Crop Science 40:498-502 (2000)
© 2000 Crop Science Society of America
TURFGRASS SCIENCE
Copper Reduces Shoot Growth and Root Development of Creeping Bentgrass
M.B. Fausta and
N.E. Christiansa
a Dep. of Horticulture, Iowa State Univ., Ames, IA 50010-1100 USA
nchris{at}iastate.edu
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ABSTRACT
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Sand-based golf course putting greens have been observed to contain elevated Cu concentrations, based on standard soil tests. Little research has been conducted that relates Cu concentration in sand media to turfgrass performance. The objectives of this study were to determine the response of greenhouse-grown creeping bentgrass (Agrostis palustris Huds. `Penncross') to treatments in rooting media that ranged from 0 to 600 mg Cu kg-1 and to provide an estimate of potentially toxic plant-available Cu levels by use of the diethylenetriaminepentaacetic acid–triethanolamine (DTPA–TEA) soil test. Calcareous and silica sands were mixed individually with reed sedge peat in a 9:1 (v/v) ratio. Penncross sod plugs were placed on the top of pots containing the premixed sand–peat media and allowed to grow for 12 wk. The silica sand medium pH decreased from 6.8 to 5.4, while the pH of the calcareous medium remained between 7.2 to 7.3 as cupric sulfate (CuSO4 · 5H2O) concentrations increased. The average dry weight of clippings for plants grown in silica sand decreased 16% as Cu treatments increased from 0 to 600 mg kg-1. At 600 mg kg-1 Cu, dry root mass was 56 and 48% lower than the control treatments for plants grown in silica and calcareous sand, respectively. The DTPA–TEA soil test extracted, on average, 19% more Cu from the calcareous sand when compared to the silica sand. However, plant roots contained an average of 34% more Cu when grown in silica sand. These results indicate that the DTPA–TEA soil test was not a good predictor of potentially toxic plant-available Cu in sand-based media, and alternative soil test methods should be investigated.
Abbreviations: DTPA, diethylenetriaminepentaacetic acid • ICAP/IRIS, inductively coupled argon plasma spectrometry • TEA, triethanolamine
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INTRODUCTION
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COPPER IS AN ESSENTIAL ELEMENT for plant growth and development. The Cu ion is influential in plant enzymatic activity, and it performs key functions in plant respiration and photosynthesis (Woolhouse and Walker, 1981). Although the presence of Cu in plant tissue is necessary, an excess may be harmful. Therefore, Cu should be applied in discrete dosages or relatively insoluble forms to prevent phytotoxicity or plant death (Ware, 1994).
Recently, turfgrass managers have developed an interest in the phytotoxic effects of Cu on plants. Elevated concentrations of Cu may develop with time in the soils of golf course greens due to frequent and prolonged use of Cu-containing fungicides, organic fertilizers composed of sewage sludge and poultry manure, and irrigation of turfgrass with sewage effluent water (Marschner, 1995). Copper is rated as a low mobility element because of the high affinity of Cu for soil colloids. Copper accumulates on the surface of contaminated soils, and it shows little downward migration (McBride, 1994).
Little research has been conducted that relates Cu concentrations in a sand root zone and shoot and root tissue to turfgrass performance. Previous research has indicated a turfgrass shoot tissue sufficiency range of 5 to 20 mg Cu kg-1 (Turner, 1993). Soil tests have shown Cu at 0.2 mg kg-1 to be the critical concentration below which Cu deficiency symptoms would be expected (Viets and Lindsay, 1973). The proposed maximum acceptable concentration of Cu in agricultural soils is 100 mg kg-1 (Kabata-Pendias and Pendias, 1992).
Riemer and Motto (1980) conducted a study on a Freehold sandy loam (fine-loamy, mixed, mesic Typic Hapludult) using `Emerald' creeping bentgrass subjected to elevated Cu concentrations. They found no differences in clipping yield for plants grown in soils with 200 or 400 mg Cu kg-1 and limed to a pH of 6.5. Clipping yield was reduced at 200 mg kg-1 Cu on the same sandy loam soil when pH was reduced to 4.8, and plants grown at 400 mg Cu kg-1 died. Sanders et al. (1986) conducted a study on the effects of Cu on ryegrass (Lolium spp.) grown in sewage sludge–contaminated soils of different pH. Clipping yield of ryegrass tissue was reduced from 10.6 g to 5.3 g as pH decreased from 5.5 to 4.9, respectively. Davis and Beckett (1978) grew perennial ryegrass (Lolium perenne L. `S.23') in a sand culture and applied cupric sulfate (CuSO4 · 5H2O) in a nutrient solution. They found the minimum Cu concentration in plant tissue necessary to cause toxicity was 21 mg kg-1, and at this concentration, plant yield was reduced. Lee et al. (1996) showed that Kentucky bluegrass (Poa pratensis L. `Touchdown') was damaged at a tissue Cu concentration of 63.5 mg kg-1.
