High survival frequencies at low herbicide use rates in populations of Lolium rigidum result in rapid evolution of herbicide resistance

The frequency of phenotypic resistance to herbicides in previously untreated weed populations and the herbicide dose applied to these populations are key determinants of the dynamics of selection for resistance. In total, 31 Lolium rigidum populations were collected from sites with no previous history of exposure to herbicides and where there was little probability of gene flow from adjacent resistant populations. The mean survival frequency across all 31 populations following two applications of commercial rates (375 g ha(-1)) of the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicide, diclofop-methyl was 0.43%. Survivors from five of these populations were grown to maturity and seed was collected. Dose-response experiments compared population level resistance to diclofop-methyl in these selected lines with their original parent populations. A single cycle of herbicide selection significantly increased resistance in all populations (LD(50) R:S ratios ranged from 2.8 to 23.2), confirming the inheritance and genetic basis of phenotypic resistance. In vitro assays of ACCase inhibition by diclofop acid indicated that resistance was due to a non-target-site mechanism. Following selection with diclofop-methyl, the five L. rigidum populations exhibited diverse patterns of cross-resistance to ACCase and ALS-inhibiting herbicides, suggesting that different genes or gene combinations were responsible for resistance. The relevance of these results to the management of herbicide resistance are discussed.     

Herbicide multiple-resistance in a Lolium rigidum biotype is endowed by multiple mechanisms: isolation of a subset with resistant acetyl-CoA carboxylase

The development of herbicide multiple-resistance in weed species represents a major threat to current agricultural practices. The mechanistic basis for herbicide multiple-resistance has been investigated in a population of the annual grass weed Lolium rigidum Gaud. (annual ryegrass) resistant to herbicides affecting 6 target sites. A subset of the resistant population (R₂ subset) has been isolated by germination on a medium containing the acetyl-CoA carboxylase (ACCase, EC 6.4.1.2) inhibiting herbicide, sethoxydim ((2-[1-(ethoxyimino)butyl]-5-[2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one)). This 12% R₂ subset of the population is 600 times more resistant to sethoxydim and between 30 to 200 times more resistant to other ACCase inhibitors than the bulk of the R population. The subset has a form of ACCase which is 6 to 55 times less sensitive to inhibition by these herbicides than the enzyme present in the bulk of the resistant or in the susceptible population. There was no difference in the uptake and metabolic degradation of [4-¹⁴C]sethoxydim between the R₂ subset and the unselected R population. These results show the accumulation of different resistance mechanisms in that single population. Furthermore we propose that this accumulation of multiple resistance mechanisms is the basis for herbicide multiple-resistance in this biotype.     

Occurrence of a herbicide-resistant acetyl-coenzyme A carboxylase mutant in annual ryegrass (Lolium rigidum) selected by sethoxydim

The spectrum of herbicide resistance was determined in an annual ryegrass (Lolium rigidum Gaud.) biotype (SLR 3) that had been exposed to the grass herbicide sethoxydim, an inhibitor of the plastidic enzyme acetylcoenzyme A carboxylase (ACCase, EC 6.4.1.2), for three consecutive years. This biotype has an 18-fold resistance to sethoxydim and enhanced resistance to other cyclohexanedione herbicides compared with a susceptible biotype (VLR 1). The resistant biotype also has a 47- to >300-fold cross-resistance to the aryloxyphenoxypropanoate herbicides which share ACCase as a target site. No resistance is evident to herbicide with a target site different from ACCase. The absorption of [4-¹⁴C]sethoxydim, the rate of metabolic degradation and the nature of the herbicide metabolites are similar in the resistant and susceptible biotypes. While the total activity of the herbicide target enzyme ACCase is similar in extracts from the two biotypes, the kinetics of herbicide inhibition differ. The concentrations of sethoxydim and tralkoxydim required to inhibit the activity of ACCase by 50% are 7.8 and >9.5 times higher, respectively, in the resistant biotype. The activity of ACCase from the resistant biotype was also less sensitive to aryloxyphenoxypropanode herbicides than the susceptible biotype. The spectrum of resistance at the whole-plant level is correlated with resistance at the ACCase level and confirms that a less sensitive form of the target enzyme endows resistance in biotype SLR 3.Abbreviations ACCase acetyl-coenzyme A carboxylase - AOPP aryloxyphenoxypropanoate - CHD cyclohexanedione - GR₅₀ dose giving 50% reduction of growth - IG₅₀ dose giving 50% reduction of germination - LD₅₀ lethal dose 50 This work was partially supported by The Grains Research and Development Corporation of Australia through a grant to Dr. R. Knight, Department of Plant Science, Waite Agricultural Research Institute. The encouragement and generous support of Dr. R. Knight is gratefully acknowledged.     

