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A wild radish population (R) has been recently confirmed to be cross-resistant to 4-hydroxyphenylpyruvate dioxygenase (HPPD)-inhibiting herbicides without previous exposure to these herbicides. This cross-resistance is endowed by enhanced metabolism. Our study identified one 2-oxoglutarate/Fe(II)-dependent dioxygenase gene ( Rr2ODD1 ) and two P450 genes ( RrCYP704C1 and RrCYP709B1 ), which were significantly more highly expressed in R versus susceptible (S) plants. Gene functional characterization using Arabidopsis transformation showed that overexpression of RrCYP709B1 conferred a modest level of resistance to mesotrione. Ultra-performance liquid chromatography-tandem mass spectrometry analysis showed that tissue mesotrione levels in RrCYP709B1 transgenic Arabidopsis plants were significantly lower than that in the wild type. In addition, overexpression of Rr2ODD1 or RrCYP704C1 in Arabidopsis endowed resistance to tembotrione and isoxaflutole. Structural modeling indicated that mesotrione can bind to CYP709B1 and be easily hydroxylated to form 4-OH-mesotrione. Although each gene confers a modest level of resistance, overexpression of the multiple herbicide-metabolizing genes could contribute to HPPD-inhibiting herbicide resistance in this wild radish population.

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

Background: Eleusine indica (L.) Gaertn. (goosegrass) is a major weed in global cropping systems. It has evolved resistance to glyphosate due to single Pro-106-Ser (P106S) or double Thr-102-Ile + Pro-106-Ser (TIPS) EPSPS target site mutations. Here, experiments were conducted to evaluate the single effect of soybean competition and its combined effect with a glyphosate field dose (1080 g ae ha -1 ) on the growth and fitness of plants carrying these glyphosate resistance endowing target site mutations., Results: TIPS E. indica plants are highly glyphosate-resistant but the double mutation endows a substantial fitness cost. The TIPS fitness penalty increased under the effect of soybean competition resulting in a cost of 95%, 95% and 96% in terms of, respectively, vegetative growth, seed mass and seed number investment. Glyphosate treatment of these glyphosate-resistant TIPS plants showed an increase in growth relative to those without glyphosate. Conversely, for the P106S moderate glyphosate resistance mutation, glyphosate treatment alone reduced survival rate, vegetative growth, aboveground biomass (34%), seed mass (48%) and number (52%) of P106S plants relative to the glyphosate nontreated plants. However, under the combined effects of both soybean competition and the field-recommended glyphosate dose, vegetative growth, aboveground biomass, seed mass and number of P106S and TIPS plants were substantially limited (by ≤99%)., Conclusion: The ecological environment imposed by intense competition from a soybean crop sets a significant constraint for the landscape-level increase of both the E. indica single and double glyphosate resistance mutations in the agroecosystem and highlights the key role of crop competition in limiting the population growth of weeds, whether they are herbicide-resistant or susceptible. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Glufosinate is an important and widely used non-selective herbicide active on a wide range of plant species. Evolution of resistance to glufosinate in weedy plant species (including the global weed Eleusine indica) is underway. Here, we established the molecular basis of target site glufosinate resistance in Eleusine indica. Full-length E. indica glutamine synthetase (GS) iso-genes (EiGS1-1, 1-2, 1-3, and EiGS2) were cloned, and expression of EiGS1-1 and EiGS1-2 was higher than that of EiGS2. A novel point mutation resulting in a Ser59Gly substitution in EiGS1-1 was identified in glufosinate-resistant plants. Rice calli and seedlings transformed with the mutant EiGS1-1 gene were resistant to glufosinate. Purified mutant EiGS1-1 expressed in yeast was more tolerant to glufosinate than the wild-type variant. These transgenic results correlate with a more glufosinate-resistant GS in the crude tissue extract of resistant versus susceptible E. indica plants. Structural modelling of the mutant EiGS1-1 revealed that Ser59 is not directly involved in glufosinate binding but is in contact with some important binding residues (e.g. Glu297) and especially with Asp56 that forms an intratoroidal contact interface. Importantly, the same Ser59Gly mutation was also found in geographically isolated glufosinate-resistant populations from Malaysia and China, suggesting parallel evolution of this resistance mutation., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

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., (© 2022 Society of Chemical Industry.)

Background: Trifluralin is widely used in Australia as one of the important pre-emergence herbicides to control annual ryegrass (Lolium rigidum Gaud.) populations. Trifluralin resistance evolution and mechanisms have been identified in some ryegrass populations., Results: In this study, 21 putative resistant field survey populations from Western Australian were screened with trifluralin, and 90% (19 of 21) contained individuals surviving 480 g ha -1 trifluralin treatment. Twelve populations contained individuals possessing the known α-tubulin resistance mutations at Val-202, Thr-239 and Arg-243 in TUA4 (alpha-tubulin 4 n), plus multiple potential resistance mutations in TUA4 pending genetic confirmation. Three populations had only individuals carrying newly identified (but uncharacterized) mutations in TUA3/TUA4. Radioactive work found that six populations evolved metabolic resistance to trifluralin, and at least four of them also possessed the known and/or putative target-site mutations., Conclusion: These results confirm that a high incidence of resistance to the dinitroaniline herbicide (trifluralin) is present, and target-site tubulin mutations make a major contribution to resistance in these annual ryegrass populations. Co-evolution of both target-site and non-target-site resistance to per-emergence herbicides warrants diverse management tactics., (© 2021 Society of Chemical Industry.)

