Thesis (M.S.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1961. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Aphanomyces spp. are water moulds, eukaryotic fungus-like organisms, belonging to the class Oomycota. This genus contains primary pathogens of plants and animals as well as opportunistic and saprotrophic species. One of the animal parasites (A. astaci) is the causal agent of the crayfish plague, a disease listed by the World Organisation for Animal Health (OIE). It is believed that A. astaci was first introduced into Italy from the US in the late 19th century and rapidly spread in Europe causing the decline of native crayfish. It currently threatens to wipe out the UK native white-clawed crayfish (Austropotamobius pallipes). Random amplified polymorphic DNA (RAPD-PCR) on pure isolates of A. astaci distinguished five genotypes (A, B, C, D, and E). This distinction proved to be a useful tool for epidemiological studies aimed at understanding the history and spread of the disease in Europe; furthermore, there are differences in virulence among genotypes. No discriminatory morphological or physiological characters are available and widely used markers such as the internal transcribed spacer (ITS), the divergent domains regions (D1-D2) of nuclear large subunit (LSU) rDNA, and cytochrome c oxidase subunit I (COI) also fail to discriminate between A. astaci genotypes. There are some practical drawbacks to genotype by the currently available genotyping methods. Whole genome sequencing (WGS) was used to catalogue DNA single nucleotide variants and genotype-unique genomic regions that could be exploited as phylogenetic markers. These newly developed molecular markers were tested both on pure cultures and historical samples derived from outbreaks and carrier crayfish available in our laboratories, validating these genotyping methods, which represent new diagnostic tools aiding the detection and prevention of crayfish plague.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Wisconsin, 1967. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 18 (1958) no. 3, p. 751-752. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 116-119).

Dans l’objectif de redynamiser la culture des protéagineux, il est primordial d’améliorer la gestion du risque lié à Aphanomyces euteiches, agent responsable de la pourriture racinaire du pois. Le manque de connaissances sur les mécanismes d’infection d’A. euteiches constitue un frein à l’élaboration de stratégies de contrôle durable. Dansl’optique d’étudier les premières étapes du processus infectieux d’A. euteiches, laconstruction d’une souche A. euteiche-GFP a été entreprise. Transfecter A. euteiches aura permis d’assurer (i) la production de protoplastes par digestion enzymatique du mycélium puis (ii) d’insérer le gène gfp par la méthode de transfection chimique PEG-CaCl2 et (iii) de constater l’insertion stable du gène gfp dont l’expression s'est avérée transitoire. En parallèle, dans l’objectif d’apporter des éléments de réponses quant aux rôles des exsudats racinaires et du Root Extracellular Trap (RET) dans les réactions de défense du pois, une étude comparée des interactions entre Pisum sativum (plante sensible) et Vicia Faba (plante tolérante) au pathogène A.euteiches a été réalisée durant les premières phases de l’infection. Alors que de nombreuses variations au niveau de la composition polysaccharidique du RET et des exsudats ont été observées chez le pois, la féverole a présenté des modifications marginales. Chez le pois, l’infection est intense et rapide alors qu’elle semble réduite chez la féverole. La féverole repousse les zoospores tandis que le pois infecté les attire davantage. La féverole semble pouvoir protéger le pois au travers de mécanismes de communication qu’il convient de caractériser. L’ensemble de ces résultats semblent prometteurs dans le développement de méthode de lutte contre la pourriture racinaire du pois. Enfin, l’étude des propriétés bio-physico-chimiques des sols susceptibles de conditionner l’apparition de la maladie ont permis de confirmer la corrélation positive entre la densité d’inoculum du pathogène et le potentiel infectieux (PI) dans des sols naturellement infestés : les sols possédant des teneurs élevées en sable ou en calcium s'avèrent défavorables au développement de la maladie. Cette étude a aussi permis de mettre en évidence une influence possible des communautés microbiennes des sols, susceptibles d’influencer le processus infectieux d’A.euteiches / Aphanomyces euteiches is a pathogenic oomycete considered to be the most damaging root disease of pea crops in the world and there is currently no registered pesticide for its control. Crop management is the most efficient tool to control root rot, and avoidance of infested soil seems to be the optimal solution. Mechanisms related to A. euteiches root colonization remain poorly understood. In order to better understand A.euteiches infectious cycle, a polyethylene glycol (PEG) – calcium chloride (CaCl2) transformation protocol has been perfected in order to stably express the reporter gene GFP. The data show for the first a transient expression of green fluorescent protein (GFP) which can be observed in A.euteiches mycelium, a Saprolegnia oomycete. Vector pGFPN, containing the ham34 promoter and terminator of the Peronospora oomycete Bremia Lactucae, was introduced in A.euteiches protoplasts. Transient expression of GFP could be observed in A. euteiches mycelium by confocal microscopy. qPCR analyses confirmed the actual gfp gene insertion in its genome. Meanwhile, the influence of both pea and faba bean root extracellular trap (RET) and root exudates has been explored for A. euteiches zoospores by chemotaxis assays, microscopic observations and oomycete DNA quantification. Reciprocally pea and faba bean roots responses to A. euteiches infection have been studied at early stage of infection by biochemical analysis of cell wall polymer content in the RET and root exudates. Whereas infected pea root exudates stimulated A.euteiches zoospores attraction, faba bean exudates had a repellent effect on zoospores. In response to infection, arabinogalactan protein content of root pea exudates was altered. Interestingly, A.euteiches colonization was less intense on faba bean root surface and protect pea root at early stage of infection. Finally, the correlation between inoculum quantity in infested and the inoculum potential (IP) in field has been confirmed by qPCR. Analyses of the influence of abiotic soil parameters on the disease showed that a high calcium concentration or sand content negatively impact the IP. Furthermore, microbial communities proved to play a role in the expression of the disease in some soils. Metagenomics could be applied in order to provide new directions in managing this disease.

