Managing thatch with fungal laccase

In greenhouse studies, nine months of laccase applications significantly reduced thatch in creeping bentgrass maintained at putting green height.

Cores were removed from pots and then tested for saturated hydraulic conductivity.
Photos by Rashmi Singh

By Sudeep S. Sidhu, Ph.D.; Qingguo Huang, Ph.D.; Robert N. Carrow, Ph.D.; and Paul L. Raymer, Ph.D.
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High organic matter accumulation in the form of thatch or mat is one of the major problems in modern turfgrass greens. Thatch is a layer of organic matter containing tightly intermingled living and dead plant tissue that accumulates between the soil and green turfgrass. It consists of stolons, rhizomes, roots, leaf sheaths and blades (5). Thatch accumulation causes problems like decreased movement of oxygen through the thatch or mat zone, decreased saturated hydraulic conductivity and increased water retention (3,6). These primary problems may lead to secondary problems like wet wilt, a soft surface, black layer, limited rooting and extra-and intra-cellular freezing damage (4,8).

The major problems in the thatch or mat layer are excessive accumulation of organic matter over time and the rapid change from the nature of the structured organic matter seen in live plant root tissues to the unstructured organic matter in dead root tissues. A high density of living roots near the soil surface may adversely affect the soil’s physical properties. However, a more serious problem is rapid root death in summer, which creates dead gelatinous organic matter that swells in the presence of water during decomposition and plugs the soil macropores (air-filled pores), causing low oxygen levels in the root zones (4,8).

High organic matter accumulation occurs when organic matter degrades more slowly than it accumulates. It is believed that rate of degradation of organic matter is related to the presence of lignin content in the organic residue. Lignin is an abundant source of carbon second only to cellulose and acts as a protective physical matrix that makes readily decomposable cellulosic and hemicellulosic sugars unavailable for microbial degradation. Lignin is extremely resistant to degradation because it has a complex structure derived from oxidative coupling of lignin monomers that limits microbial degradation of organic matter (7). For this reason, turfgrass species high in lignin content are resistant to decomposition (2).

We studied the use of fungal laccase, a lignolytic enzyme, for enhancing the rate of organic matter degradation in thatch layer. Oxidative enzymes such as laccases produced by white rot fungi are recognized for their ability to attack the aromatic components of lignin and lead to its effective degradation (1).

Materials and methods

A greenhouse experiment was established at the University of Georgia’s Griffin Campus using Crenshaw creeping bentgrass (Agrostis stolonifera) acquired from East Lake Country Club, Atlanta. The sod, which was approximately 1.18 inch (3 centimeters) thick and consisted of existing thatch and mat but not the underlying soil, was cut to fit the pots and placed on the 85:15 sand and organic matter mix. All the pots were irrigated daily, fertilized monthly with a 1.7-fluid-ounce (50-milliliter) solution of 0.4% (w/v) Macron water-soluble 28-7-14 fertilizer, and maintained at a height of 0.24 inch (0.6 centimeter). The refrigerated air-conditioned greenhouse was maintained at 77±4/64±4 F (25±2/18 ±2 C) day/night temperature.

The experimental design was a randomized complete block design with five replications. The treatment design was a 4 × 2 factorial with all combinations of four levels of laccase and two levels of guaiacol, a compound that may enhance laccase activity. The four levels of laccase were 0 (control), 0.206, 2.06 and 20.6 units/square centimeter and the two levels of guaiacol were 0 (control) and 0.1 Molar solution. After two months, the 20.6 units/square centimeter treatment application was discontinued. Laccase treatments were applied as a 1.35-ounce (40-milliliter) solution for each of the different activity levels, and the control was applied as 1.35 ounces of distilled water. Guaiacol was applied as 0.34 ounce (10 milliliters) of solution.

Laccase activity assay

The laccase enzyme used in the experiment was from Trametes versicolor, a white rot fungus (Sigma-Aldrich). The activity of laccase was quantified by a colorimetric assay using a UV/VIS-spectrophotometer. The amount of laccase that causes an absorbance change at 468 nanometers at a rate of 1.0 unit/minute in 3.4 milliliters of 1 millimolar 2,6-dimethoxyphenol in citratephosphate buffer at pH 3.8 corresponds to one activity unit (9).

