Wheat is one of the most essential food sources in the world. However, there has been serious yield loss of wheat production due to stripe rust disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici. The cost-effective and long-lasting defense to the disease can be achieved by generating genetically resistant crops against the disease forming pathogens. To accomplish this, first step is to acquire knowledge in the plant pathogen interactions of the crop and the pathogen of interests at the cellular and the molecular level. In this thesis research, PstHa2a5 candidate effector gene from Puccinia striiformis f. sp. tritici is investigated to identify its role and interaction between host factors in yellow rust infected Triticum aestivum L. The gene construct was engineered with FLAG-tag fusion at its N-terminus, and synthesized. This construct was cloned into pJL48-TRBO vector for an expression in Nicotiana benthamiana via agrobacterium-mediated gene transformation. The expressed protein structure with FLAG-tag was purified, and immunoprecipitated with one putative N. benthamiana interactor by immunoprecipitation experiments. This candidate interactor protein will be identified with Mass Spectroscopy. In addition to this, subcellular localization of the effector candidate was examined in N. benthamiana plant. This was achieved by cloning PstHa2a5 gene construct in pK7WGF2 gateway destination vector and localization is determined by GFP expression in N. benthamiana after agrobacterium-mediated gene transformation.

Les pratiques culturales modifient l'architecture des couverts de manière à augmenter ou diminuer le développement des épidémies mais les processus mis en jeu sont complexes ; des modèles mécanistes simulant l'interaction entre plante et pathogène devraient aider à les clarifier. Les modèles de Plantes Virtuelles, qui permettent de décrire explicitement la structure tridimensionnelle de la plante, semblent particulièrement prometteurs pour exprimer les effets de l'architecture de la plante sur le développement des épidémies. L'objectif de cette étude est d'examiner la possibilité de simuler l'effet de l'architecture des plantes sur le développement de la maladie en utilisant un modèle Plante Virtuelle. Dans ce travail, nous nous intéressons au pathosystème blé-Septoria tritici, dans lequel l'architecture joue un rôle important. En effet, les spores de Septoria tritici sont propagées par les éclaboussures de pluie depuis les feuilles infectées du bas du couvert vers les nouvelles feuilles saines. Notre travail s'est appuyé sur un modèle pré-existant d'épidémie de la septoriose, Septo3D. L'architecture du blé a été étudiée pour une gamme de densités et de date de semis. Les différences de phyllochrone entre traitements ont été dans une gamme susceptible de modifier le développement de la septoriose. Ces variations ont été représentées par un modèle descriptif qui tient compte du nombre de feuilles final et de la photopériode. Une description détaillée des variables d'architecture à l'échelle des organes et du couvert a fourni une documentation originale et complète sur la plasticité de l'architecture du blé. Ces données ont été utilisées pour paramétrer la description du blé dans Septo3D. Globalement, les traitements étudiés ont conduit à de fortes différences de la densité de végétation au cours du temps. Les dynamiques de développement de la septoriose ont été suivies pour trois traitements de densités contrastées. Les cinétiques de la maladie simulées par le modèle étaient conformes aux mesures expérimentales. Bien que, l'approche nécessite davantage de validation, les résultats confirment que l'approche Plante Virtuelle apporte un nouvel éclairage sur les processus et les caractéristiques des plantes qui impactent les épidémies. En conclusion, nous proposons quelques perspectives en vue de nouvelles applications et améliorations de l'approche.

