Acting pre-emptively reduces the long-term costs of managing herbicide resistance.

Globally, pesticides improve crop yields but at great environmental cost, and their overuse has caused resistance. This incurs large financial and production losses but, despite this, very diversified farm management that might delay or prevent resistance is uncommon in intensive farming. We asked farmers to design more diversified cropping strategies aimed at controlling herbicide resistance, and estimated resulting weed densities, profits, and yields compared to prevailing practice. Where resistance is low, it is financially viable to diversify pre-emptively; however, once resistance is high, there are financial and production disincentives to adopting diverse rotations. It is therefore as important to manage resistance before it becomes widespread as it is to control it once present. The diverse rotations targeting high resistance used increased herbicide application frequency and volume, contributing to these rotations' lack of financial viability, and raising concerns about glyphosate resistance. Governments should encourage adoption of diverse rotations in areas without resistance. Where resistance is present, governments may wish to incentivise crop diversification despite the drop in wheat production as it is likely to bring environmental co-benefits. Our research suggests we need long-term, proactive, food security planning and more integrated policy-making across farming, environment, and health arenas.

The role of interspecific variability and herbicide pre-adaptation in the cinmethylin response of Alopecurus myosuroides.

BACKGROUND: Cinmethylin is an inhibitor of plant fatty acid biosynthesis, with in-plant activity caused by its binding to fatty acid thioesterases (FATs). The recent registration of cinmethylin for pre-emergence herbicidal use in the UK represents a new mode-of-action (MOA) for control of the grassweed blackgrass (Alopecurus myosuroides). To date there is little published information on the extent of blackgrass' inter-population variability in sensitivity to cinmethylin, nor on any potential effect of existing non-target-site resistance (NTSR) mechanisms on cinmethylin efficacy. RESULTS: Here we present a study of variability in cinmethylin sensitivity amongst 97 UK blackgrass populations. We demonstrate that under controlled conditions, a UK field-rate dose of 500 g ha-1 provides effective control of the tested populations. Nevertheless, we reveal significant inter-population variability at doses below this rate, with populations previously characterised as strongly NTSR displaying the lowest sensitivity to cinmethylin. Assessment of paired resistant 'R' and sensitive 'S' lines from standardised genetic backgrounds confirms that selection for NTSR to the acetyl-CoA-carboxylase inhibitor fenoxaprop, and the microtubule assembly inhibitor pendimethalin, simultaneously results in reduced sensitivity to cinmethylin at doses below 500 g ha-1 . Whilst we find no resistance to the field-rate dose, we reveal that cinmethylin sensitivity can be further reduced through experimental selection with cinmethylin. CONCLUSION: Cinmethylin therefore represents a much-needed further MOA for blackgrass control, but needs to be carefully managed within a resistance monitoring and integrated weed management (IWM) framework to maximise the effective longevity of this compound. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

RNA and protein biomarkers for detecting enhanced metabolic resistance to herbicides mesosulfuron-methyl and fenoxaprop-ethyl in black-grass (Alopecurus myosuroides).

BACKGROUND: The evolution of non-target site resistance (NTSR) to herbicides leads to a significant reduction in herbicide control of agricultural weed species. Detecting NTSR in weed populations prior to herbicide treatment would provide valuable information for effective weed control. While not all NTSR mechanisms have been fully identified, enhanced metabolic resistance (EMR) is one of the better studied, conferring tolerance through increased herbicide detoxification. Confirming EMR towards specific herbicides conventionally involves detecting metabolites of the active herbicide molecule in planta, but this approach is time-consuming and requires access to well-equipped laboratories. RESULTS: In this study, we explored the potential of using molecular biomarkers to detect EMR before herbicide treatment in black-grass (Alopecurus myosuroides). We tested the reliability of selected biomarkers to predict EMR and survival after herbicide treatments in both reference and 27 field-derived black-grass populations collected from sites across the UK. The combined analysis of the constitutive expression of biomarkers and metabolism studies confirmed three proteins, namely, AmGSTF1, AmGSTU2 and AmOPR1, as differential biomarkers of EMR toward the herbicides fenoxaprop-ethyl and mesosulfuron in black-grass. CONCLUSION: Our findings demonstrate that there is potential to use molecular biomarkers to detect EMR toward specific herbicides in black-grass without reference to metabolism analysis. However, biomarker development must include testing at both transcript and protein levels in order to be reliable indicators of resistance. This work is a first step towards more robust resistance biomarker development, which could be expanded into other herbicide chemistries for on-farm testing and monitoring EMR in uncharacterised black-grass populations. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Sequence Characterization of Extra-Chromosomal Circular DNA Content in Multiple Blackgrass (Alopecurus myosuroides) Populations.

