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.

Influence of temperature on the retention, absorption and translocation of fomesafen and imazamox in Euphorbia heterophylla.

Climate change will be an additional issue to the challenge to manage herbicide resistant weeds. This work investigated the impact of three temperature regimes (10/5, 20/15 and 30/25 °C) on the efficacy, foliar retention, absorption and translocation of fomesafen, protoporphyrinogen oxidase (PPO) inhibitor, and imazamox, acetolactate synthase (ALS) inhibitor, between two Euphorbia heterophylla populations, one susceptible (S) and one multiple PPO and ALS resistant (R). The R population went from 5 (fomesafen) and 12 (imazamox) times more resistant than the S population at 10/5 °C to more than 100 times to both herbicides at 20/15 and 30/25 °C. Leaf retention of fomesafen was not affected by temperature; however, imazamox retention was less at 10/5 and 20/15 °C than at 30/25 °C, and the R population always retained less imazamox than the S population. 14C-fomesafen absorption was similar between populations, but lower amounts were absorbed at 10/5 °C regardless of the evaluation time. Recovered 14C-imazamox rates decreased in both populations as the evaluation time increased, ranging from 82 to 92% at 6 h after treatment (HAT), and from 47 to 76% at 48 HAT, depending on the temperature regime. The 14C-imazamox losses were greater from 24 HAT in R plants grown at 30/25 °C and in all temperature regimes at 48 HAT. Although both populations translocated large amounts of imazamox, the S population distributed it in the rest of the plant (33%) and roots (15%), while the R population kept it mainly on the treated leaf (24%) or lost ~20% more herbicide than S population at 48 HAT, indicating the need for further studies on root exudation between these populations. Low temperatures reduced resistance levels to fomesafen and imazamox in E. heterophylla, suggesting that temperature influences the expression of the mechanisms that govern this multiple resistance.

Distribution of Glyphosate-Resistance in Echinochloa crus-galli Across Agriculture Areas in the Iberian Peninsula.

The levels of resistance to glyphosate of 13 barnyard grass (Echinochloa crus-galli) populations harvested across different agriculture areas in the Southern Iberian Peninsula were determined in greenhouse and laboratory experiments. Shikimate accumulation fast screening separated the populations regarding resistance to glyphosate: susceptible (S) E2, E3, E4, and E6 and resistant (R) E1, E5, E7, E8, E9, E10, E11, E12, and E13. However, resistance factor (GR50 E1-E13/GR50 E6) values separated these populations into three groups: (S) E2, E3, E4, and E6, (R) E1, E5, E7, E8, and E9, and very resistant (VR) E10, E11, E12, and E13. 14C-glyphosate assays performed on two S populations (E2 and E6) showed greater absorption and translocation than those found for R (E7 and E9) and VR (E10 and E12) populations. No previous population metabolized glyphosate to amino methyl phosphonic acid (AMPA) and glyoxylate, except for the E10 population that metabolized 51% to non-toxic products. The VR populations showed two times more 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity without herbicide than the rest, while the inhibition of the EPSPS activity by 50% (I50) required much higher glyphosate in R and VR populations than in S populations. These results indicated that different target-site and non-target-site resistance mechanisms were implicated in the resistance to glyphosate in E. crus-galli. Our results conclude that resistance is independent of climate, type of crop, and geographic region and that the level of glyphosate resistance was mainly due to the selection pressure made by the herbicide on the different populations of E. crus-galli studied.

First Case of Glyphosate Resistance in Bromus catharticus Vahl.: Examination of Endowing Resistance Mechanisms.

Bromus catharticus Vahl. has been used as a valuable forage crop, but it has also been noted as a weed of winter crops and an invader in several countries. In Argentina, a putative glyphosate-resistant population of B. catharticus was identified as a consequence of the lack of effective control with glyphosate in the pre-sowing of wheat. Plant survival and shikimate accumulation analysis demonstrated a lower glyphosate-sensitivity of this population in comparison to a susceptible B. catharticus population. The resistant population was 4-fold more resistant to glyphosate than its susceptible counterpart. There was no evidence of target-site mechanisms of glyphosate resistance or an enhanced capacity to metabolize glyphosate in the resistant population. However, the resistant plants showed a lower foliar retention of glyphosate (138.34 µl solution g-1 dry weight vs. 390.79 µl solution g-1 dry weight), a reduced absorption of 14C-glyphosate (54.18 vs. 73.56%) and lower translocation of 14C-glyphosate from the labeled leaf (27.70 vs. 62.36%). As a result, susceptible plants accumulated a 4.1-fold higher concentration of 14C-glyphosate in the roots compared to resistant plants. The current work describes the first worldwide case of glyphosate resistance in B. catharticus. A reduced foliar retention of herbicide, a differential rate of glyphosate entry into leaves and an altered glyphosate translocation pattern would be the most likely mechanisms of glyphosate exclusion.