Glyphosate is the most widely used herbicide in world agriculture and for general vegetation control in a wide range of situations. Global and often intensive glyphosate selection of very large weedy plant populations has resulted in widespread glyphosate resistance evolution in populations of many weed species. Here, working with a glyphosate-resistant (GR) Echinochloa colona population that evolved in a Western Australia agricultural field, we identified an ATP-binding cassette (ABC) transporter ( EcABCC8 ) that is consistently up-regulated in GR plants. When expressed in transgenic rice, this EcABCC8 transporter endowed glyphosate resistance. Equally, rice, maize, and soybean overexpressing the EcABCC8 ortholog genes were made resistant to glyphosate. Conversely, CRISPR/Cas9-mediated knockout of the EcABCC8 ortholog gene OsABCC8 increased rice susceptibility to glyphosate. Subcellular localization analysis and quantification of glyphosate cellular levels in treated ABCC8 transgenic rice plants and isolated leaf protoplasts as well as structural modeling support that EcABCC8 is likely a plasma membrane-localized transporter extruding cytoplasmic glyphosate to the apoplast, lowering the cellular glyphosate level. This is a report of a membrane transporter effluxing glyphosate in a GR plant species, and its function is likely conserved in crop plant species., Competing Interests: The authors declare no competing interest.

Concurrent natural evolution of glyphosate resistance single- and double-point EPSPS mutations in weed species provides an opportunity for the estimation of resistance fitness benefits and prediction of equilibrium resistance frequencies in environments under glyphosate selection. Assessment of glyphosate resistance benefit was conducted for the most commonly identified single Pro-106-Ser and less-frequent double TIPS mutations in the EPSPS gene evolved in the global damaging weed Eleusine indica . Under glyphosate selection at the field dose, plants with the single Pro-106-Ser mutation at homozygous state (P106S-rr) showed reduced survival and compromised vegetative growth and fecundity compared with TIPS plants. Whereas both homozygous (TIPS-RR) and compound heterozygous (TIPS-Rr) plants with the double TIPS resistance mutation displayed similar survival rates when exposed to glyphosate, a significantly higher fecundity in the currency of seed number was observed in TIPS-Rr than TIPS-RR plants. The highest plant fitness benefit was associated with the heterozygous TIPS-Rr mutation, whereas plants with the homozygous Pro-106-Ser and TIPS mutations exhibited, respectively, 31% and 39% of the fitness benefit revealed by the TIPS-Rr plants. Populations are predicted to reach stable allelic and genotypic frequencies after 20 years of glyphosate selection at which the WT allele is lost and the stable genotypic polymorphism is comprised by 2% of heterozygous TIPS-Rr, 52% of homozygous TIPS-RR and 46% of homozygous P106S-rr. The high inbreeding nature of E .  indica is responsible for the expected frequency decrease in the fittest TIPS-Rr in favour of the homozygous TIPS-RR and P106S-rr. Mutated alleles associated with the glyphosate resistance EPSPS single EPSPS Pro-106-Ser and double TIPS mutations confer contrasting fitness benefits to E .  indica under glyphosate treatment and therefore are expected to exhibit contrasting evolution rates in cropping systems under recurrent glyphosate selection., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

Dinitroanilines are microtubule inhibitors, targeting tubulin proteins in plants and protists. Dinitroaniline herbicides, such as trifluralin, pendimethalin and oryzalin, have been used as pre-emergence herbicides for weed control for decades. With widespread resistance to post-emergence herbicides in weeds, the use of pre-emergence herbicides such as dinitroanilines has increased, in part, due to relatively slow evolution of resistance in weeds to these herbicides. Target-site resistance (TSR) to dinitroaniline herbicides due to point mutations in α-tubulin genes has been confirmed in a few weedy plant species (e.g., Eleusine indica , Setaria viridis , and recently in Lolium rigidum ). Of particular interest is the resistance mutation Arg-243-Met identified from dinitroaniline-resistant L. rigidum that causes helical growth when plants are homozygous for the mutation. The recessive nature of the TSR, plus possible fitness cost for some resistance mutations, likely slows resistance evolution. Furthermore, non-target-site resistance (NTSR) to dinitroanilines has been rarely reported and only confirmed in Lolium rigidum due to enhanced herbicide metabolism (metabolic resistance). A cytochrome P450 gene (CYP81A10) has been recently identified in L. rigidum that confers resistance to trifluralin. Moreover, TSR and NTSR have been shown to co-exist in the same weedy species, population, and plant. The implication of knowledge and information on TSR and NTSR in management of dinitroaniline resistance is discussed., Competing Interests: CS is employed by the company Nufarm Limited. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chen, Yu, Patterson, Sayer and Powles.)

Background: Tubulin, the target site of dinitroaniline herbicides, is encoded by small gene families in plants. To better characterize the mechanisms of target-site resistance to dinitroaniline herbicides in the globally important weedy species Lolium rigidum, attempts were made to amplify and sequence α-tubulin transcripts., Results: Four α-tubulin isoforms (TUA1, TUA2, TUA3 and TUA4) were identified in L. rigidum. Variations in the number and sequence of transcripts encoding these α-tubulin proteins were found in individuals from the two L. rigidum populations examined. Within and among populations, differences in the 5'- and 3'-untranslated regions of cDNA in TUA3 and TUA4 were identified. Furthermore, a novel double mutation, Arg-390-Cys+Asp-442-Glu, in the TUA3 transcript was identified and has the potential to confer dinitroaniline resistance., Conclusion: This research reveals the complexity of the α-tubulin gene family in individuals/populations of the cross-pollinated weedy species L. rigidum, and highlights the need for better understanding of the molecular architecture of tubulin gene families for detecting resistance point mutations. Although TUA4 is a commonly expressed α-tubulin isoform containing most frequently reported resistance mutations, other mutant tubulin isoforms may also have a role in conferring dinitroaniline resistance., (© 2020 Society of Chemical Industry.)

