Glufosinate Bioassay

All A. palmeri populations were controlled 100% by glufosinate (data not shown). Poirier et al. (Reference Poirier, York, Jordan, Chandi, Everman and Whitaker2014) observed similar results with 134 A. palmeri populations collected predominantly from the NC Coastal Plain during 2010. In 2018, glufosinate-tolerant cotton and soybean cultivars accounted for approximately 65% and 20% of all U.S. plantings, respectively (Unglesbee Reference Unglesbee2018; USDA 2018). When considering the large genetic diversity in A. palmeri and extensive gene flow between populations, one can deduce that great stewardship is required to reduce selection pressure, contain the dispersal of emerging resistant genotypes, and maintain those SOAs that are still effective long-term (Chandi et al. Reference Chandi, Milla-Lewis, Jordan, York, Burton, Zuleta, Whitaker and Culpepper2013; Franssen et al. Reference Franssen, Skinner, Al-Khatib, Horak and Kulakow2001; Powles and Yu Reference Powles and Yu2010). However, the data suggest that with proper herbicide rate and application timing, glufosinate can effectively manage resistant biotypes within these populations that escape other herbicide SOAs as discussed later. Additionally, while no survivors were observed following glufosinate applications, the data can be utilized as a benchmark for future screenings of A. palmeri from the region.

Glyphosate and Thifensulfuron-Methyl Bioassay

Amaranthus palmeri survival frequency from glyphosate treatment differed across populations (P < 0.0001; F = 15.6) with only one population being completely controlled by glyphosate (Figure 1A). Of the 110 populations tested, 9 had relatively low survival (1% to 30%), with 20 having moderate survival (31% to 60%). However, six of the nine low-survival populations (2% to 14% range) were not significantly different from the control (P > 0.05), suggesting lack of genetic homogeneity, thus causing high error variance. Most populations (72) had high survival (61% to 90%), with the remaining eight populations surviving at >90%. Overall, 80 of the 110 populations, or roughly 73%, had survival greater than 60%. The authors acknowledge that sampling plants in the fall may bias survival in those plants from cotton and soybean fields, as glyphosate was most likely used. The survival frequencies from glyphosate treatment differed significantly among cropping systems (P < 0.0001; F = 39.3), being highest in soybean (83%), followed by cotton and sweetpotato (70% and 69%, respectfully), then peanut (62%). Thus, a high number of survivors were found in populations from every cropping system. This is expected, because glyphosate-resistant cotton, corn (Zea mays L.), and soybean cultivars are widely grown in rotation by farmers across this region of NC (USDA-NSS 2018). Crop rotation may be practiced, but in the majority of cases, the base chemical weed control across crops contains glyphosate. Therefore, there is generally no relief from glyphosate selection pressure. Amaranthus palmeri allowed to produce seed at the end of the season, like those selected for this research, may occur in many systems, thus adding seed to the soil seedbank that may remain viable for years after burial (Sosnoskie et al. Reference Sosnoskie, Culpepper and Webster2011).

Figure 1. Survival frequencies for the 110 screened Amaranthus palmeri populations from the North Carolina Coastal Plain following an application of (A) glyphosate (840 g ha⁻¹), (B) thifensulfuron-methyl (17.5 g ha⁻¹), (C) fomesafen (280 g ha⁻¹), and (D) mesotrione (105 g ha⁻¹). All herbicides were applied at a full label rate. Thifensulfuron-methyl rate was based on sulfonylurea-tolerant soybean. Plants were designated survivors if the plants had functional green leaves and apical meristems.

Resistance to ALS inhibitors was high, as indicated by high survival frequencies following thifensulfuron-methyl across populations, with only four populations being completely controlled. Most survival fell into the low to moderate categories (Figure 1B); although 37 populations (2% to 16% range) were not significantly different from the control (P > 0.05), again suggesting genetic heterogeneity within these populations. Excluding those, 32 populations had low survival, with 31 categorized as moderately resistant, all of which had significantly more survivors than the designated control. While survival was relatively low compared with glyphosate survival, the rate applied was four times the manufacturer’s recommended rate for non-sulfonylurea-tolerant soybean, suggesting that these survivors are resistant (Anonymous 2012). The remaining six populations had high survival of 61% to 80%.

