Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: evaluation using clethodim

Abstract

The acetyl-coenzyme A carboxylase (ACCase)-inhibiting cyclohexanedione herbicide clethodim is used to control grass weeds infesting dicot crops. In Australia clethodim is widely used to control the weed Lolium rigidum. However, clethodim-resistant Lolium populations have appeared over the last 5 years and now are present in many populations across the western Australian wheat (Triticum aestivum) belt. An aspartate-2078-glycine (Gly) mutation in the plastidic ACCase enzyme has been identified as the only known mutation endowing clethodim resistance. Here, with 14 clethodim-resistant Lolium populations we revealed diversity and complexity in the molecular basis of resistance to ACCase-inhibiting herbicides (clethodim in particular). Several known ACCase mutations (isoleucine-1781-leucine [Leu], tryptophan-2027-cysteine [Cys], isoleucine-2041-asparagine, and aspartate-2078-Gly) and in particular, a new mutation of Cys to arginine at position 2088, were identified in plants surviving the Australian clethodim field rate (60 g ha(-1)). Twelve combination patterns of mutant alleles were revealed in relation to clethodim resistance. Through a molecular, biochemical, and biological approach, we established that the mutation 2078-Gly or 2088-arginine endows sufficient level of resistance to clethodim at the field rate, and in addition, combinations of two mutant 1781-Leu alleles, or two different mutant alleles (i.e. 1781-Leu/2027-Cys, 1781-Leu/2041-asparagine), also confer clethodim resistance. Plants homozygous for the mutant 1781, 2078, or 2088 alleles were found to be clethodim resistant and cross resistant to a number of other ACCase inhibitor herbicides including clodinafop, diclofop, fluazifop, haloxyfop, butroxydim, sethoxydim, tralkoxydim, and pinoxaden. We established that the specific mutation, the homo/heterozygous status of a plant for a specific mutation, and combinations of different resistant alleles plus herbicide rates all are important in contributing to the overall level of herbicide resistance in genetically diverse, cross-pollinated Lolium species.

Figure 1.

In vitro inhibition of ACCase activity by ACCase herbicides for susceptible plants (S₁, •), resistant plants homozygous for 1781-Leu (⋄), 2078-Gly (▵), and 2088-Arg (▿) from population R₇, R₁₂, and R₁₄, respectively. Data are pooled from two extractions per population per herbicide and each was assayed in duplicate.

Figure 2.

dCAPS analysis of individual L. rigidum plants homozygous susceptible 2078-Asp (S), homozygous-resistant 2078-Gly (R), or heterozygous 2078-Gly/Asp (R/S). The sizes of restriction enzyme (EcoRV) digested fragments are 353 and 323 bp, respectively.

Figure 3.

Clethodim dose response of the known susceptible population S₁ (wild type, •) and purified homozygous-resistant populations R₇P (1781-Leu, ⋄), R₁₂P (2078-Gly, ▵), and R₁₄P (2088-Arg, ▿). Data for the susceptible population S₁ were pooled from two experiments and data for the purified populations was each from a single experiment.

Figure 4.

Alignment of partial amino acid sequences of chloroplastic homomeric ACCases from 28 grass species that are putatively susceptible to ACCase herbicides. Numbers above the sequences indicate amino acid positions within the A. myosuroides full ACCase sequence (GenBank accession AJ310767). Amino acid residues 2078, 2088, and 2096 are in bold and conserved amino acids are indicated by dots. The boxed 2088 residue was conserved as a Cys (C) among most grass species except for a few species as a Phe (F).