1. Monitoring After a Herbicide Application and Confirming Herbicide Resistance
Lesson 4
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Welcome to Lesson 4 of the herbicide-resistant weeds training lessons, provided by the Weed Science Society of America. In lessons 1 through 3, we covered the growing problem of herbicide-resistant weeds, including the need for better management strategies, defined several terms in herbicide use, defined herbicide resistance, and learned several factors that contribute to the evolution of herbicide resistance in the field. We now begin Lesson 4, “Scouting After a Herbicide Application and Confirming Herbicide Resistance.”
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2. Objectives By the end of this lesson, you will:
Understand the importance of scouting for herbicide-resistant weeds.
Above: Mature Palmer amaranth, a weed known to be resistant to several herbicides.
Photo: Jason Bond, Mississippi State University
Know the factors that can contribute to weeds being present after a herbicide application.
Know how to identify herbicide resistance in the field.
Lesson 4 presents several practical concepts related to herbicide resistance in the field. This lesson covers <mouse click> the importance of monitoring for herbicide-resistant weeds; <mouse click> the factors that can contribute to weeds being present in the field after a herbicide application; <mouse click> how to identify herbicide resistance in the field; <mouse click> knowledge of when to suspect and test for herbicide resistance in the field; and <mouse click> procedures for confirming herbicide resistance in the greenhouse and in the field.
Before we begin, the reader is advised that this lesson, by design, is intended to provide a description of the most commonly used practices to monitor and identify herbicide resistance in weeds. Please consult your local extension specialist for information on specific weeds and herbicides.
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Understand when to suspect and test for herbicide resistance in the field.
Know procedures for confirming herbicide resistance.

3. The Importance of Monitoring
Many factors can contribute to the presence of weeds in the right of way after a herbicide application and later in the season. Monitoring or scouting is the only way to know which weeds are present, and their patterns in treated areas can help to understand why they are present.
Changes in weed management practices based on a monitoring program help to:
Reduce weed seed production
Maximize remedial management tactics within the same growing season
Plan alternative management strategies for future applications.
Close monitoring will be helpful in documenting changes in the weed population that occur over time in response to land management practices, including the evolution of herbicide-resistant weeds.
Many factors can contribute to the presence of weeds after a herbicide application and later in the growing season. Careful monitoring or scouting is the only way to know which weeds are present, and their patterns in treated areas can help explain why they are present.
<mouse click> Monitoring will help document changes in the weed population that occur over time in response to land management practices, including the evolution of herbicide-resistant weeds.
<mouse click> Changes in weed management practices based on monitoring help to: <mouse click> reduce weed seed production, and <mouse click> maximize the effectiveness of remedial management tactics within the same growing season.
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4. Monitoring Procedure for Resistant Weeds After a Herbicide Application
Begin inspecting treated sites 21 to 28 days after each herbicide application, and continue at regular intervals.
Move across the treated area in a systematic pattern covering the area.
Observe and record:
Weed species (proper identification is important)
Spatial patterns (if present) of weeds
Weed densities
Presence of live and dead weeds
Herbicide symptoms on live weeds
Begin inspecting treated areas 21 to 28 days after each herbicide application and continue at regular intervals until the end of the season (frost for warm-season weeds; early summer for cool-season weeds). <mouse click> Monitoring should be performed in a pattern covering the entire treated area. <mouse click> While inspecting, it is important to correctly identify and record the weed species present, <mouse click> any spatial patterns of weeds across the treated site, <mouse click> weed densities, <mouse click> the presence of live and dead weeds, and <mouse click> herbicide symptoms on live weeds.
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Above: Example of a scouting pattern in a field.

