Resistance detection using herbicide sensitivity bioassays

Seeds were stratified at 4 °C during one month to break dormancy and transferred to a growth chamber for germination (photoperiod of 16 h, 20 °C/15 °C day/night). After germination, seedlings at the cotyledon stage were transplanted into plastic trays (17 × 12.5 × 5.5 cm) filled with 60% silt-loam soil, 15% sand, 15% perlite and 10% peat by volume and placed in a greenhouse (20 °C/15 °C day/night with a 16 h photoperiod). Plants were watered as needed until the two-to-four leaf stage, when they were subjected to herbicide treatment. Each tray contained 20 seedlings. Two trays per population (40 seedlings) were sprayed per herbicide dose, and one tray per population (20 seedlings) was sprayed with water and served as the untreated control. Herbicides were applied using a custom-built, single-nozzle (nozzle AXI 110°06; Albuz, France) sprayer delivering herbicide in 300 L ha⁻¹ water at 400 kPa under a temperature of 23 °C and a relative humidity of 80%. The two major ALS inhibitors used to control common ragweed in France were sprayed as commercial formulations at their respective, maximum recommended French field rate. The commercial herbicides used were Express SX^® (FMC, 50% w/w tribenuron, field rate of 30 g tribenuron ha⁻¹) with 1 L ha⁻¹ surfactant (Actirob B^®, Bayer CropScience), and Pulsar 40^® (BASF, 40 g L⁻¹ imazamox, field rate of 50 g imazamox ha⁻¹) with 1.25 L ha⁻¹ surfactant (Dash^®, BASF).

Every spraying experiment included one batch of 20 plants from the reference population P08 (100% plants sensitive to any herbicide used to control ragweed and applied at the recommended French field rate) for each of the herbicides and herbicide rates assayed, as a check for herbicide application efficiency. Phenotype rating based on visual injury was performed four weeks after treatment, when all plants from the reference population were clearly dead and plant phenotype rating could be performed without ambiguity. Preliminary experiments demonstrated that plants with visually estimated biomass ranging from unaffected by herbicide application (identical to the untreated control plants, 100%) down to ≥ 50% of that of the untreated control plants ultimately recovered and were able to produce viable seeds. They were thus considered resistant. Plants injured and with a visually estimated biomass < 50% of that of the untreated control plants ultimately died. These plants and the plants dead at the time of rating were thus considered sensitive. After phenotype rating, one leaf fragment was collected on plants rated resistant and stored at − 20 °C prior to ALS gene Sanger sequencing.