Crop Science 40:1350-1357 (2000)
© 2000 Crop Science Society of America
TURFGRASS SCIENCE
Mechanisms of Wear Tolerance in Seashore Paspalum and Bermudagrass
L.E. Trenholma,
R.N. Carrowb and
R.R. Duncanb
a Dep. of Environmental Horticulture, Univ. of Florida, Gainesville, FL 32611 USA
b Crop and Soil Sci. Dep., Georgia Exp. Stn., Univ. of Georgia, Griffin, GA 30223-1797 USA
letr{at}gnv.ifas.ufl.edu
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ABSTRACT
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Traffic causes shoot injury to turfgrass, with resulting inhibition of growth and reduction of quality. Turfgrasses in high traffic venues are generally selected for tolerance to traffic or for an ability to quickly outgrow the injury. However, limited knowledge exists on the mechanisms that impart wear tolerance to turfgrass, particularly for warm-season grasses. This field research was undertaken to assess overall wear tolerance within and between seashore paspalum (Paspalum vaginatum Swartz.) ecotypes and bermudagrass hybrids (Cynodon dactylon L. x C. transvaalensis Burtt-Davy) and to determine the mechanisms that contribute to wear tolerance for both species. The research was conducted in two consecutive field trials during 1997 on seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars established on a native Appling (fine, kaolinitic, thermic Typic Kanhapludult) soil at the University of Georgia Experiment Station in Griffin, GA. Regression analysis determined that the most important potential mechanism related to enhanced wear tolerance of seashore paspalum was reduced leaf total cell wall (TCW) content, which accounted for 51% of the variation. Other factors that enhanced wear tolerance in this species were low leaf strength, low stem TCW, greater leaf moisture, greater shoot density, and higher K shoot tissue concentration. In bermudagrass, high stem moisture (40.9% of variation) and reduced stem cellulose content (31.5% of variation) were associated with better wear tolerance. Other factors that enhanced wear tolerance were greater stem and leaf moisture, shoot density, leaf lignin, stem and leaf lignocellulose, and concentration of K, Mn, and Mg. Knowledge of these characteristics will assist in developing screening protocols for selection of future wear tolerance cultivars within these species.
Abbreviations: DAT, days after treatment STI, shoot tissue injury TCW, total cell wall
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INTRODUCTION
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TRAFFIC IS A SOURCE OF ABIOTIC STRESS that can impose two distinct forms of injury on turf. Wear injury to shoot tissue is manifested by abrasion, tearing, or stripping of the leaf tissue. Physical injury to the shoots results in chlorophyll degradation and subsequent reduction in photosynthesis. Secondary effects can include increased susceptibility to insect or fungal attack at the sites of injury or increased weed pressure due to loss of turf density. The second type of traffic damage, soil compaction, is initially expressed at the root system level. Roots suffer from reduced growth and viability in response to low soil O2 and increased soil strength. The overall effect of either of these injuries is an inhibition of turf growth, loss of density, and premature senescence of shoot or root tissue. Rates of recovery vary based on inherent growth rate and degree of severity of the injury.
Interspecific wear tolerance differences have been observed (Shearman and Beard, 1975a), implying that the mechanism imparting wear tolerance differs between species. Warm-season turfgrass is typically more wear tolerant than cool-season turf (Youngner, 1961; Beard, 1973). Cultural practices, such as increased mowing height (Beard, 1973; Youngner, 1961), moderate N fertilization levels (Kohlmeier and Eggens, 1983), and a thatch layer (Beard, 1973) can also influence wear tolerance.
While some research has looked at wear tolerance at the intra-specific level (Beard, 1973), the data are very limited as to the degree of severity in wear tolerance within a species, and the specific mechanisms responsible for enhanced wear tolerance. Anatomical, morphological, or physiological plant characteristics correlating with wear tolerance across species may not be the same within a particular species.
Shearman and Beard (1975a) evaluated interspecies wear tolerance with four measurements: visual ratings, percentage TCW content, percentage verdure, and percentage chlorophyll per unit area. They found that visual wear estimates were well correlated
with quantitative evaluations when evaluating wear tolerance between species. Canaway (1981) determined that percentage of ground cover remaining after wear was negatively correlated with modified acid detergent fiber per unit area.
