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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Non-Target-Site Resistance Mechanisms Endow Multiple Herbicide Resistance to Five Mechanisms of Action in Conyza bonariensis.

The repeated use of herbicides can lead to the selection of multiple resistance weeds. Some populations of Conyza bonariensis occurring in olive groves from southern Spain have developed resistance to various herbicides. This study determined the resistance levels to 2,4-D, glyphosate, diflufenican, paraquat, and tribenuron-methyl in a putative resistant (R) C. bonariensis population, and the possible non-target-site resistance (NTSR) mechanisms involved were characterized. Resistance factors varied as follows: glyphosate (8.9), 2,4-D (4.8), diflufenican (5.0), tribenuron-methyl (19.6), and paraquat (85.5). Absorption of 14C-glyphosate was up to 25% higher in the susceptible (S) population compared to the R one, but 14C-paraquat absorption was similar (up to 70%) in both populations. S plants translocated more than 60% of both 14C-glyphosate and 14C-paraquat toward shoots and roots, while R plants translocated less than 10%. The R population was able to metabolize 57% of the 2,4-D into nontoxic metabolites and 68% of the tribenuron-methyl into metsulfuron-methyl (10%), metsulfuron-methyl-hydroxylate (18%), and conjugate-metsulfuron-methyl (40%). Among the NTSR mechanisms investigated, absorption and translocation could be involved in glyphosate resistance, but only translocation for paraquat. Proofs of the presence of enhanced metabolism as a resistance mechanism were found for tribenuron-methyl and 2,4-D, but not for diflufenican. This research informs the first occurrence of multiple resistance to five herbicide classes (acetolactate synthase inhibitors, 5-enolpyruvylshikimate-3-phosphate synthase inhibitors, photosystem I electron diverters, photosystem II inhibitors, and synthetic auxin herbicides) in C. bonariensis.

Non-Target Site Mechanisms Endow Resistance to Glyphosate in Saltmarsh Aster (Aster squamatus).

Of the six-glyphosate resistant weed species reported in Mexico, five were found in citrus groves. Here, the glyphosate susceptibility level and resistance mechanisms were evaluated in saltmarsh aster (Aster squamatus), a weed that also occurs in Mexican citrus groves. The R population accumulated 4.5-fold less shikimic acid than S population. S plants hardly survived at 125 g ae ha-1 while most of the R plants that were treated with 1000 g ae ha-1, which suffered a strong growth arrest, showed a vigorous regrowth from the third week after treatment. Further, 5-enolpyruvylshikimate-3-phosphate basal and enzymatic activities did not diverge between populations, suggesting the absence of target-site resistance mechanisms. At 96 h after treatment, R plants absorbed ~18% less glyphosate and maintained 63% of the 14C-glyphsoate absorbed in the treated leaf in comparison to S plants. R plants metabolized twice as much (72%) glyphosate to amino methyl phosphonic acid and glyoxylate as the S plants. Three non-target mechanisms, reduced absorption and translocation and increased metabolism, confer glyphosate resistance saltmarsh aster. This is the first case of glyphosate resistance recorded for A. squamatus in the world.

Point Mutations and Cytochrome P450 Can Contribute to Resistance to ACCase-Inhibiting Herbicides in Three Phalaris Species.

Species of Phalaris have historically been controlled by acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides; however, overreliance on herbicides with this mechanism of action has resulted in the selection of resistant biotypes. The resistance to ACCase-inhibiting herbicides was characterized in Phalaris brachystachys, Phalaris minor, and Phalaris paradoxa samples collected from winter wheat fields in northern Iran. Three resistant (R) biotypes, one of each Phalaris species, presented high cross-resistance levels to diclofop-methyl, cycloxydim, and pinoxaden, which belong to the chemical families of aryloxyphenoxypropionates (FOPs), cyclohexanediones (DIMs), and phenylpyrazolines (DENs), respectively. The metabolism of 14C-diclofop-methyl contributed to the resistance of the P. brachystachys R biotype, while no evidence of herbicide metabolism was found in P. minor or P. paradoxa. ACCase in vitro assays showed that the target sites were very sensitive to FOP, DIM, and DEN herbicides in the S biotypes of the three species, while the R Phalaris spp. biotypes presented different levels of resistance to these herbicides. ACCase gene sequencing confirmed that cross-resistance in Phalaris species was conferred by specific point mutations. Resistance in the P. brachystachys R biotype was due to target site and non-target-site resistance mechanisms, while in P. minor and P. paradoxa, only an altered target site was found.