Pollen Expression of Herbicide Target Site Resistance Genes in Annual Ryegrass (Lolium rigidum)

Herbicide resistance can occur either through target-site insensitivity or by nontarget site-based mechanisms. Two herbicide-resistant biotypes of Lolium rigidum Gaud., one resistant to acetolactate synthase (ALS)-inhibiting herbicides (biotype WLR1) and the other resistant to acetyl CoA carboxylase (ACCase)-inhibiting herbicides (biotype WLR96) through target-site insensitivity at the whole plant and enzymic levels, were found to express this resistance in the pollen. Pollen produced by resistant biotypes grew uninhibited when challenged with herbicide, whereas that from a susceptible biotype was inhibited. A third biotype, SLR31, resistant to ACCase-inhibiting and certain ALS-inhibiting herbicides at the whole plant level through nontarget site-based mechanisms, did not exhibit this expression in the pollen. The technique described may form the basis for a rapid screen for certain nuclear-encoded, target site-based herbicide-resistance mechanisms.     

Graminicide resistance in a blackgrass (Alopecurus myosuroides) population correlates with insensitivity of acetyl-CoA carboxylase

The appearance of biotypes of the annual grass weed black‐grass (Alopecurus myosuroides L. Huds), which are resistant to certain graminicides, is the most significant example of acquired resistance to herbicides seen so far in European agriculture. An investigation was perfomed into the basis of the specific cross‐resistance to cyclohexanedione (CHD) and aryloxyphenoxypropionoic acid (AOPP) herbicides in the ‘Notts A1’ population of A. myosuroides, which survived treatment of fields with recommended rates of AOPP herbicides. In comparison with the wild‐type ‘Rothamsted’ population, the resistant biotype showed over 100‐fold resistance to these herbicides in a hydroponic growth system. Biosynthesis of fatty acids and activity of crude extracts of acetyl‐CoA carboxylase (ACCase) were commensurately less sensitive to these herbicides in Notts A1 compared with the Rothamsted biotype. These data are consistent with the hypothesis that the highly resistant population has arisen through selection of a mutant ACCase which is much less sensitive to the AOPP and CHD graminicides. Rapidly growing cell suspension cultures established from the Notts A1 population also showed high resistance indices for CHD or AOPP herbicides compared with cultures from the Rothamsted biotype. Fatty acid biosynthesis and ACCase activity in the cell suspensions were similarly sensitive towards the graminicides to those in the foliar tissue counterparts of the resistant and sensitive populations. Moreover, purification of the main (chloroplast) isoform of acetyl‐CoA carboxylase showed that this enzyme from the Notts A1 population was over 200‐fold less sensitive towards the AOPP herbicide, quizalofop, than the equivalent isoform from the Rothamsted population. These data again fully supported the proposal that resistance in the Notts biotype is due to an insensitive acetyl‐CoA carboxylase isoform. Overall, cell suspensions were also demonstrated to be excellent tools for further investigation of the molecular basis of the high level herbicide resistance which is prone to occur in A. myosuroides.     