5. If Weeds are Present after an Application, Determine the Reason
Consider the following factors:
1. Application history
2. Weed biology
Investigate and rule out all other factors affecting herbicide performance before suspecting herbicide resistance.
3. Environment
4. Application problems
There are several factors that can contribute to the presence of weeds after a herbicide application. The general factors include <mouse click> application history, <mouse click> weed biology, <mouse click> environment, <mouse click> application problems, <mouse click> and herbicide resistance. <mouse click> It is extremely important to investigate and rule out all other factors affecting herbicide performance before suspecting herbicide resistance. We will now discuss each of these in more detail.
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5. Herbicide resistance

6. If Weeds are Present After an Application, Determine the Reason
Right of Way History
Previous cultural, chemical, and mechanical weed management practices have all influenced the current weed community. These practices can also provide insight into the likelihood that weed populations may become herbicide-resistant. Some important information to consider includes:
The probability for herbicide-resistant weed populations to evolve is increased as diversity in weed management practices is reduced.
Keeping records on weed populations, including density and distribution, will help you to note important changes that may be underway.
Number of herbicide mechanisms of action used
Typical number of applications to that site each year
Use of mechanical and cultural weed management practices
Presence of weed species, including density and distribution
Previous cultural, chemical, and mechanical weed management practices have all influenced the current weed community. These practices can also provide insight into the likelihood that weed populations may become herbicide-resistant. Some important information to consider includes:
<mouse click> Number of herbicide mechanisms of action used across a rotation, number and kinds of applications each year, use of mechanical and cultural weed management practices, and the presence of weed species, including density and distribution, over time.
<mouse click> The probability for herbicide-resistant weed populations to evolve is increased as diversity in weed management practices is reduced. This can occur when there is a reduction in the number of herbicide mechanisms of action used with or without concurrent reductions in the use of mechanical and cultural management options.
<mouse click> Keeping records on weed populations, including their density and distribution, will help you to note important changes that may be underway in the field, and then make the necessary adjustments to future management strategies.
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7. Weed Biology Time of emergence in relation to application
If Weeds are Present After an Application, Determine the Reason
Weed Biology
Time of emergence in relation to application
Weed seed size and depth in soil
Size and age of plants at application
Weed biology can also influence the presence of weeds. As you inspect treated sites, consider if weeds may have emerged before the application of a preemergence product or after application of a postemergence foliar product. Both scenarios could mean that the weed escaped application of the herbicide.
<mouse click> Weeds from larger seeds and seeds found deeper in the soil may avoid exposure to a herbicide due to their placement below the treated layer.
<mouse click> If plants are too large and are older, or hardened off, they may not respond well to a herbicide. Weed seeds germinate over an extended period of time, which may vary from year to year, depending on the environment. Postemergence herbicide applications must be timed to allow as many weeds as possible to emerge, yet not allow those that emerged early get too large for control.
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An example of weed seeds that were below the treated layer of a soil-residual herbicide product (shown in orange)
An example of weeds that were too large for the application of a postemergence foliar product

