Waterhemp was a sporadic weed in Kentucky soybean production since the 1970’s. Waterhemp’s presence was not significant until the 1990’s after a widespread adoption of imazaquin and imazethapyr herbicides in the late 1980’s by Kentucky farmers which resulted in ALS-resistant waterhemp in some Kentucky areas. The introduction of glyphosate resistant soybeans in 1996 resulted in glyphosate-containing products being widely used by Kentucky farmers. Waterhemp populations resistant to glyphosate have occurred in Kentucky in the past few years. The majority of Kentucky soybeans are produced in some type of conservation tillage system, primarily to conserve soil and water, which is advantageous on Kentucky’s rolling topography. Glyphosate controls a wide range of weeds and popular with farmers because of this characteristic. However, waterhemp resistant to glyphosate developed in some fields with the continuous glyphosate usage. Waterhemp control research trials were conducted in Union and Hancock Counties in Western Kentucky in an attempt to find herbicide combinations to provide season-long control. Waterhemp populations in these studies were resistant and susceptible to glyphosate but the resistant populations were great enough to cause soybean yield loss if not controlled. KEYWORDS: Amaranthus tuberculatus, Herbicide Resistance, EPSPS, PPO, ALS

Renewed interest in hemp (Cannabis sativa L.) for cannabinoid production has highlighted critical knowledge gaps for growers. The impacts of weed interference on hemp yield are largely unstudied despite causing serious economic losses in most cropping systems. Furthermore, understanding factors determining cannabinoid content of hemp will be crucial for effective production. To evaluate the effects of weed competition on hemp yield and cannabinoid content, hemp growing in plasticulture was subjected to competition from 5, 3, 1, or 0 waterhemp (Amaranthus tuberculatus (Moq.) Sauer) plants. Hemp biomass and cannabinoid content were not significantly affected by weed competition. Yields in weedy and weed-free treatments were generally comparable, although unexpectedly high variation limited the ability to detect subtle differences between treatments. Waterhemp biomass was significantly reduced by competition from hemp in comparison to hemp-free controls. Suppression of weed growth by hemp and lack of significant yield loss from weeds suggests that hemp can be highly competitive and grown successfully without herbicides in certain circumstances. Abundance and documented phytotoxicity of hemp secondary metabolites suggest a potential for allelopathic activity. While incorporated hemp residue was not more effective than a maize control at reducing plant growth, hemp residue and extracts effectively inhibit seed germination. A laboratory assay showed that crude extracts of hemp can reduce the germination of a Brassica napus L., a bioindicator. Furthermore, a greenhouse experiment showed that small amounts of hemp residue on the soil surface can effectively reduce and delay the germination of waterhemp. Together, these results show that hemp residue has the potential to be incorporated with a practice such as chaff-lining for enhanced control of germinating weed seeds.

<p>Tall waterhemp management in agronomic crops continues to be an increasing problem due to widespread resistance to herbicides, including protoporphyrinogen oxidase (PPO)-inhibitors. With limited effective postemergence herbicides, especially in soybeans, research to further understand the selection of PPO-resistant (PPO-R) tall waterhemp and identification of new herbicide resistance mechanisms is crucial for improving weed management decisions in order to slow selection for herbicide resistance and prolong the effectiveness of PPO-inhibiting herbicides.</p> <p> Previous research has shown that soil-applied applications of PPO-inhibiting herbicides can increase the frequency of the PPO resistance trait (∆G210) in surviving tall waterhemp plants, even when applied in combination at the same ratio with the very long chain fatty acid inhibitor (VLCFA), <i>s-</i>metolachlor. Field experiments were conducted to determine if selection for tall waterhemp resistant individuals to PPO-inhibitors could be reduced when the soil residual activity of <i>s</i>-metolachlor persisted longer than the PPO-inhibitor herbicide. The frequency of ∆G210 in surviving individual plants increased as the fomesafen rate increased, but was independent of the rate of <i>s</i>-metolachlor. Additionally, heterozygosity of ∆G210 in surviving individuals did not change with any rate or combination of fomesafen and <i>s</i>-metolachlor. However, saflufenacil, standard PPO-inhibitor with relatively short soil residual