Field and greenhouse studies were conducted in 2007 and 2008 to evaluate the foliar efficacy of saflufenacil on horseweed (Conyza canadensis (L.) Cronq.). In the field, saflufenacil applied alone at the lowest rate (25 g/ha) resulted in less control than all other herbicide treatments that included saflufenacil. The addition of glyphosate to 25 g/ha of saflufenacil increased the level of control over either herbicide applied alone. However, the addition of glyphosate to 50 g/ha of saflufenacil or greater was not beneficial because saflufenacil alone provided at least 95% control. Overall, horseweed height at the time of herbicide application had very little effect on the efficacy of saflufenacil applied alone or in combination with glyphosate. Application variables can enhance the foliar activity of saflufenacil. In the greenhouse, saflufenacil combined with glyphosate provided greater control than saflufenacil applied alone on both glyphosate-susceptible and -resistant horseweed populations. Regardless of horseweed population or glyphosate, saflufenacil had greater activity when crop oil concentrate rather than nonionic surfactant was used as the adjuvant. Decreasing light level within 24 hours of herbicide application resulted in greater saflufenacil activity. Applying saflufenacil in a pH 5 spray solution resulted in greater activity than pH 7 or pH 9. Although effects from saflufenacil applied under different temperatures were evident in early timings, there were no lasting effects on the efficacy of saflufenacil. Saflufenacil had significant activity on both glyphosate-susceptible and -resistant horseweed. Under certain conditions when complete control of horseweed is not achieved, such as low application rates, large target weeds, and varying environmental conditions, application variables including glyphosate tank-mixtures, crop oil concentrate, low spray solution pH, and low light level may increase the level of horseweed control from saflufenacil.

Onarm research was conducted in 2011 and 2012 to determine the postemergence and residual control by dicamba of glyphosate-resistant (GR) Palmer amaranth (Amaranthus palmeri S. Wats.). Preemergence dicamba at 0, 0.28, 0.56, and 1.1 kg ae ha-1 and 0.07 kg ae ha-1 flumioxazin was applied at 30, 15 and 0 days prior to planting. Postemergence dicamba at 0.28, 0.56, and 1.1 kg ae ha-1 with and without 0.84 kg ae ha-1 glyphosate was applied to 5, 10 and 15 cm Palmer amaranth. In addition, a greenhouse experiment was conducted in 2012 to evaluate and confirm the optimum rate for control of Palmer amaranth with a new formulation of dicamba (BAS 18322H). In the greenhouse, dicamba at 0.14, 0.28, 0.56, 1.1, and 2.2 kg ae ha-1 was applied to 5, 10, and 15 cm Palmer amaranth.

Adoption of glyphosate-resistant crops has resulted in increased glyphosate usage and decreased use of residual herbicides thus resulting in weed pressure shifts. Weeds that display multiple-resistance to glyphosate and other herbicide modes of action have become a concern in many parts of the United States. Incorporation of multiple herbicide resistance traits into multiple cropping systems, may facilitate weed resistance to additional herbicides. Furthermore, controlling volunteer crop stands containing multiple herbicide-resistance traits may be problematic in herbicide resistant crops. These volunteer crops will compete with the currently growing crop qualifying them as a weed. Therefore, this research was conducted to determine control options for: failed glyphosate resistant corn stands, and volunteer glyphosate resistant corn stands in glyphosate resistant cotton. Furthermore, research was conducted to determine what densities of glyphosate-resistant corn will cause cotton yield loss and if time of removal of these densities impacts cotton yield loss.