An isoleucine to leucine mutation in acetyl-CoA carboxylase confers herbicide resistance in wild oat.

Wild oat (Avena fatua L.) populations resistant to herbicides that inhibit acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) represent an increasingly important weed control problem. The objective of this study was to determine the ACCase mutation responsible for herbicide resistance in a well-studied wild oat biotype (UMI). A 2039-bp region encompassing the carboxybiotin and acetyl-CoA binding domains of multifunctional plastidic ACCase was analyzed. DNA sequences representing three plastidic ACCase gene loci were isolated from both the resistant UMI and a herbicide-susceptible biotype, consistent with the hexaploid nature of wild oat. Only one nonsynonymous point mutation was found among the resistant wild oat sequences, inferring an isoleucine to leucine substitution. The position of this substitution corresponds to residue 1769 of wheat (Triticum aestivum L.) plastidic ACCase (GenBank accession No. AF029895). Analysis of an F2 population derived from a cross between a herbicide-resistant and a susceptible biotype confirmed co-segregation of herbicide resistance with the mutated ACCase. We conclude that the isoleucine to leucine mutation is responsible for herbicide resistance in UMI wild oat based on a comparison of the substitution site across species and ACCase types. While isoleucine is conserved among plastidic ACCases of herbicide-susceptible grasses, leucine is found in plastidic and cytosolic forms of multifunctional herbicide-resistant ACCase.     

DNA Analysis of Herbarium Specimens of the Grass Weed Alopecurus myosuroides Reveals Herbicide Resistance Pre-Dated Herbicides

Acetyl-CoA carboxylase (ACCase) alleles carrying one point mutation that confers resistance to herbicides have been identified in arable grass weed populations where resistance has evolved under the selective pressure of herbicides. In an effort to determine whether herbicide resistance evolves from newly arisen mutations or from standing genetic variation in weed populations, we used herbarium specimens of the grass weed Alopecurus myosuroides to seek mutant ACCase alleles carrying an isoleucine-to-leucine substitution at codon 1781 that endows herbicide resistance. These specimens had been collected between 1788 and 1975, i.e., prior to the commercial release of herbicides inhibiting ACCase. Among the 734 specimens investigated, 685 yielded DNA suitable for PCR. Genotyping the ACCase locus using the derived Cleaved Amplified Polymorphic Sequence (dCAPS) technique identified one heterozygous mutant specimen that had been collected in 1888. Occurrence of a mutant codon encoding a leucine residue at codon 1781 at the heterozygous state was confirmed in this specimen by sequencing, clearly demonstrating that resistance to herbicides can pre-date herbicides in weeds. We conclude that point mutations endowing resistance to herbicides without having associated deleterious pleiotropic effects can be present in weed populations as part of their standing genetic variation, in frequencies higher than the mutation frequency, thereby facilitating their subsequent selection by herbicide applications.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum): I. Properties of the Herbicide Target Enzymes Acetyl-Coenzyme A Carboxylase and Acetolactate Synthase

Lolium rigidum biotype SR4/84 is resistant to the herbicides diclofop-methyl and chlorsulfuron when grown in the field, in pots, and in hydroponics. Similar extractable activities and affinities for acetyl-coenzyme A of carboxylase (ACCase), an enzyme inhibited by diclofop-methyl, were found for susceptible and resistant L. rigidum. ACCase activity from both biotypes was inhibited by diclofop-methyl, diclofop acid, haloxyfop acid, fluazifop acid, sethoxydim, and tralkoxydim but not by chlorsulfuron or trifluralin. Exposure of plants to diclofop-methyl did not induce any changes in either the extractable activities or the herbicide inhibition kinetics of ACCase. It is concluded that, in contrast to diclofop resistance in L. multiflorum and diclofop tolerance in many dicots, the basis of resistance to diclofop-methyl and to other aryloxyphenoxypropionate and cyclohexanedione herbicides in L. rigidum is not due to the altered inhibition characteristics or expression of the enzyme ACCase. The extractable activities and substrate affinity of acetolactate synthase (ALS), an enzyme inhibited by chlorsulfuron, from susceptible and resistant biotypes of L. rigidum were similar. ALS from susceptible and resistant plants was equally inhibited by chlorsulfuron. Prior exposure of plants to 100 millimolar chlorsulfuron did not affect the inhibition kinetics. It is concluded that resistance to chlorsulfuron is not caused by alterations in either the expression or inhibition characteristics of ALS.     

