Can allele-specific loop-mediated isothermal amplification be used for rapid detection of target-site herbicide resistance in Lolium spp.?

BACKGROUND: Herbicide resistance is one of the threats to modern agriculture and its early detection is one of the most effective components for sustainable resistance management strategies. Many techniques have been used for target-site-resistance detection. Allele-Specific Loop-Mediated Isothermal Amplification (AS-LAMP) was evaluated as a possible rapid diagnostic method for acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibiting herbicides resistance in Lolium spp. RESULTS: AS-LAMP protocols were set up for the most frequent mutations responsible for herbicide resistance to ALS (positions 197, 376 and 574) and ACCase (positions 1781, 2041 and 2078) inhibitors in previously characterized and genotyped Lolium spp. POPULATIONS: A validation step on new putative resistant populations gave the overview of a possible use of this tool for herbicide resistance diagnosis in Lolium spp. Regarding the ACCase inhibitor pinoxaden, in more than 65% of the analysed plants, the LAMP assay and genotyping were in keeping, whereas the results were not consistent when ALS inhibitors resistance was considered. Limitations on the use of this technique for herbicide resistance detection in the allogamous Lolium spp. are discussed. CONCLUSIONS: The LAMP method used for the detection of target-site resistance in weed species could be applicable with target genes that do not have high genetic variability, such as ACCase gene in Lolium spp.

Diversity and Spread of Acetolactate Synthase Allelic Variants at Position 574 Endowing Resistance in Amaranthus hybridus in Italy.

Poor control of Amaranthus spp. with herbicides inhibiting acetolactate synthase (ALS) has been observed for several years in soybean fields in north-eastern Italy, but to date only a few ALS-resistant populations have been confirmed. An extensive sampling of putatively resistant Amaranthus accessions was completed in the Friuli Venezia Giulia region, across an arable land area of about 3000 km2. In total, 58 accessions were tested to confirm their resistance status, recognize the Amaranthus species, identify the mutant ALS alleles endowing the resistance and determine the efficacy of 3 pre-emergence herbicides. Most accessions resulted in cross-resistance to thifensulfuron-methyl and imazamox. Genomic DNA were extracted from single seeds with a newly developed protocol; an allele-specific PCR assay revealed the presence of the 574-leucine in 20 accessions, of the 574-methionine in 22, and of both alleles in 9 accessions. The two variants showed a different spatial distribution. All resistant populations were ascribed to A. hybridus. A. hybridus resistant to ALS herbicides is well-established in this Italian region and its resistance is due to two ALS mutant alleles. Metribuzin, clomazone and metobromuron can be used as alternative herbicides to be applied in pre-emergence and they should be integrated into the management strategies to limit the spread of resistance.

Target-Site Mutations and Expression of ALS Gene Copies Vary According to Echinochloa Species.

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.

Population structure and evolution of resistance to acetolactate synthase (ALS)-inhibitors in Amaranthus tuberculatus in Italy.

BACKGROUND: Before 2010, Amaranthus tuberculatus (Moq.) J. D. Sauer was barely known to farmers and stakeholders in Italy. Since then, several populations resistant to acetolactate synthase (ALS)-inhibiting herbicides have been collected. In most populations, a known target site resistance-endowing mutation was found, a Trp to Leu substitution at position 574 of the ALS gene, but it was unclear whether they had evolved resistance independently or not. The aims of the work were (i) to elucidate the population structure of Italian ALS-resistant A. tuberculatus populations, and (ii) to analyze the ALS haplotypes of the various populations to determine whether resistance arose multiple times independently. RESULTS: In order to determine the population structure of eight A. tuberculatus populations, eight previously described microsatellite loci were used. Two ancestors were found: three populations derived from one, and five from the other. In the 4-kb ALS region of the genome, including the 2-kb coding region, 389 single nucleotide polymorphisms were found. In silico haplotype estimation was used to reconstruct the sequence of three distinct haplotypes carrying the Trp574Leu mutation. In addition, no mutation was found in 83% of plants of a single population. CONCLUSIONS: (i) Resistance must have arisen independently at least three times; (ii) at least one population was already resistant to ALS inhibitors when introduced in Italy; (iii) a single haplotype with a Trp574Leu mutation was shared among six populations, probably because of broad seed dispersal; and (iv) one population likely evolved nontarget site ALS inhibitors resistance. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sensitivity Analysis of Italian Lolium spp. to Glyphosate in Agricultural Environments.

