CGAr92194 {α-[1,3-dioxolan-2-yl-methoxy)imino]benzeneacetonitrile} is a herbicide safener that is used as a seed dressing agent (1.25 g ai/kg seeds) to protect grain sorghum [<i>Sorghum bicolor</i> (L.) Moench] against metolachlor [2-chloroi-<u>N</u>-(2-ethyl-6-methylphenyl-<u>N</u>-(2-methoxy1-methylethyl)acetamide] injury. The potential adverse phytotoxic effects and the mechanisms of the protective action of this safener were studied in laboratory experiments. Adverse phytotoxicity was assessed by comparing CGA-92194 and the herbicide safeners cyometrinil {(Z)-α[(cyanomethoxy)imino]benzeneacetonitrile} and flurazole [phenylmethyl 2-chloro-4-(trifluromethyl)-5-thiazolecarboxylate] for their effects on CO₂ fixation, protein, DNA, RNA and lipid syntheses of enzymatically isolated leaf cells of soybean [<u>Clycine max</u> (L.) Merr]. At physiological concentrations of less than 10 μM, CGA-92194, cyometrinil and flurazole were stimulatory of all metabolic processes. At 100 μM, the safeners were inhibitory of the five processes with flurazole being the most potent. The mechanisms of the safening action of CGA-92194 were studied by examining the potential interactions of this safener with metolachlor at the levels of uptake and macromolecular syntheses in enzymatically isolated leaf mesophyll protoplasts of grain sorghum. The influence of CGA-92194 on the <i>in vitro</i> reactivity of metolachlor with glutathione (GSH) and it metabolism by sorghum seedlings were also examined. When CGA-92194 and metolachlor were given simultaneously, CGA-92194 enhanced the uptake of ¹⁴C-metolachlor into the sorghum protoplasts in a concentration-dependent pattern. Thus, interference with herbicide uptake is not involved in the protective action of this safener Treatments with metolachlor and CGA-92194 in combination inhibited the incorporation of ¹⁴C-uracil, ³H-thymidine and ¹⁴C-acetate into sorghum protoplast macromolecules less than metolachlor given alone, suggesting the potential involvement of a competitive antagonism in the mechanism of action of CGA-92194. The metabolic activity and growth of sorghum seedlings grown from CGA-92194-pretreated seeds was significantly lower than that of seedlings grown from untreated seeds at 10 or 20 days after planting, The relationship of these effects of CGA-92194 to its safening action is unclear at the present time. CGA-92194 increased the <i>in vitro</i> chemica1 reactivity of metolachlor for GSH in a concentration-dependent pattern, Sorghum seedlings grown from safener-pretreated seeds enhanced ¹⁴C-metolachlor absorption and stimulated its metabolism <u>via</u> conjugation to GSH. This stimulation was reduced by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethy1)- oxirane] a potent inhibitor of plant GSH-<u>S</u>-transference enzymes, These results indicate that a safener-induced stimulation of the spontaneous or enzymatic conjugation of metolachlor with GSH is most likely involved in the protective action of CGA-92194. It is suggested that the safening mechanism of action of CGA—92194 involves a sequence of multilevel interactions which together contribute to the overall protection of grain sorghum from metolachlor injury. / Ph. D. / incomplete_metadata

Plants are sessile organisms and have developed varied mechanisms to tolerate stress. One such mechanism is DNA methylation, which plays a vital role within and across generational stress adaptation. To understand the role of DNA methylation in transgenerational stress adaptation, we exposed Arabidopsis thaliana for four generations of sub-lethal doses of glyphosate, trifloxysulfuron, clipping, and shading, which we further classified into the broader categories of stress ecological (shading and clipping) and herbicides (glyphosate and trifloxysulfuron). We analyzed phenotypic and whole-genome bisulfite sequencing data and found that the Arabidopsis phenotype adapts more rapidly to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic change in the DNA methylation patterns. To understand within generation stress response, Sorghum bicolor was exposed to the same stresses at sub-lethal doses and we analyzed its phenotypic, whole genome bisulfite sequencing, and gene expression responses. Ecological stresses had higher negative impact on S. bicolor plant growth as compared to herbicide stresses. Similarly, we found higher differentially expressed genes for clipping as compared to both herbicides. All four stresses changed the methylome in a unique way; where we found 998 differentially methylated regions (DMR) for trifloxysulfuron, 193 for shading, 141 for clipping and 60 for glyphosate. Out of these DMR's some occurred genic region, which could potentially