Development ofCreeping Bentgrass withMultiple Pest ResistanceUniversity scientists adopt a team approachto improve this important turfgrass species.BY MICHAEL CASLER, G. JUNG, S. BUGHRARA,A. HAMBLIN, C. WilliAMSON, AND T. VOIGT

Creeping bentgrass (Agrostisstolonifera) is the premier grassfor golf course putting greens

and is one of the most desirable grassesfor fairways and tees for much of theUSA. Recent breeding advancesdemonstrate that genetic variationexists within creeping bentgrass for arange of pest resistances and stresstolerances. Many of these traits allowbentgrass to be grown in environmentsand under conditions that wereimpossible just a few years ago.

For many golf courses, maintenanceof a high-quality turf requires frequent,varied, and intensive pesticide applica-tions. Pesticide costs can consume up to10% or more of the total budget for ahighly managed golf course. Intensivemanagement, including frequent andlow mowing, irrigation, and heavy play,serves to enhance and/or spread thedevelopment of pest problems, particu-larly fungal diseases. In addition to theirexpense, pesticides represent a potentialhealth and environmental hazard, bothto golfers and to the surroundingenvironment, and they have limitedefficacy3,8and lead to fungal resistance.2

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Genetic resistance to disease pests is awidespread phenomenon in agriculturaland horticultural plants. Disease resist-ance has been used to protect economi-cally important plants for more than 90years. There are many examples of

genetic resistance that has been durablefor more than 30 years without anyneed for fungicidal protection orincrease in disease incidence.

While there has been much researchon genetics and breeding of creepingbentgrass for individual pest resistances,there has not been a concerted effort todevelop multiple-pest-resistant germ-plasm. Plants with multiple pest resist-ance will be required to have a signifi-cant impact on pesticide use. There isstrong evidence for genetic resistanceto snow mold and dollar spot in someclones of creeping bentgrass.1

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There are currently several bentgrassbreeding programs scattered aroundthe USA, including programs in NewJersey, Pennsylvania, Texas, RhodeIsland, Michigan, Illinois, Wisconsin,and Oregon. Many of these programsoperate somewhat independently ofeach other. While there is some collabo-ration among public and private pro-grams, particularly in the seed produc-tion and commercialization of publiclydeveloped cultivars, both public andprivate programs compete in thedevelopment of cultivars to support theindustry. As such, individual programshave difficulty in identifying anddeveloping germplasm with multiplepest resistances. Each program hasexpertise, local knowledge, and environ-mental conditions to support identifi-

cation of resistances/tolerances to asmall number of pest problems. Onlywith collaboration among several diverselocations/programs can we hope toidentify germplasm with the multiplepest/ stress resistances that will be neces-sary to meet the challenges limitingadaptation of creeping bentgrass.

PROJECTDESIGNThe objective of this project was todevelop elite clones of creeping bent-grass with multiple pest resistances andstress tolerances that can be delivered tothe seed industry for use in synthesizingnew creeping bentgrass varieties thatare broadly adapted to a range of eco-logical and environmental conditions,including reduced pesticide application.The findings reported here are the re-sult of the first three years of the Bent-grass Breeding Consortium betweenthe USDA-ARS, the University ofWisconsin, the University of Illinois,and Michigan State University, sup-ported in part by the USGA.

Three populations of creepingbentgrass clones were developed for thisstudy. The Wisconsin population con-sists of a cross between two clones thatdiffered in resistance to speckled andgray snow mold pathogens. This cross,consisting of 200 clones, also is beingutilized in genetic linkage mapping4

and disease-resistance mapping.6 The

JANUARY-FEBRUARY 2007 IS

The so-called "chamber of death" resulted in damage to creeping bentgrass clones after inoculationwith Pythium disease inAugust 2005.

Michigan population consists of 200clones collected from old golf coursesin Michigan, largely for high turfquality and large patch size. The Illinoispopulation consists of 200 clones thatrepresent two generations of randommating of a population of clones col-lected from old golf courses in Illinois.

Each plant was vegetatively propa-gated in the greenhouse, and clonalmaterial was exchanged among thethree locations in February 2003.Clones were evaluated for disease andinsect reactions as described below.Unless described otherwise, all ratingswere made on a scale of 0 to 9, whereo = plant completely brown fromdisease and 9 = no sYmptoms (plantswith high values were most resistantand desirable). The rating scales wereapproximately linear with respect tothe percentage of diseased tissue, sothat a mean rating of 4.5 wouldrepresent approximately 50% diseasedleaf tissue.