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.

Cross-resistance mechanisms to ACCase-inhibiting herbicides in short-spike canarygrass (Phalaris brachystachys).

Herbicides that inhibit acetyl-coenzyme A carboxylase (ACCase) are commonly used to control weedy grasses such as short-spike canarygrass (Phalaris brachystachys). Two resistant biotypes of P. brachystachys (R1 and R2) were found in different winter wheat fields in Iran. This study was done to confirm the suspected resistance observed in the field and to elucidate the resistance mechanisms involved. The results indicated that the both resistant biotypes showed cross-resistance to diclofop-methyl (DM), pinoxaden (PN) and cycloxydim (CD) herbicides. Based on the herbicide dose that inhibited 50% of the ACCase activity (I50), the ACCase activity of the resistant biotypes was less sensitive than the S biotype to DM, CD, and PN. No differences in translocation were detected between biotypes; most of the herbicide remained in the treated leaves. The 14C-DM metabolites were identified using thin-layer chromatography. Pre-treatment with the cytochrome P450 inhibitor ABT inhibited 14C-DM metabolism in the R1 biotype, indicating that metabolism is involved in the DM resistance in the R1 biotype. DNA sequencing studies found an Ile-1781-Thr change in both resistant biotypes, conferring cross-resistance to ACCase inhibitors. In general, in the R1 biotype which showed a higher level of resistance than that of the R2 biotype, cross-resistance was observed because of mutation and DM metabolism, while in the R2 biotype, the mutation confers resistance to ACCase-inhibiting herbicides. This is the first reported evidence of the mechanisms responsible for the resistance to ACCase herbicides in P. brachystachys. These results could be useful for improved management of resistant biotypes carrying similar mutations.

Target site as the main mechanism of resistance to imazamox in a Euphorbia heterophylla biotype.

Euphorbia heterophylla is a weed species that invades extensive crop areas in subtropical regions of Brazil. This species was previously controlled by imazamox, but the continuous use of this herbicide has selected for resistant biotypes. Two biotypes of E. heterophylla from southern Brazil, one resistant (R) and one susceptible (S) to imazamox, were compared. The resistance of the R biotype was confirmed by dose-response assays since it required 1250.2 g ai ha-1 to reduce the fresh weight by 50% versus 7.4 g ai ha-1 for the S biotype. The acetolactate synthase (ALS) enzyme activity was studied using ALS-inhibiting herbicides from five different chemical families. The R biotype required the highest concentrations to reduce this enzyme activity by 50%. A Ser653Asn mutation was found in the ALS gene of the R biotype. The experiments carried out showed that imazamox absorption and metabolism were not involved in resistance. However, greater 14C-imazamox root exudation was found in the R biotype (~70% of the total absorbed imazamox). Target site mutation in the ALS gene is the principal mechanism that explains the imazamox resistance of the R biotype, but root exudation seems to also contribute to the resistance of this biotype.

Physiological, biochemical and molecular bases of resistance to tribenuron-methyl and glyphosate in Conyza canadensis from olive groves in southern Spain.

Multiple resistance to acetolactate synthase (ALS, EC 2.2.1.6) and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, EC 2.5.1.19) inhibitor herbicides was studied in two populations of Conyza canadensis (RTG and STG) harvested in southern Spain. Dose-response and enzymatic activity studies for the ALS-inhibiting herbicides showed only cross-resistance to sulfonylureas group but not to the other ALS chemical groups in the RTG population. Regarding glyphosate, the dose-response studies showed that the RTG population was 11.8 times more resistant than the STG population, while the inhibition of EPSPS enzyme (I50) was similar for both populations. Altered/reduced absorption and translocation were the main resistance mechanisms for glyphosate but not for tribenuron-methyl. The metabolic studies to find differences in the amounts of metabolites between the two populations were carried out using thin layer chromatography (for tribenuron-methyl) and capillary electrophoresis (for glyphosate). Metabolites were significantly differed among the two populations for tribenuron-methyl but not for glyphosate. The sequencing of the target-site ALS gene from RTG plants revealed a single point mutation, Pro-197-Ala, that causes resistance to sulfonylurea herbicide in C. canadensis.