The survival and resistance observed in the tested populations were like those reported by Poirier et al. (Reference Poirier, York, Jordan, Chandi, Everman and Whitaker2014), who sampled in the same general region. Those researchers designated resistance in a population if any survivors were present following glyphosate and thifensulfuron-methyl at 840 g ae ha⁻¹ and 17.5 g ha⁻¹, respectively, which were the same rates evaluated in our study. Poirier et al. (Reference Poirier, York, Jordan, Chandi, Everman and Whitaker2014) determined that 98% and 97% of the 134 tested A. palmeri populations contained individuals resistant to glyphosate and thifensulfuron-methyl, respectively. Comparing our populations under the same metrics, 99% and 96% were resistant to these herbicides, respectively. These data suggest that resistance to glyphosate and thifensulfuron has remained relatively similar from 2010 to 2016. Whitaker (Reference Whitaker2009) screened 290 A. palmeri populations collected from NC in 2005 for resistance to glyphosate and thifensulfuron-methyl and reported that 49 (17%) and 52 (18%) populations were resistant to these herbicides, respectively. These data illustrate how in a 5- to 10-yr period there was a rapid and widespread incidence of glyphosate- and ALS-resistant A. palmeri occurring in NC.

KASP Assay for Thifensulfuron-Methyl Survivors

Analysis for mutation in the ALS gene was examined in 104 populations of A. palmeri with survivors as adequate tissue from 2 was not available. Of the total 1,167 plants surviving following the thifensulfuron-methyl application within these populations, 83% possessed at least one of the four known ALS resistance–conferring mutations (Table 4). The Trp-574-Leu mutation (53%) was the most common of the known mutations, followed by the Pro-197-Ser mutation (34%). The Pro-84-His mutation reported by Larran et al. (Reference Larran, Palmieri, Perotti, Lieber, Tuesca and Permingeat2017) was present in all of our tested samples, while the Met-601-Ile mutation was found in none. The authors noted these mutations may represent natural genetic variation and were observed outside the ALS conserved regions previously associated with target-site resistance. The third mutation reported by Larran et al. (Reference Larran, Palmieri, Perotti, Lieber, Tuesca and Permingeat2017), Ala-282-Asp, was more common in the surviving A. palmeri plants; however, it was generally associated with other known mutations. A total of 382 plants had the Ala-282-Asp mutation, but only 57 in total (5% of total survivors) had that mutation alone. Further research is needed to determine the level of resistance, if any, conferred by this mutation. Only 9% of the survivors did not possess any mutation, suggesting the resistance may be conferred by a different mutation or differing metabolism. Interestingly, no amplification was detected in 34% of survivors for the Ser-653-Asn mutation, suggesting the wild-type or known mutation was not present. Further, 2% of survivors were determined to have no known resistance-conferring mutation, but also had no amplification for the Ser-653-Asn mutation, suggesting another amino acid substitution may be conferring resistance to thifensulfuron-methyl. Other mutations (Ala-122-Thr and Pro-197-Ala) have more recently been determined to confer resistance, illustrating that other potential mutations have yet to be discovered (Heap Reference Heap2019). Further research is required to quantify the level of resistance, if any, conferred by the Ala-282-Asp mutation as well as the possibility of a new mutation at the Ser-653 site.

Table 4. Mutations detected in the acetolactate synthase (ALS) enzyme and percent survival of Amaranthus palmeri following an application of thifensulfuron-methyl at 17.5 g ha⁻¹. ^a

^a Abbreviations: Pro-84-His, proline to histidine substitution; Ala-122-Ser, alanine to serine substitution; Pro-197-Ser, proline to serine substitution; Ala-282-Asp, alanine to aspartic acid substitution; Trp-574-Leu, tryptophan to leucine substitution; Met-601-Ile, methionine to isoleucine substitution; Ser-653-Asn, serine to asparagine substitution. Positive sign (+) means the mutation was present, while the minus sign (−) denotes that the mutation was not detected.

