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whole plant and seed bioassays for resistance confirmation weed science

Place plastic dishes in a germination cabinet for about a week with light and temperature conditions depending on the optimum conditions for each weed species. For most winter species, the temperature range is 15/25 °C night/day and 12 hr photoperiod with neon tubes providing a Photosynthetic Photon Flux Density (PPFD) of 15-30 µmol m -2 sec -1 . For many summer species, the temperature range is 15/30 °C night/day. Variant: Some species, such as S. halepense, need a heat treatment. Therefore after the scarification, seeds of S. halepense are subjected to the following conditions: cycles of 4 hr at 45 °C and 20 hr at 24 °C for three days in the germination cabinet, and then three days in normal conditions.

Soak e.g., Echinochloa spp. or Sorghum halepense seeds for 20 min or 5 min, respectively, in concentrated sulfuric acid.

3. Seed Germination

Vernalization NOTE: To obtain simultaneous germination and seedling emergence, a period of seed vernalization ranging from a few days to a week is required to remove physiological dormancy from many species: e.g., Amaranthus retroflexus, Chenopodium album, Lolium spp., Avena fatua, Polygonum persicaria, Phalaris paradoxa 17-19 . A longer period of up to 15 days is required for Papaver rhoeas, Cyperus difformis and Ammania coccinea and up to 30 days for Schoenoplectus mucronatus 20 .

Treatments with pre-emergence herbicides:

Through a barcode reader, which automatically identifies each tray, record the number of plants that survived the treatment as well as the Visual Estimated Biomass (VEB). Plants are assessed as being dead if they show no active growth regardless of color or other appearance.

biorxiv;2021.04.08.438914v1/FIG2 F2 fig2 Figure 2.

Glyphosate (1 mM in water) was derivatised with FMOC according to Ibáñez et al. (2005) and 10 μL injections were separated at a flow rate of 1 mL min −1 on the same C18 column as above. Column temperature was held at 35°C and samples in the autosampler kept at 15°C. The mobile phase was a gradient of ammonium acetate (pH 5.02) and acetonitrile adapted from Ibáñez et al. (2005) , with initial conditions of 10% acetonitrile followed by immediate change to 35% acetonitrile after 0.1 min, then held at 35% for 10 min before immediate change back to 10% for column equilibration prior to the next injection. Derivatised glyphosate was detected with a 996 photodiode array detector (Waters) at 265 nm. Under these conditions, the retention time for glyphosate was 7.3 min. Positive identification of derivatised glyphosate was made by comparing standard retention time and PDA peak spectral analyses across 210 – 350 nm.

15%) loss of trifluralin and clethodim was observed (Supplementary Table S4). When diluted in agar, clethodim remained stable when stored in the dark at room temperature, but 50 – 70% was lost between 5 and 10 d in the other two treatments ( Figure 4 ). Glyphosate remained stable when stored in the dark at either room temperature or 4°C, but the concentration in plates stored under ambient light and temperature decreased by around 10% over the 10 d ( Figure 4 ). There was no significant change in pyroxasulfone concentration over time under any conditions. In contrast, there was an almost complete loss of trifluralin in the first 5 d in plates stored at room temperature in the light or dark, but a much smaller decrease (around 25%) in the samples stored at 4°C ( Figure 4 ).

Populations of known resistance status used to optimize the agar test.

Correlation coefficients (r) of herbicide survival response obtained in pot-based versus agar-based studies by screening 100 populations of annual ryegrass. ns , not significant; **, P < 0.01; ***, P < 0.001.

2.5 Investigation of interference from seed dormancy

The effect of dark stratification in the presence of herbicides was assessed using population S ( Table 1 ) as a susceptible, non-dormant control; the very-dormant population (VD4) as a susceptible, dormant control; and freshly-harvested (not after-ripened) samples from five populations with known resistance to clethodim (population CR), glyphosate (GR), pyroxasulfone (PR) or trifluralin (TR) ( Table 1 ). Seeds (10 per treatment per population) were imbibed on agar as previously described and placed under environmental conditions of 25/15°C in a growth cabinet or on the laboratory window sill for 42 d, or first dark-stratified for 21 d (dishes wrapped in foil and placed in a controlled 20°C room or on the laboratory window sill) before being transferred to germination conditions for a further 21 d. The agar contained herbicide during stratification and germination, or during germination only, as indicated in the experimental scheme presented in Figure 3A . Sub-lethal herbicide treatments were included because of the longer incubation time of the seeds with the herbicides. Germination was recorded every 7 d. For the purposes of this experiment, which assessed herbicide resistance as well as seed germination, a coleoptile length of ≥ 4 cm was required for a seed to be counted as germinated. There were three replicates of each treatment. The experiment was repeated once.