Use of Fungal Laccases to Facilitate Biodethatching: A New Approach

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  • 1 Department of Crop and Soil Sciences, The University of Georgia, Griffin Campus 1109 Experiment Street, Griffin, GA 30223

Accumulation of excessive organic matter as thatch restricts permeability of putting greens and is one of the most difficult problems in turfgrass management. A greenhouse experiment using potted bentgrass (Agrostis stolonifera L.) determined the efficacy of a ligninolytic enzyme, laccase, in reducing organic matter accumulation in the thatch-mat layer. Laccase was added biweekly at 0, 0.206, 2.06, and 20.6 units of activity/cm2 with and without guaiacol (2-methoxyphenol), a mediator of laccase, and sampling was performed after two and nine months. Parameters investigated included thickness of the organic layer, thatch layer and mat layer, organic matter content, saturated hydraulic conductivity, and lignin content. Organic matter and thatch layer increased between the two sampling dates in all treatments. Laccase was shown to be effective in slowing the rate of accumulation of organic matter and thatch layer. After two months, application of 20.6 units/cm2 of laccase reduced organic layer thickness by 8.7% and extractive-free total lignin content by 8.4% when compared with non-treated control. After nine months, laccase application rates of 2.06 units/cm2 reduced organic matter and thatch layer thickness by 15.6% and 45.0%, respectively, below levels observed in the non-treated control. Applications using 0.206 units/cm2 of laccase were ineffective. Laccase applications had no influence on turf quality. These positive responses suggest laccase treatments could be a non-disruptive option for thatch and/or mat control in bentgrass.

Abstract

Accumulation of excessive organic matter as thatch restricts permeability of putting greens and is one of the most difficult problems in turfgrass management. A greenhouse experiment using potted bentgrass (Agrostis stolonifera L.) determined the efficacy of a ligninolytic enzyme, laccase, in reducing organic matter accumulation in the thatch-mat layer. Laccase was added biweekly at 0, 0.206, 2.06, and 20.6 units of activity/cm2 with and without guaiacol (2-methoxyphenol), a mediator of laccase, and sampling was performed after two and nine months. Parameters investigated included thickness of the organic layer, thatch layer and mat layer, organic matter content, saturated hydraulic conductivity, and lignin content. Organic matter and thatch layer increased between the two sampling dates in all treatments. Laccase was shown to be effective in slowing the rate of accumulation of organic matter and thatch layer. After two months, application of 20.6 units/cm2 of laccase reduced organic layer thickness by 8.7% and extractive-free total lignin content by 8.4% when compared with non-treated control. After nine months, laccase application rates of 2.06 units/cm2 reduced organic matter and thatch layer thickness by 15.6% and 45.0%, respectively, below levels observed in the non-treated control. Applications using 0.206 units/cm2 of laccase were ineffective. Laccase applications had no influence on turf quality. These positive responses suggest laccase treatments could be a non-disruptive option for thatch and/or mat control in bentgrass.

Formation of thatch and mat layers is one of the major problems in management of modern turfgrass golf greens. Thatch is a layer of organic matter that accumulates between the soil and green turfgrass and contains both living and dead plant tissues intermingled tightly with each other. Thatch consists of stolons, rhizomes, roots, crown tissue, leaf sheaths, and blades (Engel, 1954; Roberts and Bredakis, 1960). The mat layer is generally below the thatch layer and is distinguished from thatch by the presence of sand or soil intermingled with thatch as a result of cultural practices like core aeration and topdressing (McCarty, 2005). A small amount of organic matter reduces surface hardness, moderates soil temperature extremes, increases the resilience, and improves wear tolerance of the turfgrass surface (Beard, 1973); however, excessive thatch and mat layers are undesirable in turfgrass.

High organic matter accumulation in the form of thatch-mat causes problems such as decreased movement of oxygen through the thatch or mat zone, decreased saturated hydraulic conductivity, and excessive water retention (Carrow, 2003; Hartwiger, 2004; McCarty et al., 2007). These primary problems may further lead to secondary problems like wet wilt, soft surface, black layer, limited rooting, and extra- and intracellular freezing damage (Beard, 1973; Carrow, 2004; O’Brien and Hartwiger, 2003). Although structured organic matter, present in live underground plant tissues, is thought to have no adverse effect on the soil physical properties, rapid root death that results in dead gelatinous organic matter swells in the presence of water during decomposition and plugs the soil macropores (air-filled pores), causing low oxygen levels in the root zones (Carrow, 2004; O’Brien and Hartwiger, 2003). Excessive accumulation of organic matter causes anaerobic conditions, further reducing the rate of organic matter decomposition (McCoy, 1992). Grasses also generally produce more adventitious roots (surface roots) during anaerobic conditions, again further increasing organic matter content (Carrow, 2004).

Thatch management techniques such as core aeration, vertical mowing, grooming, and topdressing are currently the most effective strategies to manage thatch-mat buildup but have shown contrasting results (Barton et al., 2009; Carrow et al., 1987; Dunn et al., 1981; McCarty et al., 2005; McWhirter and Ward, 1976; Weston and Dunn, 1985; White and Dickens, 1984). These cultural practices are intensive in terms of cost, energy, and labor and have adverse effects on turfgrass quality (Barton et al., 2009; Landreth et al., 2008; McCarty et al., 2007). Several non-destructive thatch control studies using glucose, cellulase solutions (Ledeboer and Skogley, 1967), and commercial inocula containing various microorganisms were ineffective in reducing the amount of thatch (McCarty et al., 2005; Murdoch and Barr, 1976). Reduction in cellulose content and total oxidizable organic matter of bermudagrass (Cynodon dactylon L.) and centipedegrass (Eremochloa ophiuroides) (Sartain and Volk, 1984) and weight loss of bermudagrass pellets, St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze], and zoysiagrass (Zoysia japonica Stued., ‘Meyer’) stolons (Martin and Dale, 1980) was observed when inoculated with different wood-decaying fungi under controlled greenhouse and laboratory conditions. However, field inoculation experiments on bermudagrass showed no thatch degradation (Martin and Dale, 1980).

