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

Abstract

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.

Fig 1

Preemergence soil-applied herbicides, S-metolachlor (A) and pyroxasulfone (B).

Fig 2. Preemergence resistance identification method (PRIM) uses basic greenhouse supplies, access to greenhouse space and small amounts of chemicals.

Fig 3. Responses of four waterhemp (Amaranthus tuberculatus) populations to S-metolachlor applied preemergence.

S-metolachlor concentrations ranged from 0.015 to 15 μM. Seedlings are shown at 14 days after treatment (DAT). Non-treated inserts appear on the left for each population and treated inserts are arranged from left-to-right with increasing herbicide concentrations. Herbicide treatments using the PRIM assay are described in Materials and methods.

Fig 4. Quantitative survival analysis of CHR, SIR, ACR, and SEN populations in response to S-metolachlor.

Dose-response analysis of four waterhemp (Amaranthus tuberculatus) populations in herbicide-treated vermiculite using PRIM. Data were collected 14 days after treatment (DAT) by counting the number of surviving plants. Results are presented as a percentage of the untreated control for each population. Dose-response curves were fitted using the equation $y=\frac{d}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(LD50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, solid line and solid triangle; SIR, solid line and solid inverted triangle; ACR, solid line and solid circle; SEN, solid line and solid square.

Fig 5. Quantitative growth reduction analysis of CHR, SIR, ACR, and SEN populations in response to S-metolachlor.

Dose-response analysis of four waterhemp (Amaranthus tuberculatus) populations in herbicide-treated vermiculite using PRIM. Plants were harvested 14 days after treatment (DAT), dried in an oven, and aboveground dry biomass of surviving plants is expressed as a percentage of the untreated control. Dose-response curves were fitted using the equation $y=\frac{d-c}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(LD50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, discontinuous line and solid circle; SIR, discontinuous line and solid triangle; ACR, solid line and solid square; SEN, discontinuous line and solid diamond.

Fig 6. Responses of four waterhemp (Amaranthus tuberculatus) populations to pyroxasulfone applied preemergence.

Pyroxasulfone concentrations ranged from 0.0005 to 1.5 μM. Seedlings are shown at 14 days after treatment (DAT). Non-treated inserts appear on the left for each population and treated inserts are arranged from left-to-right with increasing herbicide concentrations. Herbicide treatments are described in Materials and Methods.

Fig 7. Quantitative survival analysis of CHR, SIR, ACR, and SEN populations in response to pyroxasulfone.

Dose-response analysis of four waterhemp (Amaranthus tuberculatus) populations in herbicide-treated vermiculite using PRIM. Data were collected 14 days after treatment (DAT) by counting the number of surviving plants. Results are presented as a percentage of the untreated control for each population. Dose-response curves were fitted using the equation $y=\frac{d}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(LD50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, discontinuous line and solid circle; SIR, discontinuous line and solid triangle; ACR, solid line and solid square; SEN, discontinuous line and solid diamond.

Fig 8. Quantitative growth reduction analysis of CHR, SIR, ACR, and SEN populations populations in response to pyroxasulfone.

Dose-response analysis of four waterhemp (Amaranthus tuberculatus) populations in herbicide-treated vermiculite using PRIM. Plants were harvested 14 days after treatment (DAT), dried in an oven, and aboveground dry biomass of surviving plants is expressed as a percentage of the untreated control. Dose-response curves were fitted using the equation $y=\frac{d}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(GR50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, discontinuous line and solid circle; SIR, discontinuous line and solid triangle; ACR, solid line and solid square; SEN, discontinuous line and solid diamond.

Fig 9. Quantitative survival analysis of CHR and ACR populations in response to S-metolachlor versus S-metolachlor plus malathion.

Dose-response analysis of two waterhemp (Amaranthus tuberculatus) populations in S-metolachlor-treated and S-metolachlor plus malathion-treated vermiculite using PRIM. Data were collected 14 days after treatment (DAT) by counting the number of surviving plants. Results are presented as a percentage of the untreated control for each population. Dose-response curves were fitted using the equation $y=\frac{d}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(LD50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, solid line and solid square; CHR+M, discontinuous line and hollow square; ACR, solid line and solid circle; ACR+M, discontinuous line and hollow circle. +M, treatment includes 2 μM malathion.

Fig 10. Quantitative growth reduction analysis of CHR and ACR populations in response to S-metolachlor versus S-metolachlor plus malathion.

Dose-response analysis of two waterhemp (Amaranthus tuberculatus) populations in S-metolachlor-treated and S-metolachlor plus malathion-treated vermiculite using PRIM. Plants were harvested 14 days after treatment (DAT), dried in an oven, and aboveground dry biomass of surviving plants is expressed as a percentage of the untreated control. Dose-response curves were fitted using the equation $y=\frac{d}{1+\exp \left\{b\right[\log \left(x\right)-\log \left(GR50)\right]\}}$ and each symbol’s error bar represents ±SE. CHR, solid line and solid square; CHR+M, discontinuous line and hollow square; ACR, solid line and solid circle; ACR+M, discontinuous line and hollow circle. +M, treatment includes 2 μM malathion.