PCR-based detection of resistance to acetyl-CoA carboxylase-inhibiting herbicides in black-grass (Alopecurus myosuroides Huds) and ryegrass (Lolium rigidum gaud)

A simple method based upon allele-specific PCR was developed to detect an isoleucine-leucine substitution in the gene encoding chloroplastic acetyl-coenzyme A carboxylase (ACCase) in two gramineous weeds: Lolium rigidum Gaud and Alopecurus myosuroides Huds. Analysis of 1800 A myosuroides and 750 L rigidum seedlings showed that the presence of ACCase leucine allele(s) conferred cross-resistance to the cyclohexanedione herbicide cycloxydim and to the aryloxyphenoxypropionate herbicides fenoxaprop-P-ethyl and diclofop-methyl. Seedlings containing ACCase leucine allele(s) could be either sensitive or resistant to the aryloxyphenoxypropionate herbicides haloxyfop-P-methyl and clodinafop-propargyl. Successful detection of resistant plants in a field population of A myosuroides was achieved using this PCR assay. Using it with basic molecular biology laboratory equipment, the presence of resistant leucine ACCase allele(s) can be detected within one working day.     

Purification,characterization and comparison of glutathione S-transferases from black-grass (Alopecurus myosuroides Huds) biotypes

In the UK biotypes of black-grass (Alopecurus myosuroides Huds) showing resistance to both chlorotoluron (CTU) and aryloxyphenoxypropionate graminicides are increasingly being observed. Although the precise mechanisms involved in this resistance have yet to be identified, increased herbicide metabolism has been implicated as being involved in at least some cases of resistance. Glutathione S-transferases (GSTs) are a group of enzymes which have been demonstrated to metabolise herbicides in some plants, and the resistant black-grass biotype Peldon contains approximately double the GST activity towards 1-chloro-2,4-dinitrobenzene (CDNB) of susceptible biotypes. To investigate further the possible role of GSTs in herbicide resistance in black-grass, a purification procedure has been developed for these enzymes. A 27.5 kDa polypeptide possessing GST activity was purified from the susceptible biotype Herbiseed. Purification of GSTs from the resistant biotype Peldon also identified this polypeptide along with an additional 30 kDa polypeptide. An in-vitro kinetic study of both crude and purified GST extracts, and western blot analysis using antisera raised against the 27.5 kDa polypeptide, suggest that the 30 kDa polypeptide may possess GST activity, and is not a precursor of the 27.5 kDa polypeptide. These results are discussed and compared to GST profiles for other weeds and crops demonstrating herbicide resistance or tolerance. © 1999 Society of Chemical Industry     

A pollen test to detect ACCase target‐site resistance within Alopecurus myosuroides populations

This study was conducted to determine a suitable medium for in vitro germination of Alopecurus myosuroides pollen and to develop a reliable test for the rapid screening of ACCase target site‐resistant plants within populations. The assay is based upon germination of pollen in a medium supplemented with ACCase inhibitors. A 0.25% agar medium, containing 200 mg L^–1 CaNO₃, 100 mg L^–1 H₃BO₃, 200 g L^–1 sucrose, was selected as a suitable medium for in vitro pollen germination. At 25 °C, this medium supported a mean germination rate of 85% within two hours. Plants highly resistant (Rh) to aryloxyphenoxypropionate (APP), owing to the expression of an insensitive ACCase, were found to express this resistance in their pollen. In contrast, plants moderately resistant (Rm) to APP herbicides, owing to an enhanced capacity to detoxify herbicides, did not exhibit this resistance in their pollen. Concentrations of 120 μM fenoxaprop and 1000 μM clodinafop were selected as the best for reliable discrimination of the target‐site‐resistant biotypes. At these concentrations there was more than 50% germination of the Rh pollen grains whereas less than 10% of the S and Rm pollen grains germinated. This test, using haploid material, may also permit distinction between homozygous‐ and heterozygous‐resistant individuals.     

