Leptosphaeria maculans and L. biglobosa co-infect winter oilseed rape plants to cause phoma stem canker disease. The sexual spores of both species are produced in pseudothecia on infected winter oilseed rape stem debris after harvest and this is the most important source of inoculum for infection of newly-emerged plants in autumn. Field experiments investigated the effects of host resistance on proportions of pathogens Leptosphaeria maculans and L. biglobosa in most leaf and stem tissues during 2011/2012, 2012/2013 and 2013/2014 growing seasons and on the pseudothecial development on crop debris on nine winter oilseed rape cultivars; cvs Adriana (Rlm4 + quantitative resistance (QR)), Bilbao (Rlm4), Capitol (Rlm1), Drakkar (no R gene against L. maculans), DK Cabernet (Rlm1 + (QR), Es-Astrid (QR), Excel (Rlm7), Roxet (Rlm7) and NK Grandia (QR). Cultivars with a combination of R-gene resistance and QR [Adriana (Rlm4 + QR), DK Cabernet (Rlm1 + QR)] or cultivars with only QR [(Es-Astrid and NK Grandia)] had more numbers of L. maculans leaf spots than other cultivars in autumn but less stem canker damage. There was greater number of L. biglobosa leaf spots on leaves of cvs Roxet and Excel with resistance gene Rlm7 than those of other cultivars and later more L. biglobosa DNA was detected in their stems than in those of other cultivars. In all cultivars in the three growing seasons, there was a greater amount of L. biglobosa DNA than L. maculans DNA in basal stem canker and upper stem lesions. The cv. Drakkar (no R gene against L. maculans) was susceptible in all three growing seasons, with a great number of L. maculans and L. biglobosa leaf spots and severe stem cankers. There were four cultivars (Adriana, Bilbao, Drakkar and NK Grandia) selected for the study of pseudothecial development under natural conditions with different times of exposure and in controlled environment conditions (20oC, continuous wetness). The fastest development was on the susceptible cv. Drakkar (no R gene against L. maculans), followed by Bilbao (Rlm4), Adriana (Rlm4 + QR) and NK Grandia (QR) for stem base cankers and upper stem lesions in controlled conditions. Results for pseudothecial development on stems of the nine winter oilseed rape cultivars that were exposed in natural conditions at Bayfordbury support the controlled environment results, with pseudothecia on stems of cultivars with a combination R-gene and QR consistently maturing later than those on other cultivars, regardless of the weather conditions in three growing seasons. Ascospores produced in pseudothecia are the primary inoculum that initiate phoma stem canker epidemics in autumn. Ascospore release was later in autumn 2011 than in autumn/winter 2012/2013 or 2013/2014 because of dry weather. The pattern of ascospore release had a peak, or maximum in autumn/winter 2011/2012 (4958 spores/m-3 on 22 Jan 2012) and several maxima in autumn/winter 2012/2013 (1307 spores/m-3 on 5 Nov 2012, 1291 spores/m-3 on 15 Nov 2012, 1306 spores/m-3 on 25 Dec 2012) and 2013/2014 (4575 spores/m-3 on 27 Oct 2013, 4619 spores/m-3 on 3 Nov 2013, 3674 spores/m-3 on 9 Nov 2013, 3521 spores/m-3 on 12 Dec 2013). Results from the qPCR showed that ascospores of L. maculans were released earlier than ascospores of L. biglobosa at Bayfordbury in the 2013/2014 growing season. There were differences in phenotype of isolates amongst ninety-five isolates of L. maculans and forty-eight isolates of L. biglobosa obtained from different sources (phoma leaf spots, upper stem lesions or basal stem cankers) on different cultivars. Cotyledon tests showed that the resistance genes Rlm4, Rlm5, Rlm6 and Rlm7 are still effective in England. Most isolates from phoma leaf spots carried avirulent AvrLm4 (39 isolates; 97.5%), AvrLm5 (39 isolates; 97.5%) and AvrLm6 alleles (36 isolates; 90%) and all 40 isolates carried the avirulent allele AvrLm7 (100%). Fewer isolates from basal stem cankers carried avirulent AvrLm4 (4 isolates, 16.7%) or AvrLm6 alleles (16.7%) but all 24 isolates carried the avirulent AvrLm7 (100%). Fewer isolates from upper stem lesions carried the avirulent AvrLm4 allele (5 isolates; 16.1%), but 15 isolates carried avirulent AvrLm5 (48.4%), 21 isolates carried AvrLm6 (67.7%) and all 31 isolates carried AvrLm7 (100%). By contrast, all isolates were virulent against Rlm1, Rlm2, Rlm3 and Rlm9. This knowledge, together with knowledge about R genes present in current winter oilseed rape cultivars, should be useful to provide recommendations on cultivar selection to growers based on regional frequencies of avirulent alleles of Avr allele genes in the L. maculans populations (races) and improved understanding of the race structure of L. maculans. There is a need to further investigate any R genes that operate against L. biglobosa (possibly from wild brassicas) and to study if any R genes or QR can provide resistance against both L. maculans and L. biglobosa.