8. Environment Soil moisture before and after application
If Weeds are Present After an Application, Determine the Reason
Environment
Soil moisture before and after application
Rain soon after application
Soil characteristics (e.g., pH, texture, organic matter)
Weeds under stress before and/or after herbicide application
Herbicide must have time to translocate within plant before senescence
Freeze soon after application
An example of a weed under stress
The environment can affect the performance of herbicides, and also the growth and development of weeds. In evaluating the presence of weeds after a herbicide application, consider the following factors:
The presence of soil moisture before and after herbicide application can affect both soil-residual and postemergence foliar herbicides. <mouse click> Plant material on the soil surface may intercept a soil-residual herbicide. <mouse click> A rain event soon after the application of a postemergence foliar herbicide may limit herbicide movement through the leaf cuticle and into the plant. <mouse click> Soil characteristics (such as pH, texture, or organic matter) can affect the performance of soil-residual herbicides. <mouse click> Systemic herbicides need adequate time to translocate within the plant to the site of action prior to senescence to work properly. Freezing temperatures soon after an application may cause the plant to stop growth processes, therefore the herbicide does not move to the site of action.
All of the above factors can limit the effectiveness of herbicides.
Weeds are sensitive to the environment. <mouse click> Weeds under environmental stress before and/or after a herbicide application may not respond well to a postemergence foliar herbicide.
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9. If Weeds are Present After an Application, Determine the Reason
Application Problems
Problems during a herbicide application can reduce the performance of herbicides and therefore, increase the presence of weeds. Problems can be related to the equipment (E), choice of herbicide (H) and related products, herbicide rate (R), and soil conditions (S).
Examples
Sprayer skips (E)
Poor sprayer calibration and/or equipment failures (E)
Herbicide rate too low (R)
Dust/soil on plant leaves at application (R)
Poor spray coverage (R)
Selection of ineffective herbicide for weed spectrum (H)
Improper choice of adjuvant (H)
Inadequate agitation of herbicide solution (E)
Soil conditions at time of application (S)
Application problems during a herbicide application can reduce the performance of herbicides and therefore, increase the presence of weeds. It is particularly important, however, to make a note of any problems with application of the herbicide, as these problems can be related to and/or confused with symptoms on herbicide-resistant weeds, especially weeds with low-level herbicide resistance.
Problems can be related to the equipment (E), choice of herbicide (H) and related products, herbicide rate (R), poorly agitated solution in spray tank (E) and soil conditions (S).
<mouse click> Use of lower-than-recommended rates has been associated with shifts to weeds that are more difficult to control, including herbicide-resistant weeds.
<mouse click> Dust or soil on plant leaves during the application of a postemergence foliar herbicide, and poor spray coverage, can both result in poor weed control by not allowing the weeds to intercept enough herbicide for control.
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10. Patterns: Observations in the Absence of Herbicide Resistance
Multiple weed species are present.
A uniform response of individuals within a population is observed.
The following are some observations that you may encounter after an application that are unlikely to be related to herbicide resistance:
<mouse click> Multiple weed species are present; <mouse click> a uniform response of individuals within a population is observed; and <mouse click> the spatial pattern of plants remaining in the treated site can be correlated with the herbicide application, such as the example of sprayer skips.
In general, when these conditions are observed, the initial conclusion is that the failure in herbicide performance is not related to herbicide resistance. There may be exceptions to this conclusion.
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The spatial pattern of plants remaining in the treated area can be correlated with the herbicide application.

11. Weed Shifts
With the repeated use of the same herbicide active ingredient, certain weed species may become dominant due to selection for those that are tolerant to that herbicide.
These populations are not herbicide-resistant.
Weed shifts due to herbicide use can be caused by:
Using a herbicide to which the species is tolerant
Using postemergence herbicides when weeds are too large or too small
Weed shifts are another situation where you may encounter the presence of weeds, but their presence is unlikely to be related to herbicide resistance. Weed shifts occur through the repeated use of the same herbicide active ingredient and where a certain species becomes dominant due to selection for species that are tolerant to that herbicide. Some herbicide labels claim “partial control” or “suppression” of certain weed species, or may claim control only of very small weeds. Repeated applications of that herbicide where these weeds exist, will cause a population shift.
These populations are not herbicide-resistant.
Weed shifts due to herbicide use can be caused by<mouse click> using a herbicide to which the species is tolerant, <mouse click> using rates that are lower than recommended, and <mouse click> using postemergence herbicides when weeds are too large or too small for effective and consistent control. Poor performance due to weed shifts can be corrected by using the appropriate herbicide product at the labeled rate and at the correct time.
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Using rates that are lower than recommended

12. Patterns: Field Observations Related to Herbicide Resistance
A single weed species is present, especially late in the season. All other weed species on the label have been controlled.
The response of individual surviving weeds can range from little or no injury to death.
When observing weeds in the right of way, there are some trends that can suggest the presence of herbicide-resistant weeds and justify further investigation:
<mouse click> A single weed species labeled for control by a herbicide remains after treatment. All other weed species on the label are controlled. This observation applies to both soil-residual and postemergence foliar herbicides.
<mouse click> The response of individual surviving weeds ranges from little or no injury to death.
<mouse click> The spatial pattern of surviving weeds is random or consists of multiple plants within a patch.
Let’s take a closer look at the responses of individual weeds and spatial patterns in the field.
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The spatial pattern of surviving weeds is random or consists of multiple plants within a patch.
Upper and lower photographs courtesy of Jason Bond, Mississippi State University