Genetic variation and population structure in black-grass (Alopecurus myosuroides Huds.), a successful, herbicide-resistant, annual grass weed of winter cereal fields

Black‐grass (Alopecurus myosuroides) is an allogamous grass weed common in cereal fields of northern Europe, which developed resistance to a widely used family of herbicides, the ACCase‐inhibiting herbicides. Resistance is caused by mutations at the ACCase gene and other, metabolism‐based, mechanisms. We investigated the genetic structure of 36 populations of black‐grass collected in one region of France (Côte d’Or), using 116 amplified fragment length polymorphism (AFLP) loci and sequence data at the ACCase gene. The samples were characterized for their level of herbicide resistance and genotyped for seven known ACCase mutations conferring resistance. All samples contained herbicide‐resistant plants, and 19 contained ACCase mutations. The genetic diversity at AFLP loci was high (H_T = 0.246), while differentiation among samples was low (F_ST = 0.023) and no isolation by distance was detected. Genetic diversity within samples did not vary with the frequency of herbicide resistance. A Bayesian algorithm was used to infer population structure. The two genetic clusters inferred were not associated with any geographical structure or with herbicide resistance. A high haplotype diversity (H_d = 0.873) and low differentiation (G_ST = 0.056) were observed at ACCase. However, haplotype diversity within samples decreased with the frequency of ACCase‐based resistance. We suggest that the genetic structure of black‐grass is affected by its recent expansion as a weed. Our data demonstrate that the strong selection imposed by herbicides did not modify the genome‐wide genetic structure of an allogamous weed that probably has large effective population sizes. Our study gives keys to a better understanding of the evolution of successful, noxious weeds in modern agriculture.     

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.     

A Novel W1999S Mutation and Non-Target Site Resistance Impact on Acetyl-CoA Carboxylase Inhibiting Herbicides to Varying Degrees in a UK Lolium multiflorum Population

Background

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21).

Methodology/Principal Findings

The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here.

Conclusion/Significance

This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance.     

Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides

Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion. In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase (ACCase) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of C?te d'Or) by genotyping 13 151 plants originating from 243 fields. Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated. These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides.     

The molecular bases for resistance to acetyl co-enzyme A carboxylase (ACCase) inhibiting herbicides in two target-based resistant biotypes of annual ryegrass (Lolium rigidum)

Acetyl-CoA carboxylase (ACCase) (EC.6.4.1.2) is an essential enzyme in fatty acid biosynthesis and, in world agriculture, commercial herbicides target this enzyme in plant species. In nearly all grass species the plastidic ACCase is strongly inhibited by commercial ACCase inhibiting herbicides [aryloxyphenoxypropionate (APP) and cyclohexanedione (CHD) herbicide chemicals]. Many ACCase herbicide resistant biotypes (populations) of L. rigidum have evolved, especially in Australia. In many cases, resistance to ACCase inhibiting herbicides is due to a resistant ACCase enzyme. Two ACCase herbicide resistant L. rigidum biotypes were studied to identify the molecular basis of ACCase inhibiting herbicide resistance. The carboxyl-transferase (CT) domain of the plastidic ACCase gene was amplified by PCR and sequenced. Amino acid substitutions in the CT domain were identified by comparison of sequences from resistant and susceptible plants. The amino acid residues Gln-102 (CAG codon) and Ile-127 (ATA codon) were substituted with a Glu residue (GAG codon) and Leu residue (TTA codon), respectively, in both resistant biotypes. Amino acid positions 102 and 127 within the fragment sequenced from L. rigidum corresponded to amino acid residues 1756 and 1781, respectively, in the A. myosuroides full ACCase sequence. Allele-specific PCR results further confirmed the mutations linked with resistance in these populations. The Ile-to-Leu substitution at position 1781 has been identified in other resistant grass species as endowing resistance to APP and CHD herbicides. The Gln-to-Glu substitution at position 1756 has not previously been reported and its role in herbicide resistance remains to be established.     

