Theses and Dissertations

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Buffering the effects of a changing climate: Salsola tragus as a potential source of stress tolerance genes
(Colorado State University. Libraries, 2024) Lemas, John M., author; Gaines, Todd, advisor; Brown, Cynthia, committee member; Henriksen, James, committee member
The tumbleweed Salsola tragus is an allotetraploid C4 weedy member of the Salsola polyploid complex. Commonly referred to as Russian thistle, it develops a thorny habit during inflorescence, and commonly separates at an abscission layer near the soil to form a tumbleweed. This species is economically important to all land use types and is especially impactful in the Northwestern United States where it affects spring cereal production. The International Weeds Genomics Consortium recently completed a fully annotated reference genome assembly for each of the sub genomes in the somatic cells of this allotetraploid. Polyploids, in general, are overrepresented in the most troublesome weeds globally, and Salsola tragus is no exception. Recurrent formation of polyploids, increased activity of transposable elements, and increased mutation rates that follow genome duplication may lead to the de novo formation and selection of novel highly adapted alleles over time. We utilized the reference genome assembly for this species to align a stress-response transcriptome to investigate how this species responded to two selected abiotic stressors. Many expected response pathways are represented, including response to stress phytohormones, sodium-proton antiporters, calcium exchangers, and cold-responsive binding factors. In addition, several uncharacterized proteins were differentially overexpressed in the shoot and root tissues of this species. Identified genes from this species may present novel alleles for osmotic and temperature stress tolerance. Uncharacterized genes may represent novel stress response genes and can be used to improve the provided reference annotation for this species. These genes of interest may provide the scientific community with additional genomic resources to bolster crop production in this era of climate change.
Open Access
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.
Embargo
Species distribution models for and policy approaches to invasive plant ecology and management
(Colorado State University. Libraries, 2024) Teich, Nathan Benjamin, author; Brown, Cynthia S., advisor; Jarnevich, Catherine, committee member; Pearse, Ian, committee member; Evangelista, Paul, committee member
The ability of abundance-based Species Distribution Models (SDMs) to predict where invasive plants can be abundant, and to what degree, is a powerful research and management tool. Often, invasive plant abundance-based SDMs are created using similar inputs and approaches as occurrence SDMs. However, invasive plant ecology literature suggests that the factors found to control invasive plant abundance are more diverse and contextual, and therefore not entirely interchangeable with factors that control invasive plant occurrence. To ensure invasive plant abundance-based SDMs are leveraging the robust body of knowledge, this paper aims to highlight and summarize the ecological factors underpinning invasive plant abundance and reviews how those factors can be represented within abundance-based SDMs. I find that while the inclusion of invasive plant abundance governing factors often improves abundance-based SDM performance, certain governing factors are ubiquitously represented while others are less commonly accounted for in model creation despite their ecological importance. Barriers to incorporating invasive plant abundance governing factors into abundance-based SDMs often include data limitations or methodological uncertainty. Finally, we provide future research directions that would help address certain barriers and improve our ability to integrate abundance governing factors into SDMs. Invasive plants, when they become dominant components of a plant community, threaten native species and ecosystem processes. Abundance-based SDMs are gaining traction as a geospatial tool to predict where invasive plants can become abundant and have negative impacts. Biotic interactions influence invasive plant abundance locally but are often not included within the abundance-based SDM creation process. At present, it is unknown to what degree local-scale biotic interactions with other plant species determine locations where invasive plant species can become abundant. Using data from large-scale abundance observations of the invasive plant cheatgrass (Bromus tectorum) paired with data from plant communities in the western United States, we quantified the degree to which biotic interactions explain where cheatgrass is abundant beyond what would be anticipated from an abundance-based SDM created with abiotic and landscape context predictors alone. To this end, we fit Generalized Linear Models (GLMs) for different categories of cheatgrass abundance and used the predicted suitability SDM outputs alongside biotic variables, representing known competitive and facilitative interactions, to determine if including biotic interactions improved a model's explanatory power. The addition of biotic variables marginally improved GLMs for low (5-25%) and medium (25+-50%) cheatgrass abundance but displayed greater improved performance for high (50+%) cheatgrass abundance. Most notable amongst the specific biotic variables was the cover of perennial graminoid cover, representing known competitors of cheatgrass, which interacted with SDM environmental suitability to strongly reduce the probability of high cheatgrass abundance. These findings suggest that considering biotic interactions alongside SDM predicted suitability may indeed improve our ability to predict abundance locations of invasive plant species, but potentially only in specific contexts such as where that species can already achieve high abundance. Invasive plants cost the US billions of dollars each year due to ecological and economic impacts as well as management costs. One of the most common pathways of introduction and spread of invasive plants is through ornamental plant sales. While solutions such as regulations and voluntary self-bans have been implemented in some instances to mitigate this problem, widespread adoption has not occurred. As such, new alternatives should be explored. Opt-in labeling programs are commonly used throughout the agricultural industry to better inform customers about the products they are purchasing. An opt-in labeling program that consists of a partnership between retailers and governments or non-profit organizations could help reduce the spread of invasive plants by influencing customer behavior. This approach would be less costly to retailers than regulations, create new invasive plant prevention opportunities for governments and non-profits, and better inform consumers about specific invasive plant species.
