proteína Hsp90 está presente durante todo o ciclo de vida do fungo e os níveis máximos de acúmulo foram observados aos 90 minutos da esporulação, indicando um controle transcricional do gene. Tanto a proteína quanto o mRNA são altamente induzidos quando as células são submetidas a choque térmico e a cádmio. As proteínas Hsp60 e Hsp10 são chaperones moleculares mitocondriais (chaperoninas). Os cDNAs completos destas proteínas foram isolados e totalmente seqüenciados. A seqüência de aminoácidos deduzida da Hsp60 corresponde a uma proteína de 559 resíduos, com massa molecular calculada em 58.741 Da e um pl médio de 8, 7. Experimentos de \"Northern blot\" revelaram que o mRNA para Hsp60 tem níveis máximos de expressão aos 90 minutos da esporulação. Análise por \"western blot\" mostrou que a Hsp60 está presente durante todo o ciclo de vida do fungo, com níveis máximos da proteína 90 minutos após a indução da esporulação. Tanto a proteína quanto o mRNA são bastante induzidos quando as células são submetidas ao choque térmico. A seqüência de aminoácidos deduzida da Hsp10 corresponde um polipeptídeo de 101 resíduos com massa molecular calculada em 10.688 Da e um pl médio de 6,25. Experimentos de \"Northern blot\" revelaram que o mRNA para Hsp10 tem níveis máximos de expressão aos 120 minutos da germinação e é bastante induzido quando as células são submetidas ao choque térmico.
The heat shock protein 90 (Hsp90) is a cytosolic molecular chaperone. The incomplete cDNA of this protein was isolated by immunoblot screening of a heat shock cDNA expression library. The complete genomic clone was also isolated and completely sequenced and characterized. The coding sequence is interrupted by a single intron with 184 nucleotides. The deduced amino acid sequence corresponds to a 710-residue polypeptide with a calculated molecular mass of 80,792 Da and an average pl of 4.85. Primer extension and RACE-PCR experiments demonstrated a single transcription start site localized -65 and -70 nucleotides from de ATG of the initiator methionine, respectively. Sequence motifs resembling the standard eukaryotic heat shock element (HSE) and the stress responsive element (STRE) were evident in the regulatory region -395 and -98 nucleotides from de ATG, respectively. Northern blot analysis revealed that the Hsp90 mRNA presents maximum levels by 90 minutes of the sporulation stage. Immunoblot analysis indicated that the Hsp90 is present during the entire life cycle of the fungus and maximum levels were observed 90 minutes after the induction of sporulation, indicating a transcriptional control. During heat shock both the mRNA and the Hsp90 protein are highly induced. Proteins Hsp60 and Hsp10, are mitochondrial molecular chaperones (chaperonines). The complete cDNAs encoding these proteins were and completely sequenced. The deduced amino acid sequence for Hsp60 corresponds to a 559-residue polypeptide with a calculated molecular mass of 58,741 Da and an average pl of 8.7. Immunoblot analysis showed that Hsp60 is present during the entire life cycle of the fungus and presents maximum levels by 90 minutes of the sporulation. Northern blot analysis indicated maximum levels of the Hsp60 mRNA by 90 minutes of sporulation too. Both mRNA and the protein are highly induced during heat shock. The deduced amino acid sequence for Hsp10 corresponds to a 101-residue polypeptide with a calculated molecular mass of 10,688 Da and an average pl of 6.25. Northern blot analysis indicated maximum mRNA levels by 120 minutes of germination and high levels of expression when the cells are exposed to heat shock.