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F 2 populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)

Background: Barnyardgrass (Echinochloa spp.) is a global weed in rice fields. Quinclorac is commonly used to control barnyardgrass. However, due to persistent use, quinclorac resistance has evolved. We obtained quinclorac-susceptible (QS) and -resistant (QR1, QR2) lines from the progeny of a single resistant E. crus-pavonis for a resistance mechanism study., Results: Line QR1 exhibited resistance to high quinclorac rates (up to 6400 g ha -1 ), whereas line QR2 exhibited a resistance/susceptibility segregation ratio of 3:1 at the field or lower rates (400, 100 g ha -1 ). Intriguingly, a lower level of 14 C-quinclorac metabolism and hence a higher level of 14 C-quinclorac translocation was observed in QR1 than QS plants. The basal expression levels of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS) and ACC oxidase 2 (ACO2) genes did not differ significantly between the QR1 and QS lines. However, more expression of ACS and ACO genes was induced by quinclorac treatment in QS than in QR1. Basal levels of β-cyanoalanine synthase (β-CAS) gene expression were similar in QS and QR1 plants, but a greater level of down-regulation was detected in QS than in QR1 plants after quinclorac treatment., Conclusion: These results indicate QR plants are less responsive to quinclorac than QS plants in terms of up-regulating quinclorac metabolism and ethylene synthesis. Resistance in this E. crus-pavonis line is likely controlled by a single major gene, involving possibly an alteration in auxin signal perception/transduction to the ethylene biosynthesis pathway. The β-CAS is unlikely to play a major role in quinclorac resistance in this particular population., (© 2020 Society of Chemical Industry.)

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.)

The combination of herbicides with different modes of action has been adopted not only to improve weed control but also to increase the environmental sustainability of plant-protection products. In this study, we showed a synergistic effect of the auxin herbicide 2,4-D amine with the PSII-inhibiting herbicide metribuzin to control the global grass weed wild oat ( Avena sterilis ) population and investigated the underlying mechanisms. Pretreatment with 2,4-D amine did not change the foliar absorption of metribuzin but did increase metribuzin translocation to the roots and new leaves, although enhancement of the metribuzin metabolism was also observed. Considering that the expression level of the target site psbA gene is significantly higher in leaves than in roots, increased metribuzin translocation to new leaves is likely the major cause of the observed synergism, even though enhanced metribuzin metabolism may offset the metribuzin efficacy. This is the first report on the synergistic mechanism between 2,4-D amine and metribuzin in weed control., (© Pesticide Science Society of Japan 2020. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) License (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Background: The photosystem II (PSII)-inhibiting herbicides are important for Australian farmers to control Lolium rigidum Gaud. and other weed species in trazine tolerant (TT)-canola fields. A L. rigidum population (R) collected from a TT-canola field from Western Australia showed multiple resistance to PSII, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS) inhibitors. The mechanisms of multiple resistance in this R population were determined., Results: The R population showed a low-level (about 3.0-fold) resistance to the PSII-inhibiting herbicides metribuzin and atrazine. Sequencing of the psbA gene revealed no differences between the R and susceptible (S) sequences. Furthermore, [ 14 C]-metribuzin experiments found no significant difference in metribuzin foliar uptake and translocation between the R and S plants. However, [ 14 C]-metribuzin metabolism in R plants was 2.3-fold greater than in S plants. The cytochrome P450 monooxygenase inhibitor piperonyl butoxide (PBO) enhanced plant mortality response to metribuzin and atrazine in both R and S populations. In addition, multiple resistance to ALS and ACCase inhibitors are due to known resistance mutations in ALS and ACCase genes., Conclusion: The results demonstrate that enhanced metribuzin metabolism likely involving cytochrome P450 monooxygenase contributes to metribuzin resistance in Lolium rigidum. This is the first report of metabolic resistance to the PSII-inhibiting herbicide metribuzin in Australian Lolium rigidum. © 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.)

Background: Diflufenican resistance has been reported in wild radish populations since 1998, but the resistance mechanisms have not been investigated. Recently, we identified a wild radish population (H2/10) from the Western Australian grain belt that is resistant (R) to the phytoene desaturase (PDS)-inhibiting herbicide diflufenican., Results: Dose-response results showed this R population is 4.9-fold more resistant than the susceptible (S) population based on the LD 50 R/S ratio. In addition, the R population also exhibits cross-resistance to the PDS-inhibiting herbicide fluridone. The cytochrome P450 inhibitor malathion reversed diflufenican resistance and partially reversed fluridone resistance in the R population. The full coding sequences of the PDS gene were cloned from the S and R plants and there are natural variations in the PDS gene transcripts/alleles with no correlation to resistance. In addition, the R plants had a level of PDS gene expression that is not significantly different from the S plants., Conclusion: These results demonstrated that diflufenican resistance in this R wild radish population is likely due to non-target-site based enhanced herbicide metabolism involving cytochrome P450s. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Background: Relatively new herbicides that target 4-hydroxyphenylpyruvate dioxygenase (HPPD) are now available for use on the world's great grain crops (rice, wheat, corn and soybean) and for other uses. With widespread and persistent use of HPPD-inhibiting herbicides, the evolution of HPPD-inhibiting herbicide resistant weeds is inevitable. Currently, resistance to HPPD-inhibiting herbicides is known in two weed species, waterhemp and Palmer amaranth. Here, we report a HPPD-inhibiting herbicide resistant wild radish population from the Western Australia grain belt. This population was not selected with HPPD-inhibiting herbicides, rather it evolved resistance to earlier used herbicides with different modes of action and exhibits cross-resistance to HPPD-inhibiting herbicides., Results: Dose-response experiments showed the resistant (R) population exhibits 4 to 6.5-fold resistance to the HPPD-inhibiting herbicides mesotrione, tembotrione and isoxaflutole, compared to the susceptible (S) population. This resistance is not target-site based as cloning of full coding sequences of the HPPD genes from S and R plants did not reveal resistance-endowing single nucleotide polymorphisms. The HPPD gene expression levels are similar in S and R plants. In addition, no differences in [ 14 C]-mesotrione uptake and translocation were observed in the S and R plants. However, the time required for R plants to metabolise 50% [ 14 C]-mesotrione is 7.7-fold faster than for the S plants., Conclusion: We confirm resistance to HPPD-inhibiting herbicides exists in a population of the economically damaging global weed wild radish. The resistance in this population is due to a non-target-site based enhanced rate of herbicide metabolism. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Background and Aims: Resistance to the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in wild radish (Raphanus raphanistrum) appears to be due to a complex, multifaceted mechanism possibly involving enhanced constitutive plant defence and alterations in auxin signalling. Based on a previous gene expression analysis highlighting the plasma membrane as being important for 2,4-D resistance, this study aimed to identify the components of the leaf plasma membrane proteome that contribute to resistance., Methods: Isobaric tagging of peptides was used to compare the plasma membrane proteomes of a 2,4-D-susceptible and a 2,4-D-resistant wild radish population under control and 2,4-D-treated conditions. Eight differentially abundant proteins were then targeted for quantification in the plasma membranes of 13 wild radish populations (two susceptible, 11 resistant) using multiple reaction monitoring., Key Results: Two receptor-like kinases of unknown function (L-type lectin domain-containing receptor kinase IV.1-like and At1g51820-like) and the ATP-binding cassette transporter ABCB19, an auxin efflux transporter, were identified as being associated with auxinic herbicide resistance. The variability between wild radish populations suggests that the relative contributions of these candidates are different in the different populations., Conclusions: To date, no receptor-like kinases have been reported to play a role in 2,4-D resistance. The lectin-domain-containing kinase may be involved in perception of 2,4-D at the plasma membrane, but its ability to bind 2,4-D and the identity of its signalling partner(s) need to be confirmed experimentally. ABCB19 is known to export auxinic compounds, but its role in 2,4-D resistance in wild radish appears to be relatively minor., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2019 Society of Chemical Industry.)