Bermudagrass hybrids are typically the warm-season turf of choice for use on golf courses, athletic fields, and highly managed landscaped areas. These grasses provide high quality and dense coverings for athletic activities, but require high levels of fertilizer and chemical inputs to maintain these standards under high traffic regimes. In view of increasing environmental concerns regarding turfgrass management, alternative species that may require fewer chemical and fertility inputs are being evaluated for use in high-profile turf areas. Determining the mechanisms that impart tolerance at the anatomical, morphological, or cellular levels can assist in the development and selection of appropriate cultivars for high-use sites. Currently undergoing testing is seashore paspalum, a prostrate growing, salt-tolerant turfgrass that is indigenous to tropical coastal areas worldwide. A collection of this species includes
300 ecotypes and exhibits extreme intraspecific diversity (Duncan, 1999). Some ecotypes display a wide range of tolerance to environmental stresses, particularly salinity (Dudeck and Peacock, 1985; Harivandi et al., 1984; Marcum and Murdoch, 1990, 1994), drought (Beard et al., 1991b), and flooding (Malcolm, 1969). Additionally, some ecotypes maintain high quality and growth levels with minimal N inputs (Beard et al., 1982, 1991b; Duble, 1989) and tolerate close (<13-mm) mowing heights (Beard et al., 1991a, 1991b). Quality and density ratings of selected ecotypes growing on a native clay soil have been shown to be equal or superior to `Tifway' and `TifSport' (Trenholm et al., 1999). In addition, the grass is well suited to a wide range of soil conditions and pH levels (4.09.8) and tolerates both low and high temperature extremes (Duncan, 1999) similar to the hybrid bermudagrasses.
As part of a larger project to determine stress tolerances of seashore paspalum, the objectives of this study were (i) to evaluate the overall wear tolerance within and between bermudagrass and seashore paspalum species and (ii) to determine the specific mechanisms responsible for imparting wear tolerance to the two species.
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Materials and methods
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This research consisted of two consecutive studies on seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars. These grasses were fully established on a native soil at the University of Georgia Experiment Station in Griffin, GA. Study 1 was conducted from 23 June to 13 July, while study 2 ran from 13 August to 10 September, 1997. The paspalum entries included four fine-leaved ecotypes: Temple 1, HI-1, SIPV-2, and K1, and three medium-coarse-leaved ecotypes: AP 8, PI 509022, and the cultivar `Adalayd'. Bermudagrasses included were Tifway, TifSport, and a Sea Island, GA selection believed to be closely related to `Tifgreen'. Soil type was an Appling sandy clay loam with pH 6.0. Fertilizer and cultural regime consisted of 172 kg N ha-1 applied as NH4NO3 in three split applications across the growing season, 22 kg P ha-1, 41 kg K ha-1, and micronutrients applied at recommended rates in April 1997. Plots were mowed three times weekly at a 3.2-cm clipping height with a reel mower. Glyphosate [N-(phosphonomethyl)glycine] was used as necessary to control alleyways between plots. The turf was maintained under well-irrigated conditions during the treatment period.
Treatments consisted of wear applied in a strip within replications as 90 total passes at one time by a differential slip wear device. This wear simulator had two rubber-covered rollers and applied 0.90 kg cm-1 of force. The differential slip arrangement allowed the rollers to turn at different speeds so that abrasion and scuffing actions were created. It was designed to apply minimal pressure to soil, thereby imparting wear as a primary stress with little soil compaction. Wear treatments for Study 1 were applied on 30 June and on 18 August for Study 2.
Evaluations taken the week prior to application of wear treatment included leaf turgidity, moisture percentage of leaf and stem tissue, leaf tensile strength, verdure tissue quantity, and number of shoots per unit area. Leaf tissue was collected for determination of tissue nutrient concentration, and leaf and stem tissue was collected for quantification of cell wall components. Percentage of shoot tissue injury was evaluated following wear treatment. Leaf turgidity was measured by the following formula: [(fresh weight - dry weight)/(turgid weight - dry weight)100]. Moisture percentage was determined as [100 - (dry weight/fresh weight)] for both leaf and stem tissue. Leaf tensile strength was measured by attaching leaf blades from either the first or second most recently expanded blade to both the arm and base of a triple beam balance. A beaker was placed on the platform of the balance and no. 7 lead shot was added to the beaker until a tissue breaking point was reached. Grams required to tear the leaf blade in half were recorded as leaf tensile strength.
