Clopyralid Dose Response

There was no interaction between clopyralid dose and measurement timing on the number of leaves (P=1.0), flowers (P=1.0), immature berries (P=0.97), the total number of reproductive organs (P=1.0), or plant heights (P=0.80). Clopyralid doses between 35 and 2,400 g ha⁻¹ did not reduce growth rates or development over time. Measurement timing influenced the number of leaves (P<0.0001), flowers (P<0.0001), immature berries (P<0.0001), total reproductive organs, (P<0.0001), and plant heights (P<0.0001). The number of flowers, immature fruit, and total reproductive organs increased over time, while shoot height decreased (Table 1). Results were expected given the typical increased growth and reproductive output of plants over time.

Table 1 Effect of time after clopyralid treatment on plant heights and reproductive parameters of plasticulture-grown strawberry plants at Balm, FL, in 2016.Footnote ^a

^a Data presented are least-square estimates and are averaged across all clopyralid treatments. Different lowercase letters indicate a significant difference using Tukey’s honest significant difference means test (α=0.05).

Clopyralid dose did not affect crop damage (P=0.77) or plant height (P=0.15) but did affect leaf number (P=0.0009). The maximal leaf number was at 560 g ha⁻¹, which was higher than the 35 or 1,120 g ha⁻¹ doses (Figure 1). Even so, no clopyralid treatments differed from the controls (Figure 1). Clopyralid-induced strawberry leaf number reductions are due in part to cultivar selection and application timing (Boyd and Dittmar Reference Boyd and Dittmar2015; Hunnicutt et al. Reference Hosek, Kubes, Lankova, Dobrev, Klima, Kohoutova, Petrasek, Hoyerova, Jirina and Zazimalova2013a).

Figure 1 Effect of clopyralid dose on the number of strawberry leaves produced within a plasticulture setting in Balm, FL. Data were pooled over the 2015 and 2016 harvest seasons and averaged across all timing measurements. Different letters between means indicate a significant difference using Tukey’s honest significant difference (α=0.05). Error bars represent the standard error of the mean.

Plant height results were inconsistent with previous findings, in which higher doses of clopyralid induced 6% damage, which was attributed to reductions in plant heights (McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996). Growth cessations, such as reductions in leaf number or plant height, are symptomatological of the lethal action of auxinic herbicides (Grossman Reference Figueroa and Doohan2010). This is often not observed in strawberry plants due to natural tolerance, but clopyralid damage may manifest due cultivar differences and application timing (Boyd and Dittmar Reference Boyd and Dittmar2015; Hunnicutt et al. Reference Hosek, Kubes, Lankova, Dobrev, Klima, Kohoutova, Petrasek, Hoyerova, Jirina and Zazimalova2013a, Reference Hunnicutt, MacRae, Dittmar, Noling, Ferrell, Alves and Jacoby2013b).

Clopyralid applications across a range of doses (35 to 2,400 g ha⁻¹) did not affect the number of crowns per plant (P=0.76) (3 plant⁻¹), shoot dry weight (P=0.0797) (45.5 g plant⁻¹), or root dry weight (P=0.726) (17.0 g plant⁻¹). The influence of clopyralid on season-long strawberry plant biomass accumulation has not been previously reported. The crown is a compressed stem that gives rise to inflorescence, leaves, and auxiliary buds at the nodes (Darnell Reference Darnell2003). The effect of clopyralid on the crown has not been examined in a plasticulture system. Results were consistent with previous findings for two application timings in the maiden year for a matted-row system (Clay and Andrews Reference Clay and Andrews1984). A third timing had reduced the crown number by 0.3 plant⁻¹ (Clay and Andrews Reference Clay and Andrews1984).