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 α-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 α-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 α-tubulin mutation and non-target-site enhanced trifluralin metabolism co-exist in this dinitroaniline-resistant L. rigidum population. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

Glyphosate, the most commonly used herbicide in the world, controls a wide range of plant species, mainly because plants have little capacity to metabolize (detoxify) glyphosate. Massive glyphosate use has led to world-wide evolution of glyphosate-resistant (GR) weed species, including the economically damaging grass weed Echinochloa colona An Australian population of E colona has evolved resistance to glyphosate with unknown mechanisms that do not involve the glyphosate target enzyme 5-enolpyruvylshikimate-3-P synthase. GR and glyphosate-susceptible (S) lines were isolated from this population and used for resistance gene discovery. RNA sequencing analysis and phenotype/genotype validation experiments revealed that one aldo-keto reductase (AKR) contig had higher expression and higher resultant AKR activity in GR than S plants. Two full-length AKR ( EcAKR4 - 1 and EcAKR4 - 2 ) complementary DNA transcripts were cloned with identical sequences between the GR and S plants but were upregulated in the GR plants. Rice ( Oryza sativa ) calli and seedlings overexpressing EcAKR4 - 1 and displaying increased AKR activity were resistant to glyphosate. EcAKR4-1 expressed in Escherichia coli can metabolize glyphosate to produce aminomethylphosphonic acid and glyoxylate. Consistent with these results, GR E colona plants exhibited enhanced capacity for detoxifying glyphosate into aminomethylphosphonic acid and glyoxylate. Structural modeling predicted that glyphosate binds to EcAKR4-1 for oxidation, and metabolomics analysis of EcAKR4 - 1 transgenic rice seedlings revealed possible redox pathways involved in glyphosate metabolism. Our study provides direct experimental evidence of the evolution of a plant AKR that metabolizes glyphosate and thereby confers glyphosate resistance., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

We reviewed the literature to understand the effects of glyphosate resistance on plant fitness at the molecular, biochemical and physiological levels. A number of correlations between enzyme characteristics and glyphosate resistance imply the existence of a plant fitness cost associated with resistance-conferring mutations in the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). These biochemical changes result in a tradeoff between the glyphosate resistance of the EPSPS enzyme and its catalytic activity. Mutations that endow the highest resistance are more likely to decrease catalytic activity by reducing the affinity of EPSPS for its natural substrate, and/or slowing the velocity of the enzyme reaction, and are thus very likely to endow a substantial plant fitness cost. Prediction of fitness costs associated with EPSPS gene amplification and overexpression can be more problematic. The validity of cost prediction based on the theory of evolution of gene expression and resource allocation has been cast into doubt by contradictory experimental evidence. Further research providing insights into the role of the EPSPS cassette in weed adaptation, and estimations of the energy budget involved in EPSPS amplification and overexpression are required to understand and predict the biochemical and physiological bases of the fitness cost of glyphosate resistance., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The increasing number of weedy species resistant to dinitroaniline herbicides warrants studies on the evolutionary factors contributing to resistance evolution, including genetic inheritance of resistance traits. In this study, we investigated the genetic control of trifluralin resistance in a well-characterised Lolium rigidum Gaud. population from Western Australia. This population was purified to contain plants homozygous for the Val-202-Phe α-tubulin mutation, and used as the resistant (R) parents and crossed with susceptible (S) parents to produce eight reciprocal F1 families. Trifluralin dose response curves of the eight F1 families indicate that trifluralin resistance in this population is inherited as an incomplete recessive nuclear trait. The F1 plants were crossed within each families to establish eight pseudo-F2 (ψ-F2) families. Segregation ratio of resistance and susceptibility in ψ-F2 families were determined using the discriminating trifluralin rates of 120 and 480 g a.i. ha -1 . At 480 g a.i. ha -1 trifluralin, the segregation ratio in almost all ψ-F2 families (except one) was fit to 1:3 (resistance: susceptibility) one recessive gene control model. However, at 120 g a.i. ha -1 trifluralin, the segregation ratios in half of the families did not fit this model, indicating involvement of one or more genes in resistance at the lower rate. These results showed complexity of genetic inheritance of trifluralin resistance in this L. rigidum population possessing the Val-202-Phe α-tubulin mutation., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

There have been many studies on target-site resistance (TSR) to PSII-inhibiting herbicides, but only a few on the non-target-site resistance (NTSR). Here, we reported both TSR and NTSR to metribuzin in a wild radish population. Dose-response studies revealed a higher level of resistance to metribuzin in the resistant (R) compared to the susceptible (S) population. Sequencing of the target psbA gene revealed the known Ser-264-Gly mutation in R plants. In addition, a higher level of [ 14 C]-metribuzin metabolism and, consequently, a lower level of [ 14 C] translocation were also detected in the R plants. These results demonstrated that both psbA gene mutation and enhanced metabolism contribute to metribuzin resistance in this wild radish population. Furthermore, this resistant population showed resistance to ALS-inhibiting herbicides due to multiple ALS gene mutations. This is the first report in wild radish of metabolic herbicide resistance, in addition to the target-site psbA gene mutation.