New, quick tests for herbicide resistance in black-grass (Alopecurus myosuroides Huds) based on increased glutathione S-transferase activity and abundance

Black-grass (Alopecurus myosuroides Huds) is a major grass weed in winter cereals in Europe. It reduces yields and can act as a secondary host for a range of diseases. Herbicide resistance in this species was first detected in the UK in the early 1980s, and has now been reported in thirty counties. To successfully manage herbicide resistance it is vital that suspect populations are tested so that appropriate action can be taken. Ideally, a test will be quick, cheap and easy to use. Furthermore, it should provide an unequivocal result before post-emergence herbicides are to be applied, allowing alternative strategies to be adopted where necessary. This paper reports the development of new tests for herbicide resistance based on our observation that the resistant black-grass biotype Peldon contains approximately double the activity of the enzyme glutathione S-transferase (GST) compared with susceptible biotypes. Data are presented on the production of a monoclonal antiserum to a novel 30 kDa GST polypeptide purified from the biotype Peldon. An ELISA using this antiserum is described and the utility of this assay to detect resistant black-grass biotypes in plants grown under glass and in the field is presented. In addition, a microtitre assay for GST activity is described, which allows the rapid assessment of GST activities of plants. Both abundance and activity of GSTs are discussed as markers for herbicide resistance in black-grass.     

Modelling different cultivation and herbicide strategies for their effect on herbicide resistance in Alopecurus myosuroides

A single dominant mutation conferring resistance to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides was incorporated into a quantitative model for the population development of Alopecurus myosuroide s Huds. The model predicts that from an initial seedbank of 100 seed m^–2, 10^–6 of which mutate to resistance each generation, and annual use of AOPP/CHD herbicides which kill 90% of susceptible but no resistant plants, a threshold of 10 plants m^–2 surviving herbicides ('field resistance') will develop: in 9–10 years if all tillage is by tine cultivation to 10 cm deep; after 28–30 years of annual ploughing; in 12 years if tine cultivations are interspersed with ploughing once every 4 years. If AOPP/CHD herbicides are alternated with herbicides with different modes of action, outcomes depend on the annual kill rate: with 95% kill (of susceptible plants by AOPP/CHDs and all plants by alternative herbicides) and tine cultivation, field resistance develops in 22 years; however, resistance can be delayed for 45 years if AOPP/CHDs are rotated with two additional herbicides, each with a different mode of action. The model predictions on the number of years required for field resistance to develop are not highly sensitive to the density of the seedbank or the initial frequency of resistance.     

Cross-resistance patterns to ACCase-inhibiting herbicides conferred by mutant ACCase isoforms in Alopecurus myosuroides Huds. (black-grass), re-examined at the recommended herbicide field rate

BACKGROUND: Target‐site‐based resistance to acetyl‐CoA carboxylase (ACCase) inhibitors in Alopecurus myosuroides Huds. is essentially due to five substitutions (Isoleucine‐1781‐Leucine, Tryptophan‐2027‐Cysteine, Isoleucine‐2041‐Asparagine, Aspartate‐2078‐Glycine, Glycine‐2096‐Alanine). Recent studies suggested that cross‐resistance patterns associated with each mutation using a seed‐based bioassay may not accurately reflect field resistance. The authors aimed to connect the presence of mutant ACCase isoform(s) in A. myosuroides with resistance to five ACCase inhibitors (fenoxaprop, clodinafop, haloxyfop, cycloxydim, clethodim) sprayed at the recommended field rate. RESULTS: Results from spraying experiments and from seed‐based bioassays were consistent for all mutant isoforms except the most widespread, Leucine‐1781. In spraying experiments, Leucine‐1781 ACCase conferred resistance to clodinafop and haloxyfop. Some plants containing Leucine‐1781 or Alanine‐2096 ACCase, but not all, were also resistant to clethodim. CONCLUSION: Leucine‐1781, Cysteine‐2027, Asparagine‐2041 and Alanine‐2096 ACCases confer resistance to fenoxaprop, clodinafop and haloxyfop at field rates. Leucine‐1781 ACCase also confers resistance to cycloxydim at field rate. Glycine‐2078 ACCase confers resistance to all five herbicides at field rates. Only Glycine‐2078 ACCase confers clethodim resistance under optimal application conditions. It may be that Leucine‐1781 and Alanine‐2096 ACCases may also confer resistance to clethodim in the field if the conditions are not optimal for herbicide efficacy, or at reduced clethodim field rates. Copyright © 2008 Society of Chemical Industry     

