Running head: ACCase mutations and herbicide resistance in Lolium species

The acetyl-CoA 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 five years and now are present in many populations across the Western Australian wheat belt (Owen et al. 2007). An Asp-2078-Gly mutation in the plastidic ACCase enzyme has been identified as the only known mutation endowing clethodim resistance (Délye et al. 2005). Here, with 14 clethodim resistant Lolium populations we revealed diversity and complexity in the molecular basis of resistance to ACCaseinhibiting herbicides (clethodim in particular). Several known ACCase mutations (Ile-1781-Leu, Trp2027-Cys, Ile-2041-Asn and Asp-2078-Gly) and in particular, a new mutation of Cys to Arg at position 2088, were identified in plants surviving the Australian clethodim field rate (60 g ha). 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-Arg 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-Asn), 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. 4 www.plantphysiol.org on October 1, 2017 Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. INTRODUCTION Acetyl-coenzyme A carboxylase (ACCase, EC.6.4.1.2) is a key enzyme involved in the first step of fatty acid biosynthesis. In plants, ACCase is also the target enzyme for important herbicides used in world agriculture. Three chemically distinct classes of herbicides that are known to inhibit ACCase are aryloxyphenoxypropionates (APP), cyclohexanediones (CHD) and the more recent (Hofer et al., 2006) phenylpyrazolin class herbicide pinoxaden (hereinafter referred to as ACCase herbicides). In plants, there are two isoforms of ACCase: the plastid ACCase is essential in biosynthesis of primary fatty acids and the cytosolic ACCase is involved in biosynthesis of long chain fatty acids. The homomeric ACCase in the cytosol of nearly all plant species and the heteromeric ACCase in the chloroplasts of dicots are insensitive to APP, CHD and pinoxaden herbicides. In contrast, the plastidic homomeric ACCase in nearly all grass species is herbicide-sensitive, and this is the basis for selective control of grass-weeds by ACCase herbicides. All ACCase isoforms contain three catalytic domains: the biotin carboxyl-carrier, the biotin carboxylase and the carboxyl transferase (CT) domains (Nikolau et al., 2003). Molecular and biochemical studies have clearly established that the CT domain of the plastidic homomeric ACCase is the primary target site for APP and CHD herbicides, and two regions of the CT domain of the plastidic ACCase are critical for sensitivity to these herbicides (Zhang et al., 2004; reviewed by Délye, 2005). The obligate cross-pollinated grass weed Lolium rigidum has demonstrated in Australia an ability to rapidly evolve resistance to ACCase herbicides and other herbicide groups. Within a few years of initial ACCase herbicide use (1978), the first ACCase herbicide resistant L. rigidum population was evident (Heap & Knight 1986). Since then, ACCase herbicide resistant L. rigidum populations have evolved across huge areas of Australian croplands (Llewellyn and Powles, 2001, Owen et al., 2007). The biochemical basis of ACCase herbicide resistance has been revealed in several populations to involve resistant ACCase (Matthews et al., 1990; Holtum et al., 1991; Tardif et al., 1993; Tardif et al., 1996). Many resistant populations can also have a non target site based resistance mechanism of enhanced rates of ACCase herbicide metabolism (Tardif and Powles, 1994; Preston et al., 1996; Preston and Powles, 1998). L. rigidum is an obligate cross-pollinated plant and it is emphasised that individual plants and populations can accumulate resistance mechanisms. Recently, we have identified molecular mutations in the ACCase gene endowing target site based herbicide resistance in some ACCase herbicide resistant L. rigidum populations. We have identified mutations causing resistance-endowing amino acid substitutions at amino acids 1781 and 2041 (Zhang 5 www.plantphysiol.org on October 1, 2017 Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. and Powles 2006a, b). Both of these amino acid substitutions have been previously identified to endow ACCase herbicide resistance in studies by others. Thus far, six distinct amino acid substitutions in the CT domain of the plastidic ACCase gene that individually endow resistance to certain ACCase herbicides have been characterized in Alopecurus myosuroides and other grass weed species, as reviewed by Délye et al (2005) and updated in Table 1. Specific ACCase mutations confer specific cross resistance patterns to ACCase herbicides (reviewed by Délye, 2005). The Ile-1781-Leu mutation is associated with resistance to APP and some CHD herbicides (not including clethodim). The Trp2027-Cys, Ile-2041-Asn or Gly-2096-Ala mutations confer resistance only to APP herbicides. The Asp-2078-Gly mutation confers resistance to many APP and CHD herbicides including clethodim. The Trp-1999-Cys mutation confers resistance only to the APP herbicide fenoxaprop (Liu et al., 2007). Despite widespread resistance to certain ACCase herbicides, our 1998 survey across 300 Western Australian crop fields confirmed that the CHD herbicide clethodim was still effective on many otherwise ACCase herbicide resistant L. rigidum populations (Llewellyn and Powles, 2001). Five years later, however, our random survey of 452 ryegrass populations from the same region revealed clethodim resistance to be present in 8% of these populations (Owen et al., 2007). Thus far, the Asp-2078-Gly mutation in the plastidic ACCase enzyme is the only known mutation endowing clethodim resistance (Delye et al., 2005). As L. rigidum is a highly genetically variable species we expect that all possible herbicide resistance endowing mechanisms can be present and enriched in large populations of this species under herbicide selection (Powles and Matthews 1992). Thus, we expect that a number of different mutations endowing ACCase herbicide resistance (Table 1) could be enriched both within and between different resistant populations. Our hypothesis tested here is that field evolved ACCase herbicide resistant L. rigidum populations would be comprised of individuals carrying a diverse range of resistance endowing mutations and that individuals would be heterozygous or homozygous for one or any two possible combinations of different mutations. To examine this in depth we selected 12 clethodim resistant Australian L. rigidum populations, together with two resistant Italian Lolium populations. We reveal diversity and complexity in ACCase mutations in this weed species. We identify five ACCase mutations and reveal 12 combination patterns of mutant alleles (genotypes) from these 14 clethodim resistant Lolium populations. We demonstrate that a new mutation, Cys-2088-Arg, and the known mutations, Ile-1781-Leu and Asp-2078-Gly, endow resistance to clethodim and other ACCase herbicides. We establish and emphasize that the specific mutation, the homo/heterozygous status of a plant for a specific mutation, and combinations of different resistant alleles plus herbicide rates are all important in contributing to the overall level of herbicide resistance. In addition, we developed (derived) 6 www.plantphysiol.org on October 1, 2017 Published by Downloaded from Copyright © 2007 American Society of Plant Biologists. All rights reserved. cleaved amplified polymorphic sequence ((d)CAPS) markers for the 2041, 2078 and 2088 mutations to enable rapid detection of these mutations in the Lolium populations.