- ABSTRACT (IN ENGLISH) - Biological invasions by crustaceans represent a serious threat for native species in Europe. In my thesis I focus on non-indigenous freshwater crayfish introduced to Europe and their parasite Aphanomyces astaci, the pathogen of the crayfish plague. The thesis consists of four already published first-author papers (chapters I, II, IV and V), two first-author manuscripts (chapters III and VI), and one paper which I co-authored (chapter VII). The first part (chapters I-V) focuses on genetic variation in North American crayfish introduced to Europe. We showed that in two crayfish species, both successful invaders in Europe, genetic variation differs significantly, reflecting their different colonization histories on the continent. The spiny-cheek crayfish Orconectes limosus was likely introduced to Europe just once, in small numbers (90 individuals). Variation at the mitochondrial DNA (mtDNA) level in the spiny-cheek crayfish in Europe is much lower compared to North America (chapter I), although some variation was revealed by nuclear markers in its Central European populations (chapter II). In contrast, the signal crayfish Pacifastacus leniusculus was introduced to Europe several times, in large numbers. Its European populations are highly diverse genetically and belong to a single...

The most widespread of invasive crayfish in the Czech Republic is the spiny-cheek crayfish (Orconectes limosus). It is a major disease carrier of crayfish plague, caused by the pathogen Aphanomyces astaci. The infection is lethal to European native species of crayfish. Transmission of this disease is usually associated with the spread of crayfish, which is dependent on their movement. The main aim of this work was to evaluate the differences in crayfish migratory activity in the Pšovka brook among seasons, and assess the factors that may affect it, by the use of radiotelemetry and trapping. We tested the hypothesis that crayfish use brook in the period from spring to autumn and then return back into a pond to overwinter. Furthermore, we evaluated how far from the pond crayfish migrate, and whether there is any relationship between migration and the season or water temperature. The results demonstrate that the activity varies among seasons (being strongly affected by breeding season) but in most studied periods did not significantly depend on water temperature. According to our monitoring, the spiny-cheek crayfish does not spread substantially to upstream parts of the Pšovka. Seasonal migration between the pond and the brook was monitored using two-way flow-through traps installed at the mouth of...

Chez les plantes, le RET (Root Extracellular Trap) est une structure cellulo-moléculaire jouant un rôle central dans la défense racinaire face aux stress abiotiques et biotiques. De nombreuses similitudes de composition ont été observées entre le RET et le NET (Neutrophil Extracellular Trap) du système immunitaire des mammifères, connu pour capturer et tuer certains microorganismes bactériens et fongiques. Le RET est composé de cellules frontières et de leurs sécrétions (composés de haut et de bas poids moléculaire) comprenant des polysaccharides de la paroi cellulaire, des protéoglycannes et des métabolites secondaires. Il contient également des protéines antimicrobiennes et de l'ADN extracellulaire, tout comme le NET. Dans le cadre de mon projet de thèse, nous avons caractérisé la composition moléculaire et la structuration de cette entité de défense chez deux légumineuses, le soja (Glycine max (Merr) L.) et le pois (Pisum sativum L.), par des approches d’imagerie cellulaire photonique et électronique. Nous avons également étudié l’impact du RET du soja sur des pathogènes telluriques, à savoir Phytophthora parasitica et Aphanomyces euteiches. Nous avons ainsi pu mettre en évidence la présence de différents morphotypes de cellules frontières et de mucilage au sein du RET de soja et de pois. Pour la première fois, nous avons montré la présence d’hétéromannanes, de xyloglucane et de cellulose dans le RET, formant une ossature stabilisant le mucilage et reliant les cellules frontières entre elles. Ces polysaccharides structuraux semblent être essentiels à l’intégrité structurale et fonctionnelle du RET. Enfin, nos résultats ont montré que le RET de soja était impliqué dans la défense précoce de la racine contre P. parasitica. Cette étude apporte de nouvelles connaissances relatives à la composition moléculaire et la structure du RET, nous amenant ainsi à comparer le RET à d’autres modèles que le NET des mammifères, tels que les biofilms bactériens et les mucilages de graines. En effet, de nombreuses similitudes existent entre ces différents complexes en termes de composition et de fonctionnement, qui méritent d’être explorer plus en détail dans l’avenir. / In higher plants, the RET (Root Extracellular Trap) is a complex made up of border cells and secretions, released by root tips and believed to play a central role in biotic and abiotic stress tolerance. This structure is quite similar to the Neutrophil Extracellular Trap (NET) known as one of the first lines of defense in mammals, able to trap and kill microbial pathogens. RET secretions consist of high and low-molecular weight compounds including cell wall polysaccharides, proteoglycans and secondary metabolites. They also contain a variety of anti-microbial proteins and extracellular DNA much like the NET. During my thesis work, we investigated the release and morphology of root border cells in soybean (Glycine max (Merr) L.) using light and scanning electron microscopy. The molecular composition of the mucilage was also investigated using immunocytochemistry, anti-cell wall glycan antibodies and confocal microscopy. Immunocytochemistry was also applied to pea (Pisum sativum L.) border cells and secretions to examine the occurrence of specific polysaccharides. We also studied the impact of soybean RET on the soilborne pathogens, Phytophthora parasitica and Aphanomyces euteiches. Our findings showed that root tips of soybean released three border cell morphotypes all of which secreted substantial amounts of mucilage. Immunocytochemical data showed that mucilage was enriched in pectin and the two hemicellulosic polysaccharides xyloglucan and heteromannan. Mucilage also contained cellulose, histone and extracellular DNA. Interestingly, the structural polysaccharides formed a fibrous network surrounding the cells and holding them together, supporting their role in maintaining mucilage architecture and integrity. In addition, we found that xyloglucan and cellulose were also secreted into the mucilage of pea, connecting border cells together. Finally, our findings revealed that RET prevented P. parasitica zoospores from colonizing soybean root tip, by stopping their penetration and inducing their death. Overall the study revealed novel insights into the composition, structure and function of plant RETs. Currently, the RET is much less studied than its mammal counterpart, the NET, but structural and functional similarities exist between these two traps. Interestingly, similarities do also exist between the RET and other important biological complexes, including bacterial biofilms and seed mucilage, that deserve to be further investigated and compared in the context of immunity.

