Theses and Dissertations

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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
A new paradigm in rangeland restoration: using a pre-emergent herbicide to assist in native plant establishment and release
(Colorado State University. Libraries, 2019) Clark, Shannon Lee, author; Nissen, Scott, advisor; Dayan, Franck, committee member; Meiman, Paul, committee member; Bjostad, Lou, committee member
Invasive winter annual grasses (IWAG), especially downy brome (Bromus tectorum L.), are a significant threat to rangeland ecosystems in the western United States. Invasions in natural ecosystems can cause severe negative impacts by reducing native plant diversity and lowering community productivity, increasing fire frequency, and displacing native vegetation that is critical wildlife and pollinator habitat. Herbicides currently used for IWAG management can provide adequate short-term control; however, results can be inconsistent and injury to desirable species can occur. Indaziflam (Esplanade®, Bayer CropScience) is a new herbicide option for long-term IWAG control in natural areas and rangeland. As a cellulose biosynthesis inhibitor, indaziflam stops root growth in newly germinated seedlings by preventing cellulose formation. Field studies were conducted to assess native plant tolerance, revegetation and broadleaf weed seedling control with indaziflam applications. At two sites, indaziflam did not impact perennial grass cover, native species richness, or the community composition of forbs and shrubs at one and two years after treatment (YAT). However, the abundance of native forbs and shrubs was reduced by treatments containing picloram and aminocyclopyrachlor. In a separate field study at two sites, a glyphosate dose response showed approximately three times more glyphosate was needed for a 50% reduction (GR50) in feral rye biomass (GR50 = 126.0 g ae ha-1) compared to downy brome biomass (GR50 = 40.4 g ae ha-1). Indaziflam treatments still resulted in reduced downy brome and feral rye biomass compared to the non-treated check 3 YAT, while imazapic and glyphosate did not provide control beyond the first year. Establishment of cool-season grasses (C3), warm-season grasses (C4) and forb/shrubs through drill seeding was only successful across all three functional groups in treatments which included indaziflam. C3 grasses had greater establishment, with an average frequency of 61 ± 1.7% (mean ± SE) at Site 1 and 46 ± 2.6% SE at Site 2 at 3 YAT. In a third field study, treatments containing indaziflam had increased Dalmatian toadflax [Linaria dalmatica (L.) P. Mill], diffuse knapweed (Centaurea diffusa Lam.), and common mullein (Verbascum thapsus L.) control 2 YAT compared to treatments without indaziflam. A laboratory assay was conducted to evaluate the impact of litter on imazapic, rimsulfuron, and indaziflam availability. Downy brome litter at 2,600 kg ha-1 intercepted 84.3 ± 1.0% SE of the applied herbicide. Simulated rainfall at 0 days (d) after application was able to recover 100% of the intercepted rimsulfuron and imazapic, while recovery decreased to 65 ± 1.7% at 1 d and 7 d. Only 54 ± 1.9% of indaziflam could be recovered at 0 d, and recovery decreased to 33 ± 1.1% when simulated rain was applied at 1d or 7 d after application. The multi-year winter annual grass control provided by indaziflam could provide a new strategy for rangeland restoration, allowing enough time for the release of the remnant native plant community or the establishment of native species through revegetation. Indaziflam could potentially be incorporated into management systems to manage the weed seed bank and extend biennial and perennial weed control.
Open Access
A new synopsis of the mosquitoes (Diptera: Culicidae) of Colorado
(Colorado State University. Libraries, 2016) Rose, Dominic Anthony, author; Kondratieff, Boris, advisor; Weissmann, Michael, advisor; Savage, Harry, committee member; Foy, Brian, committee member
Harmston and Lawson (1967) provided the most recent comprehensive review of the mosquitoes of Colorado, reporting 42 species in six genera. Over the last 48 years, a number of important studies that included mosquitoes of Colorado have been completed enhancing the knowledge of this medically important group of flies. To date, the number of Colorado mosquitoes has increased to 54 species in seven genera. Additionally, mosquito vectored pathogens of humans and animals have shifted in Colorado from primarily Western equine encephalitis virus and St. Louis encephalitis virus to primarily West Nile virus. The objective of the current project is to provide an up-to-date synopsis of the species and genera known to occur in Colorado including distributions at a county scale. The study also provides up-to-date illustrated keys to the adult females, fourth instar larvae, and a summary of the important taxonomic characteristics that allow separation of species for both adult females and fourth instar larvae. The species summary includes relevant biological notes and comments on each species such as its phenology, overwintering stage, larval habitat, host preference, medical importance and unique behaviors.
Open Access
A review of the broad nosed weevil genus Thecesternus Say found in the continental United States (Coleoptera: Curculionidae: Entiminae)
(Colorado State University. Libraries, 2011) McCollum, Shiloh Rae, author; Kondratieff, Boris, advisor; Bright, Donald, committee member; Black, William, committee member; Mueller, Rachel, committee member
The taxonomy and nomenclature of the North American weevil genus Thecesternus Say was reviewed. Five previously described species are recognized as valid: affinis, foveolatus, hirsutus, humeralis, and maculosus. One new species, tumulosus, from Texas is described as new. The following new synonymy is proposed: longior LeConte 1856 (= affinis LeConte 1856) and albidus Pierce 1909 (= maculosus Pierce 1909). A neotype is designated for T. humeralis (Say). A key to identify the species is provided, with various illustrations of key morphological features characterizing these species. Additionally, distribution maps, species descriptions, and species differentiation for each species is provided. A cladistic hypothesis of the included species is presented.
