Widespread resistance to selective postemergence herbicides has led to increased use of preemergence herbicides to control rigid ryegrass (Lolium rigidum Gaudin), the major weed of southern Australian cropping systems. Seeds of L. rigidum are dormant at maturity, leading to staggered germination across the growing season and avoidance of pre-sowing knockdown herbicides by the later-germinating cohorts. Although it is well known that this selects for higher seed dormancy in intensively cropped areas, there is less information on whether dormant seeds respond differently to preemergence herbicides applied at sowing. To address this, seeds of field-collected L. rigidum populations were divided into dormant and nondormant (afterripened) subsamples and treated with sublethal rates of three preemergence herbicides in order to monitor seedling emergence and seed persistence over 6 mo. The presence of prosulfocarb and pyroxasulfone eliminated the nearly 4-fold increase in seedling emergence that typically results from afterripening, while trifluralin was partially inhibitory. In all treatments, the proportion of viable seeds remaining in the soil after 6 mo was negligible (<= 3% of the viable seeds originally sown) for both the dormant and nondormant seeds. Application of radiolabeled herbicides to soil and seeds showed that the herbicides persisted in the seed tissue for longer than in the bulk soil. Therefore, the presence of dormant L. rigidum seeds in the soil seedbank is unlikely to result in cohorts that can avoid preemergence herbicides.

Bixlozone, a proherbicide that requires in planta activation to its phytotoxic form, 5-ketobixlozone, is used to selectively control annual ryegrass (Lolium rigidum) in Australian cereal and canola crops. Bixlozone resistance has been detected in southern Australian annual ryegrass populations, and this can be increased with recurrent selection. The metabolic fate of bixlozone in young weed and crop seedlings was investigated by liquid chromatography-mass spectrometry to determine if differential metabolism can explain differences in bixlozone response. The observed tolerance of canola and wild radish (Raphanus raphanistrum) was due to the reduced activation of bixlozone to 5-ketobixlozone. In contrast, a resistant annual ryegrass population and tolerant wheat and barley showed preferential conversion of bixlozone to hydroxylated derivatives, whereas susceptible annual ryegrass populations produced more 5-ketobixlozone. Direct application of 5-ketobixlozone to seedlings resulted in an unexpected conversion to hydroxylated metabolites, potentially implicating plant reductases in 5-ketobixlozone metabolism.

BACKGROUND:Cross-resistance between pre-emergence herbicides is developing in Australian populations of annual ryegrass (Lolium rigidum Gaud.). A previous study has reported that selection with prosulfocarb (a pro-herbicide requiring bioactivation to its phytotoxic sulfoxide) can decrease metabolic resistance to trifluralin. Metabolism of prosulfocarb and trifluralin was investigated in L. rigidum populations with different levels of resistance to prosulfocarb, trifluralin and also pyroxasulfone, which is detoxified by glutathione (GSH) conjugation. RESULTS:Coleoptiles and radicles of herbicide-treated seedlings responded differently to the same herbicide. Radicles had a lower capacity for bioactivation of prosulfocarb, and this was correlated with a lower ability to metabolise trifluralin within and among populations. Coleoptile resistance to prosulfocarb sulfoxide was negatively correlated with abundance of a major polar metabolite. There was no evidence of GSH conjugation with the sulfoxide, making any potential links between prosulfocarb and pyroxasulfone resistance less obvious. CONCLUSIONS:Activation and metabolism of prosulfocarb in L. rigidum is complex and differentially regulated in different tissues. Selection with prosulfocarb may ameliorate trifluralin metabolism in the radicles, but the relationship between prosulfocarb and pyroxasulfone resistance is not GSH-mediated. When applying pre-emergence herbicides, care should be taken with the composition of mixtures and rotations to avoid selection of cross-resistance between pyroxasulfone and prosulfocarb. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

