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Glufosinate resistance in Palmer amaranth (Amaranthus palmeri S. Watson) was recently detected in three accessions from Arkansas, USA. Amaranthus palmeri is the first and only broadleaf weed species resistant to this herbicide, and the resistance mechanism is still unclear. A previous study characterized the glufosinate resistance level in the accessions from Arkansas. A highly glufosinate-resistant accession was further used to investigate the mechanism conferring glufosinate resistance in A. palmeri. Experiments were designed to sequence the herbicide target enzyme cytosolic and chloroplastic glutamine synthetase isoforms (GS1 and GS2, respectively) and quantify copy number and expression. Absorption, translocation, and metabolism of glufosinate using the 14C-labeled herbicide were also evaluated in the resistant and susceptible accessions. The glufosinate-resistant accession had an increase in copy number and expression of GS2 compared with susceptible plants. All accessions showed only one GS1 copy and no differences in expression. No mutations were identified in GS1 or GS2. Absorption (54% to 60%) and metabolism (13% to 21%) were not different between the glufosinate-resistant and glufosinate-susceptible accessions. Most residues of glufosinate (94% to 98%) were present in the treated leaf. Glufosinate translocation to tissues above the treated leaf and in the roots was not different among accessions. However, glufosinate translocation to tissues below the treated leaf (not including roots) was greater in the resistant A. palmeri (2%) compared with the susceptible (less than 1%) accessions. The findings of this paper strongly indicate that gene amplification and increased expression of the chloroplastic glutamine synthetase enzyme are the mechanisms conferring glufosinate resistance in the A. palmeri accession investigated. Thus far, no additional resistance mechanism was observed, but further investigations are ongoing.

Benzobicyclon tolerance in rice (Oryza sativa L.) is dependent on the presence of a functional HIS1 gene, but the level of sensitivity might vary among different cultivars. Greenhouse, laboratory, and field experiments were conducted to further explore the role of HIS1 in cultivated rice tolerance and to exploit findings toward optimizing benzobicyclon activity on weedy rice (unwanted rice; Oryza sativa L.). In a heredity experiment, benzobicyclon tolerance was confirmed to be a semidominant trait conferred by HIS1 based on the intermediate response (ED50 values) of HIS1 heterozygous F1 plants. The spatial–temporal expression of HIS1 was next studied in tissue types (blades, sheaths, and whorls) across tolerant cultivars (‘Roy J’, ‘Diamond’, ‘LaKast’, ‘CLXL745’, and ‘XL753’) and growth stages (2- to 3- compared with 5- to 6-leaf). The relative expression of HIS1 was tissue specific and highest in whorls, followed by blades and then sheaths. Minimal differences in expression across cultivars and growth stages were observed. Furthermore, HIS1 was not largely upregulated at 6 h after benzobicyclon treatment. In the same experiment, cultivar tolerance to benzobicyclon at the label rate of 371 g ha−1 was found to be growth stage dependent. Plant growth was reduced by ∼35% when rice plants were at the 2- to 3- compared with 5- to 6-leaf growth stages. These results show that differences in benzobicyclon tolerance among HIS1 homozygous cultivars is likely not directly correlated with the expression of HIS1. In this research a model was proposed and supported by a field proof of concept study, indicating benzobicyclon efficacy on weedy rice is a function of HIS1 zygosity by growth stage at application. Prior research indicates HIS1 is the dominant allele in weedy rice accessions in Arkansas, and thus, based on our model, benzobicyclon should be applied to weedy rice with ≤2 leaves for suppression.