Use of fluorescence induction to diagnose resistance of Alopecurus myosuroides Huds. (black-grass) to chlorotoluron

The inhibition of photosynthesis of detached black-grass leaves by chlorotoluron could be quantified by measuring the fluorescence induction using a sensor that almost fully suppressed the reflection component. The induction curve was measured twice on the same leaf spots, first after putting the leaves in the herbicide solution for 16 h, and again after placing the leaves in water for 1 day. Leaves from resistant plants showed partial to full recovery from the inhibition, while susceptible plants did not. These results for fluorescence induction were compared with the inhibition of photosynthesis rate in intact black-grass plants by chlorotoluron in the nutrient solution, and the recovery that occurred when the roots were placed again in herbicide-free nutrient solution. The similarity of the results indicates that the procedure for fluorescence induction can replace the more labour-intensive and complicated demonstration of inactivation by measuring photosynthesis rate. Each plant must be measured separately to avoid suppressing the differences between plants within a population. The method was used for samples from 22 fields in The Netherlands in which resistance of black-grass to chlorotoluron was suspected. The results indicate that substantial resistance was present in only two populations, but that weak resistance occurred in all fields. Utilisation de l'induction de fluorescence pour diagnostiquer la résistance au chlorotoluron chez le vulpin, Alopecurus myosuroides Huds. L'inhibition de la photosynthèse de feuilles de vulpin par le chorotoluron pourrait être quantifiee par la mesure de l'induction de fluorescence en utilisant un récepteur qui supprime quasiment la reflexion. La courbe d'induction était mesuree 2 fois, sur les memes fragments de feuille, la première après avoir mise pendant 16 h les feuilles dans la solution herbicide, et de nouveau après avoir placé les feuilles dans l'eau pendant un jour, Les feuilles des plantes resistantes se recuperaient partiellement à totalement de l'inhibition, tandis que les plantes sensibles ne le faisaient pas. Ces résultats sur l'induction de fluorescence ont été comparés avec le taux d'inhibition de la photosynthèse dans des plantes intactes de vulpin par le chlorotoluron dans la solution nutritive et avec la récupération qui apparaissait quand les racines étaient de nouveau placées dans une solution exempte d'herbicide, Les résultats similaires montrent que le procédé de I'induction de fluorescence peut remplacer la démonstration de l'inactivation, beaucoup plus laborieuse et compliquée par la mesure du taux de photosynthèse, Chaque plante doit être mesurée séparément pour éviter la suppression des différences entre piantes dans une population. La méthode a été utilisée pour les échantillons venant de 22 champs aux Pays-Bas dans lesquels on suspectait une résistance du vulpin au chlorotoluron. Les résultats montrent qu'une resistance réelle était seulement présente dans deux populations, mais qu'une faible résistance existait dans tous les champs. Fluorimetrische Diagnose der Resistenz von Alopecurus myosuroides Huds. (Acker-Fuchsschwanz) gegenüber Chlortoluron Die Hemmung der Photosynthese in isolierten Blättern von Acker-Fuchsschwanz durch Chlortoluron konnte durch Messung der Fluoreszenz-induktion bestimmt werden, wobei ein Sensor benutzt wurde, der die Reflektion fast vollständig ehminierte. Die Induktionskurve wurde zweimal gemessen, zum einen nach dem Einstetzen der Blätter für 16 h in die Herbizidlösung und zum andern nochmals nach einem Tag auf Wasser, Blätter resistenter Pflanzen erholten sich teilweise oder ganz von der Hemmung, solche empfindlicher Pflanzen nicht. Die Ergebnisse der Fluoreszenzinduktion wurden mit der Hemmung der Photosyntheserate intakter Pflanzen durch Chlortoluron in der Nährlösung und der Erholung nach Überführen der Wurzeln in herbizidfreie Nährlösung verglichen. Die Ähnlichkeit der Ergebnisse zeigt, daß man die arbeitsintensivere und komplizierte Bestimmung der Inaktivierung mittels Messung der Photosyntheserate durch die Fluoreszenzinduktion ersetzen kann. Jede Pflanze muß für sich gemessen werden, um ein Verwischen der Unterschiede zwischen den Pflanzen einer Population zu vermeiden. Die Methode wurde zur Untersuchung von Proben aus 22 Feldern in den Niederlanden angewandt, wo Resistenz von Acker-Fuchsschwanz gegenüber Chlortoluron vermutet wurde. Nur in 2 Populationen wurde eine volle Resistenz festgestellt, aber eine schwache Resistenz lag bei allen Fällen vor.     

