Standing genetic variation fuels rapid evolution of herbicide resistance in blackgrass

Repeated herbicide applications exert enormous selection on blackgrass (Alopecurus myosuroides), a major weed in cereal crops of the temperate climate zone including Europe. This inadvertent large-scale experiment gives us the opportunity to look into the underlying genetic mechanisms and evolutionary processes of rapid adaptation, which can occur both through mutations in the direct targets of herbicides and through changes in other, often metabolic, pathways, known as non-target-site resistance. How much either type of adaptation relies on de novo mutations versus pre-existing standing variation is important for developing strategies to manage herbicide resistance. We generated a chromosome-level reference genome for A. myosuroides for population genomic studies of herbicide resistance and genome-wide diversity across Europe in this species. Bulked-segregant analysis evidenced that non-target-site resistance has a complex genetic architecture. Through empirical data and simulations, we showed that, despite its simple genetics, target-site resistance mainly results from standing genetic variation, with only a minor role for de novo mutations.

First Detection and Characterization of Cross- and Multiple Resistance to Acetyl-CoA Carboxylase (ACCase)- and Acetolactate Synthase (ALS)-Inhibiting Herbicides in Black-Grass (Alopecurus myosuroides) and Italian Ryegrass (Lolium multiflorum) Populations from Ireland

Understanding the resistance spectrum and underlying genetic mechanisms is critical for managing herbicide-resistant populations. In this study, resistance to acetyl CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors was investigated in four suspected resistant populations of Alopecurus myosuroides (ALOMY-001 to ALOMY-004) and Lolium multiflorum (LOLMU-001 to LOLMU-004), collected from cereal production fields in Ireland. Glasshouse assays with three ALOMY-active herbicides [propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron (ALS)] or five LOLMU-active herbicides [pinoxaden, propaquizafop, cycloxydim (ACCase) and mesosulfuron + iodosulfuron, pyroxsulam (ALS)], and target-site resistance mechanism studies, based on pyrosequencing, were carried out in each of those populations. For A. myosuroides, Ile-1781-Leu ACCase mutation contributed to propaquizafop and cycloxydim resistance (shoot dry weight GR50 resistance factor (RF) = 7.5–35.5) in all ALOMY populations, and the independent Pro-197-Thr or Pro-197-Ser ALS mutation contributed to mesosulfuron + iodosulfuron resistance (RF = 3.6–6.6), in ALOMY-002 to ALOMY-004. Most of the analyzed plants for these mutations were homo/heterozygous combinations or only heterozygous. For L. multiflorum, phenotypic resistance to mesosulfuron + iodosulfuron (RF = 11.9–14.6) and pyroxsulam (RF = 2.3–3.1) was noted in all LOLMU populations, but the Pro-197-Gln or Pro-197-Leu ALS mutation (mostly in homozygous status) was identified in LOLMU-001, LOLMU-002 and LOLMU-004 only. Additionally, despite no known ACCase mutations in any LOLMU populations, LOLMU-002 survived pinoxaden and propaquizafop application (RF = 3.4 or 1.3), and LOLMU-003 survived pinoxaden (RF = 2.3), suggesting the possibility of non-target-site resistance mechanisms for ACCase and/or ALS resistance in these populations. Different resistance levels, as evidenced by a reduction in growth as dose increased above field rates in ALOMY and LOLMU, were due to variations in mutation rate and the level of heterozygosity, resulting in an overall resistance rating of low to moderate. This is the first study confirming cross- and multiple resistance to ACCase- and ALS-inhibiting herbicides, highlighting that resistance monitoring in A. myosuroides and L. multiflorum in Ireland is critical, and the adoption of integrated weed management strategies (chemical and non-chemical/cultural strategies) is essential.

