North Dakota farmers and agriculture professionals report new cases of weeds overcoming herbicides each year. As herbicide resistance (HR) continues to spread, identifying where and what HR traits are lurking in local weed populations can inform control strategies. Genetic testing is a newer method to rapidly screen for HR in field-collected weed samples, which reduces the demands on greenhouse trials that are often limited by space, time, and funding. Last year, our collaborative project used both greenhouse trials and genetic testing to investigate HR in pigweeds collected from 16 counties in North Dakota. We confirmed widespread resistance to imazamox (Group 2) and glyphosate (Group 9), but a patchy distribution of resistant weed populations to fomesafen (Group 14). Importantly, genetic testing for markers associated with resistance to glyphosate and fomesafen strongly agreed with the greenhouse work in both waterhemp and Palmer amaranth. Here, we propose to expand this survey by 1) piloting a farmer- and agronomist-driven sampling program, and 2) validating genetic tests in kochia to include in future surveys. This new program answers the call for increased awareness to the growing HR crisis and offers new diagnostic technologies for farmers to identify the HR potential of weeds in their fields.

1. Increase genetic screening for herbicide resistance in pigweeds by distributing pre-labeled envelopes and cover testing fees.

2. Generate distribution map showing counties where genetic tests indicate possible pigweed resistance to specific groups of herbicides.

3. Develop genetic screening tests for herbicide resistance in kochia to add to the sampling program.

1. North Dakota submitters will receive electronic, personalized reports with the HR results from pigweed samples they collected.

2. County-level maps showing where HR genotypes were located.

3. Up to three genetic tests for HR screening in kochia for future use in the program.

Updated January 5, 2024:
Project Title: Sampling Program to Screen for Herbicide Resistance in ND Weeds
Principal Investigator: Zack Bateson (NAGC)
Co-Investigators: Joseph Ikley (NDSU), Quincy Law (NDSU), Brian Jenks (NDSU), Michael Christoffers (NDSU), Missy Berry (NAGC), and Megan O’Neil (NAGC)

Research Overview:
Herbicides have been essential in weed control for the last 70 years. However, widespread use has allowed many weed species to evolve resistance to multiple classes of herbicides. The rapid spread of herbicide resistance (HR) has motivated researchers to find mutations in the target sites associated with resistance. As target-site mutations become known, rapid DNA-based tests can be created to search for these mutations in other weed populations and species. This project makes DNA-based tests accessible to soybean growers to screen for HR pigweeds and kochia across North Dakota. The National Agricultural Genotyping Center (NAGC) has partnered with weed scientists from North Dakota State University to launch a new statewide sampling program to evaluate the distribution of specific mutations that result in resistance to Group 2, 9, and 14 herbicides in pigweeds. The project also provided initial funds to validate new screening tests for HR in kochia. We have distributed mail-in kits across North Dakota for participants to collect and ship leaves for DNA testing at NAGC. Confidential results are provided to participants that are useful for discussions for in-season and future weed control strategies. Lastly, compiled results are used to create a county-level map that highlights herbicide resistance issues across the state.
Objectives:
1. Increase genetic screening for herbicide resistance in pigweeds by distributing pre-labeled envelopes and cover testing fees.
2. Generate distribution map showing counties where genetic tests indicate possible pigweed resistance to specific groups of herbicides.
3. Develop genetic screening tests for herbicide resistance in kochia to add to the sampling program.

Completed Work: Deliverables and/or Milestones:
In August and September, we distributed 400 collection kits across North Dakota (Objective 1). Kits were shipped primarily to county extension offices for pickup, but several agronomists and farmers received kits directly. Each sampling kit consisted of one large pre-paid envelope containing four smaller envelopes to collect leaf samples from three pigweeds and one kochia. All participants have received HR results (Groups 2, 9, and 14) for pigweed samples submitted to NAGC. The kochia samples submitted remain in storage until DNA tests have been validated.
Progress of Work and Results to Date:
Submitted Samples - In the first three months of the sampling program, 34 collection kits have been returned to NAGC. In total, NAGC has received 61 pigweed and 50 kochia samples from 24 counties in North Dakota (Fig.1).
The pigweed samples originated from 17 counties and were tested at three DNA markers linked to resistance to ALS-inhibitors (Group 2), EPSPS-inhibitors (Group 9), and PPO-inhibitors (Group 14). We found 59% (10/17) of the surveyed counties had pigweed samples with genetic markers associated with HR (Fig. 2; Objective 2).