Clopyralid applications had no effect on flower number (P=0.99), immature berry number (P=0.63), total reproductive organs (P=0.92), total yield by weight (P=0.491) (216 g plant⁻¹), and berry number (P=0.66) (10 plant⁻¹). The safety of clopyralid on flower number and yield was consistent with previous findings (Boyd and Dittmar Reference Boyd and Dittmar2015; Figueroa and Doohan Reference Dittmar, Dufault, Noling, Stansly, Boyd, Paret and Webb2006; McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996). The influence of clopyralid on the immature fruit number and the total reproductive load on the plant has not been previously reported. Current results demonstrate the safety of clopyralid on both parameters. Clopyralid is safe to apply across a wide range of doses (35 to 2,240 g ha⁻¹) when the plants are fully mature with no effect on the production, development, and harvest of reproductive organs of the strawberry plant.

This study was the first to conduct a thorough examination on the effect of a wide range of clopyralid doses (35 to 2,400 g ha⁻¹) on the mature strawberry plant body. No negative effects of clopyralid dose were found on the number of strawberry crowns and leaves, the vegetative biomass both above- and belowground, the number of reproductive organs, or the reproductive output over time. Strawberry is naturally tolerant to clopyralid across a wide range of doses (35 to 2,250 g ha⁻¹) and generally only demonstrates clopyralid symptomology with leaf curling and malformations. Such symptomology is typical of an alternative pathway induced by auxinic herbicides (Grossman Reference Figueroa and Doohan2010). Strawberry plants may experience a mild growth cessation in response to clopyralid for both plant height and the number of leaves (Boyd and Dittmar Reference Boyd and Dittmar2015; McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996), but inconsistencies in strawberry tolerance are infrequent.

Clopyralid Absorption

The total radioactivity recovered was 91% and 106% for trial run 1 and 2, respectively. Maximal strawberry absorption (A _max) was 84% and reached peak absorbance (t ₉₀) in 15 h (Figure 2). Water was used to rinse the unabsorbed clopyralid from the leaf, so it was assumed that the cuticle was intact, and cuticular absorption was counted with the treated leaf.

Considering other tolerant species, total clopyralid absorption for strawberry was less then rapeseed (>97% at 24 HAT) and greater than A. cannabinum when applied at the vegetative (47% of applied radiation at 72 HAT) and early reproductive stages (56% of applied radiation at 144 HAT) (Hall and Vanden Born Reference Grossman1988; Orfanedes et al. Reference Nandula and Vencill1993). Species differences may be due to cuticle structure, as clopyralid is a lipophilic, ionic herbicide and is absorbed in foliage by nonfacilitated diffusion (Sterling Reference Sharpe, Boyd, Dittmar, MacDonald, Darnell and Ferrell1994). Given the high degree of radioactivity recovered within the plant (84%), strawberry tolerance is not due to absorption.

Figure 2 Radiolabeled clopyralid absorption and translocation into strawberry. Radioactivity expressed as a percentage of the total applied. Error bars are the standard error of the mean. The model for clopyralid absorption ( ${\rm R}_{{{\rm adj}}}^{2} \,{\equals}\,0.79$ ) was: ${\rm Absorption}\,{\equals}\,82{\times}\left\{ {1{\minus}\exp \left[ {\left( {{\rm log}0.1} \right){\times}\left( {{t \over {15}}} \right)} \right]} \right\}$ ,

while the model for clopyralid translocation from the treated leaf ( ${\rm R}_{{{\rm adj}}}^{2} \,{\equals}\,0.50$ ) was: ${\rm Translocation}\,{\equals}\,17{\times}\left\{ {1{\minus}\exp \left[ {\left( {{\rm log}0.1} \right){\times}\left( {{t \over {51}}} \right)} \right]} \right\}$ .

Total Translocation

Clopyralid translocation out of the strawberry treated leaf reached a maximum of 17% of recovered radiation by 51 HAT (Figure 2), approximately 14% of absorbed radiation. This was dramatically lower than results for rapeseed (65% of recovered radiation at 144 HAT) (Hall and Vanden Born Reference Grossman1988) and A. cannabinum for applications to the vegetative (81% of absorbed radiation at 72 HAT) and early reproductive stages (76% of absorbed radiation at 144 HAT). Limited translocation has been demonstrated in L. arundinaceum with aminocyclopyrachlor, wherein 37% of the recovered radioactivity was translocated by 96 HAT, and the parent molecule remained intact with no degradation (Lewis et al. Reference Kniss, Vassios, Nissen and Ritz2013). Carboxylic acid herbicides such as clopyralid and aminocyclopyrachlor are known to remain biologically active in decaying tissue that was exposed to the herbicide (Anonymous 2010).