Multiple Resistance to Synthetic Auxin Herbicides and Glyphosate in Parthenium hysterophorus Occurring in Citrus Orchards.

Dominican farmers have started to apply synthetic auxin herbicides (SAHs) as the main alternative to mitigate the impacts of the occurrence of glyphosate-resistant (GR) Parthenium hysterophorus populations in citrus orchards. A GR P. hysterophorus population survived field labeled rates of glyphosate, 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, and picloram, which showed poor control (<50%). In in vivo assays, resistance levels were high for glyphosate and moderate for picloram, dicamba, and 2,4-D. Sequencing the 5-enolpyruvylshikimate-3-phosphate synthase gene revealed the double Thr-102-Ile and Pro-106-Ser amino acid substitution, conferring resistance to glyphosate. Additionally, reduced absorption and impaired translocation contributed to this resistance. Regarding SAH, impaired 2,4-D transport and enhanced metabolism were confirmed in resistant plants. The application of malathion improved the efficacy of SAHs (control >50%), showing that metabolism of these herbicides was mediated by cytochrome P450 enzymes. This study reports, for the first time, multiple resistance to SAHs and glyphosate in P. hysterophorus.

Low temperatures enhance the absorption and translocation of 14C-glyphosate in glyphosate-resistant Conyza sumatrensis.

Influence of low temperatures on the glyphosate efficacy was studied in glyphosate-resistant (R) and -susceptible (S) Conyza sumatrensis biotypes. For this purpose, the physiological and enzymatic aspects involved were characterized under two growing temperature regimes [high (30/20 °C) and low 15/5 °C temperatures day/night]. The R biotype was 5.5 times more resistant than the S biotype at high temperatures; however, this R-to-S ratio decreased to 1.6 at low temperatures. At 96 h after treatment (HAT), the shikimic acid accumulation was higher in the S biotype in both temperature regimes (4.6 and 1.9 more shikimic acid at high and low temperatures, respectively), but the accumulation of the R biotype increased 2.6 times at low temperatures compared to high ones. From 24 to 96 HAT, the 14C-glyphosate absorption ranged from 28 to 65% (percentage reached from 48 HAT) at low temperatures, and from 20 to 50% at high temperatures (gradual increase), but there were no differences between C. sumatrensis biotypes within each temperature regime. At high temperatures, the 14C-glyphosate translocation was different between biotypes, where the R one retained at least 10% more herbicide in the treated leaves than the S biotype at 96 HAT. So, the S biotype translocated 40% of 14C-glyphosate absorbed to roots, and the R biotype translocated only 28% of herbicide at the same period. At low temperatures, there were no differences between biotypes, and at 96 HAT, the 14C-glyphosate found in treated leaves was ˜47% and up to ˜42% reached the roots, i.e., the resistance mechanism was suppressed. The basal and enzymatic activities of the 5-enolpyruvyishikimate 3-phosphate synthase were different between temperature regimes, but there was no differences between biotypes within each temperature regime, showing that target-site resistance mechanisms did not contribute in the glyphosate resistance of the R biotype. Low temperatures enhanced the absorption and translocation of glyphosate by suppressing the resistance mechanisms improving its efficacy on resistant plants. This is the first characterization about the role of temperatures in the glyphosate efficacy on C. sumatrensis.

The Triple Amino Acid Substitution TAP-IVS in the EPSPS Gene Confers High Glyphosate Resistance to the Superweed Amaranthus hybridus.

