Weed management options in agronomic crop production have been severely limited by widespread populations of weeds resistant to herbicides, including waterhemp [Amaranthus tuberculatus (Moq.) Sauer (syn. rudis)] resistant to foliar applications of herbicides that inhibit protoporphyrinogen oxidase (PPO; EC 1.3.3.4) activity (PPO-R). Herbicides within this site of action (WSSA site of action #14) remain efficacious when soil-applied to PPO-R waterhemp populations. Therefore, the continued use of these herbicides for soil-residual control of PPO-R waterhemp, especially in soybean production, is paramount with limited postemergence herbicides that remain effective. An improved understanding of the selection for PPO-R waterhemp would provide information to help minimize future loss of residual PPO-inhibiting herbicide activity. Five studies, consisting of 14 experiments, were conducted to improve our understanding of the selection for herbicide-resistant individuals. Soil-residual herbicides have been suspected to select for herbicide-resistant individuals; however, this phenomenon has never been observed experimentally in field conditions. This dissertation provides direct evidence from greenhouse and field experiments that significant selection pressure can occur from soil-residual herbicides; however, this selection for resistance could be delayed when using full commercial herbicide rates and effective herbicides from multiple sites of action. Also, the frequency of heterozygous individuals (RS) and PPO-inhibiting herbicide efficacy on RS individuals is a factor in the selection for herbicide resistance; however, current information on the these topics is limited. To provide additional information on RS individuals, a large-scale genotypic and phenotypic screen of multiple PPO-R waterhemp populations was conducted. It was determined that RS individuals were less frequent than expected and PPO-inhibiting herbicide efficacy on RS individuals was population-dependent. Finally, the hormetic effects of soil-residual herbicides have been paradoxically implicated as a means of both mitigating and exacerbating the selection for herbicide resistant biotypes; however, limited information was available on the hormetic effects of soil-residual PPO inhibitors. Greenhouse and growth chamber experiments were conducted to improve understanding of hormesis and soil-residual PPO-inhibiting herbicides. Experiments indicated that PPO-inhibiting herbicides may exert a limited hormetic effect on waterhemp germination below doses that cause a phytotoxic effect of the emerging seedling, indicating this effect may exacerbate the issue of selection for PPO-R waterhemp. Overall, data presented in this dissertation provides important information on the under-studied interaction between PPO-inhibiting herbicides and PPO-R waterhemp to safeguard the sustained efficacy of herbicides within this site of action.

Annual bluegrass (Poa annua L.) is a problematic weed in turfgrass that has evolved resistance to twelve different herbicide sites of action. The mitotic-inhibiting herbicide pronamide has both pre- and post-emergence activity on susceptible annual bluegrass populations. Still, post-emergence activity may be compromised in some resistant populations due to the lack of root uptake or an unknown foliar resistance mechanism. Spray droplet size may affect foliar and soil deposition of pronamide, thus potentially explaining variation in population control or differential foliar and root uptake. Pronamide, flazasulfuron, and pronamide + flazasulfuron deposition were quantified on annual bluegrass as affected by spray-droplet size. The efficacy of these herbicide treatments in resistant (R) and susceptible (S) annual bluegrass populations was then evaluated with two droplet sizes (400 and 1000 μm). Absorption and translocation of pronamide were investigated in R and S populations following foliar-only and soil-only pronamide applications.