^b County codes: BER, Bertie; BLA, Bladen; CHO, Chowan; CRA, Craven; DUP, Duplin; EDG, Edgecombe; GRE, Greene; HAL, Halifax; HAR, Harnett; HER, Hertford; JOH, Johnston; LEN, Lenoir; MAR, Martin; NAS, Nash; PIT, Pitt; ROB, Robeson; SAM, Sampson; UPC, Upper Coastal Plain Research Station; WAK, Wake; WAS, Washington; WAY, Wayne; WIL, Wilson.

^c Mutations reported by Larran et al. (Reference Larran, Palmieri, Perotti, Lieber, Tuesca and Permingeat2017) in ALS-resistant A. palmeri populations.

^d Known resistance-conferring mutations.

Fomesafen Bioassay

The resistance to ALS inhibitors and glyphosate has created limited PRE and POST herbicide control options for A. palmeri in crops such as cotton, peanut, and soybean (NCSU 2019). Growers have increased their reliance on PPO-inhibiting herbicides, such as fomesafen, in these crops to manage the resistant biotypes. While 106 of the 110 populations were completely controlled with fomesafen, 4 populations had 1% to 10% survivors (Figure 1C). Three of the populations were from Edgecombe County while the remainder was from Halifax County. Three of the four populations had very low survival of 1% to 2%, yet one population (EDG16), was in the 10% range. The populations with 2% (HAL16) and 10% survival were statistically different from the susceptible control (P = 0.0222 and < 0.0001, respectively). Survivors for the four populations (1% to 10%) were less than the Arkansas resistant standard (36% survival; P < 0.0001). While this is seemingly very low survival, when one considers the immense fecundity of A. palmeri, these frequencies can quickly increase if selection pressure remains high. Research in NC documented the average seed production from A. palmeri in competition with corn, cotton, peanut, and soybean was approximately 51,000, 534,000, 443,000, and 273,000 seeds plant⁻¹, respectively (Mahoney Reference Mahoney2020). Additionally, plants in the greenhouse are generally considered more susceptible to herbicides than field-established plants, suggesting more control may be observed in this setting compared with a production field. The fact that survivors were present following a field use rate of fomesafen in the greenhouse at the proper application timing (with respect to weed size) is concerning and suggests these populations may be in the early stage of resistance development.

KASP Assay for Fomesafen Survivors

Other states in the midsouthern and midwestern United States have documented that genetic mutations (Giacomini et al. Reference Giacomini, Umphres, Nie, Mueller, Steckel, Young, Scott and Tranel2017; Salas et al. Reference Salas, Burgos, Rangani, Singh, Refatti, Piveta, Tranel, Mauromoustakos and Scott2017) and enzymatic herbicide metabolism (Varanasi et al. Reference Varanasi, Brabham, Norsworthy, Nie, Young, Houston, Barber and Scott2018b) are the mechanisms conferring resistance to PPO inhibitors in their A. palmeri populations. In NC, common ragweed (Ambrosia artemisiifolia L.) was determined to have a single nucleotide polymorphism at the Arg-98 position (synonymous with Arg-128 in A. palmeri) conferring resistance to PPO inhibitors (Heap Reference Heap2019). Figure 2A–C illustrates KASP analysis for the ΔG210, Arg-128-Gly, and Arg-128-Met mutations, which are known to confer resistance to PPO-inhibiting herbicides (Giacomini et al. Reference Giacomini, Umphres, Nie, Mueller, Steckel, Young, Scott and Tranel2017; Salas et al. Reference Salas, Burgos, Rangani, Singh, Refatti, Piveta, Tranel, Mauromoustakos and Scott2017). Survivors from the Arkansas populations all contained the ΔG210 mutation (Figure 2A), which has been previously reported in this population (Salas et al. Reference Salas, Burgos, Rangani, Singh, Refatti, Piveta, Tranel, Mauromoustakos and Scott2017). Survivors from the NC populations did not express the ΔG210 mutation (Figure 2A), with no population expressing the Arg-128-Gly (Figure 2B) or Arg-128-Met mutations (Figure 2C). While no resistance cluster occurred for the Arg-128 mutations to visualize this, the primers for these mutations were designed such that fluorescein (FAM) fluorescence would be expressed for the wild-type or nonmutant DNA. Fluorescence readings suggested amplification occurred and was confined to the FAM fluorescence region. Further research to characterize these populations will be discussed later.

