From the March 2016 issue of GCM magazine:
Thatch collapse in golf course turf
Sphaerobolus stellatus has been identified as the causal agent of this turf disease on golf courses in the U.S. and New Zealand.
Figure 1. (A) Symptoms of thatch collapse appear as circular patches of dark green turf ranging from 3 to 18 inches (8 to 46 cm); (B) degraded organic matter creates sunken, depressed areas; (C) decomposing thatch contains a fawn color and a mushroom odor; (D) signs of thatch collapse include profuse mycelia within the upper (2.5 cm) of the soil-thatch interface; (E) clamp connections on mycelia; and (F) fruiting bodies develop within thatch and the turfgrass canopy. Photos by Amy Baetsen-Young
Amy M. Baetsen-Young, M.S., John E. Kaminski, Ph.D., Matthew T. Kasson, Ph.D. and Donald D. Davis, Ph.D.
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In 2010, a new disease thought to be caused by an unknown basidiomycete (that is, fungus with typical mushroom fruiting bodies) was reported on Festuca species on a golf course in Scotland (5). Symptoms of thatch collapse appear as 3- to 18-inch (8- to 46-cm) circular patches of dark green turf with a fawn-colored thatch profile (Figure 1) and a mushroom-like odor (5). Thatch collapse symptoms are distinct from those caused by other mushroom- or puffball-forming basidiomycetes commonly associated with turfgrass, such as fairy ring. Typical of other basidiomycete fungi, mycelial characteristics of the fungus associated with affected areas include clamp connections. The depressed thatch collapse symptoms disturb play by affecting consistency, ball roll and firmness. Identification of the causal agent, therefore, was imperative to better assess and understand management options for this new disease.
What causes thatch collapse?
In late 2011 and 2012, the basidiomycete Sphaerobolus stellatus (Tode) Persoon was found in association with thatch collapse symptoms in New Zealand, California, Michigan, Montana and South Dakota (1). This consistent association has identified S. stellatus as one possible causal agent of thatch collapse. Sphaerobolus stellatus is an agaricomycete that decomposes lignin in thatch, mulch and other sources of organic matter. It is classified in the family Geastraceae, whose members are distinguished by the fact that spore-bearing tissues are produced within a fruiting body (that is, puffball) and not on the exposed surfaces of gills. Upon maturity, the outer wall of the fruiting body splits and ejects a sticky spore mass known as glebae. The spore masses may be projected distances of a few inches to up to several yards, hence the common name “artillery fungus” (2). Landscaping mulch has provided an optimal substrate for S. stellatus, making this fungus common in home landscapes (3,4).
Figure 2. Evolutionary relationships between ITS sequences from S. stellatus isolates S. iowensis and S. ingoldii conducted in MEGA5. Evolutionary history was inferred using the Neighbor-Joining method. Percentages of the replicate trees in which the associated taxa are clustered together in the bootstrap test (1,000 replicates) and are shown next to the branches. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distances to infer the phylogenetic tree. Evolutionary distances were computed using the P-distance method and are the units of the number of base substitutions per site. The analysis involved 14 nucleotide sequences. All gaps and missing data were eliminated. A total of 656 positions were included in the final data set.
No published reports have described the basic biology of S. stellatus colonizing managed turfgrass systems or causing thatch collapse. The purpose of this study was to morphologically and molecularly identify and confirm S. stellatus as a causal agent of thatch collapse; document and compare S. stellatus isolates from distinct geographic areas; elucidate temperatures favoring mycelia growth of S. stellatus; and determine optimal temperatures for glebae production.
