Browsing by Author "Westra, Philip, advisor"
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Item Unknown Development and characterization of wheat mutants resistant to acetyl co-enzyme A carboxylase inhibitors(Colorado State University. Libraries, 2012) Ostlie, Michael H., author; Westra, Philip, advisor; Haley, Scott, committee member; Lapitan, Nora, committee member; Shaner, Dale, committee memberWeed management in bread wheat (Triticum aestivum L.) is currently limited when undesirable grass species are present due to limited options for herbicidal selectivity between the species. The incorporation of an acetyl co-enzyme A carboxylase (ACCase) inhibitor tolerance trait in wheat would allow growers to expand the arsenal and effectiveness available for managing troublesome grass species. To develop this new trait, ethyl methanesulfonate mutagenesis (EMS) was utilized to induce point mutations in the ACCase gene. Seeds mutagenized with 0.75% EMS for 2.5 hours were grown and seed was collected. Lethal applications of clethodim and quizalofop were applied to plants in the M2 generation. Approximately 200 surviving plants were collected out of the total of 2.5 million M2 seeds planted. M3 greenhouse screening revealed roughly half the plants selected with quizalofop in the M2 generation contained an increased level of resistance to the herbicide. Dose response studies confirmed a two- to- eight fold increase in resistance over wild-type wheat and limited cross-resistance across other ACCase inhibiting herbicides. DNA sequencing of the ACCase gene in quizalofop mutants uncovered a novel point mutation resulting in an Ala - > Val substitution at position 2004 from the Alopecurus myosuroides numbering scheme. A 14C-based enzyme assay established a three- to- tenfold increase in ACCase activity in the presence of quizalofop from plants containing the new mutation. The experiment successfully generated quizalofop resistant mutant wheat and led to the discovery of a previously unknown point mutation. This mutation has the potential for future implementation in a wheat cultivar resistant to quizalofop.Item Open Access EPSPS gene duplication in Palmer amaranth: relative fitness, inheritance, and duplication mechanism of the glyphosate resistance trait(Colorado State University. Libraries, 2015) Giacomini, Darci A., author; Westra, Philip, advisor; Ward, Sarah, advisor; Leach, Jan, committee member; Preston, Christopher, committee member; Bedinger, Patricia, committee memberGlyphosate resistant (GR) Palmer amaranth (Amaranthus palmeri S. Wats.) is a weedy plant species that has invaded agricultural fields in at least 25 states, raising the cost of weed control to more than 4x the original cost. In most areas, the resistance is conferred through a gene amplification mechanism in which the target gene of glyphosate, 5-enolpyruvylshikimate-3-phosphate (EPSPS) is duplicated in the genome 100+ times, resulting in an overproduction of the EPSPS protein. With so much EPSPS enzyme available in each cell, glyphosate only inhibits a fraction of the proteins, leaving the rest to function as normal and ensuring plant survival. Understanding how this increase in EPSPS gene copy number and EPSPS protein production impacts relative fitness of the resistant plants was one objective of this research. Through greenhouse studies comparing high EPSPS copy GR plants with single copy sensitive plants, no difference was observed for any of the fitness characteristics measured. Both biotypes yielded similar numbers of offspring with no significant differences in germination or growth rate, revealing a complete lack of a fitness cost associated with the resistance trait. The second objective of this research was to quantify the stability of this resistance trait via multigenerational inheritance studies and within-plant EPSPS copy number variance measurements in the absence of glyphosate selection. The inheritance work found a complex pattern of EPSPS copy number transmission through the generations, a result that could be explained at least partially by the mosaic of EPSPS gene copy numbers patterns observed in both male and female Palmer amaranth plants. Copy numbers were inherited in a non-Mendelian pattern with transgressive segregation of the trait seen in both directions (more and fewer EPSPS copies found in the offspring than expected). This retention of high EPSPS copy number in the absence of a glyphosate selection pressure and no evidence of a fitness cost associated with the resistance trait possibly indicates a long-term loss of glyphosate as a control