change gene expression and help plants withstand stress. Overall, DNA methylation can potentially help plants to withstand stress due to their dynamic and specific response to a variety of stresses both transgenerational and within generation. This information to better understand stress adaptation mechanisms in plants and used to develop stress-resilient crops. / Doctor of Philosophy / Environmental and anthropogenic stresses can negatively impact plant growth and development. Plants can have stress memory through epigenetic changes which helps them withstand stress in future generations. Epigenetics is the field of science where changes on the DNA and not sequence, that can be an addition or deletion of a methyl group, modification of histones, or production of small RNAs. We wanted to understand short and long-term effects of common anthropogenic and ecological stresses on how DNA methylation changes can help plants to withstand stress. We used the model plant Arabidopsis thaliana and the non-model crop/weed Sorghum bicolor. We exposed plants to sub-lethal doses of two herbicides, clipping, and shade stress, at levels high enough to cause significant visible injury but still allowed them to recover and reproduce for a single generation for S. bicolor and four generations for A. thaliana. We found that A. thaliana rapidly more adapts to herbicide stress as compared to ecological stresses. DNA methylation changes for glyphosate were minimal after four generations of stress whereas the other three stresses showed dynamic changes in the DNA methylation patterns. Each stressed impacted S. bicolor phenotype, DNA methylation, and gene expression in unique ways. We found ecological stresses greatly affected the phenotype of the S. bicolor plants as compared to herbicide stresses. Overall, our results showed that stress can cause DNA methylation changes and in transgenerational stress DNA methylation can potentially play a role in stress adaptation. This information could be useful for scientists to further understand stress resilience in plants.

Italian ryegrass (Lolium multiflorum Lam.) is a competitive weed in small grain production areas throughout the northwestern and southeastern US. In small grains, Italian ryegrass has generally been controlled with postemergence treatments of diclofop, or diclofop-methyl, a member of the subfamily of the aromatic carboxylic acid family, the aryloxyphenoxypropionates. The first incidence of diclofop resistance in Italian ryegrass was reported in Virginia in 1995. Experiments to characterize diclofop resistance in several Virginia biotypes of Italian ryegrass included the following objectives: (1) evaluation of the presence of diclofop resistance in several Italian ryegrass biotypes collected across Virginia, (2) evaluation of alternative herbicide efficacy for diclofop resistant Italian ryegrass control, and (3) characterization of the aryloxyphenoxypropionate (APP) resistance mechanism in resistant Italian ryegrass biotypes. The response of 32 biotypes to diclofop collected from various locations statewide with varying histories of diclofop applications confirmed diclofop resistance in Virginian Italian ryegrass populations. At 4-times the label-recommended application rate, only 50% of biotypes previously exposed to diclofop in a cropping situation were adequately controlled versus 94% of the biotypes not previously treated with diclofop. Tralkoxydim provided the most effective control of four of the biotypes. No postemergence treatment effectively controlled one biotype previously exposed to diclofop applications. Effective preemergence herbicide treatments for Italian ryegrass control in the greenhouse included acetochlor (two formulations) and flufenacet plus metribuzin. In the field, flufenacet plus metribuzin resulted in excellent Italian ryegrass control, little crop injury, and acceptable barley yields. Acetyl-coenzyme A carboxylase (ACCase) assays and herbicide absorption, translocation, and metabolism studies were conducted to investigate resistant mechanism(s) to two APP herbicides, diclofop and quizalofop. ACCase assays indicated no differences in enzyme activity between the two biotypes of Italian ryegrass evaluated. Furthermore, no significant differences in the specific activity of ACCase were detected between the two biotypes in the absence of diclofop. [14C]Quizalofop-P absorption, translocation, and metabolism did not differ between resistant and susceptible Italian ryegrass biotypes. Lack of a significant biotype effect suggests that differential metabolism does not explain the differential response to diclofop treatments observed in the herbicide dose-plant response experiment. / Ph. D.