Measurements

Parameters used to determine the effectiveness of treatments were total organic matter content for a depth of 0-2 inches (0-5 centimeters), saturated hydraulic conductivity, organic layer thickness, extractive-free acid-soluble lignin, acid-insoluble lignin and total lignin content after two months of treatment application. Total lignin content was obtained by addition of acidsoluble and acid-insoluble lignin contents. When sampled after nine months of treatment, organic layer thickness was subdivided into thatch layer thickness and mat layer thickness, while total organic matter content was subdivided into 0 to 1-inch (0- to 2.5-centimeter) depth and 1- to 2-inch (2.5-5 centimeters) depth to more accurately reflect the effectiveness of laccase on thatch layer thickness and organic matter reduction in thatch layer.

Organic matter content

Two soil cores (0.78 inch [2.0 centimeters] in diameter) were dried in an oven at 212± 9 F (100±5 C) for 48 hours, weighed and ashed in a muffle furnace at 1,112±18 F (600±10 C) for 24 hours and weighed again. Total organic matter content was the difference in the two readings; percent total organic matter was calculated.

The organic layer, thatch layer and mat layer of each pot were measured at seven different locations and averaged.

Saturated hydraulic conductivity

Intact cores 2 inches (5 centimeters) in diameter and 3 inches (7.6 centimeters) long were collected in the brass cylinders from the center of each pot using a soil corer. The saturated hydraulic conductivity of the cores was measured.

Organic layer and thatch-mat layer thickness

Plants were removed from pots and distinct separations among the thatch, mat and soil interface were clearly visible. The organic layer, thatch layer and mat layer were measured from seven different locations around the edges of the plant/root mass and averaged.

Extractive free lignin content

Thatch layer samples were extracted with water and ethanol to remove water- and alcohol-soluble impurities. Laboratory Analytical Procedure developed by National Renewable Laboratory was used to determine extractive free acid-soluble lignin and acid-insoluble lignin content in the thatch layer in a two-step sulfuric acid hydrolysis procedure. The acid-insoluble residue solids remaining after acid hydrolysis were dried in an oven at 212±9 F (100±5 C) for 24 hours, weighed, ashed in a muffle furnace at 1,112±18 F (600±10 C) for 24 hours, and weighed again. The weight difference was used to calculate the acid-insoluble lignin content.

Results and discussion

Organic and thatch layer reduction

A slight (8.7%) reduction in total organic layer thickness was observed after two months of treatment with a laccase activity level of 20.6 units/square centimeter (Table 1). No other laccase activity level had a significant effect on organic layer thickness. However, after nine months of laccase applications, a laccase activity level of 2.06/square centimeter significantly decreased total organic layer thickness by 14.5% (with guaiacol) and by 13.0% (without guaiacol) in comparison to the control (Table 1).

After nine months of treatment, a laccase activity level of 2.06/square centimeter reduced thatch layer thickness 45% (with guaiacol) and 35% (without guaiacol) in comparison to the control (Figure 1). No significant reduction in mat layer was observed with any of the treatments (Figure 1). No significant effect of guaiacol or interaction of laccase and guaiacol was observed for organic layer thickness, thatch layer thickness and mat layer thickness.

Total organic matter content

At all laccase activity levels, total organic matter content (0- to 2-inch depth) was not significantly reduced after two months of enzyme application. However, nine months of treatment at the laccase activity level of 2.06/square centimeter significantly reduced total organic matter content (0- to 2-inch depth) by 15.4% (with guaiacol) and by 15.8% (without guaiacol) in comparison to the control (Table 1). Nine months of the same treatment application significantly reduced total organic matter content at a lower depth (0-1 inch) by 27.4% (with guaiacol) and by 32.1% (without guaiacol) in comparison to the control (data not shown) (10). However, at the 1- to 2-inch depth, no laccase treatment significantly decreased total organic carbon content compared to the control.