Embargo
Candidate gene identification for glyphosate resistance and rapid cell death in Ambrosia trifida
(Colorado State University. Libraries, 2024) Sparks, Crystal Devona, author; Gaines, Todd, advisor; Dayan, Franck, committee member; Beffa, Roland, committee member; Nishimura, Marc, committee member; Westra, Phil, committee member
Glyphosate is one of the most widely used herbicides worldwide due to favorable chemical characteristics and availability of compatible transgenic biotechnology in crops. Resistance to glyphosate has evolved in many weed species capable of significant yield reduction in top production systems globally. One such species is Ambrosia trifida (giant ragweed), a monoecious broadleaf with imperfect flowers native to North America where it is highly competitive in corn, soybean, and cotton production. Some glyphosate resistant populations of A. trifida also display a rapid response with cell death in the mature leaves within 24-48 hours after treatment with glyphosate. Transcriptomic analysis revealed differential expression of multiple gene families associated with known glyphosate resistance mechanisms such as ATP-binding cassette (ABC) transporters and aldo-keto reductases. Gene ontology analysis showed an enrichment of many genes related to phytohormone response to biotic and abiotic stress in the differentially expressed genes. This could be related to a novel glyphosate resistance mechanism or a signaling cascade involved in the rapid cell death response. The A. trifida genome contains two loci of the glyphosate target site gene 5-enolpyruvylshikimate-3-phosphate-synthase (EPSPS), with a previously reported Pro106Ser mutation in EPSPS2. This locus showed up-regulation by three hours after treatment. Trait mapping revealed three genomic regions associated with glyphosate resistance and a single interval associated with the rapid response. Along with phenotypic segregation ratios, this indicates that resistance and rapid response traits are genetically independent and multiple genes likely contribute to resistance.
Open Access
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.
Open Access
Investigating the resistance status to permethrin and temephos in Aedes aegypti (the yellow fever mosquito)
(Colorado State University. Libraries, 2024) Arthur, Nicholas Wynne, author; Norton, Andrew, advisor; Camper, Matt, committee member; Kading, Rebekah, committee member; Saavedra-Rodriguez, Karla, committee member
Aedes aegypti (Ae. aegypti) is the principle urban vector of several viruses of high medical significance which carry a disease burden on a global scale. Ae. aegypti is anthropophilic and lives in close association with humans. This places nearly half of the global population at risk of becoming infected with an arboviral pathogen every year. Therefore, emphasis must be placed on investigating methods for controlling this vector to combat and reduce the spread of human disease. This is especially true in areas where socioeconomic factors promote sustained transmission cycles. While vector control programs use a variety of strategies, the primary method of reducing vector populations is through insecticide use. Widespread use of insecticides has placed intense selection pressures on Ae. aegypti populations and resistance mechanisms have developed. Target site modifications and the expression of detoxifying enzymes are the most significant resistance mechanisms to date. Several single nucleotide polymorphisms resulting in amino acid changes within the voltage-gated sodium channel (VGSC) have been shown to reduce binding site sensitivity and confer resistance to pyrethroids. Specifically, mutations at the knockdown-resistant (kdr) 410, 1,016, and 1,534 sites have been associated with a reduction in pyrethroid sensitivity. I investigated the resistance status to permethrin and temephos at five locations in Hidalgo County, Texas. I determined the presence of permethrin resistance using a well-characterized susceptible colony as a reference for insecticide sensitivity. The resistant allele C1,534 reached fixation at all sites and L410 and I1,016 were found at high frequencies. The permethrin resistance was over 40-fold when compared to the reference colony. The sites were less resistant to temephos at approximately 6-fold to 12-fold, which I attributed to cessation of this insecticide in the continental United States since 2016. In the absence of selection pressures mosquito populations trend towards susceptibility, which suggests that there are potential fitness costs associated with insecticide resistance. Studying these associations is important to public health as they may support different strategies to reduce vector populations. I used two collections from Tapachula, Mexico, that were free of pyrethroid exposure since 2013, to determine the presence of two previously described fitness cost metrics: wing length and egg production. I found that the average wing length of V410L and V1,016I homozygous resistant individuals were significantly smaller compared to homozygous susceptible individuals. The interaction between wing length and genotype had no effect on egg production. Wing length had no significant effect on egg production. Most notably, L410 and I1,016 resistant alleles had no effect on egg production.
Open Access
Factors contributing to herbicide response in CoAXium wheat
(Colorado State University. Libraries, 2024) Pelon, Amber L., author; Dayan, Franck, advisor; Gaines, Todd, committee member; Schipanski, Meagan, committee member
Compared to other pests, weed competition has the most significant negative impact on wheat grain yield. Understanding the contribution of metabolism in overall tolerance to herbicides can lead to new methods for controlling weeds in wheat. Glutathione S-transferase's (GSTs) role in the detoxification of herbicides has been studied since 1970. Previous literature reported increased resistance to herbicides with higher GST activity in black grass (Alopecurus myosuroides) and Asia minor bluegrass (Polypogon fugax). Resistance could be reversed by inhibiting GST activity. This research assesses the role of Phase 2 plant cell metabolism by testing (GST) inhibition to see if it influences the metabolism of quizalofop P-ethyl (QPE) in winter wheat (Triticum aestivum). We hypothesized that the addition of a safener would make the wheat more tolerant to the herbicide while the addition of a GST inhibitor would make the wheat more sensitive to QPE. Experiments were conducted analyzing the QPE effect on whole-plant biomass and an LC-MS/MS analysis of the amount of quizalofop acid (QZA) found in plant extracts. Safeners enhanced herbicide metabolism which increased CoAXium wheat tolerance to QPE. GST inhibitors, conversely, decreased herbicide metabolism causing CoAXium wheat to be more sensitive to QPE. Understanding the contribution of metabolism in overall resistance to herbicides can lead to breeding improvements for more herbicide-tolerant wheat varieties and new methods for controlling weeds in wheat.
