While there is universal recognition of the dangers of antimicrobial resistance (AMR) to human health, far less attention has been directed towards the steady growth of resistance to the pesticides and herbicides that safeguard global food security. As a major constraint on crop productivity, weed competition causes greater losses than invertebrate pests and fungal pathogens combined, with the development of herbicide resistance now a primary agronomic threat to arable agriculture and horticulture. Here in the UK, our dominant crop, winter wheat, is now subject to annual losses of 1 million tons of grain equating to an estimated £0.5 billion, primarily due to the mass evolution of herbicide resistance in the highly competitive weed blackgrass (Alopecurus myosuroides). Informed by strategies being developed in healthcare to combat AMR through its rapid identification, we now look to new tools to combat herbicide and pesticide resistance informed by molecular diagnostics.

Crop protection agents are a cornerstone of modern agriculture, preventing up to 40% losses in yield due to weed competition, pest damage and disease. Widely used since the 1940s, their deployment was integral to the success of Norman Borlaug’s green revolution, which saw 10% yield increases year on year in many staple crops up to the 1990s. However, from the 1970s onwards, the paradigm of perfect crops free of disease, pest damage and competing weeds has proven unsustainable, with resistance to pesticides and herbicides now widespread. Contributing factors have included the limited crop rotations used in many advanced agricultural systems, the steady loss of chemical control agents due to regulation and environmental concerns, a tailing off in the discovery of new agrochemicals and in all too many cases, poor stewardship in the use of existing products.

Here in the UK, the most visible example of resistance to agrochemicals can be seen in the explosion of herbicide resistance in grass weeds, notably blackgrass, in winter cereal crops (Figure 1). Rising from relative obscurity in the early 1980s, blackgrass is now widespread across the main grain-producing areas of central and eastern England (Figure 1). A similar story has unfolded on the continent, with herbicide-resistant blackgrass now a major problem across northern Europe. While blackgrass is theUK’s demon weed, we are by no means unique, with a steady rise of herbicide resistance reported in multiple non-domesticated plant species in different cropping systems across the world (Table 1).

Herbicide-resistant blackgrass as a major problem in arable crops of the UK. (a) Herbicide-resistant blackgrass in a barley crop. (b) A national audit of herbicide resistant blackgrass in England as reported in: https://projectblue.blob.core.windows.net/media/Default/Research%20Papers/Cereals%20and%20Oilseed/pr601-final-report-summary-v2.pdf

Figure 1
Herbicide-resistant blackgrass as a major problem in arable crops of the UK. (a) Herbicide-resistant blackgrass in a barley crop. (b) A national audit of herbicide resistant blackgrass in England as reported in: https://projectblue.blob.core.windows.net/media/Default/Research%20Papers/Cereals%20and%20Oilseed/pr601-final-report-summary-v2.pdf
Figure 1
Herbicide-resistant blackgrass as a major problem in arable crops of the UK. (a) Herbicide-resistant blackgrass in a barley crop. (b) A national audit of herbicide resistant blackgrass in England as reported in: https://projectblue.blob.core.windows.net/media/Default/Research%20Papers/Cereals%20and%20Oilseed/pr601-final-report-summary-v2.pdf
Table 1
The global incidence of herbicide-resistant grass weeds
SpeciesCommon nameArea affectedCrop
Alopecurus myosuroides Blackgrass Europe Wheat, barley 
Alopecurus aequalis Shortawn foxtail Asia Wheat, barley 
Avena fatua Wild oat Europe, North America, Australia, Asia Wheat, barley, canola 
Avena sterilis Sterile oat Australian, Europe Wheat, barley, canola 
Apera spica-venti Silky windgrass Europe Wheat, barley 
Bromus diandrus Riggut brome Australia Wheat 
Bromus sterilis Poverty brome Europe Wheat, oilseed rape 
Echinochloa colona Junglerice South America, USA Rice, maize, soybean 
Lolium perenne spp. multiflorum Italian ryegrass South America, USA, Europe Wheat, maize 
Lolium rigidum Rigid ryegrass Australia, Europe Wheat, barley, canola 
Setaria viridis Green foxtail North America, Europe Wheat, barley, canola 
SpeciesCommon nameArea affectedCrop
Alopecurus myosuroides Blackgrass Europe Wheat, barley 
Alopecurus aequalis Shortawn foxtail Asia Wheat, barley 
Avena fatua Wild oat Europe, North America, Australia, Asia Wheat, barley, canola 
Avena sterilis Sterile oat Australian, Europe Wheat, barley, canola 
Apera spica-venti Silky windgrass Europe Wheat, barley 
Bromus diandrus Riggut brome Australia Wheat 
Bromus sterilis Poverty brome Europe Wheat, oilseed rape 
Echinochloa colona Junglerice South America, USA Rice, maize, soybean 
Lolium perenne spp. multiflorum Italian ryegrass South America, USA, Europe Wheat, maize 
Lolium rigidum Rigid ryegrass Australia, Europe Wheat, barley, canola 
Setaria viridis Green foxtail North America, Europe Wheat, barley, canola 