The formation of the thatch-mat layer is the result of a greater rate of organic matter accumulation than degradation (Beard, 1973). Most microbial degradation mechanisms are restricted by the presence of lignin, a plant cell wall constituent. The slow decomposition of soil lignin has long been recognized (Kirk and Farrell, 1987). Lignin limits the accessibility of microbial degraders to more biodegradable plant materials such as cellulose and hemicelluloses (Ledeboer and Skogley, 1967). Lignin is formed in plants by oxidative coupling of monolignols of three primary hydroxycinnamyl alcohols: p-coumaryl, coniferyl, and sinapyl alcohols. The corresponding lignin monomers are known as p-hydroxy phenyl, guaiacyl, and syringyl units, respectively (Wong, 2009). Lignification is achieved by crosslinking of monomers with a growing polymer through polymer–polymer coupling. Based on the random coupling theory, several models of lignin molecular structure have been proposed but these models do not imply any particular sequence of monomeric units in the lignin macromolecule (Chen and Sarkanen, 2003; Davin and Lewis, 2003).

Natural degradation of lignin occurs in the environment by certain white-rot fungi, which solubilize and mineralize lignin with the help of lignolytic enzymes (Kirk et al., 1975, 1976). White-rot fungi preferentially attack lignin more than cellulose or hemicellulose in the wood tissue (Blanchette, 1984; Mester et al., 2004). This process of selective delignification exposes cellulosic materials for further bacterial degradation in the environment (Otjen and Blanchette, 1987). The presence of naturally occurring (guaiacol) and synthetic (1-hydroxybenzotriazole) chemicals, known as mediators, have been shown to enhance the activity of the lignolytic enzyme laccase (Kang et al., 2002; Roper et al., 1995). Because lignin content in thatch layer is higher than that of live grass tissues, the thatch layer in turfgrass species with high lignin content is more resistant to microbial decomposition (Beard, 1973; Ledeboer and Skogley, 1967).

We hypothesize that the use of lignin-degrading enzymes such as fungal laccases can effectively reduce the rate of thatch layer accumulation in golf greens. The objectives of our study were: 1) to determine if degradation of soil organic matter can be enhanced by laccase application; 2) to determine if addition of guaiacol enhances laccase efficacy in organic matter decomposition; and 3) to determine if application of laccase enzyme and guaiacol adversely affect turf quality.

Materials and Methods

A greenhouse experiment was conducted using ‘Crenshaw’ creeping bentgrass Agrostis stolonifera L. (Engelke et al., 1995), established in pots (top diameter 15 cm, height 11.5 cm) at The University of Georgia, Griffin Campus, from Oct. 2008 to July 2009. The bentgrass was acquired from East Lake Country Club, Atlanta, GA. Pots were partially filled with 85:15 sand and organic matter mix and sod ≈3 cm in thickness was cut to fit the pots and placed on top of the mix. All pots were established in June 2008 and grown under management conducive to thatch development in a controlled environment greenhouse for about four months before initiation of treatments. Pots were irrigated daily, fertilized monthly with a 50 mL solution of 0.4% (w/v) Macron water soluble 28N–7P–14K fertilizer (Lesco, Strongsville, OH), and maintained by hand clipping weekly at a height of 0.6 cm with clippings removed. The refrigerated air-conditioned greenhouse was maintained at 25 ± 2/18 ± 2 °C day/night temperature by a Wadsworth Step 50 controller (Wadsworth Control System, Arvada, CO) under natural lighting (≈85% ambient light).

The treatment design was as a four-by-two factorial with all combinations of four levels of laccase and two levels of guaiacol (2-methoxyphenol). The four laccase activity levels were 0 (control), 0.206, 2.06, and 20.6 units/cm2 and guaiacol levels were 0 and 0.1 M solution. The experimental design was a randomized complete block with five replications and sampling times of two and nine months. Forty-milliliter solutions of the different laccase activity levels and 10 mL of guaiacol solutions were applied uniformly every 2 weeks to each pot using a handheld sprayer. The control pots were treated with equivalent amounts of distilled water. After the two months of treatment applications, the 20.6 units/cm2 treatments with and without guaiacol were discontinued as a result of limited availability of laccase enzyme. As a result of unexpected problems in developing protocols for measurement of saturated hydraulic conductivity, one replication was rendered unusable for measurement of other variables. Therefore, only four replications were used for analysis after nine months’ treatment duration.

Laccase activity assay.

The laccase enzyme from Trametes versicolor, a white-rot fungus, was purchased from Sigma-Aldrich (Product 53739; Sigma Aldrich Inc., St. Louis, MO). Laccase solutions were standardized based on active units, which were quantified using a ultraviolet/VIS-spectrophotometer by a colorimetric assay. One activity unit of laccase corresponds to the amount of enzyme that causes an absorbance change at 468 nm at a rate of 1.0 unit/min in 3.4 mL of 1 mm 2,6-dimethoxyphenol, a specific substrate for laccase, in citrate–phosphate buffer at pH 3.8 (Park et al., 1999). Laccase activity treatments of 0 (control), 0.206, 2.06, and 20.6 units/cm2 actually correspond to laccase solutions with activity levels of 0, 0.912, 9.12, and 91.2 units/mL, respectively. The activity level of laccase applied per unit area was calculated by dividing the total number of units of laccase in 40 mL laccase solution by the top surface area of the pot.

Measurements.

Effectiveness of treatments was determined by measuring organic matter content (OM) for a depth of 0 to 5.0 cm, organic layer thickness (OL), extractive-free acid-soluble lignin (LS), and acid-insoluble lignin (LI) content after two and nine months of treatment application. Total lignin was obtained by addition of acid-soluble and-insoluble lignin contents.

After nine months of treatment application, some additional variables were measured. OL was subdivided into thatch layer thickness (OLT) and mat layer thickness (OLM), whereas OM was subdivided into 0 to 2.5 and 2.5 to 5.0 cm depths to more accurately reflect the effectiveness of laccase on the thatch and mat layers. Saturated hydraulic conductivity (SHC) was also measured after nine months.

