Aldo-keto reductase may contribute to glyphosate resistance in Lolium rigidum.

BACKGROUND: We have previously demonstrated that an aldo-keto reductase (AKR) from Echinochloa colona (EcAKR4-1) can metabolize glyphosate and confers glyphosate resistance. This study aims to investigate if the EcAKR4-1 orthologs from Lolium rigidum also play a role in glyphosate resistance in non-target-site based, glyphosate-resistant (R) L. rigidum populations from Western Australia. RESULTS: The full-length L. rigidum AKR gene (LrAKR4C10) orthologous to EcAKR4-1, together with a distinct LrAKR1, were cloned from plants of a glyphosate-susceptible (S) (VLR1) and three glyphosate R L. rigidum populations (WALR50, WALR60 and WALR70). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) results showed that basal expression levels of the two LrAKR genes did not differ between the R and S populations, but their expression was significantly induced by glyphosate (up to 4.3-fold) or 2,4-D treatment (up to 3.4-fold) in R populations. Escherichia coli cells transformed respectively with LrAKR4C10 and LrAKR1 were more tolerant to glyphosate. Rice (Oryza sativa) seedlings overexpressing each of the two LrAKR gene survived glyphosate rates that were lethal to the green fluorescence protein (GFP) control plants. Structural modeling predicts a similar way of glyphosate binding and detoxification by LrAKR4C10 and EcAKR4-1, but an alternative way of glyphosate binding by LrAKR1. Relatively lower capacity of the two LrAKRs in conferring glyphosate resistance than the known EcAKR4-1 was discussed in relation to structural interaction. CONCLUSION: Glyphosate-induced higher expression of the two LrAKR genes in L. rigidum populations contributes to a moderate level of glyphosate resistance likely through enhanced glyphosate metabolism. The herbicide 2,4-D can also induce the LrAKR expression, indicating the potential antagonistic effect of 2,4-D to glyphosate. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Enhanced production of water-soluble cinmethylin metabolites by Lolium rigidum populations with reduced cinmethylin sensitivity.

BACKGROUND: Cinmethylin, a pre-emergence herbicide inhibiting fatty acid thioesterase activity, has recently been introduced to Australian cereal cropping for the control of Lolium rigidum Gaud. (annual ryegrass). To date, there have been no confirmed cases of cinmethylin resistance identified in this species, but some populations exhibit reduced sensitivity to this herbicide. To explore the mechanism which contributes to reduced sensitivity of annual ryegrass to cinmethylin, the extent and nature of cinmethylin metabolism, using carbon-14 (14 C)-labelled herbicide, were analysed in three reduced-sensitivity annual ryegrass populations, alongside a susceptible population and cinmethylin-tolerant wheat as controls. RESULTS: All samples showed the same metabolite profile, with the extent of production of a specific water-soluble metabolite being correlated to the level of herbicide sensitivity. Application of the cytochrome P450 inhibitor phorate caused a decrease in water-soluble metabolite production as well as seedling growth in the presence of cinmethylin, indicating that reduced cinmethylin sensitivity in annual ryegrass could be wholly or partially due to oxidative modification of cinmethylin. CONCLUSION: Because annual ryegrass has the potential to metabolize cinmethylin in the same way as wheat, careful stewardship is required to ensure the longevity of this herbicide. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A dinitroaniline herbicide resistance mutation can be nearly lethal to plants.

BACKGROUND: Lolium rigidum is the most important weed in Australian agriculture and pre-emergence dinitroaniline herbicides (e.g., trifluralin) are widely and persistently used for Lolium control. Consequently, evolution of resistance to dinitroaniline herbicides has been increasingly reported. Resistance-endowing target-site α-tubulin gene mutations are identified with varying frequency. This study investigated the putative fitness cost associated with the common resistance mutation Val-202-Phe and the rare resistance mutation Arg-243-Met causing helical plant growth. RESULTS: Results showed a deleterious effect of Arg-243-Met on fitness when plants are homozygous for this mutation. This was evidenced as high plant mortality, severely diminished root and aboveground vegetative growth (lower relative growth rate), and very poor fecundity compared with the wild-type, which led to a nearly lethal fitness cost of >99.9% in competition with a wheat crop. A fitness penalty in vegetative growth was evident, but to a much lesser extent, in plants heterozygous for the Arg-243-Met mutation. By contrast, plants possessing the Val-202-Phe mutation exhibited a fitness advantage in vegetative and reproductive growth. CONCLUSION: The α-tubulin mutations Arg-243-Met and Val-202-Phe have contrasting effects on fitness. These results help understand the absence of plants homozygous for the Arg-243-Met mutation and the high frequency of plants carrying the Val-202-Phe mutation in dinitroaniline-resistant L. rigidum populations. The α-tubulin Arg-243-Met mutation can have an exceptional fitness cost with nearly lethal effects on resistant L. rigidum plants. © 2022 Society of Chemical Industry.

