Mechanism of Resistance to S-metolachlor in Palmer amaranth

Abstract

Herbicides are major tools for effective weed management. The evolution of resistance to herbicides in weedy species, especially contributed by non-target-site-based resistance (NTSR) is a worrisome issue in crop production globally. Glyphosate-resistant Palmer amaranth (Amaranthus palmeri) is one of the extremely difficult weeds in southern US crop production. In this study, we present the level and molecular basis of resistance to the chloroacetamide herbicide, S-metolachlor, in six field-evolved A. palmeri populations that had survivors at the recommended field-dose (1.1 kg ai ha^-1). These samples were collected in 2014 and 2015. The level of resistance was determined in dose-response assays. The effective dose for 50% control (ED₅₀) of the susceptible population was 27 g ai ha^-1, whereas the ED₅₀ of the resistant populations ranged from 88 to 785 g ai ha^-1. Therefore, A. palmeri resistance to S-metolachlor evolved in Arkansas as early as 2014. Metabolic-inhibitor and molecular assays indicated NTSR in these populations, mainly driven by GSTs. To understand the mechanism of resistance, selected candidate genes were analyzed in leaves and roots of survivors (with 1 × S-metolachlor). Expression analysis of the candidate genes showed that the primary site of S-metolachlor detoxification in A. palmeri is in the roots. Two GST genes, ApGSTU19 and ApGSTF8 were constitutively highly expressed in roots of all plants across all resistant populations tested. The expression of both GSTs increased further in survivors after treatment with S-metolachlor. The induction level of ApGSTF2 and ApGSTF2like by S-metolachlor differed among resistant populations. Overall, higher expression of ApGSTU19, ApGSTF8, ApGSTF2, and ApGSTF2like, which would lead to higher GST activity in roots, was strongly associated with the resistant phenotype. Phylogenetic relationship and analysis of substrate binding site of candidate genes suggested functional similarities with known metolachlor-detoxifying GSTs, effecting metabolic resistance to S-metolachlor in A. palmeri. Resistance is achieved by elevated baseline expression of these genes and further induction by S-metolachlor in resistant plants.

Keywords: GST; NTSR; Palmer amaranth; S-metolachlor; gene expression; resistance; tolerance.

Figure 1

Amaranthus palmeri survival (%) relative to non-treated plants in response to S-metolachlor. SS is susceptible standard; all others are resistant.

Figure 2

Effect of GST inhibitor, NBD-Cl, and S-metolachlor on root growth of Amaranthus palmeri in agar-based assays. 15CRI-A and 14CRI-G are resistant populations. NBD-Cl was used at 0.25 μM. Root lengths were measured at 14 days of incubation in a growth chamber set at 30/28°C day/night temperature. The experiment was conducted twice. Each data point is the average of 25–30 plants.

Figure 3

Effect of GST inhibitor, NBD-Cl, and S-metolachlor on seedling growth of Amaranthus palmeri in agar-based assays using a representative resistant [14CRI-G(A)] and susceptible [SS (B)] population. Root lengths were measured at 14 days of incubation in a growth chamber set at 30/28°C day/night temperature. The experiment was conducted twice. The upper and lower left panels of (A) and (B) are the non-treated and NBD-Cl treated checks of resistant and susceptible populations, respectively.

Figure 4

Expression profile of candidate GST genes in leaves (A) of S- metolachlor-resistant and -susceptible population of A. palmeri and roots (B). Each bar represents the relative expression (fold change) of each gene in non-treated and treated resistant plants compared to non-treated susceptible plants (SS). Data are means ± SE of two independent experiments consisting of three biological replicates except roots of treated plants.

Figure 5

Phylogenetic and active site analysis of selected GSTs from different species. The selected GSTs were analyzed from Arabidopsis thaliana, Zea mays, Lolium rigidum, Aloperucrus myosuroides, and Amaranthus palmeri. The evolutionary history was inferred using the Maximum Likelihood method and JTT matrix-based model (Jones et al., 1992). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 19 amino acid sequences, and the final dataset was composed of 285 positions. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018). Active site in candidate ApGSTU19 and ApGSTF8 was identified by comparing the sequence with Z. mays. Highlighted in yellow is the conserved and green is the polymorphic compared to Z. mays active site residue. Multiple sequence alignment was carried out using uniport align tool using ApGSTU19, ApGSTF8, ApGSTF2, and ApGSTF2like from A. palmeri, AtGSTU19 (AT1G78380), AtGSTF2 (AT4G02520), and AtGSTF8 (AT1G02920) in A. thaliana and ZmGST5 (CAA73369), ZmGST6 (CAB38120), ZMGST12 (AAG34820), and ZmGST13 in Z. mays (AAG34821).