Pro197Ser and the new Trp574Leu mutations together with enhanced metabolism contribute to cross-resistance to ALS inhibiting herbicides in Sinapis alba.

White mustard, (Sinapis alba), a problematic broadleaf weed in many Mediterranean countries in arable fields has been detected as resistant to tribenuron-methyl in Tunisia. Greenhouse and laboratory studies were conducted to characterize Target-Site Resistance (TSR) and the Non-Target Site Resistance (NTSR) mechanisms in two suspected white mustard biotypes. Herbicide dose-response experiments confirmed that the two S. alba biotypes were resistant to four dissimilar acetolactate synthase (ALS)-pinhibiting herbicide chemistries indicating the presence of cross-resistance mechanisms. The highest resistance factor (>144) was attributed to tribenuron-methyl herbicide and both R populations survived up to 64-fold the recommended field dose (18.7 g ai ha-1). In this study, the metabolism experiments with malathion (a cytochrome P450 inhibitor) showed that malathion reduced resistance to tribenuron-methyl and imazamox in both populations, indicating that P450 may be involved in the resistance. Sequence analysis of the ALS gene detected target site mutations in the two R biotypes, with amino acid substitutions Trp574Leu, the first report for the species, and Pro197Ser. Molecular docking analysis showed that ALSPro197Ser enzyme cannot properly bind to tribenuron-methyl's aromatic ring due to a reduction in the number of hydrogen bonds, while imazamox can still bind. However, Trp574Leu can weaken the binding affinity between the mutated ALS enzyme and both herbicides with the loss of crucial interactions. This investigation provides substantial evidence for the risk of evolving multiple resistance in S. alba to auxin herbicides while deciphering the TSR and NTSR mechanisms conferring cross resistance to ALS inhibitors.

Target-site and non-target-site resistance mechanisms confer multiple resistance to glyphosate and 2,4-D in Carduus acanthoides.

Carduus acanthoides L. is mainly a range-land weed, but in the 2010s has begun to invade GM crop production systems in Córdoba (Argentina), where glyphosate and 2,4-D have been commonly applied. In 2020, C. acanthoides was found with multiple resistance to these two herbicides. In this study, the mechanisms that confer multiple resistance to glyphosate and 2,4-D, were characterized in one resistant (R) population of C. acanthoides in comparison to a susceptible (S) population. No differences in 14C-herbicide absorption and translocation were observed between R and S populations. In addition, 14C-glyphosate was well translocated to the shoots (∼30%) and roots (∼16%) in both R and S plants, while most of 14C-2,4-D remained restricted in the treated leaf. Glyphosate metabolism did not contribute to resistance of the R population; however, as corroborated by malathion pretreatment, the mechanism of resistance to 2,4-D was enhanced metabolism (63% of the herbicide) mediated by cytochrome P450 (Cyt-P450). No differences were found in baseline EPSPS activity, copy number, and/or gene expression between the R and S populations, but a Pro-106-Ser mutation in EPSPS was present in the R population. Multiple resistances in the R population of C. acanthoides from Argentina were governed by target site resistance (a Pro-106 mutation for glyphosate) and non-target site resistance (Cyt-P450-based metabolic resistance for 2,4-D) mechanisms. This is the first case of resistance to glyphosate and 2,4-D confirmed for this weed in the world.

Tribenuron-methyl metabolism and the rare Pro197Phe double mutation together with 2,4-D metabolism and reduced absorption can evolve in Papaver rhoeas with multiple and cross herbicide resistance to ALS inhibitors and auxin mimics.

Multiple resistance mechanisms to ALS inhibitors and auxin mimics in two Papaver rhoeas populations were investigated in wheat fields from Portugal. Dose-response trials, also with malathion (a cytochrome P450 inhibitor), cross-resistance patterns for ALS inhibitors and auxin mimics, alternative herbicides tests, 2,4-D and tribenuron-methyl absorption, translocation and metabolism experiments, together with ALS activity, gene sequencing and enzyme modelling and ligand docking were carried out. Results revealed two different resistant profiles: one population (R1) multiple resistant to tribenuron-methyl and 2,4-D, the second (R2) only resistant to 2,4-D. In R1, several target-site mutations in Pro197 and enhanced metabolism (cytochrome P450-mediated) were responsible of tribenuron-methyl resistance. For 2,4-D, reduced transport was observed in both populations, while cytochrome P450-mediated metabolism was also present in R1 population. Moreover, this is the first P. rhoeas population with enhanced tribenuron-methyl metabolism. This study reports the first case for P. rhoeas of the amino acid substitution Pro197Phe due to a double nucleotide change. This double mutation could cause reduced enzyme sensitivity to most ALS inhibitors according to protein modelling and ligand docking. In addition, this study reports a P. rhoeas population resistant to 2,4-D, apparently, with reduced transport as the sole resistance mechanism.