Rapid On-Farm Testing of Resistance in Lolium rigidum to Key Pre- and Post-Emergence Herbicides.

Overreliance on herbicides for weed control is conducive to the evolution of herbicide resistance. Lolium rigidum (annual ryegrass) is a species that is prone to evolve resistance to a wide range of herbicide modes of action. Rapid detection of herbicide-resistant weed populations in the field can aid farmers to optimize the use of effective herbicides for their control. The feasibility and utility of a rapid 7-d agar-based assay to reliably detect L. rigidum resistant to key pre- and post-emergence herbicides including clethodim, glyphosate, pyroxasulfone and trifluralin were investigated in three phases: correlation with traditional pot-based dose-response assays, effect of seed dormancy, and stability of herbicides in agar. Easy-to-interpret results were obtained using non-dormant seeds from susceptible and resistant populations, and resistance was detected similarly as pot-based assays. However, the test is not suitable for trifluralin because of instability in agar as measured over a 10-d period, as well as freshly-harvested seeds due to primary dormancy. This study demonstrates the utility of a portable and rapid assay that allows for on-farm testing of clethodim, glyphosate, and pyroxasulfone resistance in L. rigidum, thereby aiding the identification and implementation of effective herbicide control options.

Herbicide resistance across the Australian continent.

BACKGROUND: Lolium rigidum is the weed of greatest economic impact in Australia due to its formidable capacity to evolve herbicide resistance. In this study, 579 field-sampled L. rigidum populations were tested for resistance to 21 herbicides applied at the recommended rate. Nine herbicide treatments were binary mixtures. RESULTS: A total of 15 876 individual resistance tests were conducted by screening two million seeds at the recommended label rate. The overall frequency of resistant populations was 31%, 14%, 71%, 6% and 0% in response to the post-emergence herbicide treatments clethodim, clethodim + butroxydim, imazamox + imazapyr, glyphosate and paraquat, respectively. The resistance frequency to stand-alone pre-emergence wheat-selective herbicides ranged from 10% to 34%. Conversely, the levels of resistance to pre-emergence mixtures or stand-alone propyzamide were significantly lower, ranging from 6% to 0%. In winter, the responses to glyphosate, paraquat, cinmethylin, prosulfocarb, pyroxasulfone and trifluralin were reassessed, with 7%, 0%, 0%, 21%, 21% and 28% as the respective resistance frequencies. South Australia and Victoria are identified as epicenters for L. rigidum population resistance to pyroxasulfone, whereas populations in New South Wales have the greatest resistance to glyphosate and in Western Australia to clethodim. CONCLUSIONS: For the first time, resistance levels to stand-alone herbicides and binary mixtures are geographically ranked across the Australian continent by benchmark statistical analysis of resistance frequencies and distribution. The extension of these results will raise awareness of rapidly emerging patterns of herbicide resistance, encouraging the adoption of cost-effective modes of action and integration of diverse strategies for weed resistance management.

Pyroxasulfone-Resistant Annual Ryegrass (Lolium rigidum) Has Enhanced Capacity for Glutathione Transferase-Mediated Pyroxasulfone Conjugation.