Figure 2. Graphical representation of the Kompetitive Allele Specific PCR (KASP) assays completed for the ΔG210 (A), Arg-128-Gly (B), and Arg-128-Met (C) mutations. Populations are color coded according to the legend in A. Four populations from North Carolina are represented (EDG16, HAL16, PNL16, and UPC16) along with the herbicide-resistant standard from Arkansas (AR). Abbreviations: FAM, fluorescein; HEX, hexachlorofluorescein.

Mesotrione Bioassay

Sixty-eight populations were completely controlled by mesotrione (Figure 1D). Of the remaining 42 surviving populations, 41 had survival in the 1% to 10% range, with 37 of these having less than 5% survival. Unlike the populations surviving fomesafen, these populations were located in many counties across the NC Coastal Plain. Populations having 2% survival or lower (30 total) were not significantly different from the SS (P > 0.05), likely due to lack of genetic homogeneity, thus causing large error variance. One population from Bladen County, NC, consistently had higher survival, at 17% on average. These A. palmeri populations surviving mesotrione are the first documented incidence of resistance to an HPPD inhibitor in NC. Currently, HPPD-resistant A. palmeri has been reported in Kansas, Nebraska, and Wisconsin (Heap Reference Heap2019). Nakka et al. (Reference Nakka, Godar, Wani, Thompson, Peterson, Roelfos and Jugulam2017) reported that rapid detoxification of mesotrione within an A. palmeri population from Kansas compared with a susceptible (2.45-fold faster) was one mechanism conferring resistance. Additionally, this population had a 12-fold increase in HPPD gene expression compared with the susceptible, which also contributed to the observed resistance. Corn production area in NC averaged approximately 359,000 total ha over the past 10 yr (USDA-NSS 2018). Mesotrione is labeled for weed control in corn (Anonymous 2018). With the oncoming release of HPPD-tolerant cotton and soybean, the area treated with mesotrione could potentially increase nearly 3.4-fold or reach 1,215,000 ha in NC alone (Unglesbee Reference Unglesbee2018; USDA-NSS 2018). Proper stewardship for this technology is a must; otherwise, evolution of HPPD-resistant A. palmeri populations will occur quickly and become widespread.

Of the 110 A. palmeri populations screened, none were completely controlled by all five tested SOAs and only three were controlled by all but one SOA (Figure 3). Most of these populations (65) were controlled by all but two SOAs, which were largely glyphosate and thifensulfuron-methyl. Greater cause for concern lies with A. palmeri populations with individuals surviving three (40) or four (2) SOAs. Based on our survey, growers can utilize glufosinate as one tool to manage these populations and mitigate further spread; however, great care should be taken to ensure the optimal application timing and rate are used to mitigate any survivors and reduce selection pressure. These populations illustrate the importance of proper herbicide stewardship in order to protect our current chemical weed management tools as well as to ensure the longevity of coming technologies. Farm managers must ensure that an integrated weed management approach is implemented and that escapes are minimized to mitigate additions of potentially resistant weed seeds to the soil seedbank.

Figure 3. Number of mechanisms of action survived by the 110 total Amaranthus palmeri populations tested from the North Carolina Coastal Plain region.