Strawberry tolerance to clopyralid is due, in part, to limited translocation. Why strawberry plants demonstrate limited clopyralid translocation is still unknown but may be due to several factors and requires further study. Movement into the endoplasmic reticulum may occur due to its role in auxin binding protein 1 storage (Henderson et al. Reference Hall and Vanden Born1997). Clopyralid interaction with strawberry auxin influx and efflux proteins may be limiting (Hosek et al. Reference Henderson, Bauly, Ashford, Oliver, Hawes, Lazarus, Venis and Napier2012). Clopyralid may be limited by strawberry plant phloem loading and unloading (Devine and Hall Reference Devine and Hall1990). The auxin receptor AFB5, which is a site of action for carboxylic acids but not aryloxyacetates or benzoates, may be missing or mutated in strawberry plants (Walsh et al. Reference Valenzuela-Valenzuela, Lownds and Sterling2006). Tolerance may also be linked to the strawberry industry’s reliance on propagating physiologically mature daughter plants for transplants, as differential tolerance to 2,4-D based on physiological maturity has been demonstrated (Pazmiño et al. Reference Orfanedes, Wax and Liebl2011). Strawberry leaves exposed to clopyralid generally only experience slight growth malformation such as cupping (Boyd and Dittmar Reference Boyd and Dittmar2015), which may be due to an alternate pathway in the action of auxinic herbicides that stimulates cell expansion (Grossman Reference Figueroa and Doohan2010), and subsequent metabolism may prevent the slower phytotoxic pathway, though further study is required.

Translocation to Plant Parts

Within the strawberry plant, the organs that contained the most radioactivity were the leaves younger than the treated leaf (A _max=6%) and the inflorescence (A _max=4%) (Figures 3 and 4). Time to reach peak translocation was 18 and 66 h for the younger leaves and inflorescence, respectively. This was expected, as the younger leaves and inflorescence represent strong sinks for resources and thus dictate the direction of phloem bulk flow and subsequent long-distance transport of radioactivity.

Figure 3 Translocation over time of radiolabeled clopyralid based on radioactivity found in the strawberry leaves. Error bars are the standard error of the mean. The model for leaves younger than the treated leaf ( ${\rm R}_{{{\rm adj}}}^{2} {\equals}0.11$ ) was: ${\rm Translocation}\,{\equals}\,6{\times}\left\{ {1{\minus}\exp \left[ {\left( {{\rm log}0.1} \right){\times}\left( {{t \over {18}}} \right)} \right]} \right\}$ .

Due to the low degree of translocation and high variability, ARF functions were not able to be fit to the data for the crowns, roots, and older leaves. At 196 HAT, 4% of the recovered radioactivity was found within the older leaves (Figure 3). This was similar to the amount found within the reproductive structures, although the reproductive structures represented far less biomass than the older leaves (unpublished data). Only 2% of recovered radioactivity was within the crowns at 196 HAT (Figure 4). With very low translocation to the crown, the reproductive potential and new growth would likely be unaffected, especially given the overall time frame for translocation (Figure 2). Only 1% of radioactivity was recovered within the roots at 196 HAT (Figure 4). With minimal translocation to the roots, nutrient uptake was also likely unaffected. This was consistent with previous findings that clopyralid does not consistently suppress vegetative or reproductive growth and yield (Boyd and Dittmar Reference Boyd and Dittmar2015).