Sample collection and isolation
A total of nine isolates of S. stellatus were obtained from seven golf courses in the United States and New Zealand. Samples consisting of plugs containing living plants, thatch and soil were collected from putting greens and approaches and incubated in a plastic container lined with moist paper towels. To isolate the fungus, fruiting bodies were allowed to grow on antibiotic oatmeal agar. When fruiting bodies were not present, samples were incubated within a sealed plastic container lined with moist paper towels and incubated under fluorescent light for two weeks to induce the development of fruiting bodies. Isolates obtained from New Zealand (n = 4) were provided by M. Cushnahan of the New Zealand Sports Turf Institute, and the isolate from South Dakota (n = 1) was provided by L. Miller of the University of Missouri.
DNA sequencing
Isolates were analyzed to determine their genetic makeup, and their DNA sequences were compared with sequences of known fungal isolates. Sequencing of the DNA of isolates revealed a 98% to 99% sequence similarity to a known S. stellatus mulch isolate. The nine isolates evaluated fell within two distinct clades within the S. stellatus clade (Figure 2). The first clade included S. stellatus from New Zealand (ICMP 18280, 18281 and 18279), South Dakota (SS-SD), California (SS-CA-1) and Michigan (SS-MI). The second clade included isolates from Montana (SS-MT) and California (SS-CA-2). A second phylogenetic analysis confirmed the results.
Figure 3. Organic matter depth of Penn A-1 creeping bentgrass plugs after six weeks of incubation at 54 F (12 C). Seeded treatments included a non-infested control, sterilized organic matter and Sphaerobolus stellatus-infested thatch. Treatment means followed by the same letter are not significantly different.
Colony morphology and S. stellatus characteristics
Sphaerobolus stellatus was isolated from fruiting bodies in either the turf canopy or thatch layer from all symptomatic samples. Hyphae containing clamp connections were observed intertwining with the organic matter and on the outside of leaf tissue. White mycelia colonizing the organic matter produced an orange hue in the thatch along the outer edges of patches (Figure 1). However, on microscopic examination of stained tissues within samples, no hyphae bearing clamp connections were observed inside leaves, roots, crowns or stolons.
Colony morphology varied among isolates. South Dakota (SD) and California (CA) isolates produced creamy white, stringy, aerial mycelia that extended upward toward the lid of the petri dish. The Michigan (MI) and Montana (MT) isolates formed creamy white, appressed mycelia. All isolates produced concentric rings of mycelia across the media.
Gleba and basidiospore morphology were assessed using isolates obtained from a golf course in California (SS-CA-1) and South Dakota (SS-SD). Thirty glebae and 50 basidiospores from within crushed glebae were randomly collected. Length and width of both structures were measured and compared with known species within the Sphaerobolus genus. All glebae produced had a dark brown to black hue and were generally spherical to rectangular in shape. Average length and width of all glebae and their respective basidiospores fit within two species of Sphaerobolus, including S. ingoldii and S. stellatus. Slight variations within the speciation of Sphaerobolus may be attributed to the limited sporulation of isolates in this study.
Confirmation of the causal agent
Figure 4. Total organic matter (%) of Penn A-1 creeping bentgrass samples as determined by the loss-on-ignition method following incubation at 54 F (12 C) for six weeks. Seeded treatments included a non-infested control and Sphaerobolus stellatus-infested organic matter. Means followed by the same letter are not significantly different.
To confirm a cause-and-effect relationship between thatch collapse and S. stellatus, Koch’s postulates were completed with S. stellatus isolated from turf in a lab experiment. A thatch-infested inoculum was developed and used for the procedure. In May and July 2012, 12 cup-cutter plugs of mature Penn A-1 creeping bentgrass were removed from a research green with no prior history of thatch collapse at our field research facility. Four plugs were inoculated with 0.6 gram of S. stellatus-infested organic matter (isolate SS-SD) at the soil/thatch interface. Eight plugs were inoculated with non-infested organic matter or not inoculated to serve as the untreated controls. All plugs were incubated at 53 F (12 C) under 24 hours of light and monitored for fruiting body development and degradation of the thatch layer. At the conclusion of the experiment, S. stellatus was re-isolated, and thatch depth and total organic matter were assessed.