Empirical observations generally indicate a shifting and decreased Lolium spp. susceptibility to glyphosate in Italy. This is likely due to the long history of glyphosate use and to the sub-lethal doses commonly used. There is, therefore, a need to determine the variability of response of Lolium spp. to glyphosate and identify the optimum field dose. To perform a sensitivity analysis on Lolium spp. populations in an agriculture area, collection sites were mainly chosen where glyphosate had not been applied intensely. Known glyphosate-resistant or in-shifting populations were included. Two outdoor dose-response pot experiments, including eleven doses of glyphosate, were conducted. The dose to control at least 93%-95% of susceptible Lolium spp. was around 450 g a.e. ha-1. However, to preserve its efficacy in the long term, it would be desirable not to have survivors, and this was reached at a glyphosate dose of 560 ± 88 g a.e. ha-1. Taking into account the variability of response among populations, it was established that the optimal dose of glyphosate to control Lolium spp. in Italy up to the stage BBCH 21 has to be at least 700 g a.e. ha-1. As a consequence, it is recommended to increase the label recommended field rate for Lolium spp. control in Italy to a minimum of 720 g a.e. ha-1.

Diversified Resistance Mechanisms in Multi-Resistant Lolium spp. in Three European Countries.

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.

A family affair: resistance mechanism and alternative control of three Amaranthus species resistant to acetolactate synthase inhibitors in Italy.

BACKGROUND: Several soybean fields in Italy were found to be infested by multiple species of Amaranthus spp. not adequately controlled by acetolactate (ALS) inhibitor herbicides. The objectives of this research were (i) to create a simplified botanical key to identify weedy amaranths; (ii) to determine the number and type of sites of action the accession are resistant to, i.e. resistance pattern; and (iii) to determine the main resistance mechanisms involved d) to evaluate the efficacy of herbicides with different site of action. RESULTS: An easy-to-use botanical key was devised and successfully used in the infested sites and results were confirmed through a species-specific molecular marker. Amaranthus retroflexus L. (redrood pigweed) was found in three sites; plants with Asp376 Glu substitution at the ALS gene were resistant to thifensulfuron-methyl. Amaranthus tuberculatus (Moq.) J.D.Sauer (waterhemp) and Amaranthus hybridus L. (smooth pigweed) accessions were cross-resistant to thifensulfuron-methyl and imazamox; most ALS-resistant plants had a point mutation at position 574. One A. hybridus accession had the substitution Trp574 Met, new for Amaranthus genus. All ALS-resistant accessions were controlled by glyphosate and metribuzin. A. retroflexus accessions were controlled by bentazon, instead an A. hybridus and some A. tuberculatus accession were not. CONCLUSIONS: The simplified botanical key proposed herein could be a useful tool for farmers and weed scientists to reliably identify Amaranthus species in the field. The main resistance mechanism in the three Amaranthus species is target-site mediated. This is the first evidence of ALS-resistant A. tuberculatus outside its native North American range. © 2019 Society of Chemical Industry.

A New Ala-122-Asn Amino Acid Change Confers Decreased Fitness to ALS-Resistant Echinochloa crus-galli.

Gene mutations conferring herbicide resistance may cause pleiotropic effects on plant fitness. Knowledge of these effects is important for managing the evolution of herbicide-resistant weeds. An Echinochloa crus-galli population resistant to acetolactate synthase (ALS) herbicides was collected in a maize field in north-eastern Italy and the cross-resistance pattern, resistance mechanism and fitness costs associated to mutant-resistant plants under field conditions in the presence or absence of intra-specific competition were determined. The study reports for the first time the Ala-122-Asn amino-acid change in the ALS gene that confers high levels of cross-resistance to all ALS inhibitors tested. Results of 3-year growth analysis showed that mutant resistant E. crus-galli plants had a delayed development in comparison with susceptible plants and this was registered in both competitive (3, 7, and 20 plants m-2) and non-competitive (spaced plants) situations. The number of panicles produced by resistant plants was also lower (about 40% fewer panicles) than susceptible plants under no-intraspecific competition. Instead, with the increasing competition level, the difference in panicle production at harvest time decreased until it became negligible at 20 plants m-2. Evaluation of total dry biomass as well as biomass allocation in vegetative parts did not highlight any difference between resistant and susceptible plants. Instead, panicle dry weight was higher in susceptible plants indicating that they allocated more biomass than resistant ones to the reproductive organs, especially in no-competition and in competition situations at lower plant densities. The different fitness between resistant and susceptible phenotypes suggests that keeping the infestation density as low as possible can increase the reproduction success of the susceptible phenotype and therefore contribute to lowering the ratio between resistant and susceptible alleles. If adequately embedded in a medium or long-term integrated weed management strategy, the presence of R plants with a fitness penalty provides an opportunity to minimize or reverse herbicide resistance evolution through the implementation of integrated weed management, i.e., all possible control tools available.