Open Access
A microbiome approach to cultivation and management of sugar beet
(Colorado State University. Libraries, 2024) Gaylord, Margaret, author; Trivedi, Pankaj, advisor; Charkowski, Amy, committee member; Wallenstein, Matthew, committee member
The world's population is projected to reach 9.8 billion by 2050, while the urgent threat of climate change is expected to impact crop physiology and pest dynamics. Understanding, preserving and leveraging the plant-associated microbiome can result in enhanced agroecosystem functioning and disease resistance for agricultural crops, thus improving food security. Sugar beet, an economically important sugar producer in the northern hemisphere, offers insights into plant-microbiome dynamics due to its susceptibility to pathogenic microbes and its association with disease suppressive soils. Cultural and chemical management practices of sugar beet are a persistent debate due to the potential negative effects on the essential microbiome and the emergence of resistant populations. To investigate the impact of weed control strategies on the soil microbiome, we conducted a long-term field study at two locations. Using next-generation sequencing and in vitro assays, we assessed the effects of glyphosate, a mix of selective herbicides and tillage treatments on the structure and function of the soil microbiome. Furthermore, we isolated 136 bacteria from the sugar beet agroecosystem and evaluated their antagonistic abilities against key diseases of sugar beet. Through in vitro and greenhouse assays, we identified effective microbial consortia for disease reduction. Additionally, we investigated the interactions between a single antagonistic isolate and an important fungal disease of sugar beet using transcriptomic analysis to reveal underlying mechanisms for biological control and pathogen response. This comprehensive understanding of the impact of various management strategies on the microbiome provides crucial insights for future crop management and highlights the potential for exploiting beneficial microbes to enhance disease control.
Open Access
Exploring the hemp virome and assessing hemp germplasm for resistance to emerging pathogens
(Colorado State University. Libraries, 2024) Hackenberg, Laine, author; Nachappa, Punya, advisor; Roberts, Robyn, committee member; Stenglein, Mark, committee member
Hemp (Cannabis sativa L.), commonly grown for its seeds, fiber, and non-psychoactive cannabinoids, has been experiencing a resurgence in the United States with its recent legalization. While farmers across the nation have readily adopted this crop, resources for pest management are still lacking, particularly regarding the diversity and distribution of pathogens. As production increases and the crop diversifies, the emergence and spread of these pathogens are certain. To circumvent loss due to disease, research is needed understand the threats and to identify sustainable management options. The goal of this study is to describe the diversity and distribution of viruses/viroids infecting hemp in Colorado and to determine if there is genetic resistance to pathogens in hemp. The objectives of this study are to 1) characterize the virome of different hemp cultivars throughout the growing season across different locations and 2) screen a panel of genetically unique genotypes of hemp for resistance to emerging viruses/viroids of hemp. Throughout 2021 and 2022, the hemp virome was examined in four major hemp producing regions of Colorado. In total, nine fields were sampled, and each field was visited during three phenological stages (early vegetative, late vegetative, and mature flowering) in order to characterize the virome throughout the growing season. Leaf tissue samples were collected from two cultivars of hemp from each field site. These tissue samples were submitted for High Throughput Sequencing (HTS) and upon bioinformatic analysis, candidate virus/viroid sequences were validated. Across both years, a total of seven viruses were identified: Alfalfa mosaic virus (AMV), Beet curly top virus (BCTV), Cannabis cryptic virus (CanCV), Cannabis sativa mitovirus (CasaMV1), Grapevine line pattern virus (GLPV), Opuntia umbra-like virus (OULV), and Tomato bushy stunt virus (TBSV). All viruses identified had >97% nucleotide identity to the nearest GenBank accession. Between individual cultivars isolated from the same field, both similar and unique viromes were observed. Viral diversity and incidence increased as the growing season progressed for both years. The three viruses that were most commonly found across all regions were CasaMV1, GLPV, and BCTV. Dominating the virome in viral load were CasaMV1 and GLPV. Given the prevalence of BCTV in the virome, in addition to its prevalence in hemp across the western United States, 13 genotypes of hemp were screened for resistance to this pathogen. These genotypes of hemp are genetically diverse, which will aide in the discovery of candidate genes involved with resistance. BCTV is the causal agent of curly top disease which can have drastic symptomology in hemp plants, causing malformed growth, stunted plants, and crop loss up to 100%. Varying BCTV copy number was observed across the hemp genotypes. Additionally, percent disease index (PDI) was analyzed to determine the frequency of infection of individual genotypes. Two of the genotypes were observed to have a lower PDI than the others, 4587 and 4710. Hop latent viroid (HLVd) has been emerging as a threat to the cannabis industry. It has been described across North America but is believed to be worldwide due to its global distribution in hops. HLVd has been documented to cause drastic reduction in cannabinoid content in mature inflorescences and therefore has the potential for substantial economic losses. Although not identified within the 2021 or 2022 virome, HLVd was determined to be an important threat facing hemp production therefore it was included in the screening. Similarly to BCTV, a panel of 14 genetically unique genotypes of hemp were analyzed for resistance to HLVd. Resistance was identified in a single genotype, 517, which had a lower frequency of infection than the others. However, no varying viroidal loads were observed between genotypes. Throughout this study, viruses associated with hemp were described as well as the identification of genetic resistance to emerging pathogens. This work will help to further integrated pest management strategies and promote sustainable agriculture.