Influence of temperature on the retention, absorption and translocation of fomesafen and imazamox in Euphorbia heterophylla.

Climate change will be an additional issue to the challenge to manage herbicide resistant weeds. This work investigated the impact of three temperature regimes (10/5, 20/15 and 30/25 °C) on the efficacy, foliar retention, absorption and translocation of fomesafen, protoporphyrinogen oxidase (PPO) inhibitor, and imazamox, acetolactate synthase (ALS) inhibitor, between two Euphorbia heterophylla populations, one susceptible (S) and one multiple PPO and ALS resistant (R). The R population went from 5 (fomesafen) and 12 (imazamox) times more resistant than the S population at 10/5 °C to more than 100 times to both herbicides at 20/15 and 30/25 °C. Leaf retention of fomesafen was not affected by temperature; however, imazamox retention was less at 10/5 and 20/15 °C than at 30/25 °C, and the R population always retained less imazamox than the S population. 14C-fomesafen absorption was similar between populations, but lower amounts were absorbed at 10/5 °C regardless of the evaluation time. Recovered 14C-imazamox rates decreased in both populations as the evaluation time increased, ranging from 82 to 92% at 6 h after treatment (HAT), and from 47 to 76% at 48 HAT, depending on the temperature regime. The 14C-imazamox losses were greater from 24 HAT in R plants grown at 30/25 °C and in all temperature regimes at 48 HAT. Although both populations translocated large amounts of imazamox, the S population distributed it in the rest of the plant (33%) and roots (15%), while the R population kept it mainly on the treated leaf (24%) or lost ~20% more herbicide than S population at 48 HAT, indicating the need for further studies on root exudation between these populations. Low temperatures reduced resistance levels to fomesafen and imazamox in E. heterophylla, suggesting that temperature influences the expression of the mechanisms that govern this multiple resistance.

Characterization of three glyphosate resistant Parthenium hysterophorus populations collected in citrus groves from Mexico.

Continuous use of glyphosate in citrus groves in the Gulf of Mexico region has selected for resistant Parthenium hysterophorus L. populations. In this study, the target-site and non-target-site resistance mechanisms were characterized in three putative glyphosate-resistant (GR) P. hysterophorus populations, collected in citrus groves from Acateno, Puebla (GR1 and GR2) and Martínez de la Torre, Veracruz (GR3), and compared with a susceptible population (GS). Based on plant mortality, the GR populations were 9.2-17.3 times more resistant to glyphosate than the GS population. The low shikimate accumulation in the GR population confirmed this resistance. Based on plant mortality and shikimate accumulation, the GR3 population showed intermediate resistance to glyphosate. The GR populations absorbed 15-28% less 14C-glyphosate than the GS population (78.7% absorbed from the applied) and retained 48.7-70.7% of 14C-glyphosate in the treated leaf, while the GS population translocated ~68% of absorbed herbicide to shoots and roots. The GR3 population showed the lowest translocation and absorption rates, but was found to be susceptible at the target site level. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequence of the GR1 and GR2 populations showed the Pro106-Ser mutation, conferring 19- and 25-times more resistance in comparison to the GS population, respectively. Reduced absorption and impaired translocation conferred glyphosate resistance on the GR3 population, and contributed partially to the resistance of the GR1 and GR2 populations. Additionally, the Pro-106-Ser mutation increased the glyphosate resistance of the last two P. hysterophorus populations.

The Triple Amino Acid Substitution TAP-IVS in the EPSPS Gene Confers High Glyphosate Resistance to the Superweed Amaranthus hybridus.

The introduction of glyphosate-resistant (GR) crops revolutionized weed management; however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an Amaranthus hybridus population (GRH)-a superweed collected in a GR-soybean field from Cordoba, Argentina-as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more 14C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA102, GTC103 and TCA106 instead of ACA102, GCG103, and CCA106, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 Å (wild type) to 2.93 Å (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH A. hybridus population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance.