Open Access
A revision of the genus Pachyrhinus Schoenherr 1823 (Coleoptera: Curculionidae) in the Nearctic region
(Colorado State University. Libraries, 2016) Benzel, Joseph, author; Kondratieff, Boris, advisor; Bright, Donald, committee member; Gilligan, Todd, committee member; Wei, Yu, committee member
This paper presents a revision of the North American species of the broad nosed weevil genus Pachyrhinus (Coleoptera: Curculionidae) Schönherr, 1823, which includes eight currently recognized species. Pachyrhinus is considered a minor pest of Pinus spp. Three species of Pachyrhinus are now recognized in North America: P. elegans (Couper, 1865), P. californicus (Horn, 1876), and P. cinereus (Casey, 1888). Pachyrhinus lateralis (Casey, 1888) and P. miscix (Fall, 1901) are considered synonyms of P. elegans. Pachyrhinus crassicornis (Casey, 1888) and P. albidus (Fall, 1901) are synonyms of P. cinereus. The previous synonymy of P. ferrugineus (Casey, 1888) with P. californicus was confirmed. The revision includes detailed images of diagnostic characters as well as scanning electron micrographs of scale morphology for all species.
Open Access
A survey of the arthropod fauna associated with hemp (Cannabis sativa L.) grown in eastern Colorado
(Colorado State University. Libraries, 2019) Schreiner, Melissa, author; Cranshaw, Whitney, advisor; Uchanski, Mark, committee member; Peairs, Frank, committee member
Industrial hemp was found to support a diverse complex of arthropods in the surveys of hemp fields in eastern Colorado. Seventy-three families of arthropods were collected from hemp grown in eight counties in Colorado in 2016, 2017, and 2018. Other important groups found in collections were of the order Diptera, Coleoptera, and Hemiptera. The arthropods present in fields had a range of association with the crop and included herbivores, natural enemies, pollen feeders, and incidental species. Hemp cultivars grown for seed and fiber had higher insect species richness compared to hemp grown for cannabidiol (CBD). This observational field survey of hemp serves as the first checklist of arthropods associated with the crop in eastern Colorado. Emerging key pests of the crop that are described include: corn earworm (Helicoverpa zea (Boddie)), hemp russet mite (Aculops cannibicola (Farkas)), cannabis aphid (Phorodon cannabis (Passerini)), and Eurasian hemp borer (Grapholita delineana (Walker)). Local outbreaks of several species of grasshoppers were observed and produced significant crop injury, particularly twostriped grasshopper (Melanoplus bivittatus (Say)). Approximately half (46%) of the arthropods collected in sweep net samples during the three year sampling period were categorized as predators, natural enemies of arthropods. These findings are of significance considering the expanding cultivation of hemp both within the region of study and across the United States. A website developed to extend the information developed in this project has been accessed over 20,000 times in 28 months, indicating there is wide interest in this subject area.
Open Access
Abiotic and biotic factors influencing western United States coniferous forests
(Colorado State University. Libraries, 2019) Lalande, Bradley, author; Stewart, Jane, advisor; Stromberger, Mary, committee member; Tinkham, Wade, committee member; Trivedi, Pankaj, committee member
In the next decade, climate models suggest that global temperatures will continue to rise. In the western United States, increases in temperatures and changes in precipitation patterns will escalate the risk of drought conditions. These potentially warmer, drier conditions could induce physiological changes within trees, subsequently increasing stress on coniferous forests that are adapted to cool, wet environments. The abiotic stress accompanied by drought conditions can predispose susceptible hosts to biotic stress of insect and disease populations. In particular, high elevation subalpine fir (Abies lasiocarpa) have encountered higher than average mortality rates throughout the western United States in association with abiotic and biotic agents. Chapter 2 of this thesis investigated the potential drivers of subalpine fir mortality and determined how climatic factors and site and stand characteristics influenced the presence of mortality and biotic agents. The objectives were to identify factors driving subalpine fir mortality in Colorado and included 1) determine abiotic and biotic factors that directly and indirectly affect subalpine fir mortality, 2) determine factors associated with the presence of D. confusus or Armillaria spp., and 3) determine if climate variables were correlated to subalpine fir mortality or the presence of D. confusus and Armillaria spp. I hypothesized that sites with a higher density (i.e. basal area, trees per hectare, or canopy closure) would experience greater mortality due to decreased growth rates from competition and that D. confusus or Armillaria spp. prevalence would be a function of tree stress (i.e. increased density), elevation, slope, and departures from normal precipitation (i.e. drought), and minimum and maximum temperatures. Stand health monitoring plots found that the most relevant factors to subalpine fir mortality are the presence of D. confusus (p = 0.003) and the percent subalpine fir on plot (p = <0.0001). I identified that stand density (p = 0.0038), elevation (p = 0.0581), and Armillaria spp. (p = 0.0006) were the greatest influences on the presence of D. confusus, while the largest influences on the presence of Armillaria spp. are warmer maximum summer temperatures (p = 0.0136) and the presence of D. confusus (p = 0.0289). Results indicated that increased subalpine fir mortality was attributed to high stand density as a predisposing factor, warming temperatures as an inciting factor, and bark beetles (Dryocoetes confusus) and root disease (Armillaria spp.) as contributing factors. The combination of predisposing, inciting, and contributing factors suggests that subalpine mortality can be defined as subalpine fir decline. Management strategies used to reduce the impact of subalpine fir decline will need to address ways to improve stand health, while decreasing populations of both, D. confusus and Armillaria spp. In regards to Armillaria, the inability to successfully manage the disease using current techniques highlights the need to find novel management strategies to minimize its impacts. Since this disease is a root pathogen, soil microbes likely influence its growth and survival. Utilizing soil microbial communities as biocontrols may assist in management of Armillaria. Field sampling within the Priest River Experimental Forest in northern Idaho provided the opportunity to observe how soil microbial communities are associated with two species of Armillaria, A. solidipes (primary pathogen) and A. altimontana (weak pathogen). My research objective for Chapter 3 