UNIVERSITY OF WISCONSINAND USDA-ARSSpeckled Snow Mold(qphula ishikariensis)The 600 clones were transplanted tothe practice fairway at Gateway GolfCourse, Land 0' Lakes, Wis., in July2003. Natural infection by T ishikariensiswas sufficiently severe and uniform toallow ratings to be made in spring2004. Ratings were made in mid-Aprilimmediately after snow melt and againin early May, following recovery. InOctober 2004, plots were inoculatedwith a mixture of isolates representingall three biological varieties of thisfungus - all known to be particularlyvirulent on creeping bentgrass. Plotswere rated again in early spring 2005,shortly after snow melt.

Pythium Blight (Pythium spp.)The 600 clones were transplanted intoa perennial rye grass fairway at the 0.].N oer Turfgrass Research and EducationFacility new Verona, Wis., in June 2003.The fairway was covered with a metal-

16 GREEN SECTION RECORD

framed hoop house with the plasticcovering normally removed. Plots wereinoculated with a mixture of isolates ofPythium spp. in early August 2004, andthey were covered with plastic to in-crease the temperature and humidity ofthe local environment. Plants were ratedfollowing two weeks of exposure to thepathogen under these conditions.

Pink Snow Mold(Microdochium nivale)The 600 clones were transplanted intoanother perennial ryegrass fairway at0.]. Noer in August 2003. In October2004, plots were inoculated with anisolate of this fungus that had previouslyshown virulence against creeping bent-grass. Due to lack of snow cover andmild winter conditions, there were nopink snow mold symptoms in spring2005 and 2006.

Dollar Spot (Sclerotinia homoeocarpa)In August 2004, the speckled snowmold experiment at Land 0' Lakes,Wis., was inoculated with an isolate ofthe dollar spot pathogen that has beenshown to be higWy virulent againstcreeping bentgrass.5 Ratings were made

approximately four weeks afterinoculation.

Black Cutworm (Agrostis ipsilon)Two replicates of the pink snow moldexperiment were inoculated withsecond-instar black cutworm larvae. Inthe middle of each bentgrass plant, aPVC pipe, 10 cm in diameter and 10cm long, was driven approximately 1cm into the ground. Five larvae wereplaced in the pipe, and the pipe wascovered with nylon mesh to preventbirds from eating the larvae. After 10days, pipes were removed and the num-ber of surviving larvae was counted.The plots were mowed and two dayslater damage was scored on a 0 to 9scale, where 0 = no feeding damageand 9 = plant completely brown (noregrowth). One replicate was inoculatedand scored in mid-August and onereplicate was inoculated and scored inmid-September.

UNIVERSITY OF ILLINOISDollar Spot (Sclerotinia homoeocarpa)The 600 clones were transplantedinto a perennial ryegrass fairway atChampaign, Ill., in June 2003. The

experimental design and plantingarrangement were as described abovefor each of the University of Wisconsinfield trials. Plots were mowed at Yz inch,allowing the clones to grow laterally forthe 2004 growing season. Plots wereinoculated with the dollar spot patho-gen in early June, and ratings weremade in late June and mid-July.

MICHIGAN STATE UNIVERSITYGray Snow Mold (1Yphula incarnata)The 600 clones were transplanted intoa perennial ryegrass fairway at EastLansing, Mich., in June 2003. Each plantwas rated for reaction to gray snowmold in April 2004 and 2005, based oninfections from natural inoculum.

Dollar Spot (Sclerotinia homoeocarpa)Three isolates of the dollar spot patho-gen were mixed and used to inoculatethe entire trial at East Lansing in earlyJuly 2004. Plots were rated for dollarspot reaction two weeks later using thesame rating scale as for snow mold.Recovery from dollar spot infection wasrated five weeks after inoculation. Thisdisease was rated again in 2005.

that there are environmental and/orcultural factors that obscure geneticdifferences among clones.

Variation among clones withinpopulations was significant for allmeasures of snow molds and dollarspot reaction. There was a large rangeamong clones for all variables, and LSDvalues were all small relative to therange among clone means. Repeat-ability was moderate to high for alldollar spot and snow mold ratings.These results demonstrated that thereare large and consistent differencesamong clones for both dollar spot andsnow mold reaction.

Gray and Speckled Snow Moldand Dollar SpotThe three populations of creepingbentgrass clones differed for mostmeasures of snow mold and. dollar spotreaction. For snow mold, the Wisconsinpopulation had the highest ratings forr ishikariensis, while the Michiganpopulation had the highest ratings forr incarnata. Similarly for dollar spot, theWisconsin population had nearly the

highest mean rating in Wisconsin, theMichigan population generally hadmean ratings in Michigan, and theIllinois population had the highestmean ratings in Illinois.