Organic matter content.

The measurement of OM was performed as described by Carrow et al. (1987). Soil cores (2.0 cm diameter) were dried in an oven at 100 ± 5 °C for 48 h and weighed. Soil cores were ashed in a muffle furnace at 600 ± 10 °C for 24 h and weighed again. Organic matter content was determined as the difference in the two readings and percent OM was calculated.

Saturated hydraulic conductivity.

Intact cores (diameter 4.7 cm and length 7.7 cm) were obtained from the center of each pot using a soil corer. The cores were collected in brass cylinders. The bottom of the core was covered with a double layer of cheesecloth held in place with a rubber band. The core was saturated overnight in a 0.05 N CaCl2 solution to minimize dispersion. A clear plastic cylinder of the same diameter as of the brass cylinder was fastened above the brass cylinder with paraffin wax tape. The SHC of the cores was measured by a constant hydraulic head method using a Marriott tube apparatus. A time of 10 min was allowed for the establishment of steady-state flow through the samples. The volume of water that passed through the core was measured for 1 min and repeated three times. Saturated hydraulic conductivity was calculated using Darcy's equation.

Organic layer thickness and thatch-mat layer thickness.

After cores were removed for measurement of OM content and SHC, plants were removed from pots and distinct separations among the thatch, mat, and soil interface were clearly visible. The OL, OLT, and OLM were measured from seven different locations around the edges of the plant/root mass and averaged.

Extractive-free lignin content.

Thatch was collected from each pot from the top 2.5 cm after sampling for OM and SHC. Extractive-free acid-soluble and -insoluble lignin content in the thatch layer was determined in a two-step hydrolysis procedure according to the laboratory analytical procedure developed by The National Renewable Energy Laboratory (NREL, 2008). In the first step, extractive-free thatch samples were hydrolyzed for 60 min with 72% H2SO4 at 30 °C. In the second step, H2SO4 was diluted to 4% and the samples were autoclaved at 121 °C for 1 h. Acid-soluble lignin was determined by measuring the absorbance of this hydrolysis liquid at 240 nm in a ultraviolet/VIS spectrophotometer. The solids remaining after acid hydrolysis were dried in an oven at 100 ± 5 °C for 24 h, weighed, ashed in a muffle furnace at 600 ± 10 °C for 24 h, and weighed again. Weight difference was used to calculate the acid-insoluble lignin content.

Turf quality.

Turf quality was determined biweekly for the first three months and again for the last two months of the experiment to document the potential for initial and long-term phytotoxicity associated with laccase application. The turf quality of each treatment was recorded every 2 weeks by rating both visual turf quality and canopy spectral reflectance. Visual turf quality ratings were rated on the basis of color, shoot density, and uniformity on a numerical scale where 1 equals no live turf and 9 equals ideal dark green, uniform turf (Johnson et al., 1987). Grass index was determined using a TCM 500 turf color meter (Spectrum Technologies, Plainfield, IL). Grass index is a numerical score of the color and density of grass based on the spectral reflectance at 660 and 850 nm. Three grass index readings were recorded from each pot and averaged for statistical analysis.

Statistical analysis.

Analysis of variance (ANOVA) was performed to evaluate the main effects of treatment duration, laccase, and guaiacol and interaction effects of these three factors using the general linear model (SAS Institute Inc., 1994). Strong treatment duration effects (P ≤ 0.001) were observed in the initial analysis and therefore each treatment duration was analyzed separately using ANOVA as a two-factor study consisting of four levels of laccase enzyme and two levels of guaiacol for the two months’ treatment duration and three levels of laccase enzyme and two levels of guaiacol for the nine months’ treatment duration. Fisher’s protected least significant difference test with α = 0.05 was used for determining statistical differences among treatment means after each ANOVA.

Results

The full statistical model was used to compare common parameters at the two- and nine-month sampling dates (Table 1). The model included the main and interaction effects of treatment duration, three levels of laccase, and two levels of guaiacol for OM (0 to 5.0 cm), OL, LS, andLI. Only three levels of laccase were used because the 20.6 units/cm2 laccase treatment was discontinued after two months of application.

Table 1.

Analysis of variance table showing the effects of treatment duration, laccase application, guaiacol application, and their interactions on organic layer thickness (OL), organic matter (OM), acid-soluble lignin (LS), acid-insoluble lignin (LI), total lignin (LT), thatch layer thickness (OLT), mat layer thickness (OLM), and saturated hydraulic conductivity (SHC) on creeping bentgrass maintained in a greenhouse.

Table 1.

Treatment duration strongly affected OM (0 to 0.5 cm; P ≤ 0.001), OL (P ≤ 0.001), and LS (P ≤ 0.001) (Table 1). Laccase application significantly affected OM (0 to 5.0 cm), OL, LS, and LI. The very strong interactions for treatment duration and laccase treatment observed in OM (0 to 0.5 cm; P ≤ 0.001), OL (P ≤ 0.001), and LS (P ≤ 0.01) were largely the result of the lack of response to laccase treatments after two months as opposed to a strong response seen after nine months. Guaiacol treatment as well as the interaction of guaiacol and treatment duration had no effect on any of the parameters (Table 1).

We observed an overall increase in OM (0 to 5.0 cm) and OL in all treatments between the two sampling dates. However, accumulation of OM (0 to 5.0 cm) and OL was significantly lower in treatments containing laccase when compared with the control. When compared with the control, the rate of accumulation of OM (0 to 5.0 cm) was reduced from 15.8 mg·g−1 in control pots to 9.0 mg·g−1 in pots treated with 2.06 units/cm2 laccase (43%) (Table 2). Similarly, application of 2.06 units/cm2 laccase reduced OL accumulation from 15.5 mm to 11.7 mm (24%) when compared with the control (Table 2). A reduction in lignin content between the two sampling dates was observed at laccase activity levels of 2.06 units/cm2 with and without guaiacol (Table 3).