Glyphosate,paraquat and ACCase multiple herbicide resistance evolved in a <Emphasis Type="Italic">Lolium rigidum</Emphasis> biotype

Glyphosate is the world’s most widely used herbicide. A potential substitute for glyphosate in some use patterns is the herbicide paraquat. Following many years of successful use, neither glyphosate nor paraquat could control a biotype of the widespread annual ryegrass (Lolium rigidum), and here the world’s first case of multiple resistance to glyphosate and paraquat is confirmed. Dose–response experiments established that the glyphosate rate causing 50% mortality (LD₅₀) for the resistant (R) biotype is 14 times greater than for the susceptible (S) biotype. Similarly, the paraquat LD₅₀ for the R biotype is 32 times greater than for the S biotype. Thus, based on the LD₅₀ R/S ratio, this R biotype of L. rigidum is 14-fold resistant to glyphosate and 32-fold resistant to paraquat. This R biotype also has evolved resistance to the acetyl-coenzyme A carboxylase (ACCase) inhibiting herbicides. The mechanism of paraquat resistance in this biotype was determined as restricted paraquat translocation. Resistance to ACCase-inhibiting herbicides was determined as due to an insensitive ACCase. Two mechanisms endowing glyphosate resistance were established: firstly, a point mutation in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene, resulting in an amino acid substitution of proline to alanine at position 106; secondly, reduced glyphosate translocation was found in this R biotype, indicating a co-occurrence of two distinct glyphosate resistance mechanisms within the R population. In total, this R biotype displays at least four co-existing resistance mechanisms, endowing multiple resistance to glyphosate, paraquat and ACCase herbicides. This alarming case in the history of herbicide resistance evolution represents a serious challenge for the sustainable use of the precious agrochemical resources such as glyphosate and paraquat.     

Resistant Acetyl-CoA Carboxylase is a Mechanism of Herbicide Resistance in a Biotype of Avena sterilis ssp. ludoviciana

A biotype of Avena sterilis ssp. ludoviciana is highly resistantto a range of herbicides which inhibit a key enzyme in fattyacid synthesis, acetyl-CoA carboxylase (ACCase). Possible mechanismsof herbicide resistance were investigated in this biotype. Acetyl-CoAcarboxylase from the resistant biotype is less sensitive toinhibition by herbicides to which resistance is expressed. I₅₀values for herbicide inhibition of ACCase were 52 to 6 timesgreater in the resistant biotype than in the susceptible biotype.This was the only major difference found between the resistantand susceptible biotypes. The amount of ACCase in the meristemsof the resistant and susceptible is similar during ontogenyand no difference was found in distribution of ACCase betweenthe two biotypes. Uptake, translocation and metabolism of [¹⁴C]diclofop-methylwere not different between the two biotypes. In vivo, ACCaseactivity in the meristems of the susceptible biotype was greatlyinhibited by herbicide application whereas only 25% inhibitionoccurred in the resistant biotype. Depolarisation of plasmamembrane potential by 50 µM diclofop acid was observedin both biotypes and neither biotype showed recovery of themembrane potential following removal of the herbicide. Hence,a modified form of ACCase appears to be the major determinantof resistance in this resistant wild oat biotype. (Received February 10, 1994; Accepted March 11, 1994)     

Resistance mechanism to acetyl coenzyme A carboxylase inhibiting herbicides in Phalaris paradoxa collected in Mexican wheat fields

Background and aims

In this study, we describe the molecular, physiological and agronomic aspects involved in the resistance to acetyl coenzyme A carboxylase inhibiting herbicides (ACCase) observed in one biotype of Phalaris paradoxa from Mexico.