Physiological, biochemical and molecular characterization of an induced mutation conferring imidazolinone resistance in wheat.

The Clearfield(®) wheat cultivars possessing imidazolinone (IMI)-resistant traits provide an efficient option for controlling weeds. The imazamox-resistant cultivar Pantera (Clearfield(®) ) was compared with a susceptible cultivar (Gazul). Target and non-target mechanisms of resistance were studied to characterize the resistance of Pantera and to identify the importance of each mechanism involved in this resistance. Pantera is resistant to imazamox as was determined in previous experiments. The molecular study confirmed that it carries a mutation Ser-Asn627 conferring resistance to imazamox in two out of three acetolactate synthase (ALS) genes (imi1 and imi2), located in wheat on chromosomes 6B and 6D, respectively. However, the last gene (imi3) located on chromosome 6A does not carry any mutation conferring resistance. As a result, photosynthetic activity and chlorophyll content were reduced after imazamox treatment. Detoxification was higher in the resistant biotype as shown by metabolomic study while imazamox translocation was higher in the susceptible cultivar. Interestingly, imazamox metabolism was higher at higher doses of herbicide, which suggests that the detoxification process is an inducible mechanism in which the upregulation of key gene coding for detoxification enzymes could play an important role. Thus, the identification of cultivars with a higher detoxification potential would allow the development of more resistant varieties.

Ile-1781-Leu and Asp-2078-Gly Mutations in ACCase Gene, Endow Cross-resistance to APP, CHD, and PPZ in Phalaris minor from Mexico.

Herbicides that inhibit acetyl coenzyme A carboxylase (ACCase) are commonly used in Mexico to control weedy grasses such as little seed canarygrass (Phalaris minor). These herbicides are classified into three major families (ariloxyphenoxypropionates (APP), cyclohexanodiones (CHD), and, recently, phenylpyrazolines (PPZ)). In this work, the resistance to ACCase (APP, CHD, and PPZ) inhibiting herbicides was studied in a biotype of Phalaris minor (P. minor) from Mexico, by carrying out bioassays at the whole-plant level and investigating the mechanism behind this resistance. Dose-response and ACCase in vitro activity assays showed cross-resistance to all ACCase herbicides used. There was no difference in the absorption, translocation, and metabolism of the (14)C-diclofop-methyl between the R and S biotypes. The PCR generated CT domain fragments of ACCase from the R biotype and an S reference were sequenced and compared. The Ile-1781-Leu and Asp-2078-Gly point mutations were identified. These mutations could explain the loss of affinity for ACCase by the ACCase-inhibing herbicides. This is the first report showing that this substitution confers resistance to APP, CHD, and PPZ herbicides in P. minor from Mexico. The mutations have been described previously only in a few cases; however, this is the first study reporting on a pattern of cross-resistance with these mutations in P. minor. The findings could be useful for better management of resistant biotypes carrying similar mutations.

Ultrasound-assisted extraction with LC-TOF/MS identification and LC-UV determination of imazamox and its metabolites in leaves of wheat plants.

INTRODUCTION: imazamox is a herbicide used in many legominous and cereal crops. There are few methods in the literature for determination of imazamox and its metabolites in plants because of the lack of commercial standards or owing to expensive and/or complex synthesis. OBJECTIVE: To develop a method based on liquid chromatography and ultraviolet absorption detection for simultaneous determination of imazamox and its metabolites in plants. METHODS: Sample preparation was based on ultrasound-assisted extraction (70 W power and duty cycle of 0.7 s/s for 10 min) with subsequent filtration of the extracts and clean-up and concentration prior to chromatographic separation and detection at 240 nm. The chromatographic analysis was completed in 30 min using a Luna® HILIC column. Identification and confirmatory analysis of the presence of imazamox and its metabolites in extracts from treated plants was performed by LC-TOF/MS in high resolution mode for precursor ions. The metabolites were quantified using a surrogate approach based on an imazamox standard. The method was validated by analysing wheat samples treated with 200 g per hectare of active ingredient imazamox. RESULTS: The linear dynamic range of the calibration curve was within 0.27-600 µg/mL, with a correlation coefficient of 0.998 and precision--studied at 0.1 and 2 µg/mL--of 2.9% and 5.0% for repeatability, and 4.7% and 6.9% for reproducibility, respectively. CONCLUSION: The analytical characteristics of the method make it recommendable for evaluating the metabolism of imazamox in plants.

