Cross resistance to accase herbicide in Lolium rigidum

Lolium rigidum is a cross-pollinating grass weed present in Europe and occurring in winter wheat and orchard crops. Several graminicides such as chlorotoluron and/or isoproturon and diclofop-methyl in mixtures or alone have been used successfully to control this weed in Spain during the past decade. However, several L rigidum populations have developed resistance to these herbicides following selection due to their continuous use. Four resistance mechanisms have been found in this grass weed, an enhanced metabolic detoxiflcation of the herbicides and an insensitive isoform of ACCase being the most important ones. The extent of cross-resistance depends on the type of mechanism. The biotype with an enhanced metabolic detoxification showed cross-resistance to ACCase-, ALS-, PSII- and tubuline-inhibiting herbicides, while the biotype resistant due to a mutation of the target site (ACCase) presented cross-resistance to ACCase-inhibiting herbicides only.     

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.     

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.     

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.     

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.     

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)     

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.     

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.     

Role of a Novel I1781T Mutation and Other Mechanisms in Conferring Resistance to Acetyl-CoA Carboxylase Inhibiting Herbicides in a Black-Grass Population

Background

Knowledge of the mechanisms of herbicide resistance is important for designing long term sustainable weed management strategies. Here, we have used an integrated biology and molecular approach to investigate the mechanisms of resistance to acetyl-CoA carboxylase inhibiting herbicides in a UK black-grass population (BG2).

Methodology/Principal Findings

Comparison between BG2 phenotypes using single discriminant rates of herbicides and genotypes based on ACCase gene sequencing showed that the I1781L, a novel I1781T, but not the W2027C mutations, were associated with resistance to cycloxydim. All plants were killed with clethodim and a few individuals containing the I1781L mutation were partially resistant to tepraloxydim. Whole plant dose response assays demonstrated that a single copy of the mutant T1781 allele conferred fourfold resistance levels to cycloxydim and clodinafop-propargyl. In contrast, the impact of the I1781T mutation was low (Rf = 1.6) and non-significant on pinoxaden. BG2 was also characterised by high levels of resistance, very likely non-target site based, to the two cereal selective herbicides clodinafop-propargyl and pinoxaden and not to the poorly metabolisable cyclohexanedione herbicides. Analysis of 480 plants from 40 cycloxydim resistant black grass populations from the UK using two very effective and high throughput dCAPS assays established for detecting any amino acid changes at the 1781 ACCase codon and for positively identifying the threonine residue, showed that the occurrence of the T1781 is extremely rare compared to the L1781 allele.

Conclusion/Significance

This study revealed a novel mutation at ACCase codon position 1781 and adequately assessed target site and non-target site mechanisms in conferring resistance to several ACCase herbicides in a black-grass population. It highlights that over time the level of suspected non-target site resistance to some cereal selective ACCase herbicides have in some instances surpassed that of target site resistance, including the one endowed by the most commonly encountered I1781L mutation.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : II. Chlorsulfuron Resistance Involves a Wheat-Like Detoxification System

Lolium rigidum Gaud. biotype SLR31 is resistant to the herbicide diclofop-methyl and cross-resistant to several sulfonylurea herbicides. Wheat and the cross-resistant ryegrass exhibit similar patterns of resistance to sulfonylurea herbicides, suggesting that the mechanism of resistance may be similar. Cross-resistant ryegrass is also resistant to the wheat-selective imidazolinone herbicide imazamethabenz. The cross-resistant biotype SLR31 metabolized [phenyl-U-¹⁴C]chlorsulfuron at a faster rate than a biotype which is susceptible to both diclofop-methyl and chlorsulfuron. A third biotype which is resistant to diclofop-methyl but not to chlorsulfuron metabolized chlorsulfuron at the same rate as the susceptible biotype. The increased metabolism of chlorsulfuron observed in the cross-resistant biotype is, therefore, correlated with the patterns of resistance observed in these L. rigidum biotypes. During high performance liquid chromatography analysis the major metabolite of chlorsulfuron in both susceptible and cross-resistant ryegrass coeluted with the major metabolite produced in wheat. The major product is clearly different from the major product in the tolerant dicot species, flax (Linium usitatissimum). The elution pattern of metabolites of chlorsulfuron was the same for both the susceptible and cross-resistant ryegrass but the cross-resistant ryegrass metabolized chlorsulfuron more rapidly. The investigation of the dose response to sulfonylurea herbicides at the whole plant level and the study of the metabolism of chlorsulfuron provide two independent sets of data which both suggest that the resistance to chlorsulfuron in cross-resistant ryegrass biotype SLR31 involves a wheat-like detoxification system.     