13. Response of Herbicide-Resistant Individuals
The response of individual plants is usually different between high-level and low-level resistance.
High-level Resistance: Plants are slightly injured to uninjured with few plants having intermediate herbicide responses. Susceptible plants can be present in the population.
Above: In this example of high-level resistance, the common ragweed is uninjured.
Low-level Resistance: A continuum of plant responses from slightly injured to nearly dead is observed with the majority of plants displaying an intermediate herbicide response. Susceptible plants will be present in the population, especially when resistance is determined early.
The response of individual plants within a weed population is usually different between high-level and low-level herbicide resistance, especially when observed early in the evolution of the population. <mouse click>
In high-level herbicide resistance, plants are either dead (susceptible) or uninjured (resistant) with few plants having intermediate responses. Susceptible plants can be present in the population. <mouse click>
In low-level herbicide resistance, a continuum of plant responses from slightly injured to nearly dead is observed with the majority of plants displaying an intermediate herbicide response. Susceptible plants will be present in the population, especially when resistance is determined early. The plant in the lower photograph circled in white is dead, the plant circled in yellow appears normal, but is stunted compared with an untreated plant, and the plants circled in blue have an intermediate response. Plants that are not dead will survive and may reproduce before the end of the growing season.
<mouse click> To learn more about scouting for low-level herbicide resistance, click the box with the small green speaker for an example with glyphosate-resistant common ragweed.
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To learn more about scouting for low-level resistance, click the green button.
= dead = intermediate
= slightly injured

14. Patterns of Herbicide-Resistant Weeds
Early years of herbicide resistance
Later years of herbicide resistance
When only a few plants survive a herbicide application and during the early years of herbicide resistance, consider hand-removing them and making adjustments to future weed management strategies. Waiting until numerous dense weed patches evolve during the later years of herbicide resistance can contribute to profit losses because of increased input costs.
In the early years of herbicide resistance evolution, herbicide-resistant weeds may be scattered as single plants or present in single or multiple, small patches.
<mouse click> In later years, after the herbicide-resistant weed population has grown, small to moderate, or large and dense patches of weeds may be present.
<mouse click> When only a few plants survive a herbicide application and during the early years of herbicide resistance, consider hand-removing them and making adjustments to future weed management strategies. Waiting until numerous dense weed patches evolve during the later years of herbicide resistance can contribute to increased management costs. Weed management strategies will be discussed further in Lesson 5.
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15. Check List for Proper Monitoring
Inspect treated areas 21 to 28 days after each herbicide application and near season end (spring and fall).
Identify and record the weed species present.
Determine the distribution pattern of survivors.
Correlate with application pattern or identify as random
Determine if plants present survived a previously applied herbicide or emerged after the last herbicide application.
Observe individual plant responses, especially if plants survived a herbicide application.
Review previous application history to understand what changes may be occurring.
Let’s review the recommended procedures for scouting weeds after application of a herbicide.
<mouse click> Monitor treated sites 21 to 28 days after each herbicide application and near the end of the season (spring for winter weeds or fall for summer weeds).
<mouse click> Identify and record the weed species present.
<mouse click> Determine the distribution pattern of survivor weeds in the right of way. If possible, correlate with an application pattern or identify as random.
<mouse click> Determine if the plants that are present survived a previously applied herbicide or emerged after the last herbicide application.
<mouse click> Observe individual plant responses, especially if plants survived a herbicide application.
<mouse click> Carefully review previous application history to understand what changes may be occurring.
We have now completed the first half of the lesson on monitoring for weeds after a herbicide application. Let’s begin the topic of confirming herbicide resistance.
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16. Confirming Herbicide Resistance
Two methods for confirming herbicide resistance:
Field
Greenhouse
There are two general methods for confirming herbicide resistance: in the field and in the greenhouse.
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Above: Screening for herbicide resistance in the greenhouse. Photo credit: Alan York, North Carolina State University.
Above: Highway right of way. Photo credit: John Byrd, Mississippi State University.