Verdure tissue was measured by pulling three 5.7-cm-diam. cores per plot following mowing. All tissue was removed from cores with shears and collectively dried in a forced-air drier at 70°C and dry weights measured. Number of shoots per core was then determined. Shoot tissue injury (STI) was defined as the percentage of leaves injured or killed due to wear treatment. This was determined by visual ratings at 2, 4, 7, and 14 d after treatment (DAT) (Trenholm et al., 1999).
Tissue nutrient concentration was determined by inductively coupled plasma emission spectrometry analysis for P, K, Ca, Mg, B, Cu, Fe, Mn, Mo, and Zn. Total N was determined by the methodology of Kirsten (1979). Cell wall components (total cell wall, lignin, lignocellulose, hemicellulose, and cellulose) were measured separately by leaf and stem tissue following the procedures outlined by Goering and Van Soest (1970). Due to lack of tissue for analysis, Tifway bermudagrass was not analyzed for stem TCW, lignocellulose, hemicellulose, or cellulose during Study 2.
Grasses were arranged in a randomized block design with three replications. Wear treatments provided a strip plot arrangement. Analysis of variance (SAS Institute, 1987) determined differences between grasses at the 0.10 probability level. For interspecific comparisons, measurements from all seven paspalum entries were averaged together and compared with average measurements from the three bermudagrass entries. Multiple regression analysis used a stepwise regression procedure with significance determined at the 0.15 probability level. All regression analysis was developed from data in both studies. Morphological and anatomical regression equations were developed by regressing all variables against the average STI taken at 4 and 7 DAT. Nutrient regression equations were developed by regressing data against the average of STI taken at 2, 4, and 7 DAT. Wear tolerance was then defined as 100 - STI; therefore, as STI increased, wear tolerance declined. Because differences between species occurred for the majority of responses, all data were analyzed separately by species Means separation was performed using Waller-Duncan k ratio t test at the 0.05 probability level. Significant study x study interactions occurred for a high number of parameters; therefore, analysis of variance data are presented separately by study.
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Results
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Analysis of variance indicated that differences existed for numerous variables among all 10 turfgrass entries as well as between species in these studies. Since these variables were used to develop multiple regression analysis for interspecific wear tolerance, differences in responses for these measurements can be important in determining wear tolerance mechanisms within the two species. Analysis of variance indicated that differences in STI between species at 7 DAT (
and 0.01 in Studies 1 and 2, respectively) were significant.
Shoot Moisture, Strength, Density, and Verdure
Leaf and stem moisture and leaf strength differed between turfgrass entries in both studies (Table 1)
. TifSport bermudagrass ranked in the lowest grouping for leaf moisture in both studies, while all three bermudagrass entries ranked in the lowest category in Study 2 stem moisture. Adalayd paspalum, which has an intermediate leaf texture, had the greatest leaf strength in both studies. Comparisons between species showed significant differences for the majority of variables. Leaf turgor and strength were greater in paspalum in both studies; leaf (Studies 1 and 2) and stem (Study 2 only) moisture were also greater in paspalum. As previously reported, but repeated here for inclusion in regression analysis, verdure and shoot density per unit area differed significantly among all 10 entries (Trenholm et al., 1999, unpublished data) (Table 2)
. Interspecific differences in amount of verdure tissue were observed, with greater amounts in bermudagrass in both studies. No differences in mean shoot density were found between species, but ranged from 1.3 to 4.0 shoots cm-2 for paspalum and from 2.1 to 3.4 shoots cm-2 for bermudagrass.
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Table 1 Shoot measurements taken prior to application of wear on seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars in two consecutive studies
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Table 2 Verdure and shoot density prior to application of wear on seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars in two consecutive studies***
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Leaf Tissue Cell Wall Constituents
In Study 1, differences occurred among entries for leaf TCW content, lignocellulose, hemicellulose, and cellulose (Table 3) . Adalayd paspalum ranked in the highest statistical category for cell wall constituents five out of five times, followed by AP 8 paspalum with four out of five highest rankings. All three bermudagrass cultivars ranked in the highest category for leaf TCW and hemicellulose. Interspecific differences were found for all components except cellulose. Paspalum entries had significantly higher leaf lignin and lignocellulose components, while bermudagrass entries ranked higher for leaf TCW and hemicellulose.
