A novel W1999S mutation and non-target site resistance impact on acetyl-CoA carboxylase inhibiting herbicides to varying degrees in a UK Lolium multiflorum population.

Acetyl-CoA carboxylase (ACCase) inhibiting herbicides are important products for the post-emergence control of grass weed species in small grain cereal crops. However, the appearance of resistance to ACCase herbicides over time has resulted in limited options for effective weed control of key species such as Lolium spp. In this study, we have used an integrated biological and molecular biology approach to investigate the mechanism of resistance to ACCase herbicides in a Lolium multiflorum Lam. from the UK (UK21).The study revealed a novel tryptophan to serine mutation at ACCase codon position 1999 impacting on ACCase inhibiting herbicides to varying degrees. The W1999S mutation confers dominant resistance to pinoxaden and partially recessive resistance to cycloxydim and sethoxydim. On the other hand, plants containing the W1999S mutation were sensitive to clethodim and tepraloxydim. Additionally population UK21 is characterised by other resistance mechanisms, very likely non non-target site based, affecting several aryloxyphenoxyproprionate (FOP) herbicides but not the practical field rate of pinoxaden. The positive identification of wild type tryptophan and mutant serine alleles at ACCase position 1999 could be readily achieved with an original DNA based derived cleaved amplified polymorphic sequence (dCAPS) assay that uses the same PCR product but two different enzymes for positively identifying the wild type tryptophan and mutant serine alleles identified here.This paper highlights intrinsic differences between ACCase inhibiting herbicides that could be exploited for controlling ryegrass populations such as UK21 characterised by compound-specific target site and non-target site resistance

Tepraloxydim dose response tests on four plant groups: homozygous wild type WW1999, heterozygous WS1999 and homozygous mutant SS1999 from the mixed resistant population UK21 and standard sensitive STD1 plants (WW1999) for comparison.

<p>Observed values represent dry weight relative to untreated (%) averaged across mother plants. The curves are constructed based on the average GR50 across biological and technical replicates.</p

ACCase gene alignment around the critical nucleotide position 5996 (second base of the 1999 codon) among eight plants sensitive and resistant to pinoxaden.

<p>All sensitive plants contained two copies of guanine (gg) whilst resistant plant had one (cg  = s) or two copies of cytosine (cc) at this position.</p

Haloxyfop-P-methyl dose response tests on four plant groups: homozygous wild type WW1999, heterozygous WS1999 and homozygous mutant SS1999 from the mixed resistant population UK21 and standard sensitive STD1 plants (WW1999) for comparison.

<p>Observed values represent dry weight relative to untreated (%) averaged across biological and technical replicates.</p

Pinoxaden dose response tests on four plant groups: homozygous wild type WW1999, heterozygous WS1999 and homozygous mutant SS1999 from the mixed resistant population UK21 and standard sensitive STD1 plants (WW1999) for comparison.

<p>Observed values represent dry weight relative to untreated (%) averaged across biological and technical replicates.</p

Diclofop-methyl dose response tests on four plant groups: homozygous wild type WW1999, heterozygous WS1999 and homozygous mutant SS1999 from the mixed resistant population UK21 and standard sensitive STD1 plants (WW1999) for comparison.

<p>Observed values represent dry weight relative to untreated (%) averaged across mother plants. The curves are constructed based on the average GR50 across biological and technical replicates.</p

dCAPS procedures for the detection of the wild type tryptophan and mutant serine amino acid residues at ACCase codon position 1999: (a) <i>Xcm</i>I restricted (134 bp) and unrestricted (164 bp) fragments correspond to sensitive W1999 and resistant S1999 ACCase alleles respectively.

<p>(b) <i>Mnl</i>I restricted fragment is indicative of the mutant serine allele (120 bp) while the undigested band (164 bp) corresponds to the wild type tryptophan allele. Heterozygous plants display one each of the restricted and unrestricted PCR fragment in both assays.</p