Plant Material. Experiments were conducted at the University of Georgia in Griffin, GA. ‘Midnight' Kentucky bluegrass (Preferred Seed, Buffalo, NY 14227) and annual bluegrass were seeded in 3.8-cm-diam by 20-cm-deep pots in a greenhouse set for 23/17 C (day/night). Annual bluegrass seed was collected from seedheads of indigenous populations in Griffin, GA. Soil was a mixture of sand and peat moss (80 : 20 v/v). Grasses received fertigation biweekly (MacroN 28-7-14 Sprayable Fertilizer, LESCO Inc., Cleveland, OH 44114) and were watered as needed to promote growth. Plants were selected based on growth stage including: 3- to 5-leaf stage (pretiller), 1-tiller, and 5- to 7-tiller (multitiller).

Greenhouse Experiments. Experiments were conducted to evaluate injury and tillering of annual and Kentucky bluegrass at three growth stages following single or sequential mesotrione applications. Annual bluegrass and Kentucky bluegrass at the three growth stages were treated with single or sequential applications of mesotrione (Tenacity 4SC, Syngenta Crop Protection, Greensboro, NC 27419) at 280 g ha−1. A nonionic surfactant (Activator 90, Loveland Products, Inc., Greeley, CO 80632) at 0.25% v/v was included in the herbicide treatments. A nontreated check for all growth stages was included. Treatments were applied with a CO2-pressured sprayer calibrated to deliver 374 L ha−1 with a single 9504E flat-fan nozzle (TeeJet Spraying Systems, Co., Roswell, Ga 30075). The sequential treatment was applied at 2 wk after initial treatment (WAIT). Grasses were not irrigated for 24 h after treatments (HAT), but received irrigation thereafter to prevent moisture stress. Injury was visually evaluated at 2 and 4 WAIT on a percent scale where 0 equaled no injury and 100 equaled complete desiccation. Tillers were counted at 2 and 4 WAIT.

Root Absorption and Translocation. Experiments were conducted to investigate root absorption of 14C-mesotrione and radioactivity translocation in annual and Kentucky bluegrass at three growth stages. Plants were removed from greenhouse pots, roots were rinsed to remove soil, and plants were grown hydroponically in a 10-L plastic tank filled with a quarter-strength Hoagland solution (Hoagland and Arnon 1950). Grasses were placed through holes in the plastic lid that facilitated root submergence in the solution. The tank was covered with aluminum foil to shield roots from light and placed in a growth chamber (Percival Scientific, Inc. 505 Research Drive, Perry, IA 50220) set for 24/14 C (day/night) with a 12 h photoperiod of 350 μmol m−2 s−1. An aquarium pump was used to provide oxygen to the solution and plants were acclimated to hydroponic culture in the growth chamber for 72 h. This acclimation period and nutrient load were chosen for plants to resume active growth in hydroponic solution without stimulating development beyond the target growth stage.

The solution of the tank was then spiked with 170 kBq of ¹⁴C-mesotrione (109 μCi mg⁻¹, phenyl-ring labeled, 99% chemical purity) plus 1 μM of nonlabeled mesotrione. Plants were harvested 72 HAT. Roots were rinsed for 20 s under a stream of tap water and blotted dry with paper towels. Roots were then separated from shoots with shears and samples were oven-dried for 7 d at 40 C. Samples were then oxidized for 2 min in a biological oxidizer (OX-500, R. J. Harvey Instrument Corp., 11 Jane St., Tappan, NY 10983) and radioactivity was quantified with liquid scintillation spectroscopy (LSC) (Beckman LS 6500®, Beckman Coulter Inc., Fall River, MA 02720). Root absorption was determined by dividing the radioactivity recovered by sample dry weight. Translocation was determined by dividing the ¹⁴C recovered in shoots by the total radioactivity in the plant (roots and shoots).

