Pigweeds are considered some of the most destructive and resilient weeds for soybean growers. Over the last decade, 33 states have reported glyphosate-resistant populations of Palmer amaranth and waterhemp with nearly half of those states also reporting plants with resistance to multiple herbicides (Heap 2019). Infestation of Palmer amaranth is a recent discovery in North Dakota, and its known distribution has increased following visual surveys and NAGC’s genetic testing during the 2019 growing season. To best slow the spread of Palmer amaranth, detection of populations must occur at the earliest stages of infestation. This is especially critical when herbicide-resistant plants are present. Whether newly discovered Palmer amaranth populations are herbicide resistant can be difficult to assess in the field because emergence can extend outside the herbicide application schedule and weeds can persist outside the margins of a field. Further challenges arise due to the difficulty of distinguishing Palmer amaranth shortly after emergence when herbicide treatments are most effective. Here, we propose to extend NAGC’s pigweed identification panel with the addition of two genetic tests that are linked to mechanisms of resistance to glyphosate and PPO-inhibitor herbicides. These new tests will help inform growers, advisors, and weed scientists whether specific herbicides will likely be effective prior to spraying so that the most appropriate herbicides will be deployed. Ultimately, slowing the invasion front of current and potential new populations requires a better understanding of the factors (herbicide resistance) influencing the spread of this highly invasive weed.

Create and develop a herbicide resistance panel to rapidly detect pigweed populations that have markers linked to resistance of glyphosate and PPO-inhibiting herbicide.

1. Faster service and reporting on the herbicide resistance characteristics of pigweeds in North Dakota, with focus on combatting newly discovered Palmer amaranth populations.

2. Provide critical information to weed scientists and county extension agents on the distribution and mode of herbicide resistance in pigweed populations of North Dakota.

3. Incorporate the herbicide panel into NAGC’s genetic workflow for use in the Palmer Amaranth Action Plan facilitated by NDSU weed scientists, county agents, and NDDA.

Updated November 23, 2020:
Name of Organization: National Agricultural Genotyping Center (NAGC)

Project Title: Enhancement to a high-throughput genetic test for identification of herbicide resistance traits in Palmer amaranth and related pigweed species.

PI: Zack Bateson
Co-PIs: Michael Christoffers & Lisa Piche

Objectives of the Research
Late-season pigweeds (Amaranthus spp) that escaped herbicide applications are considerable threats to future crop production. Herbicide resistant (HR) populations have been rapidly emerging across the US for Palmer amaranth and waterhemp, the two out-crossing pigweed species. Palmer amaranth has been recently discovered in North Dakota, but little is known about the genetic HR potential of these populations. Likewise, the distribution of waterhemp is growing in the state as well as the number of complaints from producers about ineffective herbicides. There are several genetic markers that are linked to HR traits, particularly for glyphosate and PPO-inhibitors, the two common herbicides used to combat pigweeds in bean fields. The objective of this project was to expand NAGC’s genotyping panel by including two additional tests for genetic markers linked to glyphosate and PPO-inhibitor resistance in pigweeds. Increasing the availability of these genetic tests will provide the opportunity to better characterize the HR potential of new and established pigweed populations in the region. Once HR characteristics of local pigweeds have been described, soybean growers can use this information to avoid ineffective herbicides and adjust their integrative pest management towards optimal pigweed control.

Completed work
Due to the need for rapid confirmation of new Palmer amaranth populations and the challenges of visually identifying pigweed species, NAGC developed an Amaranth Species ID test for leaf and seed samples. The test has already benefited the North Dakota soybean community through gaining a better understanding of the distribution of Palmer amaranth in North Dakota. The additional HR genotyping tests provide a deeper understanding of the chemical management options that soybean growers have to control pigweeds.

The genetic mechanisms for glyphosate and PPO-inhibitor resistances are fundamentally different, yet can be detected in the same DNA extracts validated for the Amaranth Species ID test. The first test for glyphosate resistance quantifies the EPSPS gene copy number in waterhemp and Palmer amaranth leaf samples. Elevated EPSPS gene copies is associated with reduced effectiveness of glyphosate in pigweeds. The second HR test is for PPO-inhibitor resistance that detects a mutation (amino acid deletion) within the PPX2 gene, which codes for the PPO enzyme in pigweeds. By changing the shape of the PPO enzyme, this mutation reduces the ability of the PPO-inhibitor herbicide (e.g., fomesafen) to interact with its intended target. These two tests were selected for the HR panel at NAGC because multiple laboratories have used them for statewide surveys of pigweed populations over the last few years.

Most of the validation work has been completed for the HR tests at NAGC. For the glyphosate resistance test, we have optimized assay parameters to closely match the Amaranth Species ID test. After performing specificity tests and further examined archived pigweed samples, the assay appears specific to the pigweed family. Likewise, the PPO-inhibitor test has been optimized so it can be co-amplified with the Amaranth Species ID test, and it appears to show high specificity to the pigweed family in specificity tests. Unfortunately, due to the difference in analyses between the EPSPS (copy number) and PPO-inhibitor (genotyping) assays, it is not possible to co-amplify all three tests on a single plate. We expect little delay in completing the validation under NAGC’s ISO 17025:2017 standards, which provides robust quality assurance and performance.