Palmer amaranth is one of the most difficult-to-control weeds in row crop systems and has evolved resistance to several herbicide sites of action (SOAs). A late-season weed-escape survey had been conducted earlier to determine the distribution of protoporphyrinogen oxidase–inhibitor resistant Palmer Amaranth in Arkansas. The objective of this study was to evaluate the susceptibility of Arkansas Palmer amaranth accessions to commonly used herbicide SOAs. The SOAs evaluated were group 2 + 9, 3, 4, 5, 10, 14, 15, and 27, and the representative herbicide from each group was imazethapyr + glyphosate (79 + 860 g ha−1), trifluralin (1,120 g ha−1), dicamba (280 and 560 g ha−1), atrazine (560 g ha−1), glufosinate (594 g ha−1), fomesafen (395 g ha−1), S-metolachlor (1,064 g ha−1), and tembotrione (92 g ha−1), respectively. Palmer amaranth mortality varied among accessions across SOAs. Averaged across accessions, the mortality rates, by treatment in order from lowest to highest, were as follows: glyphosate + imazethapyr (16%), tembotrione (51%), dicamba at 280 g ha−1 (51%), fomesafen (76%), dicamba at 560 g ha−1 (82%), atrazine (85%), trifluralin (87%), S-metolachlor (96%), and glufosinate (99.5%). This study provides evidence that Palmer amaranth accessions with low susceptibility to glyphosate + imazethapyr, fomesafen, and tembotrione are widespread throughout Arkansas. Of the remaining SOAs, most Palmer amaranth accessions were sensitive; however, within each herbicide SOA, except glufosinate, control of some accessions was less than expected and resistance is suspected.

Amaranthus palmeri S. Watson (Palmer amaranth) is considered a problematic and troublesome weed species in many crops in the USA, partly because of its ability to evolve resistance to herbicides. In this study, we explored the mechanism of resistance in a trifluralin-resistant A. palmeri accession collected from Arkansas, USA. Dose-response assays using agar plates demonstrated an EC50 (effective concentration that reduces root length by 50%) of 1.02 µM trifluralin compared to 0.39 µM obtained in the susceptible accession. Thus, under these conditions, the resistant accession required 2.6 times more trifluralin to inhibit root length by 50%. Seeds in the presence or absence of the cytochrome P450-inhibitior malathion displayed a differential response with no significant influence on root length, suggesting that resistance is not P450-mediated. In addition, application of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a glutathione S-transferase (GST) inhibitor, showed significant differences in root length, indicating that GSTs are most likely involved in the resistance mechanism. Sequencing of α- and β-tubulin genes revealed no single nucleotide polymorphisms (SNPs) previously described between accessions. In addition, relative gene copy number of α- and β-tubulin genes were estimated, however, both resistant and susceptible accessions displayed similar gene copy numbers. Overall, our results revealed that GST-mediated metabolism contributes to trifluralin resistance in this A. palmeri accession from Arkansas.

Managing emerged weeds that have evolved resistance to acetyl CoA carboxylase (ACCase)-inhibiting herbicides is a challenging task. A dose-response experiment was conducted on barnyardgrass biotypes resistant (R) and susceptible (S) to three aryloxyphenoxypropionate herbicides cyhalofop-butyl (CyB), fenoxaprop-ethyl (FeE), and quizalofop-ethyl (QuE) along with investigations into the potential resistance mechanism of these biotypes. The tested R barnyardgrass biotypes had strong resistance to CyB and FeE (resistant/susceptible ratio: 7.9-14.4) but weak resistance to QuE (resistant/susceptible ratio: 2.4-3.1). Absorption, translocation, and total metabolism of CyB and QuE were not associated with differences among S and R barnyardgrass biotypes. However, differences between S and R barnyardgrass were observed in production of active acid forms of each herbicide (cyhalofop-acid and quizalofop-acid). Production of cyhalofop-acid was1.6-fold less in R barnyardgrass (3-8%) for 24 h after herbicide application than in the S barnyardgrass (8-16%). Meanwhile, production of quizalofop-acid was less in R barnyardgrass (14%) throughout the study period than in the S barnyardgrass (22%). Sequencing results of ACCase gene showed no difference between S and R barnyardgrass. Overall results show that a non-target-site resistance mechanism altering metabolism of CyB and QuE likely contributes to resistance of the barnyardgrass biotypes to these herbicides.