Characterisation of target-site resistance to ACCase-inhibiting herbicides in the weed Alopecurus myosuroides (black-grass)

Resistance to aryloxyphenoxypropionate (AOPP), cyclohexanedione (CHD) and phenylurea herbicides was determined in UK populations of Alopecurus myosuroides Huds. Two populations (Oxford AA1, Notts. A1) were highly resistant (Resistance indices 13-->1000) to the AOPP and CHD herbicides fenoxaprop, diclofop, fluazifop-P and sethoxydim, but only marginally resistant to the phenylurea, chlorotoluron. Analyses of acetyl coenzyme A carboxylase (ACCase) activity showed that an insensitive ACCase conferred resistance to all the AOPP/CHD herbicides investigated. Another population, Oxford S1, showed no resistance to sethoxydim at the population level, but contained a small proportion of plants (<10%) with an insensitive ACCase. Genetic studies on the Notts A1 and Oxford S1 populations demonstrated that target site resistance conferred by an insensitive ACCase is monogenic, nuclearly inherited with the resistant allele showing complete dominance. Investigations of the molecular basis of resistance in the Notts A1 population showed that sethoxydim resistance in A myosuroides was associated with the substitution of an isoleucine in susceptible with a leucine in resistant plants, which has also been found in three other resistant grass-weed species (Setaria viridis (L) Beauv, Avena fatua L, Lolium rigidum Gaud).     

'Universal' primers for PCR-sequencing of grass chloroplastic acetyl-CoA carboxylase domains involved in resistance to herbicides

Primers were designed to amplify two regions involved in sensitivity to herbicides inhibiting the plastidic acetyl-CoA carboxylase (ACCase) from grasses (Poaceae). The first primer pair amplified a 551-bp amplicon containing a variable Ile/Leu codon at position 1781 in Alopecurus myosuroides sequence. The second primer pair amplified a 406-bp amplicon containing four variable codons (Trp/Cys, Ile/Asn, Asp/Gly, Gly/Ala) at positions 2027, 2041, 2078 and 2096, respectively, in A. myosuroides sequence. Both primer pairs amplified the targeted fragments from genes encoding plastidic ACCases, but not from the very similar genes encoding cytosolic ACCases. Clear DNA sequences were obtained from fresh or dried plant material from the field, and from 29 various grass species. Sequences revealed that the gene encoding plastidic ACCase in Poa annua and Festuca rubra contained a Leu₁₇₈₁ codon, in agreement with both species being inherently tolerant to herbicides inhibiting ACCase. Sequencing confirmed the hybrid origin of P. annua. Compared with ACCase enzyme assay, polymerase chain reaction is faster, can be performed from a single plant and suppresses the need for radioactive experiments. It can be completed with basic molecular biology laboratory equipment. It is the tool of choice for diagnosing resistance caused by alteration(s) of the plastidic ACCase.     

Prevalence of cross‐ or multiple resistance to the acetyl‐coenzyme A carboxylase inhibitors fenoxaprop,clodinafop and pinoxaden in black‐grass (Alopecurus myosuroides Huds.) in France

BACKGROUND: Repeated use of acetyl‐CoA carboxylase (ACCase) inhibitors, especially fenoxaprop and clodinafop, since the late 1980s has selected for resistance in Alopecurus myosuroides Huds. (black‐grass) in France. We investigated whether resistance to pinoxaden, a phenylpyrazoline ACCase inhibitor to be marketed in France, was present in French black‐grass populations. We investigated pinoxaden resistance conferred by five mutant ACCase isoforms. Using 84 French black‐grass field samples, we also compared the frequencies of other mechanisms endowing resistance to fenoxaprop, clodinafop or pinoxaden. RESULTS: ACCase mutant isoforms Leu‐1781, Gly‐2078 and, likely, Cys‐2027 conferred cross‐resistance to pinoxaden, while isoform Asn‐2041 possibly conferred moderate resistance. Other mechanisms of resistance to fenoxaprop, clodinafop and pinoxaden were detected in 99, 68 and 64% of the samples investigated, respectively. Cross‐ or multiple resistance to fenoxaprop or clodinafop and pinoxaden was not systematically observed, suggesting a diversity of mechanisms exist. CONCLUSION: Pinoxaden resistance was observed before pinoxaden release in France. Only a fraction of the mechanisms endowing fenoxaprop or clodinafop resistance also confer pinoxaden resistance. Pinoxaden resistance was likely mostly selected for by ACCase inhibitors, and, in some cases, possibly by herbicides with other modes of action. This illustrates the necessity to use metabolisable herbicides cautiously where black‐grass has evolved non‐target‐site‐based resistance. Copyright © 2009 Society of Chemical Industry     