Source: Heap, I. The international Herbicide-Resistant Weed Database (www.weedsciece.org: Accessed 27 April 2020)

To understand what is happening in blackgrass, we need to understand some more about the weed and the herbicides used to control it. Blackgrass has a growth habit that closely follows that of winter wheat: germinating in the autumn, over-wintering and then rapidly developing in the spring. As an obligately out-crossing species composed of genetically diverse populations, blackgrass is well placed to evolve to pretty much any selective pressure. Highly competitive, just 12 plants per square metre can reduce wheat yield by 5%. As such, farmers are anxious to control blackgrass using herbicide applications both before (pre-emergence) its germination and after (post-emergence). Over the years, the range of selective herbicides available for use in broad-acre cereal crops has been reduced due to environmental concerns, and as such, only a relatively small number of chemistries with a limited range of modes of action (MoA) remain available for use (Table 2). Typically, these herbicides are normally detoxified more rapidly in wheat than in blackgrass, allowing them to be used to control the weeds without crop damage.

Table 2
Selective herbicides used in Europe to control grass weeds in arable crops
HerbicideMode of actionHerbicide chemistry
Pre-germination   
Pendimethalin Microtubule assembly inhibition Dinitroaniline 
Flufenacet Inhibition of very long-chain fatty acid Oxyacetamide 
Post-germination   
Mesosulfuron-methyl+iodosulfuron-methyl-sodium Inhibition of acetolactate synthase (ALS) Sulfonylurea 
Isoproturon Inhibition of photosynthesis at photosystem II Urea 
Chlorotoluron Inhibition of photosynthesis at photosystem II Urea 
Fenoxaprop-ethyl Inhibition of acetyl CoA carboxylase (ACCase) Aryloxyphenoxy-propionate 
Clodinafop-propagyl Inhibition of ACCase Aryloxyphenoxy-propionate 
Pinoxaden Inhibition of ACCase Phenylpyrazoline 
Cycloxydim Inhibition of ACCase Cyclohexanedione 
HerbicideMode of actionHerbicide chemistry
Pre-germination   
Pendimethalin Microtubule assembly inhibition Dinitroaniline 
Flufenacet Inhibition of very long-chain fatty acid Oxyacetamide 
Post-germination   
Mesosulfuron-methyl+iodosulfuron-methyl-sodium Inhibition of acetolactate synthase (ALS) Sulfonylurea 
Isoproturon Inhibition of photosynthesis at photosystem II Urea 
Chlorotoluron Inhibition of photosynthesis at photosystem II Urea 
Fenoxaprop-ethyl Inhibition of acetyl CoA carboxylase (ACCase) Aryloxyphenoxy-propionate 
Clodinafop-propagyl Inhibition of ACCase Aryloxyphenoxy-propionate 
Pinoxaden Inhibition of ACCase Phenylpyrazoline 
Cycloxydim Inhibition of ACCase Cyclohexanedione 

Following repeated application, herbicide resistance can arise from two distinct mechanisms (Figure 2), termed target site resistance (TSR) and non-target site resistance (NTSR).