Embargo
The need for new inhibitors of photosynthesis in agricultural settings, and the novel herbicidal compound AS9057
(Colorado State University. Libraries, 2024) Twitty, Alyssa, author; Dayan, Franck, advisor; Abdel-Ghany, Salah, committee member; Phillip, Yael, committee member
Due to increased food demand, the need for use of herbicides is both necessary and on the rise. Several herbicide classes target photosynthetic electron transport: HRAC Groups 5, 6, and 22. These herbicides are used in large amounts in many different cropping systems to control several species of broadleaf and grass weeds. The first chapter provides a comprehensive review of what these photosynthesis inhibitors are, how they are used and their mode of action. Presently, commercial herbicides only inhibit electron flow at two different sites (PSII and PSI). Those which inhibit electron flow at PSII block the movement of electrons down the electron transport chain, while those which inhibit at PSI accept electrons. Necrosis developing on the leaves of plants treated with PSII and PSI inhibitors is due to the accumulation of reactive oxygen species. Evolution of resistance, toxicity concerns, and other limitations of these herbicides call for the exploration of new chemistries that can be used to target this pathway. One of these new chemistries has been identified as AS9057. AS9057 is a natural product identified as a novel herbicide with a potentially new mode of action using AI4AI, an AI platform for herbicide discovery developed by Agrematch. Greenhouse trials demonstrated that the herbicidal activity of AS9057 was light-dependent. The rapid burndown symptoms-developing on treated plants, combined with its chemical structure, suggested that AS9057 may target photosystem II. Measurements of photosynthetic electron transport rates in treated plants alongside data from oxygen evolution assays did not support this hypothesis. Further experiments suggested the AS9057 may instead act as an electron diverter. Oxygen consumption assays in isolated thylakoid membranes using a variety of electron transport inhibitors revealed that AS9057 likely acts on photosystem I in a similar manner to paraquat, but at a potentially new step between P700 and NADP+. This is consistent with other reports that AS9057 can act as an electron acceptor for flavoproteins. Ferredoxin-NAPD+ reductase is a flavoprotein with a redox potential similar to that of AS9057. Thus, it is currently hypothesized that AS9057 acts as an electron acceptor at or near the ferredoxin to form a radical and generate reactive oxygen species which causes the light-dependent herbicidal effect which is observed in treated plants from greenhouse trials.
Open Access
Population genetics prior to biological control: Ceutorhynchus weevils proposed for managing garlic mustard
(Colorado State University. Libraries, 2009) Rauth, Steven J., author; Hufbauer, Ruth, advisor
I studied the population genetic structure of three weevil species, Ceutorhynchus alliariae, C. roberti, and C. scrobicollis, being considered for the biological control of garlic mustard, Alliaria petiolata, in North America. My first objective was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay which could be used to identify the morphologically indistinguishable larvae. This assay was developed for use in the analysis of population genetic structure of the three species and to aid scientists in evaluating host-specificity test results where larval development was incomplete or adults failed to emerge. The resulting assay provides a fast and inexpensive means of identifying otherwise indistinguishable larvae. My second objective was to study the population genetic structure of C. scrobicollis, to evaluate whether the areas where individuals were being collected for host-specificity testing consisted of one or more populations, to estimate the numbers of individuals needed during host-specificity testing and later introduction to adequately represent the diversity of the population, and to evaluate dispersal potential. Results suggest that C. scrobicollis in the area of Berlin, Germany constitute a network of subpopulations with low but significant differentiation among sites and movement of individuals between sites. I estimated that the number of individuals that would need to be sampled to capture 90% or 99% of the genetic diversity in the Berlin area was 10 and 27, respectively. The estimated average dispersal distance based on assignment tests for C. scrobicollis was 28 km. My third objective was to compare the differences in population genetic structure between C. alliariae and C. roberti to determine whether differences in genetic diversity or dispersal potential might aid in prioritizing one species over the other. These two species have similar life histories, distributions, and effects on garlic mustard. Results showed that, over a comparable region in central Europe, total gene diversity was significantly higher in C. roberti, though the difference between the two species was relatively small. Assignment tests suggest there is substantial gene flow among sites for both species. Overall, the results were similar for both species, and I recommend prioritizing based on biological or methodological attributes.
Open Access
Molecular genetics of glyphosate resistance in Palmer amaranth (Amaranthus palmeri L.)