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Take-all, caused by the soilborne fungus, Gaeumannomyces graminis var. tritici (Ggt), is an important root disease of wheat that can be reduced by take-all decline (TAD) in successive wheat crops, due to general and/or specific suppression. A study of 112 New Zealand wheat soils in 2003 had shown that Ggt DNA concentrations (analysed using real-time PCR) increased with successive years of wheat crops (1-3 y) and generally reflected take-all severity in subsequent crops. However, some wheat soils with high Ggt DNA concentrations had low take-all, suggesting presence of TAD. This study investigated 26 such soils for presence of TAD and possible suppressive mechanisms, and characterised the microorganisms from wheat roots and rhizosphere using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). A preliminary pot trial of 29 soils (including three from ryegrass fields) amended with 12.5% w/w Ggt inoculum, screened their suppressiveness against take-all in a growth chamber. Results indicated that the inoculum level was too high to detect the differences between soils and that the environmental conditions used were unsuitable. Comparison between the Ggt DNA concentrations of the same soils collected in 2003 and in 2004 (collected for the pot trial), showed that most soils cropped with 2, 3 and 4 y of successive wheat had reduced Ggt DNA concentrations (by 195-2911 pg g-1 soil), and their disease incidences revealed 11 of the 29 test soils with potential take-all suppressiveness. Further pot trials improved the protocols, such that they were able to differentiate the magnitudes of suppressiveness among the soils. The first of the subsequent trials, using 4% w/w Ggt inoculum level, controlled conditions at 16°C, 80% RH with alternate 12 h light/dark conditions, and watering the plants twice weekly to field capacity (FC), screened 13 soils for their suppressiveness against take-all. The 13 soils consisted of 11 from the preliminary trial, one wheat soil that had been cropped with 9 y of wheat (considered likely to be suppressive), and a conducive ryegrass soil. The results revealed that 10 of these soils were suppressive to take-all. However, in only four of them were the effects related to high levels of microbial/biological involvement in the suppression, which were assessed in an experiment that first sterilised the soils. In a repeat trial using five of the soils H1, H3, M2, P7 (previously cropped with 3, 3, 4 and 9 y successive wheat, respectively) and H15 (previously cropped with 5 y of ryegrass), three of them (H1, H3 and M2) had reduced Ggt DNA concentrations (>1000 pg g-1 soil reductions), and were confirmed to be suppressive to take-all. A pot trial, in which 1% of each soil was transferred into a γ-irradiated base soil amended with 0.1% Ggt inoculum, indicated that soils H1 and H3 (3 y wheat) were specific in their suppressiveness, and M2 (4 y wheat) was general in its suppressiveness. The microbial communities within the rhizosphere and roots of plants grown in the soils, which demonstrated conduciveness, specific or general suppressiveness to take-all, were characterised using PCR-DGGE, and identities of the distinguishing microorganisms (which differentiated the soils) identified by sequence analysis. Results showed similar clusters of microorganisms associated with conducive and suppressive soils, both for specific and general suppression. Further excision, re-amplification, cloning and sequencing of the distinguishing bands showed that some actinomycetes (Streptomyces bingchengensis, Terrabacter sp. and Nocardioides sp.), ascomycetes (Fusarium lateritium and Microdochium bolleyi) and an unidentified fungus, were associated with the suppressive soils (specific and general). Others, such as the proteobacteria (Pseudomonas putida and P. fluorescens), an actinomycete (Nocardioides oleivorans), ascomycete (Gibberella zeae), and basidiomycete (Penicillium allii), were unique in the specific suppressiveness. This indicated commonality of some microorganisms in the take-all suppressive soils, with a selected distinguishing group responsible for specific suppressiveness. General suppressiveness was considered to be due to no specific microorganisms, as seen in soil M2. An attempt to induce TAD by growing successive wheat crops in pots of Ggt-infested soils was unsuccessful with no TAD effects shown, possibly due to variable Ggt DNA concentrations in the soils and addition of nutrients during the experiment. Increasing numbers of Pseudomonas fluorescens CFU in the rhizosphere of plants, during successive wheat crops was independent of the Ggt DNA concentrations and disease incidence, suggesting that increases in P. fluorescens numbers were associated with wheat monoculture. This study has demonstrated that TAD in New Zealand was due to both specific and general suppressiveness, and has identified the distinguishing microorganisms associated with the suppression. Since most of these distinguishing microorganisms are known to show antagonistic activities against Ggt or other soilborne pathogens, they are likely to act as antagonists of Ggt in the field. Future work should focus on validating their effects either individually, or interactively, on Ggt in plate and pot assays and under field conditions.