Metabolic Pathways for S-Metolachlor Detoxification Differ Between Tolerant Corn and Multiple-Resistant Waterhemp.

Herbicide resistance in weeds can be conferred by target-site and/or non-target-site mechanisms, such as rapid metabolic detoxification. Resistance to the very-long-chain fatty acid-inhibiting herbicide, S-metolachlor, in multiple herbicide-resistant populations (CHR and SIR) of waterhemp (Amaranthus tuberculatus) is conferred by rapid metabolism compared with sensitive populations. However, enzymatic pathways for S-metolachlor metabolism in waterhemp are unknown. Enzyme assays using S-metolachlor were developed to determine the specific activities of glutathione S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) from CHR and SIR seedlings to compare with tolerant corn and sensitive waterhemp (WUS). GST activities were greater (∼2-fold) in CHR and SIR compared to WUS but much less than corn. In contrast, P450s in microsomal extracts from CHR and SIR formed O-demethylated S-metolachlor, and their NADPH-dependent specific activities were greater (>20-fold) than corn or WUS. Metabolite profiles of S-metolachlor generated via untargeted and targeted liquid chromatography-mass spectrometry from CHR and SIR differed from WUS, with greater relative abundances of O-demethylated S-metolachlor and O-demethylated S-metolachlor-glutathione conjugates formed by CHR and SIR. In summary, our results demonstrate that S-metolachlor metabolism in resistant waterhemp involves Phase I and Phase II metabolic activities acting in concert, but the initial O-demethylation reaction confers resistance.

Resistance to a nonselective 4-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide via novel reduction-dehydration-glutathione conjugation in Amaranthus tuberculatus.

Metabolic resistance to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides is a threat in controlling waterhemp (Amaranthus tuberculatus) in the USA. We investigated resistance mechanisms to syncarpic acid-3 (SA3), a nonselective, noncommercial HPPD-inhibiting herbicide metabolically robust to Phase I oxidation, in multiple-herbicide-resistant (MHR) waterhemp populations (SIR and NEB) and HPPD inhibitor-sensitive populations (ACR and SEN). Dose-response experiments with SA3 provided ED50 -based resistant : sensitive ratios of at least 18-fold. Metabolism experiments quantifying parent SA3 remaining in excised leaves during a time course indicated MHR populations displayed faster rates of SA3 metabolism compared to HPPD inhibitor-sensitive populations. SA3 metabolites were identified via LC-MS-based untargeted metabolomics in whole plants. A Phase I metabolite, likely generated by cytochrome P450-mediated alkyl hydroxylation, was detected but was not associated with resistance. A Phase I metabolite consistent with ketone reduction followed by water elimination was detected, creating a putative α,ß-unsaturated carbonyl resembling a Michael acceptor site. A Phase II glutathione-SA3 conjugate was associated with resistance. Our results revealed a novel reduction-dehydration-GSH conjugation detoxification mechanism. SA3 metabolism in MHR waterhemp is thus atypical compared to commercial HPPD-inhibiting herbicides. This previously uncharacterized detoxification mechanism presents a unique opportunity for future biorational design by blocking known sites of herbicide metabolism in weeds.

Quantifying resistance to very-long-chain fatty acid-inhibiting herbicides in Amaranthus tuberculatus using a soilless assay.

Resistance to preemergence (PRE) soil-applied herbicides, such as inhibitors of very-long-chain fatty acid (VLCFA) elongases, was documented in two waterhemp [Amaranthus tuberculatus (Moq.) J.D. Sauer] populations (SIR and CHR) from Illinois, USA. To limit the spread of resistant weed populations, rapid detection measures are necessary. Soil-based resistance assays are limited by edaphic factors, application timing, variable seeding depth and rainfall amount. Therefore, cost-effective techniques mitigating effects of edaphic factors that are appropriate for small- to large-scale assays are needed. Our research goal was to identify and quantify resistance to the VLCFA-inhibiting herbicides, S-metolachlor and pyroxasulfone, using a soilless greenhouse assay. Dose-response experiments were conducted under greenhouse conditions with pre-germinated waterhemp seeds planted on the vermiculite surface, which had been saturated with S-metolachlor (0.015-15 µM), pyroxasulfone (0.0005-1.5 µM), or S-metolachlor plus the cytochrome P450 (P450) inhibitor, malathion. Lethal dose estimates of 50% (LD50) and growth reduction of 50% (GR50) were calculated for S-metolachlor and pyroxasulfone PRE and used to determine resistance indices (RI) for resistant populations (CHR and SIR) relative to sensitive populations, SEN and ACR. RI values for S-metolachlor using LD50 values calculated relative to SEN and ACR were 17.2 and 15.2 (CHR) or 11.5 and 10.1 (SIR), while RI values for pyroxasulfone using LD50 values calculated relative to SEN and ACR were 3.8 and 3.1 (CHR) or 4.8 and 3.8 (SIR). Malathion decreased the GR50 of S-metolachlor to a greater degree in CHR compared to ACR, consistent with P450 involvement in S-metolachlor resistance in CHR. Results from these soilless assays are in accord with previous findings in soil-based systems that demonstrate CHR and SIR are resistant to S-metolachlor and pyroxasulfone. This method provides an effective, reproducible alternative to soil-based systems for studying suspected PRE herbicide-resistant populations and will potentially assist in identifying non-target-site resistance mechanisms.

Metabolic resistance to S-metolachlor in two waterhemp (Amaranthus tuberculatus) populations from Illinois, USA.

BACKGROUND: Two waterhemp (Amaranthus tuberculatus) populations from Illinois demonstrating multiple-resistance to acetolactate synthase (ALS)-, 4-hydroxyphenylpyruvate dioxygenase, and photosystem II (PSII)-inhibiting herbicides (designated CHR and SIR) also displayed reduced sensitivity to very-long-chain fatty acid-inhibiting herbicides, including S-metolachlor. We hypothesized that a physiological mechanism, such as enhanced metabolism, could be responsible for the reduced efficacy of S-metolachlor. RESULTS: Metabolism experiments indicated that resistant populations degraded S-metolachlor more rapidly than sensitive populations and equally as rapidly as corn 2-24 h after treatment (HAT). Resistant waterhemp and corn metabolized 90% (DT90 ) of absorbed S-metolachlor in less than 3.2 h whereas DT90 values for sensitive waterhemp exceeded 6 h. The glutathione S-transferase inhibitor 4-chloro-7-nitrobenzofurazon and cytochrome P450-inhibitor malathion decreased the amount of S-metolachlor metabolized in resistant waterhemp at 4 HAT but not in sensitive waterhemp or corn, and altered the abundance of certain metabolites in resistant waterhemp. CONCLUSION: Results from this research demonstrate that resistance to S-metolachlor in these waterhemp populations is due to enhanced herbicide metabolism relative to sensitive populations. In addition, our results indicate that resistant waterhemp might utilize metabolic pathway(s) more intricate than either sensitive waterhemp or corn based on their metabolite profiles. © 2020 Society of Chemical Industry.