Preliminary Results
During the validations, we tested a set of archived DNA samples of Palmar amaranth (n=26) and waterhemp (n=29) originally submitted by clients in North Dakota and Minnesota for NAGC’s Amaranth Species ID test. In total, 15% (4/26) Palmer amaranth and 34% (10/29) waterhemp samples had elevated EPSPS gene copies to be considered glyphosate resistant. There were large difference in the number of gene copies between the two species, where Palmer amaranth and waterhemp had individuals with maximum EPSPS copy number of 50 and 8, respectively. This was expected because Palmer amaranth has a unique duplication mechanism that results in dozens of more gene copies than waterhemp in HR individuals. Notably, several waterhemp samples showed elevated EPSPS copies just below the threshold (four gene copies), signifying the growing potential for glyphosate resistance, which may appear in offspring from these individual plants. For PPO-inhibitor resistance, there were three waterhemp samples (10%, 3/29) containing the deletion that confers resistance. No Palmer amaranth samples in the small subset contained the deletion, but additional North Dakota populations should be tested.

Work to be Completed
Written validations for both HR tests are nearing completion. The final sensitivity and mixture analyses must be performed for glyphosate resistance test, and sequence confirmation of the expected genotypes for the PPO-inhibitor test from the archived samples. Once these analyses are completed, each validation will be critically reviewed by two NAGC scientists. If warranted, additional optimization tests may be requested and further explored. Following validation approval, two standard operation procedures (SOPs) will be created for use by scientists at NAGC. Before NAGC scientists can provide HR test results to collaborators or soybean growers, each NAGC scientist must pass a blinded proficiency test. Lastly, these HR tests will be integrated into the NAGC’s ISO 17025 accreditation scope in 2021. The completion of these tests were expedited so that both test would be available to soybean growers early in the 2021 growing season.

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Updated June 30, 2021:

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Research conducted
The National Agricultural Genotyping Center (NAGC) has expanded its genetic panel for Palmer amaranth and waterhemp surveillance in North Dakota. Specifically, NAGC validated two genetic tests to detect markers linked to glyphosate and PPO-inhibitor resistances in submitted pigweed samples. Together with NAGC’s pigweed species ID test, this genetic panel can identify important traits in pigweeds found in North Dakota fields.

Why the research is important to ND soybean farmers
Late-season pigweeds that escaped herbicide applications are threats to present and future soybean production. Unfortunately, herbicide resistant (HR) populations of Palmer amaranth and waterhemp are increasing across the soybean belt. In North Dakota, Palmer amaranth has begun expanding its distribution and there are increasing reports of waterhemp escaping herbicides. Both pigweed species are known to carry resistance to multiple herbicide groups in other states, but little is known about North Dakota populations. Genetic tests provide a first look into the HR potential of pigweeds and help pre-select seed stock for greenhouse herbicide trials that are limited by both space and time.

Final findings of the research
The two HR tests were successfully validated on leaf tissue and seed samples, and are available at NAGC for all soybean farmers across the US. Confirming pigweed species is important prior to HR tests because the PPO-inhibitor (PPO-210) and glyphosate (EPSPS copies) resistance markers have only been found in Palmer amaranth and waterhemp. Research to find genetic markers for HR in other pigweed species (Powell amaranth) are currently underway.

A blinded study showed 90% agreement between the HR test (EPSPS copies) and glyphosate outcomes from greenhouse trials. Additionally, the PPO-210 genotype that carries resistance in Palmer amaranth was found in plants collected in North Dakota. Thus, these two HR tests have indicated the presence of these markers in North Dakota pigweeds and will be used in an upcoming statewide survey.

Benefits/Recommendations to North Dakota soybean farmers and industry
Palmer amaranth infestations are relatively low in North Dakota, in part, to its recent arrival. Slowing its spread requires diligence in monitoring and finding effective methods to manage newly discovered populations. NAGC’s genetic panel can quickly confirm the pigweed species and which herbicides to avoid if broad-scale chemical treatment is necessary. These tests will help limit the overuse of ineffective herbicides as well as inform farmers about the HR potential of local weed populations throughout the growing season.

Control of pigweeds, especially Palmer amaranth, has become increasingly difficult over the past decade because of the widespread distribution of biotypes resistant to herbicides. Resistance to glyphosate and PPO-inhibitors have been documented in multiple pigweed populations. However, populations of Palmer amaranth, redroot pigweed, Powell amaranth, and smooth pigweed may still be susceptible to certain herbicides (Heap 2019) or have yet to be directly tested for herbicide resistance. Thus, the expansion of a genetic test that can quickly confirm the presence of multiple herbicide resistant traits in pigweed species from a small amount of tissue would provide more immediate answers to effective herbicide options during early detection and rapid response.