Studies were conducted to determine the distribution of s-triazine resistant biotypes of common lambsquarters (Chenopodium album L.) and smooth pigweed (Amaranthus hybridus L.) in Virginia. Collections of seed were made from suspected triazine-resistant biotypes of common lambsquarters and smooth pigweed from counties in Virginia which had reported having triazine resistance problems. Triazine resistance was confirmed by measuring chlorophyll fluorescence in the presence of atrazine. For further confirmation of triazine resistance in collected common lambsquarters and smooth pigweed biotypes, greenhouse testing of whole plants and a sinking leaf disc assay were performed. Cross-resistance to another s-triazine, as-triazine, and substituted urea herbicide was also determined for s-triazine-resistant biotypes. These studies have shown triazine- resistant smooth pigweed to be present in 19 counties and common lambsquarters to be present in 8 counties in Virginia. s-Triazine resistant biotypes were found to be resistant to another s-triazine and as-triazine herbicide, but were susceptible to the substituted urea herbicide. Additional studies were initiated to determine the effects of different temperature regimes on triazine-resistant and -susceptible biotypes of common lambsquarters and smooth pigweed from different geographical locations. These studies were conducted at the North Carolina State University Phytotron facility in controlled environment growth chambers. Triazine-resistant common lambsquarters biotypes from Virginia, Maryland, and Switzerland as well as a smooth pigweed biotype from Virginia were examined. Triazine-susceptible biotypes of common lambsquarters and smooth pigweed were included as controls. Shoot height, weight, chlorophyll a and b content, and whole leaf fatty acid content of common lambsquarters and smooth pigweed were determined at 18°/14° C, 26°/22° C, and 36°/26° C. Measurements of shoot height were made at 30 and 63 days after planting. The shoot weight, chlorophyll a and b content, and fatty acid content was determined from plants harvested at 63 days after planting. These data indicate common lambsquarters biotypes from different geographical regions exhibited a differential response to temperature. There was no difference between triazine-resistant and -susceptible biotypes in response to temperature. Differences were detected between triazine-resistant smooth pigweed biotypes which indicated that the susceptible biotypes were more vigorous as indicated by shoot height and weight at lower temperatures than triazine-resistant biotypes of smooth pigweed. / M.S.