Alopecurus myosuroides (blackgrass) is a problematic weed of Western European winter wheat, and its success is largely due to widespread multiple-herbicide resistance. Previous analysis of F2 seed families derived from two distinct blackgrass populations exhibiting equivalent non-target site resistance (NTSR) phenotypes shows resistance is polygenic and evolves from standing genetic variation. Using a CIDER-seq pipeline, we show that herbicide-resistant (HR) and herbicide-sensitive (HS) F3 plants from these F2 seed families as well as the parent populations they were derived from carry extra-chromosomal circular DNA (eccDNA). We identify the similarities and differences in the coding structures within and between resistant and sensitive populations. Although the numbers and size of detected eccDNAs varied between the populations, comparisons between the HR and HS blackgrass populations identified shared and unique coding content, predicted genes, and functional protein domains. These include genes related to herbicide detoxification such as Cytochrome P450s, ATP-binding cassette transporters, and glutathione transferases including AmGSTF1. eccDNA content was mapped to the A. myosuroides reference genome, revealing genomic regions at the distal end of chromosome 5 and the near center of chromosomes 1 and 7 as regions with a high number of mapped eccDNA gene density. Mapping to 15 known herbicide-resistant QTL regions showed that the eccDNA coding sequences matched twelve, with four QTL matching HS coding sequences; only one region contained HR coding sequences. These findings establish that, like other pernicious weeds, blackgrass has eccDNAs that contain homologs of chromosomal genes, and these may contribute genetic heterogeneity and evolutionary innovation to rapidly adapt to abiotic stresses, including herbicide treatment.

Novel and holistic approaches are required to realize allelopathic potential for weed management.

Allelopathy, that is, plant-plant inhibition via the release of secondary metabolites into the environment, has potential for the management of weeds by circumventing herbicide resistance. However, mechanisms underpinning allelopathy are notoriously difficult to elucidate, hindering real-world application either in the form of commercial bioherbicides or allelopathic crops. Such limited application is exemplified by evidence of limited knowledge of the potential benefits of allelopathy among end users. Here, we examine potential applications of this phenomenon, paying attention to novel approaches and influential factors requiring greater consideration, with the intention of improving the reputation and uptake of allelopathy. Avenues to facilitate more effective allelochemical discovery are also considered, with a view to stimulating the identification of new compounds and allelopathic species. Synthesis and Applications: We conclude that tackling increasing weed pressure on agricultural productivity would benefit from greater integration of the phenomenon of allelopathy, which in turn would be greatly served by a multi-disciplinary and exhaustive approach, not just through more effective isolation of the interactions involved, but also through greater consideration of factors which may influence them in the field, facilitating optimization of their benefits for weed management.

The blackgrass genome reveals patterns of non-parallel evolution of polygenic herbicide resistance.

