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Browsing by Author "Kim, Mee-Sook, committee member"

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    Community structure and pathogenomics of Pinaceae-infecting Fusarium spp.
    (Colorado State University. Libraries, 2024) Dobbs, John, author; Stewart, Jane E., advisor; Kim, Mee-Sook, committee member; Sloan, Daniel B., committee member; Heuberger, Adam L., committee member
    Due to a warming climate, the need for nursery grown conifer seedlings is continually increasing. However, several Fusarium spp. that cause pre- and post-emergent damping-off and root disease can hinder production of conifer seedlings. These soil or seed-borne Fusarium pathogens of conifers infect seedlings through the developing roots, and their similar effects on conifer hosts suggests that these pathogens may share a common evolutionary history. The shared ecological function of these Fusarium pathogens is likely associated with lineage-specific (LS) chromosomes or virulence gene(s) that are shared among these species. Identifying these potentially shared chromosomes or gene(s) and their functionality is best approached through the use of multiple 'omics technologies. Taken together, genomics, transcriptomics, proteomics, and metabolomics provide a comprehensive overview of the plant-microbial interactions at the time of Fusarium infection. This research accentuates how a combination of these technologies, such as genomics and transcriptomics, can be used to elucidate the biology of Fusarium pathogens and identify the presence of virulence-associated LS chromosomes or virulence gene(s) that facilitate the development of tools to rapidly identify and track these important pathogens. Chapter two, published in Frontiers in Plant Science, presents the observed regional effect on community structure of Fusarioid fungi collected from conifer seedlings among nurseries across the contiguous USA. The need for a global consensus to establish and maintain databases based on Fusarioid species type strains as references due to the continuing taxonomic disputes about the appropriate classification of Fusarium spp. designations was also discussed. For this reason, phylogenetic placement of the isolates was used for species identification; however, it is recognized that more research, such as whole genome sequencing, is needed to further validate the taxonomic identify of the isolates used in this study. Chapter three presents the whole genome comparisons of 17 Fusarium spp. isolates collected from conifer seedlings. Based on phylogenetic analyses of 16 conserved loci and composition of predicted genes, species were shown similar within and among Fusarium species complexes. Putative profiles of pathogenicity/virulence genes, including secreted in xylem (SIX) genes 2, 3, 9, and 14, and secondary metabolites, including the mycotoxins fumonisin and deoxynivelanol, were identified among the species complexes, but validation of expression of these genes is needed to demonstrate their functionality. Chapter four explores the mechanisms of pathogenicity and/or virulence of two understudied Fusarium spp., F. commune and F. annulatum, on conifer and non-conifer hosts and the differential gene expression in a susceptible conifer species. Further, the putative secretome profiles of Fusarium spp. within species complexes were identified, containing secreted carbohydrate-active enzymes, major facilitator supergroup transporters, apoplastic effectors, and gene products involved in secondary metabolite biosynthesis such as prolipyrone B/gibepyrone D, aurofusarin, and deoxinivelanol. Results from this study showed F. annulatum and F. commune caused disease on young conifer and non-conifer seedlings and identified putative genes associated with broad pathogenicity, and possibly indicating age-related resistance within the conifer host to certain upregulated pathogenicity genes. Due to the threat of spreading fungal pathogens from nurseries to field sites through latent infected seedlings and seed, this research highlights the need for robust early detection methods, while also providing insight into the biology of 17 Fusarium spp. that are potentially pathogenic to conifer seedlings. This research will help further develop technologies that aid managers for controlling Fusarium damping-off and root disease and mitigating the spread of novel haplotypes across regions.