The onion (Allium cepa L.) is the most agronomically important vegetable crop to New Zealand. In temperate climates, such as New Zealand, the most prominent problem associated with onion production is weed control. To address this problem, glyphosate-resistant onions were generated in a range of germplasm backgrounds via Agrobacterium-mediated transformation of immature embryos (Eady et al. 2003a). This thesis contains an analysis of these onions. PCR and Southern analysis were used to create molecular profiles for individual transgenic events. Of the 17 putatively transgenic onion lines tested, 16 contained a single copy of the CP4 EPSPS gene integrated into their genomes, and one line had two copies of the gene. The spectinomycin resistance gene, located on the backbone of the vectors used in the transformation process, was detected in two of the glyphosate-resistant onion lines tested. Ten different glyphosate-resistant onion lines were subjected to field trial evaluation over the 2004/2005 growing season in Canterbury. Glyphosate-resistant onion seedlings were transplanted into the field in a randomised block design and a subset of plants, chosen to represent selected lines, were sampled. Tissue was harvested from young leaves, old leaves, and bulbs. Quantitative multiplex RT-PCR was used to assess expression of the CP4 EPSPS transcript, and protein levels were determined using an ELISA-based assay. The transgene appears to be highly transcribed in some lines, as expression of the CP4 EPSPS gene was in the same order of magnitude as the highly transcribed 18S internal control. The CP4 EPSPS protein levels of the glyphosate resistant onion lines presented in this thesis ranged between 0.36–7.44 µg CP4 g-1FW in the leaf samples, which is a little lower than, but still similar to, values reported for glyphosate resistant corn and poplar. This project reports the first in-depth analysis into the expression of the CP4 EPSPS gene in field-grown glyphosate-resistant onion lines.

The onion (Allium cepa L.) is the most agronomically important vegetable crop to New Zealand. In temperate climates, such as New Zealand, the most prominent problem associated with onion production is weed control. To address this problem, glyphosate-resistant onions were generated in a range of germplasm backgrounds via Agrobacterium-mediated transformation of immature embryos (Eady et al. 2003a). This thesis contains an analysis of these onions. PCR and Southern analysis were used to create molecular profiles for individual transgenic events. Of the 17 putatively transgenic onion lines tested, 16 contained a single copy of the CP4 EPSPS gene integrated into their genomes, and one line had two copies of the gene. The spectinomycin resistance gene, located on the backbone of the vectors used in the transformation process, was detected in two of the glyphosate-resistant onion lines tested. Ten different glyphosate-resistant onion lines were subjected to field trial evaluation over the 2004/2005 growing season in Canterbury. Glyphosate-resistant onion seedlings were transplanted into the field in a randomised block design and a subset of plants, chosen to represent selected lines, were sampled. Tissue was harvested from young leaves, old leaves, and bulbs. Quantitative multiplex RT-PCR was used to assess expression of the CP4 EPSPS transcript, and protein levels were determined using an ELISA-based assay. The transgene appears to be highly transcribed in some lines, as expression of the CP4 EPSPS gene was in the same order of magnitude as the highly transcribed 18S internal control. The CP4 EPSPS protein levels of the glyphosate resistant onion lines presented in this thesis ranged between 0.36–7.44 µg CP4 g-1FW in the leaf samples, which is a little lower than, but still similar to, values reported for glyphosate resistant corn and poplar. This project reports the first in-depth analysis into the expression of the CP4 EPSPS gene in field-grown glyphosate-resistant onion lines.

Research was conducted in 2013 and 2014 to evaluate the postemergence control of Palmer amaranth [Amaranthus palmeri (S.) Wats.] with mesotrione alone and in mixtures with fomesafen and/or glyphosate and to evaluate the impact of lactofen and planting date on growth, development, and yield of indeterminate soybean [Glycine max (L.) Merr.]. Studies included a greenhouse evaluation of different rates of mesotrione on the control of 5- and 10-cm Palmer amaranth and field studies evaluating the control of 5- to 10-cm Palmer amaranth with three rates of mesotrione applied alone and in mixtures with fomesafen and/or glyphosate. Lactofen studies include a planting date study evaluating one rate of lactofen applied at V2 soybean stage with planting dates of April 15, May 1, May 15, and June 1 and a lactofen timing study where one rate of lactofen was applied at soybean growth stages ranging from V1 to R5.