Secretion of pathogen proteins is crucial for establishment of disease in animals and plants. Typically, early interactions between host and pathogen trigger regulated secretion of pathogenicity factors that function in pathogen adhesion and host penetration. During the onset of plant infection by spores of the Oomycete, Phytophthora nicotianae, proteins are secreted from three types of cortical vesicles. Following induction of spore encystment, two vesicle types undergo full fusion, releasing their entire contents onto the cell surface. However, the third vesicle type, so-called large peripheral vesicles, selectively secretes a small Sushi domain-containing protein, PnCcp, while retaining a large glycoprotein, PnLpv, before moving away from the plasma membrane. The experiments described in this thesis aim to characterize the differential secretion and retention of proteins from the large peripheral vesicles in Phytophthora spores and to investigate the possible mechanisms underlying the selective secretion of large peripheral vesicle proteins. Green fluorescent protein (GFP)-tagged PnCcp proteins were expressed in P. nicotianae by genetic transformation in order to study the subcellular localization and secretion of PnCcp in living cells. Analysis of drug-resistant transformants indicated that sometimes the PnCcp-GFP transgene was not expressed and at other times the fusion protein was not fluorescent or was mislocalized to the cytoplasm or the extracellular space other than the large peripheral vesicles. Double-immunofluorescence labelling of P. nicotianae spores both in vitro and in planta confirmed that PnCcp was secreted from the large peripheral vesicles while PnLpv was retained during encystment. Immunolocalization studies of P. nicotianae mycelia revealed that the synthesis of PnLpv precedes that of PnCcp and PnCcp is added to large peripheral vesicles after PnLpv. By immunofluorescence labelling and enzyme-linked immunosorbent assays (ELISAs), the majority of the PnCcp protein was detected to be secreted within 1-2 min of the onset of encystment and secretion of PnCcp occurred at a similar time period as that of ventral and dorsal vesicle proteins. Selective secretion of PnCcp is associated with its compartmentalization within the vesicle periphery. Pharmacological inhibition of dynamin function, purportedly in vesicle fission, by dynasore treatment provides evidence that selective secretion of PnCcp utilizes a kiss-and-run, transient fusion mechanism similar to that described in animal cells. This is the first report of transient fusion and selective protein secretion outside mammalian cells. Results also show that full or transient fusion of different populations of vesicles is triggered simultaneously by the same signal. The research in this thesis expanded our understanding of regulated protein secretion in Phytophthora and other systems.

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

Plants and their pathogens are continually co-evolving in what has been referred to as an evolutionary arms race. In this struggle between pathogen and host, the adaptation of one can lead to a selective pressure and in turn a counter-adaptation of the other. The selective pressure on secreted pathogen proteins is particularly high as these proteins have functions vital to successful infection and maintaining a parasitic relationship. In this thesis a proteomics approach was taken to investigate the proteins secreted during the initial stages of infection of Phytophthora nicotianae. This pathogen belongs to the Oomycetes, which includes many destructive plant pathogenic species. The diseases caused by these species affect many different plant hosts and impact not only agricultural production but also native ecosystems. During the initial stages of plant infection by P. nicotianae, motile zoospores locate an appropriate infection site and undergo encystment where they detach both flagella, form a cell wall and secrete proteins from three types of peripheral vesicles found in the zoospore cortex. In Chapter 2, the timing of synthesis of four proteins (Cpa, Lpv, PnCcp and Vsv) found in these vesicles was investigated during asexual sporulation. The results showed differences in the time at which Cpa, Lpv, PnCcp and Vsv were synthesised. In addition, the timing of expression of Lpv, PnCcp and Vsv genes was examined in sporulating hyphae, zoospores and 3h germinated cysts. It was found that these genes had different expression patterns. In preparation for the identification and characterisation of P. nicotianae transformants in which one of these secreted proteins, namely PnCcp, was silenced, assays for screening and analysing transformants were developed. While sequence motifs in PnCcp and Vsv indicate that these two proteins are likely to have adhesive properties, their function has not been directly demonstrated. In Chapter 3, an RNAi gene silencing approach was used to investigate the function of PnCcp. Chapter 4 focused on an investigation of protein secretion during zoospore encystment. Firstly, spatial and temporal patterns of secretion of the four peripheral vesicle proteins were determined. Lpv was found not to be secreted and there were differences in the timing of secretion of the other three proteins with Cpa being secreted earlier than Vsv and PnCcp. Secondly, proteomic and bioinformatic approaches were used to investigate proteins secreted during zoospore encystment. This analysis led to the identification of a number of proteins in extracts solubilised from encysted zoospores and 29 of these were predicted to have N-terminal secretion signals by SignalP. The majority of these proteins had unknown functions, highlighting the need for more research in this area. Other putative secreted proteins were involved in carbohydrate and protein metabolism. Together, the research reported in this thesis sheds new light on the synthesis and secretion of zoospore vesicle proteins and provides a foundation for further research aimed at characterising the function of these proteins.