Reduced Glyphosate Movement and Mutation of the EPSPS Gene (Pro106Ser) Endow Resistance in Conyza canadensis Harvested in Mexico.

Glyphosate has been the most widely used herbicide for decades providing a unique tool, alone or in mixtures, to control weeds on citrus in Veracruz. Conyza canadensis has developed glyphosate resistance for the first time in Mexico. The level and mechanisms of resistance of four resistant populations Rs (R1, R2, R3, and R4) were studied and compared with that of a susceptible population (S). Resistance factor levels showed two moderately resistant populations (R2 and R3) and two highly resistant populations (R1 and R4). Glyphosate translocation through leaves to roots was ∼2.8 times higher in the S population than in the four R populations. A mutation (Pro106Ser) in the EPSPS2 gene was identified in the R1 and R4 populations. Mutation in the target site associated with reduced translocation is involved in increased glyphosate resistance in the R1 and R4 populations; whereas for the R2 and R3 populations, it was only mediated by reduced translocation. This is the first study of glyphosate resistance in C. canadensis from Mexico in which the resistance mechanisms involved are described in detail and control alternatives are proposed.

Divergence in Glyphosate Susceptibility between Steinchisma laxum Populations Involves a Pro106Ser Mutation.

The characterization of the mechanisms conferring resistance to herbicides in weeds is essential for developing effective management programs. This study was focused on characterizing the resistance level and the main mechanisms that confer resistance to glyphosate in a resistant (R) Steinchisma laxum population collected in a Colombian rice field in 2020. The R population exhibited 11.2 times higher resistance compared to a susceptible (S) population. Non-target site resistance (NTSR) mechanisms that reduced absorption and impaired translocation and glyphosate metabolism were not involved in the resistance to glyphosate in the R population. Evaluating the target site resistance mechanisms by means of enzymatic activity assays and EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) gene sequencing, the mutation Pro106Ser was found in R plants of S. laxum. These findings are crucial for managing the spread of S. laxum resistance in Colombia. To effectively control S. laxum in the future, it is imperative that farmers use herbicides with different mechanisms of action in addition to glyphosate and adopt Integrate Management Programs to control weeds in rice fields of the central valleys of Colombia.

Constitutive overexpression of EPSPS by gene duplication is involved in glyphosate resistance in Salsola tragus.

BACKGROUND: Glyphosate-resistant Salsola tragus accessions have been identified in the USA and Argentina; however, the mechanisms of glyphosate resistance have not been elucidated. The goal of this study was to determine the mechanism/s of glyphosate resistance involved in two S. tragus populations (R1 and R2) from Argentina. RESULTS: Both glyphosate-resistant populations had a six-fold lower sensitivity to glyphosate than the S population (i.e. resistance index). No evidence of differential absorption, translocation or metabolism of glyphosate was found in the R1 and R2 populations compared to a susceptible population (S). No 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) mutations were detected, but S. tragus R1 and R2 plants had ≈14-fold higher EPSPS gene relative copy number compared to the S counterpart. In R1 and R2, EPSPS duplication entailed a greater constitutive EPSPS transcript abundance by approximately seven-fold and a basal EPSPS activity approximately three-fold higher than the S population. CONCLUSION: The current study reports EPSPS gene duplication for the first time as a mechanism of glyphosate resistance in S. tragus populations. The increase of glyphosate dose needed to kill R1 and R2 plants was linked to the EPSPS transcript abundance and level of EPSPS activity. This evidence supports the convergent evolution of the overexpression of the EPSPS gene in several Chenopodiaceae/Amaranthaceae species adapted to drought environments and the role of gene duplication as an adaptive advantage for plants to withstand stress. © 2022 Society of Chemical Industry.

Enhanced detoxification via Cyt-P450 governs cross-tolerance to ALS-inhibiting herbicides in weed species of Centaurea.

