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.

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Resistance to herbicides occurs in weeds as the result of evolutionary adaptation (Jasieniuk et al. 1996). Basically, two types of mechanisms are involved in resistance (Beckie and Tardif 2012; Délye 2013). Target-site resistance (TSR) is caused by changes in the tridimensional structure of the herbicide target protein that decrease herbicide binding, or by increased activity (e.g., due to increased expression or increased intrinsic activity) of the target protein. Nontarget-site resistance (NTSR) is endowed by any mechanism not belonging to TSR, e.g., reduction in herbicide uptake or translocation in the plant, or enhanced herbicide detoxification (reviewed in Délye 2013; Yuan et al. 2007).

Délye C, Pernin F & Michel S (2011). ‘Universal’ PCR assays detecting mutations in acetyl‐coenzyme A carboxylase or acetolactate synthase that endow herbicide resistance in grass weeds. Weed Research51, 353–362. Summary Herbicides inhibiting acetyl‐coenzyme A carboxylase (ACCase) or acetolactate synthase (ALS) are key for grass weed control. Yet, numerous cases of resistance have evolved. Using the derived cleaved amplified polymorphic sequence method, we developed molecular assays to detect amino acid replacements at the seven ACCase codons (1781, 1999, 2027, 2041, 2078, 2088 and 2096) and at two ALS codons (197 and 574) known to play a role in herbicide resistance in grass weeds. For each codon, one assay detecting all known amino acid replacements endowing herbicide resistance was developed. The nine assays were successfully used to genotype ACCase and ALS in 39 grass species. Their flexible design enables easy detection of new mutations at the targeted codons. Because they can be implemented with basic molecular biology facilities and no previous knowledge of the ACCase or ALS sequence of the grass weed of interest, these assays are tools of choice to easily detect resistance caused by alteration(s) of ACCase or ALS in such species.

BackgroundNon-target-site based resistance to herbicides is a major threat to the chemical control of agronomically noxious weeds. This adaptive trait is endowed by differences in the expression of a number of genes in plants that are resistant or sensitive to herbicides. Quantification of the expression of such genes requires normalising qPCR data using reference genes with stable expression in the system studied as internal standards. The aim of this study was to validate reference genes in Alopecurus myosuroides, a grass (Poaceae) weed of economic and agronomic importance with no genomic resources.ResultsThe stability of 11 candidate reference genes was assessed in plants resistant or sensitive to herbicides subjected or not to herbicide stress using the complementary statistical methods implemented by NormFinder, BestKeeper and geNorm. Ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase were identified as the best reference genes. The reference gene set accuracy was confirmed by analysing the expression of the gene encoding acetyl-coenzyme A carboxylase, a major herbicide target enzyme, and of an herbicide-induced gene encoding a glutathione-S-transferase.ConclusionsThis is the first study describing a set of reference genes (ubiquitin, beta-tubulin and glyceraldehyde-3-phosphate dehydrogenase) with a stable expression under herbicide stress in grasses. These genes are also candidate reference genes of choice for studies seeking to identify stress-responsive genes in grasses.

Summary Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate‐synthase (ALS) inhibitors, the most resistance‐prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929‐bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non‐synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro‐197‐Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.

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