Globally, weedy plants are a major constraint to sustainable crop production. Much of the success of weeds rests with their ability to rapidly adapt in the face of human-mediated management of agroecosystems. Alopecurus myosuroides (blackgrass) is a widespread and impactful weed affecting agriculture in Europe. Here we report a chromosome-scale genome assembly of blackgrass and use this reference genome to explore the genomic/genetic basis of non-target site herbicide resistance (NTSR). Based on our analysis of F2 seed families derived from two distinct blackgrass populations with the same NTSR phenotype, we demonstrate that the trait is polygenic and evolves from standing genetic variation. We present evidence that selection for NTSR has signatures of both parallel and non-parallel evolution. There are parallel and non-parallel changes at the transcriptional level of several stress- and defence-responsive gene families. At the genomic level, however, the genetic loci underpinning NTSR are different (non-parallel) between seed families. We speculate that variation in the number, regulation and function of stress- and defence-related gene families enable weedy species to rapidly evolve NTSR via exaptation of genes within large multi-functional gene families. These results provide novel insights into the potential for, and nature of plant adaptation in rapidly changing environments.

Dissecting weed adaptation: Fitness and trait correlations in herbicide-resistant Alopecurus myosuroides.

BACKGROUND: Unravelling the genetic architecture of non-target-site resistance (NTSR) traits in weed populations can inform questions about the inheritance, trade-offs and fitness costs associated with these traits. Classical quantitative genetics approaches allow study of the genetic architecture of polygenic traits even where the genetic basis of adaptation remains unknown. These approaches have the potential to overcome some of the limitations of previous studies into the genetics and fitness of NTSR. RESULTS: Using a quantitative genetic analysis of 400 pedigreed Alopecurus myosuroides seed families from nine field-collected populations, we found strong heritability for resistance to the acetolactate synthase and acetyl CoA carboxylase inhibitors (h2  = 0.731 and 0.938, respectively), and evidence for shared additive genetic variance for resistance to these two different herbicide modes of action, rg  = 0.34 (survival), 0.38 (biomass). We find no evidence for genetic correlations between life-history traits and herbicide resistance, indicating that resistance to these two modes of action is not associated with large fitness costs in blackgrass. We do, however, demonstrate that phenotypic variation in plant flowering characteristics is heritable, h2  = 0.213 (flower height), 0.529 (flower head number), 0.449 (time to flowering) and 0.372 (time to seed shed), demonstrating the potential for adaptation to other nonchemical management practices (e.g. mowing of flowering heads) now being adopted for blackgrass control. CONCLUSION: These results highlight that quantitative genetics can provide important insight into the inheritance and genetic architecture of NTSR, and can be used alongside emerging molecular techniques to better understand the evolutionary and fitness landscape of herbicide resistance. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Population genomics of selectively neutral genetic structure and herbicide resistance in UK populations of Alopecurus myosuroides.

BACKGROUND: Alopecurus myosuroides (blackgrass) is a major weed in Europe with known resistance to multiple herbicide modes of action. In the UK, there is evidence that blackgrass has undergone a range expansion. In this paper, genotyping-by-sequencing and population-level herbicide resistance phenotypes are used to explore spatial patterns of selectively neutral genetic variation and resistance. We also perform a preliminary genome-wide association study (GWAS) and genomic prediction analysis to evaluate the potential of these approaches for investigating nontarget site herbicide resistance. RESULTS: Blackgrass was collected from 47 fields across the British Isles and up to eight plants per field population (n = 369) were genotyped by Restriction site-associated DNA (RAD)-sequencing. A total of 20 426 polymorphic loci were identified and used for population genetic analyses. Phenotypic assays revealed significant variation in herbicide resistance between populations. Population structure was weak (FST = 0.024-0.048), but spatial patterns were consistent with an ongoing westward and northward range expansion. We detected strong and consistent Wahlund effects (FIS = 0.30). There were no spatial patterns of herbicide resistance or evidence for confounding with population structure. Using a combination of population-level GWAS and genomic prediction we found that the top 20, 200, and 2000 GWAS loci had higher predictive abilities for fenoxaprop resistance compared to all markers. CONCLUSION: There is likely extensive human-mediated gene flow between field populations of the weed blackgrass at a national scale. The lack of confounding of adaptive and neutral genetic variation can enable future, more extensive GWAS analyses to identify the genetic architecture of evolved herbicide resistance. © 2020 Society of Chemical Industry.

