Protocols for Robust Herbicide Resistance Testing in Different Weed Species

The overall goal of this procedure is to test and confirm herbicide resistance in a wide range of weed species and herbicides, as well as to infer the herbicide resistance levels. This is accomplished by first breaking the dormancy of seeds collected from plants that had survived an herbicide treatment to obtain simultaneous germination and seedling emergence. A period of seed vernalization raging from a few days to more than a week is required.

The second step is to pour an auger medium with potassium nitrate into plastic dishes. The Vern seeds are then placed on the solidified auger and the dishes are transferred into a germination cabinet with the optimum conditions for each weed species. Next 15 to 20 seedlings are transplanted into plastic trays filled with a standard potting mix.

A barcode is assigned to each tray, including all information for its unique identification, and the trays are placed in a heated greenhouse until seedlings reach the two to three leaf stage. The final step is the herbicide treatment using a precision bench sprayer, all herbicides are applied as commercial formulations with recommended surfactants at two doses recommended field dose, and three times that three to four weeks after the treatment, the final assessment is done. A barcode reader is used to record the number of survived plants and the visual estimated biomass per experimental unit.

Data are then downloaded into a computer and analyzed to determine the herbicide resistance status. The main advantage of this method over either in vivo or in vitro diagnostic screening tests is related to its robustness and flexibility. In other words, the method is reliable and can be easily adapted to a wide range of species through minor adaptations.

This method helps answer key questions related to the study and management of herbicide resistant weed populations in CRO fields, such as the pattern and level of resistance. It also allows further experimental steps such as those response experiments to be properly planned. Generally researchers new to herbicide resistant testing will struggle because of the many steps and aspects that need to be considered and adapted to the specific experimental conditions.

Begin by collecting a seed sample from one species at a time and assigning a unique code. Clean the seeds by removing the chaff and de hauling the seeds. 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 herbicides used during the season and historical records of the field. Store the seeds in unsealed paper bags labeled with the unique code assigned. Allow moisture to evaporate, but do not expose the seeds to high temperature or to extreme temperature fluctuations.

To avoid induction of secondary dormancy. Store the cleaned seeds at four degrees Celsius in a dark room to allow for vernalization. Put some deionized water in plastic dishes.

Cut two discs and a strip of filter paper, which through capillary will form a bridge between the water and the two discs. Soak the filter paper in water and place them in the plastic dishes. Then place the air dried seeds on the paper.

Transfer the plastic dishes to four degrees Celsius for the required period of time, depending on the species. Next, prepare a solution of auger at 0.6%containing 0.1%potassium nitrate using deionized water, dissolve the auger in a microwave oven and pour the auger solution into plastic dishes. Once the substrate cools, place the Vern seeds to be germinated onto the solidified auger.

Place the plastic dishes in a germination cabinet duration of vernalization, as well as light and temperature conditions vary depending on the weed species. Transplanting instead of direct sewing allows a uniform stand of plants at the same growth stage to be obtained, which is an important precondition to optimize the performance of the herbicide treatment Transplant 15 to 20 seedlings into plastic trays filled with a standard potting mix of 60%silty loam, soil, 15%sand, 15%agri perlite, and 10%peat. Identify each tray with a barcode, including all information for the unique identification population code herbicide being tested, replicate number and progressive tray number.

Place the trays in a heated greenhouse and water the plants as needed to maintain the substrate at or near field capacity. The growth temperature varies depending on the weed species for post emergence herbicides. Spray plants, when they reach the two to three leaf stage, prepare the surfactant solution in bulk according to the label instructions, the final concentration is usually expressed as a percentage of the final volume or is the volume to be distributed per unit area?

Calculate the quantity of commercial product to be dissolved in the surfactant solution. Using this equation, herbicide dose is calculated by multiplying the recommended field dose by the maximum tested dose and the final volume of the solution divided by the volume delivered by the bench sprayer. Prepare the most concentrated herbicide solution First, dilute the herbicide solution three times to prepare the less concentrated solution.

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.

Start the sequence of treatment with a lower herbicide dose. In this way, there is no need to wash the spraying cabinet between two treatments with the same herbicide. Distribute the herbicide solution using a precision bench sprayer, delivering 300 liters per hectare at a pressure of 215 kilo pascals and a speed of 0.75 meters per second with a boom equipped with three flat fan hydraulic nozzles.

Wash the spraying cabinet twice when the herbicide is changed, using 1%bleach and then rinse. Make the assessment three or four weeks after treatment depending on the herbicides tested record, the number of plants that survive the treatment as well as the visual estimated biomass or VEB using a barcode reader, which automatically identifies each tray. Plants are assessed as being dead if they show no active growth regardless of color or other appearance.

The VEB is obtained through a visual comparison of plant biomass between treated and a not treated check of the same population. A score ranging from 10 for plants not affected by the herbicide to zero when the plants are clearly dead is given to each treated tray. Evaluate the general treatment efficacy by including a susceptible population in all experiments, such as a population collected in a site which was never or seldom treated with herbicides.

Express plant survival as percentage of the number of treated plants counted just before the herbicide treatment and calculate the standard error. Perme value ascribe populations to four categories based on the results obtained from treatments with two herbicide doses susceptible when less than 5%of plants survive the herbicide dose. One x slightly resistant when survivors ranged from 5%to 20%at herbicide dose.

One x resistant when more than 20%of plants survive the herbicide dose. One x and highly resistant when the survivors are more than 20%at herbicide dose one x and more than 10%at herbicide dose. Three x representative results of an herbicide treatment with panolam on four iopa populations are shown.

The susceptible check was completely controlled while population 1410 was categorized as slightly resistant because less than 20%of the plants survived. Population 1411 was controlled at dose three x, but plant survival at dose one x resulted as higher than 20%and therefore it was ascribed to our status. Finally, population 1412 was not controlled at either recommended field dose one x or three times that so it was assigned to the highly resistant category.

A second example on visual estimation biomass in pop of or rehas plants treated with trien Ron methyl is shown here. A score of 10 is assigned to the not treated control, whereas trays with plants completely controlled have a VEB of zero. An intermediate score of five is assigned to trays.

Having a total plant biomass, which is half of the not treated plants trays with plants having a biomass comparable to the not treated control where plants were not affected by herbicide treatments and therefore resulted as highly resistant have a VEB of 10. While attempting this procedure, it is important to remember that the quality of weed seeds, which should have been collected when mature and then restored, has a strong impact on the success of the experiments Following this procedure. Other methods, like those response byas can be performed in order to precisely answer additional questions related to the level of resistance.

After watching this video, you should have a good understanding of how to approach herbicide resistant testing and how to adapt different steps to different weed species. In particular, seed CY braking and seed germination. Don't forget that working with herbicides or concentrated acids can be extremely hazardous in precautions such as wearing masks, gloves, and other personal protective gear should always be taken while performing this procedure.