Chlorotoluron Metabolism in Leaves of Resistant and Susceptible Biotypes of the Grass Weed Alopecurus myosuroides

The metabolism of the herbicide chlorotoluron by susceptible and resistant biotypes of the grass weed, Alopecurus myosuroides, was examined. After administration of radiolabelled herbicide to leaves, metabolites were extracted and analysed. The metabolites identified consisted of mono-demethylated-, di-demethylated- and ring methyl-hydroxylated chlorotoluron. Metabolism was more extensive in the resistant biotype, yielding principally the non-phytotoxic ring methyl-hydroxylated metabolite. The metabolites observed are characteristic of the activity of cytochrome P450 mixed-function oxygenase action. The specific cytochrome P450 inhibitor, 1-aminobenzotriazole, reduced accumulation of the ring methyl-hydroxylated metabolite in the resistant biotype.     

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     

An approach to investigate the costs of herbicide‐resistant Alopecurus myosuroides

Herbicide resistance in Alopecurus myosuroides causes severe problems in Western European cropping systems. Costs of herbicide resistance were investigated in this study by analysing variable production costs and sales revenues. Three farms were selected for this study, with winter wheat as the dominating crop in all farms. Resistance in A. myosuroides populations was verified at all locations. Four farming approaches were simulated over a period of 20 years: (i) continuing the actual cropping system without increase of resistance, (ii) continuing the actual cropping system with increase of resistance, (iii) changing cropping practice to overcome resistance and (iv) changing cropping practice to prevent resistance. Contribution margins representing the proportion of sales revenue that is not consumed by variable costs were calculated for all approaches. Comparative static simulations showed that average contribution margins in a cropping system with more than 60% winter cereals and reduced tillage practice dropped from 807 € ha^?1 a^?1 without herbicide resistance to 307 € ha^?1 a^?1 with herbicide resistance. Alopecurus myosuroides population densities increased to more than 1000 plants m^?2. Diverse crop rotations, including spring crops, clover–grass leys and intensive tillage, suppressed A. myosuroides populations, and average contribution margin was 630 € ha^?1 a^?1. Preventive methods with rotations of winter cereals and spring crops with less clover–grass leys resulted in an average contribution margin of 691 € ha^?1 a^?1. In conclusion, rotations of winter cereals and spring crops combined with inversion tillage and herbicides provide stable yields and can prevent weed population increase.     

Localized origins of herbicide resistance in Alopecurus myosuroides

The process of evolution of resistance to acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides was investigated in four distinct patches of Alopecurus myosuroides Huds. (black-grass) that occur within adjacent fields on a cereal farm in Nottinghamshire, UK. In one field, there was a `main' patch containing 96% resistant plants and two `satellite' patches containing ≈2.9% and 4.4% resistant plants, and in an adjacent field another patch contained 25% resistant plants. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction (PCR) was used to analyse variation at 30 genetic loci in at least 20 resistant and 20 sensitive individual plants from each patch, from additional resistant populations from Essex and Lincolnshire, and from a sensitive reference population. Banding patterns were found to be highly repeatable. Each patch contained a high level of genetic diversity, regardless of its resistance status, and there was evidence for genetic differences between the patches (Gst = 0.14, Nei's distances up to 0.26). There was no evidence that resistance had spread from the `main' patch to the others, as resistant and sensitive plants in the same patch were more closely related on average than were resistant plants from neighbouring patches. The most likely explanations of this distribution, and their implications, are discussed.     

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).     

Multiple origins for black-grass (Alopecurus myosuroides Huds) target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase

We have investigated the process of evolution of target-site-based resistance to herbicides inhibiting acetyl-CoA carboxylase (ACCase) in nine French populations of black-grass (Alopecurus myosuroides Huds). To date, two different ACCase resistant alleles are known. One contains an isoleucine-to-leucine substitution at position 1781, the second contains an isoleucine-to-asparagine substitution at position 2041. Using phylogenetic analysis of ACCase sequences, we showed that 1781Leu ACCase alleles evolved from four independent origins in the nine black-grass populations studied, while 2041Asn ACCase alleles evolved from six independent origins. No geographical structure of black-grass populations was revealed. This implies that these populations, although geographically distant, are, or have until recently been, connected by gene flows. Comparison of biological data obtained from herbicide sensitivity bioassay and molecular data showed that distinct resistance mechanisms often exist in a single black-grass population. Accumulation of different resistance mechanisms in a single plant was also demonstrated. We conclude that large-scale evolution of resistance to herbicides in black-grass is a complex phenomenon, resulting from the independent selection of various resistance mechanisms in local black-grass populations undergoing contrasted herbicide and agronomical selection pressures, and connected by gene flows whose parameters remain to be determined.     

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.     