Centaurea is a genus of winter weeds with a similar life cycle and competitive traits, which occurs in small-grains production fields in the central-southern of the Iberian Peninsula. However, most of herbicides recommended for weed management in wheat show poor control of Centaurea species. This study summarizes the biology, herbicide tolerance to acetolactate synthase (ALS) inhibitors, and recommended chemical alternatives for the control of Centaurea species. Four species (C. cyanus L., C. diluta Aiton, C. melitensis L. and C. pullata L. subsp. baetica Talavera), taxonomically characterized, were found as the main important broadleaf weeds in small-grains production fields of the Iberian Peninsula. These species showed innate tolerance to tribenuron-methyl (TM), showing LD50 values (mortality of 50% of a population) higher than the field dose of TM (20 g ai ha-1). The order of tolerance was C. diluta (LD50 = 702 g ha-1) ≫ C. pullata (LD50 = 180 g ha-1) ≫ C. cyanus (LD50 = 65 g ha-1) > C. melitensis (LD50 = 32 g ha-1). Centaurea cyanus and C. melitensis presented higher foliar retention (150-180 µL herbicide solution), absorption (14-28%) and subsequent translocation (7-12%) of TM with respect to the other two species. Centaurea spp. plants were able to metabolize 14C-TM into non-toxic forms (hydroxylated OH-metsulfuron-methyl and conjugated-metsulfuron-methyl), with cytochrome P450 (Cyt-P450) monooxygenases being responsible for herbicide detoxification. Centaurea cyanus and C. mellitensis metabolized up to 25% of TM, while C. diluta and C. pullata metabolized more than 50% of the herbicide. Centaurea species showed 80-100% survival when treated with of florasulam, imazamox and/or metsulfuron-methyl, i.e., these weeds present cross-tolerance to ALS inhibitors. In contrast, auxin mimics herbicides (2,4-D, clopyralid, dicamba, fluroxypir and MCPA) efficiently controlled the four Centaurea species. In addition, the mixture of ALS-inhibitors and auxin mimics also proved to be an interesting alternative for the control of Centaurea. These results show that plants of the genus Centaurea found in the winter cereal fields of the Iberian Peninsula have an innate tolerance to TM and cross-resistance to other ALS-inhibiting herbicides, governed by reduced absorption and translocation, but mainly by the metabolization of the herbicide via Cyt-P450.

Convergent Adaptation of Multiple Herbicide Resistance to Auxin Mimics and ALS- and EPSPS-Inhibitors in Brassica rapa from North and South America.

Herbicide-resistant weeds have been identified and recorded on every continent where croplands are available. Despite the diversity of weed communities, it is of interest how selection has led to the same consequences in distant regions. Brassica rapa is a widespread naturalized weed that is found throughout temperate North and South America, and it is a frequent weed among winter cereal crops in Argentina and in Mexico. Broadleaf weed control is based on glyphosate that is used prior to sowing and sulfonylureas or mimic auxin herbicides that are used once the weeds have already emerged. This study was aimed at determining whether a convergent phenotypic adaptation to multiple herbicides had occurred in B. rapa populations from Mexico and Argentina by comparing the herbicide sensitivity to inhibitors of the acetolactate synthase (ALS), 5-enolpyruvylshikimate-3-phosphate (EPSPS), and auxin mimics. Five B. rapa populations were analyzed from seeds collected in wheat fields in Argentina (Ar1 and Ar2) and barley fields in Mexico (Mx1, Mx2 and MxS). Mx1, Mx2, and Ar1 populations presented multiple resistance to ALS- and EPSPS-inhibitors and to auxin mimics (2,4-D, MCPA, and fluroxypyr), while the Ar2 population showed resistance only to ALS-inhibitors and glyphosate. Resistance factors ranged from 947 to 4069 for tribenuron-methyl, from 1.5 to 9.4 for 2,4-D, and from 2.7 to 42 for glyphosate. These were consistent with ALS activity, ethylene production, and shikimate accumulation analyses in response to tribenuron-methyl, 2,4-D, and glyphosate, respectively. These results fully support the evolution of the multiple- and cross-herbicide resistance to glyphosate, ALS-inhibitors, and auxinic herbicides in B. rapa populations from Mexico and Argentina.

An Asp376Glu substitution in ALS gene and enhanced metabolism confers high tribenuron-methyl resistance in Sinapis alba.

