Thesis (M.Sc.) - Carleton University, 2008. / Includes bibliographical references (p. 119-124). Also available in electronic format on the Internet.

Sixty-two natural cankers on branches and main stems of three 16-year-old grafted American chestnut trees at the Lesesne State Forest were sampled for Cryphonectria parasitica. Cankers were sampled in 1996 and 1997 at various distances from the main stem zone on the grafts (ground to 183 cm) that was inoculated in 1982 and 1983 with a mixture of dsRNA-containing white and pigmented hypovirulent strains. Grafted trees exhibited a high level of blight control, and all bark cores extracted from cankers on the grafted trees showed superficial necrosis. Bark cores extracted from these cankers yielded 156 isolates of C. parasitica. Fifty-three of these isolates were white, and 103 were pigmented. The farthest canker containing a white isolate was located 564 cm from the zone inoculated with hypovirulent strains (H-inoculated zone). The number of white isolates recovered per canker on the grafted trees near the H-inoculated zone (< 0.5 maximum sampling distance) was significantly greater (P=0.0039) than the number of white isolates recovered per canker on the grafted trees far from the H-inoculated zone (>0.5 maximum sampling distance). Lloyd's index of patchiness value for the frequency of white isolates in cankers was 1.36, indicating that white isolates were slightly aggregated in cankers. White isolates of C. parasitica were found in two of seven artificially established cankers 5 months after inoculation with a pigmented virulent strain (WK). Thirteen of 14 pigmented isolates collected from these cankers after 5 months were compatible with WK in vegetative compatibility (VC) tests. Eight of 25 white isolates recovered 5, 11, and 50 months after WK inoculation converted the pigmented WK strain to the white hypovirulent phenotype in vitro. Sixty-five pigmented isolates collected from natural cankers were paired in VC assays, revealing 28 VC groups. All 11 white isolates of C. parasitica assayed contained a 12.7 kb dsRNA in high concentrations. None of 48 pigmented isolates assayed contained dsRNA. All white isolates tested in virulence trials on American chestnut stems in a forest clearcut were hypovirulent, based on low canker severity indices. Little or no dissemination of white strains to cankers on the American chestnut stump sprout clusters, which surround the grafted trees, was found. In the future, to maximize spread of white hypovirulent strains on American chestnut trees, it may be beneficial to re-inoculate trees with hypovirulent strains farther up the main stem after substantial tree growth has occurred. / Master of Science

Thesis (M. Sc.)--Carleton University, 2004. / Includes bibliographical references (p. 137-145). Also available in electronic format on the Internet.

Cryphonectria parasitica is the causal agent of chestnut blight, which devastated the American Chestnut tree population in the early 20th century. The discovery of hypoviruses that reduce the severity of the chestnut blight infection offers the potential for biological control. However, the spread of the hypoviruses is hampered by a diverse genetically controlled nonself-recognition system, vegetative incompatibility (vic). CPVIB-1 was identified as a transcription regulator playing an important role in the programmed cell death response to this stimulus. In this study, we have found that CPVIB-1 is ubiquitin-decorated which might lead to its degradation in the proteasome pathway. RNA-Seq and ChIP-Seq were used to further explore the downstream targets of CPVIB-1 that mediate the various metabolic changes that lead to the altered phenotype of the Δcpvib-1 mutant. Due to inaccuracies in the prior annotation, we performed a genome re-annotation to improve the accuracy using a MAKER2-two-pass pipeline. To validate the improvement a second pipeline, PEPA, was developed to compare quality metrics between the old and new annotations. Approximately 1/3 of the original annotations from 2009 were found to be inaccurate. Experimental confirmation by testing 27 predicted genes using a diagnostic PCR protocol to differentiate between prior and new transcript structures showed that over 80 % of tested genome locations supported for the new annotation. Using rapamycin treatment to mimic stimulation of the vic response and applying the RNA-seq and ChIP-seq data to this new information, we found that CPVIB-1 is related to TOR signaling pathways, promoting autophagy and the proteasome pathway, but repressing carbon metabolism, protein and lipid biosynthesis. In depth analysis of CPVIB-1-bound DNA targets showed that this protein is a member of the GAGA regulator family, a group of multifaceted transcription factors with diverse roles in gene activation and repression, maintenance of mitosis, and cell development. Following treatment with rapamycin the recognition sequence bound by CPBVIB-1 was altered leading to the regulation of different suite of genes with diverse metabolic functions. Ultimately, we have developed a revised model of TOR signaling pathway where TORC1 and TORC2 signaling pathways are connected by the action of CPVIB1.

