Cytochrome P450 metabolism-based herbicide resistance to imazamox and 2,4-D in Papaver rhoeas.

Papaver rhoeas biotypes displaying multiple herbicide resistance to ALS inhibitors and synthetic auxin herbicides (SAH) are spreading across Europe. In Spain, enhanced metabolism to imazamox was confirmed in one population, while cytochrome-P450 (P450) based metabolism to 2,4-D in another two. The objectives of this research were to further confirm the presence of P450 mediated enhanced metabolism and, if so, to confirm whether a putative common P450 is responsible of metabolizing both 2,4-D and imazamox. Metabolism studies were undertaken in five P. rhoeas populations with contrasted HR profiles (herbicide susceptible, only HR to ALS inhibitors, only HR to SAH, or multiple HR to both), and moreover, three different P450 inhibitors were used. The presence of enhanced metabolism to these SoA was confirmed in three more HR P. rhoeas populations. This study provides the first direct evidence that imazamox metabolism in these biotypes is P450-mediated, also in one population without an altered target site. Additionally, it was further confirmed that enhanced metabolism of 2,4-D in biotypes only HR to SAH or multiple HR to ALS inhibitors and SAH involves P450 as well. No metabolism was detected using the three inhibitors in all the herbicide-metabolizing P. rhoeas biotypes, suggesting that a common metabolic system involving P450s is responsible of degrading herbicides affecting both SoAs. Thus, selection pressure with either SAH or imidazolinone ALS inhibitors can select not only for resistance to each of them, but it can also confer cross-resistance between them in P. rhoeas.

The First Case of Glyphosate Resistance in Johnsongrass (Sorghum halepense (L.) Pers.) in Europe.

Six Johnsongrass populations suspected of being glyphosate resistant were collected from railways and freeways near Cordoba (SW Spain), where glyphosate is the main weed control tool. The 50% reduction in shoot fresh weight (GR50) values obtained for these six populations ranged from 550.4 to 1169 g ae ha-1, which were 4.2 to 9 times greater than the value obtained for the susceptible population. Glyphosate was equally metabolized to the same extent in both resistant and susceptible populations, with no significant differences in either 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibition or basal activity. No amino acid substitutions were observed in any of the resistant populations. Slight but significant differences in glyphosate penetration were observed among some but not all of the resistant populations and for the times of incubation assayed, although these differences were not considered further. The proposed primary mechanism of resistance in these six glyphosate-resistant Johnsongrass populations is reduced herbicide translocation, because the amount of glyphosate that translocated from treated leaves to shoots and roots in the susceptible population was double that observed in the resistant populations. As glyphosate multiple resistance due to more than one mechanism is not uncommon, this is the first time that glyphosate-resistant Johnsongrass populations have been fully described for all known mechanisms.

Capillary electrophoresis and herbicide analysis: present and future perspectives.

The aim of this article is to provide an overview on the potential of the CE-herbicides binomial. To this end, the methods proposed so far are discussed: their characteristics, types of samples and analytes to which the methods have been applied, sample preparation steps, if required (e.g. cleanup-preconcentration, derivatization steps), and type of detection in each case. Also, the methods are compared with counterparts based on LC, when appropriate. The role of MS detection in present and future analytical research in this field (both for identification and quantitation) are commented. The foreseeable and desirable trends in analysis of herbicides are also outlined in the light of the present trends in metabolomics as a way of knowing the pathways, the intermediate and final degradation products that can influence the crops and the food chain of humans and other animals, as a result.

Liquid chromatography-diode array detection to study the metabolism of glufosinate in Triticum aestivum T-590 and influence of the genetic modification on its resistance.

The resistance to glufosinate of two lines-genetically modified (GM) and unmodified (T-590 and T-549, respectively)-of Triticum aestivum has been studied. In the GM line, the bar gene was introduced to increase the resistance to glufosinate. Experiments in a controlled growth chamber showed that line T-590 presented a high resistance to glufosinate with an ED50 value of 478.59 g active ingredient per hectare (g ai ha(-1)) versus 32.65 g ai ha(-1) for line T-549. The activity of glutamine synthetase (GS) in leaf extracts from both lines was investigated. The I50 for line T-590 was 694.10 µM glufosinate versus 55.46 µM for line T-549, with a resistance factor of 12.51. Metabolism studies showed a higher and faster penetration of glufosinate in line T-549 than in line T-590. LC-TOF/MS analysis of glufosinate metabolism at 48 h after herbicide treatment (300 g ai ha(-1)) revealed an 83.4% conversion of the herbicide (66.5% in N-acetyl-glufosinate metabolite), while in line T-549 conversion of the herbicide was about 40% (0% to N-acetyl-glufosinate). These results suggest that metabolism of glufosinate by the bar gene is a key mechanism of resistance in line T-590 that explains such high levels of herbicide tolerated by the plant, together with other mechanisms due to unmodified pathway, absorption and loss of glufosinate affinity for its 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.

Limited uptake, translocation and enhanced metabolic degradation contribute to glyphosate tolerance in Mucuna pruriens var. utilis plants.

Velvet bean (Mucuna pruriens, Fabaceae) plants exhibits an innate, very high resistance (i.e., tolerance) to glyphosate similar to that of plants which have acquired resistance to this herbicide as a trait. We analyzed the uptake of [(14)C]-glyphosate by leaves and its translocation to meristematic tissues, and used scanning electron micrographs to further analyze the cuticle and 3D capillary electrophoresis to investigate a putative metabolism capable of degrading the herbicide. Velvet bean exhibited limited uptake of glyphosate and impaired translocation of the compound to meristematic tissues. Also, for the first time in a higher plant, two concurrent pathways capable of degrading glyphosate to AMPA, Pi, glyoxylate, sarcosine and formaldehyde as end products were identified. Based on the results, the innate tolerance of velvet bean to glyphosate is possibly a result of the combined action of the previous three traits, namely: limited uptake, impaired translocation and enhanced degradation.

Determination of glyphosate and its metabolites in plant material by reversed-polarity CE with indirect absorptiometric detection.

A simple CE method for simultaneous determination of glyphosate and its metabolites (i.e. aminomethylphosphonic acid, glyoxylate, sarcosine and formaldehyde) in plants is reported here. A BGE of pH 7.5, 10% ACN, 7.5 mM phthalate, containing 0.75 mM hexadecyltrimethylammonium bromide as an electro-osmotic flow modifier, an applied voltage of -20 kV and absorptiometric monitoring at 220 nm were the optimal chemical and instrumental parameters. The method, with development time 20 min, shows linear calibrations within the range 5-500 microg/mL (for all target analytes) with correlation coefficients between 0.999 and 0.998. It has been validated by application to samples of Lolium spp. The electroinjection mode hinders most interferents to enter the capillary, thus providing a clean electropherogram and making unnecessary long sample-preparation steps.