Glyphosate resistant crops were rapidly adopted by farmers since their introduction in 1996 and currently, greater than 90% of cotton and soybean crops are glyphosate resistant. Glyphosate has been an effective mean for controlling Palmer amaranth, however overreliance on glyphosate based systems resulted in weeds that can no longer be controlled with glyphosate. Palmer amaranth resistance to glyphosate has been confirmed in ten US states including Virginia's bordering neighbor North Carolina. The objectives of this study were to i) determine the spread of Palmer amaranth and evaluate awareness among farmers and agribusinesses of herbicide resistant weeds in Virginia; ii) determine the efficacy of commonly used cotton and soybean herbicides programs for Palmer amaranth control; and iii) conduct greenhouse experiments to quantify the level of glyphosate resistance in a Greensville County, Virginia population. Using a communication network of Virginia county extension agents and crop advisers, Palmer amaranth was found in 15 Virginia counties. A survey was conducted to evaluate awareness of herbicide resistance and management of weeds in Virginia. Ninety percent of producers had fields planted to Roundup Ready® crops for each of the last 3 years. One hundred percent of the responders claimed awareness of the potential for weeds to develop resistance to glyphosate, but when asked about how serious they consider weed resistance to herbicides, the responders average rating was of 7.9 (on a scale of 1 to 10 where 1 is "not at all serious" and 10 is "very serious" ). Eighteen percent of the responder population claimed no awareness of glyphosate resistant weeds documented in Virginia. Herbicide efficacy experiments were established in soybean and cotton fields infested with Palmer amaranth. In soybean, experiments were established in a field where Palmer amaranth was not adequately controlled with glyphosate in the previous year. Glyphosate applied at 0.87 + 0.87 + 1.74 kg ae ha⁻¹ at 1, 3, and 5 weeks after planting (WAP) provided 82 to 85% control in 2009, but only 23 to 30% control in 2010, a hot and dry year. Glyphosate applied after preemergence (PRE) herbicides improved control to 90 percent. Programs that included s-metolachlor + metribuzin applied preemergence and followed by glyphosate + fomesafen applied postemergence provided the best control (93%) at 8 WAP. Glufosinate based herbicide programs provided greater than 85% control when applied alone, and control increased to 95% when preceded by PRE herbicides. Many conventional control systems integrating different modes of action provided more than 80% control at final evaluation of Palmer amaranth in 2009 and 2010. In soybean, the most consistent and effective program was flumioxazin applied PRE followed by chlorimuron + thifensulfuron, which provided 99 and 82% control at final evaluation in 2009 and 2010, respectively. Cotton fields were heavily infested with Palmer amaranth, but control with glyphosate had historically been good. Glyphosate applied early postemergence, late postemergence, and late post-directed provided more than 95 percent control at final evaluation of Palmer amaranth. Preemergence applications of fomesafen, fluometuron, or pendimethalin + fomesafen provided 77 to 99 percent early-season control and control was complete with an additional postemergence glyphosate application. Glufosinate applied at 0.45 kg ha⁻¹ at 1 and 3 WAP or applied at 0.45 kg ha⁻¹ following a preemergence herbicide provided greater than 95% control. Greenhouse experiments confirmed Palmer amaranth resistance in a population collected from Greensville County, Virginia. In the first experiment, the resistant biotype's I₅₀ value (rate necessary for 50% inhibition) for dry weight was 1.47 kg ae ha⁻¹, which is 4.6 times greater than the susceptible biotype and 1.7 times the recommended use rate of glyphosate. For fresh weight, the I₅₀ value of the resistant biotype was 1.60 kg ae ha⁻¹, 4.7 times that of the susceptible biotype of 0.34 kg ae ha⁻¹. In the second experiment, the I₅₀ value for the susceptible population could not be determined because all glyphosate rates resulted in complete control. However, the resistant population required 1.01 and 1.30 kg ae ha⁻¹ of glyphosate to reduce the fresh and dry weight by 50%. / Master of Science

