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

This thesis represents a critical taxonomical review of the fungal genus Cryphonectria sensu lato. An appropriate taxonomy for this group is of great importance because it includes many well known tree pathogens such as the chestnut blight fungus Cryphonectria parasitica and the Eucalyptus canker pathogen Cryphonectria cubensis. The many taxonomic changes introduced in studies presented in this thesis have largely arisen as a result of DNA sequence comparisons for Cryphonectria spp. that show that Cryphonectria sensu lato is comprised of different lineages, strongly supported by robust morphological characteristics. New taxa, of which many are pathogenic, have also been discovered. The expanded number of species of Cryphonectria and related genera as well as the consideration of large numbers of isolates has furthermore made it possible to establish a broad view of the group at the super-generic level. The first part of the thesis deals with studies on Cryphonectria cubensis. A new genus Chrysoporthe is described for C. cubensis sensu lato. Two additional species are also described for phylogenetic sub-clades previously known as C. cubensis. These include Chrysoporthe austroafricana, representing all isolates from South Africa, and an anamorphic species described in the new genus Chrysoporthella as Chrysop. hodgesiana, which is currently only known from Colombia on native Tibouchina spp. Isolate collections from several new host genera for Chr. cubensis are also characterized. Collections from Eucalyptus in Cuba, now representing the epitype of Chr. cubensis, also define the type of Chr. cubensis as residing in the South American sub-clade. Another new species, Chrysoporthe inopima from Tibouchina lepidota in Colombia is described as well as a new species Chrysoporthe doradensis for isolates from Eucalyptus spp. in Ecuador. A new family Cryphonectriaceae is described in this thesis for Cryphonectria, Chrysoporthe and Endothia. Genera in this family are united by orange stromatic tissue, with the pigments colouring purple in 3% KOH and yellow in lactic acid. The existence of this new family confirms the close relationship of Cryphonectria and morphologically similar genera. A proposal to conserve the name Cryphonectria against the new type C. parasitica is presented. This is required because Cryphonectria gyrosa, the currently accepted type, was erroneously used as type. The conservation of Cryphonectria against C. parasitica made it possible to describe the new genus Amphilogia for C. gyrosa. Amphilogia also includes a second species from New Zealand described as Amphilogia major, although no isolates currently exist for this species. New genera for existing Cryphonectria spp., as well as newly discovered fungi are presented in this thesis. The new genus Rostraureum is established for a fungus pathogenic on Terminalia ivorensis in Ecuador. This fungus also represents a new species, Rostraureum tropicale. Cryphonectria longirostris, originating from Puerto Rico, Trinidad and Tabago, is also transferred to Rostraureum. A fungus morphologically similar to Chrysoporthe on native Tibouchina, Miconia and exotic Eucalyptus spp. in Colombia, is described as Aurapex penicillata gen. sp. nov. Cryphonectria havanensis is transferred to the new genus Microthia. Cryphonectria coccolobae also resides in this genus based on morphology, although its phylogenetic relationship to C. havanensis could not be confirmed due the absence of isolates. A new fungus was discovered during surveys for C. coccolobae on Coccoloba uvifera in Florida, which is described in the new genus Ursicollum as U. fallax. Phylogenetic analyses in this study also clearly distinguish Cryphonectria eucalypti from Cryphonectria, and this fungus is thus transferred to the new genus Holocryphia. A minireview is presented at the end of the thesis and discusses the new taxonomic concepts developed for Cryphonectria during this thesis, and recent studies by other authors. The review describes how this new taxonomic scheme has changed our view and understanding of the distribution and ecology of Cryphonectria sensu stricto from what it has traditionally been seen. The final part of the thesis is written in the form of a monograph. It contains background information of all the species, including many pathogens, currently known in Cryphonectria and allied genera. The majority of these have recently been described, some in this thesis, and this chapter thus contains all recent information pertaining to them. It is intended that this monograph should be useful as a manual, enabling users to work with and isolate these fungi and to identify the different taxa based on morphology and phylogenetic relationships. The studies presented in this thesis greatly change the taxonomy of Cryphonectria sensu lato, which is now seen as representing a large number of genera and species in a new family. Many would argue that Cryphonectria is still monophyletic, but the different lineages shown by DNA sequence comparisons are morphologically inordinately diverse, and clearly represent different genera. Studies presented in this thesis further suggest that additional genera await description from diverse geographical areas and ecological niches. The studies presented in this thesis will hopefully provide a foundation against which these new taxa can be compared and will improve our understanding of tree diseases. / Thesis (PhD)--University of Pretoria, 2009. / Microbiology and Plant Pathology / Unrestricted

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

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.