Glyphosate resistance in Chloris radiata from colombian rice fields involves one target-site mechanism.

At present, appearance of herbicide resistant weeds is not new because repeated herbicide treatments per agricultural year/cycle are usual in both perennial and annual crops worldwide. Characterizing resistance mechanisms implied in each herbicide resistant weed is the best tool and the basis to develop integrated weed management (IWM) strategies. The main resistance mechanisms which confer low sensibility to glyphosate in a previously confirmed glyphosate-resistant Chloris radiata population (ChrR), occurring in Colombian rice fields, were characterized. Pure line selection by clone plants showed high resistance levels in ChrR. Comparing with GR50 and LD50 values, ChrR was 9.6 and 10.8 times more resistant with respect to a representative susceptible population (ChrS). The nontarget site mechanisms reduced glyphosate absorption and translocation did not contribute to the glyphosate resistance of the ChrR population. However, enzyme activity assays and DNA sequencing demonstrated that at least one target-site resistance mechanism is involved in such resistance. All ten ChrR plants tested had the amino acid substitution Pro-106-Ser. The results may be crucial to decrease the resistance distribution of C. radiata in Colombia by implementing IWM programs. The change in weed control strategies in rice fields from Colombia must include herbicides with different mode of action from glyphosate and non chemical methods to preserve the useful life of glyphosate longer for weed control in the country.

Comparison of premix glyphosate and 2,4-D formulation and direct tank mixture for control of Conyza canadensis and Epilobium ciliatum.

Premix or tank mix of glyphosate and 2,4-D are a good alternative to control glyphosate-resistant and -tolerant weeds; however, the combination of herbicides may increase the environmental impacts, since mixtures often have higher toxicity than a single herbicide. In addition, antagonism between these herbicides has also been reported. We compared the efficacy of a premix glyphosate+2,4-D formulation with respect to the tank mix of both herbicides on glyphosate-resistant Conyza canadensis and -tolerant Epilobium ciliatum populations in laboratory and field experiments. 2,4-D suppressed the glyphosate-resistance/tolerance of both species, whose populations presented similar responses to their susceptible counterparts (LD50 ≥ 480+320 g ha-1 glyphosate + 2,4-D, respectively). Plants of both species treated with the premix formulations retained ∼100-µL more herbicide solution, accumulated 20-25% and 28-38% more shikimate and ethylene, respectively, and presented greater 14C-glyphosate absorption and translocation, depending on the species, compared to plants treated with the tank mix treatment. Although doubling the field dose (720 + 480 g ha-1) improved (5-22%) the control of these weeds in the field, split applications of both premix and tank mix provided the best control levels (≤70%), but premix treatments maintained control levels above 85% for longer (120-d). No antagonism between glyphosate and 2,4-D was found. The addition of 2,4-D controlled both broadleaf species. For all parameters evaluated on the C. canadensis and E. ciliatum populations in the laboratory and in the field, the premix treatments showed better performance than the tank mix treatments. Premix formulations could reduce the environmental impact of herbicides used to control glyphosate resistant/tolerant weeds by decreasing the herbicide amount needed to achieve an acceptable weed control level.

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.

Multiple Herbicide Resistance Evolution: The Case of Eleusine indica in Brazil.

The occurrence of multiple herbicide resistant weeds has increased considerably in glyphosate-resistant soybean fields in Brazil; however, the mechanisms governing this resistance have not been studied. In its study, the target-site and nontarget-site mechanisms were characterized in an Eleusine indica population (R-15) with multiple resistance to the acetyl-CoA carboxylase (ACCase) inhibitors, glyphosate, imazamox, and paraquat. Absorption and translocation rates of 14C-diclofop-methyl14C-imazamox and 14C-glyphosate of the R-15 population were similar to those of a susceptible (S-15) population; however, the R-15 population translocated ∼38% less 14C-paraquat to the rest of plant and roots than the S-15 population. Furthermore, the R-15 plants metabolized (by P450 cytochrome) 55% and 88% more diclofop-methyl (conjugate) and imazamox (imazamox-OH and conjugate), respectively, than the S-15 plants. In addition, the Pro-106-Ser mutation was found in the EPSPS gene of this population. This report describes the first characterization of the resistance mechanisms in a multiple herbicide resistant weed from Brazil.

Accumulation of Target Gene Mutations Confers Multiple Resistance to ALS, ACCase, and EPSPS Inhibitors in Lolium Species in Chile.