Foliar Absorption, Translocation, and Metabolism. Experiments were conducted to evaluate foliar uptake, translocation, and metabolism of 14C-mesotrione in annual and Kentucky bluegrass at three growth stages. Plants were established in greenhouse pots as previously described and acclimated in the aforementioned growth chamber for 72 h. Grasses were prepared for radiolabeled treatments by covering the second, fully expanded leaf with flexible film (Parafilm, Bemis Company Inc., Neenah, WI 54956). A broadcast treatment of mesotrione at 280 g ha−1 was applied as previously described. Immediately after the broadcast application, two 1 μL droplets of 14C-mesotrione containing 1.3 kBq each were applied to the second fully expanded leaf with a 10 μL microsyringe (Hamilton Co., Reno, NV 89502). Radioactive droplets were applied in the middle of the leaf and to the sides of the midrib. The spotting solution contained 0.75μg μL−1 of mesotrione to simulate droplets of spray solution. A nonionic surfactant at 0.25% v/v was added to the broadcast and radioactive solutions to facilitate droplet deposition on the leaf surface.

Plants (roots and shoots) were harvested 6 d after treatment (DAT). This harvest timing was chosen from previous research with ¹⁴C-mesotrione (Abit and Al-Khatib 2009; Armel et al. 2005) and from pilot experiments that indicated turfgrasses require at least 3 d to show significant levels of metabolism (PE McCullough, personal observation). The treated leaf was excised and then rinsed with 10 ml of methanol inside a 20-ml scintillation vial. The base of the leaf was held with forceps and rinsate was applied towards the leaf tip with a 5-ml pipette on the leaf surface. This methodology was chosen from pilot experiments that completely removed adsorbed ¹⁴C immediately after treatment and previous research with ¹⁴C-mesotrione on grain sorghum [Sorghum bicolor (L.) Moench.] (Abit and Al-Khatib 2009). Roots were then separated from nontreated shoots with shears and samples were stored at −20 C until analysis.

The treated leaf and nontreated parts (nontreated shoots and roots) were minced and homogenized separately in 20 ml of methanol (FSH 125, Fisher Scientific LLC, 300 Industry Drive, Pittsburg, PA 15275) for 30 s. Samples were then sonicated in water for 8 h at room temperature, centrifuged for 10 min, and the supernatant was transferred to separate tubes. This procedure was repeated with an additional 20 ml of methanol and the supernatants were combined. Plant residue was then placed in water sonication for approximately 6 h in 20 ml of methanol plus 1% formic acid, centrifuged, and the supernatants were combined. Radioactivity of the supernatant for the treated leaf and the rest of the plant were quantified from 4-ml aliquots using LSC. Residue was dried for 72 h in the hood and then combusted in the oxidizer to quantify extraction efficiency. The supernatant from the treated leaf and nontreated parts were then combined to evaluate whole plant metabolism.

The supernatant was then evaporated in a forced-air hood. Samples were resuspended in 40 μL of methanol and spotted on 20- by 20-cm thin layer chromatography (TLC) plates. The TLC plates were developed to 16 cm in a glass chamber using chloroform:methanol (1 : 9). The plates were air-dried and metabolites were detected with a radiochromatogram scanner (BioScan System 200 Imaging Scanner, Bioscan, 4590 MacArthur Boulevard NW, Washington, DC 20007) connected to a computer equipped with Laura Chromatography Data Collection and Analysis Software® (LabLogic System, Inc. 1040 E Brandon Blvd Brandon, FL 33511).

Residue from the treated leaf and nontreated parts were oxidized separately for 2 min in a biological oxidizer and radioactivity was quantified with LSC. Foliar absorption was quantified by dividing the total radioactivity recovered in the supernatant and residue by the total ¹⁴C applied. Translocation was determined by dividing the total radioactivity recovered in nontreated shoots and roots from the total radioactivity recovered in the plant.

Experimental Design and Data Analysis. The design for the greenhouse experiment was a randomized complete block with five replications. The design for all laboratory experiments was completely randomized with five replications. Two runs were conducted for all experiments. Data were subjected to analysis of variance using the General Linear Model Procedure in SAS (SAS v. 9.3, SAS Institute Inc., Cary, NC 27513) and means were separated with Fisher's LSD test at α = 0.05. When experiment-by-treatment interactions were not detected, results were pooled over runs.