Kochia HR tests – We have ordered the necessary diagnostic reagents to explore DNA-based tests for known mutations associated with resistance to Groups 2, 4, 9, and 14 herbicides. Most of the work to date has focused on a test to detect resistance to PPO-inhibitors (Group 14). So far, we have a series of positive controls from North Dakota kochia populations with known resistance to PPO-inhibitors as well as a very promising assay to detect two distinct mutations recently discovered.
Work to be Completed:
Collection kits – We will create 100 - 200 more collection kits prior to spring planting season (Objective 1). These kits will be distributed to county extension offices and given to anyone who may request kits during the winter meetings and conventions.
Reporting and Maps – We will test and report HR results on pigweed samples that are shipped to NAGC by mid-June 2024 (Objective 2). Reports are usually released in less than 10 days from arrival to NAGC. An updated county-level map will be created in June that contains results from new submissions.
Kochia HR Test – We will continue to develop and validate new HR tests in kochia (Objective 3). Currently, we are working to obtain known positive controls for kochia resistant to Groups 2, 4, and 9 herbicides from researchers that have previously published their HR tests for kochia. Once positive controls are found and assays are validated, we will provide HR results to those that submitted kochia to NAGC.
Other relevant information: potential barriers to achieving objectives, risk mitigation strategies, or breakthroughs: The success and future growth of the sampling program is contingent on Ag professionals’ willingness to collect leaf samples across the state. While we have had relatively few kits returned (8.5% return rate), we anticipate that collection will be at its greatest during the second half of the project period. HR screening results are more useful early in the planting season when shifts in weed control strategies may still be possible. Thus, we plan to continue advertising the sampling program to reach our quota of free tests to North Dakota soybean growers.
Summary: Weed populations are evolving resistance to an increasing number of herbicides. There is an HR crisis and new surveillance tools are needed to describe the distribution of HR weed populations across the landscape. NAGC and NDSU scientists have organized a sampling program to detect potential HR weeds using DNA-based tests. So far, we have released 24 individual reports to participants that collected a total of 61 pigweeds. The preliminary maps provide a broader view of where possible HR hotspots occur in the state. As the sampling program continues, we hope results will help growers develop more effective weed control strategies.

View uploaded report

Updated June 28, 2024:
Research Overview and Objectives: background information and research gaps

Herbicides have helped farmers control weeds for more than 70 years. However, their widespread use has provided strong selection pressures for an unintentional, yet predictable outcome: the rise of herbicide resistant weeds. To date, U.S. weed scientists have reported 132 unique herbicide resistance (HR) cases, the highest recorded by any country1. The impacts of complete HR on the future of agriculture have been quantified in studies with uncontrolled weed infestations. Even with conservative estimates, the results have been alarming. For example, waterhemp can reduce yields by 40% to 70% in corn and soybean fields, respectively2,3. Other weeds, such as kochia, can reduce yields in multiple crops by 50% or more with near complete crop failures reported under extreme cases4. Yield losses begin early in the growing season, where irreversible damage caused by weed interference can start at the first trifoliate stage in soybeans and V6 stage in corn3,5.

An important step in combating HR weeds is identifying where they exist across the landscape. Healthy, late-season weeds are primary suspects of HR, but their survival alone does not confirm HR because there are other explanations for their persistence after herbicide application. For example, susceptible plants may survive if there were unfavorable weather conditions during application or weed species (e.g., waterhemp) with delayed emergence times allow them to escape herbicide control. New suspected cases of HR are confirmed by collecting seed and performing controlled herbicide trials in the greenhouse. These trials can take up to two months to perform and are often limited by space availability within the greenhouse. As a result, many researchers are forced to be highly selective on what populations to test, which makes it challenging for both researchers and farmers to better understand the HR distribution and impacts at the regional and local scales.

To help identify HR populations, new methods of genetic testing provide an indirect but rapid and convenient method to detect known mechanisms for HR weeds. Studies in waterhemp and Palmer amaranth have found genetic markers associated with HR to multiple site-of-action groups, including Groups 2, 4, 5, 9, and 14 herbicides6. Similar genetic studies have screened kochia and found genetic markers for resistance associated to Groups 2, 4, and 9 herbicides7. More recently, a new genetic marker was discovered for Group 14 resistance in kochia. All these markers are associated with a target-site mechanism, the specific way in which the active ingredient of herbicides inhibit plant growth. Most herbicides disrupt critical biosynthesis pathways within plants. The genetic tests detect specific mutations or variants at known target sites that allow certain weed species to escape the herbicide action. Previous surveys in other U.S. states have used genetic tests to evaluate the prevalence of HR genetics in pigweed populations, which indicated widespread but patchy distribution of HR populations. We observed a similar finding in our preliminary genetic survey during the 2021 growing season in North Dakota.