Brassica napus (AACC, 2n = 38) is an allotetraploid species derived from the parentel diploid species Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18). The aim of thesis was to carry out the genetic and epigenetic analysis of high-copy rRNA genes (or rDNA) in several varieties of hybrid species B. napus. The experiments involved determining the ratio of parental genes in hybrids, sequencing and methylation analysis of the promoter region of rDNA. Using Southern hybridization, it was revealed significant variability in the number of parental rDNA units between each variety. Data from sequence analysis were in good agreement with the results of Southern blot. Genetic recombination between parental rDNA units was revealed in one variety by DNA sequencing of promotor region. To study methylation, bisulfite sequencing was performed. It was found out that rDNA units of B. rapa origin have a higher value of methylation than units originated from B. oleracea.

This thesis follow up influence of fertilization on yield and oil content of oilseed rape (Brassica napus L.). There were used fertilizers based on ammonium sulfate in the regeneration (BBCH 26, spring) and production fertilization (BBCH 31) during vegetation phase of oilseed rape. The issue was solved as a two-year small-plot field experiment carried out in the vegetation seasons 2016/2017 and 2017/2018 at the Field Experimental Station in Žabčice. The following variants were included in the experiment: Control (CAN), Ammonium sulfate (AS), Ammonium sulfate with boron (AS + B), Ammonium sulfate with nitrification inhibitor (AS + IN). Each fertilization variant was applied either as regenerative fertilization (BBCH 26) or first production fertilization (BBCH 31). The yield of seeds and also their oil content was significantly influenced by the vegetation season. Yield in the vegetation season 2017/2018 were higher by 41 % than in the vegetation season 2016/2017 because of weather conditions. The oil content was higher by 4 % in the vegetation season 2017/2018. The yield of seeds and also their oil content were not significantly influenced by the fertilization variant or by the vegetation phase of application. The highest average yield 4,23 t/ha was variant Control. The Control also was the highest average oil content of 40,2 %. High temperatures during both years reduced the efficiency of nitrification inhibitor. Fertilization with a boron-containing fertilizer had no effect because of drought and pH.

This dissertation contains three research chapters conducted on Sclerotinia stem rot (SSR) of canola (Brassica napus L.). This disease is caused by the fungus Sclerotinia sclerotiorum and is considered endemic in canola-producing areas of North Dakota. The first research chapter presents results of a study that evaluated the role of eight phenotyping scoring systems and nine variant calling and filtering methods in detection of QTL associated with response to SSR. The study, conducted on two doubled-haploid mapping populations, showed that using multiple phenotypic data sets derived from lesion length and plant mortality and imputing missing genotypic data increased the number of QTL detected without negatively affecting the effect (R2) of QTL. Nineteen QTL were detected on chromosomes A02, A07, A09, C01, and C03 in this study. The second research chapter presents results of a work that assessed the role of temperature regimes and wetness duration on S. sclerotiorum ascospore germination and ascosporic infection efficiency. This study showed that optimum ascospore germination occurred at 21 °C while it significantly decreased at 10 and 30 °C. Infection efficacy experiments indicated that extreme temperatures and interrupting wet periods were detrimental for the disease development. A logistic regression model with 75% accuracy was developed for the disease perdition. The third research chapter presents results of a study that evaluated the role of temperature on mycelial growth of 19 S. sclerotiorum isolates collected from different geographical regions and on SSR development on plant introduction (PI) lines with different levels of resistance. Mycelial growth and disease development peaked at 25 °C. While lesion expansion on resistant cultivars and the susceptible check was negatively affected at 30 °C, the disease developed significantly on the PI with a high level of susceptibility. Results of these studies provide insights into integrated management strategies of SSR.

