Field trials were conducted to assess the impact of aminocyclopyrachlor on green ash (Fraxinus pennsylvanica Marshall) and honey locust (Gleditsia triacanthos L.) trees in an urban environment. Aminocyclopyrachlor is a relatively new, selective, plant-growth-regulator herbicide in the pyrimidine carboxylic acid family. Treatments were applied to Kentucky bluegrass (Poa pratensis L.) sod growing with and without trees present. Evaluations included determination of a safe spraying distance from target trees and the effect of application timing on tree response. This multi-year study showed that green ash was highly tolerant to aminocyclopyrachlor while honey locust developed severe injury in trees closest to applications. Honey locust trees up to 7 m (23 ft) from the tree trunk to the edge of the application displayed moderate to severe injury symptoms and fall treatment in October and November had the lowest tree injury compared to all other application timings. Honey locust trees exhibiting moderate to severe cosmetic injury would not be acceptable to landowners; recovery over time was minimal. Trees located 13 m (43 ft) away displayed no injury for any treatment timing. Soil analysis demonstrated that aminocyclopyrachlor dissipation was the same underneath green ash and honey locust trees, and that dissipation was faster in the presence of growing trees. Taken together, these results provide a basic groundwork necessary for improving aminocyclopyrachlor labels, and a better understanding of this herbicide's effect on certain woody species.
Index words: Herbicide injury, aminocyclopyrachlor, herbicide fate, tree safety.
Species used in this study: Green ash, Fraxinus pennsylvanica Marshall, honey locust, Gleditsia triacanthos L.
Chemicals used in this study: Aminocyclopyrachlor.
Significance to the Horticulture Industry
Aminocyclopryachlor (ACP) was developed and originally marketed as a significant new lawn care herbicide for the control of dandelions (Taraxacum sp.), ground ivy (Glechoma hederacea L.), wild violet (Viola sp.), and other troublesome weeds in turf. Its broad spectrum of control of annual and perennial weeds was well studied and characterized. This study was conducted in a large stand of established green ash (F. pennsylvanica) and honey locust (G. triacanthos) trees planted in a pattern in a stand of Kentucky bluegrass (Poa pratensis) sod. Green ash exhibited excellent ACP tolerance while honey locust required 13 m (43 ft) distance from treated sod to exhibit clear tolerance. This large study demonstrated that different tree species can exhibit huge differences in tolerance to ACP, showing that tree response in targeted treatment areas should be carefully evaluated prior to treating large areas with ACP. Recovery from honey locust injury was modest at best and would not satisfy the concerns of tree owners. Tree response was from soil uptake of ACP as no ACP was applied to any part of tested trees. Care should be taken in applying ACP in the proximity of desirable trees that may be sensitive to this herbicide.
Green ash (Fraxinus pennsylvanica Marshall) (GA) and honey locust (Gleditsia triacanthos L.) (HL) are two popular and widespread urban trees planted near turf areas settings in cities around the United States of America. Green ash is a relatively fast-growing tree valued for its shade and attractive conformation, while honey locust is a long-lived hardy species that does well in many soils in the central Great Plains of the U.S. Both species are considered medium to large-size urban trees that are readily available for new plantings (Arbor Day Foundation 2020).
Aminocyclopyrachlor (6-amino-5-chloro-2-cyclopropyl-4-pyrimidinecarboxylic acid; ACP) is a selective, auxin mimic herbicide in the pyrimidine carboxylic acid family (Turner 2009). Its structure resembles the pyridine carboxylic acids, another class of auxinic herbicides that includes active ingredients such as clopyralid, aminopyralid, and picloram (Bukun et al. 2010). It is characterized by a pyrimidine ring, with reactive groups that include a carboxylic acid group and an amine group. With a pKa of 4.65, ACP is often in its anionic form in soil, leading to a higher degree of polarity (Oliveira et al. 2011, Oliveira et al. 2013) with a log Kow of -1.12 at a pH of 4, and -2.48 at a pH of 7 (Shaner 2014). Based on those parameters, ACP has a relatively high predicted leaching potential. Its reported half-life ranges from 32.5 d under field conditions (Lindenmayer et al. 2013) up to 433 d in a sandy loam soil (Shaner 2014).