BACKGROUND: Brome grass (Bromus diandrus Roth) is prevalent in the southern and western cropping regions of Australia, where it causes significant economic damage. A targeted herbicide resistance survey was conducted in 2020 by collecting brome grass populations from 40 farms in Western Australia and subjecting these samples to comprehensive herbicide screening. One sample (population 172-20), from a field that had received 12 applications of clethodim over 20 years of continuous cropping, was found to be highly resistant to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides clethodim and quizalofop, and so the molecular basis of resistance was investigated.RESULTS: All 31 individuals examined from population 172-20 carried the same resistance-endowing point mutation causing an aspartate-to-glycine substitution at position 2078 in the translated ACCase protein sequence. A wild-type susceptible population and the resistant population had similar expression levels of plastidic ACCase genes. The level of resistance to quizalofop, either standalone or in mixture with clethodim, in population 172-20 was lower under cooler growing conditions.CONCLUSION: Target-site resistance to ACCase-inhibiting herbicides, conferred by one ACCase mutation, was selected in all tested brome plants infesting a field with a history of repeated clethodim use. This mutation appears to have been fixed in the infesting population. Notably, clethodim resistance in this population was not detected by the farmer, and a high future incidence of quizalofop resistance is anticipated. Herbicide resistance testing is essential for the detection of evolving weed resistance issues and to inform effective management strategies. (c) 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

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.

The synthetic auxin 2,4-D and the 4-hydroxyphenylpyruvate dioxygenase inhibitor pyrasulfotole are phloem-mobile post-emergence herbicides, the latter applied in co-formulation with either bromoxynil (a contact herbicide causing leaf desiccation) or MCPA (another synthetic auxin). Previous studies have shown a wide range of 2,4-D translocation phenotypes in resistant populations of the agricultural weed Raphanus raphanistrum, but it was hypothesised that enhanced movement out of the apical meristem could contribute to resistance. Little is known about pyrasulfotole translocation or the effect of bromoxynil on pyrasulfotole movement. Therefore, the behaviour of pyrasulfotole and 2,4-D applied to the growing point of susceptible and resistant R. raphanistrum seedlings was assessed, along with the effect of bromoxynil on pyrasulfotole translocation. The small amount of herbicide directly contacting the growing point after spraying was sufficient to induce herbicide symptoms, and there was no enhancement of translocation away from the growing point in either pyrasulfotole- or 2,4-D-resistant populations. Bromoxynil had a slightly inhibitory effect on pyrasulfotole translocation in some populations, somewhat negating the minor differences observed among populations when pyrasulfotole was applied alone. Resistance to pyrasulfotole could not explained by enhanced metabolism or vacuolar sequestration of the herbicide. Overall, differential translocation in either the treated leaves or apical meristems does not appear to be a major determinant of resistance to pyrasulfotole or 2,4-D.

The pre-emergence herbicide trifluralin is widely used in the minimum-tillage cropping systems of Australia, with the result that resistance to trifluralin is increasing in the major weed of the region, annual ryegrass ( Lolium rigidum ). Repeated exposure to low herbicide rates is also known to result in the rapid evolution of resistance in weed populations. As trifluralin is highly volatile, readily photo-decomposed, metabolised by soil microbes and to bind strongly to soil organic matter, there are many factors that could result in weed populations receiving reduced (even sub-lethal) rates of the herbicide. To investigate whether trifluralin dissipation could play a role in the increasing levels of trifluralin resistance in annual ryegrass, resistance levels of populations from 18 Western Australian farms were compared with the dissipation rate of trifluralin applied to soil collected from these farms. Although there was no direct correlation between resistance level and trifluralin half-life, there were links between resistance and soil properties which suggest that higher rates of trifluralin dissipation could make a minor contribution to the development of resistance. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND Raphanus raphanistrum causes $40 million total revenue losses annually in Western Australia partly due to its historically-documented ability to evolve herbicide resistance to multiple modes of action. In this study, 376 field-sampled populations of R. raphanistrum were tested for resistance to 21 herbicides applied at the recommended label rate. Eight treatments were herbicide mixtures with two, three or four modes of action. RESULTS A total of 7199 individual resistance tests were conducted across 4 years by screening approximately 104 000 individual seeds and seedlings. The mean survival of individuals within a population for all standalone herbicides was 9%, whereas survival was significantly decreased to 3.5% with a herbicide mixture. Some herbicides such as triasulfuron (herbicide Group 2), 2,4-D (Group 4) or diflufenican (Group 12) were highly impacted by resistance, with frequencies of resistant populations being > 50%. Conversely, there was negligible resistance to glyphosate (Group 9) or protoporphyrinogen oxidase (PPO) inhibitors (tiafenacil, saflufenacil + trifludimoxazin, fomesafen: Group 14), and pre-emergence herbicides (i.e., atrazine or mesotrione: Groups 5 and 27, respectively) remained largely effective. Binary, ternary or quaternary mixtures of Groups 4, 6, 12 and 27 herbicides reduced the frequency of high-level resistant populations to 7.1%, 3.8% or 0%, respectively. CONCLUSIONS The cost-effective control of R. raphanistrum remains a challenge due to herbicide resistance. Raphanus raphanistrum management relies heavily on herbicide uses not yet compromised by resistance, such as pre-emergence herbicides (atrazine, fomesafen, mesotrione), glyphosate, and mixtures of two, three or four modes of action including bromoxynil, diflufenican, MCPA, picolinafen, pyrasulfotole and topramezone. Strategies that integrate effective herbicide use patterns, novel modes of action and efficiently-mechanized non-chemical weed control options (i.e., seed destructors) can completely constrain the selection of herbicide resistance in this highly adaptable species. (c) 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Synthetic auxin herbicides were developed and commercialized 60 yr before their mode of action was definitively elucidated. Although evolution of resistance to auxinic herbicides proceeded more slowly than for some other herbicide chemistries, it has become a major problem in the dicotyledonous weeds of many cropping areas of the world. With the molecular characterization of the auxin perception and signaling pathway in the mid-2000s came a greater understanding of how auxinic herbicides work, and how resistance may develop in weeds subjected to repeated selection with these herbicides. In wild radish (Raphanus raphanistrum L.) populations in southern Australia, resistance to multiple herbicides, including synthetic auxins such as 2,4-D, has reduced the number of chemical control options available. The aim of this study was to determine whether compounds involved in auxin biosynthesis, transport, and signaling are able to synergize with 2,4-D and increase its ability to control 2,4-D-resistant R. raphanistrum populations. Although some mild synergism was observed with a few compounds (abscisic acid, cyclanilide, tryptamine), the response was not large or consistent enough to warrant further study. Similarly, alternative auxinic herbicides applied pre- or postemergence were no more effective than 2,4-D. Therefore, while use of auxinic herbicides continues to increase due to the adoption of transgenic resistant crops, nonchemical control techniques will become more important, and chemical control of 2,4-D-resistant R. raphanistrum should be undertaken with alternative modes of action, using mixtures and good stewardship to delay the development of resistance for as long as possible.