(Colorado State University. Libraries, 2009) Gaines, Todd A., author; Westra, Philip, advisor; Leach, Jan, advisor
Glyphosate resistant Palmer amaranth populations were identified in Georgia in 2004. Studies were undertaken to characterize inheritance, the molecular basis of resistance, and the potential for gene transfer to related Amaranthus species. Dose response results support rejecting a monogenic inheritance hypothesis in favor of an alternative polygenic, additive inheritance model. Apomixis in genetic populations used for inheritance studies is probably occurring and makes interpretation of inheritance difficult. Glyphosate resistance in Palmer amaranth appears to be incompletely dominant and may be polygenic. No target site mutations known to confer resistance were identified in resistant alleles of the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, the target of glyphosate. Estimation of gene copy numbers of EPSPS relative to acetolactate synthase (ALS) in gDNA by quantitative PCR (qPCR) suggested that resistant plant genomes contain 64 to 128 times more copies of EPSPS than susceptible plants. qPCR on cDNA revealed that EPSPS was expressed approximately 35 times higher in resistant plants. Elevated EPSPS copy number is heritable and correlates with expression level and resistance in F2 populations. The molecular basis of resistance is likely due to increased production of EPSPS due to gene amplification. This is the first documented occurrence of EPSPS gene amplification in a weed population under glyphosate selection pressure. The risk of resistance gene transfer was measured with field studies and hand crosses with A. hybridus, A. retroflexus, A. powellii, A. spinosus, and A. tuberculatus. Glyphosate application (0.4 kg ha-1) was used to screen for resistant progeny from the crosses. Hybridization with A. spinosus occurred in both years of the field study and in hand crosses, with average frequency ranging from <0.01% to 1.4%. Hybrids with A. spinosus were either monoecious or dioecious. Monoecious plants produced seed through self-pollination, and the F2 progeny were segregating for resistance. Hybridization occurred in the 2007 field study with A. hybridus (<0.01%) and A. tuberculatus (0.08% and 0.19% for two accessions), all of the hybrid plants were dioecious, and none produced seed. The highest risk for glyphosate resistance gene transfer from A. palmeri is to A. spinosus.
Open Access
Iris yellow spot virus in Colorado onions: a survey of its spatial distribution and techniques to manage the pest
(Colorado State University. Libraries, 2007) Fichtner, Scott M., author; Schwartz, Howard F., advisor; Hill, Joe, advisor
The Iris yellow spot virus (IYSV) is a new, and at times devastating pathogen of alliums throughout the U.S. as well as many other countries. Since its discovery in Colorado in 2001, IYSV has been identified in nearly all of the major onion producing regions within the state. The severity of this virus disease appears to fluctuate from one year to the next but incidence continues to increase with newly infested fields identified each year. With the number of outbreaks on the rise and the inadequacy of current control strategies, new management techniques as well as novel chemistry pesticides have become a major focus for the management of this virus disease and its thrips vector. Our trials demonstrated the use of reflective materials such as straw or silver reflective mulch can result in a reduction in thrips populations by as much as 69% on onions and a reduction of nearly 9% in IYSV disease incidence. Additionally, Entrust (Spinosad) and Aza-Direct (Neem extract) were found to work as well as or better than conventional materials such as Warrior (Pyrethroid) and Lannate (Carbamate). To better understand the epidemiology of the IYSV pathogen, we also conducted an extensive survey at several locations along the Colorado Front Range and Western Slope. In our surveys, we collected information including thrips populations and incidence of IYSV using 0.2 ha grids developed using mapping software (MapInfo) creating several randomly chosen plots in each field. With data collected on several sampling dates, we attempted to identify a spatial correlation of within field spread of the virus during the growing season. The levels of positive spatial autocorrelation from our survey locations were minimal, this leads us to believe that secondary outbreaks of the disease are occurring in a random fashion across the field.
Open Access
Functional characterization of germin family genes contributing to broad-spectrum, quantitative disease resistance in rice
(Colorado State University. Libraries, 2009) Davidson, Rebecca M., author; Leach, Jan E., advisor
Quantitative trait loci (QTL) are predicted to confer broad-spectrum and durable disease resistance. Application of disease resistance QTL in crop improvement programs has been hindered because we lack an understanding of (1) the genes contributing to the QTL-governed phenotype and (2) why certain alleles are more effective than others in conferring resistance. In this study, QTL-associated genes in the germin protein family, germin-like proteins (GLP) and oxalate oxidases (OXO), were identified in the rice genome and their functions were tested. Paralogous multi-gene families underlie the physical QTL regions, with twelve OsGLP members on chromosome (chr) 8 and four OsOXO members on chr 3. Based on shared motifs in 5' regulatory regions and/or protein sequence similarities to cereal orthologues, rice OsGLP genes belong to two germin subfamily groups (GER3 and GER4), and OsOXOs belong to the GER1 group. Conserved sequences for each gene family were used in RNA-interference gene silencing experiments. As more OsGLP genes were silenced, the more susceptible the plants were to two distinct fungal pathogens, Magnaporthe oryzae (Mo) and Rhizoctonia solani (Rs). Similarly, OsOXO-RNAi plants showed enhanced susceptibility to Mo, Rs and the broad host range pathogen, Sclerotinia sclerotiorum. OsGLP alleles were compared in resistant (+chr8 QTL) and susceptible (-chr8 QTL) parental rice lines. Cultivar-specific combinations of OsGLP genes were constitutively expressed and transiently induced by both wounding and Mo infection. In agreement with the silencing data, expression profiles suggest that GER4 subfamily members are involved in rice defense response. Transient induction occurred before fungal penetration of the plant cuticle, and differential expression between resistant and susceptible cultivars correlated with differential hydrogen peroxide accumulation after fungal infection and abiotic stresses. Gene silencing data confirms the roles of OsGLP and OsOXO as contributors to broad-spectrum, basal disease resistance in rice. Studies of allelic diversity among rice varieties suggest that regulation of OsGLPs may explain the effectiveness of resistant alleles compared to susceptible. Germin family proteins are encoded by developmentally regulated gene families in rice and across plant taxa. The germin subfamily members studied here have acquired functions in broad-spectrum defense responses and are important loci for crop improvement.