activity, applied alone increased the number of homozygous PPO-R tall waterhemp by 15% compared to the high rate of <i>s</i>-metolachlor and the combination of saflufenacil and <i>s</i>-metolachlor. Furthermore, this research demonstrated that end of season control of tall waterhemp plays a more vital role in delaying a large-scale shift towards herbicide resistance through reduced seed production. This can be achieved through the combination of multiple effective herbicide sites of action, including soil residual PPO-inhibitors. Tall waterhemp control and density were greatest with the high rates of fomesafen plus <i>s</i>-metolachlor, which resulted in the lowest number of PPO-R tall waterhemp that survived herbicide treatment at the end of season.</p> <p> Prior to the research conducted in this thesis, the only known resistance mechanism to PPO-inhibiting herbicides in tall waterhemp has been the ∆G210 target site mutation. A previously developed TaqMan assay used to determine the presence or absence of the ∆G210 mutation has allowed accurate, high throughput screening of this mutation. However, suspected PPO-R tall waterhemp do not always receive positive confirmation indicating the presence of an alternative resistance mechanism. Identification of additional resistance mechanisms can provide valuable insight in regards to resistance to PPO-inhibiting herbicides as well as cross resistance to other herbicide modes of action, which can lead to improved tall waterhemp management decisions. Of 148 tall waterhemp populations collected across the Midwestern U.S., 84% of the populations sampled contained at least one PPO-R biotype with the ∆G210 mutation, although several individual plants across the Midwest U.S. exhibited phenotypic resistance to fomesafen that could not be explained by ∆G210. The percentage of PPO-R tall waterhemp without ∆G210 was 19, 5, 2, 1, and 2% for Iowa, Illinois, Indiana, Minnesota, and Missouri, respectively. Following the initial greenhouse screening, subsequent tall waterhemp populations were selected that exhibited low-, mid-, and high-level resistance to fomesafen that resulted in resistance ratios from 0.6 to 17X in response to fomesafen. This research documents the variability in fomesafen response to multiple tall waterhemp populations in addition to revealing the presence of additional resistance mechanism(s), other than the previously known ∆G210 mutation that has been the benchmark for resistance to PPO-inhibiting herbicides in tall waterhemp.</p> <p> Lastly, greenhouse and lab experiments were conducted to investigate the role of antioxidant enzymes with PPO-R tall waterhemp via ∆G210. The objectives of this research were to determine if the variability in resistance ratios for PPO-R tall waterhemp documented in greenhouse and field scenarios could be due to an enhanced antioxidant enzyme pathway. Basal levels of antioxidant enzymes in PPO-S populations were not different from PPO-R populations when pooled together by respective phenotype. However, enzyme activity of tall waterhemp populations varied at the individual level, but independent of the ∆G210 mutation. This indicates that an inherent enhanced antioxidant enzyme pathway does not cause the variability in fomesafen response in tall waterhemp. With the exception of glutathione reductase, antioxidant enzyme activity following fomesafen application was generally the same for PPO-R and PPO-S populations by increasing, decreasing, or remaining unchanged. Glutathione reductase activity in PPO-S populations decreased compared to PPO-R populations from 9 to 36 HAT. By 36 HAT, all antioxidant enzyme activity for PPO-S populations was lower compared to PPO-R populations most likely a consequence of more lipid peroxidation. This research shows that antioxidant enzyme activity correlated with fomesafen application and documents the variability observed within tall waterhemp populations with and without the ∆G210 mutation. </p>

<p>PPO inhibitors are a valuable group of herbicides that provide soil-residual and foliar control of glyphosate resistant <em>Amaranthus</em> species. The ΔG210 mutation in the <em>PPX2</em> gene confers PPO-inhibitor resistance and has been present in the Midwest for more than a decade. Until recently, PPO-inhibitor resistance in waterhemp was attributable to just the ΔG210 mutation in the <em>PPX2</em> gene, but recently, several new PPO-resistant biotypes have been discovered in waterhemp and Palmer amaranth. A possible explanation is a change in PPO-inhibitor use patterns and commonly used active ingredients.