Background: Wild radish (Raphanus raphanistrum) is a globally important weed of crops. Two atrazine-resistant wild radish populations (R1 and R2), collected from the Western Australia grain belt, were investigated for resistance to photosystem II (PSII) herbicides., Results: Sequencing of the full-length psbA gene revealed the well-known Ser264-Gly substitution in population R1, whereas population R2 displayed a novel Phe274-Val substitution. Herbicide dose-response studies confirmed that the population with the Ser264-Gly mutation exhibited high-level resistance to atrazine, but super-sensitivity to bromoxynil. Plants possessing the novel Phe274-Val mutation exhibited a modest level of resistance to atrazine, metribuzin and diuron, and were bromoxynil susceptible. Structural modelling of the mutant D1 proteins predicts that the Ser264-Gly mutation endows atrazine resistance by abolishing H-bonds, but confers bromoxynil super-sensitivity by enhancing hydrogen bonding. The Phe274-Val substitution provides resistance to atrazine and diuron by indirectly affecting H-bond formation between the Ser264 residue and the herbicides., Conclusion: The results demonstrate that the Phe274-Val mutation is likely responsible for resistance to PSII-inhibiting triazine and urea herbicides. To our knowledge, this is the first evidence of the psbA Phe274-Val mutation in wild radish conferring resistance to PSII herbicides. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Synthetic auxin herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D), are widely used for selective control of broadleaf weeds in cereals and transgenic crops. Although the troublesome weed wild radish ( Raphanus raphanistrum) has developed resistance to 2,4-D, no populations have yet displayed an enhanced capacity for metabolic detoxification of the herbicide, with both susceptible and resistant wild radish plants readily metabolizing 2,4-D. Using mass spectrometry and nuclear magnetic resonance, the major 2,4-D metabolite was identified as the glucose ester, and its structure was confirmed by synthesis. As expected, both the endogenous and synthetic compounds retained auxin activity in a bioassay. The lack of detectable 2,4-D hydroxylation in wild radish and the lability of the glucose ester suggest that metabolic 2,4-D resistance is unlikely to develop in this species.

Background and Aims: Resistance to synthetic auxin herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D) is increasing in weed populations worldwide, which is of concern given the recent introduction of synthetic auxin-resistant transgenic crops. Due to the complex mode of action of the auxinic herbicides, the mechanisms of evolved resistance remain largely uncharacterized. The aims of this study were to assess the level of diversity in resistance mechanisms in 11 populations of the problem weed Raphanus raphanistrum, and to use a high-throughput, whole-genome transcriptomic analysis on one resistant and one susceptible population to identify important changes in gene expression in response to 2,4-D., Methods: Levels of 2,4-D and dicamba (3,6-dichloro-2-methoxybenzoic acid) resistance were quantified in a dose-response study and the populations were further screened for auxin selectivity, 2,4-D translocation and metabolism, expression of key 2,4-D-responsive genes and activation of the mitogen-activated proein kinase (MAPK) pathway. Potential links between resistance levels and mechanisms were assessed using correlation analysis., Key Results: The transcriptomic study revealed early deployment of the plant defence response in the 2,4-D-treated resistant population, and there was a corresponding positive relationship between auxinic herbicide resistance and constitutive MAPK phosphorylation across all populations. Populations with shoot-wide translocation of 2,4-D had similar resistance levels to those with restricted translocation, suggesting that reduced translocation may not be as strong a resistance mechanism as originally thought. Differences in auxin selectivity between populations point to the likelihood of different resistance-conferring alterations in auxin signalling and/or perception in the different populations., Conclusions: 2,4-D resistance in wild radish appears to result from subtly different auxin signalling alterations in different populations, supplemented by an enhanced defence response and, in some cases, reduced 2,4-D translocation. This study highlights the dangers of applying knowledge generated from a few populations of a weed species to the species as a whole.

This study confirmed the first case of glyphosate resistance in Tridax procumbens and investigated the glyphosate-resistance mechanisms. Sequencing and cloning of the full 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) coding sequences revealed a point mutation (ACC to TCC) at amino acid position 102, resulting in a novel Thr-102-Ser substitution. Other possible resistance mechanisms (i.e., target-site EPSPS-gene overexpression, nontarget-site differential glyphosate uptake and translocation) were also examined and were unlikely to be involved in resistance in this population. Structural modeling of the wild-type and mutant EPSPS in complex with glyphosate and phosphoenolpyruvate (PEP) revealed that the Thr-102-Ser substitution weakly decreased EPSPS affinity to glyphosate, but sharply increased EPSPS affinity to the natural substrate, PEP. Therefore, this novel mutation is very likely responsible for the observed glyphosate resistance in this tetraploid weed species via dual mechanisms of reducing glyphosate binding and favoring PEP binding to EPSPS.

