Phytophthora lateralis is the causal agent of cedar root rot, a fatal forest pathogen whose principal host is Chamaecyparis lawsoniana (Port-Orford-cedar), a predominantly riparian-restricted endemic tree species of ecological, economical, and cultural importance to coastal Oregon and California. Local scale distribution of P. lateralis is thought to be associated with timber harvest and road-building disturbances. However, knowledge of the landscape-scale factors that contribute to successful invasions of P. lateralis is also important for effective land management of Port-Orford-cedar. P. lateralis is able to infest in wet conditions via stream networks (zoospore) and dry conditions via road networks (resting spore). This study tested the hypothesis that vehicles spread P. lateralis by relating its distribution to traffic intensive, anthropogenic disturbances (i.e. a road network, timber harvest) over a 31-yr period in a 3,910-km² portion of the Rogue River-Siskiyou National Forest in the Siskiyou Mountains of Oregon. Indices of road disturbance (presence/absence, configuration, length, density, road-stream network connectivity) and timber harvest (presence/absence, area, density, frequency) were related to locations of infested cedar populations from a USFS survey dataset using a geographic information system (GIS). About 40% of 934 7th-field catchments were infested with the pathogen. Total road length of the study site was 5,070 km; maximum road density was 8.2 km/km2 and averaged 1.6 km/km² in roaded catchments (n = 766). Timber activities extracted 17,370 ha (2,338 cutting units) of forest across 509 catchments; 345 catchments were cut ≥ twice. Maximum harvest density was 0.92 km²/km² ([mean] = 0.04). Both road networks and timber harvest patchworks were significantly related to cedar root rot heterogeneity. Chi-squared contingency tables showed that infestation rates were 2.2 times higher in catchments with roads compared to roadless catchments and 1.4 times higher in catchments with road-stream intersections compared to those that were unconnected. Infestation was twice as likely in catchments with both harvest and road presence than road presence alone. Single-variable logistic regression showed that a one percent increase in harvest density increased infestation odds 25% and a one-unit (km/km²) increase in road density increased infestation odds 80%. Road and stream network configuration was also important to pathogen distribution: 1) uninfested catchments are most likely to be spatially removed from infested, roaded catchments, 2) only 11% of 287 roaded catchments downstream of infested, roaded catchments were uninfested, and 3) only 12% of 319 catchments downstream of infested catchments were uninfested. Road networks and timber harvest patchworks appear to reduce landscape heterogeneity by providing up-catchment and down-catchment access to host populations by linking pathogenic materials to the stream network. Timber harvest data suggest that while infestation risk to Port-Orford-cedar populations remains high, management policies may have curbed infestation risk in timber-harvested catchments; if this is a result of specific P. lateralis mitigation policies adopted in the late 1980's or broader, region-wide conservation policies (i.e. the Northwest Forest Plan) is yet unclear. / Graduation date: 2012

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.

Thesis (M. Tech. (Agriculture)) -- Central University of Technology, Free state, 2011 / Quality assessment of agricultural water used for fertigation in the Boland district. The study was undertaken to determine the influence of seasonal changes on the water quality (chemical and microbial analyses) of five different rivers in the Boland. The data was collected in the five most important water sources that are used for irrigation purposes in the district, i.e. Berg river, Eerste river, Klapmuts River, Klippies river and Krom river. The samples were collected from all sites once every three weeks for a period of six months. The sampling was carried out during specific periods in summer (December, 2006 to February, 2007) and winter (June to August, 2007). The results of the study showed that Klapmuts river recorded the highest levels of chloride and iron, especially in summer. However, chloride levels were far below the levels set by the Department of Water Affairs and Forestry. Results obtained from this study show the presence of pathogens in some of the rivers assessed which may have resulted from the leaching of these pathogens from nearby agricultural land, livestock watering or informal settlements in the catchment areas. The bigger rivers recorded low levels of micro-elements and this might have been affected by winter rainfall. In the Berg River, many sources of nitrate pollution seem to be present in the catchment area. The levels of iron in all the rivers assessed were far more than the levels set by the Department of Water Affairs and Forestry in all rivers assessed and these might be due to the pH levels and interaction between the rivers and seasons. Iron and manganese levels should be kept low as this may cause production problems by blocking irrigation drippers. The water samples tested for bacterial and fungal density showed Klapmuts and Eerste rivers were positive for Phytophthora cinnamomi during winter. Phytophthora citricola and Phytophthora cactorum were detected in the Klapmuts and Klippies rivers in summer. The Berg-, Klapmuts-, Krom- and Eerste rivers tested positive for species of the genera Pythium and Fusarium. Similar organisms were detected in the Eerste river mainly during summer on the fourth sampling date, while Krom river only tested positive for Pythium during summer. The total bacterial and algal density differed significantly between the seasons and was highest in winter. This might be due to high rain water influx and efflux and/or moist and aerobic conditions and air temperature. There is an increased need for farmers to sterilize feeding water (chlorination) due to high microbial count.

