Protocols for Robust Herbicide Resistance Testing in Different Weed Species

1. Seed Sampling and Storage

1. Monitor cultivated fields for unjustified poor herbicide performance, i.e., not due to adverse climatic conditions or low quality herbicide treatments.
2. Collect a seed sample from one species at a time and assign a unique code. Mature seeds are usually collected before crop harvesting from plants that had survived the herbicide treatment(s). Timely monitor to observe if the seeds are shed by mother plant when mature.
3. Fill in a form for each sample indicating the assigned unique code, name of species, collection date, GPS coordinates, municipality, farmer’s name, field size, infestation level, crop, herbicide(s) used during the season and historical records of the field.
4. Collect seeds from at least 30 randomly selected plants that are representative of the field infestation. Ensure that the seed sample contains at least 5,000 mature seeds. For an obligate outcrossing weed species (e.g., Lolium spp. or Amaranthus spp.), reduce the number of plants to 10-15, keeping the total number of seeds around 5,000 ¹¹.
5. Sub-sample the field if patches of weeds are scattered over large areas (more than a hectare) as different herbicide resistant biotypes are selected.
6. Store seeds in unsealed paper bags labeled with the unique code assigned.
7. Allow moisture to evaporate but do not expose seeds to high temperature (i.e., avoid leaving them in a car under the sun) or to extreme temperature fluctuations to avoid induction of secondary dormancy.
8. Clean (remove chaff, de-hull seeds, etc.) and store them at ambient temperature in a dry room. After conducting the first resistance tests, store the seeds for long periods of time in a dark room at 4 °C, preferably in vacuum-sealed plastic bags. In this way seeds preserve their viability for a significantly longer time.

2. Seed Dormancy Breaking

NOTE: Seed dormancy provides a flexible and efficient mechanism that enables weeds to adapt and persist in agro-ecosystems. To break dormancy and allow seed germination, different protocols have to be used depending on weed species, i.e., the type of dormancy ¹⁶.
There are three main ways to remove dormancy:

1. 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 mucronatus²⁰.
   1. Put some deionized water in plastic dishes. Cut two layers of filter paper and soak them in the water, remove any excess. Place the air-dried seeds on the paper. Transfer the plastic dishes to a fridge at 4 °C for the required period of time.
2. Scarification
   NOTE: Some weed species are more recalcitrant to germination than others due to mechanical dormancy, i.e. seed coat characteristics, and necessitate the use of a chemical scarification using sulfuric acid to germinate ²¹.
   1. Prepare a beaker with concentrated sulfuric acid (95-98%). Prepare a beaker full of water. Put the seeds in an envelope of non-woven fabric.
   2. Soak e.g., Echinochloa spp. or Sorghum halepense seeds for 20 min or 5 min, respectively, in concentrated sulfuric acid.
   3. Take the envelope out of the beaker using a pair of tweezers and put it in the beaker full of water. Open the envelope, put the seeds in a small colander and rinse them thoroughly under running water.
   Variant: A. plantago-aquatica needs a different protocol ^{22, 23}
   1. Soak the seeds for 2 min in chloroform. Rinse the seeds with deionized water and dry them with absorbent paper. Dunk the seeds in 80% sulfuric acid for 5 min.
   2. Put the seeds in a small colander and rinse them thoroughly under running water.
3. Post-harvest seed maturation
   NOTE: Seeds of other weed species do not germinate at all for a few months after maturity, regardless of the method used to break dormancy.
   1. Store the seeds for a period of at least 3-4 months at RT and low humidity and then follow the above protocols for dormancy breaking (e.g., Oryza sativa var. sylvatica or P. rhoeas).

3. Seed Germination

1. Place seeds to be germinated in plastic dishes containing 0.6% (m/v) agar with 0.1% potassium nitrate (KNO₃) added:
   1. Prepare a solution of agar at 0.6% + 0.1% KNO₃ using deionized water. Dissolve the agar in a microwave oven.
   2. Pour the agar solution into plastic dishes. Cool the substrate and then put in the seeds.
   Variant: Soil can also be used as a substrate in plastic dishes. This practice is particularly efficient for Echinochloa spp., S. halepense and Lolium spp.
2. 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.

4. Seedling Transplanting and Growth

1. Transplant fifteen to twenty seedlings into plastic trays (325 x 265 x 95 mm) filled with a standard potting mix (60% silty loam soil, 15% sand, 15% agriperlite and 10% peat — by volume).
   NOTE: Transplanting, instead of direct sowing, allows a uniform stand of plants at the same growth stage to be obtained, which is an important pre-condition to optimize the performance of the herbicide treatment.
2. Identify each tray with a barcode including all information for the unique identification: population code, herbicide being tested, replicate number and progressive tray number.
3. Place trays in a heated greenhouse and water plants as needed to maintain the substrate at or near field capacity.
   NOTE: The growth temperature varies depending on the weed species. Often tests are done during autumn/winter/spring, so light is supplemented using 400 W metal-halide lamps, which provide a PPFD of about 150 µmol m^-2 sec^-1 and a 12 hr photoperiod ^{24, 19}. Summer weed species with C₄ photosynthetic cycle usually require higher light intensity and therefore tests are done in late spring-summer or the supplemented light intensity is about 400 µmol m^-2 sec^-1 with a 14 hr photoperiod.
4. Use a different protocol for some weed species infesting paddy rice, e.g., A. plantago-aquatica, S. mucronatus and C. difformis as described in ²².
   1. Transplant the seedlings into polystyrene trays with 24 round cells (55 mm diameter, 64 mm deep) filled with 60% silty loam soil, 30% sand and 10% peat (by volume).
   2. Set the trays in 12 cm deep plastic containers filled with water and battened down by screwed stainless steel rods to prevent them floating (Figure 1).
   3. Maintain water level in the containers at 1-2 cm below the level of the soil surface and add 1.5 g of copper sulfate to each container (which contains 10-12 L of water) to avoid the proliferation of algae.