Phytophthora pini Leonian, recently re-established from P. citricola I, is a pathogen with a wide range of forest and nursery hosts. It causes foliar infections in horticultural nurseries in Oregon, where recirculating irrigation systems are common. Increased use of recirculating irrigation systems may contribute to disease caused by waterborne plant pathogens. Simulated nursery chamber experiments were utilized to investigate the relationship between Phytophthora pini zoospore inoculum dose and disease on Rhododendron. Disease incidence in this system was unexpectedly low despite high inoculum levels tested, so further experiments under lab conditions were conducted to explore possible causes. Detached leaf assays were conducted to determine how inoculum dose, leaf wounding, and agitation of zoospore inoculum affected foliar infection of Rhododendron. Wounded and nonwounded leaves were dipped into suspensions of zoospores that were either untreated, mechanically agitated by vortexing to cause encystment, or pumped through an irrigation sprayer system. Disease severity (lesion area) and incidence (number of lesions per leaf area) were measured over seven days. At inoculum levels of ���10,000 propagules/mL, motile zoospores infected both wounded and nonwounded leaves. Vortexing or pumping resulted in zoospore encystment, and inoculation with these treatments caused disease almost exclusively on wounded leaves. No disease symptoms were observed following inoculation with any inocula at ��� 2,000 propagules/mL. Scanning electron microscopy of leaves inoculated with encysted propagules showed germinated cysts with hyphae growing over and around stomata without entering leaf tissue until reaching a wound site. Nonwounded leaves inoculated with motile spores showed stomata penetrated by hyphae. These findings indicate the importance of zoospore motility in reaching suitable infection sites, and demonstrate the impact of zoospore encystment on disease development. This has implications for disease management in nurseries where pruning wounds are common and the pumping of infested irrigation water may influence zoospore motility and infectivity.
Graduation date: 2013

碩士
國立臺灣海洋大學
海洋生物研究所
101
Thraustochytrids are unicellular, eukaryotic marine protists and belong to the Kingdom Chromista. Sodium is an obligate requirement for growth of thraustochytrids. They are widely distributed in the ocean, estuary, inland salt lakes and mangroves. They possess: (1) sagenogenetosome, (2) multilamellate cell wall, and (3) biflagellate zoospores. In the environment, thraustochytrids can produce extracellular enzymes that decompose leafy tissues for nutrition. No sexual reproduction has been reported for thraustochytrids. In Taiwan, high level of heavy metals has been detected in many mangroves and it has been proven to affect morphology and physiological of thraustochytrids. In this study, we investigated the effects of various concentration of Cu (II) ion on growth and production of zoospores of thraustochytrids. Fifteen Schizochytrium isolates from four mangroves, including Tamsui in New Taipei City, Xinfeng in Hsinchu, Chunan in Miaoli, and Beimen in Taiwan. Results showed that the growth of Schizochytrium spp. ( IMB176、177、178、179、180、182、183、184、185、186、187、188 ) was inhibited at 32 ppm Cu (II), while two other isolates of Schizochytrium spp. IMB174 and, IMB181 were inhibited at 16 ppm Cu (II). Production of zoospores under the influence of Cu (II) by different Schizochytrium islolates varied. Schizochytrium spp. IMB174、IMB182、IMB183、IMB185 only produced zoospores at concentration below 2 ppm Cu (II). Schizochytrium spp. IMB176、IMB179、IMB181、IMB184、IMB186、IMB188 were capable of producing zoospores up to 128 ppm Cu (II). Schizochytrium spp. IMB175、IMB177、IMB178、IMB180、IMB187 could produce zoospores up to 256 ppm Cu (II). Results suggested that there were different responses on growth and sporulation by the fifteen Schizochytrium isolates under various Cu (II) concentrations. The results of this study will contribute to the understanding of heavy metal pollutants on the ecology of this important group of organisms.

碩士
國立中央大學
化學工程與材料工程學系
107
In the past few decades, elastic modulus, surface free energy and zwitterionic materials have been a key element of the development of marine antifouling coatings. This study combines a low elastic modulus material (polydimethylsiloxane, PDMS) and a zwitterionic material (sulfobetaine silane, SBSi) as a new anti-marine biofouling. Barnacle cypris larva (Amphibalanus amphitrite) and Ulva zoospores (Ulva lactuca, Ulva fasciata) are used as research species against marine biofouling. This study mainly carried out video tracking observation of cyprids (barnacle cypris larva), cyprids settlement assay and Ulva zoospores settlement assay, which used glass, fluorosilane, PDMS and SBSi. In the video tracking, the film showed that it had no tendency to adhere or could not adhere to the surface of PDMS-SBSI. At the same time, only 2% of the cyprids settled on the surface of the PDMS-SBSI for two days. In the settlement assay of Ulva zoospores, the attached number of zoospores is lower on the surface of PDMS-SBSI and the 92% of the attached zoospores removed after water flow impact. The study result indicate that the combination of PDMS and SBSI does have anti-contamination against barnacle cypris larva and Ulva zoospores, also, this combination exhibits superior fouling release characteristics when against Ulva zoospores. Based on these measurements, we will further investigate the stain release of PDMS-SBSI on barnacle cypris larva, or in-depth study of surface chemistry, using surface plasma resonator (SPR) to detect adhesion-induced protein complex (SIPC) adsorption on PDMS-SBSI.