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Table 3 Leaf cell wall constituents of seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars in two consecutive studies
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During Study 2, differences among entries existed for leaf TCW and lignocellulose (Table 3). TifSport had the greatest leaf TCW content, while Tifgreen had the lowest content. Hemicellulose differed between species, with the highest content found in bermudagrass.
Stem tissue, like leaf tissue, differed among entries for all cell wall measurements except lignin during Study 1 (Table 4)
. Paspalum PI 509022 ranked in the highest statistical category for all five measurements, followed by AP 8, K1, and HI-1 paspalums and TifSport bermudagrass. Species comparisons showed highest stem TCW and hemicellulose in paspalum, while levels of lignocellulose and cellulose were greatest in the bermudagrasses.
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Table 4 Stem cell wall constituents of seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars in two consecutive studies
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In Study 2, entries exhibited differences for all cell wall measurements (Table 4). Tifgreen bermudagrass had the lowest stem TCW content; TifSport and Tifgreen bermudagrasses both ranked lowest for hemicellulose content. TifSport had the highest stem lignin content, while Adalayd was lowest in lignin content. Interspecies comparisons were as in Study 1, with the highest stem TCW and hemicellulose levels in paspalum. Bermudagrass had higher levels of lignocellulose and cellulose.
Tissue Nutrient Concentration
Tissue macronutrient concentration differed among grasses in both studies (Tables 5 and 6)
. Although differences in N and P concentration were minimal between grasses, K concentration was significantly greater in the paspalums. In Study 1, the coarse-leaved paspalums generally had lower K levels than the fine-leaved ecotypes, with less distinction among the paspalums in Study 2.
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Table 5 Leaf tissue nutrient concentration of seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars taken 14 d after wear treatment during Study 1*
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Table 6 Leaf tissue nutrient concentration of seven seashore paspalum ecotypes and three hybrid bermudagrass cultivars taken 14 d after wear treatment during Study 2
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Comparisons between species indicated greater levels of both K and Mg in paspalum in both studies, while Ca was consistently higher in the bermudagrasses. In Study 2, P concentration was also greater in paspalum.
Multiple Regression Analyses
Morphological and Anatomical Mechanisms
The morphological and anatomical factors shown to correlate with wear tolerance in seashore paspalum and bermudagrass, the ranges of these units, and their contribution to the regression models are listed in Table 7
. Multiple regression analysis for paspalum wear tolerance using the units presented in the data tables for various parameters resulted in the following equation:
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Table 7 Morphological and anatomical factors influencing wear tolerance of seashore paspalum and bermudagrass as determined in two consecutive studies
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The r2 value for this equation was 0.73.
Multiple regression analysis for bermudagrass wear tolerance resulted in the following equation:
The r2 value for this equation was 0.99.
Nutrient Mechanisms
The nutrients shown to influence wear tolerance in seashore paspalum and bermudagrass, the ranges of these units, and their contribution to the regression models are listed in Table 8
. Multiple regression analysis for paspalum wear tolerance resulted in the following equation:
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Table 8 Nutrient values influencing wear tolerance of seashore paspalum and bermudagrass as determined in two consecutive studies
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The r2 value for this equation was 0.47.
Multiple regression analysis for bermudagrass wear tolerance resulted in the following equation:
The r2 value for this equation was 0.83.
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Discussion
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Regression analysis indicated that factors that enhanced wear tolerance generally differed between species. This implies that, to accurately screen for wear tolerance, anatomical, morphological, and physiological factors must be determined at the species level. Knowledge of the most important characteristics enhancing wear tolerance within species allows breeders to use the primary features as screening criteria and may permit application of genetic or biotechnological approaches for enhancement of specific mechanisms important for wear tolerance in desirable species.
The factor that explained the greatest amount of variation in wear tolerance of seashore paspalum (i.e., the most important mechanism) was leaf TCW content (Table 7). Wear tolerance of paspalum was decreased as leaf TCW increased, and to a lesser extent, was decreased by an increase in stem TCW content. This is in contrast to the findings of Shearman and Beard on cool-season grasses (1975b), but similar to findings of Canaway (1981).