was to identify the soil fungal communities associated with tree health status (healthy, moderate and dead) and each Armillaria species, A. solidipes and A. altimontana, both of which have differing ecological behaviors (virulent pathogen and non-pathogen, respectively) on western white pine. I hypothesized that soil microbial communities associated with virulent A. solidipes and non-pathogenic A. altimontana would differ in fungal richness and diversity with the latter having a greater richness and diversity due to its beneficial qualities to tree health. While richness and diversity is likely to shift among tree health with a greater diversity and richness for soils associated with healthy trees due to root exudate production near the rhizosphere. Soil samples were collected alongside western white pine (Pinus monticola), while Armillaria rhizomorphs were excavated near the roots. The most abundant fungal taxon was Mortierella spp., which functions as saprophyte decomposing dead and down wood. No significant differences in fungal diversity or richness were found in soils associated with Armillaria species, but, although not significant, there where slight differences between soils associated with moderate and dead trees with a greater diversity and richness in soils with dead trees (p = 0.18). Additionally, soil pH was significantly influenced by soil carbon, nitrogen, and organic matter, while moisture significantly influenced soil carbon, nitrogen, and organic matter, acting as indicators to overall health in the stand. Although not significantly different, more Hypocreaceae (Trichoderma), a known biocontrol for root pathogens, were found within soils associated with A. altimontana and healthy trees. More research is needed to solidify differences, yet these factors give insight into potential beneficial aspects of soil fungal communities in association with Armillaria species and tree health. Changing climates regimes outside of 30-year averages cause increased stress to forests. This stress may predispose trees to a greater abundance biotic agents such as bark beetles and secondary pathogens, such as Armillaria root disease specifically in association with subalpine fir in Colorado. Understanding the role that soil fungal communities play in association to Armillaria root disease and tree health may assist in forest management practices to increase the health of high elevation forests.
Open Access
Advances in tortricid systematics and identification (Lepidoptera: Tortricidae)
(Colorado State University. Libraries, 2012) Gilligan, Todd Michael, author; Opler, Paul, advisor; Kondratieff, Boris, advisor; Walters, Terrence, committee member; Brown, John, committee member; Simmons, Mark, committee member
The Tortricidae is a large family in the microlepidoptera, consisting of more than 10,300 species in 1,070 genera. Many tortricids are considered to be economically important, and the family contains the highest percentage of pest species in the Lepidoptera. This dissertation provides significant advances in tortricid systematics and identification through the implementation of modern technologies such as matrix-based identification keys and molecular phylogenetics. Chapter 2 focuses on Tortricidae threatening U.S. agriculture. The resulting resource, Tortricids of Agricultural Importance (TortAI - http://www.tortai.org), is designed to aid in the identification of tortricid adults encountered during domestic surveys and tortricid larvae encountered during quarantine inspections at U.S. ports of entry. Chapters 3-5 provide details on the three tortricid pests currently of greatest importance to U.S. agriculture: Epiphyas postvittana (Walker), Lobesia botrana ([Denis and Schiffermüller]), and Thaumatotibia leucotreta (Meyrick). Chapter 6 details the discovery of a new tortricid species with the potential to threaten avocado production in California. Chapter 7 describes the benefits and disadvantages to using DNA barcoding for identification purposes and outlines a novel DNA sequence search tool developed for use in TortAI. Phylogenetic relationships surrounding Eucosma, the largest genus within the Tortricidae, are examined in chapters 8-9. A molecular phylogeny and revised classification for Eucosma and related genera provide the groundwork for future study of these highly diverse taxa.
Open Access
Aspects of weed resistance to auxinic herbicides
(Colorado State University. Libraries, 2020) Rodrigues Alves de Figueiredo, Marcelo, author; Gaines, Todd A., advisor; Argueso, Cristiana T., committee member; Dayan, Franck E., committee member; Reddy, Anireddy S. N., committee member
Synthetic auxins have been widely used for selective control of broadleaf weeds since the mid-1940s. After more than 70 years using synthetic auxin herbicides, there are 41 different resistant species reported. Weed resistance to auxin herbicides is poorly understood and in most reported cases, no studies have been done to investigate the mechanistic changes that occur in resistant populations. The mechanisms of herbicide resistance in weeds are classified as 1) target-site when mutations reduce the interaction of the herbicide molecule to its binding site and/or changes of gene expression of the targeted enzyme to compensate for herbicide inhibition; and as 2) non-target-site mechanisms, which include any genetic mutations that will prevent or reduce the herbicide reaching its site of action. In this present research, two 2,4-D resistant weed species were studied, and the mechanisms of resistance were elucidated, where one species evolved metabolic non-target-site resistance to 2,4-D and the second species evolved a novel mechanism of target side modification. In 2009, an Amaranthus tuberculatus (common waterhemp) population with ten-fold resistance to 2,4-D was found in Nebraska, USA. Using the same 2,4-D-resistant and a known susceptible A. tuberculatus population from Indiana, the mechanism of 2,4-D resistance was examined by conducting [14C] 2,4-D absorption, translocation and metabolism experiments. No differences were found in 2,4-D absorption, but resistant plants translocated more of the radioactive material than susceptible A. tuberculatus. Resistant plants metabolized [14C] 2,4-D more rapidly than susceptible plants. The main metabolites were purified and their structures were solved by NMR and HRMS. Susceptible plants conjugate 2,4-D to 2,4-D Aspartic Acid (2,4-D-Asp). Resistant plants showed a distinct metabolic profile where 2,4-D is hydroxylated into 5-OH-2,4-D, conjugated in a sugar metabolite (5-OH-2,4-D-Glucoside) and malonylated into 5-OH-2,4-D-(6-O-Malonyl)-Glucoside. Pre-treatment with the cytochrome P450 inhibitor malathion inhibited 2,4-D hydroxylation. Toxicological studies in waterhemp and Arabidopsis confirmed that the hydroxylated metabolite lost its auxinic action and toxicity. In contrast, the 2,4-D-Asp metabolite induced