Greenhouse Experiments. Growth stage-by-treatment interactions were detected for annual bluegrass and Kentucky bluegrass injury; thus, results are presented across all combinations. At 2 WAIT, mesotrione injured annual bluegrass 32, 20, and 15% at the pretiller, 1-tiller, and multitiller growth stage, respectively (Table 1). Kentucky bluegrass was injured no more than 5% at all growth stages. At 4 WAIT, single mesotrione applications injured annual bluegrass 54, 33, and 11% at the pretiller, 1-tiller, and multitiller growth stage, respectively. Sequential applications increased injury 20 and 17% from single treatments on pretiller and 1-tiller annual bluegrass, respectively. The sequential treatment only injured multitiller annual bluegrass 14% at 4 WAIT and was similar to injury from the single application. The sequential treatment injured pretiller Kentucky bluegrass more than tillered plants, but injury did not exceed 12%.

Table 1.

Injury of annual bluegrass and ‘Midnight' Kentucky bluegrass with mesotrione at 280 g ai ha⁻¹ in two greenhouse experiments, Griffin, GA. Results were pooled over experimental runs.

Growth stage-by-treatment interactions were detected for tiller counts at both dates; thus, results are presented across all combinations. At 2 WAIT, mesotrione reduced annual bluegrass tiller count 32% compared to the nontreated when applied at the 1-tiller growth stage (Table 2), but other growth stages were similar to the nontreated. At 4 WAIT, differences between single and sequential applications were not detected for tiller counts. Mesotrione inhibited tillering of annual bluegrass 58 and 38% compared to the nontreated when applied at the pretiller and 1-tiller growth stage, respectively (Table 2). Mesotrione did not inhibit tillering of multitiller annual bluegrass plants compared to the nontreated. Kentucky bluegrass tillering was not inhibited following single and sequential mesotrione applications at all growth stages 2 and 4 WAIT.

Table 2.

Tiller count for annual bluegrass and ‘Midnight' Kentucky bluegrass treated with mesotrione at 280 g ai ha⁻¹ in two greenhouse experiments, Griffin, GA. Results were pooled over experimental runs.

Results suggest mesotrione has the greatest efficacy on annual bluegrass prior to reaching a multitiller growth stage. Kentucky bluegrass has superior tolerance to mesotrione compared to annual bluegrass, and treatments did not inhibit tillering from the nontreated at any growth stage. These results support previous observations on the differential tolerance levels of annual bluegrass to mesotrione at various stages of maturity in field experiments (Reicher et al. 2011; Skelton et al. 2012). POST applications of mesotrione at 140 and 210 g ha⁻¹ applied 4 wk after emergence did not reduce Kentucky bluegrass cover from the nontreated (Venner 2011). Similarly, Askew and Beam (2002) reported no injury to Kentucky bluegrass following sequential mesotrione applications. Annual bluegrass injury and growth inhibition from mesotrione would likely give Kentucky bluegrass a competitive growth advantage during seedling establishment.

Mesotrione efficacy for controlling immature annual bluegrass is also consistent with the susceptibility of other weeds at various growth stages. Canada thistle [Cirsium arvense (L.) Scop.] was more susceptible to mesotrione at the rosette stage compared to the bolting stage (Armel et al. 2005). Researchers have also noted that mesotrione efficacy on smooth [Digitaria ischaemum (Schreb.) ex Muhl.] and large crabgrass [D. sanguinalis (L.) Scop.] was reduced on multitiller plants compared to seedlings (McCurdy et al. 2008; Whaley et al. 2006). The effects of growth stage also influence the susceptibility of weeds to other herbicides. For example, Chism et al. (1992) reported that southern crabgrass [Digitaria ciliaris (Retz.) Koel.] at pretiller and two- to four-tiller stages were more susceptible to quinclorac than at the flowering stage. Kells et al. (1984) noted that fluazifop caused greater injury to quackgrass [Elytrigia repens (L.) Desv. ex B.D. Jackson, now Elymus repens (L.) Gould] at 2- to 3-leaf stage than at a 5- to 6-leaf stage.

The tolerance of annual and Kentucky bluegrass to mesotrione at the multitiller stage suggests that applications could be used for selectively controlling other weeds, such as crabgrass (Digitaria spp.), in mixed stands. However, annual bluegrass exhibits significantly more susceptibility to mesotrione prior to tillering, compared to after establishment, and applications could be injurious. The selectivity for controlling pretiller annual bluegrass suggests mesotrione has differential behavior in these species that is influenced by growth stage.