Methods

Dose–response Assays: The herbicide rate inhibiting plant growth of each biotype by 50% with respect to the untreated control, ED₅₀. Enzyme purification and ACCase assays to determine herbicide rate inhibiting the enzyme of each biotype by 50% with respect to the untreated control, I₅₀. Absorption and Translocation Assays with [¹⁴C]diclofop-methyl. Metabolism of diclofop-methyl and its metabolites were identified by thin-layer chromatography. Study of target site resistance mechanism at enzyme and molecular levels.

Results

In this work, it has been studied the whole-plant response of Phalaris paradoxa biotypes from Mexico resistant (R) and susceptible (S) to ACCase-inhibiting herbicides: aryloxyphenoxypropionate (APP), cyclohexanedione (CHD) and phenylpyrazoline (PPZ), and the mechanism behind their resistance were studied. To analyse the resistance mechanism, the enzyme ACCase activity was investigated. Results from biochemical assays indicated a target-site resistance as the cause of reduced susceptibility to ACCase inhibitors. The absorption, translocation and metabolism were similar between R and S biotypes. A point mutation never described before was detected within the triplet of glycine at the amino acid position 2096 (referring to EMBL accession no. AJ310767) and resulted in the triplet of serine. This new mutation could explain the loss of affinity for the ACCase-inhibiting herbicides.

Conclusions

We found a new mutation, which had never been described before. This mutation was detected within the triplet of glycine at the amino acid position 2096. This new mutation confers cross-resistance to three different chemical groups of ACCase-inhibiting herbicides.     

Herbicide resistance-endowing ACCase gene mutations in hexaploid wild oat (Avena fatua): insights into resistance evolution in a hexaploid species

Many herbicide-resistant weed species are polyploids, but far too little about the evolution of resistance mutations in polyploids is understood. Hexaploid wild oat (Avena fatua) is a global crop weed and many populations have evolved herbicide resistance. We studied plastidic acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicide resistance in hexaploid wild oat and revealed that resistant individuals can express one, two or three different plastidic ACCase gene resistance mutations (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg). Using ACCase resistance mutations as molecular markers, combined with genetic, molecular and biochemical approaches, we found in individual resistant wild-oat plants that (1) up to three unlinked ACCase gene loci assort independently following Mendelian laws for disomic inheritance, (2) all three of these homoeologous ACCase genes were transcribed, with each able to carry its own mutation and (3) in a hexaploid background, each individual ACCase resistance mutation confers relatively low-level herbicide resistance, in contrast to high-level resistance conferred by the same mutations in unrelated diploid weed species of the Poaceae (grass) family. Low resistance conferred by individual ACCase resistance mutations is likely due to a dilution effect by susceptible ACCase expressed by homoeologs in hexaploid wild oat and/or differential expression of homoeologous ACCase gene copies. Thus, polyploidy in hexaploid wild oat may slow resistance evolution. Evidence of coexisting non-target-site resistance mechanisms among wild-oat populations was also revealed. In all, these results demonstrate that herbicide resistance and its evolution can be more complex in hexaploid wild oat than in unrelated diploid grass weeds. Our data provide a starting point for the daunting task of understanding resistance evolution in polyploids.     