Table 2.

Organic layer thickness (OL) and organic matter (OM) content (0 to 5.0 cm depth) after two and nine months of different treatments applied to creeping bentgrass.z

Table 2.
Table 3.

Extractive-free acid-soluble (LS), acid-insoluble (LI), and total lignin (LT) content after two and nine months of different treatments applied to creeping bentgrass.z

Table 3.

Analysis after two months’ treatment.

After two months of treatment application, laccase treatments had no effect on OM (0 to 5.0 cm) but had significant effects on OL, LS and LI (Table 1). Neither guaiacol nor the laccase by guaiacol interaction had significant effects on any of the parameters (Table 1).

Compared with the control, treatment with 20.6 units/cm2 of laccase without guaiacol for two months significantly lowered LS by 5.2, LI by 20.5, and LT content by 25.6 mg·g−1 (Table 3). Similarly, treatment at the same laccase activity with guaiacol reduced LS by 4.2, LI by 18.9, and LT by 23.4 mg·g−1 when compared with control. Treatment with 2.06 units/cm2 of laccase with guaiacol also significantly reduced LS by 1.4 mg·g−1 (Table 3).

Analysis after nine months’ treatment.

After nine months of treatment application, laccase application impacted OM (0 to 5.0 and 0 to 2.5 cm), SHC, OL, OLT, LS, and LI, and SHC (P ≤ 0.001) (Table 1). A significant effect of guaiacol was also observed for LS (P ≤ 0.05) and SHC (P ≤ 0.01) (Table 1). The interaction of laccase by guaiacol was significant (P ≤ 0.01) for SHC. However, none of the treatments affected OM (2.5 to 5.0 cm) or mat layer thickness after nine months of treatment application.

Treatment with 2.06 units/cm2 of laccase with and without guaiacol decreased OL by 10.8 and 9.5 mm, respectively, when compared with the control (Table 2). These same treatments reduced OLT by 8.3 (45%) and 6.5 mm (35%) when compared with the control (Fig. 1). Laccase applied at the same activity was effective in reducing OL and OM, whereas laccase application at 0.206 units/cm2 was not different from the control (Table 2; Fig. 2).

Fig. 1.
Fig. 1.

Thatch (OLT) and mat layer thickness (OLM) after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are standard errors. Bars with the same letter (OLM = bolded and OLT = standard) are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1536

Fig. 2.
Fig. 2.

Organic matter (OM) for 0 to 2.5 cm depth and 2.5 to 5.0 cm depth after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are standard errors. Bars with the same letter [OM (0 to 2.5 cm) = bolded and OM (2.5 to 5.0 cm) = standard] are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1536

Treatment with 2.06 units/cm2 laccase with and without guaiacol reduced OM (0 to 5.0 cm) by 7.6 and 7.8 mg·g−1 and OM (0 to 2.5) by 25.9 and 30.3 mg·g−1, respectively, as compared with the control (Table 2; Fig. 2). Similarly, treatment with 2.06 units/cm2 laccase with and without guaiacol increased SHC by 21.6 (322%) and 6.3 cm·h−1 (94%), respectively, over the control (Fig. 3). When compared with control, treatment with 2.06 units/cm2 laccase without guaiacol reduced LS by 5.1, LI by 14.0, and LT by 19.0 mg·g−1, respectively (Table 3).

Fig. 3.
Fig. 3.

Saturated hydraulic conductivity (SHC) after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are ses. Bars with the same letter are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

Citation: HortScience horts 47, 10; 10.21273/HORTSCI.47.10.1536

Turf quality.

No significant differences in visual quality ratings were observed among the treatments except for the data collected after 38 weeks when 2.06 units/cm2 of laccase exhibited a slight but significant reduction in turf quality when compared with the control treatment (Table 4). No significant differences from the control were observed for any treatment when means of the visual ratings were compared for the early (2 to 12 weeks), late (32 to 38 weeks), and all periods (Table 4). When compared with the control, pots receiving 20.6 units/cm2 laccase treatment had a small but significant decrease in grass index at 4 and 6 weeks after treatment initiation (Table 5). However, no significant differences in grass index values were observed after 6 weeks of treatment application. No visual differences among treatments for turf color or growth rate were observed over the duration of the experiment.

Table 4.

Mean visual turf quality ratings of creeping bentgrass made over time following continued treatment with different laccase and guaiacol solutions to greenhouse-grown plants.

Table 4.
Table 5.

Mean grass index values of creeping bentgrass made over time following continued treatment with different laccase and guaiacol solutions to greenhouse-grown plants.

Table 5.

Discussion

Laccase application.

This is the first study to report the direct application of laccase enzyme to manage thatch-mat accumulation on creeping bentgrass. Application of laccase, especially at the 2.06 units/cm2 activity level, proved to be effective in reducing thatch-mat depth, OM, and significantly increasing SHC. Carley et al. (2011) noted that OM accumulates rapidly near the soil surface by small annual accumulations that result in long-term effects. Our results indicate that application of 2.06 units/cm2 laccase alters OM dynamics in a positive manner by effectively reducing OM (0 to 2.5 cm) and OLT in comparison with the control. However, an increase in OM (0 to 5.0 cm) and OL was observed for all the treatments over the experiment duration. Application of laccase enzyme was effective in reducing the rate of accumulation of thatch layer thickness and OM.

The 2.06 units/cm2 laccase treatment, after nine months of application, also resulted in increased SHC in which a three- and two-fold increase in SHC was observed with applications of 2.06 units/cm2 laccase with and without guaiacol, respectively. This increase can be explained on the basis of thatch layer thickness of the corresponding treatment. Thatch layer thickness more than 1.3 cm was reported to adversely affect water infiltration (McCarty et al., 2005). Thatch layer thickness for the treatment of 2.06 units/cm2 with and without guaiacol after nine months of treatment was 11.2 and 13.0 mm, respectively.