auxin inhibition to the plants tested. These results demonstrate that resistant A. tuberculatus evolved novel detoxification reactions that rapidly metabolize 2,4-D, potentially mediated by cytochrome P450. That novel mechanism is more efficient and produces metabolites with lower toxicity compared to the innate aspartic acid conjugation. Metabolism-based herbicide resistance poses a serious challenge for weed management due to the potential for cross-resistance to other herbicides. Sisymbrium orientale (Indian hedge mustard) is an important weed species in Australia reducing yields in crops and pastures. In 2005, a 2,4-D and MCPA resistant population was reported in the Port Broughton region in South Australia. Aux/IAAs are dynamic repressor proteins that regulate Auxin Response Factors (ARFs) to activate auxin related genes and are also co-receptors for auxins and synthetic auxin herbicides. The degradation of Aux/IAAs is done by the enzyme complex E3, called SCFTIR1/AFB, which, in the presence of auxin, performs ubiquitination on Aux/IAA making it a target of proteasome 26S, an enzyme responsible for proteolysis in eukaryotes. An RNAseq study showed that a 27 bp deletion in Aux/IAA2 (IAA2) degron tail was correlated to the resistant phenotype. The mutant allele was functionally validated to confer 2,4-D resistance by transforming Arabidopsis thaliana with the wild type SoIAA2 and SoIAA2Δ27 alleles. Performing binding analysis by surface plasmon resonance, the association of TIR1 in the presence of auxin (IAA, 2,4-D and dicamba) showed slower association and faster dissociation to the resistant IAA2 peptide compared to the susceptible IAA2 peptide. Our results suggest that the loss of 9 amino acids located in the degron tail may reduce the capacity of IAA2 to "embrace" TIR1 in the presence of auxin, reducing ubiquitination rate, resulting in higher stability to repress auxin response factors and ultimately conferring resistance to 2,4-D.
Open Access
Aspen mortality in the Colorado and southern Wyoming Rocky Mountains: extent, severity, and causal factors
(Colorado State University. Libraries, 2011) Dudley, Megan, author; Jacobi, William R., advisor; Tisserat, Ned A., committee member; Negron, Jose, committee member; Martin, Patrick H., committee member
Quaking aspen (Populus tremuloides Michx) is a deciduous hardwood tree widely distributed throughout North America. In Colorado, quaking aspen is found on a wide variety of sites, from the lower-elevation foothills of the eastern edge of the Rockies to moderate- and high- elevation montane sites throughout the Rocky Mountains. Aspen dieback has been documented throughout western North America over the past decade, resulting in stands that have either elevated proportions of overstory mortality or thin crowns, or both. Stands experiencing dieback may or may not produce regeneration cohorts. In this study, we surveyed aspen in the north-western corner of Colorado on the White River and Routt national forests, along the front range of Colorado on the Pike-San Isabel national forests, and in the south-central region of Wyoming on the Medicine Bow national forest during 2009 - 2010. We established 573 random roadside survey plots in stands that contained at least 50% aspen cover type. From these random plots, we found average standing aspen tree mortality ranged from 3.3 to 23.7 % on the four national forests and 11% on the east side of the continental divide and 4 % on the west side. The roadside plot data suggests that Colorado's aspen on these four national forests overall were healthy; mortality rates among aspen were fairly low (~3 - 8%) among all stems, and average percent live crown among adults was high (~85 - 90%), in spite of nearly ubiquitous presence of disease (~97 - 99%) and high incidence of insect damage (~50 - 75%). We also established 98 aspen stand assessment plots with half of the plots in damaged stands, as defined by U.S.D.A. Forest Service aerial detection surveys (ADS), and half in healthy aspen stands. Damaged stands were defined as those stands with (1) thinning crowns among at least 25% of adult aspen, (2) stands with moderate (<50% of stems) levels of overstory mortality, or (3) stands with high (>50% of stems) levels of overstory mortality. Healthy aspen stands were defined as having (1) a maximum mortality rate of 5 - 7% among all aspen, and/or (2) more than 75% of adult aspen with full crowns. Adult aspen in damaged stands tended to be less vigorous, based a health score index from 1 to 5, with higher scores indicating less healthy conditions (where 1=0-25% damage; 2 = 25-50% damage; 3 = >50% damage; 4 = recent dead; 5 = >5 years dead). Health scores averaged 1.7 in healthy stands, compared to 2.3 in damaged stands. Saplings in damaged stands tended to be healthier with a score of 1.7, compared to 2.2 in healthy stands. Further, there was no difference in the proportion live or total numbers of saplings per hectare between healthy and damaged stands. The prevalence of damaging organisms, such as Cytospora canker (20% in damaged, 13% in healthy), wood-boring insects (27% in damaged, 10% in healthy), and aspen bark beetles (16% in damaged, 7% in healthy) was considerably greater among damaged stands. Site conditions also influenced the prevalence of some of these damage agents: bark beetles were most common among stands at low elevations (18%, compared to 11% and 6% at moderate and high elevations, respectively); Cytospora canker was most common among stands on south- or west-facing aspects (20% and 19%, respectively); both aspen bark beetles and Cytospora canker were also most common among stands in the southernmost section of the survey area, the Pike-San Isabel national forest (41% and 36%). There was no difference in the severity of canker or decay fungal infection between healthy and damaged stands. Cytospora canker infestations were more severe on the Medicine Bow NF compared to the other three national forests, and Marssonina foliar blight infection appeared to be most severe on slope summits, concave sites, and sites with either no to low percent slope or moderately steep slopes. Based on the general state of aspen health within the study area, it appears that aspen in damaged stands were experiencing more severe environmental stress (e.g., late frost, drought, defoliation) and coupled with disease and insect infestations, resulted in greater mortality when compared to healthy stands. The severity of such conditions appears to be regional in scale, and it remains to be established that long-term or acute drought is the major factor influencing the observed conditions. Since no differences were detected in regeneration density between damaged or non-damaged stands, it is possible that there will be no long-lasting effects on aspen longevity on these sites with the relatively low incidence of overstory mortality throughout all four national forests.