Open Access
Dispersal of an invasive tumbleweed
(Colorado State University. Libraries, 2007) Baker, Dirk V., author; Beck, K. George, advisor
Dispersal is of critical importance for species conservation and reducing the spread of invasive species. I investigated the dispersal of the invasive tumbleweed, Centaurea diffusa. Tumbleweed dispersal is composed of three components; the plant stem must break free; wind must be sufficient to move plants; and seed must be retained in the plant with time and distance. Dispersal peaks were variable in space and time. Dispersal events correlated with wind gusts and variability in temperature. However, 18 and 78% of plants tumbled over 2 years at sites near Larkspur and Superior, CO, respectively. Weather did not fully explain the differences in dispersal magnitude. Raising plants from the sites in common garden revealed that Larkspur plants required 57% greater force to break than plants from the Superior site. In addition, plants under dry post-senescence soil conditions required four to six times greater force to break compared to plants under moderate to high soil moisture. Based on wind tunnel measurements, wind velocities necessary to break C diffusa stems ranged from 16 to 37 m/s. I developed a GIS-based model for the movement patterns of this plant based on topography, vegetation and wind patterns. Such modeling could provide information to help reduce the spread of this important invasive weed. However, model accuracy needs to be improved before it will be applicable to management. I conducted field and wind tunnel experiments to estimate seed dispersal with time and distance. The 95 percentile confidence limits for the half-life of seed retention in non-dispersed plants were 3 and 14 weeks suggesting that even late-dispersing plants have strong potential for longer distance dispersal of seeds. Seeds were commonly retained in plants for distances of 200 to 400 m and one still had seed in it after traveling over 1 km. Seed dispersal with distance seems linear, though exponential decay may better represent extreme dispersal events. Both models substantially overpredicted dispersal distances for field data collected during a drought year in the presence of biocontrol insects. This research has substantially increased knowledge of the dispersal of this plant and has highlighted future research needs.
Embargo
Involvement of CYP72A219 in herbicide-resistant Palmer amaranth and the role of P450 reductase in the mechanism of metabolic resistance
(Colorado State University. Libraries, 2023) Rigon, Carlos A. G., author; Gaines, Todd A., advisor; Dayan, Franck E., advisor; Beffa, Roland, committee member; Peebles, Christie, committee member
Herbicide resistance in weeds poses a major challenge to modern agriculture worldwide, impacting effective weed control strategies. Metabolic resistance stands out as the major and more complex resistance mechanism due to its ability to metabolize a wide range of herbicides within weed species. Metabolic resistance involves herbicide metabolism through three key phases: activation, conjugation, and sequestration. These phases involve the action of important enzymes such as cytochrome P450 monooxygenases, glutathione S-transferases, and ABC transporters. Metabolic resistance mechanisms have gained prominence in the past decade, posing significant challenges to sustainable agriculture and weed management practices. Amaranthus palmeri (Palmer amaranth) one of the most troublesome weeds globally has evolved metabolic resistance to HPPD inhibitor tembotrione. Understanding and addressing the mechanism are crucial for developing effective strategies to combat herbicide resistance and ensure global crop production. In the present study, four upregulated P450 genes were identified in HPPD-resistant Palmer amaranth from Nebraska (NER), a troublesome weed species. Among these genes, CYP72A219_4284 demonstrated the ability to deactivate the herbicide tembotrione in a heterologous system. This gene was also upregulated in metabolic HPPD-resistant Palmer amaranth plants from different fields across the United States, indicating its involvement in conferring herbicide resistance. Our study also investigated the regulation of these resistance genes, including the promoter sequences and transcription factors involved. Additionally, quantitative trait loci associated with herbicide resistance were identified. This work represents the first identification and validation of genes responsible for herbicide metabolism in Palmer amaranth. Validation of the metabolic resistant gene and the exploration of regulatory mechanisms contribute to a better understanding of metabolic herbicide resistance in weeds, facilitating the development of effective weed management strategies. Cytochrome P450 reductase (CPR), an essential enzyme localized in the endoplasmic reticulum, provides electrons for P450 enzymes during monooxygenase reactions. The transfer of electrons from NADPH to the P450 active site occurs through a complex CPR:P450 interaction. Despite the numerous P450 genes in plant genomes, CPR genes are limited, typically consisting of two or three copies. In Arabidopsis, the two CPR genes, ATR1 and ATR2, have distinct roles in primary and inducible metabolism, respectively. Our study investigated the function of ATR1 and ATR2 in transgenic Arabidopsis plants overexpressing the CYP81A12, which is known to metabolize a wide range of herbicides. The hypothesis was that silencing these ATR1 or ATR2 genes would lead to a reduction of P450 activity involved in herbicide metabolism. ATR1 predominantly transfers electrons to CYP81A12, as knocking down ATR1 led to a significant reduction in herbicide resistance. Knockouts of the ATR2 gene also resulted in decreased herbicide resistance, although the effect was less pronounced. Variation in the number and function of CPR genes among different weed species suggests diverse genetic pressures and potential targets for herbicide resistance management. Inhibition of CPR activity could be a promising approach to restore herbicide effectiveness against metabolic herbicide-resistant weeds. This is the first study to our knowledge that explores the involvement of CPR genes in herbicide resistance in weeds, providing valuable insights into their crucial role. The findings significantly advance our understanding of the mechanisms underlying CPR-mediated herbicide resistance and offer potential targets for the development of effective weed management strategies.
