Greenhouse Dose-Response Experiments

The effect of experimental run was not significant for any evaluation. Therefore, data from both experiments were pooled. ANOVA revealed a significant two-way interaction of biotype and 2,4-D dosage for all evaluations except fresh and dry weight measurements at 28 DAT, indicating differential responses of buckhorn plantain to doses of 2,4-D in susceptible (S) and resistant (R) populations (Table 2). In contrast, the two-way interaction of biotype and halauxifen-methyl dosage was only significant on one of 11 evaluations, suggesting similar responses to halauxifen-methyl across S and R populations.

Table 2.

Analysis of variance for epinasty, necrosis, chlorophyll content index, fresh and dry weight, and regrowth fresh and dry weight evaluations on buckhorn plantain treated with different rates of 2,4-D and halauxifen-methyl herbicides at various days after treatment in greenhouse dose response trials. ^{a b}

^a Abbreviations: CCI, chlorophyll content index; DAT, days after treatment.

^b Data presented are averaged across 2018 and 2019 trial years due to no significant trial year interaction (P > 0.05).

^c Epinasty was visually assessed on a 0% to 100% scale with 0% being no epinasty and 100% epinasty being complete twisting, bending, and curling of stems and leaf tissue.

^d Necrosis was visually assessed on a 0% to 100% scale, with 0% being no necrosis and 100% necrosis being completely dead plant tissue.

^e CCI was measured with an Apogee MC-100 Chlorophyll Concentration Meter on a fully expanded, representative leaf of each plant.

Epinasty differed between R and S populations in response to 2,4-D and halauxifen-methyl at 3 DAT (Table 2). Across all dosages of 2,4-D, epinasty averaged 35% in the R biotypes and 53% in S biotypes (data not shown). For halauxifen-methyl, epinasty averaged 25% and 36% in R and S biotypes, respectively, across all dosages.

For necrosis evaluations at 14 and 28 DAT, a significant two-way interaction of biotype and 2,4-D dose was observed in buckhorn plantain populations (Table 2; Figure 1). The two-way interaction for 2,4-D-treated plants indicates that S and R populations had significant response differences at specified dosages. Conversely, only the main effects of biotype and dosage were significant in halauxifen-methyl-treated populations (Table 2). For 2,4-D, the estimated GR₅₀ for necrosis in the R population 28 DAT was 7.13 kg ae ha⁻¹, more than 4 times greater than the labeled application rate of 1.68 kg ae ha⁻¹ (Figure 1). In comparison, the S population had a GR₅₀ for necrosis of 0.63 kg ae ha⁻¹, a dosage lower than half of the label rate. These results are similar to those reported by Patton et al. (Reference Patton, Weisenberger and Schortgen2018) in which a resistant buckhorn plantain biotype in Indiana required a 2,4-D dosage at least 6.2 times greater than the susceptible biotype.

Figure 1.

Nonlinear regression of buckhorn plantain necrosis 28 d after treatment with 2,4-D (A) or halauxifen-methyl (B) in suspected-2,4-D-resistant (R) and suspected-2,4-D-susceptible (S) biotypes in greenhouse dose-response experiments. Error bars represent the standard error of the mean. Intersection of trendline with horizontal line at 50% necrosis is representative of the estimated GR₅₀. ± indicates 95% confidence intervals of estimated GR₅₀. Dashed vertical line indicates the labeled rate of each herbicide active ingredient.

For halauxifen-methyl treatments, necrosis at 14 and 28 DAT was significantly affected by dose and biotype main effects (Table 2). Except when compared to the nontreated control, necrosis at 14 DAT did not differ among doses of halauxifen-methyl across both populations, averaging 43% necrosis (data not shown). At 28 DAT, dosages of halauxifen-methyl at or below the standard application dose of 0.010 kg ae ha⁻¹ resulted in necrosis greater than the nontreated control (67% compared to 4% necrosis, respectively), whereas dosages higher than the standard dose resulted in greater necrosis (96%; data not shown). Across all doses of halauxifen-methyl, 48% necrosis was observed in S populations 14 DAT, compared with 27% in R populations. At 28 DAT, necrosis was 80% and 66% in S and R populations, respectively, across all halauxifen-methyl doses. Despite the lack of a biotype-by-dose interaction for populations treated with halauxifen-methyl (Table 2), the GR₅₀ for necrosis of S and R biotypes at 28 DAT were 0.001 and 0.007 kg ae ha⁻¹ halauxifen-methyl, respectively, and were lower than the standard application rate of 0.010 kg ae ha⁻¹ (Figure 1).