For both the two and nine months’ sampling, no effect of guaiacol was observed and no interaction effect with laccase was observed except for SHC after nine months of application. The 2.06 units/cm2 laccase treatment was ineffective when applied with guaiacol after two months of application except for a reduction in extractive-free LS. The significant replication effect observed for LS and LI for two and nine months, respectively, may be associated with incomplete acid hydrolysis for some replications during autoclaving and unavailability of additional sample materials for reanalysis.

The lowest level of laccase application (0.206 units/cm2) proved to be an ineffective treatment even after nine months of application for all the parameters measured. The 20.6 units/cm2 treatment was applied for two months and resulted in no reduction of OM (0 to 5.0 cm). However, this treatment did result in a significant reduction in OL and extractive-free lignin content (LS, LI, and LT) of the thatch layer.

Laccase application had only minor influences on turfgrass quality. A slight reduction in turf quality was indicated by lower grass index values during the first 4 to 6 weeks in response to the 20.6 units/cm2 laccase treatment. However, visual quality ratings were not significantly different from controls except for one treatment combination at 38 weeks.

Treatment duration.

If laccase was effective in enhancing OM degradation, it would seem reasonable to expect that effects would become more apparent over time. Samples were analyzed after two and nine months of treatment application. Laccase activity levels of 0.206 and 2.06 units/cm2 area were continued for nine months. It was observed that treatment duration and the interaction of treatment duration with laccase treatments had a significant effect on OM (0 to 5.0 cm), OL, and LS content.

Why laccase application?

Studies in the past using various cultural management practices with different cultivation frequencies have reported contrasting results for reduction in thatch-mat accumulation (Callahan et al., 1998; Carrow et al., 1987; Engel and Alderfer, 1967; McCarty et al., 2005; Rieke, 1994). Degradation of thatch-mat is reported either in terms of thatch-mat depth (Smiley et al., 1985; Soper et al., 1988) or in terms of thatch-mat depth and OM content by weight (Barton et al., 2009; McCarty et al., 2007). The OM content by weight in different studies is observed for different depths further making it difficult to compare the results (Barton et al., 2009; McCarty et al., 2005; Murray and Juska, 1977). In our study, however, we observed both OL thickness (thatch layer and mat layer) and OM content to provide a better comparison of the effectiveness of laccase on thatch-mat degradation.

Cultural practices like core aeration and vertical mowing are disruptive in nature and have shown to reduce the turf quality both aesthetically and physically, further reducing the playability of the turf (Barton et al., 2009; Landreth et al., 2008; McCarty et al., 2007). However, application of laccase is not disruptive and the effective treatment of 2.06 units/cm2 laccase for nine months showed no overall reduction in turf quality of bentgrass.

Several non-destructive studies in the past using different treatments like sugars, mixtures of sugars and microbial inocula, and some enzymes like cellulase proved ineffective (Ledeboer and Skogley, 1967; Martin and Dale, 1980; McCarty et al., 2005; Murdoch and Barr, 1976). Most of these studies intended to increase microbial population to degrade OM. However, it is difficult to maintain higher microbial populations over sustained periods of time under field turfgrass management systems as a result of the inability to maintain proper microenvironment conditions required by particular microbial populations. Another reason that such studies were ineffective may be that they were focused on degradation of cellulose and hemicellulose by using cellulase enzyme and by increasing bacterial populations, whereas our hypothesis is that lignin degradation will open the cell wall structure of thatch biomass, hence making cellulose and hemicellulose more available for further microbial degradation. In our study, we used the end product from the white-rot fungi Trametes versicolor, the laccase enzyme, which is stable over a wide pH and temperature (Baldrian, 2006; Munoz et al., 1997; Stoilova et al., 2010; Thurston, 1994) to degrade lignin and to facilitate dethatching.

This greenhouse research demonstrated that biweekly application of laccase enzyme at 2.06 units/cm2 can be effective in reducing the rate of accumulation of OM in highly maintained turf. However, low activity levels of laccase (0.206 units/cm2) were ineffective in reducing the rate of thatch accumulation. Laccase application had little effect after two months but significantly reduced OM after nine months. Implications of these findings point to a novel approach to reduce OM in thatch or mat and its associated problems on golf greens. This approach can lead to the development of a new non-disruptive method for thatch management. Future research is needed to observe the effectiveness of laccase under field conditions as well as to optimize the activity level of laccase and the frequency of its application.

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    • Export Citation
  • Chen, Y.R. & Sarkanen, S. 2003 Macromolecular lignin replication: A mechanistic working hypothesis PhytomTOMhemistry Rev. 2 235 255

  • Davin, L.B. & Lewis, N.G. 2003 A historical perspective on lignin biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism Phytochem. Rev. 2 257 288

    • Search Google Scholar
    • Export Citation
  • Dunn, J.H., Sheffer, K.M. & Halisky, P.M. 1981 Thatch and quality of Meyer zoysia in relation to management Agron. J. 73 949 952

  • Engel, R.E. 1954 Thatch on turf and its control Golf Course Rpt. 22 12 14

  • Engel, R.E. & Alderfer, R.B. 1967 The effect of cultivation, top-dressing, lime, N, and wetting agent on thatch development on 1/4-inch bentgrass over a 10-year period N.J. Agr. Exp. Stn. Bull. 818 32 45

    • Search Google Scholar
    • Export Citation
  • Engelke, M.C., Lehman, V.G., Kneebone, W.R., Colbaugh, P.F., Reinert, J.A. & Knoop, W.E. 1995 Registration of ‘Crenshaw’ creeping bentgrass Crop Sci. 35 589

    • Search Google Scholar
    • Export Citation
  • Hartwiger, C. 2004 The importance of organic matter dynamics: How research uncovered the primary cause of secondary problems USGA Green Section Record 42 9 11

    • Search Google Scholar
    • Export Citation
  • Johnson, B.J., Carrow, R.N. & Burns, R.E. 1987 Bermudagrass turf response to mowing practices and fertilizer Agron. J. 79 677 680

