Plant culture.

The study was conducted in a greenhouse at the University of Tennessee–Knoxville between Aug. and Sept. 2009. Daytime air temperatures in the greenhouse averaged 29.5 °C.

Cores (6 cm diameter, 5 cm depth) of ‘Riviera’ common bermudagrass were harvested from the East Tennessee Research and Education Center, Knoxville, TN, and transplanted into 10-cm diameter pots filled with a peatmoss (55%), perlite (25%), and vermiculite (20%) growing medium (Super Fine Germinating Mix; Conrad Fafard Inc., Agawam, MA). After transplanting, plants were fertilized with a complete fertilizer biweekly (20 N:20 P₂O₅:20 K₂O; Howard Johnson's Triple Twenty Plus Minors, Milwaukee, WI) at 5.2 kg nitrogen/ha and watered as needed with overhead irrigation to maintain adequate soil moisture. Hand shears were used to clip plants three times per week to a height of 2 cm. Plants were allowed to resume active growth for 3 weeks before study initiation.

Herbicide treatments.

Plants were treated with mesotrione (Tenacity^®; Syngenta Professional Products, Greensboro, NC), topramezone (Impact^®; Amvac Chemical, Los Angeles, CA), and tembotrione (Laudis^®; Bayer CropScience, Research Triangle Park, NC) at low, medium, and high application rates. Low, medium, and high rates for mesotrione were 0.28, 0.35, and 0.42 kg·ha⁻¹, respectively. For topramezone, low, medium, and high rates were 0.018, 0.025, and 0.038 kg·ha⁻¹, respectively. Low, medium, and high rates for tembotrione were 0.092, 0.184, and 0.276 kg·ha⁻¹, respectively. Low rates represented the maximum labeled use rate for a single application of each herbicide (Anonymous, 2006, 2009a, 2009b). A non-treated control was also included for comparison. All herbicides were mixed with a methylated seed oil surfactant (MSO; Loveland Industries, Greeley, CO) at 0.25% v/v, because researchers have reported that MSO increases the herbicidal activity of mesotrione, topramezone, and tembotrione more than non-ionic surfactants or crop oil concentrates (Young et al., 2007). Treatments were applied using a CO₂-powered boom sprayer containing four flat-fan nozzles (TeeJet 8002 flat fan spray nozzle; Spraying Systems Co., Roswell, GA) calibrated to deliver 280 L·ha⁻¹ of spray volume. Nozzles were spaced 25 cm apart and maintained 25 cm above the surface while spraying. Before treatment application, plants were clipped for the last time at 2-cm height. Leaf material above 2 cm was harvested from individual plants 3, 7, 14, 21, 28, and 35 DAA. Both treated and non-treated plants were harvested at these intervals.

Data collection.

Bermudagrass tissue bleaching was assessed on each sampling date before harvest. Tissue bleaching was visually rated for each whole plant on a 0% to 100% scale, in which 0 equaled completely green leaf tissue and 100 equaled completely white leaf tissue. Tissue bleaching was assessed visually because Yelverton et al. (2009) reported that visual ratings of herbicide responses in turf were highly correlated with those measured using the line intersect method or digital image analysis.