Open Access
Developing integrated pest management tactics for alfalfa mosaic virus and its aphid vector in chile peppers
(Colorado State University. Libraries, 2023) Janecek, Taylor, author; Szczepaniec, Adrianna, advisor; Nachappa, Punya, committee member; Uchanski, Mark, committee member
Alfalfa mosaic virus (AMV, Bromoviridae: Alfamovirus) is a virus transmitted to plants by aphids in a non-persistent manner. The virus was first identified in chile peppers Capsicum annuum L. (Solanales: Solanaceae) in Southern Colorado in 2019. The goal of this research was to explore management strategies to suppress the virus given its devastating impact on the yield and quality of chile peppers. The objectives were to: 1) determine whether chile peppers have innate resistance to AMV, 2) test the effectiveness of host plant resistance and planting date modifications to suppress the virus in the field, 3) determine whether AMV is seed transmissible, and 4) survey abundance and diversity of aphids (Hemiptera: Aphididae) that likely transmit AMV in the system. In the greenhouse experiment, I found significant differences among varieties of chile peppers in the severity of AMV symptoms and identified a variety suitable for a field experiment. In the field, I found that the susceptible variety, Joe Parker, which tended to have high AMV symptoms in the greenhouse, was also highly susceptible to AMV in the field. Conversely, Mira Sol, which appeared to have resistance to the virus in the greenhouse screening assay had low incidence of AMV symptoms and low AMV titers in the field as well. Planting date also played an important role in symptom severity, where late planted peppers (mid-June) had significantly lower severity of AMV symptoms than peppers planted at conventional and early planting dates (the middle and end of May). Despite this, the yield and quality of peppers planted early was significantly greater than that of peppers planted later in the season. In addition, there was evidence of seed transmission of AMV in chile peppers, with 10% for Mira Sol and 2% for Joe Parker, from seeds collected from infected peppers had AMV. Lastly, I found high diversity of aphid species within my experimental plots (14-16 species) and lower diversity in nearby alfalfa fields (4-5 species). Moreover, severity and titers of AMV were positively correlated with earlier planting date, which was likely related to higher aphid densities early in the season. This research contributed to formulating integrated tactics that chile pepper producers can implement in their production to suppress the impact of AMV on the crop. Finally, this is the first report of AMV transmission through seed in peppers and is the first study describing this pathosystems in Colorado.
Embargo
Sweet surprise: the search for genes conferring beet curly top virus resistance
(Colorado State University. Libraries, 2023) Withycombe, Jordan, author; Nachappa, Punya, advisor; Nalam, Vamsi, committee member; Nishimura, Marc, committee member; Dorn, Kevin, committee member
Sugar beets (Beta vulgaris L.) are grown across the western United States and suffer economic loss annually to curly top disease. Curly top disease is caused by the beet curly top virus (BCTV) and is spread by the only known insect vector the beet leafhopper, Circulifer tenellus Baker (BLH). Current management strategies for BCTV include chemical control using neonicotinoid seed treatments and foliar insecticidal sprays, as well as the use of BCTV-resistant sugar beet varieties. However, the underlying genetic mechanism surrounding resistance in sugar beet is unknown. The overarching goal of this study was to identify the mechanism of resistance in sugar beet to BCTV and identify potential genes conferring resistance. The objectives for this study were: 1) classify the nature of BCTV resistance in a resistant (EL10) and susceptible (FC709-2) genotype of sugar beet using host suitability and host preference insect assays, as well as assess viral load within each genotype and 2) characterize the transcriptional response to BCTV infection using RNA-sequencing. To classify the nature of BCTV resistance in each genotype of sugar beet, host suitability and preference assays were conducted using virus infected and uninfected BLH. In host suitability assays, the percentage of surviving BLH adults and the number of nymphs produced when reared on a single plant of either genotype was determined over a 3-week period. There was no difference in adult survival, or the number of nymphs produced on either genotype for the virus infected or uninfected leafhoppers. Host preference assays were used to assess settling behavior of BLH over time when given a choice between the two genotypes. It was concluded that virus infected leafhoppers had a clear choice to settle on the susceptible genotype at all timepoints after 4 hours, while uninfected leafhoppers did not make as strong of a settling choice. Average viral load for each genotype across three timepoints was estimated using qPCR. The results showed that the average viral load increased in each genotype over time, yet there was no difference in the average viral load between the genotypes at any individual timepoint. The global transcriptional response to BCTV infection over time for a resistant and susceptible genotype of sugar beet was conducted using RNA-sequencing technology. Mock-inoculated and BCTV-inoculated plants from each genotype were sampled on day 1, 7 or 14 post inoculation resulting in the preparation of 36 mRNA sequencing libraries. Comparison between mock-inoculated and BCTV-inoculated plants of each genotype and timepoint were conducted separately to generate six list of differentially expressed transcripts (DETs). Each transcript was annotated with a description and further classified for its role in the plant biological, cellular or molecular processes. The results showed that both genotypes of sugar beet had a dynamic response to BCTV infection over time, although there was minimal overlap between the responses to one another. EL10, the resistant genotype, had DETs associated with phytohormone production including jasmonic acid and abscisic acid, along with proteins linked to stress reduction and the downregulation of plant primary metabolic processes. In contrast FC709-2, the susceptible genotype, was found to produce opposing phytohormones like salicylic acid and auxins, as well as the production of volatile organic compounds and an increase of primary plant metabolic processes. These opposing responses shed light on the differences in the transcriptional response of a resistant and susceptible genotype of sugar beet. Understanding and classifying the mechanisms of resistance or susceptibility to BCTV infection in sugar beet is beneficial to researchers and plant breeders as it provides a basis for further exploration of the host plant-virus-vector interactions.