Spongospora subterranea infection diminishes the quality and quantity of harvested potatoes wherever they are grown around the world. Disease control is largely ineffective, at least partially due to the abundance of the highly resistant and long-lived pathogen inoculum within cropping soils present as agglomerations of resting spores. Resting spore germination is induced by root exudate metabolites in a staggered manner over multiple years to release short-lived zoospores that facilitate infection of host plant roots. Premature germination of resting spores in absence of host plants has been suggested to reduce pathogen soil inoculum. Methods to synchronize germination of dormant spores that could be applied within a commercial field setting are however lacking. Movement of zoospores to host roots has been speculated to occur via chemical attraction processes (chemotaxis) but evidence for this has been lacking. Knowledge of zoospore attractants and/or repellents, and the communication channels that facilitate perception of these compounds may be useful for the development of novel disease control. This thesis developed a microcapillary assay to investigate the role of individual potato metabolites in the attraction or inhibition of S. subterranea zoospores. In relation to this, the effect of solution pH, pH buffer composition and compound concentration on zoospore chemotaxis were assessed. Zoospore attraction or inhibition towards a metabolite was determined by the number of zoospores that swam into a cavitated microcapillary containing the test compound. The potato metabolome was found to contain both zoospore attractants and inhibitors. Glutamine was the strongest attractant, whilst spermine was the strongest inhibitor. Strongly acidic and alkaline medium with pH < 5.3 and > 8.5 respectively were found inhibitory towards zoospore motility and chemotaxis. Total swimming distance was found to be the most important swimming parameter that determined zoospore chemotaxis. The role of Ca2\(^+\) signalling in transducing signals from these organic compounds across the cell membrane to stimulate or inhibit chemotaxis of the zoospores was also investigated. This study used a combination of glass slide assay, cavitated microcapillary assay, hydroponics set up and video microscopy to investigate the effect of Ca2\(^+\) antagonism on zoospore chemotaxis, attachment, root infection, and swimming patterns. Zoospore swimming patterns were altered, whilst chemotaxis, root attachment and zoosporangia root infection were constrained by inhibition of cytosolic Ca2\(^+\) fluxes with Ca2\(^+\) antagonist. LaCl3 and GdCl3 at concentrations ≥ 50 µM showed complete inhibition of zoospore chemotaxis, root attachment and zoosporangia root infection. High concentration of extracellular Ca2\(^+\) (≥18 mM Ca(OH)\(_2\)) had similar chemotactic and motility impairments effects as the Ca2\(^+\) channel blockers. Helical swimming patterns observed in the control were always associated with high speed, acceleration, longer distance, high chemotaxis, and disease intensity. The further the deviation from this pattern with Ca2\(^+\) antagonist treatments the slower the speed, acceleration, distance travelled, and the lower the chemotaxis and disease. In a series of field and pot trials, the effects of pre-plant Fe-EDTA, fluazinam and their combination treatments on pathogen inoculum load and disease reduction were studied. Pathogen DNA load and S. subterranea disease intensities and prevalence in tomato and potato were tracked over time to ascertain treatment effect. Soil treatments appeared to have a beneficial effect on pathogen inoculum persistence and disease, with disease incidence diminished to a greater extent than detectable inoculum load. For example, fluazinam (4.5 kg/ha) applied four times a year reduced root infection, galling, powdery scab intensity and prevalence by over 70% but reduced inoculum load by only 11%. Similarly, Fe-EDTA had a positive, but marginal effect on disease control but was relatively ineffective at inoculum load reduction. This study provides key insights into the chemical ecology required for S. subterranea zoospore motility and chemoattraction and identifies potential novel breeding targets for combating Spongospora diseases. It also highlights the role of Ca2\(^+\) signalling in host:pathogen interactions and shows how disruption of Ca2\(^+\) signal transduction could control disease. Further studies into inoculum load reduction with more active and stable compounds could prove useful in the remediation of S. subterranea contaminated fields.