Occurrence of Different Resistance Mechanisms to Acetolactate Synthase Inhibitors in European Sorghum halepense.

Four Hungarian and two Italian Sorghum halepense populations harvested in maize fields were investigated to elucidate the levels and mechanisms underlying acetolactate synthase (ALS) inhibitors resistance. The two Italian populations were highly cross-resistant to all ALS inhibitors tested, and the variant ALS allele Leu574 was identified in most of the plants; no differences were observed when the plants were treated with herbicide plus malathion. This suggests that the main resistance mechanism is target-site mediated. The Hungarian populations proved to be controlled by imazamox, while they were resistant to sulfonylureas and bispyribac-Na. All Hungarian populations, but not all plants of population 12-49H, presented the variant allele Glu376. This is the first documented occurrence of the Asp-376-Glu substitution in S. halepense. ALS enzyme bioassay and treatment with malathion confirmed that at least in plants of two populations the resistance is very likely due to both target-site and enhanced metabolism of P450 enzymes.

On-farm evaluation of inundative biological control of Ostrinia nubilalis (Lepidoptera: Crambidae) by Trichogramma brassicae (Hymenoptera: Trichogrammatidae) in three European maize-producing regions.

BACKGROUND: A 2 year study was conducted to evaluate the efficacy of biological control with optimally timed Trichogramma brassicae releases as an integrated pest management tool against the European corn borer (ECB), Ostrinia nubilalis (Hübner), in on-farm experiments (i.e. real field conditions) in three European regions with dissimilar geoclimatic conditions and ECB pressure and conventional management (i.e. insecticide treated and untreated). RESULTS: Biological control with Trichogramma (1) provided ECB protection comparable with conventional management, (2) in all cases maintained mycotoxin levels below the EU threshold for maize raw materials destined for food products, (3) was economically sustainable in southern France and northern Italy, but not in Slovenia where it resulted in a significant decrease in gross margin, mainly owing to the cost of Trichogramma product, and (4) enabled avoidance of detrimental environmental effects of lambda-cyhalothrin use in northern Italy. CONCLUSION: Optimally timed mass release of T. brassicae could be considered a sustainable tool for IPM programmes against ECB in southern France and northern Italy. Better involvement of regional advisory services is needed for the successful dissemination and implementation of biological control. Subsidy schemes could also motivate farmers to adopt this IPM tool and compensate for high costs of Trichogramma product.

iMAR: An Interactive Web-Based Application for Mapping Herbicide Resistant Weeds.

Herbicides are the major weed control tool in most cropping systems worldwide. However, the high reliance on herbicides has led to environmental issues as well as to the evolution of herbicide-resistant biotypes. Resistance is a major concern in modern agriculture and early detection of resistant biotypes is therefore crucial for its management and prevention. In this context, a timely update of resistance biotypes distribution is fundamental to devise and implement efficient resistance management strategies. Here we present an innovative web-based application called iMAR (interactive MApping of Resistance) for the mapping of herbicide resistant biotypes. It is based on open source software tools and translates into maps the data reported in the GIRE (Italian herbicide resistance working group) database of herbicide resistance at national level. iMAR allows an automatic, easy and cost-effective updating of the maps a nd provides two different systems, "static" and "dynamic". In the first one, the user choices are guided by a hierarchical tree menu, whereas the latter is more flexible and includes a multiple choice criteria (type of resistance, weed species, region, cropping systems) that permits customized maps to be created. The generated information can be useful to various stakeholders who are involved in weed resistance management: farmers, advisors, national and local decision makers as well as the agrochemical industry. iMAR is freely available, and the system has the potential to handle large datasets and to be used for other purposes with geographical implications, such as the mapping of invasive plants or pests.

Protocols for Robust Herbicide Resistance Testing in Different Weed Species.