17. Field Confirmation (Postemergence Foliar Herbicides)
Flag In Treated Area
Flag a minimum of 5, but preferably 20, of the healthiest weeds that survived the herbicide application.
Flag Outside
Find an area outside of the treated site that was not previously sprayed and contains the same species. Flag the same number of weeds.
Spray
Spray the weeds in both areas with the same herbicide in two strips – to one strip apply the labeled “rate”; to the second strip, spray the area with the labeled “rate” twice and label “twice the labeled rate”1.
Testing in the field is an option for some managers when the issue is related to postemergence foliar herbicides. Field testing provides access to quick information compared with waiting for the results of a greenhouse test, but it may be less accurate because the weeds are larger and there is greater variability in the field.
Field testing should be conducted within 45 days after the application that the weeds survived.
<mouse click> Flag a minimum of 5, but preferably up to 20, of the healthiest weeds that survived the herbicide application.
<mouse click> Find a second area that was not previously sprayed, but contains the same weed species at approximately the same stage of growth as the weeds that survived the herbicide. You will most likely have to look outside the treated area for unsprayed weeds. Flag the same number of weeds in the unsprayed area.
<mouse click> Spray the weeds in both areas with the same herbicide in two strips – one strip should be treated with the labeled “rate” and another strip treated twice to apply “twice the labeled rate”1.
<mouse click> Evaluate all plants 10 to 14 days after the herbicide application.
If more than 20 to 40% of the re-sprayed plants in the field survived, while greater than 90% of plants died in the previously unsprayed area, then a herbicide-resistant population may be present in the field. Consider removing the surviving plants by hand and making adjustments to future weed management strategies.
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Evaluate
Evaluate all plants 10 to 14 days after herbicide application.

18. Greenhouse Confirmation
The best method for confirming herbicide resistance in a weed species with no history of resistance, in a weed species suspecting of having resistance to multiple herbicides, and when laboratory tests are not available.
Procedure:
Collect seeds from mature plants suspected of being herbicide-resistant and generate plants from these seeds in the greenhouse. Plants are sprayed with the herbicide of interest and the response of suspected population is compared to the responses of known herbicide- susceptible and -resistant populations.
Greenhouse testing is the best method to confirm herbicide resistance in a weed species with no history of resistance, in a weed species suspected resistant to multiple herbicides, and when laboratory tests are not available.
A greenhouse test involves <mouse click> collecting seed from mature weeds in the right of way suspected of being herbicide-resistant, and grow seedling plants from these seeds in the greenhouse. Plants are sprayed with the herbicide of interest, and the response of the suspected herbicide-resistant population is compared with the responses of a known herbicide-susceptible and a known herbicide-resistant population when available.
<mouse click> Contact your local extension specialist for instructions on seed collection procedures. Allow only those plants necessary for collection to flower and set seed, and destroy all other surviving plants.
For information on a best method for collecting seeds for greenhouse testing, please click the For-More-Information button. 
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Contact your local extension specialist for instructions on seed collection procedures.
Allow only those plants necessary for collection to flower and set seed and destroy all other surviving plants.
Information
For More

19. Greenhouse Confirmation
Known herbicide-susceptible population
Test herbicide-resistant population
These photos help illustrate the comparisons made between susceptible weeds and weeds that are suspected resistant to herbicides. The Palmer amaranth in the left photo is susceptible to the herbicide, while the Palmer amaranth on the right was suspected herbicide-resistant. In each case, the tray of plants to the left in each photo was not sprayed and used for an untreated control. The treated weeds in the right photograph survived the herbicide application, while the treated weeds on the left did not.
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Untreated
Treated
Untreated
Treated
Above: Screening for herbicide resistance in the greenhouse. Photo credit: Alan York, North Carolina State University.