On each harvest date, clippings were immediately frozen in liquid nitrogen (N) and placed on ice for transfer to storage at –80 °C. Tissue pigments were extracted and quantified according to previously published methods (Emenhiser et al., 1996; Kopsell et al., 2007). All clippings were first homogenized in liquid N using a mortar and pestle. A ≈0.25-g subsample was placed into a Potter-Elvehjem tissue grinder tube (Kontes, Vineland, NJ) with 2.5 mL of tetrahydrofuran (THF). Extraction efficiency was measured through the addition of 0.8 mL of ethyl-β-apo-8′-carotenoate (Sigma Chemical Co., St. Louis, MO) to selected samples. Recovery of the external standard averaged 72%. Each sample was vortexed and homogenized in the tube using ≈25 insertions with a Potter-Elvehjem tissue grinder pestle attached to a drill press set at 540 rpm while the tube was immersed in ice to dissipate heat. The tube was then placed into a centrifuge for 5 min at 500 g_n. Precautions were taken to keep the tissue samples on ice during extraction to decrease degradation and increase percent recovery (Kimura and Rodriguez-Amaya, 1999). The supernatant was removed with a Pasteur pipette, placed into a conical 15-mL test tube, capped, and held on ice during the remainder of the extraction. The sample pellet was resuspended in 2 mL THF and the extraction procedure was repeated. By the third or fourth extraction, the supernatant was colorless and one additional extraction was conducted. The remaining sample pellet was discarded and the combined supernatants were reduced to 0.5 mL under N stream (N-EVAP 111; Organomation Inc., Berlin, MA). Each sample was brought up to a 5-mL volume with acetone. Samples were vortexed and filtered through a 0.2-μm polytetrafluoroethylene filter (Econofilter PTFE 25/20; Agilent Technologies, Wilmington, DE) before high-performance liquid chromatography (HPLC) analysis.

HPLC pigment separation and quantification were carried out using an Agilent 1200 series HPLC unit with a photodiode array detector (Agilent Technologies, Palo Alto, CA). The column used was a 250 × 4.6 mm i.d., 5-μm analytical scale polymeric RP-C₃₀ with a 10 × 4.0-mm i.d. guard cartridge and holder (ProntoSIL; MAC-MOD Analytical Inc., Chadds Ford, PA), which allowed for effective separation of chemically similar carotenoid compounds. The column was maintained at 30 °C using a thermostatted column compartment. All separations were achieved isocratically using a binary mobile phase of 11% methyl tert-butyl ether, 88.99% MeOH, and 0.01% triethylamine (v/v/v). The flow rate was 1.0 mL·min⁻¹ with a run time of 53 min followed by a 2-min equilibration before the next injection. Eluted compounds from a 10-μL injection loop were detected at 453 nm (carotenoids, chlorophyll b, internal standard), and 652 nm (chlorophyll a) and data were collected, recorded, and integrated using ChemStation Software (Agilent Technologies) (Demmig-Adams et al., 1996; Frank and Cogdell, 1996). Carotenoids evaluated included: β-carotene, antheraxanthin, lutein, neoxanthin, violaxanthin, and zeaxanthin. These carotenoids were selected based on their active roles in photoprotection and light harvesting. Peak assignments were performed by comparing retention times to internal standards and line spectra (250 to 700 nm) obtained from photodiode array detection with authentic standards (ChromaDex Inc., Irvine, CA). Concentrations of the authentic standards were determined spectrophotometrically using quantitative spectroscopic data (Table 1; Davies and Köst, 1988). Pigments were expressed as mg/100 g fresh weight (FW) of bermudagrass leaf blade tissue.

Table 1.‘Riviera’ bermudagrass (Cynodon dactylon) tissue pigments selected for quantification, including their common and IUPAC chemical names, and information used for calibration of authentic internal standards.^z

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Statistical analysis.

Two experimental runs were conducted with treatments in each arranged as a 3 × 3 factorial randomized complete block design with three replications. Factors included herbicide (mesotrione, topramezone, and tembotrione) and rate (low, medium, and high). Data from non-treated plants were excluded from statistical analysis to stabilize variance (Corbett et al., 2004); additionally, square-root transformations were also performed before subjecting data to analysis of variance (P = 0.05; Statistical Analysis Software, Inc., Cary, NC) (Ahrens et al., 1990). Interpretations were not different from non-transformed data; therefore, non-transformed means are presented here for clarity. Fisher's protected least significant difference test (P = 0.05) was used to separate treatment means where appropriate. No significant herbicide-by-rate interactions were detected in bermudagrass carotenoid pigment data. Thus, changes in tissue pigments resulting from herbicide (pooled across rates) are plotted over harvest interval with se bars presented as a means of statistical comparison. Trend analyses (P = 0.05) were conducted using orthogonal polynomial contrasts to model significant changes in tissue pigments as a result of application rate as well.