No description available.

L’intérêt pour la dispersion des gènes via le pollen a augmenté avec les cultures de plantes génétiquement modifiées. A ce jour, les données expérimentales ainsi que la modélisation portant sur les mouvements du pollen de colza, Brassica napus L., à l’échelle du paysage ne différencie pas clairement la part du vent et des insectes dans cette dispersion. Cependant, l’estimation de la dispersion des gènes par le pollen reste une condition nécessaire pour la gestion des risques d’échappement des (trans-)gènes vers l’environnement et les cultures conventionnelles. A travers cette thèse, nous avons pu mettre en évidence qu’une grande diversité d’insectes pollinisateurs pouvait transporter du pollen viable entre différentes plantes de colza sur des distances importantes (>1.1 km). La diversité d’insectes varie d’une région à l’autre et d’une année sur l’autre. Cependant, bien que la majorité des insectes sur une zone de grande production de colza ait du pollen de cette culture sur leur corps, seulement 39,4 % des insectes capturés sur des plantes mâle-stériles transportent du pollen de colza viable. Bien que nous n’ayons pas pu déterminer avec précision la part du vent et des insectes dans le pollinisation du colza, il semblerait que les insectes participent de façon plus importante à la pollinisation de plantes présentes en bordures de champs, augmentant ainsi le taux de pollinisation croisée. Nos résultats fournissent des données fiables pour améliorer les modèles de dispersion pollinique pour des cultures entomophiles à l’échelle du paysage. Ces modèles sont essentiels pour l’aide à la gestion afin de réduire la dispersion des gènes par le pollen des cultures génétiquement modifiées vers les plantes sauvages ou les cultures conventionnelles / Interest in pollen-borne gene dispersal has grown with the cultivation of genetically modified plants. To date both experimental data and models of oilseed rape (OSR) Brassica napus pollen movement at the landscape scale do not clearly differentiate between wind- and insect-mediated dispersal. Estimations of pollen-borne gene dispersal would be valuable for managing potential escapes of transgenes. Our study provides clear evidence that a large variety of insect species can transfer viable pollen between oilseed rape plants over considerable distances (>1.1 km). Insect’s diversity according to geographical site and years. However, the majority of pollinator have OSR pollen in their body hairs, only 39.4% of the insects caught on male-sterile flowers carried OSR pollen. Although we could not determine with precision the role of the wind and the insects in the OSR pollination, it would seem that insects take part in a more important way in pollination of plants present in edges of fields, thus increasing cross pollination rate. Our results provide valuable data to improve models of pollen dispersal for entomophilous crops at the landscape scale. These models are essential to help land-managers reduce pollen-borne gene dispersal from genetically modified plants to wild relative and field planted with non-GM crops