Open Access
Bee diversity and abundance under a grazed cover cropping management system in eastern Colorado and southwestern Nebraska and evaluating the role of beekeeping education and management on honey bee hive overwintering success in Colorado
(Colorado State University. Libraries, 2021) O'Brien, Colton, author; Kondratieff, Boris, advisor; Seshadri, Arathi, advisor; Jones, Kurt, committee member
Bee pollination is essential to the production of many valuable crops in addition to facilitating the reproduction of non-crop flowering plants in the environment. Managed and wild populations of bees face unique and overlapping challenges. Wild bees have been negatively impacted by habitat and forage loss as a result of agricultural intensification. There has been headway in finding solutions that offset the environmental impact of agriculture that benefit wild bees without being a financial burden to the producer. Solutions often include the introduction or retainment of forage and habitat within the agricultural landscape. One example of this is the inclusion of bee-friendly cover crops into a crop rotation. Cover crops can promote agroecosystem services such as, nitrogen fixation, reduce erosion etc., and also provide nesting habitat and forage for pollinators. Chapter one explores bee diversity and abundance under a grazed cover cropping management system in eastern Colorado and southwestern Nebraska. Blue vane traps were used to conduct monthly collections of bees within three cover-cropped fields to evaluate diversity and abundance of bees under varying grazing conditions. There was higher diversity of bee genera in fields where grazing intensity was low but bee abundance was higher in grazed fields with the highest representation being from the ground-nesting genus, Lasioglossum. Setting aside some cover-cropped areas to remain ungrazed, allowing plants to come into bloom will provide nutrition and nesting resources for bees in this region. Pathogens and pests are another set of challenges that pollinators face in the environment. Managed bees can be a source of inoculum for wild bees if hives are not kept healthy. Managed bees often visit the same forage sites as wild bees. These communal areas where wild and managed bees interact present opportunities for pathogens to spill over from the managed populations to the wild populations. Pathogen development and spread within managed populations can often be prevented by good beekeeper practices that keep hives healthy. Chapter two explores the role that beekeeping education plays in honey bee hive health and survival among hobby beekeepers across Colorado. While most commercial pollination services are provided by professional beekeepers with 500 or more hives, the majority of beekeepers in the United States are backyard beekeepers with typical operations of fewer than 50 hives. Despite increased interest in backyard beekeeping, average hive loss in the United States is still 35%-40%. Hive survival depends on beekeeper intervention, but many backyard beekeepers lack training and are unfamiliar with the hive management techniques necessary for maintaining healthy hives. Beekeeping education could help improve overwintering survival among back yard beekeepers. To evaluate the role of education in successful beekeeping, in Summer 2018 and Summer 2019, backyard beekeepers across the state of Colorado were contacted to participate in a honey bee health survey that included a questionnaire and a hive inspection. Using hive management, beekeeper education, mite load, and experience as predictors of hive survival, this study found that hive survival may be positively related to hive management.
Open Access
Beet curly top virus-beet leafhopper dynamics in hemp in Colorado
(Colorado State University. Libraries, 2021) Chiginsky, Judith, author; Nachappa, Punya, advisor; Prenni, Jessica, committee member; Cranshaw, Whitney, committee member
Hemp (Cannabis sativa L.) production within North America has dramatically increased in recent years following legislative changes in the 2014 and 2018 Farm Bills that allowed legal paths for its production. However, due to previous restrictions on this crop it has been understudied in the U.S. since its production declined, and ultimately was eliminated. Restrictive laws largely prevented any research regarding management of this crop. Among the understudied issues were those associated with disease identification and management. One newly described disease of the crop in Colorado beet curly top virus (BCTV). This viral pathogen is transmitted by an insect vector, the beet leafhopper. To better understand the diversity and prevalence of BCTV strains infecting hemp in Colorado, beet curly top virus (BCTV) was detected at high incidence (81%) in leaf samples from 12 counties in 2019. Two different strains of BCTV, BCTV-Worland and BCTV-Colorado were found present in single or mixed infection in hemp leaf samples. Phylogenetic analysis revealed BCTV sequences from hemp formed a distinct group along with BCTV-Colorado and BCTV-Worland strains. To determine other potential viral and viroid pathogens in hemp, shotgun metagenomic analysis was performed. Virome analysis revealed the presence of seven viruses and one viroid. Of these, cannabis cryptic virus, cannabis sativa mitovirus, citrus yellow vein associated, opuntia-like virus and hop latent viroid sequences that had high sequence similarity with their corresponding sequences in GenBank. In contrast, tobacco streak virus sequence was highly variable compared to sequences in GenBank suggesting a new genotype of this virus. The data presented here has important implications for the epidemiology and management of the various diseases of hemp and will lead to the development of integrated pest management strategies designed to interrupt transmission cycles and facilitate efficient crop production. Beet leafhopper abundance was monitored throughout the hemp season to understand timing of emergence and flight patterns in the north and western regions of Colorado as well as identify timing of population peaks. Virus incidence in hemp and weed species were assessed using PCR analysis. Beet curly top virus was detected earlier in western field sites of Colorado before being detected in northern survey sites. Of the 41 different weed species surveyed, the weeds that most often tested positive for BCTV, contributing to transmission prevalence were Lactuca serriola (prickly lettuce), Taraxacum officinale (dandelion), and Cichorium intybus (chicory). Life history assays were conducted using viruliferous and non-viruliferous beet leafhoppers in both sugar beet and hemp plants to understand if there were any fitness advantages or costs associated with being a carrier of the virus. Viruliferous beet leafhoppers reared on sugar beet produced more offspring than non-viruliferous treatments. There was no difference between viruliferous, and non-viruliferous beet leafhoppers reared on hemp, suggesting that these virus mediated differences in life history are induced in host plant interactions with the vector. However, beet leafhoppers were able to survive 7-day periods and successfully oviposit and develop on hemp. Understanding migration timing and patterns will result in a more thorough understanding of the pest ecology of the beet leafhopper, which will lead to targeted control strategies to incorporate into integrated pest management tactics to interrupt BCTV transmission cycles, in turn improving yield and farming efficiency.