option in fields infested with GR Palmer amaranth. The last objective of this project was to better understand the mechanism of EPSPS gene duplication through sequence assembly of the EPSPS amplicon and chromosomal localization of this duplicated region. The amplicon was extended out to a little over 110kb and was found to contain mostly repetitive sequence including long direct repeats, microsatellites, and multiple transposable elements. A fluorescent in situ hybridization (FISH) assay found a single chromosomal location for the EPSPS genes, suggesting a tandem gene arrangement. These results further suggest that EPSPS duplication is achieved in Palmer amaranth via unequal recombination of the repeats surrounding the gene during mitosis and/or meiosis.Item Open Access Fertility and chromosome composition of wheat x jointed goatgrass backcross progeny(Colorado State University. Libraries, 2013) Beil, Craig T., author; Westra, Philip, advisor; Byrne, Patrick, advisor; Haley, Scott, committee member; Shaner, Dale, committee memberTo view the abstract, please see the full text of the document.Item 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, advisorGlyphosate 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.Item Open Access Physiological and biochemical mechanisms behind the fast action of glufosinate(Colorado State University. Libraries, 2019) Kagueyama Takano, Hudson, author; Dayan, Franck E., advisor; Westra, Philip, advisor; Reddy, Anireddy, committee member; Preston, Christopher, committee member; Gaines, Todd, committee memberGlufosinate is one of the few herbicides that are still effective for controlling herbicide resistant weeds, but its performance is often inconsistent and affected by environmental conditions. It inhibits glutamine synthetase (GS) by competing with glutamate for the active binding site. Unlike other amino acid biosynthesis inhibitors, glufosinate is a fast-acting herbicide and susceptible plants develop visual symptoms within a few hours after treatment. Inhibition of GS leads to ammonia accumulation and photosynthesis inhibition, which have traditionally been proposed as the causes of the rapid phytotoxicity. This dissertation presents a new understanding of the mechanism(s) of action of glufosinate and a biochemical approach to improve its herbicidal efficacy. Glufosinate uptake is inhibited by glutamine levels in the plant, and translocation is not affected by the rapid phytotoxicity. Glufosinate translocates primarily through the apoplast (xylem) rather than the symplast (phloem) probably due to its physicochemical properties and the absence of an effective membrane transporter. Glufosinate efficacy is proportional to the herbicide concentration in leaf tissues. Neither ammonia accumulation nor carbon assimilation inhibition are directly associated with the fast action of glufosinate. Instead, rapid phytotoxicity results from a massive light-dependent accumulation of reactive oxygen species (ROS). Inhibition of GS blocks the photorespiration pathway leading to a massive photooxidation damage. Under full sunlight, the excess of electrons is accepted by molecular oxygen leading to ROS generation. These free radicals cause lipid peroxidation, which ultimately leads to rapid cell death. The addition of protoporphyrinogen oxidase (PPO) inhibitors to glufosinate enhances ROS accumulation and herbicidal activity. This enhanced activity results from protoporphyrin formation at high levels due to a transient accumulation of glutamate, the precursor for chlorophyll biosynthesis. The herbicide combination also showed enhanced activity in the field and may help to overcome the lack of glufosinate efficacy under certain environmental conditions.Item Unknown Regional whole plant and molecular response of Kochia scoparia to glyphosate(Colorado State University. Libraries, 2012) Wiersma, Andrew, author; Westra, Philip, advisor; Leach, Jan, advisor; Reddy, Anireddy, committee member; Holtzer, Thomas, committee memberGlobally, glyphosate (Roundup®) resistant weeds pose a serious challenge to modern agricultural practices that utilize glyphosate for weed control, including Roundup Ready® cropping regimes. Locally, glyphosate resistant Kochia scoparia have been identified throughout the central Great Plains, and the infested range is expanding rapidly. Glyphosate and Roundup Ready® crops form the foundation of no-till technology, which has considerably reduced water use and soil loss in arid to semi-arid regions of North America. Unfortunately, the continued spread of glyphosate-resistant K. scoparia will jeopardize