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.     

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.     

A rapid reliable test for screening aryloxyphenoxy- propionic acid resistance within Alopecurus myosuroides and Lolium spp. populations

A reliable seedling bioassay was developed and tested for the rapid screening for resistance to aryloxyphenoxypropionic (APP) herbicides in Alopecurus myosuroides and Lolium spp. populations. It is based upon the difference in coleoptile length of resistant and susceptible A. myosuroides and Lolium seedlings, respectively, exposed to fenoxaprop-P acid and diclofop acid solution for 6 days in a plastic box. A 6 mg L⁻¹ fenoxaprop-P acid solution was selected as the best concentration for a reliable screening of resistant biotypes within A. myosuroides populations. At this concentration, coleoptile lengths of susceptible and resistant seedlings were shorter and longer than 10 mm respectively. Similarly, resistant seedlings within Lolium populations were easily detected at 10 mg L⁻¹ diclofop acid. At this concentration, coleoptile lengths of susceptible and resistant seedlings were shorter and longer than 20 mm respectively. For both populations, the coleoptile length distributions appear to discriminate between two kinds of APP-resistant biotypes (highly and slightly resistant).     

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.     

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     

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.     

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     

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.     

大穗看麦娘与日本看麦娘的DNA条形码鉴定

大穗看麦娘是我国麦田新发生的恶性杂草,与日本看麦娘苗期形态相近,导致难以识别和有效监测.本研究利用4个DNA条形码候选序列(rbcL、matK、trnH-psbA和ITS2)对13份大穗看麦娘和10份日本看麦娘叶片材料进行分子鉴定,采用Vector NTI分析扩增的DNA序列峰图质量并比对碱基差异,通过MEGA 6.0...     

An aspartate to glycine change in the carboxyl transferase domain of acetyl CoA carboxylase and non‐target‐site mechanism(s) confer resistance to ACCase inhibitor herbicides in a Lolium multiflorum population

BACKGROUND: The increasing use of ACCase‐inhibiting herbicides has resulted in evolved resistance in key grass weeds infesting cereal cropping systems worldwide. Here, a thorough and systematic approach is proposed to elucidate the basis of resistance to three ACCase herbicides in a Lolium multiflorum Lam. (Italian rye grass) population from the United Kingdom (UK24). RESULTS: Resistance to sethoxydim and pinoxaden was always associated with a dominant D2078G (Alopecurus myosuroides Huds. equivalent) target‐site mutation in UK24. Conversely, whole‐plant herbicide assays on predetermined ACCase genotypes showed very high levels of resistance to diclofop‐methyl for all three wild DD2078 and mutant DG2078 and GG2078 ACCase genotypes from the mixed resistant population UK24. This indicates the presence of other diclofop‐methyl‐specific resistance mechanism(s) yet to be determined in this population. The D2078G mutation could be detected using an unambiguous DNA‐based dCAPS procedure that proved very transferable to A. myosuroides, Avena fatua L., Setaria viridis (L.) Beauv. and Phalaris minor Retz. CONCLUSION: This study provides further understanding of the molecular basis of resistance to ACCase inhibitor herbicides in a Lolium population and a widely applicable PCR‐based method for monitoring the D2078G target‐site resistance mutation in five major grass weed species. Copyright © 2010 Society of Chemical Industry     

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     

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.     

A quantitative genetic examination of non‐target‐site resistance applied to Avena species

Quantitative genetics tools can be used to assess whether using herbicides at low doses drive selection on standing genetic variation in populations leading to non‐target‐site resistance (NTSR). These tools are particularly important for estimating the number of genes involved and the potential speed of evolution. A short cut to answering questions about the evolution of NTSR may be to measure heritability. The heritability index (H) provides a measure of the potential to develop NTSR and can be simply calculated from classical dose–response experiments. This measure and the associated experimental designs are discussed with two applied examples on Avena spp. (A. fatua and A. sterilis). In these examples, H values ranged from 0.24 to 0.73, which means that selection for NTSR is highly probable in cases with high H value. We suggest that structuring plants into genetic groups (e.g. families or populations) can contribute to our understanding of the evolutionary potential of populations and plant species to evolve resistance, without increasing experimental cost and time.     

Diclofop‐resistant Lolium rigidum from northern Greece with cross‐resistance to ACCase inhibitors and multiple resistance to chlorsulfuron

Repeated use of ACCase‐ and ALS‐inhibiting herbicides in northern Greece has resulted in the evolution of a population of Lolium rigidum resistant to diclofop and chlorsulfuron. The biotype from Athos was highly resistant to diclofop and also exhibited differential cross‐resistance to clodinafop, fluazifop, tralkoxydim and sethoxydim. Assay of ACCase activity confirmed that the resistant biotype was tenfold more resistant to diclofop than the susceptible biotype, suggesting that the resistance mechanism could involve an altered target site. The diclofop‐resistant biotype has also exhibited multiple resistance to chlorsulfuron and the mechanism for this is unknown. Seed‐bioassay was found to be a rapid, cheap and reliable method to identify populations of L rigidum resistant to ACCase inhibitors and chlorsulfuron. Moreover, root elongation in the seed bioassay was more sensitive to ACCase inhibitors and chlorsulfuron than shoot elongation. © 2000 Society of Chemical Industry     

Occurrence of sulfonylurea resistance in Sagittaria trifolia,a basal monocot species,based on target‐site and non‐target‐site resistance

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in Japan. Since the late 1990s, severe infestations of S. trifolia have occurred following applications of sulfonylurea herbicides in Akita prefecture. In this study, two accessions of S. trifolia, R1 and R2, were collected from paddy fields with severe infestations and their resistance profiles were determined in comparison to a susceptible accession, S1. R1 and R2 were highly resistant to bensulfuron‐methyl. R1 was also highly resistant to pyrazosulfuron‐ethyl, but R2 was susceptible. Relative to S1, R1 had an amino acid substitution at the Pro₁₉₇ residue of acetolactate synthase (ALS), a well‐known mutation that confers sulfonylurea resistance, suggesting that R1 has a target‐site‐based resistance (TSR) mechanism. The sequence of the ALS gene in R2 was identical to that in S1. A Southern blot analysis indicated that there was only one copy of the ALS gene in S1 and R2. These results suggest that R2 has a non‐target‐site‐based resistance (NTSR) mechanism. R2 was moderately resistant to imazosulfuron but susceptible to thifensulfuron‐methyl. R2 and S1 were susceptible to pretilachlor, benfuresate, MCPA‐ethyl and bentazon. The results reveal the occurrence of two sulfonylurea‐resistant biotypes of S. trifolia that show different mechanisms of cross‐resistance to sulfonylureas related to TSR in R1 and NTSR in R2.     

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