Palmer amaranth (Amaranthus palmeri) and common ragweed (Ambrosia artemisiifolia) are two of the most troublesome weeds in soybean. Both weeds possess widespread resistance to glyphosate and acetolactate synthase (ALS) inhibiting herbicides resulting in the use of protoporphyrinogen oxidase- (PPO) inhibitors to control these biotypes, although PPO-resistant biotypes are increasing. New soybean herbicide-resistant trait technologies enable novel herbicide combinations. Combinations of two herbicide sites-of-action (SOA) improved control 19 to 25% and 14 to 19% of Palmer amaranth and common ragweed, respectively, versus using one SOA (mesotrione, dicamba, 2,4-D, or glufosinate alone). Seed production of 5 to 10 cm Palmer amaranth and common ragweed was reduced greater than 76% by fomesafen, auxin (dicamba and 2,4-D), or glufosinate containing treatments. Some weeds survived and set seed even when treated at the proper size. As weed size increased from 10 to 30 cm, control diminished and fecundity increased, underscoring the importance of proper herbicide application timing. Effective preemergence herbicides reduced the number of weeds present at the postemergence application compared to no treatment, reducing the likelihood of herbicide resistance development. Dicamba, 2,4-D, or glufosinate applied alone or auxin + glufosinate combinations reduced Palmer amaranth seed production greater than 95% when applied at first visible female inflorescence; this first report, in addition to previous reports on individual herbicides, indicates this application timing may be useful for soil seed bank management. This research informs mitigation of herbicide resistance spread and development. / Master of Science in Life Sciences / Over 30 million hectares of soybeans were harvested in 2019 in the United States, totaling over $31 billion in value. Two of the most troublesome weeds in soybean, Palmer amaranth (Amaranthus palmeri) and common ragweed (Ambrosia artemisiifolia) can cause even greater yield reductions in soybean, up to 79 to 95%, respectively. Frequent, exclusive, and repeated use of a single herbicide has led to multiple herbicide-resistance in both of these weeds. Co-applying two effective herbicides reduces the likelihood of resistance development. New soybean varieties have been genetically modified for resistance to herbicides that were previously unusable, allowing new herbicide combinations. Research was established to investigate these herbicide options to control and reduce seed production of Palmer amaranth and common ragweed with the overarching goal of mitigating herbicide resistance, particularly resistance to protoporphyrinogen oxidase (PPO) inhibiting herbicides, which are a critical part of herbicide options in soybean production. Preemergence herbicides are vital tools in herbicide programs, reducing the number of weeds present at a postemergence application and thereby reducing the risk of herbicide resistance development to the postemergence herbicide. PPO herbicides (flumioxazin, sulfentrazone, or fomesafen) applied preemergence reduced Palmer amaranth and common ragweed density at the postemergence application 82 to 89% and 53 to 94%, respectively. The preemergence herbicide used did not affect control four weeks after the postemergence herbicides were applied. Postemergence herbicides were applied targeting three weed heights: 5 to 10 cm (ideal), 10 to 20 cm, and 20 to 30 cm. Control decreased as weed height increased and larger weeds had greater biomass and seed production, underscoring the importance of proper herbicide application timing. The single site-of-action treatments dicamba, 2,4-D, glufosinate, or fomesafen resulted in greater than 85 and 92% morality of 5 to 10 cm Palmer amaranth and common ragweed, respectively. Palmer amaranth and common ragweed control improved by 19 to 25% and 14 to 19%, respectively, when using two herbicide sites-of-action increased versus using one SOA (mesotrione, dicamba, 2,4-D, or glufosinate alone). The use of two herbicide sites of action resulted in maximum biomass reductions, depending on weed height, of 57 to 96% and 73 to 85% for Palmer amaranth and common ragweed, respectively. Dicamba, 2,4-D, glufosinate alone and in combination with fomesafen reduced seed production (relative to the nontreated) of 5 to 10 cm Palmer amaranth and common ragweed greater than 98 and 76%, respectively. Dicamba, 2,4-D, and glufosinate applied alone or auxin (dicamba and 2,4-D) and glufosinate combinations reduced Palmer amaranth seed production greater than 95% when applied at first visible female inflorescence. This indicates that these herbicides may be useful in soil weed seed bank management. This research reinforces the utility of PPO herbicides for preemergence control and their efficacy postemergence when combined with another effective herbicide, a practice known to reduce herbicide resistance development. This research also reinforces the potential for dicamba, 2,4-D, or glufosinate to reduce weed seed production when applied at a delayed timing. Future research should investigate the progeny of these weeds treated with herbicides at a delayed timing to evaluate the potential for this practice to reduce herbicide resistance development.

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

The activity and the selectivity of cycloxydim {2-[1-(ethoxyimino)butylJ-3-hydroxy- 5-(2H-tetrahydrothiopyran-3-yl)-2-cyclohexen-l-one}, code designation BAS 517, were examined flIst with etiolated seedlings of com (Zea mays L.) and soybean [Glycine max (L.) Merr.]. Etiolated soybean seedlings were not affected by cycloxydim. The degree of growth inhibition of com varied with concentration of cycloxydim and incubation time. Compared to mesocotyls and coleoptiles, radicles of corn were the most sensitive to cycloxydim. Meristematic tissues appeared to be the site of action of cycloxydim as root meristems were the first to show symptoms. A band of reddening tissue developed at meristematic tips followed by the complete cessation of root growth. In a study comparing activities of technical grade and formulated cycloxydim and sethoxydim, {2-[ l-(ethoxyimino )butyl}- 5-[2-(ethylthio )propy11-3-hydroxy-2-cyclohexen-l-one}, formulated compounds were more potent than the technical grade chemicals without formulation additives. Technical sethoxydim was more potent than technical cycloxydim. Root tips excised from com and soybean seedlings were used subsequently for cycloxydim treatments. The activity and selectivity of cycloxydim expressed at the isolated root tip level were similar to those of cycloxydim bioassayed with whole seedlings. However, root tips appeared to be more sensitive than the whole seedlings. Injury at the tissue and cell levels of the 2-mm root tips that were treated with various concentrations of cycloxydim was examined after 24 hours incubation. Concentrations of 0.1, 1, and 10 μM cycloxydim caused severe cell vacuolization. A gradient of decreasing injury from epidermal cells toward the center of roots was observed. This pattern of injury appeared to reflect the penetration of cycloxydim into roots along a concentration gradient. / Ph. D.