Absorption and Translocation. Species-by-growth stage interaction was not detected for absorption or translocation of root-applied 14C-mesotrione; thus, results are presented by main effect (Table 3). At 72 h of root exposure to nutrient solution containing 14C-mesotrione, annual bluegrass accumulated more 14C in whole plant and shoots per dry weight than Kentucky bluegrass. Annual bluegrass accumulated 479 and 444 Bq g−1, while Kentucky bluegrass accumulated 357 and 332 Bq g−1 in whole plant and shoots, respectively. For both species, differences were not detected for relative absorption (Bq g−1) of 14C-mesotrione in whole plant and shoots among growth stages. For both species, pretiller plants translocated approximately 10% less of the root-absorbed radioactivity to shoots than tillered plants, but differences between species were not detected.

Table 3.

Root absorption and translocation of radioactivity to shoots for annual bluegrass and ‘Midnight' Kentucky bluegrass at 72 h after treatment with ¹⁴C-mesotrione in two experiments, Griffin, GA. Results were pooled over experimental runs.

Uptake and subsequent transport from roots has been attributed to the selectivity of other herbicides for annual bluegrass control in turfgrass. For example, annual bluegrass had greater root absorption of ¹⁴C-amicarbazone than creeping bentgrass (Agrostis stolonifera L.) and tall fescue [Festuca arundinacea Shreb., now Lolium arundinacea (Schreb.) S. J. Darbyshire] (Yu et al. 2013). In other experiments, annual bluegrass had similar root absorption of ¹⁴C-primisulfuron-methyl to Kentucky bluegrass, but the transport of primisulfuron acid to shoots was greater in annual bluegrass due to slower metabolism (McCullough et al. 2015). Soil uptake might be critical for mesotrione selectivity on annual bluegrass seedlings during Kentucky bluegrass establishment. Seeded areas typically take several weeks for Kentucky bluegrass to reach greater than 50% ground coverage, and applications could have minimal spray retention on immature foliage.

Species-by-growth stage interactions were not detected for foliar absorption and translocation after 144 h. Foliar absorption levels averaged 31% (± 1 SEM) and 35% (± 1) of the applied radioactivity for annual and Kentucky bluegrass, respectively, but differences among growth stages were not detected (data not shown). Abit and Al-Khatib (2009) reported no differences in foliar absorption of ¹⁴C-mesotrione in tolerant and susceptible sorghum hybrids. Annual bluegrass has exhibited greater foliar uptake of ¹⁴C-amicarbazone, ¹⁴C-bispyribac-sodium, and ¹⁴C-ethofumesate than creeping bentgrass (Kohler and Branham 2009; Lycan and Hart 2006; Yu et al. 2013). Foliar absorption levels of mesotrione might be inconsequential for the selectivity between annual and Kentucky bluegrass.

Research on foliar uptake of mesotrione in grasses at various growth stages is limited. Canada thistle in the rosette stage absorbed 5 to 10% more foliar-applied ¹⁴C-mesotrione than at the bolting stage (Armel et al. 2005). The speed of mesotrione uptake was not measured in annual and Kentucky bluegrass due to the number of plants and growth stages that were evaluated. Perhaps annual bluegrass absorbs mesotrione more quickly than Kentucky bluegrass and warrants further investigation.

Species-by-growth stage interaction was not detected for translocation of foliar-absorbed ¹⁴C-mesotrione. Annual and Kentucky bluegrass translocated 27 to 32% of radioactivity out of the treated leaf (Table 4). In previous research, the majority of radioactivity was also recovered in the treated leaf of grain sorghum hybrids after 7 d (Abit and Al-Khatib 2009). Annual and Kentucky bluegrass have exhibited similar translocation of ¹⁴C-primisulfuron-methyl, despite the differential tolerance levels to applications (McCullough et al. 2015). Researchers have also noted greater translocation of radioactivity from ¹⁴C-amicarbazone and ¹⁴C-bispyribac-sodium in annual bluegrass compared to creeping bentgrass (Lycan and Hart 2006; Yu et al. 2013). Results suggest that translocation patterns do not explain the selectivity of mesotrione in these bluegrass species.