Molecular mechanisms of herbicide resistance. (a) Target site resistance (TSR) caused by spontaneous mutations results in proteins that can no longer be inhibited by herbicides. (b) Non-target site resistance (NTSR) is caused primarily through the enhanced detoxification of herbicides catalysed by cytochromes P450, glutathione transferases (GSTs), UDP-glucuronosyltransferases (UGTs), malonyl transferases (MTs) and transporter proteins.

Figure 2
Molecular mechanisms of herbicide resistance. (a) Target site resistance (TSR) caused by spontaneous mutations results in proteins that can no longer be inhibited by herbicides. (b) Non-target site resistance (NTSR) is caused primarily through the enhanced detoxification of herbicides catalysed by cytochromes P450, glutathione transferases (GSTs), UDP-glucuronosyltransferases (UGTs), malonyl transferases (MTs) and transporter proteins.
Figure 2
Molecular mechanisms of herbicide resistance. (a) Target site resistance (TSR) caused by spontaneous mutations results in proteins that can no longer be inhibited by herbicides. (b) Non-target site resistance (NTSR) is caused primarily through the enhanced detoxification of herbicides catalysed by cytochromes P450, glutathione transferases (GSTs), UDP-glucuronosyltransferases (UGTs), malonyl transferases (MTs) and transporter proteins.

In TSR, spontaneous mutations in the genes encoding proteins targeted by herbicides result in changes in polypeptide sequence and structure that reduce their ability to bind to the active agent. As such, the proteins become less sensitive to herbicide inhibition and maintain their normal essential functions in metabolism (Figure 2A). More rarely, TSR can also be caused by gene amplification leading to the massive over-expression of proteins targeted by herbicides. TSR allows weeds to acquire high levels of resistance to specific herbicide chemistries with a single MoA. TSR in weeds is typically selected for by repeated use of high doses of herbicides acting via the same MoA. Once established, TSR behaves as a single allelic trait that often imparts a fitness penalty to the host plant. However, in the field, TSR can be overcome by rotating herbicides with different MoAs (Table 2).

As its name suggests, NTSR constitutes all forms of resistance that do not result from TSR. Unlike TSR, NTSR extends to multiple herbicide classes exhibiting differing MoAs. It is caused by multiple cytoprotective mechanisms that prevent herbicide damage, through enhancing the detoxification, sequestration and exclusion of synthetic chemicals, as well as quenching the toxic events caused through the primary MoA (Figure 2B). The NTSR response is encoded by multiple genes, and it behaves as a complex multigenic and quantitative trait. In terms of conferring herbicide resistance, NTSR varies from being barely discernible to broad-ranging tolerance to multiple chemistries. It is a highly damaging form of resistance, evolving most rapidly when weeds are exposed to rotations of low doses of different classes of herbicide. There is growing evidence that the evolution of NTSR precedes that of TSR, resulting in ‘super-weeds’ that show both forms of resistance. With few new herbicides coming to market, these resistance mechanisms are now seriously challenging our ability to chemically control weeds such as blackgrass and, as a consequence, to continue to produce economically viable yields of major cereal crops.

Faced with the continuing spread of herbicide resistance in blackgrass, in 2014, a consortium of UK researchers from Rothamsted Research, the Institute of Zoology and the Universities of Sheffield and Newcastle formed the Blackgrass Resistance Initiative (BGRI; bgri.info/), with funding from the Biotechnology and Biological Sciences Research Council (BBSRC) and the Agriculture and Horticulture Development Board (AHDB). Using a combination of modelling, classical weed science and biochemical approaches, the BGRI team conducted a national audit of resistance towards post-emergence herbicides with the goals of studying their evolution, impact and underpinning molecular basis under field conditions (https://ahdb.org.uk/multiple-herbicide-resistance-in-grass-weeds). In addition to the fundamental science, the team worked with growers and agronomists to understand how the farming community was attempting to counteract herbicide resistance through different management practices.

The study quickly flagged up the difficulties of determining the types and levels of resistance in the many hundreds of weed populations encountered. Classical methods of testing for resistance consist of sampling multiple blackgrass plants, or seeds, taking them to the glasshouse or lab for propagation, then spraying with herbicides of differing MoAs at differing application rates. These assays are very useful in practically determining resistance to differing herbicides, but they have a major disadvantage in taking days and, in the case of seed samples, many weeks to yield useful results. While such tests can inform resistance management strategies for the next growing season, their use in real-time decision-making, whether to spray and, if so, which product, is limited. With this in mind, using the latest molecular detrection technologies and our knowledge of resistance mechanisms, we set ourselves the task of designing ‘point of care’ (POC) diagnostics to identify the presence of TSR and NTSR in blackgrass.

