Target-site mutations Ile1781Leu and Ile2041Asn in the ACCase2 gene confer resistance to fluazifop-p-butyl and pinoxaden herbicides in a johnsongrass accession from Arkansas, USA.

Johnsongrass [Sorghum halepense (L.) Pers.] is a troublesome weed species in different agricultural and non-agricultural areas. Because of its biology, reproductive system, and seed production, effective management is challenging. An accession with low susceptibility to the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides fluazifop-p-butyl (fluazifop) and pinoxaden was collected in eastern Arkansas. In this research, the molecular mechanisms responsible for ACCase resistance were investigated. Dose-response experiments showed a resistance factor of 181 and 133 for fluazifop and pinoxaden, respectively. Molecular analysis of both ACCase1 and ACCase2 genes was researched. Nucleotide comparison of ACCase1 between resistant and susceptible accessions showed no single nucleotide polymorphisms. Nonetheless, analysis of ACCase2 in fluazifop-resistant johnsongrass plants revealed the Ile1781Leu target-site mutation was dominant (nearly 75%), whereas the majority of pinoxaden-resistant johnsongrass plants had the Ile2041Asn (60%). Not all sequenced johnsongrass plants displayed a target-site mutation, suggesting the presence of additional resistance mechanisms. Amplification of ACCase1 and ACCase2 was not responsible for resistance because of the similar values obtained in both resistant and susceptible accessions. Experiments with malathion and NBD-Cl suggest the presence of herbicide metabolism. Outcomes of this research demonstrated that fluazifop- and pinoxaden-resistant johnsongrass plants displayed a target-site mutation in ACCase2, but also that non-target-site resistance mechanisms would be involved and require a detailed study.

Quizalofop resistance in weedy rice (Oryza sativa L.) is mainly conferred by an Ile1781Leu mutation.

Weedy rice (Oryza sativa L.) is an economically important weed species in rice (Oryza sativa L.) cropping systems. Two weedy rice samples (acc7 and acc8) suspected to be resistant to quizalofop-ethyl (quizalofop) were collected in Arkansas. In this research, susceptibility to quizalofop and resistance mechanisms have been explored. Dose-response assays displayed a resistance index of 42- and 58-fold for the acc7 and acc8, respectively. Experiments with metabolism inhibitors demonstrated that NBD-Cl (4-chloro-7-nitrobenzofurazan) increased quizalofop efficacy slightly in acc8, whereas malathion did not improve effectiveness in resistant samples. Sequencing of the ACCase gene displayed an Ile1781Leu substitution in the resistant samples, like the mutation present in Provisia™ rice. In addition, an allele-specific PCR was developed to genotype the Ile1781Leu mutation. The gene copy number of ACCase showed similar values among samples. In the resistant plants, a KASP (Kompetitive Allele Specific PCR) assay to detect the ALSS653D (acetolactate synthase) and HIS1 (HPPD Inhibitor Sensitive 1) traits revealed that 37.5% of plants carried the ALSS653D trait, whereas 25% showed the HIS1 allele. In summary, a target-site mutation is the main resistance mechanism to quizalofop in weedy rice. Results also suggest the presence of herbicide metabolism (a non-target site resistance mechanism) mediated by glutathione-S-transferases (GSTs) in one resistant sample.

Overexpression of Acetyl CoA Carboxylase 1 and 3 (ACCase1 and ACCase3), and CYP81A21 were related to cyhalofop resistance in a barnyardgrass accession from Arkansas.

Barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] is the most difficult-to-control weed species of rice production systems worldwide. It has evolved resistance to different herbicide sites of action, including the acetyl-CoA carboxylase (ACCase)-inhibiting herbicides. Target-site mutations conferring resistance to ACCase-inhibiting herbicides are well documented; however, the role of the different ACCase genes in conferring resistance to cyhalofop-p-butyl (cyhalofop), an ACCase-inhibiting herbicide, remains poorly understood. This research assessed the contribution of gene amplification and expression of ACCase genes in a cyhalofop-resistant barnyardgrass accession. Additionally, the expression of glutathione-S-transferases (GSTs) and cytochrome P450 monooxygenases (P450s) genes as possible contributors to resistance to cyhalofop were investigated. Results demonstrated that ACCase gene amplification does not contribute to cyhalofop resistance. However, ACCase1 and ACCase3 were found to be overexpressed in the cyhalofop-resistant barnyardgrass accession. At 24 h after cyhalofop treatment, an overexpression of 2.0- and 2.8-fold was detected in ACCase1 and ACCase3, respectively. In addition, CYP81A21 (a P450 gene) was found to be 2.5-fold overexpressed compared to the susceptible accession in the same time period. These results suggest that ACCase1, ACCase3, and CYP81A21 are crucial genes in contributing cyhalofop resistance in this barnyardgrass accession.