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

Cryphonectria cubensis is an ascomycetous fungus that causes a serious canker disease on Eucalyptus trees in many parts of the world. The importance of the disease has led to numerous studies involving the taxonomy, genetic diversity and the control of Cryphonectria canker. However, there remain many questions pertaining to the disease that have not been considered. The objectives of the studies presented in this thesis were, therefore, to investigate the possibility of biological control of Cryphonectria canker, to evaluate the currently used disease screening strategy in South Africa and to establish a transfection system with dsRNA elements in Diaporthe, which is closely related to Cryphonectria. The introductory chapter of this thesis provides a review of the literature pertaining to Cryphonectria cubensis. In addition literature on hypovirulence in fungi is also extensively reviewed, with a special emphasis on the genus Cryphonectria. The aim of study in the second chapter of the thesis was to screen the South African C. cubensis population for the presence of dsRNA viruses. Two viruses were identified and the full sequence of these elements showed a strong homology to the mitochondrial viruses (mitoviruses) within the family Narnaviridae. We, therefore, named the viruses Cryphonectria cubensis mitovirus 1 (CcMV1) and Cryphonectria cubensis mitovirus 2 (CcMV2). The two viral genomes are 2601 nucleotides and 2639 nucleotides in size respectively and encode for a protein that probably functions as an RNA-dependant RNA polymerase (RdRp). Pathogenicity studies indicated that the viruses do not result in a significant reduction in pathogenicity of C. cubensis. In the third chapter, results of a study to consider whether different Eucalyptus clones responded similarly to various South African C. cubensis isolates, are presented. The aim was, therefore, to evaluate the current C. cubensis resistant screening method used on Eucalyptus spp. in South Africa. The statistical analysis of the inoculation data showed a significant isolate x clone interaction. This data also suggest the possibility of vertical resistance, which is different to previous assumptions. Transfection studies (Chapter 4) involving a positive stranded RNA virus, Diaporthe RNA virus (DaRV) from a South African D. perjuncta isolate are presented here. In this study, a virus free D. perjuncta isolate, a virulent C. cubensis isolate and a hypovirulent C. cubensis isolate containing the hypovirus CHV1-EP713 were chosen to be transfected with DaRV. By using electroporation, it was possible to infect a virus free D. perjuncta isolate with the Diaporthe RNA virus, thus extending the transfection range of this virus. The resulting transfection led to altered colony morphology but did not lead to a reduction in pathogenicity. We were also not successful in attempts to transfect isolates of C. cubensis with DaRV, indicating that the virus does not replicate in this host. In a previous study a virulent South African C. cubensis isolate was transfected with the Cryphonectria parasitica hypovirus CHV1-EP713. This resulted in the fungus becoming hypovirulent. Chapter five of this thesis presents the results of a study to evaluate the potential use of this virus in the biological control of Cryphonectria canker in South Africa. A field trial was established and existing cankers were treated with the transfected isolate. The treatment of the cankers did not lead to a significant reduction in canker size, but did alter the morphology of the cankers. The virus was also shown to be transmitted via hyphal anastomosis to the virulent canker causing isolates. In addition the co-inoculation on single trees with both the virulent and virus-containing isolate, resulted in a significant reduction in the size of the lesions. This study also showed that the transfected C. cubensis isolate are characterised by significantly smaller lesions than those associated with the virulent, virus-free isolate. Cryphonectria cubensis and the associated canker disease of Eucalyptus threaten the forestry industry in South Africa. The overall aims of the studies presented in this thesis were to gain a more complete understanding of this fungus and to evaluate potential control strategies. Each of these chapters should contribute towards a better understanding of the viruses associated with C. cubensis and other important aspects of Cryphonectria canker, which will hopefully lead to enhanced control strategies of the disease in South Africa. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

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.