M.Sc. (Zoology) / Aphanomyces invadans is an oomycete associated with epizootic ulcerative syndrome (EUS). It affects more than a 100 freshwater and estuarine species of fish and is a serious threat to aquaculture and natural aquatic ecosystems. Currently, cases of EUS have been reported across Asia, Australia, North America and more recently Southern Africa. Outbreaks occur mostly during periods of sudden temperature change, such as heavy rainfall or change of seasons. These conditions favour sporulation of A. invadans, and low temperatures have been shown to delay the inflammatory response of fish to oomycete infection. Diagnosis of A. invadans is usually based on clinical signs and confirmed by demonstrating the presence of mycotic granulomas in histological section. Further diagnosis of EUS is made by isolation of A. invadans from internal tissues. Demonstrating typical asexual characteristics by inducing sporulation allows identification of the oomycete to the genus level. After inducing sporulation, the zoospores can be isolated for use in clinical infection of fish through subcutaneous injection or bath challenge systems. Standard molecular techniques such as polymerase chain reaction (PCR) have also been development for the fast and reliable diagnosis of the disease. The aim of this study was to perform different infection trials on EUS in two susceptible fish species, three-spot gourami, Trichogaster trichopterus, and rainbow trout, Oncorhynchus mykiss. Through initial trials, three-spot gourami was established as a suitable positive control species. Subsequently, hyphae were successfully re-isolated from infected fish and demonstrated as A. invadans, which was used in a further inoculation trial. Rainbow trout were challenged with A. invadans through intramuscular inoculation revealing varying degrees of susceptibility at different water temperatures. Detection of fungal hyphae and mycotic granulomas in tissue sections was achieved through histopathological examination, including the use of birefringence and fluorescents. Confirmation of A. invadans DNA in the various infection trials was done through PCR analyses. A histological grading system is proposed which will allow simplification of large scale qualitative microscopic analyses and identification of histological trends within a data set when analysing suspected cases of EUS. It is recommended that similar infection trials be applied to endemic species in Southern Africa to investigate their susceptibility to EUS.

L’azote (N) est un facteur majeur limitant la croissance des plantes. Sa disponibilité peut également avoir un impact sur la résistance des plantes aux pathogènes en régulant leur immunité. Afin de mieux comprendre les liens entre la nutrition azotée et les défenses de la plante, nous avons analysé l’impact de la disponibilité en N sur la résistance de Medicago truncatula à un pathogène racinaire, Aphanomyces euteiches, en prenant en compte la variabilité génétique de la plante. Cet oomycète est considéré comme un des facteurs limitant le plus la production des légumineuses. Deux conditions de nutrition azotée, non limitante ou carencée en N, et dix génotypes de M. truncatula ont été testés in vitro. Les résultats ont montré que la résistance est modulée par les conditions nutritionnelles, dépendament du génotype. Les analyses d’expression de gènes impliqués dans le métabolisme azoté et dans les réponses de défense ainsi que la quantification des teneurs en acides aminés et des composés métaboliques secondaires ont montré des réponses différentes selon les génotypes et la condition nutritive. Elles ont souligné en particulier le rôle potentiellement important de la glutamine dans ce pathosystème. De plus, nous avons mis en évidence l’importance de l’homéostasie du monoxyde d’azote (NO) dans la résistance de M. truncatula à A. euteiches et que la disponibilité en azote impactait l’homéostasie du NO en affectant les niveaux de S-nitrosothiols et l’activité de la S-nitrosoglutathion réductase dans les racines. Ces résultats soulignent l’importance du métabolisme azoté et de son interaction avec le génotype de la plante dans les réactions de défense chez M. truncatula. / Nitrogen (N) is a major limiting factor for plant growth. N availability can also impact plant resistance to pathogens by regulating plant immunity. To better understand the links between N nutrition and plant defense, we analyzed the impact of N availability of plant on Medicago truncatula resistance to the root pathogen, Aphanomyces euteiches, taking into account plant genetic variability. This oomycete is considered as the most limiting factor for legume production. Two conditions of N nutrition, non-limiting or deprived in N, and ten plant genotypes were tested in vitro. The results showed that the resistance is modulated by nutritional conditions, depending on plant genotype. Analysis of the expression of genes involved in N metabolism and defense and quantification of different amino-acids contents and secondary metabolic compounds showed different responses of the genotypes and highlighted a potential role of glutamine in this pathosystem. Furthermore, our work underlined the importance of nitric oxide (NO) homeostasis for M. truncatula resistance to A. euteiches and that N availability impacts NO homeostasis by affecting S-nitrosothiol levels and S-nitrosoglutathione reductase activity in roots. These studies highlight, therefore, the importance of N metabolism and its interaction with plant genotype in defense responses in M. truncatula.