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

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

Challenging glyphosate resistance EPSPS P106S and TIPS mutations with soybean competition and glyphosate: implications for management.

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.

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

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

Contrasting plant ecological benefits endowed by naturally occurring EPSPS resistance mutations under glyphosate selection.

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.

Exploring quinclorac resistance mechanisms in Echinochloa crus-pavonis from China.

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 ß-cyanoalanine synthase (ß-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 ß-CAS is unlikely to play a major role in quinclorac resistance in this particular population.

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action.

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 F2 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.

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

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

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

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

Rotations and mixtures of soil-applied herbicides delay resistance.

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

A Val-202-Phe α-tubulin mutation and enhanced metabolism confer dinitroaniline resistance in a single Lolium rigidum population.

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.

Evolution of resistance to HPPD-inhibiting herbicides in a wild radish population via enhanced herbicide metabolism.

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.

Non-target-site resistance to PDS-inhibiting herbicides in a wild radish (Raphanus raphanistrum) population.

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 LD50 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.

Plasma membrane receptor-like kinases and transporters are associated with 2,4-D resistance in wild radish.

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.

Do plants pay a fitness cost to be resistant to glyphosate?

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.

Metribuzin Resistance in a Wild Radish ( Raphanus raphanistrum) Population via Both psbA Gene Mutation and Enhanced Metabolism.

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 [14C]-metribuzin metabolism and, consequently, a lower level of [14C] 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.

A novel psbA mutation (Phe274-Val) confers resistance to PSII herbicides in wild radish (Raphanus raphanistrum).

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.

Identity and Activity of 2,4-Dichlorophenoxyacetic Acid Metabolites in Wild Radish ( Raphanus raphanistrum).

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.

Pyroxasulfone resistance in Lolium rigidum is metabolism-based.

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 [14C]-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.

2,4-D and dicamba resistance mechanisms in wild radish: subtle, complex and population specific?

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.

A double EPSPS gene mutation endowing glyphosate resistance shows a remarkably high resistance cost.

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.