Six amino acid substitutions in the carboxyl-transferase domain of the plastidic acetyl-CoA carboxylase gene are linked with resistance to herbicides in a Lolium rigidum population

The molecular basis of an acetyl-CoA carboxylase (ACCase) target-based resistant Lolium rigidum population (WLR 96) was studied here. The carboxyl-transferase domain of the plastidic ACCase gene from resistant individuals was amplified by PCR and sequenced. The DNA sequences were aligned and compared with a susceptible population. Six amino acid substitutions were identified in the resistant population. The substitution Ile-2041-Asn, known to confer resistance to ACCase-inhibiting herbicides aryloxyphenoxypropionate (APP) in Alopecurus myosuroides, was identified in most resistant plants but it is always linked with other amino acid substitutions. This was confirmed by a cleaved amplified polymorphism (CAP) marker and an allele-specific PCR. The sole amino acid substitution Ile-2041-Asn was not found in this population. It is likely this mutation evolved later among individuals already possessing the other substitutions. Three haplotypes were identified from the resistant population based on the six amino acid combinations, and two are linked with herbicide resistance in this population. The multiple amino acid substitutions including the Ile-2041-Asn form the molecular basis endowing a high degree of resistance to ACCase-inhibiting herbicides in this L. rigidum population.     

Double herbicide-resistant biotypes of wild oat (<Emphasis Type="Italic">Avena fatua</Emphasis>) display characteristic metabolic fingerprints before and after applying ACCase- and ALS-inhibitors

Plant herbicides inhibit specific enzymes of biosynthetic metabolism, such as acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS). Herbicide resistance can be caused by point mutations at the binding domains, catalytic sites and other regions within multimeric enzymes. Direct-injection electrospray mass spectrometry was used for high-throughput metabolic fingerprinting for finding significant differences among biotypes in response to herbicide application. A Mexican biotype of wild oat (Avena fatua) that displays multiple resistances to ACCase- and ALS-inhibiting herbicides was characterized. The dose–response test showed that the double-resistant biotype had a resistance index of 3.58 for pinoxaden and 3.53 for mesosulfuron-methyl. Resistance was accompanied by characteristic mutations at the site of action: an I-1781-L substitution occurred in the ACCase enzyme and an S-653-N mutation was identified within the ALS enzyme. Other mutations were also detected in the genes of the Mexican biotypes. The ionomic fingerprint showed that the multiple-resistant biotype had a markedly different metabolic pattern under control conditions and that this difference was accentuated after herbicide treatment. This demonstrates that single changes of amino acid sequences can produce several holistic modifications in the metabolism of resistant plants compared to susceptible plants. We conclude that in addition to genetic resistance, additional mechanisms of metabolic adaptation and detoxification can occur in multiple-resistant weed plants.     

Use of adjuvant-enhanced formulations to increase bipyridylium-herbicide effectiveness

Paraquat and diquat are two popular, non-selective; bipyridylium herbicides commonly used in citrus orchards and horticultural row crops as the main chemical weed control method. However, since diquat lacks of an effective spectrum against grass weeds, and paraquat mammal toxicity raises strong environmental concerns, both an increase in diquat toxicity against grasses and a reduction in paraquat rates may be desired. Using grass-weed Lolium rigidum and broad leave weed Portulaca oleracea as experimental systems, the effects of six commercial adjuvants (poly-1-p-menthene, mixture of methyl oleate and palmitate, alkylglycol ester, dodecylbenzene ammonium sulphonate, and two paraffinic oils) on paraquat and diquat effectiveness have been studied under laboratory controlled conditions. Dose-response assays showed that adjuvants failed in increasing paraquat efficacy in both broad and grass weeds, yet antagonistic effects being observed in some mixtures such as paraquat + polymentene. However, all adjuvants tested did succeed in increasing significantly diquat effectiveness in P. oleracea and (most important) L. rigidum grass weed. Formulated-diquat ED50 rates were reduced down to 15% (diquat + DBSA) and 30% (diquat + fatty acid ester, diquat + polimentene) of those obtained on non-formulated-diquat trials for P. oleracea and L. rigidum, respectively. Results showed that formulated diquat proved to be a valid alternative to paraquat, and could be used as a more environmentally friendly substitute with comparable effectiveness and herbicide rate.