Publisher Correction: Evolution of generalist resistance to herbicide mixtures reveals a trade-off in resistance management.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Evolution of generalist resistance to herbicide mixtures reveals a trade-off in resistance management.

Intense selection by pesticides and antibiotics has resulted in a global epidemic of evolved resistance. In agriculture and medicine, using mixtures of compounds from different classes is widely accepted as optimal resistance management. However, this strategy may promote the evolution of more generalist resistance mechanisms. Here we test this hypothesis at a national scale in an economically important agricultural weed: blackgrass (Alopecurus myosuroides), for which herbicide resistance is a major economic issue. Our results reveal that greater use of herbicide mixtures is associated with lower levels of specialist resistance mechanisms, but higher levels of a generalist mechanism implicated in enhanced metabolism of herbicides with diverse modes of action. Our results indicate a potential evolutionary trade-off in resistance management, whereby attempts to reduce selection for specialist resistance traits may promote the evolution of generalist resistance. We contend that where specialist and generalist resistance mechanisms co-occur, similar trade-offs will be evident, calling into question the ubiquity of resistance management based on mixtures and combination therapies.

The costs of human-induced evolution in an agricultural system.

Pesticides have underpinned significant improvements in global food security, albeit with associated environmental costs. Currently, the yield benefits of pesticides are threatened as overuse has led to wide-scale evolution of resistance. Yet despite this threat, there are no large-scale estimates of crop yield losses or economic costs due to resistance. Here, we combine national-scale density and resistance data for the weed Alopecurus myosuroides (black-grass) with crop yield maps and a new economic model to estimate that the annual cost of resistance in England is £0.4bn in lost gross profit (2014 prices), and annual wheat yield loss due to resistance is 0.8 million tonnes. A total loss of herbicide control against black-grass would cost £1bn and 3.4 million tonnes of lost wheat yield annually. Worldwide, there are 253 herbicide-resistant weeds, so the global impact of resistance could be enormous. Our research provides an urgent case for national-scale planning to combat further evolution of resistance, and an incentive for policies focused on increasing yields through more sustainable food-production systems rather than relying so heavily on herbicides.

Alterations in Life-History Associated With Non-target-site Herbicide Resistance in Alopecurus myosuroides.

The evolution of resistance to herbicides is a classic example of rapid contemporary adaptation in the face of a novel environmental stress. Evolutionary theory predicts that selection for resistance will be accompanied by fitness trade-offs in environments where the stress is absent. Alopecurus myosuroides, an autumn-germinating grass weed of cereal crops in North-West Europe, has evolved resistance to seven herbicide modes-of-action, making this an ideal species to examine the presence and magnitudes of such fitness costs. Here, we use two contrasting A. myosuroides phenotypes derived from a common genetic background, one with enhanced metabolism resistance to a commercial formulation of the sulfonylurea (ALS) actives mesosulfuron and iodosulfuron, and the other with susceptibility to these actives (S). Comparisons of plant establishment, growth, and reproductive potential were made under conditions of intraspecific competition, interspecific competition with wheat, and over a gradient of nitrogen deprivation. Herbicide dose response assays confirmed that the two lines had contrasting resistance phenotypes, with a 20-fold difference in resistance between them. Pleiotropic effects of resistance were observed during plant development, with R plants having a greater intraspecific competitive effect and longer tiller lengths than S plants during vegetative growth, but with S plants allocating proportionally more biomass to reproductive tissues during flowering. Direct evidence of a reproductive cost of resistance was evident in the nitrogen deprivation experiment with R plants producing 27% fewer seed heads per plant, and a corresponding 23% reduction in total seed head length. However, these direct effects of resistance on fecundity were not consistent across experiments. Our results demonstrate that a resistance phenotype based on enhanced herbicide metabolism has pleiotropic impacts on plant growth, development and resource partitioning but does not support the hypothesis that resistance is associated with a consistent reproductive fitness cost in this species. Given the continued difficulties associated with unequivocally detecting costs of herbicide resistance, we advocate future studies that adopt classical evolutionary quantitative genetics approaches to determine genetic correlations between resistance and fitness-related plant life history traits.

