Multiple Resistance to Glyphosate and 2,4-D in Carduus acanthoides L. from Argentina and Alternative Control Solutions

Carduus acanthoides L. is an invasive species native to Europe and distributed in other parts of the world, including North and South America. In Cordoba, Argentina, control failures of this species have been reported in Roundup Ready (RR) soybean crops where glyphosate and 2,4-D have frequently been applied, although there are no confirmed reports worldwide of resistance to glyphosate and 2,4-D in this species. Dose–response tests showed multiple-resistance to both active principles. The resistant population (R) had LD50 values of 1854.27 and 1577.18 g ae ha−1 (grams of acid equivalent per hectare), while the susceptible (S) population had LD50 values of 195.56 and 111.78 g ae ha−1 for glyphosate and 2,4-D, respectively. Low accumulations of shikimic acid (glyphosate) and ethylene (2,4-D) at different doses in the R population compared to the S population support the results observed in the dose–response curves. No significant differences in leaf retention were observed for glyphosate and 2,4-D in the R and S populations. However, the use of adjuvants increased the retention capacity of herbicides in both populations. Ten alternative herbicides with seven different action mechanisms (MOAs) were evaluated and the most effective active principles were dicamba, bromoxynil, atrazine, tembotrione, flazasulfuron, glufosinate, and paraquat. These findings are the first evidence of glyphosate and 2,4 D resistance in C. acanthoides

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

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

Glyphosate Resistance Confirmation and Field Management of Red Brome (Bromus rubens L.) in Perennial Crops Grown in Southern Spain

The excessive use of the herbicide glyphosate on annual and perennial crops grown in Southern Spain has caused an increase in resistant weed populations. Bromus rubens has begun to spread through olive and almond cultivars due to low glyphosate control over these species, whereas previously it had been well controlled with field dose (1080 g ae ha−1). Characterization using Simple Sequence Repeat (SSR) markers confirmed the presence of B. rubens collected in Andalusia. A rapid shikimic acid accumulation screening showed 17 resistant (R) populations with values between 300 and 700 µg shikimate g−1 fresh weight and three susceptible (S) populations with values between 1200 and 1700 µg shikimate g−1 fresh weight. In dose–response experiments the GR50 values agreed with previous results and the resistance factors (RFs: GR50 R/GR50 S (Br1)) were between 4.35 (Br9) and 7.61 (Br19). Foliar retention assays shown no differences in glyphosate retention in both R and S populations. The tests carried out in a resistant field (Br10) demonstrated the control efficacy of pre-emergence herbicides since flazasulfuron in the tank mix with glyphosate had up to 80% control 15 to 120 days after application (DAA) and grass weed postemergence herbicides, such as propaquizafop + glyphosate and quizalofop + glyphosate, had up to 90% control 15 to 90 DAA. Results confirm the first scientific report of glyphosate-resistant B. rubens worldwide; however, the use of herbicides with another mode of action (MOA) is the best tool for integrated weed management

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

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 characterised 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.This work was funded by the Asociación de Agroquímicos y Medioambiente. The Spanish Ministry of Science and Innovation (Project PID2019-110847RB-I00) and the European Regional Development Fund (FEDER) supported this research. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). JT acknowledges support from the Spanish Ministry of Science, Innovation and Universities (grant Ramon y Cajal RYC2018-023866-I) and RA thanks support to the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP 2018/15910-6)

Asp376Glu mutation and enhanced metabolism controlling the resistance to ALS-inhibiting herbicides in Ixophorus unisetus (J. Presl) Schltdl. from the Bajio, Mexico

A study was carried out to determine the ALS (acetolactate synthase)-inhibitor herbicide resistance in the Mexican grass Ixophorus unisetus, a troublesome weed in corn crops in Mexico. First, the resistance was confirmed in field screening assays. Eight populations that survived nicosulfuron treatment at a field rate of 40 g ai ha−1 were labeled as putative-resistant. Dose–response trials demonstrated a high resistance in the eight populations (GR50 from 140.33 to 245.46 g ai ha−1). The synergism of malathion plus nicosulfuron demonstrated that the non-target-site resistance (NTSR) mechanism based on cytochrome P450 (Cyt-P450) was involved in five populations of I. unisetus. Molecular studies revealed that a single-nucleotide change occurs in the amino acid at position 376 (from GAT to GAG), which codifies from Asp-376 to Glu-376. This is the first time that Asp-376-Glu has been reported in this species. Assays in vitro and in vivo demonstrated I. unisetus cross-resistance to flucarbazone, penoxsulam, bispyribac-Na, and imazamox. No multiple resistance was found in two resistant populations exposed to different herbicides. Our results indicate that the lack of good control over Mexican grass in corn with ALS inhibitors is due to target-site mutation and NTSR mechanisms (Cyt-P450-mediated metabolism). A strategy should be established in Mexican fields to continue controlling this weed, including mechanical control practices and a good combination of the available pre- and post-emergence herbicides