Visual bleaching.

Significant herbicide-by-rate interactions were detected in visual bleaching data. Visual bleaching increased linearly with mesotrione rate at 7 and 14 DAA (Table 2). For mesotrione, the highest level of bleaching occurred at 7 DAA (22%) with the highest rate tested (0.42 kg·ha⁻¹) (Table 2). Bleaching never exceeded 10% after treatment with the maximum labeled use rate of mesotrione (0.28 kg·ha⁻¹) (Table 2). No differences in bleaching were detected between mesotrione rates after 14 DAA, and by 28 DAA, no visual bleaching was observed for any mesotrione treatment (Table 2). Similar responses have been reported by other researchers investigating bermudagrass bleaching after mesotrione treatment (McCurdy et al., 2008; Willis et al., 2007).

Table 2.Effect of herbicide-by-rate interaction on visual bleaching of ‘Riviera’ bermudagrass treated with mesotrione, topramezone, and tembotrione.

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With the exception of 7 DAA, no significant differences were detected between topramezone rates until 28 DAA (Table 2). All rates resulted in greater than 50% visual bleaching at 14 DAA compared with only 19% bleaching for the highest (0.42 kg·ha⁻¹) rate of mesotrione (Table 2). Visual bleaching decreased for all topramezone rates after 21 DAA (Table 2). Once visual bleaching began to subside, a linear response was detected among rates with the high (0.038 kg·ha⁻¹) rate producing more bleaching than the medium (0.025 kg·ha⁻¹) and low (0.018 kg·ha⁻¹) rates (Table 2).

Bleaching responses for tembotrione were similar to topramezone. No differences were detected between tembotrione rates until 21 DAA (Table 2). All rates resulted in greater than 32% visual bleaching at 14 DAA, compared with greater than 50% for all rates of topramezone and only 19% bleaching for the high (0.42 kg·ha⁻¹) rate of mesotrione (Table 2). Similar to topramezone, visual tissue bleaching began to subside at 21 DAA. Once bleaching began to subside, linear responses were detected among tembotrione rates suggesting that single applications at higher rates may improve activity against bermudagrass (Table 2).

Chlorophyll.

Few statistically significant differences were detected among the low, medium, and high application rates of mesotrione, topramezone, and tembotrione (Table 3). Increases in bleaching at 7 DAA were accompanied by reductions in the concentration of total chlorophyll for all herbicides (Fig. 1). At 7 DAA, total chlorophyll for mesotrione-treated plants measured 127.8 mg/100 g FW compared with 192.9 mg/100 g FW for the non-treated control. By 14 DAA, concentrations of total chlorophyll for mesotrione- and non-treated plants were not significantly different. Topramezone and tembotrione reduced total chlorophyll to a greater extent than mesotrione at 7 DAA and did not rebound to non-treated levels until 28 DAA (Fig. 1).

Table 3.Effect of herbicide-by-rate interaction on total chlorophyll (chlorophyll a + chlorophyll b) concentration [mg/100 g fresh weight (FW)] of ‘Riviera’ bermudagrass treated with mesotrione, topramezone, and tembotrione.

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Lutein and β-carotene.

Changes in total carotenoids resulting from herbicide treatment are most likely related to the rise and fall of lutein and β-carotene, because these pigments comprised greater than 53% of the total carotenoids measured on each harvest date. At 7 DAA, topramezone- (5.3 mg/100 g FW) and tembotrione- (5.5 mg/100 g FW) treated plants measured lower in lutein than those treated with mesotrione (7.7 mg/100 g FW) (Fig. 2). By 14 DAA, mesotrione- and non-treated plants had equivalent lutein concentrations; topramezone- and tembotrione-treated plants were not equivalent to the non-treated controls until 28 and 35 DAA, respectively. Topramezone affected lutein to a greater extent than tembotrione; topramezone reduced lutein 67% (compared with the non-treated control) at 14 DAA, whereas tembotrione only led to a 51% reduction. A similar response was observed 21 DAA as well (Fig. 2).