Plants are fundamental to life on earth, crucially providing the basis of our food supply. As world population continues to grow, so too does the pressure on our agricultural systems, with one of the biggest challenges being the control of plant pathogens to ensure a healthy crop. The interaction between plant and pathogen is complex, with subtleties at the molecular level dictating the boundary between health and disease. This is exemplified by pathogen effectors; secreted proteins which enter the plant cell and interact with host targets to facilitate infection. AVR2 is one such effector, secreted by the oomycete pathogen Phytophthora infestans; the pathogen responsible for potato late blight. AVR2 interacts with a family of kelch-repeat containing phosphatases in potato, the BSLs, implicated in brassinosteroid pathway signalling – a major hormone signalling pathway in plants associated with growth and development. This work investigates the role of AVR2 and its host targets in pathogen virulence, with focus on the link between the brassinosteroid pathway and immunity in S. tuberosum. StBSL1 is shown to be a ‘susceptibility factor’ in P. infestans infection – a host protein with a positive effect on pathogen virulence. AVR2 stabilises BSL1 in planta, and both AVR2 and BSL1 are shown to suppress primary defence responses in the plant. Transcriptional analysis of brassinosteroid-treated S. tuberosum is used to identify a set of marker genes for active BR signalling. Strikingly, AVR2 is shown to upregulatethis pathway, and specifically upregulates the transcription factor StHBI1-like, identified as a suppressor of immunity. These findings reveal a novel mechanism in oomycete effector biology; the exploitation of crosstalk between the brassinosteroid pathway and immune signalling in plants.

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Une voie agroécologique pour améliorer la fourniture des services écosystémiques dans les agroécosystèmes est d'associer plusieurs espèces végétales au sein d'une même parcelle. Dans ce contexte, les agroforêts tropicales, caractérisées par une forte diversité végétale, sont un modèle d'étude d'intérêt croissant. Dans ce travail qui porte sur le service de régulation des bioagresseurs, notre hypothèse est que la structure des agroforêts influence la régulation naturelle des bioagresseurs de la culture principale. La composition en plante hôte de l'agroforêt est susceptible d'influencer les bioagresseurs via les variations de la ressource. La structure spatiale de la végétation est susceptible d'influencer les bioagresseurs via les variations des conditions microclimatiques. Enfin, les variations de la ressource et du microclimat ne sont pas des mécanismes indépendants puisqu'un microclimat favorable à la croissance de la plante hôte du bioagresseur peut augmenter la quantité des tissus sensibles et donc la ressource pour ce bioagresseur. Pourtant, l'importance relative de l'effet de la composition et de la structure spatiale de la végétation des agroforêts sur l'intensité l'attaque des bioagresseurs a rarement été étudiée. Les objectifs de ce travail de thèse sont (1) de caractériser la composition et la structure spatiale des peuplements végétaux d'agroforêts à cacaoyers et (2) de quantifier leurs interactions avec l'intensité d'attaque de biogresseurs du cacaoyer. Ce travail est appliqué à trois bioagresseurs du cacaoyer : au Costa Rica, la moniliose sur un réseau de parcelles installé dans la région de Talamanca ; au Cameroun, la pourriture brune des cabosses et les mirides sur un réseau de parcelles installé dans la région Centre. Premièrement, nous avons caractérisé la composition et la structure spatiale de la végétation des agroforêts à cacaoyers du Costa Rica et du Cameroun. Au Costa Rica, nous avons identifié une diversité de structure spatiale des arbres forestiers entre parcelles, allant de l'agrégation à la régularité ce qui semble indiquer un gradient croissant de gestion entre ces parcelles. Au Cameroun, nous avons également observé une diversité de structure spatiale entre peuplement d'une même parcelle. En effet, les arbres forestiers sont aléatoirement distribués ou agrégés alors que les arbres fruitiers sont aléatoirement ou régulièrement distribués à l'échelle de la parcelle ce qui semble indiquer une différence d'intensité de gestion entre ces deux peuplements. Deuxièmement, nous avons hiérarchisé les caractéristiques de composition en hôte, de quantité de tissus sensibles et de structure spatiale de la végétation des agroforêts qui influencent l'intensité (i) de la moniliose (ii) de la pourriture brune et (iii) des mirides. L'intensité de la moniliose et la densité en mirides diminuent lorsque les quantités de tis- sus sensibles diminuent et lorsque les arbres forestiers sont aléatoirement ou régulièrement distribués plutôt qu'agrégés ou en faible densité à l'échelle de la parcelle. D'autre part, l'intensité de la moniliose diminue lorsque la densité en cacaoyers augmente et l'intensité de la pourriture brune diminue lorsque l'abondance en cacaoyers augmente, ces résultats étant contraires aux hypothèses de diminution et de dilution de la ressource. Globalement, nos résultats indiquent donc que les variations de la ressource liées aux variations de la composition en hôte ne sont pas le principal effet qui explique la présence des bioagresseurs dans les agroforêts complexes. La structure spatiale des arbres d'ombrage, encore jamais décrite à notre niveau de précision, joue un rôle déterminant et son optimisation pourrait être un moyen de lutte agroécologique contre la moniliose et les mirides du cacaoyer. Dans le cadre de l'agroécologie, ce travail nous a permis d'améliorer notre compréhension des mécanismes écologiques impliqués dans la régulation des bioagresseurs du cacaoyer à l'échelle de la parcelle et ouvre des perspectives pour leur gestion agroécologique.

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