Rigid ryegrass (Lolium rigidum Gaudin) is the most problematic weed in Australia, with evolved resistance to multiple herbicide sites of action. Selection pressure by cinmethylin (Group 30, a fatty-acid thioesterase inhibitor) has been limited, because few populations have been exposed to the herbicide since its introduction in 2019. In this study, we examined the sensitivity of L. rigidum populations to this new herbicide. From a screening of almost 500 field populations in 2020, 28 potentially resistant populations were further investigated in a dose-response experiment. Seedlings from five populations surviving treatments of 250 or 375 g ai ha(-1) cinmethylin were grown to maturity and seeds were harvested. The level of resistance found among the five putative-resistant parental populations of L. rigidum was negligible. In one population, one round of selection with cinmethylin resulted in a 2-fold increase in the lethal dose causing 50% mortality in the progeny population, although this dose was still only one-sixth of the recommended field rate of cinmethylin. Having a unique site of action, cinmethylin is a viable preemergence herbicide option to control existing multiple-resistance populations of L. rigidum. Comprehensive field monitoring and recurrent selection studies under controlled environmental conditions are needed to better ascertain the risk of L. rigidum evolving a high level of resistance to cinmethylin, although current data suggest that this risk is relatively low.

AbstractBackgroundCinmethylin, 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 (14C)‐labelled herbicide, were analysed in three reduced‐sensitivity annual ryegrass populations, alongside a susceptible population and cinmethylin‐tolerant wheat as controls.ResultsAll 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.ConclusionBecause 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.

The control of multiple-resistant wild radish (Raphanus raphanistrum L.) populations in no-till Australian wheat (Triticum aestivum L.) crops has relied upon 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides over the last decade. Two R. raphanistrum populations identified as putatively resistant to pyrasulfotole + bromoxynil in an initial large-scale screening trial were characterized and confirmed to be 5- to 8-fold (comparison of LD50 values) less sensitive than the susceptible control population to the HPPD inhibitor pyrasulfotole when plants were treated at the 4-leaf stage. The two pyrasulfotole-resistant populations exhibited up to 4-fold resistance to the coformulated herbicide mixture pyrasulfotole + bromoxynil and up to 9- and 11-fold cross-resistance to mesotrione and topramezone postemergence, respectively. A small-plot trial was conducted in the field from which of one of the populations suspected of resistance was originally collected. Pyrasulfotole + bromoxynil or topramezone + bromoxynil applied postemergence delivered reduced R. raphanistrum control (79% to 87%), whereas mesotrione applied preemergence was >99% effective. We report here the first case of field resistance to HPPD-inhibiting herbicides in R. raphanistrum, caused by 12 yr of continuous reliance on that mode of action. The mitigation of herbicide resistance in continuous no-till cropping requires a constant optimization of the herbicide technology via alternation and mixtures of multiple sites of action, use of preemergence herbicides, and ensuring postemergence herbicides are applied at the most sensitive plant growth stages.

