In our previous study with a wild radish (Raphanus raphanistrum) population resistant to HPPD-inhibiting herbicides we indicated that two additional candidate P450 genes, CYP71A28 and CYP72A13-like, may also contribute to the resistance. This study investigates the role of these two P450 genes in mesotrione resistance in the wild radish population. The full-length R. raphanistrum P450 genes (thereafter named as RrCYP71A28 and RrCYP72A15) were cloned from mesotrione-resistant (R) and -susceptible (S) wild radish plants. RT-qPCR results showed that basal expression levels of the two P450 genes are significantly higher (up to 3-fold) in the R than the S plants. Escherichia coli cells transformed respectively with RrCYP71A28 and RrCYP72A15 were more tolerant to mesotrione. Transgenic Arabidopsis plants expressing RrCYP72A15 showed a modest level of resistance to mesotrione. UPLC-MS/MS analysis demonstrated that tissue mesotrione levels in RrCYP72A15 transgenic Arabidopsis plants were significantly lower (up to 2-fold) than that in the wild type. Structural modelling predicts CYP72A15 can bind to RrCYP72A15 and metabolize mesotrione likely through formation of 4-OH-mesotrione. Although the RrCYP72A15 gene confers a modest level of resistance, overexpression of the multiple herbicide-metabolizing genes could contribute to the low level of mesotrione resistance observed in the R wild radish population.

Paraquat is one of the most widely used nonselective herbicides globally. Although the emergence of weed resistance to paraquat has progressed relatively slowly since the first reported case in Japan in 1980, it has been steadily increasing. Resistance in weedy plants is predominantly associated with non-target-site resistance (NTSR), particularly via reduced uptake and translocation to target sites (i.e., chloroplasts) and/or enhanced sequestration; increased antioxidant capacity is also a common mechanism by which plants cope with various stresses, including reactive oxygen species (ROS). However, direct evidence for paraquat transport mediated by membrane transporters in weeds has not been established. Over the past decade, research, especially in model plants such as Arabidopsis thaliana, has advanced our understanding of the mechanisms underlying plant resistance to paraquat. This brief review summarized recent studies on paraquat resistance, with a particular focus on uptake, translocation, and sequestration mechanisms. For instance, three L-amino acid transporter (LAT) proteins (LAT1/3/4) and one (PDR11) belonging to the PDR (pleiotropic drug resistance) subfamily within the ABC (ATP-binding cassette) transporter family were confirmed to exhibit paraquat transporter activity; furthermore, transporters such as DTX6 (detoxification efflux carrier) can export/sequestrate paraquat inside the cell to the vacuole and apoplast, which confers stronger paraquat resistance to nearly commercial doses. In addition, the evolving perspectives in paraquat resistance research integrating big data and artificial intelligence, development of paraquat-tolerant crops, and a proposal of ryegrass (Lolium. spp.) and/or goosegrass (Eleusine indica) as a model weed species for paraquat resistance studies were also briefly discussed. Further advances in elucidating the molecular mechanisms of paraquat resistance in plants, including weeds, are anticipated.

BACKGROUND The first case of glyphosate resistance was reported in a capeweed population from Western Australia in our previous study. This current study investigates the resistance mechanisms in the population. RESULTS Target-site EPSPS gene sequencing revealed two partial sequences of the EPSPS transcripts (1001 bp and 998 bp), and the full-length sequence (1551 bp) containing the 1001-bp transcript was cloned as it was found in the resistant plants. A known resistance-endowing target-site mutation in the 1551-bp transcript was identified in the resistant plants, resulting in the Pro-106-Ser substitution. The subpopulation derived from these mutant plants exhibited >10-fold resistance to glyphosate compared to the susceptible population. Additionally, the EPSPS gene (1551 bp) was constitutively expressed at a higher level (4.3-fold) in the resistant than in the susceptible populations. However, C-14-glyphosate foliar uptake was similar with no visual difference in C-14-glyphosate translocation from leaves to other parts of a plant, between the resistant and susceptible population. CONCLUSION Glyphosate resistance in the studied population is associated with both a target-site mutation (Pro-106-Ser) and increased EPSPS gene expression.