The herbicide pyroxasulfone was widely introduced in 2012, and cases of evolved resistance in weeds such as annual ryegrass (Lolium rigidum Gaud.) and tall waterhemp [Amaranthus tuberculatus (Moq.) Sauer] have started to emerge. Pyroxasulfone is detoxified by tolerant crops, and by annual ryegrass that has been recurrently selected with pyroxasulfone, in a pathway that is hypothesized to involve glutathione conjugation. In the current study, it was confirmed that pyroxasulfone is conjugated to glutathione in vitro by glutathione transferases (GSTs) purified from susceptible and resistant annual ryegrass populations and from a tolerant crop species, wheat. The extent of conjugation corresponded to the pyroxasulfone resistance level. Pyroxasulfone-conjugating activity was higher in radicles, roots, and seeds compared to coleoptiles or expanded leaves. Among the GSTs purified from annual ryegrass radicles and seeds, an orthologue of Brachypodium distachyon GSTF13 was >20-fold more abundant in the pyroxasulfone-resistant population, suggesting that this protein could be responsible for pyroxasulfone conjugation.

Assessing the Capability and Potential of LiDAR for Weed Detection.

Conventional methods of uniformly spraying fields to combat weeds, requires large herbicide inputs at significant cost with impacts on the environment. More focused weed control methods such as site-specific weed management (SSWM) have become popular but require methods to identify weed locations. Advances in technology allows the potential for automated methods such as drone, but also ground-based sensors for detecting and mapping weeds. In this study, the capability of Light Detection and Ranging (LiDAR) sensors were assessed to detect and locate weeds. For this purpose, two trials were performed using artificial targets (representing weeds) at different heights and diameter to understand the detection limits of a LiDAR. The results showed the detectability of the target at different scanning distances from the LiDAR was directly influenced by the size of the target and its orientation toward the LiDAR. A third trial was performed in a wheat plot where the LiDAR was used to scan different weed species at various heights above the crop canopy, to verify the capacity of the stationary LiDAR to detect weeds in a field situation. The results showed that 100% of weeds in the wheat plot were detected by the LiDAR, based on their height differences with the crop canopy.

Herbicide resistance management strategies: how do they compare with those for insecticides, fungicides and antibiotics?

Herbicides are the largest category of pesticides used in global agriculture, which is reflected in the rate of increase in the number of unique cases of herbicide-resistant weed biotypes since the late 1950s. Recommended herbicide resistance management strategies and tactics have evolved over the past 50 years through cumulative research and experience and have been regularly reviewed. Nevertheless, new perspectives may be gained by viewing current recommended strategies through the lens of insecticide, fungicide, and antibiotic resistance management. What commonalities exist and what is the basis for disparate strategies? Although pesticide and antibiotic mixtures (or combinations) are generally more effective than rotations (or alternations) in mitigating or managing resistance, the latter strategy is often employed because of greater ease of implementation and other reasons. We conclude that there are more common than different strategies for mitigating or managing pesticide and antibiotic resistance. Overall, a reduction in selection pressure for resistance evolution through diverse multi-tactic management programmes, and disruption or mitigation of the dispersal or transmission of problematic genotypes are needed to sustain the longevity of current and future mode-of-action products for crop and human health protection. © 2021 Society of Chemical Industry. © 2021 Society of Chemical Industry.

Identification of the first glyphosate-resistant capeweed (Arctotheca calendula) population.

BACKGROUND: Glyphosate is routinely used in Australia to control the Arctotheca species Arctotheca calendula (L.) Levyns (referred hereinafter as capeweed). This study identifies the first global case of field-evolved glyphosate-resistant capeweed, collected from the grainbelt of Western Australia. RESULTS: In 2020, a capeweed biotype that was collected from Borden in the southern Western Australian grainbelt was confirmed to be glyphosate-resistant (referred hereinafter as Spence population). When compared to the pooled mortality of six field-collected, glyphosate susceptible capeweed populations (S1, S2, S3, S4, S5 and S6), the Spence population was found > 11-fold more resistant to glyphosate than the pooled results of the susceptible populations (S1-S6) at the lethal dose of 50% (LD50 ) level. The growth of the Spence population was also less affected, requiring > 13-fold more glyphosate to reduce growth than the pooled susceptible populations at the growth reduction of 50% (GR50 ) level. Sequencing of the plastidic 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene indicated no known single gene mutation imparting glyphosate resistance. This study, however, did not investigate any other known mechanisms that impart glyphosate resistance. When screened at the field-applied rate, this Spence population was also found to survive an inhibitor of acetolactate synthase (ALS) (metosulam) and an inhibitor of phytoene desaturase (diflufenican). CONCLUSIONS: This is the first confirmation of glyphosate resistance evolution in a capeweed population globally. With capeweed resistance already confirmed to photosystem-I inhibiting herbicides (paraquat and diquat), this study emphasizes the importance of using integrated measures that do not depend only on the use of non-selective herbicides for controlling herbicide resistance-prone capeweed populations. © 2021 Society of Chemical Industry.

