The introduction of glyphosate-resistant (GR) crops revolutionized weed management; however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an Amaranthus hybridus population (GRH)—a superweed collected in a GR-soybean field from Cordoba, Argentina—as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more ¹⁴C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA¹⁰², GTC¹⁰³ and TCA¹⁰⁶ instead of ACA¹⁰², GCG¹⁰³, and CCA¹⁰⁶, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 Å (wild type) to 2.93 Å (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH A. hybridus population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance.

Amaranthus palmeri S. Watson is probably the worst glyphosate-resistant (GR) weed worldwide. The EPSPS (5-enolpyruvylshikimate-3-phosphate-synthase) gene amplification has been reported as the major target-site-resistance (TSR) mechanism conferring glyphosate resistance in this species. In this study, TSR and non-target-site-resistance (NTSR) mechanisms to glyphosate were investigated in a putative resistant A. palmeri population (GRP), harvested in a GR-soybean crop from Argentina. Glyphosate resistance was confirmed for the GRP population by dose-response assays. No evidence of TSR mechanisms as well as glyphosate metabolism was found in this population. Moreover, a susceptible population (GSP) that absorbed about 10% more herbicide than the GRP population was evaluated at different periods after treatment. The GSP population translocated about 20% more glyphosate to the remainder of the shoots and roots at 96 h after treatment than the control, while the GRP population retained 62% of herbicide in the treated leaves. This is the first case of glyphosate resistance in A. palmeri involving exclusively NTSR mechanisms.

Glyphosate retention, absorption and translocation with and without adjuvant were examined in Lolium rigidum and Conyza canadensis in greenhouse and laboratory settings to develop an understanding of the influence of the selected adjuvant on glyphosate activity. Tests on whole plants show that the dose of herbicide needed to reduce dry weight by 50% (GR₅₀) or plant survival (LD₅₀) decreases by mixing glyphosate and adjuvant to 22%–24% and 42%–44% for both populations of L. rigidum and C. canadensis, respectively. This improvement in efficacy could be attributed to the higher herbicide retention and lower contact angle of the glyphosate + adjuvant drops on the leaf surface compared to the glyphosate solution alone. Plants of both species treated with ¹⁴C-glyphosate + adjuvant absorbed more glyphosate compared to non-adjuvant addition. Furthermore, the movement of the herbicide through the plant was faster and greater with the adjuvant. Our results reveal that the use of adjuvants improves the effectiveness of glyphosate in two of the most important weeds in agricultural crops in Mediterranean countries.

Echinochloa spp. is one of the most invasive weeds in rice fields worldwide. Acetolactate synthase (ALS) and acetyl-CoA carboxylase (ACCase) inhibiting herbicides are two of the most widely used rice herbicides. However, overuse has led to the resistance evolution of Echinochloa spp. to penoxsulam (ALS-inhibitor) and cyhalofop-methyl (ACCase-inhibitor). In this work, 137 different Echinochloa spp. populations were collected in different rice fields in Extremadura (western Spain) where lack of control was detected. Target-site based resistance (by sequencing ALS and ACCase gene) and characterization of Echinochloa species at the molecular level (based on PCR-RFLP analyses) were carried out in those populations. Most of the populations studied (111 of 137) belong to the E. oryzicola/E. oryzoides group. Three-point mutations were identified in ALS genes: Pro197Ser, Pro197Thr, and Ser653Asn, the first being the most frequent substitution in resistant plants. In the ACCase gene, the Ile1781Leu substitution was found. In both ALS and ACCase sequencing, evidence of heterozygosity was also observed. To assess whether cross-resistance patterns differed between mutations, two populations belonging to the E. oryzicola/E. oryzoides group had its most frequent mutations (Pro197Ser, population ech3-14 and Ile1781Leu, population ech114-10) chosen to be carried out in a dose-response assay. It was confirmed that Pro197Ser conferred resistance to triazolopyrimidine, imidazolinone, sulfonylurea, and pyrimidinyl benzoate families. On the other hand, the Ile1781Leu change gave resistance to aryloxyphenoxypropionate and cyclohexanedione families. Of the authorized herbicides in rice in Spain, more that 80% belong to these families. It is therefore important that farmers carry out an integrated control system that combines both chemical and non-chemical tools.

Six Johnsongrass populations suspected of being glyphosate resistant were collected from railways and freeways near Cordoba (SW Spain), where glyphosate is the main weed control tool. The 50% reduction in shoot fresh weight (GR₅₀) values obtained for these six populations ranged from 550.4 to 1169 g ae ha⁻¹, which were 4.2 to 9 times greater than the value obtained for the susceptible population. Glyphosate was equally metabolized to the same extent in both resistant and susceptible populations, with no significant differences in either 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibition or basal activity. No amino acid substitutions were observed in any of the resistant populations. Slight but significant differences in glyphosate penetration were observed among some but not all of the resistant populations and for the times of incubation assayed, although these differences were not considered further. The proposed primary mechanism of resistance in these six glyphosate-resistant Johnsongrass populations is reduced herbicide translocation, because the amount of glyphosate that translocated from treated leaves to shoots and roots in the susceptible population was double that observed in the resistant populations. As glyphosate multiple resistance due to more than one mechanism is not uncommon, this is the first time that glyphosate-resistant Johnsongrass populations have been fully described for all known mechanisms.