Materials and Methods

Two identical, independent experiments were repeated in time. Creeping bentgrass (Agrostis stolonifera L.) cv. L-93 seeds were planted at a rate of 24 kg·ha⁻¹ in 12.7-cm diameter by 11.4-cm deep pots of calcareous sand and grown in a greenhouse. Particle size distribution of sand was within U.S. Golf Association guidelines (USGA, 1993). Greenhouse temperatures were 18 to 25 °C with a photoperiod of 16 day/8 night. Natural sunlight was supplemented with high-pressure sodium lamps that provided an additional 180 μmol·m⁻²·s⁻¹ of photosynthetic active radiation (PAR). Measurements of PAR made periodically throughout the growing cycle exceeded 400 μmol·m⁻²·s⁻¹. Sand was watered daily.

Grasses were cut at 1.3 cm three times a week with an electric reel mower (Toro, Minneapolis, MN). A wood box with holes in the top the same diameter as the pots was built to hold pots of grass in place during mowing. The wood box had ramps on either side that allowed the electric reel mower to travel over pots and cut grasses. Because the sand in pots was level with the top of the wooden support, weight of the electric mower was evenly distributed between the wood box and the pots.

Sand media were fertilized once a week with 110 mL of a complete nutrient solution based on Pellet and Roberts (1963) that contained 102 mg·L⁻¹ N, 4 mg·L⁻¹ P, 63 mg·L⁻¹ K, 17 mg·L⁻¹ Ca, 19 mg·L⁻¹ Mg, 1.2 mg·L⁻¹ Fe, 0.25 mg·L⁻¹ Mn, 0.10 mg·L⁻¹ B, 0.01 mg·L⁻¹ Zn, and 0.01 mg·L⁻¹ Mo. Grasses were moved to a growth chamber at least 10 months after seeding. Grasses were mowed, watered, and fertilized like in the greenhouse. Temperature in the growth chamber was 18 to 21 °C and a photoperiod of 16 day/8 night was maintained with cool white fluorescent lamps and incandescent bulbs that provided 550 μmol·m⁻²·s⁻¹ of PAR. Grasses were acclimated in the growth chamber for at least 3 weeks before initiation of treatments. Although no formal measurements were recorded, growth rate and clipping yield of grasses were similar when grown in the greenhouse and growth chamber.

Grasses were not cut for 3 d before application of mowing treatments. Mowing treatments included rolling, single-cutting, and double-cutting. Grasses were rolled with a 108-kg electric greens mower without the reel engaged, whereas single-cut grasses were mowed once and double-cut grasses were mowed twice at 1.3 cm. Harvests of plant tissue for carbohydrate analysis and measurements of efficiency of PSII were made 0, 12, 24, 36, 48, and 60 h after treatment. Creeping bentgrass stubble was harvested frelected pots per treatment with a razor blade at each sampling time. The tissue was immediately frozen under liquid nitrogen and stored at −80 °C for analysis of carbohydrates.

A portable fluorometer (PAM-101; Walz, Effeltrich, Germany) fitted with a 2-mm fiberoptic cable was used to measure light-adapted efficiency of PSII (F_m′ − F′/F _m′) of individual leaf blades, where F_m ′ is fluorescence in the light-adapted state and F′ is initial fluorescence (Genty et al., 1989). Efficiency of PSII was measured at the adaxial tip of 10 individual light-adapted leaves selected at random from pots before tissue sampling.

A 0.5- to 1.0-g sample of frozen leaf tissue was crushed under liquid N₂ and suspended in 10 mL of high-performance liquid chromatography (HPLC)-grade water to extract soluble carbohydrates, whereas a subsample was dried in a 68 °C oven for 72 h for dry weight analysis. The mixture was heated to 95 °C for 30 min and then centrifuged at 1000 g for 10 min (Livingston, 1991). Samples were deionized by passing a 100-μL aliquot of supernatant through a 1-mL chromatography column packed from top to bottom with 120 μL cation exchange resin [Dowex (Sigma Aldrich, St. Louis) 50 ×8-400, H+-form], 100 μL insoluble polyvinylpyrrolidone, and 120 μL anion exchange resin [Amberlite (Sigma Aldrich, St. Louis) CG-400 II, formate form]. Columns were eluted twice with 250 μL water. Elutants were combined and filtered through a 0.45-μm nylon filter for HPLC analysis (Amiard et al., 2003; Bachmann et al., 1994).

Water-soluble carbohydrates were analyzed by using HPLC. Sucrose, glucose, fructose, and fructans were separated on a Sugar-Pak column (300 × 6.5 mm; Waters, Milford, MA) and were isocratically eluted at 85 °C by using 0.1 mm disodium calcium salt of EDTA at a flow rate of 0.5 mL·min⁻¹. Separated carbohydrates were identified by external standards and quantified by using a model 2410 differential refractometer (Waters).