Robust protocols to test putative herbicide resistant weed populations at whole plant level are essential to confirm the resistance status. The presented protocols, based on whole-plant bioassays performed in a greenhouse, can be readily adapted to a wide range of weed species and herbicides through appropriate variants. Seed samples from plants that survived a field herbicide treatment are collected and stored dry at low temperature until used. Germination methods differ according to weed species and seed dormancy type. Seedlings at similar growth stage are transplanted and maintained in the greenhouse under appropriate conditions until plants have reached the right growth stage for herbicide treatment. Accuracy is required to prepare the herbicide solution to avoid unverifiable mistakes. Other critical steps such as the application volume and spray speed are also evaluated. The advantages of this protocol, compared to others based on whole plant bioassays using one herbicide dose, are related to the higher reliability and the possibility of inferring the resistance level. Quicker and less expensive in vivo or in vitro diagnostic screening tests have been proposed (Petri dish bioassays, spectrophotometric tests), but they provide only qualitative information and their widespread use is hindered by the laborious set-up that some species may require. For routine resistance testing, the proposed whole plant bioassay can be applied at only one herbicide dose, so reducing the costs.

Management of an ACCase-inhibitor-resistant Lolium rigidum population based on the use of ALS inhibitors: weed population evolution observed over a 7 year field-scale investigation.

BACKGROUND: A 7 year experiment was set up in 2002 to evaluate the long-term effects of weed management strategies based on graminicidal sulfonylureas (SUs) on the evolution of a Lolium rigidum population resistant to ACCase inhibitors in a continuous wheat cropping system. The strategies included the continued use of ALS inhibitors, the continued application of ACCase inhibitors and a simple resistance management strategy based on a biennial rotation of herbicide mode of action (MoA). RESULTS: The efficacy of the tested SUs in the field decreased significantly, starting from the fourth treatment in all control strategies. Regardless of control strategy, the few survivors of the ALS treatment in the third season produced a significant number of ACCase- and ALS-resistant (multiple-resistant) progeny. Continuous ALS and biennial rotation of herbicides reduced weed densities, but L. rigidum conserved its ACCase resistance trait. Enhanced metabolism was detected in ALS-resistant plants, whereas target site was primarily involved in the ACCase-resistant individuals. CONCLUSION: At the end of the experiment, multiple-resistant individuals were found in all samples coming from the control strategies investigated. The biennial rotation between ALS and other MoA appeared to delay the development of resistance to SUs over continuous treatments, but additional measures will likely need to be taken in order to make this sustainable in the long term, whereas the field efficacy of SUs remained relatively high until the end of the experiment. Integrated weed management with more diversity should be introduced in oversimplified cropping systems in order to sustainably manage resistant L. rigidum populations.

Vacuolar glyphosate-sequestration correlates with glyphosate resistance in ryegrass (Lolium spp.) from Australia, South America, and Europe: a 31P NMR investigation.

Lolium spp., ryegrass, variants from Australia, Brazil, Chile, and Italy showing differing levels of glyphosate resistance were examined by (31)P NMR. Extents of glyphosate (i) resistance (LD(50)), (ii) inhibition of 5-enopyruvyl-shikimate-3-phosphate synthase (EPSPS) activity (IC(50)), and (iii) translocation were quantified for glyphosate-resistant (GR) and glyphosate-sensitive (GS) Lolium multiflorum Lam. variants from Chile and Brazil. For comparison, LD(50) and IC(50) data for Lolium rigidum Gaudin variants from Italy were also analyzed. All variants showed similar cellular uptake of glyphosate by (31)P NMR. All GR variants showed glyphosate sequestration within the cell vacuole, whereas there was minimal or no vacuole sequestration in the GS variants. The extent of vacuole sequestration correlated qualitatively with the level of resistance. Previous (31)P NMR studies of horseweed ( Conyza canadensis (L.) Cronquist) revealed that glyphosate sequestration imparted glyphosate resistance. Data presented herein suggest that glyphosate vacuolar sequestration is strongly contributing, if not the major contributing, resistance mechanism in ryegrass as well.

Allelic variation of the ACCase gene and response to ACCase-inhibiting herbicides in pinoxaden-resistant Lolium spp.

BACKGROUND: The repeated use of acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides to control grass weeds has selected for resistance in Lolium spp. populations in Italy. The efficacy of pinoxaden, a recently marketed phenylpyrazoline herbicide, is of concern where resistance to ACCase inhibitors has already been ascertained. ACCase mutations associated with pinoxaden resistance were investigated, and the cross-resistance pattern to clodinafop, haloxyfop, sethoxydim, clethodim and pinoxaden was established on homo/heterozygous plants for four mutant ACCase alleles. RESULTS: Seven different mutant ACCase alleles (1781-Leu, 1999-Leu, 2041-Asn, 2041-Val, 2078-Gly, 2088-Arg and 2096-Ala) and 13 combinations with two types of mutation were detected in the pinoxaden-resistant plants. The 1781-Leu allele appears to confer a dominant resistance to pinoxaden, clodinafop, haloxyfop, sethoxydim and clethodim at 60 g AI ha(-1) . The 2041-Asn and 2041-Val alleles are associated with dominant or partially dominant resistance to FOPs, no substantial resistance to DIMs and a moderate resistance to pinoxaden. The 2088-Arg allele endows a partially dominant resistance to clodinafop, sethoxydim and most likely to pinoxaden. In addition, non-target-site resistance mechanisms seem to be involved in pinoxaden resistance. CONCLUSION: Almost all the ACCase mutations selected in the field by other ACCase inhibitors are likely to confer resistance to pinoxaden. Although pinoxaden is sometimes able to control FOP-resistant populations, it should not be considered as a sustainable ACCase resistance management tool. The presence of non-ACCase-based resistance mechanisms that could confer resistance to herbicides with different modes of action further complicates the resistance management strategies.