the utility of glyphosate and the sustainability of no-till agricultural practices. In an effort to suppress glyphosate-resistant K. scoparia, more needs to be known about 1) the spread of resistance, 2) the level of resistance, and 3) the mechanism responsible for glyphosate resistance in K. scoparia. Suspected glyphosate-resistant K. scoparia accessions were collected from Kansas, Colorado, North Dakota, South Dakota, and Alberta. Whole plant glyphosate dose response and shikimate assays were used to confirm resistance and assess the level of resistance. Then PCR, quantitative PCR, sequencing, and immunoblotting techniques were used to determine the mechanism responsible for glyphosate resistance. Sequence of the EPSPS binding site proline confirmed that amino acid substitution at that residue was not responsible for resistance in K. scoparia. However, quantitative PCR estimates of EPSPS copy number revealed increased copy number in all glyphosate-resistant individuals —ranging from 3 to 9 EPSPS copies relative to the reference ALS gene. Furthermore, increased EPSPS copy number was correlated to increased transcript and protein abundance. Based on these finding, I confirm resistance for all tested accessions throughout the North American central Great Plains, and conclude that increased glyphosate rates will have little effect in controlling glyphosate-resistant K. scoparia. Furthermore, I suggest that EPSPS gene amplification may be the mechanism responsible for glyphosate resistance in K. scoparia, and that lower level increases in EPSPS expression (as compared to A. palmeri) are sufficient for glyphosate resistance. Moreover, this research, again, demonstrates the adaptability of plants and foreshadows the need for diversifying weed management practices.Item Unknown Response of desirable trees to aminocyclopyrachlor in the field and greenhouse(Colorado State University. Libraries, 2017) Hildebrandt, Curtis M., author; Westra, Philip, advisor; Borch, Thomas, committee member; Jacobi, William R., committee memberAminocyclopyrachlor is a selective, plant growth regulator herbicide in the pyrimidine carboxylic acid family. Previously, aminocyclopyrachlor was marketed for turfgrass management but was withdrawn by the EPA in light of unexpected non-target injury related to desirable tree species such as white pine (Pinus strobus), and Norway spruce (Picea abies). Field trials to assess the impact of aminocyclopyrachlor on two tree species green ash (Fraxinus pennsylvanica) and honeylocust (Gleditsia triacanthos) were conducted to determine the relative sensitivity of green ash and honeylocust to aminocyclopyrachlor, determine a minimum safe spraying distance from green ash and honeylocust trees, and to assess the effect of application timing on tree response. An additional greenhouse dose response trial on tree seedlings was used to compare green ash and honeylocust response to Norway spruce which had demonstrated high sensitivity to aminocyclopyrachlor, and blue spruce which is a common and important native species in Colorado. Field results showed that green ash was not susceptible to herbicide applications while honeylocust showed severe injury, up to 100% for trees closest to applications. Honeylocust trees up to 7 m from the edge of the application displayed moderate to severe injury symptoms, and the fall treatment timings in October and November appeared to be safer in terms of reducing injury on target trees. Soil analysis using LC-MS and HPLC demonstrated that aminocyclopyrachlor dissipation was no different in the soil underneath green ash and honeylocust trees, and that dissipation was likely aided by absorption in tree roots. A greenhouse dose response showed that honeylocust was a moderately sensitive species, about four times more tolerant to aminocyclopyrachlor than blue spruce or Norway spruce. Green ash was consistently tolerant to aminocyclopyrachlor in the greenhouse, showing only minor response at the higher doses. Taken together these results provide a basic groundwork of research necessary for the writing of better aminocyclopyrachlor herbicide labels, and a better understanding of this herbicide's effect on certain woody vegetation.Item Open Access Understanding aminocyclopyrachlor behavior in soil and plants(Colorado State University. Libraries, 2012) Lindenmayer, Richard Bradley, author; Westra, Philip, advisor; Nissen, Scott, committee member; Shaner, Dale, committee member; Hansen, Neil, committee memberMany noxious and invasive weeds are perennial species that are inherently difficult to control. Canada