CCI measurements resulted in similar trends as necrosis evaluations. At 28 DAT, there was a significant two-way biotype-by-dose interaction for buckhorn plantain populations treated with 2,4-D (Table 2). Conversely, dose was the lone significant treatment factor for plants treated with halauxifen-methyl, suggesting that there were no differences among R and S biotypes in terms of CCI when treated with this active ingredient (Table 2). Although R ² values were lower in regression analysis and more variability was present in CCI data than necrosis evaluations, the CCI GR₅₀ for the R biotype in response to 2,4-D application was still greater (11.21 kg ae ha⁻¹) than that of the S biotype (0.73 kg ae ha⁻¹) and greater than the label application 2,4-D application rate of 1.68 kg ae ha⁻¹ (Figure 2). The CCI GR₅₀ values for R and S biotypes treated with halauxifen-methyl were not different from each other and were less than or equivalent to the standard halauxifen-methyl application rate.

Figure 2.

Nonlinear regression of buckhorn plantain chlorophyll content index 28 d after treatment with 2,4-D (A) or halauxifen-methyl (B) in suspected-2,4-D-resistant (R) and suspected-2,4-D-susceptible (S) biotypes in greenhouse dose-response experiments. Error bars represent the standard error of the mean. Intersection of trendline with horizontal line at 50% chlorophyll content index is representative of the estimated GR₅₀. Dashed vertical line indicates the labeled rate of each herbicide active ingredient.

The main effect of dosage influenced fresh and dry weight 28 DAT in 2,4-D treatments, but it affected only fresh weight in halauxifen-methyl treatments (Table 2). The loss of plant moisture after drying could potentially explain why dosage did not affect dry weight in halauxifen-methyl treatments 28 DAT. However, a significant biotype-by-dose interaction was observed for 2,4-D treated plants regarding fresh and dry regrowth weight measurements taken 42 DAT after the initial harvest (Table 2). For both fresh and dry regrowth, the GR₅₀ for the S biotype was less than the label rate of 2,4-D, indicating that 2,4-D treatments are effective at controlling this biotype (Figure 3). However, R biotypes needed 2,4-D dosages greater than four times the labeled rate to exhibit a 50% reduction in fresh or dry regrowth. In both S and R biotypes, all rates of halauxifen-methyl resulted in complete death of buckhorn plantain, whereas the nontreated plants grew new leaves following the initial harvest 28 DAT (data not shown). This finding suggests that there is no perceived resistance to this active ingredient in the 2,4-D-resistant biotype.

Figure 3.

Nonlinear regression of buckhorn plantain fresh (A) and dry (B) regrowth weight from suspected 2,4-D-resistant (R) and suspected-2,4-D-susceptible (S) biotypes 42 d after 2,4-D treatment following initial plant tissue harvest 28 d after treatment in greenhouse dose-response experiments. Whole plant biomass was harvested and oven-dried at 60 C for 7 d. Error bars represent the standard error of the mean.

Resistance factors of 11.3 for necrosis and 15.4 for CCI evaluations after R and S biotypes were treated with 2,4-D indicate the differential responses in each population to the same herbicide. Resistance factors greater than 10 are often considered as being high for synthetic auxin herbicides (Beckie and Tardif Reference Beckie and Tardif2012) and quantify the level of 2,4-D resistance present in the resistant biotype. Although R and S biotypes had different resistance factors to halauxifen-methyl, the GR₅₀ for each biotype was still below the standard application rate. These resistance factors are in the same range as those observed by Patton et al. (Reference Patton, Weisenberger and Schortgen2018) on 2,4-D-resistant buckhorn plantain in Indiana and are consistent with studies on several annual broadleaf weeds and their resistance to 2,4-D (Burke et al. Reference Burke, Yenish, Pittmann and Gallagher2009; Preston et al. Reference Preston, Dolman and Boutsalis2013; Rey-Caballero et al. Reference Rey-Caballero, Menéndez, Giné-Bordonaba, Salas, Alcántara and Torra2016).

Field Herbicide Efficacy Experiments

Commercially available synthetic auxin herbicides were evaluated to determine buckhorn plantain control efficacy in field experiments at one location with a S buckhorn plantain biotype in University Park and two locations with suspected-R biotypes in Carlisle and Harrisburg. There was a significant effect of experimental run (P = 0.0445) at the University Park site. Therefore, data are presented separately by experiment. At the Harrisburg and Carlisle sites, experimental run was not a significant effect (P = 0.5899 and 0.4300, respectively) and data were pooled across experiments for each location.

Initial buckhorn plantain coverage at the S biotype site averaged 16.1% and 45.7% of plot area in 2018 and 2019, respectively (data not shown). Except for Mecomec 4 in 2018 and Banvel, all other synthetic auxin treatments reduced buckhorn plantain ≥90% across all experiments conducted on S biotypes (Figure 4). Banvel provided poor reduction (<50%) in both years, as expected based on previous reports indicating poor efficacy on buckhorn plantain (Patton et al. Reference Patton, Elmore, Hoyle, Kao-Kniffin, Branham, Voigt, Christians, Thoms, Munshaw, Hathaway, Nikolai, Horgan, Miller, Xiong, Kreuser, Gaussoin, Gardner, Raudenbush, Li, Landschoot, Soldat and Koch2019). Both low and high rates of Gordon’s 400 2,4-D reduced buckhorn plantain by >90%.