Tall fescue (Festuca arundinacea Schreb.) has great utility as a low maintenance turfgrass in the northern and transition zone regions of the United States. However, it is difficult to successfully maintain tall fescue of high quality over consecutive summers because of its susceptibility to the fungal pathogen Rhizoctonia solani, which causes the disease brown patch. Not only is brown patch aesthetically unpleasing in a stand of tall fescue but it can also thin out the turf and allow for the encroachment of undesirable weedy species. Cultivar selection, cultural practices, mixing turf species and timing of pesticide applications all can impact the epidemiology of brown patch in tall fescue. Research was conducted in tall fescue to quantify chitinase activity in different cultivars, elucidate the impact of mowing height and nitrogen fertility on brown patch and bermudagrass (Cynodon dactylon L.) encroachment, to evaluate seeding mixtures of tall fescue with hybrid bluegrass (Poa pratensis x Poa arachnifera) on diseases and weeds as well as measuring the impact of the herbicide bispyribac-sodium on brown patch. Chitinase activity was greater in the tall fescue cultivar that was less susceptible to brown patch. In the mowing-fertility studies, cutting tall fescue at 10 cm generally reduced brown patch and bermudagrass encroachment compared to 6 cm. Mixing hybrid bluegrass with tall fescue reduced disease and weed species infestations compared to tall fescue alone. Applying bispyribac-sodium earlier in April resulted in less brown patch and better weed control compared to application in May. Based on this research brown patch severity and subsequent weed species infestations can be reduced by selecting a tall fescue cultivar with a high basal level of chitinase, mowing it at 10 cm and mixing it with a hybrid bluegrass cultivar. / Ph. D.

Herbicide combinations are common in corn production in the United States to control broadleaf and grass weed species. Studies were conducted in 1995 and 1996 to: (1) investigate the interactions of 2,4-D and dicamba with halosulfuron-methyl on common lambsquarters and common ragweed control in corn, (2) determine the effect of 2,4-D on the foliar absorption, translocation, and metabolism of 14C halosulfuron-methyl in common lambsquarters, (3) examine the interactions of 2,4-D, dicamba, and ALS-inhibitor herbicides with rimsulfuron plus thifensulfuron-methyl (RT) and with sethoxydim on giant foxtail, common ragweed, and common lambsquarters control in corn. Combinations of halosulfuron-methyl with 2,4-D or dicamba were generally additive in their effects on common lambsquarters and common ragweed control, and were occasionally synergistic on common lambsquarters. Synergistic herbicide interactions in the greenhouse were observed with 2,4-D (17 g/ha ) and halosulfuron-methyl (18 g/ha) and 2,4-D (70 g/ha ) in combination with halosulfuron-methyl at 4.5 and 36 g/ha, respectively. Absorption and translocation of 14C-halosulfuron-methyl were not influenced by the addition of 2,4-D, with absorption increasing with time. Three unknown halosulfuron-methyl metabolites (M1, M2, and M3) with Rf values of 0.0, 0.97, and 0.94, respectively, were isolated. The addition of 2,4-D increased the level of M3 at the 18 g/ha halosulfuron-methyl rate, which may contribute to common lambsquarters phytotoxicity. Antagonism on giant foxtail control was observed with all combinations of RT and 2,4-D. Tank mixtures of RT with flumetsulam plus clopyralid plus 2,4-D, atrazine, 2,4-D, and dicamba plus atrazine controlled giant foxtail £ 78% 65 (DAT). RT mixed with flumetsulam plus clopyralid plus 2,4-D injured corn 26%, and yields were reduced 34% when compared to RT alone. Giant foxtail control from sethoxydim tank-mixed with bentazon plus atrazine with urea ammonium nitrate (UAN), or with ALS-inhibiting herbicides except halosulfuron-methyl in combination with 2,4-D was 24% lower when averaged over treatments. Yields of sethoxydim-resistant (SR) corn treated with sethoxydim mixed with combinations of sulfonylurea herbicides plus 2,4-D were low, with the exception of the combination halosulfuron-methyl with sethoxydim and 2,4-D. These studies indicate that thoroughly understanding postemergence (POST) corn herbicide tank mixtures is crucial for effective weed management. / Ph. D.