Open Access
Biochemical differentiation and hormonal regulation of the developing testes in Tenebrio molitor
(Colorado State University. Libraries, 1980) Alrubeai, Hussain Fadhil, author; Gorell, Thomas A. (Thomas Andrew), advisor; Seidel, George E., committee member
During differentiation, the testes of Tenebrio molitor have been found to exhibit increases in biosynthetic capacity reflected in alterations in testicular protein and RNA. This biochemical differentiation was influenced by endogenous and/or exogenous hormones. The testes underwent dramatic increases in size and weight during the prepupal stage that were continued through later developmental stages. Histological analysis revealed that the maturation process of the germ cells to produce spermatozoa proceeded from the distal end of the follicles and toward the basal region to form a "differentiation wave." Spermatozoa were found in the prepupal testes. The underlying biochemical machinery of the developmental process was found to be accelerated in manufacturing different elements for germ cell differentiation at certain stages and particularly when the endogenous level of ecdysterone rose during the late prepupal and at mid-pupal stages. Gradual increases in testicular protein and RNA content were observed during the prepupal stage. The observed increases were more dramatic for both protein and RNA content in the pupal stage. The testicular protein and RNA content reached their maximum levels between days 4 and 7 of the pupal stage as did the rate of 3H-leucine incorporation. During the adult stage, the biosynthetic processes for producing protein and RNA were apparently reduced following the first few days after adult emergence. The protein products of the mealworm testes were shown by gel electrophoresis to be many and diverse. The 27 protein products were of various molecular weights, ranging from 12,000 to 127,000 daltons. These products were present at different ages of development and persisted for various times indicating that some of these proteins may be necessary for the formation of specific germ cell types. In addition, a variety of these testicular protein components incorporated leucine at measurable levels throughout development, particularly during the pupal stage. It was ascertained that the rate of incorporation of radioactive leucine into TCA -precipitable testicular protein was not affected by the administration of exogenous juvenile hormone alone (JHI, 1 µg/animal) during the pupal stage. However, the administration of exogenous ecdysterone (0. 5 µg/animal) to pupal Tenebrio resulted in an increase in the rate of radioactive leucine incorporation into TCA -precipitable testicular proteins, particularly during the first six days after pupal ecdysis. The amount of ecdysterone injected appeared to stimulate the production of the same testicular protein components that were present during normal pupal development. Injection of a higher dose of ecdysterone (1.5 µg/animal) during some of the pupal ages appeared to alter the testicular differentiation program by enhancing the incorporation of leucine into not only the age -specific testicular protein components but also into new protein components which did not normally appear at these specific ages. Simultaneous administration of both JH and ecdysterone on mealworm pupae at specific ages indicated that there was no apparent interaction, synergistic nor antagonistic, between these two hormones. Furthermore, the incorporation rate of leucine closely resembled that rate obtained following injection of ecdysterone alone in all the pupal ages studied.
Open Access
Biology and management of blackleg disease of potato caused by Dickeya dianthicola (ME23)
(Colorado State University. Libraries, 2021) Karim, Shaista, author; Charkowski, Amy, advisor; Trivedi, Pankaj, advisor; Jahn, Courtney, committee member; Unchanski, Mark, committee member
Potato is the most commonly consumed vegetable in the United States, where people each an average of 49.2 pounds per person per year. About 80% of potatoes in the US are produced in Idaho, followed by Washington, Wisconsin, and Oregon. Potato is a vegetatively propagated crop, and progeny tubers serve as seed for cultivation the following year. Therefore, tuber-borne pathogens, such as bacteria that cause blackleg, result in serious economic losses when progeny tubers are contaminated by pathogens. Blackleg of potato is characterized by blackening of the basal part of stem and rotting of seed tubers. It is caused by Pectobacterium and Dickeya species, which are in the Pectobacteriaceae family and are collectively referred to as the soft rot Pectobacteriaceae (SRP). In 2015, multiples outbreaks of blackleg and soft rot occurred in Northeastern United Sates. This outbreak of the disease also impacted potato production all across the neighboring states, as well as other northeastern and mid-Atlantic states where Maine seed potatoes were shipped. It is most likely that prior to the Dickeya dianthicola (ME23) outbreak in the US in 2015, Dickeya was present in seed potatoes and farms in the affected states for at least a few years. However, rain in 2013 and 2014 spread the pathogen and cool temperatures caused the bacterium to remain latent in the tubers. Warm temperature in 2015 on commercial farms that used this Dickeya-infested seed resulted in significant outbreaks. The fields with outbreaks had no previous history of blackleg, the pathogen was suspected to have been present somewhere in the environment, then multiplied suddenly in response to favorable conditions, such as a heavy rain with subsequent surface pooling, and thus caused an outbreak. To prevent further spread of the disease, the primary infection source and the route of infection of the blackleg pathogen must be identified. Being able to discriminate exact subspecies of Dickeya from the others could help reduce the infection and to understand the epidemiology of the pathogen. Therefore, my research focused on development of reliable and accessible detecting tools for D. dianthicola (ME23). Unfortunately, many commercial potato varieties are susceptible to the diseases caused by SRP. Very few are tolerant, and production is compromised due to infection caused by D. dianthicola and high risk of spreading bacteria in other farms if potato seeds are infected. This led to an urgent need to screen for resistance against blackleg disease. There is insufficient information