Molecular characterisation of resistance to ALS-inhibiting herbicides in Hordeum leporinum biotypes

BACKGROUND: The acetolactate synthase (ALS)-inhibiting herbicide sulfosulfuron is registered in Australia for the selective control of Hordeum leporinum Link. in wheat crops. This herbicide failed to control H. leporinum on two farms in Western Australia on its first use. This study aimed to determine the level of resistance of three H. leporinum biotypes, identify the biochemical and molecular basis and develop molecular markers for diagnostic analysis of the resistance. RESULTS: Dose-response studies revealed very high level (>340-fold) resistance to the sulfonylurea herbicides sulfosulfuron and sulfometuron. In vitro ALS assays revealed that resistance was due to reduced sensitivity of the ALS enzyme to herbicide inhibition. This altered ALS sensitivity in the resistant biotypes was found to be due to a mutation in the ALS gene resulting in amino acid proline to serine substitution at position 197. In addition, two- to threefold higher ALS activities were consistently found in the resistant biotypes, compared with the known susceptible biotype. Two cleaved amplified polymorphic sequence (CAPS) markers were developed for diagnostic testing of the resistant populations. CONCLUSION: This study established the first documented case of evolved ALS inhibitor resistance in H. leporinum and revealed that the molecular basis of resistance is due to a Pro to Ser mutation in the ALS gene.     

Relationship between weed dormancy and herbicide rotations: implications in resistance evolution

It is suggested that selection for late germinating seed cohorts is significantly associated with herbicide resistance in some cropping systems. In turn, it is conceivable that rotating herbicide modes of action selects for populations with mutations for increased secondary dormancy, thus partially overcoming the delaying effect of rotation on resistance evolution. Modified seed dormancy could affect management strategies – like herbicide rotation – that are used to prevent or control herbicide resistance. Here, we review the literature for data on seed dormancy and germination dynamics of herbicide‐resistant versus susceptible plants. Few studies use plant material with similar genetic backgrounds, so there are few really comparative data. Increased dormancy and delayed germination may co‐occur with resistance to ACCase inhibitors, but there is no clear‐cut link with resistance to other herbicide classes. Population shifts are due in part to pleiotropic effects of the resistance genes, but interaction with the cropping system is also possible. We provide an example of a model simulation that accounts for genetic diversity in the dormancy trait, and subsequent consequences for various cropping systems. We strongly recommend adding more accurate and detailed mechanistic modelling to the current tools used today to predict the efficiency of prevention and management of herbicide resistance. These models should be validated through long‐term experimental designs including mono‐herbicide versus chemical rotation in the field. © 2017 Society of Chemical Industry     

Cross-resistance patterns to ACCase-inhibiting herbicides conferred by mutant ACCase isoforms in Alopecurus myosuroides Huds. (black-grass), re-examined at the recommended herbicide field rate

BACKGROUND: Target‐site‐based resistance to acetyl‐CoA carboxylase (ACCase) inhibitors in Alopecurus myosuroides Huds. is essentially due to five substitutions (Isoleucine‐1781‐Leucine, Tryptophan‐2027‐Cysteine, Isoleucine‐2041‐Asparagine, Aspartate‐2078‐Glycine, Glycine‐2096‐Alanine). Recent studies suggested that cross‐resistance patterns associated with each mutation using a seed‐based bioassay may not accurately reflect field resistance. The authors aimed to connect the presence of mutant ACCase isoform(s) in A. myosuroides with resistance to five ACCase inhibitors (fenoxaprop, clodinafop, haloxyfop, cycloxydim, clethodim) sprayed at the recommended field rate. RESULTS: Results from spraying experiments and from seed‐based bioassays were consistent for all mutant isoforms except the most widespread, Leucine‐1781. In spraying experiments, Leucine‐1781 ACCase conferred resistance to clodinafop and haloxyfop. Some plants containing Leucine‐1781 or Alanine‐2096 ACCase, but not all, were also resistant to clethodim. CONCLUSION: Leucine‐1781, Cysteine‐2027, Asparagine‐2041 and Alanine‐2096 ACCases confer resistance to fenoxaprop, clodinafop and haloxyfop at field rates. Leucine‐1781 ACCase also confers resistance to cycloxydim at field rate. Glycine‐2078 ACCase confers resistance to all five herbicides at field rates. Only Glycine‐2078 ACCase confers clethodim resistance under optimal application conditions. It may be that Leucine‐1781 and Alanine‐2096 ACCases may also confer resistance to clethodim in the field if the conditions are not optimal for herbicide efficacy, or at reduced clethodim field rates. Copyright © 2008 Society of Chemical Industry     

A SNaPshot assay for the rapid and simple detection of known point mutations conferring resistance to ACCase‐inhibiting herbicides in Lolium spp.