Evolutionary epidemiology predicts the emergence of glyphosate resistance in a major agricultural weed.

The evolution of resistance to herbicides is a striking example of rapid, human-directed adaptation with major consequences for food production. Most studies of herbicide resistance are performed reactively and focus on post hoc determination of resistance mechanisms following the evolution of field resistance. If the evolution of resistance can be anticipated, however, pro-active management to slow or prevent resistance traits evolving can be advocated. We report a national-scale study that combines population monitoring, glyphosate sensitivity assays, quantitative genetics and epidemiological analyses to pro-actively identify the prerequisites for adaptive evolution (directional selection and heritable genetic variation) to the world's most widely used herbicide (glyphosate) in a major, economically damaging weed species, Alopecurus myosuroides. Results highlighted pronounced, heritable variability in glyphosate sensitivity amongst UK A. myosuroides populations. We demonstrated a direct epidemiological link between historical glyphosate selection and current population-level sensitivity, and show that current field populations respond to further glyphosate selection. This study provides a novel, pro-active assessment of adaptive potential for herbicide resistance, and provides compelling evidence of directional selection for glyphosate insensitivity in advance of reports of field resistance. The epidemiological approach developed can provide a basis for further pro-active study of resistance evolution across pesticide resistance disciplines.

The factors driving evolved herbicide resistance at a national scale.

Repeated use of xenobiotic chemicals has selected for the rapid evolution of resistance, threatening health and food security at a global scale. Strategies for preventing the evolution of resistance include cycling and mixtures of chemicals and diversification of management. We currently lack large-scale studies that evaluate the efficacy of these different strategies for minimizing the evolution of resistance. Here we use a national-scale data set of occurrence of the weed Alopecurus myosuroides (black-grass) in the United Kingdom to address this. Weed densities are correlated with assays of evolved resistance, supporting the hypothesis that resistance is driving weed abundance at a national scale. Resistance was correlated with the frequency of historical herbicide applications, suggesting that evolution of resistance is primarily driven by intensity of exposure to herbicides, but was unrelated directly to other cultural techniques. We find that populations resistant to one herbicide are likely to show resistance to multiple herbicide classes. Finally, we show that the economic costs of evolved resistance are considerable: loss of control through resistance can double the economic costs of weeds. This research highlights the importance of managing threats to food production and healthcare systems using an evolutionarily informed approach in a proactive not reactive manner.

Measuring the effectiveness of management interventions at regional scales by integrating ecological monitoring and modelling.

BACKGROUND: Because of site-specific effects and outcomes, it is often difficult to know whether a management strategy for the control of pests has worked or not. Population dynamics of pests are typically spatially and temporally variable. Moreover, interventions at the scale of individual fields or farms are essentially unreplicated experiments; a decrease in a target population following management cannot safely be interpreted as success because, for example, it might simply be a poor year for that species. Here, we argue that if large-scale data are available, population models can be used to measure outcomes against the prevailing mean and variance. We apply this approach to the problem of rotational management of the weed Alopecurus myosuroides. RESULTS: We derived density-structured population models for a set of fields that were not subject to rotational management (continuous winter wheat) and another group that were (rotated into spring barley to control A. myosuroides). We used these models to construct means and variances of the outcomes of management for given starting conditions, and to conduct transient growth analysis. We show that, overall, this management strategy is successful in reducing densities of weeds, albeit with considerable variance. However, we also show that one variant (rotation to spring barley along with variable sowing) shows little evidence for additional control. CONCLUSION: Our results suggest that rotational strategies can be effective in the control of this weed, but also that strategies require careful evaluation against a background of spatiotemporal variation. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Latitudinal variation in ambient UV-B radiation is an important determinant of Lolium perenne forage production, quality, and digestibility.