Management of Glyphosate-Resistant Weeds in Mexican Citrus Groves: Chemical Alternatives and Economic Viability

Glyphosate is a cheap herbicide that has been used to control a wide range of weeds (4–6 times/year) in citrus groves of the Gulf of Mexico; however, its excessive use has selected for glyphosate-resistant weeds. We evaluated the efficacy and economic viability of 13 herbicide treatments (glyphosate combined with PRE- and/or POST-emergence herbicides and other alternative treatments), applied in tank-mixture or sequence, to control glyphosate-resistant weeds in two Persian lime groves (referred to as SM-I and SM-II) of the municipality of Acateno, Puebla, during two years (2014 and 2015). The SM-I and SM-II fields had 243 and 346 weeds/m2, respectively, composed mainly of Bidens pilosa and Leptochloa virgata. Echinochloa colona was also frequent in SM-II. The glyphosate alone treatments (1080, 1440, or 1800 g ae ha−1) presented control levels of the total weed population ranging from 64% to 85% at 15, 30, and 45 d after treatment (DAT) in both fields. Mixtures of glyphosate with grass herbicides such as fluazifop-p-butyl, sethoxydim, and clethodim efficiently controlled E. colona and L. virgata, but favored the regrowth of B. pilosa. The sequential applications of glyphosate + (bromacil + diuron) and glufosinate + oxyfluorfen controlled more than 85% the total weed community for more than 75 days. However, these treatments were between 360% and 390% more expensive (1.79 and 1.89 $/day ha−1 of satisfactory weed control, respectively), compared to the representative treatment (glyphosate 1080 g ae ha−1 = USD$29.0 ha−1). In practical and economic terms, glufosinate alone was the best treatment controlling glyphosate resistant weeds maintaining control levels >80% for at least 60 DAT ($1.35/day ha−1). The rest of the treatments, applied in tank-mix or in sequence with glyphosate, had similar or lower control levels (~70%) than glyphosate at 1080 g ae ha−1. The adoption of glufosiante alone, glufosinate + oxyfluorfen or glyphosate + (bromacil + diuron) must consider the cost of satisfactory weed control per day, the period of weed control, as well as other factors associated with production costs to obtain an integrated weed management in the short and long term

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.EEA BarrowFil: Palma Bautista, Candelario. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Cruz Hipólito, Hugo E. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Alcántara de la Cruz, Ricardo. Universidade Federal de São Carlos. Departamento de Química; BrasilFil: Vázquez García, José G. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; EspañaFil: Yanniccari, Marcos Ezequiel. Consejo Nacional de Investigaciones Científicas y Técnicas. Laboratorio de Biotecnología y Genética Vegetal; Argentina.Fil: Yanniccari, Marcos Ezequiel. Instituto Nacional de Tecnología Agropecuaria (INTA). Chacra Experimental Integrada Barrow; ArgentinaFil: de Prado, Rafael. Universidad de Córdoba. Departamento de Química Agrícola y Edafología; Españ

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

New Case of False-Star-Grass (Chloris distichophylla) Population Evolving Glyphosate Resistance

Chloris distichophylla, suspected of glyphosate resistance (GR), was collected from areas of soybean cultivation in Rio Grande do Sul, Brazil. A comparison was made with a susceptible population (GS) to evaluate the resistance level, mechanisms involved, and control alternatives. Glyphosate doses required to reduce the dry weight (GR50) or cause a mortality rate of 50% (LD50) were around 5.1–3 times greater in the GR population than in the GS population. The shikimic acid accumulation was around 6.2-fold greater in GS plants than in GR plants. No metabolized glyphosate was found in either GR or GS plants. Both populations did not differ in the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) basal activity or in vitro inhibition of EPSPS activity by glyphosate (I50). The maximum glyphosate absorption was observed at 96 hours after treatment (HAT), which was twofold higher in the GS plants than in the GR plants. This confirms the first case of glyphosate resistance in C. distichophylla. In addition, at 96 HAT, the GS plants translocated more 14C-glyphosate than the GR ones. The best options for the chemical control of both C. distichophylla populations were clethodim, quizalofop, paraquat, glufosinate, tembotrione, diuron, and atrazine. The first case of glyphosate resistance in C. distichophylla was due to impaired uptake and translocation. Chemical control using multiple herbicides with different modes of action (MOA) could be a tool used for integrated weed management (IWM) programs