Acetolactate synthase (ALS) inhibiting herbicides (group 2) have been widely applied for the last 20 years to control Sinapis alba in cereal crops from southern Spain. In 2008, a tribenuron-methyl (TM) resistant (R) S. alba population was first reported in a cereal field in Malaga (southern Spain). In 2018, three suspected R S. alba populations (R1, R2 and R3) to TM were collected from three different fields in Granada (southern Spain, 100 km away from Malaga). The present work aims to confirm the putative resistance of these populations to TM and explore their resistance mechanisms. Dose-response assays showed that the R1, R2 and R3 populations ranging between 57.4, 44.4 and 57.1 times more resistance to TM than the susceptible population (S). A mutation in the ALS gene (Asp376Glu) was detected in the Rs S. alba populations. 14C-metabolism studies show that metabolites and TM were changing significantly faster in the R than in the S plants. Alternative chemical control trials showed that 2,4-D and MCPA (auxin mimics), glyphosate (enolpyruvyl shikimate phosphate synthase,EPSPS, inhibitor-group 9), metribuzin (PSII inhibitors/Serine 264 Binders, -group 5) and mesotrione (hydroxyphenyl pyruvate dioxygenase, HPPD, inhibitor-group 27) presented a high control of the four populations of S. alba tested, both S and R. Based on these results, it is the first case described where the Asp376Glu mutation and P450-mediated metabolism participates in resistance to TM in S. alba. Comparing these results with those found in the S. alba population in Malaga in 2008, where the resistance was TSR type (Pro197Ser), we can suggest that despite the geographical proximity (over 100 km), the resistance in these cases was due to different evolutionary events.

A novel EPSPS Pro-106-His mutation confers the first case of glyphosate resistance in Digitaria sanguinalis.

BACKGROUND: Digitaria sanguinalis has been identified as a species at high risk of evolving herbicide resistance, but thus far, there are no records of resistance to glyphosate. This weed is one of the most common weeds of summer crops in extensive cropping areas in Argentina. It shows an extended period of seedling emergence with several overlapping cohorts during spring and summer, and is commonly controlled with glyphosate. However, a D. sanguinalis population was implicated as a putative glyphosate-resistant biotype based on poor control at recommended glyphosate doses. RESULTS: The field-collected D. sanguinalis population (Dgs R) from the Rolling Pampas has evolved glyphosate resistance. Differences in plant survival and shikimate levels after field-recommended and higher glyphosate doses were evident between Dgs R and the known susceptible (Dgs S) population; the resistance index was 5.1. No evidence of differential glyphosate absorption, translocation, metabolism or basal EPSPS activity was found between Dgs S and Dgs R populations; however, a novel EPSPS Pro-106-His point substitution is probably the primary glyphosate resistance-endowing mechanism. EPSPS in vitro enzymatic activity demonstrated that an 80-fold higher concentration of glyphosate is required in Dgs R to achieve similar EPSPS activity inhibition to that in the Dgs S population. CONCLUSION: This study reports the first global case of glyphosate resistance in D. sanguinalis. This unlikely yet novel transversion at the second position of the EPSPS 106 codon demonstrates the intensity of glyphosate pressure in selecting unexpected glyphosate resistance alleles if they retain EPSPS functionality. © 2022 Society of Chemical Industry.

Comparison between the mechanisms of Clearfield ® wheat and Lolium rigidum multiple resistant to acetyl CoA carboxylase and acetolactate synthase inhibitors.

Clearfield® wheat (Triticum aestivum) have helped eliminate the toughest grasses and broadleaf weeds in Spain since 2005. This crop production system includes other tolerant cultivars to the application of imidazolinone (IMI) herbicides. However, the continuous use and off-label rates of IMI herbicides can contribute to the development of resistance in Lolium rigidum and other weed species. In this research, the main objectives were to study the resistance mechanisms to acetolactate synthase (ALS) and acetyl coenzyme A carboxylase (ACCase) inhibitors in a L. rigidum accession (LrR) from a Clearfield® wheat field, with a long history rotating these IMI-tolerant crops and compare them with those present in the IMI-tolerant wheat. The resistance to ACCase inhibitors in LrR was due to point mutations (Ile1781Leu plus Asp2078Gly) of the target site gene plus an enhanced herbicide metabolism (EHM), on the other hand, in wheat accessions was due only by EHM. Mechanisms involved in the resistance to ALS inhibitors were both point mutations of the target gene and EHM in the IMI-tolerant wheat, while only evidence of mutation (Trp574Leu) was found in the multiple herbicide resistant L. rigidum accession. This research demonstrates that if crop rotation is not accompanied by the use of alternative sites of action in herbicide-tolerant crops, resistant weeds to herbicide to which crops are tolerant, can easily be selected. Moreover, repeated and inappropriate use of Clearfield® crops and herbicide rotations can lead to the evolution of multiple resistant weeds, as shown in this study, and have also inestimable environmental impacts.

Cytochrome P450 metabolism-based herbicide resistance to imazamox and 2,4-D in Papaver rhoeas.