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.     

Recurrent selection with reduced herbicide rates results in the rapid evolution of herbicide resistance in Lolium rigidum

There has been much debate regarding the potential for reduced rates of herbicide application to accelerate evolution of herbicide resistance. We report a series of experiments that demonstrate the potential for reduced rates of the acetyl-co enzyme A carboxylase (ACCase)-inhibiting herbicide diclofop-methyl to rapidly select for resistance in a susceptible biotype of Lolium rigidum. Thirty-six percent of individuals from the original VLR1 population survived application of 37.5 g diclofop-methyl ha^–1 (10% of the recommended field application rate). These individuals were grown to maturity and bulk-crossed to produce the VLR1 low dose-selected line VLR1 (0.1). Subsequent comparisons of the dose-response characteristics of the original and low dose-selected VLR1 lines demonstrated increased tolerance of diclofop-methyl in the selected line. Two further rounds of selection produced VLR1 lines that were resistant to field-applied rates of diclofop-methyl. The LD₅₀ (diclofop-methyl dose required to cause 50% mortality) of the most resistant line was 56-fold greater than that of the original unselected VLR1 population, indicating very large increases in mean population survival after three cycles of selection. In vitro ACCase inhibition by diclofop acid confirmed that resistance was not due to an insensitive herbicide target-site. Cross-resistance studies showed increases in resistance to four herbicides: fluazifop-P-butyl, haloxyfop-R-methyl, clethodim and imazethapyr. The potential genetic basis of the observed response and implications of reduced herbicide application rates for management of herbicide resistance are discussed.     

Resistance to Acetolactate Synthase-Inhibiting Herbicides in Annual Ryegrass (Lolium rigidum) Involves at Least Two Mechanisms

WLR1, a biotype of Lolium rigidum Gaud. that had been treated with the sulfonylurea herbicide chlorsulfuron in 7 consecutive years, was found to be resistant to both the wheat-selective and the nonselective sulfonylurea and imidazolinone herbicides. Biotype SLR31, which became cross-resistant to chlorsulfuron following treatment with the aryloxyphenoxypropionate herbicide diclofop-methyl, was resistant to the wheat-selective, but not the nonselective, sulfonylurea and imidazolinone herbicides. The concentrations of herbicide required to reduce in vitro acetolactate synthase (ALs) activity 50% with respect to control assays minus herbicide for biotype WLR1 was greater than those for susceptible biotype VLR1 by a factor of >30, >30, 7,4, and 2 for the herbicides chlorsulfuron, sulfometuron-methyl, imazapyr, imazathapyr, and imazamethabenz, respectively. ALS activity from biotype SLR31 responded in a similar manner to that of the susceptible biotype VLR1. The resistant biotypes metabolized chlorsulfuron more rapidly than the susceptible biotype. Metabolism of 50% of [phenyl-U-¹⁴C]chlorsulfuron in the culms of two-leaf seedlings required 3.7 h in biotype SLR31, 5.1 h in biotype WLR1, and 7.1 h in biotype VLR1. In all biotypes the metabolism of chlorsulfuron in the culms was more rapid than that in the leaf lamina. Resistance to ALS inhibitors in L. rigidum may involve at least two mechanisms, increased metabolism of the herbicide and/or a herbicide-insensitive ALS.     

