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.

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.

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.

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.

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 Resistance Evolution in Bipyridylium-Resistant Epilobium ciliatum After Recurrent Selection.

The use of herbicides with different modes of action is the primary strategy used to control weeds possessing resistance to a single mechanism of action (MOA). However, this practice can lead to selection for generalist resistance mechanisms and may cause resistance to all MOAs. In this research, we characterized the resistance to diquat/paraquat (bipyridiliums) in an Epilobium ciliatum biotype (R1) collected in an olive orchard from Chile, where alternatives herbicides (2,4-D, glyphosate, glufosinate, flazasulfuron and pyraflufen-ethyl) with different MOAs were used, but they have also showed failure in controlling this species. Because the resistance/susceptibility patterns of the R1 biotype to glufosinate, 2,4-D and pyraflufen-ethyl were not clear, a recurrent resistance selection was carried out in field and greenhouse using these herbicides on R1 plants for three generations (R2 biotype). One biotype that was never treated with herbicides (S) was included as control. Results indicated that the S biotype was controlled at the field dose of all herbicides tested. The biotype R1 exhibited resistance to diquat, paraquat and flazasulfuron and natural tolerance to glyphosate. The R2 biotype displayed resistance to glufosinate, 2,4-D and pyraflufen-ethyl with LD50 (herbicide dose to kill 50% of plants) values higher than field doses in all assays. Physiological and biochemical studies determined the resistance to diquat of the R1 biotype, which was due to impaired translocation. The resistance to flazasulfuron in the R1 and R2 biotypes was confirmed by the low sensitivity of the acetolactate synthase (ALS) activity compared to the S biotype. The similar accumulation of shikimate in treated S, R1, and R2 plants with glyphosate supported the existence of innate tolerance to this herbicide in E. ciliatum. Resistance to glufosinate, 2,4-D and pyraflufen-ethyl in the R2 biotype, acquired after recurrent selection, was determined by low sensitivity of the glutamine synthetase, low accumulation of ethylene and protoporphyrinogen IX oxidase, respectively, in comparison to the S biotype. Epilobium ciliatum from Chilean olive orchards had resistance to only two MAOs (photosystem I and ALS inhibitors), but resistance to five MOAs could occur in the next cropping seasons, if alternatives to weed management, other than herbicides, are not included.

Identifying Chloris Species from Cuban Citrus Orchards and Determining Their Glyphosate-Resistance Status.

The Chloris genus is a C4 photosynthetic species mainly distributed in tropical and subtropical regions. Populations of three Chloris species occurring in citrus orchards from central Cuba, under long history glyphosate-based weed management, were studied for glyphosate-resistant status by characterizing their herbicide resistance/tolerance mechanisms. Morphological and molecular analyses allowed these species to be identified as C. ciliata Sw., Chloris elata Desv., and Chloris barbata Sw. Based on the glyphosate rate that causes 50% mortality of the treated plants, glyphosate resistance (R) was confirmed only in C. elata, The R population was 6.1-fold more resistant compared to the susceptible (S) population. In addition, R plants of C. elata accumulated 4.6-fold less shikimate after glyphosate application than S plants. Meanwhile, populations of C. barbata and C. ciliata with or without glyphosate application histories showed similar LD50 values and shikimic acid accumulation rates, demonstrating that resistance to glyphosate have not evolved in these species. Plants of R and S populations of C. elata differed in 14C-glyphosate absorption and translocation. The R population exhibited 27.3-fold greater 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) activity than the S population due to a target site mutation corresponding to a Pro-106-Ser substitution found in the EPSPS gene. These reports show the innate tolerance to glyphosate of C. barbata and C. ciliata, and confirm the resistance of C. elata to this herbicide, showing that both non-target site and target-site mechanisms are involved in its resistance to glyphosate. This is the first case of herbicide resistance in Cuba.

Pro-106-Ser mutation and EPSPS overexpression acting together simultaneously in glyphosate-resistant goosegrass (Eleusine indica).

Glyphosate has been used for more than 15 years for weed management in citrus groves in the Gulf of Mexico, at up to 3-4 applications per year. Goosegrass (Eleusine indica (L.) Gaertn.) control has sometimes failed. In this research, the mechanisms governing three goosegrass biotypes (Ein-Or from an orange grove, and Ein-Pl1 and Ein-Pl2 from Persian lime groves) with suspected resistance to glyphosate were characterized and compared to a susceptible biotype (Ein-S). Dose-response and shikimate accumulation assays confirmed resistance of the resistant (R) biotypes. There were no differences in glyphosate absorption, but the R biotypes retained up to 62-78% of the herbicide in the treated leaf at 96 h after treatment (HAT), in comparison to the Ein-S biotype (36%). The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity in the Ein-Or and Ein-S biotypes was over 100-fold lower than the Ein-Pl1 and Ein-Pl2 ones. The latter showed a high EPSPS-basal activity, a mutation at Pro-106-Ser position in the EPSPS gene, and EPSPS overexpression. The EPSPS basal and EPSPS overexpression were positively correlated. The R goosegrass biotypes displayed poor glyphosate translocation. Furthermore, this grassweed showed, for the first time, two mechanisms at the target-site level (Pro-106-Ser mutation + EPSPS overexpression) acting together simultaneously against glyphosate.