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: The pre-emergence dinitroaniline herbicides (such as trifluralin and pendimethalin) are vital to Australian no-till farming systems. A Lolium rigidum population collected from the Western Australian grain belt with a 12-year trifluralin use history was characterised for resistance to dinitroaniline, acetyl CoA carboxylase (ACCase)- and acetolactate synthase (ALS)-inhibiting herbicides. Target-site resistance mechanisms were investigated., Results: This L. rigidum population exhibited 32-fold resistance to trifluralin, as compared with the susceptible population. It also displayed 12- to 30-fold cross-resistance to other dinitroaniline herbicides (pendimethalin, ethalfluralin and oryzalin). In addition, this population showed multiple resistance to commonly used post-emergence ACCase- and ALS-inhibiting herbicides. Two target-site α-tubulin gene mutations (Val-202-Phe and Thr-239-Ile) previously documented in other dinitroaniline-resistant weed species were identified, and some known target-site mutations in ACCase (Ile-1781-Leu, Asp-2078-Gly and Cys-2088-Arg) and ALS (Pro-197-Gln/Ser) were found in the same population. An agar-based Petri dish screening method was established for the rapid diagnosis of resistance to dinitroaniline herbicides., Conclusion: Evolution of target-site resistance to both pre- and post-emergence herbicides was confirmed in a single L. rigidum population. The α-tubulin mutations Val-202-Phe and Thr-239-Ile, documented here for the first time in L. rigidum, are likely to be responsible for dinitroaniline resistance in this population. Early detection of dinitroaniline herbicide resistance and integrated weed management strategies are needed to maintain the effectiveness of dinitroaniline herbicides. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The dinitroaniline herbicides (particularly trifluralin) have been globally used in many crops for selective grass weed control. Consequently, trifluralin resistance has been documented in several important crop weed species and has recently reached a level of concern in Australian Lolium rigidum populations. Here, we report novel mutations in the L. rigidum α-tubulin gene which confer resistance to trifluralin and other dinitroaniline herbicides. Nucleotide mutations at the highly conserved codon Arg-243 resulted in amino acid substitutions of Met or Lys. Rice calli transformed with the mutant 243-Met or 243-Lys α-tubulin genes were 4- to 8-fold more resistant to trifluralin and other dinitroaniline herbicides (e.g., ethalfluralin and pendimethalin) compared to calli transformed with the wild type α-tubulin gene from L. rigidum . Comprehensive modeling of molecular docking predicts that Arg-243 is close to the trifluralin binding site on the α-tubulin surface and that replacement of Arg-243 by Met/Lys-243 results in a spatial shift of the trifluralin binding domain, reduction of trifluralin-tubulin contacts, and unfavorable interactions. The major effect of these substitutions is a significant rise of free interaction energy between α-tubulin and trifluralin, as well as between trifluralin and its whole molecular environment. These results demonstrate that the Arg-243 residue in α-tubulin is a determinant for trifluralin sensitivity, and the novel Arg-243-Met/Lys mutations may confer trifluralin resistance in L. rigidum .

A novel glyphosate resistance double point mutation (T102I/P106S, TIPS) in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene has been recently identified for the first time only in the weed species Eleusine indica. Quantification of plant resistance cost associated with the TIPS and the often reported glyphosate resistance single P106S mutation was performed. A significant resistance cost (50% in seed number currency) associated with the homozygous TIPS but not the homozygous P106S EPSPS variant was identified in E. indica plants. The resistance cost associated with the TIPS mutation escalated to 85% in plants under resource competition with rice crops. The resistance cost was not detected in nonhomozygous TIPS plants denoting the recessive nature of the cost associated with the TIPS allele. An excess of 11-fold more shikimate and sixfold more quinate in the shikimate pathway was detected in TIPS plants in the absence of glyphosate treatment compared to wild type, whereas no changes in these compounds were observed in P106S plants when compared to wild type. TIPS plants show altered metabolite levels in several other metabolic pathways that may account for the expression of the observed resistance cost., (© 2017 John Wiley & Sons Ltd.)

Background: An Eleusine indica population has evolved resistance to glufosinate, a major post-emergence herbicide of global agriculture. This population was analysed for target-site (glutamine synthetase) and non-target-site (glufosinate uptake, translocation and metabolism) resistance mechanisms., Results: Glutamine synthetase (GS) activity extracted from susceptible (S) and resistant (R*) plants was equally sensitive to glufosinate inhibition, with IC 50 values of 0.85 mm and 0.99 mm, respectively. The extractable GS activity was also similar in S and R* samples. Foliar uptake of [ 14 C]-glufosinate did not differ in S and R* plants, nor did glufosinate net uptake in leaf discs. Translocation of [ 14 C]-glufosinate into untreated shoots and roots was also similar in both populations, with 44% to 47% of the herbicide translocated out from the treated leaf 24 h after treatment. The HPLC and LC-MS analysis of glufosinate metabolism revealed no major metabolites in S or R* leaf tissue., Conclusions: Glufosinate resistance in this resistant population is not due to an insensitive GS, or increased activity, or altered glufosinate uptake and translocation, or enhanced glufosinate metabolism. Thus, target-site resistance is likely excluded and the exact resistance mechanism(s) remain to be determined. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: Across several agricultural systems the evolution of herbicide resistance has occurred more rapidly to post-emergence than pre-emergence herbicides; however, the reasons for this are not clear. We used a new simulation model to investigate whether interactions between differences in order of application and weed cohorts affected could explain this historically observed difference between the herbicide groups., Results: A 10 year delay in resistance evolution was predicted for a shorter-acting residual pre-emergence (cf. post-emergence), when all other parameters were identical. Differences in order of application between pre- and post-emergence herbicides had minimal effect on rates of resistance evolution when similar weed cohorts were affected., Conclusion: This modelling suggested that the historically observed lower levels of resistance to pre-emergence herbicides are most likely to be due to the smaller number of weed cohorts affected by many pre-emergence herbicides. The lower number of weed cohorts affected by pre-emergence herbicides necessitated the use of additional, effective control measures, thereby reducing resistance evolution. This study highlights the advantages of applying multiple control measures to each weed cohort. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

A relatively low number of weed species have evolved resistance to auxinic herbicides despite their use for almost 70 years. This inheritance study with two Raphanus raphanistrum populations multiple-resistant 2,4-D and the ALS-inhibiting herbicide chlorsulfuron determined the number of genes and genetic dominance of 2,4-D resistance and investigated the association between traits conferring resistance to the two herbicide modes of action. Levels of 2,4-D phenotypic resistance and resistance segregation patterns were assessed in parental populations, F 1 and F 2 families., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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: Rice is a major field crop of paramount importance for global food security. However, the increased adoption of more profitable and resource-efficient direct-seeded rice (DSR) systems has contributed to greater weed infestations, including weedy rice, which has become a severe problem in several Asian regions. In this study we have developed a conceptually novel method to protect rice plants at high doses of clomazone and triallate., Results: The insecticide phorate applied to rice seeds provided a substantial level of protection against the herbicides clomazone or triallate. A quantity of 15 kg phorate ha -1 significantly increased the LD 50 values, which were more than twofold greater than for rice plants treated only with clomazone. A quantity of 20 kg phorate ha -1 in combination with 2000 g triallate ha -1 safened rice plants (80% survival) with LD 50 >3.4-fold greater than in phorate-untreated rice. Weed control efficacy was not lowered by the presence of phorate-treated rice seeds., Conclusion: Weedy rice is one of the most damaging global weeds and a major threat to DSR systems. In this study we have developed a proof-of-concept method to allow selective weedy rice control in rice crops. We call for herbicide discovery programmes and research to identify candidate safener and herbicide combinations to achieve selective herbicide control of weedy rice and alleviate weed infestations in global rice crops. © 2016 Society of Chemical Industry., (© 2016 Society of Chemical Industry.)