Lignin content

After two months of application of laccase activity of 20.6 units/square centimeter without guaiacol, significant reductions were observed for acid-soluble lignin content (11.9%), acid-insoluble lignin content (7.8%) and total lignin content (8.4%) (Table 2). Similarly, significant reductions of 12.2% for acid-soluble lignin content, 5.4% for acid-insoluble lignin content and 6.4% for total lignin content were observed with laccase activity of 2.06 units/square centimeter without guaiacol after nine months of application (Table 2).

Thatch layer and organic matter degradation was enhanced by laccase enzyme due to removal of lignin from the thatch biomass, making readily degradable cellulose and hemicellulose available for microbial decomposition.

Saturated hydraulic conductivity

After nine months of laccase application with guaiacol at the rate of 2.06 units/square centimeter, saturated hydraulic conductivity increased by 322% compared to the control; the same treatment without guaiacol increased saturated hydraulic conductivity 94% over the control (Figure 2). The presence of high organic matter content in the thatch layer (thatch layer thickness greater than 1.3 centimeters) hinders water infiltration. Applying laccase for nine months reduced the thatch layer below this threshold level and thereby significantly increased saturated hydraulic conductivity.

Summary

In this experiment, applying laccase to creeping bentgrass with an existing thatch/mat layer under favorable greenhouse conditions for further thatch/mat development slowed the rate of accumulation of organic layer and total organic matter between two months after treatment and nine months after treatment. Although an overall thatch layer buildup was observed in all treatments, the rate of organic matter accumulation and thatch layer thickness buildup was greatly reduced in the pots treated with the laccase enzyme.

Conclusions

Applying laccase once every two weeks proved effective in reducing buildup of organic matter and thatch layer in highly maintained turf. Duration of laccase application had a significant effect on thatch-mat management, with best results after nine months. However, low activity levels of laccase (0.206 unit/square centimeter) were ineffective in reducing thatch buildup even after nine months of application.

These findings point to a novel approach to reduce organic matter 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 continues under field conditions to observe the effectiveness of laccase as well as to optimize the activity level of laccase and the frequency of its application. Results to date in the field studies have been positive and support the greenhouse results.

Acknowledgments

We thank the Georgia Golf Environmental Foundation (GGEF) and the Environmental Institute for Golf for funding this research.

Literature cited

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2. Beard, J.B. 1973. Turfgrass: Science and Culture. Prentice Hall, Englewood Cliffs, N.J.

3. Carrow, R.N. 2003. Surface organic matter in bentgrass greens. USGA Turfgrass Environmental Research Online 2(17):1-10.

4. Carrow, R.N. 2004. Surface organic matter in bentgrass greens. Golf Course Management 72(5):96-101.

5. Engel, R.E. 1954. Thatch on turf and its control. The Golf Course Reporter 22 (5):12-14.

6. Hartwiger, C. 2004. The importance of organic matter dynamics: How research uncovered the primary cause of secondary problems. USGA Green Section Record 42(3):9-11.

7. Ledeboer, F.B., and C.R. Skogley. 1967. Investigations into the nature of thatch and methods for its decomposition. Agronomy Journal 59:320-323.

8. O’Brien, P., and C. Hartwiger. 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(2):1-7.

9. Park, J.W., J. Dec, J.E. Kim and J.M. Bollag. 1999. Effect of humic constituents on the transformation of chlorinated phenols and anilines in the presence of oxidoreductive enzymes or birnessite. Environmental Science & Technology 33:2028-2034.

10. Sidhu, S.S., Q. Huang, R.N. Carrow and P.L. Raymer. 2012. Use of fungal laccases to facilitate biodethatching: a new approach. HortScience 47(10):1536-1542.

Sudeep S. Sidhu is a postdoctoral research associate, Qingguo (Jack) Huang (qhuang@uga.edu) is an associate professor, and Robert N. Carrow and Paul L. Raymer are professors in the department of crop and soil sciences at the University of Georgia’s Griffin campus.