ACP provides control of selected broadleaf weeds as well as invasive tree species such as Russian olive (Eleagnus angustifolia L.) (Getts 2015). Auxin mimic herbicides cause growth regulator-type symptoms such as leaf and petiole epinasty, chlorosis, and necrosis (Feucht 1988). For auxinic herbicides with longer soil persistence, there is increased risk of root uptake, resulting in non-target injury to nearby tree species. This has been seen with the use of aminopyralid near ponderosa pine (Pinus ponderosa L.) (Wallace et al. 2012).
Early ACP label language mentioned that extra care should be taken within the dripline of desirable trees; however, it is known that tree roots may reach extensively past the dripline in the absence of restrictive soil or substrate characteristics, and that these extensive roots play a large role in water and nutrient uptake for trees (Stone and Kalisz 1991). Since tree root growth is influenced by temperature, water availability, and other abiotic factors, maximum radial root growth can differ under variable environmental conditions. Thus, radial tree root architecture can differ widely among species (Coutts 1989, Stone and Kalisz 1991). Golf courses increasingly are designed to promote a healthy interface between native plants, including trees, and highly managed greens and fairways where chemical use can impact native vegetation.
Field trials were conducted from 2012 to 2015 to assess the impact of ACP on GA and HL growing in a Kentucky bluegrass sod irrigated with urban pop-up sprinklers (Fig. 1). The objectives were to determine the sensitivity of GA and HL to ACP, determine a minimum no-damage safe spraying distance from GA and HL trees, assess ACP application timing on tree injury, and to determine if ACP dissipation was similar underneath GA and HL trees.
Material and Methods
Test site description
Field research with GA and HL trees was conducted at the Colorado State University Agricultural Research Development and Education Center's (ARDEC) Tree and Turf Research Area, which was originally established in the mid 1990's. In a Kentucky bluegrass turf, GA and HL were arranged into nine blocks watered with an underground irrigation system with pop-up sprinkler heads used in urban lawns. All blocks contained GA and HL trees growing in columns of three with nine rows in each block, for a total of 27 GA and 27 HL trees in each block. HL and GA were either placed adjacent to one another or separated by a strip of Kentucky bluegrass (Fig. 1). Each block was irrigated independently, allowing post-application herbicide incorporation. Rows were spaced 6 m (20 ft) apart with trees within rows spaced 3.5 m (11 ft) apart. At the time of the first herbicide applications in 2012, the trees were 16 years old ranging in height from 6 to 10 m (20 to 33 ft). Soil at the site was a Fort Collins loam with a taxonomic class listed as fine-loamy, mixed, superactive, mesic Aridic Haplustalf with a pH of 8 and 2.2% organic matter.
Tree response trials
To determine herbicide application timing injury effects, different ACP (aminocyclopyrachlor, Imprelis, E.I. Dupont de Nemours and Company, 1007 Market Street, Wilmington, DE 19898) application timings corresponding to spring, summer, and fall were used during 2012, 2013 and 2014. Applications in 2012 were made on April 14, June 22 and October 20. Applications in 2013 were made on July 1, October 10 and November 27, and applications in 2014 were made on April 21 and July 28. Applications were made only once (there were no sequential applications on any plots) and observations were made annually. Plots were arranged by spraying two separate 3 m (10 ft) wide strips of ACP at a rate of 210 g ai. ha−1 (0.2 lb ai.A-1) within each block with a CO2 powered backpack sprayer. These strips were located halfway between rows of HL and GA trees such that the tree trunks were 1 m away from the edge of the application, the next row(s) at seven m away, and a third row 13 m away (Fig. 1). No spray solution was ever applied directly to the trees.