POC tests, delivering accurate and reliable diagnosis of disease and medical conditions, are routinely used in healthcare and have more recently been adopted in plant health applications. POC diagnostics typically detect DNA/RNA, proteins or small molecules. In each case, detection needs to be rapid, reliable and preferably low cost. Based on our knowledge of herbicide resistance, we chose two detection platforms: DNA for TSR and proteins for NTSR. In the case of the TSR DNA diagnostic, we needed a portable technology that could detect point mutations in genes encoding proteins such as acetolactate synthase (ALS) or acetyl- CoA carboxylase (ACCase) that are targeted by selective herbicides used in blackgrass control. Thus, specific point mutations in ALS result in TSR towards sulphonylureas and imidazolinones, while changes in ACCase sequences result in resistance towards aryloxyphenoxypropionate, phenylpyrazoline and cyclohexanedione herbicides. Conventionally, polymerase chain reaction (PCR) can be used in the laboratory to amplify potentially mutated gene sequences, with the single-nucleotide changes then identified by sequencing. This process can take at least 3 weeks from weed sampling to receiving results. Most recently, loop-mediated isothermal amplification (LAMP) has been developed for rapid POC applications, using a single-step reaction that utilizes four to six probes that bind to DNA regions around the mutation site. Through the quantification of fluorescent products arising from their specific melting temperatures, LAMP generates results within 30–45 minutes. Without the need for temperature cycling, LAMP instruments are portable and can be adapted to multiple applications using the same reagents while varying the probes, with a minimal need for sample clean-up (Figure 3). LAMP was adapted to detect mutations in ALS or ACCase genes in blackgrass, with the shifting of melting temperature of the amplification product being an indication of specific mutation events. This has allowed for the detection of gene mutations at two positions in ALS and five in ACCase. Based on this technology, it is now possible to diagnose the presence and nature of the major types of TSR in blackgrass on the farm within an hour of field sampling, with the technology adaptable to identify these defined resistance mechanisms in any grass weed.

Overview of DNA and protein molecular diagnostic tests for TSR and NTSR in grass weeds. TSR detection (left panel): DNA extracted from the leaf segments is transferred to reaction tubes, each containing mutation-specific probes. It undergoes LAMP amplification on a portable Genie III machine (Optigene Ltd.), with polymorphisms conferring TSR detected by the shift in melting temperature. NTSR detection (right panel): Following extraction of total protein from the leaves and sample application on the lateral flow device (LFD), the intensity of the test line due to the recognition of the NTSR protein biomarker is then quantified using an optical reader.

Figure 3
Overview of DNA and protein molecular diagnostic tests for TSR and NTSR in grass weeds. TSR detection (left panel): DNA extracted from the leaf segments is transferred to reaction tubes, each containing mutation-specific probes. It undergoes LAMP amplification on a portable Genie III machine (Optigene Ltd.), with polymorphisms conferring TSR detected by the shift in melting temperature. NTSR detection (right panel): Following extraction of total protein from the leaves and sample application on the lateral flow device (LFD), the intensity of the test line due to the recognition of the NTSR protein biomarker is then quantified using an optical reader.
Figure 3
Overview of DNA and protein molecular diagnostic tests for TSR and NTSR in grass weeds. TSR detection (left panel): DNA extracted from the leaf segments is transferred to reaction tubes, each containing mutation-specific probes. It undergoes LAMP amplification on a portable Genie III machine (Optigene Ltd.), with polymorphisms conferring TSR detected by the shift in melting temperature. NTSR detection (right panel): Following extraction of total protein from the leaves and sample application on the lateral flow device (LFD), the intensity of the test line due to the recognition of the NTSR protein biomarker is then quantified using an optical reader.