Considerable changes have occurred in recent years, regarding the taxonomy and ecology of Eucalyptus fungal pathogens previously treated in the genera Cryphonectria and Endothia. Cryphonectria cubensis now resides in Chrysoporthe with two species, which are very distinct from Cryphonectria. The fungus previously known as E. gyrosa was moved to C. eucalypti and will soon be known as Holocryphia eucalypti. It is very likely that C. eucalypti and Chr. cubensis were introduced onto the African continent, but the hypothesis remains to be tested, while Chr. austroafricana seems native to the African continent. The aim of studies contained in this thesis was to consider the distribution, taxonomy and diversity of Chrysoporthe spp. and Cryphonectria eucalypti on the African continent. This was achieved through surveys in southern and eastern Africa, of both Eucalyptus spp. and native tree species belonging to the Myrtales. The intention was that the results of the studies in this thesis should aid in a better understanding of the taxonomy, origin, distribution, host range, as well as pathogenicity of various Cryphonectria and Chrysoporthe species in eastern and southern Africa. Various new hosts, new areas of occurrence and taxonomic changes have occurred for species of Cryphonectria sensu lato, previously known only on Eucalyptus spp. Chapter one of this thesis presented an overview of the most recent findings regarding the taxonomy, host range and distribution of C. cubensis sensu lato and C. eucalypti. The background to the description of a new genus, Chrysoporthe Gryzenhout&M.J. Wingf. and three new species namely; Chr. cubensis, Chr. austroafricana and Chrysopothella hodgesiana, previously considered to represent C. cubensis was also considered. Furthermore, the wide host range of Chrysoporthe spp. has been reviewed. The fungi are known on various genera in the order Myrtales in both tropical and subtropical areas, worldwide. Emphasis was placed on these Eucalyptus pathogens in Africa. Chrysoporthe cubensis and Chr. austroafricana, collectively known as Cryphonectria cubensis in the past, are important canker pathogens of Eucalyptus spp. worldwide. In chapter two of this thesis I have shown, for the first time, that Chr. cubensis occurs in Kenya, Malawi and Mozambique on non-native Eucalyptus spp. and Chr. austroafricana occurs in Mozambique, Malawi and Zambia on non-native Eucalyptus spp. and native S. cordatum. I was also able to show that Chr. austroafricana causes cankers at the base and higher up on stems of Eucalyptus trees in South Africa and Malawi, which is contrary to prior knowledge. Likewise, the sexual state of this fungus has been shown to be equally abundant as the asexual state in countries north of South Africa, contrary to the situation in southern Africa where the asexual state predominates. The known distribution range of Chr. austroafricana within South Africa was also expanded through this study. Chrysoporthe cubensis is an important fungal pathogen of Eucalyptus spp., worldwide. The fungus is also known on many other hosts all residing in the order Myrtales. Previous surveys conducted in eastern and southern Africa to assess the distribution of Chrysoporthe spp. in this region, revealed the occurrence of Chr. cubensis on Eucalyptus spp. in Kenya, Malawi and Mozambique. In chapter three of this thesis, the population structure of Chr. cubensis isolates from Eucalyptus spp. from Kenya, Malawi and Mozambique was considered for the first time. This represents a first attempt to consider the genetic structure of the fungus from eastern Africa. Results show that there is a very low genetic diversity within the populations of Chr. cubensis from Kenya, Malawi and Mozambique, implying that the fungus is probably newly introduced in these areas. Based on phylogenetic analyses, the origin of eastern African Chr. cubensis is most likely Asia. In chapter four of this thesis, polymorphic microsatellite DNA markers were developed from a single spore isolate of C. eucalypti collected from Eucalyptus stem canker in South Africa. Markers were obtained using the enrichment technique known as FIASCO (Fast Isolation by AFLPs of Sequences Containing Repeats). Ten polymorphic markers were isolated, of which 2 were discarded due to their high polymorphism in the flanking region. These markers will consequently provide useful tools for future investigations considering the population biology and especially the global spread of C. eucalypti. Cryphonectria eucalypti is a fungal pathogen considered opportunistic in South Africa, while in Australia it has been associated with sporadic but serious disease problems. Chapter five of this thesis presents results on the population structure of C. eucalypti from South Africa, eastern and western Australia. Nei's gene diversity (H) showed that the eastern Australian population is most genetically diverse and the western Australian populations from Corymbia and Eucalyptus somewhat less diverse. The South African population displayed the lowest genetic diversity. The high genetic diversity in the Australian populations supports the view that C. eucalypti is native to that region. This is consistent with the fact that Eucalyptus species are also native to the Australian continent. In chapter six of this thesis, I have shown that the fungus isolated from H. canescens, S. cordatum and T. granulosa in South Africa represents a new genus and species related to, but distinctly different from Chrysoporthe. Celoporthe dispersa gen. et sp. nov. is, therefore, described to accommodate this fungus. This description was supported by both morphological characteristics and DNA sequence data. These have clearly shown that isolates of C. dispersa form a clade distinct from Chrysoporthe, Holocryphia and other taxa, which it resembles morphologically. Pathogenicity tests showed that C. dispersa is not pathogenic to H. natalensis, but a potential pathogen of Eucalyptus and Tibouchina spp. The collection of studies included in this thesis demonstrated that Chrysoporthe spp. occur in Malawi, Mozambique, Zambia, Kenya and Tanzania on both Eucalyptus and native Syzygium cordatum trees. This significantly expands the geographical distribution of these important pathogens. The studies have also shown that Chrysoporthe cubensis has recently been introduced on the continent. It is my hope that new knowledge emerging from studies in this thesis will aid in quarantine measure to control the spread of these important fungal pathogens including the new species Celoporthe dispersa. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

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.

No description available.