Cross-Resistance to Herbicides in Annual Ryegrass (Lolium rigidum) : III. On the Mechanism of Resistance to Diclofop-Methyl

Annual ryegrass (Lolium rigidum) biotype SLR 31 is resistant to the postemergent graminicide methyl-2-[4-(2,4-dichlorophenoxy)phenoxy]-propanoate (diclofop-methyl). Uptake of [¹⁴C](U-phenyl)diclofop-methyl and root/shoot distribution of radioactivity in susceptible and resistant plants were similar. In both biotypes, diclofop-methyl was rapidly demethylated to the biocidal metabolite diclofop acid which, in turn, was metabolized to ester and aryl-O-sugar conjugates. Susceptible plants accumulated 5 to 15% more radioactivity in dicloflop acid than did resistant plants. Resistant plants had a slightly greater capacity to form nonbiocidal sugar conjugates. Despite these differences, resistant plants retained 20% of ¹⁴C in the biocidal metabolite diclofop acid 192 hours after treatment, whereas susceptible plants, which were close to death, retained 30% in diclofop acid. The small differences in the pool sizes of the active and inactive metabolites are by themselves unlikely to account for a 30-fold difference in sensitivity to the herbicide at the whole plant level. Similar high-pressure liquid chromatography elution patterns of conjugates from both susceptible and resistant biotypes indicated that the mechanisms and the products of catabolism in the biotypes are similar. It is suggested that metabolism of diclofop-methyl by the resistant biotype does not alone explain resistance observed at the whole-plant level. Diclofop acid reduced the electrochemical potential of membranes in etiolated coleoptiles of both biotypes; 50% depolarization required 1 to 4 μm diclofop acid. After removal of diclofop acid, membranes from the resistant biotype recovered polarity, whereas membranes from the susceptible biotype did not. Internal concentrations of diclofop acid 4 h after exposing plants to herbicide were estimated to be 36 to 39 micromolar in a membrane fraction and 16 to 17 micromolar in a soluble fraction. Such concentrations should be sufficient to fully depolarize membranes. It is postulated that differences in the ability of membranes to recover from depolarization are correlated with the resistance response of biotype SLR 31.     

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.     

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.     

The dicotyledonous species Erodium moschatum (L) L'Hér. ex Aiton is sensitive to haloxyfop herbicide due to herbicide-sensitive acetyl-coenzyme A carboxylase

Most plants are resistant to herbicides which inhibit acetyl-coenzyme A carboxylase (ACCase) because they have both eukaryotic ACCase and herbicide-insensitive, prokaryotic ACCase. Members of the Gramineae are killed because they have only herbicide-sensitive, eukaryotic ACCase. Here we report that a dicot, Erodium moschatum, is sensitive to the ACCase-inhibiting herbicide haloxyfop because it has herbicide-sensitive ACCase. Erodium moschatum was controlled by haloxyfop application at rates which controlled the gramineous species Digitaria ciliaris and a susceptible Lolium rigidum biotype but did not control the dicot Nicotiana tabacum or a haloxyfop-resistant L. rigidum biotype WLR96. Similarly, the haloxyfop acid concentration required to inhibit activity by 50% in E. moschatum ACCase assays (1.0 μM) was similar to that required for D. ciliaris (2.3 μM) and susceptible L. rigidum (0.4 μM) but much less than that for the resistant L. rigidum biotype WLR96 (353 μM) or the dicots  N. tabacum (182 μM) and Pisum sativum (150 μM). Leaf protein extracts from  N. tabacum and P. sativum contained both eukaryotic ACCase and prokaryotic subunits of ACCase, but E. moschatum, D. ciliaris and both L. rigidum biotypes exhibited only the eukaryotic ACCase. Thus, the dicot  E. moschatum is sensitive to haloxyfop because it lacks the herbicide-insensitive prokaryotic ACCase, a protein that has been considered ubiquitous in dicot species. Received: 12 May 1998 / Accepted: 27 June 1998     

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.     

Fitness costs associated with acetyl‐coenzyme A carboxylase mutations endowing herbicide resistance in American sloughgrass (Beckmannia syzigachne Steud.)

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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.