Resistance to Amino Acid Biosynthesis Inhibiting-Herbicides in Amaranthus palmeri Populations from Aragon (Spain)

Abstract

Amaranthus palmeri is a highly problematic agricultural weed due to its rapid growth, high seed production, and strong tendency to develop herbicide resistance. In Spain, the initial colonization of A. palmeri began in 2007, when populations were detected at various locations in the province of Lleida (Catalonia). Since then, new infestations have been reported in other regions of the country, primarily infesting maize fields. Although resistance to glyphosate or to acetolactate synthase (ALS) inhibitors has been documented in several populations from Catalonia and Extremadura, little is known about the resistance profile of populations from Aragon. The main objective of this study was to characterize the putative resistance of five populations from Aragon to 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (glyphosate) and ALS inhibitors (nicosulfuron and imazamox). Sensitivity to both mechanisms of action was measured by root growth in vertical plates and shikimate accumulation for glyphosate. Target-site resistance was evaluated by analyzing EPSPS and ALS gene copy numbers and ALS gene mutations. The populations showed high variability, with no multiple resistance detected. The Bujaraloz population showed moderate resistance to glyphosate due to EPSPS gene amplification. In three populations, mutations in the ALS gene conferring resistance were detected. The Trp574Leu mutation was detected in approximately half of the individuals from the Albelda, Tamarite de Litera, and Caspe populations. In the latter, the Pro197Thr mutation was also present. This study reveals significant genetic variability within each population and provides evidence for the spread of herbicide resistance across different regions of Spain.

Keywords: ALS mutation; EPSPS gene amplification; glyphosate; imazamox; nicosulfuron; target-site.

Figure 1

Root length in populations A, B, C, and R of Amaranthus palmeri from Aragon. Box plots show 1st, 2nd, and 3rd quartiles; the minimum and maximum; and the mean (red line) (n = 15–20). Root length (Y axis) is expressed as percentage of non-treated samples of each population. For each population, significant differences between treated roots (0.4 mg L⁻¹ of glyphosate) and non-treated are highlighted with * (Student’s t test; p value < 0.05).

Figure 2

Visual appearance of the five Amaranthus palmeri populations (A, B, C, D, and R) 5 days after the treatment: non-treated (control) and glyphosate treatment at the recommended field rate (1×; 0.84 kg a.e. ha⁻¹) and at three times the recommended field rate (3×; 2.52 kg a.e. ha⁻¹). For each treatment, 12 plants were used.

Figure 3

Shikimate content in leaves of populations A, B, C, D, and R. Plants were untreated (control, 0) or sampled 5 days after treatment with glyphosate, applied at 1× (0.84 kg a.e. ha⁻¹) and 3× (2.52 kg a.e. ha⁻¹) the field rate. Box plots show 1st, 2nd, and 3rd quartiles; the minimum and maximum; and the mean (red line) (n =4). In each population, different letters indicate significant differences between treatments (ANOVA; HSD Tukey; p value < 0.05).

Figure 4

(A) Amaranthus palmeri genomic copy number of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) relative to carbamoylphosphate synthetase (CPS), expressed as 2^ΔCt in the populations of Aragon: A, B, C, D, and R. Box plots show 1st, 2nd, and 3rd quartiles; the minimum and maximum; and the mean (red line) (n = 6–12). Different letters indicate significant differences between treatments (ANOVA; HSD Tukey; p value < 0.05). (B) Correlation between genomic copy number of EPSPS and shikimate accumulation in treated individuals of all populations (Pop). Shikimate accumulation was measured after five days of treatment with 1× (0.84 kg a.e. ha⁻¹) or 3× (2.52 kg a.e. ha⁻¹) glyphosate field rates.

Figure 5

Root length in populations A, B, C, and R of Amaranthus palmeri from Aragon, treated with nicosulfuron (A) or imazamox (B). Box plots show 1st, 2nd, and 3rd quartiles; the minimum and maximum; and the mean (red line) (n = 15–20). Root length (Y axis) is expressed as percentage of non-treated samples of each population. For each population, significant differences between treated and non-treated samples are highlighted with * (Student’s t test; p value < 0.05). Representative response of each population is shown (top of the figure).

Figure 6

Amaranthus palmeri genomic copy number of acetolactate synthase (ALS) relative to carbamoylphosphate synthetase (CPS) in populations of Aragon: A, B, C, D, and R. Box plots show 1st, 2nd, and 3rd quartiles; the minimum and maximum; and the mean (red line) (n = 6–10). No significant differences in ALS gene copy numbers were detected among populations (ANOVA; p value < 0.05).