thistle (Cirsium arvense) and field bindweed (Convolvulus arevensis) are two species of particular interest as they are capable of spreading quite rapidly through creeping underground reproductive structures and are able to continually regenerate from carbohydrate reserves stored in the roots. These weed species infest both cropland and non-cropland, including rangeland, pasture, natural areas, and rights-of-way, causing yield loss in crops from competition for soil resources and by harboring crop insect and disease pests as well as reducing ecosystem diversity in natural areas by displacing desirable or native vegetation with monocultures. Based on long-term weed control observed in the field with aminocyclopyrachlor (Lindenmayer et al. 2009), a better understanding of the herbicide's behavior in soil as well as within the plants was necessary. The objectives of this research were to (1) compare soil and foliar activity of aminocyclopyrachlor on Canada thistle to that of aminopyralid; (2) determine the dissipation rates of aminocyclopyrachlor, aminopyralid, and clopyralid under field conditions as well as evaluate their adsorption in six North American soils; and (3) evaluate aminocyclopyrachlor absorption, translocation, and metabolism in field bindweed. Results of the first study indicated that aminocyclopyrachlor was just as effective when applied to the soil as it was when applied to Canada thistle foliage and was similar to aminocyclopyrachlor for up to one year after treatment. The study also revealed that Canada thistle biomass was reduced to a far greater extent when either aminocyclopyrachlor or aminopyralid was absorbed via root tissue than by emerging shoot tissue. Overall, these results suggest that Canada thistle control can be achieved even through dormant season applications, reversing the tradition of spring or fall applied herbicides to actively growing foliage and that xylem mobility throughout Canada thistle plants from root absorption may contribute to more effective weed control. Results of the second study revealed that aminocyclopyrachlor, aminopyralid, and clopyralid all had similar dissipation rates under field conditions with soil half-lives of 32.5, 28.9, and 26.6 d, respectively. Mobility of aminocyclopyrachlor and aminopyralid was limited for the first 14 d with some downward movement after 28 d, while clopyralid had more significant leaching by 14 d. Adsorption in the six soils tested was greatest with aminocyclopyrachlor, followed by aminopyralid, and clopyralid had the least soil adsorption with average Kd values across the six soils of 0.503, 0.378, and 0.236 mL g-1, respectively. Adsorption was generally correlated with soil organic matter or texture, but not with pH. These results agreed with previously published information about aminopyralid and clopyralid and shed new light on aminocyclopyrachlor soil behavior. Results of the third study showed that aminocyclopyrachlor absorption in field bindweed was maximized at 48.3% of the applied radioactivity by 48 hours after treatment (HAT). A translocation pattern of movement out of the treated leaf into the other plant tissues was revealed, with nearly equivalent aminocyclopyrachlor distribution between the treated leaf, above-ground tissue, and below-ground tissue at 192 HAT. Over the 192 h, no soluble metabolites were observed, but an increasing portion of the radioactivity was found in the fraction bound to the plant tissue. These results indicate that aminocyclopyrachlor has greater translocation to below-ground tissue in field bindweed compared with other herbicides and other weed species and aminocyclopyrachlor is not rapidly metabolized in any field bindweed plant tissue.Item Open Access Vegetation management and restoration species safety with aminocyclopyrachlor(Colorado State University. Libraries, 2015) Getts, Thomas Jordan, author; Westra, Philip, advisor; Jacobi, William, committee member; Meiman, Paul, committee memberAminocyclopyrachlor is a synthetic auxin herbicide in the pyrimidine carboxylic acid family, and is the only herbicide within the family. Aminocyclopyrachlor exhibits excellent herbicide activity offering multiple year control of many broadleaf noxious weeds and many non-desirable tree species (DuPont 2009). A non-native tree, Russian olive is the fourth most common woody species in the western United States and has been shown to cause many detrimental ecological impacts. Removing Russian olive allows native species to reestablish within certain areas. Where the soil seed bank is depleted and there is not a native seed source