In summary, strawberry absorption reached a maximum of 82% and reached peak absorption at 15 HAT. Only 16.5% of the recovered radiation was translocated out of the strawberry treated leaf and reached peak translocation at 52 HAT. Translocation out of the strawberry treated leaf appeared preferential toward dominant sinks such as leaves younger than the treated leaf (6%) and reproductive organs (4%) but also translocated to the older leaves, crowns, and roots. Limited herbicide translocation and dispersal throughout the plant factors into the mechanism of strawberry tolerance to clopyralid but does not entirely explain it. Further study is required to determine the involvement of auxin-related reception and metabolism.

Figure 4 Translocation over time of radiolabeled clopyralid based on radioactivity found in strawberry reproductive organs, crowns, and roots. Error bars are the standard error of the mean. The model for translocation to the reproductive organs in strawberry ( ${\rm R}_{{{\rm adj}}}^{2} {\equals}0.40$ ) was: ${\rm Translocation}\,{\equals}\,4.0{\times}\left\{ {1{\minus}\exp \left[ {\left( {{\rm log}0.1} \right){\times}\left( {{t \over {66}}} \right)} \right]} \right\}$ .

Early Clopyralid Application

There was a three-way interaction between clopyralid dose, application timing, and the measurement date (P<0.0001) on clopyralid damage to strawberry plants. Clopyralid damage primarily manifested as leaf cupping. When applied at 14 DATr, clopyralid doses of 140 and 280 g ha⁻¹ induced a maximum of 4% and 15% leaf cupping, respectively (Table 2). Repressed translocation from limited water availability during root hair reestablishment may affect clopyralid safety on strawberry plants at 14 DATr. Strawberry plant root hair reestablishment has taken 3 to 4 wk in a growth chamber (Borkowska Reference Borkowska2001). Alternatively, the limited clopyralid translocation demonstrated in mature strawberries (Figure 2) may provide safety in immature strawberry plants, though further study is required.

Table 2 Induced damage to plasticulture-grown strawberry plants by clopyralid when applied in combinations of two doses and three timings in Balm, FL, in 2016.Footnote ^{a ,} Footnote ^b

^a Abbreviations: DATr, days after planting of the strawberry transplants; WAT, weeks after clopyralid treatment.

^b Data were averaged across both trial runs and back-transformed means are presented.

^c Damage was determined on a percentage scale of the degree of leaf cupping and malformation of leaves per experimental unit (whole plot). Different lowercase letters indicate a significant difference using Tukey’s honest significant difference means test (α=0.05).

Leaf cupping induced by 140 g ha⁻¹ reached a maximum of 17% at 1 WAT when applied at 21 DATr (Table 2). Leaf cupping induced by 280 g ha⁻¹ reached a maximum of 56% at 2 WAT when applied at 21 DATr (Table 2). The degree of clopyralid-induced leaf cupping at 21 DATr was higher than previous studies on mature strawberry plants in Florida (~40%) (Boyd and Dittmar Reference Boyd and Dittmar2015) and North Carolina plasticulture (0% malformations) (McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996). By 8 WAT, there was no leaf cupping (Table 2). Such recovery was consistent with previous results with leaf malformations (Boyd and Dittmar Reference Boyd and Dittmar2015). The 140 g ha⁻¹ dose was safe across all application timings examined, while the 280 g ha⁻¹ induced substantial leaf cupping post-14 DATr.

Strawberry plant height was affected by clopyralid dose (P=0.027) but not application timing (P=0.55) or the measurement timing (P=0.22). There was slight stunting for the 280 g ha⁻¹ dose (97.2% of the nontreated control) compared with 140 g ha⁻¹ (100.0%). Results were consistent with previous research on mature strawberry plants (McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996), although inconsistent with findings in the clopyralid dose–response study. Size-based tolerance to clopyralid has been demonstrated in M. lupulina (Sharpe et al. Reference Sharpe2016) and is possibly a result of the sheer degree of tissue available to alter the auxin stream (Zazimalova et al. Reference White, Boyd and Van Acker2014), though further study is required.