The introduction of glyphosate-resistant (GR) crops revolutionized weed management; however, the improper use of this technology has selected for a wide range of weeds resistant to glyphosate, referred to as superweeds. We characterized the high glyphosate resistance level of an Amaranthus hybridus population (GRH)-a superweed collected in a GR-soybean field from Cordoba, Argentina-as well as the resistance mechanisms that govern it in comparison to a susceptible population (GSH). The GRH population was 100.6 times more resistant than the GSH population. Reduced absorption and metabolism of glyphosate, as well as gene duplication of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) or its overexpression did not contribute to this resistance. However, GSH plants translocated at least 10% more 14C-glyphosate to the rest of the plant and roots than GRH plants at 9 h after treatment. In addition, a novel triple amino acid substitution from TAP (wild type, GSH) to IVS (triple mutant, GRH) was identified in the EPSPS gene of the GRH. The nucleotide substitutions consisted of ATA102, GTC103 and TCA106 instead of ACA102, GCG103, and CCA106, respectively. The hydrogen bond distances between Gly-101 and Arg-105 positions increased from 2.89 Å (wild type) to 2.93 Å (triple-mutant) according to the EPSPS structural modeling. These results support that the high level of glyphosate resistance of the GRH A. hybridus population was mainly governed by the triple mutation TAP-IVS found of the EPSPS target site, but the impaired translocation of herbicide also contributed in this resistance.

Characterization of three glyphosate resistant Parthenium hysterophorus populations collected in citrus groves from Mexico.

Continuous use of glyphosate in citrus groves in the Gulf of Mexico region has selected for resistant Parthenium hysterophorus L. populations. In this study, the target-site and non-target-site resistance mechanisms were characterized in three putative glyphosate-resistant (GR) P. hysterophorus populations, collected in citrus groves from Acateno, Puebla (GR1 and GR2) and Martínez de la Torre, Veracruz (GR3), and compared with a susceptible population (GS). Based on plant mortality, the GR populations were 9.2-17.3 times more resistant to glyphosate than the GS population. The low shikimate accumulation in the GR population confirmed this resistance. Based on plant mortality and shikimate accumulation, the GR3 population showed intermediate resistance to glyphosate. The GR populations absorbed 15-28% less 14C-glyphosate than the GS population (78.7% absorbed from the applied) and retained 48.7-70.7% of 14C-glyphosate in the treated leaf, while the GS population translocated ~68% of absorbed herbicide to shoots and roots. The GR3 population showed the lowest translocation and absorption rates, but was found to be susceptible at the target site level. The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene sequence of the GR1 and GR2 populations showed the Pro106-Ser mutation, conferring 19- and 25-times more resistance in comparison to the GS population, respectively. Reduced absorption and impaired translocation conferred glyphosate resistance on the GR3 population, and contributed partially to the resistance of the GR1 and GR2 populations. Additionally, the Pro-106-Ser mutation increased the glyphosate resistance of the last two P. hysterophorus populations.

Reduced Absorption and Impaired Translocation Endows Glyphosate Resistance in Amaranthus palmeri Harvested in Glyphosate-Resistant Soybean from Argentina.

Amaranthus palmeri S. Watson is probably the worst glyphosate-resistant (GR) weed worldwide. The EPSPS (5-enolpyruvylshikimate-3-phosphate-synthase) gene amplification has been reported as the major target-site-resistance (TSR) mechanism conferring resistance to glyphosate in this species. In this study, TSR and non-target-site-resistance (NTSR) mechanisms to glyphosate were characterized in a putative resistant A. palmeri population (GRP), harvested in a GR soybean crop from Argentina. Glyphosate resistance was confirmed for the GRP population by dose-response assays. No evidence of TSR mechanisms, as well as glyphosate metabolism, was found in this population. Moreover, a susceptible population (GSP) that absorbed about 10% more herbicide than the GRP population was evaluated at different periods after treatment. The GSP population translocated about 20% more glyphosate to the remainder of the shoots and roots at 96 h after treatment than the control, while the GRP population retained 62% of herbicide in the treated leaves. This is the first case of glyphosate resistance in A. palmeri involving exclusively NTSR mechanisms.

Stacked traits conferring multiple resistance to imazamox and glufosinate in soft wheat.