Root rot diseases of bean (Phaseolus vulgaris L.) are a problem wherever they are grown, and are a major constraint to dry edible and snap bean production. Root rot is a primary yield limitation of snap bean production in the US, especially within the top three snap bean producing states of Wisconsin, Oregon and New York. Bean root rot pathogens will be present by the end of the first season even when starting with clean ground. The decline in yield can be relatively slow, so growers might not notice or appreciate the hidden yield cost associated with root rot disease. Traditional methods for disease control such as fungicides, crop rotations, cover crops, seedbed preparations have been proven ineffective (either physically ineffective or economically unviable) against root rot. Therefore, genetic resistance is needed. In order to address the need for genetic resistance to root rot in snap beans, the highly root rot resistant line RR6950, a small seeded black indeterminate type IIIA accession of unknown origin, was crossed with OSU5446, a highly root rot susceptible determinate type I blue lake four-sieve breeding line to produce the RR138 recombinant inbred mapping population. In this study we evaluated the RR138 RI population in the F₆ generation for resistance to Fusarium solani root rot in Oregon and Aphanomyces euteiches root rot in Wisconsin. We also evaluated this population for morphological traits and root structural traits including pod height, pod width, pod length, pod wall thickness, strings, seed color, flower color, tap and basal root diameter, and root angle measurements. The RR138 population was also genotyped on the 10K BeanCAP Illumina Beadchip. The Single Nucleotide Polymorphism (SNP) data was used to assemble a high-density linkage map and Quantitative Trait Loci (QTL) for phenotypic data were evaluated. The linkage map produced from this study contained 1,689 SNPs across 1,196cM. The map was populated with 1 SNP for every 1.4cM, spanning across 11 linkage groups. Three QTL associated with A. euteiches root rot resistance were consistently expressed in 2011 and 2012 trials. A. euteiches QTL were found on Pv02, Pv04, and Pv06 and accounted for 7-17% of total genetic variation. Two QTL associated with F. solani were found in 2011 trial on Pv03 and Pv07, account for 9 and 22% of total genetic variation, respectively. We also found several QTL for morphological traits and root structural traits including QTL for pod fiber and pod height on Pv04, pod length on Pv01, strings on Pv01, taproot diameter on Pv05, and shallow basal root angle on Pv05, accounting for 21, 26, 12, 20, 11, and 19% of total genetic variation, respectively. QTL discovered from Oregon data for F. solani resistance did not cluster with QTL for A. euteiches root rot resistance. "SNP0928_7", was highly associated with F. solani resistance on Pv07 and "SNP0508_2", was highly associated with A. euteiches on Pv02. QTL and markers associated with QTL from this study will be of value to snap bean breeders developing root rot resistant lines with processing traits, and provide more information about targeting the mechanism of resistance. / Graduation date: 2013

Aphanomyces euteiches est un oomycète parasite racinaire des légumineuses causant des pertes de rendement récurrentes. La paroi d'A.euteiches contient 10% de N-acétylglucosamine (NAG) sous la forme de chitosaccharides non cristallins, associés aux glucanes pariétaux. Afin de pouvoir étudier leur activité biologique, un bioessai d'élicitation du système racinaire de la plante hôte Medicago truncatula a été mis au point en utilisant une préparation de fragments de chitine comme éliciteur témoin. La purification de fractions de parois hydrolysées, enrichies en NAG, a donné des fragments de glycanes composés de glucose et de NAG. Ces hétéropolymères présentent une structure nouvelle jamais décrite à ce jour. Le bioessai d'élicitation racinaire a révélé une activité biologique des fractions de paroi différente de celle des fragments de chitine chez M.truncatula. De façon intéressante, l'une des fractions induit des oscillations calciques nucléaires dans les cellules épidermiques de cultures de racines de M.truncatula, qui sont différentes de la réponse provoquée par des chitotétramères purs.