Dose-response experiments were conducted to assess the sensitivity of one susceptible and three putative resistant (R1, R2, and R3) barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] biotypes to florpyrauxifen-benzyl and cyhalofop-butyl alone and as a formulated premix. Subsequently, potential resistance mechanisms of the barnyardgrass were evaluated. Based on biomass reduction results, resistant/susceptible ratios were calculated for R1 (7.0–50), R2 (7.0–150), and R3 (18–214) biotypes. Absorption and translocation of [14C]-florpyrauxifen-benzyl decreased in R1 and R3 biotypes, but not for [14C]-cyhalofop-butyl. The metabolism of [14C]-florpyrauxifen-benzyl to [14C]-florpyrauxifen-acid was >2-fold less in resistant biotypes (9–11%) than in the susceptible biotype (23%). Moreover, the production of [14C]-florpyrauxifen-acid in susceptible barnyardgrass (not in the R biotypes) increased 3-fold when florpyrauxifen-benzyl and cyhalofop-butyl were applied in mixture compared to florpyrauxifen-benzyl applied alone. The tested barnyardgrass biotypes had no mutation in the Transport Inhibitor Response1, auxin-signaling F-box, and acetyl coenzyme A carboxylase genes. Although further studies on cyhalofop-butyl resistance with respect to analysis of specific metabolites are needed, our findings in this study demonstrates that the evolution of florpyrauxifen-benzyl resistance in multiple resistant barnyardgrass can be related to non-target-site resistance mechanisms reducing absorption and translocation of the herbicide and causing reduced conversion or rapid degradation of florpyrauxifen-acid.

Emergence of glyphosate-resistant horseweed (Conyza canadensis) biotypes is an example of how unrelenting use of a single mode of action herbicide in agricultural weed control drives genetic adaptation in targeted species. While in other weeds glyphosate resistance arose from target site mutation or target gene amplification, the resistance mechanism in horseweed uses neither of these, being instead linked to reduced herbicide uptake and/or translocation. The molecular components underpinning horseweed glyphosate-resistance remain unknown. Here, we used an in vitro leaf disc system for comparative analysis of proteins extracted from control and glyphosate-treated tissues of glyphosate-resistant and glyphosate-susceptible biotypes. Analysis of shikimic acid accumulation, ABC-transporter gene expression, and cell death were used to select a suitable glyphosate concentration and sampling time for enriching proteins pivotal to glyphosate resistance. Protein gel analysis and mass spectrometry identified mainly chloroplast proteins differentially expressed between the biotypes before and after glyphosate treatment. Chloroplasts are the organelles in which the shikimate pathway, which is targeted by glyphosate, is located. Calvin cycle enzymes and proteins of unknown function were among the proteins identified. Our study provides candidate proteins that could be pivotal in engendering resistance and implicates chloroplasts as the primary sites driving glyphosate-resistance in horseweed.

Tribenuron-methyl has been used widely for the last 15 yr to control white mustard in cereal crops from southern Spain. Since 2007, several cases of tribenuron-methyl resistance have been reported in wheat fields. Greenhouse and laboratory studies were conducted to characterize the mechanism of suspected tribenuron-methyl resistance in a white mustard biotype (hereafter AR16) from Malaga (southern Spain). Under greenhouse conditions, the dose (g ai ha−1) inhibiting fresh weight by 50% (ED50) was 5.20 and 0.57 for the AR16and AR3(known susceptible) biotypes, respectively. With the use of14C-tribenuron-methyl, absorption and translocation from treated leaves were similar between biotypes. Thin-layer chromatography indicated foliar metabolism of tribenuron-methyl was low in both R and S biotypes. Assays on the binding affinity of tribenuron-methyl on acetolactate synthase (ALS) revealed enzyme activity was reduced by 50% (I50value) at 638.7 and 0.23 nM for the AR16and AR3biotypes, respectively. This resulted in 2,777-fold greater resistance to tribenuron-methyl for the AR16compared to AR3biotype. Sequencing the gene encoding ALS, a proline/serine amino-acid substitution, was detected in position 197 of the A domain. Based on these results, it is concluded that tribenuron-methyl resistance in the AR16biotype is due to a target-site mutation in the ALS enzyme, resulting in a lack of affinity to tribenuron-methyl.