Papaver rhoeas biotypes displaying multiple herbicide resistance to ALS inhibitors and synthetic auxin herbicides (SAH) are spreading across Europe. In Spain, enhanced metabolism to imazamox was confirmed in one population, while cytochrome-P450 (P450) based metabolism to 2,4-D in another two. The objectives of this research were to further confirm the presence of P450 mediated enhanced metabolism and, if so, to confirm whether a putative common P450 is responsible of metabolizing both 2,4-D and imazamox. Metabolism studies were undertaken in five P. rhoeas populations with contrasted HR profiles (herbicide susceptible, only HR to ALS inhibitors, only HR to SAH, or multiple HR to both), and moreover, three different P450 inhibitors were used. The presence of enhanced metabolism to these SoA was confirmed in three more HR P. rhoeas populations. This study provides the first direct evidence that imazamox metabolism in these biotypes is P450-mediated, also in one population without an altered target site. Additionally, it was further confirmed that enhanced metabolism of 2,4-D in biotypes only HR to SAH or multiple HR to ALS inhibitors and SAH involves P450 as well. No metabolism was detected using the three inhibitors in all the herbicide-metabolizing P. rhoeas biotypes, suggesting that a common metabolic system involving P450s is responsible of degrading herbicides affecting both SoAs. Thus, selection pressure with either SAH or imidazolinone ALS inhibitors can select not only for resistance to each of them, but it can also confer cross-resistance between them in P. rhoeas.

Point Mutations as Main Resistance Mechanism Together With P450-Based Metabolism Confer Broad Resistance to Different ALS-Inhibiting Herbicides in Glebionis coronaria From Tunisia.

Resistance to acetolactate synthase (ALS) inhibiting herbicides has recently been reported in Glebionis coronaria from wheat fields in northern Tunisia, where the weed is widespread. However, potential resistance mechanisms conferring resistance in these populations are unknown. The aim of this research was to study target-site resistance (TSR) and non-target-site resistance (NTSR) mechanisms present in two putative resistant (R) populations. Dose-response experiments, ALS enzyme activity assays, ALS gene sequencing, absorption and translocation experiments with radiolabeled herbicides, and metabolism experiments were carried out for this purpose. Whole plant trials confirmed high resistance levels to tribenuron and cross-resistance to florasulam and imazamox. ALS enzyme activity further confirmed cross-resistance to these three herbicides and also to bispyribac, but not to flucarbazone. Sequence analysis revealed the presence of amino acid substitutions in positions 197, 376, and 574 of the target enzyme. Among the NTSR mechanisms investigated, absorption or translocation did not contribute to resistance, while evidences of the presence of enhanced metabolism were provided. A pretreatment with the cytochrome P450 monooxygenase (P450) inhibitor malathion partially synergized with imazamox in post-emergence but not with tribenuron in dose-response experiments. Additionally, an imazamox hydroxyl metabolite was detected in both R populations in metabolism experiments, which disappeared with the pretreatment with malathion. This study confirms the evolution of cross-resistance to ALS inhibiting herbicides in G. coronaria from Tunisia through TSR and NTSR mechanisms. The presence of enhanced metabolism involving P450 is threatening the chemical management of this weed in Tunisian wheat fields, since it might confer cross-resistance to other sites of action.

Glyphosate ban in Mexico: potential impacts on agriculture and weed management.

Since glyphosate was classified as potentially carcinogenic by the International Agency for Research on Cancer, public debate regarding the environmental impact and health risks from its use has intensified. Almost all regulatory agencies throughout the world have concluded that the judicious use of glyphosate does not pose risks to the environment and human health. However, on the last day of 2020 the Mexican government decreed a ban of this herbicide beginning January, 2024. In current Mexican agriculture there are no safer chemical and/or other weed management technologies that allow for the economical substitution of glyphosate for weed control. Many Mexican weed scientists agree that glyphosate use should be reduced, but not banned outright. This decree could have more negative economic and social consequences as well as environmental and human health risks than benefits, which could compromise the country's food and public security. Crop yields are projected by some to decline by up to 40% with this ban, increasing food prices, making food less accessible to low-income consumers. In addition, a black market for the smuggling and illegal sale of glyphosate is possible. The possible environmental, economic and social impacts caused by the glyphosate ban in Mexico are discussed, emphasizing the impact on weed management. © 2021 Society of Chemical Industry.