20. Conclusions
Monitor rights of way to determine the reasons for weed survival after a herbicide application is important.
Herbicide application history, weed biology, environment, application parameters, and herbicide resistance are factors that can contribute to weeds surviving the application of a herbicide.
Symptomology may differ between the observations of low-level and high-level herbicide resistance.
This concludes the lesson on monitoring and confirming herbicide resistance. In summary, please remember that inspecting treated sites to determine the reasons for weed survival after a herbicide application is important; <mouse click> past herbicide application history, weed biology, environment, application parameters, and herbicide resistance are factors that can contribute to weeds surviving the application of a herbicide; <mouse click> symptoms may differ between the observations of low-level and high-level resistance; and <mouse click> confirming herbicide resistance early and removing herbicide-resistant weeds by hand can decrease the spread of herbicide-resistant weeds, thereby reducing the costs required to manage them.
The final training lesson is next. It covers the management of herbicide resistance. You may continue with Lesson 5 now, or save your work and return at a later time.
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Confirming herbicide resistance early, when just a few weeds are present, and removing them by hand can decrease the spread of herbicide-resistant weeds, thereby reducing the costs required to manage them.

21. Credits:
This lesson was developed by a WSSA sub-committee and reviewed by the WSSA Board of Directors and other WSSA members before being released. The sub-committee was composed of the following individuals.
Wes Everman, PhD (North Carolina State University)
Les Glasgow, PhD (Syngenta Crop Protection)
Lynn Ingegneri, PhD (Consultant)
Jill Schroeder, PhD (New Mexico State University)
David Shaw, PhD (Mississippi State University)
John Soteres, PhD (Monsanto Company) (Sub-committee chairman)
Jeff Stachler, PhD (North Dakota State University and University of Minnesota)
François Tardif, PhD (University of Guelph)
The module was adapted for noncropland applicators by John D. Byrd, Jr., Ph. D. (Mississippi State University) and William W. Witt, Ph. D. (University of Kentucky, retired).
Financial support for this was provided by Global HRAC, North America HRAC, and WSSA.
Our thanks are extended to the National Corn Growers Association for allowing us to use training materials posted on their website as the starting point for these training lessons.

22. Methods for Seed Collection
Choosing the plants:
Choose plants from areas known to have been sprayed.
Soil-residual herbicides: Areas where weeds emerged first, assuming the herbicide was adequately moved into the soil solution.
Postemergence foliar active herbicides: Areas with multiple herbicide applications.
Choose the healthiest plants, especially those with an intact main meristem. If there is no main meristem, choose plants with the greatest number of large branches.
Choose weeds from areas known to have been sprayed with the herbicide.
For soil-residual herbicides, assuming the herbicide was adequately moved into the soil solution, these are areas where weeds emerged first. For postemergence foliar active herbicides, these are generally areas with multiple herbicide applications.
Choose the healthiest weeds, especially those with an intact main meristem. If there is no main meristem, choose weeds with the greatest number of large branches.
It is also important to choose weeds where 10 to 15% of seeds have fallen from the plant or greater than 50% of fruits have matured, and seeds are firm and dark in color.
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Choose plants in which –
10 to 15% of seeds have fallen from the plant or greater than 50% of fruits have matured
Seeds are firm and dark in color

23. Methods for Seed Collection
To return
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Example
Collecting the plants:
Cut the plants below the lowest fruiting structures (90% of plant having seeds) and carefully place them in a large paper bag. Staple shut in the field to contain all seeds.
Mark the bag to clearly identify sample location, weed species, herbicide(s) and rates applied, and collection date.
Mature seed
Male flowers
Female flowers of common ragweed in leafy bracts
When collecting the plants, cut the plants immediately below the lowest fruiting structures (90% of plant having seeds) and carefully place in a large paper bag. Staple shut in the field to contain all seeds.
Mark the bags to clearly identify the sample location, weed species, herbicides and rates applied, and collection date.
Harvest a minimum of 5 to 10 plants with larger quantities preferred. Place a single plant in a single bag to allow single and multiple plant testing.
Open the bags in a building to allow the plant material to dry in the bag before shipment. Restaple the bags prior to shipment.
For a dioecious species, such as Palmer amaranth or waterhemp, collect only the female plants (plants with seeds).
Please click on the “click here to return” button at the top of the slide to continue with the lesson.
Harvest a minimum of 5 to 10 plants with larger quantities preferred. Place a single plant in a single bag.
Upon return from the field, open bags to allow plants to dry before shipment. Re-staple the bags prior to shipment.
For a dioecious species, such as Palmer amaranth or waterhemp, collect only the female plants (plants with seeds).