Les agroécosystèmes sont des mosaïques d’espaces cultivés, d’espaces naturels et semi-naturels et d’infrastructures humaines fortement imbriqués et donc intrinsèquement liés et dépendants. Les espaces semi-naturels sont confinés généralement aux bordures de champs et aux bordures de route où se côtoient biodiversités végétales cultivées et sauvages. Cette thèse se concentre principalement sur la dispersion des graines de colza (Brassica napus L.) vers ces espaces qui peut conduire à la formation de populations de colza dites férales. A l’échelle d’un agroécosystème, l’étude de données génotypiques couplées à des méthodes d’assignations aux variétés commerciales existantes, a permis de mettre en évidence un lien entre la diversité variétale des champs de colza cultivés et la diversité variétale des populations férales de l’année suivante. De surcroît, l’étude de ces diversités variétales a montré que les champs ne sont pas des entités uniformes comportant des plantes d’une seule variété et que les populations férales accumulent les variétés au fil des années grâce aux apports annuels des champs récoltés, à la survie dans la banque de graines et à l’autorecrutement au sein des populations férales. La modélisation des flux efficaces de graines par une méthode de maximum de vraisemblance a permis d’identifier des dynamiques de dispersion locales au sein des agroécosystèmes. Suivant la zone considérée et les axes de circulation vers le silo de récolte, les sources locales de graines varient et les apports extérieurs de graines sont plus ou moins importants. Nos données nous ont permis d’estimer que les semis de la même année (n), les champs récoltés l’année antérieure ou même les années précédentes (jusqu’à n-2) pouvaient contribuer de manière significative à la présence de populations férales (l’année n). Les distances moyennes de dispersion estimées varient de la dizaine de mètres au kilomètre. Enfin, une expérimentation in-situ nous a permis de quantifier les pertes de graines pendant la récolte liées aux bennes de récolte. Nous avons évalué ces pertes à 400 graines par m2 et nous avons mis en évidence de rares évènements de pertes massives de graines. L’analyse statistique des résultats de ces pertes nous a permis de les mettre en relation avec des caractéristiques du paysage, notamment les surfaces des champs et les axes de circulations principaux et secondaires. Dans le contexte de mise en culture de plantes transgéniques, ces résultats impliquent de prendre en compte la complexité du paysage dans les modèles qui prédisent les flux de transgènes à l’échelle des agroécosystèmes. / Agroecosystems are mosaics of cultivated areas where natural and semi-natural areas and human infrastructures are strongly nested and intrinsically linked and dependent. Semi-natural areas are generally confined to field edges and roadsides where wild and cultivated plants biodiversity are combined. This thesis focuses mainly on seed dispersal of oilseed rape (Brassica napus L.) towards semi-natural areas which can lead to establishment of feral oilseed rape populations. At the agroecosystem scale, the study of genotypic data coupled with assignment methods to existing commercial cultivars has highlighted the link between diversity of cultivars of oilseed rape cultivated fields with diversity of cultivar of feral populations the following year. Furthermore, the study of cultivar diversity revealed that fields are not uniformly composed of plants of a unique cultivar and that feral populations accumulate different cultivars over years consecutive of annual seeds rain of harvested fields, survival in the seed bank and self-recruitment in feral populations. Modeling the effective seed flow with maximum likelihood method revealed local dispersal dynamics within the agroecosystem. Local and extern sources of seeds differ according the area studied and the traffic roads to the silo. Our data have shown that both sowing of the same year (n), the fields harvested the previous year or even in previous years (up to n-2) could significantly contribute to the presence of feral populations (year n). The average dispersal distances estimated range from ten meters to kilometers. Finally, an in-situ experiment enabled us to quantify seed losses during harvest related to grain trailers. We evaluated these losses to 400 seeds per m2 and we highlighted rare events of massive seed deposition. Statistical analysis of these losses enabled us to relate them with landscape elements, including the traffic roads and. In the context of GM crops cultivation in agroecosystems, these results emphasize the need to introduce the landscape complexity in models predicting the presence and persistence of GM OSR feral populations.

