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Resistance to the herbicide pyroxasulfone has slowly but steadily increased in agricultural weeds. The evolved resistance of one Lolium rigidum population has been attributed to the conjugation of pyroxasulfone to reduced glutathione, mediated by glutathione transferase (GST) activity. To determine if GST-based metabolism is a widespread mechanism of pyroxasulfone resistance in L. rigidum , a number of putative-resistant populations were screened for GST activity toward pyroxasulfone, the presence of GSTF13-like isoforms (previously implicated in pyroxasulfone conjugation in this species), tissue glutathione concentrations, and response to inhibitors of GSTs and oxygenases. Although there were no direct correlations between pyroxasulfone resistance levels and these individual parameters, a random forest analysis indicated that GST activity was of primary importance for L. rigidum resistance to this herbicide.

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., (© 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

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., (© 2021 Society of Chemical Industry.)

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.

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., (© 2020 Society of Chemical Industry.)

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 LD 50 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 LD 50 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., (© 2020 Society of Chemical Industry.)

Resistance to the pre-emergence herbicide trifluralin is increasing in Australian annual ryegrass ( Lolium rigidum) populations. Three L. rigidum populations (R1, R2, and R3) collected from Australian grain fields were identified with trifluralin resistance. Both target-site and nontarget-site resistance mechanisms were investigated. No target-site α-tubulin mutations were detected in populations R1 and R3, while an Arg-243-Lys mutation was found in R2. Compared with the three trifluralin-susceptible populations, enhanced [ 14 C]-trifluralin metabolism, quantified by measuring the amount of [ 14 C] label partitioning into the polar phase of a hexane:methanol system, was identified in all the three resistant populations. This is the first report of metabolic resistance to trifluralin. Coevolution of target-site and nontarget-site resistance to trifluralin is occurring, and metabolic resistance is not rare in L. rigidum populations in Australia. A method was established for trifluralin metabolic resistance detection, overcoming the difficulties of quantifying this highly volatile herbicide by chromatographic methods.

The evolution of resistant weed populations in response to intensive herbicide selection pressure is a global issue. Resistance to post-emergence herbicides is widespread, whereas soil-applied pre-emergence herbicides can often remain effective. For example, in Australia pyroxasulfone is a new pre-emergence soil-applied herbicide which provides control of Lolium rigidum populations resistant to multiple post-emergence herbicide modes of action. A fundamental knowledge of the genetic basis of metabolic resistance in weeds is important for understanding plant evolution pathways under herbicide selection and sustaining long-term weed resistance management. In this study we define the mechanistic basis of resistance to pyroxasulfone in a L. rigidum population. TLC provides evidence that pyroxasulfone resistance is metabolism-based with approximately 88% of parental [ 14 C]-labelled pyroxasulfone metabolized in resistant plants 24 h after the herbicide treatment. HPLC-MS allowed identification of several metabolites of pyroxasulfone formed via a glutathione (GSH) conjugation pathway in pyroxasulfone-resistant L. rigidum plants. However, the initial pyroxasulfone-glutathione conjugate was not found likely due to its labile nature. The observed constitutive over-expression from six to nine-fold of two putative resistance-endowing GST genes was associated with the pyroxasulfone resistance phenotype. The most logical conclusion, based on the data thus far available, is that rapid detoxification of pyroxasulfone mediates pyroxasulfone resistance in L. rigidum plants. Future research is warranted to confirm the hypothesis advanced by this study of rapid pyroxasulfone metabolism due to GSH conjugation mediated by GST over-expressed in pyroxasulfone-resistant plants which similarly leads to the production of distinctive GSH-pyroxasulfone metabolites in L. rigidum and wheat., (Copyright © 2018. Published by Elsevier Inc.)