Other factors influencing wear tolerance of seashore paspalum were leaf moisture, leaf strength, and shoot density. Higher leaf moisture was associated with greater wear tolerance, as previously noted by Beard (1973). However, Shearman and Beard (1975c) observed no significant correlation between leaf and stem moisture (r = -0.51 and -0.26, respectively) and wear tolerance in cool-season turfgrass.
Increased leaf strength was correlated with reduced wear tolerance. The coarse-leaved paspalums, with the lowest wear tolerance of any entry, had consistently high leaf strength scores. These findings are in contrast to correlation data obtained on cool-season turf species (Shearman and Beard, 1975c), although Sun and Liddle (1993a) found that stem flexibility was more important in imparting resistance to trampling than was leaf tensile strength in nine grass and herb species. Results of the work reported here would presumably be caused by decreased elasticity in leaf blades of the coarse-leaved grasses, which in this case resulted in reduced wear tolerance due to both increased rigidity and lower inherent growth rate.
In keeping with observations of Beard (1973), wear tolerance of seashore paspalum was enhanced by greater shoot density. This could be due to the greater quantity of tissue available to absorb the impact of the injury, or it may be indicative of a greater number of meristematic growth points and therefore greater inherent growth rate. Although Shearman and Beard (1975c) reported that amount of verdure tissue did not influence wear tolerance, it is generally recognized that greater quantity of shoot tissue reduces wear injury (Youngner, 1961; Beard et al., 1981; Sun and Liddle, 1993b; Carrow, 1995).
In a separate regression analysis to determine the nutrients that influence wear tolerance, wear tolerance of seashore paspalum was enhanced in response to increased K concentration in leaf tissue. According to Beard (1973), high K levels increase wear tolerance due to increased turgidity and reduced tissue succulence. Creeping bentgrass (Agrostis stolonifera L.) cv. Toronto had significantly improved wear tolerance due to application of 270 or 360 kg ha-1 K (Shearman and Beard, 1975d). They reported increased K tissue concentration, mat accumulation, load-bearing capacity, and leaf tensile strength at higher K rates; however, in contrast, Hawes and Decker (1977) and Carroll and Petrovic (1991) found no K influence on wear tolerance or recovery from wear in Kentucky bluegrass (Poa pratensis L.) or creeping bentgrass. Likewise, bermudagrass recovery from coring and verticutting was not enhanced by K (Carrow et al., 1987).
An overview of factors associated with wear tolerance within seashore paspalum ecotypes would suggest that (i) less rigid leaf and stem cell walls and reduced individual leaf strength favor greater wear tolerance. As leaf and stem TCW content decrease, cell wall elasticity should be enhanced, while low leaf blade strength implies a more elastic (i.e., less rigid) canopy. Sun and Liddle (1993a) reported that leaf flexibility was of greater importance than tensile strength for imparting wear tolerance. Further considerations from these results suggest that wear tolerance is enhanced by (ii) greater shoot density, which provides a cushioning effect associated with increased turf canopy, and (iii) maintenance of high turgor pressure, as indicated by positive correlations with leaf moisture and leaf K concentration. To maximize wear tolerance of an individual ecotype, turfgrass managers can adjust their cultural practices to favor high shoot density and adequate K uptake and to avoid drought stress and loss of tissue moisture content. Breeders may wish to focus on enhancement of leaf K concentration and avoidance of high leaf TCW content as means of improving wear tolerance of this species.
The factors that explained the greatest amount of variation in wear tolerance of bermudagrass (i.e., the most important mechanisms) were stem moisture and stem cellulose. As in paspalum, high stem moisture (and, to a lesser degree, high leaf moisture) increased wear tolerance. Improved wear tolerance was associated with reduced stem cellulose content. Canaway (1981) also reported a negative correlation between wear tolerance and percentage of cellulose content in seven cool-season grasses. However, cellulose content per unit area was positively correlated
with wear tolerance of several cool-season species, while cellulose on a dry weight basis was not
(Shearman and Beard, 1975b).