Use of adjuvants to improve control of black-grass (Alopecurus myosuroides Huds.) by diclofop-methyl

In pot and field experiments the addition of Ethylan D256 surfactant to spray solutions improved control of black-grass (Alopecurus myosuroides Huds.) by diclofop-methyl. Various other surfactants and oil adjuvants enhanced phytotoxicity but, generally, to a lesser extent. Ethylan D256 had most effect with applications made at early growth stages. Control of older plants was generally poor, whether or not the surfactant was added. Addition of Ethylan D256 had little effect on selectivity between A. myosuroides and cereals from lower than recommended doses of diclofop-methyl, but there was a suggestion of yield reduction from higher doses. Tank mixing with esters of ioxynil and bromoxynil sometimes improved control by diclofop-methyl but in other circumstances either had no effect or reduced phytotoxicity. Applications of diclofop-methyl in 60 or 120 1 ha^?1 spray volume were as effective as applications in 240 1 ha^?1.     

Glutathione transferases in herbicide-resistant and herbicide-susceptible black-grass (Alopecurus myosuroides)

Glutathione transferase (GST) activities toward the selective herbicide fenoxaprop-ethyl, together with thiol contents, have been compared in seedlings of wheat (Triticum aestivum) and two populations of black-grass (Alopecurus myosuroides) which are resistant to a range of herbicides (Peldon and Lincs E1), and a black-grass population which is susceptible to herbicides (Rothamsted). GST activities toward the non-cereal herbicides metolachlor and fluorodifen were also determined. On the basis of enzyme specific activity, GST activities toward fenoxaprop-ethyl in the leaves were in the order wheat>Peldon=Lincs E1>Rothamsted, while with fluorodifen and metolachlor the order was Peldon=Lincs E1>Rothamsted>wheat. Using an antibody raised to the major GST from wheat, which is composed of 25-kDa subunits, it was shown that the enhanced GST activities in both Peldon and Lincs E1 correlated with an increased expression of a 25-kDa polypeptide and the appearance of novel 27-kDa and 28-kDa polypeptides. Leaves of both wheat and black-grass contained glutathione and hydroxymethylglutathione, with the concentrations of glutathione being in the order Peldon>Lincs E1=Rothamsted=wheat. However, in glasshouse dose-response assays, the Lincs E1 population showed much greater resistance to fenoxaprop-ethyl than Peldon. We conclude that high GST activities and the availability of glutathione may contribute partially to the relative tolerance of black-grass to herbicides detoxified by glutathione conjugation. Although herbicide-resistant populations show enhanced GST expression, in the case of fenoxaprop-ethyl the associated increased detoxifying activities alone cannot explain the differences between populations in the degree of resistance seen at the whole plant level. ©1997 SCI     

Phytotoxicity of trifluoromethyl- and methyl-substituted dinitroaniline herbicides on resistant and susceptible populations of black-grass (Alopecurus myosuroides)

A Petri-dish assay was used to determine the phytotoxicities of the dinitroaniline herbicides pendimethalin, trifluralin, benfluralin and ethalfluralin, and the methyl-substituted analogues of the last three herbicides, on susceptible (Rothamsted) and multiple-herbicide resistant (Peldon) populations of blackgrass Alopecurus myosuroides Huds. The Peldon population showed a high degree of resistance to pendimethalin, which possesses a 3,4-dimethyl substitution. The two populations were equally sensitive to trifluralin, benfluralin and ethalfluralin which do not possess ring-methyl groups but contain a 4-trifluoromethyl group. Substitution of the 4-trifluoromethyl with a methyl group, as demonstrated by the analogues, reduced phytotoxicity, but to a much greater degree in Peldon than in the Rothamsted population. The study indicates that resistance to pendimethalin in the Peldon population is attributable to an oxidative degradation of the 4-methyl group, analogous to that which occurs with resistance to chlorotoluron. The lack of cross-resistance to the other dinitroaniline herbicides appears to be due to the absence of ring-methyl or other groups which are vulnerable to oxidative degradation, trifluoromethyl groups being particularly resistant to this form of reaction.     