The general expression of Cu toxicity in plants is based on the interaction of several factors; however, the basic deleterious effect of Cu on growth is related to the root system. Studies conducted by Wainwright and Woolhouse (1975, 1977) showed Cu to be inhibitory to root elongation of colonial bentgrass (Agrostis capillaris L.) at all Cu concentrations used. Jarvis (1978) found the dry weight of perennial ryegrass roots to decrease from 4.5 g at 0 mg Cu kg-1 to 2.4 g for plants subjected to 953 mg kg-1 Cu. In a study conducted by Karataglis (1980), colonial bentgrass plants were grown for 8 d in a solution containing only 0.5 g L-1 Ca(NO3)2 and transferred to a solution containing 0.25 mg kg-1 Cu. As soon as Cu was applied, nontolerant plants showed inhibition of root growth.
This research was initiated to determine the sensitivity of creeping bentgrass to applied concentrations of Cu in both silica and calcareous sand–peat media. The specific objectives were (i) to measure changes in shoot and root tissue Cu concentration in response to increasing soil Cu concentrations, (ii) to determine the effect of elevated soil Cu concentrations on shoot growth and root development, and (iii) to use the DTPA–TEA soil test to predict potentially toxic concentrations of plant-available Cu in calcareous and silica sand media.
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Materials and methods
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Growing Conditions
An experiment was initiated 16 July 1997 and was repeated on 3 Dec. 1997. Research was conducted in a glass-glazed greenhouse in which air temperature was maintained at 27 ± 2°C during the day and 21 ± 2°C at night. Natural light was used in the first experiment. An overhead artificial light source with an average photosynthetically active radiation of 384 µmol s-1 m-2 (Quantum/Radiometer/ Photometer Model LI-185A, LICOR, Lincoln, NE) was used in Exp. 2. The duration of each experiment was 12 wk.
Sand Media
Two sand-based media were used. One sand was calcareous, contained 95 g kg-1 CaCO3, and had a pH of 7.4. The calcareous sand was sieved to remove all particles larger than 2 mm. The remaining sand fractions were as follows: 3% very coarse (2.0–1.0 mm), 27% coarse (1.0–0.5 mm), 56% medium (0.5–0.25 mm), 11% fine (0.25–0.15 mm), and 3% very fine ( 0.15–0.05 mm). The other rooting medium was a silica sand with a pH of 6.8, and screening was not necessary. Sand fractions for the silica material were as follows: 25% coarse, 67% medium, 7% fine, and 2% very fine. Both sand types were mixed separately with Dakota peat (Dakota Peat & Equipment, Grand Forks, ND) in a 9:1 sand/Dakota peat (v/v) ratio. Dry cupric sulfate (CuSO4 · 5H2O) was hand mixed at 0, 200, 400, and 600 mg Cu kg-1 sand with 1200 g of each separate sand and peat medium. The rooting media then were placed into 32 separate, 12.7-cm-diameter plastic pots with a volume of 876 cm3 (16 pots for each type of sand).
Turfgrass Culture
Penncross creeping bentgrass sod was cut into 10.2 cm diameter by 7.6 cm deep circular plugs. A cut was then made just below the thatch layer to remove sand, peat, and roots from each plug. Sand and peat remaining on the plugs were washed from the remaining roots, and the plug was placed on top of the sand–peat mix. The pots were placed on a greenhouse mist bench for 2 d and then moved to a climate-controlled greenhouse. Each experiment was arranged in a split-plot design with sand type as main plots and Cu levels as subplots. Both experiments had four replications (pots) per treatment. Each pot received 160 mL of distilled water and 50 mL of an N, P, and K fertilizer solution weekly. The fertilizer solution was mixed to provide 2.5, 1.2, and 1.2 g m-2 N, P, and K per 4 wk, respectively. Ammonium nitrate, H3PO4, and KNO3 supplied the N, P, and K in the fertilizer solution.