Le système d'incompatibilité végétative a été décrit chez tous des champignons (Eumycètes) comme intervenant dans la limitation des fusions somatiques entre conspécifiques. Chez les champignons la fusion somatique est uniquement possible entre individus de même GCV (Groupe de Compatibilité Végétative). Comme tous les systèmes de reconnaissance du soi, le fonctionnement du système d'incompatibilité végétative des champignons est basé sur une grande diversité allélique. Cette thèse propose d'étudier la relation qui semble exister entre cette diversité des gènes impliqués dans l’incompatibilité végétative des champignons et la pression parasitaire exercée par des éléments cytoplasmiques délétères (ou DCE) transmis lors des fusions somatiques. Trois problématiques ont été abordées, avec trois approches différentes : (1) une approche conceptuelle générale portant sur l’évolution des systèmes de reconnaissance du soi, (2) une approche de modélisation sur le maintien de la diversité en GCV de la population de champignon par un DCE et (3) une approche expérimentale, pour étudier d’une part la perméabilité de la barrière d‘incompatibilité végétative et d’autre part l’interaction C. parasitica/CHV et les liens existant entre transmission et virulence du CHV. Ces études ont permis de montrer l'importance de la perméabilité de la barrière d'incompatibilité végétative à la fois au niveau du maintien de la diversité génétique de la population d'hôte et au niveau de la prévalence des DCE. Il semblerait donc que les DCE évoluent vers des niveaux de virulence faible du fait de la limitation de leur transmission par le système d'incompatibilité végétative de leur hôte. Nos résultats expérimentaux suggèrent que lorsque la diversité en GCV de la population d'hôte est faible, la virulence des DCE pourrait évoluer suivant le modèle du trade-off impliquant une évolution vers un niveau de virulence intermédiaire optimal. Ces travaux permettent donc de mieux comprendre les mécanismes agissant sur l'écologie et l'évolution des interactions champignon/DCE qui, au vu de cette étude, apparaissent comme de bon modèles pour l’étude des systèmes hôtes/parasites. / Vegetative incompatibility systems have been described in Fungi as controlling somatic fusion between conspecifics. For fungi, only fungi of the same vc type can fuse together. As other self recognition systems, this system involved high allelic diversity at specific genes. The issue of this work is to study the cause and effect relationship between the evolution of vegetative incompatibility systems and the selective pressure drove by cytoplasmic deleterious elements, transmitted during somatic fusion. Three problematics with three different approach were done : (1) a conceptual general framework on the evolution of self recognition systems (2) a theoretical work on the maintenance of vc type diversity by DCE and (3) an experimental work on the study of relationship between transmission and virulence in the C. parasitica/CHV host-parasite system. Ours results showed the key role of the permeability of the vegetative incompatibility barrier both for vc type diversity maintenance and on DCE prevalence. DCE would evolve toward avirulence in response to the transmission limitation by host incompatibility systems. Experimental work suggested a positive link between virulence and transmission in some population of CHV when host present a low vc type diversity, which could allow the evolution of the DCE toward an intermediate optimal virulence. This study would shed some light on mechanisms acting on the ecology and the evolution of fungi/DCE interaction which, according to our results, would be good study models for works on host-parasite systems.

No description available.

In the early 1900's the American chestnut (Castanea dentata (Marsh.) Borkh.) was nearly destroyed by the introduction of the orange-pigmented, chestnut blight fungus (Cryphonectria parasitica (Murr.) Barr). Chestnut blight is less severe in Europe, where hypovirulent (= reduced virulence) strains of the fungus are found to be associated with healing cankers. These European hypovirulent strains are infected with a dsRNA virus, Cryphonectria hypovirus 1 (CHV1), and have a white phenotype when grown in culture. Transmission of CHV1 in C. parasitica is limited by incompatibility between isolates in different vegetative compatibility (vc) types. In 1982-83, naturally formed blight cankers on American chestnut grafts, derived from large survivors, were inoculated with a mixture of four European (white) hypovirulent strains of C. parasitica. After 14 years the white strains were recovered throughout the inoculated grafts, which had low levels of blight damage. CHV1 had infected at least 45 new vc types, and was present in four different fungal colony morphology groups, including one type that had intermediate or partial pigmentation. However, CHV1 was unable to move throughout a single vc type within a natural canker. The objectives of this study were: 1) to determine the frequency and phenotypic diversity of CHV1-infected C. parasitica isolates recovered from stromata and canker tissue from natural cankers on the grafted American chestnut trees and artificially established cankers on forest American chestnuts; 2) to determine the presence or absence of CHV1 in intermediate-pigmented isolates recovered from the American chestnut research plots; 3) to investigate the roles of colony age, resistance to hypovirus infection, and functional mycelial units in the failure of CHV1 to move throughout a vc type of C. parasitica in vitro, and; 4) to examine the role of low temperatures and a high elevation topographic site on CHV1 survival within C. parasitica colonies in vivo and in vitro. The results indicated that there was no direct correlation between the amount of colony pigmentation and the presence of dsRNA. Within each of the three colony phenotype categories (pigmented, intermediate and white), several C. parasitica isolates tested positive for the presence of CHV1. This presence of CHV1 in intermediate isolates, coupled with the relatively large number of intermediate isolates collected from stromata on cankers, indicates that intermediate isolates may perform an important, and previously overlooked, function in biological control of chestnut blight. In this study, all CHV1 movement trials indicated that the age of the C. parasitica colony limited the movement of CHV1 throughout the colony. The majority of the CHV1 movement through a C. parasitica colony occurred between 0 and 7 days following challenge with an isogenic CHV1-infected strain. Isolation data using a lattice grid did not indicate a consistent pattern of CHV1 movement throughout a C. parasitica colony. Low temperatures associated with high altitude had no effect on hypovirus survival in vivo or in vitro. Additionally, no long-term C. parasitica resistance to CHV1 infection or movement was identified in this study. This research has identified new insights into CHV1 spread and survival that may be important in understanding the role of CHV1 in the biological control of chestnut blight. / Ph. D.