The survival of Alopecurus myosuroides Huds. seeds in soil

The survival of Alopecurus myosuroides seeds was studied in soil under arable cropping and short term grass leys in which seed return was prevented. At two winter wheat sites, where weed seeds were sown, the mean annual seed decline was 73–83% over a 2- or 3-year period. The rate of decline was similar with all the cultivation systems studied: ploughing, tine cultivation and direct drilling. Seeds buried initially by ploughing, and then not disturbed by cultivation, were slightly more persistent. At five arable sites with natural populations of A. myosuroides, seed numbers declined to an average of 3% of the original amount present after 3 years, and to 1% after 4 years. Initial populations of over 50 000 seeds m^?2 were recorded. Plant populations were not always proportional to the total seed content of the soil, especially on ploughed land. Seed decline in two grass fields was similar to that under arable cropping. A. myosuroides plants were recorded in a wheat crop following a 2-year grass ley. Weed plants did not persist in the vegetative state in grass used for conservation and grazing. At all sites, appreciable quantities of seeds were still present in the soil after 2–4 years. Although a relatively small proportion of seeds survived, the actual number of seeds surviving was substantial. For this reason, it was concluded that any eradication policy is unlikely to be effective in a cropping system dominated by winter cereals.     

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     

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.     

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.     

Interpopulation variability and adaptive potential for reduced glyphosate sensitivity in Alopecurus myosuroides

Glyphosate use in the United Kingdom has more than doubled in the last 20 years. Much of this increase is driven by efforts to control herbicide resistant weeds, particularly Alopecurus myosuroides, prior to crop drilling. There is precedent for evolution of glyphosate resistance in similar situations, raising concerns over the sustainability of glyphosate use in the UK. We used dose–response experiments to examine variation in glyphosate sensitivity amongst 40 field‐collected A. myosuroides populations. No populations were resistant to glyphosate, but ED₉₀ values ranged between 354 and 610 g a.i. ha^?1. Five populations had ED₉₀ values significantly higher than the unexposed control population collected from a site at Rothamsted Research with no previous glyphosate exposure. Recurrent selection experiments were performed to determine whether variation in glyphosate sensitivity had a heritable basis. Following two rounds of selection, five of six field populations evolved significantly reduced sensitivity to glyphosate, with R/S ratios, based on estimated ED₅₀ values, ranging from 1.2 to 1.5. These results confirm that there is a heritable basis to variation in glyphosate sensitivity. The response to selection was modest. Evolved populations were not highly resistant to glyphosate, although some twice‐selected individuals survived recommended field rates. These results do not represent definitive proof of the potential of A. myosuroides to evolve glyphosate resistance, although they do indicate caution is needed when considering the sustainability of increased glyphosate use to control this herbicide resistance‐prone species.     

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.     

Variation in seed dormancy and light sensitivity in Alopecurus myosuroides and Apera spica-venti

Variation in seed dormancy and light sensitivity was studied in Alopecurus myosuroides and Apera spica-venti . Seeds were collected from different populations, at different dates and from plants emerging in autumn or spring, and used in four experiments. In the first experiment, initial dormancy was investigated in light and darkness. In Expt 2, buried seeds were exhumed on 16 occasions, from September 1997 to March 2000, and germinated in light, in darkness and after a 5-s light exposure. In Expt 3, emergence was recorded for seeds sown in pots outdoors. In Expt 4, stratified seeds of A. myosuroides only were exposed to photon irradiance ranging from 0.1 to 25 600 μmol m⁻². Variation was high among seed collections, but both species showed winter annual dormancy patterns. Apera spica-venti germinated to high percentages in autumn but negligibly in spring. Alopecurus myosuroides germinated less in spring when tested in darkness and after a short light exposure and emerged poorly in spring, which reflected photo-desensitisation during cold stratification. We conclude that the peak of emergence in A. myosuroides , and to some extent in A. spica-venti , is largely regulated by exposure to light interacting with low-level dormancy. This offers valuable information regarding optimal timing of weed control measures.     

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     

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.     

Segregation of non‐target‐site resistance to herbicides in multiple‐resistant Alopecurus myosuroides plants

Non‐target‐site resistance (NTSR) comprises a set of mechanisms conferring resistance to multiple modes of action. Investigation of the number of loci involved in NTSR will aid in the understanding of these resistance mechanisms. Therefore, six different multiple herbicide‐resistant Alopecurus myosuroides plants with different herbicide history were crossed in two generations with a susceptible wild type. Seeds from the backcrossing generation were studied for their segregation rate for resistance to five herbicides with four different modes of action (HRAC groups C₂, A, B and K₃). Taking into account that NTSR is a set of quantitative traits, the numbers of loci controlling NTSR were estimated using a normal mixture model fitted by the NLMIXED procedure of SAS. Each herbicide was controlled by a different number of loci comparing the six plants. In most of the cases, chlorotoluron resistance was controlled by one locus, whereas resistance to fenoxaprop‐P‐ethyl needed one or two loci. Resistance to pinoxaden was in all plants conferred by two loci. Cross‐resistance of fenoxaprop‐P‐ethyl and pinoxaden was found in all backcrossings, indicating that at least one of the two loci is responsible for both resistances. Resistance to mesosulfuron + iodosulfuron was conferred by a minimum of two loci. Results indicated that a minimum of five different loci can be involved in a multiple NTSR plant. Furthermore, the plant‐specific accumulation of NTSR loci was demonstrated. Such behaviour should be taken into account when evaluating the development and further spread of herbicide resistance.