BACKGROUND: Conventional crossing of soft wheat cultivars resistant to imazamox and glufosinate resulted in two (Rados and Helter) lines resistant to both herbicides. Stacked traits conferring this dual herbicide resistance in these lines, compared with a susceptible (S) cultivar, were characterized. RESULTS: Rados and Helter lines were ∼ 18-fold more resistant (R) to glufosinate, and between 15.1 and 19.8-fold more resistant to imazamox than the S cultivar. Resistance to glufosinate and imazamox decreased up to 12% and 50%, respectively, when the herbicides were applied sequentially. The basal activities of the acetolactate and glutamine synthases were similar between R and S plants. Rados and Helter lines were 11.7- and 17.7-fold more resistant to imazamox than the S cultivar, due to the Ser653-Asn mutation in their imi-ALS genes. R lines, susceptible to glufosinate at the target site level, showed lower ammonia accumulation evidencing the activity of the phosphinothricin acetyl transferase. Absorption and translocation patterns for 14 C-imazamox and 14 C-glufosinate were similar between R and S cultivars and so do not contribute to resistance. CONCLUSION: Stacked traits conferring dual herbicide resistance to the lines Rados and Helter come from the resistant parents. These R lines are potential tools for weed management in wheat production, mainly via herbicide rotation. © 2018 Society of Chemical Industry.

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.

Multiple mechanisms are involved in new imazamox-resistant varieties of durum and soft wheat.

Weed control in wheat is one of the major goals of farmers in their efforts toward obtaining the highest crop yields for human foods. Several studies (dose-response, enzyme activity, absorption-translocation and metabolism) were conducted to characterize the resistance level of two new wheat cultivars called Rafalín (Triticum aestivum) and Antoñín (T. durum) that were obtained by conventional breeding based on Clearfield® technology; they are resistant (R) to imazamox compared to their sensitive (S) counterparts (Gazul and Simeto, respectively). The R-cultivars were 93.7-fold (Rafalín) and 43.7-fold (Antoñín) more resistant than their respective S-cultivars. The acetolactate synthase (ALS) enzyme activity revealed high resistance to imidazolinone (IMI) herbicides in R-cultivars, but no cross-resistance to other ALS herbicides was found. The Ser653Asn mutation that confers resistance to IMI herbicides was identified in the imi1 and imi2 genes of Rafalín and only in the imi1 gene of Antoñín. The 14C-imazamox absorption did not differ between the R- and S-cultivars. Imazamox was metabolized by Cyt-P450 into imazamox-hydroxyl and imazamox-glucoside in the R-cultivars, altering their translocation patterns. The differential sensitivity to imazamox between R-cultivars was due to the number of resistance genes that carry each genotype. The R-cultivars Rafalín and Antoñín could be excellent weed control tools.

Enhanced 2,4-D Metabolism in Two Resistant Papaver rhoeas Populations from Spain.

Corn poppy (Papaver rhoeas), the most problematic broadleaf weed in winter cereals in Southern Europe, has developed resistance to the widely-used herbicide, 2,4-D. The first reported resistance mechanism in this species to 2,4-D was reduced translocation from treated leaves to the rest of the plant. However, the presence of other non-target site resistance (NTSR) mechanisms has not been investigated up to date. Therefore, the main objective of this research was to reveal if enhanced 2,4-D metabolism is also present in two Spanish resistant (R) populations to synthetic auxins. With this aim, HPLC experiments at two 2,4-D rates (600 and 2,400 g ai ha-1) were conducted to identify and quantify the metabolites produced and evaluate possible differences in 2,4-D degradation between resistant (R) and susceptible (S) plants. Secondarily, to determine the role of cytochrome P450 in the resistance response, dose-response experiments were performed using malathion as its inhibitor. Three populations were used: S, only 2,4-D R (R-703) and multiple R to 2,4-D and ALS inhibitors (R-213). HPLC studies indicated the presence of two hydroxy metabolites in these R populations in shoots and roots, which were not detected in S plants, at both rates. Therefore, enhanced metabolism becomes a new NTSR mechanism in these two P. rhoeas populations from Spain. Results from the dose-response experiments also showed that pre-treatment of R plants with the cytochrome P450 (P450) inhibitor malathion reversed the phenotype to 2,4-D from resistant to susceptible in both R populations. Therefore, it could be hypothesized that a malathion inhibited P450 is responsible of the formation of the hydroxy metabolites detected in the metabolism studies. This and previous research indicate that two resistant mechanisms to 2,4-D could be present in populations R-703 and R-213: reduced translocation and enhanced metabolism. Future experiments are required to confirm these hypotheses, understand the role of P450, and the relationship between both NTSR mechanisms. On this basis, selection pressure with synthetic auxins bears the risk of promoting the evolution enhanced metabolism in Papaver rhoeas.

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.