Evolution of resistance to herbicides in weeds is becoming an increasing problem worldwide. To develop effective strategies for weed control, a thorough knowledge of the basis of resistance is required. Although non‐target‐site‐based resistance is widespread, target site resistance, often caused by a single nucleotide change in the gene encoding the target enzyme, is also a common factor affecting the efficacies of key herbicides. Therefore, fast and relatively simple high‐throughput screening methods to detect target site resistance mutations will represent important tools for monitoring the distribution and evolution of resistant alleles within weed populations. Here, we present a simple and quick method that can be used to simultaneously screen for up to 10 mutations from several target site resistance‐associated codons in a single reaction. As a proof of concept, this SNaPshot multiplex method was successfully applied to the genotyping of nine variable nucleotide positions in the CT domain of the chloroplastic ACCase gene from Lolium multiflorum plants from 54 populations. A total of 10 nucleotide substitutions at seven of these nine positions (namely codons 1781, 1999, 2027, 2041 2078, 2088 and 2096) are known to confer resistance to ACCase‐inhibiting herbicides. This assay has several advantages when compared with other methods currently in use in weed science. It can discriminate between different nucleotide changes at a single locus, as well as screening for SNPs from different target sites by pooling multiple PCR products within a single reaction. The method is scalable, allowing reactions to be carried out in either 96‐ or 384‐well plate formats, thus reducing work time and cost.     

Modelling spatio-temporal dynamics of herbicide resistance

A mathematical model has been developed for the risk assessment of the spread of genes conferring herbicide resistance in plant populations. The model combines an age-and-stage-structured population dynamic model, a population genetic model and a model of spatial spread. This is achieved by embedding a local matrix population model into a cellular automaton model with raster cells as spatial units. The dynamics of each cell is determined by both its local dynamics and the interaction with neighbouring cells. The model is applied to the evaluation of management strategies to delay or even to prevent long-term evolution of resistance in an annual grass weed. The results show that the appearance and spread of resistant genes is a highly non-linear process exhibiting threshold phenomena, which occur for a wide range of parameters. The properties of the seed survival curve constitute the `genetic memory' of the system and thus determine its long-term dynamics. It is possible to delay the evolution of resistance by suspension of treatment, reduction in herbicide application rate and introducing fallow periods. Spatial spread from an infested plot is inhibited by leaving untreated strips between adjacent fields.     

Cytochrome P450‐mediated herbicide metabolism in plants: current understanding and prospects

Cytochrome P450s (P450s) have been at the center of herbicide metabolism research as a result of their ability to endow selectivity in crops and resistance in weeds. In the last 20 years, ≈30 P450s from diverse plant species have been revealed to possess herbicide‐metabolizing function, some of which were demonstrated to play a key role in plant herbicide sensitivity. Recent research even demonstrated that some P450s from crops and weeds metabolize numerous herbicides from various chemical backbones, which highlights the importance of P450s in the current agricultural systems. However, due to the enormous number of plant P450s and the complexity of their function, expression and regulation, it remains a challenge to fully explore the potential of P450‐mediated herbicide metabolism in crop improvement and herbicide resistance mitigation. Differences in the substrate specificity of each herbicide‐metabolizing P450 are now evident. Comparisons of the substrate specificity and protein structures of P450s will be beneficial for the discovery of selective herbicides and may lead to the development of crops with higher herbicide tolerance by transgenics or genome‐editing technologies. Furthermore, the knowledge will help design sound management strategies for weed resistance including the prediction of cross‐resistance patterns. Overcoming the ambiguity of P450 function in plant xenobiotic pathways will unlock the full potential of this enzyme family in advancing global agriculture and food security. © 2020 Society of Chemical Industry     