Doctor of Philosophy / Department of Agronomy / Tesfaye T. Tesso / The lack of effective post-emergence weed control options is often highlighted as one of the major factors behind dwindling acreage under sorghum (Sorghum bicolor (L.) Moench) in the United States. The discovery of herbicide resistance sources in wild sorghum population and subsequent efforts to incorporate them into cultivated sorghum was received with much optimism to change weed management practices in sorghum. As the development of the technology advances, especially of the Acetolactate synthase (ALS) resistance, concerns over the temporary interveinal chlorosis and reduced seedling vigor in some of the resistant families became heightened. This thesis research is designed to shed light on the genetic basis of seedling chlorosis and assess its impacts on yield potential. The study has three parts; the first part is focused on identifying the genetic causes and plant mechanisms associated with the chlorotic phenotype. ALS herbicide resistant sister-lines expressing normal and chlorotic phenotypes were analyzed via RNA sequencing at four time points during seedling growth. The study identified several variants of genes coding chloroplast precursors and those that cause epigenetic modifications. Once confirmed, genetic markers can be developed to track these gene variants in the breeding population and eliminate segregates genetically prone to chlorosis/yellowing. The second part of the study focuses on assessing the effect of ALS resistance associated chlorosis on agronomic and nutritional parameters of sorghum inbred lines. A set of ALS resistant lines expressing different levels of the chlorotic phenotype were evaluated in replicated field trials and laboratory methods. Results showed that interveinal chlorosis delays flowering but does not have negative effect on yield and nutritional parameters with and without herbicide treatment. The last part addresses whether there is any yield drag that may be associated with herbicide resistance traits and foliar interveinal chlorosis. For this, we synthesized a large set (182) of hybrids from ALS resistant, ACCase resistant and regular (susceptible) seed and pollinator parents. The hybrids were then evaluated in three sets at multiple locations during the 2014 and 2015 crop seasons along with commercial checks. The results revealed that resistance to both herbicides do not cause any drag to grain yield. The traits also do not have any negative impact on grain and nutritional quality of resistant hybrids.

The objective of this Ph.D. research was to identify new and novel mechanisms of wild radish (Raphanus raphanistrum L.) resistance to photosystem II (PSII) inhibitors, auxinics, and acetohydroxyacid synthase (AHAS) inhibitors. PSIIinhibitor resistance was demonstrated to be target-site based, and conferred by a Ser264 to Gly substitution of the D1 protein. Auxinic resistance was associated with reduced herbicide translocation to the meristematic regions of resistant wild radish plants. Two new resistance mutations of wild radish AHAS were discovered, including one encoding the globally rare Asp376 to Glu substitution, and another encoding an Ala122 to Tyr substitution, which has never been identified or assessed for resistance in plants previously. Characterization of the frequency and distribution of AHAS resistance mutations in wild radish from the WA wheatbelt revealed that Glu376 was widespread, and that some mutations of AHAS are more common than others. Computer simulation was used to examine the molecular basis of resistance-endowing AHAS target-site mutations. Furthermore, through the computer-aided analysis, residues were identified with the potential to confer resistance upon substitution, but which have not previously been assessed for this possibility. Results from this Ph.D. research demonstrate that diverse, unrelated mechanisms of resistance to PSII inhibitors, auxinics, and AHAS inhibitors have evolved in wild radish of the WA wheatbelt, and that these mechanisms have accumulated in some populations.