  • Kang, K.H., Dec, J., Park, H. & Bollag, J.M. 2002 Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in presence of phenolic mediators and humic acid Water Res. 36 4907 4915

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K., Connors, W.J., Bleam, R.D., Hackett, W.F. & Jeikus, J.G. 1975 Preparation and microbial decomposition of synthetic [14C] lignins Proc. Natl. Acad. Sci. USA 72 2515 2519

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K., Connors, W.J., Bleam, R.D. & Jeikus, G. 1976 Requirements for a growth substrate during lignin decomposition by two wood-rotting fungi Appl. Environ. Microbiol. 32 192 194

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K. & Farrell, R.L. 1987 Enzymatic ‘combustion’: The microbial degradation of lignin Annu. Rev. Microbiol. 41 465 501

  • Landreth, J., Karcher, D. & Richardson, M. 2008 Cultivating to manage organic matter in sand based putting greens: University of Arkansas researchers provide important insight for managing organic buildup on putting greens USGA Turfgrass Environ. Res. Online 46 16 19

    • Search Google Scholar
    • Export Citation
  • Ledeboer, F.B. & Skogley, C.R. 1967 Investigations into the nature of thatch and methods for its decomposition Agron. J. 59 320 323

  • Martin, S.B. & Dale, J.L. 1980 Biodegradation of turf thatch with wood-decay fungi Phytopathology 70 297 301

  • McCarty, L.B. 2005 Best golf course management practices. 2nd Ed. Prentice Hall Inc., Upper Saddle River, NJ

  • McCarty, L.B., Gregg, M.F. & Toler, J.E. 2007 Thatch and mat management in an established creeping bentgrass green Agron. J. 99 1530 1537

  • McCarty, L.B., Gregg, M.F., Toler, J.E., Camberato, J.J. & Hill, H.S. 2005 Minimizing thatch and mat development in a newly seeded creeping bentgrass golf green Crop Sci. 45 1529 1535

    • Search Google Scholar
    • Export Citation
  • McCoy, E.L. 1992 Quantitative physical assessment of organic materials used in sports turf rootzone mixes Agron. J. 84 375 381

  • McWhirter, E.L. & Ward, C.Y. 1976 Effect of vertical mowing and aerification on golf green quality Mississippi Agr. For. Exp. Stn. Res. Rpt. 2 1 2

  • Mester, T., Varela, E. & Tien, M. 2004 Wood degradation by brown-rot and white-rot fungi. The Mycota II: Genetics and biotechnology. 2nd Ed. Springer-Verlag, Berlin, Heidelberg, Germany

  • Munoz, C., Guillen, F., Martinez, A.T. & Martinez, M.J. 1997 Laccase isozymes of Pleurotus eryngii: Characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation Appl. Environ. Microbiol. 63 2166 2174

    • Search Google Scholar
    • Export Citation
  • Murdoch, C.L. & Barr, J.P. 1976 Ineffectiveness of commercial microorganism inoculums in breaking down thatch in common bermudagrass in Hawaii HortScience 11 488 489

    • Search Google Scholar
    • Export Citation
  • Murray, J.J. & Juska, F.V. 1977 Effect of management practices on thatch accumulation, turf quality, and leaf spot damage in common kentucky bluegrass Agron. J. 69 365 369

    • Search Google Scholar
    • Export Citation
  • National Renewable Energy Laboratory 2008 Determination of structural carbohydrates and lignin in biomass. 9 Aug. 2012. <http://www.nrel.gov/biomass/pdfs/42618.pdf>. NREL, Golden, CO

  • O’Brien, P. & Hartwiger, C. 2003 Aeration and topdressing for the 21st century: Two old concepts are linked together to offer up-to-date recommendations USGA Green Section Record 41 1 7

    • Search Google Scholar
    • Export Citation
  • Otjen, L. & Blanchette, R. 1987 Assessment of 30 white rot basidiomycetes for selective lignin degradation Holzforschung 41 343 349

  • Park, J.W., Dec, J., Kim, J.E. & Bollag, J.M. 1999 Effect of humic constituents on the transformation of chlorinated phenols and anilines in the presence of oxidoreductive enzymes or birnessite Environ. Sci. Technol. 33 2028 2034

    • Search Google Scholar
    • Export Citation
  • Rieke, P.E. 1994 Sand topdressing: Where are we going? Golf Course Mgt. 62 36 38

  • Roberts, E.C. & Bredakis, E.J. 1960 What, why and how of turfgrass root development Golf Course Rpt. 28 13 24

  • Roper, J.C., Sarkar, J.M., Dec, J. & Bollag, J.M. 1995 Enhanced enzymatic removal of chlorophenols in the presence of co-substrates Water Res. 29 2720 2724

    • Search Google Scholar
    • Export Citation
  • Sartain, J.B. & Volk, B.G. 1984 Influence of selected white-rot fungi and topdressings on the composition of thatch components of four turfgrasses Agron. J. 76 359 362

    • Search Google Scholar
    • Export Citation
  • SAS Institute Inc 1994 The SAS system for Windows. Release 9.2. SAS Inst., Cary, NC

  • Smiley, R.W., Crawen Fowler, M., Kane, R.T., Petrovic, A.M. & White, R.A. 1985 Fungicide effects on thatch depth, thatch decomposition rate, and growth of kentucky bluegrass Agron. J. 77 597 602

    • Search Google Scholar
    • Export Citation
  • Soper, D.Z., Dunn, J.H., Minner, D.D. & Sleper, D.A. 1988 Effects of clipping disposal, nitrogen, and growth retardants on thatch and tiller density in zoysiagrass Crop Sci. 28 325 328

    • Search Google Scholar
    • Export Citation
  • Stoilova, I., Krastanov, A. & Stanchev, V. 2010 Properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation Adv. Bioscience Biotech. 1 208 215

    • Search Google Scholar
    • Export Citation
  • Thurston, C.F. 1994 The structure and function of fungal laccases Microbiol. 140 19 26

  • Weston, J.B. & Dunn, J.H. 1985 Thatch and quality of Meyer zoysia in response to mechanical cultivation and nitrogen fertilization, p. 449–458. In: Lemaire, F. (ed.). Proc. 5th Int. Turfgrass Res. Conf., Avignon, France. 1–5 July 1985. Institut National de la Recherche Agronomique, Paris, France

  • White, R.H. & Dickens, R. 1984 Thatch accumulation in bermudagrass as influenced by cultural practices Agron. J. 76 19 22

  • Wong, D.W.S. 2009 Structure and action mechanism of ligninolytic enzymes Appl. Biochem. Biotechnol. 157 174 209

Contributor Notes

The research was supported by funding from the Golf Course Superintendents Association of America, Georgia Golf Environmental Foundation, and Georgia Agricultural Experiment Stations.