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    Fire-associated shifts in the soil microbiome in western conifer forests: implications for Armillaria root disease biocontrol and management
    (Colorado State University. Libraries, 2024) Fitz Axen, Ada J., author; Stewart, Jane E., advisor; Kim, Mee-Sook, committee member; Charkowski, Amy O., committee member; Abdo, Zaid, committee member
    The research presented in this thesis integrates the current understanding of environmental disturbances, plant associated microbiomes, and microbial biological control of fungal forest pathogens to contribute to improved disease management. In Chapter 2, I examined how fire disturbances affect soil microbial communities in areas where Armillaria root disease, caused by the pathogen Armillaria solidipes, is prevalent by documenting changes to bacterial and fungal community diversity and composition following three distinct levels of burn severity (high, low, and unburned) in a conifer forest in northern Idaho, United States. Expected reductions in bacterial community alpha diversity were observed when comparing burned communities with unburned communities; however, fungal communities showed a lack of significant change in alpha diversity in response to burn severity at the sampling time of 15-months post-fire. However, in both bacterial and fungal soil communities, compositional changes corresponding to burn severity levels were observed. Further examinations characterized similarities and differences between burn severity-associated communities and Armillaria species-associated communities to determine how these microbial changes might influence Armillaria root disease. At high-severity burn sites, colonization by A. solidipes and the associated microbial community was prevalent when compared with low-severity burn and unburned sites. In contrast, the presence and abundance of the weakly pathogenic species A. altimontana and its associated microbial community, including beneficial ectomycorrhizal fungi, appeared to be negatively impacted by high-severity burns. Further research is needed to determine which microbial taxa are critical for promoting or suppressing A. solidipes activity, yet the results from this study suggest that high-severity burns may create environments hospitable to this pathogen and thus monitoring for increased disease pressure following severe burns may be warranted. Chapter 3 focuses specifically on beneficial members of the native soil microbial community that exhibit antagonistic activity against A. solidipes. Because these native species are adapted to the environmental conditions and community interactions, they are more likely than foreign microbial species to successfully establish a stable population required for effective biological control. I isolated putative native biological control agents from soil samples collected under different burn severity conditions and tested their in vitro capabilities to inhibit the growth of A. solidipes with dual culture confrontation tests. Effective in vitro pathogen inhibition was observed with 10 microbial isolates, including five bacterial isolates from the genera Bacillus and Caballeronia and five fungal isolates from the genera Trichoderma and Mortierella. Further examination of the sites these microbes and communities originated from and their compositional changes documented in Chapter 2 revealed that the presence or abundance of our effective biological control organisms did not differ based on burn severity. Importantly, this suggests that fire disturbances may not directly influence the use of these species in management methods for Armillaria root disease in similar conifer forests. However, considering the increased presence of A. solidipes observed following a high-severity burn, there may be additional biotic or abiotic influences apart from biological control agents that are influencing the activity of A. solidipes after fire. These studies enhance our understanding of how abiotic and biotic influences interact to affect the presence of virulent soilborne forest pathogens and associated soil microbes. Considering the effects of these interactions is critical for the development of sustainable long-term management strategies that will help to preserve these ecosystems facing increasing environmental and pathogen-related stressors. The overall goals of this research are to build upon the growing body of research examining how the soil microbiome contributes to disease development and to provide tangible results that can be incorporated by forest managers to help reduce damage caused by Armillaria root disease in fire-prone conifer forests.