Since the site consisted of established large trees arranged in a specific pattern, the experiment was designed so there were six trees of each species 1 m from the edge of each application, and at least three trees at 7 m and 13 m from the edge. In some cases, there were six trees located 7 m from the edge of the application, depending on where the application was made within the block (on the edge, or more toward the middle). Each tree represented a biological replication. Between the two application strips in each block there were 12 replications of trees 1 m (3 ft) from the edge of the application, at least six replications at 7 m and six replications at 13 m. To ensure that treatments at a given application timing received similar amounts of moisture, the two strips of ACP were applied inside of the same block. The two ACP applications in each block were separated by at least 4 rows of trees to ensure that ACP from one application strip did not affect trees closest to the other. After application, plots received 1.25 cm (0.5 in) of moisture utilizing the on-site irrigation system to help move the herbicide into the soil.
Trees treated in 2012 were assessed one, two, and three years after treatment (YAT). Trees treated in 2013 were assessed one, and two YAT. Trees treated in 2014 were assessed one YAT. Assessments consisted of measuring from the edge of application to the furthest trees exhibiting injury symptoms (Table 1). Fisher's LSD at p < 0.05 in SAS (SAS Institute Inc., Cary, NC 27513-2414, USA) was used to compare injury ratings.
Tree recovery was evaluated by pooling treated trees based on 1 or 7 m from the edge of the application. Next, these pools were sorted into groups corresponding to the treatment timing for a specific application year. Recovery was assessed by taking subsequent ratings for specific trees and subtracting the rating for a previous year. Groups assessed for trees treated in 2012 were as follows: change in rating between 2013 and 2015 and change in rating between 2014 and 2015. The only group assessed for trees treated in 2013 was the change in percent injury between 2014 and 2015. Since trees treated in 2014 were only rated one year after treatment, no recovery data could be assessed. Individual trees that showed a negative value indicated recovery, while positive values indicated a worsening of symptoms. Groups of trees were plotted for each timing, and two-sided t-tests were performed using SAS (SAS, 820 SAS Campus Drive Cary, NC 27513-2414) in order to determine whether the mean change in injury rating for each timing was different from zero. Means tested were comprised of varying numbers of trees, depending on available biological replications for each timing and distance.
ACP soil analysis. To investigate the extent to which tree root absorption might be contributing to ACP dissipation, a separate study was established in the spring of 2015. Two ACP treatments of 210 g ai.ha−1 were placed corresponding to a 90-foot strip underneath rows of GA and HL trees, and a 90-foot long strip set away from nearby trees with only sod cover in the treated plot. Prior to application, six petri dishes containing filters were placed in each strip using a random pattern to capture 0 DAT data. Next, a total of twelve bare soil cores were randomly pulled with a zero-contamination soil probe down to 30 cm (12 in) from within plots 7 DAT, 30 DAT, and 45 DAT. Groups of three randomly assigned cores were mixed together to get a total of four reps for each time point of the experiment. These mixed samples were stored in the freezer at -20 C (-4 F) until analysis using an LC-MS/MS.
Soil cores were separated into four 7.5 cm (3 in) segments representing depths of 0-7.5, 7.5-15, 15-22.5, and 22.5-30 cm. Two g of well-mixed soil from each sample were placed into 15 mL centrifuge tubes followed by the addition of 5 mL of deionized water. Tubes were placed on a shaker for 2.5 hours to complete extraction. Tubes were then spun in a centrifuge at 4,000 × g for 15 min to settle suspended solids from the extracted solution. A 1.5 mL aliquot of the extract was filtered into a 1.5 mL UPLC vial using an Acrodisc® 13 mm syringe filter with a 0.25 μm nylon membrane (PALL Corporation, Port Washington, NY 11050). The vials were capped and then analyzed using a Shimadzu LC-MS/MS 8040 (Shimadzu Scientific Instruments, Columbia, MD 21046).