Rosebay rhododendron (Rhododendron maximum L.) is an evergreen ericaceous shrub that plays a dynamic role in the southern Appalachian forests. Commonly located on mesic sites, this understory shrub forms dense thickets that greatly reduce the amount of light available to herbaceous and woody plants found on the forest floor. Past research has shown that silvicultural methods can be used to eradicate R. maximum, however it is unclear which of these methods is most efficient and what effects other than stem mortality may occur. In this study, treatments involving prescribed fire, mechanical cutting, and herbicide applications were applied to R. maximum dominated forests in southwestern Virginia to determine what effect seven different silvicultural treatments had on 1) controlling of R. maximum as a forest weed 2) fuel loading inside of a R. maximum thicket, and 3) canopy tree seedling regeneration. Mechanical cutting treatments were successful in reducing R. maximum basal area per acre; however stump sprouting and increased fuel loading occurred. Herbicide applications were successful in controlling only the smallest diameter class of R. maximum stems. Prescribed fire reduced litter layers and caused delayed mortality on R. maximum stems three years following treatment. Hemispherical photographs taken within each plot showed that silvicultural treatments that successfully increased the amount of light entering each plot were influential in seedling establishment three years following treatments. Results from this study can be used to further perfect silvicultural applications that alleviate R. maximum cover on the forest landscape. / Master of Science

<p dir="ltr">Cover crops and soil residual herbicides are some of the essential tools within the integrated weed management. When used at cover crop termination, residual herbicides can extend the period of weed control and reduce the selection pressure for herbicide resistance. However, previous research has indicated that the use of cover crops can increase the microbial activity of the soil which, in turn, is the primary route for herbicide degradation in the soil. In addition to the potential effect on the microbial breakdown of herbicides, cover crops can also alter the fate of herbicides by interception. Three field projects were established to (1) investigate the influence of cover crop [cereal rye (<i>Secale cereale</i> L.) and crimson clover (<i>Trifolium incarnatum</i> L.)] use on soil enzyme activities [β-glucosidase (BG) and dehydrogenase (DHA)], its effect on the concentration of residual herbicides (sulfentrazone, <i>s</i>-metolachlor, cloransulam-methyl, atrazine, and mesotrione) in the soil, and the interception of herbicides by cover crop residue; (2) to investigate the effect of cover crop termination strategies (fallow, standing, and roller crimped 1 d prior to application) and simulated rainfall volumes (0, 4.2, and 8.3 mm simulated over 20 min; equivalent to 0, 12.5, and 25 mm h^-1) on atrazine wash off from cereal rye biomass onto the soil; (3) to investigate the effect of cereal rye termination strategies on the concentration of sulfentrazone, <i>s</i>-metolachlor, and cloransulam-methyl in the soil, weed control, and soybean [Glycine max (L.) Merr.] yield.</p><p dir="ltr">Results from the first project suggests that the use of cover crops occasionally resulted in increased BG and DHA activities relative to the fallow treatment. However, even when there was an increase in the activity of these two enzymes, increased degradation of the residual herbicides was not observed. Furthermore, the use of cereal rye or crimson clover as cover crops did not reduce the efficacy of the residual herbicides in controlling weeds early in the growing season.</p><p dir="ltr">In regards to the fate of atrazine after simulated rainfall, the results from the second project indicated that cereal rye biomass accumulation negatively impacted the amount of herbicide reaching the soil at the time of application. Although the roller crimped cereal rye reduced the amount of herbicide reaching the soil relative to the standing cereal rye, it also reduced atrazine leaching below the 0-5 cm of soil. Furthermore, in cover cropping systems with high levels of cereal rye biomass, more than 8.3 mm of rain are required to wash most of the atrazine off of the biomass.</p><p dir="ltr">Lastly, the adoption of the planting green systems resulted in up to 84% interception of residual herbicides by the roller crimped cereal rye biomass. The use of cereal rye as cover crop did not improve the weed control relative to the fallow during the two years of field research. Soybean yield losses ranged from 10 to 44% with the adoption of the planting green system, primarily due to stand losses. However, there are strategies and proper equipment available that can be used to minimize soybean stand losses.</p>