Impact of a Novel W2027L Mutation and Non-Target Site Resistance on Acetyl-CoA Carboxylase-Inhibiting Herbicides in a French Lolium multiflorum Population

Herbicides that inhibit acetyl-CoA carboxylase (ACCase) are among the few remaining options for the post-emergence control of Lolium species in small grain cereal crops. Here, we determined the mechanism of resistance to ACCase herbicides in a Lolium multiflorum population (HGR) from France. A combined biological and molecular approach detected a novel W2027L ACCase mutation that affects aryloxyphenoxypropionate (FOP) but not cyclohexanedione (DIM) or phenylpyraxoline (DEN) subclasses of ACCase herbicides. Both the wild-type tryptophan and mutant leucine 2027-ACCase alleles could be positively detected in a single DNA-based-derived polymorphic amplified cleaved sequence (dPACS) assay that contained the targeted PCR product and a cocktail of two discriminating restriction enzymes. Additionally, we identified three well-characterised I1781L, I2041T, and D2078G ACCase target site resistance mutations as well as non-target site resistance in HGR. The non-target site component endowed high levels of resistance to FOP herbicides whilst partially impacting on the efficacy of pinoxaden and cycloxydim. This study adequately assessed the contribution of the W2027L mutation and non-target site mechanism in conferring resistance to ACCase herbicides in HGR. It also highlights the versatility and robustness of the dPACS method to simultaneously identify different resistance-causing alleles at a single ACCase codon.

The analysis of reference genes expression stability in susceptible and resistant Apera spica-venti populations under herbicide treatment

Weed resistance to herbicides constitutes a serious problem to world crop production. One of the weeds that are significantly threatening the crops’ yield and quality is Apera spica-venti. The target-site resistance (TSR) mechanism of A. spica-venti has been widely studied, though, little is known about its non-target-site resistance (NTSR) mechanisms at the molecular level. Molecular examination of NTSR is, to a great extent, based on the expression profiles of selected genes, e.g. those participating in detoxification. However, to obtain reliable results of gene expression analysis, the use of a normalizer is required. The aim of this study was to select the best reference genes in A. spica-venti plants of both populations, susceptible and resistant to ALS inhibitor, under treatment with herbicide. Eleven housekeeping genes were chosen for their expression stability assessment. The efficiency correction of raw quantification cycles (Cq) was included in the gene expression stability analyses, which resulted in indicating the TATA-box binding protein (TBP), glyceraldehyde-3-phosphate dehydrogenase, cytosolic (GAPC), and peptidyl-prolyl cis–trans isomerase CYP28 (CYP28) genes as the most stably expressed reference genes. The obtained results are of vital importance for future studies on the expression of genes associated with the non-target-site resistance mechanisms in the A. spica-venti populations susceptible and resistant to herbicides.

A high diversity of mechanisms endows ALS-inhibiting herbicide resistance in the invasive common ragweed (Ambrosia artemisiifolia L.)

Ambrosia artemisiifolia L. (common ragweed) is a globally invasive, allergenic, troublesome arable weed. ALS-inhibiting herbicides are broadly used in Europe to control ragweed in agricultural fields. Recently, ineffective treatments were reported in France. Target site resistance (TSR), the only resistance mechanism described so far for ragweed, was sought using high-throughput genotyping-by-sequencing in 213 field populations randomly sampled based on ragweed presence. Additionally, non-target site resistance (NTSR) was sought and its prevalence compared with that of TSR in 43 additional field populations where ALS inhibitor failure was reported, using herbicide sensitivity bioassay coupled with ALS gene Sanger sequencing. Resistance was identified in 46 populations and multiple, independent resistance evolution demonstrated across France. We revealed an unsuspected diversity of ALS alleles underlying resistance (9 amino-acid substitutions involved in TSR detected across 24 populations). Remarkably, NTSR was ragweed major type of resistance to ALS inhibitors. NTSR was present in 70.5% of the resistant plants and 74.1% of the fields harbouring resistance. A variety of NTSR mechanisms endowing different resistance patterns evolved across populations. Our study provides novel data on ragweed resistance to herbicides, and emphasises that local resistance management is as important as mitigating gene flow from populations where resistance has arisen.