Cross-resistance of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] to aryloxyphenoxypropionate herbicides.

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 was >1.6-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.

Non-target-site resistance mechanism of barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.] to florpyrauxifen-benzyl.

BACKGROUND: Florpyrauxifen-benzyl (FPB) is an arylpicolinate herbicide (Group IV) for barnyardgrass control in rice. One susceptible (Sus) and three putative FPB-resistant (R1, R2, and R3) barnyardgrass biotypes were selected based on resistant/susceptible (R/S) ratios obtained from dose-response tests and used to investigate the potential resistance mechanisms. RESULTS: Based on visual control results, the R/S ratios of barnyardgrass biotypes R1, R2, and R3 were 60-, 33-, and 16-fold greater than the Sus standard, respectively. Sequencing results of TIR1 and AFB genes in the tested barnyardgrass revealed no difference between Sus and R barnyardgrass biotypes. Absorption of [14 C]-FPB in Sus barnyardgrass increased over time and reached 90%, which was >10 percentage points greater than that in R biotypes. The [14 C]-FPB absorption in all R barnyardgrass equilibrated after 48 h. For both Sus and R barnyardgrass, most [14 C]-FPB absorbed was present in the treated leaf (79.8-88.8%), followed by untreated aboveground (9.5-18.6%) and belowground tissues (1.3-2.2%). No differences in translocation were observed. Differences between Sus and R barnyardgrass biotypes were found for FPB metabolism. Production of the active metabolite, florpyrauxifen-acid, was greater in Sus barnyardgrass (21.5-52.1%) than in R barnyardgrass (5.5-34.9%). CONCLUSION: In conclusion, reductions in FPB absorption and florpyrauxifen-acid production may contribute to the inability to control barnyardgrass with FPB. © 2021 Society of Chemical Industry.

Absorption, translocation, and metabolism of florpyrauxifen-benzyl and cyhalofop-butyl in cyhalofop-butyl-resistant barnyardgrass [Echinochloa crus-galli (L.) P. Beauv.].

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.

Understanding Resistance Mechanisms to Trifluralin in an Arkansas Palmer Amaranth Population.

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 ß-tubulin genes revealed no single nucleotide polymorphisms (SNPs) previously described between accessions. In addition, relative gene copy number of α- and ß-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.

Presence of the HPPD Inhibitor Sensitive 1 Gene and ALSS653N Mutation in Weedy Oryza sativa Sensitive to Benzobicyclon.

Benzobicyclon has shown varying results in controlling weedy rice, including those with imidazolinone (IMI) resistance. Tolerance to benzobicyclon in cultivated japonica rice, but not indica or aus-like cultivars, is conferred by a fully functional HPPD Inhibitor Sensitive 1 (HIS1) gene. Herein, a diagnostic Kompetitive Allele Specific PCR (KASP) assay was developed to predict the HIS1 genotype of weedy rice plants from 37 accessions and correlated to their response to benzobicyclon in the field. Two-thirds of the 693 weedy rice plants screened were tolerant to benzobicyclon (371 g ai ha-1, SC formulation) at 30 days after treatment (DAT). Thirty-four percent of plants were homozygous for the HIS1 allele and 98% of these plants exhibited field tolerance. However, the his1 genotype did not always correlate with field data. Only 52% of his1 plants were considered sensitive, indicating that the single nucleotide polymorphisms (SNPs) chosen in the KASP assay are not a reliable tool in predicting his1 homozygous plants. In an additional experiment, 86% of the 344 plants with at least one copy of the ALSS653N trait harbored a HIS1 allele, suggesting fields infested with IMI herbicide-resistant weedy rice are unlikely to be controlled with benzobicyclon.

Comparative proteomic analysis of horseweed (Conyza canadensis) biotypes identifies candidate proteins for glyphosate resistance.

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.

Two non-target mechanisms are involved in glyphosate-resistant horseweed (Conyza canadensis L. Cronq.) biotypes.

The physiological and biochemical bases for glyphosate resistance and susceptibility in horseweed (Conyza canadensis L. Cronq.) populations collected from Córdoba, Huelva, Málaga, Jaén 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.

Target site mutation and reduced translocation are present in a glyphosate-resistant Lolium multiflorum Lam. biotype from Spain.

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 (14)C-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 (14)C-glyphosate to roots, while in the S population this value was 24.79%. Visualization of (14)C-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.

Pool of resistance mechanisms to glyphosate in Digitaria insularis.

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 (14)C-glyphosate up to 48 h after treatment (HAT) than resistant biotypes. High differential (14)C-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.

Differential susceptibility to glyphosate among the Conyza weed species in Spain.

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.