Caliciopsis pinea is the causal agent of caliciopsis canker disease. The fungus is an emerging pathogen affecting Pinus strobus (eastern white pine) across its native range in North America. Infections result in canker formation, reduced tree vigor, and economic losses due to downgraded lumber quality. Despite increasing reports of severe disease outbreaks, many aspects of C. pinea’s life cycle, pathogenicity, and mechanisms of spread remain poorly understood. Current diagnostic challenges stem from the pathogen’s morphological similarity to closely related species and its ability to infect hosts without production of visible fruiting structures, necessitating more precise molecular detection methods. This dissertation addresses gaps in the existing literature of this pathosystem by first presenting a comprehensive guide on the diagnostic options available to study the conifer-infecting members of the Caliciopsis genus. Microscopic and molecular tools for diagnosis are reviewed and methods for studying Caliciopsis spp. in the laboratory setting are discussed (Chapter 1). This dissertation also investigates species diversity in the Caliciopsis genus through multigene phylogenetic analyses and the description of new species in Michigan. Microscopic and molecular techniques were combined to create a three-locus concatenated phylogenetic tree using sequences generated in the study and culled from GenBank for an analysis that showed with strong statistical support, the presence of three, previously undescribed Caliciopsis species present in Michigan. These findings are supported by microscopy which demonstrated that morphological differences were present between novel species and other known species of Caliciopsis including length and width of mazaedia, placement of the locule, and ascospore size. Specific epithets are proposed for species described in this study, and a review of the taxonomic history and documentation on the existence of type specimens for all known species of Caliciopsis is offered (Chapter 2). Phylogenetic studies are followed by the development of a highly sensitive and specific quantitative polymerase chain reaction (qPCR) assay targeting the internal transcribed spacer (ITS) region of C. pinea. Several members of the Caliciopsis genus are known pathogens, but C. pinea is the most aggressive on its preferred host. Diagnostic options for this species are limited and are expanded on here with the development of new molecular techniques for detection, quantification, and diagnosis. The assay was validated against 51 target and non-target isolates from across the P. strobus native range, achieving a detection limit of 10 fg of C. pinea DNA. To ensure reproducibility, the assay was tested in multiple laboratories and across different thermocycling platforms. In all cases the reaction detected only C. pinea when screened against non-target species and the target was picked up in the background of plant DNA from infected plant material (Chapter 3). The diagnostic assay developed in these studies was then utilized to investigate the environmental factors influencing C. pinea spore dispersal. To discover optimal conditions for inoculum detection, a novel approach combining qPCR with rotating-arm air sampling was implemented over a six-month sampling period in 2021 and 2022. These air sampling devices were deployed in natural ecosystems to monitor airborne inoculum levels, providing critical insight into spore release patterns. By integrating advanced molecular diagnostics with field-based spore monitoring, this study enhances our ability to detect, diagnose, and understand the epidemiology of C. pinea. The findings have significant implications for forest management, disease mitigation, and the long-term health of eastern white pine (Chapter 4). Cumulatively, this dissertation provides the most comprehensive examination of C. pinea to date, addressing key gaps in its diagnosis, taxonomy, and epidemiology. By integrating phylogenetic analyses, molecular diagnostics, and environmental monitoring, this research enhances our ability to detect, classify, and understand C. pinea and its impact on P. strobus. Understanding spore dispersal dynamics and optimal conditions for inoculum detection provides critical insights for predicting and managing disease outbreaks. Future research should build upon these findings by exploring host-pathogen interactions at the molecular level, assessing environmental factors driving disease progression, and expanding monitoring efforts across different regions and climate conditions. As forest ecosystems face increasing stress from climate change, invasive species, and emerging pathogens, proactive disease management is essential for maintaining forest populations and overall resilience. This dissertation contributes valuable tools and foundational knowledge toward that goal, providing a framework for future studies and informing strategies to mitigate the impact of caliciopsis canker disease on North American forests.
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