BACKGROUND: Chlorsulfuron resistance and genetic dominance was evaluated in Raphanus raphanistrum genotypes homozygous (122-RR, 376-RR), heterozygous (122-RS, 376-RS) and compound heterozygous (122-R/376-R) for the target-site resistance mutations Ala-122-Tyr and Asp-376-Glu in the AHAS (acetohydroxyacid synthase) gene. RESULTS: At the AHAS level, 122-RR and 122-RS plants exhibited significantly higher I50 values than 376-RR and 376-RS plants, respectively. However, plants of the compound heterozygous genotype (122-R/376-R), showed no difference in AHAS activity compared to the 122-RS genotype but lower activity than the 122-RR genotype, and showed a nearly 400-fold greater I50 value than both genotypes (376-RR and 376-RS) carrying the 376-Glu allele. At the whole-plant level, 100% survival was observed for 122-RR plants at the highest chlorsulfuron dose of 640 g ha-1, yet 376-RR plants showed no survival at 380 g ha-1. Thus, this survival difference resulted in different median lethal dose (LD50) estimates [>640 (122-RR) versus 330 g ha-1(376-RR)]. The effect of chlorsulfuron in reducing aboveground growth of surviving plants also was markedly lower for the homozygous 122-RR (GR50 = 566 g ha-1) than for 376-RR plants (GR50 = 66). Heterozygous plants carrying the 122-Tyr allele (122-RS) exhibited two- and five-fold higher LD50 values than both homozygous and heterozygous plants carrying the 376-Glu allele (376-RR, 376-RS), respectively. Along the difference in plant survival, 122-RS plants also showed four-fold higher GR50 than both 376-RR and 376-RS plants. Survival of plants with the compound heterozygous genotype (122-R/376-R) under increasing chlorsulfuron doses was similar to 122-RR or 122-RS genotypes. However, this compound heterozygous genotype showed two- and six-fold higher LD50 values than 376-RR or 376-RS genotypes, respectively. However, both resistance 122-Tyr and 376-Glu alleles were dominant or nearly dominant over the wild-type susceptible alleles (ALA-122 and ASP-376), and the resistance 122-Tyr allele was dominant over the 376-Glu allele. CONCLUSIONS: These results broaden our understanding of AHAS target-site resistance in R. raphanistrum and strengthens the hypothesis that the AHAS 122-Tyr allele corresponds to a stronger target-site resistance allele than the 376-Glu allele. (c) 2024 Society of Chemical Industry.

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.

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.

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.

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 alpha-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.

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.

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 alpha-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 alpha-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 alpha-tubulin Arg-243-Met mutation can have an exceptional fitness cost with nearly lethal effects on resistant L. rigidum plants. (C) 2022 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.

BACKGROUND Glyphosate is routinely used in Australia to control the Arctotheca species Arctotheca calendula (L.) Levyns (referred hereinafter as capeweed). This study identifies the first global case of field-evolved glyphosate-resistant capeweed, collected from the grainbelt of Western Australia. RESULTS In 2020, a capeweed biotype that was collected from Borden in the southern Western Australian grainbelt was confirmed to be glyphosate-resistant (referred hereinafter as Spence population). When compared to the pooled mortality of six field-collected, glyphosate susceptible capeweed populations (S1, S2, S3, S4, S5, and S6), the Spence population was found >11-fold more resistant to glyphosate than the pooled results of the susceptible populations (S1-S6) at the LD50 level. The growth of the Spence population was also less affected, requiring >13-fold more glyphosate to reduce growth than the pooled susceptible populations at the GR50 level. Sequencing of the plastidic EPSPS gene indicated no known single gene mutation imparting glyphosate resistance. This study, however, did not investigate any other known mechanisms that impart glyphosate resistance. When screened at the field-applied rate, this Spence population was also found to survive an inhibitor of acetolactate synthase (ALS) (metosulam) and an inhibitor of phytoene desaturase (diflufenican). CONCLUSIONS This is the first confirmation of glyphosate resistance evolution in a capeweed population globally. With capeweed resistance already confirmed to photosystem-I inhibiting herbicides (paraquat and diquat)1 , this study emphasises the importance of using integrated measures that do not depend only on the use of non-selective herbicides for controlling herbicide resistance-prone capeweed populations. This article is protected by copyright. All rights reserved.

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.

Abstract 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.

BACKGROUND Barnyardgrass (Echinochloaspp.) 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 resistantE. crus-pavonisfor 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 ofACSandACOgenes was induced by quinclorac treatment in QS than in QR1. Basal levels of beta-cyanoalanine synthase (beta-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 thisE. crus-pavonisline is likely controlled by a single major gene, involving possibly an alteration in auxin signal perception/transduction to the ethylene biosynthesis pathway. The beta-CAS is unlikely to play a major role in quinclorac resistance in this particular population.

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.