There are multiple benefits of incorporating genetic markers to assist with HR weed identification and surveillance. First, rapid genetic tests help to reduce the demands for conducting multiple herbicide trials in the greenhouse. For instance, multiple resistances can be determined with just a single leaf sample without the need for growing many individuals from a population for multiple herbicide trials. Second, genetic testing can be performed at any life stage of the weed, essentially offering year-round testing of either vegetative plant tissue or seed. Lastly, genetic tests provide near real-time results that can be used in management discussions on subsequent herbicide applications during the active growing season.

Few laboratories have the capabilities or desire to provide genetic tests for HR as a service. Those that can are plant diagnostic laboratories associated with universities outside of North Dakota. Through a multi-year collaboration and multi-commodity sponsorship, the National Agricultural Genotyping Center (NAGC) and weed scientists at NDSU aim to increase the availability of HR genetic testing services for farmers and other Ag professionals. After preliminary studies comparing genetic testing results and NDSU greenhouse trials, NAGC now provides the agricultural community additional genetic tools to aid in control strategies for specific HR traits in pigweed and kochia. Our collaborative project continues to build one of the first, statewide sampling programs that uses genetic screening of leaf samples to 1) provide near real-time explanations for herbicide failures, 2) create statewide maps to inform farmers on the HR genetics found within their county, and 3) determine the distribution of the recently discovered resistance to Group 14 herbicides in kochia.

Measurable objectives
1. Increase genetic screening for herbicide resistance in pigweeds by distributing pre-labeled envelopes and cover testing fees.
2. Generate distribution map showing counties where genetic tests indicate possible pigweed resistance to specific groups of herbicides.
3. Develop genetic screening tests for herbicide resistance in kochia to add to the sampling program.

Materials and Methods

Collection kits - This project distributed collection kits for ND-residents to submit leaf samples to NAGC for genetic testing of known HR markers in pigweeds and kochia. Briefly, the collection kits consisted of one large pre-paid mailing envelope containing four smaller envelopes for pigweed or kochia leaf samples, a submission form, and collection instructions. Once samples arrived at NAGC, they were logged into our laboratory information management system (LIMS) for internal traceability and automated reporting to submitters. All samples were provided with a unique NAGC ID that was linked back to the submitter’s sample label.

DNA Extraction & Testing - Samples were processed in batches with each sub-sample consisting of an approximately 6mm2 piece of leaf added to a 2mL tube containing one glass bead. After homogenizing the sub-sample using the Qiagen Powerlyzer, DNA extractions were performed using NAGC’s validated protocols on a KingFisher Flex Purification System (ThermoFisher Scientific). Purified DNA extracts were subjected to the appropriate series of genetic tests, which depended on weed species. All genetic markers were identified from previous studies, except for PPO-inhibitor resistance (Group 14) in kochia, where a novel test was created and validated at NAGC. We used quantitative PCR using probe-based chemistry to detect target-site variants associated with each herbicide resistance. For most published markers, the PCR tests were modified from their original protocols for better resolution on NAGC’s QuantStudio7 Flex Real-Time PCR System (Applied Biosystems).

For Group 2 and Group 14 resistances, results were based on the presence of specific nucleotide changes in the genome (and subsequent amino acid substitutions) that allow weeds to escape the full effects of herbicides. For Group 9 resistance, pigweeds and kochia may have elevated copies of the EPSPS gene, which allows them to produce excessive quantities of EPSPS proteins to escape the major effects of glyphosate. In all cases, there are nontarget-site and alternative target-site mechanisms that may lead to resistance, so reporting was based on whether a very specific target-site was present or absent in each leaf sample.

Reporting – Following genetic testing, each submitter was provided a personalized report containing HR genetic results from their samples. At the start of the program, NAGC provided a personalized report containing HR results for pigweed samples with an approximate 7-day turnaround. For kochia, the genetic tests had their validations finalized in April 2024, allowing NAGC to release results for leaves collected in Fall 2023. Additionally, new submissions of kochia during Spring 2024 had results released with a 7-day turnaround after arrival to NAGC. Results were provided in a table that stated whether samples were either susceptible or resistant as indicated by the specific target site. All reports were compliant with ISO/IEC 17025:2017 standards and were only released via email to the individual that submitted the sample or others that were included on the submission form.