Table 4.

Translocation of radioactivity for annual bluegrass and ‘Midnight' Kentucky bluegrass at 144 h after foliar-applied of ¹⁴C-mesotrione in experiments, Griffin, GA. Results were pooled over experimental runs.

Tillered grasses translocated 24% of the absorbed radioactivity to nontreated shoots and roots. Pretiller plants translocated 41% of the ¹⁴C absorbed, which was greater than both tillered growth stages tested. The efficacy of mesotrione for controlling seedling annual bluegrass could be attributed to translocation after foliar uptake. Armel et al. (2005) noted higher levels of radioactivity translocation from ¹⁴C-mesotrione in rosette Canada thistle compared to the bolting stage. Although Kentucky bluegrass had less injury to mesotrione than annual bluegrass, pretiller plants had more injury than tillered plants for both species. Greater translocation following the foliar absorption could contribute to the susceptibility of pretiller plants to mesotrione injury, but results do not explain the differential tolerance levels between species.

Metabolism. Extraction of radioactivity from plants averaged 87% (± 0.6) (data not shown). The Rf of parent mesotrione was 0.7 and two major metabolites were identified in both species at Rf 0.05 and 0.6 after foliar uptake (Figure 1). Growth stage-by-species interaction was not detected for metabolism of 14C-mesotrione. Annual bluegrass and Kentucky bluegrass had similar metabolism of 14C-mesotrione (65 to 68%) (Table 5). This range in mesotrione degradation is similar to previous reports in grain sorghum after 7 d (Abit and Al-Khatib 2009). However, parent herbicide levels in pretiller and 1-tiller plants were approximately two times greater than multitiller plants (40 vs. 21%). Compared to the multitiller stage, pretiller and 1-tiller plants averaged 26% fewer polar metabolites and approximately three times greater nonpolar metabolite levels.

Figure 1.

Radiochromatogram scans of the major metabolites detected in pretiller and multitiller Kentucky bluegrass.

Table 5.

Metabolism of ¹⁴C-mesotrione in annual bluegrass and ‘Midnight' Kentucky bluegrass at 144 h after treatment in two experiments, Griffin, GA. Results were pooled over experimental runs.

Metabolism was noted as the primary basis for differential response of crops to mesotrione (Abit and Al-Khatib 2009; Ma et al. 2013; Mitchell et al. 2001). Abit and Al-Khatib (2009) reported a tolerant sorghum hybrid metabolized 7% more foliar-absorbed mesotrione compared to a susceptible hybrid. Enhanced metabolism of mesotrione has been reported in corn (Zea mays L.) from cytochrome P450 monooxygenase-mediated detoxification mechanisms (Hawkes et al. 2001). Pataky et al. (2009) reported the mutation of a cytochrome P450 gene, referred to as nsf1 or ben1, resulted in reduced enzyme activity and increased sensitivity of corn to cytochrome P450-metabolized herbicides, including mesotrione and nicosulfuron. Ma et al. (2013) noted corn is naturally tolerant to mesotrione due to a rapid rate of P450-catalyzed ring hydroxylation. This supposition was supported by increased corn injury by mesotrione following the addition of malathion, a cytochrome P450 monooxygenase inhibitor. Perhaps multitiller plants of annual and Kentucky bluegrass have higher concentrations of P450 enzymes than pre- and 1-tiller plants, which contribute to a more rapid degradation of mesotrione. The role of metabolism does not explain the relative differences in tolerance to mesotrione between annual and Kentucky bluegrass.

Mesotrione can be used for selective annual bluegrass control during Kentucky bluegrass establishment from seed. However, efficacy is significantly limited for controlling mature annual bluegrass plants. The selectivity of mesotrione between these species is attributed to differential root-absorption levels. Metabolism is probably inconsequential for mesotrione selectivity between these species, but multitiller annual bluegrass might have greater degradation than pretiller or 1-tiller plants. Further research is needed to analyze differences in the susceptibility levels of HPPD enzymes to inhibition by mesotrione. Research is also warranted to quantify differential levels of uptake between Kentucky bluegrass cultivars with varying levels of susceptibility to mesotrione injury.