Rosebay rhododendron (Rhododendron maximum L.) is an ericaceous shrub commonly found in riparian areas of the Appalachian Mountains. After more than a century of fire exclusion in the U.S., the distribution of R. maximum and its dominance of forest understories have increased. Rhododendron expansion has caused a decline in overstory regeneration and the potential for dangerous fuel conditions around suburban structures near the wildland-urban interface. The purpose of this study was to determine the effects of seven silvicultural treatments on both the fuel loading within an R. maximum thicket and the control of R. maximum as a forest weed. The final objective of the project was to determine the cost effectiveness of each implemented treatment. Due primarily to moisture conditions, a single prescribed burn was relatively ineffective in reducing fuel loading and causing R. maximum mortality. Mechanical cutting caused a drastic shift in the size-class distribution of R. maximum but resulted in heavy sprouting and increased fuel loading. Herbicide application did not reduce or increase fuel loading and was important in R. maximum control only when combined with other treatments. The prescribed burning treatment was the least expensive individual treatment while mechanical cutting was the most expensive. Combination treatments showed increased effectiveness in controlling R. maximum but were more expensive than the individual treatments. The results of the treatments from this study will be used over the long term to demonstrate to land managers the effects of vegetation control on rhododendron. / Master of Science

The introduction of herbicide resistant crops offers producers many more options for weed control systems. These crops allow environmentally safe, non-selective herbicides to be used as selective herbicides, broadening the spectrum of weeds controlled, while not harming the crop. As these crops are very new on the market, investigation of their performance under various environmental conditions as well as in various weed control programs is needed. Liberty-link &reg; soybeans are resistant to the herbicide glufosinate, because of the incorporation of a gene encoding phosphinothricin acetyl-transferase (pat), which is able to detoxify glufosinate. Roundup-Ready &reg; soybeans are transformed with an altered, non-sensitive form of 5-enolpyruvylshikimate- 3-phosphate synthase (EPSPS), which confers glyphosate resistance. Field and greenhouse studies were conducted to determine the efficacy of glufosinate and glyphosate on annual and perennial weeds. Also to determine whether the use of ammonium sulfate (AMS) or pelargonic acid (PA), a 9-carbon fatty acid, as additives of glufosinate or glyphosate would increase their efficacy, while maintaining their safety on the transgenic soybeans. Three annual weeds: common lambsquarters, giant foxtail, sicklepod, as well as two perennial weeds: common milkweed and horsenettle were included in studies. Uptake, translocation, and metabolism of 14C-glufosinate + AMS or PA, were studied in the five weeds in order to determine the basis for their differential weed sensitivity to glufosinate, and the effect of the two additives. The effect of temperature on Liberty-Link &reg; and Roundup-Ready &reg; soybeans after application of glufosinate or glyphosate was investigated. Injury was quantified by measuring chlorophyll content of herbicide treated soybean trifoliolates. Uptake, translocation, and metabolism studies of 14C-glufosinate and 14C-glyphosate in transgenic soybeans were conducted to determine the potential cause for the observed temperature-dependent sensitivity. Since glufosinate is a synthetic analog of a naturally occurring bacterial toxin, it was tested for possible bactericidal activity on the soybean pathogen Pseudomonas syringae. Greenhouse and field-studies showed that the 5 weeds responded differently to glufosinate and glyphosate. Common milkweed was the most tolerant to glufosinate and common lambsquarters to glyphosate while giant foxtail was the most sensitive species to both herbicides. Some interactions between AMS or PA and glufosinate or glyphosate were also observed. Uptake and translocation studies showed that AMS increased the uptake of 14C-glufosinate in some weeds, whereas PA had only minimal effects on absorption and translocation of glufosinate. Metabolism of glufosinate was detected only in common lambsquarters. A rate dependent loss of chlorophyll in Liberty-Link &reg; soybeans treated with glufosinate was observed that was greater at 15° C than at 25° or 35° C. Metabolism studies showed a decrease in the rate of glufosinate metabolism 3 hours after treatment in Liberty-Link &reg; soybeans grown at 15° C versus 25° C. Conversely, chlorophyll loss in glyphosate-treated