Mapping – At the end of the first year of genetic testing (August 2023 to June 2024), we removed submitter and exact location data to create a statewide maps to show county-level participation as well as prevalence of each HR genetic marker across North Dakota. All test summaries and maps were created using R programing with packages tidyverse, dyplr, tigris, viridis, maps, usmap, and ggforce.

Kochia HR Test Validations - While the focus of this report is on summarizing submitter samples, NAGC performed extensive validations on the three HR tests associated with resistance to Groups 2, 9, and 14. Validations included optimizing test reagents and conditions as well as performing sensitivity and specificity analyses. Final performance checks included samples from routine herbicide trials performed at NDSU greenhouses during winter and early spring of 2023-24. In total, NAGC’s complete evaluation of Groups 2, 9, and 14 HR tests prior to use on submitter samples consisted of 309, 435, and 1,332 total reactions, respectively. The additional validation reactions were necessary for the multi-dimensional characteristics of the Group 14 resistance marker, which requires three individual tests to obtain the final genotype. We also explored other genetic markers associated with resistance to Groups 1 and 4 herbicides, but plants demonstrating resistance in the NDSU greenhouse trials did not contain any of the known target-site mutations.

Research Results/Outcomes

Kit distribution - With the help of NDSU collaborators and multiple stakeholders, we distributed over 1000 collection kits across North Dakota, mostly to NDSU County Extension Offices. This has allowed many farmers in rural areas to participate in the sampling program.

Survey Samples – NAGC has received 59 collection kits containing a total of 200 samples, which represented weed populations from 34 counties (Fig 1). Overall, we performed 574 genetic tests to detect HR variants for Group 2, 9, and 14 herbicides. Kochia was the most common weed submitted followed by waterhemp (n = 134 and 40, respectively).

HR genetics by species – Across all weed samples, 44% (253/574) of the tests revealed HR genetics. Most cases of HR genetics were elevated EPSPS copy numbers, which confers resistance to glyphosate (Group 9) herbicides. We found near equal cases of the Group 2 and Group 14 resistances during this survey. The newly discovered Group 14 resistance marker was found in 50% (66/134) of the kochia samples tested by NAGC.

HR genetics by county – We received the most samples from Williams County (n = 59), followed by Benson (14), Barnes (12), McClean (12), Divide (11) Counties (Table 2). Most of the focus was on kochia, which represented 67% (134/200) of the total samples submitted to NAGC. For the largest group of samples from Williams County, 46% (81/177) of the kochia HR tests were positive for a resistant variant. For the next five counties with more than 10 samples, HR positives ranged from 28 – 73% of the total tests performed (Table 2). There were three counties (Traill, Mercer, and Renville) that had samples with no detected HR genetics, but these were small sample sizes (range: 2 – 3 samples/county).

When combining results from pigweed and kochia samples, we found widespread distribution of HR genetics across North Dakota (Table 1, Fig. 2). There were 11 counties where all resistant-associated genetic markers were detected (Fig 2., black counties). Across counties, we found 22 samples (2 waterhemp and 20 kochia) that had resistance-related markers to all three herbicide groups. Foster County had the only pigweed samples (i.e., waterhemp) that contained resistance markers to all three herbicide groups. The remaining counties were all kochia populations, with the northwestern counties of Williams (n = 5) and Divide (n = 4) containing the most populations with stacked resistances to all three herbicide groups.

Discussion

We found widespread herbicide resistant (HR) variants across North Dakota in the first year of this genetic sampling program. Through NDSU extension, most farmers are aware of extensive Group 2 and Group 9 resistances in waterhemp and kochia across the state. However, the patchy distribution and annual changes in weed populations can shift the prevalence of resistant individuals within the field. Genetic testing helps assess these changes in real-time as well as determine whether stacked resistances are present at the local and regional levels.

The new resistance to Group 14 herbicides in kochia has been a concerning discovery for the Ag community for the last two years. Since greenhouse trials on multiple populations is time-consuming and space limiting, NAGC quickly validated a new genetic test to assist with tracking resistant variants across the state. Thus far, the Group 14 genetic test matched the outcomes of greenhouse herbicide trials, providing a new tool for weed scientists, agronomists, and farmers to monitor this new trait. Unfortunately, our sampling program showed half of all submitted samples contained at least one allele associated with Group 14 resistance. There were 11 counties where the Group 14 resistance markers were not detected, but many counties had low sample sizes. A combination of farmer reports, greenhouse trials, and genetic testing will continue to help us understand the distribution and dispersion capabilities of these weed populations.