There was no effect of clopyralid dose (P=0.108) or application timing (P=0.137) on the strawberry plant aboveground biomass (43.9 g plant⁻¹). Results were consistent with the dose–response study. Neither clopyralid dose (P=0.20) nor application timing (P=0.50) affected the number of crowns (3 plant⁻¹). Results were consistent with the dose–response study and two timings by Clay and Andrews (Reference Clay and Andrews1984), but contrasted with results from a third application timing in a matted-row system (0.3 plant⁻¹). With only slight stunting in plant heights and no difference in aboveground biomass or the crown number, clopyralid applications shortly after transplant appear safe on the season-long development of the vegetative plant body.

For berry weight, there was an interaction between the clopyralid dose and the date of harvest (P=0.016). This was likely due to the natural variability of the yield over time and the small amount of harvest in the growing season. There was no indication of a delayed harvest induced by clopyralid application, with no differences between treatments within each timing, including controls (unpublished data). There was no interaction for the number of berries per plant between the treatments and the date of harvest (P=0.20), but there was an effect of treatment (P=0.017). Delayed harvest due to clopyralid typically has not occurred when applied to fully developed strawberries at labeled doses in annual plasticulture (Boyd and Dittmar Reference Boyd and Dittmar2015; McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996) or matted-row systems (Figueroa and Doohan Reference Dittmar, Dufault, Noling, Stansly, Boyd, Paret and Webb2006).

There was no effect of application timing or dose on the berry number (P=0.0543) (5 berries plant⁻¹) or the total harvested weight of the berries (P=0.056) (83.6 g plant⁻¹). Results were consistent with previous literature for clopyralid applications to fully mature strawberry in annual plasticulture (Boyd and Dittmar Reference Boyd and Dittmar2015; McMurray et al. Reference Lewis, Roten, Everman, Gannon, Richardson and Yelverton1996) and matted-row systems (Figueroa and Doohan Reference Dittmar, Dufault, Noling, Stansly, Boyd, Paret and Webb2006). There was no increase in yield with the high labeled dose compared with controls, which was contrary to previous findings in matted-row systems (Figueroa and Doohan Reference Dittmar, Dufault, Noling, Stansly, Boyd, Paret and Webb2006).

Clopyralid applied at 140 g ha⁻¹ to strawberry plants at 14 to 28 DATr induced a maximum of 17% damage (Table 2). This dose and timing did not reduce plant heights, aboveground biomass, number of crowns, or yield. Clopyralid applied at 280 g ha⁻¹ at 14 to 28 DATr induced maximal damage of 56%, with recovery by 8 WAT (Table 2). The 280 g ha⁻¹ dose induced 3% stunting compared with controls. Although there were no further reductions in biomass or yield, the damage may be too severe for producer recommendation. Clopyralid applied to strawberry from 14 to 21 DATr at 140 g ha⁻¹ represents a safe application timing and dose to increase spray penetration to gain control of problematic weeds when they are of an ideal size. Results for earlier applications are promising due to the potential for reduced strawberry canopy shielding, increased coverage to M. lupulina, overcoming limited clopyralid translocation out of treated M. lupulina branches, and targeting a susceptible M. lupulina size (Sharpe Reference Pazmiño, Rodriguez-Serrano, Romero-Puertas, Archilla-Ruiz, Del Rio and Sandlio2017; Sharpe et al. Reference Sharpe2016, Reference Sharpe, Boyd, Dittmar, MacDonald and Darnell2018b, Reference Sharpe, Boyd, Dittmar, MacDonald, Darnell and Ferrell2018c). Medicago lupulina emergence began at 45 DATr (861 GDD) and reached 90% peak emergence at 78 DATr (1,416 GDD) (Sharpe Reference Pazmiño, Rodriguez-Serrano, Romero-Puertas, Archilla-Ruiz, Del Rio and Sandlio2017). Warmer temperatures both pre- and post-clopyralid application have increased strawberry leaf cupping in growth rooms, though they did not affect growth parameters (Sharpe et al. Reference Sharpe, Boyd and Dittmar2018a). Further research is required to determine whether clopyralid can control M. lupulina at this timing at doses safe for strawberry in the field.