Figure 4.

Buckhorn plantain cover reduction in a 2,4-D-susceptible population at University Park, PA in 2018 (A) and 2019 (B) 28 d after synthetic auxin herbicide treatment in field efficacy experiments. Within each year, means followed by the same letter are not significantly different according to Fisher’s protected LSD test (α = 0.05).

Across both runs of the experiment in R biotypes, initial buckhorn plantain coverage averaged 10.3% at the Harrisburg location and 66.2% at the Carlisle location (data not shown). Gordon’s 400 2,4-D applied at low and high rates were essentially equivalent to the nontreated at both resistant locations, reducing buckhorn plantain cover by less than 20% (Figure 5). Additionally, all other treatments from the phenoxycarboxylic acid and benzoic acid chemical families were not able to provide reduction greater than the nontreated, indicating resistance mechanisms to the phenoxycarboxylic acid chemical family as initially reported by Patton et al. (Reference Patton, Weisenberger and Schortgen2018). Alternatively, Switchblade treatments resulted in good (70% to 90% reduction) to excellent reduction (≥90%) at both locations (Figure 5). This treatment contained active ingredients from the pyridinecarboxylic acid (fluroxypyr) and arylpicolinate (halauxifen-methyl) chemical families in addition to a benzoic acid (dicamba) that do not have any reported cases of resistance in buckhorn plantain. Because dicamba is not considered an effective active ingredient in controlling buckhorn plantain (Patton et al. Reference Patton, Elmore, Hoyle, Kao-Kniffin, Branham, Voigt, Christians, Thoms, Munshaw, Hathaway, Nikolai, Horgan, Miller, Xiong, Kreuser, Gaussoin, Gardner, Raudenbush, Li, Landschoot, Soldat and Koch2019) and fluroxypyr has been reported to be ineffective on other 2,4-D-resistant buckhorn plantain populations (Patton et al. Reference Patton, Weisenberger and Schortgen2018), these results suggest that the effectiveness of the Switchblade treatments could be attributed to the halauxifen-methyl component. The differing binding affinity of halauxifen-methyl at the site of activity compared to that of 2,4-D and other synthetic auxins (Bell et al. Reference Bell, Schmitzer and Robinson2014; Schmitzer et al. Reference Schmitzer, Balko, Daeuble, Epp, Satchivi, Siddall, Weimer and Yerkes2015) could indicate why this treatment was successful in resistant populations, as has been suggested in other studies (McCauley et al. Reference McCauley, Johnson and Young2018). In addition to halauxifen-methyl, Patton et al. (Reference Patton, Weisenberger and Schortgen2018) also found that other pyridinecarboxylic acid herbicides such as clopyralid and triclopyr are effective in 2,4-D-resistant buckhorn plantain populations.

Figure 5.

Buckhorn plantain cover reduction within 2,4-D-resistant populations in Carlisle, PA (A) and Harrisburg, PA (B) 28 d after treatment with synthetic auxin herbicides in field efficacy experiments. Treatment bars that do not share the same letter are significantly different according to Fisher’s protected LSD test (α = 0.05).

In the present study, resistance in buckhorn plantain was restricted to herbicides from the phenoxycarboxylic acid chemical family of synthetic auxin herbicides and is in agreement with previous research in Indiana (Patton et al. Reference Patton, Weisenberger and Schortgen2018). Field evaluations in suspected-R buckhorn plantain populations further indicated resistance to phenoxycarboxylic acids in the synthetic auxin herbicides. Greenhouse and field evaluations indicate that treatments including halauxifen-methyl could be used to effectively control these resistant populations. These results confirm resistance to 2,4-D in buckhorn plantain populations in turfgrass systems and further shed light on alternative control options in R populations. Although cross-resistance to multiple chemical families within the synthetic auxins has been reported in other weed species, a majority of reports cite single chemical family resistance in synthetic auxins (Heap Reference Heap2020). Specifically in turf systems, only lawn burweed has been reported to have synthetic auxin resistance to the pyridinecarboxylic acid herbicides (Harrington et al. Reference Harrington, Ward and Wells2001). Because it is not always practical or in the research objectives of a study to evaluate resistant weed species against all classes of an herbicide group, the disparity in cross-resistance and single chemical family resistance could be artificially inflated. There are several reports of weed species having multiple resistance to a synthetic auxin herbicide and other herbicide modes of action (Heap Reference Heap2020), but that was not evaluated in the present study. Regardless, this is the first report of synthetic auxin resistance of any weed species in Pennsylvania. Despite substantial research in unravelling the molecular mechanism for synthetic auxin resistance, additional research is needed to fully understand the factors conferring 2,4-D resistance in buckhorn plantain. Further research should also focus on cultural control options and additional commercially available herbicide formulations for buckhorn plantain, especially in resistant populations, as part of an integrated weed management strategy.