Most snow mold ratings wereuncorrelated with each other. The onlyexceptions were ratings of snow moldreaction and recovery that were takenwithin a few weeks of each other ineither Wisconsin or Michigan. Despitethese results, the moderate repeatabilityof the average snow mold reaction,across all ratings, indicated the presenceof some clones with fairly consistentresults across all ratings. This is remark-able, particularly given that most snowmold symptoms at the East Lansing,Mich., and Land 0' Lakes, Wis., loca-tions were caused by two differentsnow mold pathogens.

Dollar spot ratings were considerablymore consistent across ratings made atdifferent locations or years. This wasprobably due to the use of a constantsource of inoculum at all locations andthe fact that dollar spot is caused byonly one organism.

RESULTSPink Snow Mold, Pythium Blight,and Black CutwormInoculations with pink snow mold,Pythium blight, and black cutwormfailed to provide meaningful differencesamong the creeping bentgrass clones. Inthe case of Pythium blight, the diseasepressure was so severe and uniform thatall plants were heavily or completelydamaged. All creeping bentgrass clonesin the study were highly susceptibleunder the extreme conditions of thisinoculation. For pink snow mold, therelatively few minor symptoms weredue to mild winters with little signifi-cant snow cover. For black cutworm,the lack of variation among creepingbentgrass clones could only be attributedto extreme variation in the inoculationtechnique and/or the measurement ofsymptoms. Apparent differences amongclones were not repeatable, indicating

InAugust 2005, four creeping bentgrass clones planted in a research trial on a ryegrass fairway atGateway Golf Club (Land 0' Lakes,Wis.) show differences in genetic resistance to dollar spot.

JANUARY-FEBRUARY 2007 17

This excellent-quality creeping bentgrass plant was selected for resistance to snow mold and dollarspot at Verona, Wis., in August 2005.

VALUE AND FUTURE USEOF DISEASE-RESISTANTCLONESThese results suggest that there may besome race specificity for host resistanceto these two diseases. Both results aresurprising, because studies of hostgenotypes and pathogen isolates haveshown, in both cases, a general lack ofhost genotype x pathogen isolate inter-action.5,lo These results may be an indi-cation of more long-term evolution ofrace-specific disease resistance on golfcourses, a phenomenon that may nothave been detected from evaluation ofcollections within a limited region.Particularly for snow molds, plantsresistant to snow mold from one golfcourse may not be resistant to snowmolds from all other courses. Theseresults underscore the importance ofcollaboration between researchers atdifferent locations, allowing evaluationof each disease across a wide range of

environmental conditions and potentialpathogen isolates.

Based on these results, 20 clones withthe highest disease indices and superiorturf quality were selected for potentialrelease to private companies for use inbreeding new varieties of creepingbentgrass. These clones are also beingcrossed with additional clones withsuperior dollar spot, snow mold, andbrown patch resistance to generate anew set of genetic materials for evalu-ation and selection.

LITERATURE CITED1. Bonos, S. A., M. D. Casler, and W. A. Meyer.2003. Inheritance of dollar spot resistance increeping bentgrass (Agrostis palustris Huds.).Crop Sa. 43:2189-2196.

2. Burpee, L. L. 1997. Control of dollar spot ofcreeping bentgrass caused by an isolate ofSclerotinia homoeocarpa resistant to benzomidazoleand demethylation inhibitor fungicides. PlantDis.81:1259-1263.

3. Burpee, L. L.,A. E. Mueller, and D.].Hannusch. 1990. Control of ryphula blight and

pink snow mold of creeping bentgrass andresidual suppression of dollar spot by triadi-mefon and propiconazole. Plant Dis. 74:687-689.

4. Chakraborty, N.,]. Bae, S. Warnke, T. Chang,and G.Jung. 2005. Linkage map constructionin allotetraploid creeping bentgrass (Agrostisstolonifera L.). Theor. Appl. Genet. 111:795-803.

5. Chakraborty, N., T. Chang, M. D. Casler, andG. Jung. 2006. Response of bentgrass cultivars toSclerotinia homoeocarpa isolates representing 10vegetative compatibility groups. Crop Sa.46(3):1237-1244.

6. Chakraborty, N.,]. Curley, S. Warnke, M. D.Casler, and G.Jung. 2006. Mapping QTL fordollar spot resistance in creeping bentgrass(Agrostis stolonifera L.). Theor.Appl. Genet.(in press).

7. Golembiewski, R. c., J. M.Vargas,Jr.,A. L.Jones, and A. R. Detweiler. 1995. Detectionof demethylation inhibitor (DMI) resistance inSclerotinia homoeocarpa populations. Plant Dis.79:491-493.

8. Hsiang, T., N. Matsumoto, and S. M. Millett.1999. Biology and management of ryphula snowmolds of turfgrass. Plant Dis. 83:788-798.