Roundup-Ready soybeans was greater at 35° C than at 15° or 25° C. Translocation studies showed a significantly greater percentage of absorbed 14C-glyphosate translocated to developing meristems at 35° C than at 15° C in Roundup-Ready&reg; soybeans. Glufosinate concentrations of 1 mM and higher significantly inhibited the growth of Pseudomonas syringae (L-529) in liquid media cultures. Typical field use rates of glufosinate also reduced the number of live P. syringae on Liberty-Link&reg; soybean leaves. Overall, the results of this research show that annual and perennial weeds differ in their sensitivity to glufosinate and glyphosate. Additives such as AMS and PA may enhance the efficacy of glufosinate on perennial weed species, and glyphosate in most weeds. Differences in weed sensitivity to herbicides and effects of additives can in most cases be explained by differences in absorption or metabolism. Variable temperatures may affect the engineered resistance of transgenic soybeans to the herbicides glufosinate and glyphosate. The herbicide glufosinate has some bacteriocidal activity on P. syringae. Nomenclature: Glufosinate, 2-amino-4-(hydroxymethylphosphinyl) butanoic acid; Glyphosate, N-(phosphonomethyl)glycine; PA, pelargonic acid (nanoic acid); AMS, ammonium sulfate; giant foxtail, Setaria faberi Herrm.; common lambsquarters, Chenopodium album L.; sicklepod, Cassia obtusifolia L.; horsenettle, Solanum carolinense L.; common milkweed, Ascleipias syriaca L. pat, phosphinothricin acetyl transferase; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase. / Master of Science

Chemical site preparation is used to enhance the productivity of loblolly pine plantations; however, it is most often combined with other methods and/or chemical release, and has been studied little on its own. Our study, conducted in the Virginia piedmont, compares the effects of various site preparation applications of imazapyr and their timings (July 23rd, September 3rd, and October 1st 2005) against a year-two chemical release (September 12th 2007), and an untreated check, all following a site preparation burn (June 15, 2005). Testing for additive effects, site preparation treatments were conducted with and without, sulfometuron methyl (SMAX) which targets herbaceous vegetation. Half of all treatments, including the check and release, received a year-one weed control treatment (H) of imazapyr and SMAX. Pines were measured every few years, including the latest measurement in year 14 (2019). Competing hardwood vegetation basal area was measured in year 14. All site preparation treatments reduced competition levels compared to the check, and an inverse relationship exists between competition level and pine volume. Adding SMAX resulted in less effective competition control, but had little effect on pine volumes. Adding H decreased average competition levels, and increased pine volumes. H treatments had the most effect at improving early season (July) applications. September and October treatments generally had more effect than July and the year-two release at controlling competing vegetation, and also showed higher individual pine tree volumes than all other treatments. Chemical site preparation treatments, even performed alone, appear to be valid means of improving pine stands. / Master of Science / Competing vegetation has a negative effect on planted pine production. Areas are often prepared with herbicides or mechanical removal of vegetation before crop-trees are planted (site preparation), or receive herbicide treatments after planting (release), to reduce levels of competition. Our study focuses on pre-planting herbicide (imazapyr with and without sulfometuron methyl) use at different timings (July 23rd, September 3rd, and October 1st). A chemical that targets herbaceous vegetation (sulfometuron methyl) was included in half of the pre-planting treatments, and half of all treatments, including the otherwise untreated check and year-two release, received an added weed control treatment in year-one. This was done to test for potential additive effects of chemical vegetation control options on pine productivity. September and October treatments have a greater effect at controlling competition 14 years since treatment and increasing pine volumes than the July, year-two release, and check. The added herbaceous control increased competition levels on average, without having an effect on the planted pine volumes. The year-one weed control decreased competition levels, and increased pine productivity, particularly within the July treatments. Overall, preparing sites with herbicide is beneficial to the pine stand, even when performed alone. Adding the herbaceous vegetation control is likely not necessary, and the extra year-one weed control may provide a boost to herbicide treatments applied earlier in the season.