Fomesafen Dose–Response Assay

Further characterization of populations surviving HPPD and PPO inhibitors was done. Dose–response assays were conducted to determine the level of resistance compared to a known SS population. Increasing the dose of fomesafen reduced the dry weight and survival of all populations tested. The fomesafen dose required to cause a 50% reduction of A. palmeri biomass (GR₅₀) was 6.3 to 10.7 times greater for all putative resistant populations compared with the SS (Figure 4; Table 5); however, these doses were still approximately 80 times lower than the field use rate of fomesafen. Even as the GR₉₀ for EDG16 (9.3 g ha⁻¹) and HAL16 (9.8 g ha⁻¹) were 2.1 and 2.2 times greater than the SS (4.4 g ha⁻¹), respectively, these dosages were well below the field use rate. The fomesafen dose required to kill 50% of the population (LD₅₀) was 1.4 to 2.6 times greater in the four tested A. palmeri populations compared with the SS (Figure 5; Table 5). However, these values are also far below the field use rate of fomesafen. As with the GR₉₀, the LD₉₀ of EDG16 and HAL16 were well below the field use rate. While this may suggest a decrease in susceptibility of the population, the data suggest field use rates will still adequately control these populations.

Figure 4. Dose–response curves based on biomass reduction of Amaranthus palmeri populations from North Carolina following fomesafen application. Biomass reduction (% of nontreated) was recorded 14 d after fomesafen application. Treatments means (n = 12) plus 1 SE are plotted with the regression curves. A four-parameter logistic model best described the data: y = c + {(d – c)/[1 + (x/m)exp(−b)]}, where y is the biomass reduction, c is the minimum value, d is the maximum value, m is the point in the curve halfway between the minimum and maximum values, and b is the hillslope. The dotted vertical line denotes the field use rate. See note b to Table 4 for the key to county abbreviations.

Table 5. The dose of fomesafen needed to reduce Amaranthus palmeri biomass 50% (GR₅₀) or kill 50% of the population (LD₅₀) for populations from North Carolina. ^a

^a The 1X rate for fomesafen is 280 g ai ha⁻¹.

^b See note b to Table 4 for the key to county abbreviations.

^c Value ± SE.

^d Resistance levels (R/S) calculated using the GR₅₀ or LD₅₀ values of the field-collected populations relative to the susceptible standard.

^e Susceptible standard.

Figure 5. Dose–response curves for survival of Amaranthus palmeri populations from North Carolina following fomesafen application. Survival (%) was recorded 14 d after fomesafen application. Treatments means (n = 12) plus 1 SE are plotted with the regression curves. A four-parameter logistic model best described the data: y = c + {(d – c)/[1 + (x/m)exp(−b)]}, where y is the percent survival, c is the minimum value, d is the maximum value, m is the point in the curve halfway between the minimum and maximum values, and b is the hillslope. The dotted vertical line denotes the field use rate. See note b to Table 4 for the key to county abbreviations.

Salas et al. (Reference Salas, Burgos, Rangani, Singh, Refatti, Piveta, Tranel, Mauromoustakos and Scott2017) observed greater increases in the R/S ratios of fomesafen-resistant A. palmeri compared with a susceptible population and reported the ΔG210 mutation within the PPX2 gene conferred this resistance. Additional research from Arkansas has noted the presence of Arg-128-Gly and Arg-128-Met mutations, which have been confirmed to confer resistance, are also present in the state (Varanasi et al. Reference Varanasi, Brabham, Norsworthy, Nie, Young, Houston, Barber and Scott2018b). In addition to the target-site mutations, metabolic resistance has also been indirectly implicated in one A. palmeri population from Arkansas (Varanasi et al. Reference Varanasi, Brabham and Norsworthy2018a). The authors suggested that resistance was due to an increase in cytochrome P450 monooxygenase (P450) and glutathione S-transferase activity.