We thank Rashmi Singh, Qi Luo, Lewayne White, and Rodney Connell for their excellent technical assistance.

To whom reprint requests should be addressed; e-mail praymer@uga.edu.

  • View in gallery

    Thatch (OLT) and mat layer thickness (OLM) after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are standard errors. Bars with the same letter (OLM = bolded and OLT = standard) are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

  • View in gallery

    Organic matter (OM) for 0 to 2.5 cm depth and 2.5 to 5.0 cm depth after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are standard errors. Bars with the same letter [OM (0 to 2.5 cm) = bolded and OM (2.5 to 5.0 cm) = standard] are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

  • View in gallery

    Saturated hydraulic conductivity (SHC) after nine months of treatment on creeping bentgrass with three different levels of laccase [0 (control), 0.206 and 2.06 units/cm2] with and without the mediator, guaiacol (G). Values are means of four replicates and error bars are ses. Bars with the same letter are not considered to be statistically different according to Fisher’s protected least significant difference at α = 0.05.

  • Baldrian, P. 2006 Fungal laccases—Occurrence and properties FEMS Microbiol. Rev. 30 215 242

  • Barton, L., Wan, G.G.Y., Buck, R.P. & Colmer, T.D. 2009 Effectiveness of cultural thatch-mat controls for young and mature kikuyu turfgrass Agron. J. 101 67 74

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  • Beard, J.B. 1973 Turfgrass: Science and culture. Prentice Hall Inc., Englewood Cliffs, NJ

  • Blanchette, R.A. 1984 Screening wood decayed by white rot fungi for preferential lignin degradation Appl. Environ. Microbiol. 48 647 653

  • Callahan, L.L., Sanders, W.L., Parham, J.M., Harper, C.A., Lester, L.D. & McDonald, E.R. 1998 Cultural and chemical controls of thatch and their influence on rootzone nutrients in bentgrass green Crop Sci. 38 181 187

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    • Export Citation
  • Carley, D.S., Goodman, D., Sermons, S., Shi, W., Bowman, D., Miller, G. & Rufty, T. 2011 Soil organic matter accumulation in creeping bentgrass greens: A chronosequence with implications for management and carbon sequestration Agron. J. 103 604 610

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  • Carrow, R.N. 2003 Surface organic matter in bentgrass greens USGA Turfgrass Environ. Res. Online 2 1 10

  • Carrow, R.N. 2004 Surface organic matter in bentgrass greens Golf Course Mgt. 72 96 101

  • Carrow, R.N., Johnson, B.J. & Burns, R.E. 1987 Thatch and quality of Tifway bermudagrass turf in relation to fertility and cultivation Agron. J. 79 524 530

    • Search Google Scholar
    • Export Citation
  • Chen, Y.R. & Sarkanen, S. 2003 Macromolecular lignin replication: A mechanistic working hypothesis PhytomTOMhemistry Rev. 2 235 255

  • Davin, L.B. & Lewis, N.G. 2003 A historical perspective on lignin biosynthesis: Monolignol, allylphenol and hydroxycinnamic acid coupling and downstream metabolism Phytochem. Rev. 2 257 288

    • Search Google Scholar
    • Export Citation
  • Dunn, J.H., Sheffer, K.M. & Halisky, P.M. 1981 Thatch and quality of Meyer zoysia in relation to management Agron. J. 73 949 952

  • Engel, R.E. 1954 Thatch on turf and its control Golf Course Rpt. 22 12 14

  • Engel, R.E. & Alderfer, R.B. 1967 The effect of cultivation, top-dressing, lime, N, and wetting agent on thatch development on 1/4-inch bentgrass over a 10-year period N.J. Agr. Exp. Stn. Bull. 818 32 45

    • Search Google Scholar
    • Export Citation
  • Engelke, M.C., Lehman, V.G., Kneebone, W.R., Colbaugh, P.F., Reinert, J.A. & Knoop, W.E. 1995 Registration of ‘Crenshaw’ creeping bentgrass Crop Sci. 35 589

    • Search Google Scholar
    • Export Citation
  • Hartwiger, C. 2004 The importance of organic matter dynamics: How research uncovered the primary cause of secondary problems USGA Green Section Record 42 9 11

    • Search Google Scholar
    • Export Citation
  • Johnson, B.J., Carrow, R.N. & Burns, R.E. 1987 Bermudagrass turf response to mowing practices and fertilizer Agron. J. 79 677 680

  • Kang, K.H., Dec, J., Park, H. & Bollag, J.M. 2002 Transformation of the fungicide cyprodinil by a laccase of Trametes villosa in presence of phenolic mediators and humic acid Water Res. 36 4907 4915

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K., Connors, W.J., Bleam, R.D., Hackett, W.F. & Jeikus, J.G. 1975 Preparation and microbial decomposition of synthetic [14C] lignins Proc. Natl. Acad. Sci. USA 72 2515 2519

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K., Connors, W.J., Bleam, R.D. & Jeikus, G. 1976 Requirements for a growth substrate during lignin decomposition by two wood-rotting fungi Appl. Environ. Microbiol. 32 192 194

    • Search Google Scholar
    • Export Citation
  • Kirk, T.K. & Farrell, R.L. 1987 Enzymatic ‘combustion’: The microbial degradation of lignin Annu. Rev. Microbiol. 41 465 501