Background: When used at effective doses, weed resistance to auxinic herbicides has been slow to evolve when compared with other modes of action. Here we report the evolutionary response of a herbicide-susceptible population of wild radish (Raphanus raphanistrum L.) and confirm that sublethal doses of 2,4-dichlorophenoxyacetic acid (2,4-D) amine can lead to the rapid evolution of 2,4-D resistance and cross-resistance to acetolactate synthase (ALS)-inhibiting herbicides., Results: Following four generations of 2,4-D selection, the progeny of a herbicide-susceptible wild radish population evolved 2,4-D resistance, increasing the LD 50 from 16 to 138 g ha -1 . Along with 2,4-D resistance, cross-resistance to the ALS-inhibiting herbicides metosulam (4.0-fold) and chlorsulfuron (4.5-fold) was evident. Pretreatment of the 2,4-D-selected population with the cytochrome P450 inhibitor malathion restored chlorsulfuron to full efficacy, indicating that cross-resistance to chlorsulfuron was likely due to P450-catalysed enhanced rates of herbicide metabolism., Conclusion: This study is the first to confirm the rapid evolution of auxinic herbicide resistance through the use of low doses of 2,4-D and serves as a reminder that 2,4-D must always be used at highly effective doses. With the introduction of transgenic auxinic-herbicide-resistant crops in the Americas, there will be a marked increase in auxinic herbicide use and therefore the risk of resistance evolution. Auxinic herbicides should be used only at effective doses and with diversity if resistance is to remain a minimal issue. © 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.)

Resistance to auxinic herbicides is increasing in a range of dicotyledonous weed species, but in most cases the biochemical mechanism of resistance is unknown. Using (14)C-labelled herbicide, the mechanism of resistance to 2,4-dichlorophenoxyacetic acid (2,4-D) in two wild radish (Raphanus raphanistrum L.) populations was identified as an inability to translocate 2,4-D out of the treated leaf. Although 2,4-D was metabolized in wild radish, and in a different manner to the well-characterized crop species wheat and bean, there was no difference in metabolism between the susceptible and resistant populations. Reduced translocation of 2,4-D in the latter was also not due to sequestration of the herbicide, or to reduced uptake by the leaf epidermis or mesophyll cells. Application of auxin efflux or ABCB transporter inhibitors to 2,4-D-susceptible plants caused a mimicking of the reduced-translocation resistance phenotype, suggesting that 2,4-D resistance in the populations under investigation could be due to an alteration in the activity of a plasma membrane ABCB-type auxin transporter responsible for facilitating long-distance transport of 2,4-D., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: When applied at the correct plant stage and dose, herbicides are highly toxic to plants. At reduced, low herbicide doses (below the recommended dose) plants can survive and display continuous and quantitative variation in dose-survival responses. Recurrent (directional) selection studies can reveal whether such a phenotypic variation in plant survival response to low herbicide dose is heritable and leads to herbicide resistance. In a common experimental garden study, we have subjected a susceptible population of self-pollinated hexaploid Avena fatua to low-dose recurrent selection with the ACCase-inhibiting herbicide diclofop-methyl for three consecutive generations., Results: Significant differences in response to low-dose diclofop-methyl selection were observed between the selected progenies and parent plants, with a twofold diclofop-methyl resistance and cross-resistance to ALS-inhibiting herbicides. Thus, the capacity of self-pollinated A. fatua to respond to low-dose herbicide selection is marginal, and it is much lower than in cross-pollinated L. rigidum. Lolium rigidum in the same experiment evolved 40-fold diclofop-methyl resistance by progressive enrichment of quantitative resistance-endowing traits., Conclusion: Cross-pollination rate, genetic variation and ploidy levels are identified as possible drivers affecting the contrasting capacity of Avena versus Lolium plants to respond to herbicide selection and the subsequent likelihood of resistance evolution at low herbicide dose usage., (© 2015 Society of Chemical Industry.)

Herbicides have been the primary tool for controlling large populations of yield depleting weeds from agro-ecosystems, resulting in the evolution of widespread herbicide resistance. In response, nonherbicidal techniques have been developed which intercept weed seeds at harvest before they enter the soil seed bank. However, the efficiency of these techniques allows an intense selection for any trait that enables weeds to evade collection, with early-flowering ecotypes considered likely to result in early seed shedding. Using a field-collected wild radish population, five recurrent generations were selected for early maturity and three generations for late maturity. Phenology associated with flowering time and growth traits were measured. Our results demonstrate the adaptive capacity of wild radish to halve its time to flowering following five generations of early-flowering selection. Early-maturing phenotypes had reduced height and biomass at maturity, leading to less competitive, more prostrate growth forms. Following three generations of late-flowering selection, wild radish doubled its time to flowering time leading to increased biomass and flowering height at maturity. This study demonstrates the potential for the rapid evolution in growth traits in response to highly effective seed collection techniques that imposed a selection on weed populations within agro-ecosystems at harvest.