Doctor of Philosophy / Department of Agronomy / Johanna A. Dille / Kochia (Kochia scoparia L. Schrad.), one of the most problematic weeds in the Great Plains of United States, has evolved resistance to some herbicides including glyphosate (5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor) which was first reported in western Kansas in 2007. The objectives of this research were to (1) characterize six kochia populations from western Kansas on the basis of glyphosate resistance or multiple herbicide resistance, (2) determine germination characteristics of the populations and evaluate their growth and fecundity in the field, and (3) determine if EPSPS gene amplification responsible for glyphosate resistance in kochia was associated with growth and fecundity cost in the plants. Six kochia populations were from Scott, Finney, Thomas, Phillips, Wallace, and Wichita counties. Based on shikimate assay for glyphosate and recommended field rates for four tested herbicides, three kochia populations (Scott (SC-R), Finney (FN-R), and Thomas (TH-R)) were grouped into glyphosate-resistant (GR) and three populations (Phillips (PH-S), Wallace (WA-S) and Wichita (WI-S)) were grouped into glyphosate-susceptible (GS). All populations were resistant to dicamba (synthetic auxin) and chlorsulfuron (acetolactate synthase inhibitor), however, atrazine (PS II inhibitor) resistance in FN-R was noted as exceptional among the GR populations. Across the three germination temperatures (5, 10 and 15 C), the GR populations consistently had less total cumulative germination and at 15 C, they consistently required more time to attain 50% of maximum cumulative germination than the GS populations. Both the field study and evaluation of relationship between EPSPS gene amplification and plant performance showed that differences in plant height, biomass accumulation and fecundity among populations were not in respect to glyphosate resistance but rather, differences in their inherent ability to grow and produce seeds in the presence or absence of neighbors. This research suggests that fitness differences between GR and GS kochia populations could be identified in germination characteristics but not in their growth or fecundity.

Reduced plant population in glyphosate-resistant soybean [Glycine max (L) Merr.] may influence the critical time of herbicide application. Field studies were conducted in 2007 and 2008 at two locations in Kentucky to determine the effect of four weed control programs on soybean seed yield, seed quality, crop canopy, and weed pressure when planted at three densities - 185,000, 309,000, and 432,000 plants ha-1. Plots were treated with glyphosate at either 3 weeks after planting (WAP), 5 WAP, 7 WAP, 3 & 7 WAP, representing common weed control protocols within the state. No differences in seed yield were discovered between plant densities in two of four siteyears. 254,500 plants ha-1 was sufficient for maximum yields in all site-years and was also sufficient to achieve maximum canopy amongst those populations tested in the study. Sequential applications at 3 and 7 WAP provided the highest seed yield, while the 5 WAP and 7 WAP application timings were generally the single applications that allowed for the highest yield and canopy closure, along with the highest visual estimate of weeds controlled. Plant density did not influence the critical period for weed control.

Master of Science / Department of Agronomy / Randall S. Currie / Mithila Jugulam / Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot), one of the problem weeds of the US, evolved resistance to multiple herbicides including glyphosate due to selection in Arkansas (AR). Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and amplification of EPSPS gene, the molecular target of glyphosate confers resistance to this herbicide in several weed species, including Italian ryegrass from AR. The objective of this study was to determine the expression of EPSPS gene and protein as well as distribution of EPSPS copies on the genome of glyphosate-resistant Italian ryegrass (ARR) using a known susceptible Italian ryegrass (ARS) from AR. EPSPS gene copies and expression of ARR and ARS were determined using quantitative PCR with appropriate endogenous controls. EPSPS protein expression was determined using Western blot analysis. Fluorescence in situ hybridization (FISH) was performed on somatic metaphase chromosomes to determine the location of EPSPS copies. Based on qPCR analysis, ARR plants showed a wide range of 12 to 118 EPSPS copies compared to a single copy in ARS. EPSPS gene expression correlated with the gene copy number in both ARR and ARS. Individuals with high EPSPS copies showed high protein expression in Western blot analysis. FISH analysis showed presence of brighter EPSPS signals, distributed randomly throughout the genome of ARR individuals compared to a faint signal in ARS plants. Random distribution of EPSPS copies was previously reported in glyphosate-resistant Palmer amaranth. Overall, the results of this study will help understand the origin and mechanism of EPSPS gene amplification in Italian ryegrass.