The glyphosate herbicide has been extensively used for long time periods in woody crops to control a broad range of weeds. The rapid determination of resistant weeds in different woody crops could maintain the efficacy of herbicides and could improve weed management using rotating strategies. Unfortunately Sumatran fleabane has developed a resistance to glyphosate. The mechanism of resistance of Sumatran fleabane is unknown so far. Therefore, here, we studied the resistance of a Sumatran fleabane biotype collected from a citrus orchard, under greenhouse and laboratory conditions. Our results show a resistance factor of 7.4. The resistant biotype absorbed and translocated lower amounts of 14C-glyphosate compared to the susceptible biotype. Moreover, at the molecular level, the target site sequence of the EPSPS2 gene showed a Pro-182-Thr substitution in the resistant biotype. As a consequence, this biotype uses mechanisms of reduced absorption-translocation and target site mutation to resist against glyphosate. This is the first study to report the reduced absorption and a mutation in the EPSPS2 gene in the resistance mechanism in the Conyza genus. © 2013 INRA and Springer-Verlag France., The Monsanto Company and Spain’s MICINN Project (AGL2010-16774) supported this research.

Greenhouse and laboratory experiments were conducted to investigate differences in glyphosate susceptibility among three species of the genus Conyza introduced as weeds in Spain: tall fleabane (Conyza sumatrensis), hairy fleabane (Conyza bonariensis), and horseweed (Conyza canadensis). Plant material was obtained from seeds collected in weed populations growing in olive groves and citrus orchards in southern Spain, with no previous history of glyphosate application. Dose-response curves displayed ED(50) values of 2.9, 15.7, and 34.9 g ai ha(-1), respectively, for C. sumatrensis, C. bonariensis, and C. canadensis plants at the rosette stage (6-8 leaves). Significant differences were found among the three species in the glyphosate retention on leaves as well as the leaf contact angle. The species order according to glyphosate retention was C. sumatrensis > C. bonariensis > C. canadensis, while the mean contact angles of glyphosate droplets were 59.2, 65.5, and 72.9 degrees , respectively. There were no significant differences among species in the absorption of [(14)C]glyphosate (ranged from 37.4% for C. canadensis to 52.4% for C. sumatrensis), but the order among species was the same as glyphosate retention. The amount of radioactivity translocated from treated leaves was lower in C. canadensis as compared to the other two species (C. sumatrensis > C. bonariensis > C. canadensis). Combined, all of the studied parameters identified differential susceptibility to glyphosate among the Conyza species. Each species accumulated shikimate in leaf tissues following application of glyphosate at 200 g ai ha(-1). However, C. canadensis exhibited lower shikimate levels than the other two species at 168 h after herbicide application. For hairy fleabane, a greenhouse study explored its susceptibility to glyphosate at three developmental stages: rosette, bolting (stem height, 10-15 cm), and flowering. The ED(50) was lower at the rosette stage (15.7 g ai ha(-1)) as compared to bolting (86.6 g ai ha(-1)), with the highest ED(50) values occurring at flowering (117.5 g ai ha(-1)); plants at the earlier developmental stage retained more glyphosate. These results agree with field observations that plants at early developmental stages are more sensitive to glyphosate.

The resistance mechanism of a glyphosate-resistant Lolium multiflorum Lam. biotype collected in Córdoba (Southern Spain) was examined. Resistance Factor values at three different growth stages ranged between 4.77 and 4.91. At 96 hours after treatment (HAT) the S biotype had accumulated seven times more shikimic acid than the R biotype. There were significant differences in translocation of 14C-glyphosate between biotypes, i.e. at 96 HAT, the R biotype accumulated in the treated leaf more than 70% of the absorbed herbicide, in comparison with 59.21% of the S biotype; the R biotype translocated only 14.79% of the absorbed 14C-glyphosate to roots, while in the S population this value was 24.79%. Visualization of 14C-glyphosate by phosphor imaging showed a reduced distribution in the R biotype compared with the S. Glyphosate metabolism was not involved in the resistance mechanism due to both biotypes showing similar values of glyphosate at 96 HAT. Comparison of the EPSPS gene sequences between biotypes indicated that the R biotype has a proline 182 to serine amino acid substitution. In short, the resistance mechanism of the L. multiflorum Lam. biotype is due to an impaired translocation of the herbicide and an altered target site. © 2012 Elsevier Masson SAS., This research was funded by the firm Monsanto and by Spain's MICINN (Project AGL2010-16774). Javier Gil-Humanes acknowledges financial support from the I3P Program from the Consejo Superior de Investigaciones Científicas, which is co-financed by the European Social Fund.