Doctor of Philosophy / Department of Agronomy / Kassim Al-Khatib / Mitchell R. Tuinstra / Sorghum acreage is declining throughout the United States because management options and yield have not maintained pace with maize improvements. The most extreme difference has been the absence of herbicide technology development for sorghum over the past twenty years. The objectives of this study were to evaluate the level of resistance, type of inheritance, and causal mutation of wild sorghums that are resistant to either acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides or acetohydroxyacid synthase (AHAS)-inhibiting herbicides. ACCase-inhibiting herbicides used in this study were aryloxyphenoxypropionate (APP) family members fluazifop-P and quizalofop-P along with cyclohexanedione (CHD) family members clethodim and sethoxydim. The level of resistance was very high for APP herbicides but low to nonexistent to CHD herbicides. With genetic resistance to APP herbicides, the resistance factors, the ratio of resistance to susceptible, were greater than 54 to 64 for homozygous individuals and greater than 9 to 20 for heterozygous individuals. Resistance to CHD herbicides was very low with resistance factors ranging from one to about five. Genetic segregation studies indicate a single gene is the cause of resistance to APP herbicides. Sequencing identified a single mutation that results in cysteine replacing tryptophan (Trp-2027-Cys). Trp-2027-Cys has previously been reported to provide resistance to APP but not CHD herbicides. The other wild sorghum evaluated in this study was resistant to AHAS-inhibiting herbicides including imidazolinone (IM) family member, imazapyr, and sulfonylurea (SU) family member, nicosulfuron. Resistance factors in this genotype were very high, greater than 770 for the IM herbicide and greater than 500 for the SU herbicide, for both herbicide chemical families. Genetic segregation studies demonstrate that resistance was controlled by one major locus and two modifier loci. DNA sequencing of the AHAS gene identified two mutations, Val-560-Ile and Trp-574-Leu. Val-560-Ile is of unknown importance, but valine and isoleucine are similar and residue 560 is not conserved. Trp-574 is a conserved residue and Leu-574 is a known mutation that provides strong cross resistance to IM and SU herbicides. The results of these studies suggest that these sources of APP, SU, and IM resistance may provide useful herbicide resistance traits for use in sorghum.

Italian ryegrass (Lolium perenne spp. multiflorum) is a common weed management problem in turfgrasses, cereals and non-crop areas in the United States. In Oregon, the number of populations with multiple-resistance continues to increase. To manage these resistant populations, the resistance patterns must be determined. In this study, five Italian ryegrass populations (CT, R1, R2, R3 and R4) from two cropping systems were studied for resistance patterns and mechanisms. The CT population is from a Christmas tree plantation and was resistant to at least six herbicides with four different mechanisms of action: atrazine, diuron (2.4-fold), glyphosate (7.4-fold), hexazinone (3.1-fold), imazapyr (1.8-fold), and sulfometuron. The resistant indices (RI) for sulfometuron and atrazine could not be calculated because 50% growth reduction for the CT population was not reached even with the highest rates applied, 17.6 kg ai ha⁻¹and 16 kg ai ha⁻¹, respectively, which are 16 times the recommended field application rates for this two herbicides. The CT population accumulated less shikimate than the S population. There were two mutations in the CT population, Trp591 to Leu in the ALS gene and Ser264 to Gly in the psbA gene, which explain the ALS and PII cross resistance, respectively. R1, R2, R3 and R4 were collected from annual cropping systems. All four populations were resistant to flufenacet. RIs for two populations, R2 and R4, were 8.4 and 5.9, respectively. R2 and R4 also were resistant to mesosulfuron-methyl, pinoxaden, quizalofop and clethodim. R4 was resistant to diuron, but R2 was not. An Asp-2078-Gly substitution in the ACCase gene was found in both R2 and R4 populations, while another Ile-2041-Asn substitution in the ACCase gene was found in the R4 population. These mutations explain the ACCase cross resistance in the R2 and R4 populations. The mechanisms for the glyphosate resistance in the CT population and the flufenacet resistance in R1, R2, R3 and R4 populations were not identified in this study. None of the five populations were resistant to the herbicide pyroxasulfone. / Graduation date: 2013