Agricultural Weed Assessment Calculator: An Australian Evaluation.

Weed risk assessment systems are used to estimate the potential weediness or invasiveness of introduced species in non-agricultural habitats. However, an equivalent system has not been developed for weed species that occur in agronomic cropland. Therefore, the Agricultural Weed Assessment Calculator (AWAC) was developed to quantify the present and potential future adverse impact of a weed species on crop production and profitability (threat analysis), thereby informing or directing research, development, and extension (RDE) investments or activities. AWAC comprises 10 questions related primarily to a weed's abundance and economic impact. Twenty weed species from across Australia were evaluated by AWAC using existing information and expert opinion, and rated as high, medium, or low for RDE prioritization based on total scores of 70 to 100, 40 to <70, or <40, respectively. Five species were rated as high (e.g., Lolium rigidum Gaud.), eight were rated as medium (e.g., Conyza spp.), and seven were rated as low (e.g., Rapistrum rugosum L.). Scores were consistent with the current state of knowledge of the species' impact on grain crop production in Australia. AWAC estimated the economic or agronomic threat of 20 major or minor agricultural weeds from across Australia. The next phase of development is the testing of AWAC by weed practitioners (e.g., agronomists, consultants, farmers) to verify its utility and robustness in accurately assessing these and additional weed species.

High gene flow maintains genetic diversity following selection for high EPSPS copy number in the weed kochia (Amaranthaceae).

Kochia, a major weed species, has evolved resistance to four herbicide modes of action. Herbicide resistance appears to spread quickly, which could result in diminished standing genetic variation, reducing the ability of populations to adapt further. Here we used double digest restriction enzyme associated sequencing to determine the level of gene flow among kochia populations and whether selection for glyphosate resistance reduces genetic variation. Canadian Prairie populations show little to no genetic differentiation (FST = 0.01) and no correlation between genetic and geographic distance (r2 = - 0.02 p = 0.56), indicating high gene flow and no population structure. There is some evidence that kochia populations are genetically depauperate compared to other weed species, but genetic diversity did not differ between glyphosate susceptible and resistant populations or individuals. The inbreeding coefficients suggest there are 23% fewer heterozygotes in these populations than expected with random mating, and no variation was found within the chloroplast. These results indicate that any alleles for herbicide resistance can be expected to spread quickly through kochia populations, but there is little evidence this spread will reduce the species' genetic variation or limit the species' ability to respond to further selection pressure.

Loss of trifluralin metabolic resistance in Lolium rigidum plants exposed to prosulfocarb recurrent selection.

BACKGROUND: Resistance to the dinitroaniline herbicide trifluralin in Lolium rigidum (annual ryegrass) often is mediated by the enhanced capacity to metabolize the herbicide to less toxic polar conjugates and/or by functionally recessive target-site mutations in α-tubulin. RESULTS: In two L. rigidum populations possessing enhanced trifluralin metabolism, resistance was largely reversed by recurrent selection with the thiocarbamate herbicide prosulfocarb (i.e. plant survival was two- to >20-fold lower). Their ability to metabolize trifluralin was significantly decreased (by ≈2.3-fold) following recurrent prosulfocarb selection, to levels comparable to those observed in susceptible plants or when trifluralin metabolism was inhibited by treatment with the insecticide phorate. CONCLUSIONS: This study provides evidence that trait(s) enabling efficient trifluralin metabolism in L. rigidum are purged from the population under prosulfocarb recurrent selection. The level of trifluralin metabolism in vitro and its inhibition caused by phorate action on trifluralin-metabolizing enzyme(s) is equivalent to the effect produced by prosulfocarb selection. The hypothetical link between the two phenomena is that the putative monooxygenase(s) conferring trifluralin metabolic resistance also mediate the activation of prosulfocarb to its toxic sulfoxide. Thus, we speculate that survival to prosulfocarb via a lack of metabolic herbicide activation, and survival to trifluralin conferred by enhanced herbicide metabolism, are mutually exclusive. These findings not only open up a new research direction in terms of the interaction between different herbicide resistance mechanisms in L. rigidum, but also offer strategies for immediate management of the population dynamics of metabolism-based resistance in the field. © 2020 Society of Chemical Industry.