Bermudagrass wear tolerance was increased by greater amounts of leaf lignin, the presence of which is associated with secondary cell wall formation and thickening. Lignin content is positively associated with tissue age (Akin, 1989; Jung, 1989) and is a primary factor that reduces digestibility in forage grasses (Buxton and Russell, 1988). Although differences between species for lignin content were only seen in leaf tissue during Study 1, bermudagrass had a small increase (3% of the total variation in the regression model) in wear tolerance due to the presence of lignin. This is in agreement with reports of Beard (1973) and Shearman and Beard (1975b), based on lignin expressed in weight per unit area.
Wear tolerance in bermudagrass was enhanced by greater stem and leaf lignocellulose content. Shearman and Beard (1975b) found no correlation between wear tolerance and lignocellulose content. Leaf lignocellulose content was higher in paspalum (
and 0.17 in Studies 1 and 2, respectively), while stem tissue was greater in bermudagrass in both studies (
and 0.01, respectively). As with paspalum, shoot density also increased wear tolerance of bermudagrass.
Nutrients positively associated with wear tolerance of bermudagrass were K, Mg, and Mn. The influence of K would probably again concern maintenance of cell turgor pressure. Differences in K tissue concentration were noted, both between species and within the paspalum entries. Lowest levels of K were found in the bermudagrass entries and highest concentrations in fine-leaved paspalums. Marcum and Murdoch (1990, 1994) had previously reported higher K tissue concentration in paspalum than in bermudagrass.
When considered together, factors related to wear tolerance in bermudagrasses would indicate that (i) higher tissue rigidity from leaf lignin and leaf and stem lignocellulose content was important for wear tolerance. This differs considerably from paspalum, where greater cell and tissue elasticity imparted more wear tolerance. As noted above, the relationship of lower stem cellulose content associated with better wear tolerance could be interpreted as either greater lignification decreasing cellulose content, or that low cell wall cellulose content imparts cell flexibility to pressures from wear. The former is most likely the correct interpretation since these relationships were stronger in the stem, where stem rigidity would help resist wear forces. Other considerations for bermudagrass wear tolerance include (ii) a dense turf canopy that enhances wear tolerance, (iii) adequate stem moisture aids in maintenance of turgor pressure and possible prevention of stem breakage, and (iv) ample K, which is necessary to ensure maintenance of turgor pressure. Turfgrass managers can focus on improving shoot density, plant moisture, and adequate K status to maximize wear tolerance of hybrid bermudagrass.
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Conclusions
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These studies revealed anatomical and morphological contributions to wear tolerance of seashore paspalum and bermudagrass and thus provide insight into different wear tolerance mechanisms for each species. Both species had enhanced wear tolerance due to greater shoot density, K concentration, and leaf moisture, while other factors were species specific for wear tolerance. In paspalum, reduced leaf TCW content accounted for
50 % of the variation in wear tolerance for this species, while in bermudagrass, stem moisture and stem cellulose content combined were responsible for 72% of the variation in wear tolerance. This information is important for providing rapid screening techniques for selection of wear-tolerant cultivars or ecotypes. Breeding programs can employ lab screening protocols to reliably predict and select for wear tolerance on the basis of the most prominent variables that influence wear tolerance on a species basis. Further application of this information could be isolation of the genes responsible for imparting the desirable traits that enhance a specific stress tolerance and insertion of them into a desirable turfgrass selection.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge funding from the United States Golf Association, the Golf Course Superintendents Association of America, the Georgia Turfgrass Foundation Trust, Turfgrass Producers International, and the Potash and Phosphate Institute.
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NOTES
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Florida Agric. Exp. Stn. Journal no. R-07581.
Received for publication May 14, 1999.
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H. Samaranayake, T. J. Lawson, and J. A. Murphy
Traffic Stress Effects on Bentgrass Putting Green and Fairway Turf
Crop Sci.,
May 1, 2008;
48(3):
1193 - 1202.
[Abstract]
[Full Text]
[PDF]
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J. T. Brosnan, J. S. Ebdon, and W. M. Dest
Characteristics in Diverse Wear Tolerant Genotypes of Kentucky Bluegrass
Crop Sci.,
August 26, 2005;
45(5):
1917 - 1926.
[Abstract]
[Full Text]
[PDF]
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