Resistance to ALS-inhibiting herbicides has dramatically increased worldwide due to the persisting evolution of target site mutations that reduce the affinity between the herbicide and the target. We evaluated the effect of the well-known ALS Asp-376-Glu target site mutation on different imidazolinone herbicides, including imazamox and imazethapyr. Greenhouse dose response experiments indicate that the Amaranthus retroflexus biotype carrying Asp-376-Glu was fully controlled by applying the field recommended dose of imazamox, whereas it displayed high level of resistance to imazethapyr. Likewise, Sorghum halepense, carrying Asp-376-Glu showed resistance to field recommended doses of imazethapyr but not of imazamox. Biochemical inhibition and kinetic characterization of the Asp-376-Glu mutant enzyme heterologously expressed using different plant sequence backbones, indicate that the Asp-376-Glu shows high level of insensitivity to imazethapyr but not to imazamox, corroborating the greenhouse results. Docking simulations revealed that imazamox can still inhibit the Asp-376-Glu mutant enzyme through a chalcogen interaction between the oxygen of the ligand and the sulfur atom of the ALS Met200, while imazethapyr does not create such interaction. These results explain the different sensitivity of the Asp-376-Glu mutation towards imidazolinone herbicides, thus providing novel information that can be exploited for defining stewardship guidelines to manage fields infested by weeds harboring the Asp-376-Glu mutation.

The sustainability of rice cropping systems is jeopardized by the large number and variety of populations of polyploid Echinochloa spp. resistant to ALS inhibitors. Better knowledge of the Echinochloa species present in Italian rice fields and the study of ALS genes involved in target-site resistance could significantly contribute to a better understanding of resistance evolution and management. Using a CAPS-rbcL molecular marker, two species, E. crus-galli (L.) P. Beauv. and E. oryzicola (Vasinger) Vasing., were identified as the most common species in rice in Italy. Mutations involved in ALS inhibitor resistance in the different species were identified and associated with the ALS homoeologs. The relative expression of the ALS gene copies was evaluated. Molecular characterization led to the identification of three ALS genes in E. crus-galli and two in E. oryzicola. The two species also carried different point mutations conferring resistance: Ala122Asn in E. crus-galli and Trp574Leu in E. oryzicola. Mutations were carried in the same gene copy (ALS1), which was significantly more expressed than the other copies (ALS2 and ALS3) in both species. These results explain the high resistance level of these populations and why mutations in the other ALS copies are not involved in herbicide resistance.

Annual ryegrass species (Lolium spp.) infest cereal crops worldwide. Ryegrass populations with multiple resistance to the acetyl coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors are an increasing problem in several European countries. We investigated the resistance pattern and level of resistance in ryegrass populations collected in Denmark, Greece and Italy and studied the diversity of mechanisms endowing resistance, both target-site and metabolism based. All populations showed high resistance indexes (RI) to the ALS inhibitors, iodosufuron-methyl-sodium + mesosulfuron-methyl (RI from 8 to 70), whereas the responses to the two ACCase inhibitors, clodinafop-propargyl and pinoxaden, differed. The Greek and Italian populations were moderately to highly resistant to clodinafop (RI > 8) and showed low to moderate resistance to pinoxaden (RI ranged from 3 to 13) except for one Italian population. In contrast, the Danish Lolium populations showed low to moderate resistance to clodinafop (RI ranged from 2 to 7) and only one population was resistant to pinoxaden. Different mutant ACCase alleles (Leu₁₇₈₁, Cys₂₀₂₇, Asn₂₀₄₁, Val₂₀₄₁, Gly₂₀₇₈, Arg₂₀₈₈, Ala₂₀₉₆) and ALS alleles (Gly₁₂₂, Ala₁₉₇, Gln₁₉₇, Leu₁₉₇, Ser₁₉₇, Thr₁₉₇, Val₂₀₅, Asn₃₇₆, Glu₃₇₆, Leu₅₇₄) endowing resistance were detected in the Greek and Italian populations. In several plants, no mutated ALS and ACCase alleles were found showing a great heterogeneity within and among the Greek and Italian populations. Conversely, no mutant ACCase alleles were identified in the four Danish populations and only one mutant ALS allele (Leu₅₇₄) was detected in two Danish populations. The expression level of nitronate monooxygenase (NMO), glutathione S-transferase (GST) and cytochrome P450s (CYP72A1 and CYP72A2) varied broadly among populations and individual plants within the populations. Constitutive up-regulation of GST, CYP72A1 and CYP72A2 was detected in resistant plants respect to susceptible plants in one Danish and one Italian population. It appears that the mechanisms underlying resistance are rather complex and diversified among Lolium spp. populations from the three countries, coevolution of both target-site resistance and metabolic based herbicide resistance appears to be a common feature in Denmark and Italy. This must be considered and carefully evaluated in adopting resistance management strategies to control Lolium spp. in cereal crops.

Amaranthus palmeri S. Watson (Amaranthaceae Juss.) is a dioecious noxious weed, native to the Americas, which infests summer crops. It causes high crop losses, and rapidly evolves resistance to herbicides. In Europe, A. palmeri was recorded mostly as a casual alien, but in 2018 it was reported infesting a soybean field in Italy, and the next year two more populations were found in the same area. Experiments were conducted on these three populations to evaluate the resistance to ALS-inhibiting herbicides, to determine the main resistance mechanisms involved and assess the efficacy of alternative herbicides with different sites of action than ALS. The three populations were confirmed cross-resistant to ALS-inhibiting herbicides (thifensulfuron-methyl and imazamox). Gene sequencing identified a Trp to Leu substitution at position 574 of ALS gene in resistant plants, proving that the main resistance mechanism for the three populations is target-site related. The presence of other resistance mechanisms cannot be excluded. Metobromuron, metribuzin and glyphosate are still effective on these populations.

Glyphosate-resistant biotypes of Eleusine indica (L.) Gaertn. have been detected in Asia, the Americas but not in Europe. The aim of this study was to evaluate resistance levels and possible target site resistance mechanisms of an E. indica biotype (19-1) collected from a plant nursery in Southern Italy where poor glyphosate efficacy was reported. Two dose-response experiments were conducted to evaluate the sensitivity of biotype 19-1 to glyphosate in comparison with two susceptible checks. 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) sequencing was performed to identify possible mutations conferring the resistance. The susceptible biotypes were completely controlled at the glyphosate recommended field dose of 360 g ae ha⁻¹, while 50% of the plants of biotype 19-1 survived at 1440 g ae ha⁻¹. The resistance index of biotype 19-1 ranged between 5.8 and 7.3 for the response variables of fresh weight reduction and plant survival, respectively. All the plants surviving glyphosate application and sampled for DNA analyses had the point mutation P106A. The biotype 19-1 can be confirmed as glyphosate-resistant, representing the first glyphosate-resistant population of E. indica in Europe.