5. Herbicide Treatments

1. Treatments with pre-emergence herbicides:
   1. After about three days in the germination cabinet as described in section 3, transplant the germinating seeds into plastic trays containing the substrate described above and cover with a layer of soil (about 1 cm). This is a critical step in order to ensure that seedlings will not emerge due to the herbicide effect rather than an excessive burial depth.
   2. Take the substrate to field capacity by placing the trays, which have a few holes at the bottom, on saucers filled with water.
   3. One day after transplanting, treat the trays with the pre-emergence herbicide ²⁵.
   4. Keep the substrate at or near field capacity by adding water as needed both from above and below by capillarity from the saucer. This procedure favors the permanence of the herbicide at the proper depth (i.e., where the germinating seeds are) for a good treatment efficacy.
2. Treatments with post-emergence herbicides:
   1. Spray plants when they reach the 2-3 leaf stage (i.e., growth stage 12-13 of the Extended BBCH growth scale ²⁶).
   2. Starting from the day after the treatment, set the irrigation system according to the water requirements of the weed species and the season (e.g., for Echinochloa spp. it delivers water for 3 min 4 times per day, at regular intervals from 9 am to 9 pm). Water is distributed using an automatic sprinkler irrigation system.
      Variant: glyphosate is applied at plant stage BBCH 14–21.
3. Herbicide preparation and distribution.
   NOTE: All herbicides (pre- and post-emergence) are applied as commercial formulations with recommended surfactants at two doses, recommended field dose (1x) and three times that (3x).
   1. If needed, prepare the surfactant solution in bulk according to the label instructions; the final concentration is usually expressed as a percentage of the final volume (e.g., 0.3%) or as volume to be distributed per unit area (e.g., 1 L ha^-1).
   2. Use the surfactant solution as solvent for the herbicide (solute) solution in order to keep the right concentration of the active ingredient. Prepare the most concentrated herbicide solution first (3x). Calculate the quantity of commercial product to be dissolved in the surfactant solution (or in deionized water if a surfactant is not necessary) using the following equation:
      Dose_herb = [(Dose_field x Dose_max) x V_fin] / V_del
      Where: Dose_herb = herbicide dose (ml), Dose_field = herbicide field dose (ml ha^-1), Dose_max = maximum dose delivered, V_fin = final volume of the solution (L), V_del = volume delivered by the bench sprayer (L ha^-1).
   3. Dilute (2:1, v/v) the herbicide solution 3x to prepare the less concentrated one (1x). This procedure reduces the chance of making mistakes when weighing or pipetting the herbicides. Herbicide solution concentration is expressed as volume to be distributed per unit area (L ha^-1).
   4. Start the sequence of treatment with the lower herbicide dose (1x). In this way there is no need to wash the spraying cabinet between two treatments with the same herbicide.
   5. Distribute the herbicide solution using a precision bench sprayer delivering 300 L ha^-1 (±1%), at a pressure of 215 kPa, and a speed of 0.75 m sec^-1, with a boom equipped with three flat-fan (extended range) hydraulic nozzles.
   6. Wash the spraying cabinet twice when the herbicide is changed using bleach 1% (v/v) and then rinse.
      Variant: glyphosate is applied with a spray volume of 200 L ha^{-1 27}.
      NOTE: Particular attention has to be paid when highly biological herbicides, such the sulfonylureas sulfometuron or flazasulfuron, are used. In the latter case do one washing with a bleach solution and another with ammonia (2.5% v/v), followed by a careful rinse with water.

6. Collection and Analysis of the Data

1. 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.
   1. Make the assessment three or four weeks after treatment (WAT) depending on the herbicides tested (e.g., three WAT for ACCase inhibitors and four WAT for ALS inhibitors or glyphosate).
   2. Evaluate the general treatment efficacy by including a susceptible population (check S) in all experiments, i.e., a population collected in a site which was never or seldom treated with herbicides.
   3. Express plant survival as percentage of the number of treated plants, counted just before the herbicide treatment, and calculate the standard error (S.E.) per mean value (mean value of the two replicates).
   4. The VEB is obtained through a visual comparison of plant biomass between treated and not-treated check of the same population ^{25, 28}. A score, ranging from 10 for plants not affected by the herbicide (compared with the not-treated check) to 0 when the plants are clearly dead, is given to each treated tray.
2. Ascribe populations to four categories based on the results obtained from treatments with two herbicide doses: S when less than 5% of plants survived the herbicide dose 1x, SR when survivors ranged from 5% to 20% at herbicide dose 1x, R when more than 20% of plants survived the herbicide dose 1x and RR when survivors are more than 20% at herbicide dose 1x and more than 10% at herbicide dose 3x ¹⁷.