First Resistance Mechanisms Characterization in Glyphosate-Resistant Leptochloa virgata.

Leptochloa virgata (L.) P. Beauv. is an annual weed common in citrus groves in the states of Puebla and Veracruz, Mexico limiting their production. Since 2010, several L. virgata populations were identified as being resistant to glyphosate, but studies of their resistance mechanisms developed by this species have been conducted. In this work, three glyphosate-resistant populations (R8, R14, and R15) collected in citrus orchards from Mexico, were used to study their resistance mechanisms comparing them to one susceptible population (S). Dose-response and shikimic acid accumulation assays confirmed the glyphosate resistance of the three resistant populations. Higher doses of up to 720 g ae ha-1 (field dose) were needed to control by 50% plants of resistant populations. The S population absorbed between 7 and 13% more 14C-glyphosate than resistant ones, and translocated up to 32.2% of 14C-glyphosate to the roots at 96 h after treatment (HAT). The R8, R14, and R15 populations translocated only 24.5, 26.5, and 21.9%, respectively. The enzyme activity of 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was not different in the S, R8 and R14 populations. The R15 Population exhibited 165.9 times greater EPSPS activity. Additionally, this population showed a higher EPSPS basal activity and a substitution in the codon 106 from Proline to Serine in the EPSPS protein sequence. EPSPS gene expression in the R15 population was similar to that of S population. In conclusion, the three resistant L. virgata populations show reduced absorption and translocation of 14C-glyphosate. Moreover, a mutation and an enhanced EPSPS basal activity at target-site level confers higher resistance to glyphosate. These results describe for the first time the glyphosate resistance mechanisms developed by resistant L. virgata populations of citrus orchards from Mexico.

Target and Non-target Site Mechanisms Developed by Glyphosate-Resistant Hairy beggarticks (Bidens pilosa L.) Populations from Mexico.

In 2014 hairy beggarticks (Bidens pilosa L.) has been identified as being glyphosate-resistant in citrus orchards from Mexico. The target and non-target site mechanisms involved in the response to glyphosate of two resistant populations (R1 and R2) and one susceptible (S) were studied. Experiments of dose-response, shikimic acid accumulation, uptake-translocation, enzyme activity and 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) gene sequencing were carried out in each population. The R1 and R2 populations were 20.4 and 2.8-fold less glyphosate sensitive, respectively, than the S population. The resistant populations showed a lesser shikimic acid accumulation than the S population. In the latter one, 24.9% of 14C-glyphosate was translocated to the roots at 96 h after treatment; in the R1 and R2 populations only 12.9 and 15.5%, respectively, was translocated. Qualitative results confirmed the reduced 14C-glyphosate translocation in the resistant populations. The EPSPS enzyme activity of the S population was 128.4 and 8.5-fold higher than the R1 and R2 populations of glyphosate-treated plants, respectively. A single (Pro-106-Ser), and a double (Thr-102-Ile followed by Pro-106-Ser) mutations were identified in the EPSPS2 gene conferred high resistance in R1 population. Target-site mutations associated with a reduced translocation were responsible for the higher glyphosate resistance in the R1 population. The low-intermediate resistance of the R2 population was mediated by reduced translocation. This is the first glyphosate resistance case confirmed in hairy beggarticks in the world.

Non-target Site Tolerance Mechanisms Describe Tolerance to Glyphosate in Avena sterilis.

Sterile wild oat (Avena sterilis L.) is an autogamous grass established in warm climate regions. This species has been used as a cover crop in Mediterranean perennial crops during the spring period prior to initiating competition with the main crop for water and nutrients. However, such cover crops need to be controlled (by glyphosate or tillage) before the beginning of summer period (due to the possibility of intense drought stress). In 2011, the olive grove farmers of southern Spain expressed dissatisfaction because of the ineffective control with glyphosate on A. sterilis. Experiments were conducted to determine whether the continued use of glyphosate over a 5 year period had selected a new resistant or tolerant species. The GR50 values obtained for A. sterilis were 297.12 and 245.23 g ae ha(-1) for exposed (E) and un-exposed (UE) glyphosate accessions, respectively. The spray retention and shikimic acid accumulation exhibited a non-significant difference between the two accessions. The results of (14)C- glyphosate absorption was the same in the two accessions (E and UE), while the translocation from the treated leaf to the rest of the shoots and roots was similar in A. sterilis accessions. Glyphosate metabolism to aminomethylphosphonic acid (AMPA) and glyoxylate was similar in both accessions, but increased after treatment with glyphosate, indicating that metabolism plays an important role in tolerance. Both A. sterilis accessions, present similarity in the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity enzyme with different glyphosate concentrations and without glyphosate, confirming that both accessions present the same genomic characteristics. The above-mentioned results indicate that innate tolerance to glyphosate in A. sterilis is probably and partly due to reduced herbicide absorption and translocation and metabolism compared to the susceptibility of other grasses weeds like Chloris inflata, Eleusine indica, and Lolium rigidum.