The known resistance-conferring PPO gene mutations were not found in the NC A. palmeri populations (Figure 2A–C), which had survivors at the field use rate of fomesafen, suggesting survival may either be metabolism based, conferred by a novel (but weak) target-site mutation, or translocation based. While overall survival, GR₅₀, and LD₅₀ values were relatively low in these populations, they are still concerning, due to the immense fecundity of A. palmeri (Mahoney Reference Mahoney2020). Previous research by Norsworthy et al. (Reference Norsworthy, Griffith, Griffin, Bagavathiannan and Gbur2014) has illustrated how quickly resistance can spread throughout a field. Within 3 yr of introduction, A. palmeri infested 95% to 100% of the field. This demonstrates the critical need for a zero-tolerance policy for survivors, as one escape can become an increasing problem in a short amount of time.

Mesotrione Dose–Response Assay

As with fomesafen, increasing the dose of mesotrione reduced the dry biomass and survival of all populations tested. The dose required to cause a 50% reduction in A. palmeri biomass (GR₅₀) was 1.2- to 2.8-fold greater in all populations compared with the SS (Figure 6; Table 6). Populations BLA16 and EDG16 also had 1.6- to 2.3-fold increase in GR₅₀ compared with the other designated “susceptible” populations (DUP16 and NAS16). The GR₅₀ for populations BLA16 (13.4 g ha⁻¹) and EDG16 (11.4 g ha⁻¹) were about 12.5% of the POST field use rate. The GR₉₀ for these populations were 65.8 and 43.5 g ha⁻¹, respectively, which was 1.9 to 2.9 times greater than for the SS (23.1). The dose required to kill 50% of the A. palmeri populations (LD₅₀) was 1.2 to 3.3 times greater in all populations compared with the susceptible standard (Figure 7; Table 6) with BLA16 and EDG16 again having increased LD₅₀ values compared with DUP16 or NAS16 (1.6- to 2.7-fold). The LD₉₀ rates for HAL16 (89.8 g ha⁻¹) and EDG16 (72.8 g ha⁻¹) were increased compared with the SS (33.4 g ha⁻¹) and represent 86% and 69% of the full field use rate, respectively. Of note, 3 and 2 plants out of 36 sprayed from BLA16 and EDG16, respectively, survived the field use rate, while 2 from the BLA16 population survived the 2X rate.

Figure 6. Dose–response curves based on biomass reduction of Amaranthus palmeri populations from North Carolina following mesotrione application. Biomass reduction (% of nontreated) was recorded 21 d after mesotrione application. Treatments means (n = 16) plus 1 SE are plotted with the regression curves. A four-parameter logistic model best described the data: y = c + {(d – c)/[1 + (x/m)exp(−b)]}, where y is the biomass reduction, c is the minimum value, d is the maximum value, m is the point in the curve halfway between the minimum and maximum values, and b is the hillslope. The dotted vertical line denotes the field use rate. See note b to Table 4 for the key to county abbreviations.

Table 6. The dose of mesotrione needed to reduce Amaranthus palmeri biomass 50% (GR₅₀) or kill 50% of the population (LD₅₀) for various populations from North Carolina. ^a

^a The 1× rate for mesotrione POST is 105 g ai ha⁻¹.

^b See note b to Table 4 for the key to county abbreviations.

^c Value ± SE.

^d Resistance levels (R/S) calculated using the GR₅₀ or LD₅₀ values of the field-collected populations relative to the susceptible standard.

^e Susceptible standard.

Figure 7. Dose–response curves for survival of Amaranthus palmeri populations from North Carolina following mesotrione application. Survival (%) was recorded 21 d after mesotrione application. Treatments means (n = 16) plus 1 SE are plotted with the regression curves. A four-parameter logistic model best described the data: y = c + {(d – c)/[1 + (x/m)exp(−b)]}, where y is the percent survival, c is the minimum value, d is the maximum value, m is the point in the curve halfway between the minimum and maximum values, and b is the hillslope. The dotted vertical line denotes the field use rate. See note b to Table  4 for the key to county abbreviations.