The analytical system consisted of a Nexera X2 UPLC with 2 LC-30AD pumps, a SIL-30AC MP autosampler, a DGU-20A5 Prominence degasser, a CTO-30A column oven, and a SPD-M30A diode array detector coupled to an 8040 quadrupole mass-spectrometer. The average μg ACP applied over an area of 3.8 cm−2 was determined for treated plots at each time point, and each depth zone was analyzed independently to quantify vertical ACP movement. Unpaired t-tests with Welch's correction were performed in Prism (GraphPad, San Diego, CA 92108) to determine differences between treated plots. Changes at each depth zone over time were also analyzed using Tukey's HSD and R statistical software (R Studio, Boston, MA 02210).
Results and Discussion
Injury symptoms and observations.
There was a large difference in ACP response between GA and HL trees. GA trees were tolerant to ACP and did not develop any injury symptoms during the 3 yr of this project (Fig. 2). HL trees were very sensitive to ACP and developed various degrees of injury. Consequently, injury rating data presented are limited to injury observed on HL trees.
Injury symptoms from spring and summer application differed from fall applications. The initial April application in 2012 did not cause injury symptoms until after the first rainfall in May, indicating that rainfall and/or irrigation were required for ACP movement into soil, increasing root uptake and tree injury. When adequate moisture was available, injury symptoms appeared relatively quickly, sometimes within 1 week of the ACP application, appearing as chlorosis on the lowermost leaves of the affected tree. Injury symptoms included typical auxinic epinasty and malformed growth of new leaves and tissues; however, within two weeks after the May rainfall, the symptoms observed on the HL trees included chlorosis and necrosis. This chlorosis started at the base of the petiole, and moved out along the leaf, eventually affecting all of the leaflets (Fig. 3A). Next, necrosis started out from the base of the petiole, over-taking the leaflets, and eventual severe injury resulted in defoliation of the affected parts of trees. The chlorosis first appeared in the tops of the HL trees, with some exceptions where shoots (suckers) near the base of the trunk showed chlorosis first, typically within one to two weeks after application. Injury symptoms from the spring and summer treatments were not restricted to new growth.
Fall treatments were made after the trees dropped leaves and entered dormancy, resulting in no observable symptoms until the following spring. In these cases, herbicide injury symptoms differed. Leaf petioles were twisted, and leaflets showed pronounced curling (Fig. 3B), which was a response more typical with auxin mimic herbicides. Eventually, these epinastic symptoms turned into chlorosis that began at the tip of the leaves and developed toward the base of the petiole. Severe cases from fall treatments also resulted in tree defoliation. Where the herbicide did not cause severe injury on the tree, injury symptoms were frequently localized to the side of the tree closest to the herbicide application. Symptoms for trees treated in the spring, summer, and fall persisted into the first, and second YAT, with new growth in subsequent years showing epinastic symptoms and termination of new shoots. The most severe cases showed deformed trunk growths (e.g. callus formation), including vertical splits in trunk bark, resulting in little to no recovery. Normal HL double compound leaves are shown in Fig. 3 C.
Visual injury ratings 1 YAT (Fig. 4A) showed no differences between the April and June application for trees 1, 7, and 13 m from the application strip. Trees 1 m from the edge of the application showed less injury from the October application, relative to the April and June applications, possibly due to less root uptake during winter months. Trees 7 m from the application edge displayed lower injury from the October application as well. Trees 13 m away from the application displayed no injury symptoms from any application timing.
Visual injury ratings taken 2 YAT (Fig. 4B) showed different results. There was no injury difference between April and June application timing for trees located 1, 7, and 13 m from the edge of the application. Injury to October treated trees 1 m away still showed less injury than the April and June treatments; however, this injury was still quite severe at an average rating of 90%. October-treated trees 7 m away showed higher injury than the previous year. These trees also had a higher injury rating than June-treated trees but had similar injury to the trees in the April treatments. Trees located 13 m away displayed no injury for any treatment timing.
Visual injury ratings recorded 3 YAT (Fig. 4C) showed severe injury (>90%) for trees 1 m from the edge of the application for all three application timings. The October treatment resulted in slightly less injury compared to the June timing, but was similar to the injury observed from the April application. Tree injury for the June application was also similar to injury observed from the April timing. These findings may be associated with lower temperature and precipitation during the fall compared to the summer when root uptake is more favorable. By 3 YAT, trees 7 m from the edge of the application showed no significant differences in injury for April, June and October treatment timings. Comparison of 2012 application distances showed significantly more injury on trees 1 m from the edge of the application than trees 7 m away for 1, 2, and 3 YAT. No injury was observed on trees 13 m from the edge of the applications.