The physiological and biochemical bases for glyphosate resistance and susceptibility in horseweed (Conyza canadensis L. Cronq.) populations collected from Cordoba, Huelva, Malaga, Jaen and Seville in southern Spain were investigated. Screening 25 populations treated with glyphosate (238gacidequivalentha(-1)) at the rosette stage (BBCH 14-15) revealed reductions in fresh weight (fw) of 9-99%. The resistant biotype (R C004) was 6.1 times more resistant than the susceptible biotype (S). Shikimate accumulation in both biotypes increased until 72h after treatment (HAT), and then continued to increase (to 61.2%) in the S biotype, but decreased by 40% in the R (C004) biotype. Differential glyphosate spray retention and foliar uptake of applied (14)C-glyphosate between the R (C004) and S biotype had no effect on resistance to this herbicide. Quantitative and qualitative tests showed greater (14)C-glyphosate mobility in the S biotype than in the R (C004) biotype. Glyphosate was metabolized faster in the R (C004) biotype than in the S biotype. The herbicide disappeared completely from the R (C004) biotype by conversion into glyoxylate, sarcosine and aminomethylphosphonic acid within 96 HAT. On the other hand, 41.43nmolg(-1)fw of all glyphosate applied remained in the S biotype and glyoxylate was its only non-toxic metabolite. These results suggest that glyphosate resistance in horseweed is due to two different non-target mechanisms, namely: (a) impaired glyphosate translocation and (b) glyphosate metabolism to other compounds.

Digitaria insularis biotypes resistant to glyphosate have been detected in Brazil. Studies were carried out in controlled conditions to determine the role of absorption, translocation, metabolism, and gene mutation as mechanisms of glyphosate resistance in D. insularis. The susceptible biotype absorbed at least 12% more 14C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential 14C-glyphosate translocation was observed at 12 HAT, so that >70% of the absorbed herbicide remained in the treated leaf in resistant biotypes, whereas 42% remained in the susceptible biotype at 96 HAT. Glyphosate was degraded to aminomethylphosphonic acid (AMPA), glyoxylate, and sarcosine by >90% in resistant biotypes, whereas a small amount of herbicide (up to 11%) was degraded by the susceptible biotype up to 168 HAT. Two amino acid changes were found at positions 182 and 310 in EPSPS, consisting of a proline to threonine and a tyrosine to cysteine substitution, respectively, in resistant biotypes. Therefore, absorption, translocation, metabolism, and gene mutation play an important role in the D. insularis glyphosate resistance. © 2011 American Chemical Society.

Sourgrass is a perennial weed infesting annual and perennial crops in Brazil. Three biotypes (R1, R2, and R3) of sourgrass suspected to be glyphosate-resistant (R) and another one (S) from a natural area without glyphosate application, in Brazil, were tested for resistance to glyphosate based on screening, dose-response, and shikimic acid assays. Both screening and dose-response assays confirmed glyphosate resistance in the three sourgrass biotypes. Dose-response assay indicated a resistance factor of 2.3 for biotype R1 and 3.9 for biotypes R2 and R3. The hypothesis of a glyphosate resistance was corroborated on the basis of shikimic acid accumulation, where the S biotype accumulated 3.3, 5.0, and 5.7 times more shikimic acid than biotypes R1, R2, and R3, respectively, 168 h after treatment with 157.50 g ae ha−1of glyphosate. There were no differences in contact angle of spray droplets on leaves and spray retention, indicating that differential capture of herbicide by leaves was not responsible for resistance in these biotypes. The results confirmed resistance of sourgrass to glyphosate in Brazil.