Growth of S. stellatus was allowed to progress over six weeks on incubated plugs. The S. stellatus-infested treatment had a thatch layer depth of 0.57 inch, which was less than that of the non-infested control (0.77 inch) and of plugs that received only sterilized organic matter control (0.74 inch) treatments (Figure 3). Because of the lack of thatch-depth differences between the non-infested control and sterilized organic matter control, the percent organic matter was only determined for S. stellatus-infested samples and the non-infested control. Organic matter within the S. stellatus-infested treatment was reduced by 20.4% when compared with the control (Figure 4). In all experiments, S. stellatus was successfully re-isolated from fruiting bodies in infested plugs.
Temperatures favoring S. stellatus growth in culture
†Daily growth was determined by measuring colony diameter in two perpendicular directions biweekly and dividing by total growing days.
‡Mean daily growth values within a column that are followed by the same letter are not significantly different.
Table 1. Average daily growth of three Sphaerobolus stellatus isolates on oatmeal agar from California (SS-CA-1), Michigan (SS-MI) and South Dakota (SS-SD) in a controlled environment chamber.
Daily growth rates of three S. stellatus isolates (SS-CA-1, SS-MI and SS-SD) were examined over six temperatures. A significant effect was observed between replications of the study, and data from each study were therefore analyzed separately.
Study I. Daily growth was analyzed for each isolate over six temperatures (Figure 5A). Isolates SS-CA-1 and SS-MI had the largest daily growth at 70 F and 77 F (21 C and 25 C), and SS-SD had the largest daily growth at 70 F, 77 F and 86 F (30 C) (Table 1). All isolates had the least daily growth at 41 F (5 C).
Study II. Data revealed that SS-MI had its largest daily growth at 70 F, 77 F and 86 F; SS-CA-1 at 70 F and 77 F; and SS-SD at 70 F (Table 1). Each isolate had the smallest daily growth at 41 F (Table 1). Evaluation of daily growth among isolates at each temperature revealed generally similar results to that of Study I (Figure 5B).
Temperatures favoring glebae production in culture
The influence of temperature on S. stellatus glebae production of three isolates (SS-CA-1, SS-MI and SS-SD) was examined for 11 weeks. Significant study effects were not observed for glebae production; therefore, data were combined for statistical analyses.
Fruiting bodies developed one week after exposure to light, and glebae were discharged during the second week of the study when incubated at 70 F and 77 F. Glebae production was not observed for isolates incubated at 50 F to 59 F (10 C to 15 C) until approximately three to four weeks after incubation. The length of time that glebae were produced also varied by temperature. At 70 F, all glebae production occurred within four weeks. Glebae production at 50 F, 59 F and 77 F, however, ceased after 11, eight and six weeks, respectively. Isolate SS-SD produced glebae at all temperatures, whereas isolate SS-CA-1 only produced glebae at 50 F and 59 F, and isolate SS-MI produced glebae at 59 F. When analyzed by temperature, SS-SD produced the most glebae at all temperatures among the isolates.
Figure 5. Average daily growth of three Sphaerobolus stellatus isolates (SS-CA-1, SS-MI and SS-SD) on oatmeal agar in a controlled environment chamber at six temperatures. Duplicate experiments (A and B) were analyzed separately. Means followed by the same letter are not significantly different.
Discussion
Sphaerobolus stellatus was isolated from fruiting bodies within the turf canopy or thatch layer from all symptomatic samples collected. Fruiting bodies developed on nonliving organic matter colonized with white- to orange-colored mycelia. Although hyphae with clamp connections were observed within the organic matter, no visible signs of S. stellatus were found within living plant tissues, indicating that the organism is not pathogenic.