Overreliance on herbicides for weed control is conducive to the evolution of herbicide resistance. Annual ryegrass ( Lolium rigidum Gaud.) 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 herbicides for their control. The feasibility of a portable agar-based test to rapidly and reliably detect annual ryegrass resistance to key herbicides such as clethodim, glyphosate, pyroxasulfone and trifluralin on-farm was investigated. The three research phases of this study show that: a) easy-to-interpret results are obtained with non-dormant seed from well-characterised susceptible and resistant populations, and resistance is detected as effectively as with traditional dose-response pot-based resistance assays. However, the test may not be suitable for portable use on-farm because of b) the low stability of some herbicides such as trifluralin and clethodim in agar and c) the tendency of seed dormancy in freshly-harvested seeds to confound the results. The agar-based test is best used as a research tool as a complement to confirm results obtained in traditional pot-based resistance screenings. Comprehensive agar test and / or whole-plant resistance tests by herbicide application at the recommended label rate (whole plants grown in pots) are the current benchmark for proactive in- and off-season resistance testing and should be promoted more widely to allow early detection of resistance, optimization of herbicide technology use and deploy appropriate weed management interventions.

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.

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 inLolium 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 alpha-tubulin. RESULTS In twoL. rigidumpopulations 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 approximate to 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 inL. rigidumare purged from the population under prosulfocarb recurrent selection. The level of trifluralin metabolismin vitroand 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 inL. rigidum, but also offer strategies for immediate management of the population dynamics of metabolism-based resistance in the field.

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.

BACKGROUND A Lolium rigidum population collected from Western Australia was previously reported as highly resistant to dinitroaniline herbicides mainly due to a Val-202-Phe substitution in the target site alpha-tubulin protein. To further determine the contribution of the 202 mutation to resistance, two sub-populations, respectively comprising the 202 mutant and wild-type (WT) individuals, were isolated from within the same resistant population and subject to dinitroaniline herbicide doses. A rice transgenic study was conducted to demonstrate whether the amino acid substitution at the 202 residue confers resistance. In addition, as indicated in the phenotyping and genotyping study, non-target enhanced trifluralin metabolism was further examined in the same population. RESULTS The 202 mutants were more resistant than the wild-type plants. Rice calli transformed with the L. rigidum mutant alpha-tubulin gene (Val-202-Phe) were more resistant to dinitroaniline herbicides relative to calli transformed with the wild-type gene. Also, enhanced trifluralin metabolism was detected in the 202 mutants in comparison to the susceptible seedlings. CONLCUSION Both target-site Val-202-Phe alpha-tubulin mutation and non-target-site enhanced trifluralin metabolism co-exist in this dinitroaniline-resistant L. rigidum population. (c) 2019 Society of Chemical Industry

Wild radish (Raphanus raphanistrum L.) is a problematic and economically damaging dicotyledonous weed infesting crops in many regions of the world. Resistance to the auxinic herbicides 2,4-D and dicamba is widespread in Western Australian R. raphanistrum populations, with the resistance mechanism appearing to involve alterations in the physiological response to synthetic auxins and in plant defense. This study aimed to determine whether these alterations cause inhibition in plant growth or reproduction that could potentially be exploited to manage 2,4-D-resistant populations in cropping areas. Therefore, the morphology and seed production of resistant and susceptible populations were compared in an outdoor pot study, with plants grown in the presence and absence of competition by wheat (Triticum aestivum L.). The susceptible and resistant R. raphanistrum populations were equally suppressed by wheat competition, with plant growth and seed production being decreased by approximately 50%. Although resistant populations produced less vegetative biomass than susceptible populations, there was no negative association between resistance and seed production. Therefore, it is unlikely that any nonherbicidal management practices will be more efficacious on 2,4-D-resistant than 2,4-D-susceptible R. raphanistrum populations.