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Reductions in β-carotene were greater for tembotrione than mesotrione. Compared with the non-treated control (5.5 mg/100 g FW), tembotrione led to a 56% reduction in β-carotene (2.4 mg/100 g FW), whereas mesotrione only led to a 35% reduction (3.6 mg/100 g FW) at 7 DAA (Fig. 2). β-carotene levels for mesotrione-treated plants were not different from non-treated controls after 7 DAA (Fig. 2). No differences in β-carotene were detected between tembotrione- and topramezone-treated plants on each harvest date.

At the point of maximum visual bleaching (14 DAT), linear reductions in lutein were observed among the low, medium, and high rates of each herbicide (Table 4); lutein concentrations after treatment at low and medium rates measured 9.7 mg/100 g FW compared with 6.3 mg/100 g FW after treatment at high rates (Table 4). Comparatively, the lutein concentration of non-treated plants at 14 DAA measured 15.3 mg/100 g FW (Table 4). Linear reductions in β-carotene were also observed among the low, medium, and high application rates by 21 DAA (Table 4).

Table 4.Effects of application rate (pooled across herbicide) on lutein and β-carotene concentrations [mg/100 g fresh weight (FW)] in ‘Riviera’ bermudagrass leaf tissues 3, 7, 14, 21, 28, and 35 d after treatment with mesotrione, topramezone, and tembotrione.

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Xanthophyll cycle pigments.

Xanthophyll cycle pigments (zeaxanthin + antheraxanthin + violaxanthin) participate as antioxidants in light-harvesting complexes (Demmig-Adams et al., 1996; Niyogi et al., 1997). Mesotrione, topramezone, and tembotrione lowered the concentration of total xanthophyll cycle pigments compared with the non-treated control at 7 DAA (Fig. 3). Concentrations of total xanthophyll cycle pigments for mesotrione and non-treated controls were equivalent at 14 DAA, whereas topramezone- and tembotrione-treated plants were not equivalent to the non-treated controls until 28 and 21 DAA, respectively (Fig. 3).

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After peak visual bleaching occurred (14 DAA), topramezone and tembotrione appeared to affect xanthophyll cycle function, because the percentage of zeaxanthin + antheraxanthin in the total xanthophyll pigment pool (ZA/ZAV) increased in topramezone- and tembotrione-treated plants from 14 to 21 DAA (Fig. 3). Pooled across all herbicides, ZA/ZAV increased linearly with application rate as well (Table 5). By 21 DAA, reductions in bleaching for all herbicides were accompanied by reductions in ZA/ZAV (Table 2; Fig. 3).

Table 5.Effects of application rate (pooled across herbicide) on the concentration of violaxanthin [mg/100 g fresh weight (FW)] and the ratio of zeaxanthin + antheraxanthin to the concentration of total xanthophyll pigments (ZA/ZAV) in ‘Riviera’ bermudagrass leaf tissues 3, 7, 14, 21, 28, and 35 d after treatment with mesotrione, topramezone, and tembotrione.

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Stress induced by these herbicides from 14 to 21 DAA may have influenced violaxanthin de-epoxidase activity, thus increasing the conversion of violaxanthin to the intermediate antheraxanthin and subsequently zeaxanthin, the primary carotenoid responsible for preventing photoinhibition (Baroli et al., 2003). When peak visual bleaching occurred (14 DAT), violaxanthin was reduced by more than 50% in topramezone- and tembotrione-treated plants and linear reductions in violaxanthin were also detected among application rates (Fig. 3; Table 5). Polle et al. (2001) reported that zeaxanthin can replace violaxanthin and lutein in light-harvesting antennae of photosystem II and photosystem I. Moreover, Depka et al. (1998) reported a conversion of β-carotene to zeaxanthin under conditions of high light stress and surmised that this conversion was required to maintain the integrity of the D1 protein of photosystem II. On each date that linear increases in ZA/ZAV were observed in the current study, linear reductions in β-carotene were observed as well (Tables 2 and 3).