Sphaerobolus stellatus was isolated from several locations and turfgrass species across the United States and New Zealand. DNA sequencing revealed that the thatch collapse isolates were S. stellatus, and conclusively fulfilling Koch’s postulates confirmed S. stellatus as a causal agent of thatch collapse. Thatch collapse symptoms (that is, sunken or depressed patches) linked to S. stellatus are distinct from symptoms produced by other known basidiomycetes that inhabit soil and turfgrass thatch, including fairy ring, superficial fairy ring and yellow ring.
The degradation of organic matter by S. stellatus observed in this study is consistent with prior investigations (7). A 20% organic matter reduction from S. stellatus isolate SS-SD occurred over six weeks. The accelerated organic matter reduction in turfgrass by isolate SS-SD demonstrates the capabilities of this fungus to cause thatch collapse on golf playing surfaces.
The cultural morphology of S. stellatus was similar to two other known Sphaerobolus species, S. ingoldii and S. iowensis (6). Daily growth rates of S. stellatus increased with temperatures from 41 F to 77 F. Mean daily growth of isolates SS-CA-1 and SS-MI at 77 F were similar to daily growth rates of known isolates of S. stellatus (6). However, daily growth of isolate SS-SD was larger and more comparable to growth of S. ingoldii (6).
Glebae likely serve as an important source of inoculum for spread of S. stellatus and development of thatch collapse, since glebae were formed over a wide range of temperatures in this study. However, only isolate SS-SD produced copious amounts of glebae in our growth chamber study. Isolate SS-SD produced higher amounts of glebae at 50 F, 59 F and 68 F versus 77 F. Variation among isolates may be linked to aggressiveness of the particular strains of the organism.
This is the first formal report of the nature and biology of a thatch collapse causal agent. Other organisms may cause similar symptoms, but thus far only S. stellatus has been linked to thatch collapse. The information reported herein will advance our understanding of thatch collapse and future management strategies to minimize the development and impacts on ball roll and firmness of golf greens.
Funding
Funding for this project was provided by the Penn State University Department of Plant Science and the Pennsylvania Turfgrass Council.
Acknowledgments
We thank Lee Miller, Ph.D., and Megan Cushnahan for providing isolates used in this study. We also thank Timothy Lulis for assistance throughout this project.
Information in this paper was previously published by the authors as “Insights into the biology of Sphaerobolus stellatus” in the journal Crop Science, 55:2342–2351 (2015); (doi: 10.2135/cropsci2014.12.0821).
Literature cited
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- Brantley, E. A., D.D. Davis and L.J. Kuhns 2001a. Biological control of the artillery fungus, Sphaerobolus stellatus, with Trichoderma harzianum and Bacillus subtilis. Journal of Environmental Horticulture 19:21-23.
- Brantley, E.A., D.D. Davis and L.J. Kuhns. 2001b. Influence of mulch characteristics on the sporulation of the artillery fungus Sphaerobolus stellatus. Journal of Environmental Horticulture 19:89-95.
- Davis, D.D., and M.A. Fidanza. 2011. Fresh recycled mushroom compost suppresses artillery fungi sporulation: a 4-year field study. Journal of Environmental Horticulture 29:91-95.
- Dernoeden, P.H., J.E. Kaminski and C. Haspell. 2011. Thatch collapse: a disease of fine-leaf fescue. Golf Course Management 79(5):88.
- Geml, J. 2004. Systematics, biogeography and control of artillery fungi (Sphaerobolus spp.). Ph.D. dissertation, Penn State University, University Park, Pa.
- Winquist, E., L. Valentin, U. Moilanen, M. Leisola et al. 2009. Development of a fungal pre-treatment process for reduction of organic matter in contaminated soil. Journal of Chemical Technology and Biotechnology 84:845-850.
A.M. Baetsen-Young was a graduate student at the time of this research, J.E. Kaminski is an associate professor in the Department of Plant Science, and D.D. Davis is a professor in the Department of Plant Pathology and Environmental Microbiology at Penn State University, University Park, Pa. M.T. Kasson is an assistant professor in the Division of Plant and Soil Sciences at West Virginia University, Morgantown, W.Va.