Characterization of protoporphyrinogen oxidase (PPO) herbicide resistance in tall waterhemp (Amaranthus tuberculatus)

Abstract

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. 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), s-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 s-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 s-metolachlor. Additionally, heterozygosity of ∆G210 in surviving individuals did not change with any rate or combination of fomesafen and s-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 s-metolachlor and the combination of saflufenacil and s-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 s-metolachlor, which resulted in the lowest number of PPO-R tall waterhemp that survived herbicide treatment at the end of season. 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. 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.