  • Landreth, J., Karcher, D. & Richardson, M. 2008 Cultivating to manage organic matter in sand based putting greens: University of Arkansas researchers provide important insight for managing organic buildup on putting greens USGA Turfgrass Environ. Res. Online 46 16 19

    • Search Google Scholar
    • Export Citation
  • Ledeboer, F.B. & Skogley, C.R. 1967 Investigations into the nature of thatch and methods for its decomposition Agron. J. 59 320 323

  • Martin, S.B. & Dale, J.L. 1980 Biodegradation of turf thatch with wood-decay fungi Phytopathology 70 297 301

  • McCarty, L.B. 2005 Best golf course management practices. 2nd Ed. Prentice Hall Inc., Upper Saddle River, NJ

  • McCarty, L.B., Gregg, M.F. & Toler, J.E. 2007 Thatch and mat management in an established creeping bentgrass green Agron. J. 99 1530 1537

  • McCarty, L.B., Gregg, M.F., Toler, J.E., Camberato, J.J. & Hill, H.S. 2005 Minimizing thatch and mat development in a newly seeded creeping bentgrass golf green Crop Sci. 45 1529 1535

    • Search Google Scholar
    • Export Citation
  • McCoy, E.L. 1992 Quantitative physical assessment of organic materials used in sports turf rootzone mixes Agron. J. 84 375 381

  • McWhirter, E.L. & Ward, C.Y. 1976 Effect of vertical mowing and aerification on golf green quality Mississippi Agr. For. Exp. Stn. Res. Rpt. 2 1 2

  • Mester, T., Varela, E. & Tien, M. 2004 Wood degradation by brown-rot and white-rot fungi. The Mycota II: Genetics and biotechnology. 2nd Ed. Springer-Verlag, Berlin, Heidelberg, Germany

  • Munoz, C., Guillen, F., Martinez, A.T. & Martinez, M.J. 1997 Laccase isozymes of Pleurotus eryngii: Characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation Appl. Environ. Microbiol. 63 2166 2174

    • Search Google Scholar
    • Export Citation
  • Murdoch, C.L. & Barr, J.P. 1976 Ineffectiveness of commercial microorganism inoculums in breaking down thatch in common bermudagrass in Hawaii HortScience 11 488 489

    • Search Google Scholar
    • Export Citation
  • Murray, J.J. & Juska, F.V. 1977 Effect of management practices on thatch accumulation, turf quality, and leaf spot damage in common kentucky bluegrass Agron. J. 69 365 369

    • Search Google Scholar
    • Export Citation
  • National Renewable Energy Laboratory 2008 Determination of structural carbohydrates and lignin in biomass. 9 Aug. 2012. <http://www.nrel.gov/biomass/pdfs/42618.pdf>. NREL, Golden, CO

  • O’Brien, P. & Hartwiger, C. 2003 Aeration and topdressing for the 21st century: Two old concepts are linked together to offer up-to-date recommendations USGA Green Section Record 41 1 7

    • Search Google Scholar
    • Export Citation
  • Otjen, L. & Blanchette, R. 1987 Assessment of 30 white rot basidiomycetes for selective lignin degradation Holzforschung 41 343 349

  • Park, J.W., Dec, J., Kim, J.E. & Bollag, J.M. 1999 Effect of humic constituents on the transformation of chlorinated phenols and anilines in the presence of oxidoreductive enzymes or birnessite Environ. Sci. Technol. 33 2028 2034

    • Search Google Scholar
    • Export Citation
  • Rieke, P.E. 1994 Sand topdressing: Where are we going? Golf Course Mgt. 62 36 38

  • Roberts, E.C. & Bredakis, E.J. 1960 What, why and how of turfgrass root development Golf Course Rpt. 28 13 24

  • Roper, J.C., Sarkar, J.M., Dec, J. & Bollag, J.M. 1995 Enhanced enzymatic removal of chlorophenols in the presence of co-substrates Water Res. 29 2720 2724

    • Search Google Scholar
    • Export Citation
  • Sartain, J.B. & Volk, B.G. 1984 Influence of selected white-rot fungi and topdressings on the composition of thatch components of four turfgrasses Agron. J. 76 359 362

    • Search Google Scholar
    • Export Citation
  • SAS Institute Inc 1994 The SAS system for Windows. Release 9.2. SAS Inst., Cary, NC

  • Smiley, R.W., Crawen Fowler, M., Kane, R.T., Petrovic, A.M. & White, R.A. 1985 Fungicide effects on thatch depth, thatch decomposition rate, and growth of kentucky bluegrass Agron. J. 77 597 602

    • Search Google Scholar
    • Export Citation
  • Soper, D.Z., Dunn, J.H., Minner, D.D. & Sleper, D.A. 1988 Effects of clipping disposal, nitrogen, and growth retardants on thatch and tiller density in zoysiagrass Crop Sci. 28 325 328

    • Search Google Scholar
    • Export Citation
  • Stoilova, I., Krastanov, A. & Stanchev, V. 2010 Properties of crude laccase from Trametes versicolor produced by solid-substrate fermentation Adv. Bioscience Biotech. 1 208 215

    • Search Google Scholar
    • Export Citation
  • Thurston, C.F. 1994 The structure and function of fungal laccases Microbiol. 140 19 26

  • Weston, J.B. & Dunn, J.H. 1985 Thatch and quality of Meyer zoysia in response to mechanical cultivation and nitrogen fertilization, p. 449–458. In: Lemaire, F. (ed.). Proc. 5th Int. Turfgrass Res. Conf., Avignon, France. 1–5 July 1985. Institut National de la Recherche Agronomique, Paris, France

  • White, R.H. & Dickens, R. 1984 Thatch accumulation in bermudagrass as influenced by cultural practices Agron. J. 76 19 22

  • Wong, D.W.S. 2009 Structure and action mechanism of ligninolytic enzymes Appl. Biochem. Biotechnol. 157 174 209

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