抗精噁唑禾草灵的日本看麦娘ACCase基因突变

为明确日本看麦娘抗性种群对精噁唑禾草灵的抗性水平及抗性产生的分子机制,采用整株水平测定法测定了日本看麦娘对精噁唑禾草灵的抗性水平,扩增和比对了日本看麦娘抗性和敏感种群间乙酰辅酶A羧化酶(acetyl-Co A carboxylase,ACCase)基因的差异。结果显示,与敏感种群AH-7相比,抗性种群AH-25对精噁唑禾草灵的抗性倍数为33.82;AH-25种群ACCase基因CT区域2 078位氨基酸发生了突变,由天冬氨酸GAT突变为甘氨酸GGT;AH-25种群对炔草酯、烯草酮和烯禾啶产生了高水平的抗性,抗性倍数分别为35.66、38.64和29.14,对高效氟吡甲禾灵产生了低水平的抗性,抗性倍数为3.04,对精喹禾灵和唑啉草酯较敏感。表明ACCase基因2 078位氨基酸的突变可能是导致精噁唑禾草灵产生高水平抗性的重要原因。     

Developmental changes in glutathione S‐transferase activity in herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) in the field

Herbicide‐resistant populations of Alopecurus myosuroides Huds (black‐grass) have become widespread throughout the UK since the early 1980s. Clear evidence suggests that more than one resistance mechanism exists, and glutathione S‐transferases (GSTs) have been implicated in resistance due to enhanced metabolism. This study reports the determination of GST activity in four UK black‐grass populations from field sites situated in the East Midlands. Data demonstrate that, as untreated plants in the field mature, there is an accompanying natural elevation of GST activity with natural environmental changes from winter to spring. We speculate that this endogenous change in enzyme activity with plant development in the field contributes to reduced efficacy of some graminicides applied in the spring. These observations are discussed in relation to predicting herbicide efficacy to achieve maximum control of this important grass weed. © 2001 Society of Chemical Industry     

Recombinant glutathione transferases from flufenacet-resistant black-grass (Alopecurus myosuroides Huds.) form different flufenacet metabolites and differ in their interaction with pre- and post-emergence herbicides

BACKGROUND

Black-grass (Alopecurus myosuroides Huds.) has become a problematic weed in cereals in Europe. Besides resistance to post-emergent herbicides becoming increasingly widespread, enhanced metabolism of inhibitors of the synthesis of very-long-chain fatty acids (VLCFAs), such as flufenacet, is evolving. Yet, cross-resistance patterns and evolution of this resistance remains poorly understood.

RESULTS

The cDNA sequences of five glutathione transferases (GSTs) upregulated in flufenacet resistant black-grass were identified and used for recombinant protein expression. Moderate to slow detoxification of flufenacet was verified for all candidate GSTs expressed in E. coli, and the most active protein produced flufenacet-alcohol instead of a glutathione conjugate, in the presence of reduced glutathione (GSH). Moreover, cross-resistance to other VLCFA-inhibitors e.g., acetochlor and pyroxasulfone and the ACCase inhibitor fenoxaprop was verified in vitro. Various other herbicides of different modes of action including VLCFA-inhibitors were not detoxified by the candidate GSTs.