nearby, planting restoration species can be desirable after invasive species removal. Biodiversity of native plant species can help support larger suites of desirable species within an ecosystem. Restoration is not just important after invasive species removal, but after disturbances such as mining, fire, and floods Two studies were conducted to investigate potential restoration uses of aminocyclopyrachlor within the Northern Front Range of Colorado. The objective of the first research project was to assess restoration species herbicide tolerance. Two types of tolerance were of interest; species soil residual herbicide tolerance, and species tolerance to foliar herbicide applications. The objective of the second research project was to determine the effectiveness of cut stump applications of aminocyclopyrachlor for the control large Russian olive trees. The first study evaluated the tolerance of eight monocot species and eight broadleaf species to thirteen soil residual herbicide treatments at two pre plant application timings and two post emergence application timings of sixteen herbicides. The study was located at the Colorado State University Horticultural Research Farm from 2010 to 2012. Initial percent frequency, relative change in percent frequency, and biomass were used to evaluate the tolerance of species tested. Variables were analyzed for each species, at each application timing, looking for differences among herbicide treatments. No difference in initial establishment percent frequency was detected for any species*herbicide combination compared to the untreated check (p>05). Relative percent frequency change from 2011 to 2012 was not significant compared to the untreated for any monocot species*herbicide combination (p>.05). However, differences were detected for dicot species*herbicide combinations (p<.05). No differences in biomass occurred for any species*herbicide combination compared to the untreated check (p>.05). Generally there were numerical trends in the data, suggesting monocot species were relatively tolerant to the herbicides tested at all four application timings. Numerically, percent frequency and biomass values indicated certain dicot species establishment was inhibited by certain soil residual herbicide treatments, and were completely removed by certain foliar herbicide applications. In general monocot species tested were more tolerant than dicot species tested, especially in the foliar treatments. However, many instances of monocot and dicot species tolerances to herbicide applications tested were found. This implies that when an herbicide is used to control an invasive species, many restoration species tolerant to the soil residual herbicide could be safely planted the following year. Additionally many restoration species were tolerant to foliar herbicide applications, indicating certain applications could be made to control non-planted weedy species during restoration species establishment. The second study assessed the effectiveness of Aminocyclopyrachlor, imazapyr, triclopyr and glyphosate for cut stump application control of Russian olive. Thirty nine replications of herbicide treatments were tested at three field sites in the Northern Urban Front Range of Colorado. Treatment mortality and off-target impact were assessed every six months for thirty months. Thirty months after treatment, glyphosate, aminocyclopyrachlor, imazapyr, triclopyr, and the untreated check had 95, 92, 74, 71, and 18 percent mortality of Russian olive trees. All herbicide treatments had higher mortality compared to the untreated check (p<.05), but no herbicides were different from one another (p>.05). Herbicide applications have the potential to cause injury to non-target vegetation. The bare soil around each stump was measured (Radius of Inhibition) to capture the off target impact of herbicide applications. Thirty months after treatment there was a radius of inhibition of 4 cm, 8 cm, 13 cm, and 26 cm for glyphosate, triclopyr, imazapyr, and aminocyclopyrachlor respectively. Aminocyclopyrachlor had a larger radius of inhibition than other herbicide treatments tested thirty months after application (p<.05). In order to treat an average size Russian olive within our study, the cost of herbicide products were $1.47, $1.98, $1.16, and $5.95 for glyphosate, triclopyr, imazapyr, and aminocyclopyrachlor respectively. Overall we found aminocyclopyrachlor offered Russian olive control comparable to other herbicides tested. However, it had the largest off target impact, and was three times the cost of the second most expensive treatment.