While these GR₉₀ and LD₉₀ rates are approximately half- and three-quarters of the field use rate, respectively, it is generally accepted that plants are more susceptible to herbicides when grown in the greenhouse than in the field. Nonetheless, these rates may occur in the field, which would allow these plants to produce seed. Reduced rates can be realized if the weed is too large at application, weather conditions prohibit adequate control, or weeds are present in difficult-to-spray areas (turnarounds, field edges, etc.) where seed can then be distributed at harvest or by tillage the following season (Norsworthy et al. Reference Norsworthy, Ward, Shaw, Llewellyn, Nichols, Webster, Bradley, Frisvold, Powels, Burgos, Witt and Barrett2012). Repeated exposure to (unintentional) reduced rates can lead to resistance evolution over several generations (Busi and Powles Reference Busi and Powles2009; Busi et al. Reference Busi, Neve and Powles2013).

Mesotrione-Survivor Progeny Comparison

When comparing the BLA16 progeny population at the 0.25X, 0.5X, and 1X rate to the original field population, survival after mesotrione treatment increased in the progeny population. Both the field population (19 of 36 plants) and progeny (8 of 8 plants) had significant survival at the 0.25X rate. Survival of the progeny remained high at the 0.5X rate (7 of 8 plants) but was less for the field population (9 of 36 plants). At the 1X rate, only 3 of 36 plants survived from the field population, while 6 of 12 plants survived among the progeny population. These data demonstrate that the ability to survive mesotrione treatment was inherited by the progeny of the BLA16 population and also increased the frequency of survival; thus, we documented the first HPPD-resistant A. palmeri population in the southeastern United States.

Resistance to HPPD-inhibiting herbicides has previously been documented in Amaranthus species. Kaundun et al. (Reference Kaundun, Hutchings, Dale, Howell, Morris, Kramer, Shivrain and Mcindoe2017) reported a mesotrione-resistant waterhemp [Amaranthus tuberculatus (Moq.) Sauer] population from Nebraska having a 45.5-fold higher GR₅₀ compared with a susceptible population. The authors hypothesized that resistance could be due to increased metabolism or reduced translocation of mesotrione in the plant. Olivera et al. (Reference Olivera, Jhala, Gaines, Irmak, Amundsen, Scott and Knezevic2017) reported a 13.2-fold increase in mesotrione required to achieve 50% control of another A. tuberculatus population from Nebraska compared with a susceptible population. The authors also reported a 17.8-fold increase in GR₅₀ using dry biomass data value of the resistant versus the susceptible population. Reported R/S ratios for HPPD-resistant A. palmeri populations are generally lower than what has been observed in A. tuberculatus. Jhala et al. (Reference Jhala, Sandell, Rana, Kruger and Knezevic2014) determined a 4.6- to 4.9-fold increase in GR₅₀ values for mesotrione when comparing a resistant A. palmeri population with two susceptible populations. To achieve 50% control of the resistant population, 3.5- to 4.7-fold more mesotrione was needed. The mechanism for resistance was not determined, but cross-resistance to other HPPD-inhibiting herbicides was noted. Küpper et al. (Reference Küpper, Peter, Zöllner, Lorentz, Tranel, Beffa and Gaines2018) reported a 3.3-fold increase in LD₅₀ of an A. palmeri population from Nebraska with tembotrione. The authors hypothesized that metabolism, likely by cytochrome P450s, conferred the resistance. A higher level (10.1- to 17.8-fold) of A. palmeri resistance to mesotrione was reported in Kansas (Nakka et al. Reference Nakka, Godar, Wani, Thompson, Peterson, Roelfos and Jugulam2017). In this case, the resistant plants metabolized mesotrione faster and also produced more copies of the HPPD gene, factors that were determined to confer resistance in this population. This is the first instance of HPPD-resistant A. palmeri documented in the Southeast (Heap Reference Heap2019). Further research and characterization on the EDG16 and HAL16 populations is warranted.