First glyphosate‐resistant Lolium spp. biotypes found in a European annual arable cropping system also affected by ACCase and ALS resistance

In Europe, glyphosate‐resistant weeds have so far only been reported in perennial crops. Following farmers' complaints of poor herbicide efficacy, resistance to glyphosate as well as to ACCase and ALS inhibitors was investigated in 11 populations of Lolium spp. collected from annual arable cropping systems in central Italy. Field histories highlighted that farmers had relied heavily on glyphosate, often at low rates, as well as in a non‐registered crop. The research aimed at elucidating the resistance status, including multiple resistance, of Lolium spp. populations through glasshouse screenings and an outdoor dose–response experiment. Target‐site resistance mechanism was also investigated for the substitutions already reported for EPSPs, ALS and ACCase genes. Three different resistant patterns were identified: glyphosate resistant only, multiple resistant to glyphosate and ACCase inhibitors and multiple resistant to glyphosate and ALS inhibitors. Amino acid substitutions were found at position 106 of the EPSPs gene, at position 1781, 2088 and 2096 of the ACCase gene and at position 197 and 574 of the ALS gene. Not all populations displayed amino acid substitutions, suggesting the presence of non‐target‐site‐mediated resistance mechanisms. After 39 years of commercial availability of glyphosate, this is the first report of multiple resistance involving glyphosate selected in annual arable crops in Europe. Management implications and options are discussed.     

Leaf chlorophyll fluorescence discriminates herbicide resistance in Echinochloa species

Timely detection of herbicide resistance at an early stage of crop cultivation is essential to help farmers find alternative solutions to manage herbicide resistance in their fields. In this study, maximum quantum yield of PS II [F_v/F_m = (F_m – F_o)/F_m] was measured at the 4–5 leaf stage to discriminate between herbicide‐resistant and susceptible biotypes of Echinochloa species. The differences in F_v/F_m between herbicide‐resistant and susceptible Echinochloa spp. were consistent with the whole‐plant assay based on I₅₀ (herbicide doses causing a 50% inhibition of F_v/F_m) and GR₅₀ (herbicide doses causing a 50% reduction in plant fresh weight) values and R/S ratios (herbicide resistance index), regardless of the mode of action of the tested herbicides. A PS II inhibitor caused the fastest inhibition of F_v/F_m, compared with ACCase and ALS inhibitors, after herbicide treatment. The required time for discrimination between herbicide‐resistant and susceptible Echinochloa spp. was 64 h after PS II inhibitor treatment, much shorter than those of ACCase and ALS inhibitor‐treated plants, which required 168 and 192 h respectively. The leaf chlorophyll fluorescence assay provided reliable diagnostics of herbicide resistance in Echinochloa spp. with significant time savings and convenient measurement in field conditions compared with the conventional whole‐plant assay.     

Modelling different cultivation and herbicide strategies for their effect on herbicide resistance in Alopecurus myosuroides

A single dominant mutation conferring resistance to aryloxyphenoxypropionate (AOPP) and cyclohexanedione (CHD) herbicides was incorporated into a quantitative model for the population development of Alopecurus myosuroide s Huds. The model predicts that from an initial seedbank of 100 seed m^–2, 10^–6 of which mutate to resistance each generation, and annual use of AOPP/CHD herbicides which kill 90% of susceptible but no resistant plants, a threshold of 10 plants m^–2 surviving herbicides ('field resistance') will develop: in 9–10 years if all tillage is by tine cultivation to 10 cm deep; after 28–30 years of annual ploughing; in 12 years if tine cultivations are interspersed with ploughing once every 4 years. If AOPP/CHD herbicides are alternated with herbicides with different modes of action, outcomes depend on the annual kill rate: with 95% kill (of susceptible plants by AOPP/CHDs and all plants by alternative herbicides) and tine cultivation, field resistance develops in 22 years; however, resistance can be delayed for 45 years if AOPP/CHDs are rotated with two additional herbicides, each with a different mode of action. The model predictions on the number of years required for field resistance to develop are not highly sensitive to the density of the seedbank or the initial frequency of resistance.