Because there appeared to be less injury observed on fall-treated trees vs spring and summer for 2012, fall 2013 applications were made to determine whether October or November treatments were safer for HL trees. A July treatment was included to compare summer treatments to fall treatments.
One YAT (Fig. 5A), average injury for trees treated in July 1 m from the application was near 100%, with average injury for October and November-treated trees significantly lower at 40 and 44%, respectively. Trees 7 m from the application showed a similar trend. July-treated trees showed average injury ratings of 73% while October and November-treated trees had injury ratings of 8% and 18% respectively. There were no significant differences between average injury rating of the fall treatments at either 1 or 7 m from the application, and trees at 13 m did not show injury symptoms.
The 2 YAT (Fig. 5B) ratings showed largely similar results. The two fall treatments still showed significantly reduced injury compared to the July treatment for trees both 1 and 7 m from the application, and there were still no significant differences in average injury rating between October and November treatments. Comparison of 1 YAT ratings for trees located 1 m from the edge of the application showed significantly higher injury ratings than trees located 7 m away from the edge of the application (Fig. 5A). There were no injury symptoms on trees 13 m from the edge of the application. The 2 YAT ratings (Fig. 5B) showed the same result. Average injury rating for July treated trees 7 m from the application were lower than 2012 applications, and October and November treated trees both 1 and 7 m from the application were also lower than 2012 applications.
Injury ratings 1 YAT for HL trees treated in 2014 showed a higher level of injury on trees 1 m from the application for the April timing than the July timing (100% and 40% respectively) (Fig. 6). (April and July). This same trend was seen for trees 7 m away from the application, with April application averaging 73% injury while July application averaged only 8%, possibly due to more rainfall in May and June. Trees 13 m away showed no injury for April or July treated trees. With respect to distance, there was a significant decrease in injury between 1 and 7 m from the application, with averages dropping from 100% to 73% for the April timing, and dropping from 40% to 7% for the July timing.
In general, trees treated in 2012 did not show recovery between the 2013-2015 ratings. October-treated trees 7 m from the application also showed no major recovery. The data summed in Table 2 shows trees 7 m away from the application treated in October had significantly higher injury in 2015 than the rating taken during 2013, showing on average an injury rating of 38% higher than the initial rating (p-value=0.0022). Moderate to minor recovery could be seen on trees treated in 2012 during the year between 2014 and 2015 with 7-m trees treated in both April and October showing significant decreases in injury between 2014 and 2015 (p values = 0.0153 and 0.0024 respectively). Trees treated in 2013 also showed signs of recovery between 2014 and 2015. Trees treated in November at both 1 and 7 m from the edge of the application showed significant recovery (p-values 0.0001 and 0.0006) between years. In general, trees 7 m from the application showed more recovery, and fall-treated trees showed more recovery than spring- or summer-treated trees. In spite of visible modest recovery in some cases, in general, cosmetic honey locust injury from ACP would not be acceptable to tree owners.
Analysis of ACP residues in soils
The impact of presence or absence of trees on the fate of ACP in soil was determined by LC MS/MS analysis. There was no difference in the amount of ACP applied over the Kentucky bluegrass ground cover next to GA or HL trees and away from trees (data not shown). By 10 DAT, there was a significant difference (p-value = 0.0097) in total recovered ACP between the ‘tree' and ‘no tree' treatments, with the plots without trees having 30% more ACP residues than in the soil samples near the trees (Fig. 7), averaged across soil sampling depths . Both the 30 and 45 DAT samples showed this same trend (p-values = 0.0040 and 0.0012, respectively), with the plots without trees showing a significantly higher amount of ACP than the plots with trees present, possibly due to herbicide uptake by the tree roots (Fig. 7).