Herbicide Resistance in Plants.

Herbicide resistance in weeds is perhaps the most prominent research area within the discipline of weed science today. Incidence, management challenges, and the cost of multiple-resistant weed populations are continually increasing worldwide. Crop cultivars with multiple herbicide-resistance traits are being rapidly adopted by growers and land managers to keep ahead of the weed resistance tsunami. This Special Issue of Plants comprises papers that describe the current status and future outlook of herbicide resistance research and development in weedy and domestic plants, with topics covering the full spectrum from resistance mechanisms to resistance management. The unifying framework for this Special issue, is the challenge initially posed to all of the contributors: what are the (potential) implications for herbicide resistance management?

Cinmethylin controls multiple herbicide-resistant Lolium rigidum and its wheat selectivity is P450-based.

BACKGROUND: Multiple-herbicide resistance in Lolium rigidum and other weed species is increasingly exerting pressure on herbicide discovery research for solutions against resistance-prone weeds. In this study we investigate: (i) the responses of L. rigidum populations and wheat to the new herbicide cinmethylin in comparison with other pre-emergence herbicides, (ii) the effect of seed burial depths on cinmethylin efficacy and crop selectivity, and (iii) the basis of cinmethylin selectivity in wheat. RESULTS: Cinmethylin at 400 g ha-1 controls herbicide-susceptible and multiple-resistant L. rigidum, with a reduction of >85% in plant emergence and 90% in aboveground biomass. Cinmethylin provides effective control of a large number of field populations of L. rigidum with evident resistance to trifluralin. When the wheat seed is buried ≥1 cm below the cinmethylin-treated soil surface, the emergence of crop seedlings is not different from the untreated control. The organophosphate insecticide phorate synergizes cinmethylin toxicity in wheat, with an LD50 of 682 g ha-1 in the absence of phorate versus 109 g ha-1 in the presence of phorate (84% reduction). The synergistic effect of phorate with cinmethylin on herbicide-susceptible L. rigidum appears smaller (a 44% reduction in the LD50 of cinmethylin). CONCLUSIONS: Cinmethylin is effective in controlling multiple-resistant L. rigidum and appears safe for wheat when the seed is separated at depth from the herbicide applied to the soil surface. The basis of this metabolism-based selectivity is likely regulated by cytochrome P450 monooxygenases. © 2020 Society of Chemical Industry.

Farming without Glyphosate?

Recent statements from scientific organisations and court decisions have resulted in widespread public interest and concern over the safety of glyphosate, the most popular and effective herbicide used worldwide. Consequently, glyphosate-based products are under intense scrutiny from governments at all levels. Some jurisdictions have already banned or restricted its use, which will adversely impact international trade in bulk grain commmodities if glyphosate residues are detected. The possibility of farming without glyphosate is becoming an important issue facing the agri-food research and development sector. Contingency plans need to be formulated if that scenario becomes a reality. In this review, we briefly summarize international events that have led to this possible situation, describe current glyphosate usage in major agronomic field crops worldwide, outline possible alternatives to glyphosate in two agroregions and perform bioeconomic model scenarios of southern Australian broadacre cropping systems without the herbicide. Model predictions suggest that we can farm profitably without glyphosate by consistently utilizing key non-herbicidal weed management practices combined with robust pre-emergence soil residual herbicide treatments. However, maintaining low weed seed banks will be challenging. If the social license to use glyphosate is revoked, what other pesticides will soon follow?

Rotations and mixtures of soil-applied herbicides delay resistance.