</p> <p>A direct comparison of the ΔG210 mutation and a new mutation, R128G, was conducted in the greenhouse. Results indicate that the R128G mutation in waterhemp is not substantially better than the ΔG210 mutation with respect to conferring resistance to PPO inhibitors applied preemergence. Furthermore, there is no evidence that the utility of PPO inhibitors applied preemergence will diminish any further as a result of the R128G mutation increasing in frequency. A set of field trials was conducted to investigate how a new PPO inhibitor, trifludimoxazin, will select for resistant biotypes in the field. Overall, a greater number of resistant plants survived the foliar herbicide applications than emerged through soil applications. Trifludimoxazin did not increase the frequency of PPO-resistant individuals when applied to soil, but when applied to foliage, increased the frequency of PPO-resistant individuals by 2.5 to 2.6-fold, similar to other PPO inhibitors applied to foliage. In other experiments, investigations of waterhemp populations with unique resistance phenotypes were conducted. In populations that were more resistant than others, yet had the same ΔG210 mutation, there was no evidence of herbicide metabolism contributing to the greater resistance phenotype. In resistant populations that had no target site mutations, target site expression experiments and lipid peroxidation experiments were inconclusive. However, there was some evidence of increased target-site expression or increased antioxidant capacity as being causal mechanisms, although no mechanisms have been fully ruled out.</p>

<p dir="ltr">Palmer amaranth (Amaranthus palmeri S. Watts) and waterhemp [Amaranthus tuberculatus (Moq.) J. D. Sauer] are two of the most troublesome weeds in U.S. soybean production and the auxin herbicides dicamba and 2,4-D, are currently used extensively for postemergence control of these species. In some cases, complete control of weeds at the time of auxin application is not achieved due to adverse environmental conditions, plant factors or misapplications. In these instances, a subsequent postemergence herbicide may be required to control any plant that survived the initial auxin herbicide application. This research was conducted to determine the efficacy and optimal successive time interval between applications of viable postemergence herbicides in soybean on Palmer amaranth and waterhemp plants surviving a previous application of 2,4-D or dicamba. Results from this research indicated that respraying a failed auxin herbicide application with a subsequent auxin herbicide, especially dicamba, was less effective than respraying with glufosinate or fomesafen to control waterhemp in addition to being a less desirable approach for resistance management. Additionally, respray herbicide applications should target 7 to 14 d after the initial failed herbicide application on waterhemp for optimal overall efficacy. When dicamba was the initial herbicide sprayed on Palmer amaranth, 94% or greater control was achieved with glufosinate in 2019 on a 7- and 14-d respray interval, which was greater than the efficacy observed with either dicamba or 2,4-D (< 82%). In 2020, these differences in herbicide efficacy were not evident within these time intervals. These general trends in treatment differences were also manifested in the data for plant height, biomass and viable apical meristems. When 2,4-D was the initial herbicide sprayed on Palmer amaranth, fomesafen and glufosinate applications on a 7-d respray interval and glufosinate on a 14-d respray interval resulted in greater control than 2,4-D in 2019. All other trends in herbicide treatments and time intervals were consistent with dicamba applied as the initial herbicide. Another part of his research was conducted with the objective of quantifying herbicide spray solution deposition and herbicide efficacy on waterhemp growing in different densities plant densities: low, high-thinned and high densities. In both field and greenhouse experiments, spray deposition (µl cm-2) on waterhemp leaves was up to 53% less on plants grown in high density compared to the other density treatments. Even though no differences in herbicide deposition between low and the high-thinned densities were observed, there were differences in herbicide efficacy. When applied to plants growing in low density, applications of glufosinate, fomesafen and topramezone reduced weed biomass to a greater extent than plants growing in the high-thinned density. Following herbicide damage to the apical meristem, plants growing in high-density produced new branches from axillary buds that were previously dormant, whereas plants growing in the low density already had axillary branches initiated from these buds and did not provide an opportunity for new shoot growth. In general, no differences in herbicide efficacy were observed across weed densities following dicamba applications. Source-to-sink translocation of dicamba to previously active meristems, or axillary buds that broke dormancy after the herbicide application, may have compensated for less spray solution interception on plants growing in high density. Lower levels of postemergence herbicide efficacy in high density weed populations are not only influenced by spray deposition differences, but also by changes in plant growth and apical dominance. This research provides further evidence that justifies the need for weed managers to reduce weed densities as much as possible, via non-chemical or soil residual herbicides (preemergence), as much as possible to optimize the efficacy of foliar herbicide applications.