available for potato breeders on relative resistance or tolerance of commercial potato varieties to Dickeya and Pectobacterium spp. For the purpose of our work with SRP, we use the term resistance for plants that remain asymptomatic, or nearly so, after inoculation with Dickeya or Pectobacterium in typical temperature, humidity, and oxygen-level conditions. In addition, there is almost zero evidence of single gene resistance against this pathogen. Rather, disease resistance is quantitative and multigenetic, making it difficult for plant breeders to select for resistance. In addition, blackleg development is highly sensitive to multiple environmental factors including, plant age, availability of favorable environmental conditions and other bacterial pathogen present in the environment, making it difficult to screen varieties for resistance. The molecular and biochemical mechanisms underlying these quantitative resistances are also poorly understood. Therefore, are not efficiently utilized in potato breeding programs, altogether this makes it difficult to achieve true blackleg disease resistance. Nevertheless, it has been previously reported that plant resistant relies on production of small molecules such as phytoalexins or phytoanticipins associated with core resistant pathways. For example, these pathways may induce plant hormones associated with resistance, or antimicrobial peptides or enhance cell wall modifications as a physical barrier against plant pathogens. Interestingly, some accessions of the wild diploid species of potato (Solanum chacoense) are resistant to blackleg and soft rot diseases caused by SRP. My research focuses on identification of resistant lines of wild diploid potato relatives using physiological, biochemical and metabolic profile. In my work, I found that the metabolic profile of resistant stem extracts of S. chacoense consists of small molecules including phenolics, alkaloids, lipids, amino acids and organic acids, some of which may play a significant role in antimicrobial and anti-virulence properties. I found that the biochemical assays including quorum sensing (QS) and plant cell wall degrading enzymes (PCWDE) correlated with metabolites identified in metabolic profile of resistant accessions. Hence, these assays can be used as a less time consuming and easy tool for screening resistant lines against SRP. From these findings, I hypothesize that QS inhibiting molecules are responsible for triggering resistance against blackleg in S. chacoense and can be used as a potential tool in future breeding programs to achieve maximum resistance in our commercially grown potato varieties.
Open Access
Biology and over-winter survival of Iris yellow spot virus in Colorado
(Colorado State University. Libraries, 2014) Szostek, Stephanie Aspen, author; Schwartz, Howard F., advisor; Bartolo, Michael, committee member; Cranshaw, Whitney, committee member; Tisserat, Ned, committee member
Iris yellow spot virus (IYSV) (Family: Bunyaviridae, Genus: Tospovirus) and its insect vector, Thrips tabaci Lindeman, are of economic concern in onion (Allium cepa L.) growing regions worldwide. IYSV symptoms appear on onion foliage as tan or straw colored, elongate diamond shaped lesions. Accumulated lesions may coalesce on the foliage or girdle the scape, causing lodging and loss of seed. There is no evidence that Tospoviruses, including IYSV, are seed transmitted. Onion seed included in double antibody sandwich enzyme linked immunosorbent assays (DAS-ELISA) to detect IYSV occasionally yielded a positive result. IYSV was detected in the pedicels, petals, anthers, and fruits of onion flowers by reverse transcriptase polymerase chain reaction (RT-PCR). Onion seed collected from several cultivars of IYSV symptomatic plants was grown out under greenhouse and growth chamber conditions. IYSV was not detected in the six week old seedlings. Further investigation of onion seeds revealed IYSV could be detected in the seed coat, but not the emerging radicle. It is highly unlikely that IYSV can pass from the seed coat to the new plant during germination, and seeds remain an unlikely source of IYSV inoculum. Several weed species have been described as additional hosts and likely green bridges for IYSV survival, however, there is little work regarding the overwintering habits of T. tabaci and its potential to act as a source of inoculum during the following season. The results presented in this work close the loop, and show that both T. tabaci and IYSV are present near onion fields throughout the winter, T. tabaci will reproduce on several weed species, and larvae can acquire IYSV from non-allium sources. Thrips activity was monitored via sticky trap during the winter months from 2011 to 2013. Thrips activity appeared to cease once the average temperature fell below 0°C and resumed once the average temperature rose above 0°C. Onion cull piles were constructed, and while these piles provided an environment conducive to thrips survival, few live thrips were recovered from the piles after the onset of bulb decay. IYSV was detected by RT-PCR in live adult and larval thrips recovered from onion, Malva neglecta Wallr. (common mallow), Taraxacum officinale Weber in Wiggers (dandelion), Descurainia sophia (L.) Webb. Ex Prantl (flixweed), Lactuca serriola L. (prickly lettuce), and Tragopogon dubius Scop. (salsify) during the winters from 2010 to 2013. Of these plants, IYSV was detected in prickly lettuce and flixweed. These five weed species were grown from seed in the greenhouse and exposed to viruliferous thrips to further elucidate their potential role as green bridges. Of the five, IYSV was detected in salsify and the thrips larvae reared on this plant. Results indicate winter annuals play a role in onion thrips and IYSV over-winter survival, providing inoculum the next growing season, and that weed management during the winter may be warranted. IYSV distribution throughout onion leaves is uneven and patchy. A reverse transcription quantitative real time PCR (RT-qPCR) was developed to compare relative amounts of IYSV within leaves and between cultivars. The amount of IYSV was greatest at the lesion site itself and decreased as distance from the lesion increased. No statistically significant differences were found in the amount of IYSV between susceptible cultivar Granero and tolerant cultivar Advantage. This assay may be useful for additional comparative studies with other crops and viruses.