Dry Weight of Clippings
Clippings were taken weekly beginning 6 Aug. 1997 and 23 Dec. 1997 for Exp. 1 and 2, respectively. The grass was trimmed to a height of 0.5 cm. Clippings were dried at 67°C for 48 h. Dry weights of clippings were recorded for each pot.
Dry Root Mass
Root mass measurements were taken after 12 wk for both experiments. The sod plugs and rooting medium were removed from the greenhouse pots. A cut was made just below the thatch layer to remove the shoot, crown, and thatch of the sod. Sand, peat, and roots were placed in a water-filled catch pan, and floating roots were removed. The remaining material was poured through a 2-mm screen into a 53-µm screen. Roots caught by the 2-mm screen were removed. The material in the 53-µm screen was removed and placed again in the catch pan. The catch pan was filled with water, and the material was poured back through the 2-mm screen. This screening process was done three times for each pot. Washed roots were dried in an oven at 67°C for 48 h, and oven-dry mass was recorded. Roots were ashed at 500°C for 12 h to remove C from each root sample. The final root mass was determined by subtracting the ashed root mass from the oven-dry root mass. This was done to insure that no mineral soil was being weighed in the root samples.
Shoot and Root Tissue Copper Concentration
Shoot tissue samples of plants were collected during Weeks 3 to 12, and the 10 samples were combined for analysis. Clippings were ground to pass a 40-µm screen. Clipping analysis was conducted by dry ashing the tissue samples, digesting the samples with 5 mL of 5 M aqua regia (HCl and HNO3, 3:1 v/v), and filtering the material through no. 42 Whatman filter paper. Hot deionized water was used to rinse the material through the filter paper. The filtrate was diluted to a 35-mL volume with deionized water and analyzed by inductively coupled argon plasma spectrometry (ICAP/IRIS) (Thermo Jarrel-Ash, Franklin, MA).
Root tissue samples collected from Exp. 2 only were analyzed for Cu content. Ashed root material was digested with 5 mL of 5 M aqua regia. Copper concentration in the root tissue was derived using the same procedure as described above for shoot tissue. Plant standards and quality control checks were within 100 ± 5% of the known value.
pH Determination
Samples of the sand and peat rooting medium were removed from each pot after each experiment was terminated. Ten milliliters of 0.01 M CaCl2 · 2H2O were added to 10 g of each control and Cu-treated sand media. The sand–CaCl2 · 2H2O mixture was stirred periodically, and pH was measured after 2 h with a glass calomel electrode as described by Thomas (1996).
Copper Analysis of Sand Media
A portion of sand rooting medium was removed from each pot at the end of both experiments and was air dried. Sand particles were sieved at the initiation of the two experiments to pass a 2-mm stainless steel screen. The extractant contained 0.005 M DTPA, 0.01 M TEA, 0.01 M CaCl2 · 2H2O, and was adjusted to a pH of 7.3 with HCl. Extractant to soil ratio was 2:1 (v/w), and the shaking time was 2 h (Lindsay and Norvell, 1978). After extraction, the solution was filtered through Whatman no. 42 filter paper and the filtrate was analyzed for Cu by ICAP/IRIS.
Statistical Analysis
Data from experiments one and two were pooled for statistical analysis. Data were analyzed by using the ANOVA procedure of SAS (SAS Institute, 1990). The sand x block effect was used as an error term to test the significance of the whole unit, sand type. Regression analysis was completed by using the general linear model (proc GLM) procedure of SAS. Linear and quadratic models were used to determine the best fit of the data. Fisher's least significant difference (LSD) test was used to compare treatment means. Statistical significance was determined at P 
0.05.
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Results
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Dry Weight of Clippings
Increasing Cu treatments had no effect on the dry clipping weight of plants grown in the calcareous sand. However, in the silica sand, an increase in Cu concentration from control to 600 mg Cu kg-1 decreased average dry clipping weight by 16% (Table 1) . Dry weight of clippings differed significantly between the two sand media. The calcareous medium produced plants having an 8% higher average dry clipping yield than plants grown in silica sand at all Cu treatments. Dry weight of clippings was significantly reduced for plants grown in silica sand when Cu concentration was greater than 200 mg kg-1.
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Table 1 Mean dry weight of clippings of `Penncross' creeping bentgrass grown in two sand media and at four Cu levels
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Shoot and Root Tissue Copper Concentration
Plants grown in the silica sand control pots had a mean Cu concentration of 10.3 mg kg-1 (Fig. 1)
. Tissue Cu concentration of plants increased 46% as Cu treatments increased from the control to the 400 mg Cu kg-1 level. Concentration of Cu in the shoot tissue of plants grown on calcareous sand increased 30% from the control to the 400 mg Cu kg-1 sand level. Tissue Cu concentrations were on average 25% higher for plants grown in silica sand compared with plants grown in the calcareous sand medium.
