New Sources of Sclerotinia Stalk Rot Resistance in Cultivated Sunflower Tom Gulya (Pathologist) and Brent Hulke (Geneticist) USDA-ARS Sunflower & Plant Biology Research Unit, Fargo, ND Nikolay Balbyshev1, Zahirul Talukder1 , Laura F. Marek2, Robert Harveson3, Robert Schafer4 1 North Dakota State University, Fargo, 2USDA-NCR Plant Introduction Station/ISU, Ames, IA; 3University Nebraska Ext. Service, Scottsbluff, NE; 4Central Lakes College Ag Center, Staples, MN Figure 1. Two integral people responsible for success in this, and other sunflower/Sclerotinia projects: Dr. Nikolay Balbyshev (NDSU) on the left and Dr. Mike Boosalis (UNL-emeritus) on right. Nikolay, who has worked since 2001 on sunflower, is responsible for producing all of the mycelial and ascospore inoculum used each year by USDA sunflower researchers (in the 100’s of pounds). He will retire as of May 30, 2012. Dr. Boosalis, who was the Plant Pathology department chair at University of Nebraska, Lincoln, has been producing ascospores for Sclerotinia researchers across the country for more than 25 years since his retirement. His willingness to share his expertise and details of the ascospore production method will benefit all future Sclerotinia researchers. Project Objectives Identify new sources of resistance to Sclerotinia head rot and stalk rot in diverse cultivated sunflower germplasm, and integrate this resistance into agronomically superior lines for public release. Produce Sclerotinia inoculum (mycelium grown on millet for stalk rot, and ascospores for head rot) for this and three other sunflower projects, and handle all field evaluations and ratings. With colleagues in multiple states, document the established protocol of Dr. Boosalis for ascospore production, and compare fungal isolates and variations in methodology, with the end goal a publication in a refereed journal crediting Dr. Boosalis. 2011 Progress In 2010 a severe insect infestation caused the loss of a majority of our head rot testing, so in 2011 we relocated our head rot testing effort to another site (Staples, MN) which was also 120 miles from our lab in Fargo, ND. The Staples site and a companion one in Sabin, MN both produced good head rot data on breeding material for this and other unit projects. We augmented a previous effort to phenotype 260 USDA Plant Introductions for stalk rot resistance by evaluating the same germplasm for head rot resistance (2 sites) and Phomopsis stem canker (4 sites). All of this data will be used by a companion project dealing with Association Mapping. 2011 saw the first release of germplasm developed by Dr. Hulke and four oilseed R-lines with superior head rot resistance, combined with herbicide resistance or high oleic oil content and high yield were made publically available. A team of pathologists including Michael Wunsch (Carrington, ND), Scott Halley (Langdon, ND), Khalid Rashid (Morden, MB, Canada), Bob Harveson (Scottsbluff, NE), Nikolay Balbyshev and Tom Gulya (Fargo, ND) and the technical staff of Jim Steadman (Lincoln, NE) visited with Dr. Mike Boosalis in 2009 and again in 2010 to learn the details of the “Boosalis method” of efficiently producing ascospores in large quantity. A coordinated study is underway this winter to document this procedure and investigate its application to other fungal isolates and in multiple laboratories. 2012 Plans Breeding efforts by Dr. Hulke will continue, with emphasis on combining both head rot and stalk rot resistance into both oilseed and confection germplasm, and to pyramid other disease resistance (rust, downy mildew, Phomopsis) on top of Sclerotinia resistance. We will generate additional head rot data on the 260 Plant Introductions, at two sites, to produce reliable phenotype data for Association Mapping. Dr. Balbyshev will retire in May, 2012, and we hope to have his replacement hired by early fall. We also intend to have the “Boosalis ascospore” study completed and a manuscript submitted by this fall. A B C D E F H G J I Figure 2. Head rot ratings of testcross hybrids, using four USDA oilseed R-line inbreds released in 2011. RHA 472 is the highest yielding, RHA 473 has high oleic content, and RHA 474 and 475 have IMI herbicide resistance. Data is the average from six inoculated trials from 2007 to 2010. Resistant hybrid checks are Croplan 305 and Croplan 343. Figure 3. Histogram showing the distribution of Sclerotinia head rot ratings of 226 USDA Plant Introductions and 34 USDA inbred lines from two inoculated trials in 2011 (Sabin and Staples, MN) Figure 4. Steps involved in Sclerotinia ascospore production. A: Dr. Boosalis preparing sand/cornmeal/vermiculite/glucose media. B: Preparing the autoclaved media for inoculation. C: Tray of colonized media after 30 days growth. D: Rinsing sclerotia to remove media. E: Sclerotia after cleaning, with penny for size reference. F: Sclerotia are gently rubbed against sieve to scarify the rind, leaving tiny areas of pink medulla tissue visible. G: Scarified sclerotia are placed into Petri dishes with sterile sand, moistened and incubated, without any vernalization period. H: Plates showing abundant and uniform production of apothecia. I: Lid of Petri dish with aluminum foil disk covered with discharged ascospores. J: Foil disks of ascospores are sandwiched in between filter paper disks and stored in a dessicator at 4C until needed. Figure 5. Part of the USDA and NDSU technical staff whose season-long work made this research possible. From left, Marjorie Olson, Nikolay Balbyshev, Leonard Cook, Megan Ramsett, Lisa Brown and students Ridhima Katyal and Hannah Worral. Technicians not pictured: Angelia Hogness and Theresa Gross. Acknowledgements The authors thank all USDA Sunflower Unit technicians for assistance in this project, plus the many undergraduate students who help in greenhouse and field experiments. We also acknowledge the help of the NDSU Carrington Research and Extension Center, Central Lakes Community College Ag Center (Staples, MN), Bayer Crop Science (Sabin, MN), CHS Sunflowers (Grandin, ND), and Croplan Genetics (Crookston, MN).