In both experiments, treatments were completely randomized and three independent replicates were used to determine concentrations of carbohydrates at each sampling time. The general linear model of SAS (SAS Institute, Cary, NC) was used for analysis of variance of experiment, treatment, time, and their interactions. Values reported in the text and figures are representative of the highest-order interaction or main effects significant at the 0.05 level. Treatment means were separated by using Fisher's least significant difference.

Results and Discussion

Efficiency of PSII of single-cut and rolled grasses was reduced by 5% compared with not-cut grasses 12 h after treatment in experiment one (Fig. 1A). Double-cut grasses had a reduction in efficiency of PSII of 9% compared with not-cut grasses 12 h after treatment in experiment one (Fig. 1A). Twenty-four hours after treatment, efficiency of PSII of single- and double-cut grasses was reduced by 4% in Expt. 1 (Fig. 1A). In Expt. 2, single- and double-cut grasses had reductions in efficiency of PSII of 2% and 2.5%, respectively, compared with not-cut grasses 12 h after treatment (Fig. 1B). Efficiency of PSII of single- and double-cut grasses was reduced by 3% compared with not-cut grasses 36 h after treatment in Expt. 2 (Fig. 1B).

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A common response of many plants to wounding is to increase formation of reactive oxygen species (Orozco-Cárdenas and Ryan, 1999), which can limit the efficiency of PSII. Decreases in efficiency of PSII often are thought to be caused by an applied stress that has damaged the photosynthetic apparatus. This results in reduced efficiency of light transfer in photosynthetic electron transport (Maxwell and Johnson, 2000). Although reductions of efficiency of PSII in single- and double-cut grasses were likely too small and transient to be considered sustained photoinhibition of PSII, they nonetheless indicate a wound response. In this instance, the magnitude of reduction in efficiency of PSII indicates that double-cutting is more injurious than single-cutting.

Values reported for levels of fructan are means averaged over both experiments. Fructan level data were pooled because no interaction between experiment and treatment existed. Fructan levels were 33% lower in double-cut grasses compared with not-cut grasses 24 h after treatment. Single-cut grasses had 22% less fructan than not-cut grasses 24 h after treatment (Fig. 2). Thirty-six hours after treatment, fructan levels of single- and double-cut grasses were 52% and 45%, respectively, lower than not-cut grasses. Fructan levels were 38% lower in double-cut grasses compared with not-cut grasses 48 h after treatment (Fig. 2).

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Reduced levels of fructans also have been observed in mown perennial ryegrass (Lolium perenne L.) (Donaghy and Fulkerson, 1998; Sullivan and Sprague, 1943) and tall fescue (Festuca arundinacea Screb.) (Volenec, 1986). In these experiments, the magnitude and duration of reduction in the level of fructan were greatest in double-cut grasses indicating that multiple cuttings likely cause greater stress than single cuttings.

Changes in levels of fructose, glucose, and sucrose in response to mowing treatments were variable. No differences in levels of sucrose or fructose were observed among treatments in either study (data not shown), whereas glucose levels were reduced in single- and double-cut grasses compared with rolled and not-cut grasses (Fig. 3). Because only the main treatment effect was significant, levels of glucose were pooled over experiment and time. Levels of glucose were 31% lower in double-cut grasses compared with not-cut grasses (Fig. 3).

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It is likely that hexose sugars liberated by hydrolysis of fructan after mowing maintained levels of fructose in single- and double-cut grasses (Prud'homme et al., 1992). Likewise, sucrose likely also was resynthesized from free fructose for transport to elongating leaves to provide energy for formation of new leaf tissue (Pollack and Cairns, 1991). Amiard et al. (2003) and de Visser (1997) have demonstrated that products of fructan hydrolysis are used for respiration and development of new leaf tissue until foliage has regrown and rate of CO₂ assimilation is sufficient to support plant growth and metabolism. However, steady-state labeling experiments and assays of carbohydrate metabolism enzyme rates would be needed to confirm this hypothesis.

Mowing reduced efficiency of PSII and resulted in transient decreases in the levels of fructan and glucose in creeping bentgrass. The duration and magnitude of reduction in efficiency of PSII and fructan levels were greatest in double-cut grasses, indicating that multiple cuttings may be more damaging to plant growth and development. Mowed grasses, regardless of the number of cuttings, were able to regrow sufficient leaf tissue to support plant metabolism and restore levels of reserve carbohydrates. Narra et al. (2004) often observed similar carbohydrate levels in creeping bentgrass cut at different heights in clippings collected 4 to 6 d after mowing. However, additional stresses like high temperature (Xu and Huang, 2000) that also reduce levels of reserve carbohydrates may limit recuperative potential of mown creeping bentgrass, especially when grasses are double-cut. Additional field trials should be performed that monitor levels of carbohydrates and turfgrass quality over time to support this hypothesis.