Due to its multigenic nature, NTSR is less amenable to DNA-based detection; so instead, functional protein biomarkers of this type of resistance in blackgrass were identified using proteomics. Using this approach, a single protein identified as a glutathione transferase (GST) of the phi (F) class, termed AmGSTF1, was identified as being strongly upregulated in all NTSR populations of blackgrass studied to date. Homologous proteins were also identified as being associated with NTSR in other grass weeds. In each case, AmGSTF1 abundance positively correlated with the level of NTSR, suggesting it is a universal and quantitative biomarker of this class of resistance. Using classical immunology, specific antibodies were raised against AmGSTF1 and used in enzyme-linked immunosorbent assay (ELISA) to AmGSTF1 protein in blackgrass extracts. In the POC style, using two distinct antibodies, the ELISA assay was adapted to work in a lateral flow format to detect AmGSTF1 in blackgrass within 10 minutes of sampling (Figure 3). The portable lateral flow devices (LFDs) based on this technology are cheap, robust and work on the same basis as home pregnancy testing kits. Since AmGSTF1 is a quantitative marker for NTSR, the intensity of the immunoreaction on the test line on the LFDs provides information on the levels of resistance that can be quantified using optical reader technology.

By bringing together the DNA diagnostics for TSR and the immunodiagnostics for NTSR, we now have the potential to accurately define all known herbicide resistance mechanisms in blackgrass, and potentially in other problem grass weeds within 1 hour of sampling problem populations in the field. Using the POC medical analogy, we can now build a decision tool that characterizes individual populations of weeds as to optimal resistance management strategies (Figure 4). Looking forward, our ambition is to use this type of technology to identify the evolution of resistance before it becomes an agronomic problem. Working with agronomists is already revealing new ways in which access to these real-time field-based diagnostics could revolutionize crop protection by giving us new tools to resist resistance to herbicides in blackgrass and other problem weeds.

Using molecular diagnostics as a decision tool to resist herbicide resistance. The digital agronomist mobile phone application combines TSR and NTSR results and integrates the information to direct the optimal selection of herbicides to restore weed control.

Figure 4
Using molecular diagnostics as a decision tool to resist herbicide resistance. The digital agronomist mobile phone application combines TSR and NTSR results and integrates the information to direct the optimal selection of herbicides to restore weed control.
Figure 4
Using molecular diagnostics as a decision tool to resist herbicide resistance. The digital agronomist mobile phone application combines TSR and NTSR results and integrates the information to direct the optimal selection of herbicides to restore weed control.

Further reading

  • Heap, I. (2014) Global perspective of herbicide-resistant weeds. Pest. Manag. Sci. 70, 1306–1315, 10.1002/ps.3696

  • Yu, Q and Powles, S.B. (2014) Metabolism-based herbicide resistance and cross-resistance in crop weeds: A threat to herbicide sustainability and global crop production. Plant Physiol. 166, 1106–1118, 10.1104/pp.114.242750

  • Tétard-Jones, C., Sabbadin, F., Moss, S., et al. (2018) Changes in the proteome of the problem weed blackgrass correlating with multiple-herbicide resistance. Plant J. 94, 709–720, 10.1111/tpj.13892

  • Hicks, H.L., Comont, D., Coutts, S.R., et al. (2018) The factors driving evolved herbicide resistance at a national scale. Nat. Ecol. Evol. 2, 529–536, 10.1038/s41559-018-0470-1

  • Varah, A., Ahodo, K., Coutts, S.R., et al. (2020) The costs of human-induced evolution in an agricultural system. Nat. Sustain. 3, 63–71, 10.1038/s41893-019-0450-8

Authors information

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Robert Edwards is the Head of the School of Natural and Environmental Sciences at Newcastle University and holds the Chair in Crop Protection with over 30 years of experience of working in both private and public sectors on the action and selectivity of herbicides. Email: Robert.edwards@newcastle.ac.uk

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Nawaporn Onkokesung is a plant molecular biologist with a specific interest in plant interactions with environmental conditions and adaptive response to environmental stress. She is a research associate in Professor Robert Edwards’ group. Her researches focus on deciphering molecular mechanisms of herbicide resistance in grass weeds. Email: nawaporn.onkokesung@newcastle.ac.uk

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