CONCLUSIONS

As several in planta upregulated GSTs detoxified flufenacet in vitro, the shift in sensitivity observed in black-grass populations, is likely a result of an additive effect. The polygenic character and the relatively low turnover rate of the individual GSTs may explain the slow evolution of flufenacet resistance. In addition, flufenacet resistance was accompanied by cross-resistance with some, but not all, herbicides of the same mode of action, and furthermore to the ACCase inhibitor fenoxaprop-ethyl. Hence, not only the rotation of herbicide modes of action, but also of individual active ingredients is important for resistance management. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Molecular bases for resistance to acetyl-coenzyme A carboxylase inhibitor in Japanese foxtail (Alopecurus japonicus)

BACKGROUND: Haloxyfop‐R‐methyl is a widely used herbicide to control Poaceae weeds. Alopecurus japonicus, a widespread annual grass, can no longer be controlled by haloxyfop‐R‐methyl after continuous use of this herbicide for several years. RESULTS: Dose‐response experiments have established that the Js‐R biotype of A. japonicas has evolved resistance to aryloxyphenoxypropionates (APPs). Target‐site enzyme sensitivity experiments have established that the haloxyfop (free acid) rate causing 50% inhibition of acetyl‐CoA carboxylase (ACCase) activity (I₅₀) for the resistant (Js‐R) biotype is 11 times higher than that for the susceptible (Js‐S) biotype. In many cases, resistance to ACCase‐inhibiting herbicides is due to a resistant ACCase enzyme. Full‐length DNA and mRNA sequences of the plastidic ACCase gene were amplified. Eight single‐nucleotide differences were detected in this region. Four of the nucleotide changes were silent mutations. However, the other four nucleotide mutations caused four amino acid substitutions, replacing Arg‐1734 with Gly, Met‐1738 with Leu, Thr‐1739 with Ser and Ile‐2041 with Asn in the R biotype respectively; the substitution at position 2041 had been reported, while the other three had not. CONCLUSION: The ACCase in the Js‐R biotype was less susceptible to haloxyfop‐R‐methyl than that in the Js‐S biotype. Moreover, the amino acid substitution of Ile‐2041 with Asn might confer resistance to haloxyfop‐R‐methyl in A. japonicas. Copyright © 2012 Society of Chemical Industry     

Status of black grass (Alopecurus myosuroides) resistance to acetyl-coenzyme A carboxylase inhibitors in France

We assessed the contributions of target site‐ and non‐target site‐based resistance to herbicides inhibiting acetyl‐coenzyme A carboxylase (ACC) in Alopecurus myosuroides (black grass). A total of 243 A. myosuroides populations collected across France were analysed using herbicide sensitivity bioassay (24 300 seedlings analysed) and ACC genotyping (13 188 seedlings analysed). Seedlings resistant to at least one ACC‐inhibiting herbicide were detected in 99.2% of the populations. Mutant, resistant ACC allele(s) were detected in 56.8% of the populations. Among the five resistant ACC alleles known in A. myosuroides, alleles containing an isoleucine‐to‐leucine substitution at codon 1781 were predominant (59.5% of the plants containing resistant ACC alleles). Comparison of the results from herbicide sensitivity bioassays with genotyping indicated that more than 75% of the plants resistant to ACC‐inhibiting herbicides in France would be resistant via increased herbicide metabolism. Analysis of herbicide application records suggested that in 15.9% of the populations studied, metabolism‐based resistance to ACC‐inhibiting herbicides was mostly selected for by herbicides with other modes of action. Our study revealed the importance of non‐target site‐based resistance in A. myosuroides. Using herbicides with alternative modes of action to control populations resistant to ACC‐inhibiting herbicides, the recommended management approach, may thus be jeopardised by the widespread occurrence of metabolism‐based resistance mechanisms conferring broad‐spectrum cross‐resistance.     

A molecular assay for the proactive detection of target site-based resistance to herbicides inhibiting acetolactate synthase in Alopecurus myosuroides

Acetolactate synthase (ALS) inhibitors are the most resistance‐prone herbicide group. Rapid resistance diagnosis is thus of importance for their optimal use. We formulate rules to use the derived cleaved amplified polymorphic sequence method to develop molecular tools detecting a change at a given codon, the nature of which is unknown. We applied them to Alopecurus myosuroides (black grass) to develop assays targeting ALS codons A122, P197, A205, W574 and S653 that are crucial for herbicide sensitivity. These assays detected W574L or P197T, or both substitutions, in most plants analysed from a field where ALS inhibitors failed after 3 years of use. Similar assays can easily be set up for any species. Given the rapidity of selection for resistance to ALS inhibitors, these assays should be very useful in proactive herbicide resistance diagnosis.