Background: Organophosphate insecticides can inhibit specific cytochrome P450 enzymes involved in metabolic herbicide resistance mechanisms, leading to synergistic interactions between the insecticide and the herbicide. In this study we report synergistic versus antagonistic interactions between the organophosphate insecticide phorate and five different herbicides observed in a population of multiple herbicide-resistant Lolium rigidum., Results: Phorate synergised with three different herbicide modes of action, enhancing the activity of the ALS inhibitor chlorsulfuron (60% LD 50 reduction), the VLCFAE inhibitor pyroxasulfone (45% LD 50 reduction) and the mitosis inhibitor trifluralin (70% LD 50 reduction). Conversely, phorate antagonised the two thiocarbamate herbicides prosulfocarb and triallate with a 12-fold LD 50 increase., Conclusion: We report the selective reversal of P450-mediated metabolic multiple resistance to chlorsulfuron and trifluralin in the grass weed L. rigidum by synergistic interaction with the insecticide phorate, and discuss the putative mechanistic basis. This research should encourage diversity in herbicide use patterns for weed control as part of a long-term integrated management effort to reduce the risk of selection of metabolism-based multiple herbicide resistance in L. rigidum. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Background: Weeds can be a greater constraint to crop production than animal pests and pathogens. Pre-emergence herbicides are crucial in many cropping systems to control weeds that have evolved resistance to selective post-emergence herbicides. In this study we assessed the potential to evolve resistance to the pre-emergence herbicides prosulfocarb + S-metolachlor or pyroxasulfone in 50 individual field Lolium rigidum populations collected in a random survey in Western Australia prior to commercialisation of these pre-emergence herbicides., Results: This study shows for the first time that in randomly collected L. rigidum field populations the selection with either prosulfocarb + S-metolachlor or pyroxasulfone can result in concomitant evolution of resistance to both prosulfocarb + S-metolachlor and pyroxasulfone after three generations., Conclusions: In the major weed L. rigidum, traits conferring resistance to new herbicides can be present before herbicide commercialisation. Proactive and multidisciplinary research (evolutionary ecology, modelling and molecular biology) is required to detect and analyse resistant populations before they can appear in the field. Several studies show that evolved cross-resistance in weeds is complex and often unpredictable. Thus, long-term management of cross-resistant weeds must be achieved through heterogeneity of selection by effective chemical, cultural and physical weed control strategies that can delay herbicide resistance evolution. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Weed control failures due to herbicide resistance are an increasing and worldwide problem that significantly affect crop yields. Metabolism-based herbicide resistance (referred to as metabolic resistance) in weeds is not well characterized at the genetic level. An RNA-Seq transcriptome analysis was used to find candidate genes that conferred metabolic resistance to the herbicide diclofop in a diclofop-resistant population (R) of the major global weed Lolium rigidum. A reference cDNA transcriptome (19 623 contigs) was assembled and assigned putative annotations. Global gene expression was measured using Illumina reads from untreated control, adjuvant-only control, and diclofop treatment of R and susceptible (S). Contigs that showed constitutive expression differences between untreated R and untreated S were selected for further validation analysis, including 11 contigs putatively annotated as cytochrome P450 (CytP450), glutathione transferase (GST), or glucosyltransferase (GT), and 17 additional contigs with annotations related to metabolism or signal transduction. In a forward genetics validation experiment, nine contigs had constitutive up-regulation in R individuals from a segregating F2 population, including three CytP450, one nitronate monooxygenase (NMO), three GST, and one GT. Principal component analysis using these nine contigs differentiated F2 -R from F2 -S individuals. In a physiological validation experiment in which 2,4-D pre-treatment induced diclofop protection in S individuals due to increased metabolism, seven of the nine genetically validated contigs were induced significantly. Four contigs (two CytP450, NMO, and GT) were consistently highly expressed in nine field-evolved metabolic resistant L. rigidum populations. These four contigs were strongly associated with the resistance phenotype and are major candidates for contributing to metabolic diclofop resistance., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Agricultural weeds have rapidly adapted to intensive herbicide selection and resistance to herbicides has evolved within ecological timescales. Yet, the genetic basis of broad-spectrum generalist herbicide resistance is largely unknown. This study aims to determine the genetic control of non-target-site herbicide resistance trait(s) that rapidly evolved under recurrent selection of the novel lipid biosynthesis inhibitor pyroxasulfone in Lolium rigidum. The phenotypic segregation of pyroxasulfone resistance in parental, F1 and back-cross (BC) families was assessed in plants exposed to a gradient of pyroxasulfone doses. The inheritance of resistance to chemically dissimilar herbicides (cross-resistance) was also evaluated. Evolved resistance to the novel selective agent (pyroxasulfone) is explained by Mendelian segregation of one semi-dominant allele incrementally herbicide-selected at higher frequency in the progeny. In BC families, cross-resistance is conferred by an incompletely dominant single major locus. This study confirms that herbicide resistance can rapidly evolve to any novel selective herbicide agents by continuous and repeated herbicide use. The results imply that the combination of herbicide options (rotation, mixtures or combinations) to exploit incomplete dominance can provide acceptable control of broad-spectrum generalist resistance-endowing monogenic traits. Herbicide diversity within a set of integrated management tactics can be one important component to reduce the herbicide selection intensity., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Plants can rapidly evolve resistance to herbicide in response to repeated selection. This study focuses on cross-resistance patterns observed in Lolium rigidum following pyroxasulfone recurrent selection., Results: The parental MR (multiresistant) population following four generations of pyroxasulfone recurrent selection evolved cross-resistance to prosulfocarb and triallate. At the recommended label rate of prosulfocarb or triallate (2000 g ha(-1) ), the progeny selected four times with pyroxasulfone (MR4) displayed 58 and 35% plant survival respectively. One additional cycle of prosulfocarb selection increased the resistance level to both prosulfocarb and triallate in the population MR4-P1. Prosulfocarb resistance is yet to be reported in L. rigidum field populations., Conclusions: This study suggests that L. rigidum plants can rapidly evolve cross-resistance to several wheat-selective herbicides under recurrent selection of a single mode of action. Weed populations displaying broad-spectrum cross-resistance to several herbicide modes of action are increasing in frequency in intensive world agriculture. Proactive and integrated measures for resistance management need to be developed globally on appropriate herbicide use in crop rotations., (© 2013 Society of Chemical Industry.)

Gene flow promotes genetic exchange among plant populations mediating evolutionary dynamics; yet, the importance of gene flow at distance via pollen movement is poorly understood. A field experiment at the landscape level was conducted with Lolium rigidum herbicide-susceptible individuals (population VLR1) placed into an otherwise Lolium-free bushland environment at increasing distances from adjacent large commercial crop fields infested with herbicide-resistant L. rigidum. Herbicide resistance was used as a marker to quantify the distance and the rate of pollen-mediated gene flow. About 21,245 seeds were produced on the isolated, susceptible mother plants of which 3,303 seedlings were tested for herbicide resistance and 664 seedlings were found to be resistant. Pollen-mediated gene flow occurred at 3,000 m (maximum tested distance). Both Mendelian and molecular analyses (sequencing and CAPS markers) confirmed the introgression of herbicide resistance genes. This is the first documented case of long-distance gene flow in L. rigidum. The results are important for future modeling simulations of herbicide resistance evolution and subsequent mobility. The adoption of integrated agronomic strategies, the control of potential receptor plants on fields' margins and conservative use of herbicides can be realistic options to minimize herbicide resistance spread.