BACKGROUND: Weed resistance to foliar herbicides has dramatically increased worldwide in the last two decades. As a consequence, current practices of weed management have changed, with an increased adoption of soil-applied herbicides to restore control of herbicide-resistant weeds. We foresee metabolism-based resistance and cross-resistance to soil-applied herbicides as a potential global consequence to the increased and widespread adoption of new and old soil-applied herbicides. Thus, the aim of this study is to use computer simulation modelling to quantify and rank the risk of weeds evolving resistance to soil-applied herbicides under different usage strategies (single herbicide use, rotations and mixtures) and population genetic hypotheses. RESULTS: Simulations indicate that without rotation it takes twice as long to select for resistance to a particular soil-applied herbicide - trifluralin - than to any other herbicide option considered. Relative to trifluralin-only use, simple herbicide rotation patterns have no effect in delaying resistance, whereas more complex rotation patterns can delay resistance two- or three-fold. Herbicide mixtures further delay resistance up to six-fold in comparison to single use or simple herbicide rotations. CONCLUSION: By computer modelling simulations we demonstrate that mixtures maximize herbicide effectiveness and the selection heterogeneity of soil-applied herbicides, and delay herbicide resistance evolution in weedy plants. Our study is consistent with previous state-of-art scientific evidence (i.e. epidemiological and modelling studies across different systems and pests) and extension efforts (i.e. 'rotate herbicide mixtures') to provide insight to manage the selection and evolution of weed resistance. © 2019 Society of Chemical Industry.

Herbicide Resistance Management: Recent Developments and Trends.

This review covers recent developments and trends in herbicide-resistant (HR) weed management in agronomic field crops. In countries where input-intensive agriculture is practiced, these developments and trends over the past decade include renewed efforts by the agrichemical industry in herbicide discovery, cultivation of crops with combined (stacked) HR traits, increasing reliance on preemergence vs. postemergence herbicides, breeding for weed-competitive crop cultivars, expansion of harvest weed seed control practices, and advances in site-specific or precision weed management. The unifying framework or strategy underlying these developments and trends is mitigation of viable weed seeds into the soil seed bank and maintaining low weed seed banks to minimize population proliferation, evolution of resistance to additional herbicidal sites of action, and spread. A key question going forward is: how much weed control is enough to consistently achieve the goal of low weed seed banks? The vision for future HR weed management programs must be sustained crop production and profitability with reduced herbicide (particularly glyphosate) dependency.

Opportunities and challenges for harvest weed seed control in global cropping systems.

The opportunity to target weed seeds during grain harvest was established many decades ago following the introduction of mechanical harvesting and the recognition of high weed-seed retention levels at crop maturity; however, this opportunity remained largely neglected until more recently. The introduction and adoption of harvest weed seed control (HWSC) systems in Australia has been in response to widespread occurrence of herbicide-resistant weed populations. With diminishing herbicide resources and the need to maintain highly productive reduced tillage and stubble-retention practices, growers began to develop systems that targeted weed seeds during crop harvest. Research and development efforts over the past two decades have established the efficacy of HWSC systems in Australian cropping systems, where widespread adoption is now occurring. With similarly dramatic herbicide resistance issues now present across many of the world's cropping regions, it is timely for HWSC systems to be considered for inclusion in weed-management programs in these areas. This review describes HWSC systems and establishing the potential for this approach to weed control in several cropping regions. As observed in Australia, the inclusion of HWSC systems can reduce weed populations substantially reducing the potential for weed adaptation and resistance evolution. © 2017 Society of Chemical Industry.

Glyphosate resistance reduces kochia fitness: Comparison of segregating resistant and susceptible F2 populations.

Glyphosate is considered the world's most important herbicide, but widespread and continual use has resulted in the evolution of resistance. Kochia scoparia (kochia) has evolved resistance via tandem gene amplification of glyphosate's target, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and resistant populations have been reported from the Canadian Prairies and the Northern Great Plains. Here, we evaluated the fitness costs of EPSPS amplification in kochia by comparing susceptible and resistant full siblings from segregating F2 populations generated from within six populations. Kochia was expected to be highly diverse because of strong gene flow; however, six of the seven field-collected parents with higher EPSPS copy number were homozygous. Under competitive greenhouse conditions, the EPSPS type of the line's maternal parent showed persistent effects: delayed emergence, delayed flowering, and reductions in viable seed count and weight overall. High EPSPS copy number individuals had reduced seed count and weight, reduced competitive ability, and reduced final height in mixed stands, but better germination of the F3. However, all characteristics were highly variable and fitness costs were not constant across genetic backgrounds. In the absence of selection from glyphosate, kochia with increased EPSPS copy number will be at a competitive disadvantage in some genetic backgrounds.