Project description:Resistance to herbicides in weeds can be due to alteration(s) in the gene encoding the herbicide target site, or to herbicide degradation via a deviation in plant general metabolism. If target-site-based resistance is easy to study, the multigenic control of metabolism-based resistance renders it much more complex to study. Metabolism-based resistance to herbicides represents the major part of herbicide resistance in black-grass. Its most likely basis is an overexpression of genes encoding enzymes degrading herbicides. We thus seek to identify such overexpressed genes by comparing the transcriptomes of resistant and sensitive black-grass individuals belonging to an F2 line segregating for two resistance genes. Given there are no genomic tools developed for black-grass, this approach will use heterologous hybridisation onto a wheat Affymetrix microarray. Comparison using heterologous hybridisation onto a wheat whole-genome microarray of transcriptome of three pools of black-grass plants obtained 2h30 after herbicide spraying at field rate. The three pools correspond to: · Sensitive plants (killed by herbicide). · Moderately resistant plants (growth impaired by herbicide but plants still alive) · Resistant plants (growth unimpaired by herbicide) 6 arrays - wheat
Project description:Resistance to herbicides in weeds can be due to alteration(s) in the gene encoding the herbicide target site, or to herbicide degradation via a deviation in plant general metabolism. If target-site-based resistance is easy to study, the multigenic control of metabolism-based resistance renders it much more complex to study. Metabolism-based resistance to herbicides represents the major part of herbicide resistance in black-grass. Its most likely basis is an overexpression of genes encoding enzymes degrading herbicides. We thus seek to identify such overexpressed genes by comparing the transcriptomes of resistant and sensitive black-grass individuals belonging to an F2 line segregating for two resistance genes. Given there are no genomic tools developed for black-grass, this approach will use heterologous hybridisation onto a wheat Affymetrix microarray. Comparison using heterologous hybridisation onto a wheat whole-genome microarray of transcriptome of three pools of black-grass plants obtained 2h30 after herbicide spraying at field rate. The three pools correspond to: · Sensitive plants (killed by herbicide). · Moderately resistant plants (growth impaired by herbicide but plants still alive) · Resistant plants (growth unimpaired by herbicide)
Project description:Identification of the mechanisms that led to two different subtypes of multiple herbicide resistance in blackgrass using RNA-Seq data.
Project description:Alopecurus myosuroides Huds. is an important pinoxaden-resistant grass weed in many countries of Europe. Recently, the low efficacy of pinoxaden was reported in winter cereals in Croatia, but a preliminary dose-response trial showed no herbicide resistance for the investigated weed population. Therefore, a two-year experiment was conducted under greenhouse conditions to determine the efficacy of various pinoxaden doses (20, 40 and 80 g a.i. ha-1) on weed visual injuries and biomass reduction after herbicide application at different growth stages. As expected, the maximum weed biomass reduction (97.3%) was achieved by applying the highest dose (80 g a.i. ha-1) at the earliest growth stage (ZCK 12-14). A pinoxaden dose of 20 g a.i. ha-1 resulted in satisfactory weed biomass reduction (88.9%) only when applied at ZCK 12-14. The recommended dose (40 g a.i. ha-1) also provided sufficient weed control up to the growth stage ZCK 21-25. Slightly delayed (ZCK 31-32) application of the recommended dose brought about a low weed biomass reduction (60.1%). Double than the recommended dose also failed to provide satisfactory weed control at the advanced weed growth stages (ZCK 31-32 and ZCK 37-39). Thus, reported low efficacy of pinoxaden is most likely because of delayed herbicide application when A. myosuroides is overgrown.
Project description:The distribution of Alopecurus myosuroides (black-grass) in fields is patchy. The locations of these patches can be influenced by the environment. This presents an opportunity for precision management through patch spraying. We surveyed five fields on various types of soil using a nested sampling design and recorded both A. myosuroides seedlings in autumn and seed heads in summer. We also measured soil properties at those sampling locations. We found that the patches of seed heads within a field were smaller than the seedling patches, suggesting that techniques for patch spraying based on maps of heads in the previous season could be inherently risky. We also found that the location of A. myosuroides patches within fields can be predicted through their relationship with environmental properties and that these relations are consistent across fields on different soil types. This improved understanding of the relations between soil properties and A. myosuroides seedlings could allow farmers to use pre-existing or suitably supplemented soil maps already in use for the precision application of fertilisers as a starting point in the creation of herbicide application maps.
Project description:Enhanced detoxification is a prominent mechanism protecting plants from toxic xenobiotics and endows resistance to diverse herbicide chemistries in grass weeds such as blackgrass (Alopecurus myosuroides). The roles of enzyme families which impart enhanced metabolic resistance (EMR) to herbicides through hydroxylation (phase 1 metabolism) and/or conjugation with glutathione or sugars (phase 2) have been well established. However, the functional importance of herbicide metabolite compartmentalisation into the vacuole as promoted by active transport (phase 3), has received little attention as an EMR mechanism. ATP-binding cassette (ABC) transporters are known to be important in drug detoxification in fungi and mammals. In this study, we identified three distinct C-class ABCCs transporters namely AmABCC1, AmABCC2 and AmABCC3 in populations of blackgrass exhibiting EMR and resistance to multiple herbicides. Uptake studies with monochlorobimane in root cells, showed that the EMR blackgrass had an enhanced capacity to compartmentalize fluorescent glutathione-bimane conjugated metabolites in an energy-dependent manner. Subcellular localisation analysis using transient expression of GFP-tagged AmABCC2 assays in Nicotiana demonstrated that the transporter was a membrane bound protein associated with the tonoplast. At the transcript level, as compared with herbicide sensitive plants, AmABCC1 and AmABCC2 were positively correlated with EMR in herbicide resistant blackgrass being co-expressed with AmGSTU2a, a glutathione transferase (GST) involved in herbicide detoxification linked to resistance. As the glutathione conjugates generated by GSTs are classic ligands for ABC proteins, this co-expression suggested AmGSTU2a and the two ABCC transporters delivered the coupled rapid phase 2/3 detoxification observed in EMR. A role for the transporters in resistance was further confirmed in transgenic yeast by demonstrating that the expression of either AmABCC1 or AmABCC2, promoted enhanced tolerance to the sulfonylurea herbicide, mesosulfuron-methyl. Our results link the expression of ABCC transporters to enhanced metabolic resistance in blackgrass through their ability to transport herbicides, and their metabolites, into the vacuole.