Phenotypic and biochemical characterization of atrazine and HPPD-resistant waterhemp (amaranthus tuberculatus) populations

Waterhemp (Amaranthus tuberculatus) is a dioecious, summer annual, broadleaf species that is native to the Midwest. Two decades ago it was not considered a major agricultural pest, but with the adoption of glyphosate-only postemergence (POST) herbicide programs without soil residual activity combined with no-till practices, it has become one of the most important weeds in Illinois and across the Midwest in regards to corn and soybean production. Research has indicated that multiple herbicide resistances can be found within one waterhemp plant, with one population in Champaign Co., IL exhibiting resistance to five different herbicide sites of action. Concerns exist about how to control waterhemp, especially as more populations are found to be resistant to most herbicides farmers use today. Chapter 1 of this thesis includes a literature review of waterhemp biology, the history and use of atrazine, a photosystem II inhibitor, and carotenoid biosynthesis inhibitors, as well as the synergistic activity that has been observed when these two classes of herbicides are combined in a tank mixture. Chapter 2 covers previous research indicating that two atrazine-resistant populations of waterhemp (Amaranthus tuberculatus) from Illinois (designated ACR and MCR) displayed enhanced rates of atrazine metabolism via glutathione conjugation. Elevated constitutive expression levels of a single phi-class GST, named AtuGSTF2, correlated with atrazine resistance in ACR and MCR populations. Using this information, a discriminatory rate of 14.4 kg/ha was determined, and a POST study was conducted in the greenhouse to phenotype a segregating F2 population (derived from an MCR x WCS cross). Genotypes falling into three distinct categories (RR, Rr, or rr) were tentatively assigned based on varying phenotypic responses. Basal AtuGSTF2 expression levels were quantified and compared via RT-qPCR. Results indicated that each atrazine-resistant line (RR and Rr) tested displayed high AtuGSTF2 expression levels, ranging from 200- to 1140-fold greater than the low baseline levels detected in atrazine-sensitive lines (rr). Sequence analysis of RT-PCR products revealed several putative allelic variants of the AtuGSTF2 gene among F2 lines and their parent populations. These results demonstrate that constitutive AtuGSTF2 expression correlates strongly with phenotype and may therefore represent the predominant GST that confers atrazine resistance in ACR and MCR. Chapter 3 provides research that was conducted to explore the options for control of two HPPD- and atrazine-resistant waterhemp populations, SIR and NEB. The first objective was to determine the level of resistance to two HPPD-inhibiting herbicides; one that both populations had been exposed to previously (mesotrione) and another that had not been applied to either population (isoxaflutole). A dose-response experiment was conducted in the greenhouse at two different postemergence (POST) timings. Overall our findings did not indicate a consistent pattern in fold-resistance levels to isoxaflutole in taller plants. However, mesotrione applied POST to SIR showed a clear decrease in fold-resistance levels relative to EPOST. To further investigate potential management strategies of HPPD- and atrazine-resistant waterhemp populations in the field, our second objective was to conduct a POST herbicide interaction study and evaluate combinations of metribuzin and either isoxaflutole or mesotrione. This objective was designed to test two interconnected hypotheses: (1) HPPD-inhibitor activity contributes to synergism in a tank mix with metribuzin, and (2) metabolic atrazine resistance can be overcome by using a different PSII inhibitor (metribuzin). Results indicated that mesotrione at 52.5 g ai ha-1 combined with 191 g ai ha-1 of metribuzin displayed a synergistic effect on biomass reduction in SIR plants. However, all other combinations of either mesotrione or isoxaflutole and metribuzin resulted in an additive effect on biomass reduction in both the SIR and the NEB populations. These results give insight into how the joint activity between HPPD- and PSII-inhibitors can be used to control metabolism-based, multiple herbicide-resistant waterhemp populations. Chapter 4 summarizes the discussion and conclusions from Chapters 2 and 3 and identifies current limitations and future research goals for utilizing the herbicides we currently have available to control waterhemp, as herbicide resistance continues to evolve and no new modes of action are coming to market