KENTUCKY BLUEGRASS BLEND ECOLOGY BY DARIN WAYNE LICKFELDT B.S., Michigan State University, 1991 M.S., Michigan State University, 1994 THESIS Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Natural Resources and Environmental Sciences in the Graduate College of the University of Illinois at Urbana-Champaign, 2001 Urbana, IllinoisUNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGNGRADUATE COLLEGEMARCH 2001dateWE HEREBY RECOMMEND THAT THE THESIS BYDARIN WAYNE LICKFELDTENTITLEDKENTUCKY BLUEGRASS BLEND ECOLOGYBE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUlREl\1ENTS FORTHE DEGREE OFDOCTOR OF PHILOSOPHY//~Director of Thesis ResearchHead of DepartmentChairperson0-517KENTUCKY BLUEGRASS BLEND ECOLOGYDarin Wayne Lickfeldt, Ph.D.Department of Natural Resources and Environmental SciencesUniversity of Illinois at Urbana-Champaign, 2001Thomas B. Voigt and Andrew M. Hamblin, AdvisorsBlending turfgrass varieties to combine desirable characteristics is a commonmanagement strategy, but determining the varietal composition of blended Kentucky bluegrass(Poa pratensis L.) stands has not been possible prior to the advent of molecular markers. Thisresearch was conducted to efficiently determine varietal composition of blended stands by DNAfingerprinting and study factors that may dictate the varietal composition of blended turf.Three DNA extraction methods were compared for cost, efficiency, and reproducibilityalong with comparisons between laboratory investigators. In a second group of experiments thecomposition of several blended stands was determined. Genetic markers were used to determinethe varietal composition on two golf courses and in replicated plots containing blends of'Blacksburg', 'Midnight', and 'Unique' Kentucky bluegrass. Spatial patterns were identified usingindices of dispersion and the number of samples required to characterize blends was estimated.Lastly, relationships between disease severity, seed characteristics and percentage of varietieswere quantified.Of the three DNA extraction methods compared, the DNAzor~ reagent method was anaffordable and efficient procedure. The PEX/CT AB method was the least expensive, butextracted the lowest amount of quality DNA. The DNeasyTMkit extracted the most high quality111DNA, but at a greater cost. Spending more time with the DNA extraction procedures led to moreDNA isloated.Independent of management, location, time of seeding, or the intended varietalcomposition, blends of 'Blacksburg', 'Midnight', and 'Unique' Kentucky bluegrass had similarcompositions. Aggressiveness or competitive ability, not environment or management, maydictate the prevalence of a variety in a blend. The varieties were distributed randomly alongtransects on the golf courses.Seed size and germination differed between varieties and seedlots within varieties.Blending seed using seed weight resulted in an actual composition similar to the intendedcomposition. Including a susceptible variety in a blend resulted in more severe rust and powderymildew than when not included. Likewise, including resistant varieties as a large percentage of ablend reduced rust and powdery mildew severity.ivTO MY FAMILY - MELISSA, JASON AND GRIFFINvACKNOWLEDGEMENTSThe author thanks the following for their contributions to this research, my educationalexperience at the University of Illinois and my professional development:The Illinois Turfgrass Foundation.The Illinois Agricultural Experiment Station.The Department of Natural Resources & Environmental Sciences.Dr. Tom Voigt for his willingness to advise me, his applicable advice and guidance.Dr. Andy Hamblin for his guidance, motivation and basic research expertise.Dr. Bruce Branham for his understanding and professional guidance.Dr. Tom Fermanian for his guidance and leadership.Dr. Torbert Rocheford for his DNA marker expertise and professional guidance.At the University of Illinois - academic professionals, friends, students, and faculty -especially Joyce Jones, John Gadient, Emily Heaton, Nicolle Hofmann, JimWilsey, Darrel Miller, Luke Cella, Brian Horgan, Joe Meyer, Shelby Henning,Sam Schmitz, Carol Preston, Susan Witt, Bette Leach, Dianne Pedersen, Dr. JackJuvik, Dr. Margaret Norton, Dr. Don Bulloch, Dr. German Bollero, Dr. HenryWilkinson, Dr. Art Spomer, Dr. David Seigler, Dr. John Swaider.From other institutions - Todd Zimmerman (Aspen Ridge G.C.), Rick Kroeger and JimHorton (Alpine Hills G.C.), Ed Lee (Summit Seed Inc.), Crystal Rose-Fricker(Pure Seed Testing), Dr. Frank Rossi (Cornell Univ.), Dr. Scott Warnke (OregonState Univ.), Dr. David Gardner (Ohio State Univ.), Dr. John Stier (UW-Madison), Dr. Genwha Jung (UW-Madison) Dr. James Baird (USGA), Dr. ClarkThrossell (GCSAA), Dr. Randy Kane, (CDGA), Ron Calhoun (Michigan StateUniv.), Eric Kohler (Purdue Univ.), Dr. S. Bruce Martin (Clemson Univ.), and Dr.James Breuninger (Dow AgroSciences LLC).VITABLE OF CONTENTSCHAPTER 1- REVIEW OF LITERATURE 1Blending Kentucky Bluegrass Varieties 1DNA Extraction and Fingerprinting 3Ecological Methods for Characterizing Plant Communities 6Research Objectives 8Literature Cited 9CHAPTER 2 -COMPARING THREE DNA EXTRACTION PROCEDURES FOR COSTEFFICIENCY, AND DNA YIELD 13Abstract 13Introduction 14Materials and Methods 17ResuIts 19Discussion 22Conclusion 25Literature Cited 26CHAPTER 3 -VARIET AL COMPOSITION AND SPATIAL PATTERNS INKENTUCKY BLUEGRASS BLENDS 29Abstract 29Introduction 30Materials and Methods 36Resul ts 42Discussi on 48ConcIusion 5 1Literature Cited 52CHAPTER 4 -VARIET AL COMPOSITION OF BLENDED KENTUCKY BLUEGRASSSTANDS AND CHARACTERISTICS OF INCLUDED VARIETIES 55Abstract 55Introduction 56Materials and Methods 59Res uIts , 63VllTABLE OF CONTENTS (continued)Discussion 71ConeIusion 74Literature Cited 75CHAPTER 5 - SUMMARY, CONCLUSIONS, AND FUTURE RESEARCH 78APPENDIX - DNA EXTRACTION ,PURIFICATION, AMPLIFICATION, ANDVISUALIZATION PROCEDURES FOR KENTUCKY BLUEGRASS 83DNeasy™Plant Mini Kit Method 83Plant DNAzol@Reagent Method 84The PEX/CT AB Buffer Method 85Quantification, Amplification and Visualization 87Literature Cited 88VIT A 89VlllTableLIST OF TABLESPageComparison of material costs per sample (US$) for three DNA extractionmethods. All costs are based on recent catalog pricing (Dec. 2000) 202 DNA concentration and extraction time for three methods and three investigatorsfollowing DNA extraction from Kentucky bluegrass. Study was a full factorialarrangement in a randomized complete block design blocked by