Embargo
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.
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
Characterization of Sorghum bicolor genotypes under varying water regimes
(Colorado State University. Libraries, 2019) Person, Taylor, author; Jahn, Courtney E., advisor; Byrne, Pat, committee member; Gaines, Todd, committee member
Agriculture is the largest consumer of fresh water, accounting for 70% of use globally. The availability of water is expected to decrease in future climate models. It is imperative to develop crop varieties with improved performance under targeted limited water environments. One approach to address this aim is capitalizing on inherent variability for drought tolerance traits in crops. Depending on the nature of water stress and other environmental factors, specific root morphology, shoot morphology, and the harvest index are selectable traits that can distinguish performance of different genotypes in crops. These aforementioned parameters discriminate relative water-stressed performance in many studied crop species. Among these is sorghum (Sorghum bicolor), a grain crop adapted to many different environments, particularly where limited rainfall is an agricultural constraint. The specific objectives of the following thesis were to 1) characterize the morphology of a sorghum recombinant inbred line (RIL) population (n = 430) of Sorghum bicolor for response to varying moisture regimes and 2) evaluate 10 selected genotypes for below-ground morphology in the greenhouse and field for response to varying moisture regimes. This population expresses a wide range of phenotypes in response to varying moisture regimes in panicle weight, shoot weight, harvest index, plant height, stem diameter, tillering and days to flowering. Ten selected RILs, including the two population parents, BTx623 and IS3620C, represent the population distribution and respond uniquely to different moisture environments. When subjected to drought stress and subsequent recovery when re-watered in the greenhouse, root system response varied at both time points. Root system and stem size are indicators of this response and vascular tissue variation may play a role in these varied responses. In the field, the root system of these ten RILs, as quantified by crown root angle, had a tendency to grow more vertically than horizontally under dryland conditions while high moisture environments showed no association with root system orientation. Taken together, this study provides foundational knowledge on above and below-ground responses of RIL population BTx623 X IS3620C to varying moisture environments.
Open Access
Cheliceral morphology of Solifugae (Arachnida): primary homology, terminology, and character survey
(Colorado State University. Libraries, 2015) Bird, Tharina Louise, author; Kondratieff, Boris, advisor; Cushing, Paula, advisor; Prendini, Lorenzo, committee member; Wharton, Robert, committee member; Laituri, Melinda, committee member
Arachnids of the order Solifugae (solifuges, false spiders, sunspiders, camelspiders, Walzenspinne, windspiders) possess the largest jaws for body size in the Chelicerata. The chelicerae provide the most important character systems for solifuge systematics, including dentition and the male flagellum, both used extensively in species delimitation and diagnosis. However, the terminology used for cheliceral characters is unstandardized and often contradictory, in part because it fails to represent homologous structures among taxa. Misinterpretation of character homology may introduce errors in phylogenetic analyses concerning relationships within Solifugae and among the orders of Chelicerata. This contribution presents the first comprehensive analysis of cheliceral morphology across the order Solifugae, the aims of which were to provide a broad survey of cheliceral characters for solifuge systematics, to identify and reinterpret structures based on primary homology, revise the terminology to be consistent with homology hypotheses, and provide a guide to synonyms of terminology and character interpretations in the literature. Chelicerae were studied in 188 exemplar species (17 % of the total), representing all twelve solifuge families, 17 of the 19 subfamilies, 64 genera (46 % of the total), and the full range of variation in cheliceral morphology across the order. In total, 157 species representing 49 genera and 17 subfamilies are illustrated. Hypotheses of character transformation, particularly concerning the male flagellum, and a standardized terminology, are presented. The functional morphology of the chelicerae is discussed and the role of sexually dimorphic modifications to the male chelicerae in mating behavior emphasized. The revised terminology, based on hypotheses of primary homology, will facilitate solifuge revisionary systematics and provide a stronger basis for reconstructing phylogenetic relationships within the order and evaluating its position within Chelicerata.
Open Access
Chemical control of Cytospora leucostoma, a major limiting factor of peach production in western Colorado
(Colorado State University. Libraries, 2017) Miller, Stephan, author; Stewart, Jane, advisor; Minas, Ioannis, advisor; Norton, Andrew, committee member; Uchanski, Mark, committee member
Cytospora canker, Cytospora leucostoma, is a major limiting factor of peach production in Colorado, accounting for 15-20% of crop production loss annually. Given the unique environmental factors of the western slope region of Colorado, C. leucostoma has developed into a severe fungal disease, reducing yields annually. Chemical measures are important for Cytospora control as few options currently exist for preventing new infections. The specific objectives of the following thesis are to: (1) evaluate the efficacy of conventional and organic fungicides for C. leucostoma control, (2) test wound sealing fungicides, embedded in paint or kaolin clay, to develop preventive and containment approaches in existing orchards. Topsin, Topsin amended in 50% latex, Captan, Captan amended in 50% latex, 50% latex, lime sulfur, and lime sulfur amended in kaolin clay (Surround WP) showed evidence of efficacy from laboratory and field trials. Of these treatments, 50% latex, Topsin amended in 50% latex, and Captan amended in 50% latex, were shown to limit pathogen growth most effectively on pruning wounds during field trials in the summer season. In all field trials, however, NuCop showed absolutely no efficacy and should be avoided for C. leucostoma control in western Colorado. When various chemicals were tested on existing cankers to reduce spore inoculum loads, efficacy could not be statistically confirmed. The results of this study were compicated by variable field conditions and a large range of spore produced by each canker. Thus, larger sample sizes should be used in future studies to tease a part chemical efficacy and abiotic influences. Further, kaolin clay alone may shield fungal cankers from extreme temperatures, enhancing its growth in the field.