Embargo
Integrated weed management: insights from a weed resistance survey and non-chemical weed seed control in the Central Great Plains
(Colorado State University. Libraries, 2023) Simões Araujo, André Lucas, author; Gaines, Todd, advisor; Dayan, Franck, committee member; Fonte, Steven, committee member
With the impending release of genetically engineered sugar beet varieties with resistance to glyphosate, dicamba, and glufosinate, significant changes are expected in weed management practices, particularly with regards to in-crop weed control. Glyphosate is used during fallow and in-crop periods, while dicamba is commonly employed in fallow applications, specifically targeting glyphosate-resistant weed species. This study provides insights into the resistance status and frequency of resistance in problematic weed species to the three active ingredients in sugar beet systems across Colorado, Nebraska, and Wyoming. While numerous studies have highlighted the widespread prevalence of glyphosate-resistant kochia and Palmer amaranth across the United States, there is limited research focusing on these species within the context of a sugar beet system. Additionally, our findings reveal the first occurrence of glyphosate-resistant and dicamba-resistant Palmer amaranth populations in Colorado, and dicamba-resistant kochia populations within a sugar beet system. Furthermore, we report that all dicamba-resistant kochia populations tested in Colorado lack a known target-site resistance mechanism, suggesting the involvement of a novel resistance mechanism. Surveys assessing glufosinate resistance in the sugar beet system have not been conducted until now, and we provide valuable baseline information on the resistance frequency for this herbicide prior to an anticipated increase in glufosinate use. To address the widespread issue of herbicide resistance in various crop systems, it is crucial to adopt alternative strategies that mitigate resistance evolution and maintain the long-term effectiveness of available herbicides. One promising approach is chaff lining, a harvest weed seed control method that has gained popularity in Australia due to its effectiveness in reducing populations of herbicide-resistant ryegrass, especially when combined with other weed control methods. However, the efficacy of chaff lining may be influenced by several factors, including crop and environmental factors, as reported in Australian literature. Scientific studies assessing the applicability and effectiveness of chaff lining in the United States are limited. Recognizing this research gap and aiming to explore the potential of chaff lining, our study investigated its applicability in field settings within the Central Great Plains region of the United States. Through our research, we provide insights into chaff lining efficacy of and highlight the potential inconsistencies that may arise in suppressing weed seeds using this method. Notably, we demonstrate that various factors, including location and environmental conditions, may be involved and impact the effectiveness of chaff lining as a weed management strategy. These findings underscore the importance of integrating chaff lining with other weed management methods to achieve effective and sustainable weed control. Chaff lining, like any other weed management strategy, should not be solely relied upon. Instead, it should be implemented as part of an integrated approach to ensure its long-term effectiveness.
Embargo
Uncoupling plant growth and defense through phytohormone crosstalk modification
(Colorado State University. Libraries, 2023) Johnston, Grace Allen, author; Argueso, Cris, advisor; Leach, Jan, committee member; Prasad, Ashok, committee member
Phytohormones are essential regulators of development and response to biotic and abiotic stresses. Activation of the plant immune system by pathogen attack often results in changes in plant growth, frequently leading to smaller plants with reduced seed set. Previously, we discovered that cytokinin (CK), a hormone known for its role in the regulation of cell division and plant growth, also has an important role in the activation of defense against pathogens through a synergistic interaction with the defense hormone salicylic acid (SA). Here, we address whether these two phytohormones also regulate the negative effect of immune activation on plant growth. Differential gene expression analysis and physiological assays were used to characterize the crosstalk between CK and SA in growth and defense in Arabidopsis thaliana plants with altered states of immunity. We show that the interplay between the phytohormones CK and SA regulates both defense responses to pathogens and plant development. Endogenous levels of these two hormones were modulated in the snc1 ckx3 ckx5 (s35) triple mutant. The three mutations result in increased CK and SA content simultaneously and yields a novel reproductive growth phenotype. When challenged with pathogens from diverse lifestyles, the s35 mutant conserves an autoimmune phenotype. Transcriptome analysis of s35 reproductive tissue reveals differential regulation of genes associated with nitrogen response and regulation of redox status. Our data suggests that the increased content of both CK and SA hormones contributes to a rebalancing of redox homeostasis and perception of nutrient availability within the shoot apical meristem (SAM), resulting in the uncoupling of reproductive growth and pathogen defense. Further experimentation and investigation into the mechanistic interactions mediating the balance between plant growth and defense could lead to implementation of phytohormone crosstalk engineering to target specific advancements in crop species.