Background: This study confirms and characterises glyphosate resistance in two polyploid Echinochloa colona populations from north-eastern Australia., Results: Glyphosate dose response revealed that the two resistant populations were marginally (up to twofold) resistant to glyphosate. Resistant plants did not differ in non-target-site foliar uptake and translocation of (14) C-glyphosate, but contained the known target-site 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutation Pro-106-Thr and/or Pro-106-Leu. Although plants carrying either a single or two EPSPS mutations were glyphosate resistant relative to the susceptible population, they were still controlled at the field rate of glyphosate (450 g a.e. ha(-1) ) when treated under warm conditions (25/20 °C). However, when treated in hot conditions (35/30 °C), most mutant resistant plants (68%) can survive the field rate, and an increase (2.5-fold) in glyphosate LD50 was found for both the R and S populations., Conclusions: This study shows that one or two EPSPS Pro-106 mutations are insufficient to confer field-rate glyphosate resistance in polyploidy E. colona at mild temperatures. However, control of these mutant plants at the glyphosate field rate is poor at high temperatures, probably owing to reduced glyphosate efficacy. Therefore, glyphosate should be applied during relatively mild (warm) temperature periods in the summer growing season to improve E. colona control., (© 2015 Society of Chemical Industry.)

Background: Lolium rigidum populations in Australia and globally have demonstrated rapid and widespread evolution of resistance to acetyl coenzyme A carboxylase (ACCase)-inhibiting and acetolactate synthase (ALS)-inhibiting herbicides. Thirty-three resistant L. rigidum populations, randomly collected from crop fields in a most recent resistance survey, were analysed for non-target-site diclofop metabolism and all known target-site ACCase gene resistance-endowing mutations., Results: The HPLC profile of [(14) C]-diclofop-methyl in vivo metabolism revealed that 79% of these resistant L. rigidum populations showed enhanced capacity for diclofop acid metabolism (metabolic resistance). ACCase gene sequencing identified that 91% of the populations contain plants with ACCase resistance mutation(s). Importantly, 70% of the populations exhibit both non-target-site metabolic resistance and target-site ACCase mutations., Conclusions: This work demonstrates that metabolic herbicide resistance is commonly occurring in L. rigidum, and coevolution of both metabolic resistance and target-site resistance is an evolutionary reality. Metabolic herbicide resistance can potentially endow resistance to many herbicides and poses a threat to herbicide sustainability and thus crop production, calling for major research and management efforts., (© 2015 Society of Chemical Industry.)

Background: A population of Echinochloa colona infesting agricultural fields in the northern region of Western Australia evolved glyphosate resistance after 10 years of glyphosate selection. This study identified two phenotypic (susceptible S versus resistant R) lines from within a segregating glyphosate-resistant population. Estimation of survival, growth and reproductive rates of the phenotypes in response to glyphosate selection helped to characterise the level of resistance, fitness and the selection intensity for glyphosate in this species., Results: Estimations of LD(50) (lethal dose) and GR(50) (growth rate) showed an eightfold glyphosate resistance in this population. The resistant index based on the estimation of seed number (SY(n50)) showed a 13-fold resistance. As a result of linear combination of plant survival and fecundity rates, plant fitness values of 0.2 and 0.8 were estimated for the S and R phenotypes when exposed to the low dose of 270 g glyphosate ha(-1). At the recommended dose of 540 g glyphosate ha(-1) , fitness significantly decreased (fivefold) in S plants but remained markedly similar (0.7) in plants of the R phenotype. Thus, the calculated selection intensity (SI) at 540 g glyphosate ha(-1) was much greater (SI = 17) than at 270 g glyphosate ha(-1) (SI = 4)., Conclusions: The assessment of plant survival and fecundity in response to glyphosate selection in the S and R phenotypes allowed a greater accuracy in the estimation of population fitness of both phenotypes and thus of glyphosate selection intensity in E. colona. The estimation of seed number or mass of phenotypes under herbicide selection is a true ecological measure of resistance with implications for herbicide resistance evolution., (© 2015 Society of Chemical Industry.)

The rate of herbicide resistance evolution in plants depends on fitness traits endowed by alleles in both the presence and absence (resistance cost) of herbicide selection. The effect of two Lolium rigidum spontaneous homozygous target-site resistance-endowing mutations (Ile-1781-Leu, Asp-2078-Gly) on both ACCase activity and various plant growth traits have been investigated here. Relative growth rate (RGR) and components (net assimilation rate, leaf area ratio), resource allocation to different organs, and growth responses in competition with a wheat crop were assessed. Unlike plants carrying the Ile-1781-Leu resistance mutation, plants homozygous for the Asp-2078-Gly mutation exhibited a significantly lower RGR (30%), which translated into lower allocation of biomass to roots, shoots, and leaves, and poor responses to plant competition. Both the negligible and significant growth reductions associated, respectively, with the Ile-1781-Leu and Asp-2078-Gly resistance mutations correlated with their impact on ACCase activity. Whereas the Ile-1781-Leu mutation showed no pleiotropic effects on ACCase kinetics, the Asp-2078-Gly mutation led to a significant reduction in ACCase activity. The impaired growth traits are discussed in the context of resistance costs and the effects of each resistance allele on ACCase activity. Similar effects of these two particular ACCase mutations on the ACCase activity of Alopecurus myosuroides were also confirmed., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Background: Lolium rigidum (annual ryegrass) is a widespread annual crop weed that has evolved high levels of resistance to selective herbicides. Anecdotal evidence suggests that intensive cropping also leads to higher seed dormancy in L. rigidum. This was quantified by measuring dormancy levels in L. rigidum populations collected from paired sites (one with nil to low cropping intensity, the other intensively cropped) located throughout the Western Australian grain belt., Results: Populations from non-cropped fields or those with low cropping intensity showed higher and faster germination than populations from fields with a medium- or high-intensity cropping regime. Resistance to selective herbicides was also higher in the medium- and high-intensity cropping fields than in the low-intensity cropping fields., Conclusion: High-intensity cropping systems are likely to impose greater selection pressures for seed dormancy and selective herbicide resistance, because late-emerging seedlings avoid preplanting weed control practices (tillage and non-selective herbicide application) but are exposed to selective in-crop herbicides., (© 2014 Society of Chemical Industry.)