Different Lolium species, common weeds in cereal fields and fruit orchards in Chile, were reported showing isolated resistance to the acetyl CoA carboxylase (ACCase), acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibiting herbicides in the late 1990s. The first case of multiple resistance to these herbicides was Lolium multiflorum found in spring barley in 2007. We hypothesized that other Lolium species may have evolved multiple resistance. In this study, we characterized the multiple resistance to glyphosate, diclofop-methyl and iodosulfuron-methyl-sodium in Lolium rigidum, Lolium perenne and Lolium multiflorum resistant (R) populations from Chile collected in cereal fields. Lolium spp. populations were confirmed by AFLP analysis to be L. rigidum, L. perenne and L. multiflorum. Dose-response assays confirmed multiple resistance to glyphosate, diclofop-methyl and iodosulfuron methyl-sodium in the three species. Enzyme activity assays (ACCase, ALS and EPSPS) suggested that the multiple resistance of the three Lolium spp. was caused by target site mechanisms, except the resistance to iodosulfuron in the R L. perenne population. The target site genes sequencing revealed that the R L. multiflorum population presented the Pro-106-Ser/Ala (EPSPS), Ile-2041-Asn++Asp-2078-Gly (ACCase), and Trp-574-Leu (ALS) mutations; and the R L. rigidum population had the Pro-106-Ser (EPSPS), Ile-1781-Leu+Asp-2078-Gly (ACCase) and Pro-197-Ser/Gln+Trp-574-Leu (ALS) mutations. Alternatively, the R L. perenne population showed only the Asp-2078-Gly (ACCase) mutation, while glyphosate resistance could be due to EPSPS gene amplification (no mutations but high basal enzyme activity), whereas iodosulfuron resistance presumably could involve non-target site resistance (NTSR) mechanisms. These results support that the accumulation of target site mutations confers multiple resistance to the ACCase, ALS and EPSPS inhibitors in L. multiflorum and L. rigidum from Chile, while in L. perenne, both target and NTSR could be present. Multiple resistance to three herbicide groups in three different species of the genus Lolium in South America represents a significant management challenge.

Multiple mutations in the EPSPS and ALS genes of Amaranthus hybridus underlie resistance to glyphosate and ALS inhibitors.

Amaranthus hybridus is one of the main weed species in Córdoba, Argentina. Until recently, this weed was effectively controlled with recurrent use of glyphosate. However, a population exhibiting multiple resistance (MR2) to glyphosate and imazamox appeared in a glyphosate resistant (GR) soybean field, with levels of resistance up to 93 and 38-fold higher to glyphosate and imazamox, respectively compared to the susceptible (S) population. In addition to imidazolinones, MR2 plants showed high resistance levels to sulfonylamino-carbonyl (thio) benzoates and moderate resistance to sulfonylureas and triazolopyrimidines. Multiple amino acid substitutions were found in both target genes, acetolactate synthase (ALS) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), responsible for conferring high herbicides resistance levels in this A. hybridus population. In the case of EPSPS, the triple amino acid substitution TAP-IVS was found. In addition, MR2 plants also showed increased EPSPS gene expression compared to susceptible plants. A Ser653Asn substitution was found in the ALS sequence of MR2, explaining the pattern of cross-resistance to the ALS-inhibitor herbicide families found at the ALS enzyme activity level. No other mutations were found in other conserved domains of the ALS gene. This is the first report worldwide of the target site resistance mechanisms to glyphosate and ALS inhibitors in multiple herbicide resistance Amaranthus hybridus.

The First Case of Glyphosate Resistance in Johnsongrass (Sorghum halepense (L.) Pers.) in Europe.

Six Johnsongrass populations suspected of being glyphosate resistant were collected from railways and freeways near Cordoba (SW Spain), where glyphosate is the main weed control tool. The 50% reduction in shoot fresh weight (GR50) values obtained for these six populations ranged from 550.4 to 1169 g ae ha-1, which were 4.2 to 9 times greater than the value obtained for the susceptible population. Glyphosate was equally metabolized to the same extent in both resistant and susceptible populations, with no significant differences in either 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibition or basal activity. No amino acid substitutions were observed in any of the resistant populations. Slight but significant differences in glyphosate penetration were observed among some but not all of the resistant populations and for the times of incubation assayed, although these differences were not considered further. The proposed primary mechanism of resistance in these six glyphosate-resistant Johnsongrass populations is reduced herbicide translocation, because the amount of glyphosate that translocated from treated leaves to shoots and roots in the susceptible population was double that observed in the resistant populations. As glyphosate multiple resistance due to more than one mechanism is not uncommon, this is the first time that glyphosate-resistant Johnsongrass populations have been fully described for all known mechanisms.