<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>

To combat herbicide resistance among weeds, non-herbicide methods of control, such as cover crops, are becoming widely adapted. Experiments were conducted to determine how to effectively establish and manage cover crops in order to suppress tall waterhemp and Italian ryegrass and to assess their overall impact on soybean growth and yield. Various cover crop establishment methods were evaluated, and it was determined that interseeding at the R7 growth stage of soybean was the least effective method for proper cover crop establishment. Biomass data demonstrated that interseeding created the least amountof cover crop biomass, with no differences found among the other establishment methods that included drilling and sowing broadcast. At soybean planting timing, treatments with tillage had greater control of tall waterhemp than those without tillage. Wheat was shown to have the greatest weed suppressive capabilities, largely due to its ability to create high levels of residual biomass. Daikon radish produced the least biomass residue and had the poorest tall waterhemp control. The termination experiment of Elbon rye determined that treatments with rolling could impact soybean emergence and plant height largely due to dense biomass that lay flat onto the soil surface

<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>Acetolactate synthase (ALS) inhibitors are a widely used class of selective herbicides used to control grass and broadleaf weeds. The repeated use of ALS-inhibiting herbicides has selected for biotypes of weeds resistant to ALS inhibitors, especially in the weeds most problematic to growers in the Midwest. While ALS inhibitor use seems futile, new mechanisms of herbicide action are not predicted to be commercialized in the near future to solve this problem. This leads to the main objective of this research, determining what value ALS inhibitors provide in controlling populations of weeds with resistance to ALS inhibitors. </p> <p>Field experiments with soil-applied (PRE) applications of ALS inhibitors on horseweed (<i>Erigeron canadensis</i>) and tall waterhemp (<i>Amaranthus tuberculatus </i>var. <i>rudis</i>) exhibited higher efficacy than would be expected given the frequency of the ALS resistance trait in the population. Whereas control of these species with POST-applied applications was similar or less than the proportion of the population characterized as susceptible using molecular techniques. Soil-applied applications, therefore, resulted in relatively greater control than POST applications in populations with known ALS-inhibitor-resistance mechanisms.</p> <p>Greenhouse experiments showed that overall resistance ratios were higher for PRE applications of ALS inhibitors in horseweed, tall waterhemp, and Palmer amaranth (<i>Amaranthus palmeri</i>). However, GR₅₀ values decreased for both susceptible and resistant biotypes for the PRE applications compared to POST, suggesting the biologically effective dose of these herbicides is lower in soil residual applications. This research found that PRE applications of ALS inhibitors resulted in some level of control on horseweed and tall waterhemp classified as resistant to ALS inhibitors due to the higher efficacy of PRE herbicide applications.</p> <p>Genetic analysis assessing the amino acid substitutions that confer resistance to ALS inhibitors in tall waterhemp confirmed a difference in selection pressure between PRE and POST applications and between ALS active ingredients in tall waterhemp. Applications of chlorimuron PRE at 11 g ai ha^-1selected for 35% homozygous W574L genotypes and at 44 g ha^-1 selected for 70% homozygous W574L genotypes. An increase of homozygous W574L individuals along with a decrease in heterozygous individuals from 65 (11 g ha^-1) to 29% (44 g ha^-1) suggests that W574L is semi-dominant in tall waterhemp and that high labeled rates of chlorimuron applied PRE can partially overcome the heterozygous W574L-resistance mechanism. In horseweed, no difference in selection pressure was observed between application timing or between chlorimuron or cloransulam. A new mutation conferring ALS-inhibitor resistance in horseweed was discovered, a Pro197Leu amino acid substitution, with resistance ratios of 21X to chlorimuron and 8.6X to cloransulam. These resistance ratios are slightly less than those reported for the Pro197Ala and Pro197Ser amino acid substitutions in conferring ALS-inhibitor resistance in horseweed. </p> <p>Finally, a survey of 42 populations of tall waterhemp in Indiana counties with confirmed ALS-inhibitor resistant populations of tall waterhemp found that all populations contained at least 16% individuals with the W574L amino acid substitution, 35 populations contained at least 1% individuals with the S653N substitution, and 9 populations contained at least 1% individuals with the S653T substitution. Taking into consideration the three mutations tested, 8 of the 42 populations tested contained <50% ALS-inhibitor resistant individuals within the population. Using the same tall waterhemp populations as collected in the survey, Next-Generation Sequencing was used to determine if other amino acid substitutions conferring resistance to ALS inhibitors existed. Results from WideSeq revealed that 10 other amino acid substitutions in the ALS protein may be conferring resistance in tall waterhemp in Indiana: A122T, A122N, A122S, P197T, P197L, P197H, D376E, and G654F. Further research from this survey also suggests that metabolic resistance to ALS inhibitors is likely a contributor to resistance in tall waterhemp in Indiana.</p> <p>This research suggests that ALS-inhibiting herbicides, more specifically chlorimuron, would provide the greatest contribution to management of tall waterhemp. Chlorimuron would perform best when used in soil residual applications and in populations of tall waterhemp containing either individuals susceptible to chlorimuron or individuals heterozygous for ALS inhibitor resistance conferred by the W574L mutation. This research also demonstrates the specificity of the amino acid substitutions in the ALS protein and by weed species to realize the benefit of these herbicides for management of weeds resistant to ALS inhibitors. Molecular characterization of target site resistance to ALS inhibitors has traditionally been considered relatively simple. However, we found 11 new amino acid substitutions that confer resistance to ALS inhibitors in horseweed and tall waterhemp. The complexity of ALS inhibitor resistance calls for the use of methods such as NGS to detect all potential resistance mutations in a timely manner and for the use of tests detecting metabolic resistance. Overall, this research demonstrates that ALS inhibitors still provide some utility for management of weed populations classified as resistant to ALS inhibitors and that the resistance mechanisms in horseweed and tall waterhemp are more numerous than previously reported. </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>