Injury symptoms and observations summary
The difference in response between the two tree species to ACP was extreme. While information is scarce regarding ACP's effect on specific tree species, ACP applications resulted in only 28% injury in loblolly pine (Pinus taeda L.) trees one year after treatment (Roten 2011). It is not known why GA is tolerant to ACP, although several different factors can affect overall sensitivity to a given herbicide. Differences in absorption or translocation of the herbicide (Adu-Yeboah et al. 2014, Goggin et al. 2016), enhanced metabolism (Han et al. 2013), and changes to how the herbicide interacts with the target site (Powles and Yu 2010) can all influence whether or not an herbicide is effective in altering plant growth or function. Future studies investigating this in GA could help in the development of markers, to help determine whether given trees will be tolerant to ACP.
ACP injury symptoms differed depending on application timing with fall and spring applications caused twisting and epinasty of affected leaves. Treatment during summer months after foliage had reached full size included chlorosis, necrosis, and defoliation. This may be due to less actively growing foliage, whereas in spring treatments are made after bud burst when leaves are actively growing. Fall applications occurred after dormancy, so injury was not visible until the following spring during new growth. In addition, root uptake can be significantly lower in the fall compared to the winter, which explains the lower injury levels in the colder season. The fact that herbicide injury tended to exist on the side of the tree closest to the application is consistent with a mechanism of root uptake, as only portions of the root system would have been exposed to the herbicide (Feucht 1988). Generally, trees that were severely injured in the first year did not recover in subsequent years. After the initial treatment of ACP trees in April of 2012, it was also noted that no injury symptoms were observed until a week after the first major rainfall of the spring, which was 1.2 cm (0.5 in) of rain. Because of this, later treatments utilized the irrigation system at the site to ensure at least 1.2 (0.5 in) cm of moisture was available to activate the herbicide.
One goal of this experiment was to determine whether injured trees would recover over time. In general, trees treated in 2012 did not show any recovery or got worse between 2013 and 2015. However, between 2014 and 2015, October-treated trees 7 m from the edge of the application showed recovery, which suggests that the trees got worse in the initial year after application, and then started to recover in the second year. Overall, results of the change in rating for individual trees year to year suggested that fall months and being farther from the application were more likely to result in tree recovery. This coupled with lower injury ratings compared to spring and summer application timings indicates that applications during the dormant season of the tree in the fall may be the best time to apply ACP. This was also suggested for the use of aminopyralid in ponderosa pine and for the establishment of pine forests as a method of plant growth regulator herbicide selectivity to avoid pine injury (Paley and Radosevich 1984, Wallace et al. 2012).
Outdoor studies were conducted over 3 yr to evaluate the response of green ash and honey locust to ACP applied at 210 g ai ha−1 when measured at 1, 7, and 13 m from the edge of a 3 m wide strip sprayed on Kentucky bluegrass sod. At no time did green ash develop injury symptoms irrespective of year or distance from the target spray strip. In contrast, honey locust developed moderate to severe injury symptoms which lessened with increasing distance from the target spray strip. Symptoms varied depending on the application time of year in relation to honey locust annual growth pattern. Symptoms included epinasty, leaf chlorosis and death, and in severe cases, vertical splitting of dying bark. Such symptoms were generally irreversible and would not be tolerated by tree owners. Honey locust trees located 13 or m away from a spray area appear to be safe from ACP injury. Treated areas containing Kentucky bluegrass sod and trees showed greater ACP dissipation compared to Kentucky sod with no trees. ACP exhibits both foliar and soil activity on sensitive plants making it a chemical that must be used with caution to limit potential for injury to desired trees.
Professor, Agricultural Biology, Colorado State University, Campus Delivery 1177, Fort Collins, CO 80523-1177 USA.
Graduate Student, Agricultural Biology, Colorado State University, Campus Delivery 1177, Fort Collins, CO 80523-1177 USA.
Associate Professor, Agricultural Biology, Colorado State University, Campus Delivery 1177, Fort Collins, CO 80523-1177 USA.