</p>

<p dir="ltr">Waterhemp (<i>Amaranthus tuberculatus</i>)<i> </i>is one of the most problematic weeds in soybean production in the United States. The ability of waterhemp to quickly evolve resistance threatens the utility of many herbicides. The introduction of Xtend^®and Xtendflex^®soybeans allow for the in-season application of dicamba and glufosinate. With an increase in dicamba use in soybeans plus its continued use in corn, there have been many reports of dicamba failure on waterhemp.</p><p dir="ltr">Greenhouse dose response experiments were conducted to screen six populations of waterhemp for resistance to dicamba. Each population was compared to a known sensitive and known resistant population, with 50% growth reduction (GR₅₀) values of 39 g ae ha^-1and 226 g ae ha^-1, respectively. Low-level dicamba resistance was identified in all populations, as they had GR₅₀values that were different from the known sensitive and R:S ratios that varied from 1.7 to 4.4. Additionally, all six populations exhibited at least 50% survival at a 1/2X rate of dicamba where the sensitive only had 30% survival. Therefore, we can conclude that dicamba resistance in waterhemp is present in multiple counties in Indiana.</p><p dir="ltr">In addition to characterizing populations from Indiana, a growth monitoring study was conducted to determine how emergence timing impacted waterhemp growth. In 2021, plants that emerged in the earliest cohort were taller, had more branches, and accumulated more biomass in comparison to later emerging plants at six weeks after flagging. In 2022, drought conditions throughout the month of June impacted growth of earlier emerging plants, and waterhemp that emerged in the latest cohort were taller, had more branches, and accumulated more biomass than earlier emerging cohorts at six weeks after flagging. Seed yields per plant were low in both years, but all cohorts were able to produce seed. This research concludes that in years when soil sufficient moisture is available, earlier emerging waterhemp plants are competitive with crops and later emerging plants can still produce seed.</p><p dir="ltr">Additionally, field trials were conducted to determine herbicide programs in the Enlist^® and Xtendflex^® soybean systems that would best control a waterhemp population with multiple herbicide resistance. At 21 days after the second postemergence application, waterhemp control was highest in two pass systems. The addition of pyroxasulfone to the second postemergence application increased control of waterhemp in the Xtendflex^® system. Waterhemp densities were the lowest and soybean yield was the highest in two pass herbicide programs for both systems. The results indicate that waterhemp resistant to chemistries in HRAC Groups #2, #4, #5, #9, #14, and #27 was most effectively controlled by programs with two herbicides applications, either a preemergence followed by postemergence or two pass postemergence, and included 2,4-D and glufosinate in the postemergence application(s).</p><p dir="ltr">Finally, a waterhemp population from Francesville, IN was characterized for herbicide resistance via a series of field, greenhouse, and laboratory experiments. Preliminary laboratory analysis confirmed resistance to herbicide actives in the HRAC Groups #2 and #14 via target site mutations and to Group #9 via gene amplification. Field research trials indicated inadequate waterhemp control with preemergence applications of pendimethalin and atrazine and postemergence applications of herbicide actives from Groups #2, #9, #14, and #27 as well as glufosinate and dicamba. Greenhouse dose response experiments revealed GR₅₀values for the Francesville population that were significantly higher for dicamba, mesotrione, and topramezone than the known sensitive. R:S ratios of 4.4, 3.3, and 1.8, were documented for dicamba, mesotrione, and topramezone, respectively. Data from all experiments demonstrated that the Francesville population is resistant to herbicide actives in Groups #2, #4, #5, #9, #14, and #27.</p>