Few studies to date have considered the responses of agriculturally important forage grasses to UV-B radiation. Yet grasses such as Lolium perenne have a wide current distribution, representing exposure to a significant variation in ambient UV-B. The current study investigated the responses of L. perenne (cv. AberDart) to a simulated latitudinal gradient of UV-B exposure, representing biologically effective UV-B doses at simulated 70, 60, 50, 40, and 30° N latitudes. Aspects of growth, soluble compounds, and digestibility were assessed, and results are discussed in relation to UV-B effects on forage properties and the implications for livestock and bio-ethanol production. Aboveground biomass production was reduced by approximately 12.67% with every 1 kJ m(-2) day(-1) increase in biologically weighted UV-B. As a result, plants grown in the highest UV-B treatment had a total biomass of just 13.7% of controls. Total flavonoids were increased by approximately 76% by all UV-B treatments, while hydroxycinnamic acids increased in proportion to the UV-B dose. Conversely, the digestibility of the aboveground biomass and concentrations of soluble fructans were reduced by UV-B exposure, although soluble sucrose, glucose, and fructose concentrations were unaffected. These results highlight the capacity for UV-B to directly affect forage productivity and chemistry, with negative consequences for digestibility and bioethanol production. Results emphasize the need for future development and distribution of L. perenne varieties to take UV-B irradiance into consideration.

Acclimation and interaction between drought and elevated UV-B in A. thaliana: Differences in response over treatment, recovery and reproduction.

Here, a factorial experiment was used to investigate the interactive effects of a UV-B episode and concurrent progressive drought on the growth, chemistry, and reproductive success of A. thaliana. Both drought and UV-B negatively affected rosette growth, although UV-B had the greater effect. Acclimation to UV-B involved adjustment of leaf morphology, while drought induced accumulation of soluble sugars and phenolics. All plants recovered from treatments, but the cost of recovery was a developmental delay resulting in alteration in phenological timings. Combined treatments interacted causing additive negative effects on growth following exposure. This may be linked with inhibition of soluble sugar accumulation by UV-B, restricting the capacity for osmotic adjustment in response to drought. Following cessation of treatments, relative growth rate (RGR) and net assimilation rate (NAR) were significantly stimulated in plants treated with combined drought and UV-B. This interaction alleviated subsequent impacts of elevated UV-B on silique yield and reproductive timings. This study demonstrates the potential for interaction between these two common environmental factors. Furthermore, it shows the changeable nature of these interactions over the course of exposure and recovery through to reproduction, highlighting the need for sustained assessment of such interactions over a plant's lifecycle.

Enhanced UV-B and elevated CO(2) impacts sub-arctic shrub berry abundance, quality and seed germination.

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO(2) on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO(2) stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO(2) increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B × CO(2) interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B × elevated CO(2) on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.

UV responses of Lolium perenne raised along a latitudinal gradient across Europe: a filtration study.

Lolium perenne (cv. AberDart) was grown at 14 locations along a latitudinal gradient across Europe (37-68°N) to study the impact of ultraviolet radiation (UV) and climate on aboveground growth and foliar UV-B absorbing compounds. At each location, plants were grown outdoors for 5 weeks in a replicated UV-B filtration experiment consisting of open, UV-B transparent (cellulose diacetate) and UV-B opaque (polyester) environments. Fourier transform-infrared spectroscopy was used to compare plant metabolite profiles in relation to treatment and location. UV radiation and climatic parameters were determined for each location from online sources and the data were assessed using a combination of anova and multiple regression analyses. Most of the variation in growth between the locations was attributable to the combination of climatic parameters, with minimum temperature identified as an important growth constraint. However, no single environmental parameter could consistently account for the variability in plant growth. Concentrations of foliar UV-B absorbing compounds showed a positive trend with solar UV across the latitudinal gradient; however, this relationship was not consistent in all treatments. The most striking experimental outcome from this study was the effect of presence or absence of filtration frames on UV-absorbing compounds. Overall, the study demonstrates the value of an European approach in studying the impacts of natural UV across a large latitudinal gradient. We have shown the feasibility of coordinated UV filtration at multiple sites but have also highlighted the need for open controls and careful interpretation of plant responses.