Characterisation of ALS genes in the polyploid species Schoenoplectus mucronatus and implications for resistance management.

BACKGROUND: The polyploid weed Schoenoplectus mucronatus (L.) Palla has evolved target-site resistance to ALS-inhibiting herbicides in Italian rice crops. Molecular and genetic characterisation of the resistance mechanism is relevant to the evolution and management of herbicide resistance. The authors aimed (a) to study the organisation of the target-site loci in two field-selected S. mucronatus populations with different cross-resistance patterns, (b) to identify the mutations endowing resistance to ALS inhibitors and determine the role of these mutations by using transgenesis and (c) to analyse the implications for the management of the S. mucronatus populations. RESULTS: Two complete ALS genes (ALS1 and ALS2) having an intron and a third partial intronless ALS gene (ALS3) were identified. The presence of multiple ALS genes was confirmed by Southern blot analyses, and ALS loci were characterised by examining cytosine methylation. In S. mucronatus leaves, the transcripts of ALS1, ALS2 and ALS3 were detected. Two mutations endowing resistance (Pro(197) to His and Trp(574) to Leu) were found in both resistant populations, but at different frequencies. Tobacco plants transformed with the two resistant alleles indicated that the Pro(197)-to-His substitution conferred resistance to SU and TP herbicides, while the allele with the Trp(574)-to-Leu substitution conferred cross-resistance to SU, TP, IMI and PTB herbicides. CONCLUSION: Schoenoplectus mucronatus has multiple ALS genes characterised by methylated sites that can influence the expression profile. The two mutated alleles proved to be responsible for ALS resistance. At population level, the resistance pattern depends on the frequency of various resistant genotypes, and this influences the efficacy of various ALS-inhibiting herbicides.

Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: evaluation using clethodim.

The acetyl-coenzyme A carboxylase (ACCase)-inhibiting cyclohexanedione herbicide clethodim is used to control grass weeds infesting dicot crops. In Australia clethodim is widely used to control the weed Lolium rigidum. However, clethodim-resistant Lolium populations have appeared over the last 5 years and now are present in many populations across the western Australian wheat (Triticum aestivum) belt. An aspartate-2078-glycine (Gly) mutation in the plastidic ACCase enzyme has been identified as the only known mutation endowing clethodim resistance. Here, with 14 clethodim-resistant Lolium populations we revealed diversity and complexity in the molecular basis of resistance to ACCase-inhibiting herbicides (clethodim in particular). Several known ACCase mutations (isoleucine-1781-leucine [Leu], tryptophan-2027-cysteine [Cys], isoleucine-2041-asparagine, and aspartate-2078-Gly) and in particular, a new mutation of Cys to arginine at position 2088, were identified in plants surviving the Australian clethodim field rate (60 g ha(-1)). Twelve combination patterns of mutant alleles were revealed in relation to clethodim resistance. Through a molecular, biochemical, and biological approach, we established that the mutation 2078-Gly or 2088-arginine endows sufficient level of resistance to clethodim at the field rate, and in addition, combinations of two mutant 1781-Leu alleles, or two different mutant alleles (i.e. 1781-Leu/2027-Cys, 1781-Leu/2041-asparagine), also confer clethodim resistance. Plants homozygous for the mutant 1781, 2078, or 2088 alleles were found to be clethodim resistant and cross resistant to a number of other ACCase inhibitor herbicides including clodinafop, diclofop, fluazifop, haloxyfop, butroxydim, sethoxydim, tralkoxydim, and pinoxaden. We established that the specific mutation, the homo/heterozygous status of a plant for a specific mutation, and combinations of different resistant alleles plus herbicide rates all are important in contributing to the overall level of herbicide resistance in genetically diverse, cross-pollinated Lolium species.