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    Lophodermella needle cast pathosystem: the phylogenetic relationships, host-mycobiota interactions, and molecular diagnosis of Lophodermella pathogens on Pinus
    (Colorado State University. Libraries, 2022) Ata, Jessa Pude, author; Stewart, Jane E., advisor; Abdo, Zaid, committee member; Kim, Mee-Sook, committee member; Mondo, Stephen J., committee member; Norton, Andrew P., committee member
    The impact of needle diseases in conifer stands has increased worldwide due to regional variations of warmer and wetter climates that spur the activity of needle pathogens. Heavy needle cast infection results in loss of growth among pine stands which can lead to losses in biomass production and decline in ecosystem goods and services. Despite this threat, a well-informed disease management strategy is lacking due to limited research on many needle pathogens that remain to have unclear taxonomy, uncharacterized fungal biology, and unknown trophic lifestyles and interactions. Thus, this research applied molecular tools to understand conifer needle pathosystems, particularly Lophodermella needle casts that have caused epidemics on Pinus contorta stands in Colorado, USA. Specifically, this research aims to analyze the phylogeny of Lophodermella species using molecular data and identify shared derived characters for taxa delimitation; investigate the interaction of the mycobiota and the P. contorta host in healthy versus diseased states; and develop molecular tools for the rapid diagnosis of Lophodermella needle cast. To achieve these objectives, this research is divided into five chapters. The first chapter gives an overview of the emerging needle diseases worldwide and the needle cast epidemics on P. contorta in Colorado caused by Lophodermella concolor and L. montivaga. It discusses current knowledge on the Lophodermella pathogens and management strategies for needle diseases. The second chapter highlights the relationship of Lophodermella species from North America (L. arcuata, L. concolor and L. montivaga) and Europe (L. sulcigena and L. conjuncta), and their potential synapomorphic characters. It also revealed a newly identified, genetically unique rhytismataceous species on Pinus flexilis that is morphologically similar to L. arcuata. The third chapter discusses the adverse impact of the diseases to needle mycobiota and the defense strategies of the P. contorta host. It further shows, for the first time, the endophytic lifestyle of Lophodermella pathogens on P. contorta. The fourth chapter details the efficiency of the PCR- based markers developed from multi-copy and single-copy gene regions to identify and detect L. concolor and L. montivaga on P. contorta, and L. arcuata and Bifusella linearis on P. flexilis. And lastly, the fifth chapter summarizes the important results of this research and discusses their potential implications on the management of emerging needle diseases. My dissertation closes with recommendations on future research that will address further questions of needle diseases caused by Lophodermella species and other pathogens.
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    Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and development of molecular tools for early detection and monitoring
    (Colorado State University. Libraries, 2019) Dobbs, John, author; Stewart, Jane, advisor; Kim, Mee-Sook, committee member; Sloan, Daniel, committee member
    Pathogenic and non-pathogenic Fusarium oxysporum are morphologically indistinguishable from each other. Pathogenic F. oxysporum f. sp. koae (Fo koae) is a limiting factor for low to mid elevation, below 610m (2000 ft), Acacia koa forests and timber stands. These warmer, lower elevation sites are best suited for optimal growth of Acacia koa, but Fo koae hinders efforts to establish stands at these elevations. Detection of pathogenic isolates is necessary for informing land managers and disease resistance breeding programs. Current methods to distinguish pathogenic isolates are conducted through costly, extensive greenhouse virulence assays. Genomic comparisons of pathogens and non-pathogens such as amplified fragment length polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs), Random Amplification of Polymorphic DNA (RAPDs), pathogenicity-related genes, and microsatellites have been effective for detecting pathogens, and these genomic comparisons are more time and cost effective than traditional greenhouse virulence assays. Chapter two of this thesis examined whole genomic comparisons of a pathogenic F. oxysporum f. sp. koae (Fo koae 44) and a F. oxysporum (Fo 170) isolate found to be non-pathogenic to Acacia koa, for the identification of genomic features that could be used to distinguish pathogenicity. Genome sizes were comparable at 48Mb and 50Mb, respectfully. Fo koae 44 and Fo 170 shared an average nucleotide identity of 96%. Eleven syntenic putative core chromosomes and one unique putative lineage-specific chromosome were identified when compared to a reference strain of F. oxysporum f. sp. lycopersici. Pathogenicity-related genes, including the secreted in xylem (SIX) genes, Fusarium transcription factors, and Fusarium transporters, and unique sequences were identified as exclusive to Fo koae 44 when compared to Fo 170. These variants were used to develop pathogen-specific primers. When tested on previously characterized pathogenic (highly and moderate virulence) Fo koae and low virulent or non-pathogenic Fo isolates, six primers only amplified moderate and highly virulent isolates of Fo koae. Haplotype networks were constructed based on sequencing data of previously characterized Fo koae and Fo isolates and field collected isolates with no pathogenicity data at the translation elongation factor 1-α and RNA polymerase II second largest subunit to determine the genetic relationships of these two groups. Some field collected isolates grouped with highly virulent Fo koae isolates. These results suggested that these field collected isolates might be highly virulent and contain the putative lineage-specific chromosome.