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.     

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.     

Characterisation and molecular basis of ALS inhibitor resistance in the grass weed Alopecurus myosuroides

Several UK populations of the grass weed Alopecurus myosuroides were identified where high proportions of individuals showed resistance to the acetolactate synthase (ALS)‐inhibiting herbicides, mesosulfuron‐methyl + iodosulfuron‐methyl sodium mixture and sulfometuron‐methyl. Screening with sulfometuron, followed by DNA sequencing of the ALS gene from resistant and susceptible individuals, led to the identification of eight populations where a single point mutation segregated with resistance to sulfometuron. All highly resistant individuals from seven of eight populations showed a single‐nucleotide polymorphism (SNP) in the first position of the Pro197 codon of an A. myosuroides ALS gene, conferring a predicted proline to threonine target‐site change. One population showed resistant individuals with single‐nucleotide polymorphism in the second position of the Trp574 codon, conferring a predicted tryptophan to leucine substitution. No other mutations segregating with resistance were found. Enzyme assays confirmed that resistance was due to an altered form of ALS enzyme, which was less susceptible to inhibition by sulfonylureas, making this one of the first fully characterised cases of ALS target‐site resistance in a European grass weed. Increased information regarding the nature and distribution of ALS target‐site mutation may help support sustainable management strategies, allowing continued use of mesosulfuron + iodosulfuron against this weed in the UK.     

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     

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.     

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.     

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.     

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.     

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.     

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.