9. Vincelli, P.,]. C. Doney, and A.]. Powell. 1997.Variation among creeping bentgrass cultivars inrecovery from epidemics of dollar spot. PlantDis. 81 :99-102.

10. Wang, Z., M. D. Casler,]. C. Stier,]. G.Gregos, D. P. Maxwell, and S. M. Millett.2005. Genotypic variation for snow moldreaction among creeping bentgrass clones.Crop Sa. 45:399-406.

Editor's Note: A more completeversion of this research can befound at USGA Turfgrass andEnvironmental Research Online:http://usgatero.msu.edu/vOS/n18.

tllif

MICHAELCASLER,PH.D., ResearchGeneticist, USDA-ARS, Madison,Wis.; GEUNHWA]UNG, PH.D.,Assistant Prcifesso~University ifMassachusetts,Amherst, Mass.;SULEIMANBUGHRARA,PH.D.,Assistant Prcifesso~Michigan StateUniversity, East Lansing, Mich.;ANDREW HAMBLIN,PH.D., Us.Army CERL, Champaign, nz.;CHRIS WILLIAMSON,PH.D.,Assistant Prcifessor,University ifWisconsin, Madison, Wis.; TOM

VOIGT,PH.D., Associate Prcifesso~University ifnlinois, Urbana, nz.

18 GREEN SECTION RECORD

A Q&A with DR. MICHAEL CASLER, University ofWisconsin, regarding development of creepingbentgrass with multiple pest resistance.

Q: How common is it for universities located indifferent states to be cooperatively funded forlong-term projects such as the BentgrassBreeding Consortium? Do you think this is atrend that will increase as funding becomesmore competitive?

A: Cooperative funding for long-term projects amonguniversities is very rare. Short-term funding for cooperativeprojects is more common, usuallyfor two or three years.I don't see this trend changing,as most opportunities forlong-term funding have disappeared. In fact, as universitiesbecome more competitive, I see this trend continuing.Most of these decisions are made on a case-by-casebasis - ifthe scientists make an excellent proposal, theycan be funded, whether at one or more universities.

Q: Do you think grant administrators in mostuniversities are open to interstate teamworkapproaches such as yours, or do you feel that theywould be reluctant to readily adopt this approachbased on current grant funding and reportingprocedures?

A: As long as the funding comes from outside theuniversity, I don't think they care one way or the other.Administrators are interested in professors bringing fundsinto the university and in accomplishing research results.Ifa collaborative project accomplishes these two things,then it's not viewed any differently.The main impedimentis intellectual property, such as improved germplasm -who owns it and who benefits from its development. Inour case, this is all shared equally,but it required manymonths to work out wording that all three universitiescould accept.

Q: Spreading research dollars over a broad geo-graphic area for a project such as the BentgrassBreeding Consortium seems to make a lot ofsense in that germplasm for multiple resistancescan be identified and tested. Are there not-so-obvious drawbacks to this approach, as well?

A: The only drawback we observed was the distancesamong the collaborators and the difficulty in gettingtogether to discuss the research. Most of these can beovercome by email and professional meetings.

Q: In your research, you focus on germplasmimprovement for snow mold and dollar spotresistance in creeping bentgrass. What otherresistance( s) do you feel would be priorities forcreeping bentgrass, and would this requireexpanding the number of participating univer-sities in the Bentgrass Breeding Consortium(i.e., a more southern location for bentgrassheat tolerance)?

A: Ideally,we would like to include several other loca-tions, such as Rutgers,Texas A&M,and perhaps someothers. This would allow us to cover a wider rangeof stresses and pests. Complicating this would be twothings - introducing more traits to evaluate drasticallyreduces the probability of success, and adding morecollaborators increases the political and administrativeproblems in creating the consortium and negotiating itsrules and boundaries. In the long term, our vision is anationwide bentgrass breeding consortium, but we feelthat it's important to build slowly,rather than try to geteveryone on board at once. Funding and organizationallimitations are the main reasons for this.

Q:You stated that your objective for theBentgrass Breeding Consortium is to developmultiple-resistance germplasm in creepingbentgrass. How much feedback have you gottenfrom seed producers for your germ plasm thatthey could incorporate into new bentgrasscultivars?

A: There is quite a bit of interest among the privatebentgrass breeders. However, because the clones havenot yet been released to the breeders, they have notbeen able to provide any feedback at this time. They willhave their first access to the clones in spring 2007.

Q:When more resistant germplasm is identified,how soon can superintendents expect it to showup in improved bentgrass cultivars?

A: This process will probably require a number ofyears to evaluate the clones, incorporate them into newvarieties, test the varieties, and conduct seed increases ofthe new varieties.This process may require a minimum ofsix to eight years before the first materials reachcommercialization.

JEFF Nus, PH.D., manager, Green Seaion Research.

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