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Fig. 1 Regression analysis of Penncross creeping bentgrass shoot tissue Cu concentration. Tissue samples collected during Weeks 3 to 12 were combined and analyzed by inductively coupled argon plasma spectrometry (ICAP/IRIS) for Cu. Data from both experiments were combined for analysis. Vertical bars represent the standard error of mean values. Regression equations describing these relationships for silica and calcareous sands are: y = 10.31 + 0.07x - 0.0002x2, r2 = 0.74, and y = 9.14 + 0.03x - 0.00008x2, r2 = 0.52, respectively
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Copper concentration in root tissue increased with increasing amounts of applied Cu (Fig. 2)
. Plants grown in silica sand control pots had a mean root tissue Cu concentration of 19.3 mg kg-1. The mean Cu concentration of roots reached a maximum of 4727 mg kg-1 at the 450 mg kg-1 sand level. Root tissue Cu concentration of plants grown in calcareous sand increased linearly as Cu was applied to the sand (Fig. 2). Mean root tissue Cu concentration for plants grown in calcareous sand control pots was 12.3 mg kg-1. Root tissue Cu concentration increased to 3758 mg kg-1 at the 600 mg kg-1 Cu level. Root tissue Cu concentrations averaged 34% higher for plants grown in silica sand. Note that at the highest treatment level, 720000 µg of Cu were added to the sand in each pot and that total Cu removed by roots and shoots represents <1% of the applied Cu.
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Fig. 2 Regression analysis of Penncross creeping bentgrass root tissue Cu concentration. Tissue samples were collected during Week 12 and analyzed for Cu by inductively coupled argon plasma spectrometry (ICAP/IRIS) for plants grown in Exp. 2 only. Vertical bars represent the standard error of mean values. Regression equations describing these relationships for silica and calcareous sands are: y = 177.59 + 20.01x - 0.022x2, r2 = 0.93, and y = 257.20 + 6.10x, r2 = 0.95, respectively
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Dry Root Mass
Mean dry root mass of plants grown in silica sand decreased as sand Cu concentration increased (Fig. 3)
. Dry root mass declined by 56% for plants grown in control pots compared with those grown at the 600 mg Cu kg-1 sand level. Plants grown in the calcareous sand medium also showed decreased root development as sand Cu concentration increased (Fig. 3). Dry root mass declined by 50% at 400 mg Cu kg-1 when compared with the control. Root development of plants grown in both sand media was significantly reduced as sand Cu concentrations increased from the control to the 200 mg kg-1 Cu treatment level.
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Fig. 3 Regression analysis of Penncross creeping bentgrass dry root mass for plants grown in either calcareous or silica sand in the presence of increasing concentrations of Cu. Root mass measurements were taken after 12 wk, and data from both experiments were pooled. Vertical bars are standard errors of the mean. Regression equations showing the relationships for silica and calcareous sands are: y = 793.4 - 0.7x, r2 = 0.79, and y = 842.1 - 1.6x + 0.001x2, r2 = 0.67, respectively
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pH Determination
The silica sand medium had lower pH values than the calcareous sand medium (P > F = 0.001). The pH of the silica medium decreased as more CuSO4 · 5H2O was added to the sand. Mean pH values of the 0, 200, 400, and 600 mg kg-1 Cu silica sand treatments were 6.8, 5.8, 5.6, and 5.4 (LSD0.05 = 0.12), respectively, whereas the mean pH values of the 0, 200, 400, and 600 mg kg-1 Cu calcareous sand treatments were 7.3, 7.3, 7.3, and 7.2, respectively.
Copper Analysis of Sand Media
The DTPA–TEA-extractable Cu concentrations increased as higher concentrations of Cu were applied to each sand medium. Copper extracted from the calcareous sand increased linearly as more Cu was added (Fig. 4)
. Copper concentrations in calcareous sand ranged from 1.2 mg kg-1 in the control to 491.3 mg kg-1 in the 600 mg Cu kg-1 treatment. Silica sand samples had mean DTPA–TEA-extractable Cu concentrations that ranged from 0.7 mg kg-1 in control pots to 355.5 mg Cu kg-1 in pots treated with 600 mg Cu kg-1 sand (Fig. 4). The DTPA–TEA-extractable Cu concentrations were similar for the two media at the control, 200, and 400 mg kg-1 Cu treatment levels. Extractable Cu was 28% higher for the calcareous sand mixture at the 600 mg kg-1 sand Cu level when compared with Cu extracted from the silica sand.