Conclusion/Benefits to the North Dakota Soybean Farmers and the Industry

Identifying HR populations through greenhouse and genetic testing is beneficial at multiple levels. At the county level, reporting HR populations helps agencies monitor the spread of these troublesome traits and provides data on where resources are needed for eradication. Creating a statewide distribution map increases awareness of encroaching HR populations and intensifies scouting efforts. At the farm level, knowing the exact HR traits in the field helps with decisions on herbicides to include (or avoid). Herbicide mixes are used to combat multiple weed species, but the returns diminish when local weeds have multiple resistances to herbicides in the mix. Lastly, growers rely on NDSU Extension for recommendations on effective herbicides, which are based on data from greenhouse or test plot research. NAGC’s ability to provide genetic testing for HR alleviates some of the demands for greenhouse work and further helps researchers select weed seed sources for herbicide trials when space and time are limited.

Listings of any disclosures of inventions or plant varieties

No inventions or plant varieties were created for this sampling program.

References
[1] Heap 2022. http://www.weedscience.org. (Accessed 25 June 2024).
[2] Hager et al. 2002. Weed Sci, 50: 607-610.
[3] Steckel & Sprague 2004. Weed Sci, 52: 359-364.
[4] Geddes & Shape 2022. Crop Protection, 157: 105981.
[5] Green-Tracewicz et al. 2012. Weed Sci, 60: 86-91.
[6] Tranel 2021. Pest Manag Sci, 77: 43-54.
[7] Gaines et al. 2020. J Biol Chem, 295: 10307-10330.

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View uploaded report 2

Why the research is important to ND Soybean Farmers
Herbicides have been essential for weed control for more than 70 years. However, the rise of herbicide resistant pigweed and kochia populations has made herbicide selection more complicated. An important step towards managing herbicide resistant (HR) weeds is identifying where they exist across the landscape. To help screen for HR populations, genetic tests provide an indirect but rapid method to detect known mechanisms for resistance. Our collaborative project is one of the first, statewide sampling programs that distributes collection kits for genetic testing. The goals are to: 1) provide near real-time explanations for herbicide failures, 2) create statewide maps to show where HR populations exist, and 3) survey for the newly described Group 14 herbicide resistance in kochia.

Research Conducted
We distributed 1000 collection kits with the help of NDSU Extension collaborators and multiple stakeholders across North Dakota. Each kit allowed submitters in North Dakota to send four pigweed and/or kochia leaf samples and test for genetic markers associated with resistance to Groups 2, 9, and 14 herbicides. NAGC provided a personalized report containing genetic results for kochia and pigweeds with an approximate 7-day turnaround time during the 2024 field season. Submitter information and exact sample locations were removed to create a county-level map that showed the distribution of HR genetics in North Dakota weed populations.

Findings of the Research
Since the release of collection kits in August 2023, we have received 200 leaf samples from 34 counties (Fig.1). Most were kochia samples, which represented 67% (134/200) of the total samples collected. From these samples, we performed 574 genetic tests and 44% (253/574) of the tests revealed HR variants (Fig. 2). Most cases of HR variants in pigweeds and kochia had elevated EPSPS copy numbers (108 samples), which confers resistance to glyphosate (Group 9). We also found near equal cases of the Group 2 and Group 14 resistant variants (72 and 73 samples, respectively). The newly described Group 14 resistance marker was found in half (66/134) of all kochia samples tested by NAGC. We found 11% (22/200) of samples had genetic markers associated with stacked resistance to all three herbicide groups.

Benefits and Recommendations to North Dakota Soybean Farmers and the Industry
There were widespread herbicide resistant variants across North Dakota. Through NDSU extension, most farmers are aware of extensive Group 2 and Group 9 resistances in waterhemp and kochia across the state. However, the patchy distribution and annual changes in weed populations or introductions can shift the prevalence of resistance individuals within fields. Genetic testing helps assess these changes in real-time as well as determine whether stacked resistances are present at the local and regional levels.

Our proposed weed sampling program will be the first in North Dakota where collection is farmer-driven and supported by multiple commodity groups. The annual program will provide farmers a unique opportunity to test both existing and newly discovered populations in their fields. Collection can be performed at any time during the growing season, including before or after the timing of herbicide applications. Genetic results will help participating farmers improve their current herbicide practices. For instance, if particular HR traits are detected by genetic testing, then farmers can tailor herbicide mixes to provide the maximum return, potentially on a field-to-field basis. The statewide map will show county-level hotspots to share with the agriculture community, and provide clarity on where more resources, such as extension and outreach, are needed to combat HR weeds in North Dakota.