Dans cette thèse, j'ai étudié le rôle des sRNAs dérivés de l'hôte et du virus lors de l'infection du colza (Brassica napus, Canola) par la souche UK1 du virus de la mosaïque du navet (TuMV-UK1). En utilisant un dérivé de TuMV fusionné avec un gène codant pour la protéine fluorescente verte (TuMV-GFP), deux cultivars de colza (‘Drakkar’ et ‘Tanto’) qui diffèrent par leur susceptibilité à ce virus ont été identifiés. Le profil transcriptionnel des foyers d'infection locale, dans les feuilles de Drakkar et de Tanto, par séquençage nouvelle génération (NGS) a révélé de nombreux gènes exprimés de manière différentielle. Les mêmes échantillons d'ARN provenant de feuilles de Drakkar et de Tanto, traitées par des virus ou utilisées en contrôle, ont également servi à établir le profil NGS des sRNAs (sRNAseq) et de leurs cibles potentielles d'ARN (PAREseq). Les analyses bioinformatiques et leur validation in vivo, ont permis d’identifier les événements de clivage de transcrits impliquant des micro ARN (miRNA) connus et encore inconnus. Fait important, les résultats indiquent que TuMV détourne la voie du RNA silencing de l’hôte avec des siRNAs issus de son propre génome (vsiRNA) pour cibler les gènes de l’hôtes. Le virus déclenche également le ciblage à grande échelle des ARN messagers (ARNm) de l’hôte par l’activation de la production de siRNAs secondaires en phase, à partir de locus PHAS. À leur tour, les vsiRNAs et les siRNAs dérivés de l'hôte (hsRNAs) ciblent et clivent l'ARN viral par le complexe RISC. Ces observations éclairent le rôle des siRNAs dérivés de l'hôte et du virus dans la coordination de l'infection virale. Un autre chapitre de cette thèse est consacré à l'analyse des maladies induites par des virus en utilisant comme modèle de plante Arabidopsis, infectée par un tobamovirus, le virus de la mosaïque du colza (ORMV). De plus, ces observations ont permis de proposer un modèle dans lequel cette guérison dépend d’un adressage important de vsiRNAs secondaires antiviraux depuis leur source de production jusqu’à leurs tissus de destination, et l'établissement d'un apport en vsiRNAs capable de bloquer l'activité VSR impliquée dans la formation des feuilles symptomatiques. / In this thesis, I investigated the role of host- and virus-derived sRNAs during infection of Rapeseed (Brassica napus, Canola) by the UK1 strain of Turnip mosaic virus (TuMV-UK1). By using a TuMV derivative tagged with a gene encoding green fluorescent protein (TuMV-GFP), two rapeseed cultivars (‘Drakkar’ and ‘Tanto’) that differ in susceptibility to this virus were identified. Transcriptional profiling of local infection foci in Drakkar and Tanto leaves by next generation sequencing (NGS) revealed numerous differentially expressed genes. The same RNA samples from mock- and virus- treated Drakkar and Tanto leaves were also used for the global NGS profiling of sRNAs (sRNAseq) and their potential RNA targets (PAREseq). The bioinformatic analysis and their in vivo validation led to the identification of transcript cleavage events involving known and yet unknown miRNAs. Importantly, the results indicate that TuMV hijacks the host RNA silencing pathway with siRNAs derived from its own genome (vsiRNAs) to target host genes. The virus also triggers the widespread targeting of host messenger RNAs (mRNAs) through activation of phased, secondary siRNA production from PHAS loci. In turn, both vsiRNAs and host-derived siRNAs (hsRNAs) target and cleave the viral RNA by the RISC-mediated pathway. These observations illuminate the role of host and virus-derived sRNAs in the coordination of virus infection. Another chapter of this thesis is dedicated to the analysis of virus-induced diseases by using Arabidopsis plants infected with the Oilseed rape mosaic tobamovirus (ORMV) as a model. Initially, the infected plants develop leaves with strong disease symptoms. However, at a later stage, disease-free, “recovered” leaves start to appear. Analysis of symptoms recovery led to the identification of a mechanism in which the VSR and virus derived-siRNAs play a central role. I used Arabidopsis mutants impaired in transcriptional and post-transcriptional silencing pathways (TGS and PTGS respectively) and a plant line carrying a promoter-driven GFP transgene silenced by PTGS (Arabidopsis line 8z2). Using various techniques able to monitor virus infection, small and long viral RNA molecules, VSR activity, as well as phloem-mediated transport with in these lines, this study led to the identification of genes required for disease symptoms and disease symptom recovery. Moreover, the observations allowed to propose a model in which symptoms recovery occurs upon robust delivery of antiviral secondary vsiRNAs from source to sink tissues, and establishment of a vsiRNA dosage able to block the VSR activity involved in the formation of disease symptoms.

No description available.

No description available.