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Fig. 4 Regression analysis of diethylenetriaminepentaacetic acid– triethanolamine (DTPA–TEA)–extractable Cu as influenced by the incorporation of Cu in two sand media. Data from both experiments were combined for analysis. Vertical bars are standard errors of the mean. Regression equations for silica and calcareous sands are: y = -5.04 + 1.05x - 0.0001x2, r2 = 0.91, and y = 4.53 + 0.821x, r2 = 0.97, respectively
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Discussion
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Dry weight of clippings from plants grown in calcareous sand was not significantly reduced at any Cu concentration, whereas the clipping yield was reduced by increased Cu for plants grown in silica sand. Similar patterns in clipping yield reduction of turfgrass with increased Cu availability due to decreased soil pH have been reported (Jarvis, 1978; Riemer and Motto, 1980; Sanders et al., 1986). Besides the effect of pH on the availability of Cu to the plant, an interaction between Cu and CaCO3 present in the calcareous sand may have prevented the reduction in clipping yield noted for plants grown in silica sand. The affinity of carbonates to precipitate Cu is the most common reaction rendering a lower activity of Cu in soils within the alkaline range of pH, and thereby reducing the potential toxicity of the metal (Kabata-Pendias and Pendias, 1992).
Root development of Penncross creeping bentgrass grown in calcareous and silica sand media decreased as Cu concentration increased. The reduction in root growth of turfgrasses as influenced by Cu concentrations has been shown by others (Wainwright and Woolhouse, 1975, 1977; Jarvis, 1978; Karataglis, 1980). Inhibition of root elongation and damage to the plasma membrane of root cells, as reflected by enhanced K efflux, are immediate responses to high Cu supply (Baker and Walker, 1989).
The root tissue of plants grown in both sand media contained extremely high Cu concentrations. Plants grown in silica sand had an average of 34% more Cu in root tissue than plants grown in calcareous sand. Lindon and Henriques (1992) and Ouzounidou et al. (1995) reported similar excessive root tissue Cu concentrations in rice (Oryza sativa L.) and maize (Zea mays L.), respectively. The upper shoot tissue Cu sufficiency level of 20 mg kg-1 dry tissue as reported by Turner (1993) for grasses was never exceeded in this study. In plants receiving a large supply of Cu, the Cu content in the root tissue rises proportionally to the concentration of Cu in the external medium, whereas transport to the shoot is highly restricted (Marschner, 1995). Our research has shown that shoot tissue Cu concentration may not be a conclusive indicator of Cu toxicity in Penncross creeping bentgrass.
The DTPA soil test was developed to identify inadequate and excessive concentrations of plant-available micronutrients (Cu, Fe, Mn, Zn) in slightly acid and alkaline soils (Korcak and Fanning, 1978; Lindsay and Norvell, 1978; Norvell, 1984; O'Connor, 1988). The DTPA–TEA method extracted Cu that was highly related to applied Cu. However, the extractant did not correctly predict the amount of Cu available for plant uptake. Similar and frequently higher concentrations of Cu were extracted from the calcareous sand as from the silica medium. These results indicate that plants grown in the calcareous medium should have had equal or more Cu in shoot and root tissue than plants grown in silica sand. This research has shown higher shoot and root tissue Cu concentrations for plants grown in silica sand. The limitations of the DTPA–TEA micronutrient soil test to adequately predict plant-available Cu suggests that an alternative soil extractant might be more suitable.
An accumulation of Cu in a sand-based golf course green has the potential to harm creeping bentgrass. Developmental problems such as reduced shoot growth and root elongation of plants may cause concern for the turfgrass manager. Shoot tissue Cu concentration was not a good indication of toxicity in Penncross creeping bentgrass; therefore, it is necessary to find or develop a soil test to predict potentially toxic plant-available Cu in high sand content greens.O'Connor 1998
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NOTES
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Contribution as Journal Paper no. J-18252 of Iowa Agric. and Home Econ. Exp. Stn., Project no. 3601 and supported by Hatch Act and State of Iowa funds.
Received for publication March 29, 1999.
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ABSTRACT
INTRODUCTION
